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At least 217 records · Page 12Linked to original sources
PubMed2001

T cell receptor dynamism of mucosal and systemic CD4+ T cells in the course of an immune response to Escherichia coli heat-labile enterotoxin.

The changes in T cell receptor (TCR) Vbeta expression, use, and clonality in mice orally challenged with Escherichia coli heat-labile enterotoxin (LT) were assessed. Use of the TCR Vbeta family and clonality were significantly changed at the single-cell level. In Peyer's patches of treated mice, use of TCR Vbeta6, Vbeta8, and Vbeta14 increased in CD4(+)CD44(+) T cells, compared with use in nontreated mice. On the other hand, use of TCR Vbeta1 and Vbeta8 was enhanced in splenic CD4(+)CD44(+) T cells. Intraepithelial lymphocytes isolated from LT-challenged mice showed expanded clonality (e.g., Vbeta1, Vbeta2, Vbeta9, and Vbeta18) and altered TCR Vbeta use (e.g., Vbeta15, Vbeta16, and Vbeta17). These findings reveal that oral administration of LT has distinct effects on mucosal versus systemic alphabeta T cells for induction of CD4(+) T cells with selected Vbeta use. This most likely reflects the function of LT as a mucosal modulator.

Administration, Oral↗
PubMed2005

The mitochondrial ribosome-specific MrpL55 protein is essential in Drosophila and dynamically required during development.

We report on the essential Drosophila mRpL55 gene conserved exclusively in metazoans. Null mRpL55 mutants did not grow after hatching, moved slowly and died as first instar larvae. MrpL55 is similar to mammalian MRPL55, a protein that, in a large-scale mass spectrometry study, has been found as a mitoribosome-specific large subunit protein. We showed that MrpL55 was localised to the mitochondrion in S2 cells and tissues and was enriched in cells with a higher protein synthesis activity. The MrpL55 protein contains a KOW-like motif present in proteins with a role in transcriptional anti-termination and regulation of translation. Modulation of mRpL55 expression level is critical for development. Somatic clonal analysis showed that MrpL55 was not required in larval eye imaginal discs but required in pupal discs apparently during the second mitotic wave. Therefore, our results showed that the MrpL55 protein acts dynamically in the cell during development. We propose that MrpL55 is involved in Drosophila mitochondrial biogenesis and G2/M phase cell cycle progression.

Amino Acid Motifs↗
PubMed2004

Normal telomere erosion rates at the single cell level in Werner syndrome fibroblast cells.

The aim of this study was to investigate whether the accelerated replicative senescence seen in Werner syndrome (WS) fibroblasts is due to accelerated telomere loss per cell division. Using single telomere length analysis (STELA) we show that the mean rate of telomere shortening in WS bulk cultures ranges between that of normal fibroblasts [99 bp/population doubling (PD)] and four times that of normal (355 bp/PD). The telomere erosion rate in the fastest eroding strain slows in the later stages of culture to that observed in normal fibroblasts, and appears to be correlated with a reduction in the heterogeneity of the telomere-length distributions. Telomere erosion rates in clones of WS cells are much reduced compared with bulk cultures, as are the variances of the telomere-length distributions. The overall lack of length heterogeneity and the normal erosion rates of the clonal populations are consistent with simple end-replication losses as the major contributor to telomere erosion in WS cells. We propose that telomere dynamics at the single cell level in WS fibroblasts are not significantly different from those in normal fibroblasts, and suggest that the accelerated replicative decline seen in WS fibroblasts does not result from accelerated telomere erosion.

Cells, Cultured↗
PubMed2000

Viral impacts on total abundance and clonal composition of the harmful bloom-forming phytoplankton Heterosigma akashiwo.

