Biomarkers for vascular ageing in aorta tissues and blood samples.

OBJECTIVES: Functional and quantitative alterations and senescence of circulating and expanded endothelial progenitor cells (EPC), as well as systemic and tissue modifications of angiogenetic and inflammatory molecules, were evaluated for predicting age-related vessel wall remodeling, correlating them to intima media thickness (IMT) in the common carotid artery (CCA), a biomarker of early cardiovascular disease and aortic root dilation. POPULATIONS AND METHODS: A homogenous Caucasian population was included in the study, constituted by 160 healthy subjects (80 old subjects, mean age 72 ±â€¯6.4, range 66-83 years; and 80 younger blood donors, mean age 26.2 ±â€¯3.4, range 21-33 years), and 60 old subjects (mean age 73 ±â€¯1.4 (range 66-83) years) with aortic root dilatation and hypertension, and 60 old people (70 ±â€¯2.8 (age range 66-83)) with sporadic ascending aorta aneurysm (AAA). In addition, 20 control individuals (10 men and 10 women, mean age: 65 ±â€¯8), were also included in the study for evaluating the gene expression's levels, in aorta tissues. Appropriate techniques, practises, protocols, gating strategies and statistical analyses were performed in our evaluations. RESULTS: Interestingly, old people had a significantly reduced functionality and a high grade of senescence (high SA-ß-Gal activity and high levels of TP53, p21 and p16 genes) of EPC expanded than younger subjects. The values of related parameters progressively augmented from the old subjects, in good healthy shape, to subjects with hypertension and aorta dilation, and AAA. Moreover, they significantly impacted the endothelium than the alterations in EPC number. No changes, but rather increased systemic levels of VEGF and SDF-1 were also assessed in old people vs. younger donors. Old people also showed significantly increased systemic levels of inflammatory cytokines, and a reciprocal significant reduction of systemic s-Notch 1 than younger subjects. These parameters, also including the number EPC alterations, resulted to be significantly sustained in old people bearers of an inflammatory combined genotype. Consistent with these data, a reduced expression of Notch-1 gene, accompanied by a sustained expression of inflammatory genes (i.e. TLR4, IL-1ß, IL-6 and IL-17) were detected in aortic tissues from old control people and AAA cases. Finally, we detected the biological effects induced by all the detected alterations on vessel wall age-related remodeling, by evaluating the IMT in the population studied and correlating it to these alterations. The analysis demonstrated that the unique independent risk predictors for vascular ageing are age, the EPC reduced migratory activity and senescence, high grade of expression of genes inducing EPC senescence and chronic tissue and systemic inflammation. CONCLUSIONS: Thus, we propose these parameters, of easy determination in biological samples (i.e. blood and tissue samples) from alive human population, as optimal biomarkers for vascular ageing.

A Typical Immune T/B Subset Profile Characterizes Bicuspid Aortic Valve: In an Old Status?

Bicuspid valve disease is associated with the development of thoracic aortic aneurysm. The molecular mechanisms underlying this association still need to be clarified. Here, we evaluated the circulating levels of T and B lymphocyte subsets associated with the development of vascular diseases in patients with bicuspid aortic valve or tricuspid aortic valve with and without thoracic aortic aneurysm. We unveiled that the circulating levels of the MAIT, CD4+IL-17A+, and NKT T cell subsets were significantly reduced in bicuspid valve disease cases, when compared to tricuspid aortic valve cases in either the presence or the absence of thoracic aortic aneurysm. Among patients with tricuspid aortic valve, these cells were higher in those also affected by thoracic aortic aneurysm. Similar data were obtained by examining CD19+ B cells, naïve B cells (IgD+CD27-), memory unswitched B cells (IgD+CD27+), memory switched B cells (IgD-CD27+), and double-negative B cells (DN) (IgD-CD27-). These cells resulted to be lower in subjects with bicuspid valve disease with respect to patients with tricuspid aortic valve. In whole, our data indicate that patients with bicuspid valve disease show a quantitative reduction of T and B lymphocyte cell subsets. Future studies are encouraged to understand the molecular mechanisms underlying this observation and its pathophysiological significance.

Bone marrow B lymphocytes in multiple myeloma and MGUS: Focus on distribution of naïve cells and memory subsets.

