Circulating monocytes expressing senescence-associated features are enriched in COVID-19 patients with severe disease.

Accurate biomarkers for predicting COVID-19 severity have remained an unmet need due to an incomplete understanding of virus pathogenesis and heterogeneity among patients. Cellular senescence and its pro-inflammatory phenotype are suggested to be a consequence of SARS-CoV-2 infection and potentially drive infection-dependent pathological sequelae. Senescence-associated markers in infected individuals have been identified primarily in the lower respiratory tract, while little is known about their presence in more easily accessible bio-specimens. Here, we measured the abundance of senescence-associated signatures in whole blood, plasma and peripheral blood mononuclear cells (PBMCs) of COVID-19 patients and patients without an infection. Bulk transcriptomic and targeted proteomic assays revealed that the level of senescence-associated markers, including the senescence-associated secretory phenotype (SASP), is predictive of SARS-CoV-2 infection. Single-cell RNA-sequencing data demonstrated that a senescence signature is particularly enriched in monocytes of COVID-19 patients, partially correlating with disease severity. Our findings suggest that monocytes are prematurely induced to senescence by SARS-CoV-2 infection, might contribute to exacerbating a SASP-like inflammatory response and can serve as markers and predictors for COVID-19 and its sequelae.

Senolytic Therapy to Modulate the Progression of Alzheimer's Disease (SToMP-AD): A Pilot Clinical Trial.

Preclinical studies indicate an age-associated accumulation of senescent cells across multiple organ systems. Emerging evidence suggests that tau protein accumulation, which closely correlates with cognitive decline in Alzheimer's disease and other tauopathies, drives cellular senescence in the brain. Pharmacologically clearing senescent cells in mouse models of tauopathy reduced brain pathogenesis. Compared to vehicle treated mice, intermittent senolytic administration reduced tau accumulation and neuroinflammation, preserved neuronal and synaptic density, restored aberrant cerebral blood flow, and reduced ventricular enlargement. Intermittent dosing of the senolytics, dasatinib plus quercetin, has shown an acceptable safety profile in clinical studies for other senescence-associated conditions. With these data, we proposed and herein describe the objectives and methods for a clinical vanguard study. This initial open-label clinical trial pilots an intermittent senolytic combination therapy of dasatinib plus quercetin in five older adults with early-stage Alzheimer's disease. The primary objective is to evaluate the central nervous system penetration of dasatinib and quercetin through analysis of cerebrospinal fluid collected at baseline and after 12 weeks of treatment. Further, through a series of secondary outcome measures to assess target engagement of the senolytic compounds and Alzheimer's disease-relevant cognitive, functional, and physical outcomes, we will collect preliminary data on safety, feasibility, and efficacy. The results of this study will be used to inform the development of a randomized, double-blind, placebo-controlled multicenter phase II trial to further explore of the safety, feasibility, and efficacy of senolytics for modulating the progression of Alzheimer's disease. Clinicaltrials.gov registration number and date: NCT04063124 (08/21/2019).

Senolytic drugs: from discovery to translation.

Senolytics are a class of drugs that selectively clear senescent cells (SC). The first senolytic drugs Dasatinib, Quercetin, Fisetin and Navitoclax were discovered using a hypothesis-driven approach. SC accumulate with ageing and at causal sites of multiple chronic disorders, including diseases accounting for the bulk of morbidity, mortality and health expenditures. The most deleterious SC are resistant to apoptosis and have up-regulation of anti-apoptotic pathways which defend SC against their own inflammatory senescence-associated secretory phenotype (SASP), allowing them to survive, despite killing neighbouring cells. Senolytics transiently disable these SCAPs, causing apoptosis of those SC with a tissue-destructive SASP. Because SC take weeks to reaccumulate, senolytics can be administered intermittently - a 'hit-and-run' approach. In preclinical models, senolytics delay, prevent or alleviate frailty, cancers and cardiovascular, neuropsychiatric, liver, kidney, musculoskeletal, lung, eye, haematological, metabolic and skin disorders as well as complications of organ transplantation, radiation and cancer treatment. As anticipated for agents targeting the fundamental ageing mechanisms that are 'root cause' contributors to multiple disorders, potential uses of senolytics are protean, potentially alleviating over 40 conditions in preclinical studies, opening a new route for treating age-related dysfunction and diseases. Early pilot trials of senolytics suggest they decrease senescent cells, reduce inflammation and alleviate frailty in humans. Clinical trials for diabetes, idiopathic pulmonary fibrosis, Alzheimer's disease, COVID-19, osteoarthritis, osteoporosis, eye diseases and bone marrow transplant and childhood cancer survivors are underway or beginning. Until such studies are done, it is too early for senolytics to be used outside of clinical trials.

