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1.
Br J Cancer ; 128(6): 1148-1154, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36572732

RESUMEN

BACKGROUND: Targeted RNA sequencing (RNA-seq) from FFPE specimens is used clinically in cancer for its ability to estimate gene expression and to detect fusions. Using a cohort of NSCLC patients, we sought to determine whether targeted RNA-seq could be used to measure tumour mutational burden (TMB) and the expression of immune-cell-restricted genes from FFPE specimens and whether these could predict response to immune checkpoint blockade. METHODS: Using The Cancer Genome Atlas LUAD dataset, we developed a method for determining TMB from tumour-only RNA-seq and showed a correlation with DNA sequencing derived TMB calculated from tumour/normal sample pairs (Spearman correlation = 0.79, 95% CI [0.73, 0.83]. We applied this method to targeted sequencing data from our patient cohort and validated these results against TMB estimates obtained using an orthogonal assay (Spearman correlation = 0.49, 95% CI [0.24, 0.68]). RESULTS: We observed that the RNA measure of TMB was significantly higher in responders to immune blockade treatment (P = 0.028) and that it was predictive of response (AUC = 0.640 with 95% CI [0.493, 0.786]). By contrast, the expression of immune-cell-restricted genes was uncorrelated with patient outcome. CONCLUSION: TMB calculated from targeted RNA sequencing has a similar diagnostic ability to TMB generated from targeted DNA sequencing.


Asunto(s)
Antineoplásicos Inmunológicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , RNA-Seq , Mutación , Antineoplásicos Inmunológicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Análisis de Secuencia de ARN , ARN , Biomarcadores de Tumor/genética
2.
Br J Haematol ; 195(1): 113-118, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34426978

RESUMEN

Richter syndrome (RS), an aggressive lymphoma occurring in the context of chronic lymphocytic leukaemia/small lymphocytic lymphoma, is associated with poor prognosis when treated with conventional immunochemotherapy, therefore, improved treatments are required. Immune checkpoint blockade has shown efficacy in some B-cell malignancies and modest responses in early clinical trials for RS. We investigated the immune checkpoint profile of RS as a basis to inform rational therapeutic investigations in RS. Formalin-fixed, paraffin-embedded biopsies of RS (n = 19), de novo diffuse large B-cell lymphoma (DLBCL; n = 58), transformed indolent lymphomas (follicular [tFL], n = 16; marginal zone [tMZL], n = 24) and non-transformed small lymphocytic lymphoma (SLL; n = 15) underwent gene expression profiling using the NanoString Human Immunology panel. Copy number assessment was performed using next-generation sequencing. Immunohistochemistry (IHC) for LAG3 and PD-1 was performed. LAG3 gene expression was higher in RS compared to DLBCL (P = 0·0002, log2FC 1·96), tFL (P < 0·0001, log2FC 2·61), tMZL (P = 0·0004, log2FC 1·79) and SLL (P = 0·0057, log2FC 1·45). LAG3 gene expression correlated with the gene expression of human leukocyte antigen Class I and II, and related immune genes and immune checkpoints. IHC revealed LAG3 protein expression on both malignant RS cells and tumour-infiltrating lymphocytes. Our findings support the investigation of LAG3 inhibition to enhance anti-tumour responses in RS.


Asunto(s)
Antígenos CD/fisiología , Inhibidores de Puntos de Control Inmunológico , Leucemia Linfocítica Crónica de Células B/inmunología , Linfoma de Células B de la Zona Marginal/tratamiento farmacológico , Linfoma Folicular/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/inmunología , Terapia Molecular Dirigida , Proteínas de Neoplasias/fisiología , Antígenos CD/biosíntesis , Antígenos CD/genética , Linfocitos B/metabolismo , Variaciones en el Número de Copia de ADN , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Linfocitos Infiltrantes de Tumor/metabolismo , Linfoma de Células B de la Zona Marginal/genética , Linfoma Folicular/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Síndrome , Proteína del Gen 3 de Activación de Linfocitos
3.
Proc Natl Acad Sci U S A ; 111(17): 6377-82, 2014 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-24733943

RESUMEN

Stochastic variation in cell cycle time is a consistent feature of otherwise similar cells within a growing population. Classic studies concluded that the bulk of the variation occurs in the G1 phase, and many mathematical models assume a constant time for traversing the S/G2/M phases. By direct observation of transgenic fluorescent fusion proteins that report the onset of S phase, we establish that dividing B and T lymphocytes spend a near-fixed proportion of total division time in S/G2/M phases, and this proportion is correlated between sibling cells. This result is inconsistent with models that assume independent times for consecutive phases. Instead, we propose a stretching model for dividing lymphocytes where all parts of the cell cycle are proportional to total division time. Data fitting based on a stretched cell cycle model can significantly improve estimates of cell cycle parameters drawn from DNA labeling data used to monitor immune cell dynamics.


