Untersuchungen zum mTOR-Signalweg in humanen Kontroll- und Hutchinson-Gilford-Progerie-Fibroblasten

Altern ist ein Prozess, der sich durch einen Verlust der Funktionen des Körpers, der Organe und der Zellen mit zunehmender Lebenszeit äußert und früher oder später zum Tod der meisten lebenden Organismen führt. Beschleunigt wird das Altern durch verschiedene Faktoren, wie oxidativer Stress, energiereiche Strahlung, Lebensgewohnheiten oder genetische Defekte. Zu den genetischen Erkrankungen gehört die Hutchinson-Gilford-Progerie (HGP), eine monogenetische Erkrankung, die sich durch verfrüht einsetzende und beschleunigte Alterung auszeichnet. Als möglicher therapeutischer Ansatz sowohl bei der Progerie als auch beim normalen Alternsprozess wird das machanistic Target of Rapamycin (mTOR) und dessen Signalweg untersucht. Der mTOR-Signalweg ist in die Regulation von wichtigen Stoffwechselprozessen und die Regulation von Zellwachstum involviert. Dabei ist dessen Rolle bei Alterungsprozessen und bei der Pathogenese der Progerie nur unzureichend geklärt. Der mTOR-Signalweg wurde auf Proteinebene mittels Western-Blot-Analyse semi-quantitativ analysiert. Dazu wurden das up-stream-Protein Akt, mTOR selbst und die down-stream-Proteine p70S6K und ULK1 betrachtet. Als zelluläres Modell wurden Kontroll- und HGP-Fibroblastenkulturen so lange vermehrt, bis sie durch zunehmende Telomerverkürzung das Stadium der replikativen Seneszenz erreichten. Der Einfluss der Telomerlänge wurde durch den Vergleich von Zellen mit kurzen und langen Telomeren (nach Immortalisierung mit humaner Telomerase Reverse Trankriptase (hTERT)) untersucht. Wasserstoffperoxid diente als nicht-replikativer Stressor. Es wurde gezeigt, dass bei HGP-Fibroblasten eine um ca. 53% (±35%) beschleunigte Proliferationsrate vorlag sowie ein frühzeitiger Wachstumsstopp (im Mittel 226 Tage eher) bei gleichzeitig erhöhtem Anteil an seneszenten Zellen (HGP 73 ±18% vs. CON 40±3%) zu verzeichnen war. Während in gesunden, replikativ gealterten Zellen mit geringerer Proliferation die genannten Schlüsselproteine des mTOR-Signalweges um mindestens 50% supprimiert waren, konnte in gealterten Progerie-Zellen mit Ausnahme von p70S6K eine verstärkte Proteinexpression des mTOR-Signalweges gezeigt werden. Die Immortalisierung mit hTERT, verbunden mit einer Telomerverlängerung, wirkte den Effekten des replikativen Alterns sowohl in Kontrollzellen als auch in den Progerie-Zellen in unterschiedlichem Ausmaß entgegen. Wasserstoffperoxid hatte keinen signifikanten Effekt auf den mTOR-Signalweg und konnte die Effekte des replikativen Alterns nicht imitieren. Zusammenfassend zeigten die Ergebnisse bei Progerie-Zellen, dass eine verstärkte Proliferation mit einer veränderten Aktivierung des mTOR-Signalweges als ein möglicher Pathomechanimus der verstärkten Alterungsprozesse diskutiert werden kann.Aging is a process in almost all living organisms, which is defined by an increased loss of function over time on different levels and is finally leading to death. Aging is accelerated by various factors such as oxidative stress, high-energy radiation, lifestyle and genetic defects. Hutchinson-Gilford-Progeria (HGP) is a monogenetic disease which is characterized by premature and accelerated aging and represents an important model because many aspects are also observed in normal aging. One therapeutical target of normal aging and HGP is the mechanistic target of rapamycin (mTOR) and its signaling pathway. It regulates important metabolic processes and is involved in cell growth and metabolism. Its specific role in aging and in the pathogenesis of HGP is only partially known. Here, the mTOR signaling pathway was investigated by using semi-quantitative Western-blot-analysis of up-stream-protein Akt, mTOR, and down-stream proteins p70S6K and ULK1. Control- and HGP-fibroblasts were grown until they reached replicative senescence through progressive shortening of telomeres. The effects of telomere length were examined by comparing cells with short telomeres and cells with long telomeres after immortalization with human telomerase reverse transcriptase (hTERT). Hydrogen peroxide served as a non-replicative stressor. HGP-fibroblasts revealed an increased proliferation rate by 53% (±35%) and a premature entry into arrest (on average 226 days earlier) with a higher percentage of senescent cells (HGP 73 ±18% vs. CON 40±3%). Healthy, replicatively aged cells exhibit lower proliferation rates, while key proteins of the mTOR signaling pathway were suppressed by at least 50%. HGP cells showed an impaired reduction of the mTOR pathway except for p70S6K. Immortalization, accompanied by telomere elongation, counteracted the effects of aging in both groups to varying extents. Treatment with hydrogen peroxide showed no significant effects on the mTOR pathway and could not imitate replicative aging. The results suggest that increased proliferation rates and impaired mTOR-signaling could be a pathogenetic mechanism of accelerated aging in progeria

Understanding women’s motivations to participate in MTN-003/VOICE, a phase 2b HIV prevention trial with low adherence

