Serum Of Healthy Volunteers Research Articles

Evaluation of the Concentration of Selected Elements in the Serum of Patients with Degenerative Stenosis of the Lumbosacral Spine.

In humans, 23 elements have been shown to have biological activity. The purpose of this study was to evaluate the concentrations of iron (Fe), zinc (Zn), sodium (Na), potassium (K), magnesium (Mg), phosphorus (P), and calcium (Ca) in the serum of patients diagnosed with lumbar degenerative stenosis when compared to the concentrations of those elements in the serum of healthy volunteers. The study group consisted of 60 patients who were diagnosed with degenerative stenosis of the lumbosacral spine and who qualified for hemilaminectomy. The control group included 60 healthy volunteers without degenerative spinal stenosis. The clinical specimens studied had sera collected from both groups. The quantitative analysis of the selected elements revealed statistically significant (p < 0.05) lower concentrations of Zn (740 ± 110 µg/L vs. 880 ± 160 µg/L) and Mg (22,091 ± 4256 µg/L vs. 24,100 ± 4210 µg/L) in the serum of the patients from the study group when compared to the controls. By contrast, K (16,230 µg/L ± 1210 µg/L vs. 13,210 µg/L ± 1060 µg/L) and Fe (141.87 µg/L ± 11.22 µg/L vs. 109.1 µg/L ± 26.43 µg/L) levels were significantly higher in the study group compared to the controls (p < 0.05). No statistically significant changes were detected in the concentrations of the assessed micronutrients and macronutrients in both sexes in either the study group, the control group, or those based on body mass index (p > 0.05). In the serum samples from the study group, the strongest correlations were noted between the concentrations. In the study group, we showed a significant relationship between the levels of Fe/Zn (r = 0.41), Fe/Na (r = 0.41), Fe/P (r = 0.55), Zn/P (r = 0.68), Zn/K (r = 0.48), Zn/Ca (r = 0.94), Mg/Ca (r = 0.79), and Na/K (r = 0.67). We showed that only Mg concentration varied statistically significantly with the severity of pain (p < 0.05). These findings suggest that the assessment of Fe, Zn, Mg, and K concentrations can be helpful in predicting the onset of degenerative changes in the spine.

An innovative and universal dual-signal ratiometric spectro-electrochemical imprinted sensor design for sandwich type detection of anticancer-drug, gemcitabine, in serum samples; cross validation via computational modeling

An innovative and universal imprinted sensor design for sandwich type detection of gemcitabine (GMT) in human serum samples is described. GMT is widely used in the treatment of different tumors, such as lung, ovarian, pancreatic, and breast cancer. The serum albumin-drug interaction was translated to design a multifunctional, ratiometric and dual mode silver nanoparticle based probe (BSA-Ag nanoprobe), as a read out system. Subsequently, polypyrrol imprinted drug receptor sites was engineered to selectively capture the GMT on the transducer surface. The GMT was sandwiched between imprinted receptor surface and BSA-Ag nanoprobe to generate the spectro-electrochemical signals. The formation of nanoprobe was confirmed through various characterization techniques, including X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, micro-Raman spectroscopy, Dynamic light scattering (DLS), and UV–Visible (UV–Vis) analysis, while each step of sensor fabrication was characterized via field emission scanning electron microscope (FE-SEM), Static water Contact angle measurements, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Different variable parameters were optimized to improve the analytical performance of the sensor design. Under optimal conditions, spectro-electrochemical sensor permitted linear ranges between 1 and 200 μmol L−1 and 0.5–200 μmol L−1, with limits of detection (LOD) of 0.4 μmol L−1 and 0.15 μmol L−1 respectively. Furthermore, the designed sensor successfully differentiated the serum samples of lung cancer patients and healthy volunteers. The obtained results were validated with standard Liquid chromatography-mass spectrometry (LC/MS) analysis of the patients and healthy volunteer's serum samples. Lastly, density functional theory (DFT) and molecular docking calculations revealed the enhanced GMT binding capability of molecularly imprinted polypyrrole and molecular level interaction between the GMT and BSA, to validate the sandwich sensor design.

