Antioxidant capacity of human blood plasma and human urine: simultaneous evaluation of the ORAC index and ascorbic acid concentration employing pyrogallol red as probe.

PubMed

Torres, P; Galleguillos, P; Lissi, E; López-Alarcón, C

2008-10-15

The oxygen radical absorbance capacity (ORAC) methodology has been employed to estimate the antioxidant capacity of human blood plasma and human urine using pyrogallol red (ORAC-PGR) as target molecule. Uric acid, reduced glutathione, human serum albumin, and ascorbic acid (ASC) inhibited the consumption of pyrogallol red, but only ASC generated an induction time. Human blood plasma and human urine protected efficiently pyrogallol red. In these assays, both biological fluids generated neat induction times that were removed by ascorbate oxidase. From these results, ORAC-PGR method could be proposed as a simple alternative to evaluate an ORAC index and, simultaneously, to estimate the concentration of ascorbic acid in human blood plasma or human urine.

An ex vivo approach to botanical-drug interactions: a proof of concept study.

PubMed

Wang, Xinwen; Zhu, Hao-Jie; Munoz, Juliana; Gurley, Bill J; Markowitz, John S

2015-04-02

Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodology in applied ethnopharmacological research. This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems. Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal׳s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybin A, silybin B and hydrastine and berberine on CYP2C9 and CYP3A4/5, respectively, results which more closely mirrored those generated in clinical study. Data from conventional buffer-based in vitro studies were less predictive than the ex vivo assessments. Thus, this novel ex vivo approach may be more effective at predicting clinically relevant BDIs than conventional in vitro methods. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Profiling the Serum Protein Corona of Fibrillar Human Islet Amyloid Polypeptide.

PubMed

Pilkington, Emily H; Gustafsson, Ove J R; Xing, Yanting; Hernandez-Fernaud, Juan; Zampronio, Cleidi; Kakinen, Aleksandr; Faridi, Ava; Ding, Feng; Wilson, Paul; Ke, Pu Chun; Davis, Thomas P

2018-05-16

Amyloids may be regarded as native nanomaterials that form in the presence of complex protein mixtures. By drawing an analogy with the physicochemical properties of nanoparticles in biological fluids, we hypothesized that amyloids should form a protein corona in vivo that would imbue the underlying amyloid with a modified biological identity. To explore this hypothesis, we characterized the protein corona of human islet amyloid polypeptide (IAPP) fibrils in fetal bovine serum using two complementary methodologies developed herein: quartz crystal microbalance and "centrifugal capture", coupled with nanoliquid chromatography tandem mass spectroscopy. Clear evidence for a significant protein corona was obtained. No trends were identified for amyloid corona proteins based on their physicochemical properties, whereas strong binding with IAPP fibrils occurred for linear proteins or multidomain proteins with structural plasticity. Proteomic analysis identified amyloid-enriched proteins that are known to play significant roles in mediating cellular machinery and processing, potentially leading to pathological outcomes and therapeutic targets.

Photometric flow analysis system for biomedical investigations of iron/transferrin speciation in human serum.

PubMed

Strzelak, Kamil; Rybkowska, Natalia; Wiśniewska, Agnieszka; Koncki, Robert

2017-12-01

The Multicommutated Flow Analysis (MCFA) system for the estimation of clinical iron parameters: Serum Iron (SI), Unsaturated Iron Binding Capacity (UIBC) and Total Iron Binding Capacity (TIBC) has been proposed. The developed MCFA system based on simple photometric detection of iron with chromogenic agent (ferrozine) enables a speciation of transferrin (determination of free and Fe-bound protein) in human serum. The construction of manifold was adapted to the requirements of measurements under changing conditions. In the course of studies, a different effect of proteins on SI and UIBC determination has been proven. That was in turn the reason to perform two kinds of calibration methods. For measurements in acidic medium for SI/holotransferrin determination, the calibration curve method was applied, characterized by limit of determination and limit of quantitation on the level of 3.4 μmol L -1 and 9.1 μmol L -1 , respectively. The determination method for UIBC parameter (related to apotransferrin level) in physiological medium of pH 7.4 forced the use of standard addition method due to the strong influence of proteins on obtaining analytical signals. These two different methodologies, performed in the presented system, enabled the estimation of all three clinical iron/transferrin parameters in human serum samples. TIBC corresponding to total transferrin level was calculated as a sum of SI and UIBC. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel high-throughput method for supported liquid extraction of retinol and alpha-tocopherol from human serum and simultaneous quantitation by liquid chromatography tandem mass spectrometry.

PubMed

Hinchliffe, Edward; Rudge, James; Reed, Paul

2016-07-01

Measurement of vitamin A (retinol) and E (alpha-tocopherol) in UK clinical laboratories is currently performed exclusively by high-performance liquid chromatography with ultraviolet detection. We investigated whether retinol and alpha-tocopherol could be measured simultaneously by liquid chromatography tandem mass spectrometry. Serum samples (100 μL) were extracted using Isolute + Supported Liquid Extraction plates. Chromatography was performed on a Phenomenex Kinetex Biphenyl 2.6 μm, 50 × 2.1 mm column, and liquid chromatography tandem mass spectrometry on a Waters Acquity TQD. Injection-to-injection time was 4.3 min. The assay was validated according to published guidelines. Patient samples were used to compare liquid chromatography tandem mass spectrometry and high-performance liquid chromatography with ultraviolet detection methods. For retinol and alpha-tocopherol, respectively, the assay was linear up to 6.0 and 80.0 μmol/L, and lower limit of quantification was 0.07 and 0.26 μmol/L. Intra and interassay imprecision were within desirable analytical specifications. Analysis of quality control material aligned to NIST SRM 968e, and relative spiked recovery from human serum, both yielded results within 15% of target values. Method comparison with high-performance liquid chromatography with ultraviolet detection methodology demonstrated a negative bias for retinol and alpha-tocopherol by the liquid chromatography tandem mass spectrometry method. Analysis of United Kingdom National External Quality Assurance Scheme samples yielded mean bias from the target value of +3.0% for retinol and -11.2% for alpha-tocopherol. We have developed a novel, high-throughput method for extraction of retinol and alpha-tocopherol from human serum followed by simultaneous quantitation by liquid chromatography tandem mass spectrometry. The method offers a rapid, sensitive, specific and cost-effective alternative to high-performance liquid chromatography with ultraviolet detection methodology, and is suitable for routine clinical monitoring of patients predisposed to fat-soluble vitamin malabsorption. © The Author(s) 2015.

Human hair follicle organ culture: theory, application and perspectives.

PubMed

Langan, Ewan A; Philpott, Michael P; Kloepper, Jennifer E; Paus, Ralf

2015-12-01

For almost a quarter of a century, ex vivo studies of human scalp hair follicles (HFs) have permitted major advances in hair research, spanning diverse fields such as chronobiology, endocrinology, immunology, metabolism, mitochondrial biology, neurobiology, pharmacology, pigmentation and stem cell biology. Despite this, a comprehensive methodological guide to serum-free human HF organ culture (HFOC) that facilitates the selection and analysis of standard HF biological parameters and points out both research opportunities and pitfalls to newcomers to the field is still lacking. The current methods review aims to close an important gap in the literature and attempts to promote standardisation of human HFOC. We provide basic information outlining the establishment of HFOC through to detailed descriptions of the analysis of standard read-out parameters alongside practical examples. The guide closes by pointing out how serum-free HFOC can be utilised optimally to obtain previously inaccessible insights into human HF biology and pathology that are of interest to experimental dermatologists, geneticists, developmental biologists and (neuro-) endocrinologists alike and by highlighting novel applications of the model, including gene silencing and gene expression profiling of defined, laser capture-microdissected HF compartments. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pro-Inflammatory wnt5a and Anti-Inflammatory sFRP5 Are Differentially Regulated by Nutritional Factors in Obese Human Subjects

PubMed Central

Schulte, Dominik M.; Müller, Nike; Neumann, Katrin; Oberhäuser, Frank; Faust, Michael; Güdelhöfer, Heike; Brandt, Burkhard; Krone, Wilhelm; Laudes, Matthias

