Sponsors of SENS5

1 A Novel Autologous Stem Cell Procedure for the Treatment of Aplastic Anaemia Using Reprogrammed Mature Adult Cells: A Pilot Clinical Study

Background and objectives: The disease, aplastic anaemia is a life threatening rare bone marrow failure. In this condition the underlying haematopoietic cellular deficit, lead to hemorrhage, infection and severe anaemia. The treatment of choice for this haematological condition is allogeneic bone marrow transplantation from fully matched HLA sibling. Though this procedure is curative in the majority of young patients with aplastic anaemia; extending this benefit to older patients or those lacking a family donor remains a major challenge. Herein, the safety and efficacy of infusing autologous haematopoietic retrodifferentiated stem cells into four patients with aplastic anaemia without the use of any pre or post-conditioning regimen including immunosuppression is described.

Methods: Un-mobilized, mononuclear cells were harvested from 4 patients with acquired aplastic anaemia by aphaeresis. Mononuclear cells of patients were cultured with purified monoclonal antibody against the monomorphic regions of the beta chain of MHC class II antigens (Clone CR3/43) for 3 hours, to obtain autologous retrodifferentiated haematopoietic stem cells (RHSC). Autologous RHSC were washed and infused into the 4 patients without the use of any pre or post-conditioning regimen. Thereafter, the efficacy (engraftment) of autologous RHSC was assessed in these four patients.

Results: Following single infusion of the autologous RHSC, 2 out of 4 patients with aplastic anaemia are now transfusion independent for more than 6 years. This mode of engraftment is similar to those obtained following transfusion of stem cells from identical twins in the absence of any pre-conditioning regimen. Most importantly, karyotyping and G-banding analysis prior and post procedure in all patients remained the same, indicating that the retrodifferentiated stem cells possess long term repopulation potentials and are safe. Interpretation and conclusion: This is the first report demonstrating the functional utility of reprogrammed fully differentiated adult cells into pluripotent stem cells with extensive repopulation potentials in a human setting and without any pre or post conditioning regimen, including immunosuppression. This autologous approach of stem cell creation may broaden the curative potentials of stem cell therapy to a wider population of patients with aplastic anaemia, including many patients suffering from other haematological and non-haematological disorders.

Keywords: Retrodifferentiation, Aplastic Anaemia, Autologous Stem Cells, Reprogrammed Mature Adult Cells, Leukocytes

2 Polymorphisms of Ship2, A Phosphatase Involved in the Modulation of Downstream Signals in Insulin Pathway, in Ageing and Age-Related Diseases

Phosphoinositides are components of the cell membrane and act as fundamental signaling molecules for regulation of cell proliferation and survival, cytoskeletal reorganization, and vesicular trafficking by recruiting effector proteins to cellular membranes. To regulate the cellular levels of lipid secondary messengers such as PtdIns(3,4,5)P3, cells use two major types of phosphoinositide phosphatases: the inositol polyphosphate 3-phosphatase PTEN and SH2 domain-containing inositol 5-phosphatases 1 and 2 (SHIP1 and SHIP2). SHIP2 is a protein that catalyzes the degradation of lipid secondary messenger phosphatidylinositol 3,4,5-triphosphate (PIP3) to produce phosphatidylinositol 3,4-diphosphate (PIP2). Thus SHIP2 is an antagonist phosphatidylinositol 3-kinase (PI3K) that takes part to the insulin signaling phosphorylating PIP2 to obtain PIP3. Because the PI3K pathway plays a key role in the biological effects of insulin, it is possible that the attenuation of the PI3K-mediated insulin signaling is associated with the insulin resistance in type 2 diabetes. Many studies underlying the role of SHIP2 as a negative regulator of insulin signaling. Its overexpression reduces both insulin-stimulated MAP kinase and AKT activation, leading to downregulation of glucose uptake towards failed recruitment of GLUT4 in cell membrane and glycogen synthesis in 3T3-L1 adipocytes and L6 myotubes Expression of SHIP2 was greatly increased in the skeletal muscle and fat tissue of diabetic mice. In addition, INPPL1, the SHIP2 gene coding, is in human chromosome 11q13-14, which is suggested to be linked to type 2 diabetes with insulin resistance and hypertension. Therefore, it is possible that SHIP2 is involved in the pathogenesis of insulin resistance of type 2 diabetes mellitus in humans. A study of 2004 has demonstrated that two SNPs of INPPL1, rs2276047 in intron 9 and rs9886 in the 3′ untranslated region and an insertion/deletion polymorphism in intron 1 found in British and French type 2 diabetes people are associated with metabolic syndrome including type 2 diabetes and hypertension. Our aim is to study the prevalence of these three SNPs in Sicilian population in a case-control study, analyzing Alzheimer's patients as case and healthy people as control. Also we are analyzing the association between these polymorphisms, singularly and as haplotype, in Alzheimer's disease patients with and without diabetes because it is known that Alzheimer's disease is associated with metabolic syndrome. Preliminary results show the presence of these SNPs in Sicilian population.

Keywords: SHIP2, SNP, ageing, age-ralated disease

3 Early Impairment of Long-Term Depression in the Perirhinal Cortex of A Mouse Model of Alzheimer's Disease

Tg2576 is one of the most widely used mouse transgenic line in Alzheimer's disease (AD) research. It carries a double missense mutation on the amyloid precursor protein gene (APP), resulting in marked increases in β-amyloid (Aβ) 1–40 and Aβ1–42 in the plasma and brain by 3–5 months of age, decrease in spine density in the outer molecular layer of the dentate gyrus at 4 months, decline in long-term potentiation of synaptic transmission in the dentate gyrus associated with impairment in contextual fear conditioning by 5 months, and plaque deposition in brain starting at 8 months. Visual recognition memory is seriously impaired in patients in the early stages of AD. The perirhinal cortex (Prh), a multimodal associative cortex of the temporal lobe, is critically involved in this form of memory; long-term depression of synaptic transmission (LTD) in this cortical region is regarded as its cellular correlate. The present work was aimed at evaluating if LTD in Prh of Tg2576 mice is already compromised at 3 months of age. Field excitatory post-synaptic potentials evoked by afferent pathways stimulation were recorded in layer II/III of Prh in horizontal brain slices obtained from 3 month old Tg2576 mice and littermate controls. We found that a single train of 3000 pulses delivered at 5 Hz induced LTD in slices from control mice, but not in those from Tg2576 mice. This result is consistent with the early visual recognition impairment observed in AD patients, and is particularly interesting because no changes in synaptic plasticity have previously been found so early in this transgenic line. Indeed, one of the major goals in AD research is to characterize the mechanisms leading to early functional impairments in order to improve diagnostic and therapeutic tools.

Keywords: Alzheimer's disease, Tg2576 mice, perirhinal cortex, long-term depression, visual recognition memory

4 Impairments in Synaptic Plasticity in Aged Animals and in Animal Models of Alzheimer's Disease

Aging is associated with a gradual decline in cognitive function, and more dramatic cognitive impairments occur in patients affected by Alzheimer's disease (AD). Studies performed in the last two decades in aged animals and in animal models of AD have revealed that deterioration of cognitive performances is associated with significant changes in synaptic plasticity. In particular, electrophysiological studies have well documented that alterations in long-term declarative memory, extremely vulnerable to age and neurodegenerative diseases, are linked with deficits in induction and maintenance of long-term potentiation of synaptic transmission (LTP) in the hippocampus and in other temporal lobe regions involved in this form of memory. Interestingly, we have found that long-term depression of synaptic transmission (LTD) in the perirhinal cortex, a cellular correlate of visual recognition memory formation, is impaired very early (at three months of age) in a mouse transgenic model of AD. Moreover, recent morphological studies performed in aged animals have revealed a clear-cut correlation between the quality of memory performance and the extent of morphologic changes (remodeling) in synaptic contacts occurring during memory consolidation. Nutritional studies performed in aged animals have documented the effect of different diets on synaptic plasticity or morphology; interestingly, some diet supplements were found to reverse age-induced deficits in LTP or alterations in synaptic morphology.

Keywords: synaptic plasticity, aging, Alzheimer's disease, declarative memory, nutrition

5 Lysosens: A Synthetic Biology Approach to Treat Age-Related Macular Degeneration

A2E is a toxic compound that is formed in the lysosomes of retinal pigment epithelial (RPE) cells, as a normal by-product of the vitamin A metabolism that is essential for photoreceptor activity. It is a major component of RPE lipofuscin, which is thought to mediate light-induced oxidative damage associated with aging due to its photosensitive nature; its presence leads to the formation of epoxides that are even more toxic to the host cell. I'm studying the potential of enzyme replacement strategies to help the RPE get rid of A2E, breaking it down into less toxic catabolic products. This can be achieved in two ways: either by systemically delivering particular enzymes that are engineered to target a receptor that will cause those enzymes to be endocytosed and directed into the cells' lysosomes; or by using a gene therapy approach to enable the RPE to degrade A2E autonomously. Both approaches are being investigated by the LysoSENS project, which is now using novel methods for gene assembly to accelerate our research, giving us the ability to study our therapeutic strategies in medium-throughput assays in cultured RPE cells.

Keywords: Macular degeneration, Lysosome, A2E, Gene assembly, LysoSENS

6 Small-Molecule Stimulators of Telomerase

Although our life expectancy has increased tremendously over the last century, there is still a 125-year theoretical limit on our lifespan, and no medical therapy available today has been able to break through this barrier. There is a clock that ticks inside every dividing cell of our bodies. This clock is found at the tips of our chromosomes, in a region of the chromosome called the telomere. When human cells divide, telomeres shorten, and the length of the telomeres correlates with the age of these cells. This telomere clock may be, at least in part, the clock of aging; controlling telomere length may be a way to break through our 125-year theoretical maximum lifespan. Sierra Sciences is working to determine whether telomere shortening really is the primary cause of aging and whether we can prevent it. Telomerase stops the telomere clock from ticking, and can give cells the potential to divide forever. The gene for telomerase is turned on only in our reproductive cells and in cancer cells, and is turned off in almost all other cells. A small-molecule compound could possibly turn it back on to prevent the shortening of our telomeres. Sierra Sciences has discovered 39 families of small-molecule compounds that induce telomerase in human cells. Our chemists have created designer compounds based on one of these families, some of which give us activity up to 16% of what we believe is needed for a human cell to become immortal. Recently, we have also begun screening natural products in a partnership with Isagenix Corporation.

Keywords: telomerase activation, telomeres, telomere shortening

7 A Bioinformatics Analysis of Lamin a Regulatory Network: A Perspective on Epigenetic Involvement in Hutchinson-Gilford Progeria Syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare human genetic disease that leads to premature ageing and ageing-associated phenotype. HGPS is caused by mutation in the lamin A (LMNA) gene that leads, in affected young individuals, to the generation of progerin, a splicing mutant of lamin A. A bioinformatics analysis of the LMNA gene network of interactions is presented. Lamin A seems to be involved in epigenetic regulation of transcription, chromatin remodelling, DNA repair, with key roles in stemness regulation, normal ageing and telomere functions. The study suggests particular relevance of chromatin remodellers and histones covalent modifiers in the LMNA network. Specifically, HTATIP histone acetylase seems to be of particular relevance in the network.

Keywords: Hutchinson-Gilford progeria syndrome, Lamin A, HTATIP, Chromatin Remodellers, Epigenetic Modifiers

8 Life Extension: Careers, Relationships, Family and Faith

Abstract not available.

9 Can the Blood Leukocyte Telomere Length Predict Sporadic Thoracic Aortic Aneurysm?

The prevalence of cardiovascular diseases (CVD) rises with ageing and is one of the main causes of mortality in Western countries. In view of the progressive population ageing, there is an urge for a better understanding of age-associated diseases, such as sporadic thoracic aortic aneurysm (S-TAA), and their underlying molecular mechanisms. Several cardiovascular risk factors (i.e. hypertension, oxidative stress, smoking, etc.) seem to explain most of risk of S-TAA in general population. However, little is known about its precise cellular, molecular mechanisms and genetic determinants. In particular, at an individual level there is wide variation in both its occurrence and complications (i.e. dissection and rupture) and age and gender manifestation, even in individuals with the same risk factor profiles. The reasons for this wide inter-individual variation in susceptibility are poorly understood. However, it is currently emerging the speculation of inter-individual variation in risk of CVDs, such as S-TAA, as result of variation in the rate of biological ageing. In particular, recent studies report blood leukocyte telomere-TTAGGG DNA repeats at the ends of chromosomes-length as potential better predictor for onset of CVDs than chronological age. In the light of this evidence, we are determining whether the mean of blood leukocyte telomere length might be a predictor for S-TAA. To this aim, peripheral blood samples were collected from 161 S-TAA patients and 128 age- and gender matched controls. Genomic DNA was extracted from leukocytes and telomere lengths determined using a chemioluminescence technique. The preliminary results, showing shorter telomeres in patients than in controls, seem to be promising confirming our hypothesis. Thus, telomere assay could contribute to identify individuals at risk for S-TAA.

Keywords: S-TAA, biological ageing, mean blood leukocyte telomere length

10 Mitochondrial Gene Transfer to Transplantable Tumours Lacking a Mitochondrial Genome

Mammalian cells contain multiple mitochondria with each mitochondrion containing several genomes. With few known exceptions, mitochondria are retained within their cell of origin. Nevertheless, intercellular mitochondrial transfer via membrane nanotubes has been demonstrated in cell culture systems in vitro and recent phylogenetic evidence has indicated that mitochondrial gene transfer from host to tumour cells occurs in a canine transmissible venereal tumour (Rebbeck et. al., Science 331: 303 2011). Intercellular mitochondrial gene transfer has not been demonstrated physiologically. Here, we present evidence that metastatic 4T1 breast carcinoma cells and B16 melanoma cells that lack a mitochondrial genome and respiratory function acquire a mitochondrial gene marker and respiratory function following injection into recipient mice. This mitochondrial genome acquisition triggers tumorigenesis after a 20–30 day lag period and subsequent metastasis to the lung in both models. Transformed cells exhibit stable properties indistinguishable from those of parental cells. The most likely explanation for these results is horizontal transfer of mitochondrial genes from the tumour microenvironment to the mitochondrial genome-knockout tumour cells by a mechanism involving membrane nanotube-mediated movement of mitochondria between cells. Intercellular mitochondrial transfer may be relevant not only to glycolytic cancers with mitochondrial genome damage, but also in other diseases involving mitochondrial dysfunction and in ageing.

Keywords: mitochondrial genome, tumorigenesis, metastasis, cell metabolism, horizontal gene transfer

11 Vascular and Neuronal Damage in Cryonics Patients

Cryonics patients can suffer varying degrees of ischemic damage, including both warm and cold ischemia. Although efforts to minimize warm ischemic damage for cryonics patients are often successful, most cryonics patients suffer lengthy cold ischemia during shipment in water ice to a cryonics facility for cryoprotectant perfusion. Vascular damage due to ischemia impedes perfusion with cryoprotectant solutions intended to eliminate ice formation. Neuronal damage due to cold ischemia is probably not so great as to prevent reconstruction in many cases, but freezing damage can be great when perfusion is poor. Forms of ischemic vascular damage will be reviewed, along with means of mitigating that damage. Finally, the probable impact on neuronal damage will be discussed.

Keywords: cryonics, ischemia, edema, vascular, damage

12 Damage Assays Rather Than Biomarkers of Aging

Aging is multiple forms of damage. Aging is primarily damage to macromolecules: proteins, lipids, carbohydrates, mitochondrial DNA and nuclear DNA (including telomeres). Aging damage is primarily due to ROS, RNS, sugars (glycation), radiation, pathogens, inflammatory cytokines, and accumulated toxins (metals, PCBs, dioxins, etc.). There is no singular aging process, but each species can be characterized by a maximum lifespan. Maximum lifespan for a species corresponds to the forms of aging damage that target the weakest link for the survival of that species. Biomarkers of aging attempt to measure biological age and thereby be a better predictor of life expectancy and future functionality than chronological age. Biomarkers of aging assume a singular underlying process, often measured by multiple components. There is no singular underlying aging process or biological age. Chronological age corresponds to biological age only for individuals of a species that have the potential to achieve maximum lifespan for that species. Endogenous and exogenous damaging agents can accelerate aging. Genetics can affect defenses against damage (antioxidant enzymes, heat shock proteins, etc.), damage repair, and molecular disposal and re-synthesis systems. Genetics can accelerate or decelerate aging. Most aging damage contributes to aging-associated diseases. The great majority of nonviolent, non-contagious deaths are due to aging-associated diseases. Different organs and tissues age (accumulate aging damage) at different rates. Death due to aging-associated disease will be caused by the most damage to the most vulnerable organ or tissue. Aging damage contributing to aging-associated disease is usually more organ- or tissue-specific than aging damage that does not contribute to aging-associated disease. Damage assays focus on the causes of aging, whereas attempts to find biomarkers of aging are a counterproductive distraction based on the misconception that there is a singular aging process. Comprehensive aging damage assays can allow for ranking forms of aging damage as contributors to morbidity and mortality, prioritizing interventions, and monitoring intervention effectiveness.

Keywords: aging, biomarkers, damage, assays, rejuventation

13 Plasmonic Photothermal Therapy of Atherosclerotic Plaque with the Use of Silica-Gold Nanoparticles and Stem Cells Versus Ferro-Magnetic Approach

Background. Some modern angioplasty techniques generally just manipulate the form of the plaque and have some clinical and technical restrictions, relatively high complication rate and restenosis risk. Methods. A total of 101 Yucatan miniature swines were assigned to the three groups (34 pigs into 60/15-70/40 nm silica-gold nanoparticles (NPs) nanogroup, 34 swines – into ferro-magnetic group with 100 nm iron-bearing NPs with delivery in hand of magnetic fields, and 33 - in sirolimus stenting control). Animals in nanogroup were split up to 4 subsets by delivery approach: (1) infused circulating progenitor cells, (2) with infused ultrasound-mediated albumin-coated gas-filled microbubbles, (3) CD73+105+ progenitor cells in the composition of bioengineered on-artery patch, (4) CD73+CD105+ progenitor cells transplanted by manual subadventitial injection. We have used a modified method for the preparation of NPs as described by Lee (2008) and Deng H (2005). Studied proximal left anterior descending arteries were observed by 30 MHz intravascular ultrasound (IVUS).

Results. A change of the PV (mm3) immediately after the laser irradiation/in 6 months in groups were −28.9/−56.8%, −30.8/−59.1% and 0/+4.3% (p < 0.01) respectively, and in the subsets in 6 mns reached −54.5/−45.9/−74.7/−61.8% (p < 0.05) respectively. Temperature inside heat-site reached 83/121/98/101 and 102° C (p < 0.01) in the subsets and ferro-magnetic group respectively on data of MRI, however entire plaque warmed up until mean 40.9° C (NS). Some cases of atherothrombosis (3 cases in S2 subset) and distal embolism (8/34, 23.5% in nanogroup, 2/33, 6% in control) were revealed while first 4 weeks. Restenosis confirmed in 3 (9%) swines of stenting group only. An impact over the non-organic part of the plaque (mineral deposits, calcium necrotic core) was predominated in nanogroup (−43.4% vs −12.1% and +3.7% respectively, p < 0.005). The histological analysis has confirmed the ‘burning’/‘melting’ effect with multiple ruptures, necrosis, homogenization of tissue, and consequent possible functional insufficiency of vessel. Anti-inflammative and anti-apoptotic effects, signs of neovascularization and restoration of artery function were predominated in subsets with progenitor cells (p < 0.01). Coronary flow-mediated vasodilation was observed after hyperemia and injection of nitroglycerine (+10.2 and +16.6%, +8.2 and +9.6%, +8.1 and +9.8% in groups respectively, p < 0.05). The highest level of the infiltration with NPs was achieved in the liver that equal to 612 units per cm3 without signs of fibrosis or allergic responses. There were 344, 201 and 99 units per cm3 in the heart, brain, and lungs respectively without any morphological dynamics.

