multiple cell populations: Topics by Science.gov

Sample records for multiple cell populations

Stationary Size Distributions of Growing Cells with Binary and Multiple Cell Division

NASA Astrophysics Data System (ADS)

Rading, M. M.; Engel, T. A.; Lipowsky, R.; Valleriani, A.

2011-10-01

Populations of unicellular organisms that grow under constant environmental conditions are considered theoretically. The size distribution of these cells is calculated analytically, both for the usual process of binary division, in which one mother cell produces always two daughter cells, and for the more complex process of multiple division, in which one mother cell can produce 2 n daughter cells with n=1,2,3,… . The latter mode of division is inspired by the unicellular algae Chlamydomonas reinhardtii. The uniform response of the whole population to different environmental conditions is encoded in the individual rates of growth and division of the cells. The analytical treatment of the problem is based on size-dependent rules for cell growth and stochastic transition processes for cell division. The comparison between binary and multiple division shows that these different division processes lead to qualitatively different results for the size distribution and the population growth rates.
A compound chimeric antigen receptor strategy for targeting multiple myeloma.

PubMed

Chen, K H; Wada, M; Pinz, K G; Liu, H; Shuai, X; Chen, X; Yan, L E; Petrov, J C; Salman, H; Senzel, L; Leung, E L H; Jiang, X; Ma, Y

2018-02-01

Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.
Related B cell clones populate the meninges and parenchyma of patients with multiple sclerosis

PubMed Central

Lovato, Laura; Willis, Simon N.; Rodig, Scott J.; Caron, Tyler; Almendinger, Stefany E.; Howell, Owain W.; Reynolds, Richard; Hafler, David A.

2011-01-01

In the central nervous system of patients with multiple sclerosis, B cell aggregates populate the meninges, raising the central question as to whether these structures relate to the B cell infiltrates found in parenchymal lesions or instead, represent a separate central nervous system immune compartment. We characterized the repertoires derived from meningeal B cell aggregates and the corresponding parenchymal infiltrates from brain tissue derived primarily from patients with progressive multiple sclerosis. The majority of expanded antigen-experienced B cell clones derived from meningeal aggregates were also present in the parenchyma. We extended this investigation to include 20 grey matter specimens containing meninges, 26 inflammatory plaques, 19 areas of normal appearing white matter and cerebral spinal fluid. Analysis of 1833 B cell receptor heavy chain variable region sequences demonstrated that antigen-experienced clones were consistently shared among these distinct compartments. This study establishes a relationship between extraparenchymal lymphoid tissue and parenchymal infiltrates and defines the arrangement of B cell clones that populate the central nervous system of patients with multiple sclerosis. PMID:21216828
Related B cell clones populate the meninges and parenchyma of patients with multiple sclerosis.

PubMed

Lovato, Laura; Willis, Simon N; Rodig, Scott J; Caron, Tyler; Almendinger, Stefany E; Howell, Owain W; Reynolds, Richard; O'Connor, Kevin C; Hafler, David A

2011-02-01

In the central nervous system of patients with multiple sclerosis, B cell aggregates populate the meninges, raising the central question as to whether these structures relate to the B cell infiltrates found in parenchymal lesions or instead, represent a separate central nervous system immune compartment. We characterized the repertoires derived from meningeal B cell aggregates and the corresponding parenchymal infiltrates from brain tissue derived primarily from patients with progressive multiple sclerosis. The majority of expanded antigen-experienced B cell clones derived from meningeal aggregates were also present in the parenchyma. We extended this investigation to include 20 grey matter specimens containing meninges, 26 inflammatory plaques, 19 areas of normal appearing white matter and cerebral spinal fluid. Analysis of 1833 B cell receptor heavy chain variable region sequences demonstrated that antigen-experienced clones were consistently shared among these distinct compartments. This study establishes a relationship between extraparenchymal lymphoid tissue and parenchymal infiltrates and defines the arrangement of B cell clones that populate the central nervous system of patients with multiple sclerosis.
Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets

PubMed Central

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-01-01

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms. PMID:24903657
Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets.

PubMed

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-06-06

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms.
SYNTHETIC BIOLOGY. Emergent genetic oscillations in a synthetic microbial consortium.

PubMed

Chen, Ye; Kim, Jae Kyoung; Hirning, Andrew J; Josić, Krešimir; Bennett, Matthew R

2015-08-28

A challenge of synthetic biology is the creation of cooperative microbial systems that exhibit population-level behaviors. Such systems use cellular signaling mechanisms to regulate gene expression across multiple cell types. We describe the construction of a synthetic microbial consortium consisting of two distinct cell types—an "activator" strain and a "repressor" strain. These strains produced two orthogonal cell-signaling molecules that regulate gene expression within a synthetic circuit spanning both strains. The two strains generated emergent, population-level oscillations only when cultured together. Certain network topologies of the two-strain circuit were better at maintaining robust oscillations than others. The ability to program population-level dynamics through the genetic engineering of multiple cooperative strains points the way toward engineering complex synthetic tissues and organs with multiple cell types. Copyright © 2015, American Association for the Advancement of Science.
A multitask clustering approach for single-cell RNA-seq analysis in Recessive Dystrophic Epidermolysis Bullosa

PubMed Central

Petegrosso, Raphael; Tolar, Jakub

2018-01-01

Single-cell RNA sequencing (scRNA-seq) has been widely applied to discover new cell types by detecting sub-populations in a heterogeneous group of cells. Since scRNA-seq experiments have lower read coverage/tag counts and introduce more technical biases compared to bulk RNA-seq experiments, the limited number of sampled cells combined with the experimental biases and other dataset specific variations presents a challenge to cross-dataset analysis and discovery of relevant biological variations across multiple cell populations. In this paper, we introduce a method of variance-driven multitask clustering of single-cell RNA-seq data (scVDMC) that utilizes multiple single-cell populations from biological replicates or different samples. scVDMC clusters single cells in multiple scRNA-seq experiments of similar cell types and markers but varying expression patterns such that the scRNA-seq data are better integrated than typical pooled analyses which only increase the sample size. By controlling the variance among the cell clusters within each dataset and across all the datasets, scVDMC detects cell sub-populations in each individual experiment with shared cell-type markers but varying cluster centers among all the experiments. Applied to two real scRNA-seq datasets with several replicates and one large-scale droplet-based dataset on three patient samples, scVDMC more accurately detected cell populations and known cell markers than pooled clustering and other recently proposed scRNA-seq clustering methods. In the case study applied to in-house Recessive Dystrophic Epidermolysis Bullosa (RDEB) scRNA-seq data, scVDMC revealed several new cell types and unknown markers validated by flow cytometry. MATLAB/Octave code available at https://github.com/kuanglab/scVDMC. PMID:29630593
High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs.

PubMed

Endo, Kei; Hayashi, Karin; Saito, Hirohide

2016-02-23

The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations.
Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries.

PubMed

Scanlon, Thomas C; Gray, Elizabeth C; Griswold, Karl E

2009-11-20

In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. The quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid.
T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention.

PubMed

van Langelaar, Jamie; van der Vuurst de Vries, Roos M; Janssen, Malou; Wierenga-Wolf, Annet F; Spilt, Isis M; Siepman, Theodora A; Dankers, Wendy; Verjans, Georges M G M; de Vries, Helga E; Lubberts, Erik; Hintzen, Rogier Q; van Luijn, Marvin M

2018-05-01

Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6+CXCR3+), and not Th17 (CCR6+CXCR3-) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6+ and CCR6-CD8+ T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6+CXCR3+CCR4-) expressed the highest very late antigen-4 levels and selectively accumulated in natalizumab-treated patients who remained free of clinical relapses. This was not found in patients who experienced relapses during natalizumab treatment. The enhanced potential of Th17.1 cells to infiltrate the central nervous system was supported by their predominance in cerebrospinal fluid of early multiple sclerosis patients and their preferential transmigration across human brain endothelial layers. These findings reveal a dominant contribution of Th1-like Th17 subpopulations, in particular Th17.1 cells, to clinical disease activity and provide a strong rationale for more specific and earlier use of T cell-targeted therapy in multiple sclerosis.
The simultaneous isolation of multiple high and low frequent T-cell populations from donor peripheral blood mononuclear cells using the major histocompatibility complex I-Streptamer isolation technology.

PubMed

Roex, Marthe C J; Hageman, Lois; Heemskerk, Matthias T; Veld, Sabrina A J; van Liempt, Ellis; Kester, Michel G D; Germeroth, Lothar; Stemberger, Christian; Falkenburg, J H Frederik; Jedema, Inge

2018-04-01

Adoptive transfer of donor-derived T cells can be applied to improve immune reconstitution in immune-compromised patients after allogeneic stem cell transplantation. The separation of beneficial T cells from potentially harmful T cells can be achieved by using the major histocompatibility complex (MHC) I-Streptamer isolation technology, which has proven its feasibility for the fast and pure isolation of T-cell populations with a single specificity. We have analyzed the feasibility of the simultaneous isolation of multiple antigen-specific T-cell populations in one procedure by combining different MHC I-Streptamers. First, the effect of combining different amounts of MHC I-Streptamers used in the isolation procedure on the isolation efficacy of target antigen-specific T cells and on the number of off-target co-isolated contaminating cells was assessed. The feasibility of this approach was demonstrated in large-scale validation procedures targeting both high and low frequent T-cell populations using the Good Manufacturing Practice (GMP)-compliant CliniMACS Plus device. T-cell products targeting up to 24 different T-cell populations could be isolated in one, simultaneous MHC I-Streptamer procedure, by adjusting the amount of MHC I- Streptamers per target antigen-specific T-cell population. Concurrently, the co-isolation of potentially harmful contaminating T cells remained below our safety limit. This technology allows the reproducible isolation of high and low frequent T-cell populations. However, the expected therapeutic relevance of direct clinical application without in vitro expansion of these low frequent T-cell populations is questionable. This study provides a feasible, fast and safe method for the generation of highly personalized MHC I-Streptamer isolated T-cell products for adoptive immunotherapy. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
Spatial and Temporal Coordination of Bone Marrow-Derived Cell Activity During Arteriogenesis: Regulation of the Endogenous Response and Therapeutic Implications

PubMed Central

Meisner, Joshua K.; Price, Richard J.

2010-01-01

Arterial occlusive disease (AOD) is the leading cause of morbidity and mortality through the developed world, which creates a significant need for effective therapies to halt disease progression. Despite success of animal and small-scale human therapeutic arteriogenesis studies, this promising concept for treating AOD has yielded largely disappointing results in large-scale clinical trials. One reason for this lack of successful translation is that endogenous arteriogenesis is highly dependent on a poorly understood sequence of events and interactions between bone marrow derived cells (BMCs) and vascular cells, which makes designing effective therapies difficult. We contend that the process follows a complex, ordered sequence of events with multiple, specific BMC populations recruited at specific times and locations. Here we present the evidence suggesting roles for multiple BMC populations from neutrophils and mast cells to progenitor cells and propose how and where these cell populations fit within the sequence of events during arteriogenesis. Disruptions in these various BMC populations can impair the arteriogenesis process in patterns that characterize specific patient populations. We propose that an improved understanding of how arteriogenesis functions as a system can reveal individual BMC populations and functions that can be targeted for overcoming particular impairments in collateral vessel development. PMID:21044213
Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo

PubMed Central

2014-01-01

Background Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Tracking the contributions of individual cells to large populations, however, has been constrained by limitations in sensitivity and complexity. Results We utilize cellular barcoding methods to simultaneously track the clonal contributions of tens of thousands of cells. We demonstrate that even with optimal culturing conditions, common cell lines including HeLa, K562 and HEK-293 T exhibit ongoing clonal dynamics. Starting a population with a single clone diminishes but does not eradicate this phenomenon. Next, we compare lentiviral and zinc-finger nuclease barcode insertion approaches, finding that the zinc-finger nuclease protocol surprisingly results in reduced clonal diversity. We also document the expected reduction in clonal complexity when cells are challenged with genotoxic stress. Finally, we demonstrate that xenografts maintain clonal diversity to a greater extent than in vitro culturing of the human non-small-cell lung cancer cell line HCC827. Conclusions We demonstrate the feasibility of tracking and quantifying the clonal dynamics of entire cell populations within multiple cultured cell lines. Our results suggest that cell heterogeneity should be considered in the design and interpretation of in vitro culture experiments. Aside from clonal cell lines, we propose that cellular barcoding could prove valuable in modeling the clonal behavior of heterogeneous cell populations over time, including tumor populations treated with chemotherapeutic agents. PMID:24886633
Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

PubMed Central

Wu, Jincheng; Tzanakakis, Emmanuel S.

2014-01-01

Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899
Impact of cladribine therapy on changes in circulating dendritic cell subsets, T cells and B cells in patients with multiple sclerosis.

PubMed

Mitosek-Szewczyk, Krystyna; Tabarkiewicz, Jacek; Wilczynska, Barbara; Lobejko, Katarzyna; Berbecki, Jerzy; Nastaj, Marcin; Dworzanska, Ewa; Kolodziejczyk, Beata; Stelmasiak, Zbigniew; Rolinski, Jacek

2013-09-15

Cladribine causes sustained reduction in peripheral T and B cell populations while sparing other immune cells. We determined two populations of dendritic cells (DCs): namely CD1c(+)/CD19(-) (myeloid DCs) and CD303(+)/CD123(+) (plasmacytoid DCs), CD19(+) B lymphocytes, CD3(+) T lymphocytes and CD4(+) or CD8(+) subpopulations in patients with multiple sclerosis after cladribine therapy. We examined 50 patients with secondary progressive multiple sclerosis (SP MS) according to McDonalds et al.'s criteria, 2001 [15]. Blood samples were collected before the initiation of cladribine therapy and after 1st, 2nd, 3th, 4th and 5th courses of treatment. DC subsets, T and B cells were analyzed by flow cytometry. During cladribine treatment the myeloid DCs CD1c(+)/CD19(-) did not change (p=0.73175), and the plasmacytoid DCs CD303(+)/CD123(+) significantly increased (p=0.00034) which resulted in significant changes in the ratio of myeloid DCs to plasmacytoid DCs (p=0.00273). During therapy, B lymphocyte CD19(+) significantly decreased (p=0.00005) and significant changes in CD4(+) cells (p=0.00191), changes in CD8(+) cells (p=0.05760) and significant changes in CD3(+) (p=0.01822) were found. We noticed significant trend to increase the CD303(+) circulating the dendritic cells. This population produces large amounts of IFN-alfa. We found significant and rapid decrease in B cells and CD4(+) Th cells. Our results suggest two possible ways of beneficial cladribine influence on immune system in MS. Induction of IFN-alfa producing cells and their predominance over BDCA-1(+) DCs, which are associated with cytotoxic response. Additionally, cladribine could influence two populations of lymphocytes: B cells and Th lymphocytes responsible for induction of immune response against myelin antigens. Copyright © 2013 Elsevier B.V. All rights reserved.
Flow cytometric discrimination of seven lineage markers by using two fluorochromes

PubMed Central

Boin, Francesco; Giardino Torchia, Maria Letizia; Borrello, Ivan; Noonan, Kimberly A.; Neil, Matthew; Soloski, Mark J.

2017-01-01

Flow cytometry is the primary immunological technique used to analyze multiple parameters on complex cell populations. We present a staining method that identifies major human mononuclear lymphoid and myeloid populations (CD4+ and CD8+ T cells, γδ T cells, B cells, NK cells and monocytes), using only two fluorochromes and a minimal number of cells. Our approach increases the number of markers recordable on most flow cytometers allowing for a deeper and more comprehensive immunophenotyping. PMID:29190813
Identification of drug-resistant subpopulations in canine hemangiosarcoma

PubMed Central

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

2017-01-01

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
Multiple bidirectional alterations of phenotype and changes in proliferative potential during the in vitro and in vivo passage of clonal mast cell populations derived from mouse peritoneal mast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kanakura, Y.; Thompson, H.; Nakano, T.

1988-09-01

Mouse peritoneal mast cells (PMC) express a connective tissue-type mast cell (CTMC) phenotype, including reactivity with the heparin-binding fluorescent dye berberine sulfate and incorporation of (35S) sulfate predominantly into heparin proteoglycans. When PMC purified to greater than 99% purity were cultured in methylcellulose with IL-3 and IL-4, approximately 25% of the PMC formed colonies, all of which contained both berberine sulfate-positive and berberine sulfate-negative mast cells. When these mast cells were transferred to suspension culture, they generated populations that were 100% berberine sulfate-negative, a characteristic similar to that of mucosal mast cells (MMC), and that synthesized predominantly chondroitin sulfate (35S)more » proteoglycans. When ''MMC-like'' cultured mast cells derived from WBB6F1-+/+ PMC were injected into the peritoneal cavities of mast cell-deficient WBB6F1-W/Wv mice, the adoptively transferred mast cell population became 100% berberine sulfate-positive. In methylcellulose culture, these ''second generation PMC'' formed clonal colonies containing both berberine sulfate-positive and berberine sulfate-negative cells, but exhibited significantly less proliferative ability than did normal +/+ PMC. Thus, clonal mast cell populations initially derived from single PMC exhibited multiple and bidirectional alterations between CTMC-like and MMC-like phenotypes. However, this process was associated with a progressive diminution of the mast cells' proliferative ability.« less
Large Population-Based Study Reveals Disparities in Myeloma Precursor Disease | Center for Cancer Research

Cancer.gov

Multiple myeloma (MM) is a cancer of plasma cells, which are antibody-producing white blood cells. Patients with MM have a characteristic excess of monoclonal antibodies, so called M proteins, in their serum, urine, or both and plasma cell infiltration into their bone marrow at multiple sites. African Americans are more than twice as likely as whites to develop MM, but the

A critical examination of the numerology of antigen-binding cells: evidence for multiple receptor specificities on single cells.

PubMed

Miller, A

1977-01-01

The data available from other laboratories as well as our own on the frequency of cells recognizing major histocompatibility antigens or conventional protein and hapten antigens is critically evaluated. The frequency of specific binding for a large number of antigens is sufficiently high to support the idea that at least part of the antigen-binding cell population must have multiple specificities. Our results suggest that these multiple specific cells result from single cells synthesizing and displaying as many as 50-100 species of receptor, each at a frequency of 10(4) per cell. A model involving gene expansion of constant-region genes is suggested and some auxilliary evidence consistent with such C-gene expansion is presented.
flowVS: channel-specific variance stabilization in flow cytometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances.
flowVS: channel-specific variance stabilization in flow cytometry

DOE PAGES

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

2016-07-28

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances.
Technology advancement for integrative stem cell analyses.

PubMed

Jeong, Yoon; Choi, Jonghoon; Lee, Kwan Hyi

2014-12-01

Scientists have endeavored to use stem cells for a variety of applications ranging from basic science research to translational medicine. Population-based characterization of such stem cells, while providing an important foundation to further development, often disregard the heterogeneity inherent among individual constituents within a given population. The population-based analysis and characterization of stem cells and the problems associated with such a blanket approach only underscore the need for the development of new analytical technology. In this article, we review current stem cell analytical technologies, along with the advantages and disadvantages of each, followed by applications of these technologies in the field of stem cells. Furthermore, while recent advances in micro/nano technology have led to a growth in the stem cell analytical field, underlying architectural concepts allow only for a vertical analytical approach, in which different desirable parameters are obtained from multiple individual experiments and there are many technical challenges that limit vertically integrated analytical tools. Therefore, we propose--by introducing a concept of vertical and horizontal approach--that there is the need of adequate methods to the integration of information, such that multiple descriptive parameters from a stem cell can be obtained from a single experiment.
Technology Advancement for Integrative Stem Cell Analyses

PubMed Central

Jeong, Yoon

2014-01-01

Scientists have endeavored to use stem cells for a variety of applications ranging from basic science research to translational medicine. Population-based characterization of such stem cells, while providing an important foundation to further development, often disregard the heterogeneity inherent among individual constituents within a given population. The population-based analysis and characterization of stem cells and the problems associated with such a blanket approach only underscore the need for the development of new analytical technology. In this article, we review current stem cell analytical technologies, along with the advantages and disadvantages of each, followed by applications of these technologies in the field of stem cells. Furthermore, while recent advances in micro/nano technology have led to a growth in the stem cell analytical field, underlying architectural concepts allow only for a vertical analytical approach, in which different desirable parameters are obtained from multiple individual experiments and there are many technical challenges that limit vertically integrated analytical tools. Therefore, we propose—by introducing a concept of vertical and horizontal approach—that there is the need of adequate methods to the integration of information, such that multiple descriptive parameters from a stem cell can be obtained from a single experiment. PMID:24874188
Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

PubMed

Gómez-Villafuertes, Rosa; Paniagua-Herranz, Lucía; Gascon, Sergio; de Agustín-Durán, David; Ferreras, María de la O; Gil-Redondo, Juan Carlos; Queipo, María José; Menendez-Mendez, Aida; Pérez-Sen, Ráquel; Delicado, Esmerilda G; Gualix, Javier; Costa, Marcos R; Schroeder, Timm; Miras-Portugal, María Teresa; Ortega, Felipe

2017-12-16

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.
Multiplicative and additive modulation of neuronal tuning with population activity affects encoded information

PubMed Central

Arandia-Romero, Iñigo; Tanabe, Seiji; Drugowitsch, Jan; Kohn, Adam; Moreno-Bote, Rubén

2016-01-01

Numerous studies have shown that neuronal responses are modulated by stimulus properties, and also by the state of the local network. However, little is known about how activity fluctuations of neuronal populations modulate the sensory tuning of cells and affect their encoded information. We found that fluctuations in ongoing and stimulus-evoked population activity in primate visual cortex modulate the tuning of neurons in a multiplicative and additive manner. While distributed on a continuum, neurons with stronger multiplicative effects tended to have less additive modulation, and vice versa. The information encoded by multiplicatively-modulated neurons increased with greater population activity, while that of additively-modulated neurons decreased. These effects offset each other, so that population activity had little effect on total information. Our results thus suggest that intrinsic activity fluctuations may act as a `traffic light' that determines which subset of neurons are most informative. PMID:26924437
Identification of two novel glial-restricted cell populations in the embryonic telencephalon arising from unique origins

PubMed Central

Strathmann, Frederick G; Wang, Xi; Mayer-Pröschel, Margot

2007-01-01

Background Considerably less attention has been given to understanding the cellular components of gliogenesis in the telencephalon when compared to neuronogenesis, despite the necessity of normal glial cell formation for neurological function. Early proposals of exclusive ventral oligodendrocyte precursor cell (OPC) generation have been challenged recently with studies revealing the potential of the dorsal telencephalon to also generate oligodendrocytes. The identification of OPCs generated from multiple regions of the developing telencephalon, together with the need of the embryonic telencephalon to provide precursor cells for oligodendrocytes as well as astrocytes in ventral and dorsal areas, raises questions concerning the identity of the precursor cell populations capable of generating macroglial subtypes during multiple developmental windows and in differing locations. Results We have identified progenitor populations in the ventral and dorsal telencephalon restricted to the generation of astrocytes and oligodendrocytes. We further demonstrate that the dorsal glial progenitor cells can be generated de novo from the dorsal telencephalon and we demonstrate their capacity for in vivo production of both myelin-forming oligodendrocytes and astrocytes upon transplantation. Conclusion Based on our results we offer a unifying model of telencephalic gliogenesis, with the generation of both oligodendrocytes and astrocytes from spatially separate, but functionally similar, glial restricted populations at different developmental times in the dorsal and ventral CNS. PMID:17439658
Genetically distinct leukemic stem cells in human CD34− acute myeloid leukemia are arrested at a hemopoietic precursor-like stage

PubMed Central

Quek, Lynn; Garnett, Catherine; Karamitros, Dimitris; Stoilova, Bilyana; Doondeea, Jessica; Kennedy, Alison; Metzner, Marlen; Ivey, Adam; Sternberg, Alexander; Hunter, Hannah; Price, Andrew; Virgo, Paul; Grimwade, David; Freeman, Sylvie; Russell, Nigel; Mead, Adam

2016-01-01

Our understanding of the perturbation of normal cellular differentiation hierarchies to create tumor-propagating stem cell populations is incomplete. In human acute myeloid leukemia (AML), current models suggest transformation creates leukemic stem cell (LSC) populations arrested at a progenitor-like stage expressing cell surface CD34. We show that in ∼25% of AML, with a distinct genetic mutation pattern where >98% of cells are CD34−, there are multiple, nonhierarchically arranged CD34+ and CD34− LSC populations. Within CD34− and CD34+ LSC–containing populations, LSC frequencies are similar; there are shared clonal structures and near-identical transcriptional signatures. CD34− LSCs have disordered global transcription profiles, but these profiles are enriched for transcriptional signatures of normal CD34− mature granulocyte–macrophage precursors, downstream of progenitors. But unlike mature precursors, LSCs express multiple normal stem cell transcriptional regulators previously implicated in LSC function. This suggests a new refined model of the relationship between LSCs and normal hemopoiesis in which the nature of genetic/epigenetic changes determines the disordered transcriptional program, resulting in LSC differentiation arrest at stages that are most like either progenitor or precursor stages of hemopoiesis. PMID:27377587
Concurrent detection of secreted products from human lymphocytes by microengraving: cytokines and antigen-reactive antibodies

PubMed Central

Bradshaw, Elizabeth M.; Kent, Sally C.; Tripuraneni, Vinay; Orban, Tihamer; Ploegh, Hidde L.; Hafler, David A.; Love, J. Christopher

2008-01-01

Cell surface determinants, cytokines and antibodies secreted by hematopoietic cells are used to classify their lineage and function. Currently available techniques are unable to elucidate multiple secreted proteins while also assigning phenotypic surface-displayed markers to the individual living cells. Here, a soft lithographic method, microengraving, was adapted for the multiplexed interrogation of populations of individual human peripheral blood mononuclear cells for secreted cytokines (IFN-γ and IL-6), antigen-specific antibodies, and lineage-specific surface-expressed markers. Application of the method to a clinical sample from a recent onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that ~0.58% of circulating CD19+ B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2–3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products. PMID:18675591
Midkine and multiple sclerosis

PubMed Central

Takeuchi, Hideyuki

2014-01-01

Multiple sclerosis (MS) is an autoimmune neurological disease characterized by inflammatory demyelination with subsequent neuronal damage in the CNS. MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have been thought as autoreactive Th1 and Th17 cell-mediated diseases. CD4+CD25+FoxP3+ regulatory T-cell (Treg) plays a pivotal role in autoimmune tolerance, and tolerogenic dendritic cells (DCreg) drive the development of inducible Treg cells. Thus, a dysfunction in the development of Treg and DCreg leads to the development of autoimmune diseases. However, the factors that regulate Treg and DCreg are largely unknown. We recently showed that removal of midkine (MK) suppressed EAE due to an expansion of the Treg cell population as well as a decrease in the numbers of autoreactive Th1 and Th17 cells. MK decreased the Treg cell population by suppressing the phosphorylation of STAT5, which is essential for the expression of Foxp3, the master transcriptional factor of Treg cell differentiation. Furthermore, MK reduces the DCreg cell population by inhibiting the phosphorylation of STAT3, which is critical for DCreg development. Blockade of MK signalling by a specific RNA aptamer significantly elevated the population of DCreg and Treg cells and ameliorated EAE without detectable adverse effects. Therefore, the inhibition of MK may provide an effective therapeutic strategy against autoimmune diseases including MS. Linked Articles This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4 PMID:24460675
The many ways to make a luminal cell and a prostate cancer cell.

PubMed

Strand, Douglas W; Goldstein, Andrew S

2015-12-01

Research in the area of stem/progenitor cells has led to the identification of multiple stem-like cell populations implicated in prostate homeostasis and cancer initiation. Given that there are multiple cells that can regenerate prostatic tissue and give rise to prostate cancer, our focus should shift to defining the signaling mechanisms that drive differentiation and progenitor self-renewal. In this article, we will review the literature, present the evidence and raise important unanswered questions that will help guide the field forward in dissecting critical mechanisms regulating stem-cell differentiation and tumor initiation. © 2015 Society for Endocrinology.
Quantification of multiple gene expression in individual cells.

PubMed

Peixoto, António; Monteiro, Marta; Rocha, Benedita; Veiga-Fernandes, Henrique

2004-10-01

Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.
T-cell stimuli independently sum to regulate an inherited clonal division fate

PubMed Central

Marchingo, J. M.; Prevedello, G.; Kan, A.; Heinzel, S.; Hodgkin, P. D.; Duffy, K. R.

2016-01-01

In the presence of antigen and costimulation, T cells undergo a characteristic response of expansion, cessation and contraction. Previous studies have revealed that population-level reproducibility is a consequence of multiple clones exhibiting considerable disparity in burst size, highlighting the requirement for single-cell information in understanding T-cell fate regulation. Here we show that individual T-cell clones resulting from controlled stimulation in vitro are strongly lineage imprinted with highly correlated expansion fates. Progeny from clonal families cease dividing in the same or adjacent generations, with inter-clonal variation producing burst-size diversity. The effects of costimulatory signals on individual clones sum together with stochastic independence; therefore, the net effect across multiple clones produces consistent, but heterogeneous population responses. These data demonstrate that substantial clonal heterogeneity arises through differences in experience of clonal progenitors, either through stochastic antigen interaction or by differences in initial receptor sensitivities. PMID:27869196
DAFi: A directed recursive data filtering and clustering approach for improving and interpreting data clustering identification of cell populations from polychromatic flow cytometry data.

PubMed

Lee, Alexandra J; Chang, Ivan; Burel, Julie G; Lindestam Arlehamn, Cecilia S; Mandava, Aishwarya; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro; Scheuermann, Richard H; Qian, Yu

2018-04-17

Computational methods for identification of cell populations from polychromatic flow cytometry data are changing the paradigm of cytometry bioinformatics. Data clustering is the most common computational approach to unsupervised identification of cell populations from multidimensional cytometry data. However, interpretation of the identified data clusters is labor-intensive. Certain types of user-defined cell populations are also difficult to identify by fully automated data clustering analysis. Both are roadblocks before a cytometry lab can adopt the data clustering approach for cell population identification in routine use. We found that combining recursive data filtering and clustering with constraints converted from the user manual gating strategy can effectively address these two issues. We named this new approach DAFi: Directed Automated Filtering and Identification of cell populations. Design of DAFi preserves the data-driven characteristics of unsupervised clustering for identifying novel cell subsets, but also makes the results interpretable to experimental scientists through mapping and merging the multidimensional data clusters into the user-defined two-dimensional gating hierarchy. The recursive data filtering process in DAFi helped identify small data clusters which are otherwise difficult to resolve by a single run of the data clustering method due to the statistical interference of the irrelevant major clusters. Our experiment results showed that the proportions of the cell populations identified by DAFi, while being consistent with those by expert centralized manual gating, have smaller technical variances across samples than those from individual manual gating analysis and the nonrecursive data clustering analysis. Compared with manual gating segregation, DAFi-identified cell populations avoided the abrupt cut-offs on the boundaries. DAFi has been implemented to be used with multiple data clustering methods including K-means, FLOCK, FlowSOM, and the ClusterR package. For cell population identification, DAFi supports multiple options including clustering, bisecting, slope-based gating, and reversed filtering to meet various autogating needs from different scientific use cases. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.
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. © 2014 John Wiley & Sons Ltd.
Multidimensional data analysis in immunophenotyping.

PubMed

Loken, M R

2001-05-01

The complexity of cell populations requires careful selection of reagents to detect cells of interest and distinguish them from other types. Additional reagents are frequently used to provide independent criteria for cell identification. Two or three monoclonal antibodies in combination with forward and right-angle light scatter generate a data set that is difficult to visualize because the data must be represented in four- or five-dimensional space. The separation between cell populations provided by the multiple characteristics is best visualized by multidimensional analysis using all parameters simultaneously to identify populations within the resulting hyperspace. Groups of cells are distinguished based on a combination of characteristics not apparent in any usual two-dimensional representation of the data.
Wavelet Imaging on Multiple Scales (WIMS) reveals focal adhesion distributions, dynamics and coupling between actomyosin bundle stability

PubMed Central

Toplak, Tim; Palmieri, Benoit; Juanes-García, Alba; Vicente-Manzanares, Miguel; Grant, Martin; Wiseman, Paul W.

2017-01-01

We introduce and use Wavelet Imaging on Multiple Scales (WIMS) as an improvement to fluorescence correlation spectroscopy to measure physical processes and features that occur across multiple length scales. In this study, wavelet transforms of cell images are used to characterize molecular dynamics at the cellular and subcellular levels (i.e. focal adhesions). We show the usefulness of the technique by applying WIMS to an image time series of a migrating osteosarcoma cell expressing fluorescently labelled adhesion proteins, which allows us to characterize different components of the cell ranging from optical resolution scale through to focal adhesion and whole cell size scales. Using WIMS we measured focal adhesion numbers, orientation and cell boundary velocities for retraction and protrusion. We also determine the internal dynamics of individual focal adhesions undergoing assembly, disassembly or elongation. Thus confirming as previously shown, WIMS reveals that the number of adhesions and the area of the protruding region of the cell are strongly correlated, establishing a correlation between protrusion size and adhesion dynamics. We also apply this technique to characterize the behavior of adhesions, actin and myosin in Chinese hamster ovary cells expressing a mutant form of myosin IIB (1935D) that displays decreased filament stability and impairs front-back cell polarity. We find separate populations of actin and myosin at each adhesion pole for both the mutant and wild type form. However, we find these populations move rapidly inwards toward one another in the mutant case in contrast to the cells that express wild type myosin IIB where those populations remain stationary. Results obtained with these two systems demonstrate how WIMS has the potential to reveal novel correlations between chosen parameters that belong to different scales. PMID:29049414
Cell purification: a new challenge for biobanks.

PubMed

Almeida, Maria; García-Montero, Andres C; Orfao, Alberto

2014-01-01

Performing '-omics' analyses on heterogeneous biological tissue samples, such as blood or bone marrow, can lead to biased or even erroneous results, particularly when the targeted cells and/or molecules are present at relatively low percentages/amounts. In such cases, whole sample analysis will most probably dilute and mask the features of the cell and/or molecules of interest, and this will negatively impact the results and their interpretation. Therefore, frequently it is critically important to have well-characterized and high-quality purified cell populations for the reliable detection of subtle variations in their specific features, such as gene expression profile, protein expression pattern and metabolic status. Biobanks are technological platforms which aim to provide researchers access to a large number of high-quality biological samples and their associated data, particularly to support high-quality scientific and clinical research projects, and such projects will benefit enormously by having access to high-quality purified cell populations or their biological components (e.g. DNA, RNA, proteins). Therefore, a clear opportunity exists for preparative cell sorting techniques in biobanks. Although multiple different cell purification approaches exist or are under development (e.g. cell purification techniques based on cell adherence, density and/or cell size properties, methods based on antibody binding as well as new lab-on-a-chip purification techniques), the choice for a specific technology depends on multiple variables, including cell recovery, purity and yield, among others. In addition, most cell purification approaches are not well suited for high-throughput (HT) purification of multiple cell populations coexisting in a sample. Here we review the most (currently) used cell sorting methods that may be applied for sample preparation in biobanks. For the different approaches, technical considerations about their advantages and limitations are highlighted, and the requirements to be met by a HT cell sorting technology to be used in biobanks are also discussed.
Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.

PubMed

Monsanto, Megan M; White, Kevin S; Kim, Taeyong; Wang, Bingyan J; Fisher, Kristina; Ilves, Kelli; Khalafalla, Farid G; Casillas, Alexandria; Broughton, Kathleen; Mohsin, Sadia; Dembitsky, Walter P; Sussman, Mark A

2017-07-07

The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm 3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit + cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit - mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit + population is further enriched by selection for a CD133 + endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients. © 2017 American Heart Association, Inc.

Cancer Stem Cells: Dynamic Entities in an Ever-Evolving Paradigm.

PubMed

Lopez-Bertoni, Hernando; Li, Yunqing; Laterra, John

2015-11-01

The cancer stem cell (CSC) hypothesis postulates that there is a hierarchy of cellular differentiation within cancers and that the bulk population of tumor cells is derived from a relatively small population of multi-potent neoplastic stem-like cells (CSCs). This tumor-initiating cell population plays an important role in maintaining tumor growth through their unlimited self-renewal, therapeutic resistance, and capacity to propagate tumors through asymmetric cell division. Recent findings from multiple laboratories show that cancer progenitor cells have the capacity to de-differentiate and acquire a stem-like phenotype in response to either genetic manipulation or environmental cues. These findings suggest that CSCs and relatively differentiated progenitors coexist in dynamic equilibrium and are subject to bidirectional conversion. In this review, we discuss emerging concepts regarding the stem-like phenotype, its acquisition by cancer progenitor cells, and the molecular mechanisms involved. Understanding the dynamic equilibrium between CSCs and cancer progenitor cells is critical for the development of novel therapeutic strategies that focus on depleting tumors of their tumor-propagating cell population.
Midkine and multiple sclerosis.

PubMed

Takeuchi, Hideyuki

2014-02-01

Multiple sclerosis (MS) is an autoimmune neurological disease characterized by inflammatory demyelination with subsequent neuronal damage in the CNS. MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have been thought as autoreactive Th1 and Th17 cell-mediated diseases. CD4(+) CD25(+) FoxP3(+) regulatory T-cell (Treg) plays a pivotal role in autoimmune tolerance, and tolerogenic dendritic cells (DCreg) drive the development of inducible Treg cells. Thus, a dysfunction in the development of Treg and DCreg leads to the development of autoimmune diseases. However, the factors that regulate Treg and DCreg are largely unknown. We recently showed that removal of midkine (MK) suppressed EAE due to an expansion of the Treg cell population as well as a decrease in the numbers of autoreactive Th1 and Th17 cells. MK decreased the Treg cell population by suppressing the phosphorylation of STAT5, which is essential for the expression of Foxp3, the master transcriptional factor of Treg cell differentiation. Furthermore, MK reduces the DCreg cell population by inhibiting the phosphorylation of STAT3, which is critical for DCreg development. Blockade of MK signalling by a specific RNA aptamer significantly elevated the population of DCreg and Treg cells and ameliorated EAE without detectable adverse effects. Therefore, the inhibition of MK may provide an effective therapeutic strategy against autoimmune diseases including MS. This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4. © 2013 The British Pharmacological Society.
Parts-Per-Billion Mass Measurement Accuracy Achieved through the Combination of Multiple Linear Regression and Automatic Gain Control in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

PubMed Central

Williams, D. Keith; Muddiman, David C.

2008-01-01

Fourier transform ion cyclotron resonance mass spectrometry has the ability to achieve unprecedented mass measurement accuracy (MMA); MMA is one of the most significant attributes of mass spectrometric measurements as it affords extraordinary molecular specificity. However, due to space-charge effects, the achievable MMA significantly depends on the total number of ions trapped in the ICR cell for a particular measurement. Even through the use of automatic gain control (AGC), the total ion population is not constant between spectra. Multiple linear regression calibration in conjunction with AGC is utilized in these experiments to formally account for the differences in total ion population in the ICR cell between the external calibration spectra and experimental spectra. This ability allows for the extension of dynamic range of the instrument while allowing mean MMA values to remain less than 1 ppm. In addition, multiple linear regression calibration is used to account for both differences in total ion population in the ICR cell as well as relative ion abundance of a given species, which also affords mean MMA values at the parts-per-billion level. PMID:17539605
Evolutionary Game Theory Analysis of Tumor Progression

NASA Astrophysics Data System (ADS)

Wu, Amy; Liao, David; Sturm, James; Austin, Robert

2014-03-01

Evolutionary game theory applied to two interacting cell populations can yield quantitative prediction of the future densities of the two cell populations based on the initial interaction terms. We will discuss how in a complex ecology that evolutionary game theory successfully predicts the future densities of strains of stromal and cancer cells (multiple myeloma), and discuss the possible clinical use of such analysis for predicting cancer progression. Supported by the National Science Foundation and the National Cancer Institute.
A high-content image-based method for quantitatively studying context-dependent cell population dynamics

PubMed Central

Garvey, Colleen M.; Spiller, Erin; Lindsay, Danika; Chiang, Chun-Te; Choi, Nathan C.; Agus, David B.; Mallick, Parag; Foo, Jasmine; Mumenthaler, Shannon M.

2016-01-01

Tumor progression results from a complex interplay between cellular heterogeneity, treatment response, microenvironment and heterocellular interactions. Existing approaches to characterize this interplay suffer from an inability to distinguish between multiple cell types, often lack environmental context, and are unable to perform multiplex phenotypic profiling of cell populations. Here we present a high-throughput platform for characterizing, with single-cell resolution, the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations both during standard growth and in response to multiple, co-occurring selective pressures. The speed of this platform enables a thorough investigation of the impacts of diverse selective pressures including genetic alterations, therapeutic interventions, heterocellular components and microenvironmental factors. The platform has been applied to both 2D and 3D culture systems and readily distinguishes between (1) cytotoxic versus cytostatic cellular responses; and (2) changes in morphological features over time and in response to perturbation. These important features can directly influence tumor evolution and clinical outcome. Our image-based approach provides a deeper insight into the cellular dynamics and heterogeneity of tumors (or other complex systems), with reduced reagents and time, offering advantages over traditional biological assays. PMID:27452732
A high-content image-based method for quantitatively studying context-dependent cell population dynamics

NASA Astrophysics Data System (ADS)

Garvey, Colleen M.; Spiller, Erin; Lindsay, Danika; Chiang, Chun-Te; Choi, Nathan C.; Agus, David B.; Mallick, Parag; Foo, Jasmine; Mumenthaler, Shannon M.

2016-07-01

Tumor progression results from a complex interplay between cellular heterogeneity, treatment response, microenvironment and heterocellular interactions. Existing approaches to characterize this interplay suffer from an inability to distinguish between multiple cell types, often lack environmental context, and are unable to perform multiplex phenotypic profiling of cell populations. Here we present a high-throughput platform for characterizing, with single-cell resolution, the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations both during standard growth and in response to multiple, co-occurring selective pressures. The speed of this platform enables a thorough investigation of the impacts of diverse selective pressures including genetic alterations, therapeutic interventions, heterocellular components and microenvironmental factors. The platform has been applied to both 2D and 3D culture systems and readily distinguishes between (1) cytotoxic versus cytostatic cellular responses; and (2) changes in morphological features over time and in response to perturbation. These important features can directly influence tumor evolution and clinical outcome. Our image-based approach provides a deeper insight into the cellular dynamics and heterogeneity of tumors (or other complex systems), with reduced reagents and time, offering advantages over traditional biological assays.
Attachment defect in mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci.

PubMed Central

Moulder, J W; Levy, N J; Zeichner, S L; Lee, C K

1981-01-01

Almost all the cells in populations of mouse fibroblasts (L cells) persistently infected with the 6BC strain of Chlamydia psittaci were immune to superinfection with high multiplicities of C. psittaci, whether or not the L cells contained visible chlamydial inclusions. As ascertained by experiments with 14C-labeled C. psittaci, immunity to superinfection resulted from the failure of added chlamydiae to attach to persistently infected host cells. However, when exogenous C. psittaci was introduced into persistently infected L cells by centrifuging the inoculum onto host cell monolayers or by pretreating the monolayers with diethylaminoethyl-dextran, these chlamydiae produced expected numbers of infectious progeny. Persistently infected L cells were associated in an unknown way with a C. psittaci population that entered the host cells only with the aid of centrifugation or pretreatment with diethylaminoethyl-dextran. Inclusion-free, persistently infected L cells appeared to present at least two separate hindrances to chlamydial activity: blockage of the attachment of exogenous elementary bodies to persistently infected host cells and prevention of the initiation of chlamydial multiplication by means of a normal developmental cycle in the absence of added C. psittaci. Images PMID:7298188
Impact of alemtuzumab treatment on the survival and function of human regulatory T cells in vitro

PubMed Central

Havari, Evis; Turner, Michael J; Campos-Rivera, Juanita; Shankara, Srinivas; Nguyen, Tri-Hung; Roberts, Bruce; Siders, William; Kaplan, Johanne M

2014-01-01

Alemtuzumab is a humanized monoclonal antibody specific for the CD52 protein present at high levels on the surface of B and T lymphocytes. In clinical trials, alemtuzumab has shown a clinical benefit superior to that of interferon-β in relapsing–remitting multiple sclerosis patients. Treatment with alemtuzumab leads to the depletion of circulating lymphocytes followed by a repopulation process characterized by alterations in the number, proportions and properties of lymphocyte subsets. Of particular interest, an increase in the percentage of T cells with a regulatory phenotype (Treg cells) has been observed in multiple sclerosis patients after alemtuzumab. Since Treg cells play an important role in the control of autoimmune responses, the effect of alemtuzumab on Treg cells was further studied in vitro. Alemtuzumab effectively mediated complement-dependent cytolysis of human T lymphocytes and the remaining population was enriched in T cells with a regulatory phenotype. The alemtuzumab-exposed T cells displayed functional regulatory characteristics including anergy to stimulation with allogeneic dendritic cells and ability to suppress the allogeneic response of autologous T cells. Consistent with the observed increase in Treg cell frequency, the CD25hi T-cell population was necessary for the suppressive activity of alemtuzumab-exposed T cells. The mechanism of this suppression was found to be dependent on both cell–cell contact and interleukin-2 consumption. These findings suggest that an alemtuzumab-mediated increase in the proportion of Treg cells may play a role in promoting the long-term efficacy of alemtuzumab in patients with multiple sclerosis. PMID:24116901
Taste Receptor Cells That Discriminate Between Bitter Stimuli

PubMed Central

Caicedo, Alejandro; Roper, Stephen D.

2013-01-01

Recent studies showing that single taste bud cells express multiple bitter taste receptors have reignited a long-standing controversy over whether single gustatory receptor cells respond selectively or broadly to tastants. We examined calcium responses of rat taste receptor cells in situ to a panel of bitter compounds to determine whether individual cells distinguish between bitter stimuli. Most bitter-responsive taste cells were activated by only one out of five compounds tested. In taste cells that responded to multiple stimuli, there were no significant associations between any two stimuli. Bitter sensation does not appear to occur through the activation of a homogeneous population of broadly tuned bitter-sensitive taste cells. Instead, different bitter stimuli may activate different subpopulations of bitter-sensitive taste cells. PMID:11222863
A novel approach to tracking antigen-experienced CD4 T cells into functional compartments via tandem deep and shallow TCR clonotyping.

PubMed

Estorninho, Megan; Gibson, Vivienne B; Kronenberg-Versteeg, Deborah; Liu, Yuk-Fun; Ni, Chester; Cerosaletti, Karen; Peakman, Mark

2013-12-01

Extensive diversity in the human repertoire of TCRs for Ag is both a cornerstone of effective adaptive immunity that enables host protection against a multiplicity of pathogens and a weakness that gives rise to potential pathological self-reactivity. The complexity arising from diversity makes detection and tracking of single Ag-specific CD4 T cells (ASTs) involved in these immune responses challenging. We report a tandem, multistep process to quantify rare TCRβ-chain variable sequences of ASTs in large polyclonal populations. The approach combines deep high-throughput sequencing (HTS) within functional CD4 T cell compartments, such as naive/memory cells, with shallow, multiple identifier-based HTS of ASTs identified by activation marker upregulation after short-term Ag stimulation in vitro. We find that clonotypes recognizing HLA class II-restricted epitopes of both pathogen-derived Ags and self-Ags are oligoclonal and typically private. Clonotype tracking within an individual reveals private AST clonotypes resident in the memory population, as would be expected, representing clonal expansions (identical nucleotide sequence; "ultraprivate"). Other AST clonotypes share CDR3β amino acid sequences through convergent recombination and are found in memory populations of multiple individuals. Tandem HTS-based clonotyping will facilitate studying AST dynamics, epitope spreading, and repertoire changes that arise postvaccination and following Ag-specific immunotherapies for cancer and autoimmune disease.
Individual motile CD4+ T cells can participate in efficient multi-killing through conjugation to multiple tumor cells

PubMed Central

Liadi, Ivan; Singh, Harjeet; Romain, Gabrielle; Rey-Villamizar, Nicolas; Merouane, Amine; Adolacion, Jay R T.; Kebriaei, Partow; Huls, Helen; Qiu, Peng; Roysam, Badrinath; Cooper, Laurence J.N.; Varadarajan, Navin

2015-01-01

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multi-killing via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that while CAR4 cells can participate in killing and multi-killing, they do so at slower rates, likely due to the lower Granzyme B content. Significantly, in both sets of T cells, a minor sub-population of individual T cells identified by their high motility, demonstrated efficient killing of single tumor cells. By comparing both the multi-killer and single killer CAR+ T cells it appears that the propensity and kinetics of T-cell apoptosis was modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multi-killing should be evaluated in the context of their ability to resist activation induced cell death (AICD). We anticipate that TIMING may be utilized to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. PMID:25711538
Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model.

PubMed

Swift, Brenna E; Williams, Brent A; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

2012-07-01

Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted.
Competitive Stem Cell Recruitment by Multiple Cytotactic Cues

PubMed Central

Mendelson, Avital; Cheung, Yukkee; Paluch, Kamila; Chen, Mo; Kong, Kimi; Tan, Jiali; Dong, Ziming; Sia, Samuel K.; Mao, Jeremy J.

2014-01-01

A multitude of cytotactic cues direct cell migration in development, cancer metastasis and wound healing. However, our understanding of cell motility remains fragmented partially because current migration devices only allow the study of independent factors. We developed a cell motility assay that allows competitive recruitment of a given cell population simultaneously by gradients of multiple cytotactic cues, observable under real-time imaging. Well-defined uniform gradients of cytotactic cues can be independently generated and sustained in each channel. As a case study, bone marrow mesenchymal stem/stromal cells (MSCs) were exposed to 15 cytokines that are commonly present in arthritis. Cytokines that induced robust recruitment of MSCs in multiple groups were selected to ‘compete’ in a final round to yield the most chemotactic factor(s) based on cell migration numbers, distances, migration indices and motility over time. The potency of a given cytokine in competition frequently differed from its individual action, substantiating the need to test multiple cytokines concurrently due to synergistic or antagonistic effects. This new device has the rare capacity to screen molecules that induce cell migration in cancer therapy, drug development and tissue regeneration. PMID:23364311
Adenoviral vaccine induction of CD8+ T cell memory inflation: Impact of co-infection and infection order.

PubMed

Lee, Lian N; Bolinger, Beatrice; Banki, Zoltan; de Lara, Catherine; Highton, Andrew J; Colston, Julia M; Hutchings, Claire; Klenerman, Paul

2017-12-01

The efficacies of many new T cell vaccines rely on generating large populations of long-lived pathogen-specific effector memory CD8 T cells. However, it is now increasingly recognized that prior infection history impacts on the host immune response. Additionally, the order in which these infections are acquired could have a major effect. Exploiting the ability to generate large sustained effector memory (i.e. inflationary) T cell populations from murine cytomegalovirus (MCMV) and human Adenovirus-subtype (AdHu5) 5-beta-galactosidase (Ad-lacZ) vector, the impact of new infections on pre-existing memory and the capacity of the host's memory compartment to accommodate multiple inflationary populations from unrelated pathogens was investigated in a murine model. Simultaneous and sequential infections, first with MCMV followed by Ad-lacZ, generated inflationary populations towards both viruses with similar kinetics and magnitude to mono-infected groups. However, in Ad-lacZ immune mice, subsequent acute MCMV infection led to a rapid decline of the pre-existing Ad-LacZ-specific inflating population, associated with bystander activation of Fas-dependent apoptotic pathways. However, responses were maintained long-term and boosting with Ad-lacZ led to rapid re-expansion of the inflating population. These data indicate firstly that multiple specificities of inflating memory cells can be acquired at different times and stably co-exist. Some acute infections may also deplete pre-existing memory populations, thus revealing the importance of the order of infection acquisition. Importantly, immunization with an AdHu5 vector did not alter the size of the pre-existing memory. These phenomena are relevant to the development of adenoviral vectors as novel vaccination strategies for diverse infections and cancers. (241 words).
Adenoviral vaccine induction of CD8+ T cell memory inflation: Impact of co-infection and infection order

PubMed Central

Bolinger, Beatrice; de Lara, Catherine; Hutchings, Claire

2017-01-01

The efficacies of many new T cell vaccines rely on generating large populations of long-lived pathogen-specific effector memory CD8 T cells. However, it is now increasingly recognized that prior infection history impacts on the host immune response. Additionally, the order in which these infections are acquired could have a major effect. Exploiting the ability to generate large sustained effector memory (i.e. inflationary) T cell populations from murine cytomegalovirus (MCMV) and human Adenovirus-subtype (AdHu5) 5-beta-galactosidase (Ad-lacZ) vector, the impact of new infections on pre-existing memory and the capacity of the host’s memory compartment to accommodate multiple inflationary populations from unrelated pathogens was investigated in a murine model. Simultaneous and sequential infections, first with MCMV followed by Ad-lacZ, generated inflationary populations towards both viruses with similar kinetics and magnitude to mono-infected groups. However, in Ad-lacZ immune mice, subsequent acute MCMV infection led to a rapid decline of the pre-existing Ad-LacZ-specific inflating population, associated with bystander activation of Fas-dependent apoptotic pathways. However, responses were maintained long-term and boosting with Ad-lacZ led to rapid re-expansion of the inflating population. These data indicate firstly that multiple specificities of inflating memory cells can be acquired at different times and stably co-exist. Some acute infections may also deplete pre-existing memory populations, thus revealing the importance of the order of infection acquisition. Importantly, immunization with an AdHu5 vector did not alter the size of the pre-existing memory. These phenomena are relevant to the development of adenoviral vectors as novel vaccination strategies for diverse infections and cancers. (241 words) PMID:29281733
Multiple zebrafish atoh1 genes specify a diversity of neuronal types in the zebrafish cerebellum.

PubMed

Kidwell, Chelsea U; Su, Chen-Ying; Hibi, Masahiko; Moens, Cecilia B

2018-06-01

A single Atoh1 basic-helix-loop-helix transcription factor specifies multiple neuron types in the mammalian cerebellum and anterior hindbrain. The zebrafish genome encodes three paralagous atoh1 genes whose functions in cerebellum and anterior hindbrain development we explore here. With use of a transgenic reporter, we report that zebrafish atoh1c-expressing cells are organized in two distinct domains that are separated both by space and developmental time. An early isthmic expression domain gives rise to an extracerebellar population in rhombomere 1 and an upper rhombic lip domain gives rise to granule cell progenitors that migrate to populate all four granule cell territories of the fish cerebellum. Using genetic mutants we find that of the three zebrafish atoh1 paralogs, atoh1c and atoh1a are required for the full complement of granule neurons. Surprisingly, the two genes are expressed in non-overlapping granule cell progenitor populations, indicating that fish use duplicate atoh1 genes to generate granule cell diversity that is not detected in mammals. Finally, live imaging of granule cell migration in wildtype and atoh1c mutant embryos reveals that while atoh1c is not required for granule cell specification per se, it is required for granule cells to delaminate and migrate away from the rhombic lip. Copyright © 2018 Elsevier Inc. All rights reserved.
Detecting cells in time varying intensity images in confocal microscopy for gene expression studies in living cells

NASA Astrophysics Data System (ADS)

Mitra, Debasis; Boutchko, Rostyslav; Ray, Judhajeet; Nilsen-Hamilton, Marit

2015-03-01

In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms.
Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epifluorescence laser scanning

NASA Astrophysics Data System (ADS)

Favicchio, Rosy; Zacharakis, Giannis; Oikonomaki, Katerina; Zacharopoulos, Athanasios; Mamalaki, Clio; Ripoll, Jorge

2012-07-01

Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.
Experimental treatment of neoplasic diseases and tumors with iono magnetic therapy

NASA Astrophysics Data System (ADS)

Rizsanyi, Elek Karsay; Quiróz, David Lavan; Huamaccto, Carlos Levano; Marroquín, Erwin Guerra

2001-10-01

The Iono Magnetic Therapy is a alternative control method for cell growth population in pancreas and cerebral cancer. The magnetic field applied to cells with cancer decrease the growth of this cells or their multiplication. We observed a potential difference opposite to cell potential and propose that the ionic interchange is very slow tampering with cell growth in cancer.
Integration of multiple determinants in the neuronal computation of economic values.

PubMed

Raghuraman, Anantha P; Padoa-Schioppa, Camillo

2014-08-27

Economic goods may vary on multiple dimensions (determinants). A central conjecture in decision neuroscience is that choices between goods are made by comparing subjective values computed through the integration of all relevant determinants. Previous work identified three groups of neurons in the orbitofrontal cortex (OFC) of monkeys engaged in economic choices: (1) offer value cells, which encode the value of individual offers; (2) chosen value cells, which encode the value of the chosen good; and (3) chosen juice cells, which encode the identity of the chosen good. In principle, these populations could be sufficient to generate a decision. Critically, previous work did not assess whether offer value cells (the putative input to the decision) indeed encode subjective values as opposed to physical properties of the goods, and/or whether offer value cells integrate multiple determinants. To address these issues, we recorded from the OFC while monkeys chose between risky outcomes. Confirming previous observations, three populations of neurons encoded the value of individual offers, the value of the chosen option, and the value-independent choice outcome. The activity of both offer value cells and chosen value cells encoded values defined by the integration of juice quantity and probability. Furthermore, both populations reflected the subjective risk attitude of the animals. We also found additional groups of neurons encoding the risk associated with a particular option, the risky nature of the chosen option, and whether the trial outcome was positive or negative. These results provide substantial support for the conjecture described above and for the involvement of OFC in good-based decisions. Copyright © 2014 the authors 0270-6474/14/3311583-21$15.00/0.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.
Generation of multiple cell types in Bacillus subtilis.

PubMed

Lopez, Daniel; Vlamakis, Hera; Kolter, Roberto

2009-01-01

Bacillus subtilis is a Gram-positive bacterium that is well known for its ability to differentiate into metabolically inactive spores that are highly resistant to environmental stresses. In fact, populations of genetically identical B. subtilis comprise numerous distinct cell types. In addition to spores, cells can become genetically competent, motile, produce extracellular matrix or degradative enzymes, or secrete toxins that allow them to cannibalize their neighbors. Many of the cell fates listed above appear to be mutually exclusive. In this review, we discuss how individual cells within a population control their gene expression to ensure that proper regulation of differentiation occurs. These different cell fates are regulated by an intricate network that relies primarily on the activity of three major transcriptional regulators: Spo0A, DegU, and ComK. While individual cells must choose distinct cell fates, the population as a whole exhibits a spectrum of phenotypes whose diversity may increase fitness.
A Molecular Census of Arcuate Hypothalamus and Median Eminence Cell Types

PubMed Central

Campbell, John N.; Macosko, Evan Z.; Fenselau, Henning; Pers, Tune H.; Lyubetskaya, Anna; Tenen, Danielle; Goldman, Melissa; Verstegen, Anne M.J.; Resch, Jon M.; McCarroll, Steven A.; Rosen, Evan D.; Lowell, Bradford B.; Tsai, Linus

2017-01-01

The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility, and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a novel leptin-sensing neuronal population, multiple AgRP and POMC subtypes, and an orexigenic somatostatin neuronal population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type-specific responses to energy status, including distinctly responsive subtypes of AgRP and POMC neurons. Finally, integrating our data with human GWAS data implicates two previously unknown neuronal subtypes in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred. PMID:28166221
MicroRNA-451 regulates stemness of side population cells via PI3K/Akt/mTOR signaling pathway in multiple myeloma.

PubMed

Du, Juan; Liu, Shuyan; He, Jie; Liu, Xi; Qu, Ying; Yan, Wenqing; Fan, Jianling; Li, Rong; Xi, Hao; Fu, Weijun; Zhang, Chunyang; Yang, Jing; Hou, Jian

2015-06-20

Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As2O3, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma.
Location and cellular stages of NK cell development

PubMed Central

Yu, Jianhua; Freud, Aharon G.; Caligiuri, Michael A

2013-01-01

The identification of distinct tissue-specific natural killer (NK) cell populations that apparently mature from local precursor populations has brought new insight into the diversity and developmental regulation of this important lymphoid subset. NK cells provide a necessary link between the early (innate) and late (adaptive) immune responses to infection. Gaining a better understanding of the processes that govern NK cell development should allow us to better harness NK cell functions in multiple clinical settings as well as to gain further insight into how these cells undergo malignant transformation. In this review, we summarize recent advances in understanding sites and cellular stages of NK cell development in humans and mice. PMID:24055329
Genetically induced cell death in bulge stem cells reveals their redundancy for hair and epidermal regeneration.

PubMed

Driskell, Iwona; Oeztuerk-Winder, Feride; Humphreys, Peter; Frye, Michaela

2015-03-01

Adult mammalian epidermis contains multiple stem cell populations in which quiescent and more proliferative stem and progenitor populations coexist. However, the precise interrelation of these populations in homeostasis remains unclear. Here, we blocked the contribution of quiescent keratin 19 (K19)-expressing bulge stem cells to hair follicle formation through genetic ablation of the essential histone methyltransferase Setd8 that is required for the maintenance of adult skin. Deletion of Setd8 eliminated the contribution of bulge cells to hair follicle regeneration through inhibition of cell division and induction of cell death, but the growth and morphology of hair follicles were unaffected. Furthermore, ablation of Setd8 in the hair follicle bulge blocked the contribution of K19-postive stem cells to wounded epidermis, but the wound healing process was unaltered. Our data indicate that quiescent bulge stem cells are dispensable for hair follicle regeneration and epidermal injury in the short term and support the hypothesis that quiescent and cycling stem cell populations are equipotent. © 2014 AlphaMed Press.
Strategic priming with multiple antigens can yield memory cell phenotypes optimized for infection with Mycobacterium tuberculosis: A computational study

DOE PAGES

Ziraldo, Cordelia; Gong, Chang; Kirschner, Denise E.; ...

2016-01-06

Lack of an effective vaccine results in 9 million new cases of tuberculosis (TB) every year and 1.8 million deaths worldwide. While many infants are vaccinated at birth with BCG (an attenuated M. bovis), this does not prevent infection or development of TB after childhood. Immune responses necessary for prevention of infection or disease are still unknown, making development of effective vaccines against TB challenging. Several new vaccines are ready for human clinical trials, but these trials are difficult and expensive; especially challenging is determining the appropriate cellular response necessary for protection. The magnitude of an immune response is likelymore » key to generating a successful vaccine. Characteristics such as numbers of central memory (CM) and effector memory (EM) T cells responsive to a diverse set of epitopes are also correlated with protection. Promising vaccines against TB contain mycobacterial subunit antigens (Ag) present during both active and latent infection. We hypothesize that protection against different key immunodominant antigens could require a vaccine that produces different levels of EM and CM for each Ag-specific memory population. We created a computational model to explore EM and CM values, and their ratio, within what we term Memory Design Space. Our model captures events involved in T cell priming within lymph nodes and tracks their circulation through blood to peripheral tissues. We used the model to test whether multiple Ag-specific memory cell populations could be generated with distinct locations within Memory Design Space at a specific time point post vaccination. Boosting can further shift memory populations to memory cell ratios unreachable by initial priming events. By strategically varying antigen load, properties of cellular interactions within the LN, and delivery parameters (e.g., number of boosts) of multi-subunit vaccines, we can generate multiple Ag-specific memory populations that cover a wide range of Memory Design Space. As a result, given a set of desired characteristics for Ag-specific memory populations, we can use our model as a tool to predict vaccine formulations that will generate those populations.« less
Strategic priming with multiple antigens can yield memory cell phenotypes optimized for infection with Mycobacterium tuberculosis: A computational study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ziraldo, Cordelia; Gong, Chang; Kirschner, Denise E.

Lack of an effective vaccine results in 9 million new cases of tuberculosis (TB) every year and 1.8 million deaths worldwide. While many infants are vaccinated at birth with BCG (an attenuated M. bovis), this does not prevent infection or development of TB after childhood. Immune responses necessary for prevention of infection or disease are still unknown, making development of effective vaccines against TB challenging. Several new vaccines are ready for human clinical trials, but these trials are difficult and expensive; especially challenging is determining the appropriate cellular response necessary for protection. The magnitude of an immune response is likelymore » key to generating a successful vaccine. Characteristics such as numbers of central memory (CM) and effector memory (EM) T cells responsive to a diverse set of epitopes are also correlated with protection. Promising vaccines against TB contain mycobacterial subunit antigens (Ag) present during both active and latent infection. We hypothesize that protection against different key immunodominant antigens could require a vaccine that produces different levels of EM and CM for each Ag-specific memory population. We created a computational model to explore EM and CM values, and their ratio, within what we term Memory Design Space. Our model captures events involved in T cell priming within lymph nodes and tracks their circulation through blood to peripheral tissues. We used the model to test whether multiple Ag-specific memory cell populations could be generated with distinct locations within Memory Design Space at a specific time point post vaccination. Boosting can further shift memory populations to memory cell ratios unreachable by initial priming events. By strategically varying antigen load, properties of cellular interactions within the LN, and delivery parameters (e.g., number of boosts) of multi-subunit vaccines, we can generate multiple Ag-specific memory populations that cover a wide range of Memory Design Space. As a result, given a set of desired characteristics for Ag-specific memory populations, we can use our model as a tool to predict vaccine formulations that will generate those populations.« less
Multiscale Modelling of Cancer Progression and Treatment Control: The Role of Intracellular Heterogeneities in Chemotherapy Treatment

NASA Astrophysics Data System (ADS)

Chaplain, Mark A. J.; Powathil, Gibin G.

Cancer is a complex, multiscale process involving interactions at intracellular, intercellular and tissue scales that are in turn susceptible to microenvironmental changes. Each individual cancer cell within a cancer cell mass is unique, with its own internal cellular pathways and biochemical interactions. These interactions contribute to the functional changes at the cellular and tissue scale, creating a heterogenous cancer cell population. Anticancer drugs are effective in controlling cancer growth by inflicting damage to various target molecules and thereby triggering multiple cellular and intracellular pathways, leading to cell death or cell-cycle arrest. One of the major impediments in the chemotherapy treatment of cancer is drug resistance driven by multiple mechanisms, including multi-drug and cell-cycle mediated resistance to chemotherapy drugs. In this article, we discuss two hybrid multiscale modelling approaches, incorporating multiple interactions involved in the sub-cellular, cellular and microenvironmental levels to study the effects of cell-cycle, phase-specific chemotherapy on the growth and progression of cancer cells.
Multiscale Modelling of Cancer Progression and Treatment Control: The Role of Intracellular Heterogeneities in Chemotherapy Treatment

NASA Astrophysics Data System (ADS)

Chaplain, Mark A. J.; Powathil, Gibin G.