BACKGROUND/AIMS: Multiple myeloma (MM) is caused by proliferation of clonal plasma cells (cPCs) in bone marrow (BM), associated with numerical and functional defects in immune subsets. An impairment of B cell compartment is involved in onset/progression of the disease. METHODS: By flow cytometry, we studied distribution of naïve/transitional (IgD(+)CD27(-)), memory unswitched (IgD(+)CD27(+)), memory switched (IgD(-)CD27(+)) and double negative (DN) (IgD(-)CD27(-)) B lymphocytes in BM of control subjects, and responding and relapsing patients. RESULTS: We observed an increased percentage of IgD(+)CD27(+) B cells in healthy controls vs responding patients (p<0.05). Treated non complete responders exhibited an expanded DN compartment vs stringent complete responders (p=0.011); in turn IgD(+)CD27(-) subpopulation was larger in stringent complete responders vs other responding patients (p=0.006). None of the studied B cell subsets showed clonal restriction. Correlation analysis revealed negative correlations between naïve/transitional and DN B cells in all groups, except in newly diagnosed subjects. CONCLUSIONS: This may be considered a feasible start point to explore the importance of B cells in the immunosuppressive MM BM microenvironment, correlating these findings with immunosenescence and therapy related increased risk of infection. Moreover, we propose a possible role of naïve/transitional and DN B cells as predictive markers in treated patients.

Are Endothelial Progenitor Cells the Real Solution for Cardiovascular Diseases? Focus on Controversies and Perspectives.

Advanced knowledge in the field of stem cell biology and their ability to provide a cue for counteracting several diseases are leading numerous researchers to focus their attention on "regenerative medicine" as possible solutions for cardiovascular diseases (CVDs). However, the lack of consistent evidence in this arena has hampered the clinical application. The same condition affects the research on endothelial progenitor cells (EPCs), creating more confusion than comprehension. In this review, this aspect is discussed with particular emphasis. In particular, we describe biology and physiology of EPCs, outline their clinical relevance as both new predictive, diagnostic, and prognostic CVD biomarkers and therapeutic agents, discuss advantages, disadvantages, and conflicting data about their use as possible solutions for vascular impairment and clinical applications, and finally underline a very crucial aspect of EPCs "characterization and definition," which seems to be the real cause of large heterogeneity existing in literature data on this topic.

Double negative (IgG+IgD-CD27-) B cells are increased in a cohort of moderate-severe Alzheimer's disease patients and show a pro-inflammatory trafficking receptor phenotype.

Alzheimer's disease (AD) is a progressive, irreversible, and debilitating disease for which no effective preventive or disease modifying therapies or treatments have so far been detected. The crucial step in AD pathogenesis is the production of amyloid-ß42 peptide, which causes chronic inflammation. Activated cells in the central nervous system (CNS) produce pro-inflammatory mediators that lead to the recruitment of myeloid or lymphocytic cells. As a consequence, the communication between the CNS and peripheral blood of AD subjects could influence the lymphocyte distribution and/or the expression of phenotypic markers. In the present paper, we show a significant decrease in total CD19+ B lymphocytes and a remodeling of the B cell subpopulations in moderate-severe AD patients, compared with their coeval healthy controls and mild AD subjects. In particular, we report a significant reduction in naïve B cells (IgD+CD27-) and a simultaneous increase in double negative (DN, IgD-CD27-) memory B lymphocytes. We have also evaluated the expression of the pro-inflammatory chemokine receptors CCR6 and CCR7 in total and naïve/memory B cells from mild and moderate-severe AD patients, with the aim to detect a possible relationship between the trafficking profile and the stage of the disease. Our results demonstrate that both the amount and the trafficking profile of B cells are related to the severity of AD. The results discussed in this paper suggest a well-selected antibody panel should be used as an additional test for the identification of early AD.

Double negative (CD19+IgG+IgD-CD27-) B lymphocytes: a new insight from telomerase in healthy elderly, in centenarian offspring and in Alzheimer's disease patients.

Immunosenescence is characterized by the impairment of humoral immunity with changes in the memory/naive B cell compartment. In particular we have previously reported the percentage increase of a Memory IgD(-)CD27(-) (Double Negative, DN) B cell population in aged people. In this study, we have further characterized DN B cells with the aim to better understand their contribution to immunosenescence. As DN B cells show a poor ability to proliferate in vitro, we have evaluated the expression of the inhibitory receptors CD307d and CD22 on these cells from young and old individuals. In addition we have evaluated the ability to activate DN B cells by the simultaneous use of innate (CpG) and adaptive (α-Ig/CD40) ligands. Our data demonstrate that the refractoriness to proliferate of DN B cells does not depend on the expression of inhibitory receptors, but it is due to the kind of stimulation. Indeed, when DN B cells are stimulated engaging both BCR and TLR9, they become able to proliferate and reactivate the telomerase enzyme. In the present study, we have also compared the telomerase activity in a group of people genetically advantaged for longevity as centenarian offspring (CO) and in a group of moderate-severe Alzheimer's disease (AD) patients, who represent a model of unsuccessful aging. Our study suggests that telomerase reactivation of DN B cells, as well as their number and ability in activating, depend essentially by the biological age of the subjects studied, so the evaluation of DN B cells might allow to gain insight to healthy and unsuccessful aging.