Conclusion. Nanoburning is the novel promising technique to demolish and melt the plaque especially in combination with stem cell technologies challenging functional restoration of the vessel. Plasmonics is the high-effective and safe alternative to stenting for angioplasty.

Keywords: nanoparticles, mesenchymal stem cells, plasmonics, atherosclerosis, IVUS

14 Aging Brain Mitochondrial Dysfunction: Metabolic Rejuvenation

Numerous reports document the decline in various aspects of mitochondrial function with physiological aging as well as the pathologic aging seen in Alzheimer's disease, but remains unclear whether these deficits are the immediate response to an aging environment and the degree of their reversibility. By isolating neurons from the brains of rats and mice across the age-span and culturing them in a uniform environment, we eliminate the confounds of aging hormonal, vascular and immune systems. We find that a number of physiologic parameters related to mitochondrial function remain normal at rest in neurons from old animals, but these neurons reveal marked deficits when stressed. Functions that were similar in old and middle-age neurons at rest include glucose uptake, ATP levels, mitochondrial redox state and mitochondrial basal respiration. At rest, intracellular calcium and ROS levels were higher, while mitochondrial membrane potential and redox buffer glutathione were lower in old compared to middle-age neurons. Under metabolic stress from glutamate, rotenone, or the Alzheimer peptide A-beta, old neurons showed functional deficits in glucose uptake, ATP levels, respiratory capacity, NAD(P)H, cytochrome C levels, mitochondrial membrane potential, redox state and epigenetic histone acetylation and methylation. Remarkably, a brief treatment with 17-beta-estradiol reversed the dysfunctional old neuron baseline and glutamate-evoked increases in calcium with an EC50 200 fM as well as the deficits in complex IV, respiratory capacity and oxidized cardiolipin. Additionally, glutathione and ROS deficits in old neurons could be prevented by treatment with blueberry extract. Similarly, low glutathione and high ROS levels in Alzheimer transgenic mouse neurons could be reversed by a 15 hr. treatment with the NAD precursor nicotinamide (vitamin B3). The histone marks were rapidly reversed by histone deacetylase and methyltransferase inhibitors that rejuvenated levels of memory-essential BDNF. These observations suggest that critical deficits in aging mitochondrial function may be reversible by metabolic agents that target the NADH substrate of oxidative phosphorylation, rejuvenate complex IV and restore youthful histone epigenetics.

Patel, J.R. (2003) Age-related changes in neuronal glucose uptake in response to glutamate and beta-amyloid J. Neurosci. Res.72:527–536. PM:12704814

Brewer, G.J.; Reichensperger,J.D.; Brinton,R.D. 2006. Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons, Neurobiology of Aging 27:306–317. PM:15961189

Parihar, M.S., Brewer, G.J. 2007. Simultaneous age-related depolarization of mitochondrial membrane potential and increased mitochondrial ROS production correlate with age-related glutamate excitotoxicity in rat hippocampal neurons, J. Neuroscience Res. 85:1018–1032. PM:17335078.

Parihar, M.S., Kunz, E., Brewer, G.J. 2008. Age-related decreases in NAD(P)H and glutathione cause redox declines before ATP loss during glutamate treatment of hippocampal neurons. J. Neurosci. Res. 86:2339–2352. PM:18438923.

Jones, T.T. and Brewer, G.J. 2009. Critical age-related loss of cofactors of neuron cytochrome C oxidase reversed by estrogen. Exp. Neurology, 215:212–219. PM:18930048. PMCID:3018880

Brewer, G.J. Lindsey, A.L. Kunz, E.Z. Torricelli, J.R. Neuman, A. Fisher, D.R. Joseph, J.A. 2010. Age-related toxicity of β-amyloid associated with increased pERK and pCREB in primary hippocampal neurons: reversal by blueberry extract. J. Nutr. Biochem. 21:991–998. PM:19954954 PMCID: 2891857

Jones, T.T. and Brewer, G.J. 2010. Age-related deficiencies in complex I endogenous substrate availability and reserve capacity of complex IV in cortical neuron electron transport. Biochim. Biophys. Acta Bioenergetics, 1797:167–176. PMCID: PMC2812684. PMCID: 2812684

Ghosh, D. and Brewer, G.J., in preparation.

Walker, M.D. and Brewer, G.J., submitted.

Keywords: brain, mitochondria, ROS, estrogen, epigenetics

15 How Does Our Planet Keep Safe for Future Generations?

Scientists worry state of our planet or more exactly they worry threats of life on the Earth. Global cataclysms took place in the past but then technological development did not allow to prevent these catastrophes. Other situation on our planet occurs now. Probably we can prevent local threats but we depend on the Sun in forward alternatively. Local threats are ecological, climatic, geodesic ones. The first and main threat is global warming in the result that greenhouse effect may develop. Excretion of oceanic methanhydrates can break thermal balance and if we will have gone a bifurcation point then green house effect is inevitable (it may be during nearest decades). First of all we propose four global positions: 1) scientists must take responsibility for fate of our Civilization; 2) we must adopt a long term approach and switch into psychological consumption of lasting life on the Earth; 3) ideology of agreement must be worked out between countries for cooperation in safeguarding of security of life on the Earth; 4) we must create the “Fund” for organization of the Center on protection of life as a prompt measure. The basic directions of our efforts: 1) a ecological equilibrium search on the planet of Earth (M.Bounias department); 2) ideology of agreement (G.Khozin department); 3) to prevent climate change; 4) defense of the atmosphere ozone layer from disaster; 5) conservation of biodiversity; 6) defense of forests and them resurrection; 7) solving problems of World Ocean; 8) struggle with desertification; 9) defense of the nature reservations; 10) to provide safe burial of chemical and nuclear weapon; 11) ecology of man; 12) to create a human rehabilitation department (N.Amosov`s centre); 13) investigations of cancer genesis and rejuvenation; Our nearest goals: 1) to help to serious sick children (abroad operations); 2 to save “Dead sea”; 3) to find processes decreasing of ozone holes and to realize their; 4) to learn predicting of Earthquakes; 5) the problem of Gulfstream slowdown and local climate changes.

16 Mediterranean Diet and Longevity in Sicily: A Survey in Sicani Mountain Population

Since several years, increasing evidence suggests that the Mediterranean diet has a beneficial influences on several age-related diseases as metabolic syndrome and cardiovascular disease, hence showing protective effect on health and longevity. The effect of Mediterranean diet on human health has been reported in many population based studies and randomized trials, providing evidence for the positive effect on longevity of a dietary pattern rich in some nutritional food group. The Mediterranean diet is, indeed, characterized by a high intake of vegetables, legumes, fruits, and cereals (mostly unrefined); high intake of fish; a low intake of dairy products, mostly cheese and yogurt; a very low intake of red meat; a modest intake of ethanol, mostly as red wine and, finally, by a low intake of saturated lipids but a high intake of unsaturated lipids, particularly olive oil. Actually, Mediterranean Diet refers to dietary patterns found in olive growing regions of the Mediterranean Countries. Olive oil may be considered a functional food that contains high quantity of the MUFA oleic acid that is a compound of biological membranes and that can substitute gradually poli-insatured fatty acid. Biological membranes, rich in MUFA, have a good fluidity and a better resistance to oxidative damage (lipidic peroxidation). Extra virgin olive oil contain also alfa-tocoferol, fenolic derivative and coenzime-Q that have antioxidant function, preventing the formation of reactive oxygen species. We have showed that in Sicily there are three places, around the Sicani mountains, namely Giuliana, Bisacquino and Chiusa Sclafani where there are more centenarians with respect to the average that, in Italy, is 1.6 out 10.000 individuals. Giuliana, Bisacquino and Chiusa Sclafani are placed above sea level on the slopes of the Omo Morto Hill, on the south-western edge of the Sicani Mountains. This territory lies between the Valley of the river Belice and that of the river Sosio, and it belongs to the Special Nature Reserve of Palazzo Adriano and the Valley of the Sosio and to the Nature Areas with Special Protection of the “Sant'AdrianoWood”, “Monte Triona and MonteColomba”. In these places we have found more than 15 people which age range is 100–107 years old on the total of 8000 inhabitants. Centenarians living in these places are doing very well without any sign of age related diseases. In addition lipidic, glycemic and insulinemic profile are similar to young control and far better than that of old controls. In addition, testing these people with a mini nutritional assessment (MNE) and a frequent food questionnaire tools we collected interesting group of food regularly eaten. So, close adherence to Mediterranean diet seems to play a key role in age-related disease prevention and in attaining longevity.

Keywords: Centenarian, Longevity, Mediterranean diet, Olive oil

17 Protocol of Screening of Sub Clinical Atherosclerosis by Radio Frequency (RF) Coupled with 2D Echo.

The study was carried in Athis Mons France between 2008 and the early 2011 on a cohort of 350 patients with cardiovascular risk factors. Introduction: Atherosclerotic lesions increase with aging and the changes favored by risk factors (diabetes, obesity, hypertension, dyslipidemia, smoking) and hence the interest of making an early detection of sub clinical atherosclerosis. The study of intima media thickness and speed of the wave pulse based on age, can detect patients at risk with increased threshold values, which reflects early atherosclerotic impregnation of the arterial wall. This screening at the present time is feasible and desirable for a preventive purpose, since the advent of the radio frequency coupled with 2D ultrasound.

Purpose: Early detection of sub clinical atherosclerosis by studying the changes in the arterial wall and geometric radio frequency (RF) coupled with the 2D echo, through the analysis of cut-off values of IMT (intima media thickness) and VPW (velocity of pulse wave) of after age in patients with cardiovascular risk factors. Inclusion criteria: patients with cardiovascular risk factors (dyslipidemia, type II diabetes uncomplicated, hypertension, smoking and overweight) untreated or not controlled by the treatment. Exclusion criteria: healthy patients or with a history of established cardiovascular disease and patients with pathological values of IMT ≥900 microns and a VPW ≥12 m/s.

Methods: Comparative study of 350 patients (225 men,125 women), average age of 51 years, divided into 2 main groups, with 2 methods performed (QIMT, QAS). The patients included have had a study of the remodeling of the arterial wall (functional and geometric) by RF coupled with 2D. The parameters studied, threshold values of intima media thickness (IMT) and velocity of pulse (VPW) by age group.

Techniques: The QIMT: measurement of intima-media thickness (IMT), according to the protocol of Mannheim. The QAS: measurement of the velocity of pulse (VPW), according to the Protocol of Athis-Mons.

Groups studied: Group (QAS) 200 patients between 20–69 years, divided into two subgroups: A) first sub group: 125 men (120 white/5 black race), with risk factors of dyslipidemia, diabetes and smoking. B) second sub group: 75 white women with risk factors of hypertension and overweight.

Results: The analysis of the data obtained shows that 35.3% of men and 20.1% of women have abnormal results above the threshold values. Among the different sub groups, 30.3% (men) and 12.1% (women) with dyslipidemia and smoking as risk factors predominate. The group at highest risk is between 40–59 years with a high percentage of abnormal results. A prevalence of pathological results is strongly linked with the male gender, increase in age and the presence of the risk factors such as smoking and dyslipidemia. There is no difference for abnormal results between race and gender in relation to the velocity of pulse (VPW); In the final analysis of all the groups, 55.4% of the patients included in the study had abnormal results, among them 40% with several risk factors are inadequately controlled by the treatment and 15% of remaining listed as non-treated patients.

Conclusion: The sub clinical atherosclerosis detection is possible at the present time with the emergence of new techniques such as QIMT, QAS by Radio frequency (RF), easy to use and reproducible results with a sensitivity of 95% and a specificity of 94%. They can be calculated in real time and to codify the thresholds values of IMT and VPW according to the age group of patients with risk factors. The results of each patient must be systematically associated with the technique of the FMV (flow-mediated vasodilatation) to detect Endothelial dysfunction and also associated with the biological, biochemical markers and oxidative stress. The application of the new Protocol of early detection of atherosclerosis allows us to identify patients at risk and to support targeted and tailored to each patient. This study allowed us to codify the threshold values of the arterial stiffness and ADR according to age and stratify the risk of earlier patients.

Comments: 55.4% of the patients with abnormal results above the threshold values, are patients who deserve a more targeted and aggressive support finally reduce the progression of atherosclerosis, slow the aging of the artery, standardize the threshold values and reduce the occurrence of cardiovascular events.

Keywords: Early detection, atherosclerosis, age, QIMT, QAS

18 Rescuing Aged Mesenchymal Stem Cells by Exposure to a Young Extracellular Matrix

Previously, we reported that in both mouse and human models a native extracellular matrix (ECM) generated by bone marrow cells dramatically promoted mesenchymal stem cell (MSC) proliferation, preserved the stem cell properties, and enhanced their capacity for skeletogenesis (Chen et al, 2007, JBMR, 22:1943; Lai et al, 2010, Stem Cells Dev., 19:1095). This led us to investigate whether culturing aged MSCs on an ECM could improve their number and quality. In particular, we want to take advantage of this established model to address the question of whether there are effects of age on MSC themselves (intrinsic theory), or changes to MSCs by the surrounding ECM (extrinsic theory), or both. A cell-free ECM was prepared from cultured femoral marrow cells from either 3-month old (young) or 18-month old C57BL/6 mice (young-ECM, or old-ECM, respectively). The replication and osteogenesis of young or old MSCs maintained on young-ECM vs. old-ECM as well as plastic were examined in vitro and in vivo. We found that the frequency of MSCs in marrow from old mice, measured by colony forming cells, was only marginally lower than that of young mice. In contrast, defects in the self-renewal and bone formation capacity of old MSCs were remarkable. These defects were corrected by provision of a young-ECM, but not old-ECM. In parallel cultures maintained on a young-ECM, the intracellular levels of reactive oxygen species from both old and young mice were reduced 30–50% compared to those maintained on old-ECM or plastic. We concluded that aging negatively impacts the formation of an ECM that normally preserves MSC function, and aged MSCs can be rejuvenated by culture on a young-ECM.

Keywords: Age, Mesenchymal Stem Cells, Extracellular Matrix, Reactive Oxygen Species

19 Neuroplasticity and Connectivity in the Aging Brain

While molecular and cellular strategies have a vital place in regenerative medicine to prevent the accumulating damage of aging bodies, the most important frontier is the mind, which prevails over the physical, external boundaries that confront humans. This paper describes a technique to prolong the neurological pathways in the brain that have executive control over all body processes. Operant learning of the brain with Neurofeedback has had demonstrated success in many psychological areas and this paper will describe physiological and behavioral results from single-subject studies, and a controlled study.

These results suggest that Neurofeedback should be an essential component in any anti-aging programme. Neurofeedback is direct operant training of electrical activity in the brain (EEG). Brainwaves are recorded in action from moment to moment and that information reflected back to the person undergoing the training. The brain is rewarded for changing its own activity to more efficient patterns. This is a gradual learning process in self-regulation. The Neurofeedback system used is this research study is optimized for EEG activation and connectivity training. Based on analog and digital techniques, it provides fast and accurate feedback of all EEG variables including amplitude, phase, frequency, coherence, asymmetry, and other component values. The technique will be demonstrated at the conference by qEEG.

Keywords: Neurofeedback, Aging Brain

20 …Elegant Elegans, Black-Scholes, Alchemists and Flat Earthers…! Biology, Longevity Risk and Their Economic Effects…

In recent years developmental advances in biology at the molecular level have given rise to improving mortality and increasing longevity in the human species. As a consequence there has been a significant and growing risk affecting a number of financial institutions who have traditionally insured human life or provided financial provision for retirement. These institutions can no longer warehouse this risk or successfully fund it. This has raised a serious economic spectre. This talk explores the recent developments, the level of longevity exposure and the solutions put forward in what is seen by the financial markets as the search for the holy-grail.

Keywords: insurance, longevity, risk, retirement

22 De-Novo Geroprotector Design

Geroprotector is a therapetics aiming at root causes of age-related diseases and as such capable of extending the life span of model animals and ultimately humans. The causes of aging are very ancient and evolutionary conservative. This means that the targets for the small molecule intervention can be identified using bio-informatics tools as the most conservative proteins in the pathways containing the largest number of genes responsible for the life span control. Together with the modern structure based drug discovery tools the approach should quickly lead to emergence of novel compounds readily testable in in-vitro models of age-related disorders and in life-span measuring experiments in multiple organisms. Once the preclinical efficacy and toxicity tests are finished the compounds can be commercialized as therapies against the specific sicknesses and as having a proven geroprotective effect. We report the specifics of the scheme and demonstrate the first de-novo design of potentially geroprotective compounds.

23 Cellular “Functional Replacement” with a “Cell-Free Strategy” In Early Alzheimer's and Parkinson's Diseases: Evidences from Related Animal Models

Background. Current evidence suggests that bone marrow stem cells (BMSC) contribute to lesioned/degenerated brain tissue repair by both secretion of trophic paracrine factors and functional incorporation into affected regions. Hypothesis. In vitro generated conditioned medium from cultured BMSC (CM-BMSC) replaces the stem cell grafting benefits in early Alzheimer's and Parkinson's diseases models respectively.

Methods. BMSC-derived conditioned medium (BMSC-CM) was obtained from culture expanded BMSC (10 passages). BMSC cellular suspensions were processed to allow fluorescent detection after grafting. Classical stains and BrU labeling/counting cells were used to determine neurogenic activity. Other immunohistochemical markers were used to evaluated neural phenotype. Motor, sensorimotor and learning/memory functions were evaluated through a multiple behavioral battery. Neurochemical studies (aminoacid levels −AAs- and glutathione content −GSH- determinations) were carried out before/after or after treatment, respectively (time treatment: 12/24 weeks). Results. All treated subjects improved their initial motor or cognitive impairment as well as compared to respective controls. Nevertheless, the most significant long term behavioral recovery was detected in BMSC-CM rats. Similarly, in this group GSH and AAs levels exhibit a marked and favourable modifications comparing with those rats from other groups. Furthermore, IVI of BMSC-CM stimulated proliferation/migration events at the subventricular zone (SVZ) or dentate gyrus (DG)differentially in each model. Conclusions: Results suggests that paracrine signals from stem and progenitor cells are decisive players in the cell-mediated restorative therapy. Their acting as a “signals whole integrator” relying on neurogenesis stimulation; through neuromodulatory and neuroprotective influences in the target brain, inducing a significant/sustained behavioral recovery. Considering the technical, ethical and practical limitations of cell therapy, cell free conditioned media could be a potent alternative for therapeutic neurogenesis in neurodegenerative diseases.

Keywords: animal model, neurodegenerative disease, stem cells, mild cognitive impairment, preclinical parkinsonism

24 Neurogenesis as Therapeutic Target in Early Alzheimer's Disease: Evidences from an Animal Model

Background. Neurogenic events and associated behavioral improvements have been reported after housing under enrichment environment conditions (EE), demonstrating the presence of newborn cells from neurogenic niches. Previous studies have demonstrated the bone marrow stem cells (BMSC) possibilities as source of tissue in brain grafting studies. Objectives: To compare functional effects induced by EE (12 weeks, 6 hours/day) and/or hippocampal BMSC grafting (H-Graft) in an early Alzheimer's disease rat model.

