Identification of universal biomarkers to predict systemic lupus erythematosus (SLE) flares is challenging due to the heterogeneity of the disease. Several biomarkers have been reported. However, the ...data of validated biomarkers to use as a predictor for lupus flares show variation. This study aimed to identify the biomarkers that are sensitive and specific to predict lupus flares.
One hundred and twenty-four SLE patients enrolled in this study and were prospectively followed up. The evaluation of disease activity achieved by the SLE disease activity index (SLEDAI-2K) and clinical SLEDAI (modified SLEDAI). Patients with active SLE were categorized into renal or non-renal flares. Serum cytokines were measured by multiplex bead-based flow cytometry. The correlation and logistic regression analysis were performed.
Levels of IFN-α, MCP-1, IL-6, IL-8, and IL-18 significantly increased in active SLE and correlated with clinical SLEDAI. Complement C3 showed a weakly negative relationship with IFN-α and IL-18. IL-18 showed the highest positive likelihood ratios for active SLE. Multiple logistic regression analysis showed that IL-6, IL-8, and IL-18 significantly increased odds ratio (OR) for active SLE at baseline while complement C3 and IL-18 increased OR for active SLE at 12 weeks. IL-18 and IL-6 yielded higher sensitivity and specificity than anti-dsDNA and C3 to predict active renal and active non-renal, respectively.
The heterogeneity of SLE pathogenesis leads to different signaling mechanisms and mediates through several cytokines. The monitoring of cytokines increases the sensitivity and specificity to determine SLE disease activity. IL-18 predicts the risk of active renal SLE while IL-6 and IL-8 predict the risk of active non-renal. The sensitivity and specificity of these cytokines are higher than the anti-dsDNA or C3. We propose to use the serum level of IL-18, IL-6, and IL-8 to monitor SLE disease activity in clinical practice.
Discovery of urinary biomarkers Pisitkun, Trairak; Johnstone, Rose; Knepper, Mark A
Molecular & cellular proteomics
5, Številka:
10
Journal Article
Recenzirano
Odprti dostop
A myriad of proteins and peptides can be identified in normal human urine. These are derived from a variety of sources including glomerular filtration of blood plasma, cell sloughing, apoptosis, ...proteolytic cleavage of cell surface glycosylphosphatidylinositol-linked proteins, and secretion of exosomes by epithelial cells. Mass spectrometry-based approaches to urinary protein and peptide profiling can, in principle, reveal changes in excretion rates of specific proteins/peptides that can have predictive value in the clinical arena, e.g. in the early diagnosis of disease, in classification of disease with regard to likely therapeutic responses, in assessment of prognosis, and in monitoring response to therapy. These approaches have potential value, not only in diseases of the kidney and urinary tract but also in systemic diseases that are associated with circulating small protein and peptide markers that can pass the glomerular filter. Most large scale biomarker discovery studies reported thus far have used one of two approaches to identify proteins and peptides whose excretion in urine changes in specific disease states: 1) two-dimensional electrophoresis with mass spectrometric and/or immunochemical identification of proteins and 2) top-down mass spectrometric methods (SELDI-TOF-MS and capillary electrophoresis-MS). These studies have been chiefly in the areas of nephrology, urology, and oncology. We review these applications, focusing on two areas of progress, viz. in bladder cancer and in acute rejection of renal transplants. Progress has been limited so far. However, with the advent of powerful LC-MS/MS methods along with methods for quantifying LC-MS/MS output, there is hope for an accelerated discovery and validation of disease biomarkers in urine.
