Giuseppina Colonna-Romano1biosci

Arne N Akbar2biosci

Alessandra Aquino1phd

PhD Matteo Bulati1

Giuseppina Candore

Domenico Biosci

Lio

MD Pietro Ammatuna

MD Jean M Fletcher

PhD Calogero Caruso

MD Graham Pawelec

PhD Ma

Prof Calogero Caruso

Impact of CMV and EBV seropositivity on CD8 T lymphocytes in an old population from West-Sicily

Keywords: CD8, CMV, EBV, Elderly, Immune senescence

Herpes viruses (particularly CMV and to some extent EBV) might play a role in accelerating the deterioration of immune functions with age. Indeed, it has been demonstrated that chronic infection with CMV causes an expansion of specific CD8 T lymphocytes and that this is related to a shrinkage of the T cell repertoire in very elderly people, predicting mortality. We have analysed CD8 T cells in young and old healthy Sicilians who were both CMV-and EBV-seropositive. Our data confirm expansions of T cells specific for the HLA-A2-restricted pp65 (495-503) CMV epitope up to nearly 14% of total peripheral CD8 cells in certain elderly individuals (range 0 -14%). However, the mean percentage of CMV-specific cells in the elderly was not greater than the young (range 0.2 -3%)., The CMV-specific CD8 cells in the elderly were predominantly CD45RA+, but in the young they were mostly CD45RO+. Our findings are somewhat different from published reports from Northern European populations, both in terms of mean numbers and surface phenotypes.These findings may reflect disparate hygienic and nutritional conditions 70 -90 years ago, which were very different in Northern and Southern Europe at that time, as well as a different genetic background.

INTRODUCTION

Many alterations of both innate and clonotypic immunity have been described in the elderly, and are generally considered to represent a deterioration of the immune system, designated immunosenescence [START_REF] Effros | Roy Walford and the immunologic theory of aging[END_REF][START_REF] Vasto | Immunity & Ageing: a new journal looking at ageing from an immunological point of view[END_REF]. A well-functioning immune system in the elderly is thought to be tightly correlated to health status, and some immunological parameters are often notably reduced especially in the frail elderly (Pawelec et al., 2002a). The OCTO longitudinal study of very elderly Swedes selected for good health at >85 yr of age has shown that a cluster of immunological parameters can be used to predict mortality. Increased numbers of CD8 cells, decreased CD4 cells, (CD4/CD8 ratio <1), poor T cell proliferation to mitogens, low B cell numbers and low IL-2 production are related to two year mortality in this very old population (Wikby et al., 1998). This led to the concept of an "immunological risk profile" (IRP) [START_REF] Olsson | Age-related change in peripheral blood T-lymphocyte subpopulations and cytomegalovirus infection in the very old: the Swedish longitudinal OCTO immune study[END_REF]Pawelec et al., 2002b), which was later found to include seropositivity for CMV (Wikby et al., 2002;[START_REF] Pawelec | Is immunosenescence infectious?[END_REF]. The fraction of 85 yearolds assigned to the IRP at baseline has been remarkably constant at ca. 20% of subjects at each wave.

Retrospectively, several years ago oligoclonal expansions of T cells, especially of CD8 cells, had been reported as a characteristic of human and murine ageing (Pawelec et al., 2002a). Later it was established that CMV seropositivity was associated with many of the same phenotypic and functional alterations to T cell immunity as were being reported as biomarkers associated with ageing (Wang et al., 1995;Wedderburn et al., 2001).

With the advent of tetramer and other technologies, it was discovered that CMV was the driving force behind many or most of the oligoclonal expansions and the altered phenotypes and functions of CD8 cells observed in the elderly [START_REF] Khan | Comparative analysis of CD8+ T cell response against human Cytomegaloirus proteins pp65 and Immediate Early 1 shows similarities in precursor frequency, oligoclonality, and phenotype[END_REF]. Importantly, later longitudinal studies in Sweden, examining a representative sample of the very elderly not selected for good health, the majority of whom were actually frail, revealed that the IRP, including CMV seropositivity, still predicted mortality [START_REF] Pawelec | Human immunosenescence: is it infectious?[END_REF]. More recent studies using this longitudinal "NONA" population, now over 90 yr of age, suggested that the number of different clones expanding in the elderly, and particularly the later clonal attrition observed to occur in IRP individuals, was highly correlated with 2, 4 and 6 year survival in this population [START_REF] Hadrup | Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly[END_REF]. However, it is currently not known whether these data are applicable to all populations or whether they are unique to the different environment/genetic 4 background of the Swedish population. Because old Sicilian populations include very few people with an inverted CD4/CD8 ratio (Colonna-Romano et al., 2002), a surrogate for the IRP also observed in other elderly N. European populations (eg. in Britain, ref. Huppert et al., 2005) we asked whether CMV had an over-riding effect on specific immunity in this population as well. We also examined a second persistent herpes virus, EBV, which was known to have a similar, but lesser effect, in the Swedish population [START_REF] Pawelec | Human immunosenescence: is it infectious?[END_REF].

Thus, in the present paper, we have evaluated some parameters of immunity to herpes viruses (CMV and EBV) in a sample of elderly people from the Sicilian population, whose exposure to infective agents over most of their lifetime has presumably been higher than in Sweden. This is due to the general hygienic conditions (eg. bacterial and viral contaminants in the water) in Sicily at that time, which was rather underdeveloped compared to northern European countries until more recently. The results reveal some subtle differences in the two populations, but in general are in agreement.

Differences may be due to the cross-sectional rather than longitudinal study design employed here, to the inclusion of less elderly individuals and to different historical, environmental and genetic factors that influence ageing and longevity [START_REF] Candore | Immunogenetics, gender and longevity[END_REF].

MATERIALS AND METHODS

Subjects

The donors studied were healthy subjects. None of them was affected by neoplastic, infectious or autoimmune disease, neither were they receiving any drug influencing immune functions at the time of the study. The health status of donors was checked on the basis of both clinical history and blood tests (complete blood cell count, erythrocyte sedimentation rate, glucose, urea nitrogen, creatinine, electrolytes, C reactive protein, liver function tests, iron, proteins, cholesterol, triglycerides). All the subjects gave informed consent according to Italian law. A total of 86 young and 54 old samples were selected for this study. Mean age of old people was 82.8±5.8 years (range 75-92); control donors were volunteers engaged from student and laboratory employees aged 20-61 years (mean 39.6±22.5). Both young and old samples were screened for CMV and EBV seropositivity. A CMV IgG EIA (Roche, Monza, Italy) test and an Elisa test were performed to determine respectively the CMV and EBV status using serum samples. As expected, the percentage of people seropositive for CMV was higher in the old than in the young group, but nearly a third of the old population remained CMV-seronegative.

