Pooled Serum Samples Research Articles

Nationwide human biomonitoring strategy in Korea: Prioritization of novel contaminants using GC/TOF-MS with suspect and non-target screening

According to global regulations on hazardous chemicals, numerous alternatives have been manufactured and used in various consumer products. Suspect and non-target analyses are advanced analytical techniques used for identifying novel contaminants. In the present study, suspect and non-target analytical approaches using a gas chromatography coupled to a time-of-flight mass spectrometer were applied to identify novel contaminants in 40 pooled serum samples from a sub-population (n = 400) of the 2015–2017 national biomonitoring program. Suspect screening analysis was performed using an in-house library based on retention times and quantifier and qualifier ions for 222 contaminants, including persistent organic pollutants and emerging contaminants. Non-target analysis was performed by matching deconvoluted mass spectra to the spectral library from the National Institute of Standards and Technology. The suspect screening analysis identified organochlorinated pesticides, organophosphate esters, phthalate esters, and alternative plasticizers. Among the 68 compounds identified in the non-target analysis, siloxanes, novel organophosphate esters, and UV ink photoinitiators were considered candidates for future inclusion in the biomonitoring program based upon significant human exposure. Our findings demonstrate the feasibility of suspect and non-target analysis to identify novel contaminants to prioritize for inclusion within a national human biomonitoring program.

Novel Strategy for Screening Target Proteins by the Common Drugs─Sofosbuvir-Specific Profiling of HCV Patient Serum.

It is the scientific basis of precision medicine to study all of the targets of drugs based on the interaction between drugs and proteins. It is worth paying attention to unknown proteins that interact with drugs to find new targets for the design of new drugs. Herein, we developed a protein profiling strategy based on drug-protein interactions and drug-modified magnetic nanoparticles and took hepatitis C virus (HCV) and its corresponding drug sofosbuvir (SOF) as an example. A SOF-modified magnetic separation medium (Fe3O4@POSS@SOF) was prepared, and a gradient elution strategy was employed and optimized to profile specific proteins interacted with SOF. A series of proteomic analyses were performed to profile proteins based on SOF-protein interactions (SPIs) in the serum of HCV patients to evaluate the specificity of the profiling strategy. As a result, five proteins were profiled with strong SPIs and exhibited high relevance with liver tissue, which were potentially new drug targets. Among them, HSP60 was used to confirm the highly specific interactions between the SOF and its binding proteins by Western blotting analysis. Besides, 124 and 29 differential proteins were profiled by SOF material from three HCV patient serum and pooled 20 HCV patient serum, respectively, by comparing with healthy human serum. In comparison with those profiled by the polyhedral oligomeric silsesquioxane (POSS) material, differential proteins profiled by the SOF material were highly associated with liver diseases through GO analysis and pathway analysis. Furthermore, four common differential proteins profiled by SOF material but not by POSS material were found to be identical and expressed consistently in both pooled serum samples and independent serum samples, which might potentially be biomarkers of HCV infection. Taken together, our study proposes a highly specific protein profiling strategy to display distinctive proteomic profiles, providing a novel idea for drug design and development.

Validation of the efficacy of pooled serum for serum glucose inhouse quality control material in comparison with commercial internal quality control in clinical chemistry laboratory

BackgroundThis study aimed to create an in-house glucose quality control material for humans, addressing the challenge of obtaining high-cost commercially prepared quality control materials in developing countries. MethodsAn in-house quality control material for glucose was prepared using a pooled serum sample and analyzed using a fully automated chemistry analyzer following the ISO 80 guidelines. The mean, standard deviation (SD), and coefficient of variance were calculated from the first 30 days of measurement, and the variability was checked over eight months using SPSS software. The study used Pearson's correlation with a 95% confidence interval and a P-value less than 0.05, which was statistically significant. ResultsThe average mean ± SD of human serum glucose was 185.2 ± 8.4 mg/dL, indicating that the precision between each measurement was better. The prepared in-house quality control material was stable for approximately five months without any significant change in the serum glucose concentration (mg/dl) (p-value<0.05). ConclusionsThe study suggests that room-temperature, 2–8 °C, and −20 °C to −30 °C storage of human serum samples for glucose analysis is a viable option, with stable glucose concentrations for up to 30 days. Pooled serum is a cost-effective method for in-house quality control, especially in resource-limited laboratories.