Abstract The complexity of systemic lupus erythematosus (SLE) arises from intricate genetic and environmental interactions, with STING playing a pivotal role. This study aims to comprehend the ...function of STING using the pristane-induced lupus (PIL) model in Sting missense mutant mice ( Goldenticket or Sting Gt ), which contrasts with previous research using Sting knockout mice. Investigating two-month-old Sting Gt mice over six months post-PIL induction, we observed a profound reduction in autoimmune markers, including antinuclear and anti-dsDNA antibodies, germinal center B cells, and plasma cells, compared to their wild-type counterparts. A pivotal finding was the marked decrease in IL-17-producing T cells. Notably, the severity of lupus nephritis and pulmonary hemorrhages was significantly diminished in Sting Gt mice. These findings demonstrate that different genetic approaches to interfere with STING signaling can lead to contrasting outcomes in SLE pathogenesis, which highlights the need for a nuanced understanding of the role of STING in drug development for SLE. In summary, the loss of Sting function in Goldenticket mutant mice rescued autoimmune phenotypes in PIL. This study reveals the critical nature of STING in SLE, suggesting that the method of STING modulation significantly influences disease phenotypes and should be a key consideration in developing targeted therapies.
Aquaporin-2 (AQP2) is regulated in part via vasopressin-mediated changes in protein half-life that are in turn dependent on AQP2 ubiquitination. Here we addressed the question, "What E3 ubiquitin ...ligase is most likely to be responsible for AQP2 ubiquitination?" using large-scale data integration based on Bayes' rule. The first step was to bioinformatically identify all E3 ligase genes coded by the human genome. The 377 E3 ubiquitin ligases identified in the human genome, consisting predominant of HECT, RING, and U-box proteins, have been used to create a publically accessible and downloadable online database (https://hpcwebapps.cit.nih.gov/ESBL/Database/E3-ligases/). We also curated a second database of E3 ligase accessory proteins that included BTB domain proteins, cullins, SOCS-box proteins, and F-box proteins. Using Bayes' theorem to integrate information from multiple large-scale proteomic and transcriptomic datasets, we ranked these 377 E3 ligases with respect to their probability of interaction with AQP2. Application of Bayes' rule identified the E3 ligases most likely to interact with AQP2 as (in order of probability): NEDD4 and NEDD4L (tied for first), AMFR, STUB1, ITCH, ZFPL1. Significantly, the two E3 ligases tied for top rank have also been studied extensively in the reductionist literature as regulatory proteins in renal tubule epithelia. The concordance of conclusions from reductionist and systems-level data provides strong motivation for further studies of the roles of NEDD4 and NEDD4L in the regulation of AQP2 protein turnover.
The absence of stimulator of interferon genes (STING) in 129.B6.Fcgr2b-deficient mice rescue lupus phenotypes. The administration of a STING inhibitor (ISD017) into the young 129.B6.Fcgr2b-deficient ...mice prevents lupus nephritis development. This study mainly aimed to evaluate the effects of STING inhibition (ISD107) on established SLE in mice to prove that ISD017 could be a good therapeutic drug to reverse the already set-up autoimmunity and kidney impairment. Twenty-four-week-old Fcgr2b-deficient mice were treated with cyclophosphamide (25 mg/kg, intraperitoneal, once per week), ISD017 (10 mg/kg, intraperitoneal, three times per week), or control vehicle for 8 weeks, and were analyzed for phenotypes. Both ISD017 and cyclophosphamide treatment increased long-term survival and reduced the severity of glomerulonephritis in Fcgr2b-deficient mice. While cyclophosphamide reduced activated B cells (B220
GL-7
), ISD017 decreased activated T cells (CD4
CD69
) and neutrophils (Ly6c
Ly6g
) in Fcgr2b-deficient mice. In addition, ISD017 reduced IL-1β and interferon-inducible genes. In summary, ISD017 treatment in symptomatic 129.B6.Fcgr2b-deficient mice reduced the severity of glomerulonephritis and increased long-term survival. ISD017 worked comparably to cyclophosphamide for treating lupus nephritis in 129.B6.Fcgr2b-deficient mice. ISD017 reduced activated T cells and neutrophils, while cyclophosphamide targeted activated B cells. These results suggested that STING inhibitors can potentially be a new therapeutic drug for treating lupus.
Background
When severe, COVID-19 shares many clinical features with bacterial sepsis. Yet, secondary bacterial infection is uncommon. However, as epithelium is injured and barrier function is lost, ...bacterial products entering the circulation might contribute to the pathophysiology of COVID-19.