2015-04-01

Cancer is a complex, multiscale process involving interactions at intracellular, intercellular and tissue scales that are in turn susceptible to microenvironmental changes. Each individual cancer cell within a cancer cell mass is unique, with its own internal cellular pathways and biochemical interactions. These interactions contribute to the functional changes at the cellular and tissue scale, creating a heterogenous cancer cell population. Anticancer drugs are effective in controlling cancer growth by inflicting damage to various target molecules and thereby triggering multiple cellular and intracellular pathways, leading to cell death or cell-cycle arrest. One of the major impediments in the chemotherapy treatment of cancer is drug resistance driven by multiple mechanisms, including multi-drug and cell-cycle mediated resistance to chemotherapy drugs. In this article, we discuss two hybrid multiscale modelling approaches, incorporating multiple interactions involved in the sub-cellular, cellular and microenvironmental levels to study the effects of cell-cycle, phase-specific chemotherapy on the growth and progression of cancer cells.
Fundamental limits on dynamic inference from single-cell snapshots

PubMed Central

Weinreb, Caleb; Tusi, Betsabeh K.; Socolovsky, Merav

2018-01-01

Single-cell expression profiling reveals the molecular states of individual cells with unprecedented detail. Because these methods destroy cells in the process of analysis, they cannot measure how gene expression changes over time. However, some information on dynamics is present in the data: the continuum of molecular states in the population can reflect the trajectory of a typical cell. Many methods for extracting single-cell dynamics from population data have been proposed. However, all such attempts face a common limitation: for any measured distribution of cell states, there are multiple dynamics that could give rise to it, and by extension, multiple possibilities for underlying mechanisms of gene regulation. Here, we describe the aspects of gene expression dynamics that cannot be inferred from a static snapshot alone and identify assumptions necessary to constrain a unique solution for cell dynamics from static snapshots. We translate these constraints into a practical algorithmic approach, population balance analysis (PBA), which makes use of a method from spectral graph theory to solve a class of high-dimensional differential equations. We use simulations to show the strengths and limitations of PBA, and then apply it to single-cell profiles of hematopoietic progenitor cells (HPCs). Cell state predictions from this analysis agree with HPC fate assays reported in several papers over the past two decades. By highlighting the fundamental limits on dynamic inference faced by any method, our framework provides a rigorous basis for dynamic interpretation of a gene expression continuum and clarifies best experimental designs for trajectory reconstruction from static snapshot measurements. PMID:29463712
HPV-Induced Field Cancerisation: Transformation of Adult Tissue Stem Cell Into Cancer Stem Cell.

PubMed

Olivero, Carlotta; Lanfredini, Simone; Borgogna, Cinzia; Gariglio, Marisa; Patel, Girish K

2018-01-01

Field cancerisation was originally described as a basis for multiple head and neck squamous cell carcinoma (HNSCC) and is a pre-malignant phenomenon that is frequently attributable to oncogenic human papillomavirus (HPV) infection. Our work on β-HPV-induced cutaneous squamous cell carcinomas identified a novel Lrig1+ hair follicle junctional zone keratinocyte stem cell population as the basis for field cancerisation. Herein, we describe the ability for HPV to infect adult tissue stem cells in order to establish persistent infection and induce their proliferation and displacement resulting in field cancerisation. By review of the HPV literature, we reveal how this mechanism is conserved as the basis of field cancerisation across many tissues. New insights have identified the capacity for HPV early region genes to dysregulate adult tissue stem cell self-renewal pathways ensuring that the expanded population preserve its stem cell characteristics beyond the stem cell niche. HPV-infected cells acquire additional transforming mutations that can give rise to intraepithelial neoplasia (IEN), from environmental factors such as sunlight or tobacco induced mutations in skin and oral cavity, respectively. With establishment of IEN, HPV viral replication is sacrificed with loss of the episome, and the tissue is predisposed to multiple cancer stem cell-driven carcinomas.
Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model

PubMed Central

Swift, Brenna E.; Williams, Brent A.; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A.; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

2012-01-01

Background Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. Design and Methods The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Results Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89–99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. Conclusions This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted. PMID:22271890
Discrimination of taste qualities among mouse fungiform taste bud cells.

PubMed

Yoshida, Ryusuke; Miyauchi, Aya; Yasuo, Toshiaki; Jyotaki, Masafumi; Murata, Yoshihiro; Yasumatsu, Keiko; Shigemura, Noriatsu; Yanagawa, Yuchio; Obata, Kunihiko; Ueno, Hiroshi; Margolskee, Robert F; Ninomiya, Yuzo

2009-09-15

Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin-expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter-best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour-best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl-best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.
Biochemistry of epidermal stem cells.

PubMed

Eckert, Richard L; Adhikary, Gautam; Balasubramanian, Sivaprakasam; Rorke, Ellen A; Vemuri, Mohan C; Boucher, Shayne E; Bickenbach, Jackie R; Kerr, Candace

2013-02-01

The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue. A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer. An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis. Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells. Copyright © 2012 Elsevier B.V. All rights reserved.
Hypothyroidism Potentially Linked to Cutaneous Squamous Cell Carcinoma: Retrospective Study at a Single Tertiary Academic Medical Center.

PubMed

Ahadiat, Omeed; Higgins, Shauna; Trodello, Cameron; Talmor, Guy; Kokot, Niels; Wysong, Ashley

2018-01-01

There are multiple known risk factors for the development of cutaneous squamous cell carcinoma (SCC). To determine whether patients with cutaneous SCC have a higher prevalence of hypothyroidism than the general US population. A retrospective review was performed for patients seen at the University of Southern California with cutaneous SCC. Chart review was performed for the presence of hypothyroidism and thyroid replacement therapy before the diagnosis of SCC for each patient. Multiple prevalence studies were gathered from the literature for comparison, reporting the prevalence of overt and subclinical hypothyroidism in the general US population and/or elderly US population. Of the 265 patients diagnosed with SCC of the skin, 61 (23%) of patients were found to have a preceding diagnosis of hypothyroidism. The prevalence of hypothyroidism among the population of SCC patients was significantly greater than the prevalence of hypothyroidism (overt and subclinical) in any general and/or elderly US population reported. Patients with SCC of skin are more likely to have a history of hypothyroidism than the general population. The authors conclude that hypothyroidism may be linked to the development of cutaneous SCC.
The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation

USDA-ARS?s Scientific Manuscript database

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-t...
Improving the outcome of adoptive cell transfer by targeting tumor escape

PubMed Central

Kaluza, Karen M.; Vile, Richard

2013-01-01

Adoptive T-cell transfer is among the most promising immunotherapies against cancer. To continue increasing the potential of this therapy, our studies focus on the inhibition of tumor recurrence. Recently, we have demonstrated several ways in which combination therapies involving multiple T-cell populations and immunostimulatory chemotherapy can enhance long-term survival. PMID:23483796
Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2

PubMed Central

Pei, Xin-Yan; Dai, Yun; Youssefian, Leena E.; Chen, Shuang; Bodie, Wesley W.; Takabatake, Yukie; Felthousen, Jessica; Almenara, Jorge A.; Kramer, Lora B.; Dent, Paul

2011-01-01

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138+ cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138+ primary samples, but spared normal CD138− and CD34+ cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G0/G1 arrest and increased apoptosis in all cell-cycle phases, including G0/G1. To determine whether this regimen is active against quiescent G0/G1 MM cells, cells were cultured in low-serum medium to enrich the G0/G1 population. G0/G1–enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G0/G1–enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G0) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst+), low pyronin Y (PY)–staining (2N Hst+/PY−) G0 population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition. PMID:21911831
Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2.

PubMed

Pei, Xin-Yan; Dai, Yun; Youssefian, Leena E; Chen, Shuang; Bodie, Wesley W; Takabatake, Yukie; Felthousen, Jessica; Almenara, Jorge A; Kramer, Lora B; Dent, Paul; Grant, Steven

2011-11-10

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138(+) cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138(+) primary samples, but spared normal CD138(-) and CD34(+) cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G(0)/G(1) arrest and increased apoptosis in all cell-cycle phases, including G(0)/G(1). To determine whether this regimen is active against quiescent G(0)/G(1) MM cells, cells were cultured in low-serum medium to enrich the G(0)/G(1) population. G(0)/G(1)-enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G(0)/G(1)-enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G(0)) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst(+)), low pyronin Y (PY)-staining (2N Hst(+)/PY(-)) G(0) population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.

Biochemistry of epidermal stem cells☆

PubMed Central

Eckert, Richard L.; Adhikary, Gautam; Balasubramanian, Sivaprakasam; Rorke, Ellen A.; Vemuri, Mohan C.; Boucher, Shayne E.; Bickenbach, Jackie R.; Kerr, Candace

2014-01-01

Background The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue. Scope of review A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer. Major conclusions An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis. General significance Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells. PMID:22820019
Bistability, epigenetics, and bet-hedging in bacteria.

PubMed

Veening, Jan-Willem; Smits, Wiep Klaas; Kuipers, Oscar P

2008-01-01

Clonal populations of microbial cells often show a high degree of phenotypic variability under homogeneous conditions. Stochastic fluctuations in the cellular components that determine cellular states can cause two distinct subpopulations, a property called bistability. Phenotypic heterogeneity can be readily obtained by interlinking multiple gene regulatory pathways, effectively resulting in a genetic logic-AND gate. Although switching between states can occur within the cells' lifetime, cells can also pass their cellular state over to the next generation by a mechanism known as epigenetic inheritance and thus perpetuate the phenotypic state. Importantly, heterogeneous populations can demonstrate increased fitness compared with homogeneous populations. This suggests that microbial cells employ bet-hedging strategies to maximize survival. Here, we discuss the possible roles of interlinked bistable networks, epigenetic inheritance, and bet-hedging in bacteria.
Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

PubMed Central

Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

2016-01-01

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793
Sca-1 Identifies a Distinct Androgen-Independent Murine Prostatic Luminal Cell Lineage with Bipotent Potential

PubMed Central

Kwon, Oh-Joon; Zhang, Li; Xin, Li

2016-01-01

Recent lineage tracing studies support the existence of prostate luminal progenitors that possess extensive regenerative capacity, but their identity remains unknown. We show that Sca-1 (Stem Cell Antigen-1) identifies a small population of murine prostate luminal cells that reside in the proximal prostatic ducts adjacent to the urethra. Sca-1+ luminal cells do not express Nkx3.1. They do not carry the secretory function, although they express the androgen receptor. These cells are enriched in the prostates of castrated mice. In the in vitro prostate organoid assay, a small fraction of the Sca-1+ luminal cells are capable of generating budding organoids that are morphologically distinct from those derived from other cell lineages. Histologically, this type of organoid is composed of multiple inner layers of luminal cells surrounded by multiple outer layers of basal cells. When passaged, these organoids retain their morphological and histological features. Finally, the Sca-1+ luminal cells are capable of forming small prostate glands containing both basal and luminal cells in an in vivo prostate regeneration assay. Collectively, our study establishes the androgen-independent and bipotent organoid-forming Sca-1+ luminal cells as a functionally distinct cellular entity. These cells may represent a putative luminal progenitor population and serve as a cellular origin for castration resistant prostate cancer. PMID:26418304
Game theory in the death galaxy: interaction of cancer and stromal cells in tumour microenvironment.

PubMed

Wu, Amy; Liao, David; Tlsty, Thea D; Sturm, James C; Austin, Robert H

2014-08-06

Preventing relapse is the major challenge to effective therapy in cancer. Within the tumour, stromal (ST) cells play an important role in cancer progression and the emergence of drug resistance. During cancer treatment, the fitness of cancer cells can be enhanced by ST cells because their molecular signalling interaction delays the drug-induced apoptosis of cancer cells. On the other hand, competition among cancer and ST cells for space or resources should not be ignored. We explore the population dynamics of multiple myeloma (MM) versus bone marrow ST cells by using an experimental microecology that we call the death galaxy, with a stable drug gradient and connected microhabitats. Evolutionary game theory is a quantitative way to capture the frequency-dependent nature of interactive populations. Therefore, we use evolutionary game theory to model the populations in the death galaxy with the gradients of pay-offs and successfully predict the future densities of MM and ST cells. We discuss the possible clinical use of such analysis for predicting cancer progression.
Ascl1 (Mash1) Knockout Perturbs Differentiation of Nonneuronal Cells in Olfactory Epithelium

PubMed Central

Jang, Woochan; Wildner, Hendrik; Schwob, James E.

2012-01-01

The embryonic olfactory epithelium (OE) generates only a very few olfactory sensory neurons when the basic helix-loop-helix transcription factor, ASCL1 (previously known as MASH1) is eliminated by gene mutation. We have closely examined the structure and composition of the OE of knockout mice and found that the absence of neurons dramatically affects the differentiation of multiple other epithelial cell types as well. The most prominent effect is observed within the two known populations of stem and progenitor cells of the epithelium. The emergence of horizontal basal cells, a multipotent progenitor population in the adult epithelium, is anomalous in the Ascl1 knockout mice. The differentiation of globose basal cells, another multipotent progenitor population in the adult OE, is also aberrant. All of the persisting globose basal cells are marked by SOX2 expression, suggesting a prominent role for SOX2 in progenitors upstream of Ascl1. However, NOTCH1-expressing basal cells are absent from the knockout; since NOTCH1 signaling normally acts to suppress Ascl1 via HES1 and drives sustentacular (Sus) cell differentiation during adult epithelial regeneration, its absence suggests reciprocity between neurogenesis and the differentiation of Sus cells. Indeed, the Sus cells of the mutant mice express a markedly lower level of HES1, strengthening that notion of reciprocity. Duct/gland development appears normal. Finally, the expression of cKIT by basal cells is also undetectable, except in those small patches where neurogenesis escapes the effects of Ascl1 knockout and neurons are born. Thus, persistent neurogenic failure distorts the differentiation of multiple other cell types in the olfactory epithelium. PMID:23284756
Scalable multi-sample single-cell data analysis by Partition-Assisted Clustering and Multiple Alignments of Networks

PubMed Central

Samusik, Nikolay; Wang, Xiaowei; Guan, Leying; Nolan, Garry P.

2017-01-01

Mass cytometry (CyTOF) has greatly expanded the capability of cytometry. It is now easy to generate multiple CyTOF samples in a single study, with each sample containing single-cell measurement on 50 markers for more than hundreds of thousands of cells. Current methods do not adequately address the issues concerning combining multiple samples for subpopulation discovery, and these issues can be quickly and dramatically amplified with increasing number of samples. To overcome this limitation, we developed Partition-Assisted Clustering and Multiple Alignments of Networks (PAC-MAN) for the fast automatic identification of cell populations in CyTOF data closely matching that of expert manual-discovery, and for alignments between subpopulations across samples to define dataset-level cellular states. PAC-MAN is computationally efficient, allowing the management of very large CyTOF datasets, which are increasingly common in clinical studies and cancer studies that monitor various tissue samples for each subject. PMID:29281633
Heterogeneous Structure of Stem Cells Dynamics: Statistical Models and Quantitative Predictions

PubMed Central

Bogdan, Paul; Deasy, Bridget M.; Gharaibeh, Burhan; Roehrs, Timo; Marculescu, Radu

2014-01-01

Understanding stem cell (SC) population dynamics is essential for developing models that can be used in basic science and medicine, to aid in predicting cells fate. These models can be used as tools e.g. in studying patho-physiological events at the cellular and tissue level, predicting (mal)functions along the developmental course, and personalized regenerative medicine. Using time-lapsed imaging and statistical tools, we show that the dynamics of SC populations involve a heterogeneous structure consisting of multiple sub-population behaviors. Using non-Gaussian statistical approaches, we identify the co-existence of fast and slow dividing subpopulations, and quiescent cells, in stem cells from three species. The mathematical analysis also shows that, instead of developing independently, SCs exhibit a time-dependent fractal behavior as they interact with each other through molecular and tactile signals. These findings suggest that more sophisticated models of SC dynamics should view SC populations as a collective and avoid the simplifying homogeneity assumption by accounting for the presence of more than one dividing sub-population, and their multi-fractal characteristics. PMID:24769917
Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

PubMed Central

Tschulik, Claudia; Piossek, Christine; Bet, Jeannette; Yamamoto, Tori N.; Schiemann, Matthias; Neuenhahn, Michael; Martin, Klaus; Schlapschy, Martin; Skerra, Arne; Schmidt, Thomas; Edinger, Matthias; Riddell, Stanley R.; Germeroth, Lothar; Busch, Dirk H.

2012-01-01

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research. PMID:22545138
Cytokines, hepatic cell profiling and cell interactions during bone marrow cell therapy for liver fibrosis in cholestatic mice

PubMed Central

Pinheiro, Daphne; Leirós, Luana; Dáu, Juliana Barbosa Torreão; Stumbo, Ana Carolina; Thole, Alessandra Alves; Cortez, Erika Afonso Costa; Mandarim-de-Lacerda, Carlos Alberto; de Carvalho, Lais

2017-01-01

Bone marrow cells (BMC) migrate to the injured liver after transplantation, contributing to regeneration through multiple pathways, but mechanisms involved are unclear. This work aimed to study BMC migration, characterize cytokine profile, cell populations and proliferation in mice with liver fibrosis transplanted with GFP+ BMC. Confocal microscopy analysis showed GFP+ BMC near regions expressing HGF and SDF-1 in the fibrotic liver. Impaired liver cell proliferation in fibrotic groups was restored after BMC transplantation. Regarding total cell populations, there was a significant reduction in CD68+ cells and increased Ly6G+ cells in transplanted fibrotic group. BMC contributed to the total populations of CD144, CD11b and Ly6G cells in the fibrotic liver, related to an increment of anti-fibrotic cytokines (IL-10, IL-13, IFN-γ and HGF) and reduction of pro-inflammatory cytokines (IL-17A and IL-6). Therefore, HGF and SDF-1 may represent important chemoattractants for transplanted BMC in the injured liver, where these cells can give rise to populations of extrahepatic macrophages, neutrophils and endothelial progenitor cells that can interact synergistically with other liver cells towards the modulation of an anti-fibrotic cytokine profile promoting the onset of liver regeneration. PMID:29176797
Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells.

PubMed

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J; Méndez, Ernesto; Arias, Carlos F

2015-10-01

Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

PubMed Central

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

2015-01-01

ABSTRACT Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. PMID:26246569
GM-CSF: An Immune Modulatory Cytokine that can Suppress Autoimmunity

PubMed Central

Bhattacharya, Palash; Thiruppathi, Muthusamy; Elshabrawy, Hatem A.; Alharshawi, Khaled; Kumar, Prabhakaran; Prabhakar, Bellur S.

2015-01-01

GM-CSF was originally identified as a colony stimulating factor (CSF) because of its ability to induce granulocyte and macrophage populations from precursor cells. Multiple studies have demonstrated that GM-CSF is also an immune-modulatory cytokine, capable of affecting not only the phenotype of myeloid lineage cells, but also T-cell activation through various myeloid intermediaries. This property has been implicated in the sustenance of several autoimmune diseases like arthritis and multiple sclerosis. In contrast, several studies using animal models have shown that GM-CSF is also capable of suppressing many autoimmune diseases like Crohn's disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. Knockout mouse studies have suggested that the role of GM-CSF in maintaining granulocyte and macrophage populations in the physiological steady state is largely redundant. Instead, its immune-modulatory role plays a significant role in the development or resolution of autoimmune diseases. This is mediated either through the differentiation of precursor cells into specialized non-steady state granulocytes, macrophages and dendritic cells, or through the modulation of the phenotype of mature myeloid cells. Thus, outside of myelopoiesis, GM-CSF has a profound role in regulating the immune response and maintaining immunological tolerance. PMID:26113402
Survival and multiplication of Vibrio cholerae in the upper bowel of infant mice.

PubMed

Baselski, V S; Medina, R A; Parker, C D

1978-11-01

The survival and multiplication of Vibrio cholerae strains of varying virulence in the upper bowel of orally challenged infant mice early in infection has been examined. Analysis of changes in the apparent specific activity (counts per minute per colony-forming unit) of the cell population after 4 h compared with the inoculum indicated that strain CA401 established a viable, multiplying cell population, whereas strains VB12 (a rough variant) and 569B were subject to host bactericidal and bacteriolytic mechanisms. An analysis of parameters which may affect the specific activity is included. We have defined the infective potential of the strains in terms of the changes in specific activity. The relative infective potentials are CA401 greater than 569B greater than VB12.
Survival and multiplication of Vibrio cholerae in the upper bowel of infant mice.

PubMed Central

Baselski, V S; Medina, R A; Parker, C D

1978-01-01

The survival and multiplication of Vibrio cholerae strains of varying virulence in the upper bowel of orally challenged infant mice early in infection has been examined. Analysis of changes in the apparent specific activity (counts per minute per colony-forming unit) of the cell population after 4 h compared with the inoculum indicated that strain CA401 established a viable, multiplying cell population, whereas strains VB12 (a rough variant) and 569B were subject to host bactericidal and bacteriolytic mechanisms. An analysis of parameters which may affect the specific activity is included. We have defined the infective potential of the strains in terms of the changes in specific activity. The relative infective potentials are CA401 greater than 569B greater than VB12. PMID:730364
Identification and staining of distinct populations of secretory organelles in astrocytes.

PubMed

Bezzi, Paola; Volterra, Andrea

2014-05-01

Increasing evidence indicates that astrocytes, the most abundant glial cell type in the brain, respond to an elevation in cytoplasmic calcium concentration ([Ca(2+)]i) by releasing chemical transmitters (also called gliotransmitters) via regulated exocytosis of heterogeneous classes of organelles. By this process, astrocytes exert modulatory influences on neighboring cells and are thought to participate in the control of synaptic circuits and cerebral blood flow. Studying the properties of exocytosis in astrocytes is a challenge, because the cell biological basis of this process is incompletely defined. Astrocytic exocytosis involves multiple populations of secretory vesicles, including synaptic-like microvesicles (SLMVs), dense-core granules (DCGs), and lysosomes. Here we summarize the available information for identifying individual populations of secretory organelles in astrocytes, including DCGs, SLMVs, and lysosomes, and present experimental procedures for specifically staining such populations.
CellSort: a support vector machine tool for optimizing fluorescence-activated cell sorting and reducing experimental effort.

PubMed

Yu, Jessica S; Pertusi, Dante A; Adeniran, Adebola V; Tyo, Keith E J

2017-03-15

High throughput screening by fluorescence activated cell sorting (FACS) is a common task in protein engineering and directed evolution. It can also be a rate-limiting step if high false positive or negative rates necessitate multiple rounds of enrichment. Current FACS software requires the user to define sorting gates by intuition and is practically limited to two dimensions. In cases when multiple rounds of enrichment are required, the software cannot forecast the enrichment effort required. We have developed CellSort, a support vector machine (SVM) algorithm that identifies optimal sorting gates based on machine learning using positive and negative control populations. CellSort can take advantage of more than two dimensions to enhance the ability to distinguish between populations. We also present a Bayesian approach to predict the number of sorting rounds required to enrich a population from a given library size. This Bayesian approach allowed us to determine strategies for biasing the sorting gates in order to reduce the required number of enrichment rounds. This algorithm should be generally useful for improve sorting outcomes and reducing effort when using FACS. Source code available at http://tyolab.northwestern.edu/tools/ . k-tyo@northwestern.edu. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
Decursin and Doxorubicin Are in Synergy for the Induction of Apoptosis via STAT3 and/or mTOR Pathways in Human Multiple Myeloma Cells

PubMed Central

Jang, Jinsil; Jeong, Soo-Jin; Kwon, Hee-Young; Jung, Ji Hoon; Sohn, Eun Jung; Lee, Hyo-Jung; Kim, Ji-Hyun; Kim, Sun-Hee; Kim, Jin Hyoung; Kim, Sung-Hoon

2013-01-01

Background. Combination cancer therapy is one of the attractive approaches to overcome drug resistance of cancer cells. In the present study, we investigated the synergistic effect of decursin from Angelica gigas and doxorubicin on the induction of apoptosis in three human multiple myeloma cells. Methodology/Principal Findings. Combined treatment of decursin and doxorubicin significantly exerted significant cytotoxicity compared to doxorubicin or decursin in U266, RPMI8226, and MM.1S cells. Furthermore, the combination treatment enhanced the activation of caspase-9 and -3, the cleavage of PARP, and the sub G1 population compared to either drug alone in three multiple myeloma cells. In addition, the combined treatment downregulated the phosphorylation of mTOR and its downstream S6K1 and activated the phosphorylation of ERK in three multiple myeloma cells. Furthermore, the combined treatment reduced mitochondrial membrane potential, suppressed the phosphorylation of JAK2, STAT3, and Src, activated SHP-2, and attenuated the expression of cyclind-D1 and survivin in U266 cells. Conversely, tyrosine phosphatase inhibitor pervanadate reversed STAT3 inactivation and also PARP cleavage and caspase-3 activation induced by combined treatment of doxorubicin and decursin in U266 cells. Conclusions/Significance. Overall, the combination treatment of decursin and doxorubicin can enhance apoptotic activity via mTOR and/or STAT3 signaling pathway in multiple myeloma cells. PMID:23818927
Decursin and Doxorubicin Are in Synergy for the Induction of Apoptosis via STAT3 and/or mTOR Pathways in Human Multiple Myeloma Cells.

PubMed

Jang, Jinsil; Jeong, Soo-Jin; Kwon, Hee-Young; Jung, Ji Hoon; Sohn, Eun Jung; Lee, Hyo-Jung; Kim, Ji-Hyun; Kim, Sun-Hee; Kim, Jin Hyoung; Kim, Sung-Hoon

2013-01-01

Background. Combination cancer therapy is one of the attractive approaches to overcome drug resistance of cancer cells. In the present study, we investigated the synergistic effect of decursin from Angelica gigas and doxorubicin on the induction of apoptosis in three human multiple myeloma cells. Methodology/Principal Findings. Combined treatment of decursin and doxorubicin significantly exerted significant cytotoxicity compared to doxorubicin or decursin in U266, RPMI8226, and MM.1S cells. Furthermore, the combination treatment enhanced the activation of caspase-9 and -3, the cleavage of PARP, and the sub G1 population compared to either drug alone in three multiple myeloma cells. In addition, the combined treatment downregulated the phosphorylation of mTOR and its downstream S6K1 and activated the phosphorylation of ERK in three multiple myeloma cells. Furthermore, the combined treatment reduced mitochondrial membrane potential, suppressed the phosphorylation of JAK2, STAT3, and Src, activated SHP-2, and attenuated the expression of cyclind-D1 and survivin in U266 cells. Conversely, tyrosine phosphatase inhibitor pervanadate reversed STAT3 inactivation and also PARP cleavage and caspase-3 activation induced by combined treatment of doxorubicin and decursin in U266 cells. Conclusions/Significance. Overall, the combination treatment of decursin and doxorubicin can enhance apoptotic activity via mTOR and/or STAT3 signaling pathway in multiple myeloma cells.
Insight into the number of pre-malignancies and malignancies of the skin in a hospital population in the Netherlands.

PubMed

van Rijsingen, Margit; Seubring, Inge; Maessen-Visch, Birgitte; Lavrijsen, Sjan; van Bergen, Bert; Groenewoud, Johannes; Gerritsen, Marie-Jeanne

2015-01-01

Skin cancer incidence is rising, placing a burden on healthcare systems worldwide. This problem may even be more extensive than expected, since registration of (pre)malignancies of the skin is poor. To provide insight into the numbers of (pre)malignancies in patients with actinic keratosis (AK) or basal cell carcinoma (BCC) in 2 university and 2 general hospitals. The types and numbers of previous tumours and of tumours during a two-year follow-up were collected from 574 patients. Mean time between the first diagnosed (pre)malignancy and time of inclusion was 6.6 years. Overall, 60% had multiple types of (pre)malignancies. In BCC patients, 61% had multiple BCCs, in patients with squamous cell carcinoma (SCC), 40% had multiple SCCs. The combination 'BCC and SCC' occurred in 10%, 'BCC and AK' in 47%, 'SCC and AK' in 14%. High numbers of patients with multiple (pre)malignancies were found in this patient population in university and general hospitals, which may well reflect the Dutch hospital population. We conclude that skin cancer patients are more extensively affected than was expected up till now. Consequently, the management of skin cancer may be in need of adaptation in near future and the question arises whether dermatologists have the capacity for providing care for all these patients.

Origins of extrinsic variability in eukaryotic gene expression

NASA Astrophysics Data System (ADS)

Volfson, Dmitri; Marciniak, Jennifer; Blake, William J.; Ostroff, Natalie; Tsimring, Lev S.; Hasty, Jeff

2006-02-01

Variable gene expression within a clonal population of cells has been implicated in a number of important processes including mutation and evolution, determination of cell fates and the development of genetic disease. Recent studies have demonstrated that a significant component of expression variability arises from extrinsic factors thought to influence multiple genes simultaneously, yet the biological origins of this extrinsic variability have received little attention. Here we combine computational modelling with fluorescence data generated from multiple promoter-gene inserts in Saccharomyces cerevisiae to identify two major sources of extrinsic variability. One unavoidable source arising from the coupling of gene expression with population dynamics leads to a ubiquitous lower limit for expression variability. A second source, which is modelled as originating from a common upstream transcription factor, exemplifies how regulatory networks can convert noise in upstream regulator expression into extrinsic noise at the output of a target gene. Our results highlight the importance of the interplay of gene regulatory networks with population heterogeneity for understanding the origins of cellular diversity.
Origins of extrinsic variability in eukaryotic gene expression

NASA Astrophysics Data System (ADS)

Volfson, Dmitri; Marciniak, Jennifer; Blake, William J.; Ostroff, Natalie; Tsimring, Lev S.; Hasty, Jeff

2006-03-01

Variable gene expression within a clonal population of cells has been implicated in a number of important processes including mutation and evolution, determination of cell fates and the development of genetic disease. Recent studies have demonstrated that a significant component of expression variability arises from extrinsic factors thought to influence multiple genes in concert, yet the biological origins of this extrinsic variability have received little attention. Here we combine computational modeling with fluorescence data generated from multiple promoter-gene inserts in Saccharomyces cerevisiae to identify two major sources of extrinsic variability. One unavoidable source arising from the coupling of gene expression with population dynamics leads to a ubiquitous noise floor in expression variability. A second source which is modeled as originating from a common upstream transcription factor exemplifies how regulatory networks can convert noise in upstream regulator expression into extrinsic noise at the output of a target gene. Our results highlight the importance of the interplay of gene regulatory networks with population heterogeneity for understanding the origins of cellular diversity.
A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

PubMed

Kawamura, Ryuzo; Miyazaki, Minami; Shimizu, Keita; Matsumoto, Yuta; Silberberg, Yaron R; Sathuluri, Ramachandra Rao; Iijima, Masumi; Kuroda, Shun'ichi; Iwata, Futoshi; Kobayashi, Takeshi; Nakamura, Chikashi

2017-11-08

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.
Synaptic transmission and the susceptibility of HIV infection to anti-viral drugs

NASA Astrophysics Data System (ADS)

Komarova, Natalia L.; Levy, David N.; Wodarz, Dominik

2013-07-01

Cell-to-cell viral transmission via virological synapses has been argued to reduce susceptibility of the virus population to anti-viral drugs through multiple infection of cells, contributing to low-level viral persistence during therapy. Using a mathematical framework, we examine the role of synaptic transmission in treatment susceptibility. A key factor is the relative probability of individual virions to infect a cell during free-virus and synaptic transmission, a currently unknown quantity. If this infection probability is higher for free-virus transmission, then treatment susceptibility is lowest if one virus is transferred per synapse, and multiple infection of cells increases susceptibility. In the opposite case, treatment susceptibility is minimized for an intermediate number of virions transferred per synapse. Hence, multiple infection via synapses does not simply lower treatment susceptibility. Without further experimental investigations, one cannot conclude that synaptic transmission provides an additional mechanism for the virus to persist at low levels during anti-viral therapy.
The terminator mouse: salvation for primary cell culture.

PubMed

Kabgani, Nazanin; Moeller, Marcus J

2013-11-01

The Terminator had to come back from the future already several times in an effort to bring salvation to mankind. In the present issue of Kidney International, Guo et al. brought us a novel transgenic mouse model: the terminator mouse. This highly elegant mouse may facilitate significantly the derivation of primary cultures of a specific cell type from a tissue containing multiple cell populations.
Development of Cytomegalovirus-Based Vaccines Against Melanoma

DTIC Science & Technology

2016-10-01

functional as exhibited by their ability to secrete multiple cytokines. The inflationary CD8 T cell populations are widely distributed in lymphoid and...unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The presence of tumor infiltrating CDS+ T cells is associated with tumor regression. Cytomegalovirus...fCMV) infection elicits a robust and long-lasting CDS+ T cell response, which makes CMV a potentially promising vaccine vector against cancer. In the
Fluorescent-Magnetic-Biotargeting Multifunctional Nanobioprobes for Detecting and Isolating Multiple Types of Tumor Cells

PubMed Central

Song, Er-Qun; Hu, Jun; Wen, Cong-Ying; Tian, Zhi-Quan; Yu, Xu; Zhang, Zhi-Ling; Shi, Yun-Bo; Pang, Dai-Wen

2011-01-01

Fluorescent-magnetic-biotargeting multifunctional nanobioprobes (FMBMNs) have attracted great attention in recent years due to their increasing, important applications in biomedical research, clinical diagnosis, and biomedicine. We have previously developed such nanobioprobes for the detection and isolation of a single kind of tumor cells. Detection and isolation of multiple tumor markers or tumor cells from complex samples sensitively and with high efficiency is critical for the early diagnosis of tumors, especially malignant tumors or cancers, which will improve clinical diagnosis outcomes and help to select effective treatment approaches. Here, we expanded the application of the monoclonal antibody (mAb)-coupled FMBMNs for multiplexed assays. Multiple types of cancer cells, such as leukemia cells and prostate cancer cells, were detected and collected from mixed samples within 25 minutes by using a magnet and an ordinary fluorescence microscope. The capture efficiencies of mAb-coupled FMBMNs for the above mentioned two types of cells were 96% and 97% respectively. Furthermore, by using the mAb-coupled FMBMNs, specific and sensitive detection and rapid separation of a small number of spiked leukemia cells and prostate cancer cells in a large population of cultured normal cells (about 0.01% were tumor cells) were achieved simply and inexpensively without any sample pretreatment before cell analysis. Therefore, mAb-coupled multicolour FMBMNs may be used for very sensitive detection and rapid isolation of multiple cancer cells in biomedical research and medical diagnostics. PMID:21250650
Single-cell analysis of population context advances RNAi screening at multiple levels

PubMed Central

Snijder, Berend; Sacher, Raphael; Rämö, Pauli; Liberali, Prisca; Mench, Karin; Wolfrum, Nina; Burleigh, Laura; Scott, Cameron C; Verheije, Monique H; Mercer, Jason; Moese, Stefan; Heger, Thomas; Theusner, Kristina; Jurgeit, Andreas; Lamparter, David; Balistreri, Giuseppe; Schelhaas, Mario; De Haan, Cornelis A M; Marjomäki, Varpu; Hyypiä, Timo; Rottier, Peter J M; Sodeik, Beate; Marsh, Mark; Gruenberg, Jean; Amara, Ali; Greber, Urs; Helenius, Ari; Pelkmans, Lucas

2012-01-01

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment. PMID:22531119
Egr-5 is a post-mitotic regulator of planarian epidermal differentiation

PubMed Central

Tu, Kimberly C; Cheng, Li-Chun; TK Vu, Hanh; Lange, Jeffrey J; McKinney, Sean A; Seidel, Chris W; Sánchez Alvarado, Alejandro

2015-01-01

Neoblasts are an abundant, heterogeneous population of adult stem cells (ASCs) that facilitate the maintenance of planarian tissues and organs, providing a powerful system to study ASC self-renewal and differentiation dynamics. It is unknown how the collective output of neoblasts transit through differentiation pathways to produce specific cell types. The planarian epidermis is a simple tissue that undergoes rapid turnover. We found that as epidermal progeny differentiate, they progress through multiple spatiotemporal transition states with distinct gene expression profiles. We also identified a conserved early growth response family transcription factor, egr-5, that is essential for epidermal differentiation. Disruption of epidermal integrity by egr-5 RNAi triggers a global stress response that induces the proliferation of neoblasts and the concomitant expansion of not only epidermal, but also multiple progenitor cell populations. Our results further establish the planarian epidermis as a novel paradigm to uncover the molecular mechanisms regulating ASC specification in vivo. DOI: http://dx.doi.org/10.7554/eLife.10501.001 PMID:26457503
Development of microbial genome-probing microarrays using digital multiple displacement amplification of uncultivated microbial single cells.

PubMed

Chang, Ho-Won; Sung, Youlboong; Kim, Kyoung-Ho; Nam, Young-Do; Roh, Seong Woon; Kim, Min-Soo; Jeon, Che Ok; Bae, Jin-Woo

2008-08-15

A crucial problem in the use of previously developed genome-probing microarrays (GPM) has been the inability to use uncultivated bacterial genomes to take advantage of the high sensitivity and specificity of GPM in microbial detection and monitoring. We show here a method, digital multiple displacement amplification (MDA), to amplify and analyze various genomes obtained from single uncultivated bacterial cells. We used 15 genomes from key microbes involved in dichloromethane (DCM)-dechlorinating enrichment as microarray probes to uncover the bacterial population dynamics of samples without PCR amplification. Genomic DNA amplified from single cells originating from uncultured bacteria with 80.3-99.4% similarity to 16S rRNA genes of cultivated bacteria. The digital MDA-GPM method successfully monitored the dynamics of DCM-dechlorinating communities from different phases of enrichment status. Without a priori knowledge of microbial diversity, the digital MDA-GPM method could be designed to monitor most microbial populations in a given environmental sample.
Therapeutic manipulation of natural killer (NK) T cells in autoimmunity: are we close to reality?

PubMed Central

Simoni, Y; Diana, J; Ghazarian, L; Beaudoin, L; Lehuen, A

2013-01-01

T cells reactive to lipids and restricted by major histocompatibility complex (MHC) class I-like molecules represent more than 15% of all lymphocytes in human blood. This heterogeneous population of innate cells includes the invariant natural killer T cells (iNK T), type II NK T cells, CD1a,b,c-restricted T cells and mucosal-associated invariant T (MAIT) cells. These populations are implicated in cancer, infection and autoimmunity. In this review, we focus on the role of these cells in autoimmunity. We summarize data obtained in humans and preclinical models of autoimmune diseases such as primary biliary cirrhosis, type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, psoriasis and atherosclerosis. We also discuss the promise of NK T cell manipulations: restoration of function, specific activation, depletion and the relevance of these treatments to human autoimmune diseases. PMID:23199318
The Impact of Prophage on the Equilibria and Stability of Phage and Host

NASA Astrophysics Data System (ADS)

Yu, Pei; Nadeem, Alina; Wahl, Lindi M.

2017-06-01

In this paper, we present a bacteriophage model that includes prophage, that is, phage genomes that are incorporated into the host cell genome. The general model is described by an 18-dimensional system of ordinary differential equations. This study focuses on asymptotic behaviour of the model, and thus the system is reduced to a simple six-dimensional model, involving uninfected host cells, infected host cells and phage. We use dynamical system theory to explore the dynamic behaviour of the model, studying in particular the impact of prophage on the equilibria and stability of phage and host. We employ bifurcation and stability theory, centre manifold and normal form theory to show that the system has multiple equilibrium solutions which undergo a series of bifurcations, finally leading to oscillating motions. Numerical simulations are presented to illustrate and confirm the analytical predictions. The results of this study indicate that in some parameter regimes, the host cell population may drive the phage to extinction through diversification, that is, if multiple types of host emerge; this prediction holds even if the phage population is likewise diverse. This parameter regime is restricted, however, if infecting phage are able to recombine with prophage sequences in the host cell genome.
The compartmentalized inflammatory response in the multiple sclerosis brain is composed of tissue-resident CD8+ T lymphocytes and B cells.

PubMed

Machado-Santos, Joana; Saji, Etsuji; Tröscher, Anna R; Paunovic, Manuela; Liblau, Roland; Gabriely, Galina; Bien, Christian G; Bauer, Jan; Lassmann, Hans

2018-06-04

Multiple sclerosis is an inflammatory demyelinating disease in which active demyelination and neurodegeneration are associated with lymphocyte infiltrates in the brain. However, so far little is known regarding the phenotype and function of these infiltrating lymphocyte populations. In this study, we performed an in-depth phenotypic characterization of T and B cell infiltrates in a large set of multiple sclerosis cases with different disease and lesion stages and compared the findings with those seen in inflammatory, non-inflammatory and normal human controls. In multiple sclerosis lesions, we found a dominance of CD8+ T cells and a prominent contribution of CD20+ B cells in all disease courses and lesion stages, including acute multiple sclerosis cases with very short disease duration, while CD4+ T cells were sparse. A dominance of CD8+ T cells was also seen in other inflammatory controls, such as Rasmussen's encephalitis and viral encephalitis, but the contribution of B cells in these diseases was modest. Phenotypic analysis of the CD8+ T cells suggested that part of the infiltrating cells in active lesions proliferate, show an activated cytotoxic phenotype and are in part destroyed by apoptosis. Further characterization of the remaining cells suggest that CD8+ T cells acquire features of tissue-resident memory cells, which may be focally reactivated in active lesions of acute, relapsing and progressive multiple sclerosis, while B cells, at least in part, gradually transform into plasma cells. The loss of surface molecules involved in the egress of leucocytes from inflamed tissue, such as S1P1 or CCR7, and the upregulation of CD103 expression may be responsible for the compartmentalization of the inflammatory response in established lesions. Similar phenotypic changes of tissue-infiltrating CD8+ T cells were also seen in Rasmussen's encephalitis. Our data underline the potential importance of CD8+ T lymphocytes and B cells in the inflammatory response in established multiple sclerosis lesions. Tissue-resident T and B cells may represent guardians of previous inflammatory brain disease, which can be reactivated and sustain the inflammatory response, when they are re-exposed to their specific antigen.
Tools for Genomic and Transcriptomic Analysis of Microbes at Single-Cell Level

PubMed Central

Chen, Zixi; Chen, Lei; Zhang, Weiwen

2017-01-01

Microbiologists traditionally study population rather than individual cells, as it is generally assumed that the status of individual cells will be similar to that observed in the population. However, the recent studies have shown that the individual behavior of each single cell could be quite different from that of the whole population, suggesting the importance of extending traditional microbiology studies to single-cell level. With recent technological advances, such as flow cytometry, next-generation sequencing (NGS), and microspectroscopy, single-cell microbiology has greatly enhanced the understanding of individuality and heterogeneity of microbes in many biological systems. Notably, the application of multiple ‘omics’ in single-cell analysis has shed light on how individual cells perceive, respond, and adapt to the environment, how heterogeneity arises under external stress and finally determines the fate of the whole population, and how microbes survive under natural conditions. As single-cell analysis involves no axenic cultivation of target microorganism, it has also been demonstrated as a valuable tool for dissecting the microbial ‘dark matter.’ In this review, current state-of-the-art tools and methods for genomic and transcriptomic analysis of microbes at single-cell level were critically summarized, including single-cell isolation methods and experimental strategies of single-cell analysis with NGS. In addition, perspectives on the future trends of technology development in the field of single-cell analysis was also presented. PMID:28979258
Mechanical Fluidity of Fully Suspended Biological Cells

PubMed Central

Maloney, John M.; Lehnhardt, Eric; Long, Alexandra F.; Van Vliet, Krystyn J.

2013-01-01

Mechanical characteristics of single biological cells are used to identify and possibly leverage interesting differences among cells or cell populations. Fluidity—hysteresivity normalized to the extremes of an elastic solid or a viscous liquid—can be extracted from, and compared among, multiple rheological measurements of cells: creep compliance versus time, complex modulus versus frequency, and phase lag versus frequency. With multiple strategies available for acquisition of this nondimensional property, fluidity may serve as a useful and robust parameter for distinguishing cell populations, and for understanding the physical origins of deformability in soft matter. Here, for three disparate eukaryotic cell types deformed in the suspended state via optical stretching, we examine the dependence of fluidity on chemical and environmental influences at a timescale of ∼1 s. We find that fluidity estimates are consistent in the time and frequency domains under a structural damping (power-law or fractional-derivative) model, but not under an equivalent-complexity, lumped-component (spring-dashpot) model; the latter predicts spurious time constants. Although fluidity is suppressed by chemical cross-linking, we find that ATP depletion in the cell does not measurably alter the parameter, and we thus conclude that active ATP-driven events are not a crucial enabler of fluidity during linear viscoelastic deformation of a suspended cell. Finally, by using the capacity of optical stretching to produce near-instantaneous increases in cell temperature, we establish that fluidity increases with temperature—now measured in a fully suspended, sortable cell without the complicating factor of cell-substratum adhesion. PMID:24138852
CD44 staining of cancer stem-like cells is influenced by down-regulation of CD44 variant isoforms and up-regulation of the standard CD44 isoform in the population of cells that have undergone epithelial-to-mesenchymal transition.

PubMed

Biddle, Adrian; Gammon, Luke; Fazil, Bilal; Mackenzie, Ian C

2013-01-01

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44(high) cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44(high) population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44(high) population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44(high) population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.
The epithelial-mesenchymal interactions: insights into physiological and pathological aspects of oral tissues.

PubMed

Santosh, Arvind Babu Rajendra; Jones, Thaon Jon

2014-03-17

In the human biological system, the individual cells divide and form tissues and organs. These tissues are hetero-cellular. Basically any tissue consists of an epithelium and the connective tissue. The latter contains mainly mesenchymally-derived tissues with a diversified cell population. The cell continues to grow and differentiate in a pre-programmed manner using a messenger system. The epithelium and the mesenchymal portion of each tissue have two different origins and perform specific functions, but there is a well-defined interaction mechanism, which mediates between them. Epithelial mesenchymal interactions (EMIs) are part of this mechanism, which can be regarded as a biological conversation between epithelial and mesenchymal cell populations involved in the cellular differentiation of one or both cell populations. EMIs represent a process that is essential for cell growth, cell differentiation and cell multiplication. EMIs are associated with normal physiological processes in the oral cavity, such as odontogenesis, dentino-enamel junction formation, salivary gland development, palatogenesis, and also pathological processes, such as oral cancer. This paper focuses the role EMIs in odontogenesis, salivary gland development, palatogenesis and oral cancer.
HIV-1 dynamics revisited: biphasic decay by cytotoxic T lymphocyte killing?

PubMed Central

Arnaout, R A; Nowak, M A; Wodarz, D

2000-01-01

The biphasic decay of blood viraemia in patients being treated for human immunodeficiency virus type 1 (HIV-1) infection has been explained as the decay of two distinct populations of cells: the rapid death of productively infected cells followed by the much slower elimination of a second population the identity of which remains unknown. Here we advance an alternative explanation based on the immune response against a single population of infected cells. We show that the biphasic decay can be explained simply, without invoking multiple compartments: viral load falls quickly while cytotoxic T lymphocytes (CTL) are still abundant, and more slowly as CTL disappear. We propose a method to test this idea, and develop a framework that is readily applicable to treatment of other infections. PMID:10972131
Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors.

PubMed

Haghverdi, Laleh; Lun, Aaron T L; Morgan, Michael D; Marioni, John C

2018-06-01

Large-scale single-cell RNA sequencing (scRNA-seq) data sets that are produced in different laboratories and at different times contain batch effects that may compromise the integration and interpretation of the data. Existing scRNA-seq analysis methods incorrectly assume that the composition of cell populations is either known or identical across batches. We present a strategy for batch correction based on the detection of mutual nearest neighbors (MNNs) in the high-dimensional expression space. Our approach does not rely on predefined or equal population compositions across batches; instead, it requires only that a subset of the population be shared between batches. We demonstrate the superiority of our approach compared with existing methods by using both simulated and real scRNA-seq data sets. Using multiple droplet-based scRNA-seq data sets, we demonstrate that our MNN batch-effect-correction method can be scaled to large numbers of cells.
Dynamic cell culture on porous biopolymer microcarriers in a spinner flask for bone tissue engineering: a feasibility study.

PubMed

Jin, Guang-Zhen; Park, Jeong-Hui; Seo, Seog-Jin; Kim, Hae-Won

2014-07-01

Porous microspherical carriers have great promise for cell culture and tissue engineering. Dynamic cultures enable more uniform cell population and effective differentiation than static cultures. Here we applied dynamic spinner flask culture for the loading and multiplication of cells onto porous biopolymer microcarriers. The abilities of the microcarriers to populate cells and to induce osteogenic differentiation were examined and the feasibility of in vivo delivery of the constructs was addressed. Over time, the porous microcarriers enabled cell adhesion and expansion under proper dynamic culture conditions. Osteogenic markers were substantially expressed by the dynamic cell cultures. The cell-cultured microcarriers implanted in the mouse subcutaneous tissue for 4 weeks showed excellent tissue compatibility, with minimal inflammatory signs and significant induction of bone tissues. This first report on dynamic culture of porous biopolymer microcarriers providing an effective tool for bone tissue engineering.

Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq

PubMed Central

Loeffler-Wirth, Henry; Hopp, Lydia; Schadendorf, Dirk; Schartl, Manfred; Anderegg, Ulf; Camp, Gray; Treutlein, Barbara; Binder, Hans; Kunz, Manfred

2017-01-01

Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy. PMID:27903987
Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease.

PubMed

Holmberg, Fredrik Eo; Seidelin, Jakob B; Yin, Xiaolei; Mead, Benjamin E; Tong, Zhixiang; Li, Yuan; Karp, Jeffrey M; Nielsen, Ole H

2017-05-01

Both the incidence and prevalence of inflammatory bowel disease (IBD) is increasing globally; in the industrialized world up to 0.5% of the population are affected and around 4.2 million individuals suffer from IBD in Europe and North America combined. Successful engraftment in experimental colitis models suggests that intestinal stem cell transplantation could constitute a novel treatment strategy to re-establish mucosal barrier function in patients with severe disease. Intestinal stem cells can be grown in vitro in organoid structures, though only a fraction of the cells contained are stem cells with regenerative capabilities. Hence, techniques to enrich stem cell populations are being pursued through the development of multiple two-dimensional and three-dimensional culture protocols, as well as co-culture techniques and multiple growth medium compositions. Moreover, research in support matrices allowing for efficient clinical application is in progress. In vitro culture is accomplished by modulating the signaling pathways fundamental for the stem cell niche with a suitable culture matrix to provide additional contact-dependent stimuli and structural support. The aim of this review was to discuss medium compositions and support matrices for optimal intestinal stem cell culture, as well as potential modifications to advance clinical use in IBD. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.
A regenerative approach to the treatment of multiple sclerosis.

PubMed

Deshmukh, Vishal A; Tardif, Virginie; Lyssiotis, Costas A; Green, Chelsea C; Kerman, Bilal; Kim, Hyung Joon; Padmanabhan, Krishnan; Swoboda, Jonathan G; Ahmad, Insha; Kondo, Toru; Gage, Fred H; Theofilopoulos, Argyrios N; Lawson, Brian R; Schultz, Peter G; Lairson, Luke L

2013-10-17

Progressive phases of multiple sclerosis are associated with inhibited differentiation of the progenitor cell population that generates the mature oligodendrocytes required for remyelination and disease remission. To identify selective inducers of oligodendrocyte differentiation, we performed an image-based screen for myelin basic protein (MBP) expression using primary rat optic-nerve-derived progenitor cells. Here we show that among the most effective compounds identifed was benztropine, which significantly decreases clinical severity in the experimental autoimmune encephalomyelitis (EAE) model of relapsing-remitting multiple sclerosis when administered alone or in combination with approved immunosuppressive treatments for multiple sclerosis. Evidence from a cuprizone-induced model of demyelination, in vitro and in vivo T-cell assays and EAE adoptive transfer experiments indicated that the observed efficacy of this drug results directly from an enhancement of remyelination rather than immune suppression. Pharmacological studies indicate that benztropine functions by a mechanism that involves direct antagonism of M1 and/or M3 muscarinic receptors. These studies should facilitate the development of effective new therapies for the treatment of multiple sclerosis that complement established immunosuppressive approaches.
Meningeal mast cells affect early T cell central nervous system infiltration and blood-brain barrier integrity through TNF: a role for neutrophil recruitment?

PubMed

Sayed, Blayne A; Christy, Alison L; Walker, Margaret E; Brown, Melissa A

2010-06-15

Mast cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis, a rodent model of the human demyelinating disease multiple sclerosis. Yet their site and mode of action is unknown. In both diseases, myelin-specific T cells are initially activated in peripheral lymphoid organs. However, for disease to occur, these cells must enter the immunologically privileged CNS through a breach in the relatively impermeable blood-brain barrier. In this study, we demonstrate that a dense population of resident mast cells in the meninges, structures surrounding the brain and spinal cord, regulate basal CNS barrier function, facilitating initial T cell CNS entry. Through the expression of TNF, mast cells recruit an early wave of neutrophils to the CNS. We propose that neutrophils in turn promote the blood-brain barrier breach and together with T cells lead to further inflammatory cell influx and myelin damage. These findings provide specific targets for intervention in multiple sclerosis as well as other immune-mediated CNS diseases.
The Embryonic Septum and Ventral Pallium, New Sources of Olfactory Cortex Cells

PubMed Central

de Carlos, Juan A.

2012-01-01

The mammalian olfactory cortex is a complex structure located along the rostro-caudal extension of the ventrolateral prosencephalon, which is divided into several anatomically and functionally distinct areas: the anterior olfactory nucleus, piriform cortex, olfactory tubercle, amygdaloid olfactory nuclei, and the more caudal entorhinal cortex. Multiple forebrain progenitor domains contribute to the cellular diversity of the olfactory cortex, which is invaded simultaneously by cells originating in distinct germinal areas in the dorsal and ventral forebrain. Using a combination of dye labeling techniques, we identified two novel areas that contribute cells to the developing olfactory cortices, the septum and the ventral pallium, from which cells migrate along a radial and then a tangential path. We characterized these cell populations by comparing their expression of calretinin, calbindin, reelin and Tbr1 with that of other olfactory cell populations. PMID:22984546
Comparative characterization of stromal vascular cells derived from three types of vascular wall and adipose tissue.

PubMed

Yang, Santsun; Eto, Hitomi; Kato, Harunosuke; Doi, Kentaro; Kuno, Shinichiro; Kinoshita, Kahori; Ma, Hsu; Tsai, Chi-Han; Chou, Wan-Ting; Yoshimura, Kotaro

2013-12-01

Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 μm in diameter), venous wall (VN; >500 μm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, <100 μm in diameter). These were examined for functionality and compared with adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristics of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34(+)/CD31(-) and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in the osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as arterial wall calcification and possible applications to regenerative therapies involving each vessel wall-resident stromal population.
Automated Microfluidic Filtration and Immunocytochemistry Detection System for Capture and Enumeration of Circulating Tumor Cells and Other Rare Cell Populations in Blood.

PubMed

Pugia, Michael; Magbanua, Mark Jesus M; Park, John W

2017-01-01

Isolation by size using a filter membrane offers an antigen-independent method for capturing rare cells present in blood of cancer patients. Multiple cell types, including circulating tumor cells (CTCs), captured on the filter membrane can be simultaneously identified via immunocytochemistry (ICC) analysis of specific cellular biomarkers. Here, we describe an automated microfluidic filtration method combined with a liquid handling system for sequential ICC assays to detect and enumerate non-hematologic rare cells in blood.
Clustering Single-Cell Expression Data Using Random Forest Graphs.

PubMed

Pouyan, Maziyar Baran; Nourani, Mehrdad

2017-07-01

Complex tissues such as brain and bone marrow are made up of multiple cell types. As the study of biological tissue structure progresses, the role of cell-type-specific research becomes increasingly important. Novel sequencing technology such as single-cell cytometry provides researchers access to valuable biological data. Applying machine-learning techniques to these high-throughput datasets provides deep insights into the cellular landscape of the tissue where those cells are a part of. In this paper, we propose the use of random-forest-based single-cell profiling, a new machine-learning-based technique, to profile different cell types of intricate tissues using single-cell cytometry data. Our technique utilizes random forests to capture cell marker dependences and model the cellular populations using the cell network concept. This cellular network helps us discover what cell types are in the tissue. Our experimental results on public-domain datasets indicate promising performance and accuracy of our technique in extracting cell populations of complex tissues.
Collective Motion in Bacterial Populations with Mixed Phenotypic Behaviors

NASA Astrophysics Data System (ADS)

Hoeger, Kentaro; Strickland, Ben; Shoup, Daniel; Ursell, Tristan

The motion of large, densely packed groups of organisms is often qualitatively distinct from the motion of individuals, yet hinges on individual properties and behaviors. Collective motion of bacteria depends strongly on the phenotypic behaviors of individual cells, the physical interactions between cells, and the geometry of their environment, often with multiple phenotypes coexisting in a population. Thus, to characterize how these selectively important interactions affect group traits, such as cell dispersal, spatial segregation of phenotypes, and material transport in groups, we use a library of Bacillus subtilis mutants that modulate chemotaxis, motility, and biofilm formation. By mixing phenotypes and observing bacterial behaviors and motion at single cell resolution, we probe collective motion as a function of phenotypic mixture and environmental geometry. Our work demonstrates that collective microbial motion exhibits a transition, from `turbulence' to semiballistic burrowing, as phenotypic composition varies. This work illuminates the role that individual cell behaviors play in the emergence of collective motion, and may signal qualitatively distinct regimes of material transport in bacterial populations. University of Oregon.
The Tumor Microenvironment: A Pitch for Multiple Players

PubMed Central

Schiavoni, Giovanna; Gabriele, Lucia; Mattei, Fabrizio

2013-01-01

The cancer microenvironment may be conceptually regarded as a pitch where the main players are resident and non-resident cellular components, each covering a defined role and interconnected by a complex network of soluble mediators. The crosstalk between these cells and the tumor cells within this environment crucially determines the fate of tumor progression. Immune cells that infiltrate the tumor bed are transported there by blood circulation and exert a variety of effects, either counteracting or favoring tumor outgrowth. Here, we review and discuss the multiple populations composing the tumor bed, with special focus on immune cells subsets that positively or negatively dictate neoplastic progression. In this scenario, the contribution of cancer stem cells within the tumor microenvironment will also be discussed. Finally, we illustrate recent advances on new integrated approaches to investigate the tumor microenvironment in vitro. PMID:23616948
Chemotaxis of Cell Populations through Confined Spaces at Single-Cell Resolution

PubMed Central

Tong, ZiQiu; Balzer, Eric M.; Dallas, Matthew R.; Hung, Wei-Chien; Stebe, Kathleen J.; Konstantopoulos, Konstantinos

2012-01-01

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications. PMID:22279529
Analysis of viral (zucchini yellow mosaic virus) genetic diversity during systemic movement through a Cucurbita pepo vine

PubMed Central

Holmes, E.C.; Stephenson, A.G.

2014-01-01

Determining the extent and structure of intra-host genetic diversity and the magnitude and impact of population bottlenecks is central to understanding the mechanisms of viral evolution. To determine the nature of viral evolution following systemic movement through a plant, we performed deep sequencing of 23 leaves that grew sequentially along a single Cucurbita pepo vine that was infected with zucchini yellow mosaic virus (ZYMV), and on a leaf that grew in on a side branch. Strikingly, of 112 genetic (i.e. sub-consensus) variants observed in the data set as a whole, only 22 were found in multiple leaves. Similarly, only three of the 13 variants present in the inoculating population were found in the subsequent leaves on the vine. Hence, it appears that systemic movement is characterized by sequential population bottlenecks, although not sufficient to reduce the population to a single virion as multiple variants were consistently transmitted between leaves. In addition, the number of variants within a leaf increases as a function of distance from the inoculated (source) leaf, suggesting that the circulating sap may serve as a continual source of virus. Notably, multiple mutational variants were observed in the cylindrical Inclusion (CI) protein (known to be involved in both cell-to-cell and systemic movement of the virus) that were present in multiple (19/24) leaf samples. These mutations resulted in a conformational change, suggesting that they might confer a selective advantage in systemic movement within the vine. Overall, these data reveal that bottlenecks occur during systemic movement, that variants circulate in the phloem sap throughout the infection process, and that important conformational changes in CI protein may arise during individual infections. PMID:25107623
Neural Stem Cells Derived Directly from Adipose Tissue.

PubMed

Petersen, Eric D; Zenchak, Jessica R; Lossia, Olivia V; Hochgeschwender, Ute

2018-05-01

Neural stem cells (NSCs) are characterized as self-renewing cell populations with the ability to differentiate into the multiple tissue types of the central nervous system. These cells can differentiate into mature neurons, astrocytes, and oligodendrocytes. This category of stem cells has been shown to be a promisingly effective treatment for neurodegenerative diseases and neuronal injury. Most treatment studies with NSCs in animal models use embryonic brain-derived NSCs. This approach presents both ethical and feasibility issues for translation to human patients. Adult tissue is a more practical source of stem cells for transplantation therapies in humans. Some adult tissues such as adipose tissue and bone marrow contain a wide variety of stem cell populations, some of which have been shown to be similar to embryonic stem cells, possessing many pluripotent properties. Of these stem cell populations, some are able to respond to neuronal growth factors and can be expanded in vitro, forming neurospheres analogous to cells harvested from embryonic brain tissue. In this study, we describe a method for the collection and culture of cells from adipose tissue that directly, without going through intermediates such as mesenchymal stem cells, results in a population of NSCs that are able to be expanded in vitro and be differentiated into functional neuronal cells. These adipose-derived NSCs display a similar phenotype to those directly derived from embryonic brain. When differentiated into neurons, cells derived from adipose tissue have spontaneous spiking activity with network characteristics similar to that of neuronal cultures.
Cells Respond to Distinct Nanoparticle Properties with Multiple Strategies As Revealed by Single-Cell RNA-Seq

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.

The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells “overloaded” while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. Here we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantum dots (QDs),more » showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with upregulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly downregulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong upregulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis and organelle activities. In contrast, strategies unique to carboxylated QDs showed upregulation of DNA repair and RNA activities, and decreased regulation of cell division, coupled in some cases with upregulation of stress responses and ATP related functions. Together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified, proactive defenses or repairs of the NP insults.« less
Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B-GFP transgenic mice.

PubMed

Chen, Mei-Shu; Lin, Hua-Kuo; Chiu, Hsun; Lee, Don-Ching; Chung, Yu-Fen; Chiu, Ing-Ming

2015-03-01

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B-GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (-540 to +31). Using the fresh brain sections of F1B-GFP transgenic mice, we found that the F1B-GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B-GFP(+) cells existed in the brains of F1B-GFP transgenic mice. We demonstrated that one population of F1B-GFP(+) cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B-GFP(+) cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B-GFP(+) cells and tyrosine hydroxylase indicated that a subpopulation of F1B-GFP(+) -neuronal cells was dopaminergic neurons. Importantly, these F1B-GFP(+) /TH(+) cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B-GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. © 2014 The Authors Developmental Neurobiology Published by Wiley Periodicals, Inc.
Coreceptors and Their Ligands in Epithelial γδ T Cell Biology

PubMed Central

Witherden, Deborah A.; Johnson, Margarete D.; Havran, Wendy L.

2018-01-01

Epithelial tissues line the body providing a protective barrier from the external environment. Maintenance of these epithelial barrier tissues critically relies on the presence of a functional resident T cell population. In some tissues, the resident T cell population is exclusively comprised of γδ T cells, while in others γδ T cells are found together with αβ T cells and other lymphocyte populations. Epithelial-resident γδ T cells function not only in the maintenance of the epithelium, but are also central to the repair process following damage from environmental and pathogenic insults. Key to their function is the crosstalk between γδ T cells and neighboring epithelial cells. This crosstalk relies on multiple receptor–ligand interactions through both the T cell receptor and accessory molecules leading to temporal and spatial regulation of cytokine, chemokine, growth factor, and extracellular matrix protein production. As antigens that activate epithelial γδ T cells are largely unknown and many classical costimulatory molecules and coreceptors are not used by these cells, efforts have focused on identification of novel coreceptors and ligands that mediate pivotal interactions between γδ T cells and their neighbors. In this review, we discuss recent advances in the understanding of functions for these coreceptors and their ligands in epithelial maintenance and repair processes. PMID:29686687
Multiple sclerosis in children: an update on clinical diagnosis, therapeutic strategies, and research

PubMed Central

Waldman, Amy; Ghezzi, Angelo; Bar-Or, Amit; Mikaeloff, Yann; Tardieu, Marc; Banwell, Brenda

2015-01-01

The clinical features, diagnostic challenges, neuroimaging appearance, therapeutic options, and pathobiological research progress in childhood—and adolescent—onset multiple sclerosis have been informed by many new insights in the past 7 years. National programmes in several countries, collaborative research efforts, and an established international paediatric multiple sclerosis study group have contributed to revised clinical diagnostic definitions, identified clinical features of multiple sclerosis that differ by age of onset, and made recommendations regarding the treatment of paediatric multiple sclerosis. The relative risks conveyed by genetic and environmental factors to paediatric multiple sclerosis have been the subject of several large cohort studies. MRI features have been characterised in terms of qualitative descriptions of lesion distribution and applicability of MRI aspects to multiple sclerosis diagnostic criteria, and quantitative studies have assessed total lesion burden and the effect of the disease on global and regional brain volume. Humoral-based and cell-based assays have identified antibodies against myelin, potassium-channel proteins, and T-cell profiles that support an adult-like T-cell repertoire and cellular reactivity against myelin in paediatric patients with multiple sclerosis. Finally, the safety and efficacy of standard first-line therapies in paediatric multiple sclerosis populations are now appreciated in more detail, and consensus views on the future conduct and feasibility of phase 3 trials for new drugs have been proposed. PMID:25142460
Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord.

PubMed

Keirstead, H S; Levine, J M; Blakemore, W F

1998-02-01

Elucidation of the response of oligodendrocyte progenitor cell populations to demyelination in the adult central nervous system (CNS) is critical to understanding why remyelination fails in multiple sclerosis. Using the anti-NG2 monoclonal antibody to identify oligodendrocyte progenitor cells, we have documented their response to antibody-induced demyelination in the dorsal column of the adult rat spinal cord. The number of NG2+ cells in the vicinity of demyelinated lesions increased by 72% over the course of 3 days following the onset of demyelination. This increase in NG2+ cell numbers did not reflect a nonspecific staining of reactive cells, as GFAP, OX-42, and Rip antibodies did not co-localise with NG2 + cells in double immunostained tissue sections. NG2 + cells incorporated BrdU 48-72 h following the onset of demyelination. After the onset of remyelination (10-14 days), the number of NG2+ cells decreased to 46% of control levels and remained consistently low for 2 months. When spinal cords were exposed to 40 Grays of x-irradiation prior to demyelination, the number of NG2+ cells decreased to 48% of control levels by 3 days following the onset of demyelination and remained unchanged at 3 weeks. Since 40 Grays of x-irradiation kills dividing cells, these studies illustrate a responsive and nonresponsive NG2+ cell population following demyelination in the adult spinal cord and suggest that the responsive NG2+ cell population does not renew itself.
Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

PubMed Central

Miyazaki, Kaoru; Maruyama, Tetsuo; Masuda, Hirotaka; Yamasaki, Akiko; Uchida, Sayaka; Oda, Hideyuki; Uchida, Hiroshi; Yoshimura, Yasunori

2012-01-01

Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue reconstitution. Using this assay, we demonstrated that ESP cells differentiated into multiple endometrial lineages in the niche provided by whole endometrial cells, indicating that ESP cells are genuine endometrial stem/progenitor cells. PMID:23226538
Simultaneous profiling of activity patterns in multiple neuronal subclasses.

PubMed

Parrish, R Ryley; Grady, John; Codadu, Neela K; Trevelyan, Andrew J; Racca, Claudia

2018-06-01

Neuronal networks typically comprise heterogeneous populations of neurons. A core objective when seeking to understand such networks, therefore, is to identify what roles these different neuronal classes play. Acquiring single cell electrophysiology data for multiple cell classes can prove to be a large and daunting task. Alternatively, Ca 2+ network imaging provides activity profiles of large numbers of neurons simultaneously, but without distinguishing between cell classes. We therefore developed a strategy for combining cellular electrophysiology, Ca 2+ network imaging, and immunohistochemistry to provide activity profiles for multiple cell classes at once. This involves cross-referencing easily identifiable landmarks between imaging of the live and fixed tissue, and then using custom MATLAB functions to realign the two imaging data sets, to correct for distortions of the tissue introduced by the fixation or immunohistochemical processing. We illustrate the methodology for analyses of activity profiles during epileptiform events recorded in mouse brain slices. We further demonstrate the activity profile of a population of parvalbumin-positive interneurons prior, during, and following a seizure-like event. Current approaches to Ca 2+ network imaging analyses are severely limited in their ability to subclassify neurons, and often rely on transgenic approaches to identify cell classes. In contrast, our methodology is a generic, affordable, and flexible technique to characterize neuronal behaviour with respect to classification based on morphological and neurochemical identity. We present a new approach for analysing Ca 2+ network imaging datasets, and use this to explore the parvalbumin-positive interneuron activity during epileptiform events. Copyright © 2018 Elsevier B.V. All rights reserved.