There was a similar pattern for EBV, but with lower rates of seropositivity in both groups. However, several subjects fall in the "grey zone", i.e. a borderline levels of antibodies as evaluated by EIA tests. These results, according to the manufacturer's instructions, must be interpreted in the context of the clinical course. So for the purpose of this study we have considered these subjects negative. Accordingly, in some of these subjects we have randomly performed tetramer staining with no positive cells found.

Typing.

All the CMV and/or EBV seropositive subjects were HLA class I typed by a microlymphocytotoxicity test (Biotest, Milan, Italy). The HLA-A2+ samples were selected for further analysis.

Peptide -HLA class I tetramers

The peptide-HLA class I tetramers were provided by Proimmune (Oxford, UK). To evaluate the fraction of CD8 cells specific for CMV, the epitope from pp65 corresponding to residues 495-503 (NLVPMVATV) was conjugated to HLA-0201; for EBV, peptide GLCTLVAML (from the EBV lytic protein BMLF1 corresponding to residues 280-288) was used.

Flow Cytometric Analysis

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by density gradient centrifugation on Ficoll-Lympholyte (Cedarlane Laboratories Limited, Ontario, Canada). The samples (1 x 106 PBMC) were stained with tetrameric complexes (2 l) for 15 min. at 37°C. After incubation, PBMC were washed in PBS x1, then for three colour immunofluorescence were incubated with different monoclonal antibodies to evaluate different markers. The panel included antibodies for CD8, CD27, CD28, CD45RA, CD45RO, CD57 (Caltag, Burlingame, CA, USA), and Perforin (Pharmingen, BD Bioscience, Mountain View, CA, USA). To evaluate perforin production, cells were fixed and permeabilized according the manufacturer's instructions, using Fix and Perm solution (Caltag, Burlingame, CA). Samples were analysed on a Facscan flow cytometer (Becton Dickinson, Mountain View, Ca, USA). To evaluate the reproducibility of the method we have performed preliminary tests using the same sample twice. Then, we have performed some of these initial tests using both fresh and frozen cells in two different labs (Palermo and London laboratories)

Statistical Analysis

Values, given as mean ± SD or median (SE), were compared between young and old subjects using one-way Analysis of variance (ANOVA). The phenotype marker distribution was assessed by regression analysis. Differences were considered significant at p < 0.05.

RESULTS

T cell subsets

Table 1 shows the frequency of CMV and EBV seropositivity in the subjects evaluated in this study.

None of these individuals, old or young, had an inverted CD4/CD8 ratio, regardless of CMVseropositvity (data not shown). This implies that even though some individuals were up to 92 yr of age, none fell into the IRP category defined in the OCTO/NONA studies [START_REF] Pawelec | Human immunosenescence: is it infectious?[END_REF]. Table 2 shows the surface phenotypes of the CD8 cells in the young and old donors, clearly delineating significantly lower percentages of CD27+, CD28+ and CD45RA+ cells in the elderly, as expected from previous reports (Pawelec et al., 2002a;[START_REF] Pawelec | Is immunosenescence infectious?[END_REF][START_REF] Pawelec | Human immunosenescence: is it infectious?[END_REF]. Reciprocally, the percentage of CD45RO+ cells was significantly increased in the elderly. On the other hand, although there was a tendency towards increased percentages of CD57+ cells in the elderly, this difference did not achieve significance. There was, however, a significant decrease in perforin+ cells, as previously reported [START_REF] Rukavina | Age-Related Decline of Perforin Expression in Human Cytotoxic T Lymphocytes and Natural Killer Cells[END_REF]. These data are therefore in general agreement with results previously reported in different populations (Pawelec et al., 2002a;[START_REF] Pawelec | Is immunosenescence infectious?[END_REF][START_REF] Pawelec | Human immunosenescence: is it infectious?[END_REF]. In order to examine the general effect of viral infection on T cell subset distribution, we separately analysed these data for young and old individuals, comparing those seropositive for both EBV and CMV with those seronegative. As shown in Table 3, in seronegative donors, the percentages of CD27+ CD8 cells are significantly lower in the elderly than the young; these percentages are further decreased in seropositive donors in both young and old. Thus, old seropositive subjects have very low levels of CD27+ CD8 lymphocytes. Therefore, EBV/CMV infection influences CD27 expression, that is also decreased in elderly seronegative people, due to ageing per se or to other thus far unidentified pathogens. In contrast, analyzing CD28 expression we observed that young and old seronegative donors retained the same percentages of CD28+ CD8 cells. Old donors had significantly lower percentages of CD28+ cells than young donors only when they were seropositive.

This was also true for the lower levels of CD45RA+ and higher levels of CD45RO+ CD8 cells in the elderly, but although statistically significant, these differences were not so marked as for CD28.

Therefore, only changes in CD27 expression are independent of CMV/EBV infection. Whether changes in CD27 expression can be caused by infections other than with CMV and EBV remains to be determined.

EBV-and CMV-specific CD8 T lymphocytes

In order to quantify virus-specific cells and examine in greater detail alterations in surface marker phenotypes as influenced by viral infection, we focused on HLA A2+ CMV-and EBVseropositive subjects in order to identify CD8 cells carrying receptors for viral epitopes, by using MHC/peptide multimers. The common immunodominant GLC (EBV) and NLV (CMV) epitopes were used in HLA-A2 tetramers for staining CD8 cells. Figure 1 shows the percentages and absolute numbers of CD8 lymphocytes specific for these epitopes of EBV and CMV in each individual HLA-A2+ subject (the bars show the median values). The percentage of EBV-specific CD8 cells in aged people is significantly lower than in young (p=0.02), whereas this was not the case for the CMV-specific cells.

However, the range of CMV values, both for percentage of CD8 CMV-specific cells and absolute numbers per µl of blood, is much greater in the old than young, with some elderly having large accumulations of CMV-specific cells, as seen in another Italian study (Vescovini et al., 2004) .

EBV-and CMV-specific CD8 T cell phenotypes

Finally, we have performed surface phenotyping of CD8 CMV-or EBV-specific T lymphocytes. As in the case of total CD8 T cells, here again we have evaluated the expression of CD27, CD28, CD45RA, CD45RO, CD57 and Perforin, because these markers are suggestive of the state of differentiation of the cells.

In Fig. 2, we show the results of the study on CMV-specific CD8 T cells in both young and elderly donors: the percentage of CD27+ cells within the CMV-specific CD8 cells was not significantly different in young and old subjects;. there was also no significant difference in CD28 or CD57 expression, but there was a significant increase in the percentage of CD45RA+ CMVspecific cells, and a reciprocal decrease in CD45RO (but the latter was not statistically significant). This can be partially explained by the expression of CD45RA by terminally-differentiated CMVspecific cells, as reported by others [START_REF] Appay | Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections[END_REF], but we have no evidence that this is the case also in our subjects. It is, however, also consistent with a trend towards an increase in perforin expression by the CMV-specific CD8 T lymphocytes of the elderly, again contrary to what is seen in the entire CD8 population (Table 2).