Methods. BMSCs cellular suspensions were processed to allow green or blue fluorescent detection. After it, they were grafted to the hippocampus of mild cognitive-impaired aged rats (18 months old). Classical stains and BrU labeling/counting cells were used to determine their migration and engraftment conditions. Cognitive performance was studied using the Morris Water Maze, passive avoidance and open field tests, before and after treatment (12/24 weeks). After it, animals were sacrified for morphological or neurochemical studies (aminoacid levels –Aas- and glutathione content –GSH- determinations.

Results. All treated animals improved their initial cognitive status. Both, H-Graft and EE rats exhibit a recovery in the assessed cogntive domains but a relevant and chronic recovery was detected in EE + H-Graft. Similarly, GSH and AAs levels exhibit a marked and favourable modifications comparing with control groups. Results suggest, that additionally to increased of neurogenic activity, neuroprotective events took place. “Function improved” was a key criterion for evaluate the effectiveness of neurogenesis –endogenous or exogenous- associated to neurorestorative approaches. At system level and its functional expression was observed different improvements but the exact causes-consequences relationship between function and neurogenesis-niche interactions couldn't be clarified at all. Conclusions. Brain regeneration, either with endogenous or exogenous new cells, clearly holds promise as a novel class of therapies to cognitive and neurochemical disturbances in the aged –impaired/demented brain.

Keywords: animal model, early Alzheimer's disease, stem cell grafting, cognitive impairment, environment enrichment

25 The Future of Human Lifespans, a Demographic Perspective

Since the 18th C, human life spans have increased globally from a life expectancy at birth (LE0) of 25–40y, to >80y in healthy countries; the LE70 has also more than doubled (1 –3). In essence the J-shaped mortality curve has shifted to progressively lower levels with minor changes in the accelerating (Gompertz) phase of mortality during aging (1,3). The basic contours of the J-shaped mortality curve have remained during these remarkable downward shifts in mortality. These remarkable changes are attributable to reduced loads of infections from public health and improved nutrition, starting long before immunization and antibiotics. Reduced inflammatory and infections loads are hypothesized to retard a broad suite of age-related degenerative conditions (1,2,3). Even brief exposure to infections in a well nourished population can have long-term consequences, as illustrated by the 1918 Influenza which increased later-life heart disease by 25% in the cohort that was prenatally exposed (4). Since 1975, the minimum mortality at ages 10–30 (adolescence and early adulthood) is approaching a virtual limit at 0.02% annual mortality (5). Further increases in life expectancy can only come through delay of the accelerating phase of mortality (reduced Gompertz slope), or reducing its slope. Both possibilities are documented in animal models. However, the global reality is for increases of inflammatory loads through the epidemic of obesity, rapidly spreading infections, and environmental deterioration from increased fossil fuel use and climate change (6). The life history phase of minimum mortality associated with adolescence and early adulthood warrants scrutiny for biological mechanisms accessible to anti-aging interventions.

Keywords: inflammation, Gompertz slope, minimum mortality

27 Computationally Mining Genome-Wide Drug-Response Compendia to Discover Novel Calorie Restriction Mimetics

Calorie restriction (CR) extends lifespan in mammals and can delay the onset of age-related diseases, including cancer and diabetes. Drugs that target the same genes and pathways as CR may have enormous therapeutic potential. Recently, genome-scale data on the responses of human cell lines to over 1000 drug treatments have become available. Here we integrate these data with gene expression signatures of CR, biological pathway information, and protein-protein and drug-target interaction networks to generate a prioritized list of candidate CR mimetics.

We collect previously published gene expression signatures of CR and screen them against high-throughput drug-response data to obtain sets of drugs that mimic CR at the transcriptional level. We annotate these candidate CR mimetics with information from drug-target, pathway, and protein interaction databases, and use clustering methods to characterize them in terms of their chemical properties and the proteins, pathways, and diseases that they target. We also compare candidate drug modes of action with those of known lifespan-affecting drugs such as rapamycin. Finally, we apply our analyses to rank drugs in terms of their therapeutic promise.

Keywords: Bioinformatics, Drugs, Calorie restriction

28 Approach to Assessing the Walking Motion of Elderly Males Based on Kinetic Parameters of Young Males

This study seeks to propose a method to assess the walking motion of elderly males using criterial kinetic parameters for young males during walking and to suggest how to maintain their walking capabilities at the same level as those of young males. For this purpose, we used the coefficient of variation (=SD/MEAN * 100, CV), z-score and weighted z-score (=z-score/CV) to clarify parameters that differentiate the walking of elderly males from that of young males. The subjects were 13 healthy young Japanese males and 104 healthy elderly Japanese males. They were instructed to walk about 10m at two selected speeds (Normal Walk (NW) and Maximum-speed Walk (MW)). We videotaped them during walking with a digital VTR camera at 60fps in order to analyze their motion in the sagittal plane. The ground reaction forces on the right foot were also measured by a force platform installed below the walkway. After synchronizing the video data and ground reaction forces, we calculated various kinetic data (joint torques, joint torque powers and joint work at the ankle, knee, and hip), step length, and walking velocity using a link-segment model based on the inverse-dynamics method. The CV, weighted z-score, and correlation coefficient between age and weighted z-score of each parameter were then calculated. Finally, we composed a regression equation for estimating “gait age” from several weighted z-scores that had statistically significant and high correlation coefficients with age (p < 0.05). By using this method, we were able to determine the decay in the walking motion of individual elderly males and critical factors that should be improved to maintain the average movement of youthful walkers.

Keywords: motion analysis, gait, aging assessment, inverse dynamics

29 An Amyloid-β Binding Peptide Modulates Aβ Oligomerization and is a Possible Candidate for Therapy of Alzheimer's Disease

A key feature of Alzheimer's disease (AD) is the pathogenic self-association of the amyloid-β (Aβ) peptide, leading to the formation of diffusible Aβ oligomers and extracellular amyloid plaques. Today, more than Aβ fibrils, small soluble Aβ oligomers are suspected to be the major toxic species responsible for AD development and progression. Next to extracellular Aβ, intracellular Aβ might have important pathological functions in AD. Therefore, agents that specifically interfere with early oligomerization processes either outside or inside of neurons are highly desired for the elucidation of the pathologic mechanisms of AD. Such substances maybe even pave the way for new AD gene therapeutic approaches. In a phage display selection, we identified the Aβ oligomer binding and modulating peptide “L3”. Here, we characterize L3-Aβ binding and the influence of L3 on Aβ oligomerization in vitro using various biophysical methods. Preliminary studies in cell culture demonstrate that stably expressed L3 reduces cell toxicity of externally added and co-expressed Aβ in neuroblastoma (N2a) cells.

Keywords: Alzheimer's disease, amyloid-β, therapy, peptides

30 A Guided Tour Down the Pathways of Aging

At first glance the wall chart, “Systems Biology of Human Aging Network”, looks like a complicated web. However, as a conceptual summary, in one view, we can see how most biogerontological processes relate to each other. Importantly, examination of these relationships allows us to pick out reasonably plausible causal chains of events. Within these chains, we can see age-related changes (or accumulations) that appear to be promising targets for future therapy development. Especially harmful is damage to the body's regeneration and repair systems, because they normally repair damage to other structures and systems. Repairing the repair systems should receive high priority in planning the development of new therapies. An important example is age-associated growth of lipofuscin inside the lysosomes inside neurons. We will follow a pathway from age-associated causes down through the age-associated pathologies that result.

Keywords: Network, Causes, Pathway, Lysosome, Lipofuscin

31 Systems Biology of Human Aging 2011 Network Model

This network diagram is presented to aid in conceptualizing the many processes of aging, and the interactions among them, including promising intervention points for therapy development. This diagram is maintained on the Web as a reference for researchers and students. Content is updated as new information comes to light. www.LegendaryPharma.com/chartbg.html

The many observable signs of human senescence have been hypothesized by various researchers to result from several primary causes. Close inspection of the biochemical and physiological pathways associated with age-related changes and with the hypothesized causes reveals several parallel cascades of events that involve several important interactions and feedback loops. This network model includes both intracellular and extracellular processes. It ranges in scale from the molecular to the whole-body level.

Keywords: Network, physiology, causes, pathology, pathways

32 AI Against Aging: The Present and Future Role of AI and AGI in Longevity Research

The volume and complexity of available biological data vastly exceeds the scope of any individual human brain, one consequence of which is the likelihood that the vast majority of interesting, relevant patterns in already-published data remain undiscovered. The obvious solution to this issue is the deployment of AI technology for biological data analysis, interpretation and simulation; but AI itself is a complex area of R&D, and application of AI to bioinformatics and systems biology is currently far from “plug and play.” In this talk I will discuss recent work using Biomind LLC's machine learning tools (a kind of AI) to analyze genomic data (SNP, gene expression, and mitochondrial mutation) from various organisms including humans. This work has been carried out together with biologists from various organizations including the CDC, the NIH, the University of Virginia, and Genescient Corp. I will detail how AI tools have led us to novel and important discoveries regarding age-associated diseases like Parkinsons and Alzheimers Disease, and regarding the mechanisms underlying calorie restriction and healthspan extension. The use of these tools to uncover the mechanisms allowing Genescient's Methuselah Flies to live 5x as long as control flies will be detailed. I will also mention some very recent work incorporating drug databases into the machine learning process, so as to enable automated drug target discovery (with a current focus on neuro, cardio and immune diseases). At the end of the talk I will touch on the future of the AI/bioinformatics connection, which goes far beyond deployment of machine learning as a data analysis tool. Ultimately, I believe that the only sound route to fully understanding molecular biology and the human organism is to feed the totality of biological data into a robust Artificial General Intelligence (AGI) system and allow its reasoning to assist us in designing new experiments, therapeutics, etc. There is strong potential for AI and biomedicine to advance in synergy.

Keywords: artificial intelligence, bioinformatics, AGI, drosophila

33 Gene Therapy and Nanotechnology as Interventive Strategies for the Aging Brain

Insulin-like growth factor I (IGF-I) is a powerful neurotrophic molecule which appears to be part of the physiologic self-repair mechanisms of the adult brain. Using the aging female rat as a model of age-related dopaminergic (DA) neurodegeneration, we have implemented short-term restorative IGF-I gene therapy in the hypothalamus and cerebral ventricles. Short-term (17 days) intrahypothalamic IGF-I gene therapy achieved a nearly full restoration of hypothalamic DA neuron function as determined by morphometric analysis and by correction of the chronic hyperprolactinemia that typically develops in senescent (28–30 mo.) female rats as a consequence of hypothalamic DA neuron dysfunction. Short-term intracerebroventricular (ICV) IGF-I gene therapy was able to ameliorate motor performance in senescent females which typically show a marked decline in motor function as compared to young (2 mo.) counterparts. Although our and others' studies reveal that IGF-I has a high restorative potential in the aging brain, up to now the only way to administer the therapeutic vectors is via stereotaxic injections in the target brain areas. The invasiveness of this procedure significantly limits its eventual implementation in human patients. The association of viral vector-based gene delivery with nanotechnology now offers the possibility of developing minimally invasive gene therapy strategies for the brain. This approach combines Magnetic Drug Targeting (MDT) and magnetofection, two novel methodologies based on the use of magnetic nanoparticles (MNP). The goal of MDT is to concentrate magnetically responsive therapeutic complexes in target areas of the body by means of external magnetic fields. Magnetofection is a methodology developed in the early 2000's by Christian Plank's group, in Munich, Germany. It is based on the association of MNP with nonviral or viral vectors in order to optimize gene delivery in the presence of a magnetic field. Thus, the German group could develop magnetic nanoparticle formulations that improve considerably the efficiency of adenovirally-mediated gene delivery. Based on the capabilities of the above technologies we have undertaken to develop, in collaboration with the German team, minimally invasive IGF-I gene delivery to the brain. This will be achieved by ICV administration of MNP-viral vector complexes at distal sites and magnetic trapping of the complexes at the target brain region by means of a properly focused external magnetic field. The progress of these studies will be discussed. The long-term goal of our endeavor is to use this technology to implement minimally invasive gene therapy in Alzheimer and Parkinson patients as well as in other neurological pathologies amenable to gene therapy intervention.

Keywords: Brain aging, IGF-I, Gene therapy, Magnetic nanoparticles, Magnetofection

34 Cellular Therapy for Intestinal Regeneration

Abnormal or inadequate vasculogenesis, local inflammation, and severe epithelial damage are common features of both inflammatory bowel disease (IBD) and radiation-induced injury resulting from pelvic or abdominal cancer treatment. Several studies have shown that adult bone marrow-derived stem cells, such as mesenchymal stem cells (MSC), upon transplantation, home to the damaged digestive tissue and facilitate mucosal repair in both IBD and radiation injury. Nevertheless, the levels of MSC intestinal engraftment are low, underscoring the need for finding ways to improve engraftment of these cells. It has also been shown that endothelial progenitor cells (EPC) exist in decreased numbers in the peripheral blood of IBD patients, which is in contrast to the presently held convention that the presence of an inflammatory state promotes the mobilization of EPC into the circulation. Furthermore, intestinal microvascular and endothelial cell dysfunction can lead to persistent tissue hypoperfusion and ischemia, further contributing to chronic inflammation. Therefore, here we investigated whether transplanted EPC can contribute to the intestinal vasculogenic process and/or the stem or mature epithelial cell pool of the intestine, and whether it is possible to identify and isolate a population of MSC exhibiting enhanced engraftment and/or contribution to the resident intestinal cellular pool. Using a fetal sheep model of in utero transplantation, we injected human cord blood-derived EPC or bone marrow-derived EphB2+ or EphB2- MSC and examined the ability of these cells to home to the intestine and contribute to the intestinal architecture. Recipients were evaluated at 85 days post-transplant for the presence of donor (human)-specific cell types by confocal microscopy. We found that within the intestine, EPC, as detected by DsRed positivity, localized preferentially to the mucosal layer above the muscularis mucosa in the area of the crypts of Lieberkühn. The overall levels of EPC engraftment positively correlated with the cell dose administered. Colocalization of DsRed cells with expression of Musashi, a putative marker for intestinal stem cells, was also evaluated. These analyses revealed that 10.79 ± 0.01% of the epithelial cells within the crypts were DsRed positive and thus donor derived, and all expressed Musashi. Expression of this marker was not observed in any DsRed positive cells in any other location within the intestine. In addition, DsRed positive cells were found in the stromal layer adjacent to the crypts. We also showed that the EphB2high MSC subset had an enhanced ability to repopulate the ISC pool and to generate differentiated intestinal cells at higher levels than their EphB2low counterpart. We found that MSC localized preferentially to the mucosal layer and distributed equally between the crypts and the villi, with no evidence of cell fusion. Our studies demonstrate that both EPC and MSC can play a role in intestinal regeneration, and that a cell therapy platform using a combination of these cells may be ideally suited for repopulation of the intestinal stem cell pool and generation of differentiated intestinal cells.

Keywords: Endothelial Progenitor Cells, Mesenchymal Stem cells, EphB2, Intestine, Inflamatory Bowel Disease

35 Epigenetic Drift and Aging

Recent data suggest that the epigenome is highly dynamic and serves as an interface between the environment and the inherited static genome. The large volume of epigenomic events and its continuous need of maintenance, i.e., after DNA repair or replication, suggests a high chance of errors. The question we wish to address is how unstable the epigenome really is. Do epimutations accumulate with age and do they occur in a random fashion, i.e., as ‘epigenomic drift’? Do they ever reach levels that are high enough to have functional consequences? To experimentally determine epigenetic drift we focused on DNA methylation, a major layer of epigenomic control. To study intra-organ variation in DNA methylation during aging, it is necessary to have access to procedures that allow assessing DNA methylation patterns at the level of single-cells or single-molecules. Such methodology is currently entirely lacking: to fill this void we have optimized bisulfite sequencing for single cell analysis. The procedure developed allows us to analyze DNA methylation patterns in single cells, within promoter regions of genes or genome-wide. Data on mouse fibroblasts, neuronal nuclei and hepatocytes will be presented and discussed. We are currently applying the method to test the hypothesis that random DNA methylation changes accumulate in the mouse brain during aging, contributing to functional decline of somatic cells that gives rise to chronic pathology and aging. Apart from basic studies into cellular heterogeneity in organs and tissues in relation to aging and age-related pathology, this method can be applied in epigenetic diagnostics, e.g., in analyzing circulating tumor cells for pathogenic epimutations.

Keywords: Epigenetic instability, Aging, Brain, Single-cells

36 Aging of Synaptic Circuits

Little is known about how normal aging affects the brain. Recent evidence suggests that neuronal loss is not ubiquitous in aging neocortex. Instead, subtle and still controversial, region- and layer-specific alterations of neuron morphology and/or synapses are reported during aging, leading to the notion that discrete changes in neural circuitry may underlie age-related cognitive deficits. Although deficits in sensory function suggest that primary sensory cortices are affected by aging, our understanding of the age-related cellular and molecular changes is sparse. Cognitive aspects of olfactory function, particularly acuity, can decline during aging. However, due to ongoing adult neurogenesis, the olfactory system also undergoes a profound and continuous remodeling of synaptic circuits throughout life. This raises the question of whether during remodeling of olfactory circuits a decrease in cell number or synaptic organization may contribute to cognitive deficits. To assess the effect of aging on the organization of olfactory bulb (OB) circuitry we carried out quantitative morphometric analyses in the mouse OB at 2, 6, 12, 18 and 24 months. Our data establish that the volumes of the major OB layers do not change during aging. In parallel, we demonstrate for the first time that the stereotypic glomerular convergence of M72-GFP OSN axons in the OB is preserved during aging. We then provide the first evidence of the stability of projection neurons and interneurons subpopulations in the aging mouse OB, arguing against the notion of an age-dependent widespread loss of neurons. Finally, we show ultrastructurally a significant layer-specific loss of synapses; synaptic density is reduced in the glomerular layer but not the external plexiform layer, leading to an imbalance in OB circuitry. These results suggest that reduction of afferent synaptic input and local modulatory circuit synapses in OB glomeruli may contribute to specific age-related alterations of the olfactory function.

Keywords: Olfaction, Synapse, Mitral cells, Granule cells, Glomeruli

37 Aging of Budding Yeast

In a surplus of nutrition baker's yeast cells (Saccharomyces cerevisiae) propagate parthenogenetically by budding off daughter cells. Propagation of old mother cells leads to daughter lines with exaggerated signs of aging (Lansing effect). The age of the mother cell depends mainly on the number of cell divisions (maximum about 20 buds), but to a small degree also on the chronological age (metabolic time). It is proposed that the aging may be ascribed to an accumulation of insoluble cross-linked protein, formed as a side product of protein metabolism. Much protein is synthesized at mitosis, which may lead to a large deposit of insoluble protein. The insoluble protein of the mother cell is shared with the small daughter cell. The Lansing effect may be explained by the daughter cell obtaining a large amount of insoluble protein from an old mother cell. Multicellular organisms (e.g. Drosophila) that do not exhibit a Lansing effect may have developed strategies to avoid the insoluble protein accumulation at propagation. One such mechanism could be sexual propagation: the DNA-content of the haploid egg cell is doubled by the sperm without the protein synthesis involved in DNA-replication. However, a slow rate of insoluble protein synthesis unrelated to mitosis may remain, which may be a cause of aging in higher organisms, the age being related to the metabolic time.

