Tools for mammalian glycoscience research.

Cellular carbohydrates or glycans are critical mediators of biological function. Their remarkably diverse structures and varied activities present exciting opportunities for understanding many areas of biology. In this primer, we discuss key methods and recent breakthrough technologies for identifying, monitoring, and manipulating glycans in mammalian systems.

Chemoproteomics reveals microbiota-derived aromatic monoamine agonists for GPRC5A.

The microbiota generates diverse metabolites to modulate host physiology and disease, but their protein targets and mechanisms of action have not been fully elucidated. To address this challenge, we explored microbiota-derived indole metabolites and developed photoaffinity chemical reporters for proteomic studies. We identified many potential indole metabolite-interacting proteins, including metabolic enzymes, transporters, immune sensors and G protein-coupled receptors. Notably, we discovered that aromatic monoamines can bind the orphan receptor GPRC5A and stimulate ß-arrestin recruitment. Metabolomic and functional profiling also revealed specific amino acid decarboxylase-expressing microbiota species that produce aromatic monoamine agonists for GPRC5A-ß-arrestin recruitment. Our analysis of synthetic aromatic monoamine derivatives identified 7-fluorotryptamine as a more potent agonist of GPRC5A. These results highlight the utility of chemoproteomics to identify microbiota metabolite-interacting proteins and the development of small-molecule agonists for orphan receptors.

Chronic Kidney Disease Is Characterized by Expansion of a Distinct Proinflammatory Intermediate Monocyte Subtype and by Increased Monocyte Adhesion to Endothelial Cells.

SIGNIFICANCE STATEMENT: CKD is accompanied by abnormal inflammation, which contributes to progressive loss of functional renal tissue and accelerated cardiovascular disease. Although studies have documented that dysregulation of monocyte maturation and function is associated with CKD and its complications, it is not well characterized. This study reveals that a distinctive human monocyte subtype with high propensity for releasing proinflammatory mediators and activating endothelial cells is increased in adults with CKD compared with adults with high cardiovascular risk and normal kidney function. It also demonstrates that human monocyte adhesion to endothelial layers and responses to specific inflammatory migration signals are enhanced in CKD. These findings offer insights into the mechanisms of CKD-associated intravascular and localized inflammation and may suggest potential targets for therapeutic interventions. BACKGROUND: Cardiovascular disease (CVD) in patients with CKD is associated with increased circulating intermediate monocytes (IMs). Dysregulation of monocyte maturation and function is associated with CKD and its complications, but it is incompletely characterized. METHODS: To explore monocyte repertoire abnormalities in CKD, we studied properties of monocyte subpopulations, including IM subpopulations distinguished by HLA-DR expression level, in individuals with or without CKD. Using flow cytometry, we profiled monocyte populations in blood samples from adults with CKD, healthy volunteers (HVs), and patient controls (PCs) with high CVD risk. Monocyte subpopulations were also derived from single-cell RNA-sequencing profiles of paired blood and biopsy samples from kidney transplant recipients. We quantified intracellular cytokine production, migration, and endothelial adhesion in ex vivo assays of PBMCs. RESULTS: Of four predefined blood monocyte subpopulations, only HLA-DR hi IMs were increased in individuals with CKD compared with HVs and PCs. In HVs and patients with CKD, LPS-stimulated HLA-DR hi IMs isolated from blood produced higher amounts of TNF and IL-1 ß than other monocyte populations. Single-cell analysis revealed four monocyte clusters common to blood and kidneys, including an HLA-DR hi IM-like cluster that was enriched in kidneys versus blood. Migration toward CCL5 and CX3CL1 and adhesion to primary endothelial cell layers were increased in monocyte subpopulations in individuals with CKD compared with HVs. Monocyte adhesion to endothelial cells was partly dependent on CX3CR1/CX3CL1 interaction. CONCLUSIONS: CKD is associated with an increased number of a distinctive proinflammatory IM subpopulation and abnormalities of monocyte migration and endothelial adhesion. Dysregulated monocyte maturation and function may represent targetable factors contributing to accelerated CVD in CKD.

