Cell‐free DNA (cfDNA) is present in the circulating plasma and other body fluids and is known to originate mainly from apoptotic cells. Here, we provide the first in vivo evidence of global and local chromatin changes in human aging by analyzing cfDNA from the blood of individuals of different age groups. Our results show that nucleosome signals inferred from cfDNA are consistent with the redistribution of heterochromatin observed in cellular senescence and aging in other model systems. In addition, we detected a relative cfDNA loss at several genomic locations, such as transcription start and termination sites, 5′UTR of L1HS retrotransposons and dimeric AluY elements with age. Our results also revealed age and deteriorating health status correlate with increased enrichment of signals from cells in different tissues. In conclusion, our results show that the sequencing of circulating cfDNA from human blood plasma can be used as a noninvasive methodology to study age‐associated changes to the epigenome in vivo.

Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper-resistance gene CUP1. Transcription of the tandemly repeated CUP1 gene causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2, Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant, we find that CUP1 eccDNA does not replicate efficiently, and high-copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.

Six bladder cancer patients received intralesional injections by needle, under cystoscopic control, of 1969-4046 units (U) of xenogeneic IL-2 of high biological activity (324 U/ml; 397 micrograms/ml protein; 1.22 protein/U ratio). The treatment was spread over 7-54 days and 0.5 ml was injected each time. In 3/6 patients complete tumour regression was seen 43, 60 and 105 respectively days after the first IL-2 injection. In 2 a 70% regression was observed at days 45 and 75. In the last patient massive necrosis throughout the tumour mass was recorded on day 25 at radical cystectomy. In order to evaluate the minimum IL-2 U required to obtain positive clinical results and/or to assess whether the anti-tumour effect observed could be ascribed to the foreign protein of bovine origin contained in our IL-2 preparation, 4 additional bladder cancer patients were treated in 7-14 days with 156-1404 U of a second IL-2 lot with a much lower biological activity and similar protein content (52 U/ml; 289 micrograms/ml of protein; 5.55 protein/U ratio). No clinical or histological improvement was noted over a 42- to 54-day observation period. When we evaluated the 2 groups of patients by Student's t-test for both total U injected and U/kg of body weight (bw) we found a statistically significant differences (0.0025 less than p less than 0.0005 and p less than 0.0005, respectively). In contrast, no difference was seen for the injected protein amounts. The reported observations are in favour of a dose-dependent anti-tumour action mediated by IL-2 instead of foreign proteins. In none of the patients treated were any early or late adverse clinical side effects observed. Immunological monitoring (E, EAC, E-active rosettes, mitogen lymphocyte stimulations and leukocyte migration inhibition in the presence of allogeneic bladder cancer cells) performed on the peripheral blood (PB) showed significant but contrasting modifications after IL-2 injection. There was no clear correlation with the clinical course. The patients in whom we observed complete regression are still tumour free after 2, 4 and 7 months. In addition, in all the patients of the first group we observed an increase in tumour lymphoid infiltrate after IL-2 injection and in 2 patients lymphoid pseudo-follicles were also noted. In 2 of these patients we also observed scar-like areas in the place of the tumours previously seen.

SummaryTo understand why livers from aged donors are successfully used for transplants, we looked for markers of liver aging in 71 biopsies from donors aged 12–92 years before transplants and in 11 biopsies after transplants with high donor–recipient age‐mismatch. We also assessed liver function in 36 age‐mismatched recipients. The major findings were the following: (i) miR‐31‐5p, miR‐141‐3p, and miR‐200c‐3p increased with age, as assessed by microRNAs (miRs) and mRNA transcript profiling in 12 biopsies and results were validated by RT–qPCR in a total of 58 biopsies; (ii) telomere length measured by qPCR in 45 samples showed a significant age‐dependent shortage; (iii) a bioinformatic approach combining transcriptome and miRs data identified putative miRs targets, the most informative being GLT1, a glutamate transporter expressed in hepatocytes. GLT1 was demonstrated by luciferase assay to be a target of miR‐31‐5p and miR‐200c‐3p, and both its mRNA (RT–qPCR) and protein (immunohistochemistry) significantly decreased with age in liver biopsies and in hepatic centrilobular zone, respectively; (iv) miR‐31‐5p, miR‐141‐3p and miR‐200c‐3p expression was significantly affected by recipient age (older environment) as assessed in eleven cases of donor–recipient extreme age‐mismatch; (v) the analysis of recipients plasma by N‐glycans profiling, capable of assessing liver functions and biological age, showed that liver function recovered after transplants, independently of age‐mismatch, and recipients apparently ‘rejuvenated’ according to their glycomic age. In conclusion, we identified new markers of aging in human liver, their relevance in donor–recipient age‐mismatches in transplantation, and offered positive evidence for the use of organs from old donors.