MP20-07 NPTX1 IS A NOVEL SERUM BIOMARKER INDUCED BY ARPI TREATED PROSTATE CANCER AND ENRICHED IN LINEAGE PLASTICITY WITH BRN2 HIGH

You have accessJournal of UrologyCME1 Apr 2023MP20-07 NPTX1 IS A NOVEL SERUM BIOMARKER INDUCED BY ARPI TREATED PROSTATE CANCER AND ENRICHED IN LINEAGE PLASTICITY WITH BRN2 HIGH Takeshi Namekawa, Joshua Scurll, Maxim Kobelev, Dwaipayan Ganguli, Olena Sivak, Martin Gleave, Vancouver, Canada, Eva Corey, Colm Morrissey, and Amina Zoubeidi Takeshi NamekawaTakeshi Namekawa More articles by this author , Joshua ScurllJoshua Scurll More articles by this author , Maxim KobelevMaxim Kobelev More articles by this author , Dwaipayan GanguliDwaipayan Ganguli More articles by this author , Olena SivakOlena Sivak More articles by this author , Martin GleaveMartin Gleave More articles by this author , Vancouver Vancouver More articles by this author , Canada Canada More articles by this author , Eva CoreyEva Corey More articles by this author , Colm MorrisseyColm Morrissey More articles by this author , and Amina ZoubeidiAmina Zoubeidi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen receptor (AR) pathway inhibition is the first-line therapy for metastatic prostate cancer (PCa). However, tumors invariably develop resistance to ARPI. We previously identified a transcription factor Brain-2 (BRN2), encoded by the gene POU3F2, as a master driver of NEPC which is an aggressive form of AR-independent phenotypes treatment resistant PCa. In this study, we identified a surrogate biomarker of BRN2 activity to monitor the progression of treatment resistant PCa. METHODS: To identify BRN2 serum marker, we analyzed BRN2 ChIPseq data from 2 NEPC and 2 neural-type lung squamous cell carcinoma (nLUSC) cell lines with a focus on shared BRN2 peaks. Annotated peaks from enhancers (EnhancerAtlas) and promoters (genes within 20kb) were selected for secreted proteins and were ranked by correlation with BRN2 expression in PCa patients. Validation of protein expression was performed by western blot. R-PLEX Assay kit was used to measure concentrations in culture media from NCI-H660 cells and in serum from mice with NCI-H660 xenograft or PDX tumors and from human PCa patients. RESULTS: We identified 51 candidate serum biomarkers for BRN2 activity (or expression?) using the methods described above. These biomarkers were ranked based on their expression and correlation with BRN2 expression in PCa patient samples across four public data sets. NPTX1 was identified as the top biomarker of BRN2 expression. We further validated association of NPTX1 and BRN2 expression by BRN2 overexpression in MR42D and 22Rv1, indicating that BRN2 positively regulates NPTX1 expression. Next, we showed that NPTX1 is present in culture media from NCI-H660 cells, and that NPTX1 serum concentration positively correlated with tumor volume in mice with NCI-H660 xenografts and with NPTX1 mRNA expression in LuCaP PDX models. Importantly, evaluation of patients’ samples showed that NPTX1 levels were high in 26/35 CRPC serum samples vs levels in serum of healthy volunteers, and that the NPTX1 serum levels were highest in 4/6 patients with high POU3F2 tumor expression. CONCLUSIONS: We identified and validated NPTX1 as a serum marker that can be readily detected in serum from NEPC patients with BRN2high tumours. Source of Funding: U.S. Army Medical Research and Development Command(W81XWH1810689) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e277 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeshi Namekawa More articles by this author Joshua Scurll More articles by this author Maxim Kobelev More articles by this author Dwaipayan Ganguli More articles by this author Olena Sivak More articles by this author Martin Gleave More articles by this author Vancouver More articles by this author Canada More articles by this author Eva Corey More articles by this author Colm Morrissey More articles by this author Amina Zoubeidi More articles by this author Expand All Advertisement PDF downloadLoading ...

Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, wedescribe our FT4 candidate cRMP and its validation in clinical samples. This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera. It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers. Our candidate cRMP measures FT4 accurately and performs well in serum matrix.

Tigecycline Immunodetection Using Developed Group-Specific and Selective Antibodies for Drug Monitoring Purposes.

