Similar immune responses to alpha1-oleate and Bacillus Calmette-Guérin treatment in patients with bladder cancer.

BACKGROUND: The molecular content of urine is defined by filtration in the kidneys and by local release from tissues lining the urinary tract. Pathological processes and different therapies change the molecular composition of urine and a variety of markers have been analyzed in patients with bladder cancer. The response to BCG immunotherapy and chemotherapy has been extensively studied and elevated urine concentrations of IL-1RA, IFN-α, IFN-γ TNF-α, and IL-17 have been associated with improved outcome. METHODS: In this study, the host response to intravesical alpha 1-oleate treatment was characterized in patients with non-muscle invasive bladder cancer by proteomic and transcriptomic analysis. RESULTS: Proteomic profiling detected a significant increase in multiple cytokines in the treatment group compared to placebo. The innate immune response was strongly activated, including IL-1RA and pro-inflammatory cytokines in the IL-1 family (IL-1α, IL-1ß, IL-33), chemokines (MIP-1α, IL-8), and interferons (IFN-α2, IFN-γ). Adaptive immune mediators included IL-12, Granzyme B, CD40, PD-L1, and IL-17D, suggesting broad effects of alpha 1-oleate treatment on the tumor tissues. CONCLUSIONS: The cytokine response profile in alpha 1-oleate treated patients was similar to that reported in BCG treated patients, suggesting a significant overlap. A reduction in protein levels at the end of treatment coincided with inhibition of cancer-related gene expression in tissue biopsies, consistent with a positive treatment effect. Thus, in addition to killing tumor cells and inducing cell detachment, alpha 1-oleate is shown to activate a broad immune response with a protective potential.

Proximity extension assay proteomics and renal single cell transcriptomics uncover novel urinary biomarkers for active lupus nephritis.

OBJECTIVE: To identify urinary biomarkers that can distinguish active renal involvement in Lupus Nephritis (LN), a severe manifestation of systemic lupus erythematosus (SLE). METHODS: Urine from 117 subjects, comprised of inactive SLE, active non-renal lupus, active LN, and healthy controls, were subjected to Proximity Extension Assay (PEA) based comprehensive proteomics followed by ELISA validation in an independent, ethnically diverse cohort. Proteomic data is also cross-referenced to renal transcriptomic data to elucidate cellular origins of biomarkers. RESULTS: Systems biology analyses revealed progressive activation of cytokine signaling, chemokine activity and coagulation pathways, with worsening renal disease. In addition to validating 30 previously reported biomarkers, this study uncovers several novel candidates. Following ELISA validation in an independent cohort of different ethnicity, the six most discriminatory biomarkers for active LN were urinary ICAM-2, FABP4, FASLG, IGFBP-2, SELE and TNFSF13B/BAFF, with ROC AUC ≥80%, with most correlating strongly with clinical disease activity. Transcriptomic analyses of LN kidneys mapped the likely origin of these proteins to intra-renal myeloid cells (CXCL16, IL-1RT2, TNFSF13B/BAFF), T/NK cells (FASLG), leukocytes (ICAM2) and endothelial cells (SELE). CONCLUSION: In addition to confirming the diagnostic potential of urine ALCAM, CD163, MCP1, SELL, ICAM1, VCAM1, NGAL and TWEAK for active LN, this study adds urine ICAM-2, FABP4, FASLG, IGFBP-2, SELE, and TNFSF13B/BAFF as additional markers that warrant systematic validation in larger cross-sectional and longitudinal cohorts.

Corrigendum: Urine L-selectin reflects clinical and histological renal disease activity and treatment response in lupus nephritis across multi-ethnicity.

[This corrects the article DOI: 10.3389/fimmu.2023.1200167.].

Urine L-selectin reflects clinical and histological renal disease activity and treatment response in lupus nephritis across multi-ethnicity.

