Cystatin C is glucocorticoid responsive, directs recruitment of Trem2+ macrophages, and predicts failure of cancer immunotherapy

May combine two claims.

GR is expressed in macrophages but not in monocytes.

“As GR is expressed in macrophages but not in monocytes,62”

• cites [62] Diaz-Jimenez D. Petrillo M.G. Busada J.T. Hermoso M.A. Cidlowski J.A. Glucocorticoids mobilize macrophages by transcriptionally up-regulating the exopeptidase DPP4 J. Biol. Chem. 295 2020 3213 3227 10.1074/jbc.RA119.010894 31988243 PMC7062181 sourceDisputed

  The transcriptome-analysis section reports higher GR expression in macrophage-like THP-1 cells than in monocyte-like THP-1 cells, not an absence of GR in the latter. Its qRT-PCR and Western blot results compare GR levels in both cell types (Fig. 1C–D). (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

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

The SH2B3 R262W variant is under active positive selection.

“consistent with evidence that this variant is under active positive selection.38”

• cites [38] Zhernakova A. Elbers C.C. Ferwerda B. Romanos J. Trynka G. Dubois P.C. de Kovel C.G.F. Franke L. Oosting M. Barisani D. Evolutionary and functional analysis of celiac risk loci reveals SH2B3 as a protective factor against bacterial infection Am. J. Hum. Genet. 86 2010 970 977 10.1016/j.ajhg.2010.05.004 20560212 PMC3032060 sourcePartly supported

  The results section (page unavailable) reports significant signs of positive selection for the SH2B3 rs3184504*A allele in four European populations. However, our copy does not identify that allele as R262W, and it dates the selective sweep to approximately 1,200–1,700 years ago rather than establishing that selection is currently active. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

In vivo models of cancer suggest that even low doses of GCs can impair the efficacy of checkpoint immunotherapy and suppress anti-tumor immunity, leading to enhanced metastasis and reduced survival.

“However, in vivo models of cancer also suggests that even low doses of GCs can impair the efficacy of CPI29,30 and suppress anti-tumor immunity,30 leading to enhanced metastasis and reduced survival.31”

• cites [29] Connell C.M. Raby S. Beh I. Flint T.R. Williams E.H. Fearon D.T. Jodrell D.I. Janowitz T. Cancer immunotherapy trial registrations increase exponentially but chronic immunosuppressive glucocorticoid therapy may compromise outcomes Ann. Oncol. 28 2017 1678 1679 10.1093/annonc/mdx181 28430855 PMC5834107 sourceSupported
• cites [30] Flint T.R. Janowitz T. Connell C.M. Roberts E.W. Denton A.E. Coll A.P. Jodrell D.I. Fearon D.T. Tumor-induced IL-6 reprograms host metabolism to suppress anti-tumor immunity Cell Metabol. 24 2016 672 684 10.1016/j.cmet.2016.10.010 PMC5106372 27829137 sourceSupported
• cites [31] Obradović M.M.S. Hamelin B. Manevski N. Couto J.P. Sethi A. Coissieux M.M. Münst S. Okamoto R. Kohler H. Schmidt A. Glucocorticoids promote breast cancer metastasis Nature 2019 10.1038/s41586-019-1019-4 30867597 sourceNot checked — no full text of the source

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

GCs modulate the cellular transcriptional landscape through the GC receptor, affecting up to 20% of all genes.

“Through action on the cytosolic GC receptor (GR), GCs profoundly modulate the cellular transcriptional landscape,24 affecting up to 20% of all genes25”

• cites [24] Reddy T.E. Pauli F. Sprouse R.O. Neff N.F. Newberry K.M. Garabedian M.J. Myers R.M. Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation Genome Res. 19 2009 2163 2171 10.1101/gr.097022.109 19801529 PMC2792167 sourcePartly supported

  The ‘Identification of GC-responsive genes by RNA-seq’ section reports that dexamethasone changed gene expression, supporting the claim that glucocorticoids affect transcription. But it identifies 209 differentially expressed genes among 29,673 assessed—not a figure approaching 20% of all genes. The cited paper does not support the attributed 20% figure. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [25] Galon J. Franchimont D. Hiroi N. Frey G. Boettner A. Ehrhart-Bornstein M. O’shea J.J. Chrousos G.P. Bornstein S.R. Gene profiling reveals unknown enhancing and suppressive actions of glucocorticoids on immune cells Faseb. J. 16 2002 61 71 10.1096/fj.01-0245com 11772937 sourceNot checked — no full text of the source

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

The divergent effects of CPS1 probably reflect its independent roles in creatine metabolism and immune signaling.

“probably reflecting its independent roles in creatine metabolism39 and immune signaling.40”

• cites [39] Shin S.Y. Fauman E.B. Petersen A.K. Krumsiek J. Santos R. Huang J. Arnold M. Erte I. Forgetta V. Yang T.P. An atlas of genetic influences on human blood metabolites Nat. Genet. 46 2014 543 550 10.1038/ng.2982 24816252 PMC4064254 sourceCould not verify

  Citation 39 is attached to the creatine-metabolism part of the sentence; immune signaling has a separate citation. The Disease and pharmacological relevance section describes CPS1’s role in producing carbamoyl phosphate and its association with glycine levels, but the passages found for “CPS1 creatine metabolism” and “creatine” do not address a role in creatine metabolism or explain the stated divergent effects. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [40] Park M.J. D’Alecy L.G. Anderson M.A. Basrur V. Feng Y. Brady G.F. Kim D.I. Lahann J. Constitutive release of CPS1 in bile and its role as a protective cytokine during acute liver injury Proc. Natl. Acad. Sci. USA 116 2019 9125 9134 10.1073/pnas.1822173116 30979808 PMC6500125 sourceSupported

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

CyC production is poorly defined.