Clustering single cells: a review of approaches on high-and low-depth single-cell RNA-seq data.

PubMed

Menon, Vilas

2017-12-11

Advances in single-cell RNA-sequencing technology have resulted in a wealth of studies aiming to identify transcriptomic cell types in various biological systems. There are multiple experimental approaches to isolate and profile single cells, which provide different levels of cellular and tissue coverage. In addition, multiple computational strategies have been proposed to identify putative cell types from single-cell data. From a data generation perspective, recent single-cell studies can be classified into two groups: those that distribute reads shallowly over large numbers of cells and those that distribute reads more deeply over a smaller cell population. Although there are advantages to both approaches in terms of cellular and tissue coverage, it is unclear whether different computational cell type identification methods are better suited to one or the other experimental paradigm. This study reviews three cell type clustering algorithms, each representing one of three broad approaches, and finds that PCA-based algorithms appear most suited to low read depth data sets, whereas gene clustering-based and biclustering algorithms perform better on high read depth data sets. In addition, highly related cell classes are better distinguished by higher-depth data, given the same total number of reads; however, simultaneous discovery of distinct and similar types is better served by lower-depth, higher cell number data. Overall, this study suggests that the depth of profiling should be determined by initial assumptions about the diversity of cells in the population, and that the selection of clustering algorithm(s) is subsequently based on the depth of profiling will allow for better identification of putative transcriptomic cell types. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Ethnically diverse pluripotent stem cells for drug development.

PubMed

Fakunle, Eyitayo S; Loring, Jeanne F

2012-12-01

Genetic variation is an identified factor underlying drug efficacy and toxicity, and adverse drug reactions, such as liver toxicity, are the primary reasons for post-marketing drug failure. Genetic predisposition to toxicity might be detected early in the drug development pipeline by introducing cell-based assays that reflect the genetic and ethnic variation of the expected treatment population. One challenge for this approach is obtaining a collection of suitable cell lines derived from ethnically diverse populations. Induced pluripotent stem cells (iPSCs) seem ideal for this purpose. They can be obtained from any individual, can be differentiated into multiple relevant cell types, and their self-renewal capability makes it possible to generate large quantities of quality-controlled cell types. Here, we discuss the benefits and challenges of using iPSCs to introduce genetic diversity into the drug development process. Copyright © 2012 Elsevier Ltd. All rights reserved.
CD44 Staining of Cancer Stem-Like Cells Is Influenced by Down-Regulation of CD44 Variant Isoforms and Up-Regulation of the Standard CD44 Isoform in the Population of Cells That Have Undergone Epithelial-to-Mesenchymal Transition

PubMed Central

Biddle, Adrian; Gammon, Luke; Fazil, Bilal; Mackenzie, Ian C.

2013-01-01

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed. PMID:23437366
Developmental Origin Governs CD8+ T Cell Fate Decisions during Infection.

PubMed

Smith, Norah L; Patel, Ravi K; Reynaldi, Arnold; Grenier, Jennifer K; Wang, Jocelyn; Watson, Neva B; Nzingha, Kito; Yee Mon, Kristel J; Peng, Seth A; Grimson, Andrew; Davenport, Miles P; Rudd, Brian D

2018-06-06

Heterogeneity is a hallmark feature of the adaptive immune system in vertebrates. Following infection, naive T cells differentiate into various subsets of effector and memory T cells, which help to eliminate pathogens and maintain long-term immunity. The current model suggests there is a single lineage of naive T cells that give rise to different populations of effector and memory T cells depending on the type and amounts of stimulation they encounter during infection. Here, we have discovered that multiple sub-populations of cells exist in the naive CD8 + T cell pool that are distinguished by their developmental origin, unique transcriptional profiles, distinct chromatin landscapes, and different kinetics and phenotypes after microbial challenge. These data demonstrate that the naive CD8 + T cell pool is not as homogeneous as previously thought and offers a new framework for explaining the remarkable heterogeneity in the effector and memory T cell subsets that arise after infection. Copyright © 2018 Elsevier Inc. All rights reserved.
Transcriptomic study on persistence and survival of Listeria monocytogenes following lethal treatment with Nisin.

PubMed

Wu, Shuyan; Yu, Pak-Lam; Wheeler, Dave; Flint, Steve

2018-06-19

The aim of this study was to determine the gene expression associated with the persistence of a Listeria monocytogenes stationary phase population when facing lethal nisin treatment METHODS: RNA Seq analysis was used for gene expression profiling of the persister cells in rich medium (persister TN) compared with untreated cells (non-persister).The results were confirmed using RT PCR. Functional genes associated with the persister populations were identified in multiple systems, such as heat shock related stress response, cell wall synthesis, ATP-binding cassette (ABC) transport system, phosphotransferase system (PTS system), and SOS/DNA repair. This study pointed to genetic regulation of persister cells exposed to lethal nisin and provides some insight into possible mechanisms of impeding bacterial persistence. Copyright © 2018. Published by Elsevier Ltd.
Modeling population dynamics of mitochondria in mammalian cells

NASA Astrophysics Data System (ADS)

Kornick, Kellianne; Das, Moumita

Mitochondria are organelles located inside eukaryotic cells and are essential for several key cellular processes such as energy (ATP) production, cell signaling, differentiation, and apoptosis. All organisms are believed to have low levels of variation in mitochondrial DNA (mtDNA), and alterations in mtDNA are connected to a range of human health conditions, including epilepsy, heart failure, Parkinsons disease, diabetes, and multiple sclerosis. Therefore, understanding how changes in mtDNA accumulate over time and are correlated to changes in mitochondrial function and cell properties can have a profound impact on our understanding of cell physiology and the origins of some diseases. Motivated by this, we develop and study a mathematical model to determine which cellular parameters have the largest impact on mtDNA population dynamics. The model consists of coupled ODEs to describe subpopulations of healthy and dysfunctional mitochondria subject to mitochondrial fission, fusion, autophagy, and mutation. We study the time evolution and stability of each sub-population under specific selection biases and pressures by tuning specific terms in our model. Our results may provide insights into how sub-populations of mitochondria survive and evolve under different selection pressures. This work was supported by a Grant from the Moore Foundation.
Identical bacterial populations colonize premature infant gut, skin, and oral microbiomes and exhibit different in situ growth rates

PubMed Central

Olm, Matthew R.; Brown, Christopher T.; Brooks, Brandon; Firek, Brian; Baker, Robyn; Burstein, David; Soenjoyo, Karina; Thomas, Brian C.; Morowitz, Michael; Banfield, Jillian F.

2017-01-01

The initial microbiome impacts the health and future development of premature infants. Methodological limitations have led to gaps in our understanding of the habitat range and subpopulation complexity of founding strains, as well as how different body sites support microbial growth. Here, we used metagenomics to reconstruct genomes of strains that colonized the skin, mouth, and gut of two hospitalized premature infants during the first month of life. Seven bacterial populations, considered to be identical given whole-genome average nucleotide identity of >99.9%, colonized multiple body sites, yet none were shared between infants. Gut-associated Citrobacter koseri genomes harbored 47 polymorphic sites that we used to define 10 subpopulations, one of which appeared in the gut after 1 wk but did not spread to other body sites. Differential genome coverage was used to measure bacterial population replication rates in situ. In all cases where the same bacterial population was detected in multiple body sites, replication rates were faster in mouth and skin compared to the gut. The ability of identical strains to colonize multiple body sites underscores the habit flexibility of initial colonists, whereas differences in microbial replication rates between body sites suggest differences in host control and/or resource availability. Population genomic analyses revealed microdiversity within bacterial populations, implying initial inoculation by multiple individual cells with distinct genotypes. Overall, however, the overlap of strains across body sites implies that the premature infant microbiome can exhibit very low microbial diversity. PMID:28073918
Universal Temporal Profile of Replication Origin Activation in Eukaryotes

NASA Astrophysics Data System (ADS)

Goldar, Arach

2011-03-01

The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.
Development of a population of cancer cells: Observation and modeling by a Mixed Spatial Evolutionary Games approach.

PubMed

Świerniak, Andrzej; Krześlak, Michał; Student, Sebastian; Rzeszowska-Wolny, Joanna

2016-09-21

Living cells, like whole living organisms during evolution, communicate with their neighbors, interact with the environment, divide, change their phenotypes, and eventually die. The development of specific ways of communication (through signaling molecules and receptors) allows some cellular subpopulations to survive better, to coordinate their physiological status, and during embryonal development to create tissues and organs or in some conditions to become tumors. Populations of cells cultured in vitro interact similarly, also competing for space and nutrients and stimulating each other to better survive or to die. The results of these intercellular interactions of different types seem to be good examples of biological evolutionary games, and have been the subjects of simulations by the methods of evolutionary game theory where individual cells are treated as players. Here we present examples of intercellular contacts in a population of living human cancer HeLa cells cultured in vitro and propose an evolutionary game theory approach to model the development of such populations. We propose a new technique termed Mixed Spatial Evolutionary Games (MSEG) which are played on multiple lattices corresponding to the possible cellular phenotypes which gives the possibility of simulating and investigating the effects of heterogeneity at the cellular level in addition to the population level. Analyses performed with MSEG suggested different ways in which cellular populations develop in the case of cells communicating directly and through factors released to the environment. Copyright © 2016 Elsevier Ltd. All rights reserved.
Immune Response to a Variable Pathogen: A Stochastic Model with Two Interlocked Darwinian Entities

PubMed Central

Kuhn, Christoph

2012-01-01

This paper presents the modeling of a host immune system, more precisely the immune effector cell and immune memory cell population, and its interaction with an invading pathogen population. It will tackle two issues of interest; on the one hand, in defining a stochastic model accounting for the inherent nature of organisms in population dynamics, namely multiplication with mutation and selection; on the other hand, in providing a description of pathogens that may vary their antigens through mutations during infection of the host. Unlike most of the literature, which models the dynamics with first-order differential equations, this paper proposes a Galton-Watson type branching process to describe stochastically by whole distributions the population dynamics of pathogens and immune cells. In the first model case, the pathogen of a given type is either eradicated or shows oscillatory chronic response. In the second model case, the pathogen shows variational behavior changing its antigen resulting in a prolonged immune reaction. PMID:23424603
Immune response to a variable pathogen: a stochastic model with two interlocked Darwinian entities.

PubMed

Kuhn, Christoph

2012-01-01

This paper presents the modeling of a host immune system, more precisely the immune effector cell and immune memory cell population, and its interaction with an invading pathogen population. It will tackle two issues of interest; on the one hand, in defining a stochastic model accounting for the inherent nature of organisms in population dynamics, namely multiplication with mutation and selection; on the other hand, in providing a description of pathogens that may vary their antigens through mutations during infection of the host. Unlike most of the literature, which models the dynamics with first-order differential equations, this paper proposes a Galton-Watson type branching process to describe stochastically by whole distributions the population dynamics of pathogens and immune cells. In the first model case, the pathogen of a given type is either eradicated or shows oscillatory chronic response. In the second model case, the pathogen shows variational behavior changing its antigen resulting in a prolonged immune reaction.
Interrogation of inhibitor of nuclear factor κB α/nuclear factor κB (IκBα/NF-κB) negative feedback loop dynamics: from single cells to live animals in vivo.

PubMed

Moss, Britney L; Elhammali, Adnan; Fowlkes, Tiffanie; Gross, Shimon; Vinjamoori, Anant; Contag, Christopher H; Piwnica-Worms, David

2012-09-07

Full understanding of the biological significance of negative feedback processes requires interrogation at multiple scales as follows: in single cells, cell populations, and live animals in vivo. The transcriptionally coupled IκBα/NF-κB negative feedback loop, a pivotal regulatory node of innate immunity and inflammation, represents a model system for multiscalar reporters. Using a κB(5)→IκBα-FLuc bioluminescent reporter, we rigorously evaluated the dynamics of ΙκBα degradation and subsequent NF-κB transcriptional activity in response to diverse modes of TNFα stimulation. Modulating TNFα concentration or pulse duration yielded complex, reproducible, and differential ΙκBα dynamics in both cell populations and live single cells. Tremendous heterogeneity in the transcriptional amplitudes of individual responding cells was observed, which was greater than the heterogeneity in the transcriptional kinetics of responsive cells. Furthermore, administration of various TNFα doses in vivo generated ΙκBα dynamic profiles in the liver resembling those observed in single cells and populations of cells stimulated with TNFα pulses. This suggested that dose modulation of circulating TNFα was perceived by hepatocytes in vivo as pulses of increasing duration. Thus, a robust bioluminescent reporter strategy enabled rigorous quantitation of NF-κB/ΙκBα dynamics in both live single cells and cell populations and furthermore, revealed reproducible behaviors that informed interpretation of in vivo studies.
Multiple joint metastasis of a transitional cell carcinoma in a dog.

PubMed

Colledge, Sarah L; Raskin, Rose E; Messick, Joanne B; Tiffany Reed, L; Wigle, William L; Balog, Kelley A

2013-06-01

An 8-year-old castrated male hound mix was referred to the Purdue University Veterinary Teaching Hospital for severe lameness, pollakiuria, and dyschezia. On presentation, the dog was nonweight bearing on the right rear limb and the right carpus was diffusely swollen. Synovial fluid analysis from the right carpus revealed a population of epithelial cells displaying marked anisocytosis, anisokaryosis, multinucleation, and prominent, variably sized nucleoli. A metastatic carcinoma with presumed prostatic or urothelial origin was diagnosed based on cytomorphology. Subsequent cytologic evaluation of peripheral lymph nodes revealed the presence of a similar neoplastic population. The dog was euthanized and synovial fluid from both stifle joints, as well as impression smears of the prostate gland, were collected. Carcinoma cells were identified in each stifle joint and in the prostate gland. Immunocytochemistry was performed on synovial fluid smears from 2 of the joints (right stifle and right carpus) and on impression smears of the prostate gland. The neoplastic population in the joints and prostate gland showed strong immunoreactivity to uroplakin III, a urothelial marker, indicating metastasis of a transitional cell carcinoma to multiple joints. In addition, evidence for epithelial to mesenchymal transition was identified using cytokeratin, an epithelial marker, and vimentin, a mesenchymal marker. A necropsy was performed and histopathology confirmed the presence of metastatic transitional cell carcinoma in various tissues. This case illustrates the importance of considering metastatic disease when a patient is presented with severe lameness and joint pain, and the clinical utility of synovial fluid cytology for diagnosis of metastasis in these cases. © 2013 American Society for Veterinary Clinical Pathology.
Inertial-ordering-assisted droplet microfluidics for high-throughput single-cell RNA-sequencing.

PubMed

Moon, Hui-Sung; Je, Kwanghwi; Min, Jae-Woong; Park, Donghyun; Han, Kyung-Yeon; Shin, Seung-Ho; Park, Woong-Yang; Yoo, Chang Eun; Kim, Shin-Hyun

2018-02-27

Single-cell RNA-seq reveals the cellular heterogeneity inherent in the population of cells, which is very important in many clinical and research applications. Recent advances in droplet microfluidics have achieved the automatic isolation, lysis, and labeling of single cells in droplet compartments without complex instrumentation. However, barcoding errors occurring in the cell encapsulation process because of the multiple-beads-in-droplet and insufficient throughput because of the low concentration of beads for avoiding multiple-beads-in-a-droplet remain important challenges for precise and efficient expression profiling of single cells. In this study, we developed a new droplet-based microfluidic platform that significantly improved the throughput while reducing barcoding errors through deterministic encapsulation of inertially ordered beads. Highly concentrated beads containing oligonucleotide barcodes were spontaneously ordered in a spiral channel by an inertial effect, which were in turn encapsulated in droplets one-by-one, while cells were simultaneously encapsulated in the droplets. The deterministic encapsulation of beads resulted in a high fraction of single-bead-in-a-droplet and rare multiple-beads-in-a-droplet although the bead concentration increased to 1000 μl -1 , which diminished barcoding errors and enabled accurate high-throughput barcoding. We successfully validated our device with single-cell RNA-seq. In addition, we found that multiple-beads-in-a-droplet, generated using a normal Drop-Seq device with a high concentration of beads, underestimated transcript numbers and overestimated cell numbers. This accurate high-throughput platform can expand the capability and practicality of Drop-Seq in single-cell analysis.
Intra-genomic variation in symbiotic dinoflagellates: recent divergence or recombination between lineages?

PubMed

Wilkinson, Shaun P; Fisher, Paul L; van Oppen, Madeleine J H; Davy, Simon K

2015-03-14

The symbiosis between corals and the dinoflagellate alga Symbiodinium is essential for the development and survival of coral reefs. Yet this fragile association is highly vulnerable to environmental disturbance. A coral's ability to tolerate temperature stress depends on the fitness of its resident symbionts, whose thermal optima vary extensively between lineages. However, the in hospite population genetic structure of Symbiodinium is poorly understood and mostly based on analysis of bulk DNA extracted from thousands to millions of cells. Using quantitative single-cell PCR, we enumerated DNA polymorphisms in the symbionts of the reef-building coral Pocillopora damicornis, and applied a model selection approach to explore the potential for recombination between coexisting Symbiodinium populations. Two distinct Symbiodinium ITS2 sequences (denoted C100 and C109) were retrieved from all P. damicornis colonies analysed. However, the symbiont assemblage consisted of three distinct Symbiodinium populations: cells featuring pure arrays of ITS2 type C109, near-homogeneous cells of type C100 (with trace ITS2 copies of type C109), and those with co-dominant C100 and C109 ITS2 repeats. The symbiont consortia of some colonies consisted almost entirely of these putative C100 × C109 recombinants. Our results are consistent with the occurrence of sexual recombination between Symbiodinium types C100 and C109. While the multiple-copy nature of the ITS2 dictates that the observed pattern of intra-genomic co-dominance may be a result of incomplete concerted evolution of intra-genomic polymorphisms, this is a less likely explanation given the occurrence of homogeneous cells of the C109 type. Conclusive evidence for inter-lineage recombination and introgression in this genus will require either direct observational evidence or a single-cell genotyping approach targeting multiple, single-copy loci.
Harnessing the apoptotic programs in cancer stem-like cells

PubMed Central

Wang, Ying-Hua; Scadden, David T

2015-01-01

Elimination of malignant cells is an unmet challenge for most human cancer types even with therapies targeting specific driver mutations. Therefore, a multi-pronged strategy to alter cancer cell biology on multiple levels is increasingly recognized as essential for cancer cure. One such aspect of cancer cell biology is the relative apoptosis resistance of tumor-initiating cells. Here, we provide an overview of the mechanisms affecting the apoptotic process in tumor cells emphasizing the differences in the tumor-initiating or stem-like cells of cancer. Further, we summarize efforts to exploit these differences to design therapies targeting that important cancer cell population. PMID:26253117
Basal cell carcinoma preferentially arises from stem cells within hair follicle and mechanosensory niches.

PubMed

Peterson, Shelby C; Eberl, Markus; Vagnozzi, Alicia N; Belkadi, Abdelmadjid; Veniaminova, Natalia A; Verhaegen, Monique E; Bichakjian, Christopher K; Ward, Nicole L; Dlugosz, Andrzej A; Wong, Sunny Y

2015-04-02

Basal cell carcinoma (BCC) is characterized by frequent loss of PTCH1, leading to constitutive activation of the Hedgehog pathway. Although the requirement for Hedgehog in BCC is well established, the identity of disease-initiating cells and the compartments in which they reside remain controversial. By using several inducible Cre drivers to delete Ptch1 in different cell compartments in mice, we show here that multiple hair follicle stem cell populations readily develop BCC-like tumors. In contrast, stem cells within the interfollicular epidermis do not efficiently form tumors. Notably, we observed that innervated Gli1-expressing progenitors within mechanosensory touch dome epithelia are highly tumorigenic. Sensory nerves activate Hedgehog signaling in normal touch domes, while denervation attenuates touch dome-derived tumors. Together, our studies identify varying tumor susceptibilities among different stem cell populations in the skin, highlight touch dome epithelia as "hot spots" for tumor formation, and implicate cutaneous nerves as mediators of tumorigenesis. Copyright © 2015 Elsevier Inc. All rights reserved.
Basal cell carcinoma preferentially arises from stem cells within hair follicle and mechanosensory niches

PubMed Central

Peterson, Shelby C.; Eberl, Markus; Vagnozzi, Alicia N.; Belkadi, Abdelmadjid; Veniaminova, Natalia A.; Verhaegen, Monique E.; Bichakjian, Christopher K.; Ward, Nicole L.; Dlugosz, Andrzej A.; Wong, Sunny Y.

2015-01-01

SUMMARY Basal cell carcinoma (BCC) is characterized by frequent loss of PTCH1, leading to constitutive activation of the Hedgehog pathway. Although the requirement for Hedgehog in BCC is well-established, the identity of disease-initiating cells and the compartments in which they reside remain controversial. By using several inducible Cre drivers to delete Ptch1 in different cell compartments in mice, we show here that multiple hair follicle stem cell populations readily develop BCC-like tumors. In contrast, stem cells within the interfollicular epidermis do not efficiently form tumors. Notably, we observed that innervated Gli1-expressing progenitors within mechanosensory touch dome epithelia are highly tumorigenic. Sensory nerves activate Hedgehog signaling in normal touch domes, while denervation attenuates touch dome-derived tumors. Together, our studies identify varying tumor susceptibilities among different stem cell populations in the skin, highlight touch dome epithelia as “hot spots” for tumor formation, and implicate cutaneous nerves as mediators of tumorigenesis. PMID:25842978
Early detection of disease program: Evaluation of the cellular immune response

NASA Technical Reports Server (NTRS)

Criswell, B. S.; Knight, V.; Martin, R. R.; Kasel, J. A.

1974-01-01

The early cellular responses of specific components of the leukocyte and epithelial cell populations to foreign challenges of both an infectious and noninfectious character were evaluated. Procedures for screening potential flight crews were developed, documented, and tested on a control population. Methods for preparing suitable populations of lymphocytes, polymorphonuclear leukocytes, macrophages, and epithelial cells were first established and evaluated. Epithelial cells from viral infected individuals were screened with a number of anti-viral antisera. This procedure showed the earliest indication of disease as well as providing a specific diagnosis to the physicians. Both macrophages and polymorphonuclear leukocytes were studied from normal individuals, smokers, and patients with viral infections. Newer techniques enabling better definition of lymphocyte subpopulations were then developed, namely the E and EAC rosette procedures for recognition of T (thymus-derived) and B (bone-marrow-derived) lymphocyte subpopulations. Lymphocyte and lymphocyte subpopulation response to multiple mitogens have been evaluated.
The Development of Ciprofloxacin Resistance in Pseudomonas aeruginosa Involves Multiple Response Stages and Multiple Proteins ▿ † ‡

PubMed Central

Su, Hsun-Cheng; Ramkissoon, Kevin; Doolittle, Janet; Clark, Martha; Khatun, Jainab; Secrest, Ashley; Wolfgang, Matthew C.; Giddings, Morgan C.

2010-01-01

Microbes have developed resistance to nearly every antibiotic, yet the steps leading to drug resistance remain unclear. Here we report a multistage process by which Pseudomonas aeruginosa acquires drug resistance following exposure to ciprofloxacin at levels ranging from 0.5× to 8× the initial MIC. In stage I, susceptible cells are killed en masse by the exposure. In stage II, a small, slow to nongrowing population survives antibiotic exposure that does not exhibit significantly increased resistance according to the MIC measure. In stage III, exhibited at 0.5× to 4× the MIC, a growing population emerges to reconstitute the population, and these cells display heritable increases in drug resistance of up to 50 times the original level. We studied the stage III cells by proteomic methods to uncover differences in the regulatory pathways that are involved in this phenotype, revealing upregulation of phosphorylation on two proteins, succinate-semialdehyde dehydrogenase (SSADH) and methylmalonate-semialdehyde dehydrogenase (MMSADH), and also revealing upregulation of a highly conserved protein of unknown function. Transposon disruption in the encoding genes for each of these targets substantially dampened the ability of cells to develop the stage III phenotype. Considering these results in combination with computational models of resistance and genomic sequencing results, we postulate that stage III heritable resistance develops from a combination of both genomic mutations and modulation of one or more preexisting cellular pathways. PMID:20696867

Each cell counts: Hematopoiesis and immunity research in the era of single cell genomics.

PubMed

Jaitin, Diego Adhemar; Keren-Shaul, Hadas; Elefant, Naama; Amit, Ido

2015-02-01

Hematopoiesis and immunity are mediated through complex interactions between multiple cell types and states. This complexity is currently addressed following a reductionist approach of characterizing cell types by a small number of cell surface molecular features and gross functions. While the introduction of global transcriptional profiling technologies enabled a more comprehensive view, heterogeneity within sampled populations remained unaddressed, obscuring the true picture of hematopoiesis and immune system function. A critical mass of technological advances in molecular biology and genomics has enabled genome-wide measurements of single cells - the fundamental unit of immunity. These new advances are expected to boost detection of less frequent cell types and fuzzy intermediate cell states, greatly expanding the resolution of current available classifications. This new era of single-cell genomics in immunology research holds great promise for further understanding of the mechanisms and circuits regulating hematopoiesis and immunity in both health and disease. In the near future, the accuracy of single-cell genomics will ultimately enable precise diagnostics and treatment of multiple hematopoietic and immune related diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.
SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture.

PubMed

Arhoma, A; Chantry, A D; Haywood-Small, S L; Cross, N A

2017-11-15

Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL-induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate-based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter-Glo luminescence assay, Caspase-8 and -9 activities were measured by Caspase-Glo™ assay kit. TRAIL-resistant cells were generated by culture of RPMI 8226 and NCI-H929 by acute exposure to TRAIL followed by selection of TRAIL-resistant cells. TRAIL significantly induced apoptosis in a dose-dependent manner in OPM-2, RPMI 8226, NCI-H929, U266, JJN-3 MM cell lines and ADC-1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM-2, and enhanced TRAIL responses were both via Caspase-8 and -9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co-treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI-H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL-sensitive cell populations that had been selected for TRAIL-resistance from initially TRAIL-sensitive populations. SAHA may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance. Copyright © 2017 Elsevier Inc. All rights reserved.
Cells respond to distinct nanoparticle properties with multiple strategies as revealed by single-cell RNA-Seq

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.

The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells “overloaded” while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. In this paper, we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantummore » dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with up-regulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly down-regulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong up-regulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis, and organelle activities. In contrast, strategies unique to carboxylated QDs showed up-regulation of DNA repair and RNA activities and decreased regulation of cell division, coupled in some cases with up-regulation of stress responses and ATP-related functions. Finally, together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified proactive defenses or repairs of the NP insults.« less
Cells respond to distinct nanoparticle properties with multiple strategies as revealed by single-cell RNA-Seq

DOE PAGES

Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.; ...

2016-10-27

The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells “overloaded” while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. In this paper, we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantummore » dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with up-regulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly down-regulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong up-regulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis, and organelle activities. In contrast, strategies unique to carboxylated QDs showed up-regulation of DNA repair and RNA activities and decreased regulation of cell division, coupled in some cases with up-regulation of stress responses and ATP-related functions. Finally, together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified proactive defenses or repairs of the NP insults.« less
Metabolic reprogramming as a novel regulator of skeletal muscle development and regeneration.

PubMed

Ryall, James G

2013-09-01

Adult skeletal muscle contains a resident population of stem cells, termed satellite cells, that exist in a quiescent state. In response to an activating signal (such as physical trauma), satellite cells enter the cell cycle and undergo multiple rounds of proliferation, followed by differentiation, fusion, and maturation. Over the last 10-15 years, our understanding of the transcriptional regulation of this stem cell population has greatly expanded, but there remains a dearth of knowledge with regard to the initiating signal leading to these changes in transcription. The recent renewed interest in the metabolic regulation of both cancer and stem cells, combined with previous findings indicating that satellite cells preferentially colocalize with blood vessels, suggests that satellite cell function may be regulated by changes in cellular metabolism. This review aims to describe what is currently known about satellite cell metabolism during changes in cell fate, as well as to describe some of the exciting findings in other cell types and how these might relate to satellite cells. © 2013 The Author Journal compilation © 2013 FEBS.
Roles for Hedgehog signaling in adult organ homeostasis and repair

PubMed Central

Petrova, Ralitsa; Joyner, Alexandra L.

2014-01-01

The hedgehog (HH) pathway is well known for its mitogenic and morphogenic functions during development, and HH signaling continues in discrete populations of cells within many adult mammalian tissues. Growing evidence indicates that HH regulates diverse quiescent stem cell populations, but the exact roles that HH signaling plays in adult organ homeostasis and regeneration remain poorly understood. Here, we review recently identified functions of HH in modulating the behavior of tissue-specific adult stem and progenitor cells during homeostasis, regeneration and disease. We conclude that HH signaling is a key factor in the regulation of adult tissue homeostasis and repair, acting via multiple different routes to regulate distinct cellular outcomes, including maintenance of plasticity, in a context-dependent manner. PMID:25183867
Deciphering the landscape of host barriers to Listeria monocytogenes infection.

PubMed

Zhang, Ting; Abel, Sören; Abel Zur Wiesch, Pia; Sasabe, Jumpei; Davis, Brigid M; Higgins, Darren E; Waldor, Matthew K

2017-06-13

Listeria monocytogenes is a common food-borne pathogen that can disseminate from the intestine and infect multiple organs. Here, we used sequence tag-based analysis of microbial populations (STAMP) to investigate L monocytogenes population dynamics during infection. We created a genetically barcoded library of murinized L monocytogenes and then used deep sequencing to track the pathogen's dissemination routes and quantify its founding population ( N b ) sizes in different organs. We found that the pathogen disseminates from the gastrointestinal tract to distal sites through multiple independent routes and that N b sizes vary greatly among tissues, indicative of diverse host barriers to infection. Unexpectedly, comparative analyses of sequence tags revealed that fecally excreted organisms are largely derived from the very small number of L. monocytogenes cells that colonize the gallbladder. Immune depletion studies suggest that distinct innate immune cells restrict the pathogen's capacity to establish replicative niches in the spleen and liver. Finally, studies in germ-free mice suggest that the microbiota plays a critical role in the development of the splenic, but not the hepatic, barriers that prevent L. monocytogenes from seeding these organs. Collectively, these observations illustrate the potency of the STAMP approach to decipher the impact of host factors on population dynamics of pathogens during infection.
Deciphering the landscape of host barriers to Listeria monocytogenes infection

PubMed Central

Zhang, Ting; Abel, Sören; Abel zur Wiesch, Pia; Sasabe, Jumpei; Davis, Brigid M.; Higgins, Darren E.; Waldor, Matthew K.

2017-01-01

Listeria monocytogenes is a common food-borne pathogen that can disseminate from the intestine and infect multiple organs. Here, we used sequence tag-based analysis of microbial populations (STAMP) to investigate L. monocytogenes population dynamics during infection. We created a genetically barcoded library of murinized L. monocytogenes and then used deep sequencing to track the pathogen’s dissemination routes and quantify its founding population (Nb) sizes in different organs. We found that the pathogen disseminates from the gastrointestinal tract to distal sites through multiple independent routes and that Nb sizes vary greatly among tissues, indicative of diverse host barriers to infection. Unexpectedly, comparative analyses of sequence tags revealed that fecally excreted organisms are largely derived from the very small number of L. monocytogenes cells that colonize the gallbladder. Immune depletion studies suggest that distinct innate immune cells restrict the pathogen’s capacity to establish replicative niches in the spleen and liver. Finally, studies in germ-free mice suggest that the microbiota plays a critical role in the development of the splenic, but not the hepatic, barriers that prevent L. monocytogenes from seeding these organs. Collectively, these observations illustrate the potency of the STAMP approach to decipher the impact of host factors on population dynamics of pathogens during infection. PMID:28559314
Management of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM).

PubMed

Kyle, Robert A; Rajkumar, S Vincent

2011-06-01

Monoclonal gammopathy of undetermined significance (MGUS) is defined as a serum M protein level of less than 3 g/dL, less than 10% clonal plasma cells in the bone marrow, and the absence of end-organ damage. The prevalence of MGUS is 3.2% in the white population but is approximately twice that high in the black population. MGUS may progress to multiple myeloma, AL amyloidosis, Waldenström macroglobulinemia, or lymphoma. The risk of progression is approximately 1% per year, but the risk continues even after more than 25 years of observation. Risk factors for progression include the size of the serum M protein, the type of serum M protein, the number of plasma cells in the bone marrow, and the serum free light chain ratio. Smoldering (asymptomatic) multiple myeloma (SMM) is characterized by the presence of an M protein level of 3 g/dL or higher and/or 10% or more monoclonal plasma cells in the bone marrow but no evidence of end-organ damage. The overall risk of progression to a malignant condition is 10% per year for the first 5 years, approximately 3% per year for the next 5 years, and 1% to 2% per year for the following 10 years. Patients with both MGUS and SMM must be followed up for their lifetime.
B cell biology: implications for treatment of systemic lupus erythematosus.

PubMed

Anolik, J H

2013-04-01

B cells are critical players in the orchestration of properly regulated immune responses, normally providing protective immunity without autoimmunity. Balance in the B cell compartment is achieved through the finely regulated participation of multiple B cell populations with different antibody-dependent and independent functions. Both types of functions allow B cells to modulate other components of the innate and adaptive immune system. Autoantibody-independent B cell functions include antigen presentation, T cell activation and polarization, and dendritic cell modulation. Several of these functions are mediated by the ability of B cells to produce immunoregulatory cytokines and chemokines and by their critical contribution to lymphoid tissue development and organization including the development of ectopic tertiary lymphoid tissue. Additionally, the functional versatility of B cells enables them to play either protective or pathogenic roles in autoimmunity. In turn, B cell dysfunction has been critically implicated in the pathophysiology of systemic lupus erythematosus (SLE), a complex disease characterized by the production of autoantibodies and heterogeneous clinical involvement. Thus, the breakdown of B cell tolerance is a defining and early event in the disease process and may occur by multiple pathways, including alterations in factors that affect B cell activation thresholds, B cell longevity, and apoptotic cell processing. Once tolerance is broken, autoantibodies contribute to autoimmunity by multiple mechanisms including immune-complex mediated Type III hypersensitivity reactions, type II antibody-dependent cytotoxicity, and by instructing innate immune cells to produce pathogenic cytokines including IFNα, TNF and IL-1. The complexity of B cell functions has been highlighted by the variable success of B cell-targeted therapies in multiple autoimmune diseases, including those conventionally viewed as T cell-mediated conditions. Given the widespread utilization of B cell depletion therapy in autoimmune diseases and the need for new therapeutic approaches in SLE, a better understanding of human B cell subsets and the balance of pathogenic and regulatory functions is of the essence.
T Helper1/T Helper2 Cells and Resistance/Susceptibility to Leishmania Infection: Is This Paradigm Still Relevant?

PubMed Central

Alexander, James; Brombacher, Frank

2012-01-01

Work in large part on Leishmania major in the 1980s identified two distinct apparently counter-regulatory CD4+ T cell populations, T helper (h)1 and Th2, that controlled resistance/susceptibility to infection respectively. However, the generation of IL-4−/− mice in the 1990s questioned the paramount role of this Th2 archetypal cytokine in the non-healing response to Leishmania infection. The more recent characterization of CD4+ T cell regulatory populations and further effector CD4+ T helper populations, Th17, Th9, and T follicular (f)h cells as well as the acknowledged plasticity in T helper cell function has further added to the complexity of host pathogen interactions. These interactions are complicated by the multiplicity of cells that respond to CD4+ T cell subset signatory cytokines, as well as the diversity of Leishmania species that are often subject to significantly different immune-regulatory controls. In this article we review current knowledge with regard to the role of CD4+ T cells and their products during Leishmania infection. In particular we update on our studies using conditional IL-4Rα gene-deficient mice that have allowed dissection of the cell interplay dictating the disease outcomes of the major Leishmania species infecting humans. PMID:22566961
Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase

PubMed Central

Lada, Artem G.; Stepchenkova, Elena I.; Waisertreiger, Irina S. R.; Noskov, Vladimir N.; Dhar, Alok; Eudy, James D.; Boissy, Robert J.; Hirano, Masayuki; Rogozin, Igor B.; Pavlov, Youri I.

2013-01-01

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis. PMID:24039593
Single-cell RNA-seq and computational analysis using temporal mixture modelling resolves Th1/Tfh fate bifurcation in malaria

PubMed Central

Lönnberg, Tapio; Svensson, Valentine; James, Kylie R.; Fernandez-Ruiz, Daniel; Sebina, Ismail; Montandon, Ruddy; Soon, Megan S. F.; Fogg, Lily G.; Nair, Arya Sheela; Liligeto, Urijah; Stubbington, Michael J. T.; Ly, Lam-Ha; Bagger, Frederik Otzen; Zwiessele, Max; Lawrence, Neil D.; Souza-Fonseca-Guimaraes, Fernando; Bunn, Patrick T.; Engwerda, Christian R.; Heath, William R.; Billker, Oliver; Stegle, Oliver; Haque, Ashraful; Teichmann, Sarah A.

2017-01-01

Differentiation of naïve CD4+ T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR sequences, we first demonstrated that Th1/Tfh bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with Th1 or Tfh fates, and demonstrated a T-cell intrinsic role for Galectin-1 in supporting a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards a Th1 but not a Tfh fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources, a database www.PlasmoTH.org, which facilitates discovery of novel factors controlling Th1/Tfh fate commitment, and more generally, GPfates, a modelling framework for characterizing cell differentiation towards multiple fates. PMID:28345074
Cryptosporidium as a testbed for single cell genome characterization of unicellular eukaryotes.

PubMed

Troell, Karin; Hallström, Björn; Divne, Anna-Maria; Alsmark, Cecilia; Arrighi, Romanico; Huss, Mikael; Beser, Jessica; Bertilsson, Stefan

2016-06-23

Infectious disease involving multiple genetically distinct populations of pathogens is frequently concurrent, but difficult to detect or describe with current routine methodology. Cryptosporidium sp. is a widespread gastrointestinal protozoan of global significance in both animals and humans. It cannot be easily maintained in culture and infections of multiple strains have been reported. To explore the potential use of single cell genomics methodology for revealing genome-level variation in clinical samples from Cryptosporidium-infected hosts, we sorted individual oocysts for subsequent genome amplification and full-genome sequencing. Cells were identified with fluorescent antibodies with an 80 % success rate for the entire single cell genomics workflow, demonstrating that the methodology can be applied directly to purified fecal samples. Ten amplified genomes from sorted single cells were selected for genome sequencing and compared both to the original population and a reference genome in order to evaluate the accuracy and performance of the method. Single cell genome coverage was on average 81 % even with a moderate sequencing effort and by combining the 10 single cell genomes, the full genome was accounted for. By a comparison to the original sample, biological variation could be distinguished and separated from noise introduced in the amplification. As a proof of principle, we have demonstrated the power of applying single cell genomics to dissect infectious disease caused by closely related parasite species or subtypes. The workflow can easily be expanded and adapted to target other protozoans, and potential applications include mapping genome-encoded traits, virulence, pathogenicity, host specificity and resistance at the level of cells as truly meaningful biological units.
Stochastic Cell Fate Progression in Embryonic Stem Cells

NASA Astrophysics Data System (ADS)

Zou, Ling-Nan; Doyle, Adele; Jang, Sumin; Ramanathan, Sharad

2013-03-01

Studies on the directed differentiation of embryonic stem (ES) cells suggest that some early developmental decisions may be stochastic in nature. To identify the sources of this stochasticity, we analyzed the heterogeneous expression of key transcription factors in single ES cells as they adopt distinct germ layer fates. We find that under sufficiently stringent signaling conditions, the choice of lineage is unambiguous. ES cells flow into differentiated fates via diverging paths, defined by sequences of transitional states that exhibit characteristic co-expression of multiple transcription factors. These transitional states have distinct responses to morphogenic stimuli; by sequential exposure to multiple signaling conditions, ES cells are steered towards specific fates. However, the rate at which cells travel down a developmental path is stochastic: cells exposed to the same signaling condition for the same amount of time can populate different states along the same path. The heterogeneity of cell states seen in our experiments therefore does not reflect the stochastic selection of germ layer fates, but the stochastic rate of progression along a chosen developmental path. Supported in part by the Jane Coffin Childs Fund
PHTHALATE-INDUCED LEYDIG CELL HYPERPLASIA IS ASSOCIATED WITH MULTIPLE ENDOCRINE DISTURBANCES

EPA Science Inventory

The possibility that exposures to environmental agents are associated with reproductive disorders in human populations has generated much public interest recently. Phthalate esters are used most commonly as plasticizers in the food and construction industry, and DEHP is the most ...
Does an NKT-cell-based immunotherapeutic approach have a future in multiple myeloma?

PubMed Central

Favreau, Mérédis; Vanderkerken, Karin

2016-01-01

Natural killer T (NKT) cells constitute a unique subset of innate-like T lymphocytes which differ from conventional T cells by recognizing lipid antigens presented by the non-polymorphic major histocompatibility complex (MHC) I-like molecule CD1d. Despite being a relatively infrequent population of lymphocytes, NKT cells can respond rapidly upon activation with glycosphingolipids by production of cytokines which aim to polarize different axes of the immune system. Due to their dual effector capacities, NKT cells can play a vital role in cancer immunity, infection, inflammation and autoimmune diseases. It is believed that modulation of their activity towards immune activation can be a useful tool in anti-tumor immunotherapeutic strategies. Here we summarize the characteristics of NKT cells and discuss their involvement in immunosurveillance. Furthermore, an update is given about their role and the progress that has been made in the field of multiple myeloma (MM). Finally, some challenges are discussed that are currently hampering further progress. PMID:26895468
Sympathetic Nerves in Breast Cancer: Angiogenesis and Antiangiogenic Therapy

DTIC Science & Technology

2013-02-01

directly regulate 4T1 growth through ARs. Using in vitro conditions that yielded NE-mediated functional effects in other cell lines (1), we assessed...Therefore we propose that 4T1 is a good model for examining the effects of NE-induced effects on stromal cell populations in the absence of direct NE... effects analysis or by Holm-Sidak multiple comparison test. RESULTS 4T1 Tumor Cells Do Not Respond to NE In Vitro or Signal via AR To determine
Human influenza viruses and CD8(+) T cell responses.

PubMed

Grant, Emma J; Quiñones-Parra, Sergio M; Clemens, E Bridie; Kedzierska, Katherine

2016-02-01

Influenza A viruses (IAVs) cause significant morbidity and mortality worldwide, despite new strain-specific vaccines being available annually. As IAV-specific CD8(+) T cells promote viral control in the absence of neutralizing antibodies, and can mediate cross-reactive immunity toward distinct IAVs to drive rapid recovery from both mild and severe influenza disease, there is great interest in developing a universal T cell vaccine. However, despite detailed studies in mouse models of influenza virus infection, there is still a paucity of data on human epitope-specific CD8(+) T cell responses to IAVs. This review focuses on our current understanding of human CD8(+) T cell immunity against distinct IAVs and discusses the possibility of achieving a CD8(+) T cell mediated-vaccine that protects against multiple, distinct IAV strains across diverse human populations. We also review the importance of CD8(+) T cell immunity in individuals highly susceptible to severe influenza infection, including those hospitalised with influenza, the elderly and Indigenous populations. Copyright © 2016 Elsevier B.V. All rights reserved.
PRODUCTION AND CHARACTERIZATION OF MULTIPLE-LAYERED POPULATIONS OF ANIMAL CELLS

PubMed Central

Kruse, Paul F.; Miedema, Ed

1965-01-01

Dense populations containing 129 x 106 Jensen sarcoma, 134 x 106 DON Chinese hamster, 28.9 x 106 WI-38 human diploid, 61.8 x 106 HEp-2 human carcinoma, and 67.4 x 106 WISH human amnion cells were produced from dilute inocula, 0.85 to 5.33 x 106, in 7 to 8 days in a perfusion system using replicate T-60 flasks. Perfusion rates as high as 560 ml medium/day/T-60 were required to maintain pH (to ca ±0.1 unit) and adequate nutrient supplies. The cell densities encountered are described by the term "monolayer equivalents" (M.E.), defined as number of cells per culture divided by number of cells in a monolayer. The M.E.'s for T-60 cultures containing unusually dense populations of 40 x 106 WI-38 and 250 x 106 DON cells (9-day perfusion) were 5 and 17, respectively, and numbers of cells in illustrations of stained cross-sections of membranes from these cultures were in excellent agreement. Threshold M.E.'s exist below which proliferation is the chief cellular activity and above which one or more cell functions may predominate even though proliferation persists. Cellular nutrition and metabolism may change with changes in M.E., as illustrated in different patterns of glutamic acid, proline, and glycine utilization or production in dense vs. dilute WI-38 cell populations. The results indicated that the role of contact inhibition phenomena in arresting cellular proliferation was diminished in perfusion system environments. PMID:5884626

Ocular histiocytic sarcoma in a cat.

PubMed

Scurrell, Emma; Trott, Adele; Rozmanec, Michael; Belford, Chris J

2013-07-01

A 13-year-old male neutered British blue cat presented with uveitis, hyphema, and dyscoria in the right eye. Light microscopic examination revealed that the ciliary body, iris root, drainage angle, and adjacent choroid were infiltrated by sheets of large neoplastic mononuclear and multinucleate round to polygonal cells. Neoplastic cells stained immunopositive for CD18 and HLA-DR (MHC class II) and were immunonegative for CD3, CD79a, MUM-1, CD117 (c-Kit), and S100. These findings were consistent with a histiocytic sarcoma. The cat later developed multiple cutaneous masses composed of a similar neoplastic cell population to that seen in the eye. Eight months following enucleation, the cat developed respiratory distress and was euthanized. Postmortem examination revealed multiple pulmonary tumors associated with a pleural effusion. © 2013 American College of Veterinary Ophthalmologists.
PVRL1 Variants Contribute to Non-Syndromic Cleft Lip and Palate in Multiple Populations

PubMed Central

Avila, Joseph R.; Jezewski, Peter A.; Vieira, Alexandre R.; Orioli, Iêda M.; Castilla, Eduardo E.; Christensen, Kaare; Daack-Hirsch, Sandra; Romitti, Paul A.; Murray, Jeffrey C.

2007-01-01

Poliovirus Receptor Like-1 (PVRL1) is a member of the immunoglobulin super family that acts in the initiation and maintenance of epithelial adherens junctions and is mutated in the cleft lip and palate/ectodermal dysplasia 1 syndrome (CLPED1, OMIM #225000). In addition, a common non-sense mutation in PVRL1 was discovered more often among non-syndromic sporadic clefting cases in Northern Venezuela in a previous case-control study. The present work sought to ascertain the role of PVRL1 in the sporadic forms of orofacial clefting in multiple populations. Multiple rare and common variants from all three splice isoforms were initially ascertained by sequencing 92 Iowan and 86 Filipino cases and CEPH controls. Using a family-based analysis to examine these variants, the common glycine allele of the G361V coding variant was significantly overtransmitted among all orofacial clefting phenotypes (P = 0.005). This represented G361V genotyping from over 800 Iowan, Danish, and Filipino families. Among four rare amino acid changes found within the V1 and C1 domains, S112T and T131A were found adjacent to critical amino acid positions within the V1 variable domain, regions previously shown to mediate cell-to-cell and cell-to-virus adhesion. The T131A variant was not found in over 1,300 non-affected control samples although the alanine is found in other species. The serine of the S112T variant position is conserved across all known PVRL1 sequences. Together these data suggest that both rare and common mutations within PVRL1 make a minor contribution to disrupting the initiation and regulation of cell-to-cell adhesion and downstream morphogenesis of the embryonic face. PMID:17089422
Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum

PubMed Central

Choi, Eunyoung; Roland, Joseph T.; Barlow, Brittney J.; O’Neal, Ryan; Rich, Amy E.; Nam, Ki Taek; Shi, Chanjuan; Goldenring, James R.

2014-01-01

Objective The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. Design We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. Results The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of stomach as well as in over 50% of antral glands. MIST1-expressing chief cells were predominantly observed in the body, although individual glands of the antrum also showed MIST1-expressing chief cells. While classically-described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed-type glands containing both parietal cells and G cells throughout the antrum. Conclusions Enteroendocrine cells show distinct patterns of localization in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations. PMID:24488499
Cancer heterogeneity and multilayer spatial evolutionary games.

PubMed

Świerniak, Andrzej; Krześlak, Michał

2016-10-13

Evolutionary game theory (EGT) has been widely used to simulate tumour processes. In almost all studies on EGT models analysis is limited to two or three phenotypes. Our model contains four main phenotypes. Moreover, in a standard approach only heterogeneity of populations is studied, while cancer cells remain homogeneous. A multilayer approach proposed in this paper enables to study heterogeneity of single cells. In the extended model presented in this paper we consider four strategies (phenotypes) that can arise by mutations. We propose multilayer spatial evolutionary games (MSEG) played on multiple 2D lattices corresponding to the possible phenotypes. It enables simulation and investigation of heterogeneity on the player-level in addition to the population-level. Moreover, it allows to model interactions between arbitrary many phenotypes resulting from the mixture of basic traits. Different equilibrium points and scenarios (monomorphic and polymorphic populations) have been achieved depending on model parameters and the type of played game. However, there is a possibility of stable quadromorphic population in MSEG games for the same set of parameters like for the mean-field game. The model assumes an existence of four possible phenotypes (strategies) in the population of cells that make up tumour. Various parameters and relations between cells lead to complex analysis of this model and give diverse results. One of them is a possibility of stable coexistence of different tumour cells within the population, representing almost arbitrary mixture of the basic phenotypes. This article was reviewed by Tomasz Lipniacki, Urszula Ledzewicz and Jacek Banasiak.
Most Influenza A Virions Fail To Express at Least One Essential Viral Protein

PubMed Central

Brooke, Christopher B.; Ince, William L.; Wrammert, Jens; Ahmed, Rafi; Wilson, Patrick C.; Bennink, Jack R.

2013-01-01

Segmentation of the influenza A virus (IAV) genome enables rapid gene reassortment at the cost of complicating the task of assembling the full viral genome. By simultaneously probing for the expression of multiple viral proteins in MDCK cells infected at a low multiplicity with IAV, we observe that the majority of infected cells lack detectable expression of one or more essential viral proteins. Consistent with this observation, up to 90% of IAV-infected cells fail to release infectious progeny, indicating that many IAV virions scored as noninfectious by traditional infectivity assays are capable of single-round infection. This fraction was not significantly affected by target or producer cell type but varied widely between different IAV strains. These data indicate that IAV exists primarily as a swarm of complementation-dependent semi-infectious virions, and thus traditional, propagation-dependent assays of infectivity may drastically misrepresent the true infectious potential of a virus population. PMID:23283949
Development of Multifunctional Magnetic Nanoparticles for Genetic Engineering and Tracking of Neural Stem Cells.

PubMed

Adams, Christopher; Israel, Liron Limor; Ostrovsky, Stella; Taylor, Arthur; Poptani, Harish; Lellouche, Jean-Paul; Chari, Divya

2016-04-06

Genetic modification of cell transplant populations and cell tracking ability are key underpinnings for effective cell therapies. Current strategies to achieve these goals utilize methods which are unsuitable for clinical translation because of related safety issues, and multiple protocol steps adding to cost and complexity. Multifunctional magnetic nanoparticles (MNPs) offering dual mode gene delivery and imaging contrast capacity offer a valuable tool in this context. Despite their key benefits, there is a critical lack of neurocompatible and multifunctional particles described for use with transplant populations for neurological applications. Here, a systematic screen of MNPs (using a core shown to cause contrast in magnetic resonance imaging (MRI)) bearing various surface chemistries (polyethylenimine (PEI) and oxidized PEI and hybrids of oxidized PEI/alginic acid, PEI/chitosan and PEI/polyamidoamine) is performed to test their ability to genetically engineer neural stem cells (NSCs; a cell population of high clinical relevance for central nervous system disorders). It is demonstrated that gene delivery to NSCs can be safely achieved using two of the developed formulations (PEI and oxPEI/alginic acid) when used in conjunction with oscillating magnetofection technology. After transfection, intracellular particles can be detected by histological procedures with labeled cells displaying contrast in MRI (for real time cell tracking). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
European multiple sclerosis risk variants in the south Asian population.

PubMed

Pandit, Lekha; Ban, Maria; Beecham, Ashley Harris; McCauley, Jacob L; Sawcer, Stephen; D'Cunha, Anitha; Malli, Chaitra; Malik, Omar

2016-10-01

In less than a decade, genomewide association studies have identified over 100 single-nucleotide variants that are associated with increased risk of developing multiple sclerosis. However, since these studies have focused almost exclusively on European populations, it is unclear what role these variants might play in determining risk in other ethnic groups. To assess the effects of European multiple sclerosis-associated risk variants in the south Asian population. Using a combination of chip-based genotyping and next-generation sequencing, we have assessed 109 European-associated variants in a total of 270 cases and 555 controls from the south Asian population. We found that two-thirds of the tested variants (72/109) showed over representation of the European risk allele in south Asian cases (p < 0.0003). In the rest of the Immunochip array, the most associated variant was rs7318477 which maps close to TNFSF13B, the gene for the B-cell-related protein BAFF. Our data indicate substantial overlap in genetic risk architecture between Europeans and south Asians and suggest that the aetiology of the disease may be largely independent of ethnicity. © The Author(s), 2016.
T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones.

PubMed

Theaker, Sarah M; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J; Cole, David K; Peakman, Mark; Sewell, Andrew K; Dolton, Garry

2016-03-01

Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
Rapid learning in visual cortical networks.

PubMed

Wang, Ye; Dragoi, Valentin

2015-08-26

Although changes in brain activity during learning have been extensively examined at the single neuron level, the coding strategies employed by cell populations remain mysterious. We examined cell populations in macaque area V4 during a rapid form of perceptual learning that emerges within tens of minutes. Multiple single units and LFP responses were recorded as monkeys improved their performance in an image discrimination task. We show that the increase in behavioral performance during learning is predicted by a tight coordination of spike timing with local population activity. More spike-LFP theta synchronization is correlated with higher learning performance, while high-frequency synchronization is unrelated with changes in performance, but these changes were absent once learning had stabilized and stimuli became familiar, or in the absence of learning. These findings reveal a novel mechanism of plasticity in visual cortex by which elevated low-frequency synchronization between individual neurons and local population activity accompanies the improvement in performance during learning.
Elucidation of Seventeen Human Peripheral Blood B cell Subsets and Quantification of the Tetanus Response Using a Density-Based Method for the Automated Identification of Cell Populations in Multidimensional Flow Cytometry Data

PubMed Central

Qian, Yu; Wei, Chungwen; Lee, F. Eun-Hyung; Campbell, John; Halliley, Jessica; Lee, Jamie A.; Cai, Jennifer; Kong, Megan; Sadat, Eva; Thomson, Elizabeth; Dunn, Patrick; Seegmiller, Adam C.; Karandikar, Nitin J.; Tipton, Chris; Mosmann, Tim; Sanz, Iñaki; Scheuermann, Richard H.

2011-01-01

Background Advances in multi-parameter flow cytometry (FCM) now allow for the independent detection of larger numbers of fluorochromes on individual cells, generating data with increasingly higher dimensionality. The increased complexity of these data has made it difficult to identify cell populations from high-dimensional FCM data using traditional manual gating strategies based on single-color or two-color displays. Methods To address this challenge, we developed a novel program, FLOCK (FLOw Clustering without K), that uses a density-based clustering approach to algorithmically identify biologically relevant cell populations from multiple samples in an unbiased fashion, thereby eliminating operator-dependent variability. Results FLOCK was used to objectively identify seventeen distinct B cell subsets in a human peripheral blood sample and to identify and quantify novel plasmablast subsets responding transiently to tetanus and other vaccinations in peripheral blood. FLOCK has been implemented in the publically available Immunology Database and Analysis Portal – ImmPort (http://www.immport.org) for open use by the immunology research community. Conclusions FLOCK is able to identify cell subsets in experiments that use multi-parameter flow cytometry through an objective, automated computational approach. The use of algorithms like FLOCK for FCM data analysis obviates the need for subjective and labor intensive manual gating to identify and quantify cell subsets. Novel populations identified by these computational approaches can serve as hypotheses for further experimental study. PMID:20839340
A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals

PubMed Central

Wang, Baojun; Barahona, Mauricio; Buck, Martin

2013-01-01

Cells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas. PMID:22981411
CD8 T cell memory: it takes all kinds

PubMed Central

Hamilton, Sara E.; Jameson, Stephen C.

2012-01-01

Understanding the mechanisms that regulate the differentiation and maintenance of CD8+ memory T cells is fundamental to the development of effective T cell-based vaccines. Memory cell differentiation is influenced by the cytokines that accompany T cell priming, the history of previous antigen encounters, and the tissue sites into which memory cells migrate. These cues combine to influence the developing CD8+ memory pool, and recent work has revealed the importance of multiple transcription factors, metabolic molecules, and surface receptors in revealing the type of memory cell that is generated. Paired with increasingly meticulous subsetting and sorting of memory populations, we now know the CD8+ memory pool to be phenotypically and functionally heterogeneous in nature. This includes both recirculating and tissue-resident memory populations, and cells with varying degrees of inherent longevity and protective function. These data point to the importance of tailored vaccine design. Here we discuss how the diversity of the memory CD8+ T cell pool challenges the notion that “one size fits all” for pathogen control, and how distinct memory subsets may be suited for distinct aspects of protective immunity. PMID:23230436
A diverse and intricate signalling network regulates stem cell fate in the shoot apical meristem.

PubMed

Dodsworth, Steven

2009-12-01

At the shoot apex of plants is a small region known as the shoot apical meristem (SAM) that maintains a population of undifferentiated (stem) cells whilst providing cells for developing lateral organs and the stem. All aerial structures of the plant develop from the SAM post-embryogenesis, enabling plants to grow in a characteristic modular fashion with great phenotypic and developmental plasticity throughout their lifetime. The maintenance of the stem cell population is intimately balanced with cell recruitment into differentiating tissues through intercellular communication involving a complex signalling network. Recent studies have shown that diverse regulators function in SAM maintenance, many of which converge on the WUSCHEL (WUS) gene. In this review the diverse regulatory modules that function in SAM maintenance are discussed: transcriptional and epigenetic control, hormonal regulation, and the balance with organogenesis. The central role of WUS as an integrator of multiple signals is highlighted; in addition, accessory feedback loops emerge as a feature enabling dynamic regulation of the stem cell niche.
An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xiaojun; Department of Biotechnology, Nanchang University, Nanchang, Jiangxi 330031; Chen, Yuan

2014-03-28

Highlights: • Air drying induced the transformation of cell-surface membrane vesicles into pits. • An AFM-based pit-measuring method was developed to measure cell-surface vesicles. • Our method detected at least two populations of cell-surface membrane vesicles. - Abstract: Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM)more » has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.« less
Stem cells in pharmaceutical biotechnology.

PubMed

Zuba-Surma, Ewa K; Józkowicz, Alicja; Dulak, Józef

2011-11-01

Multiple populations of stem cells have been indicated to potentially participate in regeneration of injured organs. Especially, embryonic stem cells (ESC) and recently inducible pluripotent stem cells (iPS) receive a marked attention from scientists and clinicians for regenerative medicine because of their high proliferative and differentiation capacities. Despite that ESC and iPS cells are expected to give rise into multiple regenerative applications when their side effects are overcame during appropriate preparation procedures, in fact their most recent application of human ESC may, however, reside in their use as a tool in drug development and disease modeling. This review focuses on the applications of stem cells in pharmaceutical biotechnology. We discuss possible relevance of pluripotent cell stem populations in developing physiological models for any human tissue cell type useful for pharmacological, metabolic and toxicity evaluation necessary in the earliest steps of drug development. The present models applied for preclinical drug testing consist of primary cells or immortalized cell lines that show limitations in terms of accessibility or relevance to their in vivo counterparts. The availability of renewable human cells with functional similarities to their in vivo counterparts is the first landmark for a new generation of cell-based assays. We discuss the approaches for using stem cells as valuable physiological targets of drug activity which may increase the strength of target validation and efficacy potentially resulting in introducing new safer remedies into clinical trials and the marketplace. Moreover, we discuss the possible applications of stem cells for elucidating mechanisms of disease pathogenesis. The knowledge about the mechanisms governing the development and progression of multitude disorders which would come from the cellular models established based on stem cells, may give rise to new therapeutical strategies for such diseases. All together, the applications of various cell types derived from patient specific pluripotent stem cells may lead to targeted drug and cellular therapies for certain individuals.
Phosphoinositide protein kinase PDPK1 is a crucial cell signaling mediator in multiple myeloma.

PubMed

Chinen, Yoshiaki; Kuroda, Junya; Shimura, Yuji; Nagoshi, Hisao; Kiyota, Miki; Yamamoto-Sugitani, Mio; Mizutani, Shinsuke; Sakamoto, Natsumi; Ri, Masaki; Kawata, Eri; Kobayashi, Tsutomu; Matsumoto, Yosuke; Horiike, Shigeo; Iida, Shinsuke; Taniwaki, Masafumi

2014-12-15

Multiple myeloma is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. Herein, we identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a serine threonine kinase, is expressed and active in all eleven multiple myeloma-derived cell lines examined regardless of the type of cytogenetic abnormality, the mutation state of RAS and FGFR3 genes, or the activation state of ERK and AKT. Our results revealed that PDPK1 is a pivotal regulator of molecules that are essential for myelomagenesis, such as RSK2, AKT, c-MYC, IRF4, or cyclin Ds, and that PDPK1 inhibition caused the growth inhibition and the induction of apoptosis with the activation of BIM and BAD, and augmented the in vitro cytotoxic effects of antimyeloma agents in myeloma cells. In the clinical setting, PDPK1 was active in myeloma cells of approximately 90% of symptomatic patients at diagnosis, and the smaller population of patients with multiple myeloma exhibiting myeloma cells without active PDPK1 showed a significantly less frequent proportion of the disease stage III by the International Staging System and a significantly more favorable prognosis, including the longer overall survival period and the longer progression-free survival period by bortezomib treatment, than patients with active PDPK1, suggesting that PDPK1 activation accelerates the disease progression and the resistance to treatment in multiple myeloma. Our study demonstrates that PDPK1 is a potent and a universally targetable signaling mediator in multiple myeloma regardless of the types of cytogenetic/molecular profiles. ©2014 American Association for Cancer Research.
Calcium Signaling in Taste Cells

PubMed Central

Medler, Kathryn F.

2014-01-01

The sense of taste is a common ability shared by all organisms and is used to detect nutrients as well as potentially harmful compounds. Thus taste is critical to survival. Despite its importance, surprisingly little is known about the mechanisms generating and regulating responses to taste stimuli. All taste responses depend on calcium signals to generate appropriate responses which are relayed to the brain. Some taste cells have conventional synapses and rely on calcium influx through voltage-gated calcium channels. Other taste cells lack these synapses and depend on calcium release to formulate an output signal through a hemichannel. Beyond establishing these characteristics, few studies have focused on understanding how these calcium signals are formed. We identified multiple calcium clearance mechanisms that regulate calcium levels in taste cells as well as a calcium influx that contributes to maintaining appropriate calcium homeostasis in these cells. Multiple factors regulate the evoked taste signals with varying roles in different cell populations. Clearly, calcium signaling is a dynamic process in taste cells and is more complex than has previously been appreciated. PMID:25450977
Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability

PubMed Central

Patterson, Melissa N.; Scannapieco, Alison E.; Au, Pak Ho; Dorsey, Savanna; Royer, Catherine A.; Maxwell, Patrick H.

2015-01-01

Retrotransposon expression or mobility is increased with age in multiple species and could promote genome instability or altered gene expression during aging. However, it is unclear whether activation of retrotransposons during aging is an indirect result of global changes in chromatin and gene regulation or a result of retrotransposon-specific mechanisms. Retromobility of a marked chromosomal Ty1 retrotransposon in Saccharomyces cerevisiae was elevated in mother cells relative to their daughter cells, as determined by magnetic cell sorting of mothers and daughters. Retromobility frequencies in aging mother cells were significantly higher than those predicted by cell age and the rate of mobility in young populations, beginning when mother cells were only several generations old. New Ty1 insertions in aging mothers were more strongly correlated with gross chromosome rearrangements than in young cells and were more often at non-preferred target sites. Mother cells were more likely to have high concentrations and bright foci of Ty1 Gag-GFP than their daughter cells. Levels of extrachromosomal Ty1 cDNA were also significantly higher in aged mother cell populations than their daughter cell populations. These observations are consistent with a retrotransposon-specific mechanism that causes retrotransposition to occur preferentially in yeast mother cells as they begin to age, as opposed to activation by phenotypic changes associated with very old age. These findings will likely be relevant for understanding retrotransposons and aging in many organisms, based on similarities in regulation and consequences of retrotransposition in diverse species. PMID:26298836
Determinants of public T cell responses.

PubMed

Li, Hanjie; Ye, Congting; Ji, Guoli; Han, Jiahuai

2012-01-01

Historically, sharing T cell receptors (TCRs) between individuals has been speculated to be impossible, considering the dramatic discrepancy between the potential enormity of the TCR repertoire and the limited number of T cells generated in each individual. However, public T cell response, in which multiple individuals share identical TCRs in responding to a same antigenic epitope, has been extensively observed in a variety of immune responses across many species. Public T cell responses enable individuals within a population to generate similar antigen-specific TCRs against certain ubiquitous pathogens, leading to favorable biological outcomes. However, the relatively concentrated feature of TCR repertoire may limit T cell response in a population to some other pathogens. It could be a great benefit for human health if public T cell responses can be manipulated. Therefore, the mechanistic insight of public TCR generation is important to know. Recently, high-throughput DNA sequencing has revolutionized the study of immune receptor repertoires, which allows a much better understanding of the factors that determine the overlap of TCR repertoire among individuals. Here, we summarize the current knowledge on public T-cell response and discuss future challenges in this field.
Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells.

PubMed

Novella, Isabel S; Ebendick-Corpus, Bonnie E; Zárate, Selene; Miller, Eric L

2007-06-01

Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.

Multigenerational memory and adaptive adhesion in early bacterial biofilm communities.

PubMed

Lee, Calvin K; de Anda, Jaime; Baker, Amy E; Bennett, Rachel R; Luo, Yun; Lee, Ernest Y; Keefe, Joshua A; Helali, Joshua S; Ma, Jie; Zhao, Kun; Golestanian, Ramin; O'Toole, George A; Wong, Gerard C L

2018-04-24

Using multigenerational, single-cell tracking we explore the earliest events of biofilm formation by Pseudomonas aeruginosa During initial stages of surface engagement (≤20 h), the surface cell population of this microbe comprises overwhelmingly cells that attach poorly (∼95% stay <30 s, well below the ∼1-h division time) with little increase in surface population. If we harvest cells previously exposed to a surface and direct them to a virgin surface, we find that these surface-exposed cells and their descendants attach strongly and then rapidly increase the surface cell population. This "adaptive," time-delayed adhesion requires determinants we showed previously are critical for surface sensing: type IV pili (TFP) and cAMP signaling via the Pil-Chp-TFP system. We show that these surface-adapted cells exhibit damped, coupled out-of-phase oscillations of intracellular cAMP levels and associated TFP activity that persist for multiple generations, whereas surface-naïve cells show uncorrelated cAMP and TFP activity. These correlated cAMP-TFP oscillations, which effectively impart intergenerational memory to cells in a lineage, can be understood in terms of a Turing stochastic model based on the Pil-Chp-TFP framework. Importantly, these cAMP-TFP oscillations create a state characterized by a suppression of TFP motility coordinated across entire lineages and lead to a drastic increase in the number of surface-associated cells with near-zero translational motion. The appearance of this surface-adapted state, which can serve to define the historical classification of "irreversibly attached" cells, correlates with family tree architectures that facilitate exponential increases in surface cell populations necessary for biofilm formation.
Pinched-flow hydrodynamic stretching of single-cells.

