Self-rated health in individuals with and without disease is associated with multiple biomarkers representing multiple biological domains.

Self-rated health (SRH) is one of the most frequently used indicators in health and social research. Its robust association with mortality in very different populations implies that it is a comprehensive measure of health status and may even reflect the condition of the human organism beyond clinical diagnoses. Yet the biological basis of SRH is poorly understood. We used data from three independent European population samples (N approx. 15,000) to investigate the associations of SRH with 150 biomolecules in blood or urine (biomarkers). Altogether 57 biomarkers representing different organ systems were associated with SRH. In almost half of the cases the association was independent of disease and physical functioning. Biomarkers weakened but did not remove the association between SRH and mortality. We propose three potential pathways through which biomarkers may be incorporated into an individual's subjective health assessment, including (1) their role in clinical diseases; (2) their association with health-related lifestyles; and (3) their potential to stimulate physical sensations through interoceptive mechanisms. Our findings indicate that SRH has a solid biological basis and it is a valid but non-specific indicator of the biological condition of the human organism.

Diabetes mellitus in the extreme longevity.

Epidemiological studies have revealed a progressive increase in the prevalence of diabetes mellitus in the elderly. Numerous factors are responsible for this trend, among them there are (a) the long-lasting disease due the improved therapeutic remedial (pharmacological, dietary treatments and physical activity), (b) the increased life span expectancy. The prevalence of diabetes mellitus in long living subjects is lower than in elderly people (subjects aged from 65 to 84). Senile diabetes is prevalent in long living people, and usually begins after 90 years. The incidence of neodiagnosed diabetes is higher in the oldest old than in the elderly people. Based on the results, diabetes mellitus is a negative factor for survival, and does not usually allow to achieve very old age, i.e. centenarian.

Exposure to 900 MHz radiofrequency radiation induces caspase 3 activation in proliferating human lymphocytes.

In this study, the induction of apoptosis after exposure to 900 MHz radiofrequency radiation (GSM signal) was investigated by assessing caspase 3 activation in exponentially growing Jurkat cells and in quiescent and proliferating human peripheral blood lymphocytes (PBLs). The exposure was carried out at an average specific absorption rate of 1.35 W/kg in a dual wire patch cell exposure system where the temperature of cell cultures was accurately controlled. After 1 h exposure to the radiofrequency field, a slight but statistically significant increase in caspase 3 activity, measured 6 h after exposure, was observed in Jurkat cells (32.4%) and in proliferating human PBLs (22%). In contrast, no effect was detected in quiescent human PBLs. In the same experimental conditions, apoptosis was also evaluated in Jurkat cells by Western blot analysis and in both cell types by flow cytometry. To evaluate late effects due to caspase 3 activity, flow cytometry was also employed to assess apoptosis and viability 24 h after radiofrequency-radiation exposure in both cell types. Neither the former nor the latter was affected. Since in recent years it has been reported that caspases are also involved in processes other than apoptosis, additional cell cycle studies were carried out on proliferating T cells exposed to radiofrequency radiation; however, we found no differences between sham-exposed and exposed cultures. Further studies are warranted to investigate the biological significance of our findings of a dose-response increase in caspase 3 activity after exposure to radiofrequency radiation.

Immunosenescence and immunogenetics of human longevity.

At present, individuals can live up to 80-120 years, a time much longer than that of our ancestors, as a consequence of the improvements in life conditions and medical care. Thus, the human immune system has to cope with a lifelong and evolutionarily unpredicted exposure to a variety of antigens, which are at the basis of profound age-related changes globally indicated as immunosenescence, a multifaceted phenomenon that increases morbidity and mortality due to infections and age-related pathologies. The major changes occurring during immunosenescence are the result of the accumulation of cellular, molecular defects and involutive phenomena (such as thymic involution) occurring concomitantly to a hyperstimulation of both innate and adaptive immunity (accumulation of expanded clones of memory and effector T cells, shrinkage of the T cell receptor repertoire, progressive activation of macrophages), and resulting in a low-grade, chronic state of inflammation defined as inflammaging. It is unknown whether inflammaging, which represents a risk factor for most age-related pathologies, is a cause or rather an effect of the aging process. In this complex scenario, the role of genetic background likely represents a fundamental variable to attain successful aging and longevity. Accordingly, centenarians seem to be equipped with gene variants that allow them to optimize the balance between pro- and anti-inflammatory molecules, and thus to minimize the effects of the lifelong exposure to environmental insults and stressors. The remarkable features of the genetics of aging and longevity are reviewed, stressing the unexpected and unusual results obtained regarding such a postreproductive type of genetics.