2012-01-01

Background Obesity is associated with macrophage infiltration of adipose tissue. These inflammatory cells affect adipocytes not only by classical cytokines but also by the secreted glycopeptide wnt5a. Healthy adipocytes are able to release the wnt5a inhibitor sFRP5. This protective effect, however, was found to be diminished in obesity. The aim of the present study was to examine (1) whether obese human subjects exhibit increased serum concentrations of wnt5a and (2) whether wnt5a and/or sFRP5 serum concentrations in obese subjects can be influenced by caloric restriction. Methodology 23 obese human subjects (BMI 44.1±1.1 kg/m2) and 12 age- and sex-matched lean controls (BMI 22.3±0.4 kg/m2) were included in the study. Obese subjects were treated with a very low-calorie diet (approximately 800 kcal/d) for 12 weeks. Body composition was assessed by impedance analysis, insulin sensitivity was estimated by HOMA-IR and the leptin-to-adiponectin ratio and wnt5a and sFRP5 serum concentrations were measured by ELISA. sFRP5 expression in human adipose tissue biopsies was further determined on protein level by immunohistology. Principal Findings Pro-inflammatory wnt5a was not measurable in any serum sample of lean control subjects. In patients with obesity, however, wnt5a became significantly detectable consistent with low grade inflammation in such subjects. Caloric restriction resulted in a weight loss from 131.9±4.0 to 112.3±3.2 kg in the obese patients group. This was accompanied by a significant decrease of HOMA-IR and leptin-to-adiponectin ratio, indicating improved insulin sensitivity. Interestingly, these metabolic improvements were associated with a significant increase in serum concentrations of the anti-inflammatory factor and wnt5a-inhibitor sFRP5. Conclusions/Significance Obesity is associated with elevated serum levels of pro-inflammatory wnt5a in humans. Furthermore, caloric restriction beneficially affects serum concentrations of anti-inflammatory sFRP5 in such subjects. These findings suggest a novel regulatory system in low grade inflammation in obesity, which can be influenced by nutritional therapy. PMID:22384249

In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van den Eede, Nele, E-mail: nele.vandeneede@uantwerpen.be; Erratico, Claudio; Exarchou, Vassiliki

Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites tomore » confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography–tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis–Menten model. Apparent K{sub m} values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148 μM, respectively. Apparent V{sub max} values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254 pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment. - Highlights: • First steps in the elucidation of TBOEP toxicokinetics • Quantification of TBOEP metabolites in human serum and liver microsomes • No detectable formation of BBOEP occurred with liver or serum enzymes. • Oxidative dealkylation to BBOEHEP was likely the major metabolic pathway. • 1D-NMR and 2D-NMR were used to tentatively assign structures of HO-TBOEP isomers.« less

Multi-analyte method development for analysis of brominated flame retardants (BFRs) and PBDE metabolites in human serum.

PubMed

Lu, Dasheng; Jin, Yu'e; Feng, Chao; Wang, Dongli; Lin, Yuanjie; Qiu, Xinlei; Xu, Qian; Wen, Yimin; She, Jianwen; Wang, Guoquan; Zhou, Zhijun

2017-09-01

Commonly, analytical methods measuring brominated flame retardants (BFRs) of different chemical polarities in human serum are labor consuming and tedious. Our study used acidified diatomaceous earth as solid-phase extraction (SPE) adsorbent and defatting material to simultaneously determine the most abundant BFRs and their metabolites with different polarities in human serum samples. The analytes include three types of commercial BFRs, tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) isomers, and polybrominated biphenyl ethers (PBDEs), and dominant hydroxylated BDE (OH-PBDE) and methoxylated BDE (MeO-PBDE) metabolites of PBDEs. The sample eluents were sequentially analyzed for PBDEs and MeO-BDEs on online gel permeation chromatography/gas chromatography-electron capture-negative ionization mass spectrometry (online GPC GC-ECNI-MS) and for TBBPA, HBCD, and OH-BDEs on liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method recoveries were 67-134% with a relative standard deviation (RSD) of less than 20%. Method detection limits (MDLs) were 0.30-4.20 pg/mL fresh weight (f.w.) for all analytes, except for BDE-209 of 16 pg/mL f.w. The methodology was also applied in a pilot study, which analyzed ten real samples from healthy donors in China, and the majority of target analytes were detected with a detection rate of more than 80%. To our knowledge, it is the first time for us in effectively determining BFRs of most types in one aliquot of human serum samples. This new analytical method is more specific, sensitive, accurate, and time saving for routine biomonitoring of these BFRs and for integrated assessment of health risk of BFR exposure.

Ultrasensitive quantification of serum estrogens in postmenopausal women and older men by liquid chromatography-tandem mass spectrometry

PubMed Central

Wang, Qingqing; Rangiah, Kannan; Mesaros, Clementina; Snyder, Nathaniel W.; Vachani, Anil; Song, Haifeng; Blair, Ian A.

2015-01-01

An ultrasensitive stable isotope dilution liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for multiplexed quantitative analysis of six unconjugated and conjugated estrogens in human serum. The quantification utilized a new derivatization procedure, which formed analytes as pre-ionized N-methyl pyridinium-3-sulfonyl (NMPS) derivatives. This method required only 0.1 mL of human serum, yet was capable of simultaneously quantifying six estrogens within 20 min. The lower limit of quantitation (LLOQ) for estradiol (E2), 16α-hydroxy (OH)-E2, 4-methoxy (MeO)-E2 and 2-MeO-E2 was 1 fg on column, and was 10 fg on column for 4-OH-E2 and 2-OH-E2. All analytes demonstrated a linear response from 0.5 to 200 pg/mL (5–2000 pg/mL for 4-OH-E2 and 2-OH-E2). Using this validated method, the estrogen levels in human serum samples from 20 female patients and 20 male patients were analyzed and compared. The levels found for unconjugated serum E2 from postmenopausal women (mean 2.7 pg/mL) were very similar to those obtained by highly sensitive gas chromatography-mass spectrometry (GC-MS) methodology. However, the level obtained in serum from older men (mean 9.5 pg/mL) was lower than has been reported previously by both GC-MS and LC-MS procedures. The total (unconjugated + conjugated) 4-MeO-E2 levels were significantly higher in female samples compared with males (p<0.05). The enhanced sensitivity offered by the present method will allow for a more specific analysis of estrogens and their metabolites. Our observations might suggest that the level of total 4-MeO-E2 could be a potential biomarker for breast cancer cases. PMID:25637677

Evaluation of Aspergillus PCR protocols for testing serum specimens.

PubMed

White, P Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J G; McCulloch, Elaine; Barnes, Rosemary A; Donnelly, J Peter; Loeffler, Juergen

2011-11-01

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens▿†

PubMed Central

White, P. Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J. G.; McCulloch, Elaine; Barnes, Rosemary A.; Donnelly, J. Peter; Loeffler, Juergen

2011-01-01

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance. PMID:21940479

The Navigation Guide—Evidence-Based Medicine Meets Environmental Health: Systematic Review of Human Evidence for PFOA Effects on Fetal Growth

PubMed Central

Sutton, Patrice; Atchley, Dylan S.; Koustas, Erica; Lam, Juleen; Sen, Saunak; Robinson, Karen A.; Axelrad, Daniel A.; Woodruff, Tracey J.

2014-01-01

Background: The Navigation Guide methodology was developed to meet the need for a robust method of systematic and transparent research synthesis in environmental health science. We conducted a case study systematic review to support proof of concept of the method. Objective: We applied the Navigation Guide systematic review methodology to determine whether developmental exposure to perfluorooctanoic acid (PFOA) affects fetal growth in humans. Methods: We applied the first 3 steps of the Navigation Guide methodology to human epidemiological data: 1) specify the study question, 2) select the evidence, and 3) rate the quality and strength of the evidence. We developed a protocol, conducted a comprehensive search of the literature, and identified relevant studies using prespecified criteria. We evaluated each study for risk of bias and conducted meta-analyses on a subset of studies. We rated quality and strength of the entire body of human evidence. Results: We identified 18 human studies that met our inclusion criteria, and 9 of these were combined through meta-analysis. Through meta-analysis, we estimated that a 1-ng/mL increase in serum or plasma PFOA was associated with a –18.9 g (95% CI: –29.8, –7.9) difference in birth weight. We concluded that the risk of bias across studies was low, and we assigned a “moderate” quality rating to the overall body of human evidence. Conclusion: On the basis of this first application of the Navigation Guide systematic review methodology, we concluded that there is “sufficient” human evidence that developmental exposure to PFOA reduces fetal growth. Citation: Johnson PI, Sutton P, Atchley DS, Koustas E, Lam J, Sen S, Robinson KA, Axelrad DA, Woodruff TJ. 2014. The Navigation Guide—evidence-based medicine meets environmental health: systematic review of human evidence for PFOA effects on fetal growth. Environ Health Perspect 122:1028–1039; http://dx.doi.org/10.1289/ehp.1307893 PMID:24968388

Chemometrics enhanced HPLC-DAD performance for rapid quantification of carbamazepine and phenobarbital in human serum samples.

