An inflammatory aging clock (iAge) based on deep learning tracks multimorbidity, immunosenescence, frailty and cardiovascular aging

May combine two claims.

On the test set, the average reconstruction error was 15.2 years for age prediction and 0.26 normalized for circulating immune protein data.

“The average reconstruction errors on the test set for prediction of age and circulating immune protein data were 15.2 years and 0.26 (normalized), respectively.”

May combine two claims.

The GAE method outperformed other dimensionality-reduction methods in predicting chronological age, except that it did not differ significantly from the classic neural network.

“Overall, the GAE method utilized (a two-layer fully connected neural network with five nodes in each layer) outperformed other methods in predicting chronological age (cAge) (P < 0.05) with the exception of the classic neural network (P = 0.54) (Extended Data Fig. 3b).”

May combine two claims.

Several immune parameters predicted multimorbidity: total CD8+ T cells, plasmablasts and transitional B cells were negative predictors, while effector CD8+ T cells, total lymphocytes, monocytes and central memory T cells were positive predictors.

“In addition, immune parameters such as total CD8+ T cells, plasmablasts, transitional B cells such as IgD+CD27− and IgD−CD27− B cells (negative predictors), effector CD8+ T cells, total lymphocytes, monocytes and central memory T cells (positive predictors) were predictive of multimorbidity (Extended Data Fig. 5d).”

May combine two claims.

CXCL9 knockdown completely reversed the early cellular senescence phenotype, with upregulation of proliferative pathways and downregulation of inflammatory pathways compared with Scramble hiPSC-ECs.

“CXCL9-KD showed a complete reversal of this early cellular senescence phenotype with upregulation of proliferative pathways and downregulation of inflammatory pathways when compared to Scramble hiPSC-ECs (Fig. 5c–e).”

May combine two claims.

Among controls, 77% were in the high iAge index rank group and 23% were in the low rank group.

“In contrast, there were 77% (14 out of 18) of controls in the high rank versus 23% in the low rank group (Fig. 1e)”

May combine two claims.

IL-11Rα, CXCL10 and HGF increased with age, while CXCL1 and LIF decreased.

“In addition, IL-11Rα, CXCL10 and hepatocyte growth factor (HGF) increased with age, whereas CXCL1 and LIF decreased (Extended Data Fig. 7).”

May combine two claims.

Among centenarians, 68% were in the low iAge index rank group and 31% were in the high rank group.

“Sixty-eight percent (13 out of 19) centenarians were in the low rank group (P = 0.028), whereas only 31% (6 out of 19) were in the high rank group.”

May combine two claims.

Age was the strongest contributor to changes in naive CD8+ T cells, followed by iAge and CMV as negative contributors, and sex.

“Not surprisingly, age was the strongest contributor to changes in naive CD8+ T cells followed by iAge, CMV (negative contributors) and sex (frequency of total CD8+ T cells in females was 24% versus 30% in males; Fig. 2a).”

May combine two claims.

Blood endothelial progenitor cells from older individuals showed reduced tube formation, reduced nitric oxide production and reduced acetylated LDL uptake.

“Comparing ECs from older and younger individuals, we found that BECs from older patients showed reduced capacity to form networks of tubular structures (Extended Data Fig. 8b and Fig. 4b), reduced capacity to produce NO (Fig. 4c) and a reduced capacity to incorporate Ac-LDL (Fig. 4d).”

May combine two claims.

CXCL9 transcript levels increased in a time-dependent manner up to about fourfold at passage 8 compared with day 0 cultures, and CXCL9 expression was substantially reduced in CXCL9-knockdown hiPSC-ECs.

“We observed a time-dependent increase in CXCL9 transcript levels up to ~fourfold at passage 8 compared to cells obtained from cultures at day 0 and a substantial reduction of CXCL9 expression in CXCL9-KD hiPSC-ECs (Fig. 5a).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Total CD8+ T-cell frequency was 24% in females and 30% in males.

“frequency of total CD8+ T cells in females was 24% versus 30% in males”

May combine two claims.

By passage 4, endothelial cells lost their capacity to form tubes, and this was partially restored when CXCL9 was knocked down.

“As early as passage 4, ECs lose their capacity to form tubes, which can be partially restored when CXCL9 is knocked down (Fig. 4e and Extended Data Fig. 10).”

May combine two claims.

Increasing iAge was associated with a general decrease in B-cell and T-cell responses to stimuli and an overall potentiation of monocyte response.