Age-dependent modifications of Type 1 and Type 2 cytokines within virgin and memory CD4+ T cells in humans.

Several alterations in immune function and a concomitant progressive increase in pro-inflammatory status are the major characteristics of ageing process. Cytokines play a key role during ageing acting both in regulatory communication among cells and in effector activity during an immune response. The impact of age on intracellular Type 1 (IFN-gamma and TNF-alpha) and Type 2 (IL-4) cytokines, after stimulation with PMA/ionomycin, was determined in three CD4+ T subsets, i.e. CD95- CD28+ (virgin), CD95+ CD28+ (activated/memory), and CD95+ CD28- (effector/memory) from 47 subjects aged between 21 and 99 years. The percentage of IFN-gamma positive cells significantly decreased in virgin CD4+ subset both in old and nonagenarian subjects, as well as in activated/memory T cells from old in comparison with young subjects. The percentage of TNF-alpha positive cells significantly decreased in activated/memory CD4+ subset from old people. Regarding Type 2 cytokines, IL-4 positive cells significantly increased in activated/memory CD4+ subset from nonagenarians. On the whole our data indicate that: (1) different Type 1 and Type 2 cytokine-positive CD4+ T subsets are differently affected by ageing process; (2) activated/memory T cells appear to be the most affected subset; (3) a shift towards an increased role of Type 2 cytokines and a diminished role of Type 1 cytokines emerges with ageing.

Inflamm-aging, cytokines and aging: state of the art, new hypotheses on the role of mitochondria and new perspectives from systems biology.

In this article we summarise present knowledge on the role of pro-inflammatory cytokines on chronic inflammation leading to organismal aging, a phenomenon we proposed to call "inflamm-aging". In particular, we review genetic data regarding polymorphisms of genes encoding for cytokines and proteins involved in natural immunity (such as Toll-like Receptors and Heat Shock Proteins) obtained from large population studies including young, old and very old people in good health status or affected by age-related diseases such as Alzheimer's Disease and Type II Diabetes. On the whole, despite some controversial results, the available data are in favour of the hypothesis that pro-inflammatory cytokines play an important role in aging and longevity. Further, we present a possible hypothesis to reconcile energetic dysfunction, including mitochondria, and inflamm-aging. New perspectives for future studies, including phylogenetic studies in animal models and in silico studies on mathematical and bioinformatic models inspired by the systems biology approach, are also proposed.

Genes, ageing and longevity in humans: problems, advantages and perspectives.

Many epidemiological data indicate the presence of a strong familial component of longevity that is largely determined by genetics, and a number of possible associations between longevity and allelic variants of genes have been described. A breakthrough strategy to get insight into the genetics of longevity is the study of centenarians, the best example of successful ageing. We review the main results regarding nuclear genes as well as the mitochondrial genome, focusing on the investigations performed on Italian centenarians, compared to those from other countries. These studies produced interesting results on many putative "longevity genes". Nevertheless, many discrepancies are reported, likely due to the population-specific interactions between gene pools and environment. New approaches, including large-scale studies using high-throughput techniques, are urgently needed to overcome the limits of traditional association studies performed on a limited number of polymorphisms in order to make substantial progress to disentangle the genetics of a trait as complex as human longevity.

