The Integrated European Project "GEHA - GEnetics of Healthy Aging": recruitment, health status assessment and survival of the Italian 90+ sibpairs

The present study is part of the EU Integrated Project “GEHA – Genetics of Healthy Aging” (Franceschi C et al., Ann N Y Acad Sci. 1100: 21-45, 2007), whose aim is to identify genes involved in healthy aging and longevity, which allow individuals to survive to advanced age in good cognitive and physical function and in the absence of major age-related diseases. Aims The major aims of this thesis were the following: 1. to outline the recruitment procedure of 90+ Italian siblings performed by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The procedures related to the following items necessary to perform the study were described and commented: identification of the eligible area for recruitment, demographic aspects related to the need of getting census lists of 90+siblings, mail and phone contact with 90+ subjects and their families, bioethics aspects of the whole procedure, standardization of the recruitment methodology and set-up of a detailed flow chart to be followed by the European recruitment centres (obtainment of the informed consent form, anonimization of data by using a special code, how to perform the interview, how to collect the blood, how to enter data in the GEHA Phenotypic Data Base hosted at Odense). 2. to provide an overview of the phenotypic characteristics of 90+ Italian siblings recruited by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The following items were addressed: socio-demographic characteristics, health status, cognitive assessment, physical conditions (handgrip strength test, chair-stand test, physical ability including ADL, vision and hearing ability, movement ability and doing light housework), life-style information (smoking and drinking habits) and subjective well-being (attitude towards life). Moreover, haematological parameters collected in the 90+ sibpairs as optional parameters by the Bologna and Rome recruiting units were used for a more comprehensive evaluation of the results obtained using the above mentioned phenotypic characteristics reported in the GEHA questionnaire. 3. to assess 90+ Italian siblings as far as their health/functional status is concerned on the basis of three classification methods proposed in previous studies on centenarians, which are based on: • actual functional capabilities (ADL, SMMSE, visual and hearing abilities) (Gondo et al., J Gerontol. 61A (3): 305-310, 2006); • actual functional capabilities and morbidity (ADL, ability to walk, SMMSE, presence of cancer, ictus, renal failure, anaemia, and liver diseases) (Franceschi et al., Aging Clin Exp Res, 12:77-84, 2000); • retrospectively collected data about past history of morbidity and age of disease onset (hypertension, heart disease, diabetes, stroke, cancer, osteopororis, neurological diseases, chronic obstructive pulmonary disease and ocular diseases) (Evert et al., J Gerontol A Biol Sci Med Sci. 58A (3): 232-237, 2003). Firstly these available models to define the health status of long-living subjects were applied to the sample and, since the classifications by Gondo and Franceschi are both based on the present functional status, they were compared in order to better recognize the healthy aging phenotype and to identify the best group of 90+ subjects out of the entire studied population. 4. to investigate the concordance of health and functional status among 90+ siblings in order to divide sibpairs in three categories: the best (both sibs are in good shape), the worst (both sibs are in bad shape) and an intermediate group (one sib is in good shape and the other is in bad shape). Moreover, the evaluation wanted to discover which variables are concordant among siblings; thus, concordant variables could be considered as familiar variables (determined by the environment or by genetics). 5. to perform a survival analysis by using mortality data at 1st January 2009 from the follow-up as the main outcome and selected functional and clinical parameters as explanatory variables. Methods A total of 765 90+ Italian subjects recruited by UNIBO (549 90+ siblings, belonging to 258 families) and ISS (216 90+ siblings, belonging to 106 families) recruiting units are included in the analysis. Each subject was interviewed according to a standardized questionnaire, comprising extensively utilized questions that have been validated in previous European studies on elderly subjects and covering demographic information, life style, living conditions, cognitive status (SMMSE), mood, health status and anthropometric measurements. Moreover, subjects were asked to perform some physical tests (Hand Grip Strength test and Chair Standing test) and a sample of about 24 mL of blood was collected and then processed according to a common protocol for the preparation and storage of DNA aliquots. Results From the analysis the main findings are the following: - a standardized protocol to assess cognitive status, physical performances and health status of European nonagenarian subjects was set up, in respect to ethical requirements, and it is available as a reference for other studies in this field; - GEHA families are enriched in long-living members and extreme survival, and represent an appropriate model for the identification of genes involved in healthy aging and longevity; - two simplified sets of criteria to classify 90+ sibling according to their health status were proposed, as operational tools for distinguishing healthy from non healthy subjects; - cognitive and functional parameters have a major role in categorizing 90+ siblings for the health status; - parameters such as education and good physical abilities (500 metres walking ability, going up and down the stairs ability, high scores at hand grip and chair stand tests) are associated with a good health status (defined as “cognitive unimpairment and absence of disability”); - male nonagenarians show a more homogeneous phenotype than females, and, though far fewer in number, tend to be healthier than females; - in males the good health status is not protective for survival, confirming the male-female health survival paradox; - survival after age 90 was dependent mainly on intact cognitive status and absence of functional disabilities; - haemoglobin and creatinine levels are both associated with longevity; - the most concordant items among 90+ siblings are related to the functional status, indicating that they contain a familiar component. It is still to be investigated at what level this familiar component is determined by genetics or by environment or by the interaction between genetics, environment and chance (and at what level). Conclusions In conclusion, we could state that this study, in accordance with the main objectives of the whole GEHA project, represents one of the first attempt to identify the biological and non biological determinants of successful/unsuccessful aging and longevity. Here, the analysis was performed on 90+ siblings recruited in Northern and Central Italy and it could be used as a reference for others studies in this field on Italian population. Moreover, it contributed to the definition of “successful” and “unsuccessful” aging and categorising a very large cohort of our most elderly subjects into “successful” and “unsuccessful” groups provided an unrivalled opportunity to detect some of the basic genetic/molecular mechanisms which underpin good health as opposed to chronic disability. Discoveries in the topic of the biological determinants of healthy aging represent a real possibility to identify new markers to be utilized for the identification of subgroups of old European citizens having a higher risk to develop age-related diseases and disabilities and to direct major preventive medicine strategies for the new epidemic of chronic disease in the 21st century.