Frequency of PCV-2 viremia in nursery piglets from a Spanish swine integration system in 2020 and 2022 considering PRRSV infection status.

Porcine circovirus 2 (PCV-2) poses a significant economic threat for the swine industry, causing a range of diseases collectively referred to as porcine circovirus diseases (PCVDs). Despite PCV-2 vaccine effectiveness, the need for monitoring infectious pressure remains. PCV-2 coinfection with other pathogens like porcine reproductive and respiratory syndrome virus (PRRSV) can exacerbate disease severity and lead to PCV-2-systemic disease cases. Monitoring both PRRSV and PCV-2 in co-infected farms is crucial for an effective management and vaccination programs. The present cross-sectional study aimed to determine PCV-2 antibody levels in piglets at weaning and PCV-2 and PRRSV viremia in pooled serum samples at weaning (vaccination age) and at 6 and 9weeks of age from a Spanish swine integration system in 2020 (48 farms) and in 2022 (28 out of the 48 analysed previously). The frequency of PCV-2 detection in pools of piglet sera was 2.1% (2020) and 7.1% (2022) at vaccination age but increased at the end of the nursery period (10.4% in 2020 and 39.3% in 2022)in both years. Co-infections between PCV-2 and PRRSV were detected in a significant proportion of PRRSV positive farms (15% in 2020, and 60% in 2022). PCV-2 antibody levels(ELISA S/P ratios) at weaning were lower in PCV-2 qPCR positive farms at different sampling time-points (0.361 in 2020 and 0.378 in 2022) compared to PCV-2 qPCR negative ones (0.587 in 2020 and 0.541 in 2022). The 28 farms tested both years were classified in four different epidemiological scenarios depending on their PCV-2 virological status. Those PCV-2 qPCR negative farms in 2020 that turned to be positive in 2022 had a statistically significant increase of PRRSV RT-qPCR detection and a PCV-2 antibody levels reduction, facts that were not observed in the rest of the scenarios. This epidemiological study in farms from the same integration system determined the occurrence, in 2020 and in 2022, of PCV-2 and PRRSV infections in piglets during the nursery period by using pooled serum samples.

Suspect Screening Analysis of Pooled Human Serum Samples Using GC × GC/TOF-MS.

Humans interact with thousands of chemicals. This study aims to identify substances of emerging concern and in need of human health risk evaluations. Sixteen pooled human serum samples were constructed from 25 individual samples each from the National Institute of Environmental Health Sciences' Clinical Research Unit. Samples were analyzed using gas chromatography (GC) × GC/time-of-flight (TOF)-mass spectrometry (MS) in a suspect screening analysis, with follow-up confirmation analysis of 19 substances. A standard reference material blood sample was also analyzed through the confirmation process for comparison. The pools were stratified by sex (female and male) and by age (≤45 and >45). Publicly available information on potential exposure sources was aggregated to annotate presence in serum as either endogenous, food/nutrient, drug, commerce, or contaminant. Of the 544 unique substances tentatively identified by spectral matching, 472 were identified in females, while only 271 were identified in males. Surprisingly, 273 of the identified substances were found only in females. It is known that behavior and near-field environments can drive exposures, and this work demonstrates the existence of exposure sources uniquely relevant to females.

Highly sensitive voltammetric sensor comprising CuSn(OH)6–multi-walled carbon nanotubes–β-cyclodextrin composites and poly(L-arginine) for indole-3-lactic acid analyses in alcohol use disorder serum samples