The situation with EBV-specific cells is slightly different, but none of the comparisons between young and old reached statistical significance (Fig. 3). Nonetheless, there is a trend towards decreased levels of CD27 and especially CD28, as expected from data of others. There is no increase in CD45RA, also as expected from the previously published phenotypes of EBVspecific cells [START_REF] Appay | Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections[END_REF].

DISCUSSION

CD8 T lymphocytes have been extensively studied since it was observed that in the elderly they are oligoclonally expanded [START_REF] Posnett | Clonal populations of T cells in normal elderly humans: the T cell equivalent to "benign monoclonal gammapathy[END_REF]. Moreover, longitudinal studies on a very elderly Swedish population have suggested that an inverted CD4/CD8 ratio can be considered a mortality risk factor (Wikby et al., 1998;Wikby et al., 2002). This has been confirmed in another North European elderly population [START_REF] Huppert | Survival in a population sample is predicted by proportions of lymphocyte subsets[END_REF]. However, this was not seen in a very large sample of old subjects from western Sicily (age 86-102) (Colonna-Romano et al., 2002). There are many differences between these Northern and Southern European populations, and these would have been even more noticeable during the subjects´ fetal and childhood development three quarters of a century ago. For whatever reason, whether genetic, nutritional, different pathogen exposure, or other lifestyle factors, none of the subjects studied here fell into the IRP category established from the Swedish longitudinal studies, as defined by a CD4:8 ratio < 1. It is in these individuals that the expansion of CD8 clones, predominantly driven by CMV, is most strikingly observed; monitoring these individuals indicates that following an increase in the number of different clones expanded and the absolute number of CMV-specific cells present, there is a period of clonal attrition representing a contraction of the available T cell repertoire. This is a highly significant predictor of incipient mortality [START_REF] Hadrup | Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly[END_REF]. A similar collapse of the TCR repertoire has been observed by others, without taking CMV specificity into account [START_REF] Nayor | The influence of age on T cell generation and TCR diversity[END_REF]. However, the present findings are somewhat different from published reports from Northern European populations both in terms of mean numbers and surface phenotypes of the cells. How to reconcile the data from the Sicilian population presented here with those from the Swedish longitudinal studies? First and foremost, the present study is not longitudinal. Some individuals did present with large CMV-driven clonal expansions; they may be about to enter the IRP and experience a shorter remaining survival period although none of them at the time of the blood collection shows an inverted CD4/CD8 ratio (the subject with 5% of CMV-specific cells had a CD4/CD8 ratio 1.8, the subject who has expanded the CMVspecific CD8 lymphocytes up to 13.3% had a CD4/CD8 ratio of 2.8) . Other subjects showing essentially no CMV-specific cells (Fig. 2) may already be at the stage of repertoire collapse; the loss of their CMV-specific CD8 cells predicts mortality but could also normalize the CD4:8 ratio at the terminal phase. An alternative explanation may be that CMV is the main driving force for ageassociated clonal expansions of CD8 cells in Northern European populations (or was 80 yr ago) but that this preponderance of CMV was not the case on Sicily. Rather, a different hygiene environment involving different pathogens may be critical in this population. In our subjects, as shown, the incidence of CMV infection is about 70% whereas in the North of Europe it is about 80% in the elderly. This does not seem to be a difference sufficiently large to explain these findings. We can also mention the possibility that the viral epitopes chosen for the study are not really the immunodominat epitopes in Sicily. Moreover we have studied HLA-A2 subjects as this allele is highly represented in our population, in fact it is expressed in 42% of individuals HLA-typed by us. It is to mention that The higher phenotype frequency of the HLA-A2 allele found in Scandinavian countries decreases significantly as one moves further south in Europe, but does not influence our results (except to make it more difficult to recruit subjects for the study). However, it still remains the most frequent HLA-allele in Sicily [START_REF] Piazza | The distribution of HLA antigens in Italy[END_REF].On the other hand other HLA antigens usually considered for these studies (HLA B35 and B7) were expressed respectively in 23 and 2% of the subjects that we have studied. Regarding EBV, the primary infection, in Sicily, occurs prevalently early in life as reported [START_REF] Ammatuna | Immune staus towards Epstein-Barr virus in a group of Sicilian children[END_REF], whereas few data are available on the occurrence of primary CMV infection in Sicilian population [START_REF] De Montalebert | Prevalence of markers for human immunodeficiency virus types 1 and 2, human Tlymphotropic virus type I, cytomegalovirus, and hepatitis B and C virus in multiply transfused thalassemia patients. The French Study Group On Thalassaemia[END_REF]. An increased number of subjects studied longitudinally over the coming years should provide a definitive answer to these questions.

Vescovini, R., Telera, A., Fagnoni, F.F., Biasini, C., Medici, M.C., Valcavi, P., di Pede, P., Lucchini, G., Zanlari, L., Passeri, G., Zanni, F., Chezzi, C., Franceschi, C., Sansoni, P., 2004. Different contribution of EBV and CMV infections in very long-term carriers to age-related alterations of CD8+ T cells. Exp Gerontol. 39, 1233-1243.

Wang, E.C., Moss, P.A., Frodsham, P., Lehner, P.J., Bell, J.I., Borysiewicz, L.K., 1995. CD8highCD57+ T lymphocytes in normal, healthy individuals are oligoclonal and respond to human cytomegalovirus. J Immunol 155, 5046-5056. Wedderburn, L.R., Patel, A., Varsani, H., Woo, P., 2001. The developing human immune system: T cell receptor repertoire of children and young adults shows a wide discrepancy in the frequency of persistent oligoclonal T cell expansion. Immunology 102, 301-309. Wikby, A., Maxson, P., Olsson, J., Johansson, B., Ferguson, F.G., 1998. Changes in CD8 and CD4 lymphocyte subsets, T cell proliferation responses and non-survival in the very old: the Swedish longitudinal OCTO-immune study. Mech Ageing Dev.102, 187-198 Wikby, A., Johansson, B., Olsson, J., Lofgren, S., Nilsson, B.O., Ferguson, F., 2002. Expansions of peripheral blood CD8 T-lymphocyte subpopulations and an association with cytomegalovirus seropositivity in the elderly: the Swedish NONA immune study. Exp Gerontol.37, 445-453. . Table 1 Samples evaluated in the study. At the beginning of the study a total of 140 samples from young or old donors were screened for CMV and EBV seropositivity. Only seropositive subjects were HLA typed. Thereafter HLA-A2 positive subjects were selected for tetramer staining.