Trafficking phenotype and production of granzyme B by double negative B cells (IgG(+)IgD(-)CD27(-)) in the elderly.

The impairment of humoral immune response in elderly humans has been extensively demonstrated. We have reported the increase of memory B cells (IgG(+)IgD(-)CD27(-), double negative, DN) population in the elderly, in which there is also a typical inflammatory micro-environment. In order to evaluate whether this pro-inflammatory status could influence the trafficking phenotype of naïve/memory B cells, we have assessed the expression of CCR7, CCR6, CXCR3, CXCR4, CXCR5 and CD62L on naïve/memory B cell subpopulations in young and elderly subjects. Moreover, the combination of pro-inflammatory interleukin-21 (IL-21) and B cell receptor (BCR) stimulation enables B cells to produce and secrete granzyme B (GrB), which plays a critical role in early anti-viral immune responses, in the regulation of autoimmune mechanisms and in cancer immunosurveillance. Our data demonstrate that in the elderly, naïve/memory B cell populations present a different expression of the studied receptors that could be discussed in terms of "inflamm-aging". In particular IgG(+)IgD(-)CD27(-) DN B cells show a tissue trafficking phenotype and they can be stimulated to produce GrB.

Evidence for less marked potential signs of T-cell immunosenescence in centenarian offspring than in the general age-matched population.

People may reach the upper limits of the human life span at least partly because they have maintained more appropriate immune function, avoiding changes to immunity termed "immunosenescence." Exceptionally long-lived people may be enriched for genes that contribute to their longevity, some of which may bear on immune function. Centenarian offspring would be expected to inherit some of these, which might be reflected in their resistance to immunosenescence, and contribute to their potential longevity. We have tested this hypothesis by comparing centenarian offspring with age-matched controls. We report differences in the numbers and proportions of both CD4(+) and CD8(+) early- and late-differentiated T cells, as well as potentially senescent CD8(+) T cells, suggesting that the adaptive T-cell arm of the immune system is more "youthful" in centenarian offspring than controls. This might reflect a superior ability to mount effective responses against newly encountered antigens and thus contribute to better protection against infection and to greater longevity.

Centenarian offspring: a model for understanding longevity.

A main objective of current medical research is to improve the life quality of elderly people as priority of the continuous increase of ageing population. This phenomenon implies several medical, economic and social problems because of dramatic increase in number of non autonomous individuals affected by various pathologies. Accordingly, the research interest is focused on understanding the biological mechanisms involved in determining the positive ageing phenotype, i.e. the centenarian phenotype. In achieving this goal the choice of an appropriate study models is fundamental. Centenarians have been used as an optimal model for successful ageing. However, this model shows several limitations, i.e. the selection of appropriate controls and the use itself of the centenarians as a suitable model for healthy ageing. Thus, the interest has been centered on centenarian offspring, healthy elderly people. They may represent a model for understanding exceptional longevity for the following reasons: they exhibit a protective genetic background, cardiovascular and immunological profile, as well as a reduced rate of cognitive decline than age-matched people without centenarian relatives. Several of these aspects are summarized in this review based on the literature and the results of our studies.

Expression of calpain-calpastatin system (CCS) member proteins in human lymphocytes of young and elderly individuals; pilot baseline data for the CALPACENT project.