Assessment of the resilience of coral reefs to natural and human disturbances by means of recruitment panels in Indo-Pacific

Indo-Pacific region encompasses about 75% of world's coral reefs, but hard coral cover in this region experienced a 32% region-wide decline since 1970s. This great change is primarily ascribable to natural and anthropogenic pressures, including climate change and human activities effects. Coral reef conservation requires management strategies oriented to maintain their diversity and the capacity to provide ecosystem goods and services. Coral reef resilience, i.e. the capacity to recover after disturbances, is critical to their long-term persistence. The aims of the present study were to design and to test field experiments intended to measure changes in recruitment processes, as a fundamental aspect of the coral reef resilience. Recruitment experiments, using artificial panels suspended in the water column, were carried out in two Indo-Pacific locations affected by different disturbances: a new mine in Bangka Island (Indonesia), and the increased sedimentation due to coastal dynamics in Vavvaru Island (Maldives). One (or more) putatively disturbed site(s) was selected to be tested against 3 randomly selected control sites. Panels’ arrangement simulates 2 proximities to living corals, i.e. the sources of propagules: few centimetres and 2 meters over. Panels were deployed simultaneously at each site and left submerged for about five months. Recruits were identified to the lowest possible taxonomic level and recruited assemblages were analysed in terms of percent cover. In general it was not possible to detect significant differences between the benthic assemblages recruited in disturbed and control sites. The high variability observed in recruits assemblages structure among control sites may be so large to mask the possible disturbance effects. Only few taxa showed possible effects of the disturb they undergo. The field tests have highlighted strengths and weaknesses of the proposed approach and, based on these results, some possible improvements were suggested.

