Multiple events case-control study in a prospective cohort to identify systemic, cellular, and molecular biomarkers of obesity-induced accelerated aging in 30-years-olds: the ObAGE study protocol.

BACKGROUND: Aging is characterized by a progressive loss of capacities linked to fundamental alterations/damage in multiple cellular and molecular pathways. It is the most significant risk factor for all non-communicable diseases (NCDs). Another contributing factor to the rise in NCDs is obesity. It has been suggested that obesity not only accelerates the onset of metabolic imbalances but also decreases lifespan and impacts cellular and molecular processes in a manner similar to aging. Obesity might accelerate the pace of aging. Guided by a lifecourse approach, we will explore how exposure to obesity in critical developmental stages disrupt homeostatic resilience mechanisms that preserve physiological integrity, inducing an early expression of aging phenotypes. Also, we will determine whether exposure to early psychosocial adversity influences vulnerability to obesity as a risk factor for accelerated aging. METHODS: Multiple events case-control study embedded in a prospective cohort of Chileans at 30-31y, 50% females, of low- to-middle socioeconomic status, who participated in nutrition research since birth. At 23y, 25% had obesity and cardiometabolic risk was high. We will use a multi-layer approach including: anthropometric assessment; DXA scan for body composition; abdominal ultrasound of the liver; stool samples collection and sequencing of the ribosomal RNA 16S gene to characterize the gut microbiome; determination of age-related pro-inflammatory cytokynes and anti-inflammatory miokynes. For the first time in Chile, we will address age-related epigenetic changes using the Horvath´s epigenetic clock. In a subset we will conduct a controlled physical challenge to characterize physical resilience (autophagy). DISCUSSION: ObAGE is in an excellent position to: approach aging as a process whose expression involves multiple factors from the early stages of a person's life; understand how longitudinal changes in health trajectories impact the biological mechanisms of aging; identify potential resilience mechanisms that help prevent unhealthy aging. Because SLS participants are still young, our research setting combined with advanced scientific techniques may identify individuals or groups at risk of early onset health issues. Results from ObAGE may pave the way to address the contribution of obesity to aging through lifespan from cells to systems and might be instrumental to developing interventions to improve health span in the Chilean population. TRIAL REGISTRATION: The proposed study does not consider any health care intervention on human participants.

Effects of warm ischaemia combined with cold preservation on the hypoxia-inducible factor 1α pathway in an experimental renal autotransplantation model.

BACKGROUND: The increased use of marginal donors highlights the importance of organ quality in transplantation and the identification of prognostic biomarkers. This experimental study investigated modulation of the hypoxia-inducible factor (HIF) 1α pathway in kidney grafts in relation to different degrees of ischaemia. METHODS: In a porcine autotransplantation model, two different kidney graft protocols were compared: standard 24-h cold storage (CS) and 24-h CS preceded by 1 h warm ischaemia (WI + CS). The renal HIF-1α pathway and tubular dedifferentiation were analysed in the early phase of reperfusion and at 3 months. RESULTS: There was a relationship between the degree of ischaemic injury and the outcome of the kidney graft. During the first week of reperfusion, WI + CS grafts showed a higher degree of injury. The observed tubular dedifferentiation was associated with delayed HIF-1α expression, and with loss of its role in transcription. In highly injured kidneys, deregulation of the HIF-1α pathway was also observed in the chronic phase, with reduced production of vascular endothelial growth factor (VEGF) A, and upregulation of VEGF receptor 1 (Flt-1) and thrombospondin 1. In addition, these kidneys displayed altered kidney histology and decreased function. CONCLUSION: The HIF-1α pathway appears to be abolished early in response to severe ischaemia. A high degree of ischaemic injury also results in chronic activation of the HIF-1α pathway, diverting it away from the beneficial activation of angiogenesis. Further studies on the finely tuned balance of signals in this pathway may provide diagnostic biomarkers that can determine organ quality during kidney transplantation. Surgical relevance The increased use of marginal donors has highlighted the importance of organ quality in transplantation. Renal ischaemia-reperfusion injury following transplantation induces graft dysfunction. In a porcine model of renal autotransplantation, the induction of regenerative processes, in response to graded degrees of ischaemia, was studied in the post-transplantation phase. There was early abrogation of the hypoxia-inducible factor (HIF) 1α pathway in response to severe ischaemia. High degrees of ischaemic injury induced chronic activation of the HIF-1α pathway, diverting it from the beneficial activation of angiogenesis. Identification of the mechanisms involved in renal regeneration, such as those related to the HIF-1α pathway, are important as these mechanisms can be used to identify novel therapeutic targets or develop diagnostic biomarkers to determine organ quality early in the transplantation process.