Objective: There is an urgent need for novel biomarkers in lupus nephritis (LN). We report a non-invasive urinary biomarker, L-selectin, in two independent multi-ethnic cohorts. Methods: uL-selectin was tested cross-sectionally in a Chinese cohort (n=255) and a US cohort (n=219) of SLE patients and controls using ELISA. A longitudinal cohort includes 20 active Chinese LN patients. Results: uL-selectin was significantly increased in active LN patients compared to active non-renal SLE, inactive LN, inactive non-renal SLE, chronic kidney disease patients, and healthy controls. uL-selectin positively correlated with global and renal disease activities as well as histological activity index and chronicity index (CI). Low uL-selectin was an independent predictor for high CI. During follow-up, uL-selectin levels decreased significantly in the complete renal remission group. Conclusion: uL-selectin is a novel biomarker of disease activity and renal histopathology in LN across multiple ethnicities. It also reflects treatment response in LN patients during follow up.

Integrated genomic, proteomic and cognitive assessment in Duchenne Muscular Dystrophy suggest astrocyte centric pathology.

Introduction: Documented Duchenne Muscular Dystrophy (DMD) biomarkers are confined to Caucasians and are poor indicators of cognitive difficulties and neuropsychological alterations. Materials and methods: This study correlates serum protein signatures with cognitive performance in DMD patients of South Asian origin. Study included 25 DMD patients aged 6-16 years. Cognitive profiles were assessed by Wechsler Intelligence Scale for Children. Serum proteome profiling of 1317 proteins was performed in eight DMD patients and eight age-matched healthy volunteers. Results: Among the several novel observations we report, better cognitive performance in DMD was associated with increased serum levels of MMP9 and FN1 but decreased Siglec-3, C4b, and C3b. Worse cognitive performance was associated with increased serum levels of LDH-H1 and PDGF-BB but reduced GDF-11, MMP12, TPSB2, and G1B. Secondly, better cognitive performance in Processing Speed (PSI) and Perceptual Reasoning (PRI) domains was associated with intact Dp116, Dp140, and Dp71 dystrophin isoforms while better performance in Verbal Comprehension (VCI) and Working Memory (WMI) domains was associated with intact Dp116 and Dp140 isoforms. Finally, functional pathways shared with Alzheimer's Disease (AD) point towards an astrocyte-centric model for DMD. Conclusion: Astrocytic dysfunction leading to synaptic dysfunction reported previously in AD may be a common pathogenic mechanism underlying both AD and DMD, linking protein alterations to cognitive impairment. This new insight may pave the path towards novel therapeutic approaches targeting reactive astrocytes.

Quantitative proteomic screening uncovers candidate diagnostic and monitoring serum biomarkers of ankylosing spondylitis.

BACKGROUND: We sought to discover serum biomarkers of ankylosing spondylitis (AS) for diagnosis and monitoring disease activity. METHODS: We studied biologic-treatment-naïve AS and healthy control (HC) patients' sera. Eighty samples matched by age, gender, and race (1:1:1 ratio) for AS patients with active disease, inactive disease, and HC were analyzed with SOMAscan™, an aptamer-based discovery platform. T-tests tests were performed for high/low-disease activity AS patients versus HCs (diagnosis) and high versus low disease activity (Monitoring) in a 2:1 and 1:1 ratio, respectively, to identify differentially expressed proteins (DEPs). We used the Cytoscape Molecular Complex Detection (MCODE) plugin to find clusters in protein-protein interaction networks and Ingenuity Pathway Analysis (IPA) for upstream regulators. Lasso regression analysis was performed for diagnosis. RESULTS: Of the 1317 proteins detected in our diagnosis and monitoring analyses, 367 and 167 (317 and 59, FDR-corrected q < .05) DEPs, respectively, were detected. MCODE identified complement, IL-10 signaling, and immune/interleukin signaling as the top 3 diagnosis PPI clusters. Complement, extracellular matrix organization/proteoglycans, and MAPK/RAS signaling were the top 3 monitoring PPI clusters. IPA showed interleukin 23/17 (interleukin 22, interleukin 23A), TNF (TNF receptor-associated factor 3), cGAS-STING (cyclic GMP-AMP synthase, Stimulator of Interferon Gene 1), and Jak/Stat (Signal transducer and activator of transcription 1), signaling in predicted upstream regulators. Lasso regression identified a Diagnostic 13-protein model predictive of AS. This model had a sensitivity of 0.75, specificity of 0.90, a kappa of 0.59, and overall accuracy of 0.80 (95% CI: 0.61-0.92). The AS vs HC ROC curve was 0.79 (95% CI: 0.61-0.96). CONCLUSION: We identified multiple candidate AS diagnostic and disease activity monitoring serum biomarkers using a comprehensive proteomic screen. Enrichment analysis identified key pathways in AS diagnosis and monitoring. Lasso regression identified a multi-protein panel with modest predictive ability.