Asunto(s)
Linfocitos B/citología , Ciclo Celular , Linfocitos T/citología , Animales , Bromodesoxiuridina/metabolismo , División Celular , Proliferación Celular , ADN/metabolismo , Fluorescencia , Genes Reporteros , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , Probabilidad , Factores de Tiempo
4.
Blood ; 124(17): 2725-9, 2014 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-25079356

RESUMEN

In this study, we test the assumption that the hematopoietic progenitor/colony-forming cells of the embryonic yolk sac (YS), which are endowed with megakaryocytic potential, differentiate into the first platelet-forming cells in vivo. We demonstrate that from embryonic day (E) 8.5 all megakaryocyte (MK) colony-forming cells belong to the conventional hematopoietic progenitor cell (HPC) compartment. Although these cells are indeed capable of generating polyploid MKs, they are not the source of the first platelet-forming cells. We show that proplatelet formation first occurs in a unique and previously unrecognized lineage of diploid platelet-forming cells, which develop within the YS in parallel to HPCs but can be specified in the E8.5 Runx1-null embryo despite the absence of the progenitor cell lineage.


Asunto(s)
Linaje de la Célula/genética , Diploidia , Embrión de Mamíferos/metabolismo , Células Progenitoras de Megacariocitos/metabolismo , Megacariocitos/metabolismo , Poliploidía , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Regulación del Desarrollo de la Expresión Génica , Células Progenitoras de Megacariocitos/citología , Megacariocitos/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Tiempo , Transcriptoma , Saco Vitelino/citología , Saco Vitelino/embriología , Saco Vitelino/metabolismo
5.
Blood Adv ; 6(2): 503-508, 2022 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-34861696

RESUMEN

The genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to Bruton tyrosine kinase (BTK) inhibitors and B-cell lymphoma 2 (BCL2) inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of 8 patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each individual disease context.


Asunto(s)
Antineoplásicos , Leucemia Linfocítica Crónica de Células B , Linfoma de Células del Manto , Adulto , Antineoplásicos/uso terapéutico , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células del Manto/tratamiento farmacológico , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/genética
6.
BMC Genomics ; 12: 197, 2011 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-21501528

RESUMEN

BACKGROUND: Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. To date, only one complete genomic sequence of ILTV has been reported. This sequence was generated by concatenating partial sequences from six different ILTV strains. Thus, the full genomic sequence of a single (individual) strain of ILTV has not been determined previously. This study aimed to use high throughput sequencing technology to determine the complete genomic sequence of a live attenuated vaccine strain of ILTV. RESULTS: The complete genomic sequence of the Serva vaccine strain of ILTV was determined, annotated and compared to the concatenated ILTV reference sequence. The genome size of the Serva strain was 152,628 bp, with a G + C content of 48%. A total of 80 predicted open reading frames were identified. The Serva strain had 96.5% DNA sequence identity with the concatenated ILTV sequence. Notably, the concatenated ILTV sequence was found to lack four large regions of sequence, including 528 bp and 594 bp of sequence in the UL29 and UL36 genes, respectively, and two copies of a 1,563 bp sequence in the repeat regions. Considerable differences in the size of the predicted translation products of 4 other genes (UL54, UL30, UL37 and UL38) were also identified. More than 530 single-nucleotide polymorphisms (SNPs) were identified. Most SNPs were located within three genomic regions, corresponding to sequence from the SA-2 ILTV vaccine strain in the concatenated ILTV sequence. CONCLUSIONS: This is the first complete genomic sequence of an individual ILTV strain. This sequence will facilitate future comparative genomic studies of ILTV by providing an appropriate reference sequence for the sequence analysis of other ILTV strains.