A highly sensitive voltammetric sensing platform configured via the drop-casting of composites of CuSn(OH)6–multi-walled carbon nanotubes–β-cyclodextrin (CuSn(OH)6–MWCNT–βCD) on an arginine-electropolymerized screen-printed carbon electrode (CuSn(OH)6–MWCNT–βCD/p-Arg/SPCE) was demonstrated to determine the concentrations of indole-3-lactic acid (ILA) in buffer and serum solutions. Prior to the analysis, the successful assembly of the composites and polymer layers was confirmed using field emission scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. The changes in the current due to the direct oxidation of ILA on the modified electrode were used to quantify the ILA. A dynamic range of 0.05–20 μM with a limit of detection of 9 nM for ILA under optimal conditions was achieved primarily with the combined use of CuSn(OH)6, MWCNT, and βCD, resulting in a large electrochemical active surface area (0.185 cm2), low charge transfer resistance (∼2 Ω), and fast electron transfer. Furthermore, the sensing performance, including selectivity, sensitivity, reproducibility, repeatability, and stability, for ILA detection was evaluated. Finally, the ILA concentrations in the pooled clinical serum samples of healthy control and alcohol use disorder patients were measured using our method, the results of which exhibited a notable resemblance to those obtained using liquid chromatography with tandem mass spectrometry.

Deep Sequencing of Porcine Reproductive and Respiratory Syndrome Virus ORF7: A Promising Tool for Diagnostics and Epidemiologic Surveillance.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major concern worldwide. Control of PRRSV is a challenging task due to various factors, including the viral diversity and variability. In this study, we evaluated an amplicon library preparation protocol targeting the ORF7 region of both PRRSV species, Betaarterivirus suid 1 and Betaarterivirus suid 2. We designed tailed primers for a two-step PCR procedure that generates ORF7-specific amplicon libraries suitable for use on Illumina sequencers. We tested the method with serum samples containing common laboratory strains and with pooled serum samples (n = 15) collected from different pig farms during 2019-2021 in Hungary. Testing spiked serum samples showed that the newly designed method is highly sensitive and detects the viral RNA even at low copy numbers (corresponding to approx. Ct 35). The ORF7 sequences were easily assembled even from clinical samples. Two different sequence variants were identified in five samples, and the Porcilis MLV vaccine strain was identified as the minor variant in four samples. An in-depth analysis of the deep sequencing results revealed numerous polymorphic sites along the ORF7 gene in a total of eight samples, and some sites (positions 12, 165, 219, 225, 315, 345, and 351) were found to be common in several clinical specimens. We conclude that amplicon deep sequencing of a highly conserved region of the PRRSV genome could support both laboratory diagnosis and epidemiologic surveillance of the disease.

Linear epitope mapping in the E and NS1 proteins of dengue and Zika viruses: Prospection of peptides for vaccines and diagnostics.

The arrival of the Zika virus (ZIKV) in dengue virus (DENV)-endemic areas has posed challenges for both differential diagnosis and vaccine development. Peptides have shown promise in addressing these issues. The aim of this study was to identify the linear epitope profile recognized by serum samples from dengue and Zika patients in the E and NS1 proteins of DENV and ZIKV. This cross-sectional study included individuals of all ages with laboratory-confirmed DENV and ZIKV infections, who were selected through convenience sampling. The serum samples from dengue and Zika patients detected epitopes evenly distributed across the viral proteins in a peptide microarray platform. However, several epitopes were located within "epitope hotspots", characterized by clusters of peptides recognized in more than 30% of the sub-arrays analyzed using individual or pooled serum samples. The serum samples from dengue and Zika patients showed a high level of cross-reactivity with peptides in the DENV and ZIKV proteins. Analysis using an additional peptide microarray platform, which contained peptides selected based on the results of the initial screening, revealed that two DENV and one ZIKV peptide, highly specific to their related viruses, were located within the epitope hotspots; however, they presented low detection rates (32.5, 35.0, and 28.6%, respectively). In addition, two DENV peptides detected at similarly high rates by both dengue and Zika patients were also found within the epitope hotspots. These hotspots contain several immunodominant epitopes that are recognized by a larger number of individuals when compared to 15-amino acid (aa) sequence peptides. Thus, epitope hotspots may have greater potential to serve as antigens in diagnostic tests and vaccine development than peptides composed of only 15 amino acids.