Comprehensive proteomics and platform validation of urinary biomarkers for bladder cancer diagnosis and staging.

BACKGROUND: Bladder cancer (BC) is among the most common cancers diagnosed in men in the USA. The current gold standards for the diagnosis of BC are invasive or lack the sensitivity to correctly identify the disease. METHODS: An aptamer-based screen analyzed the expression of 1317 proteins in BC compared to urology clinic controls. The top hits were subjected to systems biology analyses. Next, 30 urine proteins were ELISA-validated in an independent cohort of 68 subjects. Three of these proteins were next validated in an independent BC cohort of differing ethnicity. RESULTS: Systems biology analysis implicated molecular functions related to the extracellular matrix, collagen, integrin, heparin, and transmembrane tyrosine kinase signaling in BC susceptibility, with HNF4A and NFKB1 emerging as key molecular regulators. STEM analysis of the dysregulated pathways implicated a functional role for the immune system, complement, and interleukins in BC disease progression. Of 21 urine proteins that discriminated BC from urology clinic controls (UC), urine D-dimer displayed the highest accuracy (0.96) and sensitivity of 97%. Furthermore, 8 urine proteins significantly discriminated MIBC from NMIBC (AUC = 0.75-0.99), with IL-8 and IgA being the best performers. Urine IgA and fibronectin exhibited the highest specificity of 80% at fixed sensitivity for identifying advanced BC. CONCLUSIONS: Given the high sensitivity (97%) of urine D-dimer for BC, it may have a role in the initial diagnosis or detection of cancer recurrence. On the other hand, urine IL-8 and IgA may have the potential in identifying disease progression during patient follow-up. The use of these biomarkers for initial triage could have a significant impact as the current cystoscopy-based diagnostic and surveillance approach is costly and invasive when compared to a simple urine test.

Resistin, Elastase, and Lactoferrin as Potential Plasma Biomarkers of Pediatric Inflammatory Bowel Disease Based on Comprehensive Proteomic Screens.

Inflammatory bowel disease (IBD) is an immune-mediated chronic inflammation of the intestine, which can present in the form of ulcerative colitis (UC) or as Crohn's disease (CD). Biomarkers are needed for reliable diagnosis and disease monitoring in IBD, especially in pediatric patients. Plasma samples from a pediatric IBD cohort were interrogated using an aptamer-based screen of 1322 proteins. The elevated biomarkers identified using the aptamer screen were further validated by ELISA using an independent cohort of 76 pediatric plasma samples, drawn from 30 CD, 30 UC, and 16 healthy controls. Of the 1322 proteins screened in plasma from IBD patients, 129 proteins were significantly elevated when compared with healthy controls. Of these 15 proteins had a fold change greater than 2 and 28 proteins had a fold change >1.5. Neutrophil and extracellular vesicle signatures were detected among the elevated plasma biomarkers. When seven of these proteins were validated by ELISA, resistin was the only protein that was significantly higher in both UC and CD (p < 0.01), with receiver operating characteristic area under the curve value of 0.82 and 0.77, respectively, and the only protein that exhibited high sensitivity and specificity for both CD and UC. The next most discriminatory plasma proteins were elastase and lactoferrin, particularly for UC, with receiver operating characteristic area under the curve values of 0.74 and 0.69, respectively. We have identified circulating resistin, elastase, and lactoferrin as potential plasma biomarkers of IBD in pediatric patients using two independent diagnostic platforms and two independent patient cohorts.