PubMed

Dudani, Jaideep S; Gossett, Daniel R; Tse, Henry T K; Di Carlo, Dino

2013-09-21

Reorganization of cytoskeletal networks, condensation and decondensation of chromatin, and other whole cell structural changes often accompany changes in cell state and can reflect underlying disease processes. As such, the observable mechanical properties, or mechanophenotype, which is closely linked to intracellular architecture, can be a useful label-free biomarker of disease. In order to make use of this biomarker, a tool to measure cell mechanical properties should accurately characterize clinical specimens that consist of heterogeneous cell populations or contain small diseased subpopulations. Because of the heterogeneity and potential for rare populations in clinical samples, single-cell, high-throughput assays are ideally suited. Hydrodynamic stretching has recently emerged as a powerful method for carrying out mechanical phenotyping. Importantly, this method operates independently of molecular probes, reducing cost and sample preparation time, and yields information-rich signatures of cell populations through significant image analysis automation, promoting more widespread adoption. In this work, we present an alternative mode of hydrodynamic stretching where inertially-focused cells are squeezed in flow by perpendicular high-speed pinch flows that are extracted from the single inputted cell suspension. The pinched-flow stretching method reveals expected differences in cell deformability in two model systems. Furthermore, hydraulic circuit design is used to tune stretching forces and carry out multiple stretching modes (pinched-flow and extensional) in the same microfluidic channel with a single fluid input. The ability to create a self-sheathing flow from a single input solution should have general utility for other cytometry systems and the pinched-flow design enables an order of magnitude higher throughput (65,000 cells s(-1)) compared to our previously reported deformability cytometry method, which will be especially useful for identification of rare cell populations in clinical body fluids in the future.
Point process models for localization and interdependence of punctate cellular structures.

PubMed

Li, Ying; Majarian, Timothy D; Naik, Armaghan W; Johnson, Gregory R; Murphy, Robert F

2016-07-01

Accurate representations of cellular organization for multiple eukaryotic cell types are required for creating predictive models of dynamic cellular function. To this end, we have previously developed the CellOrganizer platform, an open source system for generative modeling of cellular components from microscopy images. CellOrganizer models capture the inherent heterogeneity in the spatial distribution, size, and quantity of different components among a cell population. Furthermore, CellOrganizer can generate quantitatively realistic synthetic images that reflect the underlying cell population. A current focus of the project is to model the complex, interdependent nature of organelle localization. We built upon previous work on developing multiple non-parametric models of organelles or structures that show punctate patterns. The previous models described the relationships between the subcellular localization of puncta and the positions of cell and nuclear membranes and microtubules. We extend these models to consider the relationship to the endoplasmic reticulum (ER), and to consider the relationship between the positions of different puncta of the same type. Our results do not suggest that the punctate patterns we examined are dependent on ER position or inter- and intra-class proximity. With these results, we built classifiers to update previous assignments of proteins to one of 11 patterns in three distinct cell lines. Our generative models demonstrate the ability to construct statistically accurate representations of puncta localization from simple cellular markers in distinct cell types, capturing the complex phenomena of cellular structure interaction with little human input. This protocol represents a novel approach to vesicular protein annotation, a field that is often neglected in high-throughput microscopy. These results suggest that spatial point process models provide useful insight with respect to the spatial dependence between cellular structures. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.
Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.

PubMed

Sequeira, Daniela P; Correia, Ricardo; Carrondo, Manuel J T; Roldão, António; Teixeira, Ana P; Alves, Paula M

2018-05-24

Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains and promising approaches based on the expression of multiple hemagglutinins (HA) in a virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can lead to its instability due to tandem repetition of similar sequences. By combining stable with transient expression systems we can rationally distribute the number of genes to be expressed per platform and thus mitigate this risk. In this work, we developed a modular system comprising stable and baculovirus-mediated expression in insect cells for production of multi-HA influenza enveloped VLPs. First, a stable insect High Five cell population expressing two different HA proteins from subtype H3 was established. Infection of this cell population with a baculovirus vector encoding three other HA proteins from H3 subtype proved to be as competitive as traditional co-infection approaches in producing a pentavalent H3 VLP. Aiming at increasing HA expression, the stable insect cell population was infected at increasingly higher cell concentrations (CCI). However, cultures infected at CCI of 3×10 6 cells/mL showed lower HA titers per cell in comparison to standard CCI of 2×10 6 cells/mL, a phenomenon named "cell density effect". To lessen the negative impact of this phenomenon, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth. Noteworthy, cultures supplemented and infected at a CCI of 4×10 6 cells/mL showed comparable HA titers per cell to those of CCI of 2×10 6 cells/mL, thus leading to an increase of up to 4-fold in HA titers per mL. Scalability of the modular strategy herein proposed was successfully demonstrated in 2L stirred tank bioreactors with comparable HA protein levels observed between bioreactor and shake flasks cultures. Overall, this work demonstrates the suitability of combining stable with baculovirus-mediated expression in insect cells as an efficient platform for production of multi-HA influenza VLPs, surpassing the drawbacks of traditional co-infection strategies and/or the use of larger, unstable vectors. Copyright © 2017 Elsevier Ltd. All rights reserved.
Cell-specific expression of neuropeptide Y Y1 receptor immunoreactivity in the rat basolateral amygdala.

PubMed

Rostkowski, Amanda B; Teppen, Tara L; Peterson, Daniel A; Urban, Janice H

2009-11-10

Activation of neuropeptide Y (NPY) Y1 receptors (Y1r) in the rat basolateral nuclear complex of the amygdala (BLA) produces anxiolysis and interferes with the generation of conditioned fear. NPY is important in regulating the output of the BLA, yet the cell types involved in mediating this response are currently unknown. The current studies employed multiple label immunocytochemistry to determine the distribution of Y1r-immunoreactivity (-ir) in glutamatergic pyramidal and GABAergic cell populations in the BLA using scanning laser confocal stereology. Pyramidal neurons were identified by expression of calcium-calmodulin dependent kinase II (CaMKII-ir) and functionally distinct interneuron subpopulations were distinguished by peptide (cholecystokinin, somatostatin) or calcium-binding protein (parvalbumin, calretinin) content. Throughout the BLA, Y1r-ir was predominately on soma with negligible fiber staining. The high degree of coexpression of Y1r-ir (99.9%) in CaMKII-ir cells suggests that these receptors colocalize on pyramidal cells and that NPY could influence BLA output by directly regulating the activity of these projection neurons. Additionally, Y1r-ir was also colocalized with the interneuronal markers studied. Parvalbumin-ir interneurons, which participate in feedforward inhibition of BLA pyramidal cells, represented the largest number of Y1r expressing interneurons in the BLA ( approximately 4% of the total neuronal population). The anatomical localization of NPY receptors on different cell populations within the BLA provides a testable circuit whereby NPY could modulate the activity of the BLA via actions on both projection cells and interneuronal cell populations.
Production and characterization of multiple-layered populations of animal cells.

PubMed

Kruse, P F; Miedema, E

1965-11-01

Dense populations containing 129 x 10(6) Jensen sarcoma, 134 x 10(6) DON Chinese hamster, 28.9 x 10(6) WI-38 human diploid, 61.8 x 10(6) HEp-2 human carcinoma, and 67.4 x 10(6) WISH human amnion cells were produced from dilute inocula, 0.85 to 5.33 x 10(6), in 7 to 8 days in a perfusion system using replicate T-60 flasks. Perfusion rates as high as 560 ml medium/day/T-60 were required to maintain pH (to ca +/-0.1 unit) and adequate nutrient supplies. The cell densities encountered are described by the term "monolayer equivalents" (M.E.), defined as number of cells per culture divided by number of cells in a monolayer. The M.E.'s for T-60 cultures containing unusually dense populations of 40 x 10(6) WI-38 and 250 x 10(6) DON cells (9-day perfusion) were 5 and 17, respectively, and numbers of cells in illustrations of stained cross-sections of membranes from these cultures were in excellent agreement. Threshold M.E.'s exist below which proliferation is the chief cellular activity and above which one or more cell functions may predominate even though proliferation persists. Cellular nutrition and metabolism may change with changes in M.E., as illustrated in different patterns of glutamic acid, proline, and glycine utilization or production in dense vs. dilute WI-38 cell populations. The results indicated that the role of contact inhibition phenomena in arresting cellular proliferation was diminished in perfusion system environments.
Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction

PubMed Central

Yoon, Young-sup; Wecker, Andrea; Heyd, Lindsay; Park, Jong-Seon; Tkebuchava, Tengiz; Kusano, Kengo; Hanley, Allison; Scadova, Heather; Qin, Gangjian; Cha, Dong-Hyun; Johnson, Kirby L.; Aikawa, Ryuichi; Asahara, Takayuki; Losordo, Douglas W.

2005-01-01

We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis. PMID:15690083
Isolation and characterization of mouse innate lymphoid cells.

PubMed

Halim, Timotheus Y F; Takei, Fumio

2014-08-01

Innate lymphoid cells (ILCs) are rare populations of cytokine-producing lymphocytes and are divided into three groups, namely ILC1, ILC2, and ILC3, based on the cytokines that they produce. They comprise less than 1% of lymphocytes in mucosal tissues and express no unique cell surface markers. Therefore, they can only be identified by combinations of multiple cell surface markers and further characterized by cytokine production in vitro. Thus, multicolor flow cytometry is the only reliable method to purify and characterize ILCs. Here we describe the methods for cell preparation, flow cytometric analysis, and purification of murine ILC2 and ILC3. Copyright © 2014 John Wiley & Sons, Inc.
A simple theoretical framework for understanding heterogeneous differentiation of CD4+ T cells

PubMed Central

2012-01-01

Background CD4+ T cells have several subsets of functional phenotypes, which play critical yet diverse roles in the immune system. Pathogen-driven differentiation of these subsets of cells is often heterogeneous in terms of the induced phenotypic diversity. In vitro recapitulation of heterogeneous differentiation under homogeneous experimental conditions indicates some highly regulated mechanisms by which multiple phenotypes of CD4+ T cells can be generated from a single population of naïve CD4+ T cells. Therefore, conceptual understanding of induced heterogeneous differentiation will shed light on the mechanisms controlling the response of populations of CD4+ T cells under physiological conditions. Results We present a simple theoretical framework to show how heterogeneous differentiation in a two-master-regulator paradigm can be governed by a signaling network motif common to all subsets of CD4+ T cells. With this motif, a population of naïve CD4+ T cells can integrate the signals from their environment to generate a functionally diverse population with robust commitment of individual cells. Notably, two positive feedback loops in this network motif govern three bistable switches, which in turn, give rise to three types of heterogeneous differentiated states, depending upon particular combinations of input signals. We provide three prototype models illustrating how to use this framework to explain experimental observations and make specific testable predictions. Conclusions The process in which several types of T helper cells are generated simultaneously to mount complex immune responses upon pathogenic challenges can be highly regulated, and a simple signaling network motif can be responsible for generating all possible types of heterogeneous populations with respect to a pair of master regulators controlling CD4+ T cell differentiation. The framework provides a mathematical basis for understanding the decision-making mechanisms of CD4+ T cells, and it can be helpful for interpreting experimental results. Mathematical models based on the framework make specific testable predictions that may improve our understanding of this differentiation system. PMID:22697466
Evolution of the amniote pallium and the origins of mammalian neocortex

PubMed Central

Butler, Ann B.; Reiner, Anton; Karten, Harvey J.

2012-01-01

Karten's neocortex hypothesis holds that many component cell populations of the sauropsid dorsal ventricular ridge (DVR) are homologous to particular cell populations in layers of auditory and visual tectofugal-recipient neocortex of mammals (i.e., temporal neocortex), as well as to some amygdaloid populations. The claustroamygdalar hypothesis, based on gene expression domains, proposes that mammalian homologues of DVR are found in the claustrum, endopiriform nuclei, and/or pallial amygdala. Because hypotheses of homology need to account for the totality of the evidence, the available data on multiple forebrain features of sauropsids and mammals are reviewed here. While some genetic data are compatible with the claustroamygdalar hypothesis, and developmental (epigenetic) data are indecisive, hodological, morphological, and topographical data favor the neocortex hypothesis and are inconsistent with the claustroamygdalar hypothesis. Detailed studies of gene signaling cascades that establish neuronal cell-type identity in DVR, tectofugal-recipient neocortex, and claustroamygdala will be needed to resolve this debate about the evolution of neocortex. PMID:21534989
Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution

PubMed Central

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D.; Rainey, Paul B.; de Visser, J. Arjan G. M.; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology. PMID:27077662
Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution.

PubMed

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D; Rainey, Paul B; de Visser, J Arjan G M; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes-via growth-over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.
Population genetics inside a cell: Mutations and mitochondrial genome maintenance

NASA Astrophysics Data System (ADS)

Goyal, Sidhartha; Shraiman, Boris; Gottschling, Dan

2012-02-01

In realistic ecological and evolutionary systems natural selection acts on multiple levels, i.e. it acts on individuals as well as on collection of individuals. An understanding of evolutionary dynamics of such systems is limited in large part due to the lack of experimental systems that can challenge theoretical models. Mitochondrial genomes (mtDNA) are subjected to selection acting on cellular as well as organelle levels. It is well accepted that mtDNA in yeast Saccharomyces cerevisiae is unstable and can degrade over time scales comparable to yeast cell division time. We utilize a recent technology designed in Gottschling lab to extract DNA from populations of aged yeast cells and deep sequencing to characterize mtDNA variation in a population of young and old cells. In tandem, we developed a stochastic model that includes the essential features of mitochondrial biology that provides a null model for expected mtDNA variation. Overall, we find approximately 2% of the polymorphic loci that show significant increase in frequency as cells age providing direct evidence for organelle level selection. Such quantitative study of mtDNA dynamics is absolutely essential to understand the propagation of mtDNA mutations linked to a spectrum of age-related diseases in humans.
Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum.

PubMed

Choi, Eunyoung; Roland, Joseph T; Barlow, Brittney J; O'Neal, Ryan; Rich, Amy E; Nam, Ki Taek; Shi, Chanjuan; Goldenring, James R

2014-11-01

The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of the stomach as well as in over 50% of antral glands. MIST1 expressing chief cells were predominantly observed in the body although individual glands of the antrum also showed MIST1 expressing chief cells. While classically described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed type glands containing both parietal cells and G cells throughout the antrum. Enteroendocrine cells show distinct patterns of localisation in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
Fitness-Balanced Escape Determines Resolution of Dynamic Founder Virus Escape Processes in HIV-1 Infection

PubMed Central

Sunshine, Justine E.; Larsen, Brendan B.; Maust, Brandon; Casey, Ellie; Deng, Wenje; Chen, Lennie; Westfall, Dylan H.; Kim, Moon; Zhao, Hong; Ghorai, Suvankar; Lanxon-Cookson, Erinn; Rolland, Morgane; Collier, Ann C.; Maenza, Janine; Mullins, James I.

2015-01-01

ABSTRACT To understand the interplay between host cytotoxic T-lymphocyte (CTL) responses and the mechanisms by which HIV-1 evades them, we studied viral evolutionary patterns associated with host CTL responses in six linked transmission pairs. HIV-1 sequences corresponding to full-length p17 and p24 gag were generated by 454 pyrosequencing for all pairs near the time of transmission, and seroconverting partners were followed for a median of 847 days postinfection. T-cell responses were screened by gamma interferon/interleukin-2 (IFN-γ/IL-2) FluoroSpot using autologous peptide sets reflecting any Gag variant present in at least 5% of sequence reads in the individual's viral population. While we found little evidence for the occurrence of CTL reversions, CTL escape processes were found to be highly dynamic, with multiple epitope variants emerging simultaneously. We found a correlation between epitope entropy and the number of epitope variants per response (r = 0.43; P = 0.05). In cases in which multiple escape mutations developed within a targeted epitope, a variant with no fitness cost became fixed in the viral population. When multiple mutations within an epitope achieved fitness-balanced escape, these escape mutants were each maintained in the viral population. Additional mutations found to confer escape but undetected in viral populations incurred high fitness costs, suggesting that functional constraints limit the available sites tolerable to escape mutations. These results further our understanding of the impact of CTL escape and reversion from the founder virus in HIV infection and contribute to the identification of immunogenic Gag regions most vulnerable to a targeted T-cell attack. IMPORTANCE Rapid diversification of the viral population is a hallmark of HIV-1 infection, and understanding the selective forces driving the emergence of viral variants can provide critical insight into the interplay between host immune responses and viral evolution. We used deep sequencing to comprehensively follow viral evolution over time in six linked HIV transmission pairs. We then mapped T-cell responses to explore if mutations arose due to adaption to the host and found that escape processes were often highly dynamic, with multiple mutations arising within targeted epitopes. When we explored the impact of these mutations on replicative capacity, we found that dynamic escape processes only resolve with the selection of mutations that conferred escape with no fitness cost to the virus. These results provide further understanding of the complicated viral-host interactions that occur during early HIV-1 infection and may help inform the design of future vaccine immunogens. PMID:26223634
Virus Resistance Is Not Costly in a Marine Alga Evolving under Multiple Environmental Stressors

PubMed Central

Heath, Sarah E.; Knox, Kirsten; Vale, Pedro F.; Collins, Sinead

2017-01-01

Viruses are important evolutionary drivers of host ecology and evolution. The marine picoplankton Ostreococcus tauri has three known resistance types that arise in response to infection with the Phycodnavirus OtV5: susceptible cells (S) that lyse following viral entry and replication; resistant cells (R) that are refractory to viral entry; and resistant producers (RP) that do not all lyse but maintain some viruses within the population. To test for evolutionary costs of maintaining antiviral resistance, we examined whether O. tauri populations composed of each resistance type differed in their evolutionary responses to several environmental drivers (lower light, lower salt, lower phosphate and a changing environment) in the absence of viruses for approximately 200 generations. We did not detect a cost of resistance as measured by life-history traits (population growth rate, cell size and cell chlorophyll content) and competitive ability. Specifically, all R and RP populations remained resistant to OtV5 lysis for the entire 200-generation experiment, whereas lysis occurred in all S populations, suggesting that resistance is not costly to maintain even when direct selection for resistance was removed, or that there could be a genetic constraint preventing return to a susceptible resistance type. Following evolution, all S population densities dropped when inoculated with OtV5, but not to zero, indicating that lysis was incomplete, and that some cells may have gained a resistance mutation over the evolution experiment. These findings suggest that maintaining resistance in the absence of viruses was not costly. PMID:28282867
Circulating innate lymphoid cells are unchanged in response to DAC HYP therapy.

PubMed

Gillard, Geoffrey O; Saenz, Steven A; Huss, David J; Fontenot, Jason D

2016-05-15

Innate lymphoid cells (ILCs) play an important role in immunity, inflammation, and tissue remodeling and their dysregulation is implicated in autoimmune and inflammatory disorders. We analyzed the impact of daclizumab, a humanized monoclonal anti-CD25 antibody, on circulating natural killer (NK) cells and ILCs in a cohort of multiple sclerosis patients. An increase in CD56(bright) NK cells and CD56(hi)CD16(intermediate) transitional NK cells was observed. No significant change in total ILCs or major ILC subpopulations was observed. These results refine our understanding of the impact of daclizumab on innate lymphoid cell populations. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
The regrowth kinetic of the surviving population is independent of acute and chronic responses to temozolomide in glioblastoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silva, Andrew Oliveira, E-mail: andrewbiomed@gmail.com; Dalsin, Eloisa, E-mail: dalsineloisa@gmail.com; Onzi, Giovana Ravizzoni, E-mail: gioonzi@gmail.com

Chemotherapy acts on cancer cells by producing multiple effects on a cell population including cell cycle arrest, necrosis, apoptosis and senescence. However, often a subpopulation of cells survives and the behavior of this subpopulation, which is responsible for cancer recurrence, remains obscure. Here we investigated the in vitro short- and long-term responses of six glioblastoma cell lines to clinically relevant doses of temozolomide for 5 days followed by 23 days of recovery, mimicking the standard schedule used in glioblastoma patient for this drug. These cells presented different profiles of sensitivity to temozolomide with varying levels of cell cycle arrest, autophagymore » and senescence, followed by a regrowth of the surviving cells. The initial reduction in cell number and the subsequent regrowth was analyzed with four new parameters applied to Cumulative Population Doubling (CPD) curves that describe the overall sensitivity of the population and the characteristic of the regrowth: the relative end point CPD (RendCPD); the relative Area Under Curve (rAUC); the Relative Time to Cross a Threshold (RTCT); and the Relative Proliferation Rate (RPR). Surprisingly, the kinetics of regrowth were not predicted by the mechanisms activated after treatment nor by the acute or overall sensitivity. With this study we added new parameters that describe key responses of glioblastoma cell populations to temozolomide treatment. These parameters can also be applied to other cell types and treatments and will help to understand the behavior of the surviving cancer cells after treatment and shed light on studies of cancer resistance and recurrence. - Highlights: • Little is known about the behavior of the glioma cells surviving to TMZ. • The short- and long-term response of six glioma cells lines to TMZ varies considerably. • These glioma cells lines recovered proliferation after therapeutic levels of TMZ. • The growth velocity of the surviving cells was different from the untreated cells. • The kinetic of regrowth was not predicted by any TMZ-triggered mechanism.« less
Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells.

PubMed

Du, Zhong-Wei; Chen, Hong; Liu, Huisheng; Lu, Jianfeng; Qian, Kun; Huang, CindyTzu-Ling; Zhong, Xiaofen; Fan, Frank; Zhang, Su-Chun

2015-03-25

Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modelling disease processes and developing new therapeutics. However, these applications are hindered by the low efficiency and heterogeneity of cell types, such as motorneurons (MNs), differentiated from hPSCs as well as our inability to maintain the potency of lineage-committed progenitors. Here by using a combination of small molecules that regulate multiple signalling pathways, we develop a method to guide human embryonic stem cells to a near-pure population (>95%) of motor neuron progenitors (MNPs) in 12 days, and an enriched population (>90%) of functionally mature MNs in an additional 16 days. More importantly, the MNPs can be expanded for at least five passages so that a single MNP can be amplified to 1 × 10(4). This method is reproducible in human-induced pluripotent stem cells and is applied to model MN-degenerative diseases and in proof-of-principle drug-screening assays.
Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters

PubMed Central

Lai, Charles P.; Kim, Edward Y.; Badr, Christian E.; Weissleder, Ralph; Mempel, Thorsten R.; Tannous, Bakhos A.; Breakefield, Xandra O.

2015-01-01

Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA. PMID:25967391

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes.

PubMed

Li, Yuwei; Ahrens, Molly J; Wu, Amy; Liu, Jennifer; Dudley, Andrew T

2011-01-01

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.
A mex3 homolog is required for differentiation during planarian stem cell lineage development

PubMed Central

Zhu, Shu Jun; Hallows, Stephanie E; Currie, Ko W; Xu, ChangJiang; Pearson, Bret J

2015-01-01

Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment. DOI: http://dx.doi.org/10.7554/eLife.07025.001 PMID:26114597
Three-dimensional epithelial tissues generated from human embryonic stem cells.

PubMed

Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

2009-11-01

The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.
Epstein-Barr virus infection and nasopharyngeal carcinoma.

PubMed

Tsao, Sai Wah; Tsang, Chi Man; Lo, Kwok Wai

2017-10-19

Epstein-Barr virus (EBV) is associated with multiple types of human cancer, including lymphoid and epithelial cancers. The closest association with EBV infection is seen in undifferentiated nasopharyngeal carcinoma (NPC), which is endemic in the southern Chinese population. A strong association between NPC risk and the HLA locus at chromosome 6p has been identified, indicating a link between the presentation of EBV antigens to host immune cells and NPC risk. EBV infection in NPC is clonal in origin, strongly suggesting that NPC develops from the clonal expansion of a single EBV-infected cell. In epithelial cells, the default program of EBV infection is lytic replication. However, latent infection is the predominant mode of EBV infection in NPC. The establishment of latent EBV infection in pre-invasive nasopharyngeal epithelium is believed to be an early stage of NPC pathogenesis. Recent genomic study of NPC has identified multiple somatic mutations in the upstream negative regulators of NF-κB signalling. Dysregulated NF-κB signalling may contribute to the establishment of latent EBV infection in NPC. Stable EBV infection and the expression of latent EBV genes are postulated to drive the transformation of pre-invasive nasopharyngeal epithelial cells to cancer cells through multiple pathways.This article is part of the themed issue 'Human oncogenic viruses'. © 2017 The Author(s).
Association between number of cell phone contracts and brain tumor incidence in nineteen U.S. States.

PubMed

Lehrer, Steven; Green, Sheryl; Stock, Richard G

2011-02-01

Some concern has arisen about adverse health effects of cell phones, especially the possibility that the low power microwave-frequency signal transmitted by the antennas on handsets might cause brain tumors or accelerate the growth of subclinical tumors. We analyzed data from the Statistical Report: Primary Brain Tumors in the United States, 2000-2004 and 2007 cell phone subscription data from the Governing State and Local Sourcebook. There was a significant correlation between number of cell phone subscriptions and brain tumors in nineteen US states (r = 0.950, P < 0.001). Because increased numbers of both cell phone subscriptions and brain tumors could be due solely to the fact that some states, such as New York, have much larger populations than other states, such as North Dakota, multiple linear regression was performed with number of brain tumors as the dependent variable, cell phone subscriptions, population, mean family income and mean age as independent variables. The effect of cell phone subscriptions was significant (P = 0.017), and independent of the effect of mean family income (P = 0.894), population (P = 0.003) and age (0.499). The very linear relationship between cell phone usage and brain tumor incidence is disturbing and certainly needs further epidemiological evaluation. In the meantime, it would be prudent to limit exposure to all sources of electro-magnetic radiation.
Embryoid body attachment to reconstituted basement membrane induces a genetic program of epithelial differentiation via jun N-terminal kinase signaling.

PubMed

Ho, Hoang-Yen; Moffat, Ryan C; Patel, Rupal V; Awah, Franklin N; Baloue, Kaitrin; Crowe, David L

2010-09-01

Embryonic stem (ES) cells are derived from early stage mammalian embryos and have broad developmental potential. These cells can be manipulated experimentally to generate cells of multiple tissue types which could be important in treating human diseases. The ability to produce relevant amounts of these differentiated cell populations creates the basis for clinical interventions in tissue regeneration and repair. Understanding how embryonic stem cells differentiate also can reveal important insights into cell biology. A previously reported mouse embryonic stem cell model demonstrated that differentiated epithelial cells migrated out of embryoid bodies attached to reconstituted basement membrane. We used genomic technology to profile ES cell populations in order to understand the molecular mechanisms leading to epithelial differentiation. Cells with characteristics of cultured epithelium migrated from embryoid bodies attached to reconstituted basement membrane. However, cells that comprised embryoid bodies also rapidly lost ES cell-specific gene expression and expressed proteins characteristic of stratified epithelia within hours of attachment to basement membrane. Gene expression profiling of sorted cell populations revealed upregulation of the BMP/TGFbeta signaling pathway, which was not sufficient for epithelial differentiation in the absence of basement membrane attachment. Activation of c-jun N-terminal kinase 1 (JNK1) and increased expression of Jun family transcription factors was observed during epithelial differentiation of ES cells. Inhibition of JNK signaling completely blocked epithelial differentiation in this model, revealing a key mechanism by which ES cells adopt epithelial characteristics via basement membrane attachment. Copyright (c) 2010 Elsevier B.V. All rights reserved.
Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

PubMed

Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.
Factors influencing the abundance of the side population in a human myeloma cell line.

PubMed

Mo, Sui-Lin; Li, Jia; Loh, Yen S; Brown, Ross D; Smith, Adrian L; Chen, Yuling; Joshua, Douglas; Roufogalis, Basil D; Li, George Q; Fan, Kei; Ng, Michelle C H; Sze, Daniel Man-Yuen

2011-01-01

Side population (SP) refers to a group of cells, which is capable to efflux Hoechst 33342, a DNA-binding dye. SP cells exist both in normal and tumor tissues. Although SP abundance has been used as an indicator for disease prognostic and drug screening in many research projects, few studies have systematically examined the factors influencing SP analysis. In this study we aim to develop a more thorough understanding of the multiple factors involved in SP analysis including Hoechst 33342 staining and cell culture. RPMI-8226, a high SP percentage (SP%) human myeloma cell line was employed here. The results showed that SP% was subject to staining conditions including: viable cell proportion, dye concentration, staining cell density, incubation duration, staining volume, and mix interval. In addition, SP% was highest in day one after passage, while dropped steadily over time. This study shows that both staining conditions and culture duration can significantly affect SP%. In this case, any conclusions based on SP% should be interpreted cautiously. The relation between culture duration and SP% suggests that the incidence of SP cells may be related to cell proliferation and cell cycle phase. Maintaining these technical variables consistently is essential in SP research.
Targeting Leukemia Stem Cells in the Bone Marrow Niche

PubMed Central

Bornhäuser, Martin

2018-01-01

The bone marrow (BM) niche encompasses multiple cells of mesenchymal and hematopoietic origin and represents a unique microenvironment that is poised to maintain hematopoietic stem cells. In addition to its role as a primary lymphoid organ through the support of lymphoid development, the BM hosts various mature lymphoid cell types, including naïve T cells, memory T cells and plasma cells, as well as mature myeloid elements such as monocyte/macrophages and neutrophils, all of which are crucially important to control leukemia initiation and progression. The BM niche provides an attractive milieu for tumor cell colonization given its ability to provide signals which accelerate tumor cell proliferation and facilitate tumor cell survival. Cancer stem cells (CSCs) share phenotypic and functional features with normal counterparts from the tissue of origin of the tumor and can self-renew, differentiate and initiate tumor formation. CSCs possess a distinct immunological profile compared with the bulk population of tumor cells and have evolved complex strategies to suppress immune responses through multiple mechanisms, including the release of soluble factors and the over-expression of molecules implicated in cancer immune evasion. This chapter discusses the latest advancements in understanding of the immunological BM niche and highlights current and future immunotherapeutic strategies to target leukemia CSCs and overcome therapeutic resistance in the clinic. PMID:29466292
Distinct retrosplenial cortex cell populations and their spike dynamics during ketamine-induced unconscious state

PubMed Central

Zhao, Fang; Tsien, Joe Z.

2017-01-01

Ketamine is known to induce psychotic-like symptoms, including delirium and visual hallucinations. It also causes neuronal damage and cell death in the retrosplenial cortex (RSC), an area that is thought to be a part of high visual cortical pathways and at least partially responsible for ketamine’s psychotomimetic activities. However, the basic physiological properties of RSC cells as well as their response to ketamine in vivo remained largely unexplored. Here, we combine a computational method, the Inter-Spike Interval Classification Analysis (ISICA), and in vivo recordings to uncover and profile excitatory cell subtypes within layers 2&3 and 5&6 of the RSC in mice within both conscious, sleep, and ketamine-induced unconscious states. We demonstrate two distinct excitatory principal cell sub-populations, namely, high-bursting excitatory principal cells and low-bursting excitatory principal cells, within layers 2&3, and show that this classification is robust over the conscious states, namely quiet awake, and natural unconscious sleep periods. Similarly, we provide evidence of high-bursting and low-bursting excitatory principal cell sub-populations within layers 5&6 that remained distinct during quiet awake and sleep states. We further examined how these subtypes are dynamically altered by ketamine. During ketamine-induced unconscious state, these distinct excitatory principal cell subtypes in both layer 2&3 and layer 5&6 exhibited distinct dynamics. We also uncovered different dynamics of local field potential under various brain states in layer 2&3 and layer 5&6. Interestingly, ketamine administration induced high gamma oscillations in layer 2&3 of the RSC, but not layer 5&6. Our results show that excitatory principal cells within RSC layers 2&3 and 5&6 contain multiple physiologically distinct sub-populations, and they are differentially affected by ketamine. PMID:29073221
Distinct retrosplenial cortex cell populations and their spike dynamics during ketamine-induced unconscious state.

PubMed

Fox, Grace E; Li, Meng; Zhao, Fang; Tsien, Joe Z

2017-01-01

Ketamine is known to induce psychotic-like symptoms, including delirium and visual hallucinations. It also causes neuronal damage and cell death in the retrosplenial cortex (RSC), an area that is thought to be a part of high visual cortical pathways and at least partially responsible for ketamine's psychotomimetic activities. However, the basic physiological properties of RSC cells as well as their response to ketamine in vivo remained largely unexplored. Here, we combine a computational method, the Inter-Spike Interval Classification Analysis (ISICA), and in vivo recordings to uncover and profile excitatory cell subtypes within layers 2&3 and 5&6 of the RSC in mice within both conscious, sleep, and ketamine-induced unconscious states. We demonstrate two distinct excitatory principal cell sub-populations, namely, high-bursting excitatory principal cells and low-bursting excitatory principal cells, within layers 2&3, and show that this classification is robust over the conscious states, namely quiet awake, and natural unconscious sleep periods. Similarly, we provide evidence of high-bursting and low-bursting excitatory principal cell sub-populations within layers 5&6 that remained distinct during quiet awake and sleep states. We further examined how these subtypes are dynamically altered by ketamine. During ketamine-induced unconscious state, these distinct excitatory principal cell subtypes in both layer 2&3 and layer 5&6 exhibited distinct dynamics. We also uncovered different dynamics of local field potential under various brain states in layer 2&3 and layer 5&6. Interestingly, ketamine administration induced high gamma oscillations in layer 2&3 of the RSC, but not layer 5&6. Our results show that excitatory principal cells within RSC layers 2&3 and 5&6 contain multiple physiologically distinct sub-populations, and they are differentially affected by ketamine.
URI regulates tumorigenicity and chemotherapeutic resistance of multiple myeloma by modulating IL-6 transcription.

PubMed

Fan, J-L; Zhang, J; Dong, L-W; Fu, W-J; Du, J; Shi, H-G; Jiang, H; Ye, F; Xi, H; Zhang, C-Y; Hou, J; Wang, H-Y

2014-03-13

Unconventional prefoldin RPB5 interactor (URI), which acts as an oncoprotein in solid tumors, is associated with RNA polymerase II subunit 5. However, its impact on multiple myeloma (MM) has not been determined. We demonstrate here that URI is overexpressed in MM compared with plasma cells derived from healthy volunteers. Side population (SP) cells sorted from MM cells showed a much higher level of URI than non-SP cells. Using lentivirus-delivered shRNA, we established stable URI knockdown MM cell lines. URI inhibition significantly attenuated the proliferation of MM cells and decreased colony formation compared with the control cells. Tumor growth assays in NOD/SCID mice further confirmed the promotion role of URI during MM development in vivo. Furthermore, URI knockdown markedly reduced the abundance of SP in MM cell lines and enhanced the chemotherapeutic sensitivity of MM towards bortezomib. Mechanically, URI appears to be critically involved in modulating STAT3 activity through regulating interleukin (IL)-6 transcription via interaction with NFκBp65. In conclusion, URI may have an important role in the development of MM and chemotherapeutic resistance through activating the IL-6/STAT3 pathway.
URI regulates tumorigenicity and chemotherapeutic resistance of multiple myeloma by modulating IL-6 transcription

PubMed Central

Fan, J-L; Zhang, J; Dong, L-W; Fu, W-J; Du, J; Shi, H-G; Jiang, H; Ye, F; Xi, H; Zhang, C-Y; Hou, J; Wang, H-Y

2014-01-01

Unconventional prefoldin RPB5 interactor (URI), which acts as an oncoprotein in solid tumors, is associated with RNA polymerase II subunit 5. However, its impact on multiple myeloma (MM) has not been determined. We demonstrate here that URI is overexpressed in MM compared with plasma cells derived from healthy volunteers. Side population (SP) cells sorted from MM cells showed a much higher level of URI than non-SP cells. Using lentivirus-delivered shRNA, we established stable URI knockdown MM cell lines. URI inhibition significantly attenuated the proliferation of MM cells and decreased colony formation compared with the control cells. Tumor growth assays in NOD/SCID mice further confirmed the promotion role of URI during MM development in vivo. Furthermore, URI knockdown markedly reduced the abundance of SP in MM cell lines and enhanced the chemotherapeutic sensitivity of MM towards bortezomib. Mechanically, URI appears to be critically involved in modulating STAT3 activity through regulating interleukin (IL)-6 transcription via interaction with NFκBp65. In conclusion, URI may have an important role in the development of MM and chemotherapeutic resistance through activating the IL-6/STAT3 pathway. PMID:24625985
Cerebrospinal Fluid B Cells Correlate with Early Brain Inflammation in Multiple Sclerosis

PubMed Central

Kuenz, Bettina; Lutterotti, Andreas; Ehling, Rainer; Gneiss, Claudia; Haemmerle, Monika; Rainer, Carolyn; Deisenhammer, Florian; Schocke, Michael; Berger, Thomas; Reindl, Markus

2008-01-01

Background There is accumulating evidence from immunological, pathological and therapeutic studies that B cells are key components in the pathophysiology of multiple sclerosis (MS). Methodology/Principal Findings In this prospective study we have for the first time investigated the differences in the inflammatory response between relapsing and progressive MS by comparing cerebrospinal fluid (CSF) cell profiles from patients at the onset of the disease (clinically isolated syndrome, CIS), relapsing-remitting (RR) and chronic progressive (CP) MS by flow cytometry. As controls we have used patients with other neurological diseases. We have found a statistically significant accumulation of CSF mature B cells (CD19+CD138−) and plasma blasts (CD19+CD138+) in CIS and RRMS. Both B cell populations were, however, not significantly increased in CPMS. Further, this accumulation of B cells correlated with acute brain inflammation measured by magnetic resonance imaging and with inflammatory CSF parameters such as the number of CSF leukocytes, intrathecal immunoglobulin M and G synthesis and intrathecal production of matrix metalloproteinase (MMP)-9 and the B cell chemokine CxCL-13. Conclusions Our data support an important role of CSF B cells in acute brain inflammation in CIS and RRMS. PMID:18596942
Smoldering Multiple Myeloma

PubMed Central

Gao, Minjie; Yang, Guang; Kong, Yuanyuan; Wu, Xiaosong; Shi, Jumei

2015-01-01

Smoldering multiple myeloma (SMM) is an asymptomatic precursor stage of multiple myeloma (MM) characterized by clonal bone marrow plasma cells (BMPC) ≥ 10% and/or M protein level ≥ 30 g/L in the absence of end organ damage. It represents an intermediate stage between monoclonal gammopathy of undetermined significance (MGUS) and symptomatic MM. The risk of progression to symptomatic MM is not uniform, and several parameters have been reported to predict the risk of progression. These include the level of M protein and the percentage of BMPC, the proportion of immunophenotypically aberrant plasma cells, and the presence of immunoparesis, free light-chain (FLC) ratio, peripheral blood plasma cells (PBPC), pattern of serum M protein evolution, abnormal magnetic resonance imaging (MRI), cytogenetic abnormalities, IgA isotype, and Bence Jones proteinuria. So far treatment is still not recommended for SMM, because several trials suggested that patients with SMM do not benefit from early treatment. However, the Mateos et al. trial showed a survival benefit after early treatment with lenalidomide plus dexamethasone in patients with high-risk SMM. This trial has prompted a reevaluation of early treatment in an asymptomatic patient population. PMID:26000300
Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain

PubMed Central

Kim, Christina K; Yang, Samuel J; Pichamoorthy, Nandini; Young, Noah P; Kauvar, Isaac; Jennings, Joshua H; Lerner, Talia N; Berndt, Andre; Lee, Soo Yeun; Ramakrishnan, Charu; Davidson, Thomas J; Inoue, Masatoshi; Bito, Haruhiko; Deisseroth, Karl

2017-01-01

Real-time activity measurements from multiple specific cell populations and projections are likely to be important for understanding the brain as a dynamical system. Here we developed frame-projected independent-fiber photometry (FIP), which we used to record fluorescence activity signals from many brain regions simultaneously in freely behaving mice. We explored the versatility of the FIP microscope by quantifying real-time activity relationships among many brain regions during social behavior, simultaneously recording activity along multiple axonal pathways during sensory experience, performing simultaneous two-color activity recording, and applying optical perturbation tuned to elicit dynamics that match naturally occurring patterns observed during behavior. PMID:26878381
Fluorescence-based detection and quantification of features of cellular senescence.

PubMed

Cho, Sohee; Hwang, Eun Seong

2011-01-01

Cellular senescence is a spontaneous organismal defense mechanism against tumor progression which is raised upon the activation of oncoproteins or other cellular environmental stresses that must be circumvented for tumorigenesis to occur. It involves growth-arrest state of normal cells after a number of active divisions. There are multiple experimental routes that can drive cells into a state of senescence. Normal somatic cells and cancer cells enter a state of senescence upon overexpression of oncogenic Ras or Raf protein or by imposing certain kinds of stress such as cellular tumor suppressor function. Both flow cytometry and confocal imaging analysis techniques are very useful in quantitative analysis of cellular senescence phenomenon. They allow quantitative estimates of multiple different phenotypes expressed in multiple cell populations simultaneously. Here we review the various types of fluorescence methodologies including confocal imaging and flow cytometry that are frequently utilized to study a variety of senescence. First, we discuss key cell biological changes occurring during senescence and review the current understanding on the mechanisms of these changes with the goal of improving existing protocols and further developing new ones. Next, we list specific senescence phenotypes associated with each cellular trait along with the principles of their assay methods and the significance of the assay outcomes. We conclude by selecting appropriate references that demonstrate a typical example of each method. Copyright © 2011 Elsevier Inc. All rights reserved.
Integration of Immune Cell Populations, mRNA-Seq, and CpG Methylation to Better Predict Humoral Immunity to Influenza Vaccination: Dependence of mRNA-Seq/CpG Methylation on Immune Cell Populations

PubMed Central

Zimmermann, Michael T.; Kennedy, Richard B.; Grill, Diane E.; Oberg, Ann L.; Goergen, Krista M.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; Poland, Gregory A.

2017-01-01

The development of a humoral immune response to influenza vaccines occurs on a multisystems level. Due to the orchestration required for robust immune responses when multiple genes and their regulatory components across multiple cell types are involved, we examined an influenza vaccination cohort using multiple high-throughput technologies. In this study, we sought a more thorough understanding of how immune cell composition and gene expression relate to each other and contribute to interindividual variation in response to influenza vaccination. We first hypothesized that many of the differentially expressed (DE) genes observed after influenza vaccination result from changes in the composition of participants’ peripheral blood mononuclear cells (PBMCs), which were assessed using flow cytometry. We demonstrated that DE genes in our study are correlated with changes in PBMC composition. We gathered DE genes from 128 other publically available PBMC-based vaccine studies and identified that an average of 57% correlated with specific cell subset levels in our study (permutation used to control false discovery), suggesting that the associations we have identified are likely general features of PBMC-based transcriptomics. Second, we hypothesized that more robust models of vaccine response could be generated by accounting for the interplay between PBMC composition, gene expression, and gene regulation. We employed machine learning to generate predictive models of B-cell ELISPOT response outcomes and hemagglutination inhibition (HAI) antibody titers. The top HAI and B-cell ELISPOT model achieved an area under the receiver operating curve (AUC) of 0.64 and 0.79, respectively, with linear model coefficients of determination of 0.08 and 0.28. For the B-cell ELISPOT outcomes, CpG methylation had the greatest predictive ability, highlighting potentially novel regulatory features important for immune response. B-cell ELISOT models using only PBMC composition had lower performance (AUC = 0.67), but highlighted well-known mechanisms. Our analysis demonstrated that each of the three data sets (cell composition, mRNA-Seq, and DNA methylation) may provide distinct information for the prediction of humoral immune response outcomes. We believe that these findings are important for the interpretation of current omics-based studies and set the stage for a more thorough understanding of interindividual immune responses to influenza vaccination. PMID:28484452
A co-culture device with a tunable stiffness to understand combinatorial cell-cell and cell-matrix interactions.

PubMed

Rao, Nikhil; Grover, Gregory N; Vincent, Ludovic G; Evans, Samantha C; Choi, Yu Suk; Spencer, Katrina H; Hui, Elliot E; Engler, Adam J; Christman, Karen L

2013-11-01

Cell behavior on 2-D in vitro cultures is continually being improved to better mimic in vivo physiological conditions by combining niche cues including multiple cell types and substrate stiffness, which are well known to impact cell phenotype. However, no system exists in which a user can systematically examine cell behavior on a substrate with a specific stiffness (elastic modulus) in culture with a different cell type, while maintaining distinct cell populations. We demonstrate the modification of a silicon reconfigurable co-culture system with a covalently linked hydrogel of user-defined stiffness. This device allows the user to control whether two separate cell populations are in contact with each other or only experience paracrine interactions on substrates of controllable stiffness. To illustrate the utility of this device, we examined the role of substrate stiffness combined with myoblast co-culture on adipose derived stem cell (ASC) differentiation and found that the presence of myoblasts and a 10 kPa substrate stiffness increased ASC myogenesis versus co-culture on stiff substrates. As this example highlights, this technology better controls the in vitro microenvironment, allowing the user to develop a more thorough understanding of the combined effects of cell-cell and cell-matrix interactions.
Processes on the emergent landscapes of biochemical reaction networks and heterogeneous cell population dynamics: differentiation in living matters

PubMed Central

Li, Fangting

2017-01-01

The notion of an attractor has been widely employed in thinking about the nonlinear dynamics of organisms and biological phenomena as systems and as processes. The notion of a landscape with valleys and mountains encoding multiple attractors, however, has a rigorous foundation only for closed, thermodynamically non-driven, chemical systems, such as a protein. Recent advances in the theory of nonlinear stochastic dynamical systems and its applications to mesoscopic reaction networks, one reaction at a time, have provided a new basis for a landscape of open, driven biochemical reaction systems under sustained chemostat. The theory is equally applicable not only to intracellular dynamics of biochemical regulatory networks within an individual cell but also to tissue dynamics of heterogeneous interacting cell populations. The landscape for an individual cell, applicable to a population of isogenic non-interacting cells under the same environmental conditions, is defined on the counting space of intracellular chemical compositions x = (x1,x2, … ,xN) in a cell, where xℓ is the concentration of the ℓth biochemical species. Equivalently, for heterogeneous cell population dynamics xℓ is the number density of cells of the ℓth cell type. One of the insights derived from the landscape perspective is that the life history of an individual organism, which occurs on the hillsides of a landscape, is nearly deterministic and ‘programmed’, while population-wise an asynchronous non-equilibrium steady state resides mostly in the lowlands of the landscape. We argue that a dynamic ‘blue-sky’ bifurcation, as a representation of Waddington's landscape, is a more robust mechanism for a cell fate decision and subsequent differentiation than the widely pictured pitch-fork bifurcation. We revisit, in terms of the chemostatic driving forces upon active, living matter, the notions of near-equilibrium thermodynamic branches versus far-from-equilibrium states. The emergent landscape perspective permits a quantitative discussion of a wide range of biological phenomena as nonlinear, stochastic dynamics. PMID:28490602

A Stochastic Framework for Modeling the Population Dynamics of Convective Clouds

DOE PAGES

Hagos, Samson; Feng, Zhe; Plant, Robert S.; ...

2018-02-20

A stochastic prognostic framework for modeling the population dynamics of convective clouds and representing them in climate models is proposed. The framework follows the nonequilibrium statistical mechanical approach to constructing a master equation for representing the evolution of the number of convective cells of a specific size and their associated cloud-base mass flux, given a large-scale forcing. In this framework, referred to as STOchastic framework for Modeling Population dynamics of convective clouds (STOMP), the evolution of convective cell size is predicted from three key characteristics of convective cells: (i) the probability of growth, (ii) the probability of decay, and (iii)more » the cloud-base mass flux. STOMP models are constructed and evaluated against CPOL radar observations at Darwin and convection permitting model (CPM) simulations. Multiple models are constructed under various assumptions regarding these three key parameters and the realisms of these models are evaluated. It is shown that in a model where convective plumes prefer to aggregate spatially and the cloud-base mass flux is a nonlinear function of convective cell area, the mass flux manifests a recharge-discharge behavior under steady forcing. Such a model also produces observed behavior of convective cell populations and CPM simulated cloud-base mass flux variability under diurnally varying forcing. Finally, in addition to its use in developing understanding of convection processes and the controls on convective cell size distributions, this modeling framework is also designed to serve as a nonequilibrium closure formulations for spectral mass flux parameterizations.« less
A Stochastic Framework for Modeling the Population Dynamics of Convective Clouds

NASA Astrophysics Data System (ADS)

Hagos, Samson; Feng, Zhe; Plant, Robert S.; Houze, Robert A.; Xiao, Heng

2018-02-01

A stochastic prognostic framework for modeling the population dynamics of convective clouds and representing them in climate models is proposed. The framework follows the nonequilibrium statistical mechanical approach to constructing a master equation for representing the evolution of the number of convective cells of a specific size and their associated cloud-base mass flux, given a large-scale forcing. In this framework, referred to as STOchastic framework for Modeling Population dynamics of convective clouds (STOMP), the evolution of convective cell size is predicted from three key characteristics of convective cells: (i) the probability of growth, (ii) the probability of decay, and (iii) the cloud-base mass flux. STOMP models are constructed and evaluated against CPOL radar observations at Darwin and convection permitting model (CPM) simulations. Multiple models are constructed under various assumptions regarding these three key parameters and the realisms of these models are evaluated. It is shown that in a model where convective plumes prefer to aggregate spatially and the cloud-base mass flux is a nonlinear function of convective cell area, the mass flux manifests a recharge-discharge behavior under steady forcing. Such a model also produces observed behavior of convective cell populations and CPM simulated cloud-base mass flux variability under diurnally varying forcing. In addition to its use in developing understanding of convection processes and the controls on convective cell size distributions, this modeling framework is also designed to serve as a nonequilibrium closure formulations for spectral mass flux parameterizations.
A Stochastic Framework for Modeling the Population Dynamics of Convective Clouds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hagos, Samson; Feng, Zhe; Plant, Robert S.

A stochastic prognostic framework for modeling the population dynamics of convective clouds and representing them in climate models is proposed. The framework follows the nonequilibrium statistical mechanical approach to constructing a master equation for representing the evolution of the number of convective cells of a specific size and their associated cloud-base mass flux, given a large-scale forcing. In this framework, referred to as STOchastic framework for Modeling Population dynamics of convective clouds (STOMP), the evolution of convective cell size is predicted from three key characteristics of convective cells: (i) the probability of growth, (ii) the probability of decay, and (iii)more » the cloud-base mass flux. STOMP models are constructed and evaluated against CPOL radar observations at Darwin and convection permitting model (CPM) simulations. Multiple models are constructed under various assumptions regarding these three key parameters and the realisms of these models are evaluated. It is shown that in a model where convective plumes prefer to aggregate spatially and the cloud-base mass flux is a nonlinear function of convective cell area, the mass flux manifests a recharge-discharge behavior under steady forcing. Such a model also produces observed behavior of convective cell populations and CPM simulated cloud-base mass flux variability under diurnally varying forcing. Finally, in addition to its use in developing understanding of convection processes and the controls on convective cell size distributions, this modeling framework is also designed to serve as a nonequilibrium closure formulations for spectral mass flux parameterizations.« less
Non-myeloablative autologous haematopoietic stem cell transplantation expands regulatory cells and depletes IL-17 producing mucosal-associated invariant T cells in multiple sclerosis

PubMed Central

Abrahamsson, Sofia V.; Angelini, Daniela F.; Dubinsky, Amy N.; Morel, Esther; Oh, Unsong; Jones, Joanne L.; Carassiti, Daniele; Reynolds, Richard; Salvetti, Marco; Calabresi, Peter A.; Coles, Alasdair J.; Battistini, Luca; Martin, Roland; Burt, Richard K.

2013-01-01

Autologous haematopoietic stem cell transplantation has been tried as one experimental strategy for the treatment of patients with aggressive multiple sclerosis refractory to other immunotherapies. The procedure is aimed at ablating and repopulating the immune repertoire by sequentially mobilizing and harvesting haematopoietic stem cells, administering an immunosuppressive conditioning regimen, and re-infusing the autologous haematopoietic cell product. ‘Non-myeloablative’ conditioning regimens to achieve lymphocytic ablation without marrow suppression have been proposed to improve safety and tolerability. One trial with non-myeloablative autologous haematopoietic stem cell transplantation reported clinical improvement and inflammatory stabilization in treated patients with highly active multiple sclerosis. The aim of the present study was to understand the changes in the reconstituted immune repertoire bearing potential relevance to its mode of action. Peripheral blood was obtained from 12 patients with multiple sclerosis participating in the aforementioned trial and longitudinally followed for 2 years. We examined the phenotype and function of peripheral blood lymphocytes by cell surface or intracellular staining and multi-colour fluorescence activated cell sorting alone or in combination with proliferation assays. During immune reconstitution post-transplantation we observed significant though transient increases in the proportion of CD4+FoxP3+ T cells and CD56high natural killer cell subsets, which are cell subsets associated with immunoregulatory function. CD8+CD57+ cytotoxic T cells were persistently increased after therapy and were able to suppress CD4+ T cell proliferation with variable potency. In contrast, a CD161high proinflammatory CD8+ T cell subset was depleted at all time-points post-transplantation. Phenotypic characterization revealed that the CD161highCD8+ T cells were mucosal-associated invariant T cells, a novel cell population originating in the gut mucosa but expressing the central nervous system-homing receptor CCR6. Detection of mucosal-associated invariant T cells in post-mortem multiple sclerosis brain white matter active lesions confirmed their involvement in the disease pathology. Intracellular cytokine staining demonstrated interferon γ and interleukin 17 production and lack of interleukin 10 production, a pro-inflammatory profile. Mucosal-associated invariant T cell frequency did not change in patients treated with interferon β; and was more depleted after autologous haematopoietic stem cell transplantation than in patients who had received high-dose cyclophosphamide (n = 7) or alemtuzumab (n = 21) treatment alone, suggesting an additive or synergistic effect of the conditioning regime components. We propose that a favourably modified balance of regulatory and pro-inflammatory lymphocytes underlies the suppression of central nervous system inflammation in patients with multiple sclerosis following non-myeloablative autologous haematopoietic stem cell transplantation with a conditioning regimen consisting of cyclophosphamide and alemtuzumab. PMID:23864273
Heterogeneity and Developmental Connections between Cell Types Inhabiting Teeth

PubMed Central

Krivanek, Jan; Adameyko, Igor; Fried, Kaj

2017-01-01

Every tissue is composed of multiple cell types that are developmentally, evolutionary and functionally integrated into the unit we call an organ. Teeth, our organs for biting and mastication, are complex and made of many different cell types connected or disconnected in terms of their ontogeny. In general, epithelial and mesenchymal compartments represent the major framework of tooth formation. Thus, they give rise to the two most important matrix–producing populations: ameloblasts generating enamel and odontoblasts producing dentin. However, the real picture is far from this quite simplified view. Diverse pulp cells, the immune system, the vascular system, the innervation and cells organizing the dental follicle all interact, and jointly participate in transforming lifeless matrix into a functional organ that can sense and protect itself. Here we outline the heterogeneity of cell types that inhabit the tooth, and also provide a life history of the major populations. The mouse model system has been indispensable not only for the studies of cell lineages and heterogeneity, but also for the investigation of dental stem cells and tooth patterning during development. Finally, we briefly discuss the evolutionary aspects of cell type diversity and dental tissue integration. PMID:28638345
Nestin- and Doublecortin-Positive Cells Reside in Adult Spinal Cord Meninges and Participate in Injury-Induced Parenchymal Reaction

PubMed Central

Decimo, Ilaria; Bifari, Francesco; Rodriguez, Francisco Javier; Malpeli, Giorgio; Dolci, Sissi; Lavarini, Valentina; Pretto, Silvia; Vasquez, Sandra; Sciancalepore, Marina; Montalbano, Alberto; Berton, Valeria; Krampera, Mauro; Fumagalli, Guido

2011-01-01

Adult spinal cord has little regenerative potential, thus limiting patient recovery following injury. In this study, we describe a new population of cells resident in the adult rat spinal cord meninges that express the neural stem/precursor markers nestin and doublecortin. Furthermore, from dissociated meningeal tissue a neural stem cell population was cultured in vitro and subsequently shown to differentiate into functional neurons or mature oligodendrocytes. Proliferation rate and number of nestin- and doublecortin-positive cells increased in vivo in meninges following spinal cord injury. By using a lentivirus-labeling approach, we show that meningeal cells, including nestin- and doublecortin-positive cells, migrate in the spinal cord parenchyma and contribute to the glial scar formation. Our data emphasize the multiple roles of meninges in the reaction of the parenchyma to trauma and indicate for the first time that spinal cord meninges are potential niches harboring stem/precursor cells that can be activated by injury. Meninges may be considered as a new source of adult stem/precursor cells to be further tested for use in regenerative medicine applied to neurological disorders, including repair from spinal cord injury. Stem Cells 2011;29:2062–2076. PMID:22038821
Large Population-Based Study Reveals Disparities in Myeloma Precursor Disease | Center for Cancer Research

Cancer.gov

Multiple myeloma (MM) is a cancer of plasma cells, which are antibody-producing white blood cells. Patients with MM have a characteristic excess of monoclonal antibodies, so called M proteins, in their serum, urine, or both and plasma cell infiltration into their bone marrow at multiple sites. African Americans are more than twice as likely as whites to develop MM, but the reason for this higher prevalence is not entirely clear. Since MM is nearly always preceded by the premalignant condition monoclonal gammopathy of undetermined significance (MGUS), Ola Landgren, M.D., Ph.D., a Senior Investigator in CCR’s Lymphoid Malignancies Branch, and colleagues from NCI’s Division of Cancer Epidemiology and Genetics, the Mayo Clinic, and the Centers for Disease Control and Prevention (CDC), wanted to determine whether there were also disparities in MGUS prevalence or in biomarkers associated with a high risk of MGUS progression to MM.
Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability.

PubMed

Patterson, Melissa N; Scannapieco, Alison E; Au, Pak Ho; Dorsey, Savanna; Royer, Catherine A; Maxwell, Patrick H

2015-10-01

Retrotransposon expression or mobility is increased with age in multiple species and could promote genome instability or altered gene expression during aging. However, it is unclear whether activation of retrotransposons during aging is an indirect result of global changes in chromatin and gene regulation or a result of retrotransposon-specific mechanisms. Retromobility of a marked chromosomal Ty1 retrotransposon in Saccharomyces cerevisiae was elevated in mother cells relative to their daughter cells, as determined by magnetic cell sorting of mothers and daughters. Retromobility frequencies in aging mother cells were significantly higher than those predicted by cell age and the rate of mobility in young populations, beginning when mother cells were only several generations old. New Ty1 insertions in aging mothers were more strongly correlated with gross chromosome rearrangements than in young cells and were more often at non-preferred target sites. Mother cells were more likely to have high concentrations and bright foci of Ty1 Gag-GFP than their daughter cells. Levels of extrachromosomal Ty1 cDNA were also significantly higher in aged mother cell populations than their daughter cell populations. These observations are consistent with a retrotransposon-specific mechanism that causes retrotransposition to occur preferentially in yeast mother cells as they begin to age, as opposed to activation by phenotypic changes associated with very old age. These findings will likely be relevant for understanding retrotransposons and aging in many organisms, based on similarities in regulation and consequences of retrotransposition in diverse species. Copyright © 2015 Elsevier B.V. All rights reserved.
Chromosomal abnormalities in HPV-16-immortalized oral epithelial cells.

PubMed

Oda, D; Bigler, L; Mao, E J; Disteche, C M

1996-09-01

Human papilloma virus (HPV) type 16 has an established association with anogenital carcinoma, and to some extent with human oral squamous cell carcinoma. We hypothesize that HPV type 16 is capable of inducing chromosomal and cell cycle changes in cultured oral epithelial cells. Normal human oral epithelia] cells were immortalized with recombinant retrovirus containing the E6/E7 open reading frames of HPV type 16. These cells have been in culture for more than 350 passages and over 4 years. Flow cytometry demonstrated an average of 42% nuclear aneuploidy in HPV 16-immortalized cells; 16% in normal controls (probably tetrasomy). Cytogenetic analysis demonstrated significant progression of chromosomal abnormalities. Cells at early passage (p10) showed trisomy 20, with no other major changes. At passage 18, trisomy 1q and monosomy 13 were seen in addition to trisomy 20. At passage 61 there were two distinct cell populations ('a' and 'b'), with multiple chromosomal changes including trisomy 5q,14,20 in one line and 7p,9q,llq in the other. Both populations had monosomy 3p, with monosomy 8p in one population and monosomy 13 in the other. At passage 136, the cells were essentially identical to population 'b' of passage 61. At this passage, mutation of the p53 gene was detected at codon 273 of exon 8, with G to T conversion (Arg to Leu). This was absent in the normal cells from which this line was developed. Passage 262 contained the two major cell populations, each with a sub-group with additional chromosomal changes such as 10p monosomy. Cells from passages 217 and 305 were injected into nude mice a year apart. Both failed to produce tumors, as did normal cells. In conclusion, we present an HPV type 16-immortalized oral epithelial cell line (IHGK) with extensive and progressive chromosomal abnormalities, invasive growth in culture and yet no tumor formation in nude mice. We suggest that the question as to whether HPV alone can induce transformation is still open.
Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles

PubMed Central

Wheeler, Richard John

2015-01-01

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point “snapshot” of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes—cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)—as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods. PMID:26543196
Blood Erythrocyte Concentrations of Cadmium and Lead and the Risk of B-Cell Non-Hodgkin’s Lymphoma and Multiple Myeloma: A Nested Case-Control Study

PubMed Central

Porta, Miquel; Bergdahl, Ingvar A.; Palli, Domenico; Johansson, Ann-Sofie; Botsivali, Maria; Vineis, Paolo; Vermeulen, Roel; Kyrtopoulos, Soterios A.; Chadeau-Hyam, Marc

2013-01-01

Background Cadmium (Cd) and lead (Pb) are hypothesised to be risk factors for non-Hodgkin’s lymphoma (NHL), a group of haematological malignancies with a suspected environmental aetiology. Within the EnviroGenoMarkers study we utilised pre-diagnostic erythrocyte concentrations of Cd and Pb to determine whether exposure was associated with risk of B-cell NHL and multiple myeloma. Methods 194 incident cases of B-cell NHL and 76 cases of multiple myeloma diagnosed between 1990 and 2006 were identified from two existing cohorts; EPIC-Italy and the Northern Sweden Health and Disease Study. Cases were matched to healthy controls by centre, age, gender and date of blood collection. Cd and Pb were measured in blood samples provided at recruitment using inductively coupled plasma-mass spectrometry. Logistic regression was applied to assess the association with risk. Analyses were stratified by cohort and gender and by subtype where possible. Results There was little evidence of an increased risk of B-cell NHL or multiple myeloma with exposure to Cd (B-cell NHL: OR 1.09 95%CI 0.61, 1.93, MM: OR 1.16 95% CI: 0.40, 3.40 ) or Pb (B-cell NHL: 0.93 95% CI 0.43, 2.02, multiple myeloma: OR 1.63 95%CI 0.45, 5.94) in the total population when comparing the highest to the lowest quartile of exposure. However, gender and cohort specific differences in results were observed. In females the risk of B-cell NHL was more than doubled in those with a body burden of Cd >1µg/L (OR 2.20 95%CI; 1.04, 4.65). Conclusions This nested case-control study does not support a consistent positive association between Cd or Pb and NHL, but there is some indication of a gender specific effect suggesting further research is warranted. PMID:24312375
An in vivo requirement for the mediator subunit med14 in the maintenance of stem cell populations.

PubMed

Burrows, Jeffrey T A; Pearson, Bret J; Scott, Ian C

2015-04-14

The Mediator complex has recently been shown to be a key player in the maintenance of embryonic and induced pluripotent stem cells. However, the in vivo consequences of loss of many Mediator subunits are unknown. We identified med14 as the gene affected in the zebrafish logelei (log) mutant, which displayed a morphological arrest by 2 days of development. Surprisingly, microarray analysis showed that transcription was not broadly affected in log mutants. Indeed, log cells transplanted into a wild-type environment were able to survive into adulthood. In planarians, RNAi knockdown demonstrated a requirement for med14 and many other Mediator components in adult stem cell maintenance and regeneration. Multiple stem/progenitor cell populations were observed to be reduced or absent in zebrafish med14 mutant embryos. Taken together, our results show a critical, evolutionarily conserved, in vivo function for Med14 (and Mediator) in stem cell maintenance, distinct from a general role in transcription. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
The significance of multiple mating in the social wasp Vespula maculifrons.

PubMed

Goodisman, Michael A D; Kovacs, Jennifer L; Hoffman, Eric A

2007-09-01

The evolution of the complex societies displayed by social insects depended partly on high relatedness among interacting group members. Therefore, behaviors that depress group relatedness, such as multiple mating by reproductive females (polyandry), are unexpected in social insects. Nevertheless, the queens of several social insect species mate multiply, suggesting that polyandry provides some benefits that counteract the costs. However, few studies have obtained evidence for links between rates of polyandry and fitness in naturally occurring social insect populations. We investigated if polyandry was beneficial in the social wasp Vespula maculifrons. We used genetic markers to estimate queen mate number in V. maculifrons colonies and assessed colony fitness by counting the number of cells that colonies produced. Our results indicated that queen mate number was directly, strongly, and significantly correlated with the number of queen cells produced by colonies. Because V. maculifrons queens are necessarily reared in queen cells, our results demonstrate that high levels of polyandry are associated with colonies capable of producing many new queens. These data are consistent with the explanation that polyandry is adaptive in V. maculifrons because it provides a fitness advantage to queens. Our research may provide a rare example of an association between polyandry and fitness in a natural social insect population and help explain why queens in this taxon mate multiply.
Bone marrow derived stem cells in joint and bone diseases: a concise review.

