Biomedical subjects

Yvonne Barnett

Publications and source records attributed to Yvonne Barnett.

10 recordsLinked to original sources

PubMed2006

Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes.

BACKGROUND: Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose) polymerases (PARPs), using NAD+ as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosyl)ation capacity, defined as the amount of poly(ADP-ribose) produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions. RESULTS: This paper reports a new, robust protocol for the measurement of cellular poly(ADP-ribosyl)ation capacity in PBMC or Jurkat T-cells using flow cytometry, based on a previously established immuno-dot-blot assay. In order to validate the new assay, we determined the dose-response curve of 3-aminobenzamide, a well-known competitive PARP inhibitor, and we derived an IC50 that is very close to the published value. When testing a set of PBMC samples taken from fifteen healthy young human donors, we could confirm the presence of a substantial interindividual variation, as previously observed using a radiometric assay. CONCLUSION: The methodology described in this paper should be generally useful for the determination of cellular poly(ADP-ribosyl)ation capacity in a wide variety of settings, especially for the comparison of large sets of samples, such as population studies. In contrast to previously published radiometric or immuno-dot-blot assays, the new FACS-based method allows (i) selective analysis of mononuclear cells by gating and (ii) detection of a possible heterogeneity in poly(ADP-ribosyl)ation capacity between cells of the same type.

Journal Article↗

PubMed2005

An investigation of DNA mismatch repair capacity under normal culture conditions and under conditions of supra-physiological challenge in human CD4+T cell clones from donors of different ages.

T cells undergo rapid clonal expansion upon antigenic stimulation to produce an effective immune response. Any defect in the DNA mismatch repair (MMR) system may have a detrimental effect on T cell proliferation. This study employed an in vitro model of human CD4+T cell ageing to investigate MMR capacity at various stages of T cell lifespan. A novel modification of the alkaline comet assay, which utilised T4 endonuclease VII to detect single base DNA mismatches, was used to assess DNA mismatch frequency. No clear pattern in DNA mismatch frequency with increasing culture age was observed. However, the ability to repair induced DNA mismatches (following treatment with acridine mutagen ICR-191) revealed an age-related decline in the efficiency of the MMR system in clones derived from a 26 and a 45-year-old donor, but not from an 80-year-old very healthy SENIEUR donor. This study suggests that unchallenged, dividing human T cell clones have variable levels of DNA mismatches throughout their lifespan, not affecting proliferation. However, when challenged with supra-physiological levels of DNA mismatches, deficiencies were found in ageing T cell clones in MMR capacity, with the exception of T cell clones from a SENIEUR donor previously shown to maintain effective DNA excision repair.

Adult↗

PubMed2005

Genetic consultations in primary care: GPs' responses to three scenarios.

OBJECTIVE: This study investigated general practitioners' responses to three scenarios in which patients consulted regarding genetic conditions. DESIGN: Self-completed postal study. Setting. Primary care in Northern Ireland. SUBJECTS: Questionnaire were distributed to all the GPs in Northern Ireland (n = 1079). A total of 541 GPs participated (50%). MAIN OUTCOME MEASURES: Responses to three scenarios in which patients consulted regarding their family history and risk of bowel cancer, breast cancer, and cystic fibrosis. RESULTS: Most GPs correctly identified the patients' risk of bowel cancer, recommended regular colonoscopy, advised lifestyle changes, and did not refer to the genetic clinic. GPs who were qualified for longer were more likely to recommend colonoscopy and less likely to advise lifestyle changes. With the breast cancer patient GPs adopted a cautious approach; most would refer to the genetic and mammography clinics. With the cystic fibrosis example, most correctly identified the patient's risk of carrying the gene, would refer to the genetic clinic, and would encourage the patient to discuss the risk with his partner. In general, doctors were unsure, but would pass on genetic information to insurance companies if requested. CONCLUSION: The study suggests that, in most cases, general practitioners correctly identify at-risk individuals but there may still be some uncertainty regarding referrals. The results suggest that ways of educating GPs should be explored. Educational interventions should be linked to a greater understanding of factors involved in referral (including the influence of gender and experience). The guidelines provided to GPs in relation to the provision of genetic information to insurance companies may need to be reviewed in some countries.

Adult↗

PubMed2004

An investigation of DNA excision repair capacity in human CD4+ T cell clones as a function of age in vitro.