Urinary CD163 is a marker of active kidney disease in childhood-onset lupus nephritis.

OBJECTIVE: The objective of this study was to evaluate the utility of urine CD163 for detecting disease activity in childhood-onset SLE (cSLE) patients. METHODS: Sixty consecutive pediatric patients fulfilling four or more ACR criteria for SLE and 20 healthy controls were recruited for testing of urinary CD163 using ELISA. SLE disease activity was assessed using the SLEDAI-2K. RESULTS: Urine CD163 was significantly higher in patients with active LN than inactive SLE patients and healthy controls, with receiver operating characteristics area under the curve values ranging from 0.93 to 0.96. LN was ascertained by kidney biopsy. Levels of CD163 significantly correlated with the SLEDAI, renal SLEDAI, urinary protein excretion and C3 complement levels. Urine CD163 was also associated with high renal pathology activity index and chronicity index, correlating strongly with interstitial inflammation and interstitial fibrosis based on the examination of concurrent kidney biopsies. CONCLUSION: Urine CD163 emerges as a promising marker for identifying cSLE patients with active kidney disease. Longitudinal studies are warranted to validate the clinical utility of urine CD163 in tracking kidney disease activity in children with lupus.

Exploring urine:serum fractional excretion ratios as potential biomarkers for lupus nephritis.

Objectives: The goal of this exploratory study is to determine if urine:serum fractional excretion ratios can outperform the corresponding urinary biomarker proteins in identifying active renal disease in systemic lupus erythematosus (SLE). Methods: Thirty-six adult SLE patients and twelve healthy controls were examined for serum and urine levels of 8 protein markers, namely ALCAM, calpastatin, hemopexin, peroxiredoxin 6 (PRDX6), platelet factor 4 (PF4), properdin, TFPI and VCAM-1, by ELISA. Fractional excretion of analyzed biomarkers was calculated after normalizing both the urine and serum biomarker levels against creatinine. A further validation cohort of fifty SLE patients was included to validate the initial findings. Results: The FE ratios of all 8 proteins interrogated outperformed conventional disease activity markers such as anti-dsDNA, C3 and C4 in identifying renal disease activity. All but VCAM-1FE were superior to the corresponding urine biomarkers levels in differentiating LN activity, exhibiting positive correlation with renal SLEDAI. ALCAMFE, PF4FE and properdinFE ratios exhibited the highest accuracy (AUC>0.9) in distinguishing active LN from inactive SLE. Four of the FE ratios exhibited perfect sensitivity (calpastatin, PRDX6, PF4 and properdin), while ALCAMFE, PF4FE and properdinFE exhibited the highest specificity values for active LN. In addition, several of these novel biomarkers were associated with higher renal pathology activity indices. In the validation cohort ALCAMFE, PF4FE and properdinFE once again exhibited higher accuracy metrics, surpassing corresponding urine and serum biomarkers levels, with ALCAMFE exhibiting 95% accuracy in distinguishing active LN from inactive SLE. Conclusions: With most of the tested proteins, urine:serum fractional excretion ratios outperformed corresponding urine and serum protein measurements in identifying active renal involvement in SLE. Hence, this novel class of biomarkers in SLE ought to be systemically evaluated in larger independent cohorts for their diagnostic utility in LN assessment.