PubMed

Marmotti, Antonio; de Girolamo, Laura; Bonasia, Davide Edoardo; Bruzzone, Matteo; Mattia, Silvia; Rossi, Roberto; Montaruli, Angela; Dettoni, Federico; Castoldi, Filippo; Peretti, Giuseppe

2014-09-01

Stem cells have huge applications in the field of tissue engineering and regenerative medicine. Their use is currently not restricted to the life-threatening diseases but also extended to disorders involving the structural tissues, which may not jeopardize the patients' life, but certainly influence their quality of life. In fact, a particularly popular line of research is represented by the regeneration of bone and cartilage tissues to treat various orthopaedic disorders. Most of these pioneering research lines that aim to create new treatments for diseases that currently have limited therapies are still in the bench of the researchers. However, in recent years, several clinical trials have been started with satisfactory and encouraging results. This article aims to review the concept of stem cells and their characterization in terms of site of residence, differentiation potential and therapeutic prospective. In fact, while only the bone marrow was initially considered as a "reservoir" of this cell population, later, adipose tissue and muscle tissue have provided a considerable amount of cells available for multiple differentiation. In reality, recently, the so-called "stem cell niche" was identified as the perivascular space, recognizing these cells as almost ubiquitous. In the field of bone and joint diseases, their potential to differentiate into multiple cell lines makes their application ideally immediate through three main modalities: (1) cells selected by withdrawal from bone marrow, subsequent culture in the laboratory, and ultimately transplant at the site of injury; (2) bone marrow aspirate, concentrated and directly implanted into the injury site; (3) systemic mobilization of stem cells and other bone marrow precursors by the use of growth factors. The use of this cell population in joint and bone disease will be addressed and discussed, analysing both the clinical outcomes but also the basic research background, which has justified their use for the treatment of bone, cartilage and meniscus tissues.
EFFECTS OF PLATING DENSITY AND CULTURE TIME ON BONE MARROW STROMAL CELL CHARACTERISTICS

PubMed Central

Neuhuber, Birgit; Swanger, Sharon A.; Howard, Linda; Mackay, Alastair; Fischer, Itzhak

2008-01-01

Objective Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations. Methods Rat MSC were plated at 20, 200 and 2000 cells/cm2 and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate and late passages, as well as between heterogeneous and cloned MSC populations. Results We found optimal cell growth at a plating density of 200 cells/cm2. Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential. Conclusion Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood. PMID:18495329
DNA Damage: A Sensible Mediator of the Differentiation Decision in Hematopoietic Stem Cells and in Leukemia

PubMed Central

Weiss, Cary N.; Ito, Keisuke

2015-01-01

In the adult, the source of functionally diverse, mature blood cells are hematopoietic stem cells, a rare population of quiescent cells that reside in the bone marrow niche. Like stem cells in other tissues, hematopoietic stem cells are defined by their ability to self-renew, in order to maintain the stem cell population for the lifetime of the organism, and to differentiate, in order to give rise to the multiple lineages of the hematopoietic system. In recent years, increasing evidence has suggested a role for the accumulation of reactive oxygen species and DNA damage in the decision for hematopoietic stem cells to exit quiescence and to differentiate. In this review, we will examine recent work supporting the idea that detection of cell stressors, such as oxidative and genetic damage, is an important mediator of cell fate decisions in hematopoietic stem cells. We will explore the benefits of such a system in avoiding the development and progression of malignancies, and in avoiding tissue exhaustion and failure. Additionally, we will discuss new work that examines the accumulation of DNA damage and replication stress in aging hematopoietic stem cells and causes us to rethink ideas of genoprotection in the bone marrow niche. PMID:25789504
Mixed endocrine gastric tumors associated with hypergastrinemia of antral origin.

PubMed Central

Larsson, L. I.; Rehfeld, J. F.; Stockbrügger, R.; Blohme, G.; Schöön, I. M.; Lundqvist, G.; Kindblom, L. G.; Säve-Söderberg, J.; Grimelius, L.; Olbe, L.

1978-01-01

A patient with atrophic gastritis and excessively raised serum gastrin concentrations (4000 to 5000 pg/ml) was found to have multiple polypous tumors of the gastric corpus mucosa. Following gastrectomy, serum gastrin concentrations decreased to undetectable levels. The tumors consisted of a mixed population of endocrine cells. The majority of tumor cells were of the ECL type, but, in addition, enterochromaffin cells of various subtypes as well as agranular cells were found. The tumors were locally invasive and invaded the walls of submucosal blood vessels. The surrounding mucosa showed a severe atrophic gastritis with intestinalization and contained numerous goblet cells, enterochromaffin cells, and cholecystokinin cells. Cholecystokinin cells do not occur in the normal oxyntic mucosa. Hence, the observation of this cell type in intestinalized gastric epithelium suggests that "intestinalization also is associated with changes in endocrine cell populations. Gastrin has been shown to affect the function of the ECL cells. Indications for a trophic action of gastrin on these cells have been obtained. It is discussed whether greatly raised serum gastrin levels in patients with atrophic gastritis may be associated with increased risks for the development of certain types of gastric tumors. Images Figure 4 Figure 5 Figure 6 Figure 7 Figure 1 Figure 2 Figure 3 PMID:696807
Application of immunogold-silver staining and immunoenzymatic methods in multiple labelling of human pancreatic Langerhans islet cells.

PubMed

Krenács, T; Lászik, Z; Dobó, E

1989-01-01

The use of immunogold-silver staining (IGSS) combined with immunoperoxidase and/or immunoalkaline phosphatase methods for the simultaneous demonstration of pancreatic islet cell hormones on routinely fixed paraffin-embedded human tissue sections was examined. If IGSS was applied first, the black colour of silver-enhanced colloidal gold on doubly immunostained sections contrasted with the colours of most of the chromogens used generally in the 2 immunoenzymatic methods. If IGSS was followed by immunoalkaline phosphatase and immunoperoxidase techniques in optional sequence, 3 different hormone-containing cell types could be stained simultaneously without non-specific cross-reactions. IGSS and immunoalkaline phosphatase methods, together with 2 kinds of non-cross-reacting immunoperoxidase systems, permitted the detection of 4 distinct antigens on the same tissue section. Multiple immunohistochemical labelling of the endocrine pancreas provides an opportunity for the correct and rapid analysis of the topographic and morphometric relationships between different hormone-producing cell populations under both normal and pathological conditions. IGSS is of great potential for the simultaneous immunolabelling of antigens situated within separate cells.
Multiple independent second-site mutations in two siblings with somatic mosaicism for Wiskott-Aldrich syndrome.

PubMed

Boztug, K; Germeshausen, M; Avedillo Díez, I; Gulacsy, V; Diestelhorst, J; Ballmaier, M; Welte, K; Maródi, L; Chernyshova, Li; Klein, C

2008-07-01

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disorder associated with microthrombocytopenia, eczema, autoimmunity and predisposition to malignant lymphoma. Although rare, few cases of somatic mosaicism have been published in WAS patients to date. We here report on two Ukrainian siblings who were referred to us at the age of 3 and 4 years, respectively. Both patients suffered from severe WAS caused by a nonsense mutation in exon 1 of the WAS gene. In both siblings, flow cytometric analysis revealed the presence of Wiskott-Aldrich syndrome protein (WASp)-positive and WASp-negative cell populations among T and B lymphocytes as well as natural killer (NK) cells. In contrast to previously described cases of revertant mosaicism in WAS, molecular analyses in both children showed that the WASp-positive T cells, B cells, and NK cells carried multiple different second-site mutations, resulting in different missense mutations. To our knowledge, this is the first report describing somatic mosaicism in WAS patients caused by several independent second-site mutations in the WAS gene.
Intracellular Protein Shuttling: A Mechanism Relevant for Myelin Repair in Multiple Sclerosis?

PubMed Central

Göttle, Peter; Küry, Patrick

2015-01-01

A prominent feature of demyelinating diseases such as multiple sclerosis (MS) is the degeneration and loss of previously established functional myelin sheaths, which results in impaired signal propagation and axonal damage. However, at least in early disease stages, partial replacement of lost oligodendrocytes and thus remyelination occur as a result of resident oligodendroglial precursor cell (OPC) activation. These cells represent a widespread cell population within the adult central nervous system (CNS) that can differentiate into functional myelinating glial cells to restore axonal functions. Nevertheless, the spontaneous remyelination capacity in the adult CNS is inefficient because OPCs often fail to generate new oligodendrocytes due to the lack of stimulatory cues and the presence of inhibitory factors. Recent studies have provided evidence that regulated intracellular protein shuttling is functionally involved in oligodendroglial differentiation and remyelination activities. In this review we shed light on the role of the subcellular localization of differentiation-associated factors within oligodendroglial cells and show that regulation of intracellular localization of regulatory factors represents a crucial process to modulate oligodendroglial maturation and myelin repair in the CNS. PMID:26151843

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed Central

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-01-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells. PMID:3855866
Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-02-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells.
Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

PubMed

Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

2016-05-15

Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. Copyright © 2015 Elsevier Inc. All rights reserved.
Nestin- and doublecortin-positive cells reside in adult spinal cord meninges and participate in injury-induced parenchymal reaction.

PubMed

Decimo, Ilaria; Bifari, Francesco; Rodriguez, Francisco Javier; Malpeli, Giorgio; Dolci, Sissi; Lavarini, Valentina; Pretto, Silvia; Vasquez, Sandra; Sciancalepore, Marina; Montalbano, Alberto; Berton, Valeria; Krampera, Mauro; Fumagalli, Guido

2011-12-01

Adult spinal cord has little regenerative potential, thus limiting patient recovery following injury. In this study, we describe a new population of cells resident in the adult rat spinal cord meninges that express the neural stem/precursor markers nestin and doublecortin. Furthermore, from dissociated meningeal tissue a neural stem cell population was cultured in vitro and subsequently shown to differentiate into functional neurons or mature oligodendrocytes. Proliferation rate and number of nestin- and doublecortin-positive cells increased in vivo in meninges following spinal cord injury. By using a lentivirus-labeling approach, we show that meningeal cells, including nestin- and doublecortin-positive cells, migrate in the spinal cord parenchyma and contribute to the glial scar formation. Our data emphasize the multiple roles of meninges in the reaction of the parenchyma to trauma and indicate for the first time that spinal cord meninges are potential niches harboring stem/precursor cells that can be activated by injury. Meninges may be considered as a new source of adult stem/precursor cells to be further tested for use in regenerative medicine applied to neurological disorders, including repair from spinal cord injury. Copyright © 2011 AlphaMed Press.
A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

PubMed Central

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-01-01

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013
A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

PubMed

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-12-05

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.
Microfluidic co-culture platform for investigating osteocyte-osteoclast signalling during fluid shear stress mechanostimulation.

PubMed

Middleton, K; Al-Dujaili, S; Mei, X; Günther, A; You, L

2017-07-05

Bone cells exist in a complex environment where they are constantly exposed to numerous dynamic biochemical and mechanical stimuli. These stimuli regulate bone cells that are involved in various bone disorders, such as osteoporosis. Knowledge of how these stimuli affect bone cells have been utilised to develop various treatments, such as pharmaceuticals, hormone therapy, and exercise. To investigate the role that bone loading has on these disorders in vitro, bone cell mechanotransduction studies are typically performed using parallel plate flow chambers (PPFC). However, these chambers do not allow for dynamic cellular interactions among different cell populations to be investigated. We present a microfluidic approach that exposes different cell populations, which are located at physiologically relevant distances within adjacent channels, to different levels of fluid shear stress, and promotes cell-cell communication between the different channels. We employed this microfluidic system to assess mechanically regulated osteocyte-osteoclast communication. Osteoclast precursors (RAW264.7 cells) responded to cytokine gradients (e.g., RANKL, OPG, PGE-2) developed by both mechanically stimulated (fOCY) and unstimulated (nOCY) osteocyte-like MLO-Y4 cells simultaneously. Specifically, we observed increased osteoclast precursor cell densities and osteoclast differentiation towards nOCY. We also used this system to show an increased mechanoresponse of osteocytes when in co-culture with osteoclasts. We envision broad applicability of the presented approach for microfluidic perfusion co-culture of multiple cell types in the presence of fluid flow stimulation, and as a tool to investigate osteocyte mechanotransduction, as well as bone metastasis extravasation. This system could also be applied to any multi-cell population cross-talk studies that are typically performed using PPFCs (e.g. endothelial cells, smooth muscle cells, and fibroblasts). Copyright © 2017 Elsevier Ltd. All rights reserved.
Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection

PubMed Central

Asher, Tedi E.; Wilson, Nancy A.; Nason, Martha C.; Brenchley, Jason M.; Metzler, Ian S.; Venturi, Vanessa; Gostick, Emma; Chattopadhyay, Pratip K.; Roederer, Mario; Davenport, Miles P.; Watkins, David I.; Douek, Daniel C.

2009-01-01

Despite the pressing need for an AIDS vaccine, the determinants of protective immunity to HIV remain concealed within the complexity of adaptive immune responses. We dissected immunodominant virus-specific CD8+ T cell populations in Mamu-A*01+ rhesus macaques with primary SIV infection to elucidate the hallmarks of effective immunity at the level of individual constituent clonotypes, which were identified according to the expression of distinct T cell receptors (TCRs). The number of public clonotypes, defined as those that expressed identical TCR β-chain amino acid sequences and recurred in multiple individuals, contained within the acute phase CD8+ T cell population specific for the biologically constrained Gag CM9 (CTPYDINQM; residues 181–189) epitope correlated negatively with the virus load set point. This independent molecular signature of protection was confirmed in a prospective vaccine trial, in which clonotype engagement was governed by the nature of the antigen rather than the context of exposure and public clonotype usage was associated with enhanced recognition of epitope variants. Thus, the pattern of antigen-specific clonotype recruitment within a protective CD8+ T cell population is a prognostic indicator of vaccine efficacy and biological outcome in an AIDS virus infection. PMID:19349463
Macroenvironmental regulation of hair cycling and collective regenerative behavior.

PubMed

Plikus, Maksim V; Chuong, Cheng-Ming

2014-01-01

The hair follicle (HF) regeneration paradigm provides a unique opportunity for studying the collective behavior of stem cells in living animals. Activation of HF stem cells depends on the core inhibitory BMP and activating WNT signals operating within the HF microenvironment. Additionally, HFs receive multilayered signaling inputs from the extrafollicular macroenvironment, which includes dermis, adipocytes, neighboring HFs, hormones, and external stimuli. These activators/inhibitors are integrated across multiple stem-cell niches to produce dynamic hair growth patterns. Because of their pigmentation, these patterns can be easily studied on live shaved animals. Comparing to autonomous regeneration of one HF, populations of HFs display coupled decision making, allowing for more robust and adaptable regenerative behavior to occur collectively. The generic cellular automata model used to simulate coordinated HF cycling here can be extended to study population-level behavior of other complex biological systems made of cycling elements.
Macroenvironmental Regulation of Hair Cycling and Collective Regenerative Behavior

PubMed Central

Plikus, Maksim V.; Chuong, Cheng-Ming

2014-01-01

The hair follicle (HF) regeneration paradigm provides a unique opportunity for studying the collective behavior of stem cells in living animals. Activation of HF stem cells depends on the core inhibitory BMP and activating WNT signals operating within the HF microenvironment. Additionally, HFs receive multilayered signaling inputs from the extrafollicular macroenvironment, which includes dermis, adipocytes, neighboring HFs, hormones, and external stimuli. These activators/inhibitors are integrated across multiple stem-cell niches to produce dynamic hair growth patterns. Because of their pigmentation, these patterns can be easily studied on live shaved animals. Comparing to autonomous regeneration of one HF, populations of HFs display coupled decision making, allowing for more robust and adaptable regenerative behavior to occur collectively. The generic cellular automata model used to simulate coordinated HF cycling here can be extended to study population-level behavior of other complex biological systems made of cycling elements. PMID:24384813
Myeloid transformation of plasma cell myeloma: molecular evidence of clonal evolution revealed by next generation sequencing.

PubMed

Gralewski, Jonathon H; Post, Ginell R; van Rhee, Frits; Yuan, Youzhong

2018-02-20

Plasma cell myeloma (PCM) is a neoplasm of terminally differentiated B lymphocytes with molecular heterogeneity. Although therapy-related myeloid neoplasms are common in plasma cell myeloma patients after chemotherapy, transdifferentiation of plasma cell myeloma into myeloid neoplasms has not been reported in literature. Here we report a very rare case of myeloid neoplasm transformed from plasma cell myeloma. A 60-year-old man with a history of plasma cell myeloma with IGH-MAF gene rearrangement and RAS/RAF mutations developed multiple soft tissue lesions one year following melphalan-based chemotherapy and autologous stem cell transplant. Morphological and immunohistochemical characterization of the extramedullary disease demonstrated that the tumor cells were derived from the monocyte-macrophage lineage. Next generation sequencing (NGS) studies detected similar clonal aberrations in the diagnostic plasma cell population and post-therapy neoplastic cells, including IGH-MAF rearrangement, multiple genetic mutations in RAS signaling pathway proteins, and loss of tumor suppressor genes. Molecular genetic analysis also revealed unique genomic alterations in the transformed tumor cells, including gain of NF1 and loss of TRAF3. To our knowledge, this is the first case of myeloid sarcoma transdifferentiated from plasma cell neoplasm. Our findings in this unique case suggest clonal evolution of plasma cell myeloma to myeloma neoplasm and the potential roles of abnormal RAS/RAF signaling pathway in lineage switch or transdifferentiation.
Optical dissection of odor information processing in vivo using GCaMPs expressed in specified cell types of the olfactory bulb

PubMed Central

Wachowiak, Matt; Economo, Michael N.; Díaz-Quesada, Marta; Brunert, Daniela; Wesson, Daniel W.; White, John. A.; Rothermel, Markus

2013-01-01

Understanding central processing requires precise monitoring of neural activity across populations of identified neurons in the intact brain. Here we used recently-optimized variants of the genetically-encoded calcium sensor GCaMP (GCaMP3 and GCaMPG5G) to image activity among genetically- and anatomically-defined neuronal populations in the olfactory bulb (OB), including two types of GABA-ergic interneurons (periglomerular (PG) and short axon (SA) cells) and OB output neurons (mitral/tufted (MT) cells) projecting to piriform cortex. We first established that changes in neuronal spiking can be accurately related to GCaMP fluorescence changes via a simple quantitative relationship over a large dynamic range. We next used in vivo two-photon imaging from individual neurons and epifluorescence signals reflecting population-level activity to investigate the spatiotemporal representation of odorants across these neuron types in anesthetized and awake mice. Under anesthesia, individual PG and SA cells showed temporally simple responses and little spontaneous activity, while MT cells were spontaneously active and showed diverse temporal responses. At the population level, response patterns of PG, SA and MT cells were surprisingly similar to those imaged from sensory inputs, with shared odorant-specific topography across the dorsal OB and inhalation-coupled temporal dynamics. During wakefulness, PG and SA cell responses increased in magnitude but remained temporally simple while those of MT cells changed to complex spatiotemporal patterns reflecting restricted excitation and widespread inhibition. These results point to multiple circuit elements with distinct roles in transforming odor representations in the OB and provide a framework for further dissecting early olfactory processing using optical and genetic tools. PMID:23516293
Neutrophils Are Central to Antibody-Mediated Protection against Genital Chlamydia.

PubMed

Naglak, Elizabeth K; Morrison, Sandra G; Morrison, Richard P

2017-10-01

Determining the effector populations involved in humoral protection against genital chlamydia infection is crucial to development of an effective chlamydial vaccine. Antibody has been implicated in protection studies in multiple animal models, and we previously showed that the passive transfer of immune serum alone does not confer immunity in the mouse. Using the Chlamydia muridarum model of genital infection, we demonstrate a protective role for both Chlamydia -specific immunoglobulin G (IgG) and polymorphonuclear neutrophils and show the importance of an antibody/effector cell interaction in mediating humoral immunity. While neutrophils were found to contribute significantly to antibody-mediated protection in vivo , natural killer (NK) cells were dispensable for protective immunity. Furthermore, gamma interferon (IFN-γ)-stimulated primary peritoneal neutrophils (PPNs) killed chlamydiae in vitro in an antibody-dependent manner. The results from this study support the view that an IFN-γ-activated effector cell population cooperates with antibody to protect against genital chlamydia and establish neutrophils as a key effector cell in this response. Copyright © 2017 Naglak et al.
flowVS: channel-specific variance stabilization in flow cytometry.

PubMed

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

2016-07-28

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances. We present a variance-stabilization algorithm, called flowVS, that removes the mean-variance correlations from cell populations identified in each fluorescence channel. flowVS transforms each channel from all samples of a data set by the inverse hyperbolic sine (asinh) transformation. For each channel, the parameters of the transformation are optimally selected by Bartlett's likelihood-ratio test so that the populations attain homogeneous variances. The optimum parameters are then used to transform the corresponding channels in every sample. flowVS is therefore an explicit variance-stabilization method that stabilizes within-population variances in each channel by evaluating the homoskedasticity of clusters with a likelihood-ratio test. With two publicly available datasets, we show that flowVS removes the mean-variance dependence from raw FC data and makes the within-population variance relatively homogeneous. We demonstrate that alternative transformation techniques such as flowTrans, flowScape, logicle, and FCSTrans might not stabilize variance. Besides flow cytometry, flowVS can also be applied to stabilize variance in microarray data. With a publicly available data set we demonstrate that flowVS performs as well as the VSN software, a state-of-the-art approach developed for microarrays. The homogeneity of variance in cell populations across FC samples is desirable when extracting features uniformly and comparing cell populations with different levels of marker expressions. The newly developed flowVS algorithm solves the variance-stabilization problem in FC and microarrays by optimally transforming data with the help of Bartlett's likelihood-ratio test. On two publicly available FC datasets, flowVS stabilizes within-population variances more evenly than the available transformation and normalization techniques. flowVS-based variance stabilization can help in performing comparison and alignment of phenotypically identical cell populations across different samples. flowVS and the datasets used in this paper are publicly available in Bioconductor.
Midkine inhibits inducible regulatory T cell differentiation by suppressing the development of tolerogenic dendritic cells.

PubMed

Sonobe, Yoshifumi; Li, Hua; Jin, Shijie; Kishida, Satoshi; Kadomatsu, Kenji; Takeuchi, Hideyuki; Mizuno, Tetsuya; Suzumura, Akio

2012-03-15

Midkine (MK), a heparin-binding growth factor, reportedly contributes to inflammatory diseases, including Crohn's disease and rheumatoid arthritis. We previously showed that MK aggravates experimental autoimmune encephalomyelitis (EAE) by decreasing regulatory CD4(+)CD25(+)Foxp3(+) T cells (Tregs), a population that regulates the development of autoimmune responses, although the precise mechanism remains uncertain. In this article, we show that MK produced in inflammatory conditions suppresses the development of tolerogenic dendritic cells (DCregs), which drive the development of inducible Treg. MK suppressed DCreg-mediated expansion of the CD4(+)CD25(+)Foxp3(+) Treg population. DCregs expressed significantly higher levels of CD45RB and produced significantly less IL-12 compared with conventional dendritic cells. However, MK downregulated CD45RB expression and induced IL-12 production by reducing phosphorylated STAT3 levels via src homology region 2 domain-containing phosphatase-2 in DCreg. Inhibiting MK activity with anti-MK RNA aptamers, which bind to the targeted protein to suppress the function of the protein, increased the numbers of CD11c(low)CD45RB(+) dendritic cells and Tregs in the draining lymph nodes and suppressed the severity of EAE, an animal model of multiple sclerosis. Our results also demonstrated that MK was produced by inflammatory cells, in particular, CD4(+) T cells under inflammatory conditions. Taken together, these results suggest that MK aggravates EAE by suppressing DCreg development, thereby impairing the Treg population. Thus, MK is a promising therapeutic target for various autoimmune diseases.
Fibro/Adipogenic Progenitors (FAPs): Isolation by FACS and Culture.

PubMed

Low, Marcela; Eisner, Christine; Rossi, Fabio

2017-01-01

Fibro/adipogenic progenitors (FAPs ) are tissue-resident mesenchymal stromal cells (MSCs). Current literature supports a role for these cells in the homeostasis and repair of multiple tissues suggesting that FAPs may have extensive therapeutic potential in the treatment of numerous diseases. In this context, it is crucial to establish efficient and reproducible procedures to purify FAP populations from various tissues. Here, we describe a protocol for the isolation and cell culture of FAPs from murine skeletal muscle using fluorescence -activated cell sorting (FACS), which is particularly useful for experiments where high cell purity is an essential requirement. Identification, isolation, and cell culture of FAPs represent powerful tools that will help us to understand the role of these cells in different conditions and facilitate the development of safe and effective new treatments for diseases.
CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma.

PubMed

Shi, Chang-Xin; Kortüm, K Martin; Zhu, Yuan Xiao; Bruins, Laura A; Jedlowski, Patrick; Votruba, Patrick G; Luo, Moulun; Stewart, Robert A; Ahmann, Jonathan; Braggio, Esteban; Stewart, A Keith

2017-12-01

Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 "resistance" genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. Mol Cancer Ther; 16(12); 2862-70. ©2017 AACR . ©2017 American Association for Cancer Research.
Neoplastic Bone Marrow Niche: Hematopoietic and Mesenchymal Stem Cells

PubMed Central

Saki, Najmaldin; Abroun, Saeid; Farshdousti Hagh, Majid; Asgharei, Farahnaz

2011-01-01

The neoplastic niche comprises complex interactions between multiple cell types and molecules requiring cell-cell signaling as well as local secretion. These niches are important for both the maintenance of cancer stem cells and the induction of neoplastic cells survival and proliferation. Each niche contains a population of tumor stem cells supported by a closely associated vascular bed comprising mesenchyme-derived cells and extracellular matrix. Targeting cancer stem cells and neoplastic niche may provide new therapies to eradicate tumors. Much progress has been very recently made in the understanding of the cellular and molecular interactions in the microenvironment of neoplastic niches. This review article provides an overview of the neoplastic niches in the bone marrow. In addition to highlighting recent advances in the field, we will also discuss components of the niche and their signaling pathways. PMID:23508881
Early events in xenograft development from the human embryonic stem cell line HS181--resemblance with an initial multiple epiblast formation.

PubMed

Gertow, Karin; Cedervall, Jessica; Jamil, Seema; Ali, Rouknuddin; Imreh, Marta P; Gulyas, Miklos; Sandstedt, Bengt; Ahrlund-Richter, Lars

2011-01-01

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart.
Joint modeling and registration of cell populations in cohorts of high-dimensional flow cytometric data.

PubMed

Pyne, Saumyadipta; Lee, Sharon X; Wang, Kui; Irish, Jonathan; Tamayo, Pablo; Nazaire, Marc-Danie; Duong, Tarn; Ng, Shu-Kay; Hafler, David; Levy, Ronald; Nolan, Garry P; Mesirov, Jill; McLachlan, Geoffrey J

2014-01-01

In biomedical applications, an experimenter encounters different potential sources of variation in data such as individual samples, multiple experimental conditions, and multivariate responses of a panel of markers such as from a signaling network. In multiparametric cytometry, which is often used for analyzing patient samples, such issues are critical. While computational methods can identify cell populations in individual samples, without the ability to automatically match them across samples, it is difficult to compare and characterize the populations in typical experiments, such as those responding to various stimulations or distinctive of particular patients or time-points, especially when there are many samples. Joint Clustering and Matching (JCM) is a multi-level framework for simultaneous modeling and registration of populations across a cohort. JCM models every population with a robust multivariate probability distribution. Simultaneously, JCM fits a random-effects model to construct an overall batch template--used for registering populations across samples, and classifying new samples. By tackling systems-level variation, JCM supports practical biomedical applications involving large cohorts. Software for fitting the JCM models have been implemented in an R package EMMIX-JCM, available from http://www.maths.uq.edu.au/~gjm/mix_soft/EMMIX-JCM/.

A Stochastic Framework for Modeling the Population Dynamics of Convective Clouds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hagos, Samson; Feng, Zhe; Plant, Robert S.

A stochastic prognostic framework for modeling the population dynamics of convective clouds and representing them in climate models is proposed. The approach used follows the non-equilibrium statistical mechanical approach through a master equation. The aim is to represent the evolution of the number of convective cells of a specific size and their associated cloud-base mass flux, given a large-scale forcing. In this framework, referred to as STOchastic framework for Modeling Population dynamics of convective clouds (STOMP), the evolution of convective cell size is predicted from three key characteristics: (i) the probability of growth, (ii) the probability of decay, and (iii)more » the cloud-base mass flux. STOMP models are constructed and evaluated against CPOL radar observations at Darwin and convection permitting model (CPM) simulations. Multiple models are constructed under various assumptions regarding these three key parameters and the realisms of these models are evaluated. It is shown that in a model where convective plumes prefer to aggregate spatially and mass flux is a non-linear function of convective cell area, mass flux manifests a recharge-discharge behavior under steady forcing. Such a model also produces observed behavior of convective cell populations and CPM simulated mass flux variability under diurnally varying forcing. Besides its use in developing understanding of convection processes and the controls on convective cell size distributions, this modeling framework is also designed to be capable of providing alternative, non-equilibrium, closure formulations for spectral mass flux parameterizations.« less
Prevalence of problematic cell phone use in an adult population in Spain as assessed by the Mobile Phone Problem Use Scale (MPPUS).

PubMed

de-Sola, José; Talledo, Hernán; Rodríguez de Fonseca, Fernando; Rubio, Gabriel

2017-01-01

Problematic cell phone use has alarmingly increased in industrialized countries in the past 10 years. For many perpetrators, it can turn into a behavioural addiction, although this is not a recognized medical condition. Although there are many tools for evaluating this use, one of the most widely used tools is the Mobile Phone Problematic Use Scale (MPPUS), which we test on a representative sample of the population in Spain to obtain an estimate of the prevalence of problematic cell phone use in our midst. The age range consists of 16-65 years, with 1,126 surveys conducted. In this population, we verify that the reliability and internal consistency of the MPPUS (α = 0.939) are maintained. Additionally, the construct validity, considering the derived factors (Abuse and Dependence, Craving and Loss of Control, and Dependence on the Social Environment) are aligned with other research and with diverse external criteria of addiction. We establish four categories of users (Casual, Regular, At Risk, and Problematic) and obtain a prevalence of 15.4% among At Risk Users and 5.1% among Problematic Users. This finding implies a total of 20.5% of Users with Problems. A binary logistic regression analysis shows that age, gender, level of education, and daily cell phone use predict problematic cell phone use. The results, based on multiple criteria, show that such problematic use shares features of recognized addictions, affecting large segments of the population and not only adolescents.
Characterization of the Population of the Sulfur-Oxidizing Symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by Single-Cell Analyses▿ †

PubMed Central

Caro, Audrey; Gros, Olivier; Got, Patrice; De Wit, Rutger; Troussellier, Marc

2007-01-01

We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 μm in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host. PMID:17259363
Human mesenchymal stem cells: a bank perspective on the isolation, characterization and potential of alternative sources for the regeneration of musculoskeletal tissues.

PubMed

Moroni, Lorenzo; Fornasari, Pier Maria

2013-04-01

The continuous discovery of human mesenchymal stem cells (hMSCs) in different tissues is stirring up a tremendous interest as a cell source for regenerative medicine therapies. Historically, hMSCs have been always considered a sub-population of mononuclear cells present in the bone marrow (BM). Although BM-hMSCs are still nowadays considered as the most promising mesenchymal stem cell population to reach the clinics due to their capacity to differentiate into multiple tissues, hMSCs derived from other adult and fetal tissues have also demonstrated to possess similar differentiation capacities. Furthermore, different reports have highlighted a higher recurrence of hMSCs in some of these tissues as compared to BM. This offer a fascinating panorama for cell banking, since the creation of a stem cell factory could be envisioned where hMSCs are stocked and used for ad hoc clinical applications. In this review, we summarize the main findings and state of the art in hMSCs isolation, characterization, and differentiation from alternative tissue sources and we attempt to compare their potency for musculoskeletal regeneration. Copyright © 2012 Wiley Periodicals, Inc.
Characterization of the Inflammatory Response in Dystrophic Muscle Using Flow Cytometry.

PubMed

Kastenschmidt, Jenna M; Avetyan, Ileen; Villalta, S A

2018-01-01

Although mutations of the dystrophin gene are the causative defect in Duchenne muscular dystrophy (DMD) patients, secondary disease processes such as inflammation contribute greatly to the pathogenesis of DMD. Genetic and histological studies have shown that distinct facets of the immune system promote muscle degeneration or regeneration during muscular dystrophy through mechanisms that are only beginning to be defined. Although histological methods have allowed the enumeration and localization of immune cells within dystrophic muscle, they are limited in their ability to assess the full spectrum of phenotypic states of an immune cell population and its functional characteristics. This chapter highlights flow cytometry methods for the isolation and functional study of immune cell populations from muscle of the mdx mouse model of DMD. We include a detailed description of preparing single-cell suspensions of dystrophic muscle that maintain the integrity of cell-surface markers used to identify macrophages, eosinophils, group 2 innate lymphoid cells, and regulatory T cells. This method complements the battery of histological assays that are currently used to study the role of inflammation in muscular dystrophy, and provides a platform capable of being integrated with multiple downstream methodologies for the mechanistic study of immunity in muscle degenerative diseases.
A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young

2013-01-25

Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24{sup −}/CD44{sup +}) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies.more » This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer.« less
HLA-E-Restricted Cross-Recognition of Allogeneic Endothelial Cells by CMV-Associated CD8 T Cells: A Potential Risk Factor following Transplantation

PubMed Central

Allard, Mathilde; Tonnerre, Pierre; Nedellec, Steven; Oger, Romain; Morice, Alexis; Guilloux, Yannick; Houssaint, Elisabeth; Charreau, Béatrice; Gervois, Nadine

2012-01-01

Although association between CMV infection and allograft rejection is well admitted, the precise mechanisms involved remain uncertain. Here, we report the characterization of an alloreactive HLA-E-restricted CD8 T cell population that was detected in the PBL of a kidney transplant patient after its CMV conversion. This monoclonal CD8 T cell population represents a sizable fraction in the blood (3% of PBL) and is characterized by an effector-memory phenotype and the expression of multiple NK receptors. Interestingly, these unconventional T cells display HLA-E-dependent reactivity against peptides derived from the leader sequences of both various HCMV-UL40 and allogeneic classical HLA-I molecules. Consequently, while HLA-E-restricted CD8 T cells have potential to contribute to the control of CMV infection in vivo, they may also directly mediate graft rejection through recognition of peptides derived from allogeneic HLA-I molecules on graft cells. Therefore, as HLA-E expression in nonlymphoid organs is mainly restricted to endothelial cells, we investigated the reactivity of this HLA-E-restricted T cell population towards allogeneic endothelial cells. We clearly demonstrated that CMV-associated HLA-E-restricted T cells efficiently recognized and killed allogeneic endothelial cells in vitro. Moreover, our data indicate that this alloreactivity is tightly regulated by NK receptors, especially by inhibitory KIR2DL2 that strongly prevents TCR-induced activation through recognition of HLA-C molecules. Hence, a better evaluation of the role of CMV-associated HLA-E-restricted T cells in transplantation and of the impact of HLA-genotype, especially HLA-C, on their alloreactivity may determine whether they indeed represent a risk factor following organ transplantation. PMID:23226431
A subset of high Gleason grade prostate carcinomas contain a large burden of prostate cancer syndecan-1 positive stromal cells.

PubMed

Sharpe, Benjamin; Alghezi, Dhafer A; Cattermole, Claire; Beresford, Mark; Bowen, Rebecca; Mitchard, John; Chalmers, Andrew D

2017-05-01

There is a pressing need to identify prognostic and predictive biomarkers for prostate cancer to aid treatment decisions in both early and advanced disease settings. Syndecan-1, a heparan sulfate proteoglycan, has been previously identified as a potential prognostic biomarker by multiple studies at the tissue and serum level. However, other studies have questioned its utility. Anti-Syndecan-1 immunohistochemistry was carried out on 157 prostate tissue samples (including cancerous, adjacent normal tissue, and non-diseased prostate) from three independent cohorts of patients. A population of Syndecan-1 positive stromal cells was identified and the number and morphological parameters of these cells quantified. The identity of the Syndecan-1-positive stromal cells was assessed by multiplex immunofluorescence using a range of common cell lineage markers. Finally, the burden of Syndecan-1 positive stromal cells was tested for association with clinical parameters. We identified a previously unreported cell type which is marked by Syndecan-1 expression and is found in the stroma of prostate tumors and adjacent normal tissue but not in non-diseased prostate. We call these cells Prostate Cancer Syndecan-1 Positive (PCSP) cells. Immunofluorescence analysis revealed that the PCSP cell population did not co-stain with markers of common prostate epithelial, stromal, or immune cell populations. However, morphological analysis revealed that PCSP cells are often elongated and displayed prominent lamellipodia, suggesting they are an unidentified migratory cell population. Analysis of clinical parameters showed that PCSP cells were found with a frequency of 20-35% of all tumors evaluated, but were not present in non-diseased normal tissue. Interestingly, a subset of primary Gleason 5 prostate tumors had a high burden of PCSP cells. The current study identifies PCSP cells as a novel, potentially migratory cell type, which is marked by Syndecan-1 expression and is found in the stroma of prostate carcinomas, adjacent normal tissue, but not in non-diseased prostate. A subset of poor prognosis high Gleason grade 5 tumors had a particularly high PCSP cell burden, suggesting an association between this unidentified cell type and tumor aggressiveness. © 2017 Wiley Periodicals, Inc.
Cholesterol Modulates CFTR Confinement in the Plasma Membrane of Primary Epithelial Cells

PubMed Central

Abu-Arish, Asmahan; Pandzic, Elvis; Goepp, Julie; Matthes, Elizabeth; Hanrahan, John W.; Wiseman, Paul W.

2015-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function. PMID:26153705
Complex neural codes in rat prelimbic cortex are stable across days on a spatial decision task

PubMed Central

Powell, Nathaniel J.; Redish, A. David

2014-01-01

The rodent prelimbic cortex has been shown to play an important role in cognitive processing, and has been implicated in encoding many different parameters relevant to solving decision-making tasks. However, it is not known how the prelimbic cortex represents all these disparate variables, and if they are simultaneously represented when the task requires it. In order to investigate this question, we trained rats to run the Multiple-T Left Right Alternate (MT-LRA) task and recorded multi-unit ensembles from their prelimbic regions. Significant populations of cells in the prelimbic cortex represented the strategy controlling reward receipt on a given lap, whether the animal chose to go right or left on a given lap, and whether the animal made a correct decision or an error on a given lap. These populations overlapped in the cells recorded, with several cells demonstrating differential firing to all three variables. The spatial and strategic firing patterns of individual prelimbic cells were highly conserved across several days of running this task, indicating that each cell encoded the same information across days. PMID:24795579
Multiple Cellular Responses to Serotonin Contribute to Epithelial Homeostasis

PubMed Central

Pai, Vaibhav P.; Horseman, Nelson D.

2011-01-01

Epithelial homeostasis incorporates the paradoxical concept of internal change (epithelial turnover) enabling the maintenance of anatomical status quo. Epithelial cell differentiation and cell loss (cell shedding and apoptosis) form important components of epithelial turnover. Although the mechanisms of cell loss are being uncovered the crucial triggers that modulate epithelial turnover through regulation of cell loss remain undetermined. Serotonin is emerging as a common autocrine-paracine regulator in epithelia of multiple organs, including the breast. Here we address whether serotonin affects epithelial turnover. Specifically, serotonin's roles in regulating cell shedding, apoptosis and barrier function of the epithelium. Using in vivo studies in mouse and a robust model of differentiated human mammary duct epithelium (MCF10A), we show that serotonin induces mammary epithelial cell shedding and disrupts tight junctions in a reversible manner. However, upon sustained exposure, serotonin induces apoptosis in the replenishing cell population, causing irreversible changes to the epithelial membrane. The staggered nature of these events induced by serotonin slowly shifts the balance in the epithelium from reversible to irreversible. These finding have very important implications towards our ability to control epithelial regeneration and thus address pathologies of aberrant epithelial turnover, which range from degenerative disorders (e.g.; pancreatitis and thyrioditis) to proliferative disorders (e.g.; mastitis, ductal ectasia, cholangiopathies and epithelial cancers). PMID:21390323
Epidemiology of Multiple Myeloma in the Czech Republic.

PubMed

Maluskova, D; Svobodová, I; Kucerova, M; Brozova, L; Muzik, J; Jarkovský, J; Hájek, R; Maisnar, V; Dusek, L

2017-01-01

Multiple myeloma (MM) is a cancer of plasma cells with an incidence of 4.8 cases per 100,000 population in the Czech Republic in 2014; the burden of MM in the Czech Republic is moderate when compared to other European countries. This work brings the latest information on MM epidemiology in the Czech population. The Czech National Cancer Registry is the basic source of data for the population-based evaluation of MM epidemiology. This database also makes it possible to assess patient survival and to predict probable short-term as well as long-term trends in the treatment burden of the entire population. According to the latest Czech National Cancer Registry data, there were 504 new cases of MM and 376 deaths from MM in 2014. Since 2004, there has been a 26.9% increase in MM incidence and an 8.3% increase in MM mortality. In 2014, there were 1,982 persons living with MM or a history of MM, corresponding to a 74.4% increase when compared to MM prevalence in 2004. The 5-year survival of patients treated in the period 2010-2014 was nearly 40%. The available data make it possible to analyse long-term trends in MM epidemiology and to predict the future treatment burden as well as treatment results.Key words: multiple myeloma - epidemiology - Czech National Cancer Registry - Registry of Monoclonal Gammopathies - Czech Republic.
Cell-assembly coding in several memory processes.

PubMed

Sakurai, Y

1998-01-01

The present paper discusses why the cell assembly, i.e., an ensemble population of neurons with flexible functional connections, is a tenable view of the basic code for information processes in the brain. The main properties indicating the reality of cell-assembly coding are neurons overlaps among different assemblies and connection dynamics within and among the assemblies. The former can be detected as multiple functions of individual neurons in processing different kinds of information. Individual neurons appear to be involved in multiple information processes. The latter can be detected as changes of functional synaptic connections in processing different kinds of information. Correlations of activity among some of the recorded neurons appear to change in multiple information processes. Recent experiments have compared several different memory processes (tasks) and detected these two main properties, indicating cell-assembly coding of memory in the working brain. The first experiment compared different types of processing of identical stimuli, i.e., working memory and reference memory of auditory stimuli. The second experiment compared identical processes of different types of stimuli, i.e., discriminations of simple auditory, simple visual, and configural auditory-visual stimuli. The third experiment compared identical processes of different types of stimuli with or without temporal processing of stimuli, i.e., discriminations of elemental auditory, configural auditory-visual, and sequential auditory-visual stimuli. Some possible features of the cell-assembly coding, especially "dual coding" by individual neurons and cell assemblies, are discussed for future experimental approaches. Copyright 1998 Academic Press.
[Multiple myeloma with significant multifocal osteolysis in a dog without a detectible gammopathy].

PubMed

Souchon, F; Koch, A; Sohns, A

2013-01-01

Description of a variant of multiple myeloma in a dog lacking the gammopathy normally associated with this type of neoplasm. A Border Collie mongrel was presented with symptoms of progressive hind-leg weakness, lethargy and tiredness, which had started to appear 6 weeks previously. Radiographic examination showed small osteolytic areas in the spinal column, but also diffuse small areas of increased opacity as well as evidence of decreased bone density in the pelvis and of both femoral necks. Moderate regenerative anaemia, hypogammopathy and hypercalcaemia were diagnosed. Computed tomography scans displayed multifocal osteolysis and bone destruction in the skull, spinal column, scapulae, proximal humeri, pelvis and femoral necks. H&E staining of the biopsies showed bone destruction and monomorphic plasmacyotid cell populations, causing infiltrative bone marrow lesions and osteolysis. In many areas neoplastic plasma cell infiltration of the bone marrow was 70% and in some areas reached 100%. The diagnosis was non-secretory multiple myeloma without apparent secretion of paraproteins into the blood.
Comprehensive molecular profiling of 718 Multiple Myelomas reveals significant differences in mutation frequencies between African and European descent cases

PubMed Central

Christofferson, Austin; Aldrich, Jessica; Jewell, Scott; Kittles, Rick A.; Derome, Mary; Craig, David Wesley; Carpten, John D.

2017-01-01

Multiple Myeloma (MM) is a plasma cell malignancy with significantly greater incidence and mortality rates among African Americans (AA) compared to Caucasians (CA). The overall goal of this study is to elucidate differences in molecular alterations in MM as a function of self-reported race and genetic ancestry. Our study utilized somatic whole exome, RNA-sequencing, and correlated clinical data from 718 MM patients from the Multiple Myeloma Research Foundation CoMMpass study Interim Analysis 9. Somatic mutational analyses based upon self-reported race corrected for ancestry revealed significant differences in mutation frequency between groups. Of interest, BCL7A, BRWD3, and AUTS2 demonstrate significantly higher mutation frequencies among AA cases. These genes are all involved in translocations in B-cell malignancies. Moreover, we detected a significant difference in mutation frequency of TP53 and IRF4 with frequencies higher among CA cases. Our study provides rationale for interrogating diverse tumor cohorts to best understand tumor genomics across populations. PMID:29166413
Amniotic-Fluid Stem Cells: Growth Dynamics and Differentiation Potential after a CD-117-Based Selection Procedure

PubMed Central

Arnhold, S.; Glüer, S.; Hartmann, K.; Raabe, O.; Addicks, K.; Wenisch, S.; Hoopmann, M.

2011-01-01

Amniotic fluid (AF) has become an interesting source of fetal stem cells. However, AF contains heterogeneous and multiple, partially differentiated cell types. After isolation from the amniotic fluid, cells were characterized regarding their morphology and growth dynamics. They were sorted by magnetic associated cell sorting using the surface marker CD 117. In order to show stem cell characteristics such as pluripotency and to evaluate a possible therapeutic application of these cells, AF fluid-derived stem cells were differentiated along the adipogenic, osteogenic, and chondrogenic as well as the neuronal lineage under hypoxic conditions. Our findings reveal that magnetic associated cell sorting (MACS) does not markedly influence growth characteristics as demonstrated by the generation doubling time. There was, however, an effect regarding an altered adipogenic, osteogenic, and chondrogenic differentiation capacity in the selected cell fraction. In contrast, in the unselected cell population neuronal differentiation is enhanced. PMID:21437196
The Evolution of the Stem Cell Theory for Heart Failure.

PubMed

Silvestre, Jean-Sébastien; Menasché, Philippe

2015-12-01

Various stem cell-based approaches for cardiac repair have achieved encouraging results in animal experiments, often leading to their rapid proceeding to clinical testing. However, freewheeling evolutionary developments of the stem cell theory might lead to dystopian scenarios where heterogeneous sources of therapeutic cells could promote mixed clinical outcomes in un-stratified patient populations. This review focuses on the lessons that should be learnt from the first generation of stem cell-based strategies and emphasizes the absolute requirement to better understand the basic mechanisms of stem cell biology and cardiogenesis. We will also discuss about the unexpected "big bang" in the stem cell theory, "blasting" the therapeutic cells to their unchallenged ability to release paracrine factors such as extracellular membrane vesicles. Paradoxically, the natural evolution of the stem cell theory for cardiac regeneration may end with the development of cell-free strategies with multiple cellular targets including cardiomyocytes but also other infiltrating or resident cardiac cells.
Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves

PubMed Central

Collins, David J.; Morahan, Belinda; Garcia-Bustos, Jose; Doerig, Christian; Plebanski, Magdalena; Neild, Adrian

2015-01-01

In single-cell analysis, cellular activity and parameters are assayed on an individual, rather than population-average basis. Essential to observing the activity of these cells over time is the ability to trap, pattern and retain them, for which previous single-cell-patterning work has principally made use of mechanical methods. While successful as a long-term cell-patterning strategy, these devices remain essentially single use. Here we introduce a new method for the patterning of multiple spatially separated single particles and cells using high-frequency acoustic fields with one cell per acoustic well. We characterize and demonstrate patterning for both a range of particle sizes and the capture and patterning of cells, including human lymphocytes and red blood cells infected by the malarial parasite Plasmodium falciparum. This ability is made possible by a hitherto unexplored regime where the acoustic wavelength is on the same order as the cell dimensions. PMID:26522429
The gamma delta T cell repertoire in Graves' disease and multinodular goitre.

PubMed Central

McIntosh, R S; Tandon, N; Pickerill, A P; Davies, R; Barnett, D; Weetman, A P

1993-01-01

gamma delta T cells are a subset of T cells with unknown function, and restriction of the gamma delta T cell receptor (TCR) repertoire has been described in rheumatoid arthritis and multiple sclerosis. Elevated numbers of gamma delta T cells have been reported in the peripheral blood and thyroids of patients with Graves' disease. We have carried out flow cytometric analysis on peripheral blood mononuclear cells (PBMC) and intrathyroidal lymphocytes (ITL) from 12 patients with Graves' disease and nine patients with multinodular goitre (MNG), a thyroid disease of unknown etiology. There was no significant difference between the proportion of gamma delta T cells in the PBMC of Graves' and MNG patients, nor between the PBMC and ITL populations in either patient group. We have also carried out polymerase chain reaction amplification on RNA prepared from matched PBMC, ITL and the activated (CD25+) subset of ITL using six TCR V delta-family specific primers. Although there were differences in the amounts of each V delta transcript amplified from PBMC and ITL, there was no difference between the two patient groups. No consistent differences were therefore found between the gamma delta T cell populations in Graves' and MNG patients, arguing against the direct involvement of this T cell subset in the pathogenesis of Graves' disease. Images Fig. 1 PMID:8252809
Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

PubMed Central

Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L.; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M.

2017-01-01

Abstract Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. PMID:28126923

Solving Navigational Uncertainty Using Grid Cells on Robots

PubMed Central

Milford, Michael J.; Wiles, Janet; Wyeth, Gordon F.

2010-01-01

To successfully navigate their habitats, many mammals use a combination of two mechanisms, path integration and calibration using landmarks, which together enable them to estimate their location and orientation, or pose. In large natural environments, both these mechanisms are characterized by uncertainty: the path integration process is subject to the accumulation of error, while landmark calibration is limited by perceptual ambiguity. It remains unclear how animals form coherent spatial representations in the presence of such uncertainty. Navigation research using robots has determined that uncertainty can be effectively addressed by maintaining multiple probabilistic estimates of a robot's pose. Here we show how conjunctive grid cells in dorsocaudal medial entorhinal cortex (dMEC) may maintain multiple estimates of pose using a brain-based robot navigation system known as RatSLAM. Based both on rodent spatially-responsive cells and functional engineering principles, the cells at the core of the RatSLAM computational model have similar characteristics to rodent grid cells, which we demonstrate by replicating the seminal Moser experiments. We apply the RatSLAM model to a new experimental paradigm designed to examine the responses of a robot or animal in the presence of perceptual ambiguity. Our computational approach enables us to observe short-term population coding of multiple location hypotheses, a phenomenon which would not be easily observable in rodent recordings. We present behavioral and neural evidence demonstrating that the conjunctive grid cells maintain and propagate multiple estimates of pose, enabling the correct pose estimate to be resolved over time even without uniquely identifying cues. While recent research has focused on the grid-like firing characteristics, accuracy and representational capacity of grid cells, our results identify a possible critical and unique role for conjunctive grid cells in filtering sensory uncertainty. We anticipate our study to be a starting point for animal experiments that test navigation in perceptually ambiguous environments. PMID:21085643
Stem/progenitor cells derived from the cochlear sensory epithelium give rise to spheres with distinct morphologies and features.

PubMed

Diensthuber, Marc; Oshima, Kazuo; Heller, Stefan

2009-06-01

Nonmammalian vertebrates regenerate lost sensory hair cells by means of asymmetric division of supporting cells. Inner ear or lateral line supporting cells in birds, amphibians, and fish consequently serve as bona fide stem cells resulting in high regenerative capacity of hair cell-bearing organs. Hair cell regeneration does not happen in the mammalian cochlea, but cells with proliferative capacity can be isolated from the neonatal cochlea. These cells have the ability to form clonal floating colonies, so-called spheres, when cultured in nonadherent conditions. We noticed that the sphere population derived from mouse cochlear sensory epithelium cells was heterogeneous, consisting of morphologically distinct sphere types, hereby classified as solid, transitional, and hollow. Cochlear sensory epithelium-derived stem/progenitor cells initially give rise to small solid spheres, which subsequently transition into hollow spheres, a change that is accompanied by epithelial differentiation of the majority of sphere cells. Only solid spheres, and to a lesser extent, transitional spheres, appeared to harbor self-renewing stem cells, whereas hollow spheres could not be consistently propagated. Solid spheres contained significantly more rapidly cycling Pax-2-expressing presumptive otic progenitor cells than hollow spheres. Islet-1, which becomes upregulated in nascent sensory patches, was also more abundant in solid than in hollow spheres. Likewise, hair cell-like cells, characterized by the expression of multiple hair cell markers, differentiated in significantly higher numbers in cell populations derived from solid spheres. We conclude that cochlear sensory epithelium cell populations initially give rise to small solid spheres that have self-renewing capacity before they subsequently convert into hollow spheres, a process that is accompanied by loss of stemness and reduced ability to spontaneously give rise to hair cell-like cells. Solid spheres might, therefore, represent the most suitable sphere type for cell-based assays or animal model transplantation studies aimed at development of cell replacement therapies.
Excitable toxin-antitoxin modules coordinated through intracellular bottlenecks

NASA Astrophysics Data System (ADS)

Mather, William

Chronic infections and pathogenic biofilms present a serious threat to the health of humans by decreasing life expectancy and quality. The resilience of these microbial communities has been attributed to the spontaneous formation of persister cells, which constitute a small fraction of the population capable of surviving a wide range of environmental stressors. Gating of bacterial persistence has recently been linked to toxin-antitoxin (TA) modules, which are operons with an evolutionarily conserved motif that includes a toxin that halts cell growth and a corresponding antitoxin that neutralizes the toxin. While many such modules have been identified and studied in a wide range of organisms, little consideration of the interactions between multiple modules within a single host has been made. Moreover, the multitude of different antitoxin species are degraded by a relatively small number of proteolytic pathways, strongly suggesting competition between antitoxins for degradation machinery, i.e. queueing coupling. Here we present a theoretical understanding of the dynamics of multiple TA modules that are coupled through either proteolytic queueing, a toxic effect on cell growth rate, or both. We conclude that indirect queueing coordination between multiple TA modules may be central to controlling bacterial persistence. NSF Award Number MCB-1330180.
Hippocampal Remapping Is Constrained by Sparseness rather than Capacity

PubMed Central

Kammerer, Axel; Leibold, Christian

2014-01-01

Grid cells in the medial entorhinal cortex encode space with firing fields that are arranged on the nodes of spatial hexagonal lattices. Potential candidates to read out the space information of this grid code and to combine it with other sensory cues are hippocampal place cells. In this paper, we investigate a population of grid cells providing feed-forward input to place cells. The capacity of the underlying synaptic transformation is determined by both spatial acuity and the number of different spatial environments that can be represented. The codes for different environments arise from phase shifts of the periodical entorhinal cortex patterns that induce a global remapping of hippocampal place fields, i.e., a new random assignment of place fields for each environment. If only a single environment is encoded, the grid code can be read out at high acuity with only few place cells. A surplus in place cells can be used to store a space code for more environments via remapping. The number of stored environments can be increased even more efficiently by stronger recurrent inhibition and by partitioning the place cell population such that learning affects only a small fraction of them in each environment. We find that the spatial decoding acuity is much more resilient to multiple remappings than the sparseness of the place code. Since the hippocampal place code is sparse, we thus conclude that the projection from grid cells to the place cells is not using its full capacity to transfer space information. Both populations may encode different aspects of space. PMID:25474570
Lgr5-positive cells are cancer stem cells in skin squamous cell carcinoma.

PubMed

Liu, Shunli; Gong, Zhenyu; Chen, Mingrui; Liu, Benli; Bian, Donghui; Wu, Kai

2014-11-01

Cancer stem cells (CSCs) in most human tumors are commonly identified and enriched using similar strategies for identifying normal stem cells, including flow cytometry assays for side population, high aldehyde dehydrogenase (ALDH) activity, and CD133 positivity. Thus, development of a method for isolating a specific cancer using cancer-specific characteristic appears to be potentially important. Here, we reported extremely high Lgr5 levels in the specimen from skin squamous cell carcinoma (SCC) in patients. Using SCC cell line A431, we detected high Lgr5 and CD133 levels in ALDH-high or side population from these cancer cells. To figure out whether Lgr5 is a marker of CSCs in SCC, we transfected A431 cells with a Lgr5-creERT-2A-DTR/Cag-Loxp-GFP-STOP-Loxp-RFP plasmid and purified transfected cells (tA431) based on GFP by flow cytometry. 4-Hydroxytamoxifen (4-OHT) was given to label Lgr5-positive cells with RFP, for comparison to GFP-positive Lgr5-negative cells. Lgr5-positive cells grew significantly faster than Lgr5-negative cells, and the fold increase in growth of Lgr5-positive vs Lgr5-negative cells is significantly higher than SP vs non-SP, or ALDH-high vs ALDH-low, or CD133-positive vs CD133-negative cells. Moreover, in Lgr5-negative population, Lgr5-positive re-appeared in culture with time, suggesting that Lgr5-positive cells can be regenerated from Lgr5-negative cells. Furthermore, the growth of tA431 cells significantly decreased upon a single dose of diphtheria toxin (DT)/4-OHT to eliminate Lgr5-positive cell lineage, while multiple doses of DT/4-OHT nearly completely inhibited tA431 cell growth. Taken together, our data provide compelling data to demonstrate that Lgr5-positive cells are CSCs in skin SCC.
Variability in seeds: biological, ecological, and agricultural implications.

PubMed

Mitchell, Jack; Johnston, Iain G; Bassel, George W

2017-02-01

Variability is observed in biology across multiple scales, ranging from populations, individuals, and cells to the molecular components within cells. This review explores the sources and roles of this variability across these scales, focusing on seeds. From a biological perspective, the role and the impact this variability has on seed behaviour and adaptation to the environment is discussed. The consequences of seed variability on agricultural production systems, which demand uniformity, are also examined. We suggest that by understanding the basis and underlying mechanisms of variability in seeds, strategies to increase seed population uniformity can be developed, leading to enhanced agricultural production across variable climatic conditions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Detecting and Characterizing Genomic Signatures of Positive Selection in Global Populations

PubMed Central

Liu, Xuanyao; Ong, Rick Twee-Hee; Pillai, Esakimuthu Nisha; Elzein, Abier M.; Small, Kerrin S.; Clark, Taane G.; Kwiatkowski, Dominic P.; Teo, Yik-Ying

2013-01-01

Natural selection is a significant force that shapes the architecture of the human genome and introduces diversity across global populations. The question of whether advantageous mutations have arisen in the human genome as a result of single or multiple mutation events remains unanswered except for the fact that there exist a handful of genes such as those that confer lactase persistence, affect skin pigmentation, or cause sickle cell anemia. We have developed a long-range-haplotype method for identifying genomic signatures of positive selection to complement existing methods, such as the integrated haplotype score (iHS) or cross-population extended haplotype homozygosity (XP-EHH), for locating signals across the entire allele frequency spectrum. Our method also locates the founder haplotypes that carry the advantageous variants and infers their corresponding population frequencies. This presents an opportunity to systematically interrogate the whole human genome whether a selection signal shared across different populations is the consequence of a single mutation process followed subsequently by gene flow between populations or of convergent evolution due to the occurrence of multiple independent mutation events either at the same variant or within the same gene. The application of our method to data from 14 populations across the world revealed that positive-selection events tend to cluster in populations of the same ancestry. Comparing the founder haplotypes for events that are present across different populations revealed that convergent evolution is a rare occurrence and that the majority of shared signals stem from the same evolutionary event. PMID:23731540
Computational design of nanoparticle drug delivery systems for selective targeting

NASA Astrophysics Data System (ADS)

Duncan, Gregg A.; Bevan, Michael A.

2015-09-01

Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues.Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues. Electronic supplementary information (ESI) available: Movie showing simulation renderings of targeted (ρL = 1820/μm2, KD = 120 μM) nanoparticle selective binding to cancer (ρR = 256/μm2) vs. healthy (ρR = 64/μm2) cell surfaces. Target membrane proteins have linear color scale depending on binding energy ranging from white when unbound (URL = 0) to red when tightly bound (URL = UM). See DOI: 10.1039/c5nr03691g
WKB theory of large deviations in stochastic populations

NASA Astrophysics Data System (ADS)

Assaf, Michael; Meerson, Baruch

2017-06-01

Stochasticity can play an important role in the dynamics of biologically relevant populations. These span a broad range of scales: from intra-cellular populations of molecules to population of cells and then to groups of plants, animals and people. Large deviations in stochastic population dynamics—such as those determining population extinction, fixation or switching between different states—are presently in a focus of attention of statistical physicists. We review recent progress in applying different variants of dissipative WKB approximation (after Wentzel, Kramers and Brillouin) to this class of problems. The WKB approximation allows one to evaluate the mean time and/or probability of population extinction, fixation and switches resulting from either intrinsic (demographic) noise, or a combination of the demographic noise and environmental variations, deterministic or random. We mostly cover well-mixed populations, single and multiple, but also briefly consider populations on heterogeneous networks and spatial populations. The spatial setting also allows one to study large fluctuations of the speed of biological invasions. Finally, we briefly discuss possible directions of future work.
Characterization and differentiation of human embryonic stem cells.

PubMed

Carpenter, M K; Rosler, E; Rao, M S

2003-01-01

Cell replacement therapies have been limited by the availability of sufficient quantities of cells for transplantation. Human ES (hES) cell lines have recently been generated by several laboratories. When maintained for over 1 year in vitro, they remain karyotypically and phenotypically stable and may therefore provide an excellent source material for cell therapies. Currently, data is available for 26 hES cell lines. Although limited characterization has been performed on most of these lines, there are remarkable similarities in expression of markers. hES cell lines derived in different laboratories show similar expression profiles of surface markers, including SSEA-4, Tra-1-60, and Tra-1-81. In addition, markers associated with pluripotent cells such as OCT-4 are expressed at in all cell lines tested. These cells express high levels of telomerase and appear to have indefinite growth potential. The generation of the large quantities of cells necessary for cell replacement therapies will require a cell population which is stable over long term culture. We have characterized the properties of multiple hES cell lines that have been maintained in culture for extended periods. Quantitative analyses demonstrate that all of the cell lines examined show consistent marker expression and retain a normal karyotype after long-term culture. hES cells have been differentiated into the derivatives of all three germ layers. Specifically this includes cardiomyocytes, neural cells, hepatocyte-like cells, endothelial cells and hematopoietic progenitor cells. These data demonstrating the karyotypic and phenotypic stability of hES cells and their extensive differentiative capacity indicate that they may be an appropriate source of cells for multiple regenerative medicine applications.
Using Movies to Analyse Gene Circuit Dynamics in Single Cells

PubMed Central

Locke, James CW; Elowitz, Michael B

2010-01-01

Preface Many bacterial systems rely on dynamic genetic circuits to control critical processes. A major goal of systems biology is to understand these behaviours in terms of individual genes and their interactions. However, traditional techniques based on population averages wash out critical dynamics that are either unsynchronized between cells or driven by fluctuations, or ‘noise,’ in cellular components. Recently, the combination of time-lapse microscopy, quantitative image analysis, and fluorescent protein reporters has enabled direct observation of multiple cellular components over time in individual cells. In conjunction with mathematical modelling, these techniques are now providing powerful insights into genetic circuit behaviour in diverse microbial systems. PMID:19369953
Role of Non-Myocyte Gap Junctions and Connexin Hemichannels in Cardiovascular Health and Disease: Novel Therapeutic Targets?

PubMed

Johnson, Robert D; Camelliti, Patrizia

2018-03-15

The heart is a complex organ composed of multiple cell types, including cardiomyocytes and different non-myocyte populations, all working closely together to determine the hearts properties and maintain normal cardiac function. Connexins are abundantly expressed proteins that form plasma membrane hemichannels and gap junctions between cells. Gap junctions are intracellular channels that allow for communication between cells, and in the heart they play a crucial role in cardiac conduction by coupling adjacent cardiomyocytes. Connexins are expressed in both cardiomyocytes and non-myocytes, including cardiac fibroblasts, endothelial cells, and macrophages. Non-myocytes are the largest population of cells in the heart, and therefore it is important to consider what roles connexins, hemichannels, and gap junctions play in these cell types. The aim of this review is to provide insight into connexin-based signalling in non-myocytes during health and disease, and highlight how targeting these proteins could lead to the development of novel therapies. We conclude that connexins in non-myocytes contribute to arrhythmias and adverse ventricular remodelling following myocardial infarction, and are associated with the initiation and development of atherosclerosis. Therefore, therapeutic interventions targeting these connexins represent an exciting new research avenue with great potential.
Neuro-immune interactions at barrier surfaces

PubMed Central

Veiga-Fernandes, Henrique; Mucida, Daniel

2016-01-01

Multidirectional interactions between the nervous and immune systems have been documented in homeostasis and pathologies ranging from multiple sclerosis to autism, and from leukemia to acute and chronic inflammation. Recent studies have addressed this crosstalk using cell-specific targeting, novel sequencing, imaging and analytical tools, shedding light on unappreciated mechanisms of neuro-immune regulation. This review focuses on neuro-immune interactions at barrier surfaces, mostly the gut, but also including the skin and the airways, areas densely populated by neurons and immune cells that constantly sense and adapt to tissue-specific environmental challenges. PMID:27153494
Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

NASA Astrophysics Data System (ADS)

Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

2018-03-01

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.
Muscle stem cell dysfunction impairs muscle regeneration in a mouse model of Down syndrome.

PubMed

Pawlikowski, Bradley; Betta, Nicole Dalla; Elston, Tiffany; Williams, Darian A; Olwin, Bradley B

2018-03-09

Down syndrome, caused by trisomy 21, is characterized by a variety of medical conditions including intellectual impairments, cardiovascular defects, blood cell disorders and pre-mature aging phenotypes. Several somatic stem cell populations are dysfunctional in Down syndrome and their deficiencies may contribute to multiple Down syndrome phenotypes. Down syndrome is associated with muscle weakness but skeletal muscle stem cells or satellite cells in Down syndrome have not been investigated. We find that a failure in satellite cell expansion impairs muscle regeneration in the Ts65Dn mouse model of Down syndrome. Ts65Dn satellite cells accumulate DNA damage and over express Usp16, a histone de-ubiquitinating enzyme that regulates the DNA damage response. Impairment of satellite cell function, which further declines as Ts65Dn mice age, underscores stem cell deficiencies as an important contributor to Down syndrome pathologies.
Amnion: a potent graft source for cell therapy in stroke.

PubMed

Yu, Seong Jin; Soncini, Maddalena; Kaneko, Yuji; Hess, David C; Parolini, Ornella; Borlongan, Cesar V

2009-01-01

Regenerative medicine is a new field primarily based on the concept of transplanting exogenous or stimulating endogenous stem cells to generate biological substitutes and improve tissue functions. Recently, amnion-derived cells have been reported to have multipotent differentiation ability, and these cells have attracted attention as a novel cell source for cell transplantation therapy. Cells isolated from amniotic membrane can differentiate into all three germ layers, have low immunogenicity and anti-inflammatory function, and do not require the destruction of human embryos for their isolation, thus circumventing the ethical debate commonly associated with the use of human embryonic stem cells. Accumulating evidence now suggests that the amnion, which had been discarded after parturition, is a highly potent transplant material in the field of regenerative medicine. In this report, we review the current progress on the characterization of MSCs derived from the amnion as a remarkable transplantable cell population with therapeutic potential for multiple CNS disorders, especially stroke.
Increasing RpoS expression causes cell death in Borrelia burgdorferi.

PubMed

Chen, Linxu; Xu, Qilong; Tu, Jiagang; Ge, Yihe; Liu, Jun; Liang, Fang Ting

2013-01-01

RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.
Cell therapies: realizing the potential of this new dimension to medical therapeutics.

PubMed

Singh, Pawanbir; Williams, David J

2008-08-01

Stem cells promise to treat conditions poorly served by conventional therapeutics. Cells from both embryonic and somatic tissues are being used to create cell therapies for genetic, traumatic and degenerative conditions. The current human, healthcare and fiscal costs of these conditions are significant. This review summarizes the use of stem cells for neurological and cardiac disorders and diabetes to determine the requirements for generic translational research to assist such therapies to be a reality. While there are multiple strategies in each disease area, with no clear favourite, there are clear opportunities in treatments that use a single cell type. A key requirement is to work with pluripotent progenitor cells to cultivate and differentiate a sufficiently large population of functioning cells. Challenges also arise in determining and achieving timely delivery of the correct dose of cells to where they can most effectively treat the disease and best benefit individual patients. (c) 2008 John Wiley & Sons, Ltd.
Cooperation in the dark: signalling and collective action in quorum-sensing bacteria.