Recent observations that viruses are very abundant and biologically active components in marine ecosystems suggest that they probably influence various biogeochemical and ecological processes. In this study, the population dynamics of the harmful bloom-forming phytoplankton Heterosigma akashiwo (Raphidophyceae) and the infectious H. akashiwo viruses (HaV) were monitored in Hiroshima Bay, Japan, from May to July 1998. Concurrently, a number of H. akashiwo and HaV clones were isolated, and their virus susceptibilities and host ranges were determined through laboratory cross-reactivity tests. A sudden decrease in cell density of H. akashiwo was accompanied by a drastic increase in the abundance of HaV, suggesting that viruses contributed greatly to the disintegration of the H. akashiwo bloom as mortality agents. Despite the large quantity of infectious HaV, however, a significant proportion of H. akashiwo cells survived after the bloom disintegration. The viral susceptibility of H. akashiwo isolates demonstrated that the majority of these surviving cells were resistant to most of the HaV clones, whereas resistant cells were a minor component during the bloom period. Moreover, these resistant cells were displaced by susceptible cells, presumably due to viral infection. These results demonstrated that the properties of dominant cells within the H. akashiwo population change during the period when a bloom is terminated by viral infection, suggesting that viruses also play an important role in determining the clonal composition and maintaining the clonal diversity of H. akashiwo populations. Therefore, our data indicate that viral infection influences the total abundance and the clonal composition of one host algal species, suggesting that viruses are an important component in quantitatively and qualitatively controlling phytoplankton populations in natural marine environments.

DNA Viruses↗
PubMed2005

Clonal evolution of stem and differentiated cells can be predicted by integrating cell-intrinsic and -extrinsic parameters.

Stem cells and their derivatives represent a renewable source of cells for therapeutic applications. However, the inability to quantitatively integrate and exploit the effects of multiple parameters on the fate of stem cells limits their use in clinical applications. To address this, we developed a computational model that combines probabilistic, individual-cell and deterministic cell-population parameters to simultaneously calculate the specific effects of exogenous and endogenous factors on the overall population-dynamics behaviour. The model tracks the progeny trajectory of individual cells over several generations as a threshold function of ligand-receptor signalling interactions. Simulations in silico were validated against an Oct 4-promoter-driven green-fluorescent-protein-expressing murine embryonic stem cell line, and used to understand the effects of key parameters on the clonal evolution of stem versus differentiated cells in this system. Our approach demonstrated the ability to distinguish between individual-cell and population-averaged parameters with respect to their effects on governing dynamic behaviour. Moreover, we could discriminate between digital versus graded regulation of the Oct 4 transcription factor in accounting for experimental observations. Finally, we showed that our approach could be generalized to other stem-cell systems, in particular the previously characterized intestinal crypt system, in elucidating relative contributions of stem and progenitor cells to population output. On the basis of all these results, we believe that our iterative experimental and computational approach has been found to be useful for the study of various stem-cell systems.

Animals↗
PubMed2026

Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality.

BACKGROUND: Gene therapy (GT) using retroviral vectors (RVs) is efficacious in treating monogenic diseases. However, there is an inherent risk for severe adverse effects due to insertional mutagenesis. Preclinical safety assessment and patient monitoring are inevitable in GT. To assess the genotoxic risk of novel RV vectors, mainly murine hematopoietic stem and progenitor cells (HPSCs) are routinely used, because human HSPCs cannot be immortalized in vitro using mutagenic vectors. In this study, we aim to identify early signs of clonal outgrowth by performing integration site analyses (ISA). METHODS: The small molecules A83-01, pomalidomide, and UM171 (APU) were used for the ex vivo expansion, lentiviral transduction, and long-term cultivation of umbilical cord blood-derived HSPCs. We determined the influence of APU on the stemness of HSPCs and their differentiation capacity via single-cell RNA sequencing (scRNA seq) and in xenotransplantation studies. To track vector insertion site dynamics, we transduced 7-day expanded HSPCs with a mutagenic or a safer RV. ISA was conducted in human HSPCs over a 5-week cultivation in vitro and compared to the bone marrow of xenotransplanted mice to assess clonal skewings. RESULTS: APU supported the expansion of CD34+CD38-CD45RA-CD90+EPCR+ HSPCs. scRNA seq confirmed the enrichment of HSC signature genes in APU-expanded HSPCs compared to the clinically used medium SFT3 (SCF, FLT3-L, TPO, IL-3). After RV transduction, APU still maintained around 30% of CD34+ cells for 5 more weeks. Without the compounds, already 2 weeks post-transduction, less than 10% of cells were CD34+. The long-term culture allowed the detection of high-risk integrations of the mutagenic SIN-LV.SF in MEIS1 or SUSD6 due to their increasing abundance over time. Bone marrow of xenotransplanted mice was less clonal but did not support the outgrowth of insertional mutants. Overall, APU increased clonal diversity. CONCLUSIONS: Our findings propose that long-term cultivation of transduced HSPC in APU allows for outgrowth of clonal integration sites. The decrease of clonality has been observed in gene therapy patient's years after treatment. Thus, the in vitro model could be used to develop novel human HSPC-based genotoxicity assays that predict insertional mutagenesis, in addition to existing preclinical biosafety assays.