PubMed

Vosough, Maryam; Ghafghazi, Shiva; Sabetkasaei, Masoumeh

2014-02-01

This paper describes development and validation of a simple and efficient bioanalytical procedure for simultaneous determination of phenobarbital and carbamazepine in human serum samples using high performance liquid chromatography with photodiode-array detection (HPLC-DAD) regarding a fast elution methodology in less than 5 min. Briefly, this method consisted of a simple deproteinization step of serum samples followed by HPLC analysis on a Bonus-RP column using an isocratic mode of elution with acetonitrile/K2HPO4 (pH=7.5) buffer solution (45:55). Due to the presence of serum endogenous components as non-calibrated components in the sample, second-order calibration based on multivariate curve resolution-alternating least squares (MCR-ALS), has been applied on a set of absorbance matrices collected as a function of retention time and wavelengths. Acceptable resolution and quantification results were achieved in the presence of matrix interferences and the second-order advantage was fully exploited. The average recoveries for carbamazepine and phenobarbital were 89.7% and 86.1% and relative standard deviation values were lower than 9%. Additionally, computed elliptical joint confidence region (EJCR) confirmed the accuracy of the proposed method and indicated the absence of both constant and proportional errors in the predicted concentrations. The developed method enabled the determination of the analytes in different serum samples in the presence of overlapped profiles, while keeping experimental time and extraction steps at minimum. Finally, the serum concentration levels of carbamazepine in three time intervals were reported for morphine-dependents who had received carbamazepine for treating their neuropathic pain. © 2013 Elsevier B.V. All rights reserved.

Chip-LC-MS for label-free profiling of human serum.

PubMed

Horvatovich, Peter; Govorukhina, Natalia I; Reijmers, Theo H; van der Zee, Ate G J; Suits, Frank; Bischoff, Rainer

2007-12-01

The discovery of biomarkers in easily accessible body fluids such as serum is one of the most challenging topics in proteomics requiring highly efficient separation and detection methodologies. Here, we present the application of a microfluidics-based LC-MS system (chip-LC-MS) to the label-free profiling of immunodepleted, trypsin-digested serum in comparison to conventional capillary LC-MS (cap-LC-MS). Both systems proved to have a repeatability of approximately 20% RSD for peak area, all sample preparation steps included, while repeatability of the LC-MS part by itself was less than 10% RSD for the chip-LC-MS system. Importantly, the chip-LC-MS system had a two times higher resolution in the LC dimension and resulted in a lower average charge state of the tryptic peptide ions generated in the ESI interface when compared to cap-LC-MS while requiring approximately 30 times less (~5 pmol) sample. In order to characterize both systems for their capability to find discriminating peptides in trypsin-digested serum samples, five out of ten individually prepared, identical sera were spiked with horse heart cytochrome c. A comprehensive data processing methodology was applied including 2-D smoothing, resolution reduction, peak picking, time alignment, and matching of the individual peak lists to create an aligned peak matrix amenable for statistical analysis. Statistical analysis by supervised classification and variable selection showed that both LC-MS systems could discriminate the two sample groups. However, the chip-LC-MS system allowed to assign 55% of the overall signal to selected peaks against 32% for the cap-LC-MS system.

Cells and biofluids analyzed in aqueous environment by infrared spectroscopy

NASA Astrophysics Data System (ADS)

Naumann, D.; Lasch, P.; Fabian, H.

2006-02-01

Infrared transmission/absorption measurements of cells and biofluids in water are restricted to very short optical pathlengths. When the amide I and amide II bands of protein constituents have to be analysed, path-lengths of less than 8 μm are necessary. Infrared spectra of cancer cells were collected from physiological buffer solutions utilizing custom-made mid-infrared compatible IR-cuvettes. The technology permitted to obtain cell-type specific spectral signatures and probe biochemical changes induced by varying temperatures or cell-drug interaction. Optical path-lengths of 8-30 μm were used on a set of microbial test strains to evaluate, whether the methodology can also be used to discriminate and identify micro-organisms. A semi-automatic methodology was developed for the analysis of liquid serum samples, which combines simple sample handling with high sample throughput and extreme measurement reproducibility. The applicability of this infrared technology to the analysis of liquid serum samples from cattle and human beings suffering from various acute viral or bacterial infections was explored testing the interrelationship between α-helical and β-sheet specific spectral signatures in the amide I band contour and total albumin and globulin content in serum. The technical details, advantages, and limitations of the new technology are described in the context of developing a routine, IR-based biodiagnostic technique for biofluids and biological cells.

An ex vivo approach to botanical-drug interactions: A proof of concept study

PubMed Central

Wang, Xinwen; Zhu, Hao-Jie; Munoz, Juliana; Gurley, Bill J.; Markowitz, John S.

2015-01-01

Ethnopharmacological relevance Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodoloy in applied ethnopharmacological research. Materials and Methods This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems. Results Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal’s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybinA, silybin B and hydrastine and berberine on CYP2C9 and CYP3A4/5, respectively, results which more closely mirrored those generated in clinical study. Conclusions Data from conventional buffer-based in vitro studies were less predictive than the ex vivo assessments. Thus, this novel ex vivo approach may be more effective at predicting clinically relevant BDIs than conventional in vitro methods. PMID:25623616

Methods of albumin estimation in clinical biochemistry: Past, present, and future.

PubMed

Kumar, Deepak; Banerjee, Dibyajyoti

2017-06-01

Estimation of serum and urinary albumin is routinely performed in clinical biochemistry laboratories. In the past, precipitation-based methods were popular for estimation of human serum albumin (HSA). Currently, dye-binding or immunochemical methods are widely practiced. Each of these methods has its limitations. Research endeavors to overcome such limitations are on-going. The current trends in methodological aspects of albumin estimation guiding the field have not been reviewed. Therefore, it is the need of the hour to review several aspects of albumin estimation. The present review focuses on the modern trends of research from a conceptual point of view and gives an overview of recent developments to offer the readers a comprehensive understanding of the subject. Copyright © 2017 Elsevier B.V. All rights reserved.

Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction.

PubMed

Chua, K H; Aminuddin, B S; Fuzina, N H; Ruszymah, B H I

2004-05-01

We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.

Role of IL-1 Beta in the Development of Human TH17 Cells: Lesson from NLPR3 Mutated Patients

PubMed Central

Lasigliè, Denise; Traggiai, Elisabetta; Federici, Silvia; Alessio, Maria; Buoncompagni, Antonella; Accogli, Andrea; Chiesa, Sabrina; Penco, Federica; Martini, Alberto; Gattorno, Marco

2011-01-01

Background T helper 17 cells (TH-17) represent a lineage of effector T cells critical in host defence and autoimmunity. In both mouse and human IL-1β has been indicated as a key cytokine for the commitment to TH-17 cells. Cryopyrin-associated periodic syndromes (CAPS) are a group of inflammatory diseases associated with mutations of the NLRP3 gene encoding the inflammasome component cryopyrin. In this work we asked whether the deregulated secretion of IL-1β secondary to mutations characterizing these patients could affect the IL-23/IL-17 axis. Methodology/Principal Findings A total of 11 CAPS, 26 systemic onset juvenile idiopathic arthritis (SoJIA) patients and 20 healthy controls were analyzed. Serum levels of IL-17 and IL-6 serum were assessed by ELISA assay. Frequency of TH17 cells was quantified upon staphylococcus enterotoxin B (SEB) stimulation. Secretion of IL-1β, IL-23 and IL-6 by monocyte derived dendritic cells (MoDCs), were quantified by ELISA assay. A total of 8 CAPS and 11 SoJIA patients were also analysed before and after treatment with IL-1β blockade. Untreated CAPS patients showed significantly increased IL-17 serum levels as well as a higher frequency of TH17 compared to control subjects. On the contrary, SoJIA patients displayed a frequency of TH17 similar to normal donors, but were found to have significantly increased serum level of IL-6 when compared to CAPS patients or healthy donors. Remarkably, decreased IL-17 serum levels and TH17 frequency were observed in CAPS patients following in vivo IL-1β blockade. On the same line, MoDCs from CAPS patients exhibited enhanced secretion of IL-1β and IL-23 upon TLRs stimulation, with a reduction after anti-IL-1 treatment. Conclusion/Significance These findings further support the central role of IL-1β in the differentiation of TH17 in human inflammatory conditions. PMID:21637346

Second generation multiple reaction monitoring assays for enhanced detection of ultra-low abundance Mycobacterium tuberculosis peptides in human serum.