Proceedings of the Canadian Frailty Network Workshop: Identifying Biomarkers of Frailty to Support Frailty Risk Assessment, Diagnosis and Prognosis. Toronto, January 15, 2018.

The Canadian Frailty Network (CFN), a pan-Canadian not-for-profit organization funded by the Government of Canada through the Networks of Centres of Excellence Program, is dedicated to improving the care of older Canadians living with frailty. The CFN has partnered with the Canadian Longitudinal Study on Aging (CLSA) to measure potential frailty biomarkers in biological samples (whole blood, plasma, urine) collected in over 30,000 CLSA participants. CFN hosted a workshop in Toronto on January 15 2018, bringing together experts in the field of biomarkers, aging and frailty. The overall objectives of the workshop were to start building a consensus on potential frailty biomarker domains and identify specific frailty biomarkers to be measured in the CLSA biological samples. The workshop was structured with presentations in the morning to frame the discussions for the afternoon session, which was organized as a free-flowing discussion to benefit from the expertise of the participants. Participants and speakers were from Canada, Italy, Spain, United Kingdom and the United States. Herein we provide pertinent background information, a summary of all the presentations with key figures and tables, and the distillation of the discussions. In addition, moving forward, the principles CFN will use to approach frailty biomarker research and development are outlined. Findings from the workshop are helping CFN and CLSA plan and conduct the analysis of biomarkers in the CLSA samples and which will inform a follow-up data access competition.

Muscle-specific differences in expression and phosphorylation of the Janus kinase 2/Signal Transducer and Activator of Transcription 3 following long-term mechanical ventilation and immobilization in rats.

AIM: Muscle wasting is one of the factors most strongly predicting mortality and morbidity in critically ill intensive care unit (ICU). This muscle wasting affects both limb and respiratory muscles, but the understanding of underlying mechanisms and muscle-specific differences remains incomplete. This study aimed at investigating the temporal expression and phosphorylation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in muscle wasting associated with the ICU condition to characterize the JAK/STAT proteins and the related changes leading or responding to their activation during exposure to the ICU condition. METHODS: A novel experimental ICU model allowing long-term exposure to the ICU condition, immobilization and mechanical ventilation, was used in this study. Rats were pharmacologically paralysed by post-synaptic neuromuscular blockade and mechanically ventilated for durations varying between 6 hours and 14 days to study muscle-specific differences in the temporal activation of the JAK/STAT pathway in plantaris, intercostal and diaphragm muscles. RESULTS: The JAK2/STAT3 pathway was significantly activated irrespective of muscle, but muscle-specific differences were observed in the temporal activation pattern between plantaris, intercostal and diaphragm muscles. CONCLUSION: The JAK2/STAT3 pathway was differentially activated in plantaris, intercostal and diaphragm muscles in response to the ICU condition. Thus, JAK2/STAT3 inhibitors may provide an attractive pharmacological intervention strategy in immobilized ICU patients, but further experimental studies are required in the study of muscle-specific effects on muscle mass and function in response to both short- and long-term exposure to the ICU condition prior to the translation into clinical research and practice.

Molecular pathology endpoints useful for aging studies.

The first clinical trial aimed at targeting fundamental processes of aging will soon be launched (TAME: Targeting Aging with Metformin). In its wake is a robust pipeline of therapeutic interventions that have been demonstrated to extend lifespan or healthspan of preclinical models, including rapalogs, antioxidants, anti-inflammatory agents, and senolytics. This ensures that if the TAME trial is successful, numerous additional clinical trials are apt to follow. But a significant impediment to these trials remains the question of what endpoints should be measured? The design of the TAME trial very cleverly skirts around this based on the fact that there are decades of data on metformin in humans, providing unequaled clarity of what endpoints are most likely to yield a positive outcome. But for a new chemical entity, knowing what endpoints to measure remains a formidable challenge. For economy's sake, and to achieve results in a reasonable time frame, surrogate markers of lifespan and healthy aging are desperately needed. This review provides a comprehensive analysis of molecular endpoints that are currently being used as indices of age-related phenomena (e.g., morbidity, frailty, mortality) and proposes an approach for validating and prioritizing these endpoints.