Humans↗
PubMed1990

Somatic mutation, affinity maturation and the antibody repertoire: a computer model.

Somatic mutation has been implicated as a significant and possibly primary factor in the maturation of antibody affinity in the humoral immune response. B cells stimulated by antigen experience a hyper-mutation in the gene segments that code for the antigen-binding site of the antibody, creating antibody specificities that did not exist at the time of immunization. Although most of the mutations are likely to be disadvantageous, new specificities with a higher affinity for the antigen are sometimes created. These higher-affinity cells are preferentially selected for proliferation and eventual antibody secretion, resulting in a progressively higher average affinity over time. In this paper we present the results of an investigation of somatic mutation through the use of a computer model. At the basis of the model is a large repertoire of discrete antibodies and antigens, having three-dimensional structures, that exhibit properties similar to those of the real populations. The key factor is that the binding strength between any antibody/antigen pair can be calculated as a function of the complementarity of the (a) size, (b) shape and (c) functional groups that comprise the two structures. The created repertoires are imbedded in a dynamical system model of the immune response to directly evaluate the affect of somatic mutation on affinity maturation. We also present an expanded hypothesis of clonal selection and development to explain how the mutational restrictions imposed by the genetic code and the structure of the antibody repertoire, along with antigen concentration, affinity, and probabilistic factors may interact and contribute to the expansion of specific clones as the response develops over time.

Animals↗
PubMed2004

On the emergence of multifocal cancers.

Several tumors can exist as multiple lesions within a tissue. The lesions may either arise independently, or they may be monoclonal. The importance of multiple lesions for tumor staging, progression, and treatment is subject to debate. Here we use mathematical models to analyze the emergence of multiple, clonally related lesions within a single tissue. We refer to them as multi-focal cancers. We find that multifocal cancers can arise through a dynamical interplay between tumor promoting and inhibiting factors. This requires that tumor promoters act locally, while tumor inhibitors act over a longer range. An example of such factors may be angiogenesis promoters and inhibitors. The model further suggests that multifocal cancers represent an intermediate stage in cancer progression as the tumor evolves away from inhibition and towards promotion. Different patterns of progression can be distinguished: (i) If tumor inhibition is strong, the initial growth occurs as a unifocal and self contained lesion; progression occurs through bifurcation of the lesion and this gives rise to multiple lesions. As the tumor continues to evolve and pushes the balance between inhibition and promotion further towards promotion, the multiple lesions eventually give rise to a single large mass which can invade the entire tissue. (ii) If tumor inhibition is weaker upon initiation, growth can occur as a single lesion without the occurrence of multiple lesions, until the entire tissue is invaded. The model suggests that the sum of the tumor sizes across all lesions is the best characteristic which correlates with the stage and metastatic potential of the tumor.

Journal Article↗
PubMed1983

Approaches to enhance proliferation of human epidermal keratinocytes in mass culture.

Because of interest in mechanisms of carcinogenesis in human epithelial cells, quantitative procedures were developed for the mass culture of human epidermal keratinocytes in the absence of feeder cells. Several approaches were used to enhance proliferation since target cells are considered most susceptible to transformation if they are treated with carcinogenic agents during DNA synthesis. Mass cultures of enzymatically dispersed human foreskin were initiated in collagen-coated flasks containing medium NCTC 168 with 10% Chelex 100-treated horse serum. Under these conditions, human keratinocytes required a higher calcium ion concentration ([Ca2+]) than that reported for suspensions plated at low cell density. Neither the cohesiveness of the epidermal sheet nor continued proliferation was maintained by 0.15 mM Ca2+; 0.3 mM Ca2+ maintained these properties in primary culture only. A concentration of 1.0 mM Ca2+ provided the highest cell yield for prolonged growth as determined by the enumeration of cell nuclei isolated by citric acid. Reproducibility of successful initiation was achieved by inoculation of cells into a medium designed for clonal growth followed by culture in medium NCTC 168. Thus the balance of nutrients and electrolytes must be adjusted to satisfy the requirements of a dynamically expanding keratinocyte population.

