A Human Skin Explant Test as a Novel In Vitro Assay for the Detection of Skin Sensitization to Aggregated Monoclonal Antibodies.

Introduction: Monoclonal antibodies (mAbs) are important therapeutics. However, the enhanced potential for aggregation has become a critical quality parameter during the production of mAbs. Furthermore, mAb aggregation may also present a potential health risk in a clinical setting during the administration of mAb therapeutics to patients. While the extent of immunotoxicity in patient populations is uncertain, reports show it can lead to immune responses via cell activation and cytokine release. In this study, an autologous in vitro skin test designed to predict adverse immune events, including skin sensitization, was used as a novel assay for the assessment of immunotoxicity caused by mAb aggregation. Material and Methods: Aggregation of mAbs was induced by a heat stress protocol, followed by characterization of protein content by analytical ultra-centrifugation and transmission electron microscopy, revealing a 4% aggregation level of total protein content. Immunotoxicity and potential skin sensitization caused by the aggregates, were then tested in a skin explant assay. Results: Aggregated Herceptin and Rituximab caused skin sensitization, as shown by histopathological damage (grade II-III positive response) together with positive staining for Heat Shock Protein 70 (HSP70). Changes in T cell proliferation were not observed. Cytokine analysis revealed a significant increase of IL-10 for the most extreme condition of aggregation (65 °C at pH3) and a trend for an overall increase of IFN-γ, especially in response to Rituximab. Conclusions: The skin explant assay demonstrated that aggregated mAbs showed adverse immune reactions, as demonstrated as skin sensitization, with histopathological grades II-III. The assay may, therefore, be a novel tool for assessing immunotoxicity and skin sensitization caused by mAb aggregation.

Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice.

Cancer survivors suffer from progressive frailty, multimorbidity, and premature morbidity. We hypothesise that therapy-induced senescence and senescence progression via bystander effects are significant causes of this premature ageing phenotype. Accordingly, the study addresses the question whether a short anti-senescence intervention is able to block progression of radiation-induced frailty and disability in a pre-clinical setting. Male mice were sublethally irradiated at 5 months of age and treated (or not) with either a senolytic drug (Navitoclax or dasatinib + quercetin) for 10 days or with the senostatic metformin for 10 weeks. Follow-up was for 1 year. Treatments commencing within a month after irradiation effectively reduced frailty progression (p<0.05) and improved muscle (p<0.01) and liver (p<0.05) function as well as short-term memory (p<0.05) until advanced age with no need for repeated interventions. Senolytic interventions that started late, after radiation-induced premature frailty was manifest, still had beneficial effects on frailty (p<0.05) and short-term memory (p<0.05). Metformin was similarly effective as senolytics. At therapeutically achievable concentrations, metformin acted as a senostatic neither via inhibition of mitochondrial complex I, nor via improvement of mitophagy or mitochondrial function, but by reducing non-mitochondrial reactive oxygen species production via NADPH oxidase 4 inhibition in senescent cells. Our study suggests that the progression of adverse long-term health and quality-of-life effects of radiation exposure, as experienced by cancer survivors, might be rescued by short-term adjuvant anti-senescence interventions.

Cancer treatments save lives, but they can also be associated with long-term side effects which greatly reduce quality of life; former patients often face fatigue, memory loss, frailty, higher likelihood of developing other cancers, and overall accelerated aging. Senescence is a change in a cell's state that follows damage and is associated with aging. When a cell becomes senescent it stops dividing, can promote inflammation and may damage other cells. Research has shown that cancer treatment increases the numbers of cells entering senescence, potentially explaining the associated long-term side effects. A new class of drugs known as senolytics can kill senescent cells, but whether they could help to counteract the damaging effects of cancer treatments remain unclear. To explore this question, Fielder et al. focused on mice having received radiation therapy, which also exhibit the long-term health defects observed in human patients. In these animals, a single, short senolytic treatment after irradiation nearly erased premature aging; frailty did not increase faster than normal, new cancers were less prevalent, and the rodents retained good memory and muscle function for at least one year after irradiation. Even mice treated later in life, after frailty was already established, showed some improvement. In addition, multiple tissues, including the brain and the liver, hosted fewer senescent cells in the animals treated with senolytics, even up to old age. Research should now explore whether these remarkable effects could also be true for humans.

Palmitate induces DNA damage and senescence in human adipocytes in vitro that can be alleviated by oleic acid but not inorganic nitrate.