Tigecycline (TGC), a third-generation tetracycline, is characterized by a more potent and broad antibacterial activity, and the ability to overcome different mechanisms of tetracycline resistance. TGC has proven to be of value in treatment of multidrug-resistant infections, but therapy can be complicated by multiple dangerous side effects, including direct drug toxicity. Given that, a TGC immunodetection method has been developed for therapeutic drug monitoring to improve the safety and efficacy of therapy. The developed indirect competitive ELISA utilized TGC selective antibodies and group-specific antibodies interacting with selected coating TGC conjugates. Both assay systems showed high sensitivity (IC50) of 0.23 and 1.59 ng/mL, and LOD of 0.02 and 0.05 ng/mL, respectively. Satisfactory TGC recovery from the spiked blood serum of healthy volunteers was obtained in both assays and laid in the range of 81-102%. TGC concentrations measured in sera from COVID-19 patients with secondary bacterial infections were mutually confirmed by ELISA based on the other antibody-antigen interaction and showed good agreement (R2 = 0.966). A TGC pharmacokinetic (PK) study conducted in three critically ill patients proved the suitability of the test to analyze the therapeutic concentrations of TGC. Significant inter-individual PK variability revealed in this limited group supports therapeutic monitoring of TGC in individual patients and application of the test for population pharmacokinetic modelling.

Evaluation of the concentration of selected elements in serum patients with intervertebral disc degeneration

Quantitative analysis of the trace element content of human intervertebral discs (IVDs) is essential because it can identify specific enzymes or metabolites that may be related to human intervertebral disc degeneration (IVDD). The goal of this study was to assess the concentrations of copper (Cu), iron (Fe), manganese (Mn), lead (Pb), zinc (Zn), sodium (Na), magnesium (Mg), potassium (K), phosphorus (P), and calcium (Ca) in serum samples obtained from patients with IVDD in comparison to healthy volunteers (a control group). The study group consisted of 113 Caucasian patients qualified by a specialist neurosurgeon for microdiscectomy. The control group consisted of 113 healthy volunteers who met the eligibility criteria for blood donors. The examined clinical material was the serum samples obtained from both groups.Based on the quantitative analysis of selected elements, there were statistically significantly (p 0.05) higher concentrations of Cu (1180 μg/L±800 μg/L vs. 1230 μg/L±750 μg/L), Zn (790 μg/L±300 μg/L vs. 850 μg/L±200 μg/L), and Mg (21730 μg/L±4360 μg/L vs. 23820 μg/L±4990 μg/L) in the serum of healthy volunteers compared to those in the study group. In addition, statistically significant changes were not detected in the concentrations of any elements among either sex in either the study or control group or in their body mass index (BMI) values (p > 0.05). In the serum samples from the study group, the strongest relationships were noted between the concentrations of Zn and Pb (r = 0.61), Zn and P (r = 0.69), Zn and Ca (r = 0.84), Zn and Cu (r = 0.83), Mg and Ca (r = 0.74), and Ca and P (r = 0.98).It has been indicated that, above all, the concentrations of Cu, Zn, Ca, and Mg depend on the advancement of radiological changes, according to the Pfirrmann scale. However, no influence on pain intensity was found, depending on the concentration of the assessed elements.The analysis indicates that the determination of serum Cu, Zn, Ca, and Mg concentrations may have diagnostic significance in predicting the onset of lumbosacral IVDD. The predictive evaluation of changes in the concentrations of selected elements in patients with degenerative lumbar IVD lesions appears to be a promising, cost-effective strategy.

Kinetics of free and ligand-bound atacicept in human serum.

BAFF (B cell activation factor of the TNF family/B lymphocyte stimulator, BLyS) and APRIL (a proliferation-inducing ligand) are targeted by atacicept, a decoy receptor consisting of the extracellular domain of TACI (transmembrane activator and calcium-modulator and cyclophilin (CAML) interactor) fused to the Fc portion of human IgG1. The purpose of the study was to characterize free and ligand-bound atacicept in humans. Total and active atacicept in serum of healthy volunteers receiving a single dose of subcutaneous atacicept or in patients treated weekly for one year were measured by ELISA, Western blot, or cell-based assays. Pharmacokinetics of free and bound atacicept were predicted based on total atacicept ELISA results. Persistence of complexes of purified atacicept bound to recombinant ligands was also monitored in mice. Results show that unbound or active atacicept in human serum exceeded 0.1 µg/ml for one week post administration, or throughout a 1-year treatment with weekly administrations. After a single administration of atacicept, endogenous BAFF bound to atacicept was detected after 8h then increased about 100-fold within 2 to 4 weeks. Endogenous heteromers of BAFF and APRIL bound to atacicept also accumulated, but atacicept-APRIL complexes were not detected. In mice receiving intravenous injections of purified complexes pre-formed in vitro, atacicept-BAFF persisted longer (more than a week) than atacicept-APRIL (less than a day). Thus, only biologically inactive BAFF and BAFF-APRIL heteromers accumulate on atacicept in vivo. The measure of active atacicept provides further support for the once-weekly dosing regimen implemented in the clinical development of atacicept.