Safety and Preliminary Efficacy of Mesenchymal Stromal Cell (ORBCEL-M) Therapy in Diabetic Kidney Disease: A Randomized Clinical Trial (NEPHSTROM).

SIGNIFICANCE STATEMENT: Mesenchymal stromal cells (MSCs) may offer a novel therapy for diabetic kidney disease (DKD), although clinical translation of this approach has been limited. The authors present findings from the first, lowest dose cohort of 16 adults with type 2 diabetes and progressive DKD participating in a randomized, placebo-controlled, dose-escalation phase 1b/2a trial of next-generation bone marrow-derived, anti-CD362 antibody-selected allogeneic MSCs (ORBCEL-M). A single intravenous (iv) infusion of 80×10 6 cells was safe and well-tolerated, with one quickly resolved infusion reaction in the placebo group and no subsequent treatment-related serious adverse events (SAEs). Compared with placebo, the median annual rate of decline in eGFR was significantly lower with ORBCEL-M, although mGFR did not differ. The results support further investigation of ORBCEL-M in this patient population in an appropriately sized phase 2b study. BACKGROUND: Systemic therapy with mesenchymal stromal cells may target maladaptive processes involved in diabetic kidney disease progression. However, clinical translation of this approach has been limited. METHODS: The Novel Stromal Cell Therapy for Diabetic Kidney Disease (NEPHSTROM) study, a randomized, placebo-controlled phase 1b/2a trial, assesses safety, tolerability, and preliminary efficacy of next-generation bone marrow-derived, anti-CD362-selected, allogeneic mesenchymal stromal cells (ORBCEL-M) in adults with type 2 diabetes and progressive diabetic kidney disease. This first, lowest dose cohort of 16 participants at three European sites was randomized (3:1) to receive intravenous infusion of ORBCEL-M (80×10 6 cells, n =12) or placebo ( n =4) and was followed for 18 months. RESULTS: At baseline, all participants were negative for anti-HLA antibodies and the measured GFR (mGFR) and estimated GFR were comparable between groups. The intervention was safe and well-tolerated. One placebo-treated participant had a quickly resolved infusion reaction (bronchospasm), with no subsequent treatment-related serious adverse events. Two ORBCEL-M recipients died during follow-up of causes deemed unrelated to the trial intervention; one recipient developed low-level anti-HLA antibodies. The median annual rate of kidney function decline after ORBCEL-M therapy compared with placebo did not differ by mGFR, but was significantly lower by eGFR estimated by the Chronic Kidney Disease Epidemiology Collaboration and Modification of Diet in Renal Disease equations. Immunologic profiling provided evidence of preservation of circulating regulatory T cells, lower natural killer T cells, and stabilization of inflammatory monocyte subsets in those receiving the cell therapy compared with placebo. CONCLUSIONS: Findings indicate safety and tolerability of intravenous ORBCEL-M cell therapy in the trial's lowest dose cohort. The rate of decline in eGFR (but not mGFR) over 18 months was significantly lower among those receiving cell therapy compared with placebo. Further studies will be needed to determine the therapy's effect on CKD progression. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrial.gov NCT02585622 .

Effect of Immunosuppression on the Immune Response to SARS-CoV-2 Infection and Vaccination.

Immunosuppressive treatment in patients with rheumatic diseases can maintain disease remission but also increase risk of infection. Their response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is frequently blunted. In this study we evaluated the effect of immunosuppression exposure on humoral and T cell immune responses to SARS-CoV-2 infection and vaccination in two distinct cohorts of patients; one during acute SARS-CoV-2 infection and 3 months later during convalescence, and another prior to SARS-CoV-2 vaccination, with follow up sampling 6 weeks after vaccination. Results were compared between rituximab-exposed (in previous 6 months), immunosuppression-exposed (in previous 3 months), and non-immunosuppressed groups. The immune cell phenotype was defined by flow cytometry and ELISA. Antigen specific T cell responses were estimated using a whole blood stimulation interferon-γ release assay. A focused post-vaccine assessment of rituximab-treated patients using high dimensional spectral cytometry was conducted. Acute SARS-CoV-2 infection was characterised by T cell lymphopenia, and a reduction in NK cells and naïve CD4 and CD8 cells, without any significant differences between immunosuppressed and non-immunosuppressed patient groups. Conversely, activated CD4 and CD8 cell counts increased in non-immunosuppressed patients with acute SARS-CoV-2 infection but this response was blunted in the presence of immunosuppression. In rituximab-treated patients, antigen-specific T cell responses were preserved in SARS-CoV-2 vaccination, but patients were unable to mount an appropriate humoral response.