Hypertrophy in white adipose tissue (WAT) can result in sustained systemic inflammation, hyperlipidaemia, insulin resistance, and onset of senescence in adipocytes. Inflammation and hypertrophy can be induced in vitro using palmitic acid (PA). WAT adipocytes have innately low ß-oxidation capacity, while inorganic nitrate can promote a beiging phenotype, with promotion of ß-oxidation when cells are exposed to nitrate during differentiation. We hypothesized that treatment of human adipocytes with PA in vitro can induce senescence, which might be attenuated by nitrate treatment through stimulation of ß-oxidation to remove accumulated lipids. Differentiated subcutaneous and omental adipocytes were treated with PA and nitrate and senescence markers were analyzed. PA induced DNA damage and increased p16INK4a levels in both human subcutaneous and omental adipocytes in vitro. However, lipid accumulation and lipid droplet size increased after PA treatment only in subcutaneous adipocytes. Thus, hypertrophy and senescence seem not to be causally associated. Contrary to our expectations, subsequent treatment of PA-induced adipocytes with nitrate did not attenuate PA-induced lipid accumulation or senescence. Instead, we found a significantly beneficial effect of oleic acid (OA) on human subcutaneous adipocytes when applied together with PA, which reduced the DNA damage caused by PA treatment.

Protective effect of argan oil on DNA damage in vivo and in vitro.

Background: Iron-overload is a well-known cause for the development of chronic liver diseases and known to induce DNA damage.Material and methods: The protective effect of argan oil (AO) from the Argania spinosa fruit and olive oil (OO) (6% AO or OO for 28 days) was evaluated on a mouse model of iron overload (3.5mg Fe2+/liter) and in human fibroblasts where DNA damage was induced via culture under hyperoxia (40% oxygen).Results: Iron treatment induced DNA damage in liver tissue while both oils were able to decrease it. We confirmed this effect in vitro in MRC-5 fibroblasts under hyperoxia. A cell-free ABTS assay suggested that improvement of liver toxicity by both oils might depend on a high content in tocopherol, phytosterol and polyphenol compounds known for their antioxidant potential. The antioxidant effect of AO was confirmed in fibroblasts by reduced intracellular peroxide levels after hyperoxia. However, we could not find a significant decrease of genes encoding pro-inflammatory cytokines (TNFα, IL-6, IL-1ß, COX-2) or senescence markers (p16 and p21) for the oils in mouse liver.Conclusion: We found a striking effect of AO by ameliorating DNA damage after iron overload in a mouse liver model and in human fibroblasts by hyperoxia adding compelling evidence to the protective mechanisms of AO and OO.

The bystander effect contributes to the accumulation of senescent cells in vivo.

Senescent cells accumulate with age in multiple tissues and may cause age-associated disease and functional decline. In vitro, senescent cells induce senescence in bystander cells. To see how important this bystander effect may be for accumulation of senescent cells in vivo, we xenotransplanted senescent cells into skeletal muscle and skin of immunocompromised NSG mice. 3 weeks after the last transplantation, mouse dermal fibroblasts and myofibres displayed multiple senescence markers in the vicinity of transplanted senescent cells, but not where non-senescent or no cells were injected. Adjacent to injected senescent cells, the magnitude of the bystander effect was similar to the increase in senescence markers in myofibres between 8 and 32 months of age. The age-associated increase of senescence markers in muscle correlated with fibre thinning, a widely used marker of muscle aging and sarcopenia. Senescent cell transplantation resulted in borderline induction of centrally nucleated fibres and no significant thinning, suggesting that myofibre aging might be a delayed consequence of senescence-like signalling. To assess the relative importance of the bystander effect versus cell-autonomous senescence, we compared senescent hepatocyte frequencies in livers of wild-type and NSG mice under ad libitum and dietary restricted feeding. This enabled us to approximate cell-autonomous and bystander-driven senescent cell accumulation as well as the impact of immunosurveillance separately. The results suggest a significant impact of the bystander effect for accumulation of senescent hepatocytes in liver and indicate that senostatic interventions like dietary restriction may act as senolytics in immunocompetent animals.

Metabolic memory of dietary restriction ameliorates DNA damage and adipocyte size in mouse visceral adipose tissue.