Legend to figures

Total Samples evaluated n=140

Figure 1 .

 1 Figure 1. Percentage and absolute number (103/µl) of EBV-and CMV-specific CD8 T lymphocytes in young (Y) (n= 18) and old (O) (n= 13) subjects. Horizontal bars represent median values.(Y % GLC Median Value 0.97 P25 0.5, P75 1.98; O % GLC Median Value 0.61, P25 0.21, P75 1.42; Y % NLV Median Value 1.01 P25 0.45, P75 1.92; O % NLV Median Value 0.85, P25 0.26, P75 1.72; Y Absolute number GLC Median Value 5.9 P25 1.18, P75 8.05; O Absolute number GLC Median Value 2.9, P25 1.44, P75 4.21; Y Absolute number NLV Median Value 4.63 P25 2.37, P75 6.8; O Absolute number NLV Median Value 3.04, P25 1.05, P75 7.48). Both for EBV and CMV specific CD8 lymphocytes differences were not significant by ANOVA .

Figure 2 Figure 3

 23 Figure 2 Evaluation of phenotypic markers on CMV-specific CD8 lymphocytes obtained from N= 16 young (Y) and N= 12 old (O) subjects. The longer horizontal bars represent median values; the shorter horizontal bars rapresent P25 and P75. Significant result, Significance was obtained by ANOVA test was obtained for CD45RA (p = 0.01). p value obtained for CD45R0 was 0.08 (marginally significant) Figure 3 Evaluation of phenotypic markers on EBV-specific CD8 lymphocytes obtained from 16 young (Y) and 12 old (O) subjects. The longer horizontal bars represent median values; the shorter horizontal bars rapresent P25 and P75. No significance was obtained by ANOVA test.

Table 2 .

 2 CD8 phenotype in blood samples of young and old subjects not selected on the basis of seropositivity to CMV and EBV (data are present as Mean + SD; P values were calculated by ANOVA *p<0.05 significant).

	Results of CMV screening					Results of EBV screening
	OLD (n=54)	YOUNG (n=86)	OLD (n=54)	YOUNG (n=67)
	CMV	CMV	CMV		CMV	EBV		EBV	EBV	EBV
	Positive	Negative	Positive	Negative	Positive	Negative	Positive	Negative
	39	15	39		47	29		25	23	44
	(72.3%)	(27.7%)	(45.3%)	(54.7%)	(53.7%)	(43.3%)	(34.3%)	(65.7%)
	13 A2+		18 A2+			13 A2+		18 A2+
		%CD27	%CD28	%CD45RA %CD45R0	%CD57	%PERF
	Young (26)	61.8±17.6	64±16.1	65.4±12.9	40.2±14.8	44.2±18.6	15.2±8.1
	Old (22)	42±17.6	54.3±18.4	55.1±18.2	49.7±16.4	53.6±21.4	10.4±7.9
	P	0.0005*	0.04*		0.02*		0.04*	0.11	0.05*

Table 3 .

 3 CD8 phenotype in blood samples of young and old subjects assembled according to the seropositivity or seronegativity to CMV and EBV (data are present as Mean + SD; P values were calculated by ANOVA *p<0.05 significant).

			SERONEGATIVE SAMPLES
		%CD27	%CD28	%CD45RA %CD45R0	%CD57	%PERF
	YOUNG(n=10) 75.8±11.4	77.3±15.3	72.8±15.5	39.3±22	33.3±23.3	12.1±6.2
	OLD (n=10)	48.1±18.5	72.9±7.01	63±14	49.6±18.4	55.8±23.2	6.4±8
	P	0.001*	0.4	0.1	0.3	0.08	0.1
			SEROPOSITIVE SAMPLES
		%CD27	%CD28	%CD45RA %CD45R0	%CD57	%PERF
	YOUNG(n=16) 53.05±14.9 57.04±11.2	60.8±8.7	40.79±7.2	49.83±13.8 17.25±8.6
	OLD (n=12)	37.9±16.45 42.33±11.9 49.91±19.4 50.25±15.8 52.16±21.1	13.16±7
	P	0.013*	0.0016*	0.05*	0.04*	0.71	0.17

ACKNOWLEDGEMENTS

Our collaboration was supported by the EU 5th Framework program through contract QLK6-CT-1999-02031, project `ImAginE´ (Immunology and Ageing in Europe, see www.medizin.unituebingen. de/imagine/) and by a grant from Palermo University to strengthen international collaboration (HLA ed altri fattori immunologici in grado di influenzare l'aspettativa di vita individuale). MB is a PhD student in the Pathobiology PhD course (directed by C.C.) of Palermo University and this work is submitted in partial fulfilment of the requirement for the PhD.


Miriam Capri

Stefano Salvioli

Daniela Monti

Calogero Caruso

Sonya Vasto

Fabiola Olivieri

Francesca Marchegiani

Paolo Sansoni

Giovannella Baggio

Daniela Mari

Giuseppe Passarino

Giovanna De Benedictis

Professor Claudio Franceschi
email: claudio.franceschi@unibo.it

Human longevity within an evolutionary perspective: the peculiar paradigm of a post-reproductive genetics

Keywords: longevity, centenarians, genetics, antagonistic pleiotropy

2

Abstract

The data we collected on the genetics of human longevity, mostly resulting from studies on centenarians, indicate that: 1. Centenarians and long-living sibpairs are a good choice for the study of human longevity, because they represent an extreme phenotype, i.e. the survival tail of the population who escaped neonatal mortality, pre-antibiotic era illnesses, and fatal outcomes of agerelated complex diseases. 2. The model of centenarians is not simply an additional model with respect to well-studied organisms, and the study of humans has revealed characteristics of ageing and longevity (geographical and sex differences, role of antigenic load and inflammation, role of mtDNA variants) which did not emerge from studies in laboratory model systems and organisms. 3.

All the phenotypic characteristics of nonagenarians and centenarians fit the hypothesis that ageing is a remodelling process where the body of survivors progressively adapts to internal and external damaging agents they are exposed to during several decades, largely unpredicted by evolution. 4. Such a remodelling process, which can be considered a Darwinian process occurring at the somatic level within the framework of the evolutionary constraints established by evolution for H. sapiens as a species may explain why the same gene polymorphism can have different (beneficial or detrimental) effects at different ages. 5. Geographic and demographic evidence suggest that longevity can be achieved by different combinations of genes, environment, and chance quantitatively and qualitatively different in different geographic areas, and that population-specific genetic factors, play a role on the longevity trait. 6. The concomitant and integrated use of new in silico and high throughput strategies will greatly accelerate the identification of new longevity genes in humans.