Evaluation of miR-429 as a novel serum biomarker for pancreatic ductal adenocarcinoma and analysis its tumor suppressor function and target genes.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer. Various microRNAs have been identified to play an important role in PDAC. The study aimed to explore the role of miR-429 in PDAC. The expression and prognostic value of miR-429 were first analyzed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Next, miR-429 expression was evaluated in the tissues and serum of 90 patients with PDAC. CCK8, SRB, wound healing and transwell assays were used to determine the effect of miR-429 on the proliferation, invasion, and migration of PDAC cells, respectively. Weighted gene co-expression network analysis (WGCNA), correlation analysis, TargetScan, and miRDB databases were used to screen and identify the target genes of miR-429. The results revealed that the expression of miR-429 was downregulated in PDAC tissues and the serum compared with those in normal tissues and the serum of healthy volunteers, respectively. The decreased expression of miR-429 was significantly associated with shorter overall survival. The overexpression of miR-429 significantly inhibited the proliferation, invasion, and migration of PDAC cells. Potential target genes of miR-429 were identified using WGCNA and bioinformatics analysis, and the results showed that cadherin 11 (CDH11), inositol polyphosphate-4-phosphatase type I (INPP4A), laminin gamma 1 (LAMC1), low density lipoprotein receptor-related protein 1 (LRP1), and quaking (QKI) were potential target genes of miR-429 in PDAC. Lower expression of CDH11 and QKI was associated with a more favorable prognosis in patients with PDAC. The overexpression of miR-429 could inhibit the expression of CDH11 and QKI. A nomogram model, involving miR-429, CDH11, and QKI, was subsequently constructed to determine their ability to accurately predict overall and disease-free survival in patients with PDAC. Taken together, miR-429 is involved in the development and progress of PDAC. MiR-429 could be recommended as a prognostic biomarker and therapeutic indicator in PDAC diagnosis.

Differential expression profiles of miRNA in granulomatous lobular mastitis and identification of possible biomarkers.

The etiology and pathogenesis of granulomatous lobular mastitis (GLM) remain largely elusive and the expression levels and regulatory roles of microRNAs (miRNAs or miRs) in GLM have remained mostly undetermined. In the present study, the miRNAs that were differentially expressed in breast biopsy samples from patients with GLM and normal tissue adjacent to fibroadenoma were analyzed, a comprehensive differential expression profile of miRNAs was provided and potential biomarkers were screened out. The expression profile of miRNAs was determined by high-throughput sequencing in the tissues of patients with GLM and healthy controls. Significantly differentially expressed miRNAs were screened by threshold setting and cluster analysis and their target genes were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, circulating differentially expressed miRNAs between the GLM and control groups were further analyzed by reverse transcription-quantitative PCR (RT-qPCR). A total of 31,077 miRNAs were detected by high-throughput sequencing. By using the cutoff criteria of |log2 fold change|>2.5 and q<0.001, 13 miRNAs that were indicated to be GLM biomarkers were screened out. The expression levels of these 13 miRNAs in the GLM group were higher than those in the control group. GO and KEGG enrichment analyses suggested that the occurrence and development of GLM may be associated with autoimmune inflammation, metabolism and pathogenic organisms. miR-451a and miR-5571-3p were confirmed to be significantly increased in the serum of patients with GLM compared with their levels in the serum of healthy volunteers, which suggests that they may be used as biomarkers of GLM. To the best of our knowledge, the present study was the first report detailing genome-wide miRNA profiling of patients with GLM compared with controls. The possible targets and pathways of GLM were evaluated by bioinformatics analysis. The present study identified 13 differentially expressed miRNAs with important theoretical significance and potential application. Furthermore, miR-451a and miR-5571-3p were verified by RT-qPCR as possible biomarkers of GLM.

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.

LncRNA MEG3 modulates hepatic stellate cell activation by sponging miR‑145 to regulate PPARγ.