“While creatinine production is well characterized and relates to muscle mass and diet,7 CyC production is poorly defined.8”

• cites [8] Stevens L.A. Schmid C.H. Greene T. Li L. Beck G.J. Joffe M.M. Froissart M. Kusek J.W. Zhang Y.L. Coresh J. Levey A.S. Factors other than glomerular filtration rate affect serum cystatin C levels Kidney Int. 75 2009 652 660 10.1038/ki.2008.638 19119287 PMC4557800 sourceSupported

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

CyC has been validated as a marker of renal function in multiple large clinical cohorts comprising patients without acute disease.

“CyC has been validated as a marker of renal function in multiple large clinical cohorts54 comprising patients without acute disease.”

• cites [54] Inker L.A. Schmid C.H. Tighiouart H. Eckfeldt J.H. Feldman H.I. Greene T. Kusek J.W. Manzi J. Van Lente F. Zhang Y.L. Estimating glomerular filtration rate from serum creatinine and cystatin C N. Engl. J. Med. 367 2012 20 29 22762315 PMC4398023 sourcePartly supported

  The Methods (Study Design and Data Sources; page not provided) describe development and validation using cystatin C data from 18 studies involving 6,471 participants. The paper does not state that those cohorts comprised patients without acute disease, so that population qualifier is unsupported. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

In a recent cortisol genome-wide meta-analysis, the SERPINA1/6 signal was thought to be mediated by altered hepatic expression of SERPINA6, which encodes cortisol-binding globulin.

“In a recent cortisol genome-wide meta-analysis, this signal was thought to be mediated by altered hepatic expression of SERPINA6,50 which encodes cortisol-binding globulin (CBG).”

• cites [50] Crawford A.A. Bankier S. Altmaier E. Barnes C.L.K. Clark D.W. Ermel R. Friedrich N. van der Harst P. Joshi P.K. Karhunen V. Variation in the SERPINA6/SERPINA1 locus alters morning plasma cortisol, hepatic corticosteroid binding globulin expression, gene expression in peripheral tissues, and risk of cardiovascular disease J. Hum. Genet. 66 2021 625 636 10.1038/s10038-020-00895-6 33469137 PMC8144017 sourceSupported

Classified as probably not a substantial claim.

Severe COVID-19 infection is characterized by persistent lung inflammation associated with concomitant recruitment of monocyte-derived macrophages.

“Severe COVID-19 infection is characterized by persistent lung inflammation associated with concomitant recruitment of monocyte-derived macrophages.65”

• cites [65] Liao M. Liu Y. Yuan J. Wen Y. Xu G. Zhao J. Cheng L. Li J. Wang X. Wang F. Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19 Nat. Med. 26 2020 842 844 10.1038/s41591-020-0901-9 32398875 sourcePartly supported

  The paper reports abundant proinflammatory monocyte-derived macrophages in severe COVID-19 and higher inflammatory cytokine levels in bronchoalveolar lavage fluid (Abstract; main text, page not provided). But persistently high cytokine levels were reported for only three patients with critical illness, and recruitment of monocytic cells was proposed as a possible mechanism rather than directly established as concomitant recruitment. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Until the release of the RECOVERY trial, patients with severe COVID-19 were not routinely treated with GC agonists such as dexamethasone.

“Until the release of the RECOVERY trial,66 patients with severe COVID-19 were not routinely treated with GC agonists such as dexamethasone.”

• cites [66] The RECOVERY Collaborative Group Dexamethasone in hospitalized patients with Covid-19 N. Engl. J. Med. 384 2021 693 704 32678530 PMC7383595 sourcePartly supported

  The Discussion says that before the trial, many COVID-19 treatment guidelines contraindicated or did not recommend glucocorticoids. But the introductory passage says practice varied widely: China recommended them for severe cases, and some series reported use in as many as 50% of patients. The source therefore supports a lack of general recommendation, not an unqualified claim that patients with severe COVID-19 were not routinely treated. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Visceral adipose fat predominantly comprises adipocytes, endothelial cells, and macrophages.

“Visceral adipose fat is known to predominantly comprise adipocytes, endothelial cells, and macrophages.53”

• cites [53] Glastonbury C.A. Couto Alves A. El-Sayed Moustafa J.S. Small K.S. Cell-type heterogeneity in adipose tissue is associated with complex traits and reveals disease-relevant cell-specific eQTLs Am. J. Hum. Genet. 104 2019 1013 1024 10.1016/j.ajhg.2019.03.025 31130283 PMC6556877 sourcePartly supported

  The Results (Estimation of Relative Cell-Type Proportions; page unavailable) identify adipocytes, endothelial cells, and macrophages as major estimated cell types, but the analyzed biopsies were subcutaneous. The Methods (GTEx RNA-Seq Dataset; page unavailable) also specifies subcutaneous samples. Visceral fat was measured as a body-fat trait, not sampled to establish its cellular composition, so the claim’s visceral-specific wording is unsupported. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Metabolic syndrome is associated with significant macrophage accumulation in adipose tissue.