“Notably, there was a general decrease of B-cell and T-cell responses to stimuli and an overall potentiation of monocyte response associated with increasing iAge (combined P < 10−5; Fig. 2b).”

May combine two claims.

FGSEA in aged cells showed upregulation of hallmark inflammatory pathways and downregulation of hallmark cell proliferation pathways.

“Fast gene set enrichment analysis (FGSEA) in aged cells revealed upregulation of genes in hallmark inflammatory pathways and downregulation of genes in hallmark cell proliferation pathways (Fig. 5b).”

May combine two claims.

Late-passaged iPSC-ECs failed to produce nitric oxide in response to acetylcholine or to take up acetylated LDL compared with early passages.

“Late-passaged iPSC-ECs failed to produce NO in response to acetylcholine or uptake Ac-LDL respectively, when compared to early passages of iPSC-ECs (Fig. 4f,g).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The best hyperparameter setting for GAE with code length 5 was depth 2, guidance ratio 0.2, and L2 0.001.

“we used fivefold cross validation to select the best hyper-parameter setting (depth = 2, guidance ratio = 0.2, L2 = 0.001) on all GAEs with a code length of 5.”

May combine two claims.

iAge predicts multimorbidity and mortality.

“we demonstrate that iAge predicts multimorbidity and mortality”

May combine two claims.

CXCL9 is mainly produced by aged endothelium and predicts subclinical cardiovascular aging in nominally healthy individuals.

“we find that CXCL9 is mainly produced by aged endothelium and predicts subclinical levels of cardiovascular aging in nominally healthy individuals.”

• AssumptionRests on an assumption: CXCL9 attracts T cells, is induced by IFN-γ, and is primarily secreted by neutrophils, macrophages, and endothelial cells.

Probably not a substantial claim: may not pass S3 (load-bearing).

The best code length for the GAE was 5.

“After obtaining the best code length as 5 (Extended Data Fig. 3a)”

May combine two claims.

iAge is directly associated with multiple disease phenotypes, including cardiovascular aging, frailty, immune decline, and exceptional longevity.

“iAge is directly associated with multiple disease phenotypes, including cardiovascular aging, frailty, immune decline and exceptional longevity.”

May combine two claims.

GAE outperformed Elastic Net, PCA, and plain auto-encoder.

“In our case, this method outperformed other commonly used linear modeling methods such as Elastic Net and PCA and also other nonlinear approaches such as plain auto-encoder89 (Extended Data Fig. 3b).”

May combine two claims.

iAge is associated with multiple diseases and immunosenescence.

“we find that iAge tracks with multiple diseases and immunosenescence.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The best hyper-parameter setting for GAE with code length 5 was depth = 2, guidance-ratio = 0.2, and L2 = 0.001, selected using 5-fold cross-validation.

“After obtaining the best code length as 5, we used the 5-fold-cross-validation to select the best hyper-parameter setting (depth = 2, guidance-ratio = 0.2, L2 = 0.001) on all GAE with code length 5.”

Probably not a substantial claim: may not pass S3 (load-bearing).

CXCR3 was not expressed in iPSC-induced cardiomyocytes, fibroblasts, or iPSC.

“CXCR3 was not expressed in iPSC induced cardiomyocytes (iPSC-CM), Fibroblast, or iPSC.”

May combine two claims.

CXCL9-knockdown hiPSC-ECs retained their capacity to form tubes even at later passages, whereas scramble hiPSC-ECs showed impaired tube formation at later passages.

“Representative images of capillary-like networks from scramble- and CXCL9-KD hiPSC-ECs show that CXCL9-KD hiPSC-ECs retain their capacity to form tubes even at later passages when compared to scramble that showed impaired tube formation towards later passages of hiPSC-ECs.”

May combine two claims.

The GAE method performed better than linear methods for reconstructing protein data and predicting chronological age.

“The GAE method outperforms linear methods for protein data reconstruction and prediction of chronological age (b).”

Probably not a substantial claim: may not pass S3 (load-bearing).

There was a significant correlation between age and CXCL9 in univariate regression analysis.

“Significant correlation between age and CXCL9 using univariate regression analysis.”

Classified as probably not a substantial claim. May combine two claims.

The Stanford 1000 Immunomes Project consists of 1001 ambulatory subjects aged 8 to 96 (34% male, 66% female) recruited between 2007 and 2016 for a longitudinal aging and vaccination study, and for an independent chronic fatigue syndrome study from which only healthy controls were included.

