Heterologous expression of the filarial nematode alt gene products reveals their potential to inhibit immune function

BACKGROUND: Parasites exploit sophisticated strategies to evade host immunity that require both adaptation of existing genes and evolution of new gene families. We have addressed this question by testing the immunological function of novel genes from helminth parasites, in which conventional transgenesis is not yet possible. We investigated two such novel genes from Brugia malayi termed abundant larval transcript (alt), expression of which reaches ~5% of total transcript at the time parasites enter the human host. RESULTS: To test the hypothesis that ALT proteins modulate host immunity, we adopted an alternative transfection strategy to express these products in the protozoan parasite Leishmania mexicana. We then followed the course of infection in vitro in macrophages and in vivo in mice. Expression of ALT proteins, but not a truncated mutant, conferred greater infectivity of macrophages in vitro, reaching 3-fold higher parasite densities. alt-transfected parasites also caused accelerated disease in vivo, and fewer mice were able to clear infection of organisms expressing ALT. alt-transfected parasites were more resistant to IFN-γ-induced killing by macrophages. Expression profiling of macrophages infected with transgenic L. mexicana revealed consistently higher levels of GATA-3 and SOCS-1 transcripts, both associated with the Th2-type response observed in in vivo filarial infection. CONCLUSION: Leishmania transfection is a tractable and informative approach to determining immunological functions of single genes from heterologous organisms. In the case of the filarial ALT proteins, our data suggest that they may participate in the Th2 bias observed in the response to parasite infection by modulating cytokine-induced signalling within immune system cells

Inactivation of the RB family prevents thymus involution and promotes thymic function by direct control of Foxn1 expression

Thymic involution during aging is a major cause of decreased production of T cells and reduced immunity. Here we show that inactivation of Rb family genes in young mice prevents thymic involution and results in an enlarged thymus competent for increased production of naive T cells. This phenotype originates from the expansion of functional thymic epithelial cells (TECs). In RB family mutant TECs, increased activity of E2F transcription factors drives increased expression of Foxn1, a central regulator of the thymic epithelium. Increased Foxn1 expression is required for the thymic expansion observed in Rb family mutant mice. Thus, the RB family promotes thymic involution and controls T cell production via a bone marrow–independent mechanism, identifying a novel pathway to target to increase thymic function in patients

Foxn1 Regulates Lineage Progression in Cortical and Medullary Thymic Epithelial Cells But Is Dispensable for Medullary Sublineage Divergence

The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development

Prevalence of childhood disability and the characteristics and circumstances of disabled children in the UK : secondary analysis of the Family Resources Survey

Background: Robust data on the prevalence of childhood disability and the circumstances and characteristics of disabled children is crucial to understanding the relationship between impairment and social disadvantage. It is also crucial for public policy development aimed at reducing the prevalence of childhood disability and providing appropriate and timely service provision. This paper reports prevalence rates for childhood disability in the United Kingdom (UK) and describes the social and household circumstances of disabled children, comparing these where appropriate to those of non-disabled children. Methods: Data were generated from secondary analysis of the Family Resources Survey, a national UK cross-sectional survey, (2004/5) which had data on 16,012 children aged 0-18 years. Children were defined as disabled if they met the Disability Discrimination Act (DDA) definition (1995 and 2005). Frequency distributions and cross-tabulations were run to establish prevalence estimates, and describe the circumstances of disabled children. To establish the association between individual social and material factors and childhood disability when other factors were controlled for, logistic regression models were fitted on the dependent variable 'DDA defined disability'. Results: 7.3% (CI 6.9, 7.7) of UK children were reported by as disabled according to the DDA definition. Patterns of disability differed between sexes with boys having a higher rate overall and more likely than girls to experience difficulties with physical coordination; memory, concentration and learning; communication. Disabled children lived in different personal situations from their non-disabled counterparts, and were more likely to live with low-income, deprivation, debt and poor housing. This was particularly the case for disabled children from black/minority ethnic/ mixed parentage groups and lone-parent households. Childhood disability was associated with lone parenthood and parental disability and these associations persisted when social disadvantage was controlled for. Conclusion: These analyses suggest that UK disabled children experience higher levels of poverty and personal and social disadvantage than other children. Further research is required to establish accurate prevalence estimates of childhood disability among different black and minority ethnic groups and to understand the associations between childhood disability and lone parenthood and the higher rates of sibling and parental disability in households with disabled children