A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases.

The universal second messenger cAMP is generated upon stimulation of Gs protein-coupled receptors, such as the ß2 -adreneoceptor, and leads to the activation of PKA, the major cAMP effector protein. PKA oscillates between an on and off state and thereby regulates a plethora of distinct biological responses. The broad activation pattern of PKA and its contribution to several distinct cellular functions lead to the introduction of the concept of compartmentalization of cAMP. A-kinase anchoring proteins (AKAPs) are of central importance due to their unique ability to directly and/or indirectly interact with proteins that either determine the cellular content of cAMP, such as ß2 -adrenoceptors, ACs and PDEs, or are regulated by cAMP such as the exchange protein directly activated by cAMP. We report on lessons learned from neurons indicating that maintenance of cAMP compartmentalization by AKAP5 is linked to neurotransmission, learning and memory. Disturbance of cAMP compartments seem to be linked to neurodegenerative disease including Alzheimer's disease. We translate this knowledge to compartmentalized cAMP signalling in the lung. Next to AKAP5, we focus here on AKAP12 and Ezrin (AKAP78). These topics will be highlighted in the context of the development of novel pharmacological interventions to tackle AKAP-dependent compartmentalization.

Ischemic pre- and post-conditioning: current clinical applications.

Ischemic conditioning is a phenomenon through which short sequences of ischemia-reperfusion applied to an organ confer some degree of protection towards future ischemic insults. This phenomenon was first observed in the mid-1980s in cardiac surgery, and has been since widely studied in different settings. Different sort of ischemic conditioning exist: local vs remote, direct or pharmacological, and with different timeframes of protection. Ischemic conditioning seems especially suited to applications in transplantation since schedules of both cold and warm ischemia, as well as reperfusion, are carefully and easily controlled, and the benefits of protecting fragile organs against ischemia-reperfusion injuries might help widen the pool of possible grafts and ensure better graft function and survival. The pathways through which ischemic conditioning work are many, offering both preservation of cell energy, protection against oxidative stress, better blood flow to organs and protection against apoptosis. In the field of pharmacological conditioning, which tries to mimic the protective effects of traditional ischemic conditioning without the potential side-effects associated with vessel clamping, many common-use drugs including anesthetics have been shown to be effective. Significant results have been obtained in small animal models, but while ischemic conditioning is successfully used in cardiac surgery, studies in large animal models and human applications in liver and kidney transplantation are still inconclusive.

Cyclodextrin curcumin formulation improves outcome in a preclinical pig model of marginal kidney transplantation.