“The Stanford 1000 Immunomes Project consist of 1001 ambulatory subjects age 8 to 96 (34% males, 66% females) recruited during the years 2007 to 2016 for a longitudinal study of aging and vaccination, and for an independent study of chronic fatigue syndrome from which only healthy controls were included.”

May combine two claims.

In the authors' experiment, the optimal code length for the GAE was 5, because a sixth code gave no significant improvement in total loss (p = 0.18).

“In our experiment, the best code length is 5 (a) as adding one more code (6) does not significantly improve the total loss (p = 0.18).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The predictive performance of gradient boosting decision tree (GBDT) was similar to that of PCA.

“In (c), we found that the predictive performance of gradient boosting decision tree (GBDT) has similar performance as PCA.”

May combine two claims.

Eighteen features were selected, including the inflammatory clock, high cholesterol, BMI, and immune parameters such as total CD8+ T cells, plasmablasts, and transitional B cells as negative predictors, and IgD+CD27- and IgD-CD27- B cells, effector CD8+ T cells, total lymphocytes, monocytes, and central memory T cells as positive predictors.

“Eighteen features are selected including inflammatory clock, high cholesterol and BMI (c) and immune parameters such as total CD8 (+) T cells, plasmablasts and transitional B cells (negative predictors) and IgD+CD27- and IgD-CD27-B cells, effector CD8 (+) T cells, total lymphocytes and monocytes, and central memory T cells (positive predictors) (d)”

May combine two claims.

Normalization and batch correction removed batch effect, as shown by a lower mean absolute Spearman correlation between all features and batch ID, indicating that sample source could not be distinguished from corrected data.

“Data normalization and batch correction removes batch effect as indicated by lower mean absolute Spearman correlation between all features and batch id (blue line), which indicates impossibility to distinguish sample source from corrected data.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

CXCR3 was highly expressed in iPSC-induced endothelial cells and Human Umbilical Vein Endothelial Cells (HUVEC).

“However, it is highly expressed in iPSC induced endothelial cells and Human Umbilical Vein Endothelial Cells (HUVEC).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Because LASSO regression is stochastic, each implementation produced a slightly different list of genes predictive of iAge.

“Due to the stochastic nature of LASSO regression, each implementation produced a slightly different list of genes that were predictive of iAge.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.

Multimorbidity is the top priority for global health research and is a gold standard in aging research because it reflects accumulated physiological damage.

“Multimorbidity is the number one priority for global health research and has become the gold standard in aging research as it represents the accumulation of physiological damage in an individual33.”

• cites [33] Barnett, K. et al. Epidemiology of multimorbidity and implications for health care, research, and medical education: a cross-sectional study. Lancet 380, 37–43 (2012). sourceNot checked yet

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May combine two claims.

There are no standard biomarkers for chronic inflammatory state, and studies have generally produced conflicting results.

“Despite the importance of this type of inflammatory reaction, there are currently no standard biomarkers to characterize a chronic inflammatory state and studies have generally yielded conflicting results18,19.”

• cites [18] Franceschi, C., Garagnani, P., Vitale, G., Capri, M. & Salvioli, S. Inflammaging and garb-aging. Trends Endocrinol. Metab. 28, 199–212 (2017). sourceNot checked yet

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• cites [19] Morrisette-Thomas, V. et al. Inflamm-aging does not simply reflect increases in pro-inflammatory markers. Mechanisms Ageing Dev. 139, 49–57 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Inflammatory components of the immune system are often chronically elevated in older people and are linked to higher incidence of cancer, cardiovascular disease, neurodegenerative disorders and other conditions.

“inflammatory components of the immune system are often chronically elevated in aged individuals and associated with an increased incidence of cancer, cardiovascular disease, neurodegenerative disorders and others1–3”

• cites [1] Kotas, M. E. & Medzhitov, R. Homeostasis, inflammation, and disease susceptibility. Cell 160, 816–827 (2015). sourceNot checked yet

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• cites [2] Liu, F. et al. Mechanosignaling through YAP and TAZ drives fibroblast activation and fibrosis. Am. J. Physiol. Lung Cell Mol. Physiol. 308, L344–L357 (2015). sourceNot checked yet

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• cites [3] Crusz, S. M. & Balkwill, F. R. Inflammation and cancer: advances and new agents. Nat. Rev. Clin. Oncol. 12, 584–596 (2015). sourceNot checked yet

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Classified as probably not a substantial claim.