The different apoptotic potential of the p53 codon 72 alleles increases with age and modulates in vivo ischaemia-induced cell death.

A common arginine to proline polymorphism is harboured at codon 72 of the human p53 gene. In this investigation, we found that fibroblasts and lymphocytes isolated from arginine allele homozygote centenarians and sexagenarians (Arg+) undergo an oxidative-stress-induced apoptosis at a higher extent than cells obtained from proline allele carriers (Pro+). At variance, the difference in apoptosis susceptibility between Arg+ and Pro+ is not significant when cells from 30-year-old people are studied. Further, we found that Arg+ and Pro+ cells from centenarians differ in the constitutive levels of p53 protein and p53/MDM2 complex, as well as in the levels of oxidative stress-induced p53/Bcl-xL complex and mitochondria-localised p53. Consistently, all these differences are less evident in cells from 30-year-old people. Finally, we investigated the in vivo functional relevance of the p53 codon 72 genotype in a group of old patients (66-99 years of age) affected by acute myocardial ischaemia, a clinical condition in which in vivo cell death occurs. We found that Arg+ patients show increased levels of Troponin I and CK-MB, two serum markers that correlate with the extent of the ischaemic damage in comparison to Pro+ patients. In conclusion, these data suggest that p53 codon 72 polymorphism contributes to a genetically determined variability in apoptotic susceptibility among old people, which has a potentially relevant role in the context of an age-related pathologic condition, such as myocardial ischaemia.

Implication of a 5' coding sequence in targeting maternal mRNA to the Drosophila oocyte.

Early accumulation of maternal mRNA in one of the cells of the cluster of 16 cystocytes is a critical event in the determination of the Drosophila oocyte. A number of developmentally important mRNAs have been shown to accumulate in the early oocyte. We report here the early expression of the yemanuclein-alpha (yem-alpha) transcript, its accumulation in the germarial oocyte and its dynamic localization in the growing oocyte. We have investigated the mechanisms involved in these processes. Microtubules are likely to be involved in both transport and localization as was shown for other maternal transcripts which behave similarly. However, unlike all the cases reported so far, transport and localization are not dependent on 3'UTR sequences. We show that the 5' coding sequence is necessary for the early accumulation of yem-alpha RNA in the oocyte and for its localization pattern during oogenesis.

The yemanuclein-alpha: a new Drosophila DNA binding protein specific for the oocyte nucleus.

The Drosophila yG 4.5 gene (now called yemanuclein-alpha gene), which maps at 98F, is a member of the yema gene cluster isolated in a search for differentially expressed maternal genes. The yemanuclein-alpha transcript (formerly yT 4.5) is specifically expressed in the female germ cells at early oogenic stages and displays a graded distribution along the antero-posterior axis of the oocyte. These provocative features are reminiscent of that of K10, bicoid and Bicaudal-D gene transcripts and lead us to hypothesize that the yemanuclein-alpha gene plays a key role in egg organization. We show in the present work that the yemanuclein-alpha is a nuclear protein highly specific for the oocyte nucleus. The sequence analysis of the 5696 bp EcoRI fragment containing the yemanuclein-alpha gene, and of 5 overlapping cDNAs, reveals a 3006 nucleotides long open reading frame (ORF) flanked by long untranslated 5' and 3' sequences. This ORF predicts a 109,215 kDa protein which is basic (pHi: 8.57), and serine rich (12.08%). It contains a 40 amino acid acidic domain in the first third of the protein with a potential alpha-helix organization; this domain has some similarity with the nucleolin acidic domain. Parts of the yemanuclein-alpha sequence are likely to form secondary structures known to interact with DNA. We demonstrate the DNA binding activity of the yemanuclein-alpha by affinity chromatography experiments. Our data indicate that the yemanuclein-alpha shares some of the features which are characteristic of genuine transcriptional activators.

Non-perturbative treatment of the linear covariant gauges by taking into account the Gribov copies.