Decreasing organ quality is prompting research toward new methods to alleviate ischemia reperfusion injury (IRI). Oxidative stress and nuclear factor kappa beta (NF-κB) activation are well-described elements of IRI. We added cyclodextrin-complexed curcumin (CDC), a potent antioxidant and NF-κB inhibitor, to University of Wisconsin (UW) solution (Belzer's Solution, Viaspan), one of the most effective clinically approved preservative solutions. The effects of CDC were evaluated on pig endothelial cells and in an autologous donation after circulatory death (DCD) kidney transplantation model in large white pigs. CDC allowed rapid and lasting uptake of curcumin into cells. In vitro, CDC decreased mitochondrial loss of function, improved viability and lowered endothelial activation. In vivo, CDC improved function recovery, lowered histological injury and doubled animal survival (83.3% vs. 41.7%). At 3 months, immunohistochemical staining for epithelial-to-mesenchymal transition (EMT) and fibrosis markers was intense in UW grafts while it remained limited in the UW + CDC group. Transcriptional analysis showed that CDC treatment protected against up-regulation of several pathophysiological pathways leading to inflammation, EMT and fibrosis. Thus, use of CDC in a preclinical transplantation model with stringent IRI rescued kidney grafts from an unfavorable prognosis. As curcumin has proved well tolerated and nontoxic, this strategy shows promise for translation to the clinic.

Organ procurement from donors deceased from cardiac death: a single-center efficiency assessment.

Organ donation after cardiac death has been used for kidney and liver procurement in France since 2006. Until recently, most teams relied on in situ cold perfusion to prepare the donor before organ retrieval. Our team has used since 2007 normothermic abdominal recirculation. While this technique is presumed to be more difficult to implement, it also ensures a lower rate of primary nonfunction when compared to in situ cold perfusion. We present the efficiency results of our organ donation after cardiac death program. After 3 years, we have been able to establish a program in which we use normothermic abdominal recirculation in 97% of donors after cardiac death. The yearly efficiency of this program is comparable to the national efficiency of organ procurement from conventional deceased donors in France.

Estimated pregnancy length from ovulation to parturition in the bitch and its influencing factors: a retrospective study in 162 pregnancies.

An accurate timing of parturition is very useful for managing canine parturition. It is generally accepted that parturition in bitches occurs between 64 and 66 days after the luteinizing hormone peak. In this retrospective study, we determined pregnancy length in different breeds and its influencing factors dating it from the estimated day of ovulation (EDO), defined as the day when peripheral plasma level of progesterone (P4) reaches 6 ng/ml. From January 2001 to December 2006, 162 pregnancies in 151 bitches of 53 different breeds were followed. Different parameters concerning the bitch, the litter, the type of semen and the type of reproduction were studied. The mean estimated pregnancy length in the bitch from EDO to parturition was 63.1±2.1 days. The main influencing factors for the pregnancies studied were the breed, the size of the bitch and the number of puppies within the litter.

[Should temperature be monitorized during kidney allograft preservation?]. / Faut-il monitorer la température pendant la phase de préservation en transplantation rénale ?

GOAL: It is generally considered that kidney grafts should be preserved at 4 degrees C during cold storage. However, actual temperature conditions are not known. We decided to study the temperature levels during preservation with the Biotainer storage can and Vitalpack transport pack. MATERIAL: Temperature was monitored using the Thermobouton probe during preservation of pig kidneys, in the same conditions used with human grafts. The probe recorded the temperature level every 10 minutes during four days. We compared the results found with the new storage can with results obtained in the same conditions with the storage can formerly used by our team. We also studied the best position of the probe for temperature monitoring and the influence of the amount of ice within the transport pack on the temperature level. We then monitored the temperature during the conservation of actual human kidney grafts harvested at our institution from August 2007 to May 2008. RESULTS: The temperature levels were the same regardless of the position of the probe within the transport pack. The lowest temperature was maintained during 15 hours, and the temperature level stayed below 5 degrees C for 57 hours with the new storage can. The former storage can maintained the lowest temperature level for 80 minutes, and temperature reached 5 degrees C after 10 hours 40 minutes. Temperature levels were similar when 2 or 4 kg of crushed ice were used. We observed similar results when monitoring the conservation of human grafts. CONCLUSION: The new storage can affords more stable temperature levels when compared to the formerly used can. Since temperature is stable during conservation, continuous monitoring in everyday practice does not seem warranted.