The multimorbidity analysis adjusted for age, BMI, sex, CMV and high cholesterol because each has a reported effect on age-related pathologies.

“For these analyses, we controlled for age, body mass index (BMI), sex, CMV and high cholesterol (also a binary category), because of the reported effect of each of these variables in the etiology of age-related pathologies.”

Classified as probably not a substantial claim.

Fivefold cross validation was used to measure predictive performance of each dimensionality-reduction method on the test set.

“We employed fivefold cross validation and measured the predictive performances of each method on the test set.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The authors' earlier cellular composite metric for immune aging, IMM-AGE, was strongly associated with all-cause mortality.

“Recent work from our group identified a cellular composite metric for immune aging (IMM-AGE), which was strongly associated with all-cause mortality16.”

• cites [16] Alpert, A. et al. A clinically meaningful metric of immune age derived from high-dimensional longitudinal monitoring. Nat. Med. 25, 487–495 (2019). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Deep immune phenotyping for the Stanford 1KIP samples was performed at the Stanford HIMC using rigorously standardized procedures.

“For all samples of the Stanford 1KIP, deep immune phenotyping was conducted at the Stanford HIMC, where peripheral blood specimens were processed and analyzed using rigorously standardized procedures31.”

• cites [31] Maecker, H. T. et al. New tools for classification and monitoring of autoimmune diseases. Nat. Rev. Rheumatol. 8, 317–328 (2012). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Chronic systemic inflammation is associated with aging, is low-grade and persistent, and ultimately causes collateral damage to tissues and organs.

“This type of inflammation is associated with aging and is characterized by being low-grade and persistent, ultimately leading to collateral damage to tissues and organs1,17.”

• cites [1] Kotas, M. E. & Medzhitov, R. Homeostasis, inflammation, and disease susceptibility. Cell 160, 816–827 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [17] Goldberg, E. L. & Dixit, V. D. Drivers of age-related inflammation and strategies for healthspan extension. Immunol. Rev. 265, 63–74 (2015). sourceNot checked yet

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May combine two claims.

Knockdown of CXCL9 rescues endothelial dysfunction by passage 8 but does not fully restore endothelial cell function to the level of healthy passage 0 cells.

“This suggests that while knockdown of CXCL9 rescues endothelial dysfunction by passage 8, it cannot restore EC function completely to the level of healthy ECs at passage 0.”

May combine two claims.

CXCL9 has a profound effect in the cardiovascular system and indicates a new role in angiogenesis and endothelial cell function during cardiovascular aging.

“Taken together these results demonstrate that CXCL9 has a profound effect in the cardiovascular system and indicates a new role for this chemokine in angiogenesis and EC function during cardiovascular aging.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The validation cohort was selected from 151 recruited participants using strict selection criteria.

“selected from a total of 151 recruited participants using strict selection criteria (Methods).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

C-reactive protein and IL-6 have been linked to immunosenescence in prior studies, but their relationship with systemic chronic inflammation had not been established.

“Canonical acute inflammation proteins such as C-reactive protein and interleukin (IL)-6 have been associated with immunosenescence in previous studies37,38, but the relationship with systemic chronic inflammation (SCI) has not yet been established.”

• cites [37] Bertsch, T. et al. C-reactive protein and the acute phase reaction in geriatric patients. Z. Gerontologie Geriatr. 48, 595–600 (2015). sourceNot checked yet

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• cites [38] Scheller, J., Chalaris, A., Schmidt-Arras, D. & Rose-John, S. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochim. Biophys. Acta 1813, 878–888 (2011). sourceNot checked yet

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Classified as probably not a substantial claim. May combine two claims.

High relative wall thickness indicates concentric cardiac remodeling, and elevated pulse wave velocity reflects organ damage and predicts future cardiovascular events and all-cause mortality better than conventional risk factors.