Calcium↗
PubMed1999

Microsatellite and morphological analysis of population structure in the parthenogenetic freshwater snail Melanoides tuberculata: insights into the creation of clonal variability.

The distribution of variability was studied at various geographical scales in the tropical freshwater snail Melanoides tuberculata, in order to analyse the role of factors shaping this distribution, including the mating system and population dynamics. This parthenogenetic polyploid species reproduces mainly asexually, with males occurring at low frequency. About 800 individuals (38 sites) were sampled from Africa and the Middle East, where the species originated, and from recently colonized habitats in South and Central America, and especially the island of Martinique. We first described variation of general aspects and ornamentation of the shells. This analysis confirms the existence of discrete morphs. Second, individuals were studied at three microsatellite loci, showing that each morph is a genetic clone with some minor variation compatible with models of microsatellite evolution. The genetic analysis also showed much more variation within than between clones. However, two populations from Africa exhibited a large amount of variability, and a mixture of sexual and asexual reproduction might explain these genetic patterns. The worldwide distribution of variability is, therefore, compatible with the African origin of the species, and the introduction of a few clones in other parts of the world. These results also suggest that the distribution of variability in Martinique is influenced by flooding events, and that two morphs from Martinique can be interpreted as hybrids between two pre-existing morphs, based on morphological, genetic and geographical arguments.

Journal Article↗
PubMed2000

The evolutionary dynamics of male-killers and their hosts.

Male-killing bacteria are cytoplasmic sex-ratio distorters that are transmitted vertically through females of their insect hosts. The killing of male hosts by their bacteria is thought to be an adaptive bacterial trait because it augments the fitness of female hosts carrying clonal relatives of those bacteria. Here we attempt to explain observations of multiple male-killers in natural host populations. First we show that such male-killer polymorphism cannot be explained by a classical model of male-killing. We then show that more complicated models incorporating the evolution of resistance in hosts can explain male-killer polymorphism. However, this is only likely if resistance genes are very costly. We also consider the long-term evolutionary dynamics of male-killers, and show that evolution towards progressively more 'efficient' male-killers can be thwarted by the appearance of host resistance. The presence of a resistance gene can allow a less efficient male-killer to outcompete its rival and hence reverse the trend towards more efficient transmission and reduced metabolic load on the host.

Animals↗
PubMed1994

Clonal dominance detected in metastases but not primary tumors of retrovirally marked human breast carcinoma injected into nude mice.

Human breast cancer cell lines which grow in athymic (nude) mice provide a model of tumor cell growth and metastasis. Marking transplanted tumor cell populations with retroviral vectors provides a means of studying the dynamics of tumor cell growth in vivo. We evaluated three human breast cancer cell lines, MDA-MB-435, MDA-MB-231 and MCF-7, and found the cells were highly susceptible to retroviral gene transfer after a single 2-h exposure (90.9%, 62.7% and 72.3%, respectively). MDA-MB-435 cells (5 x 10(5)) marked with a retroviral vector containing the beta-galactosidase gene (approximately 10(4) uniquely marked clones) were injected into the mammary fat pad of athymic mice to study clonal dominance. Primary tumors resected 10 weeks after injection expressed beta-galactosidase, demonstrating persistent vector expression in vivo. Southern blot analysis did not reveal clonal dominance in the primary tumors of the five mice studied. In contrast, pulmonary metastases in each animal were monoclonal or biclonal. These results demonstrate clonal dominance in pulmonary metastases but not primary tumors of retrovirally marked MDA-MB-435 cells. Our findings suggest that this model may also be used to introduce retroviral vectors expressing oncogenes, and anti-sense oncogenes, to determine their effect on tumor cell proliferation and metastasis in vivo.

Animals↗
PubMed2001

Lifelong hematopoiesis in both reconstituted and sublethally irradiated mice is provided by multiple sequentially recruited stem cells.