Background: In biomedical prevention trials, correct and consistent use of the investigational product is crucial to determine efficacy. Product adherence in VOICE, a phase 2B randomized trial of a vaginal gel and oral tablets for HIV prevention, was low (~ 34%), yet self-reported adherence and retention was high (\u3e 90%). This analysis from VOICE-D, a post-trial qualitative ancillary study, explores motivations to participate in VOICE, and possible sources of misalignment between the stated priorities of the trial and the participants. Methods: VOICE-D enrolled 171 former VOICE participants to investigate, among other things, reasons for joining and remaining in the trial. Local language in-depth interviews and focus groups were transcribed and translated into English and coded and analyzed using NVivo. Data on motivation to join obtained from a VOICE termination visit survey of 106 participants were also analyzed to corroborate the VOICE-D findings. Results: Participants primarily participated for personal health benefits (e.g. free healthcare and HIV testing) and reported remaining enrolled from a sense of commitment to the trial. Altruistic motivations were the most commonly stated motivation on the termination visit survey; qualitatively, many of those stating altruistic reasons also desired personal health benefits. Joining for financial reimbursement was not commonly mentioned. Social networks influenced recruitment and spread therapeutic misconception. Conclusions: Women’s participation for personal health benefits highlighted their desire to monitor their HIV risk and overall health. Helping participants view use of investigational products as improving social capital and reminding participants of their study responsibilities may improve trial outcomes. Understanding the reasons for participating in studies will help to ensure alignment between priorities of researchers and participants

Establishment and Characterization of hTERT Immortalized Hutchinson–Gilford Progeria Fibroblast Cell Lines

Hutchinson–Gilford progeria syndrome (HGPS) is a rare premature aging syndrome caused by a dominant mutation in the LMNA gene. Previous research has shown that the ectopic expression of the catalytic subunit of telomerase (hTERT) can elongate the telomeres of the patients’ fibroblasts. Here, we established five immortalized HGP fibroblast cell lines using retroviral infection with the catalytic subunit of hTERT. Immortalization enhanced the proliferative life span by at least 50 population doublings (PDs). The number of cells with typical senescence signs was reduced by 63 + 17%. Furthermore, the growth increase and phenotype improvement occurred with a lag phase of 50–100 days and was not dependent on the degree of telomere elongation. The initial telomeric stabilization after hTERT infection and relatively low amounts of hTERT mRNA were sufficient for the phenotype improvement but the retroviral infection procedure was associated with transient cell stress. Our data have implications for therapeutic strategies in HGP and other premature aging syndromes

Misreporting of product adherence in the MTN-003/VOICE trial for HIV prevention in Africa: Participants’ explanations for dishonesty

Consistent over-reporting of product use limits researchers’ ability to accurately measure adherence and estimate product efficacy in HIV prevention trials. While lying is a universal characteristic of the human condition, growing evidence of a stark discrepancy between self-reported product use and biologic or pharmacokinetic evidence demands examination of the reasons research participants frequently misrepresent product use in order to mitigate this challenge in future research. This study (VOICE-D) was an ancillary post-trial study of the vaginal and oral interventions to control the epidemic (VOICE) phase IIb trial (MTN 003). It was conducted in three African countries to elicit candid accounts from former VOICE trial participants about why actual product use was lower than reported. In total 171 participants were enrolled between December 2012 and March 2014 in South Africa (n = 47), Uganda (n = 59) and Zimbabwe (n = 65). Data suggested that participants understood the importance of daily product use and honest reporting, yet acknowledged that research participants typically lie. Participants cited multiple reasons for misreporting adherence, including human nature, self-presentation with study staff, fear of repercussions (study termination resulting in loss of benefits and experience of HIV-related stigma), a permissive environment in which it was easy to get away with misreporting, and avoiding inconvenient additional counseling. Some participants also reported mistrust of the staff and reciprocal dishonesty about the study products. Many suggested real-time blood-monitoring during trials would encourage greater fidelity to product use and honesty in reporting. Participants at all sites understood the importance of daily product use and honesty, while also acknowledging widespread misreporting of product use. Narratives of dishonesty may suggest a wider social context of hiding products from partners and distrust about research, influenced by rumors circulating in clinic waiting-rooms and surrounding communities. Prevailing power hierarchies between staff and participants may exacerbate misreporting. Participants recognized and suggested that objective, real-time feedback is needed to encourage honest reporting

Disclosure of pharmacokinetic drug results to understand nonadherence

Objectives: In VOICE, a phase IIB trial of daily oral and vaginal tenofovir for HIV prevention, at least 50% of women receiving active products had undetectable tenofovir in all plasma samples tested. MTN-003D, an ancillary study using in-depth interviews (IDIs) and focus group discussions (FGDs), together with retrospective disclosure of plasma tenofovir pharmacokinetic results, explored adherence challenges during VOICE. Methods: We systematically recruited participants with pharmacokinetic data (median six plasma samples), categorized as low (0%, N = 79), inconsistent (1–74%, N = 28) or high (≥75%; N = 20) on the basis of frequency of tenofovir detection. Following disclosure of pharmacokinetic results, reactions were captured and adherence challenges systematically elicited; IDIs and FGDs were audio-recorded, transcribed, coded and thematically analysed. Results: We interviewed 127 participants from South Africa, Uganda and Zimbabwe. The most common reactions to pharmacokinetic results included surprise (41%; low pharmacokinetic), acceptance (39%; inconsistent pharmacokinetic) and happiness (65%; high pharmacokinetic). On the basis of participants’ explanations, we developed a typology of adherence patterns: noninitiation, discontinuation, misimplementation (resulting from visit-driven use, variable taking, modified dosing or regimen) and adherence. Fear of product side effects/harm was a frequent concern, fuelled by stories shared among participants. Although women with high pharmacokinetic levels reported similar concerns, several described strategies to overcome challenges. Women at all pharmacokinetic levels suggested real-time drug monitoring and feedback to improve adherence and reporting. Conclusion: Retrospective provision of pharmacokinetic results seemingly promoted candid discussions around nonadherence and study participation. The effect of real-time drug monitoring and feedback on adherence and accuracy of reporting should be evaluated in trials