Dietary restriction (DR) is thought to exert its beneficial effects on healthspan at least partially by a senolytic and senostatic action, i.e. by reducing frequencies of cells with markers of DNA damage and senescence in multiple tissues. Due to its importance in metabolic and inflammation regulation, fat is a prime tissue for health span determination as well as a prime target for DR. We aimed to determine here whether the beneficial effects of DR would be retained over a subsequent period of ad libitum (AL) feeding. Male mice were kept under either 40% DR or AL feeding regimes from 3 to 12 months of age and then either switched back to the opposite feeding regimen or kept in the same state for another 3 months. Visceral adipose tissue from 4 to 5 mice per group for all conditions was analysed for markers of senescence (adipocyte size, γH2A.X, p16, p21) and inflammation (e.g. IL-6, TNFα, IL-1ß) using immuno-staining or qPCR. Macrophages were detected by immunohistochemistry. We found that both 9 and 12 months DR (long term) as well as 3 month (short term, mid-life onset) DR reduced the number of cells harbouring DNA damage and adipocyte size (area and perimeter) in visceral adipocytes with similar efficiency. Importantly, beneficial health markers induced by DR such as small adipocyte size and low DNA damage were maintained for at least 3 month after termination of DR, demonstrating that the previously identified 'metabolic memory' of the DR state in male mice extends to senescence markers in visceral fat.

Inorganic Nitrate Supplementation in Young and Old Obese Adults Does Not Affect Acute Glucose and Insulin Responses but Lowers Oxidative Stress.

BACKGROUND: Aging and obesity are associated with raised oxidative stress and a reduction of nitric oxide (NO) bioavailability, with subsequent decline in insulin sensitivity and endothelial function. Inorganic nitrate is converted into NO via a 2-step reduction process and may be an effective nutritional intervention to modify vascular and metabolic functions. OBJECTIVES: This study tested whether inorganic nitrate supplementation improved glucose disposal and attenuated the acute effects of hyperglycemia on oxidative stress, inflammation, and vascular function in young and old obese participants. METHODS: Ten young (aged 18-44 y) and 10 old (aged 55-70 y) obese participants consumed 75 g glucose followed by either potassium nitrate (7 mg/kg body weight) or potassium chloride (placebo) in a randomized, double-blind crossover design. Resting blood pressure (BP), endothelial function, and blood biomarkers were measured for 3 h postintervention. Biomarkers included plasma nitrate/nitrite (NOx), glucose, insulin, cyclic GMP, interleukin 6, 3-nitrotyrosine, E- and P-selectins, intercellular adhesion molecule 3 (ICAM-3), and thrombomodulin, as well as superoxide in freshly isolated peripheral blood mononuclear cells (PBMCs). RESULTS: Inorganic nitrate supplementation did not affect plasma glucose (P = 0.18) or insulin (P = 0.26) responses. The increase in plasma NOx concentrations 3 h after the administration of inorganic nitrate was significantly higher in young than in old participants (234% increase compared with 149% increase, respectively, P < 0.001). Plasma 3-nitrotyrosine concentrations declined significantly after inorganic nitrate supplementation compared with placebo (3 h postdose, 46% decrease compared with 27% increase, respectively, P = 0.04), and a similar nonsignificant trend was observed for superoxide concentrations (3 h postdose, 16% decrease compared with 23% increase, respectively, P = 0.06). Plasma cyclic GMP, ICAM-3, and thrombomodulin concentrations differed between young and old participants (P < 0.01). Inorganic nitrate supplementation did not improve BP or endothelial function. CONCLUSIONS: Oral supplementation with inorganic nitrate did not improve glucose and insulin responses but reduced oxidative stress in old individuals during acute hyperglycemia. This trial was registered at www.controlled-trials.com as ISRCTN42776917.

Telomerase Activity is Downregulated Early During Human Brain Development.

Changes in hTERT splice variant expression have been proposed to facilitate the decrease of telomerase activity during fetal development in various human tissues. Here, we analyzed the expression of telomerase RNA (hTR), wild type and α-spliced hTERT in developing human fetal brain (post conception weeks, pcw, 6-19) and in young and old cortices using qPCR and correlated it to telomerase activity measured by TRAP assay. Decrease of telomerase activity occurred early during brain development and correlated strongest to decreased hTR expression. The expression of α-spliced hTERT increased between pcw 10 and 19, while that of wild type hTERT remained unchanged. Lack of expression differences between young and old cortices suggests that most changes seem to occur early during human brain development. Using in vitro differentiation of neural precursor stem cells (NPSCs) derived at pcw 6 we found a decrease in telomerase activity but no major expression changes in telomerase associated genes. Thus, they do not seem to model the mechanisms for the decrease in telomerase activity in fetal brains. Our results suggest that decreased hTR levels, as well as transient increase in α-spliced hTERT, might both contribute to downregulation of telomerase activity during early human brain development between 6 and 17 pcw.