Asunto(s)
Genoma Viral/genética , Genómica/métodos , Iltovirus/genética , Secuencia de Aminoácidos , Animales , ADN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Vacunas Atenuadas/genética
9.
Sci Rep ; 9(1): 6426, 2019 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-31015508

RESUMEN

Next Generation Sequencing is now routinely used in the practice of diagnostic pathology to detect clinically relevant somatic and germline sequence variations in patient samples. However, clinical assessment of copy number variations (CNVs) and large-scale structural variations (SVs) is still challenging. While tools exist to estimate both, their results are typically presented separately in tables or static plots which can be difficult to read and are unable to show the context needed for clinical interpretation and reporting. We have addressed this problem with CNspector, a multi-scale interactive browser that shows CNVs in the context of other relevant genomic features to enable fast and effective clinical reporting. We illustrate the utility of CNspector at different genomic scales across a variety of sample types in a range of case studies. We show how CNspector can be used for diagnosis and reporting of exon-level deletions, focal gene-level amplifications, chromosome and chromosome arm level amplifications/deletions and in complex genomic rearrangements. CNspector is a web-based clinical variant browser tailored to the clinical application of next generation sequencing for CNV assessment. We have demonstrated the utility of this interactive software in typical applications across a range of tissue types and disease contexts encountered in the context of diagnostic pathology. CNspector is written in R and the source code is available for download under the GPL3 Licence from https://github.com/PapenfussLab/CNspector . A server running CNspector loaded with the figures from this paper can be accessed at https://shiny.wehi.edu.au/jmarkham/CNspector/index.html .


Asunto(s)
Síndrome del Nevo Basocelular/diagnóstico , Carcinoma Basocelular/diagnóstico , Variaciones en el Número de Copia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Navegador Web , Síndrome del Nevo Basocelular/genética , Síndrome del Nevo Basocelular/patología , Carcinoma Basocelular/genética , Carcinoma Basocelular/patología , Deleción Cromosómica , Duplicación Cromosómica , Exones , Genoma Humano , Humanos , Internet , Análisis de Secuencia de ADN
10.
Front Immunol ; 9: 2053, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30250473

RESUMEN

In response to external stimuli, naïve B cells proliferate and take on a range of fates important for immunity. How their fate is determined is a topic of much recent research, with candidates including asymmetric cell division, lineage priming, stochastic assignment, and microenvironment instruction. Here we manipulate the generation of plasmablasts from B lymphocytes in vitro by varying CD40 stimulation strength to determine its influence on potential sources of fate control. Using long-term live cell imaging, we directly measure times to differentiate, divide, and die of hundreds of pairs of sibling cells. These data reveal that while the allocation of fates is significantly altered by signal strength, the proportion of siblings identified with asymmetric fates is unchanged. In contrast, we find that plasmablast generation is enhanced by slowing times to divide, which is consistent with a hypothesis of competing timed stochastic fate outcomes. We conclude that this mechanistically simple source of alternative fate regulation is important, and that useful quantitative models of signal integration can be developed based on its principles.


Asunto(s)
Linfocitos B/fisiología , Células Plasmáticas/fisiología , Células Precursoras de Linfocitos B/fisiología , Animales , Relojes Biológicos , Antígenos CD40/metabolismo , Diferenciación Celular , División Celular , Linaje de la Célula , Células Cultivadas , Femenino , Inmunización , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Procesos Estocásticos
11.
PLoS One ; 11(1): e0146227, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26742110

RESUMEN

Adaptive immune responses are complex dynamic processes whereby B and T cells undergo division and differentiation triggered by pathogenic stimuli. Deregulation of the response can lead to severe consequences for the host organism ranging from immune deficiencies to autoimmunity. Tracking cell division and differentiation by flow cytometry using fluorescent probes is a major method for measuring progression of lymphocyte responses, both in vitro and in vivo. In turn, mathematical modeling of cell numbers derived from such measurements has led to significant biological discoveries, and plays an increasingly important role in lymphocyte research. Fitting an appropriate parameterized model to such data is the goal of these studies but significant challenges are presented by the variability in measurements. This variation results from the sum of experimental noise and intrinsic probabilistic differences in cells and is difficult to characterize analytically. Current model fitting methods adopt different simplifying assumptions to describe the distribution of such measurements and these assumptions have not been tested directly. To help inform the choice and application of appropriate methods of model fitting to such data we studied the errors associated with flow cytometry measurements from a wide variety of experiments. We found that the mean and variance of the noise were related by a power law with an exponent between 1.3 and 1.8 for different datasets. This violated the assumptions inherent to commonly used least squares, linear variance scaling and log-transformation based methods. As a result of these findings we propose a new measurement model that we justify both theoretically, from the maximum entropy standpoint, and empirically using collected data. Our evaluation suggests that the new model can be reliably used for model fitting across a variety of conditions. Our work provides a foundation for modeling measurements in flow cytometry experiments thus facilitating progress in quantitative studies of lymphocyte responses.


