CMV-Specific T-cell Responses at Older Ages: Broad Responses With a Large Central Memory Component May Be Key to Long-term Survival.

Cytomegalovirus (CMV) infection sometimes causes large expansions of CMV-specific T cells, particularly in older people. This is believed to undermine immunity to other pathogens and to accelerate immunosenescence. While multiple different CMV proteins are recognized, most publications on age-related T-cell expansions have focused on dominant target proteins UL83 or UL123, and the T-cell activation marker interferon-γ (IFN-γ). We were concerned that this narrow approach might have skewed our understanding of CMV-specific immunity at older ages. We have, therefore, widened the scope of analysis to include in vitro-induced T-cell responses to 19 frequently recognized CMV proteins in "young" and "older" healthy volunteers and a group of "oldest old" long-term survivors (>85 years of age). Polychromatic flow cytometry was used to analyze T-cell activation markers (CD107, CD154, interleukin-2 [IL-2], tumor necrosis factor [TNF], and IFN-γ) and memory phenotypes (CD27, CD45RA). The older group had, on average, larger T-cell responses than the young, but, interestingly, response size differences were relatively smaller when all activation markers were considered rather than IFN-γ or TNF alone. The oldest old group recognized more proteins on average than the other groups, and had even bigger T-cell responses than the older group with a significantly larger central memory CD4 T-cell component.

Functional Diversity of Cytomegalovirus-Specific T Cells Is Maintained in Older People and Significantly Associated With Protein Specificity and Response Size.

BACKGROUND: Parallel upregulation of several T-cell effector functions (ie, polyfunctionality) is believed to be critical for the protection against viruses but thought to decrease in large T-cell expansions, in particular at older ages. The factors determining T-cell polyfunctionality are incompletely understood. Here we revisit the question of cytomegalovirus (CMV)-specific T-cell polyfunctionality, including a wide range of T-cell target proteins, response sizes, and participant ages. METHODS: Polychromatic flow cytometry was used to analyze the functional diversity (ie, CD107, CD154, interleukin 2, tumor necrosis factor, and interferon γ expression) of CD4+ and CD8+ T-cell responses to 19 CMV proteins in a large group of young and older United Kingdom participants. A group of oldest old people (age >85 years) was included to explore these parameters in exceptional survivors. Polyfunctionality was assessed for each protein-specific response subset, by subset and in aggregate, across all proteins by using the novel polyfunctionality index. RESULTS: Polyfunctionality was not reduced in healthy older people as compared to young people. However, it was significantly related to target protein specificity. For each protein, it increased with response size. In the oldest old group, overall T-cell polyfunctionality was significantly lower. DISCUSSION: Our results give a new perspective on T-cell polyfunctionality and raise the question of whether maintaining polyfunctionality of CMV-specific T cells at older ages is necessarily beneficial.

Impact of Persistent Cytomegalovirus Infection on Dynamic Changes in Human Immune System Profile.

Human cytomegalovirus (HCMV) imprints the immune system after primary infection, however its effect during chronic infection still needs to be deciphered. In this study we report the variation of blood cell count along with anti-HCMV IgG and T cell responses to pp-65 and IE-1 antigens, that occurred after an interval of five years in a cohort of 25 seropositive healthy adults. We found increased anti-viral IgG antibody responses and intracellular interferon-gamma secreting CD8+ T cell responses to pp-65: a result consistent with memory inflation. With the only exception of shortage in naive CD8+ T cells most memory T cell subsets as well as total CD8+ T cells, T cells, lymphocytes, monocytes and leukocytes had increased. By contrast, none of the cell types tested were found to have increased in 14 subjects stably seronegative. Rather, in addition to a shortage in naive CD8+ T cells, also memory T cell subsets and most other cell types decreased, either in a statistically significant or non-significant manner. The trend of T cell pool representation with regard to CD4/CD8 ratio was in the opposing directions depending on HCMV serology. Globally, this study demonstrates different dynamic changes of most blood cell types depending on presence or absence of HCMV infection. Therefore, HCMV plays a continual role in modulating homeostasis of blood T cells and a broader expanding effect on other cell populations of lymphoid and myeloid origin.

Centenarians' offspring as a model of healthy aging: a reappraisal of the data on Italian subjects and a comprehensive overview.