Urine ALCAM, PF4 and VCAM-1 Surpass Conventional Metrics in Identifying Nephritis Disease Activity in Childhood-Onset Systemic Lupus Erythematosus.

Objectives: Serial kidney biopsy for repeat evaluation and monitoring of lupus nephritis (LN) in childhood-onset Systemic Lupus Erythematosus (cSLE) remains challenging, thus non-invasive biomarkers are needed. Here, we evaluate the performance of ten urine protein markers of diverse nature including cytokines, chemokines, and adhesion molecules in distinguishing disease activity in cSLE. Methods: Eighty-four pediatric patients meeting ≥4 ACR criteria for SLE were prospectively enrolled for urine assay of 10 protein markers normalized to urine creatinine, namely ALCAM, cystatin-C, hemopexin, KIM-1, MCP-1, NGAL, PF-4, Timp-1, TWEAK, and VCAM-1 by ELISA. Samples from active renal (LN) and active non-renal SLE patients were obtained prior to onset/escalation of immunosuppression. SLE disease activity was evaluated using SLEDAI-2000. 59 patients had clinically-active SLE (SLEDAI score ≥4 or having a flare), of whom 29 patients (34.5%) were classified as active renal, and 30 patients (35.7%) were active non-renal. Twenty-five healthy subjects were recruited as controls. Results: Urine concentrations of ALCAM, KIM-1, PF4 and VCAM-1 were significantly increased in active LN patients versus active non-renal SLE, inactive SLE and healthy controls. Five urine proteins differed significantly between 2 (hemopexin, NGAL, MCP1) or 3 (Cystatin-C, TWEAK) groups only, with the highest levels detected in active LN patients. Urine ALCAM, VCAM-1, PF4 and hemopexin correlated best with total SLEDAI as well as renal-SLEDAI scores (p < 0.05). Urine ALCAM, VCAM-1 and hemopexin outperformed conventional laboratory measures (anti-dsDNA, complement C3 and C4) in identifying concurrent SLE disease activity among patients (AUCs 0.75, 0.81, 0.81 respectively), while urine ALCAM, VCAM-1 and PF4 were the best discriminators of renal disease activity in cSLE (AUCs 0.83, 0.88, 0.78 respectively), surpassing conventional biomarkers, including proteinuria. Unsupervised Bayesian network analysis based on conditional probabilities re-affirmed urine ALCAM as being most predictive of active LN in cSLE patients. Conclusion: Urinary ALCAM, PF4, and VCAM-1 are potential biomarkers for predicting kidney disease activity in cSLE and hold potential as surrogate markers of nephritis flares in these patients.

Baseline Elevations of Leukotriene Metabolites and Altered Plasmalogens Are Prognostic Biomarkers of Plaque Progression in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is associated with an increased incidence of acute and chronic cardiovascular disease as compared to the general population. This study uses a comprehensive metabolomic screen of baseline sera from lupus patients to identify metabolites that predict future carotid plaque progression, following 8-9 years of follow-up. Nine patients had SLE without plaque progression, 8 had SLE and went on to develop atherosclerotic plaques (SLEPP), and 8 patients were controls who did not have SLE. The arachidonic acid pathway metabolites, leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE), and the oxidized lipids 9/13-hydroxyoctodecadienoic acid (HODE) were found to be significantly altered (p < 0.05 and fold-change >2) in SLEPP patients compared to SLE patients without plaque progression. SLEPP patients also exhibited significantly altered levels of branched chain amino acid (BCAA) metabolites and plasmalogens compared to the non-SLE controls. Taken together with the rich literature on these metabolites, these findings suggest that the identified metabolites may not only be prognostic of cardiovascular disease development in SLE patients, but they may also be active drivers of atheroma formation. Early identification of these high risk SLE patients may help institute preventive measures early in the disease course.

Aptamer-Based Screen of Neuropsychiatric Lupus Cerebrospinal Fluid Reveals Potential Biomarkers That Overlap With the Choroid Plexus Transcriptome.

