scholarly journals Proteomics Profiling to Distinguish DOCK8 Deficiency From Atopic Dermatitis

2021 ◽  
Vol 2 ◽  
Author(s):  
Minnie Jacob ◽  
Afshan Masood ◽  
Zakiya Shinwari ◽  
Mai Abdel Jabbar ◽  
Hamoud Al-Mousa ◽  
...  

Dedicator of cytokinesis 8 deficiency is an autosomal recessive primary immune deficiency disease belonging to the group of hyperimmunoglobulinemia E syndrome (HIES). The clinical phenotype of dedicator of cytokinesis 8 (DOCK8) deficiency, characterized by allergic manifestations, increased infections, and increased IgE levels, overlaps with the clinical presentation of atopic dermatitis (AD). Despite the identification of metabolomics and cytokine biomarkers, distinguishing between the two conditions remains clinically challenging. The present study used a label-free untargeted proteomics approach using liquid-chromatography mass spectrometry with network pathway analysis to identify the differentially regulated serum proteins and the associated metabolic pathways altered between the groups. Serum samples from DOCK8 (n = 10), AD (n = 9) patients and healthy control (Ctrl) groups (n = 5) were analyzed. Based on the proteomics profile, the PLS-DA score plot between the three groups showed a clear group separation and sample clustering (R2 = 0.957, Q2 = 0.732). Significantly differentially abundant proteins (p < 0.05, FC cut off 2) were identified between DOCK8-deficient and AD groups relative to Ctrl (n = 105, and n = 109) and between DOCK8-deficient and AD groups (n = 85). Venn diagram analysis revealed a differential regulation of 24 distinct proteins from among the 85 between DOCK8-deficient and AD groups, including claspin, haptoglobin-related protein, immunoglobulins, complement proteins, fibulin, and others. Receiver-operating characteristic curve (ROC) analysis identified claspin and haptoglobin-related protein, as potential biomarkers with the highest sensitivity and specificity (AUC = 1), capable of distinguishing between patients with DOCK8 deficiency and AD. Network pathway analysis between DOCK8-deficiency and AD groups revealed that the identified proteins centered around the dysregulation of ERK1/2 signaling pathway. Herein, proteomic profiling of DOCK8-deficiency and AD groups was carried out to determine alterations in the proteomic profiles and identify a panel of the potential proteomics biomarker with possible diagnostic applications. Distinguishing between DOCK8-deficiency and AD will help in the early initiation of treatment and preventing complications.

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3887-3887
Author(s):  
Christine F. Skibola