Kidney transplantation in HIV-positive patients: report of our first 7 cases.

INTRODUCTION: Infection with human immunodeficiency virus (HIV) is associated with end-stage renal disease (ESRD). Although many teams initially were reluctant to offer kidney transplantation as a therapeutic option in HIV-positive patients with ESRD, new drug regimens introduced in the late 1990s have dramatically improved the life expectancy in these patients. OBJECTIVE: To report the results of the first 7 kidney transplantation procedures in HIV-positive patients at our institution. PATIENTS AND METHODS: Patients were selected to minimize the risks of HIV disease progression, opportunistic infections, and tumors. Protease-inhibitor therapies were suspended because of possible interaction with immunosuppression drugs. The induction regimen did not include lymphocyte-depleting drugs. After undergoing transplantation, patients were monitored by the transplantation and infectious disease teams. RESULTS: To date, all patients are alive with functioning grafts. We did not observe any episodes of acute rejection, and there were few adverse events. Drug tolerance was good for both immunosuppression and antiretroviral therapies. CONCLUSION: Kidney transplantation in HIV-positive patients with ESRD is warranted. Provided that patients are carefully selected, good results can be achieved with few adverse events, episodes of acute rejection, and drug interactions. Posttransplantation, these patients must be closely monitored by both the transplantation and infectious diseases teams to ensure optimal management.

Cyclin-dependent kinase 5 activator p35 over-expression and amyloid beta synergism increase apoptosis in cultured neuronal cells.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by neuronal loss, dementia and pain. Two main protein aggregates, extracellular (senile plaques, SP) and intracellular (neurofibrillary tangles, NFT), are associated with AD. NFT are mainly composed of hyperphosphorylated microtubule-associated protein tau. Nowadays several protein kinases have been implicated in the phosphorylation of tau, including glycogen synthase kinase 3 beta (GSK3beta), MAP kinase, protein kinase A and cyclin-dependent kinase 5 (Cdk5). A deregulation in the activity of Cdk5 has been postulated to participate in the abnormal tau hyperphosphorylation in AD. Activation of Cdk5 occurs after its association with p35, a neuron-specific activator, predominantly in the nervous system. Therefore, in this study we used the tetracycline transactivator system to increase p35/GFP in neuronal cells, treated with amyloid beta 1-42 (Abeta(1-42)) peptide. These cells showed an increase of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase-3 staining, indicating increased apoptosis of neuronal cells. This effect could be reversed by the addition of tetracycline in the culture medium, suggesting synergistic effects of p35 over-expression and Abeta treatment in the apoptosis of neuronal cells. These results represent a linkage between amyloidogenic and cdk5 pathways leading to apoptosis of neuronal cells.

Systematic microbiological tests in kidney transplantation and their value in predicting posttransplantation infection.

BACKGROUND: Immunosuppressive therapy has many side effects among which is an increased infectious risk for the recipient. Transmission of pathogens from the graft to the recipient has not been well evaluated; there are no guidelines regarding the need for microbiological tests on the graft prior to transplantation. We routinely performed such tests to evaluate the risk and determine whether a patient should receive preemptive antibiotic therapy after transplantation. We herein have reported our preliminary results. MATERIALS AND METHODS: We reviewed 150 consecutive renal transplantations from cadaveric heart-beating donors. Microbiological tests were systematically performed not only on the preservation solution, but also on graft artery, vein, ureter, and perirenal fat. We reviewed the recipient's medical history for clinically significant infectious episodes in the first month after transplantation. RESULTS: Thirty-one percent of all microbiological tests were positive with 23 patients showing multiple positive tests, 74% of which were concordant. We documented 3 cases of direct graft-to-recipient pathogen transmission, all of which presented with 3 positive concordant tests. Graft culture prior to transplantation is often positive, but in more than half of the cases positive tests are either isolated or discordant. We only treated patients with concordant test results; no adverse consequence was observed among the untreated patients. Transmission occurred only in patients with at least 3 concordant tests. CONCLUSIONS: Multiple microbiological tests on the graft prior to transplantation seemed useful to determine which patients would benefit from preemptive antibiotic therapy. Further studies may help to define which microbiological tests are the most important.