The peculiar geography and demography of centenarians

Human populations are characterized by specific gene pools that arise from the particular group's history in terms of chance (genetic drift) and environment (natural selection). Furthermore, humans are unique in having linked cultural and biological inheritance. Accordingly, human longevity is a very complex trait and its study is quite difficult and still in its infancy. A useful rule when tackling the genetics of a complex trait such as longevity is to consider an extreme phenotype.

From this perspective, subjects able to reach extreme longevity, such as centenarians, have been an extraordinary model to study human longevity and healthy ageing, and to identify their biological determinants. Centenarians represent the survival tail of the population and they escaped neonatal mortality, pre-antibiotic era illnesses and they avoided or postponed most severe age-related diseases and their fatal outcomes (Franceschi et al., 1995a;[START_REF] Gueresi | The MALVA (MAntova LongeVA) study: an investi-gation on people 98 years of age and over in a province of Northern Italy[END_REF][START_REF] Leroi | What evidence is there for the existence of individual genes with antagonistic pleiotropic effects?[END_REF][START_REF] Franceschi | Genes involved in immune response/inflammation, IGF1/insulin pathway and response to oxidative stress play a major role in the genetics of human longevity: the lesson of centenarians[END_REF]. From a geographical point of view human longevity is not homogenously distributed and it seems to be clustered in families enriched in long-lived parents and ancestors [START_REF] Poulain | Identification of a geographic area characterized by extreme longevity in the Sardinia island: the AKEA study[END_REF][START_REF] Gueresi | The MALVA (MAntova LongeVA) study: an investi-gation on people 98 years of age and over in a province of Northern Italy[END_REF][START_REF] Willcox | Siblings of Okinawan centenarians share lifelong mortality advantages[END_REF]. In particular, significant differentials have been observed between Italian regions in the distribution of centenarians by sex, (femininity ratio, FR) from two women per man in the south to over eight in certain regions in the north [START_REF] Rose | The variability of the mitochondrial genome in human aging: a key for life and death[END_REF][START_REF] Gueresi | The MALVA (MAntova LongeVA) study: an investi-gation on people 98 years of age and over in a province of Northern Italy[END_REF]. A recent investigation suggests that the FR observed at the age of 100 in Italy is mainly due to mortality differentials among men and women over the age of 60 [START_REF] Robine | Differentials in the femininity ratio among centenarians: variations between northern and southern Italy from 1870[END_REF]. The high mortality of men in the north and their low mortality in the south are the main explanations of why the FRs are higher in the north and lower in the south. Mortality differentials among women have only a marginal impact.

The lowest FR is observed in certain parts of the Sardinia island such as the province of Nuoro, where the female-to-male ratio tends to be highly male-biased. This phenomenon appears to be mostly due a survival advantage of Sardinian men due to lower mortality for cardiovascular diseases after age 80 years [START_REF] Caselli | Survival difference among the oldest old in Sardinia; who, what , where and why?[END_REF]. We recently published the first results of a genealogical tree reconstruction of selected centenarians aged 105 and over, from the Nuoro province (Caselli et al., 2006). An accurate centenarian age validation was performed that required a meticulous examination of numerous civil status records and parish registers. An important finding was that longevity occurs among the ascendants of the maternal branch of the family. The data used are still provisional but, should it apply to other validated cases, it would provide additional evidence of a genetic component in longevity. A more thorough examination of the data available may yield deeper insights into the role played by endogamy and consanguinity in attaining longevity.

Familiarity and genetics play a role in attaining extreme old age in humans: evidence from centenarians and their family members

Actually, an impressive and coherent series of epidemiological data from different populations (White Americans from New England, Mormons from Utah, Ashkenazi Jewish living in USA, Icelanders, Japanese from Okinawa, Netherlanders from Leiden) suggests the presence of a strong familiar and genetic component of human longevity. All these studies demonstrate that parents, siblings and offspring of long-lived subjects have a significant survival advantage and a higher probability to have been or to become long-lived, and a lower risk to undergo major age-related diseases, such as cardiovascular diseases (CVD), diabetes and cancer, when compared to appropriate selected controls. Thus, literature indicates that longevity is present in many generations of a single family in spite of the great variations in life style and life expectancy which occurred in the last century. In particular, it is interesting to note that in the most recent studies on this topic, spouses of long-lived subjects have been added as additional control group. The results indicate that this control group does not have any advantage/benefit in terms of survival and protection from the above mentioned diseases, even if they have shared with the long-lived partner most of their adult life. On the whole, these data suggest that such a familiar component can in fact be a genetic component of longevity, and indicate that long-living subjects and families enriched in long-living members represent study groups particularly suitable to study the determinants of human longevity [START_REF] Perls | Life-long sustained mortality advantage of siblings of centenarians[END_REF][START_REF] Terry | Cardiovascular advantages among the offspring of centenarians[END_REF][START_REF] Rattan | Aging intervention, prevention, and therapy through hormesis[END_REF][START_REF] Atzmon | Clinical phenotype of families with longevity[END_REF][START_REF] Karasik | Genetic contribution to biological aging: The Framingham study[END_REF][START_REF] Ikeda | JACC study group. Parental longevity and mortality amongst Japanese men and women: The JACC Study[END_REF][START_REF] Schoenmaker | Evidence of genetic enrichment for exceptional survival using a family approach: The Leiden longevity study[END_REF] Twin studies indicate that approximately 25% of the variation in adult lifespan is caused by genetic differences between individuals, and that genetic influences on lifespan are minimal before the age of 60 but increase after this age [START_REF] Christensen | The quest for genetic determinants of human longevity: challenges and insights[END_REF]. As the number of very old, centenarian, twins is very rare, such data on the heritability on longevity in twins do not exclude that the genetic component of extreme longevity can be even greater that the indicated 25% which, in our opinion, represents the lower estimate. In any case environment, i.e. climate, food, anthropological/cultural habits regarding nutrition and exercise, economical, working and housing conditions, as well as social and familiar support, strongly contribute to achieve the extreme limits of human lifespan. We recently performed a study on the personality of centenarians living in central Italy and preliminary data indicates that centenarians have a peculiar personality and character. On that occasion we also investigated the quality of sleep in centenarians and again we found that sleep quality is remarkable good, likely contributing to the capability to survive to extreme age (Sorbi et al., unpublished results). It is interesting to note that we know practically nothing about another component such as stochasticity that human observations in twins and animal studies indicate to play a consistent role in longevity [START_REF] Rea | A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans[END_REF].