Keywords: Aging, Insoluble protein, Lansing effect

38 Neuron Replacement in the Neocortex

The cerebrum, as the substrate for our consciousness, memories, personality, and self-identity, presents unique challenges for regenerative medicine. Regenerative approaches must not only maintain general cerebral function, but also preserve as much as possible the details of the wiring and firing parameters that define each individual. A combination of molecular repair and gradual cellular replacement appears most likely to succeed. Toward this end, we are establishing paradigms in mice for replacing glutamatergic projection neurons in the neocortex, seat of our highest cognitive functions. Any strategy for using transplanted cells for neocortical cell replacement is currently hampered by the inability to get cells to disperse into the existing neural tissue. To overcome this issue, we will transplant at the edge of the neocortex highly migratory embryonic GABAergic precursor cells that are engineered with lentiviruses to transdifferentiate to a glutamatergic fate once they have dispersed throughout the neocortex. In a second set of experiments, we have successfully used two independent transgenic approaches to induce apoptosis in small numbers of neurons in the absence of major inflammation (unlike for stroke or injury for example) to determine if under these conditions endogenous stem cells can be activated to replace dying neurons. These mice also provide a potentially ideal host environment for our transplanted migratory precursors. Preliminary results will be presented. Furthermore, even if our efforts are successful in generating new glutamatergic neurons, these replacement paradigms will unlikely in themselves provide the means for complete neocortical rejuvenation. Other aspects of cortical regeneration that will also need to be addressed will be discussed.

Keywords: neocortex, glutamatergic neurons, cell migration, cell dispersion, apoptosis

39 How Can CR Inform Therapeutic Strategies?

Why organisms age and the reasons for the differences in longevity between different species is a fundamental question in biology. Changes in aging rates can be induced, most notably by increasing or decreasing calorie intake: Organisms subjected to caloric restriction (CR) live longer compared to those fed ad libitum (AL). CR robustly affects the age-related changes of several pathways: gene expression, hormones, oxidative damage, immune system, etc. All these mechanisms have a remarkable effect on longevity. Here we suggest why understanding these mechanisms in an integrated way is in the interest of all aging research fields and the SENS strategy.

Keywords: Calorie Restriction, Ad libitum, Anti-Aging

40 Thymic Tissue Regeneration Using Natural Scaffolds

Tissue engineering offers the potential for treatment of many age related diseases. Decreased immunity is a major problem in the elderly resulting in the increase of infection related problems along with a decreased effectiveness of vaccination. T cells develop and mature in the thymus. During aging, the thymus undergoes involution resulting in a decrease in T cell production. Methods to increase T cell output from the thymus have the potential to improve the immune function of older individuals. Recently, we have initiated studies using tissue engineering techniques to regenerate thymic tissue. Data on production and characterization of thymus scaffolds will be presented. Re-seeding of the scaffolds will be described as well as preliminary data on the in vitro function of the recellularized thymic tissue.

Keywords: thymus, tissue engineering, T cell, scaffold, immunity

41 Differentiation of IPS Cells into NK Cells to Combat Cancer and HIV

Rejuvenation of the immune system through induced pluripotent stem cell (iPSC) technology provides a fundamentally new approach to treat and cure malignancies and chronic viral infections. iPSCs have now been made by hundreds of labs using diverse starting cell populations and reprogramming methods. Our group has used both iPSCs and human embryonic stem cells (hESCs) as starting cell populations to produce natural killer (NK) cells, key components of the innate immune system. NK cells isolated from peripheral blood (PB-NK cells) are used for adoptive immunotherapy to treat certain refractory cancers such as leukemias. Like PB-NK cells, iPSC and hESC-derived NK cells consist of a mature, homogenous population of cells expressing typical NK cell activating and inhibitory receptors. These iPSC/hESC-derived NK cells are able to kill diverse tumor cells both in vitro and in vivo. Additionally, iPSC/hESC -derived NK cells potently inhibit growth of HIV-infected cells. Currently, we are converting the process to derive NK cells from hESCs and iPSCs to utilize conditions suitable for clinical trials.

Keywords: induced pluripotent stem cells, human embryonic stem cells, natural killer cells, immunotherapy

42 Improved Sequencing as a Sens Accelerant

We are improving DNA sequencing to achieve our goal of turning biology into an information science. Along the way, various SENS approaches will be accelerated by improved DNA sequencing, and we present here specific experimental paths for using the tool in service of SENS. As one example, sequencing offers extreme technical shortcuts in molecular directed evolution techniques, allowing larger populations to be interrogated with fewer rounds of evolution and increased stringency of selection. This will accelerate attempts to find, improve, or evolve enzymes and other catalysts targeting age-related molecular damage. As another example, sequencing will enable better quality control of stem cells in both clinical and laboratory settings. We will discuss these specific experimental strategies and others that leverage improved sequencing to hasten progress toward saving lives via SENS therapy approaches.

Keywords: sequencing, tools, directed evolution, stem cells

43 Plasticity of Aging via External Influences

Several types of interventions provide evidence that aging can be successfully controlled. Now, when numerous life-extending mutations dispelled the long-term myth on immutability of species-specific life span it is a good time to rethink rational for anti-aging strategy. The evidence is currently accumulating that age-related changes in a cell are not the cause, but the consequence of organism's senescence. The normal cells are not isolated in the body, and their functions are regulated by the factors originated outside these cells. The levels of these factors are highly dependent on the current response of living being to external challenges. Therefore the features of organisms are such that their optimal activity is shown in the most probable range of environmental factors characterizing the habitat. These external influences induce organisms to raise its resistance, or to reduce its rate of aging. But most studies of aging are conducted in humans and domestic or laboratory animals, i.e. in conditions where artificial environmental protection is applied. This yields changes in functioning including physiology and behavior, compared to those expected in the wild (i.e. in conditions for which organism was adjusted during evolutionary time). It is possible that new conditions are less adequate to the evolutionary adjusted genetic set up of an organism, which increases the aging rate and reduces longevity. The evolutionary point of view and available experimental, ecological and demographic data indicate that at the appropriate conditions full renewal of an organism is possible in human beings and in other species with the repeated cycles of reproduction.

44 Intra-Chromosomal DNA Sequences and Problem of Aging

A popular theory of aging associates it with the age-related changes of cells due to the shortening of chromosomes because of under-replication DNA in telomeric regions of linear chromosomes. However, linearity of chromosomes not necessarily means the linear organization of their DNA. Moreover, there is reason to believe that the DNA strands with free ends the double helix is an artifact of the methods of its isolation and the norm is always a ring. In super-coiled form a ring of DNA has a rhabdoid form - a simplified analogue of a linear chromosome, complete with histone and non-histone proteins etc. Even a linear molecule of DNA of some viruses has “sticky” ends, which, uniting in pre-replicative period, form circular DNA. Hairpins on the ends of linear DNA of other viruses provide a one-chained ring at a divergence of complementary chains. There are hypotheses about the circular organization of the hereditary material in higher eukaryotes, the structure of which has not yet been clarified. In any case, it is conceivable that the shortening of telomere regions of chromosomes is not a passive process associated with steric peculiarities of their structure and the process is actively regulated positive (telomerase, tankyrase, etc.) and negative (TRF1, TRF2 and other) factors, in accordance with needs of the body and under his direct control. Telomeres, as well as many structures of the body, have more than one function. For example, they attributed the function of limiting the number of cell divisions. Telomeres are crucial for proper implementation of meiosis. Chromosomes are attached to the nuclear membrane just by telomeres. Perhaps intra-chromosomal telomeric sequences contribute to the formation of macrostructure chromosome and its stability. This is evidenced by quadruplexes DNA both on the distal parts of chromosomes and on telomeric sequences at sites scattered along the entire length of the chromosome. Just the same intra-chromosomal telomeric DNA sequences can serve as additional anchoring zones giant chromosomes.

45 Super-Longevity: Reality or Myth?

We can indirectly estimate an opportunity to live up to age, which essentially exceeds a canonical centenary boundary. The regularities of statistics of mortality allow making it. The Gompertz law connects the force of mortality M(X) in some cohort with its current age X by means of formula M(X) = M*exp[aX]. Here the parameter ‘M’ is a characteristic force of mortality and the kinetic parameter ‘a’ characterize the rate of aging. Let us consider extreme age (Xe) as the age when M(X) = 1 per year. That is to say that the life expectancy at this age must be approximately only one year. For this reason Xe(a,M) = (1/a)*ln(1/M). It is underestimated estimation for Xe because the force of mortality at advanced age grows more slowly than exponent of the Gompertz law. The second regularity of mortality statistics connects characteristic force of mortality ‘M’ of some cohort with its kinetic parameter ‘a’ (rate of aging) by means of formula lnM = lnB-a*T. Here the parameter ‘B’ is a characteristic force of mortality when a=0 (hypothetical case of full non-senescence) and parameter ‘T’ is a characteristic species-specific age. That is to say that the relationship between of extreme age and the rate of aging parameter ‘a’ is Xe(a) = T+(1/a)*ln(1/B). We know that for human beings B∼0.05 per year and T∼70 years. As ln(1/0.05) = 3 the rough formula for extreme age evaluation is Xe(a)∼70+3/a. As parameter ‘a’ for contemporary developed countries is around 0.1 per year and more an underestimated extreme age for these countries must be around 100 years. We know that for underdeveloped populations (especially for previous centuries) parameter ‘a’ was around 0.03–0.06 per year. It means that the appropriate extreme age should be approximately 120–170 years. So the empirical laws of mortality are compatible with a real possibility of super-longevity. Unfortunately, this rather rare but potentially possible super-longevity is combined with low life expectancy because of high value of parameter M. The artificial combination of low values both for ‘M’, and for ‘a’ will allow increasing both life expectancy and records of super-longevity.

46 Plasmonic Photothermal Therapy of Atherosclerotic Plaque with the Use of Silica-Gold Nanoparticles and Stem Cells Versus Ferro-Magnetic Approach

Background. Some modern angioplasty techniques generally just manipulate the form of the plaque and have some clinical and technical restrictions, relatively high complication rate and restenosis risk. Methods. A total of 101 Yucatan miniature swines were assigned to the three groups (34 pigs into 60/15-70/40 nm silica-gold nanoparticles (NPs) nanogroup, 34 swines – into ferro-magnetic group with 100 nm iron-bearing NPs with delivery in hand of magnetic fields, and 33 - in sirolimus stenting control). Animals in nanogroup were split up to 4 subsets by delivery approach: (1) infused circulating progenitor cells, (2) with infused ultrasound-mediated albumin-coated gas-filled microbubbles, (3) CD73+105+ progenitor cells in the composition of bioengineered on-artery patch, (4) CD73+CD105+ progenitor cells transplanted by manual subadventitial injection. We have used a modified method for the preparation of NPs as described by Lee (2008) and Deng H (2005). Studied proximal left anterior descending arteries were observed by 30 MHz intravascular ultrasound (IVUS). Results. A change of the PV (mm3) immediately after the laser irradiation/in 6 months in groups were −28.9/−56.8%, −30.8/−59.1% and 0/+4.3% (p < 0.01) respectively, and in the subsets in 6 mns reached −54.5/−45.9/−74.7/−61.8% (p < 0.05) respectively. Temperature inside heat-site reached 83/121/98/101 and 102° C (p < 0.01) in the subsets and ferro-magnetic group respectively on data of MRI, however entire plaque warmed up until mean 40.9° C (NS). Some cases of atherothrombosis (3 cases in S2 subset) and distal embolism (8/34, 23.5% in nanogroup, 2/33, 6% in control) were revealed while first 4 weeks. Restenosis confirmed in 3 (9%) swines of stenting group only. An impact over the non-organic part of the plaque (mineral deposits, calcium necrotic core) was predominated in nanogroup (−43.4% vs −12.1% and +3.7% respectively, p < 0.005). The histological analysis has confirmed the ‘burning’/‘melting’ effect with multiple ruptures, necrosis, homogenization of tissue, and consequent possible functional insufficiency of vessel. Anti-inflammative and anti-apoptotic effects, signs of neovascularization and restoration of artery function were predominated in subsets with progenitor cells (p < 0.01). Coronary flow-mediated vasodilation was observed after hyperemia and injection of nitroglycerine (+10.2 and +16.6%, +8.2 and +9.6%, +8.1 and +9.8% in groups respectively, p < 0.05). The highest level of the infiltration with NPs was achieved in the liver that equal to 612 units per cm3 without signs of fibrosis or allergic responses. There were 344, 201 and 99 units per cm3 in the heart, brain, and lungs respectively without any morphological dynamics. Conclusion. Nanoburning is the novel promising technique to demolish and melt the plaque especially in combination with stem cell technologies challenging functional restoration of the vessel. Plasmonics is the high-effective and safe alternative to stenting for angioplasty.

Keywords: nanoparticles, mesenchymal stem cells, plasmonics, atherosclerosis, IVUS

47 Osteoblastic Phenotype Expression of MC3T3-E1 Seeded on Electrospun Poly(E-Caprolacton) Scaffolds Incorporating Poly(Ethylene Glycol)-B-Polyhedral Oligomeric Silsequioxane(POSS)

Inorganic-organic hybrid scaffold composites are being developed with the aim of increasing the mechanical scaffold stability and improving their tissue interactions in cell culture we have prepared the electrospun PCL fibrous webs incorporating amphiphilic PEG-POSS telechelic were prepared via electrospinning. The unique microstructure, morphology, thermal stability and wettability of the resulting PCL/PEG-POSS electrospun nanowebs were investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and contact angle measurement, respectively. The addition of PEG-POSS telechelic strongly affected the fiber diameters and microstructures as well as the wettability, compared to pure PCL and PCL/PEG8.0k electrospun fibrous webs. The potential biomedical applications of such nanospun webs as a scaffolding material were also evaluated in vitro using mouse osteoblast-like MC3T3-E1 cells. The cell adhesion, spreading, and interaction behavior of pure PCL, PCL/PEG-POSS and PCL/PEG nanofibers were explored. It was found that the PEG-POSS telechelics containing electrospun PCL fibrous webs showed higher initial cell attachment than PCL/PEG8.0k due to the higher surface free energy of POSS siloxanes. The prepared PCL/PEG-POSS fibrous scaffolds were found to be nontoxic and to maintain the good adhesion ratio between cells and surface (about ∼93 %) after cell culturing for 24 hrs.

Keywords: hybrid scaffold, polyhedral oligosilsesquioxane (POSS), MC3T3-E1 cell, PCL wettability, nanofiber

48 The Future of Sensfai

The SENS Foundation Academic Initiative (SENSFAI) has recently entered a period of transition. Since its establishment three years ago (at the time as the Methuselah Foundation Undergraduate Research Initiative, or MFURI), the Academic Initiative has provided students interested in doing SENS research with guidance, mentoring, and funding. However, the Initiative's activities have diminished over the last year. SENSFAI is now under new leadership, and its purpose and structure are being rethought. Some important matters will not change; for instance, the development of talented students will remain the Initiative's main focus. For students who wish to launch careers in the development of rejuvenation biotechnologies, there remains no better place to start than the SENS Foundation Academic Initiative. However, the manner in which student projects are organized is being improved. In the past individual students have been assigned to individual mentors, with each student completing his or her discrete project over a period of a few months to a year. Now, students and mentors will be organized into larger groups working in parallel on closely related projects, allowing for greatly enhanced levels of collaboration. This new system should create a small but close-knit community of young scientists involved in SENS research, and the members of this group could conceivably remain in contact for the duration of their careers. In addition to this structural change, the Academic Initiative will become more aggressive in its outreach, particularly on the internet. This will begin with the launch of a completely new website, sensfai.org, and continue with an advertising campaign that will encourage undergraduate students to rethink aging and, ultimately, to focus their efforts on its defeat through scientific endeavor. Over the coming months and years, then, SENSFAI will be restructured, grow its student base, and expand its outreach operations, always in support of the mission of the SENS Foundation.

Keywords: Academic Initiative, Outreach, SENSFAI, Students

49 Aging, Adipose Tissue, and Cellular Senescence

Adipose tissue is at the nexus of processes involved in healthspan and metabolic dysfunction. Progression of age-related fat tissue dysfunction follows different trajectories across different fat depots, with fat becoming redistributed from subcutaneous to intra-peritoneal depots and ultimately ectopic sites, such as liver, muscle, and bone marrow. This is associated with insulin resistance, hypertension, atherosclerosis, strokes, myocardial infarction, cancer, and cognitive dysfunction. The preadipocytes from which new fat cells develop throughout the lifespan switch into a pro-inflammatory, tissue-remodeling state in old age, instead of differentiating into fat cells. In cellular senescence, proliferation becomes arrested and cells acquire a pro-inflammatory senescent secretory phenotype (SASP), with release of chemokines, cytokines, and extracellular matrix proteases. We found that chronological aging, obesity, progeroid states, and serial subculturing result in accumulation of senescent preadipocytes with impaired adipogenesis and a pro-inflammatory phenotype. Senescent cells accumulate to a greater extent in subcutaneous than intraperitoneal fat. TNF alpha and IL-6, which increase in fat tissue with aging and obesity, can induce preadipocyte cellular senescence and pro-inflammatory cytokine and chemokine secretion, potentially spreading senescence from cell to cell. We found that growth hormone/IGF-1 pathway mutations that extend lifespan and delay onset of age-related dysfunction in mice also delay fat redistribution, preadipocyte dysfunction, and cellular senescence, particularly in more IGF-1-responsive subcutaneous fat. We propose a model in which metabolic stress or repeated preadipocyte replication with aging cause cellular senescence in a fat depot-dependent manner, with inflammatory cytokine and chemokine generation, immune response activation, and failure to sequester lipotoxic fatty acids. This model is consistent with recent concepts about cellular senescence as being a stress-responsive, adaptive phenotype that entails metabolic and secretory readjustments. Senescent cells in adipose tissue could have profound clinical consequences because of their pro-inflammatory secretory phenotype, the large amount of adipose tissue in humans, and its central metabolic role.

Keywords: fat tissue, cellular senescence, preadipocyte

50 What Can We Learn About Immortality from Embryonic Stem Cells?

Mouse embryonic stem (ES) cells in culture defy cellular senescence and undergo more than 250 doublings without undergoing crisis or transformation. They tend to maintain a normal karyotype and high genome integrity. Mouse ES cells are thus often considered “immortal” cells. We have recently found that Zscan4, which was originally identified for its 2-cell embryo-specific expression, plays a critical role in the maintenance of genome stability and telomerase-independent telomere elongation. The implication of this finding to stem cell biology and aging research will be discussed.

Keywords: ES cells, Zscan4, telomere, immortality, genome stability

51 Integrated System of Aging Biomarkers

Traditionally evaluation of age-related changes is performed by physiological, functional and psychological tests, by visual examination and some biochemical analyses. There is a big gap between the molecular data of aging and their implementation in practice mainly because aging data is scarce and it gets lost in the stream of bio-medical knowledge. As we know only a few databases exist that concern the molecular aspects of aging and none of them describes age-related changes and phenotype context like cell type or tissues. We propose creation of an open web-based Integrated Information System on Aging Biomarkers. The goals of the System: 1. Systematization of data on age-related changes happening on various levels of organization in humans and model animals 2. Systematization of experimantal data on interventions in aging processes in model animals 3. Integration of clinical data on the impact of various interventions on aging processes in patients 4. Creation of a basis for modeling of aging processes, therapeutic interventions and their impact on patients' health and longevity.