BACKGROUND: Ubiquitous system of regulatory, calcium-dependent, cytoplasmic proteases - calpains - and their endogenous inhibitor - calpastatin - is implicated in the proteolytic regulation of activation, proliferation, and apoptosis of many cell types. However, it has not been thoroughly studied in resting and activated human lymphocytes yet, especially in relation to the subjects' ageing process. The CALPACENT project is an international (Polish-Italian) project aiming at verifying the hypothesis of the role of calpains in the function of peripheral blood immune cells of Polish (Pomeranian) and Italian (Sicilian) centenarians, apparently relatively preserved in comparison to the general elderly population. In this preliminary report we aimed at establishing and comparing the baseline levels of expression of µ- and m-calpain and calpastatin in various, phenotypically defined, populations of human peripheral blood lymphocytes for healthy elderly Sicilians and Poles, as compared to these values observed in young cohort. RESULTS: We have found significant differences in the expression of both µ- and m-calpain as well as calpastatin between various populations of peripheral blood lymphocytes (CD4+, CD8+ and CD19+), both between the age groups compared and within them. Interestingly, significantly higher amounts of µ- and m-calpains but not of calpastatin could be demonstrated in the CD4+CD28- and CD8+CD28- lymphocytes of old subjects (but not in the cells of young individuals), as compared to their CD28+ counterparts. Finally, decreased expression of both calpains in the elderly T cells is not related to the accumulation of effector/memory (CD45RO+) cells in the latter, as the expression of both calpains does not differ significantly between the naïve and memory T cells, while is significantly lower for elderly lymphocytes if both populations are taken separately. CONCLUSIONS: Observed differences in the amounts of CCS member proteins between various populations of lymphocytes of young and elderly subjects may participate in the impaired proliferative activity of these cells in the elderly.

A novel B cell population revealed by a CD38/CD24 gating strategy: CD38(-)CD24 (-) B cells in centenarian offspring and elderly people.

The B cell arm of adaptive immunity undergoes significant modifications with age. Elderly people are characterized by impaired B cell responses reflected in a reduced ability to effectively respond against viruses and bacteria. Alterations of immunity with advancing age (immunosenescence) have been widely studied in centenarians who are considered a good example of successful aging. In recent years, attention has shifted to centenarian offspring (CO) as a model of people genetically advantaged for healthy aging and longevity. Here, we describe the preliminary characterization of a proposed new population of memory B cells, defined as CD19(+)CD38(-)CD24(-), which we find at higher frequencies in the elderly but less so in CO than healthy age-matched random controls. In addition, we found a decreased expression of RP105 (CD180), a toll-like receptor-associated molecule, on these cells. CD180 downregulation may potentially be a marker of immunosenescence. Moreover, we show that these CD19(+)CD38(-)CD24(-) B cells produce TNF and hypothesize that their observed expansion in the elderly might contribute to the increased inflammatory status sometimes designated "inflamm-aging."

Genetics of longevity. data from the studies on Sicilian centenarians.

The demographic and social changes of the past decades have determined improvements in public health and longevity. So, the number of centenarians is increasing as a worldwide phenomenon. Scientists have focused their attention on centenarians as optimal model to address the biological mechanisms of "successful and unsuccessful ageing". They are equipped to reach the extreme limits of human life span and, most importantly, to show relatively good health, being able to perform their routine daily life and to escape fatal age-related diseases, such as cardiovascular diseases and cancer. Thus, particular attention has been centered on their genetic background and immune system. In this review, we report our data gathered for over 10 years in Sicilian centenarians. Based on results obtained, we suggest longevity as the result of an optimal performance of immune system and an over-expression of anti-inflammatory sequence variants of immune/inflammatory genes. However, as well known, genetic, epigenetic, stochastic and environmental factors seem to have a crucial role in ageing and longevity. Epigenetics is associated with ageing, as demonstrated in many studies. In particular, ageing is associated with a global loss of methylation state. Thus, the aim of future studies will be to analyze the weight of epigenetic changes in ageing and longevity.

Immunosenescence, inflammation and Alzheimer's disease.

Ageing impacts negatively on the development of the immune system and its ability to fight pathogens. Progressive changes in the T-cell and B-cell systems over the lifespan of individuals have a major impact on the capacity to respond to immune challenges. The cumulative age-associated changes in immune competence are termed immunosenescence that is characterized by changes where adaptive immunity deteriorates, while innate immunity is largely conserved or even upregulated with age. On the other hand, ageing is also characterized by "inflamm-ageing", a term coined to explain the inflammation commonly present in many age-associated diseases. It is believed that immune inflammatory processes are relevant in Alzheimer's disease, the most common cause of dementia in older people. In the present paper we review data focusing on changes of some immunoinflammatory parameters observed in patients affected by Alzheimer's disease.

Immune profiling of Alzheimer patients.

Alzheimer's disease (AD) is characterized by extracellular senile plaques in the brain, containing amyloid-ß peptide (Aß). We identify immunological differences between AD patients and age-matched controls greater than those related to age itself. The biggest differences were in the CD4+ rather than the CD8+ T cell compartment resulting in lower proportions of naïve cells, more late-differentiated cells and higher percentages of activated CD4+CD25+ T cells without a Treg phenotype in AD patients. Changes to CD4+ cells might be the result of chronic stimulation by Aß present in the blood. These findings have implications for diagnosis and understanding the aetiology of the disease.

