Standard RUTF vs. locally-made RUSF for acutely malnourished children: A quasi-experimental comparison of the impact on growth and compliance in a rural community of Pakistan.

BACKGROUND: The reduction in severe and moderate acute malnutrition (SAM and MAM) rates in Pakistan has been sub-optimal compared to other low-and middle-income countries (LMICs). Specially-formulated products have been designed globally to manage SAM and MAM, such as ready-to-use therapeutic food (RUTF) and ready-to-use supplementary food (RUSF), with variable efficacies. RUTF is primarily produced and patented in industrialized countries, raising supply challenges in resource-constrained regions with a high burden of acute malnutrition. RUSF minimizes costs by using locally-available ingredients while providing similar nutritional value. In this study, we compared the efficacy, side effects, and compliance of two months of supplementation with either RUTF or RUSF. METHODS: Children aged nine months in the rural district of Matiari, Pakistan, with a weight-for-height z-score (WHZ) <-2 received either RUTF (500 kcal sachet) for two months in 2015 or RUSF (520 kcal sachet) for two months in 2018. RESULTS: The RUSF group had a higher height gain and mid-upper arm circumferences (MUAC) score. Higher compliance was noted with lower side effects in the RUSF group. A higher compliance rate did correlate with the growth parameters in respective groups. CONCLUSION: Our study found that both RUTF and RUSF partially improve the anthropometric status of acutely malnourished children, with neither being superior to the other.

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.

A novel technology for home monitoring of lupus nephritis that tracks the pathogenic urine biomarker ALCAM.

Introduction: The gold standard for diagnosis of active lupus nephritis (ALN), a kidney biopsy, is invasive with attendant morbidity and cannot be serially repeated. Urinary ALCAM (uALCAM) has shown high diagnostic accuracy for renal pathology activity in ALN patients. Methods: Lateral flow assays (LFA) for assaying uALCAM were engineered using persistent luminescent nanoparticles, read by a smartphone. The stability and reproducibility of the assembled LFA strips and freeze-dried conjugated nanoparticles were verified, as was analyte specificity. Results: The LFA tests for both un-normalized uALCAM (AUC=0.93) and urine normalizer (HVEM)-normalized uALCAM (AUC=0.91) exhibited excellent accuracies in distinguishing ALN from healthy controls. The accuracies for distinguishing ALN from all other lupus patients were 0.86 and 0.74, respectively. Conclusion: Periodic monitoring of uALCAM using this easy-to-use LFA test by the patient at home could potentially accelerate early detection of renal involvement or disease flares in lupus patients, and hence reduce morbidity and mortality.

Comprehensive Urinomic Identification of Protein Alternatives to Creatinine Normalization for Diagnostic Assessment of Lupus Nephritis.

Introduction: The current gold standard used for urine biomarker normalization, creatinine, poses a challenge to translate to the point of care because antibodies to creatinine are difficult to develop and currently available ligands to creatinine are sub-optimal for this purpose. Hence, protein alternatives to creatinine are clearly needed. To address this need, lupus nephritis was selected as a model disease where urine protein assessment is required for diagnosis. Methods: A comprehensive proteomic screen of 1129 proteins in healthy and lupus nephritis urine was executed to identify protein alternatives to creatinine for the normalization of urine biomarkers. Urinary proteins that correlated well with creatinine but did not vary with disease were further validated by ELISA in an independent cohort of lupus nephritis subjects. Results: The comprehensive proteomic screen identified 14 urine proteins that correlated significantly with urine creatinine but did not differ significantly between SLE and controls. Of the top five proteins selected for ELISA validation, urine HVEM and RELT once again showed significant correlation with urine creatinine in independent cohorts. Normalizing a lupus nephritis biomarker candidate ALCAM using urinary HVEM demonstrated comparable diagnostic ability to creatinine normalization when distinguishing active lupus nephritis from inactive SLE patients. Conclusions: The discovery of urine HVEM as a protein alternative to creatinine for biomarker normalization has applications in the engineering of antibody-based point of care diagnostics for monitoring lupus nephritis progression.