OBJECTIVE: As no gold-standard diagnostic test exists for neuropsychiatric systemic lupus erythematosus (NPSLE), we undertook this study to execute a broad screen of NPSLE cerebrospinal fluid (CSF) using an aptamer-based platform. METHODS: CSF was obtained from NPSLE patients and subjected to proteomic assay using the aptamer-based screen. Potential biomarkers were identified and validated in independent NPSLE cohorts in comparison to other neurologic diseases. RESULTS: Forty proteins out of the 1,129 screened were found to be elevated in NPSLE CSF. Based on enzyme-linked immunosorbent assay validation, CSF levels of angiostatin, α2-macroglobulin, DAN, fibronectin, hepatocellular carcinoma clone 1, IgM, lipocalin 2, macrophage colony-stimulating factor (M-CSF), and serine protease inhibitor G1 were significantly elevated in a predominantly White NPSLE cohort (n = 24), compared to patients with other neurologic diseases (n = 54), with CSF IgM (area under the curve [AUC] 0.95) and M-CSF (AUC 0.91) being the most discriminatory proteins. In a second Hong Kong-based NPSLE cohort, CSF IgM (AUC 0.78) and lipocalin 2 (AUC 0.85) were the most discriminatory proteins. Several CSF proteins exhibited high diagnostic specificity for NPSLE in both cohorts. Elevated CSF complement C3 was associated with an acute confusional state. Eleven molecules elevated in NPSLE CSF exhibited concordant elevation in the choroid plexus, suggesting shared origins. CONCLUSION: Lipocalin 2, M-CSF, IgM, and complement C3 emerge as promising CSF biomarkers of NPSLE with diagnostic potential.

Comprehensive aptamer-based screen of 1317 proteins uncovers improved stool protein markers of colorectal cancer.

BACKGROUND: To screen and validate novel stool protein biomarkers of colorectal cancer (CRC). METHODS: A novel aptamer-based screen of 1317 proteins was used to uncover elevated proteins in the stool of patients with CRC, as compared to healthy controls (HCs) in a discovery cohort. Selected biomarker candidates from the discovery cohort were ELISA validated in three independent cross-sectional cohorts comprises 76 CRC patients, 15 adenoma patients, and 63 healthy controls, from two different ethnicities. The expression of the potential stool biomarkers within CRC tissue was evaluated using single-cell RNA-seq datasets. RESULTS: A total of 92 proteins were significantly elevated in CRC samples as compared to HCs in the discovery cohort. Among Caucasians, the 5 most discriminatory proteins among the 16 selected proteins, ordered by their ability to distinguish CRC from adenoma and healthy controls, were MMP9, haptoglobin, myeloperoxidase, fibrinogen, and adiponectin. Except myeloperoxidase, the others were significantly associated with depth of tumor invasion. The 8 stool proteins with the highest AUC values were also discriminatory in a second cohort of Indian CRC patients. Several of the stool biomarkers elevated in CRC were also expressed within CRC tissue, based on the single-cell RNA-seq analysis. CONCLUSIONS: Stool MMP9, fibrinogen, myeloperoxidase, and haptoglobin emerged as promising CRC stool biomarkers, outperforming stool Hemoglobin. Longitudinal studies are warranted to assess the clinical utility of these novel biomarkers in early diagnosis of CRC.

Predicting disease course in ulcerative colitis using stool proteins identified through an aptamer-based screen.