Keywords: Biomarkers of aging, Bioinformatics, Systems Biology

52 Sens Foundation and the Rise of Rejuvenation Biotechnology

53 Postponing Aging and Prolonging Life Expectancy with Knowledge-Based Economy

Articles on human aging studies and their positive results increase the interest of the population in aging research as people see what is in store for them. Let us understand human aging as the accumulation of damage in the body that increases the probability of death. In developed countries the average life expectancy is increasing by one to three months every year. At present the average life expectancy exceeds 82 years in many developed countries: Japan, San Marino, Andorra, etc. It is a result of complex overall improvements in living and working conditions, medical service, as well as developing knowledge of these conditions and health education. Ignoring medical evidence regarding diseases by some elements of the population is a widespread theme in the media. However, most people use some knowledge for living longer.

Keywords: postponing aging, prolonging life expectancy, knowledge-based economy

54 Does Inhibition of Lipofuscin Accumulation Delay Aging?

An inverse correlation between lifespan and lipofuscin (LF) accumulation within postmitotic cells such as neurons and myocardial cells has been clearly shown. LF accumulation inside lysosomes affects the renewal of the cell components by autophagy. Therefore, it can be assumed that diminished LF accumulation would delay aging. LF forms intralysosomally secondary to iron-catalyzed peroxidation. Reduction of intralysosomal redox-active iron levels should therefore reduce LF formation. We have recently shown that apo-ferritin, metallothionein and heat shock protein 70 following autophagy temporarily bind intralysosomal iron and prevent intralysosomal oxidative stress. In the postmitotic retinal pigment epithelial (RPE) cells, heavy LF accumulation is associated with age-related macular degeneration (AMD). Initially, RPE cells disappear and secondarily the photoreceptors as well. Exposure to blue light that enhances oxidation is known to aggravate macular degeneration, while zinc intake has been seen to delay the disease. Interestingly, zinc exposure upregulates metallothionein that binds a variety of metals, including iron. As a result cells become more resistant to lysosomal permeabilization that is a severe effect of intralysosomal oxidative stress. Presumably, LF accumulation which is an effect of less drastic stress would also be diminished.

Keywords: Lipofuscin, Iron, Oxidative stress, Lysosomes

55 Computational Support for Systems Biology of Aging

Senescence arises from complex interactions among diverse sets of biological processes. In this talk I describe our efforts to encode knowledge about aging in formal computer-based models that let one visualize causal influences, revise and expand on these hypothesizes, generate explanations of phenomena, and compare predictions to observations. Users can access, examine, and utilize this content over the World Wide Web, letting it serve as a repository for the community's expanding knowledge about aging.

Keywords: systems biology, formal models

56 Trophokines: Novel Therapies for Senescence

Hepatocyte growth factor (HGF) is a pleiotropic cytokine that promotes cell proliferation, motility, survival, and morphogenesis. HGF binds to its receptor c-Met tyrosine kinase and triggers signal transduction that protects cells against apoptosis and enhances cell growth for tissue regeneration. The profound effects of HGF to prevent cell death and to promote tissue regeneration make HGF an interesting drug candidate for therapeutic use. However, the activation of c-Met by HGF also leads to enhanced tumor metastasis and invasion. This pro-invasive feature of HGF has raised concerns regarding its clinical applications. It would be ideal to separate the beneficial cell-protective signals from the pro-invasive signals of HGF. To test this possibility, Michieli et al., (Nature Biotechnology 20:488; 2002) created a recombinant single-chain chimera consisting of a truncated HGF alpha chain and a truncated MSP (macrophage-stimulating protein, a cytokine with high homology to HGF) alpha-chain linked by a short polypeptide linker, termed Metron factor-1 or MF-1. This chimeric HGF−MSP protein binds to the HGF receptor c-Met and the MSP receptor Ron independently and elicits a biological signal only through Met−Ron heterodimerization. MF-1 prevents liver or renal damage and enhances liver or kidney regeneration, without inducing tumor metastasis and growth. MF-1 has a short plasma half-life (one hour) and is not suitable for development as therapeutic agent with the common i.v. or s.c. administration routes. However, it does provide a unique approach to separate the beneficial organ-protective signaling from undesired pro-invasive signals in HGF-Met pathway. Based on this work we designed and constructed a number of novel heterodimeric Fc fusion proteins (“trophokines”) that can induce Met-Ron heterodimerization. Each of our novel trophokines contains an HGF-Fc and MSP-Fc fusion protein, which form heterodimers through the knobs-to-holes designed into the CH3 domains of their IgG1 Fc regions. Because of this unique design, each trophokine protein contains one c-Met binding site and one Ron binding site to induce c-Met and Ron heterodimerization. These newly designed molecules prevent target cell apoptosis and promote tissue regeneration. By eliminating the pro-invasive or scattering effects of HGF on cells, these novel proteins do not promote tumor metastasis or tumor growth. Most importantly, these novel trophokines have significantly improved plasma half-life and facile manufacturing, purification, and other downstream development processes. These novel proteins are likely to provide therapeutic benefit to patients in a wide range of pathological conditions, such as liver cirrhosis, liver fibrosis, renal failure, wound healing, and chemotherapy-induced liver or renal damage. We have successfully expressed four designed trophokine fusion proteins and the results of preclinical biochemical and biological studies will be presented.

Keywords: HGF, Trophokine, c-met, MSP, cytokine

57 Myostatin: Modulator of Muscle Mass, Mobility and Metabolism

The loss of skeletal muscle is one of the most dramatic changes in the human body consequent to advancing age and is referred to as sarcopenia. It is a primary cause of age-related changes in muscle performance, functional status and metabolic homeostasis. In an effort to counter sarcopenia and its consequences, we have studied strategies to inhibit the muscle-enriched TGF-β superfamily member, myostatin. The purpose of this seminar is to demonstrate how antibody-directed approaches to myostatin have not only increased muscle mass in mouse models of aging and disease, but improved physical function and whole-body metabolism. Ultimately, strategies to disrupt myostatin may provide a means to extend healthspan.

Keywords: Sarcopenia, Functional Decline, Healthspan, Frailty

58 Back to the Temperature

Lowering body temperature (Tb) still remains the most efficient and reliable way for life span extension in exotherms. Several lines of evidence suggest that a moderate decrease in Tb could have a profound longevity-promoting effect in endotherms as well. This effect is a typical example of systemic interventions, with self-rearrangement at all levels of biological organization of an organism. In mediating the effects of Tb on life span, the two major determinants of mammalian longevity, mitochondrial DNA (mtDNA) GC content and metabolic rate (Lehmann et al., Rejuvenation Res 2008; 11:409–417) appear to be of particular significance. Because of the ability of the thermoregulatory center to escape long-term suppression, a periodic decrease in Tb (for example, during sleeping) may represent an alternative and technically feasible strategy. Our calculations show that even a moderate decrease in Tb could result in a dramatic extension of human life span.

Keywords: body temperature, life span extension, mtDNA, metabolic rate, endotherms

59 Parasitic Metabolic Coupling in Cancer: Understanding Oxidative Stress and Autophagy in the Tumor Microenvironment

Cancer cells do not exist as pure homogeneous populations in vivo. Instead they are embedded in “cancer cell nests” that are surrounded by stromal cells, especially cancer associated fibroblasts. Thus, it is not unreasonable to suspect that stromal fibroblasts could influence the metabolism of adjacent cancer cells, and visa versa. In accordance with this idea, we have recently proposed that the Warburg effect in cancer cells may be due to culturing cancer cells by themselves, out of their normal stromal context or tumor microenvironment. In fact, when cancer cells are co-cultured with fibroblasts, then cancer cells increase their mitochondrial mass, while fibroblasts lose their mitochondria. An in depth analysis of this phenomenon reveals that aggressive cancer cells are “parasites” that use oxidative stress as a “weapon” to extract nutrients from surrounding stromal cells. Oxidative stress in fibroblasts induces the autophagic destruction of mitochondria, by mitophagy. Then, stromal cells are forced to undergo aerobic glycolysis, and produce energy-rich nutrients (such as lactate and ketones) to “feed” cancer cells. This mechanism would allow cancer cells to seed anywhere, without blood vessels as a food source, as they could simply induce oxidative stress wherever they go, explaining how cancer cells survive during metastasis. We suggest that stromal catabolism, via autophagy and mitophagy, fuels the anabolic growth of tumor cells, promoting tumor progression and metastasis. We have previously termed this new paradigm “The Autophagic Tumor Stroma Model of Cancer Metabolism”, or the “Reverse Warburg Effect”.

Keywords: hydrogen peroxide, oxidative stress, aging, inflammation, cancer metabolism

60 Intervall-Hypoxia-Hyperoxia-Therapy as a Non-Invasive Tool to Treat Chronic Diseases Based on Mitochondrial Dysfunction

Intervall-hypoxia-therapy (IHT) is an in western medicine so far mostly unknown non-invasive therapeutic opportunity, even so that the physiological principles are at present already quit well understood down to the molecular level. With respective devices the concentration of oxygen in the breathable air is modulated between 21% (sea level) and 9% (+6500 m altitude) for defined periods. A novel element in this concept is the use of hyperoxia. Breathing air in alternating defined cycles with either decreased or increased (36% O2) O2 saturation does not only increase the O2 amplitude, but rather implements the modulation of additional physiological mechanisms, which improve cellular viability. Two principle mechanisms are modulated by hyoxia: a) The ability of the cell to store NO as Di-S-nitrosothiol-iron-complexes (DNIC) improve the physiological NO-reactivity and decreases the production of reactive nitrogene species (RNS). b) The activation of a plethora of hypoxia dependent genes, including those for erythropoetin, VEGF, VEGFR-1 and −2, Endothelin-1, iNOS, HOx-1, glycolysis enzymes, glucose transportes GLUT-1 and −3, iron metabolism, growth factors including TGF-b, PGF, PDGF-ß, HGF, apoptosis regulation factors Bcl-2, Mcl-1, Bax through the regulation of hypoxic-inducible-factor-1a (HIF-1a). On the other side hyperoxia although regulates on the gene expression level: ARE mediated phase-2 detoxifying ant antioxidant enzymes, IL8, IGFBP-2, ICAM-1, IL6, ENaC, p21, CCSP. Intervall-Hypoxia-Hyperoxia-Therapy (IHHT) is an advancement of classical IHT which broadens the impact of controled O2 partial pressure modulation on cell metabolism and discloses new treatment opportunities. The O2 partial pressure at the cellular, even more at the mitochondrial level, is of tremendous impact for the developement/treatment of chronic multi-system ilnesses. For example: The developement of OSA (obstructive sleep apnoe) is the consequence of an unregulated hypoxia. It leads amongst others to a therapy resistant hypertension. With heart-rate-variability controlled IHHT we could reverse the OSA in a patient with long standing OSA to normal and improve blood pressure and resting heart rate significantly. In a doulble-blind placebo controlled study with 10 IHHT treatment units of 35 minutes each we could show that the plasma Q10 level increased by 43% (p < 0.01). In patients with chronic lyme borreliosis symptomatology and immunological parameters could be improved significantly. In patients with chronic diseases accompanied by high oxidative stress and imflammation, including solid cancer, colitis ulcerosa and morbus crohn, nitrosative stress and inflammation could be ameliorate significantly. Do to the regulatory impact on fundamentaly important basic cellular regulatory principles, all involved in the regulation of oxidative/nitrosative stress, chronic inflammation, and mitochondrial energy metabolism, IHHT has a very broad applicability from the treatment of serious chronic diseases to prevention and anti-aging.

Keywords: intervall hypoxia hyperoxia therapy, mitochondrial dysfunction, Coenzyme Q10, oxidative stress, anti aging

61 Association of Renal Damage and Oxidative Stress in an Experimental Fructose-Induced Metabolic Syndrome Model

The prevalence of metabolic syndrome is increasing worldwide in both developing and developed countries. Metabolic syndrome (MS) encompasses a clustering of risk factors for cardiovascular disease, including obesity, insulin resistance and dyslipidemia and have causative roles in the development of chronic kidney disease (CKD). On its turn, CKD leads to end-stage renal disease, cardiovascular disease and death. The relationship between CKD and metabolic syndrome may provide clues to better understand the role of lifestyle-related factors and the age-related decline in GFR. The aim of this study was to evaluate the influence of a DTS-phytocompound that we have recently shown to be protective in diabetic nephropathy (DTS: panax pseudoginseng, eucommia ulmoides, Kyotsu Jigyo, Tokyo, Japan) administration on oxidant-antioxidant balance, protein damage and lipid levels in kidney of rats administered high dose of fructose. Adult male Wistar rats were divided into four groups of 10 rats each. Groups A and D animals received starch-based control diet, while groups B and C animals were fed a high-fructose diet (60 g/100 g). Groups C and D rats additionally received DTS (50 mg/kg/day) for 60 days. The extent of lipid peroxidation, enzymatic and non-enzymatic antioxidants and lipid levels were measured after 60 days. The accumulation of nitrated and oxidatively modified proteins in kidney was assessed by immunohistochemical study. Fructose-fed rats showed significantly higher levels of peroxidation end products, diminished antioxidant status, increased staining for the presence of 4-hydroxy-2-nonenal, 2,4-dinitrophenol and 3-nitrotyrosine protein adducts and lipid accumulation in kidney. DTS administration significantly decreased such renal alterations as well as mineral status abnormalities (partial normalization of higher Zn and lower Cu, Mg and Mn levels in liver and kidney of untreated MS-rats). The benefits of DTS in this model suggest the therapeutic use of DTS to counter the kidney changes associated with metabolic syndrome. Gender-related analysis of this interventional model is under way.

Keywords: metabolic syndrome, renal disease, DTS

62 Poli-Mix Functional Food Enhances Steady-State Bioenergetic Status Independently Of Age: an Experimental Study

The aim of the present study was to ascertain whether a poli-mix functional food VCC1-99 (a mixture of 42 extracts of non-GMO fruits and vegetables, one ml containing 400mg carbohydrates, 0.04mg protein, 0.04mg fat, 8mg vitamin C, 1.2mg vitamin E, 0.5mg coenzyme Q10, 6mg royal jelly, 5mg L-carnitine, Vibracell, Named srl, Lesmo, Italy) could affect hepatic adenosine triphosphate (ATP) level in mice aging from 1 to 24 months. Hundreds-twenty healthy Balb-C mice raised in a controlled vivarium were divided into 3 separate age-groups (40 mice each): young (one month), middle-age (12 months) and aged (24 months). All mice were given water ad libitum and standard chow. Each age-group was further split into 2 different treatment schedule: a) 1ml of VCC1-99 into drinking water or b) 1ml of a solution containing similar quantities of vitamin C, E and coenzyme Q10 as an antioxidant control (AC). Further 5 mice gor each age-group served as untreated control (UC). Animals were supplemented for 3 weeks. Steady-state hepatic ATP was assessed by using 31P-NMR spectroscopy before treatment and at weekly intervals. Fasting mice were anaesthetized with 50mg/kg pentobarbital intraperitoneal injection and immobilized on a shielding belt embedded with paramagnetic beads to minimize signal interference from abdominal muscles. The stability of magnetic field was tailored by adjusting the shim coils according to the flee induction decay of liver water protons. A calibration of NMR peaks of metabolic phosphorus for each mouse against a reference standard was also carried out. Normal ventilation of fresh air (1.0L/min) through a face mask was also assured during the test. Out of the overall NMR spectrum, variables considered were: β-ATP/ref. peak, inorganic phosphorus (Pi)/ref peak and β-ATP/Pi. No liver pH (calculated as chemical shift difference of Pi and α-ATP) changes were recorded irrespective of the age and treatment. As compared to UC, AC mice showed a significant enhancement of β-ATP/ref. peak ratio only in aged mice starting from 2nd week of treatment (+9.3–9.6% p < 0.05) and of β-ATP/Pi ratio on the 3rd week of middle-age (+18%, p < 0.05) mice together with a significant decrease of Pi/ref peak ratio (p < 0.05). On the other hand, treatment with VCC1-99 showed to significantly enhance β-ATP/ref. peak ratio and β-ATP/Pi ratio (p < 0.01) in all age-groups and throughout the observation period. Such enhancement was significantly higher when compared to AC. Accordingly, it occurred a significant decrease of Pi/ref peak ratio (p < 0.05) but this values in middle-age and aged mice was comparable to what observed in AC mice. These findings suggest that VCC1-99 is able to markedly enhance the hepatic production of high-energy triphosphates with higher steady state energy status. These findings are likely to be extended to overall better aerobic capacity either in high-metabolism turnover conditions observed in youth and in decreased gas exchange anaerobic threshold situation taking place in the elderly.

Keywords: functional food, liver steady state energetic status, NMR, Antioxidant, elderly

63 Camelid Antibodies in Medicine

The camelids - camels, llamas and their relatives - are unique in producing antibodies that have no light chain. These single polypeptide chain antibodies bind to antigens as strongly as conventional, multi-chain antibodies, but have lower molecular weights, are easier to engineer and produce, and more stable. They are attracting a lot of interest as potential therapeutics and diagnostics. For therapeutics they can be engineered easily into small proteins that have all the potency of a conventional antibody, but with enhanced ability to penetrate the intercellular space, and so get at cellular targets. Camelid antibodies may also be better suited to penetrating the blood-brain barrier, and BBB-penetrating camelid antibodies have been developed suggesting a potential to target neurodegenerative disease. Their greater stability suggests that they would be more easily delivered orally or nasally rather than by injection. Some have speculated that their greater stability and (potential) low production costs mean they could be used outside high-tech, high-cost medicines in such applications as topical anti-bacterial agents or even in shampoo. For diagnostics and sensor uses their greater stability to denaturants and heat means that they can be used on sensors and point-of-care devices more readily. This talk will summarise the biology of these valuable biomedical tools, review the work being done at Cambridge on using them, and discuss how near we are to applying camelid antibodies as therapeutics to age-related disease.

Keywords: antibody, antibody engineering, camelid

64 Trachea and Oesophagus Tissue Engineering

The ramifications of stem cell research and therapy are enormous. We provided evidence that stem cells can be successfully applied to laryngo-tracheal and windpipe transplants for adults and children but why not imagine to use stem cell therapy in patients with otherwise untreatable end-stage diseases of the respiratory system? The debate still ranges on about the use of embryonic or adult stem cells. However, many are unaware, or ignore, that the adult body naturally carries its own stem cells (stem cell niches), and that these can be recruited or mobilized to repair or restore damaged tissues and organs. Our ongoing phase I-II clinical trials use “dormant” stem cells that are either taken or recruited directly from the patients body to repair untreatable tissues or organs, thus nearly eliminating any ethical or religious arguments to the stem cell treatment, and regulations.

Keywords: tissue engineering, trachea, larynx

65 Leucocyte Transfusions as a Novel Immunotherapy for Cancer Patients

Spontaneous regression/complete resistant (SR/CR) mice are a line of cancer-resistant mice that are capable of resisting large doses of transplanted lethal cancer cells [1 –3]. The basis for this powerful resistance to cancer cells is leukocytes that are capable of detecting, infiltrating, and killing cancer cells within a few hours of exposure [1]. The major component of this anti-cancer response resides in granulocyte, monocyte, and natural killer cell fractions that constitute innate cellular immunity [1,2]. The adoptive transfer of donor leukocytes from SR/CR mice can confer protection against future exposures to cancer cells, as well as the elimination of established malignancies without any further manipulation in cancer-sensitive wild-type (WT) recipient mice [2]. These prior studies suggested the possibility of developing a similar leukocyte transfer platform for humans if humans with anti-cancer activity similar to the SR/CR mice could serve as leukocyte donors. We obtained an FDA IND and WIRB approved Clinical trial for the novel Granulocyte/Leucocyte transfusion in Cancer Patients using young healthy donors. The development and ongoing status of this immunotherapy treatment for cancer will be presented.