Asunto(s)
Linfocitos B/citología , Linfocitos T CD8-positivos/citología , Citometría de Flujo/estadística & datos numéricos , Modelos Estadísticos , Animales , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , División Celular/inmunología , Entropía , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Distribuciones Estadísticas , Procesos Estocásticos
12.
Epigenetics Chromatin ; 7(1): 36, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25489339

RESUMEN

BACKGROUND: Transcription factors (TFs) and histone modifications (HMs) play critical roles in gene expression by regulating mRNA transcription. Modelling frameworks have been developed to integrate high-throughput omics data, with the aim of elucidating the regulatory logic that results from the interactions of DNA, TFs and HMs. These models have yielded an unexpected and poorly understood result: that TFs and HMs are statistically redundant in explaining mRNA transcript abundance at a genome-wide level. RESULTS: We constructed predictive models of gene expression by integrating RNA-sequencing, TF and HM chromatin immunoprecipitation sequencing and DNase I hypersensitivity data for two mammalian cell types. All models identified genome-wide statistical redundancy both within and between TFs and HMs, as previously reported. To investigate potential explanations, groups of genes were constructed for ontology-classified biological processes. Predictive models were constructed for each process to explore the distribution of statistical redundancy. We found significant variation in the predictive capacity of TFs and HMs across these processes and demonstrated the predictive power of HMs to be inversely proportional to process enrichment for housekeeping genes. CONCLUSIONS: It is well established that the roles played by TFs and HMs are not functionally redundant. Instead, we attribute the statistical redundancy reported in this and previous genome-wide modelling studies to the heterogeneous distribution of HMs across chromatin domains. Furthermore, we conclude that statistical redundancy between individual TFs can be readily explained by nucleosome-mediated cooperative binding. This could possibly help the cell confer regulatory robustness by rejecting signalling noise and allowing control via multiple pathways.

13.
PLoS One ; 8(9): e73954, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24069254

RESUMEN

Mycoplasma synoviae strain MS-H, developed by chemical mutagenesis of the Australian field strain 86079/7NS, is a live temperature-sensitive (ts (+)) vaccine used for control of M. synoviae infection in poultry worldwide. Genetic basis of temperature sensitivity and attenuation of MS-H has not been revealed thus far. Comparison of the complete genome sequence of MS-H, its parent strain 86079/7NS and two non-temperature sensitive (ts (-)) reisolates of MS-H revealed a mutation in a highly conserved domain of GTP binding protein Obg of MS-H, with reversion in ts (-) MS-H reisolates. Nucleotide change from G to A at position 369 of the obg gene resulted in an alteration of glycine to arginine at position 123 in Obg fold. Further analysis of the complete obg gene sequence in several MS-H reisolates revealed that a Gly123Arg substitution was associated with alteration in temperature sensitivity phenotype of MS-H. A second mutation, C to T at position 629, in obg gene was found in some of the MS-H reisolates and appeared to suppress the effects of the Gly123Arg substitution. In silico analysis of point mutations revealed that Gly123Arg has highly destabilizing effect on the MS-H Obg structure that can potentially abolish its biological functions in vivo especially at non-permissive temperature. Findings of this study implicate Obg alteration (Gly123Arg) as one of the possible causes of MS-H attenuation/temperature sensitivity and warrant further investigations into exploring the role of Obg-like proteins, an evolutionarily conserved protein from human to bacteria, in the biology of mycoplasmas.