B cell immunosenescence: different features of naive and memory B cells in elderly.

Elderly people show a reduced protection against new infections and a decreased response to vaccines as a consequence of impairment of both cellular and humoral immunity. In this paper we have studied memory/naïve B cells in the elderly, evaluating surface immunoglobulin expression, production of the pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-10, and presence of somatic hypermutation, focusing on the IgG(+)IgD(-)CD27(-) double negative (DN) B cells that are expanded in the elderly. Our results show that naïve B cells from young donors need a sufficiently strong stimulus to be activated "in vitro", while naïve B cells from old subjects are able to produce IL-10 and TNF-α when stimulated "physiologically" (α-CD40/IL-4), suggesting that these cells might play a role in the control of the immuno-inflammatory environment in the elderly. In addition, in the elderly there is an accumulation of DN B cells with a reduced rate of somatic hypermutation. Thus, DN B lymphocytes may be exhausted cells that are expanded and accumulate as a by-product of persistent stimulation or impaired germinal center formation.

B cells and immunosenescence: a focus on IgG+IgD-CD27- (DN) B cells in aged humans.

Immunosenescence contributes to the decreased ability of the elderly to control infectious diseases, which is also reflected in their generally poor response to new antigens and vaccination. It is known that the T cell branch of the immune system is impaired in the elderly mainly due to expansion of memory/effector cells that renders the immune system less able to respond to new antigens. B lymphocytes are also impaired in the elderly in terms of their response to new antigens. In this paper we review recent work on B cell immunosenescence focusing our attention on memory B cells and a subset of memory B cells (namely IgG(+)IgD(-)CD27(-)) that we have demonstrated is increased in healthy elderly.

Systemic immune responses in Alzheimer's disease: in vitro mononuclear cell activation and cytokine production.

To investigate the systemic signs of immune-inflammatory responses in Alzheimer's disease (AD), in the present study we have analyzed blood lymphocyte subsets and the expression of activation markers on peripheral blood mononuclear cells (PBMCs) from AD patients and age-matched healthy controls (HC) activated in vitro by recombinant amyloid-beta peptide (rAbeta42). Our study of AD lymphocyte subpopulations confirms the already described decrease of the absolute number and percentage of B cells when compared to HC lymphocytes, whereas the other subsets are not significantly different in patients and controls. We report the increased expression of the activation marker CD69 and of the chemokine receptors CCR2 and CCR5 on T cells but no changes of CD25 after activation. B cells are also activated by rAbeta42 as demonstrated by the enhanced expression of CCR5. Moreover, rAbeta42 induces an increased expression of the scavenger receptor CD36 on monocytes. Some activation markers and chemokine receptors are overexpressed in unstimulated AD cells when compared to controls. This is evidence of the pro-inflammatory status of AD. Stimulation by rAbeta42 also induces the production of the pro-inflammatory cytokines IL-1beta, IL-6, IFN-gamma, and TNF-alpha, and of the anti-inflammatory cytokines IL-10 and IL-1Ra. The chemokines RANTES, MIP-1beta, and eotaxin as well as some growth factors (GM-CSF, G-CSF) are also overproduced by AD-derived PBMC activated by rAbeta42. These results support the involvement of systemic immunity in AD patients. However, our study is an observational one so we cannot draw a conclusion about its contribution to the pathophysiology of the disease.

Mechanisms of immunosenescence.

On April 7,8, 2009 a Symposium entitled "Pathophysiology of Successful and Unsuccessful Ageing" took place in Palermo, Italy. Here, the lectures of G. Pawelec, D. Dunn-Walters and. G. Colonna-Romano on T and B immunosenescence are summarized. In the elderly, many alterations of both innate and acquired immunity have been described. Alterations to the immune system in the older person are generally viewed as a deterioration of immunity, leading to the use of the catch-all term immunosenescence. Indeed, many immunological parameters are often markedly different in elderly compared to young people, and some, mostly circumstantial, evidence suggests that retained function of both innate and acquired immunity in the elderly is correlated with health status. What is often not clear from studies is how far immune dysfunction is a cause or an effect. A better understanding of immunosenescence and mechanisms responsible for proven deleterious changes is needed to maintain a healthy state in later life and to design possible therapeutic interventions.