“As metabolic syndrome is associated with significant macrophage accumulation in adipose tissue,69”

• cites [69] Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante A.W. Obesity is associated with macrophage accumulation in adipose tissue J. Clin. Invest. 112 2003 1796 1808 10.1172/jci19246 14679176 PMC296995 sourcePartly supported

  The Discussion (p. 10) reports that adipose-tissue macrophage accumulation increases with measures of adiposity in mice and humans. The paper establishes this association for obesity, but the cited passages do not establish it for metabolic syndrome as a distinct condition. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

The STARNET study recruited patients with established coronary artery disease.

“The STARNET study recruited patients with established coronary artery disease,52”

• cites [52] Franzén O. Ermel R. Cohain A. Akers N.K. Di Narzo A. Talukdar H.A. Foroughi-Asl H. Giambartolomei C. Fullard J.F. Sukhavasi K. Cardiometabolic risk loci share downstream cis- and trans-gene regulation across tissues and diseases Science 2016 eaad6970 10.1126/science.aad6970 PMC5534139 27540175 sourceSupported

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

CyC levels are dynamically regulated in COVID-19 patients and correlate with in-hospital mortality.

“have recently reported that CyC levels are dynamically regulated in COVID-19 patients and correlate with in-hospital mortality.49”

• cites [49] Kleeman S.O. Cordioli M. Timmers P.R.H.J. Khan A. Tober-Lau P. Kurth F. Demichev V. Meyer H.V. Wilson J.F. Ralser M. Cystatin C is associated with adverse COVID-19 outcomes in diverse populations iScience 25 2022 105040 10.1016/j.isci.2022.105040 36062073 PMC9428108 sourcePartly supported

  The Results report longitudinal changes in the creatinine–CyC (C2) ratio and an association between a higher ratio and COVID-specific mortality. The Methods specify that in-hospital mortality was analyzed against the C2 ratio, not CyC levels alone. Thus, the mortality association is supported, but the claim overstates what the paper shows about CyC levels themselves. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

Associations between patient-level PGSs and outcomes are more robust, potentially capturing causal associations.

“we and others argue that associations between patient-level PGSs and outcomes are more robust, potentially capturing causal associations.95”

• cites [95] Richardson T.G. Harrison S. Hemani G. Davey Smith G. An atlas of polygenic risk score associations to highlight putative causal relationships across the human phenome Elife 8 2019 e43657 10.7554/eLife.43657 PMC6400585 30835202 sourcePartly supported

  The Introduction says PRS associations may highlight putative causal relationships and can have higher statistical power than the MR approach it compared. It does not establish that those associations are more robust; its simulations found substantial false-positive rates from horizontal pleiotropy, and it says robust follow-up evaluation is necessary. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

ApoE is known to be secreted by cancer cells.

“apolipoprotein E (ApoE), which is known to be secreted by cancer cells87”

• cites [87] Pencheva N. Tran H. Buss C. Huh D. Drobnjak M. Busam K. Tavazoie S.F. Convergent multi-miRNA targeting of ApoE drives LRP1/LRP8-dependent melanoma metastasis and angiogenesis Cell 151 2012 1068 1082 10.1016/j.cell.2012.10.028 23142051 PMC3753115 sourceSupported

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

Trem2-knockout mice have accelerated amyloid burden in mouse models of Alzheimer's disease.

“Trem2-knockout mice have accelerated amyloid burden in mouse models of Alzheimer’s disease (AD),92”

• cites [92] Wang Y. Cella M. Mallinson K. Ulrich J.D. Young K.L. Robinette M.L. Gilfillan S. Krishnan G.M. Sudhakar S. Zinselmeyer B.H. TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model Cell 160 2015 1061 1071 10.1016/j.cell.2015.01.049 25728668 PMC4477963 sourcePartly supported

  The Results section (“TREM2 modulates Aβ accumulation”; page unavailable) reports greater hippocampal Aβ accumulation in Trem2-knockout 5XFAD mice, but no significant cortical increase at the measured age. It does not establish faster accumulation over time or increased amyloid burden across multiple AD mouse models. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Ligation of the Trem2 receptor by ApoE is sufficient to promote phagocytosis in TREM2-expressing microglial cells and activates Apoe RNA expression.

“Ligation of the Trem2 receptor by ApoE is sufficient to promote phagocytosis in TREM2-expressing microglial (brain-resident macrophage) cells and, in turn, activates Apoe RNA expression,89”

• cites [89] Krasemann S. Madore C. Cialic R. Baufeld C. Calcagno N. El Fatimy R. Beckers L. O’Loughlin E. Xu Y. Fanek Z. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases Immunity 47 2017 566 581.e9 10.1016/j.immuni.2017.08.008 28930663 PMC5719893 sourcePartly supported

  The Discussion (page unavailable) says APOE is a TREM2 ligand that increases TREM2-mediated phagocytosis. The Results report increased Apoe expression in microglia phagocytosing apoptotic neurons and reduced Apoe expression when Trem2 is deleted. They do not establish that ligation by APOE alone is sufficient to promote phagocytosis or directly activates Apoe RNA expression. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

CyC is associated with recruitment of immunosuppressive Trem2+ macrophages, which in turn promote failure of cancer immunotherapy.