Within the scenario of an increasing life expectancy worldwide it is mandatory to identify determinants of healthy aging. Centenarian offspring (CO) is one of the most informative model to identify trajectories of healthy aging and their determinants (genetic and environmental), being representative of elderly in their 70th whose lifestyle can be still modified to attain a better health. This study is the first comprehensive investigation of the health status of 267 CO (mean age: 70.2 years) and adopts the innovative approach of comparing CO with 107 age-matched offspring of non-long-lived parents (hereafter indicated as NCO controls), recruited according to strict inclusion demographic criteria of Italian population. We adopted a multidimensional approach which integrates functional and cognitive assessment together with epidemiological and clinical data, including pro- and anti-inflammatory cytokines and adipokines, lipid profile, and insulin resistance. CO have a lower prevalence of stroke, cerebral thrombosis-hemorrhage, hypertension, hypercholesterolemia, and other minor diseases, lower BMI and waist circumference, a better functional and cognitive status and lower plasma level of FT4 compared to NCO controls. We conclude that a multidimensional approach is a reliable strategy to identify the health status of elderly at an age when interventions to modify their health trajectory are feasible.

New advances in CMV and immunosenescence.

Immunosenescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterized by impaired protective immunity and decreased efficacy of vaccines. An increasing number of immunological, clinical and epidemiological studies suggest that persistent Cytomegalovirus (CMV) infection is associated with accelerated aging of the immune system and with several age-related diseases. However, current evidence on whether and how human CMV (HCMV) infection is implicated in immunosenescence and in age-related diseases remains incomplete and many aspects of CMV involvement in immune aging remain controversial. The attendees of the 4th International Workshop on "CMV & Immunosenescence", held in Parma, Italy, 25-27th March, 2013, presented and discussed data related to these open questions, which are reported in this commentary.

Naïve and memory CD8 T cell pool homeostasis in advanced aging: impact of age and of antigen-specific responses to cytomegalovirus.

Alterations in the circulating CD8+ T cell pool, with a loss of naïve and accumulation of effector/effector memory cells, are pronounced in older adults. However, homeostatic forces that dictate such changes remain incompletely understood. This observational cross-sectional study explored the basis for variability of CD8+ T cell number and composition of its main subsets: naïve, central memory and effector memory T cells, in 131 cytomegalovirus (CMV) seropositive subjects aged over 60 years. We found great heterogeneity of CD8+ T cell numbers, which was mainly due to variability of the CD8 + CD28- T cell subset regardless of age. Analysis, by multiple regression, of distinct factors revealed that age was a predictor for the loss in absolute number of naïve T cells, but was not associated with changes in central or effector memory CD8+ T cell subsets. By contrast, the size of CD8+ T cells specific to pp65 and IE-1 antigens of CMV, predicted CD28 - CD8+ T cell, antigen-experienced CD8+ T cell, and even total CD8+ T cell numbers, but not naïve CD8+ T cell loss. These results indicate a clear dichotomy between the homeostasis of naïve and antigen-experienced subsets of CD8+ T cells which are independently affected, in human later life, by age and antigen-specific responses to CMV, respectively.

Cytomegalovirus infection modulates the phenotype and functional profile of the T-cell immune response to mycobacterial antigens in older life.

Infection with Cytomegalovirus is associated with accelerated immunosenescence. Expansions of CMV-specific T cell responses have previously been demonstrated to affect the ability of T cells to respond to other infections. Most people above 60years of age display M. tuberculosis-specific immunity because of vaccination, exposure, or both. T-cell responses can be assessed by measuring intracellular IFN-γ in vitro after tuberculin stimulation. Here we investigated tuberculin-specific CD4 T-cell responses in independently living healthy older people in the South of England using flow-cytometry. Individuals were investigated for tuberculin and CMV-specific T-cell immunity using in vitro antigen stimulation followed by intracellular staining for IFN-γ, TNF-α, IL2, as well as degranulation and CD154 upregulation. We also examined a control group of younger individuals (20-35years of age). There was no significant difference between older and young people in regards to tuberculin responsiveness of CD4 T-cells; however, older people seemed to show more outliers. Increased responsiveness to tuberculin was significantly correlated to CMV responsiveness but not age. In older donors, the memory phenotype of tuberculin-induced T-cells was significantly skewed towards a more terminal differentiation phenotype in CMV-infected compared to uninfected individuals and the degree of skewing correlated quantitatively with the size of the CMV-specific CD4 T-cell response. This is a fundamental advance over previous reports of changes of the tuberculin-specific CD4 T-cell response with CMV serostatus. Our results show that how the immune system responds to CMV has a fundamental impact on the phenotype and function of the immune response to mycobacterial antigens in older life.