Hemolysis, icterus and lipemia interfere with the determination of two oxidative stress biomarkers in canine serum

BackgroundOxidative stress has been proven to play a role in numerous human and canine diseases. Among the biomarkers of oxidative stress, Thiobarbituric Acid Reactive Substances (TBARS) and Total Antioxidant Status (TAS) are two of the most widely used. Preanalytical factors are crucial for obtaining accurate results in these assays. Hemolysis, icterus and lipemia (HIL) are common sources of preanalytical errors in the laboratory; however, limited information is available regarding the considerations for canine specimens. Therefore, the objective of this study was to evaluate the potential interferences of HIL in the determination of TBARS and TAS in canine serum.MethodsSolutions of pooled canine serum samples were prepared by adding increasing concentrations of hemolysate, bilirubin and a synthetic lipid emulsion. TBARS and TAS were determined, and biases from the control value caused by the interfering substances were calculated.ResultsHemolysis, icterus and lipemia induced significant interferences on TBARS and TAS, albeit to varying degrees depending on the specific biomarker and interfering substance. TBARS appeared to be more susceptible to interferences in this study. Slight hemolysis, moderate icterus and slight lipemia caused notable deviations in TBARS values, surpassing the acceptable threshold for interference. TAS assay was also affected by HIL, although to a lesser extent compared to TBARS. Significant biases from TAS control value were observed when icterus was moderate, and when hemolysis and lipemia were more pronounced.ConclusionsIn light of our results, we conclude that hemolyzed, icteric and lipemic specimens are not suitable for TBARS and TAS determination in canine serum. Our findings hold considerable practical utility, as a simple visual inspection would be sufficient for identifying and excluding such specimens.

Methodological considerations in determining sex steroids in children: comparison of conventional immunoassays with liquid chromatography-tandem mass spectrometry.

In laboratory medicine, external quality assessment (EQA) schemes have become versatile tools for detecting analytical flaws. However, EQA schemes are lacking for pediatric sex steroid levels. We aimed to investigate the suitability of different estradiol and testosterone immunoassays in a pediatric setting in comparison with clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays. The study was conducted by staff and the advisory group on endocrinology at Equalis, the Swedish provider of EQA schemes for laboratory medicine. The test material consisted of five pooled serum samples from children who were either prepubertal or in puberty. Clinical laboratories enrolled in Equalis EQA schemes for estradiol and testosterone were invited to participate, as were clinical laboratories using LC-MS/MS-assays. Samples were analyzed by either routine immunoassays (n=18) or in-house LC-MS/MS assays (n=3). For estradiol, LC-MS/MS assays showed a high degree of conformity with interlaboratory coefficients of variation (CV) below 24.2 %. Reported levels were between 4.9±1.2 and 33.9±1.6 pmol/L (group mean±standard deviation). The direct immunoassays had lower precision; their CVs were up to 81.4 %. Reported concentrations were between 25.3±18.1 and 45.7±19.4 pmol/L, an overestimation compared to LC-MS/MS. Testosterone LC-MS/MS also showed a high degree of conformity, CVs were below 13.4 %, and reported concentrations were from 0.06±0.00 to 1.00±0.11 nmol/L. The direct immunoassays had a larger discrepancy between results; CVs were up to 95.8 %. Concentrations were between 0.12±0.11 and 0.85±0.23 nmol/L. For the safe diagnosis and determination of sex steroids in children, analysis with mass spectrometry-based methods is recommended.

Deep Characterization of Serum Metabolome Based on the Segment-Optimized Spectral-Stitching Direct-Infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Approach.

Direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (DI-FTICR MS) shows great promise for metabolomic analysis due to ultrahigh mass accuracy and resolution. However, most of the DI-FTICR MS approaches focused on high-throughput metabolomics analysis at the expense of sensitivity and resolution and the potential for metabolome characterization has not been fully explored. Here, we proposed a novel deep characterization approach of serum metabolome using a segment-optimized spectral-stitching DI-FTICR MS method integrated with high-confidence and database-independent formula assignments. With varied acquisition parameters for each segment, a highly efficient acquisition was achieved for the whole mass range with sub-ppm mass accuracy. In a pooled human serum sample, thousands of features were assigned with unambiguous formulas and possible candidates based on highly accurate mass measurements. Furthermore, a reaction network was used to select confidently unique formulas from possible candidates, which was constructed by unambiguous formulas and possible candidates connected by the formula differences resulting from biochemical and MS transformation. Compared with full-range and conventional segment acquisition, 8- and 1.2-fold increases in observed features were achieved, respectively. Assignment accuracy was 93-94% for both a standard mixture containing 190 metabolites and a spiked serum sample with the root mean square mass error of 0.15-0.16 ppm. In total, 3534 unequivocal neutral molecular formulas were assigned in the pooled serum sample, 35% of which are contained in the HMDB. This method offers great enhancement in the deep characterization of serum metabolome by DI-FTICR MS.