Effect of Low-Dose Rapamycin on Senescence Markers and Physical Functioning in Older Adults with Coronary Artery Disease: Results of a Pilot Study.

Rapamycin, an mTOR inhibitor affects senescence through suppression of senescence-associated secretory phenotype (SASP). We studied the safety and feasibility of low-dose rapamycin and its effect on SASP and frailty in elderly undergoing cardiac rehabilitation (CR). 13 patients; 6 (0.5mg), 6 (1.0mg), and 1 patient received 2mg oral rapamycin (serum rapamycin <6ng/ml) daily for 12 weeks. Median age was 73.9±7.5 years and 12 were men. Serum interleukin-6 decreased (2.6 vs 4.4 pg/ml) and MMP-3 (26 vs 23.5 ng/ml) increased. Adipose tissue expression of mRNAs (arbitrary units) for MCP-1 (3585 vs 2020, p=0.06), PPAR-γ (1257 vs 1166), PAI-1 (823 vs 338, p=0.08) increased, whereas interleukin-8 (163 vs 312), TNF-α (75 vs 94) and p16 (129 vs 169) decreased. Cellular senescence-associated beta galactosidase activity (2.2% vs 3.6%, p=0.18) tended to decrease. We observed some correlation between some senescence markers and physical performance but no improvement in frailty with rapamycin was noted. (NCT01649960).

Inflammatory characteristics of adipose tissue collected by surgical excision vs needle aspiration.

Subcutaneous adipose tissue can be obtained for research during an elective, clinically indicated operation by standard surgical excision approaches and by needle aspiration in pure research settings. Whether measurements of inflammatory markers and cells from tissues collected in these two different ways are comparable is debatable. We sought to determine whether these two techniques yield systematically different results for measurements of inflammation, cellular senescence and adipose tissue composition. Twelve subjects undergoing surgery participated. At the time of surgery abdominal subcutaneous adipose tissue from adjacent sites was removed by excision and needle aspiration. Stromovascular cell composition (flow cytometry), the number of senescent cells (senescence-associated-ß-galactosidase staining) and interleukin (IL)-6, IL-1, TNF-α and MCP1 mRNA (reverse transcription-PCR) were measured in each sample. We found no statistically significant differences between the two sample-collection approaches for any of the parameters measured. We conclude that these two methods of obtaining adipose tissue do not systematically differ in the results of cytokine mRNA content, cellular senescence or stromovascular cell composition.

Nutritional risk in an urban homebound older population. The nutrition and healthy aging project.

PURPOSE: To establish the prevalence of nutritional problems and their related socio-demographic and health-related risk factors in the homebound elderly population. METHODS: Subjects included 239 men and women, ages 65 to 105 years. Trained, two-person field teams conducted comprehensive in-home assessments. Medical record reviews assessed co-morbidity and medication use. RESULTS: The majority of these urban study subjects are of very advanced age (mean age 81 years), female (72%), non-white (73%), living alone (51%), of low income (76%), and somewhat socially isolated (26% had no weekly social contact). More older women than men were widowed (60 vs. 33%, respectively) and poor (80 vs. 67%). The disease burden and functional dependency were both high in men and women; 77% had three or more chronic medical conditions; 76% were functionally dependent in one or more ADL's and 95% in one or more IADL's. Poor dietary quality was universal in these older men and women; half or more consumed diets that deviated from recommended standards for at least 13 of the 24 nutritional guidelines studied. Five percent of subjects were underweight (Body Mass Index (BMI) <18.5); 22% were overweight (BMI 25.0-29.9); and 33% were obese (BMI >30.0). Fasting albumin, hemoglobin, and absolute lymphocyte concentrations were borderline to very low in 18-32%. Dyslipidemia was more common in women; however, men and women had similar Total:HDL cholesterol ratios. CONCLUSIONS: Nutritional status is poor in homebound persons of very advanced age with substantial co-morbidity and functional dependency. The complexities of nutritional risk necessitate multi-disciplinary and individualized nutritional intervention strategies.