The genetics of human longevity and the results of association studies on candidate genes: longevity genes can be population-specific

We recently reviewed the data obtained in our and other laboratories suggesting that polymorphisms of genes involved in immune response/inflammation, response to stressors including oxidative stress, insulin/IGF1 signalling pathway, among others, have a different frequency in centenarians in comparison to younger controls (young and old people) (Franceschi et al., 2005). We also recently reviewed (Salvioli et al, 2006a) the data suggesting that both inherited and somatically-acquired variability of mitochondrial DNA (mtDNA) also play a role in human longevity, likely interacting with nuclear gene variants, within the theoretical framework that the cross-talk between these two genomes is essential for longevity as well as for other complex traits [START_REF] Rose | The variability of the mitochondrial genome in human aging: a key for life and death[END_REF]. It is interesting to note that, according to a large scale study, involving more than 900 subjects of different ages, the association between longevity and mtDNA haplogroup J is population specific, being evident in centenarians from northern Italy but not in those from southern Italy. In nonagenarians/centenarians from Ireland Finland and Japan mtDNA haplogroups and longevity are also associated (Niemi et al., 1998;[START_REF] Tanaka | Mitochondrial genotype associated with longevity[END_REF][START_REF] De Benedictis | Mitochondrial DNA inherited variants are associated with successful aging and longevity in humans[END_REF][START_REF] Ross | Mitochondrial DNA polymorphism: Its role in longevity of the Irish population[END_REF]Salvioli et al., 2006a) Interesting conclusions emerge from a comprehensive/critical analysis of the available data on the genetics of centenarians [START_REF] Franceschi | Genes involved in immune response/inflammation, IGF1/insulin pathway and response to oxidative stress play a major role in the genetics of human longevity: the lesson of centenarians[END_REF][START_REF] Franceschi | Inflammaging and antiinflammaging: a systemic perspective on aging and longevity emerged from studies in humans[END_REF][START_REF] Capri | The genetics of human longevity[END_REF]Salvioli et al., 2006a;[START_REF] Christensen | The quest for genetic determinants of human longevity: challenges and insights[END_REF]Candore et al., 2006a):

-most of the studies so far reported have been performed in a restricted number of populations, mostly Italians and Danish, but also Japanese, French, Ashkenazi Jewish, Germans and Finnish, among others; -in most of the studies, a limited number of polymorphisms has been considered; -sex/gender-differences reported by some studies have not been systematically investigated. A possible reason is the low number of centenarians examined in some studies which did not allow to appreciate the possible importance of sex/gender; -the number of genes which have been studied in more than one populations is relatively low, and the results are often discordant or difficult to compare.

To simplify such a very complex mass of data, two basically different interpretations are possible:

1. the first assumes that most of the results derived from the above-mentioned studies are likely false positive, owing to bias related to population stratification between cases (centenarians) and controls, lack of power, or other (trivial) reasons; 2. the second assumes that different/discordant findings in two (or more) different populations of centenarians and controls can be expected because different susceptibility genes may contribute to the longevity phenotype in genetically different populations.

In most of the cases we cannot decide which of the two alternatives is true, but in any case these considerations are crucial as regards replication studies, that is studies that aim to confirm in another population the associations between genetic loci and traits found in one population.

Assuming that longevity can be achieved by different routes, resulting from different combinations of genes, environment, and chance that vary quantitatively and qualitatively in different geographic areas, we wonder whether replication can reasonably be expected for longevity in light of the above considerations. We are not challenging the tenet that studies on complex traits must be replicated in different populations and/or geographic areas. We are challenging the simplistic expectation that a gene polymorphism cannot be considered a longevity marker unless it is associated with longevity in all the populations tested (De Benedictis and Franceschi, 2006;[START_REF] Franceschi | Inflammaging and antiinflammaging: a systemic perspective on aging and longevity emerged from studies in humans[END_REF] How to overcome the "cohort effect" and to take advantage from demographic data and the use of different age groups Most of the above-mentioned limitations are common to other type of association studies on complex traits. Indeed, in the case of association studies on human longevity another limitation is present, i.e. the so called "cohort effect", due to the unavoidable comparison of people belonging to different cohorts. The cohort effect assumes that the variation in longevity may be caused by different social and environmental factors experienced by distinct birth cohorts. We and others tried to minimize the effect of such an intrinsic limitation by: 1. using younger "controls" belonging to different age groups, including people aged around 60 years of age who are those more close to centenarians as a cohort but also those who did not underwent any significant mortality selection; 2. creating algorithms that add demographic information to gene data; this practice aims at estimating the association between gene variants and survival/mortality parameters without generating bias due to cohort effects. The advantage of such an approach is that it increases the power of genetic analysis, besides being more correct from a methodological point of view [START_REF] De Benedictis | The unusual genetics of human longevity[END_REF]Salvioli et al., 2006b;[START_REF] Franceschi | Inflammaging and antiinflammaging: a systemic perspective on aging and longevity emerged from studies in humans[END_REF].

Recently, we aimed to investigate the influence of the genetic variability of candidate genes on survival at old age in good health on the basis of a synthetic survival curve constructed using historic mortality data taken from the Italian population from 1890 onward [START_REF] Passarino | A cluster analysis to define human aging phenotypes[END_REF].

We defined three age classes ranging from 18 to 106 years and then assembled a multinomial logistic regression model to evaluate the effect of dichotomous variables (genotypes) on the probability to be assigned to a specific category (age class). By applying the regression model to a cross-sectional dataset (10 genes; 972 subjects selected for healthy status) categorized according to age and sex we found that genetic factors influence survival at advanced age in good health in a sex-and age-specific way. Furthermore, we found that genetic variability plays a stronger role in males than in females and that, in both genders, its impact is especially important at very old ages.

These results underline the age-specific effect of the gene network in modulating survival at advanced age in good health [START_REF] Passarino | A cluster analysis to define human aging phenotypes[END_REF].

The rationale of and the working hypothesis behind the association studies in centenarians

Today the available genetic methodologies allow to perform genome-wide association studies with several hundred thousands polymorphisms scanning the entire genome in order to find polymorphisms associated with a defined phenotype without any a priori hypothesis. This was not the case in previous studies performed on a limited number of candidate genes and polymorphisms.

It is interesting to remind the theoretical assumptions that led us to focus of the specific genes we have investigated in Italians centenarians.

First of all it is important to remind that the first data on possible determinants of longevity in centenarians emerged from two major hypothesis, the first regarding a general conceptualization on the possible biological determinants of the ageing process, and the second originating from studies on the immunological status of centenarians.

In the 1989 paper one of us arrived to the conclusion that the ageing process is indirectly controlled by a variety of cellular and molecular defence mechanisms, including anti-oxidant defence, heat shock proteins, DNA repair mechanisms, and proposed the concept that they constitute a network of defence mechanisms [START_REF] Franceschi | Cell proliferation, cell death and aging[END_REF]. This hypothesis was further developed by Tom Kirkwood and Claudio Franceschi [START_REF] Kirkwood | Is aging as complex as it would appear? New perspectives in aging research[END_REF]) and constituted the theoretical basis of an international conference held in Modena in 1992, devoted to "Aging and Cellular Defence Mechanisms".

