mTORC1-S6K Activation by Endotoxin Contributes to Cytokine Up-Regulation and Early Lethality in Animals

Background: mTORC1 (mammalian target of rapamycin complex 1) activation has been demonstrated in response to endotoxin challenge, but the mechanism and significance are unclear. We investigated the effect of mTORC1 suppression in an animal model of endotoxemia and in a cellular model of endotoxin signaling. Methodology/Principal Findings: Mice were treated with the mTORC1 inhibitor rapamycin or vehicle prior to lethal endotoxin challenge. Mortality and cytokine levels were assessed. Cultured macrophage-like cells were challenged with endotoxin with or without inhibitors of various pathways known to be upstream of mTORC1. Activated pathways, including downstream S6K pathway, were assessed by immunoblots. We found that mTORC1-S6K suppression by rapamycin delayed mortality of mice challenged with lethal endotoxin, and was associated with dampened circulating levels of VEGF, IL-1b, IFN-c and IL-5. Furthermore, in vitro cellular studies demonstrated that LPS (lipopolysaccharide) activation of mTORC1-S6K still occurs in the presence of PI3K-Akt inhibition alone, but can be suppressed by concurrent inhibition of PI3K-Akt and MEK-ERK pathways. Conclusions/Significance: We conclude that cellular activation of mTORC1-S6K contributes to cytokine up-regulation an

Elderly persons in the risk zone. Design of a multidimensional, health-promoting, randomised three-armed controlled trial for "prefrail" people of 80+ years living at home

Background The very old (80+) are often described as a "frail" group that is particularly exposed to diseases and functional disability. They are at great risk of losing the ability to manage their activities of daily living independently. A health-promoting intervention programme might prevent or delay dependence in activities of daily life and the development of functional decline. Studies have shown that those who benefit most from a health-promoting and disease-preventive programme are persons with no, or discrete, activity restrictions. The three-armed study "Elderly in the risk zone" is designed to evaluate if multi-dimensional and multi-professional educational senior meetings are more effective than preventive home visits, and if it is possible to prevent or delay deterioration if an intervention is made when the persons are not so frail. In this paper the study design, the intervention and the outcome measures as well as the baseline characteristics of the study participants are presented. Methods/Design The study is a randomised three-armed single-blind controlled trial with follow-ups 3 months, 1 and 2 years. The study group should comprise a representative sample of pre-frail 80-year old persons still living at home in two municipalities of Gothenburg. To allow for drop-outs, it was estimated that a total of about 450 persons would need to be included in the study. The participants should live in their ordinary housing and not be dependent on the municipal home help service or care. Further, they should be independent of help from another person in activities of daily living and be cognitively intact, having a score of 25 or higher as assessed with the Mini Mental State Examination (MMSE). Discussion We believe that the design of the study, the randomisation procedure, outcome measurements and the study protocol meetings should ensure the quality of the study. Furthermore, the multi-dimensionality of the intervention, the involvement of both the professionals and the senior citizens in the planning of the intervention should have the potential to effectively target the heterogeneous needs of the elderly. Trial registration ClinicalTrials.gov, NCT0087705

Fidelity and moderating factors in complex interventions: a case study of a continuum of care program for frail elderly people in health and social care

<p>Abstract</p> <p>Background</p> <p>Prior studies measuring fidelity of complex interventions have mainly evaluated adherence, and not taken factors affecting adherence into consideration. A need for studies that clarify the concept of fidelity and the function of factors moderating fidelity has been emphasized. The aim of the study was to systematically evaluate implementation fidelity and possible factors influencing fidelity of a complex care continuum intervention for frail elderly people.</p> <p>Methods</p> <p>The intervention was a systematization of the collaboration between a nurse with geriatric expertise situated at the emergency department, the hospital ward staff, and a multi-professional team with a case manager in the municipal care services for older people. Implementation was evaluated between September 2008 and May 2010 with observations of work practices, stakeholder interviews, and document analysis according to a modified version of The Conceptual Framework for Implementation Fidelity.</p> <p>Results</p> <p>A total of 16 of the 18 intervention components were to a great extent delivered as planned, while some new components were added to the model. No changes in the frequency or duration of the 18 components were observed, but the dose of the added components varied over time. Changes in fidelity were caused in a complex, interrelated fashion by all the moderating factors in the framework, i.e., context, staff and participant responsiveness, facilitation, recruitment, and complexity.</p> <p>Discussion</p> <p>The Conceptual Framework for Implementation Fidelity was empirically useful and included comprehensive measures of factors affecting fidelity. Future studies should focus on developing the framework with regard to how to investigate relationships between the moderating factors and fidelity over time.</p> <p>Trial registration</p> <p>ClinicalTrials.gov, <a href="http://www.clinicaltrials.gov/ct2/show/NCT01260493">NCT01260493</a>.</p

Design of a randomized controlled study of a multi-professional and multidimensional intervention targeting frail elderly people