It is important to determine the mechanism of liver fibrosis for targeted therapy and the development of targeted therapies for liver fibrosis may offer promise for patients with liver disease. Long non-coding RNAs (lncRNAs) serve a role in hepatic fibrosis. The lncRNA maternally expressed gene 3 (MEG3) has been confirmed to inhibit liver fibrosis. The present study investigated the role of the MEG3 in healthy patients and patients with liver fibrosis. The expression levels of MEG3 and microRNA (miR)-145 in the serum of healthy volunteers and patients with liver fibrosis and in LX-2 cells were detected using reverse transcription-quantitative PCR. A dual-luciferase reporter assay was used to determine the targeting relationship between MEG3 and miR-145, and the targeting relationship between miR-145 and peroxisome proliferator-activated receptor γ (PPARγ). The protein expression levels of PPARγ, α-smooth muscle actin (α-SMA) and collagen I (COL1A1) were detected using western blotting. The expression levels of α-SMA and COL1A1 were also determined using immunofluorescence. Finally, a Cell Counting Kit-8 assay was performed to assess the proliferative ability of LX-2 cells. A significantly reduced MEG3 expression level was demonstrated in serum from patients with liver fibrosis compared with serum from healthy controls. TGF-β1 induced a significantly decreased MEG3 expression level in LX-2 human hepatic stellate cells in vitro. The TGF-β1-induced increases in cell proliferation and α-SMA and COL1A1 protein expression levels were reversed following MEG3 overexpression. The results also demonstrated that MEG3 sponged miR-145 and competed endogenously with miR-145 to regulate PPARγ. In summary, the present study identified MEG3 as an anti-fibrotic lncRNA and provided new information regarding the role of MEG3 in liver fibrosis. MEG3 may therefore be a potential target in the treatment of liver fibrosis.

Decreased inhibition of exosomal miRNAs on SARS-CoV-2 replication underlies poor outcomes in elderly people and diabetic patients

Elderly people and patients with comorbidities are at higher risk of COVID-19 infection, resulting in severe complications and high mortality. However, the underlying mechanisms are unclear. In this study, we investigate whether miRNAs in serum exosomes can exert antiviral functions and affect the response to COVID-19 in the elderly and people with diabetes. First, we identified four miRNAs (miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p) through high-throughput sequencing and quantitative real-time PCR analysis, that are remarkably decreased in the elderly and diabetic groups. We further demonstrated that these miRNAs, either in the exosome or in the free form, can directly inhibit S protein expression and SARS-CoV-2 replication. Serum exosomes from young people can inhibit SARS-CoV-2 replication and S protein expression, while the inhibitory effect is markedly decreased in the elderly and diabetic patients. Moreover, three out of the four circulating miRNAs are significantly increased in the serum of healthy volunteers after 8-weeks’ continuous physical exercise. Serum exosomes isolated from these volunteers also showed stronger inhibitory effects on S protein expression and SARS-CoV-2 replication. Our study demonstrates for the first time that circulating exosomal miRNAs can directly inhibit SARS-CoV-2 replication and may provide a possible explanation for the difference in response to COVID-19 between young people and the elderly or people with comorbidities.

Epitope mapping from Mycobacterium leprae proteins: Convergent data from in silico and in vitro approaches for serodiagnosis of leprosy

Knowledge of immunodominant B-cell epitopes is essential to design powerful diagnostic strategies aiming for antibody detection. Outstanding progress in computational prediction has achieved a significant contribution to the biomedical fields, including immunodiagnosis. In silico analysis may have an even more important role when information concerning antigens from etiologic agents of neglected diseases, such as leprosy, is scarce. The aim of this study was to provide mapping of B-cell epitopes from two Mycobacterium leprae-derived antigens (Ag85B and ML2055), confirm their antigenicity, and to assess the ability of in silico immunoinformatics tools to accurately predict them. Linear B-cell epitopes predicted by ABCpred and SVMTrip servers were compared to antigenic regions of synthetic overlapping peptides that exhibited reactivity to antibodies from patients with leprosy. Our in vitro results identified several immunodominant regions that had also been indicated by in silico prediction, providing agreement between experimental and simulated data. After chemical synthesis, we used enzyme-linked immunosorbent assays to determine the effectiveness of the first identified sequence (GTNVPAEFLENFVHG) which had 72 % sensitivity and 78 % specificity (AUC = 0.79) while the second one (PVSSEAQPGDPNAPS) had 72 % sensitivity and 93.8 % specificity (AUC = 0.85). Using dot blotting, an easy-to-read visual test, both peptides could distinguish sera from patients with leprosy from those with tuberculosis and from sera of healthy volunteers. Our findings suggest that these synthetic peptides, with some refinement, may be useful as serological diagnostic antigens for leprosy. In addition, it was displayed that immunoinformatics provides reliable information for mapping potential B-cell epitopes for development of peptide-based diagnostic assays for neglected diseases.