Altered expression of C/EBP family members results in decreased adipogenesis with aging.

Fat mass, adipocyte size and metabolic responsiveness, and preadipocyte differentiation decrease between middle and old age. We show that expression of CCAAT/enhancer binding protein (C/EBP)-alpha, a key regulator of adipogenesis and fat cell function, declined substantially with aging in differentiating preadipocytes cultured under identical conditions from rats of various ages. Overexpression of C/EBP alpha in preadipocytes cultured from old rats restored capacity to differentiate into fat cells, indicating that downstream differentiation-dependent genes maintain responsiveness to regulators of adipogenesis. C/EBP alpha-expression also decreased with age in fat tissue from three different depots and in isolated fat cells. The overall level of C/EBP beta, which modulates C/EBP alpha-expression, did not change with age, but the truncated, dominant-negative C/EBP beta-liver inhibitory protein (LIP) isoform increased in cultured preadipocytes and isolated fat cells. Overexpression of C/EBP beta-LIP in preadipocytes from young rats impaired adipogenesis. C/EBP delta, which acts with full-length C/EBP beta to enhance adipogenesis, decreased with age. Thus processes intrinsic to adipose cells involving changes in C/EBP family members contribute to impaired adipogenesis and altered fat tissue function with aging. These effects are potentially reversible.

Fat depot origin affects fatty acid handling in cultured rat and human preadipocytes.

Regional differences in free fatty acid (FFA) handling contribute to diseases associated with particular fat distributions. As cultured rat preadipocytes became differentiated, FFA transfer into preadipocytes increased and was more rapid in single perirenal than in epididymal cells matched for lipid content. Uptake by human omental preadipocytes was greater than uptake by abdominal subcutaneous preadipocytes. Adipose-specific fatty acid binding protein (aP2) and keratinocyte lipid binding protein abundance was higher in differentiated rat perirenal than in epididymal preadipocytes. This interdepot difference in preadipocyte aP2 expression was reflected in fat tissue in older animals. Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform. Long-chain acyl-CoA levels were higher in perirenal than in epididymal preadipocytes and isolated fat cells. These data are consistent with interdepot differences in fatty acid flux ensuing from differences in fatty acid binding proteins and enzymes of fat metabolism. Heterogeneity among depots results, in part, from distinct intrinsic characteristics of adipose cells. Different depots are effectively separate miniorgans.

Esterification of free fatty acids in adipocytes: a comparison between octanoate and oleate.

Medium-chain triacylglycerols (MCT) are present in milk, coconut oil and other foods, and are used therapeutically in special diets for certain disorders of lipid and glucose utilization. Recently, it has become apparent that MCT are not only oxidized in the liver, but are also present in lymph and fat tissue, particularly after chronic treatment. To evaluate the influence of MCT on metabolism in fat cells, we compared incorporation of octanoate and oleate into cellular triacylglycerols of 3T3-L1 adipocytes as well as their effects on preadipocyte differentiation. We found that less octanoate than oleate was stored and that more octanoate than oleate was oxidized. Octanoate was esterified to a greater extent at the sn-1,3 position of glyceryl carbons than at the sn-2 position, whereas the opposite was true for oleate. Glycerol release from fat cells pre-treated with octanoate was also greater than from cells pre-treated with oleate, presumably related to the preferential release of octanoate from the sn-1,3 position. Octanoate was not incorporated into lipids in undifferentiated cells and did not induce differentiation in these cells, whereas oleate was readily stored and actually induced differentiation. Incorporation of octanoate into lipids increased as cells differentiated, but reached a maximum of about 10% of the total stored fatty acids. If these effects in vitro also occur in vivo, substitution of octanoate for oleate or other long-chain fatty acids could have the beneficial effect of diminishing fat-cell number and lipid content.

Improved inpatient billing metrics by physician-directed billing software.

Billing may not be prominent on academic physicians' lists of priorities because of other demands and commitments for their time. The authors developed and examined the use of an electronic inpatient billing tool that also facilitated physician rounding responsibilities. The results show that use of an electronic tool was more acceptable to physicians than their original card system, and it significantly decreased charge-processing lag time.