Development of a metabolite calculator for diagnosis of pancreatic cancer.

Carbohydrate antigen (CA) 19-9 is a known pancreatic cancer (PC) biomarker, but is not commonly used for general screening due to its low sensitivity and specificity. This study aimed to develop a serum metabolites-based diagnostic calculator for detecting PC with high accuracy. A targeted quantitative approach of direct flow injection-tandem mass spectrometry combined with liquid chromatography-tandem mass spectrometry was employed for metabolomic analysis of serum samples using an Absolute IDQ™ p180 kit. Integrated metabolomic analysis was performed on 241 pooled or individual serum samples collected from healthy donors and patients from nine disease groups, including chronic pancreatitis, PC, other cancers, and benign diseases. Orthogonal partial least squares discriminant analysis (OPLS-DA) based on characteristics of 116 serum metabolites distinguished patients with PC from those with other diseases. Sparse partial least squares discriminant analysis (SPLS-DA) was also performed, incorporating simultaneous dimension reduction and variable selection. Predictive performance between discrimination models was compared using a 2-by-2 contingency table of predicted probabilities obtained from the models and actual diagnoses. Predictive values obtained through OPLS-DA for accuracy, sensitivity, specificity, balanced accuracy, and area under the receiver operating characteristic curve (AUC) were 0.9825, 0.9916, 0.9870, 0.9866, and 0.9870, respectively. The number of metabolite candidates was narrowed to 76 for SPLS-DA. The SPLS-DA-obtained predictive values for accuracy, sensitivity, specificity, balanced accuracy, and AUC were 0.9773, 0.9649, 0.9832, 0.9741, and 0.9741, respectively. We successfully developed a 76 metabolome-based diagnostic panel for detecting PC that demonstrated high diagnostic performance in differentiating PC from other diseases.

Solid-phase immunoextraction followed by liquid chromatography-tandem mass spectrometry for the selective determination of thyroxine in human serum

In this work, an analytical method based on solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) has been developed for the selective determination of thyroxine (T4) in human serum. For this purpose, two immunosorbents (ISs) specific to T4 were synthesized by grafting two different T4-specific monoclonal antibodies on a cyanogen bromide (CNBr)-activated-Sepharose® 4B solid support. The grafting yields obtained from the immobilization of each antibody on the CNBr-activated-Sepharose® 4B were over 90%, demonstrating that most of the antibodies were covalently bound to the solid support. The SPE procedure was optimized by studying the retention capability and selectivity of the two ISs in pure media fortified with T4. Under the optimized conditions, high elution efficiencies were achieved in the elution fraction for both specific ISs (i.e., 85%), whereas low ones were obtained in the control ISs (ca. 2%), showing the selectivity of the specific ISs. The ISs were also characterized by studying extraction and synthesis repeatability (RSD <8%), and capacity (104 ng of T4 per 35 mg of ISs, i.e., 3 μg g−1). Finally, the methodology was applied to a pooled human serum sample in order to study its analytical utility and accuracy. Relative recovery (RR) values between 81 and 107% were obtained, showing no matrix effects during the global methodology. Furthermore, the need to perform the immunoextraction was evidenced by comparing the LC-MS scan chromatograms and RR values with and without applying the immunoextraction procedure on a serum sample submitted to protein precipitation. This works exploits, for the first time, the use of an IS on the selective determination of T4 in human serum samples.