PubMed

Mehaffy, Carolina; Dobos, Karen M; Nahid, Payam; Kruh-Garcia, Nicole A

2017-01-01

Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. Mtb resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins. Multiple reaction monitoring assays were optimized for the enhanced detection of 41 Mtb peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host m / z interference. The final optimized method was tested in 40 exosome samples from TB positive patients. Our enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the Mtb proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the Mtb strains circulating within each given region. An enhanced MRM method to detect ultra-low abundance Mtb peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development.

Mechanistic studies of the metabolic chiral inversion of (R)-ibuprofen in humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baillie, T.A.; Adams, W.J.; Kaiser, D.G.

1989-05-01

The metabolic chiral inversion of R-(-)-ibuprofen has been studied in human subjects by means of specific deuterium labeling and stereoselective gas chromatography-mass spectrometry methodology. After simultaneous p.o. administration of a mixture of R-(-)-ibuprofen (300 mg) and R-(-)-(3,3,3-2H3)ibuprofen (304 mg) to four adult male volunteers, the enantiomeric composition and deuterium content of the drug in serum, and of the drug and its principal metabolites in urine, were followed over a period of 24 hr. The results of these analyses indicated that: (1) conversion of R-(-)- to S-(+)-ibuprofen takes place with complete retention of deuterium at the beta-methyl (C-3) position; (2) chiralmore » inversion of R-(-)-(2H3)ibuprofen is not subject to a discernible deuterium isotope effect; and (3) replacement of the beta-methyl hydrogen atoms by deuterium has no effect on any of the serum pharmacokinetic parameters for R-(-)- or S-(+)-ibuprofen. These data indicate that the process whereby R-(-)-ibuprofen undergoes metabolic inversion in human subjects does not involve 2,3-dehydroibuprofen as an intermediate, and that the underlying mechanism cannot, therefore, entail a desaturation/reduction sequence.« less

Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative

PubMed Central

Mengoli, Carlo; Springer, Jan; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Klingspor, Lena; Lagrou, Katrien; Melchers, Willem J. G.; Morton, C. Oliver; Barnes, Rosemary A.; Donnelly, J. Peter; White, P. Lewis

2015-01-01

The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing. PMID:26085614

Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative.

PubMed

Loeffler, Juergen; Mengoli, Carlo; Springer, Jan; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Klingspor, Lena; Lagrou, Katrien; Melchers, Willem J G; Morton, C Oliver; Barnes, Rosemary A; Donnelly, J Peter; White, P Lewis

2015-09-01

The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing. Copyright © 2015 Loeffler et al.

A novel approach for estimating ingested dose associated with paracetamol overdose

PubMed Central

Zurlinden, Todd J.; Heard, Kennon

2015-01-01

Aim In cases of paracetamol (acetaminophen, APAP) overdose, an accurate estimate of tissue‐specific paracetamol pharmacokinetics (PK) and ingested dose can offer health care providers important information for the individualized treatment and follow‐up of affected patients. Here a novel methodology is presented to make such estimates using a standard serum paracetamol measurement and a computational framework. Methods The core component of the computational framework was a physiologically‐based pharmacokinetic (PBPK) model developed and evaluated using an extensive set of human PK data. Bayesian inference was used for parameter and dose estimation, allowing the incorporation of inter‐study variability, and facilitating the calculation of uncertainty in model outputs. Results Simulations of paracetamol time course concentrations in the blood were in close agreement with experimental data under a wide range of dosing conditions. Also, predictions of administered dose showed good agreement with a large collection of clinical and emergency setting PK data over a broad dose range. In addition to dose estimation, the platform was applied for the determination of optimal blood sampling times for dose reconstruction and quantitation of the potential role of paracetamol conjugate measurement on dose estimation. Conclusions Current therapies for paracetamol overdose rely on a generic methodology involving the use of a clinical nomogram. By using the computational framework developed in this study, serum sample data, and the individual patient's anthropometric and physiological information, personalized serum and liver pharmacokinetic profiles and dose estimate could be generated to help inform an individualized overdose treatment and follow‐up plan. PMID:26441245

A novel approach for estimating ingested dose associated with paracetamol overdose.

PubMed

Zurlinden, Todd J; Heard, Kennon; Reisfeld, Brad

2016-04-01

In cases of paracetamol (acetaminophen, APAP) overdose, an accurate estimate of tissue-specific paracetamol pharmacokinetics (PK) and ingested dose can offer health care providers important information for the individualized treatment and follow-up of affected patients. Here a novel methodology is presented to make such estimates using a standard serum paracetamol measurement and a computational framework. The core component of the computational framework was a physiologically-based pharmacokinetic (PBPK) model developed and evaluated using an extensive set of human PK data. Bayesian inference was used for parameter and dose estimation, allowing the incorporation of inter-study variability, and facilitating the calculation of uncertainty in model outputs. Simulations of paracetamol time course concentrations in the blood were in close agreement with experimental data under a wide range of dosing conditions. Also, predictions of administered dose showed good agreement with a large collection of clinical and emergency setting PK data over a broad dose range. In addition to dose estimation, the platform was applied for the determination of optimal blood sampling times for dose reconstruction and quantitation of the potential role of paracetamol conjugate measurement on dose estimation. Current therapies for paracetamol overdose rely on a generic methodology involving the use of a clinical nomogram. By using the computational framework developed in this study, serum sample data, and the individual patient's anthropometric and physiological information, personalized serum and liver pharmacokinetic profiles and dose estimate could be generated to help inform an individualized overdose treatment and follow-up plan. © 2015 The British Pharmacological Society.

Isolation and animal serum free expansion of human umbilical cord derived mesenchymal stromal cells (MSCs) and endothelial colony forming progenitor cells (ECFCs).

PubMed

Reinisch, Andreas; Strunk, Dirk

2009-10-08

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation. To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue. Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins. This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord. All materials and procedures will be described.

[The identification investigation of the material evidence of biological origin after multifactorial exposure].

PubMed

Iakovlev, D Iu; Solodun, Iu V; Proskurin, V N

1999-01-01

The possibility of investigating pieces of material evidence of biological origin after exposure to various factors is evaluated. The possibility of detecting proteins of liquid media of human organism by electrophoresis in polyacrylamide denatured gel is investigated. The method is intended for identification of biological material in a state of grave destruction. Methodology of such studies is proposed. The data indicate that the structural integrity and qualitative composition of the spectrum of main serum proteins are retained after combined exposure to damaging factors and complete destruction of blood cells.

Rapidly Escalating Hepcidin and Associated Serum Iron Starvation Are Features of the Acute Response to Typhoid Infection in Humans

PubMed Central

Darton, Thomas C.; Blohmke, Christoph J.; Giannoulatou, Eleni; Waddington, Claire S.; Jones, Claire; Sturges, Pamela; Webster, Craig; Drakesmith, Hal; Pollard, Andrew J.; Armitage, Andrew E.

2015-01-01

Background Iron is a key pathogenic determinant of many infectious diseases. Hepcidin, the hormone responsible for governing systemic iron homeostasis, is widely hypothesized to represent a key component of nutritional immunity through regulating the accessibility of iron to invading microorganisms during infection. However, the deployment of hepcidin in human bacterial infections remains poorly characterized. Typhoid fever is a globally significant, human-restricted bacterial infection, but understanding of its pathogenesis, especially during the critical early phases, likewise is poorly understood. Here, we investigate alterations in hepcidin and iron/inflammatory indices following experimental human typhoid challenge. Methodology/Principal Findings Fifty study participants were challenged with Salmonella enterica serovar Typhi and monitored for evidence of typhoid fever. Serum hepcidin, ferritin, serum iron parameters, C-reactive protein (CRP), and plasma IL-6 and TNF-alpha concentrations were measured during the 14 days following challenge. We found that hepcidin concentrations were markedly higher during acute typhoid infection than at baseline. Hepcidin elevations mirrored the kinetics of fever, and were accompanied by profound hypoferremia, increased CRP and ferritin, despite only modest elevations in IL-6 and TNF-alpha in some individuals. During inflammation, the extent of hepcidin upregulation associated with the degree of hypoferremia. Conclusions/Significance We demonstrate that strong hepcidin upregulation and hypoferremia, coincident with fever and systemic inflammation, are hallmarks of the early innate response to acute typhoid infection. We hypothesize that hepcidin-mediated iron redistribution into macrophages may contribute to S. Typhi pathogenesis by increasing iron availability for macrophage-tropic bacteria, and that targeting macrophage iron retention may represent a strategy for limiting infections with macrophage-tropic pathogens such as S. Typhi. PMID:26394303

Comparative analysis of EV isolation procedures for miRNAs detection in serum samples.