Abstract Background: The aim of this study was to conduct an exploratory serum proteomic profiling study of diffuse large B-cell lymphoma (DLBCL) using mass spectrometry (MS) analysis to identify potential biomarkers that may provide further clues to disease mechanisms. Methods: Serum samples from 57 chemotherapy-naive male DLBCL cases and 30 healthy controls matched by age and BMI from a San Francisco Bay Area case-control study were divided into single-use aliquots and stored at -80°C until proteomic analysis. Serum samples were depleted of the 12 most abundant proteins by filtration on immunoaffinity spin columns prior to MS analysis. Proteomic analysis was performed using a GeLC approach. Specifically, equal amounts of protein from serologic specimens were separated by one-dimensional denaturing gel electrophoresis. Each lane was cut into six equal MW fractions followed by in-gel digestion with trypsin. The resultant peptides were analyzed using nano-liquid chromatography MS. Because MS is more qualitative than quantitative, the most significant and biologically relevant proteins of interest were chosen for further confirmation by ELISA in the "sandwich" configuration to specifically and quantitatively measure protein concentrations. ELISA assays were performed on unprocessed serum samples without depletion of the major serum proteins, providing a direct, one-step measurement, thereby avoiding any artifacts due to uncontrolled parameters common in preprocessing. Statistically significant differentially expressed serum peptides between the cases and controls as detected by MS were further analyzed using Systems biology/Pathway analysis. Results: MS data computed as normalized spectral counts of peptides revealed 1,207 protein IDs with >99% confidence, and 44 statistically significant candidates that were differentially expressed between the DLBCL cases and controls. We confirmed 4 [adiponectin (AdipoQ), extracellular matrix protein 1 (ECM1), CD14, and SerpinA3 (ACT)] of the 6 top candidates chosen for further confirmation by ELISA, which were elevated by 68.8, 62.9, 33.6 and 28.8 %, respectively, in DLBCL sera compared to controls. Adiponectin is an adipocyte-derived cytokine that regulates the metabolism of lipids and glucose. There is accumulating evidence that adiponectin also exhibits pro- and anti-tumorigenic activity, depending on the tumor type. Our study confirms previous reports of significantly elevated adiponectin levels in patients with adult non-Hodgkin lymphoma (NHL) and DLBCL, childhood NHL, and Hodgkin lymphoma compared to controls (PMID: 22547160). CD14 is a myeloid differentiation marker found primarily on monocytes and macrophages. Soluble CD14 levels have been positively associated with AIDS-related NHL risk (PMID: 23169327). ECM1 regulates cell migration, invasion and stem-like properties via induction of EMT progression and cancer stem cell formation. High ECM1 levels have been detected in various epithelial cancers, including invasive breast ductal carcinoma, esophageal squamous carcinoma, and gastric and colorectal cancer. ECM1 also has been associated with invasiveness and poor prognosis in thyroid and breast cancer. ACT is a metastasis-associated protein. Elevated ACT levels in serum have been associated with poor overall survival in acute leukemia (PMID: 24179540). The proteins identified in this study were critically analyzed using pathway analysis tools, and were found to be strongly associated with FcR signaling, and response to kinase activation including MAPK, membrane PTK, and neurotrophin TRK. These activities, and the proteins identified appear to involve IL-6 (Jak1/Stat3), ESR1/ CREB1, RXR1/ PPAR1, NF-kB, and HIF-1 signaling pathways at different stages of disease vs. controls. Based on these data, the predicted downstream effectors include regulation of immune processes that involve acute-phase and inflammatory responses, and regulation of lipid metabolism and transport. Conclusions: Through serum proteomic profiling studies, we have identified 3 novel (ECM1, CD14, and ACT) and 1 previously reported biomarker (adiponectin) of DLBCL that may have potential biological relevance in the pathogenesis or progression of the disease. Prospective epidemiology and clinical studies will be needed to determine whether these markers are involved in disease etiology and their possible prognostic value. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Vol 12 ◽  
Author(s):  
Liam J. O’Neil ◽  
Pingzhao Hu ◽  
Qian Liu ◽  
Md. Mohaiminul Islam ◽  
Victor Spicer ◽  
...  

ObjectivesPatients with Rheumatoid Arthritis (RA) are increasingly achieving stable disease remission, yet the mechanisms that govern ongoing clinical disease and subsequent risk of future flare are not well understood. We sought to identify serum proteomic alterations that dictate clinically important features of stable RA, and couple broad-based proteomics with machine learning to predict future flare.MethodsWe studied baseline serum samples from a cohort of stable RA patients (RETRO, n = 130) in clinical remission (DAS28<2.6) and quantified 1307 serum proteins using the SOMAscan platform. Unsupervised hierarchical clustering and supervised classification were applied to identify proteomic-driven clusters and model biomarkers that were associated with future disease flare after 12 months of follow-up and RA medication withdrawal. Network analysis was used to define pathways that were enriched in proteomic datasets.ResultsWe defined 4 proteomic clusters, with one cluster (Cluster 4) displaying a lower mean DAS28 score (p = 0.03), with DAS28 associating with humoral immune responses and complement activation. Clustering did not clearly predict future risk of flare, however an XGboost machine learning algorithm classified patients who relapsed with an AUC (area under the receiver operating characteristic curve) of 0.80 using only baseline serum proteomics.ConclusionsThe serum proteome provides a rich dataset to understand stable RA and its clinical heterogeneity. Combining proteomics and machine learning may enable prediction of future RA disease flare in patients with RA who aim to withdrawal therapy.