Proteomic-based identification of APCS as candidate protein for diagnosis of patients exhibiting anti-tubercular drug induced liver injury

Traditional markers evaluate anti-tubercular drug-induced liver injury (AT-DILI). However, these markers have certain limitations and studies are in progress to characterize AT-DILI at an early stage. In the present study, 40 patients were categorized and equally distributed into healthy controls, newly diagnosed tuberculosis (TB), TB without hepatotoxicity and TB with hepatotoxicity groups based on their conventional liver function tests. Relative protein quantification was performed on depleted pooled serum samples of each representative group by LC–MS/MS, and validation of shortlisted protein was done by ELISA. Levels of all analysed biochemical parameters showed a statistical increment in the hepatotoxicity group compared to the other three groups, representing AT-DILI. Comparative proteomic analysis between TB with hepatotoxicity versus TB without hepatotoxicity groups highlighted 24 significant differentially expressed proteins, including PROS1, KNG1, CFH, LCAT, APCS and ADIPOQ. Identified proteins were involved in complement activation, triglyceride-rich lipoprotein particle remodelling and pathways comprising complement, coagulation cascades and cholesterol metabolism. Based on functional relevance, the serum amyloid P component (APCS) was shortlisted for validation, and it showed a similar trend as observed in the discovery phase with 100% sensitivity and 87% specificity; however, findings need exploration in larger cohorts.

Screening strategy for 1210 exogenous chemicals in serum by two-dimensional liquid chromatography-mass spectrometry

Humans are at risk of exogenous exposure to exogenous chemicals. Challenges exist for the comprehensive monitoring of residues with different physical and chemical properties in serum. Here, an on-line two-dimensional liquid chromatography (2D-LC) - high resolution mass spectrometry system (HRMS) was developed, expanding the range of the partition coefficient in octanol/water of the residue analysis from −8 to 12. A high-coverage serum residue screening strategy was further designed by integrating 2D-LC system with HRMS full MS/data independent acquisition and automatic spectral library searching. This strategy enables to simultaneously screen 1210 pesticides, veterinary/human drugs, other chemical pollutants and their metabolites in serum with a single analysis. Method validation showed 92% and 81% of 1022 residues spiked in serum could be detected at 50 ng/mL and 5 ng/mL, respectively. The developed method was applied to the analysis of 24 separately pooled serum samples, 58 suspect residues were found, some of them were detected at high frequencies over than 50%. Among them, 4,6-Dinitro-O-cresol and probable carcinogenic folpet are highly toxic, and cimaterol is banned in China. Collectively, this study developed a 2D-LC-HRMS -based screening strategy for screening pesticides, veterinary/human drugs, and other chemical pollutants in serum, it is helpful for studying the effect of exogenous exposures on human health.

Serum Protein Concentration and Serum Protein Fractions in Bottlenose Dolphins (Tursiops truncatus) under Human Care Using Agarose Gel Electrophoresis.

Serum protein electrophoresis (SPE) is the most used and reliable method to determine the percentage of serum protein subfractions. The interpretation of the kinetics of total proteins and albumin and globulin fractions is receiving increased attention in wild animals, as well as in domestic animals, due to the possibility of identifying typical pathologic patterns. However, the interpretation of these data had to be performed in light of an appropriate method-and species- specific reference intervals (RIs). In marine mammals, as well as other non-domestic species, specific attention should also be given to the different environment (free ranging vs. human managed) and the associated different exposure to environmental stimuli. The aim of this report was to establish RIs for the serum protein fractions evaluated using agarose gel electrophoresis (AGE) in bottlenose dolphins under human care. Peripheral blood samples were collected from 40 bottlenose dolphins during standard veterinary procedures to evaluate their health status. Total protein concentration was determined using the biuret method while AGE was performed using an automated system. A pooled dolphin's serum sample was used to determine the intra-assay and inter-assay imprecision of AGE. The RIs were calculated using an Excel spreadsheet with the Reference Value Advisor set of macroinstructions. The intra and inter-assay imprecisions were 1.2% and 2.5%, respectively, for albumin; 2.9% and 5.7%, respectively, for α-globulins; 3.8% and 4.0%, respectively, for β-globulins; and 3.4% and 4.8%, respectively, for γ-globulins. The total protein, albumin, α-globulin, β-globulin, and γ-globulin concentrations were 65.5 ± 5.4 g/L, 45.5 ± 4.9 g/L, 8.0 ± 1.0 g/L, 5.0 ± 2.0 g/L, and 7.0 ± 2.0 g/L, respectively. We established the RIs for the total protein and serum protein fractions using AGE in bottlenose dolphins under human care.

Sedimentation velocity FDS studies of antibodies in pooled human serum.