DNA damage has been shown to increase with age in lymphocytes of healthy humans and in human CD4+ T cell clones. Such genetic damage, if unrepaired, may have a detrimental effect on lymphocyte-mediated immune responses. This study investigated DNA excision repair capacity of human CD4+ T cell clones as a function of age in vitro. Cultures of T cell clones were treated with a range of DNA damaging agents; hydrogen peroxide, N-methyl-N'-nitro-N-nitrosoguanidine or 254 nm ultraviolet irradiation. Following treatment, the amount of DNA damage in the clones was determined over a time course using modified comet assays. The results obtained revealed a decline related to in vitro age in the DNA repair capacity of clones derived from a 26 and a 45 year old donor. This decline may represent at least a partial explanation for the age related increase in DNA damage in these clones when cultured in vitro. In contrast, there was no evidence for a decline related to in vitro age in repair capacity in the clones derived from an 80 year old SENIEUR donor. An alternative mechanism must underlie the age related increased in DNA damage in these clones when cultured in vitro.

Adult↗

PubMed2004

Effects of a reduced oxygen tension culture system on human T cell clones as a function of in vitro age.

Oxidative DNA damage has been suggested to contribute to the decline in T cell clone (TCC) function with increased age in vitro. To test this hypothesis the effect of a reduced oxygen tension culture system (6% O(2)) on TCCs was examined. Specifically, the effects of the altered culture conditions on DNA damage levels, in vitro lifespan and proliferative capacity were assessed in five independently derived human CD4+ TCCs. DNA damage levels over the entire lifespan were significantly lowered by reducing oxygen tension. Lifespan (total population doublings (PDs) achieved) and proliferative capacity (PDs/week) were reduced for all clones under reduced oxygen tension when compared to standard culture conditions. This observed tendency warrants further investigation using a greater number of clones from donors of all age groups before definitive conclusions regarding the effect of low oxygen tension on the lifespan and proliferative capacity of TCC can be made. However, these results may suggest that the reduced oxygen tension culture system has interfered with some aspect of T cell biology not yet examined within the remit of this study.

Adult↗

PubMed2004

Engineering anticancer T cells for extended functional longevity.

Like other somatic cells, human T lymphocytes have a finite replicative capacity in vitro, and, by implication and consistent with the limited data available, in vivo as well. An accumulation of dysfunctional T cells may be detrimental under conditions of chronic antigenic stress (chronic infection, cancer, autoimmunity). Using T cells from young donors to model the process of T cell clonal expansion in vitro under these conditions reveals age-associated increasing levels of oxidative DNA damage and microsatellite instability (MSI), coupled with decreasing DNA repair capacity, telomerase induction and telomere length, decreased levels of expression of the T cell costimulator CD28 and consequently reduced secretion of the T cell growth factor interleukin-2 (IL-2). However, data from similar experiments using T cell clones (TCCs) derived from extremely healthy very elderly donors ("successfully aged") indicate that DNA repair is better maintained, MSI less prevalent, and (already short) telomere lengths are maintained. Nonetheless, oxidative DNA damage is seen to the same extent, and clonal longevity is also similar in these clones. DNA damage levels are reduced by culture in 5% oxygen, but longevity is not improved. This may be because of the requirement for intermittent reactivation via receptor pathways dependent on free radical production in T cells. These recent findings from our international immunosenescence research consortium suggest that strategies other than telomere maintenance, better protection against free radicals, or improved DNA repair will be required for functional longevity extension of human TCCs. To obtain sufficient cells for adoptive immunotherapy of cancer, alternative avenues need exploration; currently, these include enforced expression of certain heat shock proteins and proteasome components, and interference with the expression of negative regulatory receptors expressed by T cells.

Antigens↗

PubMed2004

Genetics and genetic testing: are GPs likely to attend training courses?

BACKGROUND: GPs must make difficult screening and diagnostic decisions regarding genetic testing for different cancers. Educational programs may improve knowledge and enable more appropriate referral. METHODS: A postal survey of all general practitioners (GPs) in Northern Ireland (N = 534; response rate = 49.4%) asked GPs if they would attend 3 different types of training courses in genetics. RESULTS: Almost 75% indicated that they would be likely and/or very likely to attend such courses. Women and GPs who had been qualified recently were most likely to attend (P < .005, P < .05). CONCLUSION: The results suggest that GPs are interested in training courses. Male GPs and GPs who have been qualified for longer should be specifically targeted.