In this paper, a proposal for the restriction of the Euclidean functional integral to a region free from infinitesimal Gribov copies in linear covariant gauges is discussed. An effective action, akin to the Gribov-Zwanziger action of the Landau gauge, is obtained which implements the aforementioned restriction. Although originally non-local, this action can be cast in local form by introducing auxiliary fields. As in the case of the Landau gauge, dimension two condensates are generated at the quantum level, giving rise to a refinement of the action which is employed to obtain the tree-level gluon propagator in linear covariant gauges. A comparison of our results with those available from numerical lattice simulations is also provided.

Age-dependent remodeling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age.

Structural and phenotypic modifications of rat thymocytes from birth up to one year of age, i.e. during maturation and at the beginning of the involutive process of the thymus are described. Since the biological significance and the mechanisms of thymic involution are still a matter of debate, this study aims at clarifying the complexity of the compensatory events occurring during this relatively neglected period of time. Thymuses from Sprague-Dawley rats were analyzed morphologically and morphometrically by light and electron microscopy. At the same time, thymocyte subsets, isolated from the same animals, were characterized by flow cytometry according to physical parameters and phenotypic markers. Results indicate that major changes occur during the first month from birth and from six months onward. In particular, already during the first weeks after birth, thymocytes undergo a slight reduction of mitoses associated with an increased number of apoptoses. Moreover, during the same period of time, flow cytometry revealed an expansion of small thymocytes and changes in thymocyte subsets such as increase of CD4+CD8+ and CD5+alpha(beta)TCR- and a decrease of CD4-CD8-, CD4-CD8+ cells. The thymus of adult rats was characterized by time-dependent decrease of both mitoses and apoptoses, progressive physical disconnection among cells, increase of necrotic areas and fibrosis. Around one year of age tissue changes were associated with a dramatic reduction of the population of large thymocytes and the rise of numerous small thymocytes that were unexpectedly negative for all tested markers. By contrast, medium-size thymocytes exhibited a marked decrease of CD4+CD8+ and CD5+alpha(beta)TCR- subsets. In conclusion, our data indicate that thymus undergoes, with time, a complex remodeling and suggest that thymic involution is not only a simple shrinkage of the organ but rather the result of a series of compensatory mechanisms among different cell populations in a setting of progressive involution.

Earthworm coelomocytes in vitro: cellular features and "granuloma" formation during cytotoxic activity against the mammalian tumor cell target K562.

Earthworms possess specific, adaptive, cellular immunodefense as well as non-specific responses found in other complex metazoans. Here we characterized coelomocytes from the earthworm Eisenia foetida by electron microscopy and cytofluorimetric analyses, and investigated structural changes that occur when effector coelomocytes and target K562 erythromyeloid human tumor cells interact during cytotoxic activity. In in vitro cultures 1) the two earthworm cell types (i.e. small and large coelomocytes) retained their morphological features; 2) their DNA content was significantly less than that of human lymphocytes and the erythromyeloid human tumor cell line K562; 3) significant percentages of coelomocytes were found to be in S or G2/M phases of the cell cycle. When cultivated alone for up to 3 h, coelomocytes formed no aggregates. However, when mixed with K562, coelomocytes spontaneously killed tumor cells, and cytotoxic reactivity was accompanied by the formation of multiple aggregates similar to granulomas. These results are the first to describe this type of earthworm non-specific "inflammatory" response in vitro against tumor cells.

Mitochondria, aging and longevity--a new perspective.

A new perspective is emerging indicating that mitochondria play a critical role in aging not only because they are the major source and the most proximal target of reactive oxygen species, but also because they regulate stress response and apoptosis. Recent literature indicates that, in response to stress, a variety of molecules translocate to and localise in mitochondria. These molecules are likely to interact with each other, in order to mediate mitochondria/nucleus cross-talk and to regulate apoptosis. We surmise that an integration of signals in multimolecular complexes occurs at mitochondrial level. These phenomena can be of critical importance for human aging and longevity.

