MD Martina Marín-Gutzke
email: maringutzke@yahoo.com

Alberto Sánchez-Olaso

Reconstructive surgery in young women with breast cancer

Introduction

Mastectomy is associated with significant treatment-related distress and changes in body image and sexuality due to disfigurement and sense of loss. These effects may be more pronounced in younger women [START_REF] Schover | Sexuality and body image in younger women with breast cancer[END_REF][START_REF] Fobair | Body image and sexual problems in young women with breast cancer[END_REF]. Breast reconstruction is the reverse of mastectomy, and its main objective is to restore the patient's body image, psychological well-being and expectations after mastectomy.

Autologous and prosthetic (or nonautologous) reconstruction are the two main techniques used for breast reconstruction [START_REF] Disa | Postmastectomy reconstruction: an approach to patient selection[END_REF]. Because postoperative outcomes currently obtained with both methods are comparable, the key to fulfilling the primary objective is to individualize patient selection. Factors that are relevant in the process of choosing the best technique for each patient include physician counseling, type of tumor, stage, size, uni-or bilateral involvement, use of adjuvant radiotherapy, body characteristics, overall health, breast cancer treatment plan, personal preferences and age [START_REF] Nahabedian | Breast reconstruction: a review and rationale for patient selection[END_REF]. It has been shown that patients under 50 years of age have a 4.3-fold greater likelihood of undergoing mastectomy followed by breast reconstruction than older patients [START_REF] Morrow | Factors influencing the use of breast reconstruction postmastectomy: a National Cancer Database study[END_REF]. The reasons behind this include the desire to achieve adequate breast cosmesis and symmetry, as well as fear of recurrence due to the fact that breast tumors in young women are more likely to be associated with high-risk tumor features and poorer prognosis, and tend to present with a more advanced stage at diagnosis [START_REF] Peppercorn | Breast cancer in women under 40[END_REF].

Radiotherapy deserves special attention because it is being used with increasing frequency in selected patients with early breast cancer. Prosthetic reconstruction in patients receiving adjuvant radiotherapy is problematic; hence, autologous tissue is preferable to breast implants within an irradiated operative field. However even autologous tissue reconstructions can be adversely affected by postmastectomy radiotherapy [START_REF] Kronowitz | Radiation therapy and breast reconstruction: a critical review of the literature[END_REF].

Prophylactic mastectomy is also being indicated in a growing number of patients with genetic mutations, in cases of lobulillar carcinoma or due to cancer phobia, making bilateral reconstruction an emergent scenario in young women with breast cancer. In these patients it is important to choose a similar reconstructive procedure on both sides to achieve breast symmetry.

Expectations of breast reconstruction are specifically related to breast size and shape, but also to the symmetry of the reconstructed breast with respect of the contralateral nonreconstructed breast, as the latter one will probably suffer changes throughout life.

Prosthetic breast reconstruction

This method of reconstruction involves placement of silicone gel or saline breast implants. The best results are achieved in patients in whom the breast volume is moderate, there is minimal ptosis, and there is adequate and healthy soft tissue coverage over the device. Patients who have large or ptotic breasts typically require matching procedures, namely reduction mammaplasty and mastopexy, respectively. This problem is simplified in patients undergoing bilateral or prophylactic mastectomies, in which symmetry is more readily achieved.

The advantages of implant-based reconstruction are that it combines a relatively minor procedure with excellent cosmetic results, uses adjacent tissues of similar color, texture, and sensation, obviates distant donor-site-related morbidity, requires less surgical and post-operative recovery times, and saves autologous tissue for a later occasion [START_REF] Spear | Implant-based reconstruction[END_REF].

Breast reconstruction can be performed either in a single or in a two-stage reconstruction. The first approach involves the immediate placement of a permanent or adjustable implant [START_REF] Reavey | Update on breast reconstruction in breast cancer[END_REF]. Advantages of an adjustable implant over a permanent one are that it may be gradually filled to the desired volume postoperatively, and that patients do not have to undergo two surgical procedures. Nevertheless, it requires the placement of a remote port (to fill the implant) which needs to be subsequently removed [START_REF] Reavey | Update on breast reconstruction in breast cancer[END_REF].

The two-stage reconstruction involves placement of a tissue expander within a submuscular pocket during the primary procedure, followed by its replacement with a permanent implant during a second surgical procedure (Figure 1). The tissue expander is inflated at weekly intervals by percutaneous injection of saline solution, allowing the expansion of the skin covering the expander [START_REF] Djohan | Breast reconstruction options following mastectomy[END_REF]. Although the earliest prosthetic breast reconstructions were carried out in a single stage, nowadays two-stage expander-implant reconstructions are preferred because they are generally believed to produce superior results [START_REF] Spear | Implant-based reconstruction[END_REF]. Potential complications of a prosthetic breast reconstruction include hematoma, seroma, implant extrusion, infection, rupture and capsular contraction [START_REF] Reavey | Update on breast reconstruction in breast cancer[END_REF].

Autologous breast reconstruction

This method uses the patient's own tissue to reconstruct the breast, and includes several techniques. The main advantage of autologous breast reconstruction is that results are more natural in terms of appearance, long-term durability and touch. Major disadvantages are the long surgical times, arduous recovery and higher risk of complications. The most frequent donor sites are the lower abdomen and the back.

Other flap donor sites include buttocks, back and thighs.

Abdominal donor site

The lower abdomen provides enough soft tissue bulk for reconstruction of the breast mound, without the need for implants. However, particularly in young women it is essential to consider the possibility of future pregnancies and exercise as a possible cause of abdominal wall morbidity. Flaps can be either pedicled (transverse rectus abdominis muscle [TRAM] flaps) or free (free TRAM, deep inferior epigastric perforator

[DIEP] and superficial inferior epigastric artery [SIEA] flaps, which progressively spare more muscle) [START_REF] Reavey | Update on breast reconstruction in breast cancer[END_REF]. Besides patient preferences, the choice of the donor site or type of flap largely depends on the amount of abdominal fat and skin available and the presence of contraindications, relative or absolute, such as history of previous abdominal surgery, obesity, smoking, history of blood clots and other major systemic medical conditions [START_REF] Djohan | Breast reconstruction options following mastectomy[END_REF].

TRAM flap

This procedure uses abdominal muscle, fat and skin tissue vascularized by the rectus muscle pedicle to reconstruct the breast mound. Grossly, in the pedicled TRAM flap excess skin, subcutaneous fat and rectus muscle from the infraumbilical area are transferred through a subcutaneous tunnel to the ipsilateral [START_REF] Marín-Gutzke | Anatomic and clinical study of rectus abdominis musculocutaneous flaps based on the superior epigastric system: ipsilateral pedicled TRAM flap as a safe alternative[END_REF] or contralateral mastectomy site. The flap is then rotated, shaped into a breast mound, and sutured; the umbilicus and the abdominal skin are sutured into its new position and the abdomen skin is sutured as in an abdominoplasty (Figure 2) [START_REF] Cordeiro | Breast Reconstruction after Surgery for Breast Cancer[END_REF]. Despite the loss of muscle function after a pedicled TRAM flap harvest, it is still possible for patients to become pregnant and carry a pregnancy to term, as well as to achieve a normal vaginal delivery [START_REF] Chen | Successful pregnancies following TRAM flap surgery[END_REF].

Free TRAM flap

In the free TRAM flap the skin, subcutaneous fat, deep inferior epigastric artery and a small portion of the rectus muscle and fascia from the infraumbilical area are transferred to the chest defect, were epigastric vessels are reattached to either thoracodorsal or internal thoracic vessels via microsurgery (Figure 3) [START_REF] Cordeiro | Breast Reconstruction after Surgery for Breast Cancer[END_REF]. This technique allows the relocation of larger amounts of tissue with a lesser risk of fat necrosis. Hence it may be a better procedure in patients with risk factors such as smoking, diabetes mellitus and obesity [START_REF] Reavey | Update on breast reconstruction in breast cancer[END_REF].

DIEP and SIEA flaps

These two techniques involve the use of skin and subcutaneous fat free flaps. The free DIEP flap uses perforator vessels from the inferior epigastric artery for vascular supply, thereby preserving rectus muscle continuity (Figure 4) [START_REF] Reavey | Update on breast reconstruction in breast cancer[END_REF]. From an aesthetic point of view, results are essentially the same between the DIEP and the free TRAM flap, however data regarding flap-related complications or donor-site morbidity are conflicting, with some studies showing that DIEP flaps produce less abdominal morbidity and postoperative pain, a quicker recovery and a shorter hospital stay [START_REF] Blondeel | One hundred free DIEP flap breast reconstructions: a personal experience[END_REF], but others indicating no significant differences in these aspects between both techniques [START_REF] Bajaj | Comparison of donor-site complications and functional outcomes in free muscle-sparing TRAM flap and free DIEP flap breast reconstruction[END_REF]. However, important differences regarding abdominal wall function have been observed in favor of free TRAM and DIEP flaps over the pedicled TRAM flap [START_REF] Atisha | A systematic review of abdominal wall function following abdominal flaps for postmastectomy breast reconstruction[END_REF]. Computed tomography angiography is now widely used to assess perforator vessels and aid in surgical planning (Figure 5). Dominant perforators with shorter intramuscular course should be selected.

The SIEA flap is the least invasive method of all lower abdominal flap breast reconstructions. It uses the SIEA for vascular supply and does not require dissection of the rectus muscle and fascia, potentially eliminating abdominal donor site morbidity [START_REF] Wu | Comparison of donor-site morbidity of SIEA, DIEP, and muscle-sparing TRAM flaps for breast reconstruction[END_REF]. However, superficial inferior epigastric vessels are only present in approximately 50% of the population [START_REF] Reavey | Update on breast reconstruction in breast cancer[END_REF].

Dorsal donor site

Latissimus dorsi (LD) flap

Almost any patient is a potential candidate for a LD flap due to its reliable vascularization. This flap can be used in patients who would normally be at risk for complications associated with reconstructions using abdominal flaps, such as patients with diabetes mellitus, hypertension, smokers or who are obese [START_REF] Hammond | Latissimus dorsi flap breast reconstruction[END_REF], after a failed implant or TRAM reconstruction, or when previous abdominal surgeries have been carried out, including abdominoplasty. However, the amount of tissue available to reconstruct the breast is often insufficient, requiring the use of tissue expanders and implants [START_REF] Djohan | Breast reconstruction options following mastectomy[END_REF].