In the search for improved stool biomarkers for inflammatory bowel disease (IBD), an aptamer-based screen of 1129 stool proteins was conducted using stool samples from an IBD cohort. Here we report that of the 20 proteins subsequently validated by ELISA, stool Ferritin, Fibrinogen, Haptoglobin, Hemoglobin, Lipocalin-2, MMP-12, MMP-9, Myeloperoxidase, PGRP-S, Properdin, Resistin, Serpin A4, and TIMP-1 are significantly elevated in both ulcerative colitis (UC) and Crohn's disease (CD) compared to controls. When tested in a longitudinal cohort of 50 UC patients at 4 time-points, fecal Fibrinogen, MMP-8, PGRP-S, and TIMP-2 show the strongest positive correlation with concurrent PUCAI and PGA scores and are superior to fecal calprotectin. Unlike fecal calprotectin, baseline stool Fibrinogen, MMP-12, PGRP-S, TIMP-1, and TIMP-2 can predict clinical remission at Week-4. Here we show that stool proteins identified using the comprehensive aptamer-based screen are superior to fecal calprotectin alone in disease monitoring and prediction in IBD.

Urine protein biomarkers of bladder cancer arising from 16-plex antibody-based screens.

PURPOSE: The purpose of this study is to identify novel urine protein biomarkers of bladder cancer using a Luminex based screening platform. MATERIALS AND METHODS: The current study examines urine samples from 66 subjects, comprised of 31 Urology clinic controls and 35 bladder cancer patients, using a Luminex based screening platform. ELISA validation was carried out for the top 4 prospective urine biomarkers using an independent cohort of 20 Urology clinic controls and 60 bladder cancer (BC) subjects. RESULTS: Of the 16 proteins screened by Luminex, 10 showed significant elevation in BC compared to the controls. Eight of these urine proteins were able to differentiate BC from control urine with ROC AUC values exceeding 0.70 at p < 0.0001, with specificity values exceeding 0.9. Upon ELISA validation, urine IL-1α, IL-1ra, and IL-8 were able to distinguish control urine from urine drawn from various bladder cancer stages, with IL-8 being the best discriminator. Compared to members of the IL-1 cytokine family, urine IL-8 was also best at discriminating T1 and/or T2-T4 from Ta BC (ROC AUC ≥ 0.83), as well as high grade from low grade BC (ROC AUC ≥ 0.82). CONCLUSIONS: These findings suggest that urine IL-1α, IL-1ra and IL-8 are useful indicators of bladder cancer. Urine IL-8 not only distinguishes bladder cancer from controls, it also discriminates high grade from low grade disease, and the successive clinical stages of bladder cancer. While supportive of previous reports, these findings warrant further analysis in prospective cohorts.

Epigallocatechin-3-Gallate Dampens Non-Alcoholic Fatty Liver by Modulating Liver Function, Lipid Profile and Macrophage Polarization.

Epigallocatechin-3-gallate (EGCG) has been shown to attenuate obesity, fatty liver disease, hepatic inflammation and lipid profiles. Here, we validate the efficacy of EGCG in a murine model of non-alcoholic fatty liver disease (NAFLD) and extend the mechanistic insights. NAFLD was induced in mice by a high-fat diet (HFD) with 30% fructose. EGCG was administered at a low dose (25 mg/kg/day, EGCG-25) or high dose (50 mg/kg/day, EGCG-50) for 8 weeks. In HFD-fed mice, EGCG attenuated body and liver weight by ~22% and 47%, respectively, accompanied by ~47% reduction in hepatic triglyceride (TG) accumulation and ~38% reduction in serum cholesterol, resonating well with previous reports in the literature. In EGCG-treated mice, the hepatic steatosis score and the non-alcoholic steatohepatitis activity score were both reduced by ~50% and ~57%, respectively, accompanied by improvements in hepatic inflammation grade. Liver enzymes were improved ~2-3-fold following EGCG treatment, recapitulating previous reports. Hepatic flow cytometry demonstrated that EGCG-fed mice had lower Ly6C+, MHCII+ and higher CD206+, CD23+ hepatic macrophage infiltration, indicating that EGCG impactedM1/M2 macrophage polarization. Our study further validates the salubrious effects of EGCG on NAFLD and sheds light on a novel mechanistic contribution of EGCG, namely hepatic M1-to-M2 macrophage polarization. These findings offer further support for the use of EGCG in human NAFLD.