“we propose a model in which CyC is associated with recruitment of immunosuppressive Trem2+ macrophages,80,81 which in turn promote failure of cancer immunotherapy.”

• cites [80] Binnewies M. Pollack J.L. Rudolph J. Dash S. Abushawish M. Lee T. Jahchan N.S. Canaday P. Lu E. Norng M. Targeting TREM2 on tumor-associated macrophages enhances immunotherapy Cell Rep. 37 2021 109844 10.1016/j.celrep.2021.109844 34686340 sourceNot checked — no full text of the source
• cites [81] Molgora M. Esaulova E. Vermi W. Hou J. Chen Y. Luo J. Brioschi S. Bugatti M. Omodei A.S. Ricci B. TREM2 modulation remodels the tumor myeloid landscape enhancing anti-PD-1 immunotherapy Cell 182 2020 886 900.e17 10.1016/j.cell.2020.07.013 32783918 PMC7485282 sourcePartly supported

  The Results sections on macrophage subsets and checkpoint blockade report TREM2-expressing tumor macrophages with immunosuppression-associated markers and improved anti-PD-1 responses when TREM2 is absent. They do not link CyC to recruitment of those macrophages. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

TREM2 R47H mutations impair ApoE binding and increase the risk of human AD.

“TREM2 R47H mutations impair ApoE binding88 and increase the risk of human AD,93”

• cites [88] Atagi Y. Liu C.-C. Painter M.M. Chen X.-F. Verbeeck C. Zheng H. Li X. Rademakers R. Kang S.S. Xu H. Apolipoprotein E is a ligand for triggering receptor expressed on myeloid cells 2 (TREM2) J. Biol. Chem. 290 2015 26043 26050 10.1074/jbc.M115.679043 26374899 PMC4646257 sourceSupported
• cites [93] Guerreiro R. Wojtas A. Bras J. Carrasquillo M. Rogaeva E. Majounie E. Cruchaga C. Sassi C. Kauwe J.S.K. Younkin S. TREM2 variants in Alzheimer’s disease N. Engl. J. Med. 368 2013 117 127 10.1056/NEJMoa1211851 23150934 PMC3631573 sourceSupported

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

CyC knockout is associated with reduced amyloid burden.

“while CyC knockout is associated with reduced amyloid burden.94”

• cites [94] Sun B. Zhou Y. Halabisky B. Lo I. Cho S.-H. Mueller-Steiner S. Devidze N. Wang X. Grubb A. Gan L. Cystatin C-cathepsin B Axis regulates amyloid beta levels and associated neuronal deficits in an animal model of Alzheimer’s disease Neuron 60 2008 247 257 10.1016/j.neuron.2008.10.001 18957217 PMC2755563 sourceSupported

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

To assess for confounding, the attenuation ratio of each trait was determined via LD score regression and was within the expected range for polygenic traits.

“To assess for confounding we determined the attenuation ratio of each trait via LD score regression, which was within the expected range for polygenic traits (Table S3).104”

• cites [104] Loh P.R. Kichaev G. Gazal S. Schoech A.P. Price A.L. Mixed-model association for biobank-scale datasets Nat. Genet. 50 2018 906 908 10.1038/s41588-018-0144-6 29892013 PMC6309610 sourceSupported

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

Four housekeeping genes (GUSB, PPIA, RPL15, RPL19) with minimal variation on GC treatment were selected for RT-PCR normalization on the basis of a literature review and differential expression analysis in ENCODE RNA-seq data.

“Four housekeeping genes (GUSB, PPIA, RPL15, RPL19) with minimal variation on GC treatment were selected on the basis of a literature review98 and differential expression analysis in ENCODE RNA-seq data (accession ENCSR897XFT), implemented in edgeR.”

• cites [98] Franzén O. Gan L.M. Björkegren J.L.M. PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data Database 2019 10.1093/database/baz046 PMC6450036 30951143 sourceCould not verify

  The Results and discussion section describes an index of genes involved in housekeeping tasks, but does not identify GUSB, PPIA, RPL15, or RPL19 as RT-PCR normalization genes. Searches for those genes, GC-treatment stability, a literature review supporting their selection, and the specified ENCODE analysis found no passage addressing the attributed selection; the paper’s Materials and methods instead describes single-cell RNA-seq analyses. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

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

Two Cst3-specific gRNAs and one scrambled gRNA were from a pre-validated database.

“We tested two Cst3-specific gRNAs and one scrambled gRNA from a pre-validated database.99”

• cites [99] Sanjana N.E. Shalem O. Zhang F. Improved vectors and genome-wide libraries for CRISPR screening Nat. Methods 11 2014 783 784 10.1038/nmeth.3047 25075903 PMC4486245 sourcePartly supported

  The main text (page not specified) describes mouse and human GeCKOv2 libraries with six sgRNAs per gene and non-targeting control sgRNAs, supporting the citation as a source of candidate guides. It does not identify the two Cst3 guides or a scrambled guide, or say that those particular guides were pre-validated. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

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

In human cohort 1, subjects were randomized to a 22-h treatment with placebo or near-physiological glucocorticoid treatment with intravenous hydrocortisone, and after 2 weeks received the other treatment in a crossover design.