2001 ◽  
Vol 47 (1) ◽  
pp. 110-117 ◽  
Author(s):  
Magnus Jonsson ◽  
Joyce Carlson ◽  
Jan-Olof Jeppsson ◽  
Per Simonsson

Abstract Background: Electrophoresis of serum samples allows detection of monoclonal gammopathies indicative of multiple myeloma, Waldenström macroglobulinemia, monoclonal gammopathy of undetermined significance, and amyloidosis. Present methods of high-resolution agarose gel electrophoresis (HRAGE) and immunofixation electrophoresis (IFE) are manual and labor-intensive. Capillary zone electrophoresis (CZE) allows rapid automated protein separation and produces digital absorbance data, appropriate as input for a computerized decision support system. Methods: Using the Beckman Paragon CZE 2000 instrument, we analyzed 711 routine clinical samples, including 95 monoclonal components (MCs) and 9 cases of Bence Jones myeloma, in both the CZE and HRAGE systems. Mathematical algorithms developed for the detection of monoclonal immunoglobulins (MCs) in the γ- and β-regions of the electropherogram were tested on the entire material. Additional algorithms evaluating oligoclonality and polyclonal concentrations of immunoglobulins were also tested. Results: CZE electropherograms corresponded well with HRAGE. Only one IgG MC of 1 g/L, visible on HRAGE, was not visible after CZE. Algorithms detected 94 of 95 MCs (98.9%) and 100% of those visible after CZE. Of 607 samples lacking an MC on HRAGE, only 3 were identified by the algorithms (specificity, 99%). Algorithms evaluating total gammaglobulinemia and oligoclonality also identified several cases of Bence Jones myeloma. Conclusions: The use of capillary electrophoresis provides a modern, rapid, and cost-effective method of analyzing serum proteins. The additional option of computerized decision support, which provides rapid and standardized interpretations, should increase the clinical availability and usefulness of protein analyses in the future.


2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Chi Zhang ◽  
Li Li ◽  
Shiping Cheng ◽  
Debajyoti Chowdhury ◽  
Yong Tan ◽  
...  

Abstract Background Hypertension (HTN) patients who have phlegm-dampness syndrome (PDS) tend to be obese and have worse outcomes. However, the association of body weight (BW) changes and mechanisms underlying the pathophysiology of HTN-PDS are not well elucidated. This study aims to identify the longitudinal observations associated with the circulating markers discriminating BW changes of individuals with HTN-PDS. Methods An integrative approach relying on metabolomics and proteomics was applied to serum samples from HTN-PDS patients in a prospective cohort to identify the plausible mechanistic pathways underpinning HTN-PDS pathophysiology. Study participants were determined to have experienced a weight change if they showed a 5%–15% increase/reduction in BW at the end of the follow-up period. The joint pathway analysis and network analysis were performed using Ingenuity Pathway Analysis (IPA®) on the serum samples obtained from the participants over the period. Results The study involved 22 HTN-PDS patients who were overweight initially and were able to lose enough weight and 24 HTN-PDS individuals who developed overweight from normal BMI during a one-year follow-up. Our analysis suggested three types of phosphatidylcholine (PC) were altered. PC (22:2(13Z,16Z)/24:1(15Z)) and LysoPC (16:1(9Z)) were decreased in Queryweight gain samples, whereas the levels of PC (14:0/16:0) were increased in weight loss samples. The metabolomic analysis suggested 24 metabolites associated with HTN-PDS. Of them, 13 were up-regulated and 11 were down-regulated. The two-dimensional difference gel electrophoresis (2D DIGE) identified 45 phosphorylated proteins got altered in the HTN-PDS patients, wherein 23 were up-regulated and 22 were down-regulated. Integrated proteomic and metabolomics analyse acknowledged biomarkers PC, Complement C3, C4a/C4b, A2M and SERPINF1 as strong predictors for BW changes in HTN-PDS patients. Conclusion The combined serum proteomic and metabolomic profiling reveals a link between BW change and the complement system activity, altered phosphatidylcholine metabolism in HTN-PDS patients. Future studies with larger cohorts are required to strengthen and validate these findings.


2021 ◽  
Author(s):  
Seiichiro Abe ◽  
Juntaro Matsuzaki ◽  
Kazuki Sudo ◽  
Ichiro Oda ◽  
Hitoshi Katai ◽  
...  