In this technique, the LD is rotated on its thoracodorsal neurovascular pedicle and humeral insertion, tunneled subcutaneously to the mastectomy defect and sutured to the pectoralis major muscle and inframmamary fold (Figure 6) [START_REF] Cordeiro | Breast Reconstruction after Surgery for Breast Cancer[END_REF]. The advantage of this flap is that it provides padding for the mastectomy defect and softens the contours of the reconstructed breast, thus creating a natural appearance with the placement of an implant.

Current modifications in the surgical technique have significantly improved the consistency and quality of the aesthetic results, thereby making the LD flap an appealing breast reconstruction option for young women with breast cancer [START_REF] Hammond | Latissimus dorsi flap breast reconstruction[END_REF].

When carrying out a reconstruction using an LD flap, it is preferable to perform a twostage reconstruction by placing an expander, and then replacing it with an anatomical permanent implant after the expansion has been completed [START_REF] Sternberg | Latissimus dorsi flap remains an excellent choice for breast reconstruction[END_REF].

The two-stage procedure has been shown to reduce the rate of capsular contracture from 21-75% to 3,5-16% [START_REF] Sternberg | Latissimus dorsi flap remains an excellent choice for breast reconstruction[END_REF][START_REF] Mccraw | Early and late capsular "deformation" as a cause of unsatisfactory results in the latissimus dorsi breast reconstruction[END_REF][START_REF] Spear | The role of the latissimus dorsi flap in reconstruction of the irradiated breast[END_REF]. In cases of bilateral mastectomy reconstruction in young and slim women, the LD flap provides an ideal option, with an adequate symmetry (Figure 6). Ischemic complications are uncommon in reconstructions using LD flaps. Seroma formation on the back (18-34%) is treated with prolonged suction drainage and thoracic compression garments [START_REF] Sternberg | Latissimus dorsi flap remains an excellent choice for breast reconstruction[END_REF][START_REF] Spear | The role of the latissimus dorsi flap in reconstruction of the irradiated breast[END_REF]. In some cases, an outpatient percutaneous aspiration may be necessary.

Timing of breast reconstruction after mastectomy

Timing of breast reconstruction involves several factors, but largely depends on the need for postmastectomy radiotherapy. Radiotherapy increases by more than 40% the risk of complications in prosthetic-based reconstructions, such as capsular contracture and extrusion [START_REF] Kronowitz | Radiation therapy and breast reconstruction: a critical review of the literature[END_REF]. Irradiation of autologous tissue flaps can also lead to skin contracture, fat necrosis and atrophy, resulting in distortion of the reconstructed breast [START_REF] Reavey | Update on breast reconstruction in breast cancer[END_REF]. The LD flap is not resistant to the effects of radiation, and may become atrophic and firm after radiotherapy [START_REF] Hammond | Latissimus dorsi flap breast reconstruction[END_REF].

On the other hand, even with modern radiotherapy techniques prosthetic or autologous reconstructions can limit the amount of radiation delivered to target tissues, and thus decrease radiotherapy efficacy [START_REF] Kronowitz | Radiation therapy and breast reconstruction: a critical review of the literature[END_REF]. Every effort must be made to determine the need for radiotherapy before immediate breast reconstruction in order to avoid these potentially unwanted results.

The two timing options are immediate and delayed. Immediate breast reconstruction, which is performed at the time of mastectomy, produces the best aesthetic outcome, since it allows for preservation of the breast skin envelope and sometimes even the nipple-areola complex. Furthermore, there is the psychological benefit of having a reconstructed breast upon awakening from mastectomy as well as the need for fewer surgical operations [START_REF] Kronowitz | Advances and surgical decision-making for breast reconstruction[END_REF]. Immediate reconstruction is an oncologically safe option and does not delay the initiation of adjuvant chemotherapy [START_REF] Taylor | Oncological aspects of breast reconstruction[END_REF][START_REF] Ananthakrishnan | Options and considerations in the timing of breast reconstruction after mastectomy[END_REF]. This procedure is indicated in patients who will almost certainly not require adjuvant radiotherapy, i.e. women with stage I or stage II breast cancer with a low risk of requiring adjuvant radiotherapy [START_REF] Kronowitz | Advances and surgical decision-making for breast reconstruction[END_REF]. Unfortunately, this risk cannot be predicted, and an increasing number of patients with earlier stage cancer are receiving adjuvant radiotherapy due to the proven benefits in terms of loco-regional control and overall survival [START_REF] Clarke | Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials[END_REF].

Delayed breast reconstruction is indicated in women who will require adjuvant radiotherapy. In these women, the breast should be reconstructed using an autologous breast reconstruction with, for example, a TRAM or LD flap.

To optimize reconstruction in patients at risk of requiring postmastectomy radiotherapy (stage II), it is possible to carry out a delayed-immediate reconstruction. With this approach, patients who do not require radiotherapy may benefit from the advantages of a skin-sparing mastectomy while obtaining similar aesthetic outcomes to those of an immediate reconstruction. On the other hand, patients who require radiotherapy may avoid the potential aesthetic complications associated with irradiation after immediate breast reconstructions [START_REF] Kronowitz | Delayed-immediate breast reconstruction: technical and timing considerations[END_REF].

The purpose of a delayed-immediate breast reconstruction is to preserve the breast skin envelope while avoiding radiotherapy-related complications associated with breast irradiation after an immediate breast reconstruction. Hence, patients who require adjuvant radiotherapy, as well as those who do not, can achieve aesthetic results [START_REF] Kronowitz | Advances and surgical decision-making for breast reconstruction[END_REF]. This approach is also done in a two-stage fashion, but the tissue expander is placed completely filled at the time of mastectomy, pending final pathology results. If no radiotherapy is needed, replacement with an implant or autolougous tissue can be carried out within the next 1 or 2 weeks. If radiotherapy is required, the expander is deflated to optimize irradiation of the internal mammary nodes. After completion of radiotherapy, the patient undergoes reexpansion of the preserved breast skin and subsequent autologous reconstruction [START_REF] Kronowitz | Advances and surgical decision-making for breast reconstruction[END_REF].

Partial mastectomy reconstruction

Conservative surgery followed by radiotherapy has replaced modified radical mastectomy as the preferred treatment for early stage invasive breast cancer. Partial mastectomy preserves the nipple and areola complex (NAC), although in some cases breast asymmetry and distortion can occur. The effects of radiotherapy add to the complexity of these cases. Approximately 10% to 30% of patients are dissatisfied with the aesthetic following partial mastectomy and radiotherapy. The causes of aesthetic failure are tumor resection with noticeable volume changes, distortion of the position of the NAC, and the effects of radiation per se [START_REF] Hamdi | Partial Mastectomy Reconstruction[END_REF].

Partial mastectomy reconstruction can also be immediate or delayed. In the immediate reconstruction we try to avoid breast deformities by reconstructing the defect at the same surgical time as the mastectomy. The combination of partial mastectomy and immediate partial breast reconstruction is known as oncoplastic surgery. By performing a reduction mammoplasty-type procedure during the tumor resection surgery, a more aesthetic postoperative breast is obtained. The pattern can then be rotated laterally or medially to fit the location of the tumor (Figure 7) [START_REF] Hamdi | Partial Mastectomy Reconstruction[END_REF].

In a delayed partial breast reconstruction, the surgeon's efforts are directed towards correcting the breast deformity after completion of radiotherapy. The best aesthetic results are achieved when autologous tissues, such as the LD or TRAM flaps described above, are used (Figure 8) [START_REF] Hamdi | Partial Mastectomy Reconstruction[END_REF].

Conclusions

In young patients who undergo mastectomy, recovery of body image is essential for physical and mental quality of life. Young women with breast cancer have specific characteristics related to the age of onset of the disease; diagnosis is frequently established at a more advanced stage, and reconstructive expectations are largely related to breast size, shape, and symmetry of the reconstructed breast with regards to the contralateral non-reconstructed breast and the changes it may suffer throughout life.

Patient individualization is the key to a successful breast reconstruction and essential to meeting the patient's expectations after mastectomy. Each reconstructive technique has its own indications, advantages and limitations, which must be openly and thoroughly discussed with the patient. Timing of the surgery is primarily determined by the requirement for adjuvant radiotherapy. If the risk of requiring adjuvant radiotherapy is low, an immediate reconstruction is the method of choice. In patients with a high risk of requiring adjuvant radiotherapy, a delayed approach is recommended, and in patients in whom the need for adjuvant radiotherapy is in doubt, a delayed-immediate approach is the best to optimize aesthetic and oncologic outcomes.

Figure 1 .

 1 Figure 1. Two-stage breast reconstruction in a young woman after mastectomy.

Figure 2 .

 2 Figure 2. Breast reconstruction using a pedicled transverse rectus abdominis muscle (TRAM) flap.

Figure 3 .

 3 Figure 3. Breast reconstruction using a free transverse rectus abdominis muscle (TRAM) flap.

Figure 4 . 20 Figure 5 . 21 Figure 6 .

 4205216 Figure 4. Breast reconstruction using a free deep inferior epigastric perforator (DIEP) flap.

Figure 7 .

 7 Figure 7. Oncoplastic surgery.

Figure 8 .

 8 Figure 8. Delayed partial breast reconstruction.

Acknowledgements

The authors acknowledge the support of Pfizer Spain, which facilitated the necessary meetings to evaluate and discuss all the data presented in this review, and Dr. Ximena Alvira from HealthCo SL (Madrid, Spain) for assistance in the preparation of this manuscript.


Eugene M G Milne
email: eugene.milne@newcastle.ac.uk

Dynamics of Human Mortality

Keywords:

Historical human mortality curves display 5 phases, differing in dimensions with population, time and circumstance. Existing explanatory models describe some but not all of these, and modelling of entire curves has hitherto necessitated an assumption of multiple distributions.

A new distribution, shown previously to describe survival in experimental animals, postulates empirically that net mortality risk comprises two components described as 'redundancy decay' and 'interactive risk'. The former is proposed effectively to set a 'program' for the increase of mortality risk with age, and may be a better measure of the 'rate of ageing' which has previously been assumed to determine the slope of semi-logarithmic mortality curves.