Quantification of Circulating Cell Free Mitochondrial DNA in Extracellular Vesicles with PicoGreen™ in Liquid Biopsies: Fast Assessment of Disease/Trauma Severity.

The analysis of circulating cell free DNA (ccf-DNA) is an emerging diagnostic tool for the detection and monitoring of tissue injury, disease progression, and potential treatment effects. Currently, most of ccf-DNA in tissue and liquid biopsies is analysed with real-time quantitative PCR (qPCR) that is primer- and template-specific, labour intensive and cost-inefficient. In this report we directly compare the amounts of ccf-DNA in serum of healthy volunteers, and subjects presenting with various stages of lung adenocarcinoma, and survivors of traumatic brain injury using qPCR and quantitative PicoGreen™ fluorescence assay. A significant increase of ccf-DNA in lung adenocarcinoma and traumatic brain injury patients, in comparison to the group of healthy human subjects, was found using both analytical methods. However, the direct correlation between PicoGreen™ fluorescence and qPCR was found only when mitochondrial DNA (mtDNA)-specific primers were used. Further analysis of the location of ccf-DNA indicated that the majority of DNA is located within lumen of extracellular vesicles (EVs) and is easily detected with mtDNA-specific primers. We have concluded that due to the presence of active DNases in the blood, the analysis of DNA within EVs has the potential of providing rapid diagnostic outcomes. Moreover, we speculate that accurate and rapid quantification of ccf-DNA with PicoGreen™ fluorescent probe used as a point of care approach could facilitate immediate assessment and treatment of critically ill patients.

Coverage with Timely Administered Vaccination against Hepatitis B Virus and Its Influence on the Prevalence of HBV Infection in the Regions of Different Endemicity.

Universal hepatitis B vaccination of newborns was implemented in Russia starting from 1998. From 1998 to 2019, the incidence of acute hepatitis B reduced from 43.8 to 0.57 cases per 100,000 population. Here, we assessed the timely coverage of newborns with the birth dose (HepB-BD), second dose (HepB-2nd), and three vaccine doses (HepB3) in two remote regions of Russia with low (Belgorod Oblast) and high (Yakutia) levels of hepatitis B virus (HBV) endemicity. Vaccination data were obtained from the medical records of 1000 children in Yakutia and 2182 children in Belgorod Oblast. Sera of healthy volunteers from Belgorod Oblast (n = 1754) and Yakutia (n = 1072) across all age groups were tested for serological markers of HBV to assess the infection prevalence and herd immunity. Average HepB-BD coverage was 99.2% in Yakutia and 89.4% in Belgorod Oblast (p < 0.0001) and in both regions varied significantly, from 66% to 100%, between medical centers. The principal reason for the absence of HepB-BD was parent refusal, which accounted for 63.5% of cases of non-vaccination (83/123). While timely HepB-2nd coverage was only 55.4%–64.7%: HepB3 coverage by the age of one year exceeded 90% in both study regions. HBV surface antigen (HBsAg) prevalence in the 1998–2019 birth cohort was 0.2% (95% CI: 0.01–1.3%) in Belgorod Oblast and 3.2% (95% CI: 1.9–5.2%) in Yakutia. The proportion of persons testing negative for both antibodies to HBsAg (anti-HBs) and antibodies to HBV core antigen (anti-HBc) in the 1998–2019 birth cohort was 26.2% (125/481) in Belgorod Oblast and 32.3% (162/501) in Yakutia. We also assessed the knowledge of and attitude towards vaccination among 782 students and teachers of both medical and non-medical specialties from Belgorod State University. Only 60% of medical students knew that hepatitis B is a vaccine-preventable disease. Both medical and nonmedical students, 37.8% and 31.3%, respectively, expressed concerns about safety and actual necessity of vaccination. These data indicate the need to introduce a vaccine delivery audit system, improve medical education with respect to vaccination strategies and policies, and reinforce public knowledge on the benefits of vaccination.

Dietary Fatty Acids in Postprandial Triglyceride-Rich Lipoproteins Modulate Human Monocyte-Derived Dendritic Cell Maturation and Activation.