“As high RWT indicates concentric cardiac remodeling and elevated PWV is reflective of organ damage and predicts future cardiovascular events and all-cause mortality better than conventional cardiovascular disease risk factors45–47”

• cites [45] Vlachopoulos, C., Aznaouridis, K. & Stefanadis, C. Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis. J. Am. Coll. Cardiol. 55, 1318–1327 (2010). sourceNot checked yet

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• cites [46] Ben-Shlomo, Y. et al. Aortic pulse wave velocity improves cardiovascular event prediction: an individual participant meta-analysis of prospective observational data from 17,635 subjects. J. Am. Coll. Cardiol. 63, 636–646 (2014). sourceNot checked yet

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• cites [47] Chirinos, J. A. Large artery stiffness, microvascular function, and cardiovascular risk. Circ. Cardiovasc Imaging 9, e005903 (2016). sourceNot checked yet

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Classified as probably not a substantial claim.

The study used a well-established endothelial aging model by generating hiPSCs from five independent human donors and differentiating them into endothelial cells.

“we used a well-established model for endothelial aging57,58 by generating human induced pluripotent stem cells (hiPSCs) from fibroblasts obtained from five independent human donors59 and subsequently differentiated them into endothelial cells (hiPSC-ECs)60.”

• cites [57] Boisen, L., Drasbek, K. R., Pedersen, A. S. & Kristensen, P. Evaluation of endothelial cell culture as a model system of vascular ageing. Exp. Gerontol. 45, 779–787 (2010). sourceNot checked yet

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• cites [58] Brandes, R. P., Fleming, I. & Busse, R. Endothelial aging. Cardiovascular Res. 66, 286–294 (2005). sourceNot checked yet

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• cites [59] Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). sourceNot checked yet

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• cites [60] Sayed, N. et al. Clinical trial in a dish using iPSCs shows lovastatin improves endothelial dysfunction and cellular cross-talk in LMNA cardiomyopathy. Sci. Transl. Med. 12, eaax9276 (2020). sourceNot checked yet

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May combine two claims.

iAge correlates with an established marker of immune senescence, naive CD8+ T-cell frequency, and with PBMC signaling responses in vitro.

“These results demonstrate that iAge correlates with an established biomarker of immune senescence (naive CD8+ T-cell frequency) and with PBMC signaling responses in vitro.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The phospho-STAT signaling analysis used multiple regression controlling for age, CMV and sex.

“We conducted multiple regression analysis controlling age, CMV and sex (Methods).”

May combine two claims.

CXCL9 increases in endothelia and endothelial cell dysfunction are associated with aging in both humans and mice.

“These results demonstrate a concomitant increase in CXCL9 in the endothelia and EC dysfunction associated with aging both in humans and mice.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The endothelium has a role in the etiology of hypertension and arterial stiffness.

“Long-standing evidence has suggested a role for the endothelium in the etiology of hypertension and arterial stiffness48,49.”

• cites [48] Figueiredo, V. N. et al. Vascular stiffness and endothelial dysfunction: correlations at different levels of blood pressure. Blood Press. 21, 31–38 (2012). sourceNot checked yet

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• cites [49] Vanhoutte, P. M., Feletou, M. & Taddei, S. Endothelium-dependent contractions in hypertension. Br. J. Pharmacol. 144, 449–458 (2005). sourceNot checked yet

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Classified as probably not a substantial claim.

The cardiovascular analysis used multiple regression hierarchical analysis controlling for age, sex, BMI, heart rate, systolic blood pressure, fasting glucose and total cholesterol to HDL ratio.

“We then performed multiple regression hierarchical analysis using the six selected inflammatory markers associated with aging in this cohort and cardiovascular measurements (PWV and RWT) controlling for age, sex, BMI, heart rate, systolic blood pressure, fasting glucose and total cholesterol to high-density lipoprotein (HDL) ratio.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Advanced signs of cardiovascular aging such as tissue remodeling and cardiac hypertrophy are often preceded and may be initiated by malfunctioning aged endothelia.

“More recent work has also shown that advanced signs of cardiovascular aging such as tissue remodeling and cardiac hypertrophy are often preceded and may be initiated by the malfunctioning of aged endothelia50–52.”

• cites [50] Castellon, X. & Bogdanova, V. Chronic inflammatory diseases and endothelial dysfunction. Aging Dis. 7, 81–89 (2016). sourceNot checked yet

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• cites [51] Harvey, A., Montezano, A. C. & Touyz, R. M. Vascular biology of ageing: implications in hypertension. J. Mol. Cell Cardiol. 83, 112–121 (2015). sourceNot checked yet

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• cites [52] Kamo, T., Akazawa, H. & Komuro, I. Cardiac nonmyocytes in the hub of cardiac hypertrophy. Circ. Res. 117, 89–98 (2015). sourceNot checked yet

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May combine two claims.