Entire human mortality curves are shown here to be compatible with this single distribution given the assumption that individuals vary only with respect to the interactive risk parameter (k).

Historical Swedish cohort data are modelled here exclusively through changes in k values, clustered increasingly toward the higher end of their range, while redundancy values are held constant. This pattern is compatible with the hypothesis that historical changes in human mortality may be explained purely in terms of interactive risks and without changes in the underlying pattern or rate of ageing.

Introduction:

Human mortality in developed nations has fallen dramatically over the past two centuries. This has resulted largely from sanitation and other public health measures, but the latter decades of the twentieth century saw a significant contribution from increasingly effective health care interventions. [START_REF] Mackenbach | The contribution of medical care to mortality decline: McKeown revisited[END_REF][START_REF] Mckeown | Medicine in Modern Society: Medical planning based on evaluation of medical achievement[END_REF][START_REF] Wilmoth | Demography of longevity: past, present, and future trends[END_REF] The shape and dynamics of human mortality curves over this period have long been of interest not only for their demographic and actuarial implications, but also because they may provide insights into the process of ageing.

One perspective is that human ageing has slowed over this period -based upon the presumption that ageing is merely a description of many cumulative disease processes, such that if disease is reduced then, ergo, ageing is reduced also. An alternative view is that there are fundamental ageing processes -'intrinsic ageing' that proceed even in health, and that these may or may not have been changed during the great increase in human life expectancy.

In ageing research, many interpretations of mortality curves have relied upon the slope and intercept of their log projections, with the slope being interpreted as a key measure of senescence and the intercept as a measure of 'age-independent mortality'. [START_REF] Finch | Longevity, senescence and the genome[END_REF] Use of the slope of log mortality as a measure of the 'rate of ageing' in ageing studies has been challenged. [START_REF] Driver | The Gompertz function does not measure ageing[END_REF][START_REF] Masoro | Caloric restriction and aging: controversial issues[END_REF] And historical human mortality has, paradoxically, tended to show a steeper slope (i.e. apparently faster ageing) as mortality has fallen. Apparent regularities in mortality appear to have changed during the 20 th century and suggest that traditional concepts require revision. [START_REF] Yashin | The new trends in survival improvement require a revision of traditional gerontological concepts[END_REF] A recently described mathematical model of survival and mortality patterns in many species, postulates that net risk to the individual may be partitioned into two major components. [START_REF] Milne | The natural distribution of survival[END_REF] The first component is described as 'interactive risk', representing a combination of intrinsic vulnerability and extrinsic hazard which may vary widely between individuals or for the same individual in different times and environments. In traditional actuarial terms, it is loosely related to the Makeham constant. [START_REF] Makeham | On the Law of Mortality[END_REF] However, instead of representing purely external hazard, it combines external, genetic, behavioural, and other intrinsic elements. Unlike Makeham, it is not additive in nature, but is described by a parameter (k) which acts as a coefficient to the half-life of the basic units that comprise an organism's vital functions, such that the nearer parameter k is to zero, the higher is the risk to the organism. Also in distinction to the Makeham factor, changes in parameter k produce true changes in mortality gradient. [START_REF] Golubev | Does Makeham make sense?[END_REF] Overall, this component acts as a summary measure of almost all the variable risks that we recognise as threats to life, health and well-being.

The second component is defined as the 'redundancy' of functions within the organism. This is an empirical postulate, which is regarded as largely sequestered and influenced little by external factors. Redundancy, according to the model, declines linearly throughout life. This component is closely related to the aforementioned concept of 'intrinsic ageing' -causing progressive and inexorable vulnerability with increasing age even in the absence of disease. [START_REF] Hayflick | The not-so-close relationship between biological aging and age-associated pathologies in humans[END_REF][START_REF] Holliday | Debates: The Close Relationship Between Biological Aging and Age-Associated Pathologies in Humans[END_REF][START_REF] Peto | There is no such thing as ageing[END_REF] It should be noted, however, that the general physical expressions of ageing are not assumed to be confined to this component -instead, they may be manifest in either component of the model.

An important aspect of the model is that the two components are essentially independent of one another. That is, a linear progression of redundancy decay is sustained, irrespective of the level of interactive risk, or the degree to which interactive risk reflects cumulative damage or disease. This separation of components distinguishes the model from previous reliability-based approaches to animal mortality, which have posited redundancy depletion as a consequence of disease and random damage, and have characterised it as, for example, a depletion of cell numbers. [START_REF] Gavrilov | Mathematical model of aging in animals[END_REF][START_REF] Gavrilov | The reliability theory of aging and longevity[END_REF] The assumption of sequestered, relatively invariable, linear redundancy decay seems counterintuitive. It is useful, however, while recognising that it may simply be an mathematical convenience, to assume its existence as an unspecified biological process into which we have some limited insight, since real data can then be tested to see whether their behaviour is compatible with such a construction of net risk.

On this basis we can, for example, identify certain conditions which appear to exert an influence upon redundancy decay -such as altered living temperature -considered to influence 'intrinsic ageing'. The altered survival curves arising from laboratory evolution of Drosophila longevity may be modelled simply by alteration of redundancy parameters within the model . [START_REF] Milne | The natural distribution of survival[END_REF][START_REF] Rose | Laboratory Evolution of Postponed Senescence in Drosophila-Melanogaster[END_REF] Mortality and survival trajectories of Medflies, mice and C.elegans can be modelled using this approach, while mortality plateaux and decelerations are generated by an assumption of interindividual variation in rates of redundancy decay. [START_REF] Milne | The natural distribution of survival[END_REF] These findings are consistent with the model's assumption that the 'redundancy' is related closely to 'intrinsic ageing' in laboratory animals, whatever that might be, and suggest that inter-individual variation in redundancy decay rates may be significant for those species.

In contrast, the long exponential rises in mortality that are characteristic of human populations suggest, within the model, relatively invariable individual redundancy decay characteristics. In this context, 'relatively invariable' may be taken to imply that the mean state of redundancy decay of, for example, an 80 year old will be reached by 99% of the population within only a small range of years about 80 chronological years of age, since, within the model, greater variation tends to produce greater variability in mortality patterns than is observed in human populations. This is consistent with the known genetic homogeneity of humans relative to, for example, wild fruit flies, and invites the hypothesis that the dramatic changes in human mortality seen in recent centuries may be modellable exclusively by variation in interactive risk, with little or no change in redundancy decay.

This paper explores that hypothesis through modelling of historical human mortality data.

The shape of human mortality curves

The general shape of human mortality curves in those countries for which robust data are available may be considered as having five key phases. Not all of these phases are mutually exclusive and, in some cases, they overlap or may be superimposed one upon another. As a consequence, not all are readily visible in all mortality curves, so these have been illustrated in figure 1 using two example curves, the upper curve being data drawn from the Human Morality Database for British women from the cohort of 1850, and the lower curve Swiss men from the cohort of 1916. [START_REF] Hmd | Human Mortality Database[END_REF] Phase 1: Birth and childhood -For humans, birth is a uniquely traumatic event. As a consequence, the extreme left hand end of all mortality curves is a peak. The mortality rate descends rapidly in the first year and gradually slower thereafter, falling to a low point or trough that historically was seen around the time of puberty, but which now appears to emerge as early as seven or eight years of age. [START_REF] Milne | When does human ageing begin?[END_REF] Modern causes of death in this period reflect both the vulnerabilities of normal infants, and the specific deficits of those with congenital conditions. For most of history far higher mortalities have applied during this period, arising from first exposures to the great infectious diseases.

Phase 2: Sexual maturity and early adult life -From puberty onwards, mortality begins to rise, and demonstrates in both sexes, though particularly in men, an upward convexity of the mortality curve. As the earliest point at which the lifelong rise in mortality becomes apparent, the nadir has been regarded as the 'Initial Mortality Rate', [START_REF] Finch | Longevity, senescence and the genome[END_REF] or even as an indication of the onset of ageing. [START_REF] Hayflick | When does aging begin?[END_REF] The upward convexity, on the other hand, appears to be a feature that reflects a combination of external hazards and increased opportunity to encounter those hazards, exacerbated by risk-taking behaviours and psychology that may be evolutionarily programmed. Exclusion of causes of death attributable to trauma removes this feature from mortality curves almost completely. [START_REF] Dolejs | The extension of Gompertz law's validity[END_REF] Phase 3: The 'Makeham' phase -This is the first of the overlapping phases of mortality and might properly be regarded as one that applies to the entire lifespan. It is named here for William Makeham whose work on mortality contributed the eponymous constant to the well-known Gompertz-Makeham law. [START_REF] Olshansky | Ever since Gompertz[END_REF] In truth his constant stands for what might more properly be considered to be a variable factor which did not vary sufficiently to diminish its value as a descriptor of 19 th century mortality curves. The Makeham factor adds a constant level of external mortality to the life course. In the absence of exponentially increasing risk it would result in first-order (exponential) decay in population size. In the presence of exponentially increasing risk it tends to produce a shallow upward incline or even a plateau of the log-mortality curve in early to mid-life that gives way to a Gompertzian log-linear increase thereafter.

The period that is referred to in this paper as the Makeham phase is not lifelong. Instead, it represents that plateau / shallow incline seen particularly in older mortality data, as illustrated for the England & Wales female cohort of 1850 in figure 1.

Phase 4: The 'Gompertz' phase -The most famous element of the human mortality curve is that part which conforms to the Gompertz law and is log linear from middle-age to late life. [START_REF] Gompertz | On the nature of the function expressive of the law of human mortality and on a new mode of determining life contingencies[END_REF] An absolute age-range for this phenomenon has never been described, though it has been suggested to start from as early as adolescence (having allowed for phases 2 & 3 above) and to extend to around the age of 90. Unsurprisingly, its pervasiveness through the lifespan has suggested to many that it represents a fundamental law of mortality, although it was described as an actuarial rather than biological law and has been called a "law without explanation". [START_REF] Bonneux | Benjamin Gompertz revisited[END_REF] The historical pursuit of an explanation has been described elsewhere. [START_REF] Olshansky | Ever since Gompertz[END_REF] Phase 5: Late life deceleration and extreme old age -The final phase is one which has commanded great interest and attention in recent years, since it became apparent that decelerations and even plateaux or falls in mortality may be observed in a variety of species. [START_REF] Carey | Slowing of mortality rates at older ages in large medfly cohorts[END_REF][START_REF] Kannisto | Reductions in Mortality at Advanced Ages -Several Decades of Evidence from 27 Countries[END_REF][START_REF] Vaupel | Biodemographic trajectories of longevity[END_REF][START_REF] Wilmoth | Do the oldest old grow old more slowly?[END_REF] As far back as Gompertz himself it was recognised that human mortality falls away from an exponential rise in the late years of life.