Sulfated glycans engage the Ang-Tie pathway to regulate vascular development.

The angiopoietin (Ang)-Tie pathway is essential for the proper maturation and remodeling of the vasculature. Despite its importance in disease, the mechanisms that control signal transduction through this pathway are poorly understood. Here, we demonstrate that heparan sulfate glycosaminoglycans (HS GAGs) regulate Ang-Tie signaling through direct interactions with both Ang ligands and Tie1 receptors. HS GAGs formed ternary complexes with Ang1 or Ang4 and Tie2 receptors, resulting in potentiation of endothelial survival signaling. In addition, HS GAGs served as ligands for the orphan receptor Tie1. The HS-Tie1 interaction promoted Tie1-Tie2 heterodimerization and enhanced Tie1 stability within the mature vasculature. Loss of HS-Tie1 binding using CRISPR-Cas9-mediated mutagenesis in vivo led to decreased Tie protein levels, pathway suppression and aberrant retinal vascularization. Together, these results reveal that sulfated glycans use dual mechanisms to regulate Ang-Tie signaling and are important for the development and maintenance of the vasculature.

Peripheral blood immune cell profiling of acute corneal transplant rejection.

Acute rejection (AR) of corneal transplants (CT) has a profound effect on subsequent graft survival but detailed immunological studies in human CT recipients are lacking. In this multi-site, cross-sectional study, clinical details and blood samples were collected from adults with clinically diagnosed AR of full-thickness (FT)-CT (n = 35) and posterior lamellar (PL)-CT (n = 21) along with Stable CT recipients (n = 177) and adults with non-transplanted corneal disease (n = 40). For those with AR, additional samples were collected 3 months later. Immune cell analysis was performed by whole-genome microarrays (whole blood) and high-dimensional multi-color flow cytometry (peripheral blood mononuclear cells). For both, no activation signature was identified within the B cell and T cell repertoire at the time of AR diagnosis. Nonetheless, in FT- but not PL-CT recipients, AR was associated with differences in B cell maturity and regulatory CD4+ T cell frequency compared to stable allografts. These data suggest that circulating B cell and T cell subpopulations may provide insights into the regulation of anti-donor immune response in human CT recipients with differing AR risk. Our results suggest that, in contrast to solid organ transplants, genetic or cellular assays of peripheral blood are unlikely to be clinically exploitable for prediction or diagnosis of AR.

Rates of Reversal of Volume Overload in Hospitalized Acute Heart Failure: Association With Long-term Kidney Function.

RATIONALE & OBJECTIVE: Achievement of decongestion in acute heart failure (AHF) is associated with improved survival and cardiovascular outcomes but can be associated with acute declines in estimated glomerular filtration rate (eGFR). We examined whether the rate of in-hospital decongestion is associated with longer term kidney function decline. STUDY DESIGN: Post hoc analysis of trial data. SETTINGS & PARTICIPANTS: Patients with ≥2 measures of kidney function (n = 3,500) from the Efficacy of Vasopressin Antagonism in Heart Failure Outcome Study With Tolvaptan (EVEREST) trial. EXPOSURE: In-hospital rate of change in assessments of volume overload, including B-type natriuretic peptide (BNP), N-terminal pro-B-type natriuretic peptide (NT-proBNP), and clinical congestion score (0-12); and rate of change in hemoconcentration including measures of hematocrit, albumin, and total protein. OUTCOME: Incident chronic kidney disease GFR category 4 or worse (chronic kidney disease [CKD] categories G4-G5; defined by a new eGFR of <30 mL/min/1.73 m2) and eGFR decline of >40%. ANALYTICAL APPROACH: Multivariable cause-specific hazards models. RESULTS: Over median 10-month follow-up period, faster decreases in volume overload and more rapid increases in hemoconcentration were associated with a decreased risk of incident CKD G4-G5 and eGFR decline of >40%. In adjusted analyses, for every 6% faster decline in BNP per week, there was a 32% lower risk of both incident CKD G4-G5 (HR, 0.68 [95% CI, 0.58-0.79]) and eGFR decline of >40% (HR, 0.68 [95% CI, 0.57-0.80]). For every 1% faster increase per week in absolute hematocrit, there was a lower risk for both incident CKD G4-G5 (HR, 0.73 [95% CI, 0.64-0.84]) and eGFR decline of >40% (HR, 0.82 [95% CI, 0.71-0.95]), with results consistent for other biomarkers. LIMITATIONS: Possibility of residual confounding. CONCLUSIONS: These results provide reassurance that more rapid decongestion in patients with AHF does not increase the risk of adverse kidney outcomes in patients with heart failure.

