Wayne A. Mitchell

Tracing thymic output in older individuals.

As a result of age‐associated thymic atrophy, T cell production declines with age. Some studies suggest that production undergoes an exponential decline starting at birth, while others consider the decline to be in a biphasic manner with a rapid reduction in output occurring before middle age followed by a phase in which output declines at a regular, albeit much slower, rate. Both approaches provide estimations of the time of termination of thymic output, but on the basis of limited amounts of data. We have analysed blood from more than 200 individuals between the ages of 58 and 104 years to determine changes in thymic output using signal‐joint T cell receptor excision circles (sjTREC)/T cells as our measure. To reduce any potential geographical or nutritional bias we have obtained samples from five different European countries. Our results reveal that while the absolute number of T cells per microlitre of blood does not change significantly across the age range we tested, the values of sjTREC per microlitre show wide variation and reveal an age‐associated decline in thymic output. In addition we show gender differences, with notably higher thymic output in females than males at each decade. More importantly, we noted a significant decline in sjTREC/T cell levels in those more than 90 years of age in both males and females. Our results provide information about the potential end‐point for thymic output and suggest that sjTREC analysis may be a biomarker of effective ageing.

Immunosenescence: Implications for vaccination programmes in adults

Vaccination is crucially important in preventing infection and protecting vulnerable population from infectious diseases. However, a multitude of changes in the immune system occurring with advancing age, termed immunosenescence, lead to limit the protective effects of vaccination in older adults. While it is widely believed that the current immunization strategies saves many lives, vaccine preventable infectious diseases (VPDs) still place a considerable burden, not only on older individuals, but also on the adult population and healthcare systems of developed countries. This review will first examine the evidence linking the contribution of immunosenescence to a less than optimal vaccine response in aged individuals in order to demonstrate that strategy of promoting vaccination in these populations is not sufficient to reduce the burden associated with VPDs. Furthermore, based upon the side effects of the herd immunity when vaccine-policies are mainly childhood-centered, considerations will be given on the imperative necessity to frame shift our thinking and efforts away from a nearly complete childhood-centered vaccine programme toward a life-span immunization programmes.

Vaccine effectiveness in older individuals: What has been learned from the influenza-vaccine experience

Vaccination policies in most high-income countries attempt to reduce the adverse impact of influenza targeting people aged at least 60 years. However, while it is widely believed that the current immunization strategy saves many lives, influenza infection still remains a severe burden in aged individuals leading to a wide debate on the exact magnitude of the benefit of vaccination in this population. The first aim of the present review is to examine how effective current influenza-vaccine strategies are in aged adults, by analysing which are the most important factors modulating the interpretation of study results in this population. Furthermore, consideration will be given to how immune factors influence the measurement of vaccine efficacy/effectiveness, where advancing age leads to deleterious changes in the adaptive immune system, resulting in less than optimal responses to infectious agents and vaccination. Finally this review concludes with possible strategies to improve the ability of the senescent immune system to respond to vaccination.

Reversal of age-associated thymic atrophy: Treatments, delivery, and side effects

Our ability to survive infectious agents depends on making adequate immune responses, but as we get older our thymus atrophies. Production and export of T cells bearing new antigen receptor specificities to the peripheral T cell pool declines and results in shrinkage of the repertoire. Other changes in the peripheral T cell pool include an increase in cells moving closer to their replicative limit. Age related immune dysfunction, evident through the increased susceptibility to infection, follows these changes. Improvement in immune function in the elderly may require us to rejuvenate the immune system starting first with reversing the atrophy seen in the thymus. This has been achieved experimentally with interleukin 7, growth hormone, growth hormone secretagogues, keratinocyte growth factor or through chemical or surgical castration. The widespread use of one or more of these treatments will depend upon their effectiveness, their ease of delivery and the extent of any side effects.

A Fluorescence in Situ Hybridization Map of 6q Deletions in Acute Lymphocytic Leukemia: Identification and Analysis of a Candidate Tumor Suppressor Gene

With the objective of identifying candidate tumor suppressor genes, we used fluorescence in situ hybridization to map leukemia-related deletions of the long arm of chromosome 6 (6q). Twenty of 24 deletions overlapped to define a 4.8-Mb region of minimal deletion between markers D6S1510 and D6S1692 within chromosome 6 band q16. Using reverse transcription-PCR, we found evidence of expression in hematopoietic cells for 3 of 15 genes in the region (GRIK2, C6orf111, and CCNC). Comparison between our own and published deletion data singled out GRIK2 as the gene most frequently affected by deletions of 6q in acute lymphocytic leukemia (ALL). Sequence analysis of GRIK2 in 14 ALL cases carrying heterozygous 6q deletions revealed a constitutional and paternally inherited C to G substitution in exon 6 encoding for an amino acid change in one patient. The substitution was absent among 232 normal alleles tested, leaving open the possibility that heterozygous carriers of such mutations may be susceptible to ALL. Although low in all normal hematopoietic tissues, quantitative reverse transcription-PCR showed higher baseline GRIK2 expression in thymus and T cells than other lineages. Among T-cell ALL patients, 6q deletion was associated with a statistically significant reduction in GRIK2 expression (P = 0.0001). By contrast, elevated GRIK2 expression was measured in the myelomonocytic line THP-1 and in one patient with common ALL. Finally, we detected significant levels of GRIK2 expression in prostate, kidney, trachea, and lung, raising the possibility that this gene may be protective against multiple tumor types.