Method

Although various approaches have been used in modelling human mortality in the past, most have described only one or two of the phases outlined in the previous section. The Gompertz law, for example describes only phase 4, while the addition of the Makeham factor extends this to allow description of phases 3 and 4. [START_REF] Gompertz | On the nature of the function expressive of the law of human mortality and on a new mode of determining life contingencies[END_REF][START_REF] Makeham | On the Law of Mortality[END_REF]) Dolejs considered factors underlying phases 2 and 3. [START_REF] Dolejs | The extension of Gompertz law's validity[END_REF] Reliability based approaches attempt to describe phases 4 and 5, and assume a Makeham factor to permit incorporation of phase 3. [START_REF] Gavrilov | The reliability theory of aging and longevity[END_REF] Evolutionary approaches aim to describe the overall shape of the curve in terms of agespecific selective pressures and have a general explanatory value rather than providing precise modelling. [START_REF] Kirkwood | Why do we age?[END_REF][START_REF] Lee | Rethinking the evolutionary theory of aging: transfers, not births, shape senescence in social species[END_REF][START_REF] Rose | The Long Tomorrow[END_REF] Witten concluded that the whole human life course could be modelled given an assumption of three distinct distributions. [START_REF] Witten | A return to time, cells, systems, and aging: V. Further thoughts on Gompertzian survival dynamics--the geriatric years[END_REF] These he named 'neonatal failure', 'Gompertzian failure' and 'old failure', corresponding to phases 1, 4 and 5 respectively. Combined with a Makeham assumption to account for phases 2 and 3, this approach can produce a survival profile similar to the whole of the human pattern. However, the use of three different distributions was forced by his conclusion that it was not possible to model the whole life course otherwise -neonatal failures appeared to show exponential kinetics, but exponential distributions could not be combined to produce Gompertzian or Weibull patterns of later life. In consequence, a three-group survival model was proposed wherein individual risks underwent transition between groups and hence between survival distributions.

The model used here, based upon a single survival distribution, has been described elsewhere but is summarized below. [START_REF] Milne | The natural distribution of survival[END_REF] In its simplest form, this defines instantaneous per-period probability of survival (P c ) at any age (c) according to the formula:

[EQUATION 1 GOES HERE]

(1)

Where: s = theoretical redundancy level (r) at age 0; a = time taken for decay of 1 unit of redundancy in the same units as age; k = interactive risk parameter (the unit-specific half-life when r = 1); n = number of functions that comprise the organism; c = current age; and where redundancy (r) at the current age (c) is described by:

[EQUATION 2 GOES HERE] (2)
Using these formulae, age-specific mortality rates may be conveniently estimated as 1-P c. .

More complex treatments of this model introduce inter-and intra-individual variation to the parameters -that is, they are assumed to have typical biological distributions (normal or log-normal)

from which values are sampled randomly, with the exception of n which is assumed to be a speciesspecific constant.

For humans an arbitrarily assigned value of n = 300 was used in the examples for this paper. The In this progression, there are several notable features. Firstly, both the sub-population size and the spread of the phase 1 cluster progressively diminished between the cohorts. Secondly, the phase 3 sub-population fell dramatically over the same period. Thirdly, the phase 4/5 sub-population became substantially more dominant, with a steadily increasing mean value of parameter k. It is a picture of progressive polarisation of the k distribution which also poses an interesting question, namely: how is it possible for a virtually constant mean value of k to emerge in the majority of a population across more than half of the lifespan?

This question becomes even more pertinent when considering models with fewer sub-groups than in the illustrated examples. The pattern generated is sufficiently robust that even small numbers of sub-groups in each sub-population produce extremely realistic mortality curves, and close correlation with phases 4/5 may be generated by a single subgroup.

The answer appears to lie in the dynamics of the model. As noted above, it is reasonable to assume that individual values of k will fall with increasing age and its associated diseases, though it should be borne in mind that even with a constant or rising value of k, an individual would continue to become more vulnerable (in terms of equation 1) as a consequence of redundancy decay. There is, however, an additional factor that must be taken into account here, namely the influence of 'mortality selection', whereby those with the lowest k values are also those most likely to be lost from the population -a process described by Wachter as 'culling'. [START_REF] Wachter | Hazard curves and life span prospects[END_REF] If all individuals had a constant level of k, within even a modest distribution, the effect would be that the population mean value should rise with increasing age. The fact that it does not do so suggests an equilibrium to exist between the downward drift in net risk of (non-redundant) ageing and the upward pressure of mortality selection. The point at which the population becomes characterisable as an aggregate of individuals with constant k is, I suggest, the point of onset of that equilibrium state.

This would be a situation broadly analogous to the recent observation that in the over-90s

dependency becomes approximately stable as a proportion of the population, despite markedly increasing frailty at an individual level. [START_REF] Christensen | Exceptional longevity does not result in excessive levels of disability[END_REF] Phases 3-5 of human mortality curves are well-recognised to display a pattern known as Strehler-Mildvan (S-M) correlation -a linear inverse relationship between G and the negative log of A when analysed using the Gompertz equation: [START_REF] Strehler | General theory of mortality and aging[END_REF])

[EQUATION 3 GOES HERE] (3)
It has been argued that, in part, this correlation arises artefactually through neglect of those influences addressed by addition of the Makeham constant, though it persists when this is accounted for. [START_REF] Finch | Longevity, senescence and the genome[END_REF][START_REF] Gavrilov | The reliability theory of aging and longevity[END_REF][START_REF] Golubev | Does Makeham make sense?[END_REF] Figure 4 illustrates S-M correlation for the Swedish cohort models in figure 3. Gompertz analysis of the modelled curves produces a very similar correlation. However, given the three sub-population approach used here, we can also separate out that part of the effect which is attributable to the phase 4/5 cluster. In principle, this has the same logic as allowing for Makeham and produces the crisper S-M correlation at the right of figure 4.

In the latter part of the 20 th century, a new pattern appeared in human mortality curves. Visible in cohort, but markedly in period data and more prominent for women than men, this takes the form of a concavity of mortality in the later decades of life. It is illustrated in figure 5 using period data for French women from 2006 -one of the clearest examples of this pattern. It is labelled as 'phase 6' because its ubiquity across the data of many countries suggests it is a general phenomenon.

This pattern is associated with the apparent increase in age at acceleration of mortality in postmenopausal women. [START_REF] Milne | Postponement of postmenopausal mortality acceleration in low-mortality populations[END_REF] Under this analysis, the shift of that point might be better interpreted as a consequence of diminishing influence of phase 3 and increasing influence of phase 6 upon the mortality curve, such that the juncture of acceleration shifts from the proposed k equilibrium point to the trough of phase 6.

It is also a pattern that is at least partly responsible for the deviations from Strehler-Mildvan correlation reported by Yashin. [START_REF] Yashin | The new trends in survival improvement require a revision of traditional gerontological concepts[END_REF][START_REF] Yashin | New age patterns of survival improvement in Sweden: do they characterize changes in individual aging?[END_REF] These occur because traditional Gompertzian analyses of mortality curves with a prominent phase 6 produce downward, parallel deviations of lines of best fit, or even apparent reversals of rotation.

The most likely explanation for phase 6 is that it reflects the impact of health care. Its timing coincides with the era of effective interventions, and the age range within which it is manifest, from around 50 to 90 years, is that group for whom treatments are likely to be used. The age of maximum deviation from the modelled line in figure 5 is about 70 years, and we might consider the shape of this phase to reflect increasing likelihood of medical intervention, balanced against decreasing likelihood of benefit.

By virtue of its broad age-specificity, and the strong suggestion in real data that the remainder of the mortality pattern has not altered (visible clearly in figure 5), this appears to be a phase that, like phase 2, represents an exception to the assumption of constant k.

The much stronger appearance of this phase in women than men requires explanation. It seems implausible that health care has so much greater an effect in one sex. However, the populations whose data are available are also populations in which the 20 th century epidemics of smoking and heart disease occurred. These impacted far more heavily on men than women, and it may be that a pattern which would be visible in both sexes appeared first in women because it was not being obscured by those factors. Alternatively, since these were also generations with fertility patterns markedly different to any other prior period in history, it is possible that the modern prominence of this phase is a consequence.

Discussion:

The approach to modelling described here can accurately mimic phases 1, 3, 4 and 5 of human mortality. Phases 2 and 6 may be adequately explained in terms of synchronous, age-specific changes in parameter k generated by behaviour and external risk. If individual risk varies with age according to equation 1, and humans are similar in terms of initial redundancy and its rate of decay, then all that is needed to generate typical human population mortality curves is variation in interactive risk.

Curve fitting for the examples illustrated in this paper was by visual fit only. The values used were necessarily, given the nature of the model , semi-arbitrary, and in its current form, the model cannot be fitted by, for example, a least squares method. Further work would be needed to develop a more objective method of deriving best fit. However, experience of fitting curves to a wide variety of mortality curves suggests that, given certain conventions (e.g. a chosen value for n) the resolution of even a visual fit is good. For example, a variation in the mean value of parameter a of as little as 1-2 years appeared to be quite recognisable. As a consequence, had there been an increase in the mean value of parameter a in the data shown in figure 3 between 1751 and 1900 of as little as 1-2 years it would have been apparent. The absence of such a change is striking, and suggests that a key aspect of human ageing and mortality has remained unaltered through that period. We should, therefore, consider what might be the biological significance of an unchanging parameter a?