Inferior Vena Cava Diameter Measurement Provides Distinct and Complementary Information to Right Atrial Pressure in Acute Decompensated Heart Failure.

BACKGROUND: Inferior vena cava (IVC) measurements correlate only modestly with right atrial pressure (RAP). Part of this inaccuracy is due to the high compliance of the venous system, where a large change in blood volume may result in only a small change in pressure. As such, the information provided by the IVC may be different rather than redundant. METHODS AND RESULTS: We analyzed patients in the ESCAPE (Evaluation Study of Congestive Heart Failure and Pulmonary Artery Catheterization Effectiveness) trial who had both pulmonary artery catheter and IVC measurements at baseline (n = 108). There was only a modest correlation between baseline RAP and IVC diameter (r = 0.41; P < 0.001). Hemoconcentration, defined as an increase in hemoglobin levels between admission and discharge, was correlated with decrease in IVC diameter (r = 0.35; P = 0.02) but not with a decrease in RAP (r = 0.01; P = 0.95). When patients had both IVC and RAP measurements that were below the median, survival rates were superior to the rates of those who had only 1 measurement below the median, and when both rates were above the median, patients fared the worst (P = 0.002). CONCLUSION: IVC and RAP have limited correlation with each another, and changes in intravascular volume appear to correlate better with IVC diameter rather than with RAP. Furthermore, complementary information is provided by pressure and volume assessments in acute decompensated heart failure.

The Clinical Application of Urine Soluble CD163 in ANCA-Associated Vasculitis.

BACKGROUND: Up to 70% of patients with ANCA-associated vasculitis (AAV) develop GN, with 26% progressing to ESKD. Diagnostic-grade and noninvasive tools to detect active renal inflammation are needed. Urinary soluble CD163 (usCD163) is a promising biomarker of active renal vasculitis, but a diagnostic-grade assay, assessment of its utility in prospective diagnosis of renal vasculitis flares, and evaluation of its utility in proteinuric states are needed. METHODS: We assessed a diagnostic-grade usCD163 assay in (1) a real-world cohort of 405 patients with AAV and 121 healthy and 488 non-AAV disease controls; (2) a prospective multicenter study of 84 patients with potential renal vasculitis flare; (3) a longitudinal multicenter cohort of 65 patients with podocytopathy; and (4) a cohort of 29 patients with AAV (with or without proteinuria) and ten controls. RESULTS: We established a diagnostic reference range, with a cutoff of 250 ng/mmol for active renal vasculitis (area under the curve [AUC], 0.978). Using this cutoff, usCD163 was elevated in renal vasculitis flare (AUC, 0.95) but remained low in flare mimics, such as nonvasculitic AKI. usCD163's specificity declined in patients with AAV who had nephrotic-range proteinuria and in those with primary podocytopathy, with 62% of patients with nephrotic syndrome displaying a "positive" usCD163. In patients with AAV and significant proteinuria, usCD163 normalization to total urine protein rather than creatinine provided the greatest clinical utility for diagnosing active renal vasculitis. CONCLUSIONS: usCD163 is elevated in renal vasculitis flare and remains low in flare mimics. Nonspecific protein leakage in nephrotic syndrome elevates usCD163 in the absence of glomerular macrophage infiltration, resulting in false-positive results; this can be corrected with urine protein normalization.