PubMed

Andreu, Zoraida; Rivas, Eva; Sanguino-Pascual, Aitana; Lamana, Amalia; Marazuela, Mónica; González-Alvaro, Isidoro; Sánchez-Madrid, Francisco; de la Fuente, Hortensia; Yáñez-Mó, María

2016-01-01

Extracellular vesicles (EVs) are emerging as potent non-invasive biomarkers. However, current methodologies are time consuming and difficult to translate to clinical practice. To analyse EV-encapsulated circulating miRNA, we searched for a quick, easy and economic method to enrich frozen human serum samples for EV. We compared the efficiency of several protocols and commercial kits to isolate EVs. Different methods based on precipitation, columns or filter systems were tested and compared with ultracentrifugation, which is the most classical protocol to isolate EVs. EV samples were assessed for purity and quantity by nanoparticle tracking analysis and western blot or cytometry against major EV protein markers. For biomarker validation, levels of a set of miRNAs were determined in EV fractions and compared with their levels in total serum. EVs isolated with precipitation-based methods were enriched for a subgroup of miRNAs that corresponded to miRNAs described to be encapsulated into EVs (miR-126, miR-30c and miR-143), while the detection of miR-21, miR-16-5p and miR-19a was very low compared with total serum. Our results point to precipitation using polyethylene glycol (PEG) as a suitable method for an easy and cheap enrichment of serum EVs for miRNA analyses. The overall performance of PEG was very similar, or better than other commercial precipitating reagents, in both protein and miRNA yield, but in comparison to them PEG is much cheaper. Other methods presented poorer results, mostly when assessing miRNA by qPCR analyses. Using PEG precipitation in a longitudinal study with human samples, we demonstrated that miRNA could be assessed in frozen samples up to 8 years of storage. We report a method based on a cut-off value of mean of fold EV detection versus serum that provides an estimate of the degree of encapsulation of a given miRNA.

Profilin-1 Is Expressed in Human Atherosclerotic Plaques and Induces Atherogenic Effects on Vascular Smooth Muscle Cells

PubMed Central

Caglayan, Evren; Romeo, Giulio R.; Kappert, Kai; Odenthal, Margarete; Südkamp, Michael; Body, Simon C.; Shernan, Stanton K.; Hackbusch, Daniel; Vantler, Marius; Kazlauskas, Andrius; Rosenkranz, Stephan

2010-01-01

Background Profilin-1 is an ubiquitous actin binding protein. Under pathological conditions such as diabetes, profilin-1 levels are increased in the vascular endothelium. We recently demonstrated that profilin-1 overexpression triggers indicators of endothelial dysfunction downstream of LDL signaling, and that attenuated expression of profilin-1 confers protection from atherosclerosis in vivo. Methodology Here we monitored profilin-1 expression in human atherosclerotic plaques by immunofluorescent staining. The effects of recombinant profilin-1 on atherogenic signaling pathways and cellular responses such as DNA synthesis (BrdU-incorporation) and chemotaxis (modified Boyden-chamber) were evaluated in cultured rat aortic and human coronary vascular smooth muscle cells (VSMCs). Furthermore, the correlation between profilin-1 serum levels and the degree of atherosclerosis was assessed in humans. Principal Findings In coronary arteries from patients with coronary heart disease, we found markedly enhanced profilin expression in atherosclerotic plaques compared to the normal vessel wall. Stimulation of rat aortic and human coronary VSMCs with recombinant profilin-1 (10−6 M) in vitro led to activation of intracellular signaling cascades such as phosphorylation of Erk1/2, p70S6 kinase and PI3K/Akt within 10 minutes. Furthermore, profilin-1 concentration-dependently induced DNA-synthesis and migration of both rat and human VSMCs, respectively. Inhibition of PI3K (Wortmannin, LY294002) or Src-family kinases (SU6656, PP2), but not PLCγ (U73122), completely abolished profilin-induced cell cycle progression, whereas PI3K inhibition partially reduced the chemotactic response. Finally, we found that profilin-1 serum levels were significantly elevated in patients with severe atherosclerosis in humans (p<0.001 vs. no atherosclerosis or control group). Conclusions Profilin-1 expression is significantly enhanced in human atherosclerotic plaques compared to the normal vessel wall, and the serum levels of profilin-1 correlate with the degree of atherosclerosis in humans. The atherogenic effects exerted by profilin-1 on VSMCs suggest an auto-/paracrine role within the plaque. These data indicate that profilin-1 might critically contribute to atherogenesis and may represent a novel therapeutic target. PMID:21049052

Maternal serum alpha-fetoprotein and human chorionic gonadotropin levels in women with human immunodeficiency virus.

PubMed

Gross, Susan; Castillo, Wilfrido; Crane, Marilyn; Espinosa, Bialines; Carter, Suzanne; DeVeaux, Richard; Salafia, Carolyn

2003-04-01

The purpose of this study was to establish whether there is a correlation between maternal serum genetic screen analyte results in pregnant women with human immunodeficiency virus and corresponding human immunodeficiency virus index values. Medical records of all pregnant women with human immunodeficiency virus who were delivered at Bronx Lebanon Hospital Center from January 2000 through December 2001 were reviewed for maternal serum screen results, viral load, CD4 counts and percent, antiretroviral therapy, opportunistic infections, substance abuse, and other demographic data. Statistical analysis was accomplished with the chi(2) test, Mann-Whitney U test, and Spearman rank correlation test, with a probability value of <.05 considered significant. Of the 98 women with human immunodeficiency virus who were delivered, 49 women (50%) had a maternal serum genetic screen available. Screened and unscreened women had similar severity of human immunodeficiency virus disease, CD4 count and percentage, and viral loads. Serum screen results showed elevations in maternal serum human chorionic gonadotropin (1.43 +/- 1.04 multiples of the median [MoM]; range, 0.2-5.2 MoM) and maternal serum alpha-fetoprotein (1.29 +/- 0.9 MoM; range, 0.5-3.3 MoM) compared with expected values in the general obstetric population. Maternal serum human chorionic gonadotropin was correlated inversely with CD4 count (P =.002) and CD4 percent (P <.0001). Maternal serum alpha-fetoprotein varied directly with viral load (P <.0001). Increasing maternal serum human chorionic gonadotropin and maternal serum alpha-fetoprotein levels in patients with human immunodeficiency virus are correlated with increasing viral load and decreasing CD4 counts.

A validated LC-MS/MS method for the quantitative measurement of creatinine as an endogenous biomarker in human plasma.

PubMed

Zhao, Yue; Liu, Guowen; Angeles, Aida; Christopher, Lisa J; Wang, Zhaoqing; Arnold, Mark E; Shen, Jim X

2016-10-01

Creatinine is an endogenous compound generated from creatine by normal muscular metabolism. It is an important indicator of renal function and the serum level is routinely monitored in clinical labs. Results & methodology: Surrogate analyte (d3-creatinine) was used for calibration standard and quality control preparation and the relative instrument response ratio between creatinine and d3-creatinine was used to calculate the endogenous creatinine concentrations. A fit-for-purpose strategy of using a surrogate analyte and authentic matrix was adopted for this validation. The assay was the first human plasma assay using such strategy and was successfully applied to a clinical study to confirm a transient elevation of creatinine observed using an existing clinical assay.

Pathogen inactivation of human serum facilitates its clinical use for islet cell culture and subsequent transplantation.

PubMed

Ståhle, Magnus U; Brandhorst, Daniel; Korsgren, Olle; Knutson, Folke

2011-01-01

Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept® technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37°C for 3-4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

An Aspartic Protease of the Scabies Mite Sarcoptes scabiei Is Involved in the Digestion of Host Skin and Blood Macromolecules

PubMed Central

Mahmood, Wajahat; Viberg, Linda T.; Fischer, Katja; Walton, Shelley F.; Holt, Deborah C.