PubMed

Brown, S P; Johnstone, R A

2001-05-07

The study of quorum-sensing bacteria has revealed a widespread mechanism of coordinating bacterial gene expression with cell density. By monitoring a constitutively produced signal molecule, individual bacteria can limit their expression of group-beneficial phenotypes to cell densities that guarantee an effective group outcome. In this paper, we attempt to move away from a commonly expressed view that these impressive feats of coordination are examples of multicellularity in prokaryotic populations. Here, we look more closely at the individual conflict underlying this cooperation, illustrating that, even under significant levels of genetic conflict, signalling and resultant cooperative behaviour can stably exist. A predictive two-trait model of signal strength and of the extent of cooperation is developed as a function of relatedness (reflecting multiplicity of infection) and basic population demographic parameters. The model predicts that the strength of quorum signalling will increase as conflict (multiplicity of infecting strains) increases, as individuals attempt to coax more cooperative contributions from their competitors, leading to a devaluation of the signal as an indicator of density. Conversely, as genetic conflict increases, the model predicts that the threshold density for cooperation will increase and the subsequent strength of group cooperation will be depressed.
Ocrelizumab: a B-cell depleting therapy for multiple sclerosis.

PubMed

Jakimovski, Dejan; Weinstock-Guttman, Bianca; Ramanathan, Murali; Kolb, Channa; Hojnacki, David; Minagar, Alireza; Zivadinov, Robert

2017-09-01

Multiple sclerosis (MS) is the most common neurological disease responsible for early disability in the young working population. In the last two decades, based on retrospective/prospective data, the use of disease-modifying therapies has been shown to slow the rate of disability progression and prolonged the time to conversion into secondary-progressive MS (SPMS). However, despite the availability of several approved therapies, disability progression cannot be halted significantly in all MS patients. Areas covered: This article reviews the immunopathology of the B-cells, and their role in pathogenesis of MS and their attractiveness as a potential therapeutic target in MS. The review focuses on the recently published ocrelizumab phase III trials in terms of its efficacy, safety, and tolerability as well as its future considerations. Expert opinion: B lymphocyte cell depletion therapy offers a compelling and promising new option for MS patients. Nonetheless, there is a need for heightened vigilance and awareness in detecting potential long-term consequences that currently remain unknown.

Modulation by Melatonin of the Pathogenesis of Inflammatory Autoimmune Diseases

PubMed Central

Lin, Gu-Jiun; Huang, Shing-Hwa; Chen, Shyi-Jou; Wang, Chih-Hung; Chang, Deh-Ming; Sytwu, Huey-Kang

2013-01-01

Melatonin is the major secretory product of the pineal gland during the night and has multiple activities including the regulation of circadian and seasonal rhythms, and antioxidant and anti-inflammatory effects. It also possesses the ability to modulate immune responses by regulation of the T helper 1/2 balance and cytokine production. Autoimmune diseases, which result from the activation of immune cells by autoantigens released from normal tissues, affect around 5% of the population. Activation of autoantigen-specific immune cells leads to subsequent damage of target tissues by these activated cells. Melatonin therapy has been investigated in several animal models of autoimmune disease, where it has a beneficial effect in a number of models excepting rheumatoid arthritis, and has been evaluated in clinical autoimmune diseases including rheumatoid arthritis and ulcerative colitis. This review summarizes and highlights the role and the modulatory effects of melatonin in several inflammatory autoimmune diseases including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes mellitus, and inflammatory bowel disease. PMID:23727938
Inferring HIV Escape Rates from Multi-Locus Genotype Data

DOE PAGES

Kessinger, Taylor A.; Perelson, Alan S.; Neher, Richard A.

2013-09-03

Cytotoxic T-lymphocytes (CTLs) recognize viral protein fragments displayed by major histocompatibility complex molecules on the surface of virally infected cells and generate an anti-viral response that can kill the infected cells. Virus variants whose protein fragments are not efficiently presented on infected cells or whose fragments are presented but not recognized by CTLs therefore have a competitive advantage and spread rapidly through the population. We present a method that allows a more robust estimation of these escape rates from serially sampled sequence data. The proposed method accounts for competition between multiple escapes by explicitly modeling the accumulation of escape mutationsmore » and the stochastic effects of rare multiple mutants. Applying our method to serially sampled HIV sequence data, we estimate rates of HIV escape that are substantially larger than those previously reported. The method can be extended to complex escapes that require compensatory mutations. We expect our method to be applicable in other contexts such as cancer evolution where time series data is also available.« less
Feeding ducks, bacterial chemotaxis, and the Gini index

NASA Astrophysics Data System (ADS)

Peaudecerf, François J.; Goldstein, Raymond E.

2015-08-01

Classic experiments on the distribution of ducks around separated food sources found consistency with the "ideal free" distribution in which the local population is proportional to the local supply rate. Motivated by this experiment and others, we examine the analogous problem in the microbial world: the distribution of chemotactic bacteria around multiple nearby food sources. In contrast to the optimization of uptake rate that may hold at the level of a single cell in a spatially varying nutrient field, nutrient consumption by a population of chemotactic cells will modify the nutrient field, and the uptake rate will generally vary throughout the population. Through a simple model we study the distribution of resource uptake in the presence of chemotaxis, consumption, and diffusion of both bacteria and nutrients. Borrowing from the field of theoretical economics, we explore how the Gini index can be used as a means to quantify the inequalities of uptake. The redistributive effect of chemotaxis can lead to a phenomenon we term "chemotactic levelling," and the influence of these results on population fitness are briefly considered.
Postnatal extra-embryonic tissues as a source of multiple cell types for regenerative medicine applications.

PubMed

Gubar, O S; Rodnichenko, A E; Vasyliev, R G; Zlatska, A V; Zubov, D O

2017-09-01

We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73 + CD90 + CD105 + CD146 + CD166+CD34 - CD45 - HLA-DR - . HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use.
Gorlin syndrome and bilateral ovarian fibroma

PubMed Central

Pirschner, Fernanda; Bastos, Pollyana Marçal; Contarato, George Luiz; Bimbato, Anna Carolina Bon Lima; Filho, Antônio Chambô

2012-01-01

INTRODUCTION Gorlin syndrome (GS), also known as nevoid basal cell carcinoma syndrome (NBCCS), is a rare hereditary, autosomal dominant disease that affects various systems. Its prevalence is estimated at 1/57,000 to 1/256,000 of the population. It is characterized by basal cell carcinomas, multiple odontogenic keratocysts, skeletal abnormalities and ovarian fibroma, among other disorders. PRESENTATION OF CASE To report the case of a young patient with Gorlin syndrome and bilateral ovarian fibroma. DISCUSSION A 20-year old patient with Gorlin syndrome presented with facial asymmetry, broad nasal root, dental abnormalities, micrognathism, convergent strabismus, multiple pigmented lesions on the trunk and face, pectus excavatum, kyphoscoliosis and a palpable mass in the abdomen occupying the entire pelvic region. CONCLUSION Gorlin–Goltz syndrome is a hereditary pathology that includes numerous clinical manifestations. Diagnosis is clinical and genetic confirmation is unnecessary. PMID:22771908
Astrocytes in the tempest of multiple sclerosis.

PubMed

Miljković, Djordje; Timotijević, Gordana; Mostarica Stojković, Marija

2011-12-01

Astrocytes are the most abundant cell population within the CNS of mammals. Their glial role is perfectly performed in the healthy CNS as they support functions of neurons. The omnipresence of astrocytes throughout the white and grey matter and their intimate relation with blood vessels of the CNS, as well as numerous immunity-related actions that these cells are capable of, imply that astrocytes should have a prominent role in neuroinflammatory disorders, such as multiple sclerosis (MS). The role of astrocytes in MS is rather ambiguous, as they have the capacity to both stimulate and restrain neuroinflammation and tissue destruction. In this paper we present some of the proved and the proposed functions of astrocytes in neuroinflammation and discuss the effect of MS therapeutics on astrocytes. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Single Cell Profiling using Ionic Liquid Matrix-Enhanced Secondary Ion Mass Spectrometry for Neuronal Cell Type Differentiation

PubMed Central

Do, Thanh D.; Comi, Troy J.; Dunham, Sage J. B.; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

2017-01-01

A high-throughput single cell profiling method has been developed for matrix-enhanced secondary ion mass spectrometry (ME-SIMS) to investigate the lipid profiles of neuronal cells. Populations of cells are dispersed onto the substrate, their locations determined using optical microscopy, and the cell locations used to guide the acquisition of SIMS spectra from the cells. Up to 2,000 cells can be assayed in one experiment at a rate of 6 s per cell. Multiple saturated and unsaturated phosphatidylcholines (PCs) and their fragments are detected and verified with tandem mass spectrometry from individual cells when ionic liquids are employed as a matrix. Optically guided single cell profiling with ME-SIMS is suitable for a range of cell sizes, from Aplysia californica neurons larger than 75 μm to 7-μm rat cerebellar neurons. ME-SIMS analysis followed by t-distributed stochastic neighbor embedding of peaks in the lipid molecular mass range (m/z 700–850) distinguishes several cell types from the rat central nervous system, largely based on the relative proportions of the four dominant lipids, PC(32:0), PC(34:1), PC(36:1), and PC(38:5). Furthermore, subpopulations within each cell type are tentatively classified consistent with their endogenous lipid ratios. The results illustrate the efficacy of a new approach to classify single cell populations and subpopulations using SIMS profiling of lipid and metabolite contents. These methods are broadly applicable for high throughput single cell chemical analyses. PMID:28194949
Natural genetic variation profoundly regulates gene expression in immune cells and dictates susceptibility to CNS autoimmunity

PubMed Central

Bearoff, Frank; del Rio, Roxana; Case, Laure K.; Dragon, Julie A.; Nguyen-Vu, Trang; Lin, Chin-Yo; Blankenhorn, Elizabeth P.; Teuscher, Cory; Krementsov, Dimitry N.

2016-01-01

Regulation of gene expression in immune cells is known to be under genetic control, and likely contributes to susceptibility to autoimmune diseases, such as multiple sclerosis (MS). How this occurs in concert across multiple immune cell types is poorly understood. Using a mouse model that harnesses the genetic diversity of wild-derived mice, more accurately reflecting genetically diverse human populations, we provide an extensive characterization of the genetic regulation of gene expression in five different naïve immune cell types relevant to MS. The immune cell transcriptome is shown to be under profound genetic control, exhibiting diverse patterns: global, cell-specific, and sex-specific. Bioinformatic analysis of the genetically-controlled transcript networks reveals reduced cell type-specificity and inflammatory activity in wild-derived PWD/PhJ mice, compared with the conventional laboratory strain C57BL/6J. Additionally, candidate MS-GWAS genes were significantly enriched among transcripts overrepresented in C57BL/6J cells compared to PWD. These expression level differences correlate with robust differences in susceptibility to experimental autoimmune encephalomyelitis, the principal model of MS, and skewing of the encephalitogenic T cell responses. Taken together, our results provide functional insights into the genetic regulation of the immune transcriptome, and shed light on how this in turn contributes to susceptibility to autoimmune disease. PMID:27653816
Cell-type-specific expression of NFIX in the developing and adult cerebellum.

PubMed

Fraser, James; Essebier, Alexandra; Gronostajski, Richard M; Boden, Mikael; Wainwright, Brandon J; Harvey, Tracey J; Piper, Michael

2017-07-01

Transcription factors from the nuclear factor one (NFI) family have been shown to play a central role in regulating neural progenitor cell differentiation within the embryonic and post-natal brain. NFIA and NFIB, for instance, promote the differentiation and functional maturation of granule neurons within the cerebellum. Mice lacking Nfix exhibit delays in the development of neuronal and glial lineages within the cerebellum, but the cell-type-specific expression of this transcription factor remains undefined. Here, we examined the expression of NFIX, together with various cell-type-specific markers, within the developing and adult cerebellum using both chromogenic immunohistochemistry and co-immunofluorescence labelling and confocal microscopy. In embryos, NFIX was expressed by progenitor cells within the rhombic lip and ventricular zone. After birth, progenitor cells within the external granule layer, as well as migrating and mature granule neurons, expressed NFIX. Within the adult cerebellum, NFIX displayed a broad expression profile, and was evident within granule cells, Bergmann glia, and interneurons, but not within Purkinje neurons. Furthermore, transcriptomic profiling of cerebellar granule neuron progenitor cells showed that multiple splice variants of Nfix are expressed within this germinal zone of the post-natal brain. Collectively, these data suggest that NFIX plays a role in regulating progenitor cell biology within the embryonic and post-natal cerebellum, as well as an ongoing role within multiple neuronal and glial populations within the adult cerebellum.
From the cradle to the grave: activities of GATA-3 throughout T cell development and differentiation

PubMed Central

Hosoya, Tomonori; Maillard, Ivan; Engel, James Douglas

2010-01-01

Summary GATA family transcription factors play multiple vital roles in hematopoiesis in many cell lineages, and in particular, T cells require GATA-3 for execution of several developmental steps. Transcriptional activation of the Gata3 gene is observed throughout T-cell development and differentiation in stage-specific fashion. GATA-3 has been described as a master regulator of T-helper 2 (Th2) cell differentiation in mature CD4+ T cells. During T-cell development in the thymus, its roles in the CD4 vs. CD8 lineage choice and at the β-selection checkpoint are the best characterized. In contrast, its importance prior to β-selection has been obscured both by the developmental heterogeneity of double negative (DN) 1 thymocytes and the paucity of early T-lineage progenitors (ETPs), a subpopulation of DN1 cells that contains the most immature thymic progenitors that retain potent T-lineage developmental potential. By examining multiple lines of in vivo evidence procured through the analysis of Gata3 mutant mice, we have recently demonstrated that GATA-3 is additionally required at the earliest stage of thymopoiesis for the development of the ETP population. Here, we review the characterized functions of GATA-3 at each stage of T-cell development and discuss hypothetical molecular pathways that mediate these functions. PMID:20969588
Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

PubMed Central

2011-01-01

Background The recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.). Results A modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM), Phthorimaea operculella (Zeller). Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation. Conclusions A new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event. PMID:21995716
A parallel implementation of an off-lattice individual-based model of multicellular populations

NASA Astrophysics Data System (ADS)

Harvey, Daniel G.; Fletcher, Alexander G.; Osborne, James M.; Pitt-Francis, Joe

2015-07-01

As computational models of multicellular populations include ever more detailed descriptions of biophysical and biochemical processes, the computational cost of simulating such models limits their ability to generate novel scientific hypotheses and testable predictions. While developments in microchip technology continue to increase the power of individual processors, parallel computing offers an immediate increase in available processing power. To make full use of parallel computing technology, it is necessary to develop specialised algorithms. To this end, we present a parallel algorithm for a class of off-lattice individual-based models of multicellular populations. The algorithm divides the spatial domain between computing processes and comprises communication routines that ensure the model is correctly simulated on multiple processors. The parallel algorithm is shown to accurately reproduce the results of a deterministic simulation performed using a pre-existing serial implementation. We test the scaling of computation time, memory use and load balancing as more processes are used to simulate a cell population of fixed size. We find approximate linear scaling of both speed-up and memory consumption on up to 32 processor cores. Dynamic load balancing is shown to provide speed-up for non-regular spatial distributions of cells in the case of a growing population.
[Effect of the development phase and growth rate of a Shigella sonnei population on the reproduction of homologous bacteriophage].

PubMed

Voroshilova, N N; Kazakova, T B

1983-04-01

This study showed that the minimum latent period (20 minutes) of the intracellular multiplication of dysentery bacteriophage S-9 in the population of S. sonnei substrate strain under the conditions of static heterogeneous surface batch cultivation was observed at the end of the lag phase and at the growth acceleration phase, in the first and second thirds of the exponential curve, while the maximum latent period (35-40 minutes) was observed at the stationary phase. The maximum yield of phage S-9 from one infected bacterial cell (628.3 +/- 116.8) was registered during the first third of the phase of the exponential growth of the bacterial population and the minimum yield (18.66 +/- 6.6), at the beginning of the lag phase. The significant direct correlation between the specific growth rate of the bacterial population and the yield of the phage from one infected bacterial cell at the end of the lag phase, at the growth acceleration and deceleration phases, as well as the significant inverse correlation between the yield of the phage and the time of the generation of the bacterial population at the growth acceleration phase were established.
The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis

PubMed Central

2014-01-01

Background It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. Results We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. Conclusions In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae. PMID:24602211
Niches for the Long-Term Maintenance of Tissue-Resident Memory T Cells

PubMed Central

Takamura, Shiki

2018-01-01

Tissue-resident memory T cells (TRM cells) are a population of immune cells that reside in the lymphoid and non-lymphoid organs without recirculation through the blood. These important cells occupy and utilize unique anatomical and physiological niches that are distinct from those for other memory T cell populations, such as central memory T cells in the secondary lymphoid organs and effector memory T cells that circulate through the tissues. CD8+ TRM cells typically localize in the epithelial layers of barrier tissues where they are optimally positioned to act as sentinels to trigger antigen-specific protection against reinfection. CD4+ TRM cells typically localize below the epithelial layers, such as below the basement membrane, and cluster in lymphoid structures designed to optimize interactions with antigen-presenting cells upon reinfection. A key feature of TRM populations is their ability to be maintained in barrier tissues for prolonged periods of time. For example, skin CD8+ TRM cells displace epidermal niches originally occupied by γδ T cells, thereby enabling their stable persistence for years. It is also clear that the long-term maintenance of TRM cells in different microenvironments is dependent on multiple tissue-specific survival cues, although the specific details are poorly understood. However, not all TRM persist over the long term. Recently, we identified a new spatial niche for the maintenance of CD8+ TRM cells in the lung, which is created at the site of tissue regeneration after injury [termed repair-associated memory depots (RAMD)]. The short-lived nature of RAMD potentially explains the short lifespans of CD8+ TRM cells in this particular tissue. Clearly, a better understanding of the niche-dependent maintenance of TRM cells will be important for the development of vaccines designed to promote barrier immunity. In this review, we discuss recent advances in our understanding of the properties and nature of tissue-specific niches that maintain TRM cells in different tissues. PMID:29904388
The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis.

PubMed

Koziol, Uriel; Rauschendorfer, Theresa; Zanon Rodríguez, Luis; Krohne, Georg; Brehm, Klaus

2014-03-06

It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae.
Modeling mechanical inhomogeneities in small populations of proliferating monolayers and spheroids.

PubMed

Lejeune, Emma; Linder, Christian

2018-06-01

Understanding the mechanical behavior of multicellular monolayers and spheroids is fundamental to tissue culture, organism development, and the early stages of tumor growth. Proliferating cells in monolayers and spheroids experience mechanical forces as they grow and divide and local inhomogeneities in the mechanical microenvironment can cause individual cells within the multicellular system to grow and divide at different rates. This differential growth, combined with cell division and reorganization, leads to residual stress. Multiple different modeling approaches have been taken to understand and predict the residual stresses that arise in growing multicellular systems, particularly tumor spheroids. Here, we show that by using a mechanically robust agent-based model constructed with the peridynamic framework, we gain a better understanding of residual stresses in multicellular systems as they grow from a single cell. In particular, we focus on small populations of cells (1-100 s) where population behavior is highly stochastic and prior investigation has been limited. We compare the average strain energy density of cells in monolayers and spheroids using different growth and division rules and find that, on average, cells in spheroids have a higher strain energy density than cells in monolayers. We also find that cells in the interior of a growing spheroid are, on average, in compression. Finally, we demonstrate the importance of accounting for stochastic fluctuations in the mechanical environment, particularly when the cellular response to mechanical cues is nonlinear. The results presented here serve as a starting point for both further investigation with agent-based models, and for the incorporation of major findings from agent-based models into continuum scale models when explicit representation of individual cells is not computationally feasible.
Differentiation State-Specific Mitochondrial Dynamic Regulatory Networks Are Revealed by Global Transcriptional Analysis of the Developing Chicken Lens

PubMed Central

Chauss, Daniel; Basu, Subhasree; Rajakaruna, Suren; Ma, Zhiwei; Gau, Victoria; Anastas, Sara; Brennan, Lisa A.; Hejtmancik, J. Fielding; Menko, A. Sue; Kantorow, Marc

2014-01-01

The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that requires a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected more than 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, more than 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic (DUT, PDK1, SNPH), autophagy (ATG3, ATG4B, BECN1, FYCO1, WIPI1), and mitophagy (BNIP3L/NIX, BNIP3, PARK2, p62/SQSTM1) transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells. PMID:24928582
Temporal Transcriptional Profiling of Somatic and Germ Cells Reveals Biased Lineage Priming of Sexual Fate in the Fetal Mouse Gonad

PubMed Central

Jameson, Samantha A.; Natarajan, Anirudh; Cool, Jonah; DeFalco, Tony; Maatouk, Danielle M.; Mork, Lindsey; Munger, Steven C.; Capel, Blanche

2012-01-01

The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates. PMID:22438826
Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

NASA Technical Reports Server (NTRS)

Karr, Laurel J.; Marshall, Gwen; Hockett, Richard D.; Bucy, R. Pat; Curreri, Peter A. (Technical Monitor)

2002-01-01

A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

Intra-tumor heterogeneity: lessons from microbial evolution and clinical implications

PubMed Central

2013-01-01

Multiple subclonal populations of tumor cells can coexist within the same tumor. This intra-tumor heterogeneity will have clinical implications and it is therefore important to identify factors that drive or suppress such heterogeneous tumor progression. Evolutionary biology can provide important insights into this process. In particular, experimental evolution studies of microbial populations, which exist as clonal populations that can diversify into multiple subclones, have revealed important evolutionary processes driving heterogeneity within a population. There are transferrable lessons that can be learnt from these studies that will help us to understand the process of intra-tumor heterogeneity in the clinical setting. In this review, we summarize drivers of microbial diversity that have been identified, such as mutation rate and environmental influences, and discuss how knowledge gained from microbial experimental evolution studies may guide us to identify and understand important selective factors that promote intra-tumor heterogeneity. Furthermore, we discuss how these factors could be used to direct and optimize research efforts to improve patient care, focusing on therapeutic resistance. Finally, we emphasize the need for longitudinal studies to address the impact of these potential tumor heterogeneity-promoting factors on drug resistance, metastatic potential and clinical outcome. PMID:24267946
New contributions to Gruberia lanceolata (Gruber, 1884) Kahl, 1932 based on analyses of multiple populations and genes (Ciliophora, Heterotrichea, Gruberiidae).

PubMed

Chen, Xiangrui; Shazib, Shahed Uddin Ahmed; Kim, Ji Hye; Kim, Min Seok; Shin, Mann Kyoon

2018-05-09

Gruberia Kahl, 1932 is a species-poor genus comprising only seven named species. Most of these species have not been reinvestigated since the original reports. In the present work, we investigated the taxonomy and phylogeny of Gruberia lanceolata (Gruber, 1884) Kahl, 1932 based on analyses of morphology and multiple gene sequences from four South Korean populations. This species is mainly characterized by a well-developed peristome region, segmented paroral membrane, and moniliform macronucleus. Some morphological features were not stable among the four populations investigated, such as body shape and size, cell color, and the ratio of oral length to body length. However, our molecular analyses of four different genetic markers - three nuclear DNA markers (18S rDNA, ITS1-5.8S-ITS2 region, D1D2 of 28S rDNA) and one mitochondrial (mt) marker (CO1 gene) - indicated that all Korean populations examined were the same species. Based on our present findings and historic works, we propose that G. calkinsi, G. aculeata, and G. beninensis are junior synonyms of G. lanceolata. Copyright © 2018 Elsevier GmbH. All rights reserved.
Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease

PubMed Central

Tirnitz-Parker, Janina Elke Eleonore; Hamson, Elizabeth Jane; Warren, Alessandra; Maneck, Bharvi; Chen, Jinbiao; Patkunanathan, Bramilla; Boland, Jade; Cheng, Robert; Shackel, Nicholas Adam; Seth, Devanshi; Bowen, David Geoffrey; Martelotto, Luciano Gastón; Watkins, D. Neil; McCaughan, Geoffrey William

2017-01-01

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (>99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMO-dependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness. PMID:28187190
Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease.

PubMed

Grzelak, Candice Alexandra; Sigglekow, Nicholas David; Tirnitz-Parker, Janina Elke Eleonore; Hamson, Elizabeth Jane; Warren, Alessandra; Maneck, Bharvi; Chen, Jinbiao; Patkunanathan, Bramilla; Boland, Jade; Cheng, Robert; Shackel, Nicholas Adam; Seth, Devanshi; Bowen, David Geoffrey; Martelotto, Luciano Gastón; Watkins, D Neil; McCaughan, Geoffrey William

2017-01-01

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (>99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMO-dependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness.
Prevalence of problematic cell phone use in an adult population in Spain as assessed by the Mobile Phone Problem Use Scale (MPPUS)

PubMed Central

de-Sola, José; Talledo, Hernán; Rubio, Gabriel

2017-01-01

Problematic cell phone use has alarmingly increased in industrialized countries in the past 10 years. For many perpetrators, it can turn into a behavioural addiction, although this is not a recognized medical condition. Although there are many tools for evaluating this use, one of the most widely used tools is the Mobile Phone Problematic Use Scale (MPPUS), which we test on a representative sample of the population in Spain to obtain an estimate of the prevalence of problematic cell phone use in our midst. The age range consists of 16–65 years, with 1,126 surveys conducted. In this population, we verify that the reliability and internal consistency of the MPPUS (α = 0.939) are maintained. Additionally, the construct validity, considering the derived factors (Abuse and Dependence, Craving and Loss of Control, and Dependence on the Social Environment) are aligned with other research and with diverse external criteria of addiction. We establish four categories of users (Casual, Regular, At Risk, and Problematic) and obtain a prevalence of 15.4% among At Risk Users and 5.1% among Problematic Users. This finding implies a total of 20.5% of Users with Problems. A binary logistic regression analysis shows that age, gender, level of education, and daily cell phone use predict problematic cell phone use. The results, based on multiple criteria, show that such problematic use shares features of recognized addictions, affecting large segments of the population and not only adolescents. PMID:28771626
Single Cell Mass Cytometry for Analysis of Immune System Functional States

PubMed Central

Bjornson, Zach B.; Nolan, Garry P.; Fantl, Wendy J.

2013-01-01

Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection. PMID:23999316
Specific subpopulations of hypothalamic leptin receptor-expressing neurons mediate the effects of early developmental leptin receptor deletion on energy balance.

PubMed

Rupp, Alan C; Allison, Margaret B; Jones, Justin C; Patterson, Christa M; Faber, Chelsea L; Bozadjieva, Nadejda; Heisler, Lora K; Seeley, Randy J; Olson, David P; Myers, Martin G

2018-06-06

To date, early developmental ablation of leptin receptor (LepRb) expression from circumscribed populations of hypothalamic neurons (e.g., arcuate nucleus (ARC) Pomc- or Agrp-expressing cells) has only minimally affected energy balance. In contrast, removal of LepRb from at least two large populations (expressing vGat or Nos1) spanning multiple hypothalamic regions produced profound obesity and metabolic dysfunction. Thus, we tested the notion that the total number of leptin-responsive hypothalamic neurons (rather than specific subsets of cells with a particular molecular or anatomical signature) subjected to early LepRb deletion might determine energy balance. We generated new mouse lines deleted for LepRb in ARC Ghrh Cre neurons or in Htr2c Cre neurons (representing roughly half of all hypothalamic LepRb neurons, distributed across many nuclei). We compared the phenotypes of these mice to previously-reported models lacking LepRb in Pomc, Agrp, vGat or Nos1 cells. The early developmental deletion of LepRb from vGat or Nos1 neurons produced dramatic obesity, but deletion of LepRb from Pomc, Agrp, Ghrh, or Htr2c neurons minimally altered energy balance. Although early developmental deletion of LepRb from known populations of ARC neurons fails to substantially alter body weight, the minimal phenotype of mice lacking LepRb in Htr2c cells suggests that the phenotype that results from early developmental LepRb deficiency depends not simply upon the total number of leptin-responsive hypothalamic LepRb cells. Rather, specific populations of LepRb neurons must play particularly important roles in body energy homeostasis; these as yet unidentified LepRb cells likely reside in the DMH. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.
Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells.

PubMed

Abu-Arish, Asmahan; Pandzic, Elvis; Goepp, Julie; Matthes, Elizabeth; Hanrahan, John W; Wiseman, Paul W

2015-07-07

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Inhibition of cell-mediated immunity by the histone deacetylase inhibitor vorinostat: implications for therapy of cutaneous T-cell lymphoma.

PubMed

Stephen, Sasha; Morrissey, Kelly A; Benoit, Bernice M; Kim, Ellen J; Vittorio, Carmela C; Nasta, Sunita D; Showe, Louise C; Wysocka, Maria; Rook, Alain H

2012-02-01

Several histone deacetylase inhibitors (HDACi), including vorinostat, have been approved for the therapy of cutaneous T-cell lymphoma (CTCL). Emerging data suggest that HDACi may exert immune suppressive effects which would be disadvantageous for therapy of CTCL. We describe a patient with Sezary syndrome who was monitored for drug-induced immunosuppression while undergoing treatment with vorinostat. Analysis of the patient's natural killer cell function before and after initiation of treatment confirmed inhibition of this important cell-mediated immune function. In addition, the in vitro effects of vorinostat on the immunity of healthy volunteers confirmed that this class of drug can profoundly suppress multiple arms of the cellular immune response. These findings raise concerns of increased susceptibility to infection in this high-risk population.
A microfluidic platform for chemical stimulation and real time analysis of catecholamine secretion from neuroendocrine cells.

PubMed

Ges, Igor A; Brindley, Rebecca L; Currie, Kevin P M; Baudenbacher, Franz J

2013-12-07

Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped "cell traps", each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion/repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time.
A Novel Source of Cultured Podocytes

PubMed Central

Da Sacco, Stefano; Lemley, Kevin V.; Sedrakyan, Sargis; Zanusso, Ilenia; Petrosyan, Astgik; Peti-Peterdi, Janos; Burford, James; De Filippo, Roger E.; Perin, Laura

2013-01-01

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies. PMID:24349133
Gastric marginal zone lymphoma of mucosa-associated lymphoid tissue and signet ring cell carcinoma, synchronous collision tumour of the stomach: a case report.

PubMed

George, Smiley Annie; Junaid, T A

2014-01-01

To report a rare case of synchronous marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) signet ring cell carcinoma occurring as a collision tumour in the stomach. A 53-year-old man was diagnosed initially with signet ring cell carcinoma of the stomach. The microscopy of the subsequent total gastrectomy revealed a collision tumour of MALT lymphoma and signet ring cell carcinoma associated with Helicobacter pylori gastritis. This case highlighted the importance of a careful evaluation of the accompanying lymphoid population in the biopsy samples of gastric adenocarcinoma and underlined the need for multiple endoscopic biopsies to detect these rare synchronous tumours. © 2013 S. Karger AG, Basel.
Gastric Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue and Signet Ring Cell Carcinoma, Synchronous Collision Tumour of the Stomach: A Case Report

PubMed Central

George, Smiley Annie; Junaid, T.A.

2014-01-01

Objective To report a rare case of synchronous marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) signet ring cell carcinoma occurring as a collision tumour in the stomach. Clinical Presentation and Intervention A 53-year-old man was diagnosed initially with signet ring cell carcinoma of the stomach. The microscopy of the subsequent total gastrectomy revealed a collision tumour of MALT lymphoma and signet ring cell carcinoma associated with Helicobacter pylori gastritis. Conclusion This case highlighted the importance of a careful evaluation of the accompanying lymphoid population in the biopsy samples of gastric adenocarcinoma and underlined the need for multiple endoscopic biopsies to detect these rare synchronous tumours. PMID:24247357
Digital quantification of gene expression using emulsion PCR.

PubMed

Shi, Xiaolong; Tang, Chao; Wang, Wei; Zhou, Dequan; Lu, Zuhong

2010-01-01

Here we describe a single-molecule quantitative assay of mRNA levels based on mRNA mediate-ligation and BEAMing (beads, emulsion, amplification, and magnetics) technique, which allows accurate and parallel measurement of multiple genes from a small amount of cells. In this method, a pair of oligos complementary target mRNA was used to probe transcripts for each gene of interest. The ligated products of oligos pair were clonally amplified on beads in millions of parallel compartmentalized droplets in a water-in-oil emulsion. The levels of each transcript within a sample were measured by counting the number of the correspondingly amplified beads which were immobilized on a glass surface. To demonstrate its utility, this method has been applied to the quantitation of the mRNA levels for two transcription factors, Klf4 and Sox5, and a housekeeping gene, Gapdh, in human leukemia K562 cells before and after induction with phorbol 12-myristate 13-acetate. Interestingly, we found a significant downregulation of the mRNA level of Sox5 after phorbol 12-myristate 13-acetate treatment. The mRNA mediate-ligation and BEAMing technique provides an accurate and sensitive way to quantify the amount of multiple specific mRNA in a very small number of cells, which may be valuable in the studies requiring precise and parallel quantization of multiple mRNA in the defined cell populations.
Meiotic recombination and spermatogenic impairment in Mus musculus domesticus carrying multiple simple Robertsonian translocations.

PubMed

Merico, V; Pigozzi, M I; Esposito, A; Merani, M S; Garagna, S

2003-01-01

We quantitatively analyzed the spermatogenic process, including evaluation of seminiferous tubules with defective cycles, rates of germ cell death and sperm morphology, in adult male mice with standard telocentric chromosomes (2n = 40, CD1 strain), homozygous (2n = 24, Mil II population) and heterozygous (2n = 24 x 40) for Robertsonian (Rb) rearrangements. The animals were analyzed at three different ages: three, five and seven months after birth. The number and position of crossover events were also determined by chiasmata counting and immunostaining with an antibody against mouse MLH1 protein. Our analysis of spermatogenesis confirms the impairment of the spermatogenic process in multiple simple heterozygotes due to both germ cell and abnormal sperm morphology. The detrimental effects exerted by Rb heterozygosities were found to be at least partially buffered with time: the frequency of defective tubules was lower and germ cell survival and sperm morphology better in 7-month-old animals than in the 3- and 5-month-old mice. While there are previously published data on germ cell death in multiple simple heterozygotes, this is the first report of a partial rescue of spermatogenesis with time. The mean frequency of MLH1 foci was lower in Rb homozygous and heterozygous mice than in mice carrying all telocentric chromosomes. The lower number of foci in Rb mice can be ascribed to a decrease in the number of multiple chiasmata and the maintenance of single chiasmata preferentially located in the terminal region of both the telocentric and metacentric chromosomes. Copyright 2003 S. Karger AG, Basel
High-definition imaging of circulating tumor cells and associated cellular events in non-small cell lung cancer patients: a longitudinal analysis.

PubMed

Nieva, Jorge; Wendel, Marco; Luttgen, Madelyn S; Marrinucci, Dena; Bazhenova, Lyudmila; Kolatkar, Anand; Santala, Roger; Whittenberger, Brock; Burke, James; Torrey, Melissa; Bethel, Kelly; Kuhn, Peter

2012-02-01

Sampling circulating tumor cells (CTCs) from peripheral blood is ideally accomplished using assays that detect high numbers of cells and preserve them for downstream characterization. We sought to evaluate a method using enrichment free fluorescent labeling of CTCs followed by automated digital microscopy in patients with non-small cell lung cancer. Twenty-eight patients with non-small cell lung cancer and hematogenously seeded metastasis were analyzed with multiple blood draws. We detected CTCs in 68% of analyzed samples and found a propensity for increased CTC detection as the disease progressed in individual patients. CTCs were present at a median concentration of 1.6 CTCs ml⁻¹ of analyzed blood in the patient population. Higher numbers of detected CTCs were associated with an unfavorable prognosis.
Systematic tracking of altered haematopoiesis during sporozoite-mediated malaria development reveals multiple response points.

PubMed

Vainieri, Maria L; Blagborough, Andrew M; MacLean, Adam L; Haltalli, Myriam L R; Ruivo, Nicola; Fletcher, Helen A; Stumpf, Michael P H; Sinden, Robert E; Celso, Cristina Lo

2016-06-01

Haematopoiesis is the complex developmental process that maintains the turnover of all blood cell lineages. It critically depends on the correct functioning of rare, quiescent haematopoietic stem cells (HSCs) and more numerous, HSC-derived, highly proliferative and differentiating haematopoietic progenitor cells (HPCs). Infection is known to affect HSCs, with severe and chronic inflammatory stimuli leading to stem cell pool depletion, while acute, non-lethal infections exert transient and even potentiating effects. Both whether this paradigm applies to all infections and whether the HSC response is the dominant driver of the changes observed during stressed haematopoiesis remain open questions. We use a mouse model of malaria, based on natural, sporozoite-driven Plasmodium berghei infection, as an experimental platform to gain a global view of haematopoietic perturbations during infection progression. We observe coordinated responses by the most primitive HSCs and multiple HPCs, some starting before blood parasitaemia is detected. We show that, despite highly variable inter-host responses, primitive HSCs become highly proliferative, but mathematical modelling suggests that this alone is not sufficient to significantly impact the whole haematopoietic cascade. We observe that the dramatic expansion of Sca-1(+) progenitors results from combined proliferation of direct HSC progeny and phenotypic changes in downstream populations. We observe that the simultaneous perturbation of HSC/HPC population dynamics is coupled with early signs of anaemia onset. Our data uncover a complex relationship between Plasmodium and its host's haematopoiesis and raise the question whether the variable responses observed may affect the outcome of the infection itself and its long-term consequences on the host. © 2016 The Authors.
Systematic tracking of altered haematopoiesis during sporozoite-mediated malaria development reveals multiple response points

PubMed Central

Vainieri, Maria L.; Blagborough, Andrew M.; MacLean, Adam L.; Haltalli, Myriam L. R.; Ruivo, Nicola; Fletcher, Helen A.; Stumpf, Michael P. H.; Sinden, Robert E.; Lo Celso, Cristina

2016-01-01

Haematopoiesis is the complex developmental process that maintains the turnover of all blood cell lineages. It critically depends on the correct functioning of rare, quiescent haematopoietic stem cells (HSCs) and more numerous, HSC-derived, highly proliferative and differentiating haematopoietic progenitor cells (HPCs). Infection is known to affect HSCs, with severe and chronic inflammatory stimuli leading to stem cell pool depletion, while acute, non-lethal infections exert transient and even potentiating effects. Both whether this paradigm applies to all infections and whether the HSC response is the dominant driver of the changes observed during stressed haematopoiesis remain open questions. We use a mouse model of malaria, based on natural, sporozoite-driven Plasmodium berghei infection, as an experimental platform to gain a global view of haematopoietic perturbations during infection progression. We observe coordinated responses by the most primitive HSCs and multiple HPCs, some starting before blood parasitaemia is detected. We show that, despite highly variable inter-host responses, primitive HSCs become highly proliferative, but mathematical modelling suggests that this alone is not sufficient to significantly impact the whole haematopoietic cascade. We observe that the dramatic expansion of Sca-1+ progenitors results from combined proliferation of direct HSC progeny and phenotypic changes in downstream populations. We observe that the simultaneous perturbation of HSC/HPC population dynamics is coupled with early signs of anaemia onset. Our data uncover a complex relationship between Plasmodium and its host's haematopoiesis and raise the question whether the variable responses observed may affect the outcome of the infection itself and its long-term consequences on the host. PMID:27335321
A cell clone strain from Mythimna separata (Lepidoptera: Noctuidae) highly susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata NPV (MsNPV).

PubMed

Meng, Xiang-Qian; Zheng, Gui-Ling; Zhao, Chuan-De; Wan, Fang-Hao; Li, Chang-You

2017-08-01

In this study, we describe a cell line, Ms-10C, cloned from the line QAU-Ms-E-10 (simplified Ms-10), an embryonic line from Mythimna separata. The cloned cell line was significantly more sensitive to nucleopolyhedrovirus (NPV). Ms-10C cells were mainly spherical with a diameter of 14.42 ± 2.23 μm. DNA amplification fingerprinting (DAF) confirmed the profile of PCR-amplified bands of the cloned cell line was consistent with those of the parental cell line, Ms-10. The sequencing result of the mitochondrial cytochrome c oxidase I (mtCO I) fragment confirmed that the amplified 636-bps mtCOI fragment was 100% identical to that of M. separata. Its chromosomes exhibited the typical characters of lepidopteran cell lines. Its population doubling time was 42.2 h at 27°C. Ms-10C was more sensitive than Ms-10 to both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata nucleopolyhedrovirus (MsNPV). At 4 d post infection, the infection rates of two viruses reached 94.2 and 92.3%, respectively. The availability of this cell clone strain will provide a useful tool for the basic research on nucleopolyhedrovirus and for potential application in expression of recombinant proteins with baculovirus expression vector system.
Honey bee hemocyte profiling by flow cytometry.

PubMed

Marringa, William J; Krueger, Michael J; Burritt, Nancy L; Burritt, James B

2014-01-01

Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure.

Honey Bee Hemocyte Profiling by Flow Cytometry

PubMed Central

Marringa, William J.; Krueger, Michael J.; Burritt, Nancy L.; Burritt, James B.

2014-01-01

Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure. PMID:25285798
Chimeric analysis of EGFP and DsRed2 transgenic mice demonstrates polyclonal maintenance of pancreatic acini.

PubMed

Ryu, Je-Young; Siswanto, Antoni; Harimoto, Kenichi; Tagawa, Yoh-ichi

2013-06-01

The pancreatic islet is an assembly of specific endocrine cells. There are many conflicting reports regarding whether the acinus develops from single or multiple progenitor cells. This study investigated the development and maintenance clonality of the pancreatic acinus and duct using a chimeric analysis with EGFP and DsRed2 transgenic mice. Chimeric mice (G-R mice) were obtained by the aggregation method, using 8-cell stage embryos from EGFP and DsRed2 transgenic mice. The islets from the G-R mice were chimeric and mosaic, consisting of either EGFP- or DsRed2-positive populations, as in previous reports. On the other hand, most acini developed from either EGFP or DsRed2 origin, but some were chimeric. Interestingly, these chimeric acini were clearly separated into two-color regions and were not mosaic. Some large intralobular pancreatic ducts consisting of more than 10 cells were found to be chimeric, but no small ducts made up of less than 9 cells were chimeric. Our histological observations suggest that the pancreatic acinus polyclonally and directionally is maintained by multiple progenitor cells. Pancreatic large ducts also seem to develop polyclonally and might result from the assembly of small ducts that develop from a single origin. These findings provide useful information for further understanding pancreatic maintenance.
Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge

PubMed Central

Kogot, Joshua M.; Zhang, Yanting; Moore, Stephen J.; Pagano, Paul; Stratis-Cullum, Dimitra N.; Chang-Yen, David; Turewicz, Marek; Pellegrino, Paul M.; de Fusco, Andre; Soh, H. Tom; Stagliano, Nancy E.

2011-01-01

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×1010 members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS. PMID:22140433
Geographic risk modeling of childhood cancer relative to county-level crops, hazardous air pollutants and population density characteristics in Texas.

PubMed

Thompson, James A; Carozza, Susan E; Zhu, Li

2008-09-25

Childhood cancer has been linked to a variety of environmental factors, including agricultural activities, industrial pollutants and population mixing, but etiologic studies have often been inconclusive or inconsistent when considering specific cancer types. More specific exposure assessments are needed. It would be helpful to optimize future studies to incorporate knowledge of high-risk locations or geographic risk patterns. The objective of this study was to evaluate potential geographic risk patterns in Texas accounting for the possibility that multiple cancers may have similar geographic risks patterns. A spatio-temporal risk modeling approach was used, whereby 19 childhood cancer types were modeled as potentially correlated within county-years. The standard morbidity ratios were modeled as functions of intensive crop production, intensive release of hazardous air pollutants, population density, and rapid population growth. There was supportive evidence for elevated risks for germ cell tumors and "other" gliomas in areas of intense cropping and for hepatic tumors in areas of intense release of hazardous air pollutants. The risk for Hodgkin lymphoma appeared to be reduced in areas of rapidly growing population. Elevated spatial risks included four cancer histotypes, "other" leukemias, Central Nervous System (CNS) embryonal tumors, CNS other gliomas and hepatic tumors with greater than 95% likelihood of elevated risks in at least one county. The Bayesian implementation of the Multivariate Conditional Autoregressive model provided a flexible approach to the spatial modeling of multiple childhood cancer histotypes. The current study identified geographic factors supporting more focused studies of germ cell tumors and "other" gliomas in areas of intense cropping, hepatic cancer near Hazardous Air Pollutant (HAP) release facilities and specific locations with increased risks for CNS embryonal tumors and for "other" leukemias. Further study should be performed to evaluate potentially lower risk for Hodgkin lymphoma and malignant bone tumors in counties with rapidly growing population.
A Biodosimeter for Multiparametric Determination of Radiation Dose, Radiation Quality, and Radiation Risk

NASA Technical Reports Server (NTRS)

Richmond, Robert; Cruz, Angela; Jansen, Heather; Bors, Karen

2003-01-01

Predicting risk of human cancer following exposure of an individual or a population to ionizing radiation is challenging. To an approximation, this is because uncertainties of uniform absorption of dose and the uniform processing of dose-related damage at the cellular level within a complex set of biological variables degrade the confidence of predicting the delayed expression of cancer as a relatively rare event. Cellular biodosimeters that simultaneously report: 1) the quantity of absorbed dose after exposure to ionizing radiation, 2) the quality of radiation delivering that dose, and 3) the risk of developing cancer by the cells absorbing that dose would therefore be useful. An approach to such a multiparametric biodosimeter will be reported. This is the demonstration of a dose responsive field effect of enhanced expression of keratin 18 (K18) in cultures of human mammary epithelial cells irradiated with cesium-1 37 gamma-rays. Dose response of enhanced K18 expression was experimentally extended over a range of 30 to 90 cGy for cells evaluated at mid-log phase. K18 has been reported to be a marker for tumor staging and for apoptosis, and thereby serves as an example of a potential marker for cancer risk, where the reality of such predictive value would require additional experimental development. Since observed radiogenic increase in expression of K18 is a field effect, ie., chronically present in all cells of the irradiated population, it may be hypothesized that K18 expression in specific cells absorbing particulate irradiation, such as the high-LET-producing atomic nuclei of space radiation, will report on both the single-cell distributions of those particles amongst cells within the exposed population, and that the relatively high dose per cell delivered by densely ionizing tracks of those intersecting particles will lead to cell-specific high-expression levels of K18, thereby providing analytical end points that may be used to resolve both the quantity and the quality of the radiation dose absorbed by individual cells. The principal value of this reported potential multiparametric cellular biodosimeter is suggested to be that it justifies a search for similar but more robust radiogenic assays. That is, K18 is only one radiation dose-sensitive expressed protein, whereas analytical techniques of genomics and proteomics can be used to simultaneously analyze multiple gene and protein expressions resulting from radiation-dose absorption. The potential usefulness of multiparametric cellular biodosimeters will be best realized from quantitatively profiling these multiple markers using these modern techniques.
Fibroblast Growth Factor 1 (FGFR1) Modulation Regulates Repair Capacity of Oligodendrocyte Progenitor Cells Following Chronic Demyelination

PubMed Central

Zhou, Yong-Xing; Pannu, Ravinder; Le, Tuan Q.; Armstrong, Regina C.

2011-01-01

The adult mammalian brain contains multiple populations of endogenous progenitor cell types. However, following CNS trauma or disease, the regenerative capacity of progenitor populations is typically insufficient and may actually be limited by non-permissive or inhibitory signals in the damaged parenchyma. Remyelination is the most effective and simplest regenerative process in the adult CNS yet is still insufficient following repeated or chronic demyelination. Our previous in vitro studies demonstrated that fibroblast growth factor receptor 1 (FGFR1) signaling inhibited oligodendrocyte progenitor (OP) differentiation into mature oligodendrocytes. Therefore, we questioned whether FGFR1 signaling may inhibit the capacity of OP cells to generate oligodendrocytes in a demyelinating disease model and whether genetically reducing FGFR1 signaling in oligodendrocyte lineage cells could enhance the capacity for remyelination. FGFR1 was found to be upregulated in the corpus callosum during cuprizone mediated demyelination and expressed on OP cells just prior to remyelination. Plp/CreERT:Fgfr1fl/flmice were administered tamoxifen to induce conditional Fgfr1 deletion in oligodendrocyte lineage cells. Tamoxifen administration during chronic demyelination resulted in reduced FGFR1 expression in OP cells. OP proliferation and population size were not altered one week after tamoxifen treatment. Tamoxifen was then administered during chronic demyelination and mice were given a six week recovery period without cuprizone in the chow. After the recovery period, OP numbers were reduced and the number of mature oligodendrocytes was increased, indicating an effect of FGFR1 reduction on OP differentiation. Importantly, tamoxifen administration in Plp/CreERT:Fgfr1fl/fl mice significantly promoted remyelination and axon integrity. These results demonstrate a direct effect of FGFR1 signaling in oligodendrocyte lineage cells as inhibiting the repair capacity of OP cells following chronic demyelination in the adult CNS. PMID:21854849
Diversity of Human and Macaque Airway Immune Cells at Baseline and during Tuberculosis Infection

PubMed Central

Myers, Amy J.; Jarvela, Jessica; Flynn, JoAnne; Rutledge, Tara; Bonfield, Tracey

2016-01-01

Immune cells of the distal airways serve as “first responders” of host immunity to the airborne pathogen Mycobacterium tuberculosis (Mtb). Mtb infection of cynomolgus macaques recapitulates the range of human outcomes from clinically silent latent tuberculosis infection (LTBI) to active tuberculosis of various degrees of severity. To further advance the application of this model to human studies, we compared profiles of bronchoalveolar lavage (BAL) cells of humans and cynomolgus macaques before and after Mtb infection. A simple gating strategy effectively defined BAL T-cell and phagocyte populations in both species. BAL from Mtb-naive humans and macaques showed similar differential cell counts. BAL T cells of macaques were composed of fewer CD4+cells but more CD8+ and CD4+CD8+ double-positive cells than were BAL T cells of humans. The most common mononuclear phagocyte population in BAL of both species displayed coexpression of HLA-DR, CD206, CD11b, and CD11c; however, multiple phagocyte subsets displaying only some of these markers were observed as well. Macaques with LTBI displayed a marked BAL lymphocytosis that was not observed in humans with LTBI. In macaques, the prevalence of specific mononuclear phagocyte subsets in baseline BAL correlated with ultimate outcomes of Mtb infection (i.e., LTBI versus active disease). Overall, these findings demonstrate the comparability of studies of pulmonary immunity to Mtb in humans and macaques. They also indicate a previously undescribed complexity of airway mononuclear phagocyte populations that suggests further lines of investigation relevant to understanding the mechanisms of both protection from and susceptibility to the development of active tuberculosis within the lung. PMID:27509488
CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

PubMed

Sebastian, Nadia T; Zaikos, Thomas D; Terry, Valeri; Taschuk, Frances; McNamara, Lucy A; Onafuwa-Nuga, Adewunmi; Yucha, Ryan; Signer, Robert A J; Riddell, James; Bixby, Dale; Markowitz, Norman; Morrison, Sean J; Collins, Kathleen L

2017-07-01

Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.
Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

PubMed

Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M; Michor, Franziska; Fan, Rong; Pan, Xinghua

2017-06-02

Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

PubMed

Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

2014-08-01

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
Stem Cells as Drug Delivery Methods: Application of Stem Cell Secretome for Regeneration

PubMed Central

Tran, Christine; Damaser, Margot S.

2014-01-01

Mesenchymal stem cells (MSC) are a unique cell population defined by their ability to indefinitely self-renew, differentiate into multiple cell lineages, and form clonal cell populations. It was originally thought that this ability for broad plasticity defined the therapeutic potential of MSCs. However, an expanding body of recent literature has brought growing awareness to the remarkable array of bioactive molecules produced by stem cells. This protein milieu or “secretome” comprises a diverse host of cytokines, chemokines, angiogenic factors, and growth factors. The autocrine/paracrine role of these molecules is being increasingly recognized as key to the regulation of many physiological processes including directing endogenous and progenitor cells to sites of injury as well as mediating apoptosis, scarring, and tissue revascularization. In fact, the immunomodulatory and paracrine role of these molecules may predominantly account for the therapeutic effects of MSCs given that many in vitro and in vivo studies have demonstrated limited stem cell engraftment at the site of injury. While the study of such a vast protein array remains challenging, technological advances in the field of proteomics have greatly facilitated our ability to analyze and characterize the stem cell secretome. Thus, stem cells can be considered as tunable pharmacological storehouses useful for combinatorial drug manufacture and delivery. As a cell-free option for regenerative medicine therapies, stem cell secretome has shown great potential in a variety of clinical applications including the restoration of function in cardiovascular, neurodegenerative, oncologic, and genitourinary pathologies. PMID:25451858
The Use of Human Wharton's Jelly Cells for Cochlear Tissue Engineering.

PubMed

Mellott, Adam J; Detamore, Michael S; Staecker, Hinrich

2016-01-01

Tissue engineering focuses on three primary components: stem cells, biomaterials, and growth factors. Together, the combination of these components is used to regrow and repair damaged tissues that normally do not regenerate easily on their own. Much attention has been focused on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), due to their broad differentiation potential. However, ESCs and iPSCs require very detailed protocols to differentiate into target tissues, which are not always successful. Furthermore, procurement of ESCs is considered ethically controversial in some regions and procurement of iPSCs requires laborious transformation of adult tissues and characterization. However, mesenchymal stem cells are an adult stem cell population that are not ethically controversial and are readily available for procurement. Furthermore, mesenchymal stem cells exhibit the ability to differentiate into a variety of cell types arising from the mesoderm. In particular, human Wharton's jelly cells (hWJCs) are mesenchymal-type stem cells found in umbilical cords that possess remarkable differentiation potential. hWJCs are a highly desirable stem cell population due to their abundance in supply, high proliferation rates, and ability to differentiate into multiple cell types arising from all three germ layers. hWJCs are used to generate several neurological phenotypes arising from the ectoderm and are considered for engineering mechanosensory hair cells found in the auditory complex. Here, we report the methods for isolating hWJCs from human umbilical cords and non-virally transfected for use in cochlear tissue engineering studies.
Characterization and Separation of Cancer Cells with a Wicking Fiber Device.

PubMed

Tabbaa, Suzanne M; Sharp, Julia L; Burg, Karen J L

2017-12-01

Current cancer diagnostic methods lack the ability to quickly, simply, efficiently, and inexpensively screen cancer cells from a mixed population of cancer and normal cells. Methods based on biomarkers are unreliable due to complexity of cancer cells, plasticity of markers, and lack of common tumorigenic markers. Diagnostics are time intensive, require multiple tests, and provide limited information. In this study, we developed a novel wicking fiber device that separates cancer and normal cell types. To the best of our knowledge, no previous work has used vertical wicking of cells through fibers to identify and isolate cancer cells. The device separated mouse mammary tumor cells from a cellular mixture containing normal mouse mammary cells. Further investigation showed the device separated and isolated human cancer cells from a heterogeneous mixture of normal and cancerous human cells. We report a simple, inexpensive, and rapid technique that has potential to identify and isolate cancer cells from large volumes of liquid samples that can be translated to on-site clinic diagnosis.
NG2-expressing cells as oligodendrocyte progenitors in the normal and demyelinated adult central nervous system

PubMed Central

Polito, Annabella; Reynolds, Richard

2005-01-01

The mammalian adult central nervous system (CNS) is known to respond rapidly to demyelinating insults by regenerating oligodendrocytes for remyelination from a dividing precursor population. A widespread population of cells exists within the adult CNS that is thought to belong to the oligodendrocyte lineage, but which do not express proteins characteristic of mature myelinating oligodendrocytes, such as myelin basic protein (MBP) and 2,3-cyclic nucleotide 3-phosphodiesterase (CNP). Instead, these cells have phenotypic characteristics of a more immature stage of the oligodendrocyte lineage. They express the NG2 chondroitin sulphate proteoglycan, in addition to O4 and the platelet-derived growth factor α-receptor, all widely accepted as markers for oligodendrocyte progenitor cells (OPCs) throughout development. However, NG2+ cells residing in the adult CNS do not resemble embryonic or neonatal NG2+ cells in terms of their morphology or proliferation characteristics, but instead represent a unique type of glial cell that has the ability to react rapidly to CNS damage. In this review, we present the evidence that adult NG2+ cells are part of the oligodendrocyte lineage and are capable of giving rise to new oligodendrocytes under both normal and demyelinating conditions. We also review the literature that these cells may have multiple functional roles within the adult CNS, notwithstanding their primary role as OPCs. PMID:16367798
Risk of multiple myeloma following medication use and medical conditions: a case-control study in Connecticut women.

PubMed

Landgren, Ola; Zhang, Yawei; Zahm, Sheila Hoar; Inskip, Peter; Zheng, Tongzhang; Baris, Dalsu

2006-12-01

Certain commonly used drugs and medical conditions characterized by chronic immune dysfunction and/or antigen stimulation have been suggested to affect important pathways in multiple myeloma tumor cell growth and survival. We conducted a population-based case-control study to investigate the role of medical history in the etiology of multiple myeloma among Connecticut women. A total of 179 incident multiple myeloma cases (21-84 years, diagnosed 1996-2002) and 691 population-based controls was included in this study. Information on medical conditions, medications, and medical radiation was obtained by in-person interviews. We calculated odds ratios (OR) as measures of relative risks using logistic regression models. A reduced multiple myeloma risk was found among women who had used antilipid statin therapy [OR, 0.4; 95% confidence interval (95% CI), 0.2-0.8] or estrogen replacement therapy (OR, 0.6; 95% CI, 0.4-0.99) or who had a medical history of allergy (OR, 0.4; 95% CI, 0.3-0.7), scarlet fever (OR, 0.5; 95% CI, 0.2-0.9), or bursitis (OR, 0.4; 95% CI, 0.2-0.7). An increased risk of multiple myeloma was found among women who used prednisone (OR, 5.1; 95% CI, 1.8-14.4), insulin (OR, 3.1; 95% CI, 1.1-9.0), or gout medication (OR, 6.7; 95% CI, 1.2-38.0). If our results are confirmed, mechanistic studies examining how prior use of insulin, prednisone, and, perhaps, gout medication might promote increased occurrence of multiple myeloma and how antilipid statins, estrogen replacement therapy, and certain medical conditions might protect against multiple myeloma may provide insights to the as yet unknown etiology of multiple myeloma.
Reduced cellularity of bone marrow in multiple sclerosis with decreased MSC expansion potential and premature ageing in vitro.

PubMed

Redondo, Juliana; Sarkar, Pamela; Kemp, Kevin; Virgo, Paul F; Pawade, Joya; Norton, Aimie; Emery, David C; Guttridge, Martin G; Marks, David I; Wilkins, Alastair; Scolding, Neil J; Rice, Claire M

2017-05-01

Autologous bone-marrow-derived cells are currently employed in clinical studies of cell-based therapy in multiple sclerosis (MS) although the bone marrow microenvironment and marrow-derived cells isolated from patients with MS have not been extensively characterised. To examine the bone marrow microenvironment and assess the proliferative potential of multipotent mesenchymal stromal cells (MSCs) in progressive MS. Comparative phenotypic analysis of bone marrow and marrow-derived MSCs isolated from patients with progressive MS and control subjects was undertaken. In MS marrow, there was an interstitial infiltrate of inflammatory cells with lymphoid (predominantly T-cell) nodules although total cellularity was reduced. Controlling for age, MSCs isolated from patients with MS had reduced in vitro expansion potential as determined by population doubling time, colony-forming unit assay, and expression of β-galactosidase. MS MSCs expressed reduced levels of Stro-1 and displayed accelerated shortening of telomere terminal restriction fragments (TRF) in vitro. Our results are consistent with reduced proliferative capacity and ex vivo premature ageing of bone-marrow-derived cells, particularly MSCs, in MS. They have significant implication for MSC-based therapies for MS and suggest that accelerated cellular ageing and senescence may contribute to the pathophysiology of progressive MS. The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding for this study was provided by the Medical Research Council, UK (grant no. MR/K004166/1). The ACTiMuS study is sup-ported by the Silverman Family Foundation, Multiple Sclerosis Trust, Rosetree’s Trust, Catholic Bishops of England and Wales and Friends of Frenchay and SIAMMS-II by the Sir Halley Stewart Trust. C.M.R., P.S., and K.K. received support from the Burden Neurological Institute.
Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

PubMed Central

Li, Weizhe; Germain, Ronald N.

2017-01-01

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques. PMID:28808033
Bayesian multivariate Poisson abundance models for T-cell receptor data.

PubMed

Greene, Joshua; Birtwistle, Marc R; Ignatowicz, Leszek; Rempala, Grzegorz A

2013-06-07

A major feature of an adaptive immune system is its ability to generate B- and T-cell clones capable of recognizing and neutralizing specific antigens. These clones recognize antigens with the help of the surface molecules, called antigen receptors, acquired individually during the clonal development process. In order to ensure a response to a broad range of antigens, the number of different receptor molecules is extremely large, resulting in a huge clonal diversity of both B- and T-cell receptor populations and making their experimental comparisons statistically challenging. To facilitate such comparisons, we propose a flexible parametric model of multivariate count data and illustrate its use in a simultaneous analysis of multiple antigen receptor populations derived from mammalian T-cells. The model relies on a representation of the observed receptor counts as a multivariate Poisson abundance mixture (m PAM). A Bayesian parameter fitting procedure is proposed, based on the complete posterior likelihood, rather than the conditional one used typically in similar settings. The new procedure is shown to be considerably more efficient than its conditional counterpart (as measured by the Fisher information) in the regions of m PAM parameter space relevant to model T-cell data. Copyright © 2013 Elsevier Ltd. All rights reserved.
Cellular therapies for heart disease: unveiling the ethical and public policy challenges.

PubMed

Raval, Amish N; Kamp, Timothy J; Hogle, Linda F

2008-10-01

Cellular therapies have emerged as a potential revolutionary treatment for cardiovascular disease. Promising preclinical results have resulted in a flurry of basic research activity and spawned multiple clinical trials worldwide. However, the optimal cell type and delivery mode have not been determined for target patient populations. Nor have the mechanisms of benefit for the range of cellular interventions been clearly defined. Experiences to date have unveiled a myriad of ethical and public policy challenges which will affect the way researchers and clinicians make decisions for both basic and clinical research. Stem cells derived from embryos are at the forefront of the ethical and political debate, raising issues of which derivation methods are morally and socially permissible to pursue, as much as which are technically feasible. Adult stem cells are less controversial; however, important challenges exist in determining study design, cell processing, delivery mode, and target patient population. Pathways to successful commercialization and hence broad accessibility of cellular therapies for heart disease are only beginning to be explored. Comprehensive, multi-disciplinary and collaborative networks involving basic researchers, clinicians, regulatory officials and policymakers are required to share information, develop research, regulatory and policy standards and enable rational and ethical cell-based treatment approaches.
Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

PubMed

Tinkum, Kelsey L; Stemler, Kristina M; White, Lynn S; Loza, Andrew J; Jeter-Jones, Sabrina; Michalski, Basia M; Kuzmicki, Catherine; Pless, Robert; Stappenbeck, Thaddeus S; Piwnica-Worms, David; Piwnica-Worms, Helen

2015-12-22

Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

Control of cell fate by the formation of an architecturally complex bacterial community.

PubMed

Vlamakis, Hera; Aguilar, Claudio; Losick, Richard; Kolter, Roberto

2008-04-01

Bacteria form architecturally complex communities known as biofilms in which cells are held together by an extracellular matrix. Biofilms harbor multiple cell types, and it has been proposed that within biofilms individual cells follow different developmental pathways, resulting in heterogeneous populations. Here we demonstrate cellular differentiation within biofilms of the spore-forming bacterium Bacillus subtilis, and present evidence that formation of the biofilm governs differentiation. We show that motile, matrix-producing, and sporulating cells localize to distinct regions within the biofilm, and that the localization and percentage of each cell type is dynamic throughout development of the community. Importantly, mutants that do not produce extracellular matrix form unstructured biofilms that are deficient in sporulation. We propose that sporulation is a culminating feature of biofilm formation, and that spore formation is coupled to the formation of an architecturally complex community of cells.
Control of cell fate by the formation of an architecturally complex bacterial community

PubMed Central

Vlamakis, Hera; Aguilar, Claudio; Losick, Richard; Kolter, Roberto

2008-01-01

Bacteria form architecturally complex communities known as biofilms in which cells are held together by an extracellular matrix. Biofilms harbor multiple cell types, and it has been proposed that within biofilms individual cells follow different developmental pathways, resulting in heterogeneous populations. Here we demonstrate cellular differentiation within biofilms of the spore-forming bacterium Bacillus subtilis, and present evidence that formation of the biofilm governs differentiation. We show that motile, matrix-producing, and sporulating cells localize to distinct regions within the biofilm, and that the localization and percentage of each cell type is dynamic throughout development of the community. Importantly, mutants that do not produce extracellular matrix form unstructured biofilms that are deficient in sporulation. We propose that sporulation is a culminating feature of biofilm formation, and that spore formation is coupled to the formation of an architecturally complex community of cells. PMID:18381896
Detecting and characterizing genomic signatures of positive selection in global populations.

PubMed

Liu, Xuanyao; Ong, Rick Twee-Hee; Pillai, Esakimuthu Nisha; Elzein, Abier M; Small, Kerrin S; Clark, Taane G; Kwiatkowski, Dominic P; Teo, Yik-Ying

2013-06-06

Natural selection is a significant force that shapes the architecture of the human genome and introduces diversity across global populations. The question of whether advantageous mutations have arisen in the human genome as a result of single or multiple mutation events remains unanswered except for the fact that there exist a handful of genes such as those that confer lactase persistence, affect skin pigmentation, or cause sickle cell anemia. We have developed a long-range-haplotype method for identifying genomic signatures of positive selection to complement existing methods, such as the integrated haplotype score (iHS) or cross-population extended haplotype homozygosity (XP-EHH), for locating signals across the entire allele frequency spectrum. Our method also locates the founder haplotypes that carry the advantageous variants and infers their corresponding population frequencies. This presents an opportunity to systematically interrogate the whole human genome whether a selection signal shared across different populations is the consequence of a single mutation process followed subsequently by gene flow between populations or of convergent evolution due to the occurrence of multiple independent mutation events either at the same variant or within the same gene. The application of our method to data from 14 populations across the world revealed that positive-selection events tend to cluster in populations of the same ancestry. Comparing the founder haplotypes for events that are present across different populations revealed that convergent evolution is a rare occurrence and that the majority of shared signals stem from the same evolutionary event. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Exploring the cellular basis of human disease through a large-scale mapping of deleterious genes to cell types.

PubMed

Cornish, Alex J; Filippis, Ioannis; David, Alessia; Sternberg, Michael J E

2015-09-01

Each cell type found within the human body performs a diverse and unique set of functions, the disruption of which can lead to disease. However, there currently exists no systematic mapping between cell types and the diseases they can cause. In this study, we integrate protein-protein interaction data with high-quality cell-type-specific gene expression data from the FANTOM5 project to build the largest collection of cell-type-specific interactomes created to date. We develop a novel method, called gene set compactness (GSC), that contrasts the relative positions of disease-associated genes across 73 cell-type-specific interactomes to map genes associated with 196 diseases to the cell types they affect. We conduct text-mining of the PubMed database to produce an independent resource of disease-associated cell types, which we use to validate our method. The GSC method successfully identifies known disease-cell-type associations, as well as highlighting associations that warrant further study. This includes mast cells and multiple sclerosis, a cell population currently being targeted in a multiple sclerosis phase 2 clinical trial. Furthermore, we build a cell-type-based diseasome using the cell types identified as manifesting each disease, offering insight into diseases linked through etiology. The data set produced in this study represents the first large-scale mapping of diseases to the cell types in which they are manifested and will therefore be useful in the study of disease systems. Overall, we demonstrate that our approach links disease-associated genes to the phenotypes they produce, a key goal within systems medicine.
The eukaryotic cell originated in the integration and redistribution of hyperstructures from communities of prokaryotic cells based on molecular complementarity.