“subjects were randomized to a 22-h treatment (commencing at 9a.m.) with placebo (intravenous 0.9% saline) or near-physiological glucocorticoid treatment with intravenous hydrocortisone.”

• Also stated in: § Method details · plain

  “After 2 weeks, subjects were given whichever treatment they had no yet received, as part of a crossover study design.”

• cites [59] Chantzichristos D. Svensson P.-A. Garner T. Glad C.A. Walker B.R. Bergthorsdottir R. Ragnarsson O. Trimpou P. Stimson R.H. Borresen S.W. Identification of human glucocorticoid response markers using integrated multi-omic analysis from a randomized crossover trial Elife 10 2021 e62236 10.7554/eLife.62236 33821793 PMC8024021 sourceSupported

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

Immune clusters in human skin tumor scRNA-seq were annotated by comparison to reference PBMC data using clustifyR.

“Immune clusters were annotated by comparison to reference PBMC data, implemented in clustifyR package for R.60”

• cites [60] Riemondy K.A. Fu R. Gillen A.E. Sheridan R.M. Tian C. Daya M. Hao Y. Hesselberth J.R. clustifyr: an R package for automated single-cell RNA sequencing cluster classification F1000Research 2020 10.12688/f1000research.22969.2 PMC7383722 32765839 sourceSupported

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

Unannotated clusters in human skin tumor scRNA-seq were manually annotated via established marker gene expression and clonal copy number variation profiles.

“Unannotated clusters (presumed to reflect one of tumor, cancer-associated fibroblast or endothelial cells) were manually annotated via established marker gene expression98 and clonal copy number variation profiles, examined using the inferCNV114 package for R.”

• cites [98] Franzén O. Gan L.M. Björkegren J.L.M. PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data Database 2019 10.1093/database/baz046 PMC6450036 30951143 sourceSupported
• cites [114] Tickle T. Tirosh I. Georgescu C. Brown M.,H.B. inferCNV of the trinity CTAT project Klarman Cell Obs. 2019 Broad Inst. MIT HarvardNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

High-confidence Trem2+ monocytes were identified by expression of known TREM2+ monocyte markers (TREM2, APOE, CSF1R) using gene-weighted density estimation in Nebulosa.

“High-confidence Trem2+ monocytes were identified by expression of known TREM2+ monocyte markers (TREM2, APOE, CSF1R81,89), identified by gene-weighted density estimation implemented in the Nebulosa package for R.78”

• cites [81] Molgora M. Esaulova E. Vermi W. Hou J. Chen Y. Luo J. Brioschi S. Bugatti M. Omodei A.S. Ricci B. TREM2 modulation remodels the tumor myeloid landscape enhancing anti-PD-1 immunotherapy Cell 182 2020 886 900.e17 10.1016/j.cell.2020.07.013 32783918 PMC7485282 sourcePartly supported

  The Results section, “Trem2 Is Selectively Expressed in Human Tumor Macrophages” (page unavailable), reports that TREM2+ tumor macrophages co-express CSF1R. “TREM2 Correlates with Poor Disease Prognosis” reports a correlation between TREM2 and APOE expression in a colorectal-cancer cohort. These passages do not establish TREM2, APOE, and CSF1R together as markers of TREM2+ monocytes; the Nebulosa method is cited separately in the citing sentence. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [89] Krasemann S. Madore C. Cialic R. Baufeld C. Calcagno N. El Fatimy R. Beckers L. O’Loughlin E. Xu Y. Fanek Z. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases Immunity 47 2017 566 581.e9 10.1016/j.immuni.2017.08.008 28930663 PMC5719893 sourcePartly supported

  The Results section links Trem2 and Apoe to a disease-associated microglial signature, but lists Csf1r among homeostatic microglial genes rather than as a marker of Trem2+ monocytes. The Methods section identifies splenic monocytes using CD11b and Ly6C, not the claimed three-gene set. The Nebulosa method is attached to a separate citation in the citing sentence. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [78] Alquicira-Hernandez J. Powell J.E. Nebulosa recovers single-cell gene expression signals by kernel density estimation Bioinformatics 37 2021 2485 2487 10.1093/bioinformatics/btab003 33459785 sourceSupported

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

The 72 SNPs used to test association between CST3 and SNPs at the SERPINA6/SERPINA1 loci were previously shown to be significantly associated with plasma cortisol.

“72 SNPs previously shown to be significantly associated with plasma cortisol.50”

• cites [50] Crawford A.A. Bankier S. Altmaier E. Barnes C.L.K. Clark D.W. Ermel R. Friedrich N. van der Harst P. Joshi P.K. Karhunen V. Variation in the SERPINA6/SERPINA1 locus alters morning plasma cortisol, hepatic corticosteroid binding globulin expression, gene expression in peripheral tissues, and risk of cardiovascular disease J. Hum. Genet. 66 2021 625 636 10.1038/s10038-020-00895-6 33469137 PMC8144017 sourcePartly supported

  The Results (Genome-wide association meta-analysis; no page listed) identify the SERPINA6/SERPINA1 locus as significantly associated with morning plasma cortisol. The Results (Tissue-specific eQTL analyses; no page listed) report 21 SNPs that were both liver cis-eQTLs and genome-wide significant for cortisol. Our copy does not establish that the citing document’s particular set of 72 SNPs were all significantly associated with plasma cortisol. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Other loci associated with CyC production, such as SH2B3 and FLT3, identify components of immune cell signaling cascades and are strongly associated with autoimmune disease.