2013-01-01

Background Scabies is a disease of worldwide significance, causing considerable morbidity in both humans and other animals. The scabies mite Sarcoptes scabiei burrows into the skin of its host, obtaining nutrition from host skin and blood. Aspartic proteases mediate a range of diverse and essential physiological functions such as tissue invasion and migration, digestion, moulting and reproduction in a number of parasitic organisms. We investigated whether aspartic proteases may play role in scabies mite digestive processes. Methodology/Principle Findings We demonstrated the presence of aspartic protease activity in whole scabies mite extract. We then identified a scabies mite aspartic protease gene sequence and produced recombinant active enzyme. The recombinant scabies mite aspartic protease was capable of digesting human haemoglobin, serum albumin, fibrinogen and fibronectin, but not collagen III or laminin. This is consistent with the location of the scabies mites in the upper epidermis of human skin. Conclusions/Significance The development of novel therapeutics for scabies is of increasing importance given the evidence of emerging resistance to current treatments. We have shown that a scabies mite aspartic protease plays a role in the digestion of host skin and serum molecules, raising the possibility that interference with the function of the enzyme may impact on mite survival. PMID:24244770

In vitro control of human bone marrow stromal cells for bone tissue engineering.

PubMed

Anselme, Karine; Broux, Odile; Noel, Benoit; Bouxin, Bertrand; Bascoulergue, Gerard; Dudermel, Anne-France; Bianchi, Fabien; Jeanfils, Joseph; Hardouin, Pierre

2002-12-01

For the clinical application of cultured human mesenchymal stem cells (MSCs), cells must have minimal contact with fetal calf serum (FCS) because it might be a potential vector for contamination by adventitious agents. The use of human plasma and serum for clinical applications also continues to give rise to considerable concerns with respect to the transmission of known and unknown human infectious agents. With the objective of clinical applications of cultured human MSCs, we tested the ability of autologous plasma, AB human serum, FCS, and artificial serum substitutes containing animal-derived proteins (Ultroser G) or vegetable-derived proteins (Prolifix S6) to permit their growth and differentiation in vitro. To conserve as much autologous plasma as possible, we attempted to mix it at decreasing concentrations with the serum substitute containing vegetable-derived mitogenic factors. Under control conditions, by day 10 all the fibroblast colony-forming units (CFU-Fs) were alkaline phosphatase (ALP) positive. However, their number and size were highly variable among donors. Better CFU-F formation was obtained with Ultroser G, and with human AB serum and autologous plasma mixed at, respectively, 5 and 1% with Prolifix S6. The effects of these mixtures on CFU-F formation demonstrate synergy, with the human serum or plasma supplying the factors that favor differentiation of MSCs while Prolifix S6 supplies the mitogenic factors. Finally, we demonstrated the possibility of controlling human MSC growth and differentiation in vitro. Notably, by means of a minimal quantity of human serum or human plasma mixed with a new serum substitute containing vegetable-derived proteins, we displayed growth and differentiation of human MSCs comparable to that obtained with FCS or serum substitutes containing animal-derived proteins. These results will have crucial significance for future applications of cultured human MSCs in bone tissue engineering.

Human serum reduces mitomycin-C cytotoxicity in human tenon's fibroblasts.

PubMed

Crowston, Jonathan G; Wang, Xiao Y; Khaw, Peng T; Zoellner, Hans; Healey, Paul R

2006-03-01

To determine the effect of human serum factors on mitomycin-C (MMC) cytotoxicity in cultured human subconjunctival Tenon's capsule fibroblasts. Fibroblast monolayers were treated with 5-minute applications of mitomycin-C (0.4 mg/mL) and incubated in culture medium with or without additional human serum. Fibroblast apoptosis was quantified by direct cell counts based on nuclear morphology, flow cytometry with annexin-V/propidium iodide, and a lactate dehydrogenase release assay. The number of viable fibroblasts and fibroblast proliferation were measured with a colorimetric MTT assay and by bromodeoxyuridine (BrdU) labeling. Mitomycin-C induced significant levels of fibroblast apoptosis. The addition of human serum resulted in a 40% reduction in MMC-induced fibroblast apoptosis (range, 31.3%-55.3%; P = 0.021) as determined by nuclear morphology and a 32.4% reduction measured by annexin-V/PI. There was a corresponding dose-dependent increase in the number of viable fibroblasts. Serum did not restore proliferation in MMC-treated fibroblasts. Factors present in human serum reduce MMC cytotoxicity in cultured human Tenon's fibroblasts. Human serum increased the number of viable fibroblasts by inhibiting MMC-induced fibroblast apoptosis. Serum factors access aqueous humor after trabeculectomy and may therefore influence the clinical outcome of MMC treatment.

Hexons from adenovirus serotypes 5 and 48 differentially protect adenovirus vectors from neutralization by mouse and human serum

PubMed Central

Harmon, Andrew W.; Moitra, Rituparna; Xu, Zhili

2018-01-01

Adenovirus vectors are widely used in gene therapy clinical trials, and preclinical studies with these vectors are often conducted in mice. It is therefore critical to understand whether mouse studies adequately predict the behavior of adenovirus vectors in humans. The most commonly-used adenovirus vectors are derived from adenovirus serotype 5 (Ad5). The Ad5 hexon protein can bind coagulation factor X (FX), and binding of FX has a major impact on vector interactions with other blood proteins. In mouse serum, FX protects Ad5 vectors from neutralization by natural antibodies and complement. In the current study, we similarly find that human FX inhibits neutralization of Ad5 vectors by human serum, and this finding is consistent among individual human sera. We show that human IgM and human IgG can each induce complement-mediated neutralization when Ad5 vectors are not protected by FX. Although mouse and human serum had similar effects on Ad5 vectors, we found that this was not true for a chimeric Ad5 vector that incorporated hexon regions from adenovirus serotype 48. Interestingly, this hexon-chimeric vector was neutralized by human serum, but not by mouse serum. These findings indicate that studies in mouse serum accurately predict the behavior of Ad5 vectors in human serum, but mouse serum is not an accurate model system for all adenovirus vectors. PMID:29401488

Brain-Derived Neurotrophic Factor in Patients with Huntington's Disease

PubMed Central

Zuccato, Chiara; Mariotti, Caterina; Valenza, Marta; Lahiri, Nayana; Wild, Edward J.; Sassone, Jenny; Ciammola, Andrea; Bachoud-Lèvi, Anne Catherine; Tabrizi, Sarah J.; Di Donato, Stefano; Cattaneo, Elena

2011-01-01

Reduced Brain-Derived Neurotrophic Factor (BDNF) levels have been described in a number of patho-physiological conditions, most notably, in Huntington's disease (HD), a progressive neurodegenerative disorder. Since BDNF is also produced in blood, we have undertaken the measurement of its peripheral levels in the attempt to identify a possible link with HD prognosis and/or its progression. Here we evaluated BDNF level in 398 blood samples including 138 controls, 56 preHD, and 204 HD subjects. We found that BDNF protein levels were not reliably different between groups, whether measured in plasma (52 controls, 26 preHD, 105 HD) or serum (39 controls, 5 preHD, 29 HD). Our experience, and a re-analysis of the literature highlighted that intra-group variability and methodological aspects affect this measurement, especially in serum. We also assessed BDNF mRNA levels in blood samples from 47 controls, 25 preHD, and 70 HD subjects, and found no differences among the groups. We concluded that levels of BDNF in human blood were not informative (mRNA levels or plasma protein level) nor reliable (serum protein levels) as HD biomarkers. We also wish to warn the scientific community in interpreting the significance of changes measured in BDNF protein levels in serum from patients suffering from different conditions. PMID:21857974

21 CFR 866.5700 - Whole human plasma or serum immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure by...

21 CFR 866.5700 - Whole human plasma or serum immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure by...

21 CFR 866.5700 - Whole human plasma or serum immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure by...

21 CFR 866.5700 - Whole human plasma or serum immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure by...

21 CFR 866.5700 - Whole human plasma or serum immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Whole human plasma or serum immunological test... Systems § 866.5700 Whole human plasma or serum immunological test system. (a) Identification. A whole human plasma or serum immunological test system is a device that consists of reagents used to measure by...

Human dental pulp stem cells cultured in serum-free supplemented medium

PubMed Central

Bonnamain, Virginie; Thinard, Reynald; Sergent-Tanguy, Solène; Huet, Pascal; Bienvenu, Géraldine; Naveilhan, Philippe; Farges, Jean-Christophe; Alliot-Licht, Brigitte

2013-01-01

Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs. PMID:24376422

Resveratrol-loaded glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles: Preparation, characterization, and targeting effect on liver tumors.