Thymic output, ageing and zinc

The role of the thymus is vital for orchestration of T-cell development and maturation. With increasing age the thymus undergoes a process of involution which results in a reduction in thymic size, function and output. Until relatively recent it was not feasible to accurately measure the magnitude of age-related loss of thymic function. With the discovery of T-cell receptor excision circles (TRECs), which are the stable by-products of the newly generated T-cells, it is now possible to quantitatively measure the extent of thymic output. This review examines the available data on immune function and zinc deficiency and places them in the context of the aims of the ZINCAGE project which include the evaluation of the role played by zinc in maintaining thymic output in healthy elderly individuals.

Cytotoxic drugs enhance the ex vivo sensitivity of malignant cells from a subset of acute myeloid leukaemia patients to apoptosis induction by tumour necrosis factor receptor-related apoptosis-inducing ligand

Summary. We have studied the actions of tumour‐necrosis‐factor‐related apoptosis‐inducing ligand (TRAIL) on cells isolated from patients with acute myeloid leukaemia (AML). Apoptosis induction was initially assessed by quantitative morphological analysis. Only 2/19 isolates showed a > 10% increase in apoptotic cells following TRAIL treatment. However, incubation with TRAIL combined with fludarabine, cytosine arabinoside or daunorubicin resulted in additive or super‐additive apoptosis induction in approximately half of the isolates. Molecular evidence of super‐additive apoptosis induction by TRAIL and cytotoxic agents was obtained by quantification of caspase 3 activation, detected by Western blot analysis of poly (ADP ribose) polymerase cleavage. The ability of TRAIL and daunorubicin to induce super‐additive apoptosis correlated with the ability of these agents to activate caspase 8 and to augment cellular levels of the truncated pro‐apoptotic form of the BCL‐2 family member BID. Our data suggest that co‐administration of TRAIL with conventional cytotoxic drugs may be of therapeutic value in some patients with AML.

Real time-PCR assay estimating the naive T-cell pool in whole blood and dried blood spot samples: Pilot study in young adults

Because of their central role orchestrating the immune response, the decrease in repertoire number and diversity of naïve T-cells is a significant feature of immnosenescence. Reflecting the effective naive T-cell pool, quantifying the sj-TREC ratio (number of signal joint T-cell receptor excision circles/10(5) T-cells) in blood samples suffers however from constraints. The most limiting one is the absolute requirement of the flow cytometry analysis of peripheral blood samples for the T-cell numeration. In order to make this ratio more accessible for clinical and epidemiological studies addressing how changes in responsiveness of the immune system lead to an increased susceptibility to various diseases and poorer response to vaccination, we have developed a rapid and simple method for the quantification of the sj-TREC ratio in whole blood and in dried blood spot (DBS) samples. This novel method is a QPCR analysis using fluorescently labelled sequence-specific probes both for quantifying sj-TREC and T-cell count and therefore eliminating the absolute necessity of the flow cytometer analysis. In this pilot study, we have compared the sj-TREC ratio we obtained with this novel method in whole blood and in DBS samples of 10 healthy volunteers with those obtained with the technique of reference and found that they are comparable.

Pulmonary Delivery of Interleukin-7 Provides Efficient and Safe Delivery to the Aging Immune System

Age-associated atrophy of the thymus with coincident reduction in thymopoeisis, decline in thymic output, and subsequent immune dysfunction has been reversed by the use of interleukin-7 (IL-7). In the earlier studies and in clinical trials, delivery of IL-7 has been by multiple injections over several days to maintain effective activity levels in the tissues. This is unlikely to meet with high compliance rates in future clinical use, and so we tested alternate routes of delivery using a technique involving tagging IL-7 with fluorescent dye that emits in the near-infrared region and whose fluorescence can be visualized within the tissues of live animals. We have shown that intratracheal instillation, enabling transfer through the lungs, provides an effective route for delivering IL-7 into the bloodstream and from there into the tissues in older animals. Delivery is rapid and widespread tissue distribution is seen. Comparison of administration either subcutaneously or by instillation reveals that IL-7 delivery by the pulmonary route provides significantly greater transmission to lymphoid tissues when compared with injection. In functional assessment studies, pulmonary administration led to significantly improved intrathymic T cell development in older animals when compared with IL-7 delivered by injection. Furthermore, in these older animals, delivery of IL-7 by intratracheal instillation was not accompanied by any apparent adverse events when compared with controls receiving saline vehicle by instillation or animals receiving IL-7 by subcutaneous injection.

Maintenance and Restoration of Immune System Function

The immune system is our own personal biological warfare machine used to defend the body against infection. Complexity in the immune system arises because of the lack of any long term stability in the pool of pathogenic organisms. These have the ability to mutate and alter their ability to invade the host. As a consequence our immune system has to be able to cope with all possible variants that potential pathogens can produce. Central to the immune response are T cells bearing antigen receptors specific for components of potential pathogens. These T cells are produced by the thymus and circulate around the body searching for the antigen which binds to their receptor. Binding of antigen with its receptor is one of the steps leading to clonal expansion, the start of an immune response and the production of memory T cells. Failure to meet the appropriate antigen within a defined period leads to cell death. So, our ability to survive infectious agents depends on making adequate responses, but as we get older our thymus atrophies and the number of T cells bearing new T cell receptor declines and results in shrinkage of the repertoire. In addition as we age our memory T cells are moving closer to their replicative limit. Age related immune dysfunction, evident through the increased susceptibility to infection, follows these changes. Improvement in immune function in the elderly may require us to rejuvenate the immune system starting first with reversing the atrophy seen in the thymus. This has been achieved experimentally with interleukin 7 and also with keratinocyte growth factor or through chemical or surgical castration. Widespread use of one or more of these treatments will depend upon improving the ease of their delivery. Should development continue at its current rate then orally bioavailable forms of treatments to reverse thymic atrophy may be widely available within the next ten years.