Dietary fatty acids have been demonstrated to modulate systemic inflammation and induce the postprandial inflammatory response of circulating immune cells. We hypothesized that postprandial triglyceride-rich lipoproteins (TRLs) may have acute effects on immunometabolic homeostasis by modulating dendritic cells (DCs), sentinels of the immunity that link innate and adaptive immune systems. In healthy volunteers, saturated fatty acid (SFA)-enriched meal raised serum levels of granulocyte/macrophage colony-stimulating factor GM-CSF (SFAs > monounsaturated fatty acids (MUFAs) = polyunsaturated fatty acids (PUFAs)) in the postprandial period. Autologous TRL-SFAs upregulated the gene expression of DC maturation (CD123 and CCR7) and DC pro-inflammatory activation (CD80 and CD86) genes while downregulating tolerogenic genes (PD-L1 and PD-L2) in human monocyte-derived DCs (moDCs). These effects were reversed with oleic acid-enriched TRLs. Moreover, postprandial SFAs raised IL-12p70 levels, while TRL-MUFAs and TRL-PUFAs increased IL-10 levels in serum of healthy volunteers and in the medium of TRL-treated moDCs. In conclusion, postprandial TRLs are metabolic entities with DC-related tolerogenic activity, and this function is linked to the type of dietary fat in the meal. This study shows that the intake of meals enriched in MUFAs from olive oil, when compared with meals enriched in SFAs, prevents the postprandial production and priming of circulating pro-inflammatory DCs, and promotes tolerogenic response in healthy subjects. However, functional assays with moDCs generated in the presence of different fatty acids and T cells could increase the knowledge of postprandial TRLs’ effects on DC differentiation and function.

Abstract 6446: Liquid biopsy in colon cancer utilizing exosomal miRNA biomarkers: An initial analysis

Abstract Traditional clinicopathologic variables do not predict recurrence and disease progression in colorectal cancer (CRC) accurately. Development of a liquid biopsy as a means to obtain real-time, clinically meaningful biomarkers to assess progression and response to treatment is critical. Current liquid biopsy techniques utilizing circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have significant limitations. Exosomes are emerging as a promising tool for liquid biopsy due to their stability and role in cancer cell to cell communication. To that end, the purpose of this study is to describe patterns of highly abundant microRNA (miRNA) found in serum exosomes of patients with metastatic colon cancer as a step toward developing an exosomal miRNA signature for liquid biopsy. Next generation sequencing was performed on exosomes isolated from serum of stage IV colon cancer patients (n=9) to determine the miRNA read counts. miRNA data from primary tumors (n=53) of stage IV colon cancer patients and available paired peritumoral tissue (n=8) were obtained from The Cancer Genome Atlas (TCGA) database for qualitative comparison. There were 515 total miRNA shared in common between the serum exosomes and primary tumors and 277 miRNA uniquely present in serum exosomes. In our initial analysis, we identified five miRNA, two in the common pool (mir-486-1 and mir-486-2) and three in the unique group (miR-486-5p, miR-92a-3p, and miR-146a-5p) that were most abundant in serum exosomes and may serve as biomarkers. mir-486-1 and mir-486-2 were the most highly abundant miRNAs in exosomes, but were only expressed at low levels in the tissue. This inverse relationship has been demonstrated by other investigators, with decreased levels of tissue mir-486 associated with more advanced stages of disease and poorer survival. Exosomal miR-92a-3p has been shown to correlate with tumor grade and stage in CRC patients. Delivery of miR-146a-5p by exosomes has been shown to promote tumorigenesis and generate immunosuppressive microenvironment in CRC. In general, the top 10 most abundant exosomal miRNA were not among the most abundant tissue level miRNAs. Our group is currently analyzing the differential expression of serum exosomal miRNA from 52 additional patients with non-metastatic colon cancer (n=14), liver metastases (n=16) and PC (n=12) in comparison to healthy volunteer serum (n=10) to better characterize miRNA patterns at different stages of disease. In summary, highly abundant miRNA found in serum exosomes of patients with metastatic colon cancer demonstrate a different pattern of expression than miRNA found in the primary colon cancer itself. Further analysis of these miRNA patterns from serum exosomes of colon cancer patients with non-metastatic disease, visceral metastases and PC compared to normal volunteers will aid in the development of a serum exosomal miRNA signature. Citation Format: Audrey H. Choi, Paul A. Vallejos, Mei Li M. Kwong, Janviere Kabagwira, Matthew J. Selleck, Nathan R. Wall, Maheswari Senthil. Liquid biopsy in colon cancer utilizing exosomal miRNA biomarkers: An initial analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6446.