PubMed

Norris, Vic; Root-Bernstein, Robert

2009-06-04

In the "ecosystems-first" approach to the origins of life, networks of non-covalent assemblies of molecules (composomes), rather than individual protocells, evolved under the constraints of molecular complementarity. Composomes evolved into the hyperstructures of modern bacteria. We extend the ecosystems-first approach to explain the origin of eukaryotic cells through the integration of mixed populations of bacteria. We suggest that mutualism and symbiosis resulted in cellular mergers entailing the loss of redundant hyperstructures, the uncoupling of transcription and translation, and the emergence of introns and multiple chromosomes. Molecular complementarity also facilitated integration of bacterial hyperstructures to perform cytoskeletal and movement functions.
Left atrial appendages from adult hearts contain a reservoir of diverse cardiac progenitor cells.

PubMed

Leinonen, Jussi V; Emanuelov, Avishag K; Platt, Yardanna; Helman, Yaron; Feinberg, Yael; Lotan, Chaim; Beeri, Ronen

2013-01-01

There is strong evidence supporting the claim that endogenous cardiac progenitor cells (CPCs) are key players in cardiac regeneration, but the anatomic source and phenotype of the master cardiac progenitors remains uncertain. Our aim was to investigate the different cardiac stem cell populations in the left atrial appendage (LAA) and their fates. We investigated the CPC content and profile of adult murine LAAs using immunohistochemistry and flow cytometry. We demonstrate that the LAA contains a large number of CPCs relative to other areas of the heart, representing over 20% of the total cell number. We grew two distinct CPC populations from the LAA by varying the degree of proteolysis. These differed by their histological location, surface marker profiles and growth dynamics. Specifically, CD45(pos) cells grew with milder proteolysis, while CD45(neg) cells grew mainly with more intense proteolysis. Both cell types could be induced to differentiate into cells with cardiomyocyte markers and organelles, albeit by different protocols. Many CD45(pos) cells expressed CD45 initially and rapidly lost its expression while differentiating. Our results demonstrate that the left atrial appendage plays a role as a reservoir of multiple types of progenitor cells in murine adult hearts. Two different types of CPCs were isolated, differing in their epicardial-myocardial localization. Considering studies demonstrating layer-specific origins of different cardiac progenitor cells, our findings may shed light on possible pathways to study and utilize the diversity of endogenous progenitor cells in the adult heart.
The Evolution of the Stem Cell Theory for Heart Failure

PubMed Central

Silvestre, Jean-Sébastien; Menasché, Philippe

2015-01-01

Various stem cell-based approaches for cardiac repair have achieved encouraging results in animal experiments, often leading to their rapid proceeding to clinical testing. However, freewheeling evolutionary developments of the stem cell theory might lead to dystopian scenarios where heterogeneous sources of therapeutic cells could promote mixed clinical outcomes in un-stratified patient populations. This review focuses on the lessons that should be learnt from the first generation of stem cell-based strategies and emphasizes the absolute requirement to better understand the basic mechanisms of stem cell biology and cardiogenesis. We will also discuss about the unexpected “big bang” in the stem cell theory, “blasting” the therapeutic cells to their unchallenged ability to release paracrine factors such as extracellular membrane vesicles. Paradoxically, the natural evolution of the stem cell theory for cardiac regeneration may end with the development of cell-free strategies with multiple cellular targets including cardiomyocytes but also other infiltrating or resident cardiac cells. PMID:26844266
Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

NASA Astrophysics Data System (ADS)

Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

2016-06-01

Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10-100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.
Gorlin syndrome and bilateral ovarian fibroma.

PubMed

Pirschner, Fernanda; Bastos, Pollyana Marçal; Contarato, George Luiz; Bimbato, Anna Carolina Bon Lima; Filho, Antônio Chambô

2012-01-01

Gorlin syndrome (GS), also known as nevoid basal cell carcinoma syndrome (NBCCS), is a rare hereditary, autosomal dominant disease that affects various systems. Its prevalence is estimated at 1/57,000 to 1/256,000 of the population. It is characterized by basal cell carcinomas, multiple odontogenic keratocysts, skeletal abnormalities and ovarian fibroma, among other disorders. To report the case of a young patient with Gorlin syndrome and bilateral ovarian fibroma. A 20-year old patient with Gorlin syndrome presented with facial asymmetry, broad nasal root, dental abnormalities, micrognathism, convergent strabismus, multiple pigmented lesions on the trunk and face, pectus excavatum, kyphoscoliosis and a palpable mass in the abdomen occupying the entire pelvic region. Gorlin-Goltz syndrome is a hereditary pathology that includes numerous clinical manifestations. Diagnosis is clinical and genetic confirmation is unnecessary. Copyright © 2012 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.
Robust measurement of telomere length in single cells

PubMed Central

Wang, Fang; Pan, Xinghua; Kalmbach, Keri; Seth-Smith, Michelle L.; Ye, Xiaoying; Antumes, Danielle M. F.; Yin, Yu; Liu, Lin; Keefe, David L.; Weissman, Sherman M.

2013-01-01

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases. PMID:23661059
Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations.

PubMed

Bull, Marta; Learn, Gerald; Genowati, Indira; McKernan, Jennifer; Hitti, Jane; Lockhart, David; Tapia, Kenneth; Holte, Sarah; Dragavon, Joan; Coombs, Robert; Mullins, James; Frenkel, Lisa

2009-09-22

Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons. Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by >/=2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage. In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.
PKK deficiency in B cells prevents lupus development in Sle lupus mice

PubMed Central

Oleksyn, D.; Zhao, J.; Vosoughi, A.; Zhao, JC.; Misra, R; Pentland, AP; Ryan, D.; Anolik, J.; Ritchlin, C.; Looney, J.; Anandarajah, AP.; Schwartz, G.; Calvi, LM; Georger, M; Mohan, C.; Sanz, I.; Chen, L

2018-01-01

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies that can result in damage to multiple organs. It is well documented that B cells play a critical role in the development of the disease. We previously showed that protein kinase C associated kinase (PKK) is required for B1 cell development as well as for the survival of recirculating mature B cells and B- lymphoma cells. Here, we investigated the role of PKK in lupus development in a lupus mouse model. We demonstrate that the conditional deletion of PKK in B cells prevents lupus development in Sle1Sle3 mice. The loss of PKK in Sle mice resulted in the amelioration of multiple classical lupus-associated phenotypes and histologic features of lupus nephritis, including marked reduction in the levels of serum autoantibodies, proteinuria, spleen size, peritoneal B-1 cell population and the number of activated CD4 T cells. In addition, the abundance of autoreactive plasma cells normally seen in Sle lupus mice was also significantly decreased in the PKK-deficient Sle mice. Sle B cells deficient in PKK display defective proliferation responses to BCR and LPS stimulation. Consistently, B cell receptor-mediated NF-κB activation, which is required for the survival of activated B cells, was impaired in the PKK-deficient B cells. Taken together, our work uncovers a critical role of PKK in lupus development and suggests that targeting the PKK-mediated pathway may represent a promising therapeutic strategy for lupus treatment. PMID:28274793
Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

PubMed

Eikawa, Shingo; Kakimi, Kazuhiro; Isobe, Midori; Kuzushima, Kiyotaka; Luescher, Immanuel; Ohue, Yoshihiro; Ikeuchi, Kazuhiro; Uenaka, Akiko; Nishikawa, Hiroyoshi; Udono, Heiichiro; Oka, Mikio; Nakayama, Eiichi

2013-01-15

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response. Copyright © 2012 UICC.
Model for the computation of self-motion in biological systems

NASA Technical Reports Server (NTRS)

Perrone, John A.

1992-01-01

A technique is presented by which direction- and speed-tuned cells, such as those commonly found in the middle temporal region of the primate brain, can be utilized to analyze the patterns of retinal image motion that are generated during observer movement through the environment. The developed model determines heading by finding the peak response in a population of detectors or neurons each tuned to a particular heading direction. It is suggested that a complex interaction of multiple cell networks is required for the solution of the self-motion problem in the primate brain.
Altered intestinal epithelium-associated lymphocyte repertoires and function in ApcMin/+ mice.

PubMed

Marsh, Lorraine; Coletta, P Louise; Hull, Mark A; Selby, Peter J; Carding, Simon R

2012-01-01

ApcMin/+ mice spontaneously develop multiple intestinal adenomas along the length of the small intestine and colon. Currently little is known about the role of the immune system in regulating intestinal tumorigenesis in these animals. This study characterised small intestinal intraepithelial lympho-- cyte (IEL) populations in C56BL/6J ApcMin/+ mice and wild-type (Apc+/+) mice. We also determined the effect that T cells expressing either γδ or αβ encoded T cell receptors (TcR) exert on intestinal tumorigenesis. ApcMin/+ mice had significantly lower numbers of CD3+ IELs compared with Apc+/+ littermates and displayed reduced cytotoxicity against tumour target cells. Further analysis of IEL cytotoxicity revealed differences in the cytotoxic pathways utilised by IELs in ApcMin/+ and Apc+/+ mice with ApcMin/+ IELs displaying an absence of perforin/granzyme-mediated killing and increased levels of Fas-FasL-mediated cytotoxicity compared with wild-type IELs. Analysis of ApcMin/+ mice crossed with αβ T-cell deficient (TcRβ-/-) or γδ T-cell deficient (TcRδ-/-) mice on the same genetic background revealed decreased tumour multiplicity in the absence of both αβ and γδ T-cells. This study demonstrates that altered T-cell subsets play important roles in promoting tumorigenesis in ApcMin/+ mice and forms the basis for future mechanistic studies.
Multiple roles of tumor necrosis factor-alpha in fracture healing.

PubMed

Karnes, Jonathan M; Daffner, Scott D; Watkins, Colleen M

2015-09-01

This review presents a summary of basic science evidence examining the influence of tumor necrosis factor-alpha (TNF-α) on secondary fracture healing. Multiple studies suggest that TNF-α, in combination with the host reservoir of peri-fracture mesenchymal stem cells, is a main determinant in the success of bone healing. Disease states associated with poor bone healing commonly have inappropriate TNF-α responses, which likely contributes to the higher incidence of delayed and nonunions in these patient populations. Appreciation of TNF-α in fracture healing may lead to new therapies to augment recovery and reduce the incidence of complications. Copyright © 2015 Elsevier Inc. All rights reserved.
Multiple Modes of Cell Death Discovered in a Prokaryotic (Cyanobacterial) Endosymbiont

PubMed Central

Zheng, Weiwen; Rasmussen, Ulla; Zheng, Siping; Bao, Xiaodong; Chen, Bin; Gao, Yuan; Guan, Xiong; Larsson, John; Bergman, Birgitta

2013-01-01

Programmed cell death (PCD) is a genetically-based cell death mechanism with vital roles in eukaryotes. Although there is limited consensus on similar death mode programs in prokaryotes, emerging evidence suggest that PCD events are operative. Here we present cell death events in a cyanobacterium living endophytically in the fern Azolla microphylla, suggestive of PCD. This symbiosis is characterized by some unique traits such as a synchronized development, a vertical transfer of the cyanobacterium between plant generations, and a highly eroding cyanobacterial genome. A combination of methods was used to identify cell death modes in the cyanobacterium. Light- and electron microscopy analyses showed that the proportion of cells undergoing cell death peaked at 53.6% (average 20%) of the total cell population, depending on the cell type and host developmental stage. Biochemical markers used for early and late programmed cell death events related to apoptosis (Annexin V-EGFP and TUNEL staining assays), together with visualization of cytoskeleton alterations (FITC-phalloidin staining), showed that all cyanobacterial cell categories were affected by cell death. Transmission electron microscopy revealed four modes of cell death: apoptotic-like, autophagic-like, necrotic-like and autolytic-like. Abiotic stresses further enhanced cell death in a dose and time dependent manner. The data also suggest that dynamic changes in the peptidoglycan cell wall layer and in the cytoskeleton distribution patterns may act as markers for the various cell death modes. The presence of a metacaspase homolog (domain p20) further suggests that the death modes are genetically programmed. It is therefore concluded that multiple, likely genetically programmed, cell death modes exist in cyanobacteria, a finding that may be connected with the evolution of cell death in the plant kingdom. PMID:23822984
Fast and Label-Free Isolation of Circulating Tumor Cells from Blood: From a Research Microfluidic Platform to an Automated Fluidic Instrument, VTX-1 Liquid Biopsy System.

PubMed

Lemaire, Clementine A; Liu, Sean Z; Wilkerson, Charles L; Ramani, Vishnu C; Barzanian, Nasim A; Huang, Kuo-Wei; Che, James; Chiu, Michael W; Vuppalapaty, Meghah; Dimmick, Adam M; Carlo, Dino Di; Kochersperger, Michael L; Crouse, Steve C; Jeffrey, Stefanie S; Englert, Robert F; Hengstler, Stephan; Renier, Corinne; Sollier-Christen, Elodie

2018-02-01

Tumor tissue biopsies are invasive, costly, and collect a limited cell population not completely reflective of patient cancer cell diversity. Circulating tumor cells (CTCs) can be isolated from a simple blood draw and may be representative of the diverse biology from multiple tumor sites. The VTX-1 Liquid Biopsy System was designed to automate the isolation of clinically relevant CTC populations, making the CTCs available for easy analysis. We present here the transition from a cutting-edge microfluidic innovation in the lab to a commercial, automated system for isolating CTCs directly from whole blood. As the technology evolved into a commercial system, flexible polydimethylsiloxane microfluidic chips were replaced by rigid poly(methyl methacrylate) chips for a 2.2-fold increase in cell recovery. Automating the fluidic processing with the VTX-1 further improved cancer cell recovery by nearly 1.4-fold, with a 2.8-fold decrease in contaminating white blood cells and overall improved reproducibility. Two isolation protocols were optimized that favor either the cancer cell recovery (up to 71.6% recovery) or sample purity (≤100 white blood cells/mL). The VTX-1's performance was further tested with three different spiked breast or lung cancer cell lines, with 69.0% to 79.5% cell recovery. Finally, several cancer research applications are presented using the commercial VTX-1 system.
Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization

PubMed Central

Co, Mary Dawn T; Kilpatrick, Elizabeth D; Rothman, Alan L

2009-01-01

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8+ T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-γ (IFN-γ) responses of almost 1% of CD8+ T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7–9 days, before detectable IFN-γ-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA+ CCR7− CD62L−) phenotype. The T-cell receptor Vβ analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness. PMID:19740333
Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization.

PubMed

Co, Mary Dawn T; Kilpatrick, Elizabeth D; Rothman, Alan L

2009-09-01

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8(+) T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-gamma (IFN-gamma) responses of almost 1% of CD8(+) T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7-9 days, before detectable IFN-gamma-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA(+) CCR7(-) CD62L(-)) phenotype. The T-cell receptor Vbeta analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness.

Synthetic biology for microbial heavy metal biosensors.

PubMed

Kim, Hyun Ju; Jeong, Haeyoung; Lee, Sang Jun

2018-02-01

Using recombinant DNA technology, various whole-cell biosensors have been developed for detection of environmental pollutants, including heavy metal ions. Whole-cell biosensors have several advantages: easy and inexpensive cultivation, multiple assays, and no requirement of any special techniques for analysis. In the era of synthetic biology, cutting-edge DNA sequencing and gene synthesis technologies have accelerated the development of cell-based biosensors. Here, we summarize current technological advances in whole-cell heavy metal biosensors, including the synthetic biological components (bioparts), sensing and reporter modules, genetic circuits, and chassis cells. We discuss several opportunities for improvement of synthetic cell-based biosensors. First, new functional modules must be discovered in genome databases, and this knowledge must be used to upgrade specific bioparts through molecular engineering. Second, modules must be assembled into functional biosystems in chassis cells. Third, heterogeneity of individual cells in the microbial population must be eliminated. In the perspectives, the development of whole-cell biosensors is also discussed in the aspects of cultivation methods and synthetic cells.
Aurora B kinase inhibition in mitosis: strategies for optimising the use of aurora kinase inhibitors such as AT9283.

PubMed

Curry, Jayne; Angove, Hayley; Fazal, Lynsey; Lyons, John; Reule, Matthias; Thompson, Neil; Wallis, Nicola

2009-06-15

Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.
Mature B-cell neoplasms in Chernobyl clean-up workers of 1986-1987: summary of cytomorphological and immunocytochemical study in 25 years after Chernobyl accident.

PubMed

Gluzman, D F; Sklyarenko, L M; Nadgornaya, V A; Zavelevich, M P

2011-03-01

The data on the verified cases of mature B-cell neoplasms (chronic lymphocytic leukemia - CLL, B-prolymphocytic leukemia, non-Hodgkin's lymphoma in leukemization phase and multiple myeloma - MM; 146 cases in total) in the consecutive group of Ukrainian clean-up workers within 10-25 years after Chernobyl accident are summarized. B-cell neoplasms represent the most prevalent group among all diagnosed neoplasms of hematopoietic and lymphoid tissues in clean-up worker patients under study (49.4%). MM percentage in the patients of Chernobyl clean-up worker group turned out to be significantly higher than in the patients of the general populations studied at the same period. While the percentage of B-CLL is similar in clean-up worker patients and patients of general population, the trend towards younger age of patients with mature B-cell neoplasms in clean-up worker group is evident. The current concepts on the possible association between mature B-cell neoplasms (mainly B-CLL) and radiation exposure are briefly outlined. Only the precise diagnosis of hematopoietic malignancies combining with large-scale analytical epidemiological studies with careful dose assessment and long-term follow-up may represent the basis for resolving the question whether mature B-cell neoplasms may be radiogenic.
Hapl-o-Mat: open-source software for HLA haplotype frequency estimation from ambiguous and heterogeneous data.

PubMed

Schäfer, Christian; Schmidt, Alexander H; Sauter, Jürgen

2017-05-30

Knowledge of HLA haplotypes is helpful in many settings as disease association studies, population genetics, or hematopoietic stem cell transplantation. Regarding the recruitment of unrelated hematopoietic stem cell donors, HLA haplotype frequencies of specific populations are used to optimize both donor searches for individual patients and strategic donor registry planning. However, the estimation of haplotype frequencies from HLA genotyping data is challenged by the large amount of genotype data, the complex HLA nomenclature, and the heterogeneous and ambiguous nature of typing records. To meet these challenges, we have developed the open-source software Hapl-o-Mat. It estimates haplotype frequencies from population data including an arbitrary number of loci using an expectation-maximization algorithm. Its key features are the processing of different HLA typing resolutions within a given population sample and the handling of ambiguities recorded via multiple allele codes or genotype list strings. Implemented in C++, Hapl-o-Mat facilitates efficient haplotype frequency estimation from large amounts of genotype data. We demonstrate its accuracy and performance on the basis of artificial and real genotype data. Hapl-o-Mat is a versatile and efficient software for HLA haplotype frequency estimation. Its capability of processing various forms of HLA genotype data allows for a straightforward haplotype frequency estimation from typing records usually found in stem cell donor registries.
Multiple Approaches to the Investigation of Cell Assembly in Memory Research-Present and Future.

PubMed

Sakurai, Yoshio; Osako, Yuma; Tanisumi, Yuta; Ishihara, Eriko; Hirokawa, Junya; Manabe, Hiroyuki

2018-01-01

In this review article we focus on research methodologies for detecting the actual activity of cell assemblies, which are populations of functionally connected neurons that encode information in the brain. We introduce and discuss traditional and novel experimental methods and those currently in development and briefly discuss their advantages and disadvantages for the detection of cell-assembly activity. First, we introduce the electrophysiological method, i.e., multineuronal recording, and review former and recent examples of studies showing models of dynamic coding by cell assemblies in behaving rodents and monkeys. We also discuss how the firing correlation of two neurons reflects the firing synchrony among the numerous surrounding neurons that constitute cell assemblies. Second, we review the recent outstanding studies that used the novel method of optogenetics to show causal relationships between cell-assembly activity and behavioral change. Third, we review the most recently developed method of live-cell imaging, which facilitates the simultaneous observation of firings of a large number of neurons in behaving rodents. Currently, all these available methods have both advantages and disadvantages, and no single measurement method can directly and precisely detect the actual activity of cell assemblies. The best strategy is to combine the available methods and utilize each of their advantages with the technique of operant conditioning of multiple-task behaviors in animals and, if necessary, with brain-machine interface technology to verify the accuracy of neural information detected as cell-assembly activity.
Fully reduced HMGB1 accelerates the regeneration of multiple tissues by transitioning stem cells to GAlert.

PubMed

Lee, Geoffrey; Espirito Santo, Ana Isabel; Zwingenberger, Stefan; Cai, Lawrence; Vogl, Thomas; Feldmann, Marc; Horwood, Nicole J; Chan, James K; Nanchahal, Jagdeep

2018-05-08

A major discovery of recent decades has been the existence of stem cells and their potential to repair many, if not most, tissues. With the aging population, many attempts have been made to use exogenous stem cells to promote tissue repair, so far with limited success. An alternative approach, which may be more effective and far less costly, is to promote tissue regeneration by targeting endogenous stem cells. However, ways of enhancing endogenous stem cell function remain poorly defined. Injury leads to the release of danger signals which are known to modulate the immune response, but their role in stem cell-mediated repair in vivo remains to be clarified. Here we show that high mobility group box 1 (HMGB1) is released following fracture in both humans and mice, forms a heterocomplex with CXCL12, and acts via CXCR4 to accelerate skeletal, hematopoietic, and muscle regeneration in vivo. Pretreatment with HMGB1 2 wk before injury also accelerated tissue regeneration, indicating an acquired proregenerative signature. HMGB1 led to sustained increase in cell cycling in vivo, and using Hmgb1 -/- mice we identified the underlying mechanism as the transition of multiple quiescent stem cells from G 0 to G Alert HMGB1 also transitions human stem and progenitor cells to G Alert Therefore, exogenous HMGB1 may benefit patients in many clinical scenarios, including trauma, chemotherapy, and elective surgery. Copyright © 2018 the Author(s). Published by PNAS.
Collective synchronization of self/non-self discrimination in T cell activation, across multiple spatio-temporal scales

NASA Astrophysics Data System (ADS)

Altan-Bonnet, Gregoire

The immune system is a collection of cells whose function is to eradicate pathogenic infections and malignant tumors while protecting healthy tissues. Recent work has delineated key molecular and cellular mechanisms associated with the ability to discriminate self from non-self agents. For example, structural studies have quantified the biophysical characteristics of antigenic molecules (those prone to trigger lymphocyte activation and a subsequent immune response). However, such molecular mechanisms were found to be highly unreliable at the individual cellular level. We will present recent efforts to build experimentally validated computational models of the immune responses at the collective cell level. Such models have become critical to delineate how higher-level integration through nonlinear amplification in signal transduction, dynamic feedback in lymphocyte differentiation and cell-to-cell communication allows the immune system to enforce reliable self/non-self discrimination at the organism level. In particular, we will present recent results demonstrating how T cells tune their antigen discrimination according to cytokine cues, and how competition for cytokine within polyclonal populations of cells shape the repertoire of responding clones. Additionally, we will present recent theoretical and experimental results demonstrating how competition between diffusion and consumption of cytokines determine the range of cell-cell communications within lymphoid organs. Finally, we will discuss how biochemically explicit models, combined with quantitative experimental validation, unravel the relevance of new feedbacks for immune regulations across multiple spatial and temporal scales.
High-content adhesion assay to address limited cell samples†

PubMed Central

Warrick, Jay W.; Young, Edmond W. K.; Schmuck, Eric G.; Saupe, Kurt W.

2013-01-01

Cell adhesion is a broad topic in cell biology that involves physical interactions between cells and other cells or the surrounding extracellular matrix, and is implicated in major research areas including cancer, development, tissue engineering, and regenerative medicine. While current methods have contributed significantly to our understanding of cell adhesion, these methods are unsuitable for tackling many biological questions requiring intermediate numbers of cells (102–105), including small animal biopsies, clinical samples, and rare cell isolates. To overcome this fundamental limitation, we developed a new assay to quantify the adhesion of ~102–103 cells at a time on engineered substrates, and examined the adhesion strength and population heterogeneity via distribution-based modeling. We validated the platform by testing adhesion strength of cancer cells from three different cancer types (breast, prostate, and multiple myeloma) on both IL-1β activated and non-activated endothelial monolayers, and observed significantly increased adhesion for each cancer cell type upon endothelial activation, while identifying and quantifying distinct subpopulations of cell-substrate interactions. We then applied the assay to characterize adhesion of primary bone marrow stromal cells to different cardiac fibroblast-derived matrix substrates to demonstrate the ability to study limited cell populations in the context of cardiac cell-based therapies. Overall, these results demonstrate the sensitivity and robustness of the assay as well as its ability to enable extraction of high content, functional data from limited and potentially rare primary samples. We anticipate this method will enable a new class of biological studies with potential impact in basic and translational research. PMID:23426645
TP53-dependent chromosome instability is associated with transient reductions in telomere length in immortal telomerase-positive cell lines

NASA Technical Reports Server (NTRS)

Schwartz, J. L.; Jordan, R.; Liber, H.; Murnane, J. P.; Evans, H. H.

2001-01-01

Telomere shortening in telomerase-negative somatic cells leads to the activation of the TP53 protein and the elimination of potentially unstable cells. We examined the effect of TP53 gene expression on both telomere metabolism and chromosome stability in immortal, telomerase-positive cell lines. Telomere length, telomerase activity, and chromosome instability were measured in multiple clones isolated from three related human B-lymphoblast cell lines that vary in TP53 expression; TK6 cells express wild-type TP53, WTK1 cells overexpress a mutant form of TP53, and NH32 cells express no TP53 protein. Clonal variations in both telomere length and chromosome stability were observed, and shorter telomeres were associated with higher levels of chromosome instability. The shortest telomeres were found in WTK1- and NH32-derived cells, and these cells had 5- to 10-fold higher levels of chromosome instability. The primary marker of instability was the presence of dicentric chromosomes. Aneuploidy and other stable chromosome alterations were also found in clones showing high levels of dicentrics. Polyploidy was found only in WTK1-derived cells. Both telomere length and chromosome instability fluctuated in the different cell populations with time in culture, presumably as unstable cells and cells with short telomeres were eliminated from the growing population. Our results suggest that transient reductions in telomere lengths may be common in immortal cell lines and that these alterations in telomere metabolism can have a profound effect on chromosome stability. Copyright 2000 Wiley-Liss, Inc.
Sequential glycan profiling at single cell level with the microfluidic lab-in-a-trench platform: a new era in experimental cell biology.

PubMed

O'Connell, Tríona M; King, Damien; Dixit, Chandra K; O'Connor, Brendan; Walls, Dermot; Ducrée, Jens

2014-09-21

It is now widely recognised that the earliest changes that occur on a cell when it is stressed or becoming diseased are alterations in its surface glycosylation. Current state-of-the-art technologies in glycoanalysis include mass spectrometry, protein microarray formats, techniques in cytometry and more recently, glyco-quantitative polymerase chain reaction (Glyco-qPCR). Techniques for the glycoprofiling of the surfaces of single cells are either limited to the analysis of large cell populations or are unable to handle multiple and/or sequential probing. Here, we report a novel approach of single live cell glycoprofiling enabled by the microfluidic "Lab-in-a-Trench" (LiaT) platform for performing capture and retention of cells, along with shear-free reagent loading and washing. The significant technical improvement on state-of-the-art is the demonstration of consecutive, spatio-temporally profiling of glycans on a single cell by sequential elution of the previous lectin probe using their corresponding free sugar. We have qualitatively analysed glycan density on the surface of individual cells. This has allowed us to qualitatively co-localise the observed glycans. This approach enables exhaustive glycoprofiling and glycan mapping on the surface of individual live cells with multiple lectins. The possibility of sequentially profiling glycans on cells will be a powerful new tool to add to current glycoanalytical techniques. The LiaT platform will enable cell biologists to perform many high sensitivity assays and also will also make a significant impact on biomarker research.
Central nervous system demyelinating disease protection by the human commensal Bacteroides fragilis depends on polysaccharide A expression.

PubMed

Ochoa-Repáraz, Javier; Mielcarz, Daniel W; Ditrio, Lauren E; Burroughs, Ashley R; Begum-Haque, Sakhina; Dasgupta, Suryasarathi; Kasper, Dennis L; Kasper, Lloyd H

2010-10-01

The importance of gut commensal bacteria in maintaining immune homeostasis is increasingly understood. We recently described that alteration of the gut microflora can affect a population of Foxp3(+)T(reg) cells that regulate demyelination in experimental autoimmune encephalomyelitis (EAE), the experimental model of human multiple sclerosis. We now extend our previous observations on the role of commensal bacteria in CNS demyelination, and we demonstrate that Bacteroides fragilis producing a bacterial capsular polysaccharide Ag can protect against EAE. Recolonization with wild type B. fragilis maintained resistance to EAE, whereas reconstitution with polysaccharide A-deficient B. fragilis restored EAE susceptibility. Enhanced numbers of Foxp3(+)T(reg) cells in the cervical lymph nodes were observed after intestinal recolonization with either strain of B. fragilis. Ex vivo, CD4(+)T cells obtained from mice reconstituted with wild type B. fragilis had significantly enhanced rates of conversion into IL-10-producing Foxp3(+)T(reg) cells and offered greater protection against disease. Our results suggest an important role for commensal bacterial Ags, in particular B. fragilis expressing polysaccharide A, in protecting against CNS demyelination in EAE and perhaps human multiple sclerosis.
A B Cell-Driven Autoimmune Pathway Leading to Pathological Hallmarks of Progressive Multiple Sclerosis in the Marmoset Experimental Autoimmune Encephalomyelitis Model

PubMed Central

’t Hart, Bert A.; Dunham, Jordon; Faber, Bart W.; Laman, Jon D.; van Horssen, Jack; Bauer, Jan; Kap, Yolanda S.

2017-01-01

The absence of pathological hallmarks of progressive multiple sclerosis (MS) in commonly used rodent models of experimental autoimmune encephalomyelitis (EAE) hinders the development of adequate treatments for progressive disease. Work reviewed here shows that such hallmarks are present in the EAE model in marmoset monkeys (Callithrix jacchus). The minimal requirement for induction of progressive MS pathology is immunization with a synthetic peptide representing residues 34–56 from human myelin oligodendrocyte glycoprotein (MOG) formulated with a mineral oil [incomplete Freund’s adjuvant (IFA)]. Pathological aspects include demyelination of cortical gray matter with microglia activation, oxidative stress, and redistribution of iron. When the peptide is formulated in complete Freund’s adjuvant, which contains mycobacteria that relay strong activation signals to myeloid cells, oxidative damage pathways are strongly boosted leading to more intensive pathology. The proven absence of immune potentiating danger signals in the MOG34–56/IFA formulation implies that a narrow population of antigen-experienced T cells present in the monkey’s immune repertoire is activated. This novel pathway involves the interplay of lymphocryptovirus-infected B cells with MHC class Ib/Caja-E restricted CD8+ CD56+ cytotoxic T lymphocytes. PMID:28744286
Claustrum of the short-tailed fruit bat, Carollia perspicillata: Alignment of cellular orientation and functional connectivity.

PubMed

Orman, Rena; Kollmar, Richard; Stewart, Mark

2017-04-15

The claustrum is a gray-matter structure that underlies neocortex and reciprocates connections with cortical and subcortical targets. In lower mammals, the claustrum is directly adjacent to neocortex, making the definition of claustral boundaries challenging. Latexin, an endogenous inhibitor of metallocarboxypeptidases, localizes to claustral cells, enabling a clear delineation of claustrum. Given its proportionately large claustrum, we hypothesized that the short-tailed fruit bat, Carollia perspicillata, can be a useful model for claustral structure-function relations. We used latexin immunohistochemistry to identify claustral boundaries and intrinsic structure and multielectrode recordings from brain slices to explore intrinsic excitatory connectivity of the claustrum. Carollia's claustrum contains cells whose intrinsic connectivity and alignment permit the generation of spontaneous, synchronous population events and mirror their pattern of spread in disinhibited brain slices over millimeters. Carollia shows cellular alignment and spontaneous population-activity spread along both horizontal and dorsoventral axes. Carollia claustrum possesses intrinsic excitatory connectivity sufficient to: 1) generate single, spontaneous, synchronized burst discharges, 2) support activity spread along axes where claustral cells are aligned, and 3), because of multiple axes for cell alignment, support activity spread along both rostrocaudal and dorsoventral axes. The smaller event sizes in bat claustrum compared with rat claustrum are consistent with events occurring in population subsets rather than the full claustral cell population. The overall size of claustrum, its pronounced vascularity, and its more complex intrinsic connectivity than rat suggest that the bat is an animal model for claustral structure and function that will permit unique access to claustrum's processing capabilities. J. Comp. Neurol. 525:1459-1474, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Two-population model for medial temporal lobe neurons: The vast majority are almost silent

NASA Astrophysics Data System (ADS)

Magyar, Andrew; Collins, John

2015-07-01

Recordings in the human medial temporal lobe have found many neurons that respond to pictures (and related stimuli) of just one particular person of those presented. It has been proposed that these are concept cells, responding to just a single concept. However, a direct experimental test of the concept cell idea appears impossible, because it would need the measurement of the response of each cell to enormous numbers of other stimuli. Here we propose a new statistical method for analysis of the data that gives a more powerful way to analyze how close data are to the concept-cell idea. Central to the model is the neuronal sparsity, defined as the total fraction of stimuli that elicit an above-threshold response in the neuron. The model exploits the large number of sampled neurons to give sensitivity to situations where the average response sparsity is much less than one response for the number of presented stimuli. We show that a conventional model where a single sparsity is postulated for all neurons gives an extremely poor fit to the data. In contrast, a model with two dramatically different populations gives an excellent fit to data from the hippocampus and entorhinal cortex. In the hippocampus, one population has 7% of the cells with a 2.6% sparsity. But a much larger fraction (93%) respond to only 0.1% of the stimuli. This can result in an extreme bias in the responsiveness of reported neurons compared with a typical neuron. Finally, we show how to allow for the fact that some identified units correspond to multiple neurons and find that our conclusions at the neural level are quantitatively changed but strengthened, with an even stronger difference between the two populations.
Small Talk: Cell-to-Cell Communication in Bacteria

ScienceCinema

Bassler, Bonnie [Princeton University, Princeton, New Jersey, United States

2017-12-09

Cell-cell communication in bacteria involves the production, release, and subsequent detection of chemical signaling molecules called autoinducers. This process, called quorum sensing, allows bacteria to regulate gene expression on a population-wide scale. Processes controlled by quorum sensing are usually ones that are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. For example, quorum sensing controls bioluminescence, secretion of virulence factors, biofilm formation, sporulation, and the exchange of DNA. Thus, quorum sensing is a mechanism that allows bacteria to function as multi-cellular organisms. Bacteria make, detect, and integrate information from multiple autoinducers, some of which are used exclusively for intra-species communication while others enable communication between species. Research is now focused on the development of therapies that interfere with quorum sensing to control bacterial virulence.
Could a B-1 cell derived phagocyte "be one" of the peritoneal macrophages during LPS-driven inflammation?

PubMed

Popi, Ana Flavia; Osugui, Lika; Perez, Katia Regina; Longo-Maugéri, Ieda Maria; Mariano, Mario

2012-01-01

The inflammatory response is driven by signals that recruit and elicit immune cells to areas of tissue damage or infection. The concept of a mononuclear phagocyte system postulates that monocytes circulating in the bloodstream are recruited to inflamed tissues where they give rise to macrophages. A recent publication demonstrated that the large increase in the macrophages observed during infection was the result of the multiplication of these cells rather than the recruitment of blood monocytes. We demonstrated previously that B-1 cells undergo differentiation to acquire a mononuclear phagocyte phenotype in vitro (B-1CDP), and we propose that B-1 cells could be an alternative origin for peritoneal macrophages. A number of recent studies that describe the phagocytic and microbicidal activity of B-1 cells in vitro and in vivo support this hypothesis. Based on these findings, we further investigated the differentiation of B-1 cells into phagocytes in vivo in response to LPS-induced inflammation. Therefore, we investigated the role of B-1 cells in the composition of the peritoneal macrophage population after LPS stimulation using osteopetrotic mice, BALB/Xid mice and the depletion of monocytes/macrophages by clodronate treatment. We show that peritoneal macrophages appear in op/op((-/-)) mice after LPS stimulation and exhibit the same Ig gene rearrangement (VH11) that is often found in B-1 cells. These results strongly suggest that op/op((-/-)) peritoneal "macrophages" are B-1CDP. Similarly, the LPS-induced increase in the macrophage population was observed even following monocyte/macrophage depletion by clodronate. After monocyte/macrophage depletion by clodronate, LPS-elicited macrophages were observed in BALB/Xid mice only following the transfer of B-1 cells. Based on these data, we confirmed that B-1 cell differentiation into phagocytes also occurs in vivo. In conclusion, the results strongly suggest that B-1 cell derived phagocytes are a component of the LPS-elicited peritoneal macrophage population.
Automated deconvolution of structured mixtures from heterogeneous tumor genomic data

PubMed Central

Roman, Theodore; Xie, Lu

2017-01-01

With increasing appreciation for the extent and importance of intratumor heterogeneity, much attention in cancer research has focused on profiling heterogeneity on a single patient level. Although true single-cell genomic technologies are rapidly improving, they remain too noisy and costly at present for population-level studies. Bulk sequencing remains the standard for population-scale tumor genomics, creating a need for computational tools to separate contributions of multiple tumor clones and assorted stromal and infiltrating cell populations to pooled genomic data. All such methods are limited to coarse approximations of only a few cell subpopulations, however. In prior work, we demonstrated the feasibility of improving cell type deconvolution by taking advantage of substructure in genomic mixtures via a strategy called simplicial complex unmixing. We improve on past work by introducing enhancements to automate learning of substructured genomic mixtures, with specific emphasis on genome-wide copy number variation (CNV) data, as well as the ability to process quantitative RNA expression data, and heterogeneous combinations of RNA and CNV data. We introduce methods for dimensionality estimation to better decompose mixture model substructure; fuzzy clustering to better identify substructure in sparse, noisy data; and automated model inference methods for other key model parameters. We further demonstrate their effectiveness in identifying mixture substructure in true breast cancer CNV data from the Cancer Genome Atlas (TCGA). Source code is available at https://github.com/tedroman/WSCUnmix PMID:29059177
Quantification of the heterogeneity of prognostic cellular biomarkers in ewing sarcoma using automated image and random survival forest analysis.

PubMed

Bühnemann, Claudia; Li, Simon; Yu, Haiyue; Branford White, Harriet; Schäfer, Karl L; Llombart-Bosch, Antonio; Machado, Isidro; Picci, Piero; Hogendoorn, Pancras C W; Athanasou, Nicholas A; Noble, J Alison; Hassan, A Bassim

2014-01-01

Driven by genomic somatic variation, tumour tissues are typically heterogeneous, yet unbiased quantitative methods are rarely used to analyse heterogeneity at the protein level. Motivated by this problem, we developed automated image segmentation of images of multiple biomarkers in Ewing sarcoma to generate distributions of biomarkers between and within tumour cells. We further integrate high dimensional data with patient clinical outcomes utilising random survival forest (RSF) machine learning. Using material from cohorts of genetically diagnosed Ewing sarcoma with EWSR1 chromosomal translocations, confocal images of tissue microarrays were segmented with level sets and watershed algorithms. Each cell nucleus and cytoplasm were identified in relation to DAPI and CD99, respectively, and protein biomarkers (e.g. Ki67, pS6, Foxo3a, EGR1, MAPK) localised relative to nuclear and cytoplasmic regions of each cell in order to generate image feature distributions. The image distribution features were analysed with RSF in relation to known overall patient survival from three separate cohorts (185 informative cases). Variation in pre-analytical processing resulted in elimination of a high number of non-informative images that had poor DAPI localisation or biomarker preservation (67 cases, 36%). The distribution of image features for biomarkers in the remaining high quality material (118 cases, 104 features per case) were analysed by RSF with feature selection, and performance assessed using internal cross-validation, rather than a separate validation cohort. A prognostic classifier for Ewing sarcoma with low cross-validation error rates (0.36) was comprised of multiple features, including the Ki67 proliferative marker and a sub-population of cells with low cytoplasmic/nuclear ratio of CD99. Through elimination of bias, the evaluation of high-dimensionality biomarker distribution within cell populations of a tumour using random forest analysis in quality controlled tumour material could be achieved. Such an automated and integrated methodology has potential application in the identification of prognostic classifiers based on tumour cell heterogeneity.
The Role of the Gut Microbiome in Multiple Sclerosis Risk and Progression: Towards Characterization of the "MS Microbiome".

PubMed

Pröbstel, Anne-Katrin; Baranzini, Sergio E

2018-01-01

Multiple sclerosis (MS) is the prototypic complex disease, in which both genes and the environment contribute to its pathogenesis. To date, > 200 independent loci across the genome have been associated with MS risk. However, these only explain a fraction of the total phenotypic variance, suggesting the possible presence of additional genetic factors, and, most likely, also environmental factors. New DNA sequencing technologies have enabled the sequencing of all kinds of microorganisms, including those living in and around humans (i.e., microbiomes). The study of bacterial populations inhabiting the gut is of particular interest in autoimmune diseases owing to their key role in shaping immune responses. In this review, we address the potential crosstalk between B cells and the gut microbiota, a relevant scenario in light of recently approved anti-B-cell therapies for MS. In addition, we review recent efforts to characterize the gut microbiome in patients with MS and discuss potential challenges and future opportunities. Finally, we describe the international MS microbiome study, a multicenter effort to study a large population of patients with MS and their healthy household partners to define the core MS microbiome, how it is shaped by disease-modifying therapies, and to explore potential therapeutic interventions.
[Characterization of stem cells derived from the neonatal auditory sensory epithelium].

PubMed

Diensthuber, M; Heller, S

2010-11-01

In contrast to regenerating hair cell-bearing organs of nonmammalian vertebrates the adult mammalian organ of Corti appears to have lost its ability to maintain stem cells. The result is a lack of regenerative ability and irreversible hearing loss following auditory hair cell death. Unexpectedly, the neonatal auditory sensory epithelium has recently been shown to harbor cells with stem cell features. The origin of these cells within the cochlea's sensory epithelium is unknown. We applied a modified neurosphere assay to identify stem cells within distinct subregions of the neonatal mouse auditory sensory epithelium. Sphere cells were characterized by multiple markers and morphologic techniques. Our data reveal that both the greater and the lesser epithelial ridge contribute to the sphere-forming stem cell population derived from the auditory sensory epithelium. These self-renewing sphere cells express a variety of markers for neural and otic progenitor cells and mature inner ear cell types. Stem cells can be isolated from specific regions of the auditory sensory epithelium. The distinct features of these cells imply a potential application in the development of a cell replacement therapy to regenerate the damaged sensory epithelium.

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution

PubMed Central

Humphries, Adam; Cereser, Biancastella; Gay, Laura J.; Miller, Daniel S. J.; Das, Bibek; Gutteridge, Alice; Elia, George; Nye, Emma; Jeffery, Rosemary; Poulsom, Richard; Novelli, Marco R.; Rodriguez-Justo, Manuel; McDonald, Stuart A. C.; Wright, Nicholas A.; Graham, Trevor A.

2013-01-01

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO−) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis. PMID:23766371
Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution.

PubMed

Humphries, Adam; Cereser, Biancastella; Gay, Laura J; Miller, Daniel S J; Das, Bibek; Gutteridge, Alice; Elia, George; Nye, Emma; Jeffery, Rosemary; Poulsom, Richard; Novelli, Marco R; Rodriguez-Justo, Manuel; McDonald, Stuart A C; Wright, Nicholas A; Graham, Trevor A

2013-07-02

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO(-)) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis.
A microfluidic platform for chemical stimulation and real time analysis of catecholamine secretion from neuroendocrine cells

PubMed Central

Ges, Igor A.; Brindley, Rebecca L.; Currie, Kevin P.M.; Baudenbacher, Franz J.

2013-01-01

Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped “cell traps”, each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion / repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time. PMID:24126415
IgM Repertoire Biodiversity is Reduced in HIV-1 Infection and Systemic Lupus Erythematosus.

PubMed

Yin, Li; Hou, Wei; Liu, Li; Cai, Yunpeng; Wallet, Mark Andrew; Gardner, Brent Paul; Chang, Kaifen; Lowe, Amanda Catherine; Rodriguez, Carina Adriana; Sriaroon, Panida; Farmerie, William George; Sleasman, John William; Goodenow, Maureen Michels

2013-01-01

HIV-1 infection or systemic lupus erythematosus (SLE) disrupt B cell homeostasis, reduce memory B cells, and impair function of IgG and IgM antibodies. To determine how disturbances in B cell populations producing polyclonal antibodies relate to the IgM repertoire, the IgM transcriptome in health and disease was explored at the complementarity determining region 3 (CDRH3) sequence level. 454-deep pyrosequencing in combination with a novel analysis pipeline was applied to define populations of IGHM CDRH3 sequences based on absence or presence of somatic hypermutations (SHM) in peripheral blood B cells. HIV or SLE subjects have reduced biodiversity within their IGHM transcriptome compared to healthy subjects, mainly due to a significant decrease in the number of unique combinations of alleles, although recombination machinery was intact. While major differences between sequences without or with SHM occurred among all groups, IGHD and IGHJ allele use, CDRH3 length distribution, or generation of SHM were similar among study cohorts. Antiretroviral therapy failed to normalize IGHM biodiversity in HIV-infected individuals. All subjects had a low frequency of allelic combinations within the IGHM repertoire similar to known broadly neutralizing HIV-1 antibodies. Polyclonal expansion would decrease overall IgM biodiversity independent of other mechanisms for development of the B cell repertoire. Applying deep sequencing as a strategy to follow development of the IgM repertoire in health and disease provides a novel molecular assessment of multiple points along the B cell differentiation pathway that is highly sensitive for detecting perturbations within the repertoire at the population level.
Incidence and Clinical Features of Rare Cutaneous Malignancies in Olmsted County, Minnesota, 2000 to 2010.

PubMed

Tolkachjov, Stanislav N; Schmitt, Adam R; Muzic, John G; Weaver, Amy L; Baum, Christian L

2017-01-01

The incidence of rare cutaneous malignancies is unknown. Current estimates of rare cutaneous malignancy incidences are based on broad epidemiologic data or single institution experiences, not population-based data. To determine the incidence of several rare nonmelanoma skin cancers. The authors conducted a retrospective chart review of a population-based cohort between the years 2000 and 2010. Residents of Olmsted County, Minnesota, who were diagnosed with a biopsy-proven nonmelanoma skin cancer-excluding basal cell carcinoma and squamous cell carcinoma-were included in this study. The primary outcome was tumor incidence. Additionally, the authors extracted patient demographics, tumor characteristics, treatment modalities, and outcomes. The age-adjusted and sex-adjusted incidences per 100,000 persons of multiple rare cutaneous malignancies were: atypical fibroxanthoma (1.8), sebaceous carcinoma (0.8), dermatofibrosarcoma protuberans (0.4), microcystic adnexal carcinoma (0.7), eccrine carcinoma (0.4), eccrine porocarcinoma (0.2), and leiomyosarcoma (0.2). The authors report population-based incidences and clinical characteristics for these rare cutaneous malignancies. The immune status and smoking status of patients and the treatment and outcomes of these tumors are reported. Additional studies in a broader population are needed to further define the epidemiology and outcomes of these malignancies.
Pdgfrα functions in endothelial-derived cells to regulate neural crest cells and the development of the great arteries.

PubMed

Aghajanian, Haig; Cho, Young Kuk; Rizer, Nicholas W; Wang, Qiaohong; Li, Li; Degenhardt, Karl; Jain, Rajan

2017-09-01

Originating as a single vessel emerging from the embryonic heart, the truncus arteriosus must septate and remodel into the aorta and pulmonary artery to support postnatal life. Defective remodeling or septation leads to abnormalities collectively known as conotruncal defects, which are associated with significant mortality and morbidity. Multiple populations of cells must interact to coordinate outflow tract remodeling, and the cardiac neural crest has emerged as particularly important during this process. Abnormalities in the cardiac neural crest have been implicated in the pathogenesis of multiple conotruncal defects, including persistent truncus arteriosus, double outlet right ventricle and tetralogy of Fallot. However, the role of the neural crest in the pathogenesis of another conotruncal abnormality, transposition of the great arteries, is less well understood. In this report, we demonstrate an unexpected role of Pdgfra in endothelial cells and their derivatives during outflow tract development. Loss of Pdgfra in endothelium and endothelial-derived cells results in double outlet right ventricle and transposition of the great arteries. Our data suggest that loss of Pdgfra in endothelial-derived mesenchyme in the outflow tract endocardial cushions leads to a secondary defect in neural crest migration during development. © 2017. Published by The Company of Biologists Ltd.
SunRiSE - measuring translation elongation at single-cell resolution by means of flow cytometry.

PubMed

Argüello, Rafael J; Reverendo, Marisa; Mendes, Andreia; Camosseto, Voahirana; Torres, Adrian G; Ribas de Pouplana, Lluis; van de Pavert, Serge A; Gatti, Evelina; Pierre, Philippe

2018-05-31

The rate at which ribosomes translate mRNAs regulates protein expression by controlling co-translational protein folding and mRNA stability. Many factors regulate translation elongation, including tRNA levels, codon usage and phosphorylation of eukaryotic elongation factor 2 (eEF2). Current methods to measure translation elongation lack single-cell resolution, require expression of multiple transgenes and have never been successfully applied ex vivo Here, we show, by using a combination of puromycilation detection and flow cytometry (a method we call 'SunRiSE'), that translation elongation can be measured accurately in primary cells in pure or heterogenous populations isolated from blood or tissues. This method allows for the simultaneous monitoring of multiple parameters, such as mTOR or S6K1/2 signaling activity, the cell cycle stage and phosphorylation of translation factors in single cells, without elaborated, costly and lengthy purification procedures. We took advantage of SunRiSE to demonstrate that, in mouse embryonic fibroblasts, eEF2 phosphorylation by eEF2 kinase (eEF2K) mostly affects translation engagement, but has a surprisingly small effect on elongation, except after proteotoxic stress induction.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.
Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles.

PubMed

Wheeler, Richard John

2015-11-05

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point "snapshot" of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes--cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)--as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods. © 2015 Wheeler. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Elasticity of human embryonic stem cells as determined by atomic force microscopy.

PubMed

Kiss, Robert; Bock, Henry; Pells, Steve; Canetta, Elisabetta; Adya, Ashok K; Moore, Andrew J; De Sousa, Paul; Willoughby, Nicholas A

2011-10-01

The expansive growth and differentiation potential of human embryonic stem cells (hESCs) make them a promising source of cells for regenerative medicine. However, this promise is off set by the propensity for spontaneous or uncontrolled differentiation to result in heterogeneous cell populations. Cell elasticity has recently been shown to characterize particular cell phenotypes, with undifferentiated and differentiated cells sometimes showing significant differences in their elasticities. In this study, we determined the Young's modulus of hESCs by atomic force microscopy using a pyramidal tip. Using this method we are able to take point measurements of elasticity at multiple locations on a single cell, allowing local variations due to cell structure to be identified. We found considerable differences in the elasticity of the analyzed hESCs, reflected by a broad range of Young's modulus (0.05-10 kPa). This surprisingly high variation suggests that elasticity could serve as the basis of a simple and efficient large scale purification/separation technique to discriminate subpopulations of hESCs.
A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development.

PubMed

Yao, Zizhen; Mich, John K; Ku, Sherman; Menon, Vilas; Krostag, Anne-Rachel; Martinez, Refugio A; Furchtgott, Leon; Mulholland, Heather; Bort, Susan; Fuqua, Margaret A; Gregor, Ben W; Hodge, Rebecca D; Jayabalu, Anu; May, Ryan C; Melton, Samuel; Nelson, Angelique M; Ngo, N Kiet; Shapovalova, Nadiya V; Shehata, Soraya I; Smith, Michael W; Tait, Leah J; Thompson, Carol L; Thomsen, Elliot R; Ye, Chaoyang; Glass, Ian A; Kaykas, Ajamete; Yao, Shuyuan; Phillips, John W; Grimley, Joshua S; Levi, Boaz P; Wang, Yanling; Ramanathan, Sharad

2017-01-05

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. Copyright © 2017 Elsevier Inc. All rights reserved.
mBAND Analysis of Early and Late Damages in the Chromosome of Human Lymphocytes after Exposures to Gamma Rays and Fe Ions

NASA Technical Reports Server (NTRS)

Sunagawa, Mayumi; Zhang, Ye; Yeshitla, Samrawit; Kadhim, Munira; Wilson, Bobby; Wu, Honglu

2013-01-01

Stable type chromosome aberrations that survive multiple generations of cell division include translocation and inversions. An efficient method to detect an inversion is multi-color banding fluorescent in situ hybridization (mBAND) which allows identification of both inter- and intrachromosome aberrations simultaneously. Post irradiation, chromosome aberrations may also arise after multiple cell divisions as a result of genomic instability. To investigate the stable or late-arising chromosome aberrations induced after radiation exposure, we exposed human lymphocytes to gamma rays and Fe ions ex vivo, and cultured the cells for multiple generations. Chromosome aberrations were analyzed in cells collected at first mitosis and at several time intervals during the culture period post irradiation. With gamma irradiation, about half of the damages observed at first mitosis remained after 7 day- and 14 day- culture, suggesting the transmissibility of damages to the surviving progeny. At the doses that produced similar frequencies of gamma-induced chromosome aberrations as observed at first mitosis, a significantly lower yield of aberrations remained at the same population doublings after Fe ion exposure. At these equitoxic doses, more complex type aberrations were observed for Fe ions, indicating that Fe ion-induced initial chromosome damages are more severe and may lead to cell death. Detailed analysis of breaks participating in total chromosome exchanges within the first cell cycle post irradiation revealed a common hotspot located in the 3p21 region, which is a known fragile site corresponding to the band 6 in the mBand analysis. The breakpoint distribution in chromosomes collected at 7 days, but not at 14 days, post irradiation appeared similar to the distribution in cells collected within the first cell cycle post irradiation. The breakpoint distribution for human lymphocytes after radiation exposure was different from the previously published distribution for human mammary epithelial cells, indicating that interphase chromatin folding structures play a role in the distribution of radiation-induced breaks.
Early and Late Chromosome Damages in Human Lymphocytes Induced by Gamma Rays and Fe Ions

NASA Technical Reports Server (NTRS)

Sunagawa, Mayumi; Zhang, Ye; Yeshitla, Samrawit; Kadhim, Munira; Wilson, Bobby; Wu, Honglu

2014-01-01

Chromosomal translocations and inversions are considered stable, and cells containing these types of chromosome aberrations can survive multiple cell divisions. An efficient method to detect an inversion is multi-color banding fluorescent in situ hybridization (mBAND) which allows identification of both inter- and intrachromosome aberrations simultaneously. Post irradiation, chromosome aberrations may also arise after multiple cell divisions as a result of genomic instability. To investigate the stable or late-arising chromosome aberrations induced after radiation exposure, we exposed human lymphocytes to gamma rays and Fe ions ex vivo, and cultured the cells for multiple generations. Chromosome aberrations were analyzed in cells collected at first mitosis and at several time intervals during the culture period post irradiation. With gamma irradiation, about half of the damages observed at first mitosis remained after 7 day- and 14 day- culture, suggesting the transmissibility of damages to the surviving progeny. Detailed analysis of chromosome break ends participating in exchanges revealed a greater fraction of break ends involved in intrachromosome aberrations in the 7- and 14-day samples in comparison to the fraction at first mitosis. In particular, simple inversions were found at 7 and 14 days, but not at the first mitosis, suggesting that some of the aberrations might be formed days post irradiation. In contrast, at the doses that produced similar frequencies of gamma-induced chromosome aberrations as observed at first mitosis, a significantly lower yield of aberrations remained at the same population doublings after Fe ion exposure. At these equitoxic doses, more complex type aberrations were observed for Fe ions, indicating that Fe ion-induced initial chromosome damages are more severe and may lead to cell death. Comparison between low and high doses of Fe ion irradiation in the induction of late damages will also be discussed.
T Lymphocytes Do Not Directly Mediate the Protective Effect of Estrogen on Experimental Autoimmune Encephalomyelitis

PubMed Central

Polanczyk, Magdalena J.; Jones, Richard E.; Subramanian, Sandhya; Afentoulis, Michael; Rich, Cathleen; Zakroczymski, Melissa; Cooke, Paul; Vandenbark, Arthur A.; Offner, Halina

2004-01-01

Gender influences mediated by 17β-estradiol (E2) have been associated with susceptibility to and severity of autoimmune diseases such as diabetes, arthritis, and multiple sclerosis. In this regard, we have shown that estrogen receptor-α (Esr1) is crucial for the protective effect of 17β-estradiol (E2) in murine experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis. The expression of estrogen receptors among various immune cells (eg, T and B lymphocytes, antigen-presenting cells) suggests that the therapeutic effect of E2 is likely mediated directly through specific receptor binding. However, the target immune cell populations responsive to E2 treatment have not been identified. In the current study, we induced EAE in T-cell-deficient, severe combined immunodeficient mice or in immunocompetent mice with encephalitogenic T cells from wild-type Esr1+/+ or Esr1 knockout (Esr1−/−) donors and compared the protective E2 responses. The results showed that E2-responsive, Esr1+/+ disease-inducing encephalitogenic T cells were neither necessary nor sufficient for E2-mediated protection from EAE. Instead, the therapeutic response appeared to be mediated through direct effects on nonlymphocytic, E2-responsive cells and down-regulation of the inflammatory response in the central nervous system. These results provide the first demonstration that the protective effect of E2 on EAE is not mediated directly through E2-responsive T cells and raise the alternative possibility that nonlymphocytic cells such as macrophages, dendritic cells, or other nonlymphocytic cells are primarily responsive to E2 treatment in EAE. PMID:15579449
Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: carbon and energy flow contribute to the distinct biofilm growth state.

PubMed

Clark, Melinda E; He, Zhili; Redding, Alyssa M; Joachimiak, Marcin P; Keasling, Jay D; Zhou, Jizhong Z; Arkin, Adam P; Mukhopadhyay, Aindrila; Fields, Matthew W

2012-04-16

Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion.
Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: Carbon and energy flow contribute to the distinct biofilm growth state

PubMed Central

2012-01-01

Background Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. Results The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. Conclusions Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion. PMID:22507456
Fixation times in differentiation and evolution in the presence of bottlenecks, deserts, and oases.

PubMed

Chou, Tom; Wang, Yu

2015-05-07

Cellular differentiation and evolution are stochastic processes that can involve multiple types (or states) of particles moving on a complex, high-dimensional state-space or "fitness" landscape. Cells of each specific type can thus be quantified by their population at a corresponding node within a network of states. Their dynamics across the state-space network involve genotypic or phenotypic transitions that can occur upon cell division, such as during symmetric or asymmetric cell differentiation, or upon spontaneous mutation. Here, we use a general multi-type branching processes to study first passage time statistics for a single cell to appear in a specific state. Our approach readily allows for nonexponentially distributed waiting times between transitions, reflecting, e.g., the cell cycle. For simplicity, we restrict most of our detailed analysis to exponentially distributed waiting times (Poisson processes). We present results for a sequential evolutionary process in which L successive transitions propel a population from a "wild-type" state to a given "terminally differentiated," "resistant," or "cancerous" state. Analytic and numeric results are also found for first passage times across an evolutionary chain containing a node with increased death or proliferation rate, representing a desert/bottleneck or an oasis. Processes involving cell proliferation are shown to be "nonlinear" (even though mean-field equations for the expected particle numbers are linear) resulting in first passage time statistics that depend on the position of the bottleneck or oasis. Our results highlight the sensitivity of stochastic measures to cell division fate and quantify the limitations of using certain approximations (such as the fixed-population and mean-field assumptions) in evaluating fixation times. Published by Elsevier Ltd.
Statistical performance of image cytometry for DNA, lipids, cytokeratin, & CD45 in a model system for circulation tumor cell detection.

PubMed

Futia, Gregory L; Schlaepfer, Isabel R; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A

2017-07-01

Detection of circulating tumor cells (CTCs) in a blood sample is limited by the sensitivity and specificity of the biomarker panel used to identify CTCs over other blood cells. In this work, we present Bayesian theory that shows how test sensitivity and specificity set the rarity of cell that a test can detect. We perform our calculation of sensitivity and specificity on our image cytometry biomarker panel by testing on pure disease positive (D + ) populations (MCF7 cells) and pure disease negative populations (D - ) (leukocytes). In this system, we performed multi-channel confocal fluorescence microscopy to image biomarkers of DNA, lipids, CD45, and Cytokeratin. Using custom software, we segmented our confocal images into regions of interest consisting of individual cells and computed the image metrics of total signal, second spatial moment, spatial frequency second moment, and the product of the spatial-spatial frequency moments. We present our analysis of these 16 features. The best performing of the 16 features produced an average separation of three standard deviations between D + and D - and an average detectable rarity of ∼1 in 200. We performed multivariable regression and feature selection to combine multiple features for increased performance and showed an average separation of seven standard deviations between the D + and D - populations making our average detectable rarity of ∼1 in 480. Histograms and receiver operating characteristics (ROC) curves for these features and regressions are presented. We conclude that simple regression analysis holds promise to further improve the separation of rare cells in cytometry applications. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.
Application of laser-capture microdissection to analysis of gene expression in the testis.

PubMed

Sluka, Pavel; O'Donnell, Liza; McLachlan, Robert I; Stanton, Peter G

2008-01-01

The isolation and molecular analysis of highly purified cell populations from complex, heterogeneous tissues has been a challenge for many years. Spermatogenesis in the testis is a particularly difficult process to study given the unique multiple cellular associations within the seminiferous epithelium, making the isolation of specific cell types difficult. Laser-capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. This technology has enhanced our ability to directly examine gene expression in enriched testicular cell populations by routine methods of gene expression analysis, such as real-time RT-PCR, differential display, and gene microarrays. The application of LCM has however introduced methodological hurdles that have not been encountered with more conventional molecular analyses of whole tissue. In particular, tissue handling (i.e. fixation, storage, and staining), consumables (e.g. slide choice), staining reagents (conventional H&E vs. fluorescence), extraction methods, and downstream applications have all required re-optimisation to facilitate differential gene expression analysis using the small amounts of material obtained using LCM. This review will discuss three critical issues that are essential for successful procurement of cells from testicular tissue sections; tissue morphology, capture success, and maintenance of molecular integrity. The importance of these issues will be discussed with specific reference to the two most commonly used LCM systems; the Arcturus PixCell IIe and PALM systems. The rat testis will be used as a model, and emphasis will be placed on issues of tissue handling, processing, and staining methods, including the application of fluorescence techniques to assist in the identification of cells of interest for the purposes of mRNA expression analysis.
On-chip activation and subsequent detection of individual antigen-specific T cells

PubMed Central

Song, Qing; Han, Qing; Bradshaw, Elizabeth M.; Kent, Sally C.; Raddassi, Khadir; Nilsson, Björn; Nepom, Gerald T.; Hafler, David A.; Love, J. Christopher

2010-01-01

The frequencies of antigen-specific CD4+ T cells in samples of human tissue has been difficult to determine accurately ex vivo, particularly for autoimmune diseases such as multiple sclerosis or Type 1 diabetes. Conventional approaches involve the expansion of primary T cells in vitro to increase the numbers of cells, and a subsequent assessment of the frequencies of antigen-specific T cells in the expanded population by limiting dilution or by using fluorescently labeled tetramers of peptide-loaded major histocompatibility complex (MHC) receptors. Here we describe an alternative approach that uses arrays of subnanoliter wells coated with recombinant peptide-loaded MHC Class II monomers to isolate and stimulate individual CD4+ T cells in an antigen-specific manner. In these experiments, activation was monitored using microengraving to capture two cytokines (IFNγ and IL-17) released from single cells. This new method should enable direct enumeration of antigen-specific CD4+ T cells ex vivo from clinical samples. PMID:20000848
Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Trends in survival of multiple myeloma: a thirty-year population-based study in a single institution.

PubMed

Ríos-Tamayo, Rafael; Sánchez, María José; Puerta, José Manuel; Sáinz, Juan; Chang, Daysi-Yoe-Ling; Rodríguez, Teresa; López, Pilar; de Pablos, José María; Navarro, Pilar; de Veas, José Luís García; Romero, Antonio; Garrido, Pilar; Moratalla, Lucía; Alarcón-Payer, Carolina; López-Fernández, Elisa; González, Pedro Antonio; Jiménez-Moleón, José Juan; Calleja-Hernández, Miguel Ángel; Jurado, Manuel

2015-10-01

Despite the progress made in recent years, multiple myeloma is still considered an incurable disease. Most survival data come from clinical trials. Little is known about the outcome in unselected real-life patients. Overall survival was analyzed in a cohort of newly diagnosed symptomatic multiple myeloma patients, over the last three decades, in a single institution population-based study. 582 consecutive myeloma patients were included in the study. Survival increased over time in patients younger than 65 years but did not reach statistical significance in patients with 65 years or older. The prognostic factors associated with overall survival were the International Staging System, the serum lactate dehydrogenase level, the renal impairment, the realization of autologous stem cell transplantation, and the presence of concomitant amyloidosis. Overall survival shows a steady improvement over time. The survival of myeloma is improving progressively in real-life patients, particularly after the widespread use of the novel agents. A comprehensive assessment of comorbidity can help to explain the huge heterogeneity of myeloma outcome. The optimization of current therapeutic resources as well as the incorporation of new drugs will allow further improvement of survival in the coming years. Copyright © 2015 Elsevier Ltd. All rights reserved.
The Endogenous GRP78 Interactome in Human Head and Neck Cancers: A Deterministic Role of Cell Surface GRP78 in Cancer Stemness.

PubMed

Chen, Hsin-Ying; Chang, Joseph Tung-Chieh; Chien, Kun-Yi; Lee, Yun-Shien; You, Guo-Rung; Cheng, Ann-Joy

2018-01-11

Cell surface glucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, was suggested to be a cancer stem cell marker, but the influence of this molecule on cancer stemness is poorly characterized. In this study, we developed a mass spectrometry platform to detect the endogenous interactome of GRP78 and investigated its role in cancer stemness. The interactome results showed that cell surface GRP78 associates with multiple molecules. The influence of cell population heterogeneity of head and neck cancer cell lines (OECM1, FaDu, and BM2) according to the cell surface expression levels of GRP78 and the GRP78 interactome protein, Progranulin, was investigated. The four sorted cell groups exhibited distinct cell cycle distributions, asymmetric/symmetric cell divisions, and different relative expression levels of stemness markers. Our results demonstrate that cell surface GRP78 promotes cancer stemness, whereas drives cells toward a non-stemlike phenotype when it chaperones Progranulin. We conclude that cell surface GRP78 is a chaperone exerting a deterministic influence on cancer stemness.
Multiple Notch signaling events control Drosophila CNS midline neurogenesis, gliogenesis and neuronal identity

PubMed Central

Wheeler, Scott R.; Stagg, Stephanie B.; Crews, Stephen T.