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.

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.

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.

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.

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.

Assessing colitis ex vivo using optical coherence elastography in a murine model.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that causes regions of ulceration within the interior of the colon. UC is estimated to afflict hundreds of thousands of people in the United States alone. In addition to traditional colonoscopy, ultrasonic techniques can detect colitis, but have limited spatial resolution, which frequently results in underdiagnoses. Nevertheless, clinical diagnosis of colitis is still generally performed via colonoscopy. Optical techniques such as confocal microscopy and optical coherence tomography (OCT) have been proposed to detect UC with higher resolution. However, UC can potentially alter tissue biomechanical properties, providing additional contrast for earlier and potentially more accurate detection. Although clinically available elastography techniques have been immensely useful, they do not have the resolution for imaging small tissues, such as in small mammalian disease models. However, OCT-based elastography, optical coherence elastography (OCE), is well-suited for imaging the biomechanical properties of small mammal colon tissue. METHODS: In this work, we induced elastic waves in ex vivo mouse colon tissue using a focused air-pulse. The elastic waves were detected using a phase-stabilized swept source OCE system, and the wave velocity was translated into stiffness. Measurements were taken at six positions for each sample to assess regional sample elasticity. Additional contrast between the control and diseased tissue was detected by analyzing the dispersion of the elastic wave and tissue optical properties obtained from the OCT structural image. RESULTS: The results show distinct differences (P<0.05) in the stiffness between control and colitis disease samples, with a Young's modulus of 11.8±8.0 and 5.1±1.5 kPa, respectively. The OCT signal standard deviations for control and diseased samples were 5.8±0.3 and 5.5±0.2 dB, respectively. The slope of the OCT signal spatial frequency decay in the control samples was 92.7±10.0 and 87.3±4.7 dB∙µm in the colitis samples. The slope of the linearly fitted dispersion curve in the control samples was 1.5 mm, and 0.8 mm in the colitis samples. CONCLUSIONS: Our results show that OCE can be utilized to distinguish tissue based on stiffness and optical properties. Our estimates of tissue stiffness suggest that the healthy colon tissue was stiffer than diseased tissue. Furthermore, structural analysis of the tissue indicates a distinct difference in tissue optical properties between the healthy and UC-like diseased tissue.

Low dose Epigallocatechin Gallate Alleviates Experimental Colitis by Subduing Inflammatory Cells and Cytokines, and Improving Intestinal Permeability.

BACKGROUND: In this study, we investigate the impact of epigallocatechin gallate (EGCG), the most abundant and potent catechin in green tea, on a mouse model of inflammatory bowel disease (IBD) and the underlying mechanisms of action. METHODS: C57BL/6J mice were subjected to dextran sulfate sodium (DSS)-induced IBD-like disease and then randomly divided into three groups: Model group (MD), low-dose EGCG group (LE, 20 mg/kg/d), and high-dose EGCG group (HE, 50 mg/kg/d). DSS-induced clinical and macroscopic changes were monitored daily. Intestinal permeability was assessed by FITC-Dextran assay. RESULTS: Both high- and low-dose EGCG treatment alleviated clinical manifestations including body weight loss and disease activity index (DAI) of DSS-induced colitis. The DAI score was significantly improved after two days of EGCG treatment. At the end of the study, the macroscopic severity score (MSS) of HE and LE treatment groups were 2.4 ± 1.2, and 2.2 ± 1.0, respectively, significantly lower than that of the controls (5.0 ± 2.1). EGCG treatment also prevented colon shortening, and improved intestinal permeability and histopathological changes. In addition, EGCG treatment attenuated colon inflammation by suppressing colonic levels of pro-inflammatory cytokines IL-6, MCP-1, and TNF-alpha, and inhibited CD3+ T cell and CD68+ macrophage infiltration. CONCLUSION: EGCG is effective in inflammatory colitis because it reduces cellular and molecular inflammation, and reduces intestinal permeability.

Beyond Autoantibodies: Biologic Roles of Human Autoreactive B Cells in Rheumatoid Arthritis Revealed by RNA-Sequencing.