Asunto(s)
Proteínas Bacterianas/genética , Vacunas Bacterianas/genética , Proteínas de Unión al GTP/genética , Mutación , Mycoplasma synoviae/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Codón , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/inmunología , Genoma Bacteriano , Modelos Moleculares , Datos de Secuencia Molecular , Infecciones por Mycoplasma/inmunología , Infecciones por Mycoplasma/prevención & control , Mycoplasma synoviae/inmunología , Fenotipo , Polimorfismo de Nucleótido Simple , Conformación Proteica , Estabilidad Proteica , Alineación de Secuencia , Temperatura
14.
PLoS One ; 8(2): e55121, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23383306

RESUMEN

In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field.


Asunto(s)
Pollos/virología , Evolución Molecular , Genoma Viral/genética , Herpesvirus Gallináceo 1/genética , Filogenia , Recombinación Genética/genética , Secuencia de Aminoácidos , Animales , Australia , Secuencia de Bases , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Funciones de Verosimilitud , Modelos Genéticos , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia , Especificidad de la Especie
15.
Science ; 335(6066): 338-41, 2012 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-22223740

RESUMEN

In response to stimulation, B lymphocytes pursue a large number of distinct fates important for immune regulation. Whether each cell's fate is determined by external direction, internal stochastic processes, or directed asymmetric division is unknown. Measurement of times to isotype switch, to develop into a plasmablast, and to divide or to die for thousands of cells indicated that each fate is pursued autonomously and stochastically. As a consequence of competition between these processes, censorship of alternative outcomes predicts intricate correlations that are observed in the data. Stochastic competition can explain how the allocation of a proportion of B cells to each cell fate is achieved. The B cell may exemplify how other complex cell differentiation systems are controlled.


Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Activación de Linfocitos , Animales , Muerte Celular , Diferenciación Celular , División Celular , Femenino , Cambio de Clase de Inmunoglobulina , Ratones , Modelos Inmunológicos , Procesos Estocásticos
16.
Science ; 337(6091): 188, 2012 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-22798607

RESUMEN

Recombination between herpesviruses has been seen in vitro and in vivo under experimental conditions. This has raised safety concerns about using attenuated herpesvirus vaccines in human and veterinary medicine and adds to other known concerns associated with their use, including reversion to virulence and disease arising from recurrent reactivation of lifelong chronic infection. We used high-throughput sequencing to investigate relationships between emergent field strains and vaccine strains of infectious laryngotracheitis virus (ILTV, gallid herpesvirus 1). We show that independent recombination events between distinct attenuated vaccine strains resulted in virulent recombinant viruses that became the dominant strains responsible for widespread disease in Australian commercial poultry flocks. These findings highlight the risks of using multiple different attenuated herpesvirus vaccines, or vectors, in the same populations.


Asunto(s)
Pollos , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/genética , Herpesvirus Gallináceo 1/patogenicidad , Vacunas contra Herpesvirus , Enfermedades de las Aves de Corral/virología , Recombinación Genética , Animales , ADN Viral/genética , Genoma Viral , Infecciones por Herpesviridae/virología , Herpesvirus Gallináceo 1/inmunología , Herpesvirus Gallináceo 1/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Organismos Libres de Patógenos Específicos , Vacunas Atenuadas , Virulencia , Replicación Viral
17.
Vaccine ; 29(52): 9583-7, 2011 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-22044743

RESUMEN

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in poultry. Live attenuated ILTV vaccines have been used extensively to help control outbreaks of disease. Two Australian-origin attenuated vaccine strains, SA2 and A20 ILTV, are commercially available and are in frequent use in Australia. Both these vaccines are of chicken embryo origin (CEO). The A20 ILTV strain was developed from the SA2 ILTV strain by sequential passage of SA2 ILTV in tissue culture in order to reduce its residual virulence. Previous studies in our laboratories have demonstrated the greater attenuation of A20 ILTV under controlled experimental conditions, but the genetic basis of the in vivo phenotypes of A20 and SA2 ILTV has not been elucidated. In this study, the genetic differences between A20 and SA2 ILTV were examined by performing complete genome sequencing and comparative analysis. The genome sequences were also compared to a reference sequence from another CEO ILTV vaccine (Serva ILTV: GenBank accession number HQ_630064) of European-origin. Additional in ovo studies to assess cell to cell spread were performed in order to allow further comparisons of the pathogenicity of SA2 and A20 ILTV. The sequencing results showed that the genome sizes of SA2 and A20 ILTV were 152,975 and 152,978bp, respectively, while Serva ILTV had a genome size of 152,630bp. The genomes of SA2 and A20 ILTV shared 99.9% nucleotide sequence identity with each other, but only 99.2% identity with Serva ILTV. In complete genome alignments between SA2 and A20 ILTV, a total of 24 single nucleotide polymorphisms (SNPs) were identified, but only two of these were non-synonymous. These were located in the ORF B and UL15 genes. Four indels were detected in non-coding regions. The findings from this study demonstrate the general genetic stability of ILTV, but also show that non-synonymous changes in the ORF B and UL15 genes have arisen following tissue culture passage of SA2 ILTV to produce the A20 vaccine. It is likely that these non-synonymous changes are related to the greater attenuation of A20 ILTV compared to SA2 ILTV, and to the reduced ability of A20 ILTV to spread from cell to cell, as observed in this study. The results from this study also demonstrate the divergence between the genomes of the Australian-origin ILTV vaccine strains and the Serva vaccine strain.