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

The susceptibility to undergo apoptosis of fresh human peripheral blood mononuclear cells (PBMCs) from three groups of healthy donors of different ages: young people (19-40 years), old people (65-85 years) and centenarians was assessed. Apoptosis was induced by 2-deoxy-D-ribose (dRib), an agent which induces apoptosis in quiescent PBMCs by interfering with cell redox status and mitochondrial membrane potential (MMP). Our major finding is that an inverse correlation emerged between the age of the donors and the propensity of their PBMCs to undergo dRib-induced apoptosis. PBMCs from old people and centenarians also showed an increased resistance to dRib-induced glutathione depletion and a decreased tendency to lose MMP. The anti-apoptotic molecule Bcl-2 was similarly expressed in PBMCs from the three age groups. Moreover, the plasma level of the stable product of transglutaminase, epsilon(gamma-glutamyl)lysine isodipeptide, a marker of total body apoptotic rate, was decreased in centenarians compared to young and elderly people. On the whole, these findings suggest that physiological aging is characterised by a decreased tendency to undergo apoptosis, a phenomenon likely resulting from adaptation to lifelong exposure to damaging agents, such as reactive oxygen species, and may contribute to one of the major phenomena of immunosenescence, i.e. the progressive accumulation of memory/effector T cells.

Deoxyribonucleic acid of Cancer pagurus. IV. Elution behaviour on hydroxyapatite chromatographic column.

Fractionation of native DNA on hydroxyapatite columns depends, when flat and continuous gradients are used, on the base composition, GC-rich fractions being eluted in the first fractions. Crab satellite DNA behaves abnormally : the first eluted fractions are enriched in poly d(A-T).d(A-T) instead of GC as usual. It amy be suggested that these differences in the behaviour could be attributed to the fact that the secondary structure of crab DNA satellite is different from the secondary structure of the main DNA component.

Detection of the receptor for the human urokinase-type plasminogen activator using fluoresceinated uPA.

The urokinase-type plasminogen activator (uPA) is a serine protease that plays a crucial role in blood coagulation and in tumor invasion and metastasis. uPA is a relatively large polypeptide and binds the uPA receptor (uPAR) with high affinity and specificity. Therefore, it was a good candidate for direct labeling with a fluorochrome for detection of the uPAR. We have produced a fluorescein (FITC)-labeled human uPA using a conjugation procedure that did not significantly alter its binding characteristics to the uPAR. Thirty nM FITC-uPA efficiently stains 2 x 10(5) uPAR-transfected mouse cells in suspension, as determined by flow cytometric analysis. One microgram of FITC-uPA efficiently stains 2 x 10(5) uPAR transfectants grown on slides and analyzed by fluorescence optical microscopy. Human cell lines expressing the endogenous uPAR were stained with similar efficiency. Fixation in paraformaldehyde only slightly reduced the efficiency of staining of both transfectants and cell lines. These characteristics allow the use of FITC-uPA in both static and dynamic morphological studies of uPAR-expressing cells.

High-efficiency expression gene cloning by flow cytometry.

Our goal was to develop a convenient and widely applicable procedure for gene cloning based on flow cytometry. To this purpose, we have developed an efficient protocol for DNA transfection and selection of rare transfectants. Transfection by calcium phosphate co-precipitation was extensively investigated. The use of specific batches of calcium chloride, of carrier DNA purified in guanidinium thiocyanate, and of plasmid DNA banded in cesium chloride proved crucial for high efficiency of transfection. Several tissue culture parameters were also found critical. With the optimized procedure we can transfect almost 100% of the COS-7 cells with cDNA encoding cell surface antigens or green fluorescent protein. Moreover, we routinely obtain high average levels of expression. Efficient cell sorting in flow cytometry was achieved by subtracting the cell autofluorescence background, by displacing stained cells in the red dimension, and by combining fluorescein-conjugated primary and secondary antibodies. Efficient recovery of the transfected DNA constructs was obtained from 2500-3000 cells directly sorted in Hirt lysis buffer. Using the above protocol we have cloned by expression the gene encoding Trop-2, a cell surface glycoprotein expressed by human carcinomas.