2009-01-01

The study of how transcriptional control and cell signaling influence neurons and glia to acquire their differentiated properties is fundamental to understanding CNS development and function. The Drosophila CNS midline cells are an excellent system for studying these issues because they consist of a small population of diverse cells with well-defined gene expression profiles. In this paper, the origins and differentiation of midline neurons and glia were analyzed. Midline precursor (MP) cells each divide once giving rise to two neurons; here, we use a combination of single-cell gene expression mapping and time-lapse imaging to identify individual MPs, their locations, movements and stereotyped patterns of division. The role of Notch signaling was investigated by analyzing 37 midline-expressed genes in Notch pathway mutant and misexpression embryos. Notch signaling had opposing functions: it inhibited neurogenesis in MP1,3,4 and promoted neurogenesis in MP5,6. Notch signaling also promoted midline glial and median neuroblast cell fate. This latter result suggests that the median neuroblast resembles brain neuroblasts that require Notch signaling, rather than nerve cord neuroblasts, the formation of which is inhibited by Notch signaling. Asymmetric MP daughter cell fates also depend on Notch signaling. One member of each pair of MP3–6 daughter cells was responsive to Notch signaling. By contrast, the other daughter cell asymmetrically acquired Numb, which inhibited Notch signaling, leading to a different fate choice. In summary, this paper describes the formation and division of MPs and multiple roles for Notch signaling in midline cell development, providing a foundation for comprehensive molecular analyses. PMID:18701546
Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

PubMed

Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F; Shao, Wei; Cyktor, Joshua C; Cillo, Anthony R; Halvas, Elias K; Coffin, John M; Mellors, John W; Kearney, Mary F

2017-05-02

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.
In vivo detection of peripherin-specific autoreactive B cells during type 1 diabetes pathogenesis1

PubMed Central

Garabatos, Nahir; Alvarez, Raimon; Carrillo, Jorge; Carrascal, Jorge; Izquierdo, Cristina; Chapman, Harold D.; Presa, Maximiliano; Mora, Conchi; Serreze, David V.; Verdaguer, Joan; Stratmann, Thomas

2014-01-01

Summary Autoreactive B cells are essential for the pathogenesis of type 1 diabetes. The genesis and dynamics of autoreactive B cells remain unknown. Here, we analyzed the immune response in the NOD mouse model to the neuronal protein peripherin (PRPH), a target antigen of islet-infiltrating B cells. PRPH autoreactive B cells recognized a single linear epitope of this protein, in contrast to the multiple epitope recognition commonly observed during autoreactive B cell responses. Autoantibodies to this epitope were also detected in the disease-resistant NOR and C57BL/6 strains. To specifically detect the accumulation of these B cells, we developed a novel approach, octameric peptide display, to follow the dynamics and localization of anti-PRPH B cell during disease progression. Before extended insulitis established, anti-PRPH B cells preferentially accumulated in the peritoneum. Anti-PRPH B cells were likewise detected in C57BL/6 mice, albeit at lower frequencies. As disease unfolded in NOD mice, anti-PRPH B cells invaded the islets and increased in number at the peritoneum of diabetic but not pre-diabetic mice. Isotype switched B cells were only detected in the peritoneum. Anti-PRPH B cells represent a heterogeneous population composed of both B1 and B2 subsets. In the spleen, anti-PRPH B cell were predominantly in the follicular subset. Therefore, anti-PRPH B cells represent a heterogeneous population that is generated early in life but proliferates as diabetes establishes. These findings on the temporal and spatial progression of autoreactive B cells should be relevant for our understanding of B cell function in diabetes pathogenesis. PMID:24610011
Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells.

PubMed

Liu, Haisong; Yang, Huan; Zhu, Dicong; Sui, Xin; Li, Juan; Liang, Zhen; Xu, Lei; Chen, Zeyu; Yao, Anzhi; Zhang, Long; Zhang, Xi; Yi, Xing; Liu, Meng; Xu, Shiqing; Zhang, Wenjian; Lin, Hua; Xie, Lan; Lou, Jinning; Zhang, Yong; Xi, Jianzhong; Deng, Hongkui

2014-10-01

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.
Left Atrial Appendages from Adult Hearts Contain a Reservoir of Diverse Cardiac Progenitor Cells

PubMed Central

Platt, Yardanna; Helman, Yaron; Feinberg, Yael; Lotan, Chaim; Beeri, Ronen

2013-01-01

Aims There is strong evidence supporting the claim that endogenous cardiac progenitor cells (CPCs) are key players in cardiac regeneration, but the anatomic source and phenotype of the master cardiac progenitors remains uncertain. Our aim was to investigate the different cardiac stem cell populations in the left atrial appendage (LAA) and their fates. Methods and Results We investigated the CPC content and profile of adult murine LAAs using immunohistochemistry and flow cytometry. We demonstrate that the LAA contains a large number of CPCs relative to other areas of the heart, representing over 20% of the total cell number. We grew two distinct CPC populations from the LAA by varying the degree of proteolysis. These differed by their histological location, surface marker profiles and growth dynamics. Specifically, CD45pos cells grew with milder proteolysis, while CD45neg cells grew mainly with more intense proteolysis. Both cell types could be induced to differentiate into cells with cardiomyocyte markers and organelles, albeit by different protocols. Many CD45pos cells expressed CD45 initially and rapidly lost its expression while differentiating. Conclusions Our results demonstrate that the left atrial appendage plays a role as a reservoir of multiple types of progenitor cells in murine adult hearts. Two different types of CPCs were isolated, differing in their epicardial-myocardial localization. Considering studies demonstrating layer-specific origins of different cardiac progenitor cells, our findings may shed light on possible pathways to study and utilize the diversity of endogenous progenitor cells in the adult heart. PMID:23555001
AMA1-Deficient Toxoplasma gondii Parasites Transiently Colonize Mice and Trigger an Innate Immune Response That Leads to Long-Lasting Protective Immunity

PubMed Central

Lagal, Vanessa; Dinis, Márcia; Cannella, Dominique; Bargieri, Daniel; Gonzalez, Virginie; Andenmatten, Nicole; Meissner, Markus

2015-01-01

The apical membrane antigen 1 (AMA1) protein was believed to be essential for the perpetuation of two Apicomplexa parasite genera, Plasmodium and Toxoplasma, until we genetically engineered viable parasites lacking AMA1. The reduction in invasiveness of the Toxoplasma gondii RH-AMA1 knockout (RH-AMA1KO) tachyzoite population, in vitro, raised key questions about the outcome associated with these tachyzoites once inoculated in the peritoneal cavity of mice. In this study, we used AMNIS technology to simultaneously quantify and image the parasitic process driven by AMA1KO tachyzoites. We report their ability to colonize and multiply in mesothelial cells and in both resident and recruited leukocytes. While the RH-AMA1KO population amplification is rapidly lethal in immunocompromised mice, it is controlled in immunocompetent hosts, where immune cells in combination sense parasites and secrete proinflammatory cytokines. This innate response further leads to a long-lasting status immunoprotective against a secondary challenge by high inocula of the homologous type I or a distinct type II T. gondii genotypes. While AMA1 is definitively not an essential protein for tachyzoite entry and multiplication in host cells, it clearly assists the expansion of parasite population in vivo. PMID:25847964
Whole-genome and multisector exome sequencing of primary and post-treatment glioblastoma reveals patterns of tumor evolution

PubMed Central

Kim, Hoon; Zheng, Siyuan; Amini, Seyed S.; Virk, Selene M.; Mikkelsen, Tom; Brat, Daniel J.; Grimsby, Jonna; Sougnez, Carrie; Muller, Florian; Hu, Jian; Sloan, Andrew E.; Cohen, Mark L.; Van Meir, Erwin G.; Scarpace, Lisa; Laird, Peter W.; Weinstein, John N.; Lander, Eric S.; Gabriel, Stacey; Getz, Gad; Meyerson, Matthew; Chin, Lynda; Barnholtz-Sloan, Jill S.

2015-01-01

Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ∼7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity. PMID:25650244
Digital gene expression approach over multiple RNA-Seq data sets to detect neoblast transcriptional changes in Schmidtea mediterranea.

PubMed

Rodríguez-Esteban, Gustavo; González-Sastre, Alejandro; Rojo-Laguna, José Ignacio; Saló, Emili; Abril, Josep F

2015-05-08

The freshwater planarian Schmidtea mediterranea is recognised as a valuable model for research into adult stem cells and regeneration. With the advent of the high-throughput sequencing technologies, it has become feasible to undertake detailed transcriptional analysis of its unique stem cell population, the neoblasts. Nonetheless, a reliable reference for this type of studies is still lacking. Taking advantage of digital gene expression (DGE) sequencing technology we compare all the available transcriptomes for S. mediterranea and improve their annotation. These results are accessible via web for the community of researchers. Using the quantitative nature of DGE, we describe the transcriptional profile of neoblasts and present 42 new neoblast genes, including several cancer-related genes and transcription factors. Furthermore, we describe in detail the Smed-meis-like gene and the three Nuclear Factor Y subunits Smed-nf-YA, Smed-nf-YB-2 and Smed-nf-YC. DGE is a valuable tool for gene discovery, quantification and annotation. The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.
Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.

PubMed

Fatima, Soghra; Zhou, Sheng; Sorrentino, Brian P

2012-02-01

The side population phenotype is associated with the Hoechst dye efflux activity of the Abcg2 transporter and identifies hematopoietic stem cells (HSCs) in the bone marrow. This association suggests the direct use of Abcg2 expression to identify adult stem cells in various other organs. We have generated a lineage tracing mouse model based on an allele that coexpresses both Abcg2 and a CreERT2 expression cassette. By crossing these mice with lox-STOP-lox reporter lines (LacZ or YFP), cells that express Abcg2 and their progeny were identified following treatment with tamoxifen (Tam). In the liver and kidney, in which mature cells express Abcg2, reporter gene expression verified the expected physiologic expression pattern of the recombinant allele. Long-term marking of HSCs was seen in multiple peripheral blood lineages from adult mice, demonstrating that Abcg2(+) bone marrow HSCs contribute to steady-state hematopoiesis. Stem cell tracing patterns were seen in the small intestine and in seminiferous tubules in the testis 20 months after Tam treatment, proving that stem cells from these organs express Abcg2. Interstitial cells from skeletal and cardiac muscle were labeled, and some cells were costained with endothelial markers, raising the possibility that these cells may function in the repair response to muscle injury. Altogether, these studies prove that Abcg2 is a stem cell marker for blood, small intestine, testicular germ cells, and possibly for injured skeletal and/or cardiac muscle and provide a new model for studying stem cell activity that does not require transplant-based assays. Copyright © 2011 AlphaMed Press.
The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

PubMed Central

Maisnier-Patin, Sophie; Roth, John R.

2015-01-01

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac+) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac+ triggers exponential cell growth leading to a stable Lac+ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac+ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac+ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions. PMID:26134316
Analysis of cellular autofluorescence in touch samples by flow cytometry: implications for front end separation of trace mixture evidence.

PubMed

Katherine Philpott, M; Stanciu, Cristina E; Kwon, Ye Jin; Bustamante, Eduardo E; Greenspoon, Susan A; Ehrhardt, Christopher J

2017-07-01

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650-670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor's contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two-person touch mixture was separated into two fractions via fluorescence-activated cell sorting (FACS) using gating criteria based on intensity of 650-670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures. Graphical Abstract Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.
Flow cytometry: basic principles and applications.

PubMed

Adan, Aysun; Alizada, Günel; Kiraz, Yağmur; Baran, Yusuf; Nalbant, Ayten

2017-03-01

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.
Cloning higher plants from aseptically cultured tissues and cells

NASA Technical Reports Server (NTRS)

Krikorian, A. D.

1982-01-01

A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.
Epstein-Barr Viral Productive Amplification Reprograms Nuclear Architecture, DNA Replication and Histone Deposition

PubMed Central

Chiu, Ya-Fang; Sugden, Arthur U.; Sugden, Bill

2014-01-01

Summary The spontaneous transition of Epstein-Barr Virus (EBV) from latency to productive infection is infrequent, making its analysis in the resulting mixed cell populations difficult. We engineered cells to support this transition efficiently and developed EBV DNA variants that could be visualized and measured as fluorescent signals over multiple cell cycles. This approach revealed that EBV’s productive replication began synchronously for viral DNAs within a cell but asynchronously between cells. EBV DNA amplification was delayed until early S-phase and occurred in factories characterized by the absence of cellular DNA and histones, by a sequential redistribution of PCNA, and by localization away from the nuclear periphery. The earliest amplified DNAs lacked histones accompanying a decline in four histone chaperones. Thus, EBV transitions from being dependent on the cellular replication machinery during latency to commandeering both that machinery and nuclear structure for its own reproductive needs. PMID:24331459
ABCB5 maintains melanoma-initiating cells through a pro-inflammatory cytokine signaling circuit

PubMed Central

Wilson, Brian J.; Saab, Karim R.; Ma, Jie; Schatton, Tobias; Pütz, Pablo; Zhan, Qian; Murphy, George F.; Gasser, Martin; Waaga-Gasser, Ana Maria; Frank, Natasha Y.; Frank, Markus H.

2014-01-01

The drug efflux transporter ABCB5 identifies cancer stem-like cells (CSC) in diverse human malignancies, where its expression is associated with clinical disease progression and tumor recurrence. ABCB5 confers therapeutic resistance but other functions in tumorigenesis independent of drug efflux have not been described that might help explain why it is so broadly overexpressed in human cancer. Here we show that in melanoma-initiating cells ABCB5 controls IL-1β secretion which serves to maintain slow-cycling, chemoresistant cells through an IL-1β/IL8/CXCR1 cytokine signaling circuit. This CSC maintenance circuit involved reciprocal paracrine interactions with ABCB5-negative cancer cell populations. ABCB5 blockade induced cellular differentiation, reversed resistance to multiple chemotherapeutic agents, and impaired tumor growth in vivo. Together, our results defined a novel function for ABCB5 in CSC maintenance and tumor growth. PMID:24934811
Regenerative medicine using dental pulp stem cells for liver diseases.

PubMed

Ohkoshi, Shogo; Hara, Hajime; Hirono, Haruka; Watanabe, Kazuhiko; Hasegawa, Katsuhiko

2017-02-06

Acute liver failure is a refractory disease and its prognosis, if not treated using liver transplantation, is extremely poor. It is a good candidate for regenerative medicine, where stem cell-based therapies play a central role. Mesenchymal stem cells (MSCs) are known to differentiate into multiple cell lineages including hepatocytes. Autologous cell transplant without any foreign gene induction is feasible using MSCs, thereby avoiding possible risks of tumorigenesis and immune rejection. Dental pulp also contains an MSC population that differentiates into hepatocytes. A point worthy of special mention is that dental pulp can be obtained from deciduous teeth during childhood and can be subsequently harvested when necessary after deposition in a tooth bank. MSCs have not only a regenerative capacity but also act in an anti-inflammatory manner via paracrine mechanisms. Promising efficacies and difficulties with the use of MSC derived from teeth are summarized in this review.
Recent advances in the study of age-related hearing loss - A Mini-Review

PubMed Central

Kidd, Ambrose R; Bao, Jianxin

2013-01-01

Hearing loss is a common age-associated affliction that can result from the loss of hair cells and spiral ganglion neurons (SGNs) in the cochlea. Although hair cells and SGNs are typically lost in the same cochlea, recent analysis suggests that they can occur independently, via unique mechanisms. Research has identified both environmental and genetic factors that contribute to degeneration of cochlear cells. Additionally, molecular analysis has identified multiple cell signaling mechanisms that likely contribute to pathological changes that result in hearing deficiencies. These analyses should serve as useful primers for future work, including genomic and proteomic analysis, to elucidate the mechanisms driving cell loss in the aging cochlea. Significant progress in this field has occurred in the past decade. As our understanding of aging-induced cochlear changes continues to improve, our ability to offer medical intervention will surely benefit the growing elderly population. PMID:22710288
Evolution of resistance to anti-cancer therapy during general dosing schedules

PubMed Central

Foo, Jasmine; Michor, Franziska

2009-01-01

Anti-cancer drugs targeted to specific oncogenic pathways have shown promising therapeutic results in the past few years; however, drug resistance remains an important obstacle for these therapies. Resistance to these drugs can emerge due to a variety of reasons including genetic or epigenetic changes which alter the binding site of the drug target, cellular metabolism or export mechanisms. Obtaining a better understanding of the evolution of resistant populations during therapy may enable the design of more effective therapeutic regimens which prevent or delay progression of disease due to resistance. In this paper, we use stochastic mathematical models to study the evolutionary dynamics of resistance under time-varying dosing schedules and pharmacokinetic effects. The populations of sensitive and resistant cells are modeled as multi-type non-homogeneous birth-death processes in which the drug concentration affects the birth and death rates of both the sensitive and resistant cell populations in continuous time. This flexible model allows us to consider the effects of generalized treatment strategies as well as detailed pharmacokinetic phenomena such as drug elimination and accumulation over multiple doses. We develop estimates for the probability of developing resistance and moments of the size of the resistant cell population. With these estimates, we optimize treatment schedules over a subspace of tolerated schedules to minimize the risk of disease progression due to resistance as well as locate ideal schedules for controlling the population size of resistant clones in situations where resistance is inevitable. Our methodology can be used to describe dynamics of resistance arising due to a single (epi)genetic alteration in any tumor type. PMID:20004211

Characterization of Multiple Cytokine Combinations and TGF-β on Differentiation and Functions of Myeloid-Derived Suppressor Cells

PubMed Central

Lee, Cho-Rong; Lee, Wongeun; Cho, Steve K.; Park, Sung-Gyoo

2018-01-01

Myeloid-derived suppressor cells (MDSCs) regulate T cell immunity, and this population is a new therapeutic target for immune regulation. A previous study showed that transforming growth factor-β (TGF-β) is involved in controlling MDSC differentiation and immunoregulatory function in vivo. However, the direct effect of TGF-β on MDSCs with various cytokines has not previously been tested. Thus, we examined the effect of various cytokine combinations with TGF-β on MDSCs derived from bone marrow cells. The data show that different cytokine combinations affect the differentiation and immunosuppressive functions of MDSCs in different ways. In the presence of TGF-β, interleukin-6 (IL-6) was the most potent enhancer of MDSC function, whereas granulocyte colony-stimulating factors (G-CSF) was the most potent in the absence of TGF-β. In addition, IL-4 maintained MDSCs in an immature state with an increased expression of arginase 1 (Arg1). However, regardless of the cytokine combinations, TGF-β increased expansion of the monocytic MDSC (Mo-MDSC) population, expression of immunosuppressive molecules by MDSCs, and the ability of MDSCs to suppress CD4+ T cell proliferation. Thus, although different cytokine combinations affected the MDSCs in different ways, TGF-β directly affects monocytic-MDSCs (Mo-MDSCs) expansion and MDSCs functions. PMID:29543758
Single-trait and multi-trait genome-wide association analyses identify novel loci for blood pressure in African-ancestry populations

PubMed Central

Liang, Jingjing; Le, Thu H.; Edwards, Digna R. Velez; Tayo, Bamidele O.; Gaulton, Kyle J.; Lu, Yingchang; Jensen, Richard A.; Chen, Guanjie; Schwander, Karen; McKenzie, Colin A.; Fox, Ervin; Nalls, Michael A.; Young, J. Hunter; Lane, Jacqueline M.; Zhou, Jie; Tang, Hua; Fornage, Myriam; Musani, Solomon K.; Wang, Heming; Forrester, Terrence; Chu, Pei-Lun; Evans, Michele K.; Morrison, Alanna C.; Martin, Lisa W.; Wiggins, Kerri L.; Hui, Qin; Zhao, Wei; Jackson, Rebecca D.; Faul, Jessica D.; Reiner, Alex P.; Bray, Michael; Denny, Joshua C.; Mosley, Thomas H.; Palmas, Walter; Guo, Xiuqing; Polak, Joseph F.; Taylor, Ken D.; Boerwinkle, Eric; Bottinger, Erwin P.; Liu, Kiang; Risch, Neil; Hunt, Steven C.; Kooperberg, Charles; Zonderman, Alan B.; Becker, Diane M.; Cai, Jianwen; Loos, Ruth J. F.; Psaty, Bruce M.; Weir, David R.; Kardia, Sharon L. R.; Arnett, Donna K.; Won, Sungho; Edwards, Todd L.; Redline, Susan; Cooper, Richard S.; Rao, D. C.; Rotimi, Charles; Levy, Daniel; Chakravarti, Aravinda

2017-01-01

Hypertension is a leading cause of global disease, mortality, and disability. While individuals of African descent suffer a disproportionate burden of hypertension and its complications, they have been underrepresented in genetic studies. To identify novel susceptibility loci for blood pressure and hypertension in people of African ancestry, we performed both single and multiple-trait genome-wide association analyses. We analyzed 21 genome-wide association studies comprised of 31,968 individuals of African ancestry, and validated our results with additional 54,395 individuals from multi-ethnic studies. These analyses identified nine loci with eleven independent variants which reached genome-wide significance (P < 1.25×10−8) for either systolic and diastolic blood pressure, hypertension, or for combined traits. Single-trait analyses identified two loci (TARID/TCF21 and LLPH/TMBIM4) and multiple-trait analyses identified one novel locus (FRMD3) for blood pressure. At these three loci, as well as at GRP20/CDH17, associated variants had alleles common only in African-ancestry populations. Functional annotation showed enrichment for genes expressed in immune and kidney cells, as well as in heart and vascular cells/tissues. Experiments driven by these findings and using angiotensin-II induced hypertension in mice showed altered kidney mRNA expression of six genes, suggesting their potential role in hypertension. Our study provides new evidence for genes related to hypertension susceptibility, and the need to study African-ancestry populations in order to identify biologic factors contributing to hypertension. PMID:28498854
Linear viral load increase of a single HPV-type in women with multiple HPV infections predicts progression to cervical cancer.

PubMed

Depuydt, Christophe E; Thys, Sofie; Beert, Johan; Jonckheere, Jef; Salembier, Geert; Bogers, Johannes J

2016-11-01

Persistent high-risk human papillomavirus (HPV) infection is strongly associated with development of high-grade cervical intraepithelial neoplasia or cancer (CIN3+). In single type infections, serial type-specific viral-load measurements predict the natural history of the infection. In infections with multiple HPV-types, the individual type-specific viral-load profile could distinguish progressing HPV-infections from regressing infections. A case-cohort natural history study was established using samples from untreated women with multiple HPV-infections who developed CIN3+ (n = 57) or cleared infections (n = 88). Enriched cell pellet from liquid based cytology samples were subjected to a clinically validated real-time qPCR-assay (18 HPV-types). Using serial type-specific viral-load measurements (≥3) we calculated HPV-specific slopes and coefficient of determination (R(2) ) by linear regression. For each woman slopes and R(2) were used to calculate which HPV-induced processes were ongoing (progression, regression, serial transient, transient). In transient infections with multiple HPV-types, each single HPV-type generated similar increasing (0.27copies/cell/day) and decreasing (-0.27copies/cell/day) viral-load slopes. In CIN3+, at least one of the HPV-types had a clonal progressive course (R(2) ≥ 0.85; 0.0025copies/cell/day). In selected CIN3+ cases (n = 6), immunostaining detecting type-specific HPV 16, 31, 33, 58 and 67 RNA showed an even staining in clonal populations (CIN3+), whereas in transient virion-producing infections the RNA-staining was less in the basal layer compared to the upper layer where cells were ready to desquamate and release newly-formed virions. RNA-hybridization patterns matched the calculated ongoing processes measured by R(2) and slope in serial type-specific viral-load measurements preceding the biopsy. In women with multiple HPV-types, serial type-specific viral-load measurements predict the natural history of the different HPV-types and elucidates HPV-genotype attribution. © 2016 UICC.
Mechanisms of Microwave Induced Damage in Biologic Materials

DTIC Science & Technology

1988-01-25

display senescence after multiple population doublings (14). Fibroblasts of Clone 12 BE, derived from a patient with the disease xeroderma pigmentosum ...were also employed. The xeroderma pigmentosum (XP) cells are deficient in DNA repair (6), a genetic defect which produced the disease condition in the...SARs as high as 1500-1600 mW/g. 2.3.3 Xeroderma Pigmentosum (Clone 12BE) Irradiations To supplement the CW data on IMR-90 an additional set of
Cutaneous and diphtheritic avian poxvirus infection in a nestling Southern Giant Petrel (Macronectes giganteus) from Antarctica

USGS Publications Warehouse

Shearn-Bochsler, Valerie; Green, David Earl; Converse, K.A.; Docherty, D.E.; Thiel, T.; Geisz, H.N.; Fraser, William R.; Patterson-Fraser, Donna L.

2008-01-01

The Southern giant petrel (Macronectes giganteus) is declining over much of its range and currently is listed as vulnerable to extinction by the International Union for the Conservation of Nature (IUCN). Island-specific breeding colonies near Palmer Station, Antarctica, have been monitored for over 30 years, and because this population continues to increase, it is critically important to conservation. In austral summer 2004, six diseased giant petrel chicks were observed in four of these colonies. Diseased chicks were 6a??9 weeks old and had multiple proliferative nodules on their bills and skin. One severely affected chick was found dead on the nest and was salvaged for necropsy. Histopathological examination of nodules from the dead chick revealed epithelial cell hyperplasia and hypertrophy with numerous eosinophilic intracytoplasmic inclusions (B??llinger bodies). A poxvirus was isolated from multiple nodules. Poxviral infection has not been reported in this species, and the reason for its emergence and its potential impact on the population are not yet known.
CRISPR-based herd immunity can limit phage epidemics in bacterial populations

PubMed Central

Geyrhofer, Lukas; Barton, Nicholas H

2018-01-01

Herd immunity, a process in which resistant individuals limit the spread of a pathogen among susceptible hosts has been extensively studied in eukaryotes. Even though bacteria have evolved multiple immune systems against their phage pathogens, herd immunity in bacteria remains unexplored. Here we experimentally demonstrate that herd immunity arises during phage epidemics in structured and unstructured Escherichia coli populations consisting of differing frequencies of susceptible and resistant cells harboring CRISPR immunity. In addition, we develop a mathematical model that quantifies how herd immunity is affected by spatial population structure, bacterial growth rate, and phage replication rate. Using our model we infer a general epidemiological rule describing the relative speed of an epidemic in partially resistant spatially structured populations. Our experimental and theoretical findings indicate that herd immunity may be important in bacterial communities, allowing for stable coexistence of bacteria and their phages and the maintenance of polymorphism in bacterial immunity. PMID:29521625
Sexy males and sexless females: the origin of triploid apomicts.

PubMed

Muralidhar, P; Haig, D

2017-05-01

Apomixis and polyploidy are closely associated in angiosperms, but the evolutionary reason for this association is unknown. Taraxacum officinale, the common dandelion, exists both as diploid sexuals and triploid apomicts. Here, in the context of T. officinale, we provide a model of the evolution of triploid apomicts from diploid sexuals. We posit an apomictic allele that arrests female meiosis in diploids, so that the plant produces diploid egg cells that can develop without fertilization, but haploid pollen. We propose occasional fertilization of diploid egg cells by haploid pollen, resulting in triploid apomicts that produce triploid egg cells but largely nonfunctional pollen. The irreversibility of this process renders diploid partial apomicts evolutionarily short-lived, and results in fixation of triploid apomicts except when they suffer extreme selective disadvantages. Our model can account for the high genetic diversity found in T. officinale triploid populations, because recombinant haploid pollen produced by diploids allows the apomictic allele to spread onto many genetic backgrounds. This leads to multiple clonal lineages in the newly apomictic population, and thereby alleviates some of the usual pitfalls of asexual reproduction.
Resveratrol and Malignancies

PubMed Central

Bunaciu, Rodica P.

2015-01-01

Carcinogenesis is a multifactorial process, frequently encompassing 3 stages: initiation, promotion and progression. It is characterized by multiple deviations from normal both at the cell and organism levels. Although most people have a small number of cells that present deviations from normal, most of those cells will not cause cancer. However, some will. What tips the balance between normal and abnormal is the subject of intense scientific research as well as unfounded speculations. Chronic inflammation is one of the risk factors for cancer. Resveratrol is consumed by the population as a dietary supplement in the hope of decreasing the risk of inflammation and cancer and other chronic diseases such as diabetes and vascular diseases. There is a discrepancy between the doses used in the animal studies showing that resveratrol decreases all three stages of carcinogenesis, and the doses ingested by the population either as supplements or in the diet. While there is health benefit from using high resveratrol doses, it might be also of practical and scientific benefit to focus future effort in understanding the effects of normal dietary resveratrol levels. PMID:26478855
[Therapy of multiple myeloma: indications and options].

PubMed

Peest, D; Ganser, A

2007-12-01

The multiple myeloma (MM) has an incidence of 3-4/100,000 in the Caucasian population. MM has to be distinguished from smouldering MM and monoclonal gammopathy of uncertain significance (MGUS). In younger patients (<65 years) a good long-term remission is the aim of therapy, while in the elderly patients with comorbidities the aim is a good partial remission with good quality of life. In the elderly this can be achieved with a combination of melphalan and prednisone. High-dose chemotherapy, often as a tandem transplantation, is part of standard therapy of MM patients <65 years. However, allogeneic stem cell transplantation is the only curative approach. New substances approved for treatment of relapsed MM include bortezomib, thalidomide, and lenalidomide.
Innate lymphoid cells and natural killer T cells in the gastrointestinal tract immune system.

PubMed

Montalvillo, Enrique; Garrote, José Antonio; Bernardo, David; Arranz, Eduardo

2014-05-01

The gastrointestinal tract is equipped with a highly specialized intrinsic immune system. However, the intestine is exposed to a high antigenic burden that requires a fast, nonspecific response -so-called innate immunity- to maintain homeostasis and protect the body from incoming pathogens. In the last decade multiple studies helped to unravel the particular developmental requirements and specific functions of the cells that play a role in innate immunity. In this review we shall focus on innate lymphoid cells, a newly discovered, heterogeneous set of cells that derive from an Id2-dependent lymphoid progenitor cell population. These cells have been categorized on the basis of the pattern of cytokines that they secrete, and the transcription factors that regulate their development and functions. Innate lymphoid cells play a role in the early response to pathogens, the anatomical contention of the commensal flora, and the maintenance of epithelial integrity.Amongst the various innate lymphoid cells we shall lay emphasis on a subpopulation with several peculiarities, namely that of natural killer T cells, a subset of T lymphocytes that express both T-cell and NK-cell receptors. The most numerous fraction of the NKT population are the so-called invariant NKT or iNKT cells. These iNKT cells have an invariant TCR and recognize the glycolipidic structures presented by the CD1d molecule, a homolog of class-I MHC molecules. Following activation they rapidly acquire cytotoxic activity and secrete both Th1 and Th2 cytokines, including IL-17. While their specific role is not yet established, iNKT cells take part in a great variety of intestinal immune responses ranging from oral tolerance to involvement in a number of gastrointestinal conditions.
Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.

PubMed

Sharrow, Allison C; Perkins, Brandy; Collector, Michael I; Yu, Wayne; Simons, Brian W; Jones, Richard J

2016-08-01

The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDH(high)) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDH(high) cells in ovarian cancer. We compared ALDH(high) to ALDH(low) cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. ALDH(high) cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDH(high) cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDH(high) cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. Based on functional characterization, ALDH(high) ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes. Copyright © 2016 Elsevier Inc. All rights reserved.
Genetic Heterogeneity and Clonal Evolution of Tumor Cells and their Impact on Precision Cancer Medicine.

PubMed

Sabaawy, Hatem E

2013-11-18

The efficacy of targeted therapies in leukemias and solid tumors depends upon the accurate detection and sustained targeting of initial and evolving driver mutations and/or aberrations in cancer cells. Tumor clonal evolution of the diverse populations of cancer cells during cancer progression contributes to the longitudinal variations of clonal, morphological, anatomical, and molecular heterogeneity of tumors. Moreover, drug-resistant subclones present at initiation of therapy or emerging as a result of targeted therapies represent major challenges for achieving success of personalized therapies in providing meaningful improvement in cancer survival rates. Here, I briefly portray tumor cell clonal evolution at the cellular and molecular levels, and present the multiple types of genetic heterogeneity in tumors, with a focus on their impact on the implementation of personalized or precision cancer medicine.
Complex chimerism

PubMed Central

Ma, Kimberly K.; Petroff, Margaret G.; Coscia, Lisa A.; Armenti, Vincent T.; Adams Waldorf, Kristina M.

2013-01-01

Thousands of women with organ transplantation have undergone successful pregnancies, however little is known about how the profound immunologic changes associated with pregnancy might influence tolerance or rejection of the allograft. Pregnant women with a solid organ transplant are complex chimeras with multiple foreign cell populations from the donor organ, fetus, and mother of the pregnant woman. We consider the impact of complex chimerism and pregnancy-associated immunologic changes on tolerance of the allograft both during pregnancy and the postpartum period. Mechanisms of allograft tolerance are likely dynamic during pregnancy and affected by the influx of fetal microchimeric cells, HLA relationships (between the fetus, pregnant woman and/or donor), peripheral T cell tolerance to fetal cells, and fetal minor histocompatibility antigens. Further research is necessary to understand the complex immunology during pregnancy and the postpartum period of women with a solid organ transplant. PMID:23974274
Entropy production in a photovoltaic cell

NASA Astrophysics Data System (ADS)

Ansari, Mohammad H.

2017-05-01

We evaluate entropy production in a photovoltaic cell that is modeled by four electronic levels resonantly coupled to thermally populated field modes at different temperatures. We use a formalism recently proposed, the so-called multiple parallel worlds, to consistently address the nonlinearity of entropy in terms of density matrix. Our result shows that entropy production is the difference between two flows: a semiclassical flow that linearly depends on occupational probabilities, and another flow that depends nonlinearly on quantum coherence and has no semiclassical analog. We show that entropy production in the cells depends on environmentally induced decoherence time and energy detuning. We characterize regimes where reversal flow of information takes place from a cold to hot bath. Interestingly, we identify a lower bound on entropy production, which sets limitations on the statistics of dissipated heat in the cells.
Minute perivascular epithelioid cell (PEC) nests in the abdominal lymph nodes--a putative precursor of PEComa.

PubMed

Nagasaka, Toru; Murakami, Yoshiko; Sasaki, Eiichi; Hosoda, Waki; Nakanishi, Toru; Yatabe, Yasushi

2015-04-01

A perivascular epithelioid cell tumor (PEComa) is a peculiar growth defined as a mesenchymal tumor composed of histologically and immunohistochemically distinct perivascular epithelioid cells (PECs). Because neither normal counterparts nor precursor lesions of PEComa have been identified, we examined minute PEC nests, ranged from 0.8 mm to 10 mm, to investigate the possible origin of the PEComa. We examined a total of 80 677 para-aortic and pelvic lymph nodes that were systematically dissected from 1656 patients for gynecological malignancies. The identified lesions were confirmed immunohistochemically with multiple PEC markers, including smooth muscle actin, HMB45, melan-A, MiTF, ER and PgR. A total of 66 minute PEC nests were found in 21 patients (1.3% of the total population) with an average frequency of 3.1 lesions per patient. In cases of multiple involvement, 11 of 13 nests were located at the same level of multiple lymph node or on continuous levels. The lesions were preferentially distributed at the level of para-aortic and high pelvic lymph nodes. All nests were positive for actin and HMB45, whereas the other markers were positive with varying frequencies. The minute PEC nests may be associated with the possible normal counterpart of PEComas. © 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.
Myeloid-derived suppressor cells in multiple myeloma: pre-clinical research and translational opportunities.

PubMed

Botta, Cirino; Gullà, Annamaria; Correale, Pierpaolo; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

2014-01-01

Immunosuppressive cells have been reported to play an important role in tumor-progression mainly because of their capability to promote immune-escape, angiogenesis, and metastasis. Among them, myeloid-derived suppressor cells (MDSCs) have been recently identified as immature myeloid cells, induced by tumor-associated inflammation, able to impair both innate and adaptive immunity. While murine MDSCs are usually identified by the expression of CD11b and Gr1, human MDSCs represent a more heterogeneous population characterized by the expression of CD33 and CD11b, low or no HLA-DR, and variable CD14 and CD15. In particular, the last two may alternatively identify monocyte-like or granulocyte-like MDSC subsets with different immunosuppressive properties. Recently, a substantial increase of MDSCs has been found in peripheral blood and bone marrow (BM) of multiple myeloma (MM) patients with a role in disease progression and/or drug resistance. Pre-clinical models recapitulating the complexity of the MM-related BM microenvironment (BMM) are major tools for the study of the interactions between MM cells and cells of the BMM (including MDSCs) and for the development of new agents targeting MM-associated immune-suppressive cells. This review will focus on current strategies for human MDSCs generation and investigation of their immunosuppressive function in vitro and in vivo, taking into account the relevant relationship occurring within the MM-BMM. We will then provide trends in MDSC-associated research and suggest potential application for the treatment of MM.
Realgar nanoparticles versus ATO arsenic compounds induce in vitro and in vivo activity against multiple myeloma.

PubMed

Cholujova, Danka; Bujnakova, Zdenka; Dutkova, Erika; Hideshima, Teru; Groen, Richard W; Mitsiades, Constantine S; Richardson, Paul G; Dorfman, David M; Balaz, Peter; Anderson, Kenneth C; Jakubikova, Jana

2017-12-01

Multiple myeloma (MM), a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow, remains incurable despite the use of novel and conventional therapies. In this study, we demonstrated MM cell cytotoxicity triggered by realgar (REA; As 4 S 4 ) nanoparticles (NREA) versus Arsenic trioxide (ATO) against MM cell lines and patient cells. Both NREA and ATO showed in vivo anti-MM activity, resulting in significantly decreased tumour burden. The anti-MM activity of NREA and ATO is associated with apoptosis, evidenced by DNA fragmentation, depletion of mitochondrial membrane potential, cleavage of caspases and anti-apoptotic proteins. NREA induced G 2 /M cell cycle arrest and modulation of cyclin B1, p53 (TP53), p21 (CDKN1A), Puma (BBC3) and Wee-1 (WEE1). Moreover, NREA induced modulation of key regulatory molecules in MM pathogenesis including JNK activation, c-Myc (MYC), BRD4, and histones. Importantly, NREA, but not ATO, significantly depleted the proportion and clonogenicity of the MM stem-like side population, even in the context of the bone marrow stromal cells. Finally, our study showed that both NREA and ATO triggered synergistic anti-MM activity when combined with lenalidomide or melphalan. Taken together, the anti-MM activity of NREA was more potent compared to ATO, providing the preclinical framework for clinical trials to improve patient outcome in MM. © 2017 John Wiley & Sons Ltd.
Multiparameter Cell Cycle Analysis.

PubMed

Jacobberger, James W; Sramkoski, R Michael; Stefan, Tammy; Woost, Philip G

2018-01-01

Cell cycle cytometry and analysis are essential tools for studying cells of model organisms and natural populations (e.g., bone marrow). Methods have not changed much for many years. The simplest and most common protocol is DNA content analysis, which is extensively published and reviewed. The next most common protocol, 5-bromo-2-deoxyuridine S phase labeling detected by specific antibodies, is also well published and reviewed. More recently, S phase labeling using 5'-ethynyl-2'-deoxyuridine incorporation and a chemical reaction to label substituted DNA has been established as a basic, reliable protocol. Multiple antibody labeling to detect epitopes on cell cycle regulated proteins, which is what this chapter is about, is the most complex of these cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. However, because this knowledge is relatively well presented methodologically in many papers and reviews, this chapter will present a minimal Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature. Most of the presented work involves how to segment the data to produce a complete, progressive, and compartmentalized cell cycle analysis from early G1 to late mitosis (telophase). A more recent development, using fluorescent proteins fused with proteins or peptides that are degraded by ubiquitination during specific periods of the cell cycle, termed "Fucci" (fluorescent, ubiquitination-based cell cycle indicators) provide an analysis similar in concept to multiple antibody labeling, except in this case cells can be analyzed while living and transgenic organisms can be created to perform cell cycle analysis ex or in vivo (Sakaue-Sawano et al., Cell 132:487-498, 2007). This technology will not be discussed.
Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells.

PubMed

Ebeid, Michael; Sripal, Prashanth; Pecka, Jason; Beisel, Kirk W; Kwan, Kelvin; Soukup, Garrett A

2017-01-01

Over 5% of the global population suffers from disabling hearing loss caused by multiple factors including aging, noise exposure, genetic predisposition, or use of ototoxic drugs. Sensorineural hearing loss is often caused by the loss of sensory hair cells (HCs) of the inner ear. A barrier to hearing restoration after HC loss is the limited ability of mammalian auditory HCs to spontaneously regenerate. Understanding the molecular mechanisms orchestrating HC development is expected to facilitate cell replacement therapies. Multiple events are known to be essential for proper HC development including the expression of Atoh1 transcription factor and the miR-183 family. We have developed a series of vectors expressing the miR-183 family and/or Atoh1 that was used to transfect two different developmental cell models: pluripotent mouse embryonic stem cells (mESCs) and immortalized multipotent otic progenitor (iMOP) cells representing an advanced developmental stage. Transcriptome profiling of transfected cells show that the impact of Atoh1 is contextually dependent with more HC-specific effects on iMOP cells. miR-183 family expression in combination with Atoh1 not only appears to fine tune gene expression in favor of HC fate, but is also required for the expression of some HC-specific genes. Overall, the work provides novel insight into the combined role of Atoh1 and the miR-183 family during HC development that may ultimately inform strategies to promote HC regeneration or maintenance.
Intersections of lung progenitor cells, lung disease and lung cancer.

PubMed

Kim, Carla F

2017-06-30

The use of stem cell biology approaches to study adult lung progenitor cells and lung cancer has brought a variety of new techniques to the field of lung biology and has elucidated new pathways that may be therapeutic targets in lung cancer. Recent results have begun to identify the ways in which different cell populations interact to regulate progenitor activity, and this has implications for the interventions that are possible in cancer and in a variety of lung diseases. Today's better understanding of the mechanisms that regulate lung progenitor cell self-renewal and differentiation, including understanding how multiple epigenetic factors affect lung injury repair, holds the promise for future better treatments for lung cancer and for optimising the response to therapy in lung cancer. Working between platforms in sophisticated organoid culture techniques, genetically engineered mouse models of injury and cancer, and human cell lines and specimens, lung progenitor cell studies can begin with basic biology, progress to translational research and finally lead to the beginnings of clinical trials. Copyright ©ERS 2017.

Integrating single-cell transcriptomic data across different conditions, technologies, and species.

PubMed

Butler, Andrew; Hoffman, Paul; Smibert, Peter; Papalexi, Efthymia; Satija, Rahul

2018-06-01

Computational single-cell RNA-seq (scRNA-seq) methods have been successfully applied to experiments representing a single condition, technology, or species to discover and define cellular phenotypes. However, identifying subpopulations of cells that are present across multiple data sets remains challenging. Here, we introduce an analytical strategy for integrating scRNA-seq data sets based on common sources of variation, enabling the identification of shared populations across data sets and downstream comparative analysis. We apply this approach, implemented in our R toolkit Seurat (http://satijalab.org/seurat/), to align scRNA-seq data sets of peripheral blood mononuclear cells under resting and stimulated conditions, hematopoietic progenitors sequenced using two profiling technologies, and pancreatic cell 'atlases' generated from human and mouse islets. In each case, we learn distinct or transitional cell states jointly across data sets, while boosting statistical power through integrated analysis. Our approach facilitates general comparisons of scRNA-seq data sets, potentially deepening our understanding of how distinct cell states respond to perturbation, disease, and evolution.
Molecular machinery of signal transduction and cell cycle regulation in Plasmodium.

PubMed

Koyama, Fernanda C; Chakrabarti, Debopam; Garcia, Célia R S

2009-05-01

The regulation of the Plasmodium cell cycle is not understood. Although the Plasmodium falciparum genome is completely sequenced, about 60% of the predicted proteins share little or no sequence similarity with other eukaryotes. This feature impairs the identification of important proteins participating in the regulation of the cell cycle. There are several open questions that concern cell cycle progression in malaria parasites, including the mechanism by which multiple nuclear divisions is controlled and how the cell cycle is managed in all phases of their complex life cycle. Cell cycle synchrony of the parasite population within the host, as well as the circadian rhythm of proliferation, are striking features of some Plasmodium species, the molecular basis of which remains to be elucidated. In this review we discuss the role of indole-related molecules as signals that modulate the cell cycle in Plasmodium and other eukaryotes, and we also consider the possible role of kinases in the signal transduction and in the responses it triggers.
Emergence of increased frequency and severity of multiple infections by viruses due to spatial clustering of hosts

NASA Astrophysics Data System (ADS)

Taylor, Bradford P.; Penington, Catherine J.; Weitz, Joshua S.

2016-12-01

Multiple virus particles can infect a target host cell. Such multiple infections (MIs) have significant and varied ecological and evolutionary consequences for both virus and host populations. Yet, the in situ rates and drivers of MIs in virus-microbe systems remain largely unknown. Here, we develop an individual-based model (IBM) of virus-microbe dynamics to probe how spatial interactions drive the frequency and nature of MIs. In our IBMs, we identify increasingly spatially correlated clusters of viruses given sufficient decreases in viral movement. We also identify increasingly spatially correlated clusters of viruses and clusters of hosts given sufficient increases in viral infectivity. The emergence of clusters is associated with an increase in multiply infected hosts as compared to expectations from an analogous mean field model. We also observe long-tails in the distribution of the multiplicity of infection in contrast to mean field expectations that such events are exponentially rare. We show that increases in both the frequency and severity of MIs occur when viruses invade a cluster of uninfected microbes. We contend that population-scale enhancement of MI arises from an aggregate of invasion dynamics over a distribution of microbe cluster sizes. Our work highlights the need to consider spatially explicit interactions as a potentially key driver underlying the ecology and evolution of virus-microbe communities.
Generation of mammalian cells stably expressing multiple genes at predetermined levels.

PubMed

Liu, X; Constantinescu, S N; Sun, Y; Bogan, J S; Hirsch, D; Weinberg, R A; Lodish, H F

2000-04-10

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.
PDGF-responsive progenitors persist in the subventricular zone across the lifespan

PubMed Central

Moore, Lisamarie; Bain, Jennifer M.; Loh, Ji Meng; Levison, Steven W.

2013-01-01

The SVZ (subventricular zone) contains neural stem cells and progenitors of various potentialities. Although initially parsed into A, B, and C cells, this germinal zone is comprised of a significantly more diverse population of cells. Here, we characterized a subset of postnatal PRPs (PDGF-AA-responsive precursors) that express functional PDGFα and β receptors from birth to adulthood. When grown in PDGF-AA, dissociated neonatal rat SVZ cells divided to produce non-adherent clusters of progeny. Unlike the self-renewing EGF/FGF-2-responsive precursors that produce neurospheres, these PRPs failed to self-renew after three passages; therefore, we refer to the colonies they produce as spheroids. Upon differentiation these spheroids could produce neurons, type 1 astrocytes and oligodendrocytes. When maintained in medium supplemented with BMP-4 they also produced type 2 astrocytes. Using lineage tracing methods, it became evident that there were multiple types of PRPs, including a subset that could produce neurons, oligodendrocytes, and type 1 and type 2 astrocytes; thus some of these PRPs represent a unique population of precursors that are quatropotential. Spheroids also could be generated from the newborn neocortex and they had the same potentiality as those from the SVZ. By contrast, the adult neocortex produced less than 20% of the numbers of spheroids than the adult SVZ and spheroids from the adult neocortex only differentiated into glial cells. Interestingly, SVZ spheroid producing capacity diminished only slightly from birth to adulthood. Altogether these data demonstrate that there are PRPs that persist in the SVZ that includes a unique population of quatropotential PRPs. PMID:24367913
Critical and maximally informative encoding between neural populations in the retina

PubMed Central

Kastner, David B.; Baccus, Stephen A.; Sharpee, Tatyana O.

2015-01-01

Computation in the brain involves multiple types of neurons, yet the organizing principles for how these neurons work together remain unclear. Information theory has offered explanations for how different types of neurons can maximize the transmitted information by encoding different stimulus features. However, recent experiments indicate that separate neuronal types exist that encode the same filtered version of the stimulus, but then the different cell types signal the presence of that stimulus feature with different thresholds. Here we show that the emergence of these neuronal types can be quantitatively described by the theory of transitions between different phases of matter. The two key parameters that control the separation of neurons into subclasses are the mean and standard deviation (SD) of noise affecting neural responses. The average noise across the neural population plays the role of temperature in the classic theory of phase transitions, whereas the SD is equivalent to pressure or magnetic field, in the case of liquid–gas and magnetic transitions, respectively. Our results account for properties of two recently discovered types of salamander Off retinal ganglion cells, as well as the absence of multiple types of On cells. We further show that, across visual stimulus contrasts, retinal circuits continued to operate near the critical point whose quantitative characteristics matched those expected near a liquid–gas critical point and described by the nearest-neighbor Ising model in three dimensions. By operating near a critical point, neural circuits can maximize information transmission in a given environment while retaining the ability to quickly adapt to a new environment. PMID:25675497
A multiplex culture system for the long‐term growth of fission yeast cells

PubMed Central

Callens, Céline; Coelho, Nelson C.; Miller, Aaron W.; Sananes, Maria Rosa Domingo; Dunham, Maitreya J.; Denoual, Matthieu

2017-01-01

Abstract Maintenance of long‐term cultures of yeast cells is central to a broad range of investigations, from metabolic studies to laboratory evolution assays. However, repeated dilutions of batch cultures lead to variations in medium composition, with implications for cell physiology. In Saccharomyces cerevisiae, powerful miniaturized chemostat setups, or ministat arrays, have been shown to allow for constant dilution of multiple independent cultures. Here we set out to adapt these arrays for continuous culture of a morphologically and physiologically distinct yeast, the fission yeast Schizosaccharomyces pombe, with the goal of maintaining constant population density over time. First, we demonstrated that the original ministats are incompatible with growing fission yeast for more than a few generations, prompting us to modify different aspects of the system design. Next, we identified critical parameters for sustaining unbiased vegetative growth in these conditions. This requires deletion of the gsf2 flocculin‐encoding gene, along with addition of galactose to the medium and lowering of the culture temperature. Importantly, we improved the flexibility of the ministats by developing a piezo‐pump module for the independent regulation of the dilution rate of each culture. This made it possible to easily grow strains that have different generation times in the same assay. Our system therefore allows for maintaining multiple fission yeast cultures in exponential growth, adapting the dilution of each culture over time to keep constant population density for hundreds of generations. These multiplex culture systems open the door to a new range of long‐term experiments using this model organism. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:28426144
Should "Multiple Imputations" Be Treated as "Multiple Indicators"?

ERIC Educational Resources Information Center

Mislevy, Robert J.

1993-01-01

Multiple imputations for latent variables are constructed so that analyses treating them as true variables have the correct expectations for population characteristics. Analyzing multiple imputations in accordance with their construction yields correct estimates of population characteristics, whereas analyzing them as multiple indicators generally…
Femtosecond laser pulse optimization for multiphoton cytometry and control of fluorescence

NASA Astrophysics Data System (ADS)

Tkaczyk, Eric Robert

This body of work encompasses optimization of near infrared femtosecond laser pulses both for enhancement of flow cytometry as well as adaptive pulse shaping to control fluorescence. A two-photon system for in vivo flow cytometry is demonstrated, which allows noninvasive quantification of circulating cell populations in a single live mouse. We monitor fluorescently-labeled red blood cells for more than two weeks, and are also able to noninvasively measure circulation times of two distinct populations of breast cancer cells simultaneously in a single mouse. We build a custom laser excitation source in the form of an extended cavity mode-locked oscillator, which enables superior detection in whole blood or saline of cell lines expressing fluorescent proteins including the green fluorescent protein (GFP), tdTomato and mPlum. A mathematical model explains unique features of the signals. The ability to distinguish different fluorescent species is central to simultaneous measurement of multiple molecular targets in high throughput applications including the multiphoton flow cytometer. We demonstrate that two dyes which are not distinguishable to one-photon measurements can be differentiated and in fact quantified in mixture via phase-shaped two-photon excitation pulses found by a genetic algorithm. We also selectively enhance or suppress two-photon fluorescence of numerous common dyes with tailored pulse shapes. Using a multiplicative (rather than ratiometric) fitness parameter, we are able to control the fluorescence while maintaining a strong signal. With this method, we control the two-photon fluorescence of the blue fluorescent protein (BFP), which is of particular interest in investigations of protein-protein interactions, and has frustrated previous attempts of control. Implementing an acousto-optic interferometer, we use the same experimental setup to measure two-photon excitation cross-sections of dyes and prove that photon-photon interferences are the predominant mechanism of control. This research establishes the basis for molecularly tailored pulse shaping in multiphoton flow cytometry, which will advance our ability to probe the biology of circulating cells during disease progression and response to therapy.
Population PK-PD analyses of CD25 occupancy, CD56bright NK cell expansion, and regulatory T cell reduction by daclizumab HYP in subjects with multiple sclerosis.

PubMed

Diao, Lei; Hang, Yaming; Othman, Ahmed A; Mehta, Devangi; Amaravadi, Lakshmi; Nestorov, Ivan; Tran, Jonathan Q

2016-11-01

Daclizumab high yield process (HYP) is a humanized IgG1 monoclonal antibody that binds to the α-subunit of the interleukin-2 receptor and is being developed for treatment of multiple sclerosis (MS). This manuscript characterized the pharmacokinetic-pharmacodynamic (PK-PD) relationships of daclizumab HYP in subjects with MS. Approximately 1400 subjects and 7000 PD measurements for each of three biomarkers [CD25 occupancy, CD56 bright natural killer (NK) cell count, regulatory T cell (Treg) count] from four clinical trials were analyzed using non-linear mixed effects modelling. Evaluated regimens included 150 or 300 mg subcutaneous (s.c.) every 4 weeks. CD25 occupancy was characterized using a sigmoidal maximum response (E max ) model. Upon daclizumab HYP treatment, CD25 saturation was rapid with complete saturation occurring after approximately 7 h and maintained when daclizumab HYP serum concentration was ≥5 mg l -1 . After the last 150 mg s.c. dose, unoccupied CD25 returned to baseline levels in approximately 24 weeks, with daclizumab HYP serum concentration approximately ≤1 mgl -1 1L. CD56 bright NK cell expansion was characterized using an indirect response model. Following daclizumab HYP 150 mg s.c. every 4 weeks, expansion plateaus approximately at week 36, at which the average maximum expansion ratio is 5.2. After the last dose, CD56 bright NK cells gradually declined to baseline levels within 24 weeks. Treg reduction was characterized by a sigmoidal E max model. Average maximum reduction of 60% occurred approximately 4 days post 150 mg s.c. dose. After the last dose, Tregs were projected to return to baseline levels in approximately 20 weeks. Robust PK-PD models of CD25 occupancy, CD56 bright NK cell expansion and Treg reduction by daclizumab HYP were developed to characterize its key pharmacodynamic effects in the target patient population. © 2016 The British Pharmacological Society.
Stable isotope phenotyping via cluster analysis of NanoSIMS data as a method for characterizing distinct microbial ecophysiologies and sulfur-cycling in the environment

NASA Astrophysics Data System (ADS)

Dawson, K.; Scheller, S.; Dillon, J. G.; Orphan, V. J.

2016-12-01

Stable isotope probing (SIP) is a valuable tool for gaining insights into ecophysiology and biogeochemical cycling of environmental microbial communities by tracking isotopically labeled compounds into cellular macromolecules as well as into byproducts of respiration. SIP, in conjunction with nanoscale secondary ion mass spectrometry (NanoSIMS), allows for the visualization of isotope incorporation at the single cell level. In this manner, both active cells within a diverse population as well as heterogeneity in metabolism within a homogeneous population can be observed. The ecophysiological implications of these single cell stable isotope measurements are often limited to the taxonomic resolution of paired fluorescence in situ hybridization (FISH) microscopy. Here we introduce a taxonomy-independent method using multi-isotope SIP and NanoSIMS for identifying and grouping phenotypically similar microbial cells by their chemical and isotopic fingerprint. This method was applied to SIP experiments in a sulfur-cycling biofilm collected from sulfidic intertidal vents amended with 13C-acetate, 15N-ammonium, and 33S-sulfate. Using a cluster analysis technique based on fuzzy c-means to group cells according to their isotope (13C/12C, 15N/14N, and 33S/32S) and elemental ratio (C/CN and S/CN) profiles, our analysis partitioned 2200 cellular regions of interest (ROIs) into 5 distinct groups. These isotope phenotype groupings are reflective of the variation in labeled substrate uptake by cells in a multispecies metabolic network dominated by Gamma- and Deltaproteobacteria. Populations independently grouped by isotope phenotype were subsequently compared with paired FISH data, demonstrating a single coherent deltaproteobacterial cluster and multiple gammaproteobacterial groups, highlighting the distinct ecophysiologies of spatially-associated microbes within the sulfur-cycling biofilm from White Point Beach, CA.
Stable Isotope Phenotyping via Cluster Analysis of NanoSIMS Data As a Method for Characterizing Distinct Microbial Ecophysiologies and Sulfur-Cycling in the Environment

PubMed Central

Dawson, Katherine S.; Scheller, Silvan; Dillon, Jesse G.; Orphan, Victoria J.

2016-01-01

Stable isotope probing (SIP) is a valuable tool for gaining insights into ecophysiology and biogeochemical cycling of environmental microbial communities by tracking isotopically labeled compounds into cellular macromolecules as well as into byproducts of respiration. SIP, in conjunction with nanoscale secondary ion mass spectrometry (NanoSIMS), allows for the visualization of isotope incorporation at the single cell level. In this manner, both active cells within a diverse population as well as heterogeneity in metabolism within a homogeneous population can be observed. The ecophysiological implications of these single cell stable isotope measurements are often limited to the taxonomic resolution of paired fluorescence in situ hybridization (FISH) microscopy. Here we introduce a taxonomy-independent method using multi-isotope SIP and NanoSIMS for identifying and grouping phenotypically similar microbial cells by their chemical and isotopic fingerprint. This method was applied to SIP experiments in a sulfur-cycling biofilm collected from sulfidic intertidal vents amended with 13C-acetate, 15N-ammonium, and 33S-sulfate. Using a cluster analysis technique based on fuzzy c-means to group cells according to their isotope (13C/12C, 15N/14N, and 33S/32S) and elemental ratio (C/CN and S/CN) profiles, our analysis partitioned ~2200 cellular regions of interest (ROIs) into five distinct groups. These isotope phenotype groupings are reflective of the variation in labeled substrate uptake by cells in a multispecies metabolic network dominated by Gamma- and Deltaproteobacteria. Populations independently grouped by isotope phenotype were subsequently compared with paired FISH data, demonstrating a single coherent deltaproteobacterial cluster and multiple gammaproteobacterial groups, highlighting the distinct ecophysiologies of spatially-associated microbes within the sulfur-cycling biofilm from White Point Beach, CA. PMID:27303371
Agent-Based Phytoplankton Models of Cellular and Population Processes: Fostering Individual-Based Learning in Undergraduate Research

NASA Astrophysics Data System (ADS)

Berges, J. A.; Raphael, T.; Rafa Todd, C. S.; Bate, T. C.; Hellweger, F. L.

2016-02-01

Engaging undergraduate students in research projects that require expertise in multiple disciplines (e.g. cell biology, population ecology, and mathematical modeling) can be challenging because they have often not developed the expertise that allows them to participate at a satisfying level. Use of agent-based modeling can allow exploration of concepts at more intuitive levels, and encourage experimentation that emphasizes processes over computational skills. Over the past several years, we have involved undergraduate students in projects examining both ecological and cell biological aspects of aquatic microbial biology, using the freely-downloadable, agent-based modeling environment NetLogo (https://ccl.northwestern.edu/netlogo/). In Netlogo, actions of large numbers of individuals can be simulated, leading to complex systems with emergent behavior. The interface features appealing graphics, monitors, and control structures. In one example, a group of sophomores in a BioMathematics program developed an agent-based model of phytoplankton population dynamics in a pond ecosystem, motivated by observed macroscopic changes in cell numbers (due to growth and death), and driven by responses to irradiance, temperature and a limiting nutrient. In a second example, junior and senior undergraduates conducting Independent Studies created a model of the intracellular processes governing stress and cell death for individual phytoplankton cells (based on parameters derived from experiments using single-cell culturing and flow cytometry), and then this model was embedded in the agents in the pond ecosystem model. In our experience, students with a range of mathematical abilities learned to code quickly and could use the software with varying degrees of sophistication, for example, creation of spatially-explicit two and three-dimensional models. Skills developed quickly and transferred readily to other platforms (e.g. Matlab).
Concurrent CMV and EBV DNAemia is significantly correlated with a delay in the response to HAART in treatment-naive HIV type 1-positive patients.

PubMed

Panagiotakis, Simeon H; Soufla, Giannoula; Baritaki, Stavroula; Sourvinos, George; Passam, Andreas; Zagoreos, Ioannis; Stavrianeas, Nikolaos; Spandidos, Demetrios A

2007-01-01

The purpose of this study was to assess the qualitative single and multiple herpes virus DNAemia in the peripheral blood leukocytes (PBLs) of HIV-1-positive patients and its impact on the response to highly active antiretroviral therapy (HAART) and immune reconstitution. All (163) HIV-1-positive patients attending "Syngros AIDS Referral Center" from November 2000 to February 2001 were recruited. CMV, HSV-1, HSV-2, EBV, and HHV-8 DNA were detected in PBLs by polymerase chain reaction (PCR). Patients' follow-up comprised regular measurements of CD4(+) T cell count and HIV-1 viral load (VL) for an average period of 21 months. Immune reconstitution was defined as an increase in the CD4 T cell count by above 200 cells/micro l, while response to HAART was defined as a decrease in HIV-1 VL to undetectable levels. Single and multiple herpetic DNAemia in PBLs was found to be significantly higher in HIV-1-positive patients compared to healthy controls (p < 0.02) for all the viruses detected apart from HSV-2, which was not detected in the PBLs of either population. Concurrent CMV and EBV DNAemia significantly correlates with a delay in the response to HAART (p = 0.033) in treatment-naive patients. Untreated patients with a CD4(+) T cell count <200 cells/micro l, and with either CMV or EBV DNAemia, presented a delayed increase in the CD4 count after initiation of HAART (p = 0.035 and p = 0.037 respectively), while multiple herpetic DNAemia in the above patients was borderline associated with immune reconstitution (p = 0.068). Conclusively, CMV and EBV DNAemia may be poor prognostic factors for the response to HAART in treatment-naive HIV-1 patients.
Self-renewal and multilineage differentiation of mouse dental epithelial stem cells.

PubMed

Chang, Julia Yu Fong; Wang, Cong; Jin, Chengliu; Yang, Chaofeng; Huang, Yanqing; Liu, Junchen; McKeehan, Wallace L; D'Souza, Rena N; Wang, Fen

2013-11-01

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies. © 2013.
Self-renewal and Multilineage Differentiation of Mouse Dental Epithelial Stem Cells

PubMed Central

Chang, Julia Yu Fong; Wang, Cong; Jin, Chengliu; Yang, Chaofeng; Huang, Yanqing; Liu, Junchen; McKeehan, Wallace L.; D’Souza, Rena N.; Wang, Fen

2013-01-01

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49fBright population was enriched in sphere-forming cells. In addition, the CD49fBright population includes both slow-cycling and Lgr5+ DESCs. The in vitro sphere culture system and identification of CD49fBright as a DESC marker provide a novel plateform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies. PMID:23906788
Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta

2013-05-24

Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states.more » The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.« less
Pagetoid reticulosis (Woringer-Kolopp disease). An ultrastructural and immunocytological study.

PubMed

Takahashi, H; Takahashi, K; Tanno, K; Iijima, S

1982-05-01

Histopathological, immunocytological and ultrastructural observations are reported in the first case of pagetoid reticulosis (Woringer-Kolopp disease) in Japan. The patient was a 61-year-old woman with multiple skin lesions running a chronic and apparently benign clinical course. Histology of the skin biopsies revealed typical pagetoid appearance of the epidermis due to intraepidermal infiltration of abnormal cells. Ultrastructural investigation showed that the intraepidermal abnormal cells were classified into mycosis fungoides cells, Sézary cells, lymphoblast-like cells, and large blastoid cells and that the mycosis fungoides cells were a major cell population. Intermediate or transitional cells were found between these cells and large blastoid cells were mostly situated in the basal cell layer. By the rosetting assays of the free cell suspensions prepared from the epidermis of the biopsied skin lesions, 93% of the suspended cells were positive for spontaneous rosette formation with sheep erythrocytes. The immunoperoxidase technique demonstrated no cytoplasmic immunoglobulins in almost all the intraepidermal abnormal cells. These results indicate that the intraepidermal abnormal cells are T-lymphocytes. Thus, it is concluded that the present case is a cutaneous T-cell lymphoma of low-grade malignancy showing a prominent epidermotropism. This case is the first description of the disease in Japan.
Overexpression of the Cytokine BAFF and Autoimmunity Risk.

PubMed

Steri, Maristella; Orrù, Valeria; Idda, M Laura; Pitzalis, Maristella; Pala, Mauro; Zara, Ilenia; Sidore, Carlo; Faà, Valeria; Floris, Matteo; Deiana, Manila; Asunis, Isadora; Porcu, Eleonora; Mulas, Antonella; Piras, Maria G; Lobina, Monia; Lai, Sandra; Marongiu, Mara; Serra, Valentina; Marongiu, Michele; Sole, Gabriella; Busonero, Fabio; Maschio, Andrea; Cusano, Roberto; Cuccuru, Gianmauro; Deidda, Francesca; Poddie, Fausto; Farina, Gabriele; Dei, Mariano; Virdis, Francesca; Olla, Stefania; Satta, Maria A; Pani, Mario; Delitala, Alessandro; Cocco, Eleonora; Frau, Jessica; Coghe, Giancarlo; Lorefice, Lorena; Fenu, Giuseppe; Ferrigno, Paola; Ban, Maria; Barizzone, Nadia; Leone, Maurizio; Guerini, Franca R; Piga, Matteo; Firinu, Davide; Kockum, Ingrid; Lima Bomfim, Izaura; Olsson, Tomas; Alfredsson, Lars; Suarez, Ana; Carreira, Patricia E; Castillo-Palma, Maria J; Marcus, Joseph H; Congia, Mauro; Angius, Andrea; Melis, Maurizio; Gonzalez, Antonio; Alarcón Riquelme, Marta E; da Silva, Berta M; Marchini, Maurizio; Danieli, Maria G; Del Giacco, Stefano; Mathieu, Alessandro; Pani, Antonello; Montgomery, Stephen B; Rosati, Giulio; Hillert, Jan; Sawcer, Stephen; D'Alfonso, Sandra; Todd, John A; Novembre, John; Abecasis, Gonçalo R; Whalen, Michael B; Marrosu, Maria G; Meloni, Alessandra; Sanna, Serena; Gorospe, Myriam; Schlessinger, David; Fiorillo, Edoardo; Zoledziewska, Magdalena; Cucca, Francesco

2017-04-27

Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug-targetable pathways. Using case-control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus-specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence-based fine mapping, cross-population and cross-phenotype analyses, and gene-expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated. A variant in TNFSF13B, encoding the cytokine and drug target B-cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease-risk allele was also associated with up-regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion-deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up-regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria. A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.).
Progress in myeloma stem cells

PubMed Central

Cruz, Richard Dela; Tricot, Guido; Zangari, Maurizio; Zhan, Fenghuang

2011-01-01

Multiple myeloma (MM) is the second most common hematologic malignancy in the United States and affects about 4 in 100,000 Americans. Even though much progress has been made in MM therapy, MM remains an incurable disease for the vast majority of patients. The existence of MM stem cell is considered one of the major causes of MM drug-resistance, leading to relapse. This highlights the importance and urgency of developing approaches to target MM stem cells. However, very little is known about the molecular characteristics of the MM stem cells, which makes it difficult to target MM stem cells therapeutically. Evidence of the existence of a myeloma stem cell has been provided by Matsui et al. showing that the CD138- and CD20+ fraction, which is a minor population of the MM cells, has a greater clonogenic potential and has the phenotype of a memory B-cell (CD19+, CD27+). In this review, we report recent progress of cell surface markers in cancer stem cells, especially in myeloma and the molecular mechanisms related to drug resistance and myeloma disease progression. PMID:22432075

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