Abstract Background The aim of this study was to identify serum miRNAs that discriminate early gastric cancer (EGC) samples from non-cancer controls using a large cohort. Methods This retrospective case–control study included 1417 serum samples from patients with EGC (seen at the National Cancer Center Hospital in Tokyo between 2008 and 2012) and 1417 age- and gender-matched non-cancer controls. The samples were randomly assigned to discovery and validation sets and the miRNA expression profiles of whole serum samples were comprehensively evaluated using a highly sensitive DNA chip (3D-Gene®) designed to detect 2565 miRNA sequences. Diagnostic models were constructed using the levels of several miRNAs in the discovery set, and the diagnostic performance of the model was evaluated in the validation set. Results The discovery set consisted of 708 samples from EGC patients and 709 samples from non-cancer controls, and the validation set consisted of 709 samples from EGC patients and 708 samples from non-cancer controls. The diagnostic EGC index was constructed using four miRNAs (miR-4257, miR-6785-5p, miR-187-5p, and miR-5739). In the discovery set, a receiver operating characteristic curve analysis of the EGC index revealed that the area under the curve (AUC) was 0.996 with a sensitivity of 0.983 and a specificity of 0.977. In the validation set, the AUC for the EGC index was 0.998 with a sensitivity of 0.996 and a specificity of 0.953. Conclusions A novel combination of four serum miRNAs could be a useful non-invasive diagnostic biomarker to detect EGC with high accuracy. A multicenter prospective study is ongoing to confirm the present observations.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Barbara Lisowska-Myjak ◽  
Agnieszka Strawa ◽  
Hanna Zborowska ◽  
Artur Jakimiuk ◽  
Ewa Skarżyńska

AbstractEstablishing any characteristic associations between the serum parameters of thyroid function and serum proteins in pregnancy may aid in elucidating the role of the thyroid gland in the regulation of pregnancy-specific metabolic processes and in selecting candidate biomarkers for use in their clinical assessment. Concentrations of thyroid stimulating hormone (TSH), free tri-iodothyronine (fT3) and free thyroxine (fT4), six electrophoretically separated protein fractions (albumin, alpha-1-, alpha2-, beta-1-, beta-2- and gamma-globulins), representative proteins—albumin (ALB), transferrin (TRF), alpha-2-macroglobulin (AMG) and ceruloplasmin (CER) were measured in 136 serum samples from 65 women in their consecutive trimesters of pregnancy. The concentrations of TSH, fT4 and fT3 were significantly correlated (p < 0.05) with the concentrations of the albumin, alpha-2- and beta-1 globulin fractions. Significant correlations (p < 0.05) which were positive between fT4 and ALB and negative between fT4 and TRF were established throughout pregnancy. Significant negative correlations (p < 0.05) were demonstrated for fT3 with alpha-2-globulin, AMG and CER. Changes in the serum concentrations of thyroid hormones seen between the trimesters were found to correlate with the concentrations of high-abundance serum proteins. Opposite directions of correlations between fT4 and ALB and fT4 and TRF observed throughout pregnancy may indicate the shared biological role of these parameters in maintaining maternal homeostasis and they suggest their potential use in the clinic as a simple biomarker panel. A negative correlation of fT3 with CER in the second trimester possibly reflects their involvement in the active regulation of metabolic processes.


2020 ◽  
Vol 47 (12) ◽  
pp. 1760-1767
Author(s):  
Sarah M. Wade ◽  
Trudy McGarry ◽  
Siobhan C. Wade ◽  
Ursula Fearon ◽  
Douglas J. Veale

ObjectiveMicroRNA (miRNA) are small endogenous regulatory RNA molecules that have emerged as potential therapeutic targets and biomarkers in autoimmunity. Here, we investigated serum miRNA levels in patients with psoriatic arthritis (PsA) and further assessed a serum miRNA signature in therapeutic responder versus nonresponder PsA patients.MethodsSerum samples were collected from healthy controls (HC; n = 20) and PsA patients (n = 31), and clinical demographics were obtained. To examine circulatory miRNA in serum from HC and PsA patients, a focused immunology miRNA panel was analyzed utilizing a miRNA Fireplex assay (FirePlex Bioworks Inc.). MiRNA expression was further assessed in responders versus nonresponders according to the European League Against Rheumatism response criteria.ResultsSix miRNA (miR-221-3p, miR-130a-3p, miR-146a-5p, miR-151-5p, miR-26a-5p, and miR-21-5p) were significantly higher in PsA compared to HC (all P < 0.05), with high specificity and sensitivity determined by receiver-operating characteristic curve analysis. Analysis of responder versus nonresponders demonstrated higher baseline levels of miR-221-3p, miR-130a-3p, miR-146a-5p, miR-151-5p, and miR-26a-5p were associated with therapeutic response.ConclusionThis study identified a 6-serum microRNA signature that could be attractive candidates as noninvasive markers for PsA and may help to elucidate the disease pathogenesis.