PubMed

Wu, Mingfang; Lian, Bolin; Deng, Yiping; Feng, Ziqi; Zhong, Chen; Wu, Weiwei; Huang, Yannian; Wang, Lingling; Zu, Chang; Zhao, Xiuhua

2017-08-01

In this study, glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles were prepared to establish a tumor targeting nano-sized drug delivery system. Glycyrrhizic acid was coupled to human serum albumin, and resveratrol was encapsulated in glycyrrhizic acid-conjugated human serum albumin by high-pressure homogenization emulsification. The average particle size of sample nanoparticles prepared under the optimal conditions was 108.1 ± 5.3 nm with a polydispersity index (PDI) of 0.001, and the amount of glycyrrhizic acid coupled with human serum albumin was 112.56 µg/mg. The drug encapsulation efficiency and drug loading efficiency were 83.6 and 11.5%, respectively. The glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles were characterized through laser light scattering, scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry, thermogravimetric analyses, and gas chromatography. The characterization results showed that resveratrol in glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles existed in amorphous state and the residual amounts of chloroform and methanol in nanoparticles were separately less than the international conference on harmonization (ICH) limit. The in vitro drug-release study showed that the nanoparticles released the drug slowly and continuously. The inhibitory rate of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide method. The IC50 values of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles and resveratrol were 62.5 and 95.5 µg/ml, respectively. The target ability of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles for HepG2 cells was evaluated using fluorescence-modified albumin techniques. The uptake rate of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles was higher than that of pure resveratrol and increased with increased nanoparticles concentration. The in vivo body distribution of glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles labeled with the near-infrared fluorophore Cy5 was monitored in H22 tumor-bearing mice through near-infrared fluorescence imaging systems. Glycyrrhizic acid-conjugated human serum albumin nanoparticles wrapping resveratrol nanoparticles exhibited effective target orientation to liver tumor and sustained-release property.

Identification and mapping of linear antibody epitopes in human serum albumin using high-density Peptide arrays.

PubMed

Hansen, Lajla Bruntse; Buus, Soren; Schafer-Nielsen, Claus

2013-01-01

We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm(2). Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.

Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation.

PubMed

Mizuno, Mitsuru; Katano, Hisako; Otabe, Koji; Komori, Keiichiro; Kohno, Yuji; Fujii, Shizuka; Ozeki, Nobutake; Horie, Masafumi; Tsuji, Kunikazu; Koga, Hideyuki; Muneta, Takeshi; Sekiya, Ichiro

2017-06-13

In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 μL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide - and annexin V - cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. The viability and chondrogenic potential of synovial MSCs were maintained when the cells were suspended in human serum at 4 and 13 °C.

Measurement of diabetic sugar concentration in human blood using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Firdous, S.; Nawaz, M.; Ahmed, M.; Anwar, S.; Rehman, A.; Rashid, R.; Mahmood, A.

2012-06-01

This study demonstrates the use of Raman spectroscopy for the direct measurement of diabetic sugar in human blood using 532 nm laser system. Raman spectra were collected from whole blood drawn from 21 individuals. We have elicited a reliable glucose signature in diabetic patients, and measured glucose levels in blood serum of normal, healthy diabetic and diabetic patients with other malignancies like cancer and hepatitis. Quantitative predictions of glucose spectra illustrate the predictions based on molecular information carried by the Raman light in highly light-scattering and absorbing media. Raman spectrum peaks for diabetic blood serum are observed at 1168, 1531, 1463, 1021 cm-1 with intensity level 17000 to 18500 pixels attributed to carbohydrates, proteins, lipids, collagen, and skeletal C-C stretch of lipids acyl chains. Raman spectra for normal, diabetic patients having cancer and hepatitis were also recorded. This in vitro glucose monitoring methodology will lead in vivo noninvasive on-line monitoring having painless and at the same time the data will be displayed on-line and in real time. The measured Raman peaks provides detailed bio-chemical fingerprint of the sample and could confer diagnostic benefit in a clinical setting.

Quantitative Method to Investigate the Balance between Metabolism and Proteome Biomass: Starting from Glycine.

PubMed

Gu, Haiwei; Carroll, Patrick A; Du, Jianhai; Zhu, Jiangjiang; Neto, Fausto Carnevale; Eisenman, Robert N; Raftery, Daniel

2016-12-12

The balance between metabolism and biomass is very important in biological systems; however, to date there has been no quantitative method to characterize the balance. In this methodological study, we propose to use the distribution of amino acids in different domains to investigate this balance. It is well known that endogenous or exogenous amino acids in a biological system are either metabolized or incorporated into free amino acids (FAAs) or proteome amino acids (PAAs). Using glycine (Gly) as an example, we demonstrate a novel method to accurately determine the amounts of amino acids in various domains using serum, urine, and cell samples. As expected, serum and urine had very different distributions of FAA- and PAA-Gly. Using Tet21N human neuroblastoma cells, we also found that Myc(oncogene)-induced metabolic reprogramming included a higher rate of metabolizing Gly, which provides additional evidence that the metabolism of proliferating cells is adapted to facilitate producing new cells. It is therefore anticipated that our method will be very valuable for further studies of the metabolism and biomass balance that will lead to a better understanding of human cancers. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analytical Methodologies for Detection of Gamma-Valerolactone, Delta-Valerolactone, Acephate and Azinphos Methyl and Their Associated Metabolites in Complex Biological Matrices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zink, E.; Clark, R.; Grant, K.

2005-01-01

Non-invasive biomonitoring for chemicals of interest in law enforcement and similar monitoring of pesticides, together with their metabolites, can not only save money but can lead to faster medical attention for individuals exposed to these chemicals. This study describes methods developed for the analysis of gamma-valerolactone (GVL), delta-valerolactone (DVL), acephate, and azinphos methyl in saliva and serum. Liquid chromatography/mass spectrometry (LC/MS) operated in the negative and positive ion mode and gas chromatography/mass spectrometry (GC/MS) were used to analyze GVL and DVL. Although both analytical techniques worked well, lower detection limits were obtained with GC/MS. The lactones and their corresponding sodiummore » salts were spiked into both saliva and serum. The lactones were isolated from saliva or serum using newly developed extraction techniques and then subsequently analyzed using GC/MS. The sodium salts of the lactones are nonvolatile and require derivatization prior to analysis by this method. N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) was ultimately selected as the reagent for derivatization because the acidic conditions required for reactions with diazomethane caused the salts to undergo intramolecular cyclization to the corresponding lactones. In vitro studies were conducted using rat liver microsomes to determine other metabolites associated with these compounds. Azinphos methyl and acephate are classified as organophosphate pesticides, and are known to be cholinesterase inhibitors in humans and insects, causing neurotoxicity. For this reason they have both exposure and environmental impact implications. These compounds were spiked into serum and saliva and prepared for analysis by GC/MS. Continuation of this research would include analysis by GC/MS under positive ion mode to determine the parent ions of the unknown metabolites. Further research is planned through an in vivo analysis of the lactones and pesticides. These methodologies could be extended for further analysis of other similar compounds.« less

Analytical Methodologies for Detection of Gamma-valerolactone, Delta-valerolactone, Acephate, and Azinphos Methyl and their Associated Metabolites in Complex Biological Matrices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zink, Erika M.; Clark, Ryan J.; Grant, Karen E.

2005-01-01

Non-invasive biomonitoring for chemicals of interest in law enforcement and similar monitoring of pesticides together with their metabolites can not only save money but can lead to faster medical attention for individuals exposed to these chemicals. This study describes methods developed for the analysis of gamma-valerolactone (GVL), delta-valerolactone (DVL), acephate, and azinphos methyl in saliva and serum. Liquid chromatography/mass spectrometry (LC/MS) operated in the negative ion mode and in the positive ion mode and gas chromatography/mass spectrometry (GC/MS) were used to analyze GVL and DVL. Although both analytical techniques worked well, lower detection limits were obtained with GC/MS. The lactonesmore » and their corresponding sodium salts were spiked into both saliva and serum. The lactones were isolated from saliva or serum using newly developed extraction techniques and then subsequently analyzed using GC/MS. The sodium salts of the lactones are nonvolatile and require derivatization prior to analysis by this method. N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) was ultimately selected as the reagent for derivatization because the acidic conditions required for reactions with diazomethane caused the salts to undergo intramolecular cyclization to the corresponding lactones. In vitro studies were conducted using rat liver microsomes to determine other metabolites associated with these compounds. Azinphos methyl and acephate are classified as organophosphate pesticides, and are known to be cholinesterase inhibitors in humans and insects, causing neurotoxicity. For this reason they have both exposure and environmental impact implications. These compounds were spiked into serum and saliva and prepared for analysis by GC/MS. Continuation of this research would include analysis by GC/MS under positive ion mode to determine the parent ions of the unknown metabolites. Further research is planned through an in vivo analysis of the lactones and pesticides. These methodologies could be extended for further analysis of other similar compounds as well as chemical and biological warfare agents.« less

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

PubMed

Bonazza, Camila; Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J; Girão, Manoel J B C; Oliva, Maria Luiza V; Castro, Rodrigo Aquino

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

PubMed Central

Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

A new supramolecular based liquid solid microextraction method for preconcentration and determination of trace bismuth in human blood serum and hair samples by electrothermal atomic absorption spectrometry.