The biotech industry has great interest in investigating therapeutic proteins in high concentration environments like human serum. The fluorescence detection system (Aviv-FDS) allows the performance of analytical ultracentrifuge (AUC) sedimentation velocity (SV) experiments in tracer or BOLTS protocols. Here, we compare six pooled human serum samples by AUC SV techniques and demonstrate the potential of this technology for characterizing therapeutic antibodies in serum. Control FDS SV experiments on serum alone reveal a bilirubin-HSA complex whose sedimentation is slowed by solution nonideality and exhibits a Johnston-Ogston (JO) effect due to the presence of high concentrations of IgG. Absorbance SV experiments on diluted serum samples verify the HSA-IgG composition as well as a significant IgM pentamer boundary at 19s. Alexa-488 labeled Simponi (Golimumab) is used as a tracer to investigate the behavior of a therapeutic monoclonal antibody (mAb) in serum, and the sedimentation behavior of total IgG in serum. Serum dilution experiments allow extrapolation to zero concentration to extract so, while global direct boundary fitting with SEDANAL verifies the utility of a matrix of self- and cross-term phenomenological nonideality coefficients (ks and BM1) and the source of the JO effect. The best fits include weak reversible association (~ 4 × 103M-1) between Simponi and total human IgG. Secondary mAbs to human IgG and IgM verify the formation of a 10.2s 1:1 complex with human IgG and a 19s complex with human IgM pentamers. These results demonstrate that FDS AUC allows a range of approaches for investigating therapeutic antibodies in human serum.

The role of circulatory chemokines in sepsis-induced myopathy in mice

BACKGROUND: Sepsis is a condition of life-threatening organ dysfunction caused by a dysregulated host response to infection and carries a high risk of mortality. A rapidly growing number of “sepsis survivors” experience persistent and severe skeletal muscle weakness and atrophy (i.e., sepsis-induced myopathy). The myopathy is triggered by the interaction of circulating pathogens, inflammatory factors (e.g., cytokines and chemokines), and myofibers. Currently, there are no treatments for sepsis-induced myopathy partially due to a lack of therapeutic targets. Chemokines are peptides that possess chemo attractant properties and are released by immune cells during infection insult. Circulating levels of chemokines in septic patients are known to be high. However, the role these chemokines play in context of sepsis-induced myopathy is currently unknown. Our goal in this study was to identify the role of circulating chemokines on sepsis-induced myopathy. METHODS: We induced sepsis via cecal ligation and puncture (CLP) or sham control surgeries in a group of young adults (5 mo) male C57BL6 mice. Seven days after surgery, serum was collected from sham and CLP mice. The serum samples were pooled for cell culture experiments. In a subset of healthy mice, we isolated primary myoblasts. The myoblasts were co-cultured in conditioned media containing pooled serum samples collected from sham and CLP mice. We performed a multiplex assay to identify the levels of chemokines on the pooled serum samples. We measured myoblast proliferation, myotube area, and fusion index. In follow-up experiments, we exposed C2C12 myotubes to a selected group of chemokine peptides previously identified in serum samples. RESULTS: Exposure to septic serum attenuated the proliferative capacity of healthy myoblasts by 46% (p &lt; 0.05). In addition, myoblasts exposed to septic serum did not differentiate into healthy myotubes. Multiplex analyses of pooled sham and septic serum revealed a marked elevation of the following chemokines: CXCL-1 (healthy = 173.4 ± 0.1, septic = 69,630 ± 13,732 pg/ml), CXCL-2 (healthy = 1,078 ± 0.1, septic = 3,907 ± 259 pg/ml), CXCL-10 (healthy = 366 ± 0.1, septic = 3,961 ± 293 pg/ml), CCL-2 (healthy = 276 ± 0.1, septic = 1672 ± 362 pg/ml). Exposure of mature C2C12 myotubes to the chemokine peptides for 24 h increased the mRNA expression of atrophy-related genes ( FOXO1, GABARAP, BNIP3, TRIM63, MAP11A3B, FOXO32, FOXO30). CONCLUSION: Circulating chemokines play a regulatory role in sepsis-induced myopathy via attenuation of the proliferative capacity of myoblasts and by altering myotube morphology and the expression of atrophy related genes. Chemokines are potential therapeutic targets to treat sepsis-induced myopathy. Funded by NIH R21AG072011-03 to OL This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Immunoglobulin profiling with large high-density peptide microarrays as screening method to detect candidate proteins for future biomarker detection in dogs with steroid-responsive meningitis-arteritis.