Incorporation of [1-13C]oleate into cellular triglycerides in differentiating 3T3L1 cells.

Oleate is one of the most abundant dietary fatty acids, and much remains to be learned about its metabolism in fat cells. We studied the incorporation of exogenous [1-13C]oleate into triglycerides (TG) in differentiating 3T3L1 preadipocytes using 13C NMR spectroscopy. The quantity of oleate incorporated into TG was found to increase as preadipocytes differentiated into fat cells. The ratio of unesterified [1-13C]oleate to total stored fatty acids was higher in less differentiated cells, and declined at later stages of differentiation as cells accumulated fatty acids through de novo synthesis. When added as the only exogenous fatty acid, oleate was largely esterified at the sn-2 position. When equimolar unlabeled linoleate was co-provided at the same time, the ratio of [1-13C]oleate esterified at the sn-1,3 position increased, implying competition between linoleate and oleate for esterification, especially at the sn-2 position. When cells pre-enriched with [1-13C]oleate (esterified to TG) were treated with isoproterenol, a lipolytic agent, most of the [1-13C]oleate was still found in TG, despite a high rate of lipolysis determined by measuring glycerol release. This implies extensive re-esterification of the oleate released by lipolysis.

A case of a laboratory animal feed with high estrogenic activity and its impact on in vivo responses to exogenously administered estrogens.

We recently noted that immature rats failed to exhibit a normal uterine response to exogenously administered estradiol as assessed by both biochemical (induction of gene expression) and morphological (altered uterine and vaginal histology and size) end points. An initial analysis suggested that this was due to a high degree of estrogenization from a dietary source which was producing a near maximal uterotrophic response prior to hormone treatment. Subsequent chemical analysis indicated that the feed in question contained high amounts of two well-known phytoestrogens, genistein (210 mg/kg) and daidzen (14 mg/kg), and the lot of feed in question produced a large uterotrophic effect when fed to immature ovariectomized rats. These findings illustrate that, despite increased awareness of phytoestrogens, some batches of animal feed contain very high amounts of estrogenic components which have marked effects on in vivo end points of hormone action. These observations have important implications for both basic research and screening methods that utilize in vivo approaches.

A 13C nuclear magnetic resonance study of free fatty acid incorporation in acylated lipids in differentiating preadipocytes.

To understand the role of free fatty acid (FFA) incorporation in the accumulation of lipids in the adipocyte and ultimately in the development of obesity, 13C nuclear magnetic resonance was used to study lipid metabolism in differentiating preadipocytes. The incorporation of 13C=O-labeled FFA into cellular lipids in primary cultured rat preadipocytes and 3T3L1 preadipocytes at different stages of differentiation was monitored by the 13C carbonyl chemical shift. Significant incorporation of palmitic acid into phosphatidylcholine in both the alpha and beta acyl chain positions was found in cells at early stages of differentiation. At later differentiation stages or after extended incubation periods, most of the 13C=O signals were found in the triacylglycerol (TG) molecules. Unsaturated 13C=O-labeled acyl chains were detected in the TG molecules when cells were incubated with saturated 13C=O-labeled FFA, indicating that intracellular dehydrogenation had occurred in the 13C=O-labeled palmitoyl chain. By using 13C-labeled methyl myristate as an internal intensity reference, incorporation of 13C FFA into each acyl chain position of the major intracellular lipids was determined quantitatively.

Telephone charges and payments in a diabetes clinic.

OBJECTIVES: To determine reimbursement rates after initiation of charges for certain telephone calls in a pediatric diabetes care center. DESIGN: A review of charges and payments data during 1996. RESULTS: Four hundred seventy-two telephone calls initiated by patients and parents were billed during the study period. These calls regarded treatment of hypoglycemia, hyperglycemia, ketonuria, sick day treatment, and insulin dose changes. Insured patients were charged for 384 telephone calls and indigent patients were charged for 88 telephone calls. Telephone calls from insured patients generated charges of $9215 and payments of $3074. Insurance payments were $1677 (18% of charges), and patient payments were $1396 (15% of charges). Telephone calls from uninsured patients covered by Texas Medicaid or Chronically Ill and Disabled Children funding generated charges of $2193 and no payments. CONCLUSIONS: Telephone charges were reimbursed by all payors at an overall rate of 27%.