Keywords: Leucocyte, Infusions, Cancer, Young healthy donors

67 Cardio-Protective Effect of Biofermented Nutraceutical on Endothelial Function in Healthy Middle-Aged Subjects

Endothelial dysfunction is independently related to future cardiovascular events and the prognosis of cardiovascular diseases. Although the inner mechanisms remain uncertain, an imbalance between increased oxidative stress and impaired antioxidant mechanism, especially inactivation of nitric oxide (NO) by superoxide anion and other reactive oxygen species, may contribute to the promotion of atherosclerosis and the pathogenesis of cardiovascular disease. Thus the aim in this study was to test a biofermented nutraceutical (FPP) which has been previously shown to positively modulate NO. All 42 participants were healthy middle-age subjects and not taking any drugs or supplements and who maintained their usual diet and lifestyle, excluding all beverages and supplements that could influence vasomotor function. Subjects were given FPP 3g three times a day for 6 weeks and tests were repeated at 3 and 6 weeks while the control group was given a placebo. Endothelial function was measured on each individual three times, at start of treatment and twice during the four weeks of treatment with an interval of two weeks. Flow-mediated dilation (FMD) will thus be measured as the percent change in brachial artery diameter from pre-cuff inflation to 60-seconds post-cuff release as uniform as possible for each visit. Blood samples for erythrocyte content and inflammation, samples for lipids, blood glucose and inflammation were taken at each visit. Blood samples will be collected from the subjects at baseline and before FBF measurement and assessed for NO compounds (Nox: NO2− + NO3−) plasma levels and asymmetrical dimethylarginine (ADMA). In the interventional group overall FMD significantly increased from 4.2% to 7.3% (p < 0.05) in the interventional group whereas in the control group FMD was stable. A significant increase in plasma nitric oxide and a decrease in ADMA, malondealdehyde and 4-hydroxynonenal, C-reactive protein, monocyte chemotactic protein-1, and soluble CD40 ligand levels were detected after consumption of FPP (p < 0.01). Although these data need further larger studies, it appears that, at least in healthy individuals, nutraceutical intervention is likely to positively act on significant cardiovascular parameters and thus can be considered in the armamentarium of a pro-active medical strategy of age-management.

Keywords: fermented papaya preparation, endothelial dysfunction, nitric oxide, flow mediated dilation, pxidative stress

68 Lysosomal Enhancement Strategies for the Treatment of Oxysterol-Induced Cytotoxicity

7-ketocholesterol (7KC) is a cytotoxic oxysterol that plays a role in many age-related degenerative diseases. 7KC formation and accumulation occur in the lysosomes in a number of cell types, hindering enzymatic transformation, and increasing the chance for lysosomal membrane permeabilization. We assayed the potential to mitigate 7KC cytotoxicity and enhance cell viability by transiently transfecting human fibroblasts to overexpress several 7KC-active enzymes. One of our engineered constructs, a lysosomally-targeted cholesterol oxidase that lacked isomerization activity, significantly increased cell viability at concentrations up to 50 μM 7KC relative to controls. Additionally, both overexpression of the lysosomal membrane protein LAMP1 and treatment with 0.9% hydroxypropyl β-cyclodextrin attenuated 7KC-mediated cell death. The overexpression of cholesterol oxidase, sterol 27-hydroxylase, and 11β-hydroxysteroid dehydrogenase, which were localized to the cytoplasm, mitochondria, and endoplasmic reticulum respectively, resulted in either decreased cell viability or no significant change from controls. These results indicate that the lysosome should be the target for treatment of oxysterol-mediated toxicity, and support the utility of using novel lysosomally-targeted enzymes for therapeutic benefit.

Keywords: oxysterol, lysosome, 7-ketocholesterol, atherosclerosis

69 Rejuvenating the B-Lymphocyte Lineage in Aging

Aging is associated with a decline in B-lymphopoiesis in the bone marrow and accumulation of long-lived B-cells in the periphery. These changes decrease the body's ability to mount protective antibody responses. The mechanisms underlining these alterations are poorly understood. We found that the age-related alterations in the B-lineage reflect homeostatic pressures that are imposed by the accumulating long-lived B cells. Thus, a continuous demand for peripheral B cells renders the BM devoid of senescence, and depletion of B cells in old mice revives B cell production in the bone marrow and rejuvenates the peripheral B cell compartment. Collectively, our studies suggest that immunosenescence in the B-lineage can be reversed to enhance immune responsiveness in aging.

Keywords: B-lymphocytes, bone marrow, aging, homeostasis

70 The Health, Mortality, and Economic Impacts of Decelerated Aging

Scientists are now pursuing the means to slow ageing in people, but there has been comparatively less effort in understanding the health and economic consequences of success. In this paper we will address the short-term (10 years) and long-term (50 years) effects of decelerated ageing on the health and mortality of future generations of older persons, as well as the economic impact on individuals and populations. We use a dynamic simulation approach based on modeling the progression over the life course of Future American based on the Future Elderly Model. The model also tracks taxes, pensions and health care costs, which allows us to quantify the fiscal consequences of decelerating aging. We contrast a number of scenarios involving the deceleration of the incidence of various health conditions and mortality.

Keywords: deceleration aging, long-term forecasts, economic impacts

71 A Biomimetic Model Aimed at Recovering Learning in a Brain Damaged Animal: Converging Neuroscience with Technology

Life quality and life span are seriously compromised by numerous brain diseases. Currently, rehabilitation is based largely on behavioral manipulations directed at activation of brain ‘self-repair’ processes. Future advances are expected to include biological manipulations such as genetic manipulation and stem cell-based therapy that promote neuronal recovery. Another feasible strategy is replacement of defined neuronal microcircuits by synthetic analogs. Decades of iterations between scientific inquiry, technological development and escalating clinical demands have advanced the techniques of monitoring and stimulation of localized brain sites. To date, deep brain stimulation successfully ameliorates a range of Parkinson and OCD symptoms and deep brain recording is used to detect the source of epileptic fits. The hope is that these two techniques can be interfaced by a real-time processor and used as a closed loop system with the brain. Our objective was to test the feasibility of the closed-loop hybrid methodology for rehabilitation of brain functions by replacement of a damaged brain microcircuit. Specifically, we reasoned that a function lost after localized brain damage can be rehabilitated by a biomimetic device that analyzes on-line the inputs to the damaged microcircuit and sends the outcome of this analysis to the output of the damaged microcircuit. The cerebellum is a good choice for testing the feasibility of the closed-loop rehabilitation methodology. In particular, the cerebellum is well conserved across the mammalian kingdom and is comprised of recurrent microcircuits sharing similar anatomical and physiological architecture. Cortical Purkinje and deep nuclei neurons are sites of convergence of sensory signals originating at two brainstem nuclei; the pontine nucleus (PN) conveys telesensory signals and the inferior olive (IO) conveys somatosensory signals. Associative exposure to the above signals initiates cerebellar learning, expressed as long term depression (LTD) of the synapse conveying the telesensory signals to the Purkinje and as emergence of a neuronal model of the learned response at the cerebellar deep nuclei. In the present study we replaced in a rat the cerebellar microcircuit essential for acquisition of eyeblink response by a biomimetic cerebellar model that received its sensory inputs from the PN and IO precerebellar nuclei and which send its output to the brainstem motor nucleus. Results demonstrated synaptic plasticity in the model and behavior which was compatible to the dynamics of acquisition and habituation in a normal rat. We now look to advance the technologies necessary to support years long hybrids, to advance the understanding of the sensory coding that is required for the detection of the sensory events from electrophysiological records. Finally, we will test whether animals will adopt the hybrid technology or regress to alternative behavioral strategies in ecological conditions.

*This study was supported by the European Community's 7th Framework Program (216809), the Converging Technologies – ISF Grant (1709/07), and by The Center for Complexity Science (GR2004-065). PIs were del Giudice P., Guger C., Marcus M., Mintz M., Shacham Y., Silmon A., Vershure P., Yaron H.M.

Keywords: brain, damage, hybrid, rehabilitation, rat

72 Cryonic Life Extension

Cryonics involves the cryopreservation of humans as soon as possible after legal and clinical “death”. Legal and clinical death differ importantly from biological death or true (irreversible) cessation of function. It is therefore a mistake to portray cryonics as an alternative to cremation or burial. It is true that cryopreserved people are not alive but neither are they dead. Cryonics should be seen as part of the field of life extension. Cryonics enables the transport of critically ill people through time in an unchanging state to a time when more advanced medical and repair technologies are available. Even after “longevity escape velocity” has been attained and aging has been largely tamed, cryonics will continue to be needed for people who die of accidents or diseases for which there is no cure at the time.

Keywords: cryonics, longevity escape velocity, cryopreservation

73 The Effects of DNA Damage Response and Repair Genes Overexpression on Drosophila Melanogaster Lifespan

DNA is a key molecular target for age-related deterioration. DNA damages (abasic sites, oxidized bases, deaminated purines, glyoxal and methylglyoxal adducts, DNA-protein crosslinks, indigenous DNA adducts, and double-strand breaks, but not O6-methyldeoxyguanine and single-strand DNA breaks), somatic mutations (transitions and transversions, transposon excisions and insertions) and chromosome aberrations are accumulated with age simultaneously with the decrease of efficiency of some DNA repair pathways, including mismatch repair, base and nucleotide excision repair, non-homologous end joining. Age-related increase of DNA damage level leads to alterations in gene expression, cellular senescence and cancer. The mutations in genes involved in maintenance of DNA fidelity (WRN, RECQL4, BLM, lmna, XPA, -B, -C, -D, -F, -G, CSA, CSB, ATM, and NBS1) leads to accelerating aging syndromes. The dysfunction of numerous genes results in decreased lifespan, but this can be caused not only by the acceleration of healthy aging, but also by specific aging-independent pathologies. Studies of the lifespan of animals with overexpressed candidate longevity-assurance genes appear more promising. A recent our study has shown that overexpression of the stress response and DNA repair gene GADD45 in nervous system decreases the number of spontaneous single-strand DNA breaks and prolongs the maximum lifespan (27–33%) of Drosophila flies without the decreasing of life quality parameters (physical activity or reproduction). We have tested as well the life span and the health span of fruit flies with overexpression of DNA damage response genes (component of 9-1-1 complex Hus1, checkpoint kinase Chk2), homologous recombination genes (Rad51, Brca2), Werner syndrome gene Drosophila homolog WRNexo, excision nucleotide repair genes (XPG and ERCC1/XPF homologs). We would like to thank the Science for Life Extension Foundation (Mikhail Batin) and the Institute of Biology of Aging (Alex Peregudov) for financial support of the project.

Keywords: Drosophila, longevity, DNA repair, DNA damage response, genes overexpression

74 Lung Regeneration: Progress and Challenges

Because adult lung tissue has limited regenerative capacity, lung transplantation is the primary therapy for severely damaged lungs. To explore whether lung tissue can be regenerated in vitro, we treated lungs from adult rats using a procedure that removes cellular components but leaves behind a scaffold of extracellular matrix that retains the hierarchical branching structures of airways and vasculature. We then used a bioreactor to culture pulmonary epithelium and vascular endothelium on the acellular lung matrix. The seeded epithelium displayed remarkable hierarchical organization within the matrix and the seeded endothelial cells efficiently repopulated the vascular compartment. In vitro, the mechanical characteristics of the engineered lungs were similar to those of native lung tissue, and when implanted into rats in vivo for short time intervals (45–120 min), the engineered lungs participated in gas exchange. Although representing only an initial step toward the ultimate goal of generating fully functional lungs in vitro, these results suggest that further investigation of this approach is warranted.

Keywords: Lung, Tissue Engineering, Extracellular Matrix

75 Rebalancing The Aging T-Cell Pool to Improve Resistance to Infection in the Old Age

Aging is associated with an increased susceptibility of older individuals to new and emerging infections; poor responses to vaccination compound this vulnerability. These problems may be further exacerbated in individuals with lifelong persistent viral infections, and human studies have highlighted an association between the Herpesvirus infections (most notably the cytomegalovirus, CMV) and pronounced signs of immune aging. In both mouse and man, repeated interactions between reactivating viruses such as CMV and antiviral T cells leads to memory T-cell inflation (MI) with increasing accumulation of these cells over the lifespan. It was hypothesized that MI may exact a price for the immune system: competition between inflating, CMV-specific memory T-cells and naïve T-cells supposed to defend against all other infections may impair the maintenance of a diverse naïve T-cell pool, consequently leaving the individual at a disadvantage when exposed to a new pathogen. We have directly tested this hypothesis using a mouse model of lifelong persistent infection. Young mice were infected with different herpresviruses, and then monitored for MI until age 20 months before challenge with Listeria monocytogenes. Subsets of each cohort underwent therapeutic interventions including: (1) antiviral treatment; (2) partial lymphocyte depletion to make space for new T-cells, (3) Kepivance/KGF to increase new T-cell generation, or (4) young naïve T-cell infusion in attempts to “rebalance” the naïve T-cell repertoire. In this model, lifelong persistent viral infection did not further impair the reduced bacterial clearance or magnitude of the anti-Listeria response in old mice, however, it altered the effector cytokine profiles of Listeria-specific T cells. Several types of treatment modestly restored the appropriate effector cytokine profiles. Importantly, young T-cells infused into aged recipients showed a “young” cytokine profile, indicating the aged environment can support their effector development. Implications of these interventions for future strategies to improve the quality of immune responses and resistance to infection in aged individuals will be discussed.

Keywords: Immune aging, CMV, Immune rejuvenation

76 The Role of Transglutaminase-2 in Vascular Stiffness

Aging is associated with alterations in the properties of all elements of the vascular wall including endothelium, vascular smooth muscle, and matrix. These changes result in increased vascular stiffness and isolated systolic hypertension. Both dynamic and structural changes (e.g., fracturing of elastin, increased collagen content, and accumulation of advanced glycation end products) have been described in aging. Vessel structure can additionally be regulated by alterations in matrix crosslinking. Transglutaminases (TGs) are enzymes that catalyze a transamidation reaction, leading to the crosslinking of proteins through the formation of the stable N-3-(³-glutamyl) lysine isopeptide bonds. At least 3 of the 9 members of the TG superfamily are expressed in vascular systems. Tissue transglutaminase (TG2) in particular is ubiquitously expressed in vasculature. TG2 is confined mainly to the cytosol, and a portion of it is associated with the cell membrane and secreted out of the cell to the extracellular matrix (ECM) through an as yet unidentified mechanism where it is activated and catalyzes the transamidation reaction. Crosslinking activity of TG2 has been shown to be inhibited in vitro by NO through protein S-nitrosylation of key cysteine residue. It is well established that endothelial NO bioavailability diminishes with aging. Decreased endothelium-dependent NO synthesis in the aging vasculature leads to reduced TG2 S-nitrosylation and, thus, enhanced transamidation activity. This, in turn, results in increased crosslinking of matrix proteins and, consequently, to decreased compliance and increased stiffness of aging conduit blood vessels. Thus, NO regulates vascular stiffness characteristics by post-translational modification of a matrix modifying enzyme. TG2 remains a potential target for therapy in age-related systolic hypertension.

Keywords: tissue transglutaminase, S-nitrosylation, S-nitrosation, aging, vascular stiffness

77 Mitosens: Allotopic Expression of Mitochondrial Genes Using a Co-Translational Import Strategy

The mitochondrion contains its own genome and encodes 13 proteins that are essential for the respiratory chain to function properly. Congenital mutations in many of the mitochondrial genes are the cause of serious disease phenotypes including diabetes, blindness, dementia, ataxia, epilepsy, and many other neurological disorders. Somatic mutations also accumulate in the mitochondria with normal aging. Allotopic expression of mitochondrial genes in the cell's nucleus is one approach to rescuing mitochondrial mutations. In our strategy, we utilize 5′ and 3′ elements to target mRNA to the outside surface of the mitochondria; this approach is a refinement of simple allotopic expression and is hypothesized to result in the co-translational import of the encoded proteins into the mitochondria. This approach is hypothesized to overcome obstacles (such as clogging of the mitochondrial import machinery by hydrophobic proteins) that many groups have experienced. Thus far, we have stably transfected 5 of the 13 mitochondrial genes into the nuclear genome of human cell lines and are characterizing the expression and function of these exogenously expressed genes. We will discuss current progress and future plans for replacing and/or making redundant the entire mitochondrial genome. We will also discuss potential applications of the MitoSENS approach in treating mitochondrial diseases, as well as the diseases and pathologies of aging.

Keywords: Mitochondria, allotopic

78 Reclinomonas Americana mtDNA as a Source for Allotopic Expression of Human mtDNA

Age dependent accumulation of mitochondrial DNA (mtDNA) mutations is generally accepted as one of the factors involved in the mechanisms of aging. Therefore, allotopic expression of mitochondrial genes (the transfer of mitochondrial genes to nucleus) is proposed to rescue the cells from the deleterious effects of mtDNA mutations. In this study we analysed the possibility of the usage of Reclinomonas americana (R. americana) mtDNA as an evolutionary protected template for allotopic expression of human mtDNA encoded OXPHOS subunits. MtDNA of R. americana is one of the biggest mitochondrial genomes and represents very early stages of mtDNA evolution. R. americana mtDNA uses standard genetic code. In this study we compared 1) R. americana and human mtDNAs, 2) 25 subunits of OXPHOS complexes encoded by R. americana mtDNA and their both nuclear and 13 mitochondrial human orthologs. The DNA similarity was only found in one short region which is a part of COX3 gene. The protein similarity was more conserved than DNA. The similarity of human nuclear orthologs of R. americana mtDNA genes was higher than mitochondrial orthologs. Our results suggest that mtDNA has a higher mutation rate than nuclear DNA, and the evolutionary change of human mitochondrial genetic code usage had mutational consequences that created one of the barriers in the transfer of mitochondrial genome to nucleus. Therefore R. americana mtDNA can be used as a source or template for allotopic expression in humans. For example, it can provide important insights during the design of the new subunit DNAs to functionally replace the existing ones.

Keywords: Aging, Mitochondrial DNA, Reclinomonas Americana, Allotopic expression

79 Pulsed Electromagnetic Fields for Cell Injury and Repair

At the cellular level aging is a process of declining capacity for repair of cell injury. With aging there is cumulative, unrepaired or poorly repaired cell injury. This is “death by a 1000 cuts.” Cell injury results when cells can no longer adapt to stress, have unrecoverable exposure to damaging agents or suffer from intrinsic abnormalities, whether genetic or nutrient-based. Cell injury can progress from milder reversible states through more severe irreversible conditions leading to tissue and or organ failure of varying degrees, and finally senescent death. Promoting longevity requires intervention of the underlying causal conditions of cell injury where possible and facilitating cell recovery and repair at the earliest stages of cell injury. In the stages of reversible injury there is reduced oxidative phosphorylation with depletion of ATP, cellular edema caused by changes in ion and water flows, mitochondrial and cytoskeleton alterations and DNA damage. In the ideal situation, ongoing health maintenance, including mitigating known causal circumstances, and also proactive use of low intensity, low frequency, pulsed electromagnetic fields (PEMFs), is predicted to improve the slope of aging by reducing and/or reversing various degrees of cell injury. PEMFs are known to pass uninhibited through the body, while inducing charge in cells and tissues, consequently affecting biochemical and physiologic processes in the direction of reducing cell injury, and therefore aging. The biologic benefits of PEMFs are mediated through improvements in various physiologic processes including production of nitric oxide, pain and inflammation reduction, improved circulation, enhanced cellular membrane function and metabolism, communication and replication and growth and repair. It will be demonstrated that PEMFs applied clinically and for personal health maintenance can reverse many of the components of cell injury and improve longevity. Additionally the presentation will focus of the many different ways that PEMFs improve biochemical activity at the cellular level and allow nutrients and other life extending chemistry in the body to migrate more freely to be more functionally useful. Low intensity, low frequency PEMFs should be considered part of a comprehensive program to reduce suffering and enhance higher quality longevity.