“Other loci associated with CyC production, such as SH2B336 and FLT3,37 identify components of immune cell signaling cascades and are strongly associated with autoimmune disease.”

May combine two claims.

The CyC-production PGS is positively associated with multiple diseases linked to metabolic syndrome, including type 2 diabetes, obesity, hypertension, and ischemic heart disease, meeting phenome-wide significance (p < 1e-5).

“We identified positive associations meeting phenome-wide significance (p < 1e−5) between the CyC-production PGS and multiple diseases linked to metabolic syndrome, including type 2 diabetes, obesity, hypertension, and ischemic heart disease.”

May combine two claims.

The index SNP at the SH2B3 locus is a missense variant (R262W) and exhibits a markedly larger effect size than would be expected for its allele frequency.

“The index SNP at the SH2B3 locus is a missense variant (R262W) and exhibits a markedly larger effect size than would be expected for its allele frequency (minor allele fraction = 0.48;”

May combine two claims.

Myeloid-specific CST3 expression was confirmed in peripheral blood mononuclear cells with scRNA-seq and across multiple scRNA-seq datasets harmonized as part of the Human Protein Atlas.

“We confirmed myeloid-specific CST3 expression in peripheral blood mononuclear cells (PBMCs) with scRNA-seq56 (Figure 4C) and across multiple scRNA-seq datasets harmonized as part of the Human Protein Atlas57 (Figure 4D).”

May combine two claims.

Monocyte-like THP-1 cells constitutively express CyC without GC inducibility.

“We confirmed this hypothesis in monocyte-like THP-1 cells by RNA (Figure 4F) and protein level (Figure 4G).”

May combine two claims.

Dexamethasone treatment significantly increased extracellular CyC protein in M2 macrophages (p = 0.05) and increased CyC in M1 macrophages (p = 0.07).

“We measured significantly increased extracellular CyC protein in M2 macrophages (p = 0.05, two-sided t test) and increased CyC in M1 macrophages (p = 0.07, two-sided t test; Figures S4D and S4E).”

May combine two claims.

A single SNP (rs2749527) at the SERPINA1/6 locus is associated with significantly reduced plasma cortisol (p = 1.75e-13) and significantly reduced CST3 gene expression in visceral adipose fat (p = 0.0024 additive, p = 9.21e-6 recessive).

“trans-expression quantitative trait locus (trans-eQTL) analysis in the Stockholm Tartu Atherosclerosis Reverse Networks Engineering Task (STARNET)52 cohort identified a single SNP (rs2749527) at the SERPINA1/6 locus that was associated with significantly reduced plasma cortisol (p = 1.75e−13, fixed effect meta-analysis) and significantly reduced CST3 gene expression in visceral adipose fat (p = 0.”

May combine two claims.

GC-responsive CyC secretion occurs in macrophages, but not monocytes.

“GC-responsive CyC secretion occurs in macrophages, but not monocytes”

May combine two claims.

We detected high and GC-inducible CyC expression in cancer cell lines.

“we detected high and GC-inducible CyC expression in cancer cell lines (Figures 3H and 3I).”

May combine two claims.

GCs are necessary but not sufficient for induction of CyC in all biological contexts.

“our data demonstrate that GCs are necessary (Figures 5A and 5B) but not sufficient (Figures 4H–4J) for induction of CyC in all biological contexts.”

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

Out of the 309 patients in human cohort 3, 131 had available paired serum creatinine for at least one timepoint and clinical outcome data (COVID-specific mortality).

“Out of these patients, 131 had available paired serum creatinine for at least one timepoint, as well as clinical outcome data (COVID-specific mortality).”

Classified as probably not a substantial claim.

Plasma CyC levels were quantified in 309 patients in human cohort 3.

“allowing for quantification of plasma CyC levels in 309 patients.”

May combine two claims.

eGFR-Cr and/or eGFR-Cy correlate weakly with true GFR in the AFR population.

“This indicates that eGFR-Cr and/or eGFR-Cy correlate weakly with true GFR in the AFR population”

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

The enriched heritability for renal function in liver tissues is in keeping with coupled hepatic and kidney function observed clinically as hepatorenal syndrome.

“in keeping with coupled hepatic and kidney function, observed clinically as hepatorenal syndrome.43”

• cites [43] Ginès P. Solà E. Angeli P. Wong F. Nadim M.K. Kamath P.S. Hepatorenal syndrome Nat. Rev. Dis. Prim. 4 2018 23 10.1038/s41572-018-0022-7 30213943 sourceNot checked — no full text of the source

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

Inflammation is characterized by the recruitment of monocytes to diseased tissues, where they differentiate into macrophages.

“Inflammation is characterized by the recruitment of monocytes to diseased tissues, where they differentiate into macrophages.61”

• cites [61] Ley K. Laudanna C. Cybulsky M.I. Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated Nat. Rev. Immunol. 7 2007 678 689 17717539 sourceNot checked — no full text of the source

May combine two claims.

GC signaling is necessary but not sufficient for CST3 expression.

