,Wagne

an

nge

(

)In vitro senescence models for human T lymphocytes. Vaccine

:

–

Ito

,Yonehar

,Ishi

,Yonehar

,Mizushim

I,Sameshim

,Has

, Set

an

agat

(

)Thepolypeptideencodedbythe

NAforhumancell surface antigen Fas can mediate apoptosis. Cell

:

–

Cory S (

) Fascinating death factor. Nature

:

–

Ju ST, Panka DJ, Cul H, Ettinger R, El-Khatib M, Sherr DH, Stanger BZ and Marshak-Rothstein A (

) Fas(CD

)/Fa

interaction required for

The primers used for the PCR are 6247Dir (5’-AGGAAAGGACTT- CAAAGCCTA-3’) e 6231Rev (5’-TTGATGCATCACAGAATTTCGTC- 3’). In this case the reverse primer contains a mismatch (underlined and bold) that creates an artificial new restriction site for HincII in the PCR product. For each PCR reaction in 25 ml, 100 ng of DNA template were added to the reaction mixture, containing a final concentration of 200 nM of each primer, 200 mM of dNTPs, 1.5 mM MgCl , 50 mM 2 KCl , 10 mM Tris Cl (pH 8.5) and 1 U of Taq polymerase (Promega). 2 The reaction was carried out in a PE 9700 Thermal Cycler for 30 cycles, with each cycle consisting of 30 s at 948C, 30 s at 608C, and 30 s at 728C. The first cycle was preceded by a step of denaturation at 948C for 6 min, and the last one was followed by a step of extension at 728C for 10 min. Ten ml of PCR product is digested with 1 U of HincII under recommended conditions and loaded on a 3% agarose gel. If allele T is present, the digestion generates a 162 bp fragment; if delT allele is present the 185 bp band was present on the gel.

Statistical Analysis

The distribution of the 7670 and 71377 Fas genotypes, and that of IVS2nt-124 and IVS3nt169 FasL genotypes in centenarians was compared to that in controls using the w2 test (362 contingency table with Monte Carlo option); differences in allele frequency were analyzed by z-test using SPSS 10 software operating under Windows ME. Genetic analysis regarding haplotype frequency estimation was performed according to Guo and Thompson,27 using Arlequin software (by Drs. L Excoffier, S Schneider, D Roessli, University of Geneva, Switzerland, downloaded at http://lgb.unige.ch/arlequin/software/).

Acknowledgements

We acknowledge Professor R Lockshin (St. John’s University, Jamaica, NY), Dr. M Bonafe` (Univ. of Bologna) and Professor D Lio (Univ. of Palermo) for critical comments and helpful suggestions. This work has been supported by grants from MURST (Cofin 1999) to A Cossarizza.

References

Mont

, Grassill

,Troian

,Cossarizz

,Salviol

,Barbieri D,Agnesini Bettuzzi S, Ingletti MC, Corti A and Franceschi C (

) immortalization and apoptosis: an intriguing relationship. Ann. N.Y. Acad.

:

–

S, Ronchetti I, Bellomo G, Cossarizza A and Franceschi C (

) proliferation and cell death in immunosenescence. Ann. N.Y. Acad. Sci.

–

Monti D, Troiano L, Tropea F, Grassilli E, Cossarizza A, Barozzi D, Pelloni Tamassia MG and Franceschi C (

) Apoptosis - Programmed cell death: role in the aging process? Am. J. Clin. Nutr.

:

s–

s

from newborns to centenarians Int. Rev. Immunol.

:

–

deterioration or remodelling? Int. Rev. Immunol.

:

–

Franceschi C,Mont

, Sansoni Pand Cossarizza A (

) The immunology

Wack A, Cossarizza A, Heltai S, D’Addato S, Franceschi C, Dellabona P Casorati G (

) Age-related increase of clonal expansions in peripheral blood T cells: CD

+are more affected than CD

+, irrispective their CD

RA, CD

R

and CD

expression. Int. Immunol.

:

–

Scala E, Fagiolo U, Monti D, Cossarizza A, Bonafe´ M and Franceschi C Hemopoiesis in healthy old people and centenarians: responsiveness of CD

+cells to hemopoietic growth factors and of cytokine network. J. Gerontol.

A: B

–B

Gerli R, Monti D, Bistoni O, Mazzone AM, Cossarizza A, Di Gioacchino

NF-Rs and

D

serum levels in healthy aged people and Mech. Ageing Dev.

:

–

D, Franceschi C and Paganelli R (

) Increased cytokine production mononuclear cells of healthy elderly people. Eur. J. Immunol.

:

–

Zhou T, Edwards CK and Mountz JD (

) Prevention of age-related T apoptosis defect in CD

-fas-transgenic mice. J. Exp. Med.

:

–

Spaulding C, Guo W and Effros RB (

) Resistance to apoptosis in human CD

+Tcellsthat reachreplicativesenescenceaftermultipleroundsofantigen- specific proliferation. Exp. Gerontol.

:

–

Monti D, Salvioli S, Capri M, Malorni W, Straface E, Cossarizza A, Botti B, Piacentin

,Baggi

,Barb

,Valensi

,Bonafe` Man

rancesch

(

) Decreased susceptibility to oxidative stress-induced apoptosis of peripheral blood mononuclear cells from healthy elderly and centenarians. Mech. Ageing Dev.

:

–

Herndo

J,Hs

Can

ount

D(

)Increasedapoptosiso

D

RO-T cells with aging. Mech. Ageing Dev.

:

–

Aggarwal S, Gollapudi S and Gupta S (

) Increased TNF-alpha-induced apoptosis in lymphocytes from aged humans: changes in TNF-alpha receptor expression and activation of caspases. J. Immunol.

:

–

Motta L, Receputo G and Franceschi C for the Italian Multicentric Study on Centenarians. (

) I centenari in Italia: aspetti biologici e clinico- epidemiologici.In:Attide

ongressodell

ocieta` Italianad

edicin

nterna. Vol.

, pp

–

(Roma: L. Pozzi Publ.).