There are at least two sources of CXCL9-mediated inflammation with aging: one age-intrinsic in aging endothelial cells and one age-independent in the validation cohort.

“At least two sources of CXCL9-mediated inflammation can ensue with aging based on our findings; one that is age-intrinsic and observed in aging ECs and one that is independent of age (likely as a response to cumulative exposure to environmental insults) and found in the validation cohort of 97 apparently healthy adults.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

iPSC cardiomyocytes were differentiated to >85% purity and endothelial cells to >90% purity.

“These iPSCs were differentiated into cardiomyocytes to purities of >85% and ECs to purities of >90%.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The study was conducted in accordance with ethical regulations and approved by Stanford IRB, with written informed consent from all cohorts.

“This study was conducted in accordance with current relevant ethical regulations on human participant research. Written informed consent was obtained from all the study cohorts used and the study protocol was approved by the Stanford University Administrative Panels on Human Subjects in Medical Research, Institutional Review Board.”

Probably not a substantial claim: may not pass S3 (load-bearing).

CXCL9 is associated with falls in the older population.

“Notably, CXCL9 has also been shown to be associated with falls in the older population101,102”

• cites [101] Amorim, J. S. C. et al. Inflammatory markers and occurrence of falls: Bambui cohort study of aging. Rev. Saude Publica 53, 35 (2019). sourceNot checked yet

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• cites [102] Chiu, B. C. et al. Differential effects of ageing on cytokine and chemokine responses during type-1 (mycobacterial) and type-2 (schistosomal) pulmonary granulomatous inflammation in mice. Mech. Ageing Dev. 123, 313–326 (2002). sourceNot checked yet

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Probably not a substantial claim: may not pass S1 (asserted). May combine two claims.

CXCL9 acts in both paracrine and autocrine fashion, creating a positive feedback loop.

“we hypothesize that this chemokine acts both in a paracrine fashion (when it is produced by macrophages to attract T cells to the site of injury) and in an autocrine fashion (when it is produced by the endothelium) creating a positive feedback loop.”

• AssumptionRests on an assumption: CXCL9 attracts T cells, is induced by IFN-γ, and is primarily secreted by neutrophils, macrophages, and endothelial cells.

Probably not a substantial claim: may not pass S3 (load-bearing).

Endothelial cells may be one source of inflammation.

“they also suggest that ECs may be one source of inflammation”

Probably not a substantial claim: may not pass S3 (load-bearing).

The stimuli strongly activate the memory compartment of bulk CD8+ and CD4+ T cells but act weakly on naive T cells.

“while the stimuli we used strongly activate the memory compartment of bulk CD8+ and CD4+ T cells, these act relatively weakly on naive T cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The stimuli used in the cytokine stimulation assay may not fully mirror physiological stimuli acting on specific immune cell subsets in vivo.

“one limitation of the assay relates to the stimuli used here which may not completely mirror the physiological stimuli that act on specific immune cell subsets in vivo.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All echocardiographic parameters were measured in triplicate and averaged.

“All parameters were measured in triplicate and averaged.”

May combine two claims.

The lack of angiogenesis, impaired nitric oxide production and dysfunctional acetylated LDL uptake indirectly suggested a cellular senescence phenotype that could be rescued by knocking down CXCL9 during iPSC-EC passaging.

“The lack of angiogenesis, impaired production of NO and dysfunctional uptake of Ac-LDL indirectly suggested a cellular senescence phenotype that could be rescued by knocking down CXCL9 as iPSC-EC is passaged.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Echocardiographs were interpreted by one physician blinded to age and clinical/biological data.

“All studies were interpreted by one physician (F.H.) who was blinded to age as well as clinical and biological data.”

Probably not a substantial claim: may not pass S3 (load-bearing).

CXCL9 increases with age in humans.