Broad-spectrum activity of a novel antibiotic peptide against multidrug-resistant veterinary isolates.

The emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria has become a medical and veterinary problem. Antimicrobial peptides (AMPs) show potential to overcome antibiotic resistance and could be used therapeutically. A novel AMP (AMP2041) was developed in silico and its microbiocidal activity against MDR clinical strains isolated from cattle (n=6), dogs (n=8), and pigs (n=20) was evaluated. AMP2041 showed strong antimicrobial activity against all Gram-positive and Gram-negative MDR clinical strains tested. Within 20 min of incubation, there was complete killing of Pseudomonas aeruginosa ATCC 27953 and a 90% reduction of colony count for Escherichia coli ATCC 25922. For Staphylococcus aureus ATCC 25923, a 90% reduction of colony count was observed within 120 min of incubation.

CMV and Immunosenescence: from basics to clinics.

Alone among herpesviruses, persistent Cytomegalovirus (CMV) markedly alters the numbers and proportions of peripheral immune cells in infected-vs-uninfected people. Because the rate of CMV infection increases with age in most countries, it has been suggested that it drives or at least exacerbates "immunosenescence". This contention remains controversial and was the primary subject of the Third International Workshop on CMV & Immunosenescence which was held in Cordoba, Spain, 15-16th March, 2012. Discussions focused on several main themes including the effects of CMV on adaptive immunity and immunosenescence, characterization of CMV-specific T cells, impact of CMV infection and ageing on innate immunity, and finally, most important, the clinical implications of immunosenescence and CMV infection. Here we summarize the major findings of this workshop.

Endothelial dysfunction and high cardiovascular risk profile in severe alcoholics improve only partially following a medium-term alcohol withdrawal.

BACKGROUND: Little is known about brachial artery flow-mediated vasodilatation (FMD) in active and medium-term withdrawing heavy alcoholics (HA). METHODS: FMD and some parameters of cardiovascular (CV) risk were measured in 29 HA (average alcohol intake 135, range 86 to 215 g per day) at baseline and after a 9 ± 7 months withdrawal and in 35 teetotalers. RESULTS: HA showed baseline impaired maximal % FMD (8.5 ± 5.4 SD vs. 14.9 ± 7.4, <0.001 vs. teetotalers), higher systolic (SBP) and diastolic (DBP) blood pressure (+24 mm Hg, <0.001; +15 mm Hg, <0.01), uric acid (5.3 ± 1.1 vs. 4.4 ± 0.8 mg/dl, <0.05), high-sensitivity C-reactive protein (hs-CRP; 2.7 ± 2.0 vs. 1.0 ± 0.9 mg/l, <0.02), endothelin-1 (ET-1, 0.88 ± 0.36 vs. 0.37 ± 0.10 pg/ml,<0.001), asymmetric dimethylarginine (ADMA, 0.50 ± 0.21 vs. 0.41 ± 0.12 µmol/l, p < 0.001), homeostasis model assessment of insulin resistance (HOMA-IR) (2.3 ± 1.1 vs. 1.2 ± 0.4, <0.001), and urinary 8-isoprostane (U8-iso-PGF2α) (237.2 ± 172.4 vs. 168.5 ± 96.6 pg/mg creatinine, <0.05). After withdrawal, SBP fell by 15 mm Hg, DBP by 11 mm Hg (p < 0.001), and hs-CRP by 0.94 mg/l (p < 0.02), all remaining still higher than teetotalers (<0.05, 0.01, 0.05 respectively). ET-1, HOMA-IR, and U8-iso-PGF2α were unchanged (p = NS vs. baseline, <0.05 to 0.001 vs. teetotalers). Maximal % FMD rose (to 10.6 ± 6.2, p < 0.04), but it still remained impaired (<0.04 vs. teetotalers). ADMA increased further to 0.64 ± 0.15 µmol/l (<0.05 vs. baseline, <0.02 vs. teetotalers). CONCLUSIONS: HA show marked endothelial dysfunction (ED) and high BP, impaired insulin sensitivity, inflammation, increased oxidative stress, and elevated ET-1 and ADMA, which are unaffected or only partially reversed by a medium-term alcohol withdrawal. ED and related abnormalities persist in detoxified alcoholics, thus contributing to a greater CV morbidity and mortality.