OBJECTIVE: To obtain the comprehensive transcriptome profile of human citrulline-specific B cells from patients with rheumatoid arthritis (RA). METHODS: Citrulline- and hemagglutinin-specific B cells were sorted by flow cytometry using peptide-streptavidin conjugates from the peripheral blood of RA patients and healthy individuals. The transcriptome profile of the sorted cells was obtained by RNA-sequencing, and expression of key protein molecules was evaluated by aptamer-based SOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays. RESULTS: Citrulline-specific B cells, in comparison to citrulline-negative B cells, from patients with RA differentially expressed the interleukin-15 receptor α (IL-15Rα) gene as well as genes related to protein citrullination and cyclic AMP signaling. In analyses of an independent cohort of cyclic citrullinated peptide-seropositive RA patients, the expression of IL-15Rα protein was enriched in citrulline-specific B cells from the patients' peripheral blood, and surprisingly, all B cells from RA patients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production of AREG directly led to increased migration and proliferation of fibroblast-like synoviocytes, and, in combination with anti-citrullinated protein antibodies, led to the increased differentiation of osteoclasts. CONCLUSION: To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing several US Food and Drug Administration-approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases.

Detecting murine Inflammatory Bowel Disease using Optical Coherence Elastography.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that causes regions of ulceration within the interior of the colon. UC is estimated to afflict hundreds of thousands of people in the United States alone. Ultrasonic techniques can detect colitis, but have limited spatial resolution, which frequently results in underdiagnoses. Nevertheless, clinical diagnosis of colitis is still generally performed via colonoscopy. Optical techniques such as confocal microscopy and optical coherence tomography (OCT) have been proposed as higher resolution alternative imaging modalities to detect colitis. Additionally, IBD can potentially alter tissue biomechanical properties, which cannot be quantified from structural imaging alone. Elastography is a potential method to assess colon biomechanical properties to provide additional contrast for distinguishing healthy and diseased colon tissue. In this work, we induced elastic waves in ex vivo mouse colon tissue using a focused air-pulse. The elastic waves were detected using a phase-stabilized swept source optical coherence elastography system, and the wave velocity was translated into stiffness. Measurements were taken at six random positions for each sample in order to assess regional sample elasticity. The results show distinct differences ($p \lt 0.05$) in the stiffness between healthy and IBD-diseased samples, with a Young's Modulus of $10.2 \pm 3.7$ kPa and $4.9 \pm 0.3$ kPa, respectively. Dispersion analysis presents another parameter to distinguish tissue health. The high frequency components of the phase velocity dispersion curve indicate a variation between healthy and IBD colonic tissue. Our results show that OCE may be useful for detecting IBD noninvasively.

Biomarkers for sporadic Creutzfeldt-Jakob disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare but fatal type of spongiform encephalopathy with unknown cause. Unfortunately, definitive diagnosis of this disease can only be done by examination of postmortem brain tissue. Presumptive diagnosis is done through a combination of clinical manifestations, radiology results, and cerebrospinal fluid (CSF) testing for CSF 14-3-3. Even with these guidelines, premortem diagnosis of sCJD can be unreliable with high rates of misdiagnosis. This calls for more reliable biomarkers of the disease, allowing for better diagnosis as well as understanding the pathogenesis of sCJD. This review compiles potential genetic, protein, biomolecular, and imaging biomarker studies for sCJD since 2010, highlighting the promise of proteins, cytokines, and composite biomarkers for improving the diagnosis as well as understanding the pathogenesis of this mysterious ailment.

Aptamer-Based Screens of Human Body Fluids for Biomarkers.

In recent years, aptamers have come to replace antibodies in high throughput multiplexed experiments. The aptamer-based biomarker screening technology, which kicked off in 2010, is capable of interrogating thousands of proteins in a very small sample volume. With this new technology, researchers hope to find clinically appropriate biomarkers for a myriad of illnesses by screening human body fluids. In this work, we have reviewed a total of eight studies utilizing aptamer-based biomarker screens of human body fluids, and have highlighted novel protein biomarkers discovered.