Keywords: Cell injury, Pulsed electromagnetic fields, Charge induction, Enhancing longevity, Cell recovery and repair

80 Decrease of PGC-1ALPHA/PGC-1BETA Dependent Signaling Cascade of Mitochondrial Biogenesis in Old Rat Liver is Prevented by Chronic Supplementation of Acetyl-L-Carnitine

Mitochondria are a major source and target of endogenous Reactive Oxygen Species (ROS) that are produced as normal by-products of the electron transport system. Decay of mitochondrial function is a major contributor to aging process. The age-related overproduction of ROS compared to the scavenging efficiency of the antioxidant cellular system leads to the onset of an oxidative stress condition. The accumulation of oxidatively damaged macromolecules may alter the mitochondrial bioenergetic function and may play a key role in the functional decay of cells and tissues that is among the causes of aging and of the associated degenerative pathologies. Acetyl-L-carnitine (ALCAR) is a biomolecule able to limit age-linked mitochondrial decay in brain, liver, heart and skeletal muscles by increasing mitochondrial efficiency. Aim of this study was to test if long-term ALCAR supplementation to aged rats was able to activate the PGC-1alpha/PGC-1beta dependent mitochondrial biogenesis in liver tissue. Young (6-month old) and old rats (28-month old) were used. Old rats were daily supplemented with ALCAR for 1 and 2 months. MtDNA content was quantified by Real Time PCR and citrate synthase activity was determined as marker of mitochondrial mass content. The levels of PGC-1alpha, PGC-1beta, TFAM, NRF-1 as well as of ND1 (a mitochondrial DNA encoded subunit of mitochondrial respiratory chain) and of COX IV (a nuclear DNA encoded subunit of mitochondrial respiratory chain) were quantify. Furthermore, the levels of the antioxidant enzymes Peroxiredoxin III, exclusively located into mitochondria and involved in the regulation of mitochondrial H2O2 concentration, and of SOD2 as well as of Drp1 and of MFN2, two proteins implicated in the processes of mitochondrial fission and fusion, were also measured. The obtained results revealed that ALCAR administration to old rats is able to prevent in liver the age-dependent decrease of mitochondrial decay. In particular, ALCAR treatment was able to up regulate the PGC-1alpha/PGC-1beta dependent mitochondrial biogenesis activating pathway, and to increase the mtDNA content and the mitochondrial mass, reduced in the liver of old rats.

Keywords: Aging liver, Acetyl-L-carnitine, Mitochondrial biogenesis, PGC-1alpha/PGC-1beta, Peroxiredoxin III

81 Cells and Niches Transplantation as Approaches to the Immune System Rejuvenation

The manifestation of age-related changes of the immune system is one of the key factors of the whole organism aging. In contrast to many other organs and systems, the immune system aging begins in an early age and has more pronounced changes. The immune function decrease is explained by many factors, including thymus dysfunction, decrease in the proportion and function of T cells, changes in hematopoietic stem cell pool, etc. To prevent these changes we used various model approaches. The model of heterochronic parabiosis demonstrated that: i) age changes of the immune system are not passive, and regular interchange by blood factors, circulating lymphocytes and stem cells between heterochronic partners does not lead to the old system rejuvenation; ii) the main age changes relate to the cells of lymphoid organ microenvironment, which provoke disturbance of the T-cell peripheral differentiation and, consequently, decrease of the immune functions and life expectancy in young heterochronic partners. The model of neonatal lymphoid organ transplantation, allowing form a lymphoid niches with young donor origin, demonstrated that the optimal development of the neonatal spleen occurs only in the absence of its own spleen in young recipients. Additional thymus graft is capable of greater stimulation of the functional activity of splenocytes from neonatal graft versus own organ in old animals. The report discusses the possible mechanisms capable to improve immunological functions in aged animals which depend on a combination of transplanted organs and age-related interaction between them.

Keywords: Immunity, aging, parabiosis, niche transplantation

82 The Pro-Atherogenic Phenotype in Ageing – Lipoprotein B100 and A1 Leading The Dance into Decline of Vascular Health by Oxidative and Non-Oxidative Mechanisms

Lipoproteins are essential vehicles for conveyance of lipids and cholesterol in human metabolism. Apolipoprotein B100 (apoB100) is present in very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL). VLDL, IDL and chylomicrons deliver lipids to the peripheral cells and VLDL, IDL and LDL deliver cholesterol to the liver and peripheral tissue. Apolipoprotein A-1 (apoA1) is the major lipoprotein of high density lipoprotein (HDL). HDL conveys cholesterol ester from peripheral sites to the liver and thereby has anti-atherogenic activity. There are density subfractions of HDL: HDL1 (minor), HDL2 and HDL3; and also minor fractions of nascent ApoA1 and pre-β-HDL - forms that occur prior to lipid and cholesterol ester loading. Malfunction of ApoB100 and ApoA1 contribute to the development of cardiovascular disease (CVD) – the major cause of death in the human population worldwide. CVD is driven mainly by increased arterial atherosclerosis. Increased risk of atherosclerosis is associated with high levels of LDL and more particularly with high levels of small, dense LDL. Small, dense LDL has increased affinity for arterial proteoglycan and cell surface non-LDL receptor binding sites. Atherosclerosis is also driven by decreased levels of HDL. In ageing, the plasma half-life of LDL increases, leading to increased exposure of LDL to the oxidative environment of the plasma and extracellular fluid. In ageing also plasma levels of HDL decline. This is due to increased catabolism of apoA1 – mostly by destabilization of HDL2 and degradation in the kidney and liver. Atherogenicity of LDL increases and levels of HDL decrease with decline in glucose tolerance. Recent research has shown that damage to apoB100 by non-oxidative mechanisms produces atherogenic transformation of LDL to small, dense LDL which then binds with increased affinity to the arterial wall where it is susceptible to oxidation and accumulation in cells of atheromatous plaques. Similarly, non-oxidative damage to apoA1 of HDL may cause de-stabilization and increased catabolism. Such modifications increase in ageing as the rates of formation and rates of detoxification of modifying agents decline. The involvement of non-oxidative mechanisms in early-stage escalation of atherogenicity of LDL and de-stabilization of HDL may explain why antioxidant therapy has often been ineffective in early-stage prevention of CVD and point the way to new strategies for therapy.

Keywords: Cardiovascular disease, Lipoprotein, Low density lipoprotein, High density lipoprotein, Methylglyoxal

83 Very Small Embryonic Like Stem Cells (VSELs) – Our Key to Longevity and Passkey to Cancer?

Pluripotent very small embryonic/epiblast derived stem cells (VSELs) as we hypothesize are deposited at begin of gastrulation in developing tissues and play an important role as backup population of pluripotent stem cells (PSCs) for tissue committed stem cells (TCSCs). Molecular analysis of adult bone marrow (BM)-derived purified VSELs revealed that they i) express pluripotent stem cells markers e.g., Oct4, Nanog, Klf-4, SSEA-1 ii) share several markers characteristic for epiblast as well as migratory primordial germ cells (PGCs), and iii) posses a unique pattern of genomic imprinting (e.g., erasure of differently methylated regions at Igf2-H19 and Rasgrf1 loci and hypermethylation at KCNQ1 and Igf2R loci). We hypothesize that these pluripotent stem cells play an important role in tissue/organ rejuvenation, and their proliferation and potentially premature depletion is negatively controlled by epigenetic changes of imprinted genes that regulate insulin factor signaling (Igf2-H19 locus, Igf2R and RasGRF1). Since the attenuation of insulin/insulin growth factor (Ins/Igf) signaling positively correlates with longevity, we propose, based on our experimental data in animal models, that gradual decrease in the number of VSELs deposited in adult tissues, which occurs throughout life in an Ins/Igf signaling-dependent manner is an important mechanism of aging. In contrast, a decrease in Ins/Igf stimulation of VSELs as seen for example during calorie restriction extends the half life of these cells in adult organs and has a beneficial effect on life span. On other hand the same epigenetic changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues and prevent them from teratoma formation. In contrast epigenetic changes/mutations that lead to activation of imprinted genes could potentially lead to tumor formation by these cells. Mounting evidence accumulates that perturbation of expression of imprinted genes is a common phenomenon observed in developing tumors. We envision that mutated VSELs in addition to teratomas are origin of several malignancies including germinal tumors and pediatric sarcomas.

Keywords: VSELs, pluripotent stem cells and aging, insulin/insulin growth factors, cancer stem cells, imprinted genes

84 Autophagy, A Guardian Against Neurodegeneration

Intracellular protein misfolding/aggregation are features of many currently incurable late-onset neurodegenerative diseases, like Alzheimer's disease, Parkinson's disease and polyglutamine expansion diseases like Huntington's disease (HD) and various spinocerebellar ataxias. The mutations causing many of these diseases confer novel toxic functions on the target proteins. We showed that the autophagy inducer, rapamycin, reduced the levels of mutant huntingtin and attenuated its toxicity in cells, and in Drosophila and mouse HD models. We have extended the range of intracellular proteinopathy substrates that are cleared by autophagy to other related neurodegenerative disease targets and have provided proof-of-principle in cells, Drosophila and mice. In order to induce autophagy long-term, we have been striving to identify safer alternatives to the mTOR inhibitor, rapamycin. To this end, we have been trying to discover novel components of the autophagy machinery and new signalling pathways and drugs that impact on autophagy. While autophagy induction is protective in models of various neurodegenerative diseases, certain other conditions, including lysosomal storage disorders, are associated with compromised autophagy. I will review these data and then describe how impaired autophagy compromises cellular processes, including the ubiquitin-proteasome system.

Keywords: autophagy, aggregates, Huntington's disease, dementia, neurodegeneration

85 Technological Entanglement: Assessing Anti-Aging Strategies Through Assisted Death Laws

Humans are on the threshold of being able to significantly increase their life expectancy yet, in the majority of jurisdictions there is little or no consensus as to how we are permitted to die. Today life extension and anti-aging research is currently being undertaken around the world. However not all longevity technologies are equal. Indeed, such technologies present varying degrees of “entanglement” with the human body as one moves for example, through simple changes in diet through to gene therapies, regenerative medicine and nanotechnology. Given the ongoing and seemingly intractable controversies and crises with respect to assisted death, arguably also a form of technology in itself, the question to be asked is, when a particular longevity intervention or panel of therapies is pursued, what might be required in order to bring about death, should death be desired. The objective here is to begin to understand emerging anti-aging technologies from the degree of entanglement that they present to the human body such that we can begin to illuminate the social pathways forward with respect to integrated, systems-level design, implementation and ideology.

Keywords: technological entanglement, assisted death, normative impact of technology

86 Plastic Antibodies, Synthetic Receptors for Biological Macromolecules

General methods for the recognition of specific peptide sequences, proteins and related biological macromolecules remain a significant challenge. The talk will describe general protocols for creating synthetic polymer receptors for peptides and proteins with antibody-like affinity for biological macromolecules. These nanoparticles have been found to function in vivo and are now being evaluated for therapeutic applications, in bioprocessing and diagnostics.

Keywords: synthetic antibody, protein capture agent

87 Aspirin, The Oldest New Anti-Aging Drug

Although introduced over a century ago, aspirin (acetylsalicylic acid) is still one of the most frequently utilized drugs worldwide, and is employed for a remarkable variety of beneficial effects. Even at low dose (81 mg or less per day), it provides protection against cardiovascular disease, stroke, and even against a number of cancers. Aspirin appears to delay cardiovascular aging through both organ-specific and global mechanisms. Reported activities of aspirin that might attenuate senescence include (a.) inhibition of cyclo-oxygenases (COX), blocking conversion of arachidonic acid into pro-inflammatory prostaglandins, with resultant disruption of platelet activation; (b.) scavenging of reactive oxygen species (ROS); (c.) uncoupling of mitochondrial respiration leading to reduced ROS generation; and/or (d.) prevention of the aging-associated decline in telomere length. In animal models, aspirin retards the development of multiple age-associated diseases, including atherosclerosis, diabetes, and hypertension associated with endothelial senescence. Recent studies have reported life-extending effects of aspirin in male mice, delayed onset of cellular senescence in endothelial cells, and reduced total mortality for men taking aspirin in large clinical trials. Are these extensions of lifespan just the sum of its documented disease-specific effects, or might aspirin actually retard aging? If aspirin opposes one or more basic mechanisms of senescence, then the amelioration and postponement of multiple diseases could be interpreted as secondary to slowed aging. Our data show that aspirin increases C. elegans lifespan and protects worms from oxidative damage due to hydrogen peroxide. Five distinct physiological and molecular indices of aging are all abetted or delayed by aspirin, and to a lesser or similar extent by salicylate. Thus, we regard the nematode as a tractable model in which to discover mechanisms through which aspirin impacts longevity. We find that aspirin acts, in part, by activating FOXO transcription factors otherwise silenced through insulinlike signaling, but that it also induces genes involved in anti-inflammatory and oxidative-stress responses.

Keywords: aspirin, cardiovascular, senescence/aging, atherosclerosis, inflammation

88 Nanobodies Against APP

Deposition of misfolded amyloid beta (abeta) is one of the important neuropathological characteristics of Alzheimer's disease (AD). Abeta is produced from the Amyloid Precursor Protein by the sequential activity of two enzymes, β–secretase and ³-secretase. A third protease, α-secretase, cleaves APP in the middle of the abeta sequence precluding formation of Abeta. The levels of abeta generated from APP can therefore be controlled by tailoring the activity of these proteases toward APP. Inhibiting β–secretase activity is a promising approach to decrease amyloidogenic processing of APP to Abeta, however β–secretase has other preferred substrates so inhibiting the enzyme itself may have unwanted side effects. An alternative approach is to block β–secretase activity using antibodies that bind to the APP substrate at the β–secretase cleavage site. We isolated and characterized a single chain variable domain antibody fragment (scFv) that inhibit the activity of β-secretase by binding APP (iBSEC1). The iBSEC1 scFv potently reduced abeta levels in cell models of AD and reduced toxicity. We also generated a proteolytic scFv engineered (Asec1A) to cleave at the α-secretase site of APP to promote non-amyloidogenic processing of APP by increasing α-secretase activity towards APP. The Asec1A proteolytic scFv reduced aggregation and toxicity of abeta and also decreased APP levels and toxicity in cell models of AD. While the iBSEC1 and Asec1A scFvs both by themselves work effectively to reduce abeta generation from APP, the two scFvs can be combined into a bispecific recombinant antibody DIA10D. When expressed in cell models of AD, the DIA10D diabody potently reduces levels of extracellular abeta and increases the levels of the neuroprotective APP product, sAPPα. By simultaneously inhibiting β-secretase processing and increasing α-secretase processing of APP, we can direct processing of APP towards the neuroprotective non-amyloidogenic pathway.

Keywords: beta-amyloid, amyloid precursor protein, BACE inhibitor, proteolytic antibody, aggregation

89 Clearance of Pathological Tau Protein with Immunotherapy

One of the perils of aging is the accumulation of various protein/peptide aggregates throughout the body, some of which are associated with toxicity. In several age-related disorders, aggregates of certain amino acid sequences are much more prominent than under normal conditions, and define the disease. Harnessing the immune system has emerged in recent years as a promising approach to treat these conditions. My laboratory has worked in this field targeting the amyloid-β peptide, the prion protein, the tau protein, and more recently the islet amyloid polypeptide. The focus of my talk will be on our tau immunotherapy studies. We have shown in tangle mouse models that active or passive immunizations clear pathological tau aggregates from the brain with associated functional benefits (Asuni, A. et al., J. Neurosci., 2007; Boutajangout A. et al., J. Neurosci., 2010, and Boutajangout A. et al., J. Neurochem., 2011). Importantly, these include a complete prevention of cognitive deficits in three different tests. The feasibility of targeting tau aggregates by these means has now been confirmed by several groups. We are currently assessing if its efficacy of clearance and safety depend on which region of the tau protein is being targeted. We are as well clarifying the mode of action of this very promising approach, which likely involves both intra- and extracellular pathways.

Keywords: Tau, Tangles, Immunotherapy, Behavior, Amyloid

90 Tissue Engineering of the Liver Using Decellularised Scaffolds

A major roadblock to successful organ bioengineering is the need for a functional vascular network within the engineered tissue. Here, we describe the fabrication of three-dimensional, naturally derived scaffolds with an intact vascular tree. Livers from various species were perfused with detergent to selectively remove the cellular components of the tissue while preserving the extracellular matrix components and the intact vascular network. The decellularized vascular network was able to withstand fluid flow that entered through a central inlet vessel, branched into an extensive capillary bed, and coalesced into a single outlet vessel. The vascular network was used to reseed the scaffolds with human fetal liver and endothelial cells. These cells engrafted in their putative native locations within the decellularized organ and displayed typical endothelial, hepatic and biliary epithelial markers, thus creating a liver-like tissue in vitro. These results represent a significant advancement in the bioengineering of whole organs. This technology may provide the necessary tools to produce the first fully functional bioengineered livers for organ transplantation and drug discovery.

Keywords: endothelial, hepatocytes, billiary, transplantation, stem cells

91 Effect of DHEA Supplementation on Cognitive Function in Aged Premenopausal Rhesus Macaques

The adrenal steroid dehydroepiandrosterone (DHEA) is a reliable biomarker of aging in both humans and nonhuman primates. Because the brain expresses the enzymes necessary to convert DHEA to neuroactive steroids such as estradiol (E2), the age-related decline in circulating DHEA may predispose the brain to degeneration and cognitive decline. To test this hypothesis, we administered DHEA to perimenopausal rhesus macaques (Macaca mulatta) in a manner designed to mimic the daily DHEA rhythm of young animals (5 mg of oral DHEA each morning). Animals were tested on a visual delayed response and delayed matching-to-sample task before, during, and after 2 months of hormonal supplementation. While minor improvements in performance were observed at short delays on both tasks, no major overall improvement in cognition was observed. These data concur with clinical experiments showing no effect of DHEA supplementation on cognition in postmenopausal women. However, the current study enabled us to perform postmortem gene expression analysis, to investigate why DHEA was cognitively ineffective in primates, despite promising results from rodent studies. We propose two possible reasons for this lack of effect: 1) Analysis of enzymes involved in the conversion of DHEA to E2 showed an age-related decline in central gene expression of 3-beta hydroxysteroid dehydrogenase; thus these animals may have been unable to synthesize sufficient E2 within the central nervous system to produce noticeable cognitive benefits, and 2) As these animals were peri- and not post-menopausal, they may have had sufficient circulating E2 (of ovarian origin) to maintain normal cognitive function, thus masking any potential benefits of the exogenous DHEA. Further investigation into the intracrine synthesis of neuroactive hormones may lead to new insights to maintaining cognition late in life with minimal peripheral side effects.