Author(s):  
Lijuan Wang ◽  
Hong Lv ◽  
Guojun Zhang

Background The study aimed to evaluate a fully automated chemiluminescent immunoassay and compared it with a quantitative RNA assay and anti-HCV assay to verify the utility of this automated Ag assay as an alternative method for hepatitis C diagnosis. Methods A total of 229 serum samples previously tested for anti-HCV concentrations by the Architect Anti-HCV assay, were selected for HCV RNA testing by real time RT-PCR kit (Shanghai ZJ Bio-Tec Co., Ltd) and 125 specimens were tested for HCV Ag by the Architect HCV core Antigen kit. Results The log10 HCVAg and HCV RNA concentrations were highly correlated [ r = 0.834); with HCV RNA as the comparator test, HCVAg had 100% specificity, 100% positive predictive value (PPV) and 94.8% sensitivity. We found 1 pg/mL of total HCV core Ag is equivalent to approximately 6607HCV RNA international units (IU)/mL. Receiver operator characteristic curve analysis showed that the area under the curve of HCV core Ag (0.989) was greater than HCV Ab (0.871). HCV Ag concentrations and RNA-to-Ag ratio of the groups for HCV RNA concentrations ≤105 and >105 IU/mL were both significantly different from each other ( P < 0.05). Conclusion The Architect HCV core Ag assay may be an alternative method for hepatitis C diagnosis, performed on the same analytical platform and sample as the anti-HCV assay, shortening the diagnostic window period, demonstrating good correlation with HCV RNA assay with high specificity and positive predictive value.


Author(s):  
M A Jenkins ◽  
M D Guerin

Capillary electrophoresis is a technique that can be automated for the separation of charged particles. By investigating suitable sample dilution and injection time and adhering to a strict washing procedure we have been able to quantify paraproteins in serum samples. This has enabled us to use the technique of capillary electrophoresis for the provision of serum protein electrophoresis in a routine clinical laboratory. We present our findings of 260 serum samples, which included 76 samples with paraproteins analysed by both capillary electrophoresis (EC) and high resolution agarose gel electrophoresis (HRAGE). CE was able to detect all the monoclonal bands detected by HRAGE, and, in particular, better able to detect IgA monoclonal bands occurring in the beta region. The major advantages of CE over HRAGE relate to the automated nature of CE with the elimination of the need for a densitometer.


1992 ◽  
Vol 8 (6) ◽  
pp. 407-413 ◽  
Author(s):  
Adam B. Czuppon ◽  
Boleslaw Marczynski ◽  
Xaver Baur

Serum samples of 10 workers undergoing occupational type inhalative challenge tests by toluene diisocyanate (TDI) were investigated by anion-exchange fast-protein-liquid-chromatography (FPLC) and polyacrylamide-gel electrophoresis (PAGE-SDS). Their serum chromatography profiles were compared to those of 20 unexposed individuals. The peak height of the first prealbumin peak in sera of workers after inhalative challenge tests was significantly different (p > 0, 01 Chi-square test) compared to that obtained before exposure and to that of unexposed subjects. In addition, qualitative changes of these peaks were also noted in sera of workers exposed to TDI. In the cases of exposed individuals, that peak was more diffuse with some shoulders and less symmetric in appearance. Similarly, PAGE-SDS of the serum proteins, followed by silver nitrate staining, revealed a different banding pattern after in vivo TDI exposure. One of the serum components at approximately 15 kD showed an increase of staining intensity after exposure (n = 10), compared to unexposed subjects or to patients before exposure. This serum fraction has not yet been identified. The results here demonstrate that it is possible to detect changes of serum proteins in TDI-exposed individuals within a relatively short analysis time. This could be useful for biological monitoring of exposure, since no method for such is yet available.


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