Effects of lung recruitment maneuvers on splanchnic organ perfusion during endotoxin-induced pulmonary arterial hypertension

Lung recruitment maneuvers (RMs), used to reopen atelectatic lung units and to improve oxygenation during mechanical ventilation, may result in hemodynamic impairment. We hypothesize that pulmonary arterial hypertension aggravates the consequences of RMs in the splanchnic circulation. Twelve anesthetized pigs underwent laparotomy and prolonged postoperative ventilation. Systemic, regional, and organ blood flows were monitored. After 6 h (= baseline), a recruitment maneuver was performed with sustained inflation of the lungs. Thereafter, the pigs were randomly assigned to group C (control, n = 6) or group E with endotoxin-induced pulmonary arterial hypertension (n = 6). Endotoxemia resulted in a normotensive and hyperdynamic state and a deterioration of the oxygenation index by 33%. The RM was then repeated in both groups. Pulmonary artery pressure increased during lipopolysaccharide infusion from 17 ± 2 mmHg (mean ± SD) to 31 ± 10 mmHg and remained unchanged in controls (P < 0.05). During endotoxemia, RM decreased aortic pulse pressure from 37 ± 14 mmHg to 27 ± 13 mmHg (mean ± SD, P = 0.024). The blood flows of the renal artery, hepatic artery, celiac trunk, superior mesenteric artery, and portal vein decreased to 71% ± 21%, 69% ± 20%, 76% ± 16%, 79% ± 18%, and 81% ± 12%, respectively, of baseline flows before RM (P < 0.05 all). Organ perfusion of kidney cortex, kidney medulla, liver, and jejunal mucosa in group E decreased to 65% ± 19%, 77% ± 13%, 66% ± 26%, and 71% ± 12%, respectively, of baseline flows (P < 0.05 all). The corresponding recovery to at least 90% of baseline regional blood flow and organ perfusion lasted 1 to 5 min. Importantly, the decreases in regional blood flows and organ perfusion and the time to recovery of these flows did not differ from the controls. In conclusion, lipopolysaccharide-induced pulmonary arterial hypertension does not aggravate the RM-induced significant but short-lasting decreases in systemic, regional, and organ blood flows.

Observation of ventilation-induced Spo(2) oscillations in pigs: first step to noninvasive detection of cyclic recruitment of atelectasis?

High arterial partial oxygen pressure (Pao(2)) oscillations within the respiratory cycle were described recently in experimental acute lung injury. This phenomenon has been related to cyclic recruitment of atelectasis and varying pulmonary shunt fractions. Noninvasive detection of Spo(2) (oxygen saturation measured by pulse oximetry) as an indicator of cyclic collapse of atelectasis, instead of recording Pao(2) oscillations, could be of clinical interest in critical care. Spo(2) oscillations were recorded continuously in three different cases of lung damage to demonstrate the technical feasibility of this approach. To deduce Pao(2) from Spo(2), a mathematical model of the hemoglobin dissociation curve including left and right shifts was derived from the literature and adapted to the dynamic changes of oxygenation. Calculated Pao(2) amplitudes (derived from Spo(2) measurements) were compared to simultaneously measured fast changes of Pao(2), using a current standard method (fluorescence quenching of ruthenium). Peripheral hemoglobin saturation was capable to capture changes of Spo(2) within each respiratory cycle. For the first time, Spo(2) oscillations due to cyclic recruitment of atelectasis within a respiratory cycle were determined by photoplethysmography, a technology that can be readily applied noninvasively in clinical routine. A mathematic model to calculate the respective Pao(2) changes was developed and its applicability tested.