Evaluation of MiR-1908-3p as a novel serum biomarker for breast cancer and analysis its oncogenic function and target genes

BackgroundBreast cancer is one of the most common tumors for women globally. Various miRNAs have been reported to play a crucial role in breast cancer, however the clinical significance of miR-1908-3p in breast cancer remains unclear. The present study aimed to explore the role of miR-1908-3p in breast cancer.MethodsThe expression of miR-1908-3p was detected in 50 pairs of breast cancer tissues and adjacent normal tissues, 60 breast cancer patient serum and 60 healthy volunteer serum. The functional roles of miR-1908-3p in breast cancer cells such as proliferation, migration and invasion were evaluated using CCK8, SRB, wound healing and transwell chambers. In addition, bioinformatics tools were used to identify potential targets of miR-1908-3p.ResultsThe results showed that the expression of miR-1908-3p were increased in breast cancer tissues and serum compared with normal breast tissues and serum of healthy volunteers respectively. Furthermore, the young breast cancer patients and HER2-positive patients had a higher level of tissues’ miR-1908-3p than elder breast cancer patients and HER2-negative patients, respectively. The young breast cancer patients had a higher level of serum miR-1908-3p than elder breast cancer patients, ROC analysis suggested that miR-1908-3p had the potential as a promising serum diagnostic biomarker of breast cancer. Up-regulation of miR-1908-3p promoted the cells proliferation, migration and invasion while knockdown of miR-1908-3p inhibited these processes in breast cancer cell MCF-7 and MDA-MB-231. The potential target genes of miR-1908-3p in breast cancer included ID4, LTBP4, GPM6B, RGMA, EFCAB1, ALX4, OSR1 and PPARA. Higher expression of these eight genes correlated with a better prognosis for breast cancer patients.ConclusionsThese results suggest that miR-1908-3p may exert its oncogenic functions via suppression of these eight genes in breast cancer.

Serum exosomal long noncoding RNAs lnc-FAM72D-3 and lnc-EPC1-4 as diagnostic biomarkers for hepatocellular carcinoma.

Long noncoding RNAs (lncRNAs), such as LINC00462, HOTAIR, and MALAT1, are significantly upregulated in hepatocellular carcinoma (HCC) tissues. However, lncRNA expression in the serum of HCC patients is still unclear. To identify candidate lncRNAs for HCC diagnosis, we purified exosomal total RNA from the serum of healthy volunteers (controls) and hepatitis, cirrhosis, and HCC patients. To assess the function of lncRNAs, small interfering RNAs and overexpression vectors were designed and cell viability and cell apoptosis assays conducted. The exosomes of the control group had a larger number of lncRNAs with a high amount of alternative splicing compared to hepatic disease patients. qPCR assays showed that lnc-FAM72D-3, lnc-GPR89B-15, lncZEB2-19, and lnc-EPC1-4 are differentially expressed in HCC. Furthermore, the expression level of lnc-EPC1-4 correlated with age. While the expression levels of lnc-GPR89B-15 and lnc-EPC1-4 correlated with serum alpha-fetoprotein level. lnc-FAM72D-3 knockdown decreased cell viability and promoted cell apoptosis, indicating that lnc-FAM72D-3 functions as an oncogene in HCC. In contrast, lnc-EPC1-4 overexpression inhibited cell proliferation and induced cell apoptosis, indicating that it functions as a tumor suppressor gene. Collectively, these findings show that lnc-FAM72D-3 and lnc-EPC1-4 play a novel role that might contribute to hepatocarcinogenesis and identify potential candidate biomarkers for HCC diagnosis.

A Panel of CircRNAs in the Serum Serves as Biomarkers for Mycobacterium tuberculosis Infection.

Tuberculosis (TB), one of the ancient and deadliest diseases, is a chronic immune disorder caused by Mycobacterium tuberculosis (Mtb) infection. Due to the lack of ideal diagnostic and therapeutic markers, TB is still posing a major health, social, and economic burden worldwide. Circular RNA (circRNA), a newly discovered endogenous RNA, is abundant and stable in the cytoplasm and has tissue specificity. More and more studies suggested circRNA is involved in a variety of human pathological and physiological processes. Recently, several studies have confirmed circRNAs not only existed in the serum but also could serve as ideal biomarkers for detecting diseases since the circRNAs have continuous, stable, and covalently closed circular structures and are not easily degraded by nucleases. In this study, we screened the circRNA expression profiles in active TB serum samples and healthy volunteers serum samples by circRNA microarrays. Then, we performed qRT-PCR to verified the dysregulated circRNAs and ROC curve analysis to evaluate the value of circRNAs for TB diagnosis. The results showed circRNA_051239, circRNA_029965, and circRNA_404022 could serve as biomarkers for TB diagnosis.