The second hypothesis was based on the assumption that the ageing of the immune system, Immunosenescence, plays a central role in the ageing process (Franceschi et al., 1995b). Since the beginning of last decade of the last century, we systematically investigated the most parameters of the immune response and arrived to the unexpected conclusion that several parameters, such as NK cell number and activity, were remarkably conserved in centenarians and quite similar to those found in young people. We also identified the most important changes which characterize immunosenescence, i. e. a decrease of B lymphocytes and naïve T cells, a progressive increase of CD28-cytotoxic T cells and the accumulation of expanded clones of memory T cells, a shrinkage of the T cell repertoire, the lack of organ-specific autoantibodies, among others (Franceschi et al., 1995b;[START_REF] Cossarizza | Cytometric analysis of immunosenescence[END_REF]Franceschi et al., 2000a). A seminal observation we made during these investigations was that peripheral blood mononuclear cells from old donors, when stimulated with mitogens, were able to secrete higher amounts of pro-inflammatory cytokines than cells from young people (Fagiolo et al., 2003). This observation was highly unexpected as the dominant paradigm at that time was that immunosenescence was a global failure of the immune system where all the responses were negatively affected. Our observation challenged that tenet showing that inflammatory responses were higher and more active in old people than in young ones.

Accordingly, we focused our attention on pro-inflammatory cytokines showing for example that, unexpectedly, centenarians (even those in relatively good health) had high plasma level of IL-6 [START_REF] Baggio | Lipoprotein(a) and lipoprotein profile in healthy centenarians: a reappraisal of vascular risk factors[END_REF][START_REF] Passeri | Low vitamin D status, high bone turnover, and bone fractures in centenarians[END_REF]. Incidentally, in the 1998 paper we also reported the unexpected finding that centenarians have high plasma level of Lp(a) a lipoprotein under strict genetic control which is considered a risk factor for CVD. Concomitantly, another "paradox" of centenarians emerged, i.e. their pro-coagulant status without clinical signs of thrombosis [START_REF] Mari | Hypercoagulability in centenarians: the paradox of successful aging[END_REF]. These observations were made by a group working in Milan leaded by Daniela Mari, and were subsequently confirmed and extended [START_REF] Coppola | Von Willebrand factor in Italian centenarians[END_REF].

On the whole these results were a clear indication that inflammation, a fundamental response which allows animals to cope with a variety of stressors and pathogens, was up-regulated and over-stimulated in centenarians. At the same time the literature on diseases, such as atherosclerosis and Alzheimer's disease (AD) started to understand that all these major age-related diseases shared an inflammatory background which likely played a major role in their pathogenesis [START_REF] Ross | Atherosclerosis--an inflammatory disease[END_REF][START_REF] Mcgeer | Brain inflammation in Alzheimer disease and the therapeutic implications[END_REF] . How to reconcile these contrasting observations?

On one side inflammation appeared to be a prerequisite or at least a basic component of the major killers and on the other side the centenarians, i.e. those people who were able to reach a remarkable advanced age escaping or delaying those major pathologies, were clearly "inflamed" and showed several signs of inflammation, including high levels of pro-inflammatory cytokines and coagulation factors [START_REF] Mari | Hypercoagulability in centenarians: the paradox of successful aging[END_REF][START_REF] Baggio | Lipoprotein(a) and lipoprotein profile in healthy centenarians: a reappraisal of vascular risk factors[END_REF][START_REF] Coppola | Von Willebrand factor in Italian centenarians[END_REF][START_REF] Passeri | Low vitamin D status, high bone turnover, and bone fractures in centenarians[END_REF].

In order to answer this question and to solve this apparent paradox we started investigations aimed to identify a possible genetic propensity to produce high or low levels of pro-inflammatory cytokines and other pro-inflammatory molecules. Different teams of investigators were involved in this ambitious task, working in strict collaboration and sharing hypothesis and samples. In the meantime, the group of Giovanna De Benedictis, in strict collaboration with the Modena/Bologna group and the group of the Italian National Research Center on Ageing (INRCA, Ancona), pioneered the genetic approach to the longevity trait, starting a series of investigations focused on apolipoproteins, a topic which has been thoroughly analysed in subsequent years including studies on genes coding for apolipoproteins such as APOB, APOE, APOC3 but also APOJ , genes related to stress (SOD, TH, HSP70), DNA repair (PARP), sirtuins (SIRT3) and mtDNA. Apart from the original group of Milan and Modena/Bologna the enlarged team included groups working in Ancona at (INRCA) and that of the University of Palermo leaded by Calogero Caruso, who focused on the opposite role played by inflammatory gene polymorphisms in longevity and CVD. This critical mass allowed us to increase the number of candidate genes to investigate and to include genes such as paroxonase (PON1) which protect LDL from oxidation, as well as those coding for anti-inflammatory cytokines such as IL-10 and TGFbeta1, and genes coding for molecules crucial for the recognition of pathogens and the start of immune response such as toll receptors (TLR4) and other which plays an important role in inflammatory responses (PYRIN), α1-antitrypsin, connexin, CCR5, COX and LOX (Candore et al., 2006a,b;[START_REF] Capri | The genetics of human longevity[END_REF]Salvioli et al., 2006a, Franceschi et al., 2007[START_REF] Vasto | Inflammatory networks in ageing, age-related diseases and longevity[END_REF].

On the whole, these last data suggest that a pro-inflammatory genotype is unfavorable to reach extreme longevity in good health and likely favors the onset of age-related diseases such as CVD and AD which are the leading causes of mortality and disability in the elderly. Within this perspective, healthy ageing and longevity are likely the result not only of a lower propensity to mount inflammatory responses but also of an efficient anti-inflammatory network, which in unsuccessful ageing fail to fully neutralize the inflammatory processes consequent to the lifelong antigenic burden and exposure to damaging agents. Such a global imbalance can be a major driving force for frailty and common age-related pathologies, and should be addressed and studied within an evolutionary-based systems biology perspective [START_REF] Franceschi | Inflammaging and antiinflammaging: a systemic perspective on aging and longevity emerged from studies in humans[END_REF].