A cytofluorimetric study of T lymphocyte subsets in rat lymphoid tissues (thymus, lymph nodes) and peripheral blood: a continuous remodelling during the first year of life.

We previously demonstrated that the rat thymus undergoes a progressive remodelling long before the appearance of typical signs of involution [Quaglino, D., Capri, M., Bergamini, G., Euclidi, E., Zecca, L., Franceschi, C., Pasquali Ronchetti, I., 1998. Age-dependent remodelling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age. Eur. J. Cell. Biol. 76, 156-166]. To focus better on the complex remodelling that occurs in the rat immune system during the first year of life, we analysed the phenotype profile of thymocytes, and T lymphocytes from mesenteric lymph nodes and peripheral blood of the same animals by flow cytometry. Two experimental sets were performed simultaneously using the same animal strain, but starting and ending the study at different ages (15 days up to 300 days in the first experimental set, and 90 days up to 360 days of life in the second). In the rat these ages appear to be crucial not only for developmental, maturative and early involutional processes of the thymus, but also of the entire immune system. The main findings were the following: (1) in the thymus, CD8(-)CD4(-) cells increased, CD5(+)alphabeta TCR(-) and CD8(+)CD4(+) thymocytes decreased, while the most mature cell subset appeared well preserved with ageing; (2) in the lymph nodes, T helper and T cytotoxic lymphocytes decreased in the most aged animals. Memory/activated CD4(+)CD45RC(-) T cells decreased, while naive/resting CD4(+)CD45RC(+) cells increased in the youngest animals and decreased in the oldest. CD8(+)CD45RC(-) and CD8(+)CD45RC(+) lymphocytes showed a complex age-dependent trend, and (3) in peripheral blood, minor modifications were evident, such as an age-dependent increase in the alphabeta TCR(+)CD25(+) cell subset. Some of these changes were related to the developmental process, while others could likely be interpreted as early signs of immunosenescence. The role of these modifications in immune system is discussed within the framework of the remodelling hypothesis of immunosenescence. The age-dependent changes in these three lymphoid compartments, however, appear to be different and only partially overlapping, thus suggesting that the maturational, developmental and ageing processes have distinct characteristics in the central and peripheral lymphoid organs.

Anti-beta 2 glycoprotein I antibodies in centenarians.

BACKGROUND: Non-organ-specific autoantibodies are present in centenarians without evidence of autoimmune diseases but conflicting or no data on anti-phospholipid and anti-phospholipid binding proteins were reported. OBJECTIVE: To investigate the presence and antigen specificity of anti-phospholipid and anti-phospholipid binding proteins in centenarians. METHODS: Seventy-seven centenarians, 70 adult controls, 65 unselected elderly subjects, and 38 old SENIEUR volunteers were investigated. Anti-cardiolipin, anti-human beta 2 glycoprotein I, and lupus anticoagulant were detected. Antigen specificity was assayed against plates coated with anionic, neutral and cationic phospholipids and beta 2 glycoprotein I-dependence was also evaluated. RESULTS: 54.3% of the centenarians were positive for IgG and 8.6% for IgM anti-beta 2 glycoprotein I antibodies, while only 20.7% centenarians were positive for anti-cardiolipin IgG and 2.59% for IgM; none resulted positive for lupus anticoagulant. Anti-cardiolipin positive sera cross-reacted with negatively charged phospholipids and displayed decreased binding to serum-free cardiolipin-coated plates that was restored by human beta 2 glycoprotein I or fetal calf serum. CONCLUSIONS: Centenarians display high reactivity against human beta 2 glycoprotein I but low binding to the bovine molecule in the anti-cardiolipin assay. In spite of the presence of antibodies comparable to those found in patients with the anti-phospholipid syndrome, no vascular events were reported suggesting the presence of unknown protective factors and/or the lack of triggering factors.