Adjuvant and concurrent temozolomide for 1p/19q non-co-deleted anaplastic glioma (CATNON; EORTC study 26053-22054): second interim analysis of a randomised, open-label, phase 3 study.

BACKGROUND: The CATNON trial investigated the addition of concurrent, adjuvant, and both current and adjuvant temozolomide to radiotherapy in adults with newly diagnosed 1p/19q non-co-deleted anaplastic gliomas. The benefit of concurrent temozolomide chemotherapy and relevance of mutations in the IDH1 and IDH2 genes remain unclear. METHODS: This randomised, open-label, phase 3 study done in 137 institutions across Australia, Europe, and North America included patients aged 18 years or older with newly diagnosed 1p/19q non-co-deleted anaplastic gliomas and a WHO performance status of 0-2. Patients were randomly assigned (1:1:1:1) centrally using a minimisation technique to radiotherapy alone (59·4 Gy in 33 fractions; three-dimensional conformal radiotherapy or intensity-modulated radiotherapy), radiotherapy with concurrent oral temozolomide (75 mg/m2 per day), radiotherapy with adjuvant oral temozolomide (12 4-week cycles of 150-200 mg/m2 temozolomide given on days 1-5), or radiotherapy with both concurrent and adjuvant temozolomide. Patients were stratified by institution, WHO performance status score, age, 1p loss of heterozygosity, the presence of oligodendroglial elements on microscopy, and MGMT promoter methylation status. The primary endpoint was overall survival adjusted by stratification factors at randomisation in the intention-to-treat population. A second interim analysis requested by the independent data monitoring committee was planned when two-thirds of total required events were observed to test superiority or futility of concurrent temozolomide. This study is registered with ClinicalTrials.gov, NCT00626990. FINDINGS: Between Dec 4, 2007, and Sept 11, 2015, 751 patients were randomly assigned (189 to radiotherapy alone, 188 to radiotherapy with concurrent temozolomide, 186 to radiotherapy and adjuvant temozolomide, and 188 to radiotherapy with concurrent and adjuvant temozolomide). Median follow-up was 55·7 months (IQR 41·0-77·3). The second interim analysis declared futility of concurrent temozolomide (median overall survival was 66·9 months [95% CI 45·7-82·3] with concurrent temozolomide vs 60·4 months [45·7-71·5] without concurrent temozolomide; hazard ratio [HR] 0·97 [99·1% CI 0·73-1·28], p=0·76). By contrast, adjuvant temozolomide improved overall survival compared with no adjuvant temozolomide (median overall survival 82·3 months [95% CI 67·2-116·6] vs 46·9 months [37·9-56·9]; HR 0·64 [95% CI 0·52-0·79], p<0·0001). The most frequent grade 3 and 4 toxicities were haematological, occurring in no patients in the radiotherapy only group, 16 (9%) of 185 patients in the concurrent temozolomide group, and 55 (15%) of 368 patients in both groups with adjuvant temozolomide. No treatment-related deaths were reported. INTERPRETATION: Adjuvant temozolomide chemotherapy, but not concurrent temozolomide chemotherapy, was associated with a survival benefit in patients with 1p/19q non-co-deleted anaplastic glioma. Clinical benefit was dependent on IDH1 and IDH2 mutational status. FUNDING: Merck Sharpe & Dohme.

First-in-Human Experience With Peritoneal Direct Sodium Removal Using a Zero-Sodium Solution: A New Candidate Therapy for Volume Overload.