Report from the second cytomegalovirus and immunosenescence workshop.

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.

Intense antiextracellular adaptive immune response to human cytomegalovirus in very old subjects with impaired health and cognitive and functional status.

Human aging is characterized by expanded and altered adaptive immune responses to human CMV (HCMV). It is unclear whether this expansion has its origins in age-related homeostatic disturbances or viral reactivation, whether anti-CMV immune surveillance may still be effective, and what are the consequences of this expanded immune response for health and longevity. We conducted an observational cross-sectional study in groups of HCMV-seropositive subjects aged >or=65 y of variable health status to compare the intensity of Ab responses against HCMV with those against EBV and with CD4(+) and CD8(+) T cell proinflammatory effector responses directed to HCMV-derived pp65 and immediate-early protein 1 synthetic peptides. Ab responses to HCMV, but not to EBV, and anti-HCMV CD4(+), but not CD8(+), T cell responses were more intense in elderly subjects aged >or=85 y in poor health and were inversely correlated with markers of functional activity and cognitive function. Therefore, humoral and CD4(+) T cell anti-HCMV responses were specifically intensified in advanced aging associated with comorbidity and cognitive and functional impairments. Such a distinctive pattern of adaptive immunity indicates that immune responses targeting the extracellular phase of HCMV are increased in these elderly subjects and could represent an indirect effect of localized and undetectable HCMV reactivation. This study demonstrates that the oldest subjects in poor health with physical and mental impairment express intense functional immune responses to extracellular HCMV and suggests that they may be at risk for direct pathogenic effects by HCMV reactivation as well as indirect pathogenic effects linked to proinflammatory anti-HCMV effector responses.

Calcium metabolism and vitamin D in the extreme longevity.

Skeletal remodelling is a continuous process during life and is still active also in extreme senescence. In the elderly, bone resorption often prevails over bone formation, causing bone loss and fragility. Elderly subjects are exposed to the risk of fractures, and loss of self-sufficiency, if considering that the proximal femur is the most frequently involved site. Bone remodelling can maintain circulating calcium within physiological ranges, at the expense of a substantial loss of this ion from the skeleton, particularly during senescence. Calcium metabolism is regulated at cellular/molecular level by a network of cytokines, growth factors, systemic hormones that act on bone in paracrine/autocrine/systemic fashion. Among the molecules involved in bone metabolism, parathyroid hormone (PTH) and vitamin D present some peculiar aspects during senescence. The osteometabolic features in a consistent group of centenarians have been evaluated. It results that a severe hypovitaminosis D was present in 99 out of 104 centenarians (25-OH vitamin D below 5 nmol/L), and that it plays an important role as a factor inducing a vicious circle involving hypocalcemia, secondary hyperparathyroidism, together with biochemical features indicating a consistent bone loss. Serum C-terminal cross-linking telopeptide, a specific marker of bone resorption was elevated in 92% of these subjects. Moreover, it has been found that several femoral fractures had occurred after 90 years of age. These data offer a rational for the possible prevention of elevated bone turnover, bone loss and consequently the reduction of osteoporotic fractures and fractures-induced disability, in the oldest olds, through the simple supplementation with calcium and vitamin D.

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

The data we collected on the genetics of human longevity, mostly resulting from studies on centenarians, indicate that: (1) centenarians and long-living sib-pairs 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 age-related 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 Homo 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 many 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.

Massive load of functional effector CD4+ and CD8+ T cells against cytomegalovirus in very old subjects.