Methods
We studied 19 adults, severely ill patients with COVID-19 infection, who were admitted to King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between 13th March and 17th April 2020. Blood samples on days 1, 3, and 7 of enrollment were analyzed for endotoxin activity assay (EAA), (1 → 3)-β-
d
-glucan (BG), and 16S rRNA gene sequencing to determine the circulating bacteriome.
Results
Of the 19 patients, 13 were in intensive care and 10 patients received mechanical ventilation. We found 8 patients with high EAA (≥ 0.6) and about half of the patients had high serum BG levels which tended to be higher in later in the illness. Although only 1 patient had a positive blood culture, 18 of 19 patients were positive for 16S rRNA gene amplification. Proteobacteria was the most abundant phylum. The diversity of bacterial genera was decreased overtime.
Conclusions
Bacterial DNA and toxins were discovered in virtually all severely ill COVID-19 pneumonia patients. This raises a previously unrecognized concern for significant contribution of bacterial products in the pathogenesis of this disease.
Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery ...how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs). Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.
Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing ...the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.
Abstract
Bacterial extracellular vesicles (EVs) are generally formed by pinching off outer membrane leaflets while simultaneously releasing multiple active molecules into the external environment. In ...this study, we aimed to identify the protein cargo of leptospiral EVs released from intact leptospires grown under three different conditions: EMJH medium at 30 °C, temperature shifted to 37 °C, and physiologic osmolarity (EMJH medium with 120 mM NaCl). The naturally released EVs observed under transmission electron microscopy were spherical in shape with an approximate diameter of 80–100 nm. Quantitative proteomics and bioinformatic analysis indicated that the EVs were formed primarily from the outer membrane and the cytoplasm. The main functional COG categories of proteins carried in leptospiral EVs might be involved in cell growth, survival and adaptation, and pathogenicity. Relative to their abundance in EVs grown in EMJH medium at 30 °C, 39 and 69 proteins exhibited significant changes in response to the temperature shift and the osmotic change, respectively. During exposure to both stresses,
Leptospira
secreted several multifunctional proteins via EVs, while preserving certain virulence proteins within whole cells. Therefore, leptospiral EVs may serve as a decoy structure for host responses, whereas some virulence factors necessary for direct interaction with the host environment are reserved in leptospiral cells. This knowledge will be useful for understanding the pathogenesis of leptospirosis and developing as one of vaccine platforms against leptospirosis in the future.
Cholangiocarcinoma (CCA), a lethal malignancy of the biliary epithelium, is the second most common primary liver cancer. The poor prognosis of CCA is due to the high rate of tumour invasion and ...distant metastasis. We found that the RNA-binding protein LIN28B, a known regulator of microRNA biogenesis, stem cell maintenance, and oncogenesis, is expressed in a subpopulation of CCA patients. To further investigate the potential role of LIN28B in CCA pathogenesis, we studied the effect of LIN28B overexpression in the cholangiocyte cell line MMNK-1 and cholangiocarcinoma cell lines HuCCT-1 and KKU-214. Here, we show that enhanced LIN28B expression promoted cancer stem cell-like properties in CCA, including enhanced cell migration, epithelial-to-mesenchymal transition (EMT), increased cell proliferation and spheroid formation. Proteomic analysis revealed TGF-β-induced protein (TGFBI) as a novel LIN28B target gene, and further analysis showed upregulation of other components of the TGF-β signalling pathway, including TGF-β receptor type I (TGFBRI) expression and cytokine TGFB-I, II and III secretion. Importantly, the small molecule TGF-β inhibitor SB431542 negated the effects of LIN28B on both cell migration and clonogenic potential. Overexpression of TGFBI alone promoted cholangiocarcinoma cell migration and EMT changes, but not spheroid formation, suggesting that TGFBI partially contributes to LIN28B-mediated aggressive cell behaviour. These observations are consistent with a model in which TGF-β and LIN28B work together to form a positive feedback loop during cholangiocarcinoma metastasis and provide a therapeutic intervention opportunity.
PDF