OBJECTIVE: To evaluate the dynamics of stem cell production to hematopoiesis, the number of active stem cell clones and the lifespan of individual clones were studied. MATERIALS AND METHODS: The clonal contribution of primitive hematopoietic stem cells (HSC) responsible for long-term hematopoiesis was determined using two approaches. In one model, irradiated female mice were reconstituted with retrovirally marked male hematopoietic cells. In the second model, mice were irradiated sublethally without hematopoietic cell transplantation. In both models, bone marrow cells were serially sampled from the same mouse throughout a 12- to 20-month period and injected into irradiated recipients for analysis of day 10 colony-forming unit-spleen (CFU-S). The donor origin of CFU-S was determined by the presence of retrovirally marked cells or cells with chromosomal aberrations. RESULTS: The results of the two essentially different models show that 1) hematopoiesis is mainly the product of small clones of hematopoietic cells; 2) the lifespan of the majority of clones is only 1 to 2 months; 3) the clones usually function locally; and 4) the vast majority of the clones replace one another sequentially. Primitive HSCs capable of producing long-lived clones (about 10% among all clones), which exist during the entire life of a mouse, were detected by the radiation-marker technique only. CONCLUSION: Multiple short-living clones (at least on the level of CFU-S production) comprise the vast majority of the active stem cells in transplanted recipients or after endogenous recovery from sublethal irradiation.

Animals↗
PubMed1991

DNA Fingerprinting with a Dispersed Repeated Sequence Resolves Pathotype Diversity in the Rice Blast Fungus.

The poor definition of pathotype variation in the rice blast fungus has historically handicapped strategies for reducing blast disease damage to the world's rice crop. We have employed a probe for a dispersed repeated DNA sequence called MGR [Hamer et al. (1989). Proc. Natl. Acad. Sci. USA 86, 9981-9985] to construct genotype-specific, EcoRl restriction fragment length profiles (MGR-DNA fingerprints) from United States field isolates of this fungus. By using a blind-test design, we demonstrated that MGR-DNA fingerprints distinguished the major pathotypes in the United States, accurately identified the pathotypes of isolates collected over a 30-year period, and defined the organization of clonal lineages within and among pathotype groups. These results resolved a lingering controversy regarding rice blast pathotype stability and illustrated new opportunities for tracking the population dynamics and evolution of this important crop pathogen.

Journal Article↗
PubMed2002

A novel dynamic model of hematopoietic stem cell organization based on the concept of within-tissue plasticity.

OBJECTIVE: At present, no dynamic quantitative models of stem cell organization are available that fulfill all criteria of the prevalent functional definition of hematopoietic stem cells and, at the same time, provide a consistent explanation of cell kinetic and functional stem cell heterogeneity, reversibility of cellular properties, self-organized regeneration after damage, fluctuating activity and competition of stem cell clones, and microenvironment dependency of stem cell quality. To solve this problem, we propose a new, comprehensive model concept. MATERIALS AND METHODS: A single cell-based stochastic model is described. It makes the novel concept of within-tissue plasticity operational. Within a range of potential options, individual cells may reversibly change their actual set of properties depending on the influence of the local growth environment. Stochastic switching between the growth environments introduces fluctuations that eventually generate heterogeneity. Extensive model simulations are compared with experimental data. RESULTS: Although stemness is not an explicit cellular model property, the system behavior is consistent with the functional definition of stem cells and explains a large set of experimental observations on stem cell function in vivo and in vitro on the level of cell populations and individual cells. Classic results such as the colony-forming unit spleen assay, as well as recent experimental observations on stem cell kinetics, individual clone tracking, and fluctuating clonal contribution, are discussed. CONCLUSIONS: This concept introduces a fundamentally new perspective on stem cell organization treating stemness not as an explicit cellular property but as the result of a dynamic process of self-organization. The model needs to be extended to incorporate lineage specification and tissue plasticity.

Animals↗
PubMed2001

Clonal maintenance of imprinted expression of SNRPN and IPW in normal lymphocytes: correlation with allele-specific methylation of SNRPN intron 1 but not intron 7.