Asunto(s)
ADN Viral/genética , Genoma Viral , Herpesvirus Gallináceo 1/genética , Animales , Australia , Embrión de Pollo , Pollos , ADN Viral/química , Herpesvirus Gallináceo 1/patogenicidad , Mutación INDEL , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Sintenía , Vacunas Atenuadas/genética , Virulencia
18.
J R Soc Interface ; 7(48): 1049-59, 2010 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-20053654

RESUMEN

During the adaptive immune response, lymphocyte populations undergo a characteristic three-phase process: expansion through a series of cell divisions; cessation of expansion; and, finally, most of the accumulated lymphocytes die by apoptosis. The data used, thus far, to inform understanding of these processes, both in vitro and in vivo, are taken from flow cytometry experiments. One significant drawback of flow cytometry is that individual cells cannot be tracked, so that it is not possible to investigate interdependencies in the fate of cells within a family tree. This deficit in experimental information has recently been overcome by Hawkins et al. (Hawkins et al. 2009 Proc. Natl Acad. Sci. USA 106, 13 457-13 462 (doi:10.1073/pnas.0905629106)), who reported on time-lapse microscopy experiments in which B-cells were stimulated through the TLR-9 receptor. Cells stimulated in this way do not aggregate, so that data regarding family trees can be recorded. In this article, we further investigate the Hawkins et al. data. Our conclusions are striking: in order to explain the familial correlation structure in division times, death times and propensity to divide, a minimum of two distinct heritable factors are necessary. As the data show that two distinct factors are necessary, we develop a stochastic model that has two heritable factors and demonstrate that it can reproduce the key features of the data. This model shows that two heritable factors are sufficient. These deductions have a clear impact upon biological understanding of the adaptive immune response. They also necessitate changes to the fundamental premises behind the tools developed by statisticians to draw deductions from flow cytometry data. Finally, they affect the mathematical modelling paradigms that are used to study these systems, as these are widely developed based on assumptions of cellular independence that are not accurate.


Asunto(s)
División Celular , Linfocitos/citología , Animales , Apoptosis/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Proliferación Celular , Humanos , Activación de Linfocitos/inmunología , Linfocitos/inmunología
19.
Nat Protoc ; 2(9): 2057-67, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17853861

RESUMEN

Cellular proliferation is an essential feature of the adaptive immune response. The introduction of the division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has made it possible to monitor the number of cell divisions during proliferation and to examine the relationship between proliferation and differentiation. Although qualitative examination of CFSE data may be useful, substantially more information about division and death rates can be extracted from quantitative CFSE time-series experiments. Quantitative methods can reveal in detail how lymphocyte proliferation and survival are regulated and altered by signals such as those received from co-stimulatory molecules, drugs and genetic polymorphisms. In this protocol, we present a detailed method for examining time-series data using graphical and computer-based procedures available to all experimenters.


Asunto(s)
Fluoresceínas , Colorantes Fluorescentes , Activación de Linfocitos , Linfocitos/inmunología , Programas Informáticos , Succinimidas , Diferenciación Celular , División Celular , Proliferación Celular , Células Cultivadas , Simulación por Computador , Interpretación Estadística de Datos , Citometría de Flujo , Linfocitos/citología , Modelos Biológicos , Coloración y Etiquetado
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