Alpha4beta1 integrin mediates the recruitment of immature dendritic cells across the blood-brain barrier during experimental autoimmune encephalomyelitis

Dendritic cells (DCs) within the CNS are recognized to play an important role in the effector phase and propagation of the immune response in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. However, the mechanisms regulating DC trafficking into the CNS still need to be characterized. In this study, we show by performing intravital fluorescence videomicroscopy of the inflamed spinal cord white-matter microvasculature in SJL mice with EAE that immature, and to a lesser extent, LPS-matured, bone marrow-derived DCs efficiently interact with the CNS endothelium by rolling, capturing, and firm adhesion. Immature but not LPS-matured DCs efficiently migrated across the wall of inflamed parenchymal microvessels into the CNS. Blocking alpha4 integrins interfered with the adhesion but not the rolling or capturing of immature and LPS-matured DCs to the CNS microvascular endothelium, inhibiting their migration across the vascular wall. Functional absence of beta1 integrins but not of beta7 integrins or alpha4beta7 integrin similarly reduced the adhesion of immature DCs to the CNS microvascular endothelium, demonstrating that alpha4beta1 but not alpha4beta7 integrin mediates this step of immature DCs interaction with the inflamed blood-brain barrier during EAE. Our study shows that during EAE, especially immature DCs migrate into the CNS, where they may be crucial for the perpetuation of the CNS-targeted autoimmune response. Thus therapeutic targeting of alpha4 integrins affects DC trafficking into the CNS and may therefore lead to the resolution of the CNS autoimmune inflammation by reducing the number of CNS professional APCs.

Regulation of leukocyte recruitment by the long pentraxin PTX3

Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of inflammation.

Excitability and recruitment patterns of spinal motoneurons in human sleep as assessed by F-wave recordings

This study examines the excitability and recruitment of spinal motoneurons in human sleep. The main objective was to assess whether supraspinal inhibition affects the different subpopulations of the compound spinal motoneuron pool in the same way or rather in a selective fashion in the various sleep stages. To this end, we studied F-conduction velocities (FCV) and F-tacheodispersion alongside F-amplitudes and F-persistence in 22 healthy subjects in sleep stages N2, N3 (slow-wave sleep), REM and in wakefulness. Stimuli were delivered on the ulnar nerve, and F-waves were recorded from the first dorsal interosseus muscle. Repeated sets of stimuli were stored to obtain at least 15 F-waves for each state of vigilance. F-tacheodispersion was calculated based on FCVs using the modified Kimura formula. Confirming the only previous study, excitability of spinal motoneurons was generally decreased in all sleep stages compared with wakefulness as indicated by significantly reduced F-persistence and F-amplitudes. More importantly, F-tacheodispersion showed a narrowed range of FCV in all sleep stages, most prominently in REM. In non-REM, this narrowed range was associated with a shift towards significantly decreased maximal FCV and mean FCV as well as with a trend towards lower minimal FCV. In REM, the lowering of mean FCV was even more pronounced, but contrary to non-REM sleep without a shift of minimal and maximal FCV. Variations in F-tacheodispersion between sleep stages suggest that different supraspinal inhibitory neuronal circuits acting on the spinal motoneuron pool may contribute to muscle hypotonia in human non-REM sleep and to atonia in REM sleep.

The recruitment of bone marrow-derived cells to skin wounds is independent of wound size

Wounded skin recruits progenitor cells, which repair the tissue defect. These cells are derived from stem cells in several niches in the skin. In addition, bone marrow-derived cells (BMDCs) are recruited and contribute to wound repair. We hypothesized that larger wounds recruit more cells from the bone marrow. Wild-type rats were lethally irradiated and transplanted with bone marrow cells from green fluorescent protein (GFP)-transgenic rats. Seven weeks later, 4, 10, and 20 mm wounds were created. The wound tissue was harvested after 14 days. The density of GFP-positive cells in the wounds and the adjacent tissues was determined, as well as in normal skin from the flank. Bone marrow-derived myofibroblasts, activated fibroblasts, and macrophages were also quantified. After correction for cell density, the recruitment of BMDCs (23±11%) was found to be independent of wound size. Similar fractions of GFP-positive cells were also detected in nonwounded adjacent tissue (29±11%), and in normal skin (26±19%). The data indicate that BMDCs are not preferentially recruited to skin wounds. Furthermore, wound size does not seem to affect the recruitment of BMDCs.