Quantitative planar array screen of 1000 proteins uncovers novel urinary protein biomarkers of lupus nephritis.

OBJECTIVE: The goal of these studies is to discover novel urinary biomarkers of lupus nephritis (LN). METHODS: Urine from systemic lupus erythematosus (SLE) patients was interrogated for 1000 proteins using a novel, quantitative planar protein microarray. Hits were validated in an independent SLE cohort with inactive, active non-renal (ANR) and active renal (AR) patients, in a cohort with concurrent renal biopsies, and in a longitudinal cohort. Single-cell renal RNA sequencing data from LN kidneys were examined to deduce the cellular origin of each biomarker. RESULTS: Screening of 1000 proteins revealed 64 proteins to be significantly elevated in SLE urine, of which 17 were ELISA validated in independent cohorts. Urine Angptl4 (area under the curve (AUC)=0.96), L-selectin (AUC=0.86), TPP1 (AUC=0.84), transforming growth factor-ß1 (TGFß1) (AUC=0.78), thrombospondin-1 (AUC=0.73), FOLR2 (AUC=0.72), platelet-derived growth factor receptor-ß (AUC=0.67) and PRX2 (AUC=0.65) distinguished AR from ANR SLE, outperforming anti-dsDNA, C3 and C4, in terms of specificity, sensitivity and positive predictive value. In multivariate regression analysis, urine Angptl4, L-selectin, TPP1 and TGFß1 were highly associated with disease activity, even after correction for demographic variables. In SLE patients with serial follow-up, urine L-selectin (followed by urine Angptl4 and TGFß1) were best at tracking concurrent or pending disease flares. Importantly, several proteins elevated in LN urine were also expressed within the kidneys in LN, either within resident renal cells or infiltrating immune cells, based on single-cell RNA sequencing analysis. CONCLUSION: Unbiased planar array screening of 1000 proteins has led to the discovery of urine Angptl4, L-selectin and TGFß1 as potential biomarker candidates for tracking disease activity in LN.

Author Correction: Comprehensive aptamer-based screening identifies a spectrum of urinary biomarkers of lupus nephritis across ethnicities.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Upregulation of Proinflammatory Bradykinin Peptides in Systemic Lupus Erythematosus and Rheumatoid Arthritis.

Our recent study has implicated bradykinin (BK) signaling as being of pathogenic importance in lupus. This study aims to investigate the biomarker potential of BK peptides, BK and BK-des-arg-9, in lupus and other rheumatic autoimmune diseases. Sera from systemic lupus erythematosus (SLE) patients and healthy subjects were screened for BK and BK-des-arg-9 by liquid chromatography-mass spectrometry metabolomics. Serum from 6-mo-old C57BL/6 mice and three murine lupus strains were also screened for the two peptides by metabolomics. Given the promising initial screening results, validation of these two peptides was next conducted using multiple reaction monitoring in larger patient cohorts. In initial metabolomics screening, BK-des-arg-9 was 22-fold higher in SLE serum and 106-fold higher in mouse lupus serum compared with healthy controls. In validation assays using multiple reaction monitoring and quadrupole time-of-flight mass spectrometry, BK and BK-des-arg-9 showed significant elevations in SLE serum compared with controls (p < 0.0001; area under the curve = 0.79-0.88), with a similar but less pronounced increase being noted in rheumatoid arthritis serum. Interestingly, increased renal SLE disease activity index in lupus patients was associated with reduced circulating BK-des-arg-9, and the reasons for this remain to be explored. To sum, increased conversion of BK to the proinflammatory metabolite BK-des-arg-9 appears to be a common theme in systemic rheumatic diseases. Besides serving as an early marker for systemic autoimmunity, independent studies also show that this metabolic axis may also be a pathogenic driver and therapeutic target in lupus.