Is the onset of disabling chronic conditions in later childhood associated with exposure to social disadvantage in earlier childhood? a prospective cohort study using the ONS Longitudinal Study for England and Wales

Background: The aetiology of disabling chronic conditions in childhood in high income countries is not fully understood, particularly the association with socio-economic status (SES). Very few studies have used longitudinal datasets to examine whether exposure to social disadvantage in early childhood increases the risk of developing chronic conditions in later childhood. Here we examine this association, and its temporal ordering, with onset of all-cause disabling chronic later childhood in children reported as free from disability in early childhood. Methods: The study comprised a prospective cohort study, using data from the Office for National Statistics Longitudinal Study (ONSLS) for England and Wales. The study sample included 52,839 children with complete data born between 1981–1991 with no disabling chronic condition/s in 1991. Index cases were children with disability recorded in 2001. Comparison cases were children with no recorded disability in 1991. A socio-economic disadvantage index (SDI) was constructed from data on social class, housing tenure and car/van access. Associations were explored with logistic regression modelling controlling sequentially for potentially confounding factors; age, gender, ethnicity and lone parenthood. Results: By 2001, 2049 (4%) had at least one disability. Socio-economic disadvantage, age, gender and lone parenthood but not ethnicity were significantly associated with onset of disabling chronic conditions. The SDI showed a finely graded association with onset of disabling chronic conditions in the index group (most disadvantaged OR 2·11 [CI 1·76 to 2·53]; disadvantaged in two domains OR 1·45 [CI 1·20 to 1·75]; disadvantaged in one domain OR 1·14 [CI 0·93 to 1·39] that was unaffected by age, gender and ethnicity and slightly attenuated by lone parenthood. Conclusion: To our knowledge, this is the first study to identify socio-economic disadvantage in earlier childhood as a predisposing factor for onset of all-cause disabling chronic conditions in later childhood. Temporal ordering and gradation of the response indicate socio-economic disadvantage may play a causal role. This suggests that targeting preventative efforts to reduce socio-economic disadvantage in early childhood is likely to be an important public health strategy to decease health inequalities in later childhood and early adulthood

Long-Term Persistence of Functional Thymic Epithelial Progenitor Cells In Vivo under Conditions of Low FOXN1 Expression

Normal thymus function reflects interactions between developing T-cells and several thymic stroma cell types. Within the stroma, key functions reside in the distinct cortical and medullary thymic epithelial cell (TEC) types. It has been demonstrated that, during organogenesis, all TECs can be derived from a common thymic epithelial progenitor cell (TEPC). The properties of this common progenitor are thus of interest. Differentiation of both cTEC and mTEC depends on the epithelial-specific transcription factor FOXN1, although formation of the common TEPC from which the TEC lineage originates does not require FOXN1. Here, we have used a revertible severely hypomorphic allele of Foxn1, Foxn1R, to test the stability of the common TEPC in vivo. By reactivating Foxn1 expression postnatally in Foxn1R/- mice we demonstrate that functional TEPCs can persist in the thymic rudiment until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression, suggesting that manipulation of FOXN1 activity may prove a valuable method for long term maintenance of TEPC in vitro

Foxn1 Is Dynamically Regulated in Thymic Epithelial Cells during Embryogenesis and at the Onset of Thymic Involution

Thymus function requires extensive cross-talk between developing T-cells and the thymic epithelium, which consists of cortical and medullary TEC. The transcription factor FOXN1 is the master regulator of TEC differentiation and function, and declining Foxn1 expression with age results in stereotypical thymic involution. Understanding of the dynamics of Foxn1 expression is, however, limited by a lack of single cell resolution data. We have generated a novel reporter of Foxn1 expression, Foxn1G, to monitor changes in Foxn1 expression during embryogenesis and involution. Our data reveal that early differentiation and maturation of cortical and medullary TEC coincides with precise sub-lineage-specific regulation of Foxn1 expression levels. We further show that initiation of thymic involution is associated with reduced cTEC functionality, and proportional expansion of FOXN1-negative TEC in both cortical and medullary sub-lineages. Cortex-specific down-regulation of Foxn1 between 1 and 3 months of age may therefore be a key driver of the early stages of age-related thymic involution

The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus. © 2012 Nature America, Inc. All rights reserved

Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

Expression of Plet1 controls interstitial migration of murine small intestinal dendritic cells

Under homeostatic conditions, dendritic cells (DCs) continuously patrol the intestinal lamina propria. Upon antigen encounter, DCs ini