DNA methylation is a heritable and reversible modification to CpG sites in the mammalian genome. Parental allele-specific methylation is hypothesized to be important in the establishment and maintenance of imprinted gene expression; however, dynamic changes in allele-specific patterns have been observed. The upstream regulatory region of the small nuclear riboprotein N gene (SNRPN) is an important imprinting control region (ICR) for establishing and maintaining the methylation imprint in the locus on 15q11-13 associated with Prader-Willi and Angelman syndromes (PWS). To compare directly the role of allele-specific methylation patterns and the maintenance of imprinted expression in the PWS region, clonal populations of normal T lymphocytes were cultured for 22-25 generations. A novel long-range semi-nested polymerase chain reaction (PCR) strategy was utilized in order to span two different methylation sites, and a polymorphism within SNRPN was used so that allele-specific methylation of both sites could be determined. Reverse transcription/PCR followed by polymorphism analysis was also performed in order to determine parental allele-specific transcription. Exclusive paternal expression at both SNRPN and IPW was maintained in all T cell clones and correlated with maternal methylation of the intron 1 NotI site. In contrast, biallelic methylation was observed in all clones at the previously described paternally methylated HpaII site in intron 7. These results demonstrate that the maintenance of paternal expression of SNRPN and IPW correlates with a strict clonal maintenance of allele-specific methylation at the CpG-dense 5' end of SNRPN. Differential maintenance of methylation sites within imprinted genes may depend on the density and chromatin organization of surrounding CpG sites.

Alleles↗
PubMed2004

A graph model for the evolution of specificity in humoral immunity.

The immune system protects the body against health-threatening entities, known as antigens, through very complex interactions involving the antigens and the system's own entities. One remarkable feature resulting from such interactions is the immune system's ability to improve its capability to fight antigens commonly found in the individual's environment. This adaptation process is called the evolution of specificity. In this paper, we introduce a new mathematical model for the evolution of specificity in humoral immunity, based on Jerne's functional, or idiotypic, network. The evolution of specificity is modeled as the dynamic updating of connection weights in a dynamic graph whose nodes are related to the network's idiotypes. At the core of this weight-updating mechanism are the increase in specificity caused by clonal selection and the decrease in specificity due to the insertion of uncorrelated idiotypes by the bone marrow. As we demonstrate through numerous computer experiments, for appropriate choices of parameters the new model correctly reproduces, in qualitative terms, several immune functions.

Antibody Formation↗
PubMed2026

Unveiling tumor heterogeneity by single cell RNA-sequencing: From basic considerations to clinical applications.

Tumor heterogeneity-encompassing diverse cellular phenotypes, genomic alterations, and microenvironmental contexts-is a principal barrier to effective cancer therapy. Single-cell RNA sequencing (scRNA-seq) has transformed our ability to resolve this complexity by capturing transcriptomes at single-cell resolution. Here, we review the technical foundations required for high-quality scRNA-seq studies. We then trace the evolution of scRNA-seq platforms from manual micromanipulation to high-throughput systems, and describe the computational pipelines that enable reliable data interpretation. The application of scRNA-seq is exemplarily shown in the context of lung cancer, where single-cell profiling has revealed (i) the clonal and sub-clonal architecture of tumors, (ii) extensive remodeling of the immune microenvironment, iii) key mechanisms underlying resistance to targeted agents and immune-checkpoint blockade, and (iv) the dynamics of neo-antigen-specific T-cell responses. Integrating machine-learning techniques-such as deep-learning classifiers and graph-based models-with single-cell transcriptomic data has markedly sped up biomarker discovery, produced more accurate risk-stratification scores, and enabled the generation of patient-specific therapeutic predictions. We surveyed the major trial registry ClinicalTrials.gov and identified ∼380 ongoing or completed studies that explicitly incorporate scRNA-seq as a correlative or pharmacodynamic endpoint. Overall, the analysis shows that scRNA-seq becomes an increasingly important component of modern trials, providing high-resolution cellular and molecular readouts that complement conventional imaging and bulk-omics endpoints. While key challenges remain, ranging from costs, scalability and need for rigorous validation before routine clinical deployment, ongoing technological advances continue to expand the potential of scRNA-seq as a cornerstone of precision medicine.

Humans↗