“Some studies in humans have found CXCL9 to increase with age93–98”

• cites [93] Bonfante, H. L. et al. CCL2, CXCL8, CXCL9 and CXCL10 serum levels increase with age but are not altered by treatment with hydroxychloroquine in patients with osteoarthritis of the knees. Int J. Rheum. Dis. 20, 1958–1964 (2017). sourceNot checked yet

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• cites [94] Roubenoff, R. et al. Monocyte cytokine production in an elderly population: effect of age and inflammation. J. Gerontol. A Biol. Sci. Med Sci. 53, M20–M26 (1998). sourceNot checked yet

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• cites [95] Torres, K. C. L. et al. Immune senescence and biomarkers profile of Bambui aged population-based cohort. Exp. Gerontol. 103, 47–56 (2018). sourceNot checked yet

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• cites [96] Shurin, G. V. et al. Dynamic alteration of soluble serum biomarkers in healthy aging. Cytokine 39, 123–129 (2007). sourceNot checked yet

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• cites [97] Zheng, Y. et al. Age-related pro-inflammatory and pro-angiogenic changes in human aqueous humor. Int J. Ophthalmol. 11, 196–200 (2018).Not checked yet

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• cites [98] Sandovici, I. et al. Ageing is associated with molecular signatures of inflammation and type 2 diabetes in rat pancreatic islets. Diabetologia 59, 502–511 (2016). sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Inter-observer variability for PWV measurement had an intraclass correlation coefficient of 0.94.

“Inter-observer variability was calculated on 50 samples in our laboratory and the intraclass correlation coefficient was 0.94 for PWV measured by two independent observers”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All PCR reactions were performed in triplicate, normalized to GAPDH, and assessed using comparative Ct or ΔΔCt.

“All PCR reactions were performed in triplicate, normalized to the GAPDH endogenous control gene and assessed using the comparative Ct method.”

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based). May combine two claims. The quote only partly states this claim.

One root cause of CXCL9 overproduction is cellular aging itself, which can trigger metabolic dysfunction with production of DAMPs.

“We thus hypothesize that one root cause of CXCL9 overproduction is cellular aging per se, which can trigger metabolic dysfunction”

• AssumptionRests on an assumption: CXCL9 attracts T cells, is induced by IFN-γ, and is primarily secreted by neutrophils, macrophages, and endothelial cells.

Probably not a substantial claim: may not pass S3 (load-bearing).

Monotonic transformations of cAge or iAge do not affect ranks, so the findings are robust to transformations such as log and exp.

“Any monotonic transformation of cAge or iAge does not affect ranks, hence findings are robust to transformations such as log and exp.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

iPSCs were derived from five healthy individuals and passed assessments for pluripotency and genomic stability.

“We derived iPSCs from five healthy individuals and cell lines passed common assessments for pluripotency such as expression of pluripotent markers (Oct4 and Nanog) and genomic stability such as karyotyping.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Cytokine stimulation assays have proven utility for assessing overall immune competence.

“Despite the proven utility of cytokine stimulation assays used in our study with respect to an individual’s overall immune competence5,24,25”

• cites [5] Furman, D. et al. Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states. Nat. Med. 23, 174–184 (2017). sourceNot checked yet

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• cites [24] Furman, D. et al. Apoptosis and other immune biomarkers predict influenza vaccine responsiveness. Mol. Syst. Biol. 9, 659 (2013). sourceNot checked yet

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• cites [25] Furman, D. et al. Cytomegalovirus infection enhances the immune response to influenza. Sci. Transl. Med. 7, 281ra43 (2015). sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The proteomic age metric had a correlation of R = 0.92 with chronological age.

“which is lower than that of the recently reported ‘proteomic age’ metric (R = 0.92)90.”

• cites [90] Tanaka, T. et al. Plasma proteomic signature of age in healthy humans. Aging Cell. 17, e12799 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

CXCL9 has a central role in vascular dysfunction and likely contributes to arterial stiffness and premature aging in vivo.

“These results demonstrate a central role for CXCL9 in vascular dysfunction, which likely contributes to arterial stiffness and premature aging in vivo.”

May combine two claims.

CXCL9 is a master regulator of vascular function and cellular senescence.

“we demonstrate that CXCL9 is a master regulator of vascular function and cellular senescence”

• AssumptionRests on an assumption: CXCL9 attracts T cells, is induced by IFN-γ, and is primarily secreted by neutrophils, macrophages, and endothelial cells.

Probably not a substantial claim: may not pass S3 (load-bearing).

Inflammatory markers are at least partly responsible for reduced JAK-STAT response to cytokine stimulations in various leukocyte populations.

“inflammatory markers have been shown to be, at least in part, responsible for a reduced JAK–STAT response to cytokine stimulations in various leukocyte populations in our previous studies of aging28.”