Use of the SCOT solution in kidney transplantation: preliminary report.

SUBJECT: This article reports preliminary findings relating to the use of a new preservation solution, the Solution de Conservation des Organes et des Tissus (SCOT), in renal transplantation. This fourth-generation solution combines an extracellular-like composition with 20 kDa polyethylene-glycol, known for its cell-protection capacity and immunocamouflage properties. METHODS: We have reported preliminary data obtained in 29 transplantations (25 cadaveric donors and 4 living related donors) between December 2004 and November 2005. The SCOT solution was used for both in situ flush and static preservation. We compared primary organ nonfunction and delayed graft function rates as well as blood creatinine levels in recipients of grafts processed with SCOT solution, versus University of Wisconsin solution (paired for age with the previous group) and with EuroCollins solution (historic data). RESULTS: We observed delayed graft-function in 10% of the SCOT-processed group, 14% in the University of Wisconsin solution group, and 23% of the EuroCollins group. No case of primary organ nonfunction was reported. Creatinine levels in both SCOT and University of Wisconsin solution groups were similar. We did not observe any humoral or cellular graft rejection in the first 3 months after transplantation. In light of these preliminary results, the use of SCOT is safe for kidney preservation in the human setting. Further experience is warranted to assess the clinical consequences of its immunocamouflage properties as described in animal models.

Role of the JAKs/STATs pathway in the intracellular calcium changes induced by interleukin-6 in hippocampal neurons.

Recent studies show that inflammation has an active role in the onset of neurodegenerative diseases. It is known that in response to extracellular insults microglia and/or astrocytes produce inflammatory agents. These contribute to the neuropathological events in the aging process and neuronal degeneration. Interleukin-6 (IL-6) has been involved in the pathogenesis of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Here, we show that IL-6 treatment of rat hippocampal neurons increases the calcium influx via NMDA-receptor, an effect that is prevented by the specific NMDA receptor antagonist MK-801 (dizocilpine). We also show that this calcium influx is mediated by the JAKs/STATs pathway, since the inhibitor of JAKs/STATs pathway, JAK 3 inhibitor, blocks calcium influx even in the presence of IL-6. This increase in calcium signal was dependent on external sources, since this signal was not observed in the presence of EGTA. Additional studies indicate that the increase in cytosolic calcium induces tau protein hyperphosphorylation, as revealed by using specific antibodies against Alzheimer phosphoepitopes. This anomalous tau hyperphosphorylation was dependent on both the JAKs/STATs pathway and NMDA receptor. These results suggest that IL-6 would induce a cascade of molecular events that produce a calcium influx through NMDA receptors, mediated by the JAKs/STATs pathway, which subsequently modifies the tau hyperphosphorylation patterns.

Participation of structural microtubule-associated proteins (MAPs) in the development of neuronal polarity.

Several lines of evidence have indicated that changes in the structure of neuronal cytoskeleton provide the support for the dramatic morphological changes that occur during neuronal differentiation. It has been proposed that microtubule-associated proteins can contribute to the development of this phenomenon by controlling the dynamic properties of microtubules. In this report we have characterized the effect of the combined suppression of MAP1B and tau, and MAP1B and MAP2 on neuronal polarization in cultured hippocampal cells grown on a laminin-containing substrate. We have taken advantage of the use of a mouse line deficient in MAP1B expression obtained by the gene trapping approach. In addition to this engineered mice line we used the antisense oligonucleotide approach to induce the suppression of tau or MAP2, in wild type and MAP1B-deficient neurons. Together these results show a synergistic role for MAP1B/MAP2 and MAP1B/TAU.