Data Collection↗

PubMed2002

T cells and aging, January 2002 update.

Age-related changes in the immune system may contribute to morbidity and mortality due to decreased resistance to infection and, possibly, certain cancers in the aged. Many studies mostly performed in mice, rats and man but also including monkeys and dogs have established that age-associated immune decline is characterized by decreases in both humoral and cellular responses. The former may be largely a result of the latter, because observed changes both in the B cell germline-encoded repertoire and the age-associated decrease in somatic hypermutation of the B cell antigen receptors are now known to be critically affected by helper T cell aging. As antigen presenting cell (APC) function appears to be well-maintained in the elderly, this review will focus on the T cell. Factors contributing to T cell immunosenescence may include a) altered production of T cell progenitors (stem cell defects, stromal cell defects), b) decreased levels of newly-generated mature T cells (thymic involution), c) aging of resting immune cells, d) disrupted activation pathways in immune cells (stimulation via the T cell receptor for antigen, costimulation, apoptosis control), e) replicative senescence of clonally expanding cells. This review aims to consider the current state of knowledge on the scientific basis for and potential clinical relevance of those factors in immunosenescence in humans. Experiments in other species will be touched upon with the proviso that there are clearly differences between them, especially between humans and rodents, but exactly what those differences are is not completely clear. Given its potential importance and the increasing proportion of elderly people the world over, coupled with the realisation that whereas mortality is decreasing, morbidity may not be decreasing in parallel (1), a better understanding of the causes and impact of immunosenescence may offer the possibility of identifying where prevention or delay of onset, as well as therapeutic intervention, might be beneficial. Amelioration of the effects of dysregulated immune responses in the elderly by replacement therapy, supplementation therapy or other approaches may result in an enhancement of their quality of life, and significant reductions in the cost of medical care in old age.

Aging↗

PubMedDate not supplied

Human CD4+ T cell clone longevity in tissue culture: lack of influence of donor age or cell origin.

CD4+ human T cell clones were derived from activated peripheral blood lymphocytes of healthy young adults to establish cloning efficiencies (CE) and clonal longevities. These results were compared with those obtained using cells from the very elderly, also in excellent health. CE and both maximal and average longevities under appropriate culture conditions were very similar in the two groups. Moreover, CE of CD34+ hematopoietic progenitor cells and longevities of clones derived from them were also similar. Finally, CE and longevities of clones derived from a patient with chronic myelogenous leukaemia were found to be comparable as well. Hence, T cells with absolutely no antigenic exposure in vivo prior to cloning (i.e. CD34-derived) and those potentially exposed to chronic antigenic stimulation (CML-derived) and those from young or old donors all had similar cloning and propagation properties in vitro. These results imply that the longevity of T cells in culture is more likely to be dictated by cloning conditions than any intrinsic differences between the cells studied.

Adult↗

PubMedDate not supplied

Nonagenarians from the Swedish NONA Immune Study have increased plasma antioxidant capacity and similar levels of DNA damage in peripheral blood mononuclear cells compared to younger control subjects.

The results of previous work from our laboratories have suggested that free radical damage to T cells as they age may contribute to the age-related decline in the T cell-mediated immune response. The aims of this investigation were to assess the efficiency of in vivo antioxidant capacity through determining the antioxidant capacity of plasma using the ferric reducing ability of plasma assay, and to assess the levels and types of DNA damage (as a measure of in vivo antioxidant efficiency) using the alkaline comet assay and two enzymatic modifications of the comet assay, in peripheral blood mononuclear cells (PBMCs) from nonagenarian subjects drawn from the Swedish NONA Immune Study. The results obtained were compared with those from middle-aged (40-60 years) controls to identify potential anti-immunosenescent effects of in vivo antioxidants. The results revealed a significantly higher plasma antioxidant capacity in NONA subjects compared to controls, and these results support a relationship between longevity and intact immune function, which may be underpinned by antioxidant defences which reduce free radical damage to PBMC, thus helping to maintain cell function. The NONA subjects were found to have similar levels of DNA damage in their PBMCs to those found in middle aged controls.

Adult↗