BACKGROUND: Loop diuretics have well-described toxicities, and loss of response to these agents is common. Alternative strategies are needed for the maintenance of euvolemia in heart failure (HF). Nonrenal removal of sodium directly across the peritoneal membrane (direct sodium removal [DSR]) with a sodium-free osmotic solution should result in extraction of large quantities of sodium with limited off-target solute removal. METHODS: This article describes the preclinical development and first-in-human proof of concept for DSR. Sodium-free 10% dextrose was used as the DSR solution. Porcine experiments were conducted to investigate the optimal dwell time, safety, and scalability and to determine the effect of experimental heart failure. In the human study, participants with end-stage renal disease on peritoneal dialysis (PD) underwent randomization and crossover to either a 2-hour dwell with 1 L DSR solution or standard PD solution (Dianeal 4.25% dextrose, Baxter). The primary end point was completion of the 2-hour dwell without significant discomfort or adverse events, and the secondary end point was difference in sodium removal between DSR and standard PD solution. RESULTS: Porcine experiments revealed that 1 L DSR solution removed 4.1±0.4 g sodium in 2 hours with negligible off-target solute removal and overall stable serum electrolytes. Increasing the volume of DSR solution cycled across the peritoneum increased sodium removal and substantially decreased plasma volume (P=0.005). In the setting of experimental heart failure with elevated right atrial pressure, sodium removal was ≈4 times greater than in healthy animals (P<0.001). In the human proof-of-concept study, DSR solution was well tolerated and not associated with significant discomfort or adverse events. Plasma electrolyte concentrations were stable, and off-target solute removal was negligible. Sodium removal was substantially higher with DSR (4.5±0.4 g) compared with standard PD solution (1.0±0.3 g; P<0.0001). CONCLUSIONS: DSR was well tolerated in both animals and human subjects and produced substantially greater sodium removal than standard PD solution. Additional research evaluating the use of DSR as a method to prevent and treat hypervolemia in heart failure is warranted. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03801226.

Empagliflozin in Heart Failure: Diuretic and Cardiorenal Effects.

BACKGROUND: Sodium-glucose cotransporter-2 inhibitors improve heart failure-related outcomes. The mechanisms underlying these benefits are not well understood, but diuretic properties may contribute. Traditional diuretics such as furosemide induce substantial neurohormonal activation, contributing to the limited improvement in intravascular volume often seen with these agents. However, the proximal tubular site of action of the sodium-glucose cotransporter-2 inhibitors may help circumvent these limitations. METHODS: Twenty patients with type 2 diabetes mellitus and chronic, stable heart failure completed a randomized, placebo-controlled crossover study of empagliflozin 10 mg daily versus placebo. Patients underwent an intensive 6-hour biospecimen collection and cardiorenal phenotyping at baseline and again after 14 days of study drug. After a 2-week washout, patients crossed over to the alternate therapy with the above protocol repeated. RESULTS: Oral empagliflozin was rapidly absorbed as evidenced by a 27-fold increase in urinary glucose excretion by 3 hours (P<0.0001). Fractional excretion of sodium increased significantly with empagliflozin monotherapy versus placebo (fractional excretion of sodium, 1.2±0.7% versus 0.7±0.4%; P=0.001), and there was a synergistic effect in combination with bumetanide (fractional excretion of sodium, 5.8±2.5% versus 3.9±1.9%; P=0.001). At 14 days, the natriuretic effect of empagliflozin persisted, resulting in a reduction in blood volume (-208 mL [interquartile range, -536 to 153 mL] versus -14 mL [interquartile range, -282 to 335 mL]; P=0.035) and plasma volume (-138 mL, interquartile range, -379 to 154±453 mL; P=0.04). This natriuresis was not, however, associated with evidence of neurohormonal activation because the change in norepinephrine was superior (P=0.02) and all other neurohormones were similar (P<0.34) during the empagliflozin versus placebo period. Furthermore, there was no evidence of potassium wasting (P=0.20) or renal dysfunction (P>0.11 for all biomarkers), whereas both serum magnesium (P<0.001) and uric acid levels (P=0.008) improved. CONCLUSIONS: Empagliflozin causes significant natriuresis, particularly when combined with loop diuretics, resulting in an improvement in blood volume. However, off-target electrolyte wasting, renal dysfunction, and neurohormonal activation were not observed. This favorable diuretic profile may offer significant advantage in the management of volume status in patients with heart failure and may represent a mechanism contributing to the superior long-term heart failure outcomes observed with these agents. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03027960.

Targeting stromal cell Syndecan-2 reduces breast tumour growth, metastasis and limits immune evasion.