Evidence for the role of MAP1B in axon formation.

Cultured neurons obtained from a hypomorphous MAP1B mutant mouse line display a selective and significant inhibition of axon formation that reflects a delay in axon outgrowth and a reduced rate of elongation. This phenomenon is paralleled by decreased microtubule formation and dynamics, which is dramatic at the distal axonal segment, as well as in growth cones, where the more recently assembled microtubule polymer normally predominates. These neurons also have aberrant growth cone formation and increased actin-based protrusive activity. Taken together, this study provides direct evidence showing that by promoting microtubule dynamics and regulating cytoskeletal organization MAP1B has a crucial role in axon formation.

Perinatal lethality of microtubule-associated protein 1B-deficient mice expressing alternative isoforms of the protein at low levels.

Microtubule-associated protein 1B (MAP1B) has been implicated in axogenesis in cultured cells. To gain insight into the functions that MAP1B plays in vivo, we analyzed a strain of Map1B mutant mice generated by a gene trapping approach. Homozygous mice die on the first day after birth, probably due to a severe abnormal development of the nervous system. They present alterations in the structure of several brain regions. The normal Map1B gene yields different protein isoforms from alternatively spliced transcripts. The smaller isoforms were present in wild type, hetero-, and homozygous mice, but their expression was higher in the mutants than in the wild-type. Moreover, trace amounts of MAP1B protein were also observed in Map1B homozygous mutants, indicating an alternative splicing around the gene trap insertion. Thus, the Map1B gene trapped mutation reported in this work did not generated a null mutant, but a mouse with a drastic deficiency in MAP1B expression. Analyses of these mice indicate the presence of several neural defects and suggest the participation of MAP1B in neuronal migration.

Molecular genetic approaches to microtubule-associated protein function.

Protein function in vivo can be studied by deleting (knock-out) the gene that encodes it, and search for the consequences. This procedure involves different technologies, including recombinant DNA procedures, cell biology methods and histological and immunocytochemical analysis. In this work we have reviewed these procedures when they have been applied to ascertain the function of several microtubule-associated proteins. These proteins have been previously involved, through in vitro experiments, in having a role in the microtubule stabilization. Here, we will summarize the generation and characterization of different microtubule-associated protein knock-out mice. Special attention will be paid to MAP1B knock-out mice. Amongst the different MAPs knock-out mice these show the strongest phenotype, the most likely for being MAP1B, the MAP that is expressed earliest in neurogenesis. Molecular genetics could be considered as a valid and useful procedure to truly establish the in vivo functions of a protein, although it is necessary to be aware of possible artifacts such as the generation of some kinds of RNA alternative splicing. To avoid this the best strategy to be used must consider the deletion of the exon that contains the functional domains of the protein.

Immunological characterization of epitopes on tau of Alzheimer's type and chemically modified tau.

The microtubule-associated protein tau is the main structural component of paired helical filaments (PHFs), which in turn are one of the major aberrant polymers found in Alzheimer's disease. Immunological studies were carried out using site-directed monoclonal and polyclonal antibodies that recognize tubulin binding epitopes on tau, to further understand the mechanisms of tau self-association into PHFs. Tau protein was subjected to either carbamoylation with potassium cyanate (KCNO) or glycation with glucose, and the immunoreactivity of the chemically-modified protein with these antibodies was compared with tau derived from paired helical filaments and with normal brain tau. The data on the immunoblot patterns of tau isoforms and the ELISA titration curves revealed significant differences between the modified tau and normal controls. However, the Western blot patterns of immunoreactive tau from the chemically-modified protein and from Alzheimer brains were similar. The data on the differences in the electrophoretic profiles and Western blots of normal brain tau as compared with solubilized paired helical filaments, insoluble tangles and tau proteins of the Alzheimer's type, provide new clues to understand the anomalous interactions of tau in Alzheimer's disease.