Recruitment dynamics of the grove-dominant tree Microberlinia bisulcata in African rain forest: extending the light response versus adult longevity trade-off concept

In groves of ectomycorrhizal caesalpiniaceous species in the Atlantic coastal forest of Central Africa the dominant tree Microberlinia bisulcata, which is shade-intolerant as a seedling but highly light-responding as a sapling, shows very limited regeneration. M. bisulcata saplings were mapped in an 82.5-ha plot at Korup and found to be located significantly far (>40 m) away from adults, a result confirmed by direct testing in a second 56-ha plot. Sapling growth over 6 years, the distribution of newly emerging seedlings around adults, recruitment of saplings in a large opening and the outward extent of seedlings at the grove edge were also investigated. Two processes appear to have been operating: (1) a very strong and consistent restriction of the very numerous seedlings establishing after masting close to adults, and (2) a strong but highly spatially variable promotion of distant survivors by increased light from the deaths of large trees of species other than M. bisulcata (which itself has very low mortality rate). This leads to an apparent escape-from-adults effect. To maintain saplings in the shade between multiple short periods of release ectomycorrhizal connections to other co-occurring caesalp species may enable a rachet-type mechanism. The recorded sapling dynamics currently contribute an essential part of the long-term cycling of the groves. M. bisulcata is an interesting example of an important group of tropical trees, particularly in Africa, which are both highly light-demanding when young yet capable also of forming very large forest emergents. To more comprehensively explain tropical tree responses, the case is made for adding a new dimension to the trade-off concept of early tree light-response versus adult longevity.

Recruitment of platelets, white blood cells, and hematopoietic progenitor cells during high-yield plateletpheresis

Previous observations suggested recruitment of platelets (PLTs) and white blood cells (WBCs) during plateletpheresis and recruitment of hematopoietic progenitor cells (HPCs) by HPC apheresis. Quantification of recruitment helps to optimize yields and safety of these procedures; detection of WBC or HPC recruitment during plateletpheresis may further elucidate the mechanisms.

The CCR2/CCL2 interaction mediates the transendothelial recruitment of intravascularly delivered neural stem cells to the ischemic brain

The inflammatory response is a critical component of ischemic stroke. In addition to its physiological role, the mechanisms behind transendothelial recruitment of immune cells also offer a unique therapeutic opportunity for translational stem cell therapies. Recent reports have demonstrated homing of neural stem cells (NSC) into the injured brain areas after intravascular delivery. However, the mechanisms underlying the process of transendothelial recruitment remain largely unknown. Here we describe the critical role of the chemokine CCL2 and its receptor CCR2 in targeted homing of NSC after ischemia.

Platelet Recruitment during Multiple Donor Platelet Apheresis Differs between Cell Separators

SUMMARY: BACKGROUND: Recruitment of platelets (PLT) during donor PLT apheresis may facilitate the harvest of multiple units within a single donation. METHODS: We compared two PLT apheresis procedures (Amicus and Trima Accel) in a prospective, randomized, paired cross-over study in 60 donors. The 120 donations were compared for depletion of circulating PLT in the donors, PLT yields and PLT recruitment. A recruitment was defined as ratio of total PLT yield and donor PLT depletion > 1. RESULTS: Despite comparable differences of pre- and post-apheresis PLT counts (87 × 10(9)/l in Trima Accel vs. 92 × 10(9)/l in Amicus, p = 0.383), PLT yields were higher with Trima Accel (7.48 × 10(11) vs. 6.06 × 10(11), p < 0.001), corresponding to a higher PLT recruitment (1.90 vs. 1.42, p < 0.001). We observed a different increase of WBC counts after aphereses, which was more pronounced with Trima Accel than with Amicus (1.30 × 10(9)/l vs. 0.46 × 10(9)/l, p < 0.001). CONCLUSION: Both procedures induced PLT recruitment. This was higher in Trima Accel, contributing to a higher yield in spite of a comparable depletion of circulating PLT in the donors. This recruitment facilitates the harvest of multiple units within a single donation and seems to be influenced by the procedure utilized. The different increases of circulating donor white blood cells after donation need further investigation.