Tumour stromal cells support tumourigenesis. We report that Syndecan-2 (SDC2) is expressed on a nonepithelial, nonhaematopoietic, nonendothelial stromal cell population within breast cancer tissue. In vitro, syndecan-2 modulated TGFß signalling (SMAD7, PAI-1), migration and immunosuppression of patient-derived tumour-associated stromal cells (TASCs). In an orthotopic immunocompromised breast cancer model, overexpression of syndecan-2 in TASCs significantly enhanced TGFß signalling (SMAD7, PAI-1), tumour growth and metastasis, whereas reducing levels of SDC2 in TASCs attenuated TGFß signalling (SMAD7, PAI-1, CXCR4), tumour growth and metastasis. To explore the potential for therapeutic application, a syndecan-2-peptide was generated that inhibited the migratory and immunosuppressive properties of TASCs in association with reduced expression of TGFß-regulated immunosuppressive genes, such as CXCR4 and PD-L1. Moreover, using an orthotopic syngeneic breast cancer model, overexpression of syndecan-2-peptide in TASCs reduced tumour growth and immunosuppression within the TME. These data provide evidence that targeting stromal syndecan-2 within the TME inhibits tumour growth and metastasis due to decreased TGFß signalling and increased immune control.

RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp.

Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless single-stranded DNA recombineering to generate specific gene-editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic-selectable markers to generate gene deletion mutants. As a further proof of principle, we use CRISPR-Cas9-mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. IMPORTANCE Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlying its diverse activities. However, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables the rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in the genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.

Cystatin C and Muscle Mass in Patients With Heart Failure.

BACKGROUND: The estimated glomerular filtration rate (eGFR) from cystatin C (eGFRcys) is often considered a more accurate method to assess GFR compared with an eGFR from creatinine (eGFRcr) in the setting of heart failure (HF) and sarcopenia, because cystatin C is hypothesized to be less affected by muscle mass than creatinine. We evaluated (1) the association of muscle mass with cystatin C, (2) the accuracy of eGFRcys, and (3) the association of eGFRcys with mortality given muscle mass. METHODS AND RESULTS: We included 293 patients admitted with HF. Muscle mass was estimated with a validated creatinine excretion-based equation. Accuracy of eGFRcys and eGFRcr was compared with measured creatinine clearance. Cystatin C and creatinine were 31.7% and 59.9% higher per 14 kg higher muscle mass at multivariable analysis (both P < .001). At lower muscle mass, eGFRcys and eGFRcr overestimated the measured creatinine clearance. At higher muscle mass, eGFRcys underestimated the measured creatinine clearance, but eGFRcr did not. After adjusting for muscle mass, neither eGFRcys nor eGFRcr were associated with mortality (both P > .19). CONCLUSIONS: Cystatin C levels were associated with muscle mass in patients with HF, which could potentially decrease the accuracy of eGFRcys. In HF where aberrations in body composition are common, eGFRcys, like eGFRcr, may not provide accurate GFR estimations and results should be interpreted cautiously.

Non-IDH1-R132H IDH1/2 mutations are associated with increased DNA methylation and improved survival in astrocytomas, compared to IDH1-R132H mutations.

Somatic mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 occur at high frequency in several tumour types. Even though these mutations are confined to distinct hotspots, we show that gliomas are the only tumour type with an exceptionally high percentage of IDH1R132H mutations. Patients harbouring IDH1R132H mutated tumours have lower levels of genome-wide DNA-methylation, and an associated increased gene expression, compared to tumours with other IDH1/2 mutations ("non-R132H IDH1/2 mutations"). This reduced methylation is seen in multiple tumour types and thus appears independent of the site of origin. For 1p/19q non-codeleted glioma (astrocytoma) patients, we show that this difference is clinically relevant: in samples of the randomised phase III CATNON trial, patients harbouring tumours with IDH mutations other than IDH1R132H have a better outcome (hazard ratio 0.41, 95% CI [0.24, 0.71], p = 0.0013). Such non-R132H IDH1/2-mutated tumours also had a significantly lower proportion of tumours assigned to prognostically poor DNA-methylation classes (p < 0.001). IDH mutation-type was independent in a multivariable model containing known clinical and molecular prognostic factors. To confirm these observations, we validated the prognostic effect of IDH mutation type on a large independent dataset. The observation that non-R132H IDH1/2-mutated astrocytomas have a more favourable prognosis than their IDH1R132H mutated counterpart indicates that not all IDH-mutations are identical. This difference is clinically relevant and should be taken into account for patient prognostication.