“demonstrating that GC signaling is necessary but not sufficient for CST3 expression.”

May combine two claims.

CyC production is relatively constant in health and does not significantly increase in response to GC agonism.

“Altogether, these findings indicate that CyC production is relatively constant in health and, in this context, does not significantly increase in response to GC agonism”

May combine two claims.

A single genetic instrument connects CBG, plasma cortisol, and CyC, thus providing genetic evidence for a direct link between GCs and CyC.

“As such, a single genetic instrument connects CBG, plasma cortisol, and CyC, thus providing genetic evidence for a direct link between GCs and CyC.”

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

The study was registered at ClinicalTrials.gov with identifier NCT02152553.

“The study was registered at ClinicalTrials.gov with identifier NCT02152553.”

May combine two claims.

GC-inducible CyC secretion is context dependent and not detected systemically in healthy mice and humans.

“demonstrating that GC-inducible CyC secretion is context dependent and not detected systemically in healthy mice and humans.”

May combine two claims.

Knockout of CST3 in Mm1 cells was confirmed for extracellular CyC protein levels and Sanger sequencing of the predicted edit site, with 97% editing efficiency.

“Knockout was confirmed for extracellular CyC protein levels (Figure S6A) and Sanger sequencing of the predicted edit site (Figure S6B), which confirmed 97% editing efficiency.”

Classified as probably not a substantial claim.

TREM2+ macrophages play a highly immunosuppressive role in the tumor microenvironment.

“Others have shown that TREM2+ macrophages play a highly immunosuppressive role in the tumor microenvironment79”

• cites [79] Katzenelenbogen Y. Sheban F. Yalin A. Yofe I. Svetlichnyy D. Jaitin D.A. Bornstein C. Moshe A. Keren-Shaul H. Cohen M. Coupled scRNA-Seq and intracellular protein activity reveal an immunosuppressive role of TREM2 in cancer Cell 182 2020 872 885.e19 32783915 sourceNot checked — no full text of the source

May combine two claims.

GC agonists increase extracellular CyC levels, which in turn promotes recruitment or expansion of Trem2+ myeloid cells.

“This would support a stepwise model in which GC agonists increase extracellular CyC levels (Figure 5C), which in turn promotes recruitment or expansion of Trem2+ myeloid cells.”

Classified as probably not a substantial claim.

All PGS analyses were performed in either a held-out validation cohort (for the UKB) or an independent non-overlapping cohort (TCGA, panIO) to mitigate against the risk of overfitting.

“we performed all PGS analyses in either a held-out validation cohort (for the UKB) or an independent non-overlapping cohort (TCGA, panIO) to mitigate against the risk of overfitting.”

May combine two claims.

scRNA-seq profiles of cancer cells confirmed CST3 knockout in the cancer cell compartment but not in other compartments.

“scRNA-seq profiles of cancer cells confirmed CST3 knockout in this compartment (Figure S7B; p = 9.16e−30, pseudobulk likelihood ratio test) but not in other compartments (p > 0.05).”

May combine two claims.

The Mm1 cell line is derived from a liver metastasis of the autochthonous KPC model of pancreatic cancer, which recapitulates the low immunogenicity and immunotherapy responsiveness of the human pancreatic cancer.

“which is derived from a liver metastasis of the autochthonous KPC model of pancreatic cancer,73 which recapitulates the low immunogenicity and immunotherapy responsiveness of the human pancreatic cancer.”

• cites [73] Hingorani S.R. Wang L. Multani A.S. Combs C. Deramaudt T.B. Hruban R.H. Rustgi A.K. Chang S. Tuveson D.A. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice Cancer Cell 7 2005 469 483 10.1016/j.ccr.2005.04.023 15894267 sourceNot checked — no full text of the source

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

CyC levels are highest in cerebrospinal and seminal fluid.

“CyC levels are highest in cerebrospinal and seminal fluid,74”

• cites [74] Barrett A.J. Davies M.E. Grubb A. The place of human γ-trace (cystatin C) amongst the cysteine proteinase inhibitors Biochem. Biophys. Res. Commun. 120 1984 631 636 10.1016/0006-291X(84)91302-0 6203523 sourceNot checked — no full text of the source

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

All human subjects gave informed consent, and all studies were approved by the respective institutional review boards.

“All human subjects gave informed consent, and all studies were approved by the respective institutional review boards.”

Classified as probably not a substantial claim.

The Cox regression of overall and cancer-specific survival against CyC-production PGS in UKB used time from recruitment to death or data cut-off (June 2020) to account for left truncation bias.

“To account for this, the time interval used for Cox regression of overall and cancer-specific survival against CyC-production PGS in UKB referred to time from recruitment to death or data cut-off (June 2020).”

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

Written informed consent was obtained from all subjects before participation.

“Written informed consent was obtained from all subjects before participation.”

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

Each phenotype-specific Cox regression in the phenome-wide analysis was adjusted for sex, genetic ancestry (PC1-4), and year of birth to account for historical variation in disease risk.

“Each phenotype-specific Cox regression was adjusted for sex, genetic ancestry (PC1-4) and year of birth (to account for historical variation in disease risk).”

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

For each experiment all samples were harvested concurrently.

“For each experiment all samples were harvested concurrently.”

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

Each cluster in murine Mm1 tumor scRNA-seq was annotated as one of 14 cellular populations according to expression of validated marker genes.