Previous mortality models have aimed primarily to describe population patterns. Where they have worked well their parameters have then been interpreted as measures of biological characteristics of individual organisms. Some of these concepts, such as 'Initial Mortality Rate' and 'Mortality Rate Doubling Time' or slope of mortality, have already been challenged as measures of ageing. [START_REF] Hawkes | Mortality and fertility rates in humans and chimpanzees: How within-species variation complicates cross-species comparisons[END_REF][START_REF] Masoro | Caloric restriction and aging: controversial issues[END_REF] And a general failing of such analyses is that they risk generating 'ecological fallacies'. [START_REF] Last | A dictionary of epidemiology[END_REF] These are instances of population characteristics being attributed to the individual members of that population. [START_REF] Vaupel | Compositional interpretations of medfly mortality[END_REF] An important example in ageing research may be the proposition that slowing of population mortality rates in old age reflects slowed ageing in individuals, or that population mortality plateaux imply that ageing for individuals has ceased.

The approach described here aims, in contrast, to describe the lifetime risks of individuals, which are then aggregated to form population patterns. This imposes the constraint upon any proposed model of individual lifetime risk that it must, in aggregate, deliver the known population dynamics of ageing and mortality. Thus, if a model does not deliver observed population patterns it is certainly wrong -though if it does, it is not necessarily right.

In describing the risk dynamics of individuals, there are three fundamental 'shifts' of log mortality risk that must be present. These are caricatured in figure 6. Shifts of type A and type C are common to many mortality models. The Gompertz model, for example, yields these shifts through changes in its two (slope and intercept) parameters. However, in Gompertz and all other similar models, shifts of type B are only achieved by alterations of more than one parameter -often in a counter-intuitive fashion, hence the paradox in traditional analyses, that falls in human mortality have appeared to be accompanied by an increase in what has often been assumed to be the rate of ageing.

Type B shifts arising through change in a single parameter (k) appear to be unique to the Nested Binomial Model, and they can only arise because it provides a structure of increasing risk with age through a relatively constant rate of declining redundancy, determined by parameter a.

The lifetime schedule of mortality risk might, thus, be seen as being loosely programmed -not as a sequence of genetic instructions, but in a manner more comparable to sand running through the neck of an hourglass, at a rate determined by parameter a.

Such an 'hourglass program' for ageing is an attractive concept in ageing theory. It sidesteps the inherent contradiction of an evolved genetic program for ageing since selection to 'slow the flow' could be effected during reproductive life, with the consequence that it would flow similarly in the post-reproductive period. [START_REF] Vaupel | Post-Darwinian Longevity[END_REF] Such a phenomenon might be argued to exhibit 'agonistic pleiotropy' since selection for additional survival in early life would have the consequence of enhancing survival in later life, but it could also accommodate antagonistic pleiotropy and mutation accumulation if those phenomena manifest as interactive risks rather than as influences upon redundancy decline. [START_REF] Medawar | An Unsolved Problem of Biology, An Unsolved Problem of Biology[END_REF][START_REF] Willams | Pleiotropy, natural selection, and the evolution of senescence[END_REF] An hourglass program of ageing would be highly compatible with the concept of longevity genes (since 'flow' may be controlled by relatively few) and with the evolution of longevity when selection favours individuals that reproduce later in life. [START_REF] Rose | Laboratory Evolution of Postponed Senescence in Drosophila-Melanogaster[END_REF] It would sit easily with disposable soma theory, since metabolic costs may be traded between the 'rate of flow' and reproductive effort. [START_REF] Kirkwood | Evolution of ageing[END_REF][START_REF] Kirkwood | Why do we age?[END_REF] More problematic would be the contentions that the 'flow' is largely sequestered, linear in decline, and relatively invariable between humans. Of these, sequestration may be the least difficult, since this characteristic is, to some degree, fundamental to all arguments that advocate 'intrinsic ageing' as a driver of mortality risk. Dietary restriction is of interest because it is widely believed to alter a process that is implicitly sequestered and largely unaltered by other experimental treatments.

Linearity of decline is counter-intuitive. Many key findings in current ageing research relate to processes that appear variable in progression, and the most widely held paradigm of ageing is that of a process which runs more quickly in some individuals than others as a consequence of environments and behaviours. Smoking and obesity, for example, are held to accelerate ageing.

Variation in telomere length is increasingly regarded as an indicator of variable 'biological age'. If the model has biological significance, then a critical step in exploring it further will be to identify candidate processes that might fulfil the linear decline criterion.

The relative invariability of this supposed process in humans is also intriguing. In the illustrated examples the 95% confidence intervals for the distribution of parameter a embraced a range of about 3 years. The consistency of the model with real data is progressively lost as this range widens, but the similarity appears to remain reasonably good up to a 95% C.I. range of about 8 years, at which point the range encompassed by the mean 3 standard deviations is about 12 years. At this level, it is less difficult to reconcile our expectations of variability in human ageing from that which the model allows. But this addresses only the inherent distribution within populations, not the apparent absence of a shift in the mean with time. As noted above, a change in the mean value of parameter a of as little as 1-2 years in the illustrated data for Sweden would probably have been visible as deviation of the model from observed values, yet it did not happen. Further studies should examine whether this is also the case for (e.g.) affluent and deprived modern populations.

We may also predict that if there are chronologically age-specific elements to lifetime mortality risk, these are also likely to exist in relation to expressions of disease -that is, where we see large variations in disease over time or place, we should expect to find some evidence of preserved agespecificities in disease expression.

A notable feature of modelling human mortality through parameter k variation is its compatibility with 'phase 1'. In a previous paper I considered the possibility that the leftmost part of human mortality curves might arise because of an early life expansion of redundancy, resulting in diminishing mortality in the early years of life. [START_REF] Milne | The natural distribution of survival[END_REF] Although this remains a possible explanation, it seems unsatisfactory. Whatever 'intrinsic ageing' might be, it appears to be a process continuous from (and probably before) conception. [START_REF] Milne | When does human ageing begin?[END_REF] If it is closely associated with redundancy decline, then there is no good reason to suppose that redundancy does not also decline from or before conception. Moreover, a close linkage of redundancy to growth and maturation would implicitly link its nature to some physical quantity such as cell numbers or organ capacity.

Logically, one would then expect its decay to be similarly linked to their decline. Since their decline appears variable and subject to a wide range of environmental and disease factors, this would be at odds with the empirical independence of redundancy from interactive risk. Furthermore, such variability of cell volume or organ decay would be more likely to generate differences than similarity of redundancy decay, thereby preventing the long exponential rises in mortality commonly seen in human data. So, while it is possible to shape the leftmost part of modelled mortality curves with variation in redundancy decay, to do so requires new assumptions. In contrast, more accurate and robust modelling arises from simple variations in interactive risk without the need for additional assumptions.

A definitive test of the biological the basis of the model requires that we have a hypothesis for the physical nature of the quantity described as 'redundancy', such that it can be properly measured and its interactions assessed. As yet we do not have one, but closer consideration of whether , for example, cumulative damage from free radicals, progressive genetic entropy, deteriorating mitochondrial competence, or energetic capacity might fit the criteria of the model is merited.

Many researchers believe that dietary restriction alters the rate of ageing. However, evidence from Drosophila studies suggests that the effects of dietary restriction are reversible, and the displacements of slope and elevation may differ between males and females. [START_REF] Mair | Demography of dietary restriction and death in Drosophila[END_REF][START_REF] Partridge | Dietary restriction in Drosophila[END_REF] A key challenge in further exploring this model will be to determine whether and how the model mimics the observed effects of dietary restriction.

Empirical tests of the approach would include exploration of its capacity to predict future changes in mortality and life expectancy. This seems practicable, since we can calculate sequential parameter k values and extrapolate their trends to test against existing methods of forecasting.

Longitudinal analyses of data from cohorts such as that of Framingham have also used risk models (e.g. in the case of Framingham, non-proportional hazards Weibull accelerated failure time) to generate practical risk formulae for coronary heart disease and stroke risk. Yet when those formulae are used in different populations, they prove inaccurate and require adjustment. It may be that calculations of this kind can be improved by incorporating the concepts of the NBM into the risk models used.

The deviation from the pattern described here as phase 6 is also of interest, since, if it does represent the impact of good health and social care -manufactured survival -then it may provide a more objective societal measure of the effectiveness of healthcare and social systems than is currently available.

In summary, this model offers a complete and parsimonious theoretical structure for human mortality, and an explanation for those behaviours of adult mortality curves that appear to obey a schedule or program. Its analysis of historical data appears consistent with the hypothesis that historical changes in human mortality may be explained without changes in the underlying pattern of ageing, and it may have applications in ageing theory, demography, epidemiology and public health.

ResultsFigure 3

 3 Figure3shows the age-specific mortality figures (open circles) for Swedish male cohorts born in the

Figure 1 .

 1 Figure 1. Phases in human mortality curves. Five basic phases are identified and illustrated using

Figure 2 .

 2 Figure 2. The general features of human mortality curves modelled with 180 sub-groups based upon

Figure 3 .

 3 Figure3. Swedish male mortality for the cohorts born in1751, 1801, 1851 and 1901 (open circles)

Figure 4 .

 4 Figure 4. Strehler-Mildvan correlation in Swedish male cohort mortality data. Open diamonds are

Figure 5 .

 5 Figure 5. French female period mortality for 2006 (open circles) compared with a modelled mortality

Figure 6 .

 6 Figure 6. Three basic types of shift of log-mortality curve. Types A and C are described by single

Table 1 .

 1 Sub-population sizes and k parameters used in modelling Swedish male cohort mortality from four completed cohorts at 50 year intervals since 1751. In each case, 60 sub-groups were generated in each sub-population. Values for parameters held constant were s = 2.1, s.d. 1.01; a = 82, s.d. 1.01; n = 300. All s.d. values are exponentials of figures used in defining lognormal distributions.

	Phase 1		Phase 3		Phase 4/5
	Population proportion	Mean k	S.D.	Population proportion	Mean k	S.D.	Population proportion	Mean k	S.D.
	25%	6	1.35	25%	50	1.7	50%	300	1.01
	24%	6	1.35	28.5%	65	1.5	47.5%	400	1.01
	22.5%	6	1.35	19.5%	55	2	58%	520	1.01
	17%	5	1.35	8%	40	1.35	75%	620	1.01


Thi Kim

Duy Vo

Patrice Godard

Marie De Saint-Hubert

Gabriel Morrhaye

Emilie Bauwens

Florence Debacq-Chainiaux

Youri Glupczynski

Christian Swine

Vincent Geenen

Henri J Martens

Olivier Toussaint
email: olivier.toussaint@fundp.ac.