• cites [28] Shen-Orr Shai, S. et al. Defective signaling in the JAK–STAT pathway tracks with chronic inflammation and cardiovascular risk in aging humans. Cell Syst. 3, 374–384 (2016). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

High baseline inflammatory markers correlate with weaker responses to hepatitis B and herpes zoster vaccine formulations.

“previous reports showing that high levels of baseline inflammatory markers correlate with weaker responses to hepatitis B and herpes zoster vaccine formulations15,91.”

• cites [15] Thevaranjan, N. et al. Age-associated microbial dysbiosis promotes intestinal permeability, systemic inflammation, and macrophage dysfunction. Cell Host Microbe 21, 455–466 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [91] Fourati, S. et al. Pre-vaccination inflammation and B-cell signalling predict age-related hyporesponse to hepatitis B vaccination. Nat. Commun. 7, 10369 (2016). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Increasing doses of CXCL9 and expression of its receptor lead to cumulative deterioration of endothelial function in aging.

“In this model, increasing doses of CXCL9 and expression of its receptor in these cells leads to cumulative deterioration of endothelial function in aging.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The experiment was repeated three times.

“Experiment was repeated 3 times.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The study did not use randomization or blinding.

“No randomization or blinding was conducted for this study.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

PRIN06 and PRIN09 samples were measured in two batches and the datasets were bridged using SomaLogic calibrators.

“PRIN06 and PRIN09 samples were measured in two batches. Datasets were bridged to each other using SomaLogic calibrators.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Five-fold cross-validation was used to identify the best code length among lengths from 1 to 10.

“We used 5-fold cross-validation to identify the best code length, among lengths from 1 to 10.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Within each fold, nested 3-fold cross-validation was performed to select hyper-parameters (depth, weight decay, and guidance-ratio).

“Within each fold we performed nested 3-fold cross-validation to select hyper-parameters (depth, weight decay and guidance-ratio).”

Probably not a substantial claim: may not pass S3 (load-bearing).

No statistical method was used to predetermine sample size for the experiments.

“For our experiments, no statistical method was used to predetermine sample size.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For the cardiovascular study, one physician interpreted the data while blinded to age, clinical and biological data.

“For the cardiovascular study the data were interpreted by one physician (F.H.) who was blinded to age as well as clinical and biological data.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In a validation study, 97 healthy adults aged 25-90, well matched for cardiovascular risk factors, were selected from 151 recruited subjects.

“In a validation study, 97 healthy adults (aged 25–90) well matched for cardiovascular risk factors, were selected from a total of 151 recruited subjects.”

Classified as probably not a substantial claim. May combine two claims.

Differential penalty values were applied to each regressor, forcing the age variable in while imposing a stringent lasso penalty on all other features, so that selected variables would not correlate with age.

“By applying differential penalty values for each regressor, age variable is ‘forced in’, while imposing a stringent penalty (the lasso penalty) to all other features, so that selected variables do not correlate with age.”

Classified as probably not a substantial claim.

Covariates including age, BMI, CMV detection and sex were controlled by inclusion in all regression analyses.

“Covariates such as age, BMI, detection of CMV and sex were controlled by their inclusion in all regression analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the SOMAscan assay, no samples or probe data were excluded.

“Data in relative fluorescent units for 1,305 SOMAmer probes were obtained for these samples and no samples or probe data were excluded.”

Classified as probably not a substantial claim.

Serum immune protein data were normalized and batch-corrected so that data from different sources could be combined and used together.

“Immune protein data from serum samples were subjected to normalization and batch correction procedures (See Methods) to ensure data from different sources can be combined and used as a whole.”

Classified as probably not a substantial claim.

Age-adjusted cross-validation was performed on available data from all 902 subjects to select predictors of comorbidity without bias while controlling for age.

“To select for predictors of comorbidity without bias, based on available data for all 902 subjects while controlling for the age effect, age-adjusted cross-validation was performed (a).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Blinding was not performed in the mouse experiments.

“Blinding was not performed in mice experiments.”

Classified as probably not a substantial claim. May combine two claims.

Auto-encoders use a nonlinear transformation of data and can model complex processes.

“Auto-encoders use a nonlinear transformation of data and hence, can model complex processes119.”

• cites [119] Behzadi-Khormouji, H. et al. Deep learning, reusable and problem-based architectures for detection of consolidation on chest X-ray images. Comput. Methods Prog. Biomed. 185, 105162 (2020). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The analyses did not exclude any data.

“No data were excluded from the analyses.”