<p>Abstract</p> <p>Background</p> <p>Frail elderly people need an integrated and coordinated care. The two-armed study "Continuum of care for frail elderly people" is a multi-professional and multidimensional intervention for frail community-dwelling elderly people. It was designed to evaluate whether the intervention programme for frail elderly people can reduce the number of visits to hospital, increase satisfaction with health and social care and maintain functional abilities. The implementation process is explored and analysed along with the intervention. In this paper we present the study design, the intervention and the outcome measures as well as the baseline characteristics of the study participants.</p> <p>Methods/design</p> <p>The study is a randomised two-armed controlled trial with follow ups at 3, 6 and 12 months. The study group includes elderly people who sought care at the emergency ward and discharged to their own homes in the community. Inclusion criteria were 80 years and older <it>or </it>65 to 79 years with at least one chronic disease and dependent in at least one activity of daily living. Exclusion criteria were acute severely illness with an immediate need of the assessment and treatment by a physician, severe cognitive impairment and palliative care. The intention was that the study group should comprise a representative sample of frail elderly people at a high risk of future health care consumption. The intervention includes an early geriatric assessment, early family support, a case manager in the community with a multi-professional team and the involvement of the elderly people and their relatives in the planning process.</p> <p>Discussion</p> <p>The design of the study, the randomisation procedure and the protocol meetings were intended to ensure the quality of the study. The implementation of the intervention programme is followed and analysed throughout the whole study, which enables us to generate knowledge on the process of implementing complex interventions. The intervention contributes to early recognition of both the elderly peoples' needs of information, care and rehabilitation and of informal caregivers' need of support and information. This study is expected to show positive effects on frail elderly peoples' health care consumption, functional abilities and satisfaction with health and social care.</p> <p>Trial registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01260493">NCT01260493</a></p

TNF-α secretion of LPS-treated macrophages occurred after mTORC1-S6K activation.

<p>Conditioned media and cell lysates of RAW cells treated with 100 ng/ml of LPS were collected at various time points. The immunoblots probing for pS6(S240/244) (A.), and the averages of TNF-α levels in the conditioned media from triplicate experiments with error bars representing standard deviations (B.) are shown here. mTORC1-S6K activation occurred as soon as 30 min after LPS when TNF-α levels were not detectably increased at that time point.</p

Rapamycin treatment effectively suppressed mTORC1-S6K signaling.

<p>Following LPS challenge in vivo, mice were treated with vehicle or 6 mg/kg rapamycin 30 min prior to 25 mg/kg LPS challenge. Organs were harvested from mice at 2, 6, 24 hr post challenge. Immunoblots of organs extracted from 3 mice from each treatment group probed for pS6 (S240/244), a downstream target of mTORC1-S6K are shown here. Unchallenged mice with or without rapamycin served as controls (time 0 hr). Rapamycin treatment successfully suppressed mTORC1-S6K up-regulation in endotoxemic mice as reflected by the near-absent pS6 signals.</p

mTORC1-S6K up-regulation in the livers, lungs, and kidneys of mice treated with endotoxin.

<p>Representative immunoblots of normalized organ protein extracts probed for pS6 (S240/244), a downstream target of mTORC1-S6K are shown here. Mice were subjected to 25 mg/kg of LPS i.p. and sacrificed at 2,6, and 24 hr later. Untreated mice served as time 0 hr controls. mTORC1-S6K activation could be seen as early as 2 hr after LPS challenge in the livers and lungs of endotoxemic mice. mTORC1-S6K was also activated in the kidneys at 24 hr post challenge. Increased pAkt (S473) was seen only in the lung extracts post LPS challenged, occurring at 6 hr post LPS, after mTORC1-S6K activation, and was not seen in either liver or kidney.</p

Rapamycin suppression of mTORC1-S6K enhanced early survival of endotoxemic mice.

<p>Kaplan-Meier curves of LPS-challenged mice treated with vehicle control (N = 10) or rapamycin (N = 9) are shown here. Rapamycin-treated mice had significantly better survival compared to vehicle-treated (P = 0.033, logrank test). However, all mice had died by 72 hr after LPS challenge.</p

IKKβ was not required for LPS-induced mTORC1-S6K activation.

<p>IKKβ was suppressed in RAW cells by siRNA (IK) 48 hr prior to LPS stimulation. Nonsense siRNA constructs (NS) served as controls. Representative immunoblots show that IKKβ suppression had no effect on mTORC1-S6K activation in LPS-treated RAW cells as reflected by pS6 levels.</p

Rapamycin suppression of mTORC1-S6K was associated with lower cytokine levels.

<p>Following LPS treatment in vivo, circulating cytokine levels of LPS-challenged mice pre-treated with vehicle (solid bar) or rapamycin (open bar) at 0 (no LPS), 2, 6, and 24 hr post LPS are shown here. Data are expressed as mean of three mice in each group at each time point, with error bars representing standard deviations. Statistical analysis was performed using Repeated Measures ANOVA, and p values<0.05 are considered as significant (*). mTORC1-S6K suppression by rapamycin led to decreased circulation levels of IL1-β, VEGF, IFN-γ and IL-5. There was a trend toward lower TNF-α levels with a p value of 0.058.</p