Transcriptomic biomarkers of ageing 1 Transcriptomic biomarkers of human ageing in peripheral blood mononuclear cell total RNA

Keywords: Ageing, PBMC, gene expression, biomarker, immunosenescence, oxidative stress, cDNA array APC: Antigen Presenting Cell, cDNA: complementary deoxyribonucleic acid, IFN-γ: interferon-γ, IL-10: Interleukin-10, PBMC: Peripheral Blood Mononuclear Cells, RT-qPCR: retrotranscription quantitative polymerase chain reaction

Age-related changes of gene expression contribute to the physiological alteration observed with human ageing. Herein the abundance of a selection of 148 transcripts involved in immunosenescence and stress response was compared in total RNA of PBMC of healthy young to middle-age probands (35.0 ± 6.5 year old) and healthy old probands (82.5 ± 6.8 year old). This study provides a list of 16 differentially abundant transcripts species in the healthy old probands.

Thus these changes of abundance can be considered as easily accessible biomarkers of ageing. Some of these differential abundances like CD28, CD69, LCK (decreased abundance in old subjects), CD86, cathepsin D, H and S (increased abundance in old subjects) might explain biochemical and cytochemical changes observed at the protein level in the immune system and thus might correspond to regulatory processes affecting the ageing process. Indeed these changes reflect the low-grade proinflammatory status observed in old persons and suggest a hypo-responsiveness of T-cells together with an increase in antigen presentation potential. In addition, among the differentially abundant transcripts were transcripts involved in the oxidative stress response HMOX1 and HSPA6 mRNAs were found as more abundant in PBMC from elderly subjects.

Introduction

Ageing is defined as a progressive deterioration of biological functions [START_REF] Farooqui | Aging: an important factor for the pathogenesis of neurodegenerative diseases[END_REF] with progressive decreases of physiological capacity, reduced ability to cope with environmental stimuli, dysregulated inflammatory responses and changes in the immune response.

Immunosenescence refers to the gradual age-related deterioration or modification of the immune system [START_REF] Thoman | The cellular and subcellular bases of immunosenescence[END_REF]. Age-related changes of the immune system have been observed in both innate and acquired immunity [START_REF] Huppert | Aging and lymphocyte subpopulations: whole-blood analysis of immune markers in a large population sample of healthy elderly individuals[END_REF][START_REF] Plackett | Aging and innate immune cells[END_REF]). The effect of ageing on the functional capacity of T-cells was the most extensively studied. Some of the age-dependent biological changes that contribute to the onset of immunosenescence could result from the organism adaptation to the continuous challenge of adaptation to the environment. Ageing is accompanied by a chronic pro-inflammatory state, due to continuous antigenic stress that impinges upon innate immunity, throughout life, and has potential implications for the onset of inflammatory diseases [START_REF] Vasto | Inflammatory networks in ageing, age-related diseases and longevity[END_REF]).

Transcriptomic profilings have been performed to study the effect of ageing on the regulation of gene expression in several cell types or organs [START_REF] Galvin | Expression profiling in the aging brain: a perspective[END_REF][START_REF] Vasto | Inflammatory networks in ageing, age-related diseases and longevity[END_REF].

Another purpose of gene expression profilings is to identify biomarkers that can be used later to better distinguish the state of physiological ageing of humans as opposed to their chronological ageing.

Herein, we developed a low-density DNA array to study the relative abundance of a selection of 148 transcripts species involved in immunosenescence, inflammation, stress response, cell or apoptosis regulation. The selection of 32 transcripts species involved in T-cell senescence was made upon survey of the literature. The selection of 116 transcripts species associated with stress response and cell senescence was also made according to literature survey. In addition, experimental work on cell senescence allowed to disregard transcripts for which differential abundance was not found in several in vitro and in vivo experimental models (Borlon et al. 2008a;Borlon et al. 2008b;[START_REF] De Magalhaes | Gene expression and regulation in H2O2induced premature senescence of human foreskin fibroblasts expressing or not telomerase[END_REF][START_REF] Debacq-Chainiaux | Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 signaling pathway[END_REF]Debacq-Chainiaux et 4 al. 2008;[START_REF] Pascal | Comparison of replicative senescence and stress-induced premature senescence combining differential display and low-density DNA arrays[END_REF][START_REF] Zdanov | Identification of p38MAPK-dependent genes with changed transcript abundance in H2O2-induced premature senescence of IMR-90 hTERT human fibroblasts[END_REF]. The reliability of the particular technology used herein was demonstrated previously in cell senescence-related studies and other biological models such as hepatic toxicology, adipocyte differentiation and oncology [START_REF] De Longueville | Gene expression profiling of drug metabolism and toxicology markers using a low-density DNA microarray[END_REF][START_REF] Gillet | Microarray-based detection of multidrug resistance in human tumor cells by expression profiling of ATP-binding cassette transporter genes[END_REF][START_REF] Vankoningsloo | CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes[END_REF]. Inter-and intra-platform reproducibility of gene expression measurements was demonstrated with this technology [START_REF] Patterson | Performance comparison of onecolor and two-color platforms within the MicroArray Quality Control (MAQC) project[END_REF][START_REF] Shi | The MicroArray Quality Control (MAQC) project shows inter-and intraplatform reproducibility of gene expression measurements[END_REF]). Numerous verifications with real time retrotranscription quantitative polymerase chain reaction (RT-qPCR) have been done in all these models. For instance, in the emblematic model of replicative senescence of WI-38 human fibroblasts, comparison was also possible with many data previously found by other technologies like Northern blots or high density DNA arrays by many independent laboratories [START_REF] Debacq-Chainiaux | Screening of senescence-associated genes with specific DNA array reveals the role of IGFBP-3 in premature senescence of human diploid fibroblasts[END_REF].

We report the identification of age-related transcriptomic biomarkers in total RNA of PBMC of old blood donors compared to young counter parts.

Materials and methods

Population recruitment

The recruitment was conducted at the University Hospital of Mont-Godinne (UCL, Belgium). We considered two groups: young to middle-age healthy volunteers and aged healthy volunteers. Young to middle-age participants were recruited on a voluntary basis among the staff of the hospital upon general announcement. The age of the young to middle-age volunteers ranged between 25 to 48 years old. All were clinically symptom-free. Healthy aged participants were recruited on a voluntary basis from different senior associations. To be eligible, participants needed 1) to be aged 75 years and over; 2) not to be institutionalized; 3) to have no evidence of an acute medical condition, nor deterioration of a chronic condition in the previous month. Exclusion criteria were the use of steroidal or non steroidal anti-inflammatory drugs one week before the inclusion, cancer or cancer history, and haematological disease. The ethics committee of the hospital approved the study.

Isolation of PBMC and RNA expression

PBMCs were isolated from 16 ml of whole blood by centrifugation through Ficoll-Hypaque gradient centrifugation (Becton Dickinson Vacutainer CPT, USA) and were washed twice in HBSS buffer. After adjustment at 10 8 cells/ml, part of the suspension were dispatched for hemogram determination (ADVIA 120, Baker, USA) while the rest were used for total RNA extraction (Total RNAgents® Total RNA Isolation System, Promega, USA) according to the manufacturer's protocol and stored at -80°C.

The quality of total RNA samples was assessed (Agilent 2100 Bioanalyzer, Agilent Technologies, Germany). Reverse transcription, RNA amplification and labelling using the MessageAmp™ II-Biotin Enhanced kit (Ambion, USA) were performed from 250 ng of total RNA.

Low density DNA-array analysis

Design of the array

We purchased a low-density DNA array on-demand able to detect 148 different transcripts species (Eppendorf, Germany) involved in immunosenescence, and more specifically focusing the T-cell compartment. Transcripts involved in stress response and cell senescence was also considered as described in the introduction.

The method is based on a system with two assays per glass side with three identical sub-arrays per assay. The sequences of the DNA covalently linked to the glass slide were carefully chosen by sequence comparison. Checks were made to ensure that no cross-hybridization takes place. Several positive and negative hybridization controls plus detection controls were spotted on the array in order to control the reliability of the experimental data.

Hybridization and detection

Hybridization on the array was carried out as described by the manufacturer using 15 µg of complementary DNA (cDNA). The hybridization reaction was performed overnight at 60°C in a Thermoblock for DualChip® Slides used with a Thermomixer comfort (Eppendorf, Germany).

Detection was performed using a Cy3-conjugated IgG anti-biotin (Jackson Immuno Research Laboratories, USA). Fluorescence of the hybridized arrays was scanned using the Packard ScanArray (PerkinElmer, USA) at a resolution of 10 µm. To maximize the dynamic range of detection, the same arrays were scanned at three photomultiplier (PMT) gains for quantifying highand low-copy expressed genes. The scanned 16-bit images were imported into the ImaGene 4.1 software (BioDiscovery, USA). The fluorescence intensity of each DNA spot (median intensity of each pixel present within the spot) was calculated using local median background subtraction.

Intensity values from high-PMT-gain pictures were used, except in the case of saturated spots. In the latter case, intensity values from intermediate PMT-gain pictures (or from low PMT-gain pictures in case of spots also saturated in the intermediate PMT-gain pictures) were used after scale 7 correction. Only spots with median intensity after background subtraction at least 2 fold higher than their local background were taken into account. The median of the three intensity values of the triplicate DNA spots was used in further steps.

Data normalization and inter-batch correction

All micro-arrays results were normalized against a single reference applying a local weighted regression [START_REF] Smyth | Statistical issues in cDNA microarray data analysis[END_REF]. The micro-array was provided by Eppendorf in three batches. The systematic intensity bias detected between the production batches was corrected successfully. Four samples were hybridized four times onto arrays of the three different batches. For each spotted sequence, a correction factor between the first batch and each of the others was computed using these four samples. This spot and batch dependent correction factor was applied to normalize intensities of each array. These normalized and corrected intensities were used in further statistical analyses.