Keywords: cognition, primate, steroid, brain, dehydroepiandrosterone

92 Chemical Strategies for Exploring Advanced Glycation End-Products

Advanced glycation end-products are a class of natural products that form non-enzymatically on exposed protein residues in the human body. AGEs accumulate as a result of normal metabolism and aging, and significant elevations in these molecules have also been observed in the plasma of patients with chronic diseases, such as diabetes, cancer, arthritis, cardiovascular disease, and others. Our laboratory is taking an orthodox approach to studying these materials; we have initiated a synthetic program to prepare AGE- adducts on large scale and in chemically homogenous form. This talk will describe ongoing efforts along these lines, with a particular focus on exploring a class of arginine-derived AGEs. Chemical and biological insight arising from these studies will also be discussed. It is our hope that this small molecule-based strategy will serve to shed new light on the role of AGEs in both healthy and disease physiology.

Keywords: glycation, methylglyoxal, hydroimidazolones, AGEs

93 Anti Aging Oligos 5′-(TTAGGG)N-3′ Treatment Maintaining Telomere Length in vitro in Human Skin Cells Overcoming Senescence and the T-Loop Deletion Factor

Telomere shortening is thought to play a role in cellular aging contributing to human aging and longevity. Critical telomere shortening affects different genes, as the human genome varies, which is why the cascades differ, hence the different effects and organ failures. For years telomere length maintenance has been targeted. However there was no one treatment to keep the length within the normal limits. Variations of telomere shortening occur within same type of tissue, as well as different tissue types, from same and different individuals. Also very little is known about ratios comparing old cells with short telomeres to new cells with normal telomeres, among same and different tissues and individuals. Many telomere shortening factors have been considered, including the t-loop deletion factor and 5′-(TTAGGG)n-3′ oligos, in order to find treatment that works. However it still takes trial and error to adapt the dose levels and administrating the treatment frequencies, according to the individual genes and their interaction with the environment. Additional difficulties arose when the treatment was to be cheap, as well as testing it. Treating human skin cells with 5′-(TTAGGG)1-50-3′ oligos overcomes senescence and the t-loop deletion factor. The difficult part is the dose and administration frequencies. Overall as expected the longer the oligomer, the lower the administration frequencies. Doses are according to transfection efficiencies, in this experiment of DEAE-Dextran. Additional immortality control is required to take cells into a new culture and stop their treatment to see if they will immortalise, which will indicate too higher dose and/or too frequent. The opposite will be if the cells live as long as the non treated cells control, which would indicate lower dose and/or lower frequency of treatments. Cheap, easy, effective and sufficient test was simply cell counting. Different cell dyes were also tested. Culturing is efficient with both CO2 incubator and/or electric blanket. However the electric blanket after you manage to maintain the correct temperature in the dish requires also pH regulation every 3 hours. Cell counting is easier with TESCO-food-colours. Every little helps. Every microscope with eyepiece x10 and objective x20 is sufficient, OPAX-1108 was used in this experiment. Photos were obtained with 3.2MP mobile-phone-cam. Future experiments will gather more cell-lines data, target tissues, whole organs and eventually extending whole human life spans. Prediction is that telomere length maintenance is tissue independent, but ratios of old cells with short telomeres to new cells with normal telomeres remains tissue dependent. This would suggest that even tissue such as blood cannot be used as parameter predictions for relative telomere length in other tissues.

Keywords: Telomere/s maintenance, Anti Aging, 5′-(TTAGGG)1-50-3′ Oligos Treatment, T-loop deletion factor, human skin cells

94 Last Economic Recession was Good for Life Expectancy

Economic growth and a good economy are usually considered prerequisites for good health. Factually, the relationship of economic growth with life expectancy is complicated. Economic growth is connected with using human resources, sometimes with stress, with some wear-and-tear of the human body. Traditional and mostly acceptable workloads can be too high for some workers. Therefore, generally low economic activity can be good for public health. There have been studies of the influence of economic recessions on health and life expectancy. According to a University of Michigan study during the Great Depression in 1929–1933 the average life expectancy in the United States rose 6.2 years. The association between improving health and economic slowdown was true for all ages and for every major cause of death. It is a slowdown in the processes of human aging if aging is to be understood as the accumulation of damage in the human body that increases the probability of death.Our study of recent data from the European Union showed that the average life expectancy increased more rapidly in countries where gross domestic product (GDP) fell in 2009. In countries where GDP fell the average life expectancy increased by 0.36 years for men and 0.33 years for women, but where GDP rose life expectancy increased by 0.29 years for men and 0.17 years for women. In the Baltic States, which were heavily hit by the recession, the increase of the average life expectancy was for men 1.1 years and for women 0.7 years, several times more rapidly then earlier. Studies of life expectancy increasing during recession periods can give valuable information for improving economic policy, public health intervention and extending the average life expectancy without essential expenses.

Keywords: economic recession, health, life expectancy

95 The Impact of Pnpase on the Therapeutic Potential of Mitochondrial RNA Import

A decline in the function of mitochondria may contribute to the aging process and age-related disorders. A functional decline could arise from accumulated mtDNA mutations over time, leading to reduced oxidative phosphorylation and other untoward effects on mitochondrial activities. Strategies that restore mitochondrial function could potentially offset key aspects of aging decline. RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, polynucleotide phosphorylase (PNPASE), a 3′ to 5′ exoribonuclease and poly-A polymerase, was localized in the mitochondrial intermembrane space, a location lacking resident RNAs. In recent studies we have shown a role for PNPASE in regulating the import of nuclear-encoded RNAs into the mitochondrial matrix. PNPASE reduction impairs mitochondrial RNA processing and polycistronic transcripts accumulate. Augmented import of nuclear-encoded RNAs depends on PNPASE expression and PNPASE RNA processing and import activities are separable. A mitochondrial RNA targeting signal was identified that enables the import of heterologous RNAs in a PNPASE-dependent manner. Combined, our studies show an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria and provide a potential therapeutic route for halting or reversing the decline in mitochondrial function with aging.

Keywords: PNPASE, mitochondria, RNA import

96 Increased Damage to Proteins in Ageing – Wear and Tear Potentially Avoidable by Extending Preventive Maintenance of Life's Essential Machinery

Proteins undergo continual spontaneous modifications in physiological systems leading to change in their structure and function. This increases with age. Proteins are modified by glycation, oxidation and nitration leading to formation of glycation, oxidation and nitration adducts residues in proteins – including formation of non-disulfide crosslinks. Damaged proteins undergo proteolysis to form glycated, oxidised and nitrated amino acids or free adducts that are then metabolised or excreted. Protein damage adduct residues and free adducts may be robustly quantified by stable isotopic dilution analysis tandem mass spectrometry. Data of protein damage is now being combined to produce refined dynamic, multi-compartmental mathematical models of in-life protein damage. Accumulation of protein damage in ageing, that is, increased steady-state levels of damaged proteins occurs as a consequence of: (i) increased rates of protein damage – linked to increased rate of damaging agent formation and decreased anti-glycation and oxidant defences, (ii) decreased rates of repair of damaged proteins, and (iii) decreased rates of proteolysis of damaged proteins. Oxidative and non-oxidative mechanisms are involved. Antistress gene transcriptional responses also decline with age leaving the proteome particularly vulnerable in periods of oxidative, metabolic and lipogenic stress. Healthy ageing has been achieved in transgenic animals through manipulation of oxidative and non-oxidative mechanisms. Healthy ageing may be available for people through dietary supplements which prevent decline in expression in adulthood of a battery of genes protective against multi-modal stresses. Such interventions are designed to maintain vascular, metabolic, skeleto-muscular and other aspects of health, in part, through preventing increased flux of damage to proteins and increased steady-state levels of damaged proteins.

Keywords: Protein damage, Glycation, Oxidative stress, Proteolysis, Systems biology

97 Aging Theories: A New Perspective

In 1881, Weismann was the first researcher to propose a reason and a mechanism by which aging occurs. He proposed death was adapted as a program to eliminate older individuals. The view that aging could be programmed and evolved by natural selection is strongly rejected by most of the scientific community. Thus, different aging theories have been developed cautiously so as not to be classified as adaptive-programmed aging. Most researchers, if not all, classify any theory that does not evoke a program for death/aging as non-adaptive. However, theories like “mutation accumulation theory” (MA) or “antagonistic pleiotropy” (AP) assume aging is an inevitable side-effect of adaptation in the niche; MA occurs due extrinsic mortality and AP occurs due to within-species competition. In several hypotheses, the species' maximal lifespan is supposed to be indirectly adjusted by extrinsic factors. Therefore, most gerontologists agree that species senescence is somehow adaptive, although non-programmed. The definition and classification of aging theories is an important first step toward the understanding of senescence. We classified the theories in three categories: An adaptive aging theory assumes the intrinsic lifespan limit arises directly (programmed) or indirectly (non-programmed) due to adaptation in the niche. (1) An ADAPTIVE-NON-PROGRAMMED aging theory assumes the intrinsic lifespan limit is not selectively beneficial for the genotype, but evolved as a side-effect of adaptation in the niche. (2) An ADAPTIVE-PROGRAMMED aging/death theory assumes death itself confers a selective advantage on the genotype which produces it. (3) A NON-ADAPTIVE aging theory assumes the intrinsic lifespan limit arises as a side-effect of metabolism and its evolution has no relationship with adaptation in the niche. We discuss the relevance of our interpretations for aging research and SENS strategy.

Keywords: Aging, Evolution, oxidative damage, programmed death

98 The Circadian Clock Conducts Development in Mammals: Can Stopping it be a Means of Control Over Aging?

It is obvious enough that aging is a “product” of the individual development of an organism (even because development gives “material” for aging). Although there would be no aging without development, there seems to be an infinite dispute between those scientists who consider aging (or life expectancy) as a biological program and those who do not. According to our point of view, the aging of an organism begins only after its development is completed, and perhaps even later. There is an incredible quantity of examples of reversible arrest of the development in various organisms, from bacteria to mammals, caused by a “waiting” strategy. The essence of this type of strategy consists in switching the resources of an organism from reproduction to the maintenance of its own vitality, which thus makes a considerable increase in life expectancy possible. By investigating neobiosis (the reversible arrest of growth in dormice as an example of this kind strategy) and analyzing data of the various authors concerning the diapause mechanism in insects, we have reached conclusions concerning the key role that the circadian clock (localized in the SCN of the hypothalamus) plays in inducing the given condition. We have found and described cases of prolonged neobiosis (which are similar to superdiapause in some kinds of insects, for example, in the Emperor Moth). The stop and start of the molecular mechanism of the circadian clock occur only in certain phases of a daily cycle, and that can essentially facilitate the search for the factor which initiates neobiosis. Identification of biologically active substances which trigger the circadian clock-stopping mechanism can become the precondition for the creation of “non-maturing” mice and for experimental research of an interconnection between development and aging, and in the future can lead to the creation of technology aimed at total control over aging.

Keywords: neobiosis, SCN, circadian clock, superdiapause, control over aging

99 D3 – An Orally Applicable and Potent Aβ Oligomer Neutralizing Substance

Not only Aβ fibrils, but also small soluble Aβ oligomers in particular are suspected to be the major toxic species responsible for Alzheimer's disease (AD) development and progression. The aim of the study was to identify peptides consisting of the D-enantiomeric amino acids (D-peptides) that bind highly specific to Aβ(1–42) oligomers. D-peptides are thought to be protease resistant and less immunogenic than the respective L-enantiomers and can easily be identified by mirror image phage display. A mirror image phage display selection against a mixture of Aβ(1–42) monomers and oligomers yielded the D-enantiomeric dodecapeptide D3. We show that next to plaque load and inflammation reduction, oral application of D3 improved the cognitive performance of AD transgenic mice. Additional in vitro data suggest that D3 specifically precipitates toxic Aβ species and converts them into non-amyloidogenic, non-fibrillar and non-toxic amorphous aggregates. D3 exerts an interesting and novel mechanism of action that abolishes toxic Aβ oligomers, and thereby supports their decisive role in AD development and progression. Literature: Van Groen et al., A novel D-enantiomeric peptide reduces Alzheimer's disease amyloid plaque load in transgenic mice. ChemMedChem. 2008: 3(12), 1848-52 Funke et al., Oral Treatment with the D-Enantiomeric Peptide D3 Improves Pathology and Behavior of Alzheimer's disease Transgenic Mice. ACS Chem. Neurosci. 2010: 1, 639–648.

Keywords: Alzheimer's disease, amyloid-β, therapy, peptides

100 Trans-BBB Exosomes

Successful delivery of macromolecular therapeutic agents across the blood brain barrier (BBB) to the central nervous system is a major challenge in the treatment of neurological diseases of aging. Recently, the exploitation of natural nanoparticles known as exosomes has been pioneered and demonstrates their potential as therapeutic delivery vehicles. The natural macromolecular delivery and targeting properties of exosomes are well exemplified with siRNA delivery systemically to brain to target genes implicated in Alzheimer's disease. Moreover, delivery of exosomes appears to be well tolerated. This novel natural nanotechnology therefore has potential for systemic drug delivery across the BBB to treat a range of neurological and neurodegenerative conditions.

Keywords: exosomes, nanoparticles, blood-brain barrier, delivery mechanisms

101 Decline of the Nuclear Factor E2-Related Factor 2 (NRF2) and Antistress Gene Response in Ageing and Strategies to Prevent it for Healthy Ageing

Ageing is associated with accumulation of damaged proteins, decline in glucose tolerance, dyslipidaemia, oxidative stress, inflammatory disorders and increased cell senescence. The endogenous defence to protein damage, metabolic stress, inflammation and oxidative stress is coordinated by nuclear factor E2 related factor 2 (nrf2), kelch-related ECH protein-1 (keap1) and antioxidant response element (ARE) linked gene expression in the antistress gene response. The nrf2/keap1/ARE transcriptional system regulates the expression of a battery of protective genes. It was initially linked to increased expression of genes with gene products linked to antioxidant function and phase II conjugations – such as quinone reductase, glutathione transferases and others. Further studies revealed that the protective effects of this system extend beyond those of increasing endogenous antioxidant activities to detoxification of reactive aldehydes and dicarbonyl glycating agents, increasing clearance of damaged proteins by increasing expression of components of the proteasomal and autophagy systems, preventing metabolic stress by increased expression of enzymes of the pentosephosphate pathway, and preventing dyslipidaemia by down regulation of lipogenic gene expression. In ageing, however, expression of both transcription factor nrf2 and ARE-linked genes decline - leaving tissues exposed and susceptible to increased multi-model stresses and irreversible decline. Strategies to counter this are dietary supplements of bioactive compounds that activate nrf2 such as glucosinolate-derived isothiocyanates, polyphenols, flavonoids, indoles, allyl sulfides, triterpenoids and others. Potency and time course responses to bioactives at concentrations relevant to dietary exposure have been assessed and are presented. Potent synthetic analogues are now also under evaluation for pharmaceutical treatment of ageing-related disorders. Maintaining the activity of nrf2 and related ARE-linked gene expression by chronic exposure to nrf2 activators in dietary supplements is a route to achieve health ageing currently under development and evaluation.

Keywords: nrf2, Antistress gene response, Oxidative stress, Dyslipidaemia, Dietary bioactives

102 Is Accelerated Wound Healing Good for Longevity?

Wound healing (WH) is a fundamental biological process. Does the ability to heal faster have any impact on longevity? In an attempt to answer this question, we compared the established mouse models with genetically modified life span and also an altered rate of WH. Our analysis showed that the ability to preserve the rate of skin WH up to an old age, but not a high WH rate in the youngs, appears to be associated with a longevity phenotype. While the aging process is likely to cause wounds to heal slowly, an accelerated WH rate in younger animals could indicate an altered cell proliferation and/or migration capacity, which is likely to affect other major processes such as the onset and progression of cancer. In support of this is our study on the long-lived transgenic αMUPA mice, a unique model of genetically extended life span. These mice spontaneously eat less, preserve their body mass, are more resistant to spontaneous and induced tumorigenesis and live longer, thus mimicking the effects of caloric restriction. We found that in the young age, the αMUPA mice were not superior over their parental wild-type FVB/N strain with regard to WH capacity. However, the αMUPA showed a much slower age-related decline in the rate of WH, thus confirming once more that ‘slowly but steady wins the race’.

Keywords: Wound healing, Longevity, αMUPA mice

103 The Integrated Biomarkers and Systemic Targets for Clinical Use of Anti-Aging Regulatory Programs

Current research is focused on the development of the integrated biomarkers' system and anti-aging regulatory programs. The main target systems are immune, neuroendocrine, hepatobiliary and gastrointestinal. The ultimate goal of this research is to improve these systems' conditions and functions. We have revealed the usefulness of the consortium of probiotics and prebiotics for gastrointestinal tract and hepatobiliary system. In regards to immune system, DNA-protecting effect of probiotics is being analyzed. It is believed that this approach enhances the function of systemic targets on different levels and helps to develop effective protocols of anti-aging regulatory program.

Keywords: anti-aging, probiotics

104 Creation of Functional Ovarian Tissue

The human ovary is the female reproductive organ containing cells that secrete hormones (estrogen, progesterone), which maintain female sexual characteristics as well as egg (ovum) production. Loss of ovarian tissue function due to various endocrine and fertility disorders requires chronic estrogen (E) and progesterone (PG) administration. Conventional hormone replacement therapy is based on oral, injection and transdermal (skin patch) administration but these have several disadvantages, such as frequent administration, fluctuation in blood hormone levels and associated complications. In addition, these modalities are unable to restore ovarian tissue function, which includes the production of eggs. This is critical for premenopausal women who desire to bear babies. As such, a continuous and unlimited hormonal supply produced from ovarian cells would be an attractive option. More importantly, creation of ovarian tissue that produces eggs for potential fertility would provide numerous benefits to women of all ages. The overall goal of our research is to develop an ovarian cell cultivation system that provides female hormones to maintain female secondary sexual characteristics with the ultimate goal of maturing these cells into follicles that could lead to the production of eggs. Toward this goal, we have followed two approaches to address the current medical needs. Our aims are 1) to develop a cell-based therapy using the microencapsulation technology for the maintenance of female sexual characteristics, and 2) to engineer ovarian follicles that would lead to egg production. Research progress on these two approaches will be discussed in detail.

Keywords: Ovary, Female hormones, Endocrine function, Follicles

105 Agingportfolio.org Knowledge Management System for Aging Research

Aging research spans many areas of natural and social and behavioural sciences. Few data repositories aggregate data from the scientific literature, clinical trials databases, patents and grant application reports. To address this issue, we developed a flexible open-access knowledge management system for aging research the International Aging Research Portfolio (IARP) accessible via www.agingportfolio.org. Pubmed and other resources enable the scientific community to perform swift searches of the scientific literature. However, grant information, which is more difficult to track and may not be accessible to search engines is very important for research planning purposes. It may take years for a grant to result in a publication and to prevent redundancy and promote scientific cooperation it is important to browse through all available grant information worldwide to see if there are simillar projects being funded. The IARP system provieds timely access to the most recent grant data from many international sources including the US National Institutes of Health, European Commission, Canadian Institutes of Health Research, Australian National Health and Medical Health Council and publication abstract data through a license of MEDLINE database. The database is searcheable and the results can be presented as charts and graphs for trends analysis. One of the main features of the IARP system is automatic and manual classification of the research projects into a structured directory. The IARP science advisory board and volunteer expert category editors classify the projects from the database into relevant categories. These manually classified projects become training sets for the automatic classification algorithms with elements of machine learning. The IARP system is highly modular and portable and may be used as a platform for developing other knowledge management systems for aging research.

Keywords: aging research, aging database, aging system, gerontology system, knowledge management