Large high-density peptide microarrays are a suitable screening method to detect possible autoantigens which might be involved in the pathogenesis of SRMA. The IgA and IgG profile of pooled serum samples of 5 dogs with SRMA and 5 dogs with neck pain due to intervertebral disc herniation (IVDH) without ataxia or paresis were compared via commercially available high-density peptide microarrays (Discovery Microarray) containing 29,240 random linear peptides. Canine distemper virus nucleoprotein (CDVN) served as positive control as all dogs were vaccinated. Common motifs were compared to amino acid sequences of known proteins via databank search. One suitable protein was manually selected for further analysis with a smaller customized high-density peptide microarray. Pooled serum of dogs with SRMA and IVDH showed different IgA and IgG responses on Discovery Microarray. Only top IgG responses of dogs with SRMA showed a common motif not related to the control protein CDVN. This common motif is part of the interleukin 1 receptor antagonist protein (IL1Ra). On IL1Ra, dogs with SRMA displayed IgA binding to an additional epitope, which dogs with IVDH did not show. IL1Ra is an anti-inflammatory acute phase protein. Different immunoglobulin binding patterns on IL1Ra could be involved in the pathogenesis of SRMA and IL1Ra might be developed as future biomarker for SRMA.

Specific miRNAs are associated with human cancer cachexia in an organ-specific manner.

Cancer cachexia (CCx) is a complex and multi-organ wasting syndrome characterized by substantial weight loss and poor prognosis. An improved understanding of the mechanisms involved in the onset and progression of cancer cachexia is essential. How microRNAs contribute to the clinical manifestation and progression of CCx remains elusive. The aim of this study was to identify specific miRNAs related to organ-specific CCx and explore their functional role in humans. miRNA patterns in serum and in cachexia target organs (liver, muscle and adipose tissue) from weight stable (N≤12) and cachectic patients (N≤23) with gastrointestinal cancer were analysed. As a first step, a miRNA array (158 miRNAs) was performed in pooled serum samples. Identified miRNAs were validated in serum and corresponding tissue samples. Using in silico prediction, related genes were identified and evaluated. The findings were confirmed in vitro by siRNA knock-down experiments in human visceral preadipocytes and C2C12 myoblast cells and consecutive gene expression analyses. Validating the results of the array, a 2-fold down-regulation of miR-122-5p (P=0.0396) and a 4.5-fold down-regulation of miR-194-5p (P<0.0001) in serum of CCx patients in comparison with healthy controls were detected. Only miR-122-5p correlated with weight loss and CCx status (P=0.0367). Analysing corresponding tissues six muscle and eight visceral adipose tissue (VAT) cachexia-associated miRNAs were identified. miR-27b-3p, miR-375 and miR-424-5p were the most consistently affected miRNAs in tissues of CCx patients correlating negatively with the severity of body weight loss (P=0.0386, P=0.0112 and P=0.0075, respectively). We identified numerous putative target genes of the miRNAs in association with muscle atrophy and lipolysis pathways. Knock-down experiments in C2C12 myoblast cells revealed an association of miR-27b-3p and the in silico predicted atrophy-related target genes IL-15 and TRIM63. Both were up-regulated in miR-27b-3p knock-down cells (P<0.05). Concordantly, in muscle tissue of CCx individuals, significant higher expression levels of IL-15 (P=0.0237) and TRIM63 (P=0.0442) were detected. miR-424-5p was identified to regulate the expression of lipase genes. Knock-down experiments in human visceral preadipocytes revealed an inverse association of miR-424-5p with its predicted target genes LIPE, PNPLA2, MGLL and LPL (P<0.01). The identified miRNAs, in particular miR-122-5p, miR-27b-3p, miR-375 and miR-424-5p, represent features of human CCx and may contribute to tissue wasting and skeletal muscle atrophy through the regulation of catabolic signals. Further studies are needed to explore the potential of the identified miRNAs as a screening tool for early detection of cancer cachexia.