RT-qPCR

First-strand cDNA was synthesized from 1 µg of total RNA, in a 20 µL reaction mix, using 200 U Superscript II reverse transcriptase in the presence of 1 µg of oligo(dT) (Invitrogen, UK). The expression of five genes was assessed using qPCR (sequence of the primers: table 1). Amplification reaction assays were performed in 25 µL total volume containing 12.5 µL of Sybr Green PCR Mastermix, 2.5 µL of each primer at 300 mM final concentrations and 5 µL of 1000-dilution of cDNA. qPCR reactions without cDNA were performed as template-free negative controls. qPCR were performed in duplicates with a hot start at 95 °C for 5 min followed by 40 cycles at 95°C for 15 s and 65°C for 1 min using the 7000 SDS thermal cycler (Applied Biosystems, The Netherlands). Melting curves were generated after amplification. Fluorescence emission was detected for each PCR cycle and the threshold cycle values (Ct) were determined using the thermal cycler software. For each experiment and each gene the efficiency of the qPCR was assessed with three dilution factors (100; 1 000 and 10 000) of one cDNA sample. The RPS9 gene was used as a 8 house keeping gene in order to compare gene expression between samples. This gene is selected for its expression constant in all the tested samples. The expression ratio of one gene g between two samples A and B was computed as following:

E g C g,B E r C r, B x E r C r, A E g C g,
A where E g and E r are efficiencies of qPCR for genes g and RPS9, C g,A , C g,B , C r,A , and C r,B are the Ct of genes g and RPS9 in samples A and B.

Statistical analyses

The Mann Whitney test was used to compare the difference of mRNA abundances between two groups. We used p-value with the Benjamini correction (P-value × m/g, where m is the number of variables and g the rank of the variable according to p-value). The correlation between micro-array and RT-qPCR results was performed using a Pearson correlation analysis.

Results

We designed a specific low-density DNA array for studying mRNA abundance of gene involved in T-cell senescence, cell senescence in general and stress response. We explained in the introduction how this list of transcripts was designed. This list is available as supplementary materials (Table S1).

Mean age of the probands

RNA was isolated from PBMC of 25 young to middle-age healthy volunteers (14 women and 11 men, 25-48 years old, mean age 35.0 ± 6.5; m±sd), and 23 aged healthy volunteers (12 women and 11 men, 75-103 years old, mean age 82.5 ± 6.8; m±sd) subjects. The two groups were matched for gender. The health status of the probands was determined as explained in the materials and methods.

Micro-array result validation

More than 100 verifications of data obtained in bioregontological studies with a similar array have already been done using RT-qPCR. However, the results obtained herein on a random selection of 5 transcripts among our list of transcripts were used to assess the quality of micro-assay measurements using RT-qPCR. Fourteen independent samples from blood of different persons were analysed both with the array and with RT-qPCR. The comparison between RT-qPCR cycle threshold normalized and micro-array log2 ratio value using a Pearson correlation analysis yielded a p-value of 0.001. The coefficient correlation was 0.94, indicating that the micro-array results were significantly correlated with the RT-qPCR values (Figure 1). The amplitudes of the expression ratio logarithms resulting from the RT-qPCR are generally slightly higher, as reported previously. The difference can be explained by a greater sensitivity of the RT-qPCR due to exponential amplification of the signal (Borlon et al. 2008a;Borlon et al. 2008b;[START_REF] Debacq-Chainiaux | Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 signaling pathway[END_REF][START_REF] Debacq-Chainiaux | Screening of senescence-associated genes with specific DNA array reveals the role of IGFBP-3 in premature senescence of human diploid fibroblasts[END_REF].

Identification of differentially abundant transcripts

From a total of 148 transcript species including those of 13 housekeeping genes, 77 transcript species were detected in the samples of PBMC of all the subjects, 71 transcript species were either not detectable in any of the subjects or not quantifiable. Thus 77 detected transcript species were selected as eligible for further analyses. There was no situation where a transcript was detected in a condition and not detected in the other.

We did no observe any significant differences of the abundance of each transcript between men and women in each age group of probands. To elucidate differences of the abundance of PBMC transcripts between young to middle-age and aged healthy participants, mRNA level was analysed individually for the 77 detected transcripts using a Mann-Whitney test, with a Benjamini and Hochberg false-discovery rate, and a p-value ≤ 0.05. This resulted in a list of 16 differentially abundant transcripts. From these 16 transcripts, 11 were more abundant and 5 were less abundant in the PBMC of the elderly subjects. The list of these transcripts and a heat map are shown in Table 2 and Figure 2 respectively.

Age-associated differential expression of genes in PBMC

The transcripts differentially abundant in PBMC of aged versus young to middle-age subjects, might be potential markers of healthy ageing. Most of these transcripts differentially expressed in aged and young to middle-age groups can be sorted according to their variation with ageing. The transcripts less abundant with age were: CD28, CD69, LCK, PRDX6 and MAX. Transcripts more abundant in the aged group included CD86, CTSD, CTSH, CTSS, EIF4A1, GAA, HMOX1, HSPA6, TNFRSF1A, TNFRSF1B and TUBA1B. The most important variations observed were increased abundance of HSPA6 and TNFRSF1A, respectively 1.83-and 1.68-fold, and the decreased abundance of CD28 and CD69, respectively 1.61-and 1.43-fold, in the aged group compared to the young group (Table 2 and Figure 2). However, we have currently no available data to evaluate the abundance of the corresponding proteins.

Discussion

Ageing is associated with a myriad of changes in all the organs of our body. Ageing is a continuous process that affects individuals from maturity to old age, first gradually from 20 to 65 years old in humans. Abrupt changes are observed in healthy individuals over 75 years old [START_REF] Faria | Variation rhythms of lymphocyte subsets during healthy aging[END_REF].

In this work, we searched for transcriptional markers of human ageing in the total RNA of PBMC isolated from young to middle-age healthy (25-48 years old) and aged healthy (75-103 years old)

probands. The study design was to select a limited panel of transcript species and to measure their abundance in PBMC samples from subjects expected to be in a sufficient number to reach statistical significances. The selected panel contained transcripts involved in the cellular mechanism of senescence, stress response as well as specific transcripts previously proposed to be involved in immunosenescence [START_REF] Franceschi | Biomarkers of immunosenescence within an evolutionary perspective: the challenge of heterogeneity and the role of antigenic load[END_REF][START_REF] Pawelec | T cells and aging (update february 1999)[END_REF]). The comparison of data from young to middle-age and old probands showed a similar transcript abundance for about 79% of the transcripts while 21% displayed an altered abundant level in a total of 77 transcripts significantly above the detection limit.

The majority of the differential abundance is relevant to the immune system. Interestingly, this transcriptional view of immunosenescence can be linked to observed deficits and modifications of the immune system of old humans. The decreased abundance of CD28 and LCK mRNA in aged healthy is reflected at the protein level [START_REF] Boucher | CD28 expression in T cell aging and human longevity[END_REF][START_REF] Vallejo | CD28 extinction in human T cells: altered functions and the program of T-cell senescence[END_REF]. Both molecules indicate TCR signaling pathways modification occurring with ageing. Interaction of the TCR with an antigen induces a rapid early cascade of intracellular signaling events that leads to T-cell activation. Nevertheless, the binding of TCR to antigen alone is insufficient to drive immune reaction. Activation requires co-stimulation with CD28, a membrane glycoprotein, which plays multiple roles such as amplifying the cytokine production or insuring a complete and efficient Tcell activation [START_REF] Nel | T-cell activation through the antigen receptor. Part 1: signaling components, signaling pathways, and signal integration at the T-cell antigen receptor synapse[END_REF][START_REF] Riley | The CD28 family: a T-cell rheostat for therapeutic control of Tcell activation[END_REF]. TCR engagement recruits LCK and FYN, protein kinases initiating the TCR signalling pathway [START_REF] Salmond | T-cell receptor proximal signaling via the Src-family kinases, Lck and Fyn, influences T-cell activation, differentiation, and tolerance[END_REF]. Unlike LCK, expression of FYN was not affected by ageing in our study (data not shown). Decreased abundance of CD28 and LCK mRNA indicates that early signaling events following antigen stimulation are altered with ageing in T lymphocytes. This hypothesis of lowered activation was reinforced by the 30% decrease of the mRNA level of early activation surface markers CD69 found in comparison to young to middle-age subjects (Table 2 andFigure 2). The changes at the mRNA level positively correlate with biochemical and cytometric observations [START_REF] Boucher | CD28 expression in T cell aging and human longevity[END_REF][START_REF] Fulop | Age-related impairment of p56lck and ZAP-70 activities in human T lymphocytes activated through the TcR/CD3 complex[END_REF][START_REF] Lio | In vitro T cell activation in elderly individuals: failure in CD69 and CD71 expression[END_REF]. These age-related variations in the T-cell activation mechanisms probably explain the lower efficiency of vaccination procedure in old persons, where TCR antigen recognition and T-cell activation are primary phenomenon involved as well as higher infectious risk when involving adaptive immune system.

Interestingly, the ligand of CD28 is a heterodimer formed by CD80 and CD86 present on many cell types with an antigen presenting cell (APC) activity [START_REF] Lindsten | Characterization of CTLA-4 structure and expression on human T cells[END_REF][START_REF] Salomon | Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation[END_REF][START_REF] Thompson | CD28 activation pathway regulates the production of multiple T-cell-derived lymphokines/cytokines[END_REF]). Up-regulation of CD80 in older adult monocytes by Toll-like receptor signaling was recently shown to be less effective while more complex regulation of CD86, which involves a secondary down-regulation by Interleukin-10 (Il-10), also seems to be impaired in old subjects [START_REF] Van Duin | Prevaccine determination of the expression of costimulatory B7 molecules in activated monocytes predicts influenza vaccine responses in young and older adults[END_REF]). Nevertheless, a closer look at the dendritic cells from aged subjects revealed that both their CD80 and CD86 can be more efficiently up-regulated by self-antigen than in young subjects [START_REF] Agrawal | Increased reactivity of dendritic cells from aged subjects to self-antigen, the human DNA[END_REF]. We observed that, in healthy elderly, the basal level of expression of CD86 was 22% higher than in the young to middle-age group with no significant change in CD80. This higher level of CD86 could be related to the pro-inflammatory cytokine profile observed in the elderly subjects [START_REF] Fagiolo | Increased cytokine production in mononuclear cells of healthy elderly people[END_REF]). The CD86 down regulator IL-10 (Lim et al. 2002) was not evidenced significantly differences between healthy young and old probands (not shown). This suggests that the age-related potent increased co-stimulatory activity of APC is due to the elevated concentration of pro-inflammatory cytokines, which is counterbalanced by a reduced expression of CD28 receptor that could in the end lead to hyporesponsivenes of T-cell response.