A progressive, systemic, and low-grade proinflammatory status is one of the major characteristics of immunosenescence. Emerging data suggest a possible contribution of CMV, known to chronically infect a large proportion of humans, lifelong from newborns to centenarians. To test this hypothesis, we evaluated functional T cell responses to two CMV immunogenic proteins, pp65 and IE-1, in 65 chronically infected subjects aged 25-100 years. PBMC were stimulated with mixtures of peptides spanning the entire sequence of both proteins, and Ag specificity and magnitude of intracellular IFN-gamma- and TNF-alpha-positive cells were then analyzed within both CD4+ and CD8+ T cells. Results indicate that pp65 and, to a lesser extent, IE-1 constitute major Ags against which aged people target functionally efficient T cell effector responses with massive production of Th1 cytokines and exhibition of CD107a degranulation marker. As a result, the production of IFN-gamma induced in T cells by both Ags was seven to eight times greater in very old than in young subjects. The comparative analysis of pp65-specific responses in these very long-term carriers revealed a reciprocal relationship between CD4+ and CD8+ producing IFN-gamma in the same individuals. These results indicate that CMV represents an important pathogen responsible for a strong immune activation in human aging. Such a remarkable burden of effector CD4+ and CD8+ T cells may be necessary to protect the elderly from CMV endogenous reactivation, but can turn detrimental by giving a substantial contribution to the proinflammatory status that accompanies the main age-related diseases.

Pathophysiology of ageing, longevity and age related diseases.

On April 18, 2007 an international meeting on Pathophysiology of Ageing, Longevity and Age-Related Diseases was held in Palermo, Italy. Several interesting topics on Cancer, Immunosenescence, Age-related inflammatory diseases and longevity were discussed. In this report we summarize the most important issues. However, ageing must be considered an unavoidable end point of the life history of each individual, nevertheless the increasing knowledge on ageing mechanisms, allows envisaging many different strategies to cope with, and delay it. So, a better understanding of pathophysiology of ageing and age-related disease is essential for giving everybody a reasonable chance for living a long and enjoyable final part of the life.

Different contribution of EBV and CMV infections in very long-term carriers to age-related alterations of CD8+ T cells.

Aging is accompanied by a complex dynamics of CD8+ T cell subsets whose origin is unclear. To evaluate the impact of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) chronic infections on CD8+ T cells in far advanced age, we studied CD8+ T cells frequencies and phenotype in nonagenarians and centenarians by HLA-A*0201- and HLA-B*0702-tetramers incorporating epitopes specific of both viruses along with viral replication. The results demonstrate that EBV and CMV infections induce quantitatively and qualitatively different CD8+ T-cell responses in advanced aging. The frequency and absolute number of CD8+ T cells specific for one lytic and two latent EBV-epitopes, were relatively low and mostly included within CD8+ CD28+ cells. By contrast, CMV infection was characterized by highly variable numbers of CD8+ T cells specific for two differently restricted CMV-epitopes that, in some subjects, were strikingly expanded. Moreover, the great majority of anti-CMV CD8+ T cells did not bear CD28 antigen. Notwithstanding the expansion of CMV-specific CD8+ lymphocytes, CMV-DNA detection in blood samples was invariably negative. Altogether, we suggest that CMV, but not EBV, can sustain chronic activation of the HLA-class I restricted effector arm in elderly that might have detrimental effects on age-associated diseases.

Low vitamin D status, high bone turnover, and bone fractures in centenarians.

The oldest olds, including centenarians, are increasing worldwide and, in the near future, will represent a consistent part of the population. We have studied bone status and metabolism in 104 subjects over 98 yr of age to evaluate possible interventions able to avoid fragility fractures and disability. Ninety females and 14 males not affected by any acute disease were considered. After a complete clinical assessment, blood was drawn for evaluating bone turnover markers, and performance tests together with skeletal ultrasonography (either at the phalanges or at the heel) were performed. We found that 38 subjects had sustained a total of 55 fractures throughout their lives, and 75% of these were fragility fractures. Twenty-eight fractures occurred at the proximal femur, with 14 after the age of 94 yr. Serum 25-hydroxyvitamin D was undetectable in 99 of 104 centenarians. PTH and serum C-terminal fragment of collagen type I were elevated in 64 and 90% of centenarians, respectively, with a trend toward hypocalcemia. Bone alkaline phosphatase levels were close to the upper limit. Serum IL-6 was elevated in 81% of centenarians and was positively correlated with PTH and negatively correlated with serum calcium. Serum creatinine was not correlated with PTH. Bone ultrasonography showed that most centenarians had low values, and ultrasonographic parameters were correlated with resorption markers. We conclude that the extreme decades of life are characterized by a pathophysiological sequence of events linking vitamin D deficiency, low serum calcium, and secondary hyperparathyroidism with an increase in bone resorption and severe osteopenia. These data offer a rationale for the possible prevention of elevated bone turnover, bone loss, and consequently the reduction of osteoporotic fractures and fracture-induced disability in the oldest olds through the supplementation with calcium and vitamin D.