Conclusions

In spite of the growing amount of genetic data regarding longevity, genetics can account only for a part of the phenomenon. This is demonstrated by different experimental evidence: i. syngenic animals have different mortality curves, suggesting the presence of a stochastic component; ii. many genetic variants considered unfavourable for longevity and associated to an increased risk of disease are found in centenarians with the same frequency observed in the general population (Franceschi et al., 2005a;Salvioli et al., 2006a). Moreover, inside the human species, gender differences are present. It is known that women live longer than men (about 5 years more) and the major part of subjects that achieve the extreme ages is composed by females. Furthermore, a positive association between genetic variants and longevity is often found only for males and rarely for both genders (Candore et al., 2006a). This suggests that longevity trait is more dependent on genetics in men than in women (Franceschi et al., 2000b). It is hypothesised that in female longevity a non-genetic component is present which

is not yet completely appreciated. As a consequence, it is possible to affirm that longevity is a complex trait which depends on environmental, cultural and genetic factors. These factors are interacting with each other, as demonstrated by different studies [START_REF] Gueresi | The MALVA (MAntova LongeVA) study: an investi-gation on people 98 years of age and over in a province of Northern Italy[END_REF][START_REF] Carru | Association between the HFE mutations and longevity: a study in Sardinian population[END_REF] and they are also modulated by the cross talk between nuclear and mitochondrial genomes [START_REF] De Benedictis | Mitochondrial DNA inherited variants are associated with successful aging and longevity in humans[END_REF][START_REF] Bellizzi | Gene expression of cytokines and cytokine receptors is modulated by the common variability of the mitochondrial DNA in cybrid cell lines[END_REF].

Association studies can point out positive or negative association between particular allelic variants and longevity. This rather simple situation is complicated by the fact that the same variants can be associated with both longevity and age-related diseases, depending on the genetic and environmental scenario (population studied, parameters considered, etc.). This clearly indicates that it is crucial the interaction and combination among more genes and their allelic variants. Besides, available data indicate that the aging process allows the emerging of biological effects due to individual genetic differences which can be neutral or silent at younger age. This suggests that the genetics of ageing/longevity is peculiar, and likely it is not ruled by classical genetic principles, and should thus be considered a post-reproductive genetics, since it regards decades of life where the strength of selection progressively fades away [START_REF] De Benedictis | The unusual genetics of human longevity[END_REF]. From this point of view, it is much more likely that aging is a sort of side-effect of genes playing an important role during juvenile age. As an example, it is possible that a genetic variant that increases reproductive fitness can have a detrimental effect on longevity. This phenomenon is defined as "antagonistic pleiotropy" [START_REF] Williams | The dawn of Darwinian medicine[END_REF]. It is thus evident that, if a genetic variant confers a selective advantage during young age, it will be selected even if it is unfavorable for longevity (for example by conferring a higher risk for age-related diseases). This seems to be the case for the inflammatory gene polymorphisms responsible for a higher responder status that were selected to fight infections in young age [START_REF] Caruso | Inflammation and lifespan[END_REF][START_REF] Licastro | Innate immunity and inflammation in ageing: a key for understanding age-related diseases[END_REF].

In this perspective, the apparent paradoxes emerged from the studies on centenarians can be generated not only by the lack of validated scales for centenarians, but also by the fact that a genetic variant can play different roles in young age and in old age. Indeed, one of the most important indications coming from the studies on centenarians, is that extreme longevity has unusual and largely unexpected features for which most of the current concepts in biology and genetics are inadequate [START_REF] De Benedictis | The unusual genetics of human longevity[END_REF]. Actually, it is clear that centenarians are part of a special biology in which different tissue and organs could not have reached the same level of senescence (a kind of mosaicism). Moreover, it will be the balance between the amount of enduring stresses and the efficacy of stress-response systems of the body that will determine how fast a tissue or body will age (Franceschi et al., 2000c;[START_REF] Rattan | Aging intervention, prevention, and therapy through hormesis[END_REF]).

Finally, since human aging is the emerging product of a series of different factors (environment, culture, economic conditions, stochasticity, etc.) in which genetics plays only a part, we wish to stress that it is not always possible to extrapolate and predict results to be extended to humans from animal models in which most of these variables have been eliminated or are under control, as for example cultures of mutated C. elegans or KO mice. In particular, in some cases studies on animal models have led to analogous results also in humans, as in the case of Insulin/IGF-1 signaling pathway [START_REF] Franceschi | Genes involved in immune response/inflammation, IGF1/insulin pathway and response to oxidative stress play a major role in the genetics of human longevity: the lesson of centenarians[END_REF], while in other cases results obtained in animal models have not been reproducible in humans. For example, the knock out of p66 Shc gene leads to an increase in mice lifespan [START_REF] Migliaccio | The p66shc adaptor protein controls oxidative stress response and life span in mammals[END_REF] and consequently it has been hypothesised that centenarians should hypoexpress this gene [START_REF] Purdom | p66(Shc): at the crossroad of oxidative stress and the genetics of aging[END_REF], while, on the contrary we observed that dermal fibroblasts from centenarians do have higher levels of p66 Shc mRNA and protein with respect to young and middle-aged subjects [START_REF] Pandolfi | p66 shc is highly expressed in fibroblasts from centenarians[END_REF]. Thus, often it appears not sufficiently adequate the animal model for the study of human longevity (Franceschi and De Benedictis, 2006;[START_REF] Capri | The genetics of human longevity[END_REF].

Further, it is claimed that results obtained on human populations should always be replicated. Indeed, association with particular genetic polymorphisms and longevity is reported for some population but not for others (see for a recent striking example the study of [START_REF] Barzilai | Unique lipoprotein phenotype and genotype associated with exceptional longevity[END_REF][START_REF] Cellini | Cholesteryl ester transfer protein (CETP) I405V polymorphism and longevity in Italian centenarians[END_REF] on CETP gene). However, this is not strange because, as underlined in this paper, human populations are characterized by specific gene pools that arise from the particular group's history in terms of chance (genetic drift) and environment (natural selection). Hence, replication cannot reasonably be expected for longevity in light of the considerations discussed in the present study [START_REF] De Benedictis | The unusual genetics of human longevity[END_REF].

To conclude, there are new recent advances in the analysis of human genome which in the next future will open interesting perspectives on the study of human longevity. So, the concomitant and integrated use of new in silico and high throughput strategies will greatly accelerate the identification of new longevity genes in humans.

Acknowledgements

This work has been supported by grants from EU (European Union) 6 th FP Project "GEHA -Genetics of Healthy Ageing", "T-CIA -T-Cells Immunity and Ageing", EU (fondi strutturali Obiettivo 2), the PRRIITT program of the Emilia-Romagna Region, MIUR (Italian Ministry of University) Programmi di Ricerca di Interesse Nazionale (PRIN) protocol #2003068355_002, Fondo per gli Investimenti della Ricerca di Base (FIRB) protocol #RBNE018AAP and #RBNE018R89 to CF; and from Italian Ministry of Health (Ricerche Finalizzate 2002) and EU 6 th FP Project "Zincage" to DM.