“Each cluster was annotated as one of 14 cellular populations according to expression of validated marker genes (summarized in Table S7, and Figure S7A)”

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

Comparison of population structure with the 1000 Genomes cohort confirmed that all STARNET subjects had European genetic ancestry.

“Comparison of population structure with 1000 Genomes cohort confirmed that all STARNET subjects had European genetic ancestry.”

Classified as probably not a substantial claim.

The Cox regression of overall and cancer-specific survival against CyC-residual adjusted for age at blood sampling, sex, body mass index, hemoglobin, eGFR-Cr, and C-reactive protein.

“Model covariates included age (at blood sampling), sex, body mass index (BMI), hemoglobin, eGFR-Cr, C-reactive protein.”

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

Each sample was profiled in duplicate.

“with each sample profiled in duplicate.”

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

Cell viability was checked by trypan blue method and was consistently above 95% prior to seeding.

“Cell viability was checked by trypan blue method and was consistently above 95% prior to seeding.”

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

No subjects in the immunotherapy meta-cohort had >third degree relatedness, which excludes the possibility of duplicate samples in the cohort.

“No subjects had >third degree relatedness, which also excludes the possibility of duplicates samples in the cohort.”

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

UKB-specific PGS-cancer survival analyses were additionally adjusted for recruitment center and genotyping array.

“UKB-specific PGS-cancer survival analyses were additionally adjusted for recruitment center (to account for regional heterogeneity in cancer outcomes) and genotyping array.”

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

For patients with at least one creatinine measurement in human cohort 3, missing data were imputed with the most recent value.

“For patients with at least one creatinine measurement, missing data were imputed with the most recent value.”

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

In the phenome-wide time-to-event analysis in UKB, the date for each time-to-event Phecode was determined by either the most recent coded diagnosis or most recent UKB center visit to account for region-specific variability in health record linkage.

“To account for region-specific variability in health record linkage, this date was determined by either the most recent coded diagnosis or most recent UKB center visit.”

Classified as probably not a substantial claim.

Cancer-specific survival analyses with respect to CyC-production PGS were adjusted for age of diagnosis, genetic ancestry (PC1-4), sex (except sex-specific cancers), and a term reflecting whether curative surgery was performed.

“Cancer-specific survival analyses with respect to CyC-production PGS were adjusted for age of diagnosis, genetic ancestry (PC1-4), sex (except sex-specific cancers), and a term reflecting whether curative surgery was performed.”

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

All animal experiments and care were performed in accordance with the Cold Spring Harbor Laboratory (CSHL) Institutional Animal Care and Use Committee (IACUC) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

“All animal experiments and care were performed in accordance with the Cold Spring Harbor Laboratory (CSHL) Institutional Animal Care and Use Committee (IACUC) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.”

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

The study was approved by the Ethics Review Board of the University of Gothenburg, Sweden (permit no. 374-13, 8 August 2013) and conducted in accordance with the Declaration of Helsinki.

“The study was approved by the Ethics Review Board of the University of Gothenburg, Sweden (permit no. 374-13, 8 August 2013) and conducted in accordance with the Declaration of Helsinki.”

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

The Mm1 (KPC-derived) cell line behaves highly reproducibly across different experimenters over time, allowing greater confidence in findings from each experiment.

“we have found this line to behave highly reproducibly across different experimenters over time, allowing us to place greater confidence on the findings from each experiment.”

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

Plasma CyC and creatinine in human cohort 1 were measured using validated clinical assays at Sahlgrenska University Hospital.

“Plasma CyC and creatinine were measured used validated clinical assays (creatinine: Alinity c Creatinine (Enzymatic) Reagent Kit; CyC: Gentian Cystatin C Immunoassay) at the laboratory of Sahlgrenska University Hospital in Gothenburg, Sweden.”

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

The nuclei and chromogens in the Multiplex IHC analysis were detected by color deconvolution with thresholds determined by internal controls.

“The nuclei and the chromogens were detected by color deconvolution with thresholds determined by internal controls.”

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

The eQTL analysis using 72 SNPs reflecting 4 independent LD blocks was modeled as 20 independent hypotheses, and the Bonferroni-corrected significance threshold was 0.0025.

“As the 72 SNPs reflecting 4 independent LD blocks, we modeled this analysis as 20 (4 LD blocks, 5 tissues) independent hypotheses and so the Bonferroni-corrected significance threshold was 0.0025.”

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

Model fitting for each CyC-production polygenic score was confirmed by visual inspection of chain convergence.

“Model fitting was confirmed by visual inspection of chain convergence for each PGS.”

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

Graphical overlays for both compartments were used to simplify the quality control of the tissue classifier.

“Graphical overlays for both compartments simplified the quality control of the tissue classifier.”

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

The Cox regression of lifespan against CyC-production PGS in the UKB validation cohort adjusted for year of birth to account for historical increases in mean lifespan.

“Model covariates included year of birth (of subject, as parental birth years are not recorded) to account for historical increases in mean lifespan.”

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

All H&E scans were reviewed by two pathologists (DL and VHK), and the regions of viable and necrotic tissue were determined and computed.

“All H&E scans were reviewed by two pathologists (DL and VHK), and the regions of viable respectively necrotic tissue was determined and computed.”