Effects of mesenchymal stromal cells on regulatory T cells: Current understanding and clinical relevance.

The immunomodulatory potential of mesenchymal stromal cells (MSCs) and regulatory T cells (T-reg) is well recognized by translational scientists in the field of regenerative medicine and cellular therapies. A wide range of preclinical studies as well as a limited number of human clinical trials of MSC therapies have not only shown promising safety and efficacy profiles but have also revealed changes in T-reg frequency and function. However, the mechanisms underlying this potentially important observation are not well understood and, consequently, the optimal strategies for harnessing MSC/T-reg cross-talk remain elusive. Cell-to-cell contact, production of soluble factors, reprogramming of antigen presenting cells to tolerogenic phenotypes, and induction of extracellular vesicles ("exosomes") have emerged as possible mechanisms by which MSCs produce an immune-modulatory milieu for T-reg expansion. Additionally, these two cell types have the potential to complement each other's immunoregulatory functions, and a combinatorial approach may exert synergistic effects for the treatment of immunological diseases. In this review, we critically assess recent translational research related to the outcomes and mechanistic basis of MSC effects on T-reg and provide a perspective on the potential for this knowledge base to be further exploited for the treatment of autoimmune disorders and transplants.

Ultrafine Particles Emitted through Routine Operation of a Hairdryer.

Particulate matter is a large concern for human health. Fine and ultrafine particulate matter has been shown to negatively impact human health; for example, it causes cardiopulmonary diseases. Current regulation targets the size of the particles, but composition also impacts toxicity. Indoor sources of air pollution pose unique challenges for human health due to the potential for human exposure to high concentrations in confined spaces. In this work, six hairdryers were each operated within a plexiglass chamber, and their emissions were analyzed with transmission electron microscopy and energy-dispersive spectroscopy. All hairdryers were found to emit ultrafine iron, carbon, and copper. In addition, emissions from two hairdryers primarily contained silver nanoparticles in the ultrafine range (<100 nm). The ultrafine particle emission rates for the hairdryers that did not contain silver were measured and found to be lower than ultrafine particle emissions by gas stoves and electric burners. Based on their size, these particles can either remain in the lung or enter the bloodstream after inhalation and potentially cause long-term health effects.

TGF-ß1-Licensed Murine MSCs Show Superior Therapeutic Efficacy in Modulating Corneal Allograft Immune Rejection In Vivo.

Mesenchymal stromal cells (MSCs) are a promising therapeutic option for multiple immune diseases/disorders; however, efficacy of MSC treatments can vary significantly. We present a novel licensing strategy to improve the immunosuppressive capacity of MSCs. Licensing murine MSCs with transforming growth factor-ß1 (TGF-ß MSCs) significantly improved their ability to modulate both the phenotype and secretome of inflammatory bone marrow-derived macrophages and significantly increased the numbers of regulatory T lymphocytes following co-culture assays. These TGF-ß MSC-expanded regulatory T lymphocytes also expressed significantly higher levels of PD-L1 and CD73, indicating enhanced suppressive potential. Detailed analysis of T lymphocyte co-cultures revealed modulation of secreted factors, most notably elevated prostaglandin E2 (PGE2). Furthermore, TGF-ß MSCs could significantly prolong rejection-free survival (69.2% acceptance rate compared to 21.4% for unlicensed MSC-treated recipients) in a murine corneal allograft model. Mechanistic studies revealed that (1) therapeutic efficacy of TGF-ß MSCs is Smad2/3-dependent, (2) the enhanced immunosuppressive capacity of TGF-ß MSCs is contact-dependent, and (3) enhanced secretion of PGE2 (via prostaglandin EP4 [E-type prostanoid 4] receptor) by TGF-ß MSCs is the predominant mediator of Treg expansion and T cell activation and is associated with corneal allograft survival. Collectively, we provide compelling evidence for the use of TGF-ß1 licensing as an unconventional strategy for enhancing MSC immunosuppressive capacity.