13

Further involvement of ageing in the mechanism of antigen presentation is strongly suggested by a series of lysosomal proteases, which mRNA is more abundant in the elderly group. The cathepsin genes CTSD, CTSH and CTSS all code for lysosomal proteases involved in physiological protein degradation [START_REF] Kirschke | Lysosomal proteinases[END_REF]. Cathepsins are constitutively expressed in all known professional APCs and are believed to play role in antigen processing [START_REF] Chapman | Endosomal proteases in antigen presentation[END_REF].

This has been clearly demonstrated for Cathepsin S (CTSS), which degrades the invariant peptide chain associated with the exogenous peptide presenting molecule MHC class II [START_REF] Gupta | Anchor side chains of short peptide fragments trigger ligand-exchange of class II MHC molecules[END_REF][START_REF] Maubach | Expression and upregulation of cathepsin S and other early molecules required for antigen presentation in activated hepatic stellate cells upon IFN-gamma treatment[END_REF]). CTSS also generates peptides for the vacuolar pathway of antigen presentation in vivo [START_REF] Beers | Cathepsin S controls MHC class II-mediated antigen presentation by epithelial cells in vivo[END_REF][START_REF] Shen | Cellular protein is the source of cross-priming antigen in vivo[END_REF], whereas the role of Cathepsin D (CTSD) and Cathepsin H (CTSH) in presented antigen generation are still to confirm, as discrepancies appeared according to the model used [START_REF] Deussing | Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation[END_REF][START_REF] Hewitt | Natural processing sites for human cathepsin E and cathepsin D in tetanus toxin: implications for T cell epitope generation[END_REF][START_REF] Villadangos | Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism[END_REF]. From a clinical point of view, higher expression of CTSH has been correlated with malignant progression of prostate tumors [START_REF] Waghray | Analysis of a truncated form of cathepsin H in human prostate tumor cells[END_REF]) and other cancers [START_REF] Dan | Identification of candidate predictive markers of anticancer drug sensitivity using a panel of human cancer cell lines[END_REF]. As for CD86, it has been shown that interferon-γ (IFN-γ) increases the expression level of CTSS [START_REF] Maubach | Expression and upregulation of cathepsin S and other early molecules required for antigen presentation in activated hepatic stellate cells upon IFN-gamma treatment[END_REF], which is coherent with the idea that the pro-inflammatory cytokine profile of old subjects stimulates the antigen presentation processing.

The plausible consequences of increased pro-inflammatory cytokine profile of old persons are known. In this study, we observed an up-regulation of some of these cytokine receptors at the mRNA level. The TNFRSF1A and TNFRSF1B genes encode the type 1 (TNFR-1), and type 2 (TNFR-2) receptors of TNF-α, respectively and exert their biological through distinct signalling pathways [START_REF] Gupta | A role of fas-associated death domain (FADD) in increased apoptosis in aged humans[END_REF]). In addition to inflammatory cytokine induction, TNF-α can stimulate apoptosis, but also activates NF-κB transcription factor, which in turn activates transcription of genes that block TNF-induced cell death [START_REF] Xiao | Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cells[END_REF][START_REF] Xiao | Nuclear factor-kappaB-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulosa cells from tumor necrosis factor alphainduced apoptosis[END_REF]. With ageing, both TNF-α production and TNF-α-induced apoptosis are increased [START_REF] Agarwal | Tumour necrosis factor-alpha and interleukin-2 in pus aspirate and blood in patients with amoebic liver abscess[END_REF][START_REF] Fagiolo | Increased cytokine production in mononuclear cells of healthy elderly people[END_REF]). An increased expression of TNFRSF1A and a decreased expression of TNFRSF1B in lymphocytes from aged humans have been reported [START_REF] Agarwal | Tumour necrosis factor-alpha and interleukin-2 in pus aspirate and blood in patients with amoebic liver abscess[END_REF]). These changes have been associated with increased apoptosis observed in aged lymphocytes, as it was suggested that TNFRSF1A pathway is more pro-apoptotic while TNFRSF1B pathway would preferentially induce the anti-apoptotic c-IAP1 and c-IAP2 proteins (Rothe et al. 1995). In our study, however, both TNFRSF1A and TNFRSF1B are up-regulated in PBMC from aged humans. Our results, if confirmed at protein level by further studies, would be in contradiction with the hypothesis of an overall pro-apoptotic mechanism in aged T-cell and suggests that a balance might emerge between the two receptors, leading to a more subtle regulation of T-cell apoptosis.

Aside from the modifications observed in the abundance of transcripts directly involved in the immune system function, other transcripts displayed significantly modified abundance in the PBMC of the old probands.

Expression of GAA, EIF4A, MAX and TUBAI1B is altered in ageing with increased mRNA abundance of GAA, EIF4A and TUBAI1B transcripts and decreased abundance of MAX transcripts. The GAA gene encodes acid alpha-glucosidase, which is essential for the degradation of glycogen to glucose in lysosomes. The EIF4A1 gene is implied in the control of translation, and thus is involved in protein synthesis and cell proliferation [START_REF] Flynn | The role of eIF4 in cell proliferation[END_REF]. The MAX gene encodes a protein of transcription regulator. The TUBAI1B gene encodes the tubulin, which is the major constituent of microtubules. These are potential interesting transcript markers in ageing.

Ageing is associated with progressively increased oxidative stress and damage, with potential changes of expression of genes involved in stress response. In this work, HMOX1 and HSPA6 mRNAs were found as more abundant in PBMC from elderly subjects, compared with PBMC from young to middle-age control subjects. HMOX1 gene encodes heme oxygenase, which is an essential enzyme associated with oxidative stress in many cell types such as fibroblasts [START_REF] Pascal | Heme oxygenase-1 and interleukin-11 are overexpressed in stress-induced premature senescence of human WI-38 fibroblasts induced by tert-butylhydroperoxide and ethanol[END_REF]).

HSPA6 is a member of the human heat-shock protein gene family, encoding a basic 70-KDa protein [START_REF] Leung | The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B') and isolation of its cDNA and genomic DNA[END_REF]). HMOX1 and HSPA6 mRNA levels are potential functional markers of ageing, probably due to their involvement in stress response [START_REF] Calabrese | Cellular stress response: a novel target for chemoprevention and nutritional neuroprotection in aging, neurodegenerative disorders and longevity[END_REF][START_REF] Chowdhury | Oxidant stress stimulates expression of the human peroxiredoxin 6 gene by a transcriptional mechanism involving an antioxidant response element[END_REF][START_REF] Gong | Heme oxygenase-1 protects HepG2 cells against cytochrome P450 2E1-dependent toxicity[END_REF][START_REF] Hori | Gene transfection of H25A mutant heme oxygenase-1 protects cells against hydroperoxide-induced cytotoxicity[END_REF][START_REF] Lee | Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia[END_REF]. HMOX1 activity and protein levels were 15 increased in the liver with age [START_REF] Abraham | Effect of age on rat liver heme and drug metabolism[END_REF][START_REF] Kang | The effect of age and calorie restriction on HIF-1responsive genes in aged liver[END_REF]. As for HSPA6, it was reported that aged individuals of particular species develop a constitutively increased level of several chaperones, including HSP70 [START_REF] Soti | Molecular chaperones and the aging process[END_REF]. The finding that HMOX1 and HSPA6 transcript species increased in healthy old persons may be explained by compensatory induction of these genes to cope with increased oxidative stress and damage in ageing. We have no clue about the potential reasons for the decreased mRNA level of PRDX6 antioxidant enzyme.

Ageing is a multi-dimensional process. Age-related changes of gene expression contribute to the physiological alterations observed with ageing. This work provided a list of 16 transcripts differentially abundant in the PBMC of old healthy subjects, compared to subjects of young to middle age. Even with the selection bias of gene selection and PBMC examination, changes of expression of immune response-related genes appears predominant in this study. Most of these changes can be related to modification of immune functions observed in aged healthy people. Thus, the transcriptomic changes identified herein should not be regarded only as biomarkers since these alterations might also have physiological consequences.

Table and

Figure 1 :

 1 Figure 1: Comparison of the gene expression ratios obtained by RT-qPCR and micro-arrays. Grey

Figure 2 :

 2 Figure 2 : Clustered heat map of the most significantly differentially expressed genes in the PBMC

Table 1 :

 1 Primer sets used in RT-qPCR. Primers were designed based on the original probe location with the Primer3 program.

Table 2 :

 2 Genes differentially expressed in PMBC of old healthy probands (n=23) versus young to middle-age healthy probands (n=25). Statistical analysis was carried out with the Mann Whitney test.

Table 1

 1

	Gene	Forward	Reverse
	RPS9	CTGGATGAGGGCAAGATGAAG	GTCTGCAGGCGTCTCTCTAAGAA
	BCL2	AGATACTGAGTAAATCCATGCACCTAAAC	CCACTGTCACTCTTGCAAATTCTAC
	CD69	GGAAATCTGTGTCAGTGGATGCT	GTGCAGATTTTCCTGGAATTTCTT
	CLU	GGA TGA AGG ACC AGT GTG ACA AG	CAG CGA CCT GGA GGG ATT C
	CTSD	GATGAGCTGGAAGGAGTGAGAG	CCACCTCCAGGCCAATACAT
	IL1R1	TTAAAGATGCCCTAAGTGTTGAAGA	GGCTCAATACCTTTTATTGCAAATAAG

Gene symbol, gene name and GenBank accession number of all transcripts which abundance was determined in this study.

Acknowledgements

We thank all the patients included in this study for their participation. This work was supported by the Walloon Region of Belgium (DGTRE Reseaux 2-SENEGENE n° 05/1/6192). O. Toussaint and F. Debacq-Chainiaux are respectively Senior Research Associate and Research Associate of the FNRS Belgium.