PubMed

Kahe, Hadi; Chamsaz, Mahmoud

2016-11-01

A simple and reliable supramolecule-aggregated liquid solid microextraction method is described for preconcentration and determination of trace amounts of bismuth in water as well as human blood serum and hair samples. Catanionic microstructures of cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) surfactants, dissolved in deionized water/propanol, are used as a green solvent to extract bismuth (III)-diethyldithiocarbamate complexes by dispersive microextraction methodology. The extracted solid phase is easily removed and dissolved in 50 μL propanol for subsequent measurement by electrothermal atomic absorption spectrometry (ET-AAS). The procedure benefits the merits of supramolecule aggregates' properties and dispersive microextraction technique using water as the main component of disperser solvent, leading to direct interaction with analyte. Phase separation behavior of extraction solvent and different parameters influencing the extraction efficiency of bismuth ion such as salt concentration, pH, centrifugation time, amount of chelating agent, SDS:CTAB mole ratio, and solvent amounts were thoroughly optimized. Under the optimal experimental conditions, the calibration curve was linear in the range of 0.3-6 μg L -1 Bi (III) with a limit of detection (LOD) of 0.16 μg L -1 (S/N = 3). The relative standard deviations (RSD) of determination were obtained to be 5.1 and 6.2 % for 1 and 3 μg L -1 of Bi (III), respectively. The developed method was successfully applied as a sensitive and accurate technique for determination of bismuth ion in human blood serum, hair samples, and a certified reference material.

The solvatochromic effects of side chain substitution on the binding interaction of novel tricarbocyanine dyes with human serum albumin.

PubMed

Beckford, Garfield; Owens, Eric; Henary, Maged; Patonay, Gabor

2012-04-15

The effects of solvatochromism on protein-ligand interactions have been studied by absorbance and near-infrared laser induced fluorescence (NIR-LIF) spectroscopy. The utility of three novel classes of cyanine dyes designed for this purpose illustrates that the affinity interactions of ligands at the hydrophobic binding pockets of Human Serum Albumin (HSA) are not only dependent on the overall hydrophobic characteristics of the molecules but are highly influenced by the size of the ligands as well. Whereas changes to the chromophore moiety exhibited slight to moderate changes to the hydrophobic nature of these molecules, substitution at the alkyl indolium side chain has enabled us to vary the binding affinity towards serum albumin. Substitution at the indolium side chain among an ethyl to butyl group results in improved binding characteristics and an almost three-fold increase in affinity constant. In addition, replacement of the ethyl side chain with a phenylpropyl group also yielded unique solvotachromic patterns such as increased hydrophobicity and subsequent biocompatibility with the HSA binding regions. Ligand interaction was however inhibited by steric hindrance associated with the bulky phenyl ring system thus affecting the increased binding that could be realized from the improved hydrophobic nature of the molecules. This characteristic change in binding affinity is of potential interest to developing a methodology which reveals information on the hydrophobic character and steric specificity of the binding cavities. Copyright © 2012 Elsevier B.V. All rights reserved.

Assay of amoxicillin and clavulanic acid, the components of Augmentin, in biological fluids with high-performance liquid chromatography.

PubMed

Foulstone, M; Reading, C

1982-11-01

Augmentin is a new antibacterial formulation comprised of amoxicillin and the beta-lactamase inhibitor clavulanic acid. In the present paper, the use of high-performance liquid chromatography (HPLC) to provide a rapid assay of the components of Augmentin in body fluids is described. Clavulanic acid was assayed by reacting the sample with imidazole, which readily produces a derivative absorbing at 311 nm. This derivative chromatographs on reverse-phase HPLC columns clear of interfering components in both human serum and urine. Concentrations of clavulanic acid as low as 0.1 microgram/ml were readily detectable in human serum with this procedure. There was no interference from amoxicillin, amoxicillin penicilloic acid, or the acid and alkali degradation products of clavulanic acid when this assay system was used. Amoxicillin in body fluids was assayed directly by HPLC without derivatization. The same chromatographic conditions were employed for the assay of amoxicillin and the clavulanic acid derivative, simplifying the methodology. Amoxicillin, however, was determined of the antibiotic per ml. An alkali blanking procedure for amoxicillin and clavulanic acid is also described which allows the detection of any underlying peaks which may cochromatograph. The use of ultrafiltration to remove protein from serum samples before HPLC was successfully applied to the assay of clavulanic acid and amoxicillin. Ultrafiltration is not an essential procedure for these assays, but it prolongs column life and reduces interference in the amoxicillin assay. Results obtained by HPLC were compared with those obtained by using microbiological assays.

Deficiency of the Chemotactic Factor Inactivator in Human Sera with α1-Antitrypsin Deficiency

PubMed Central

Ward, Peter A.; Talamo, Richard C.

1973-01-01

As revealed by appropriate fractionation procedures, human serum deficient in α1-antitrypsin (α1-AT) is also deficient in the naturally occurring chemotactic factor inactivator. These serum donors had severe pulmonary emphysema. Serum from patients with clinically similar pulmonary disease, but with presence of α1-AT in the serum, showed no such deficiency of the chemotactic factor inactivator. When normal human serum and α1-AT-deficient human sera are chemotactically activated by incubation with immune precipitates, substantially more chemotactic activity is generated in α1-AT-deficient serum. These data indicate that in α1-AT-deficient serum there is an imbalance in the generation and control of chemotactic factors. It is suggested that the theory regarding development of pulmonary emphysema in patients lacking the α1-antitrypsin in their serum should be modified to take into account a deficiency of the chemotactic factor inactivator. PMID:4683887

Multiple free-radical scavenging (MULTIS) capacity in cattle serum.

PubMed

Sueishi, Yoshimi; Kamogawa, Erisa; Kimura, Anna; Kitahara, Go; Satoh, Hiroyuki; Asanuma, Taketoshi; Oowada, Shigeru

2017-01-01

Multiple free-radical scavenging (MULTIS) activity in cattle and human sera was evaluated with electron spin resonance spectroscopy. Scavenging rates against six active species, namely hydroxyl radical, superoxide anion, alkoxyl radical, alkylperoxyl radical, methyl radical, and singlet oxygen were quantified. The difference in the electron spin resonance signal intensity in the presence and absence of the serum was converted into the scavenging rates. Comparative MULTIS measurements were made in sera from eight beef cattle, three fetal calves and fifteen healthy human volunteers. Further, we determined the MULTIS value of albumin, the most abundant component in serum. MULTIS values in cattle sera indicated higher scavenging activity against most free radical species tested than human sera. In particular, cattle serum scavenging activities against superoxide and methyl radical were higher than human serum by 2.6 and 3.7 fold, respectively. In cattle serum, albumin appears to play a dominant role in MULTIS activity, but in human serum that is not the case. Previous data indicated that the abundance of uric acid in bovine blood is nearly 80% less than humans; however, this difference does not explain the deviation in MULTIS profile.

Preliminary crystallographic studies of four crystal forms of serum albumin

NASA Technical Reports Server (NTRS)

Carter, D. C.; Chang, B.; Ho, J. X.; Keeling, K.; Krishnasami, Z.

1994-01-01

Several crystal forms of serum albumin suitable for three-dimensional structure determination have been grown. These forms include crystals of recombinant and wild-type human serum albumin, baboon serum albumin, and canine serum albumin. The intrinsic limits of X-ray diffraction for these crystals are in the range 0.28-0.22 nm. Two of the crystal forms produced from human and canine albumin include incorporated long-chain fatty acids. Molecular replacement experiments have been successfully conducted on each crystal form using the previously determined atomic coordinates of human serum albumin illustrating the conserved tertiary structure.

Experimental Study on the Efficacy of Site-Specific PEGylated Human Serum Albumins in Resuscitation From Hemorrhagic Shock.

PubMed

Song, Xinlei; Zhang, Shu; Cheng, Yanna; Zhao, Ting; Lian, Qianqian; Lu, Lu; Wang, Fengshan

2016-11-01

To evaluate the resuscitative efficacy and the effect on reperfusion injury of two site-specific PEGylated human serum albumins modified with linear or branched PEG20kDa, compared with saline, 8% human serum albumin and 25% human serum albumin, in a hemorrhagic shock model. Laboratory. Male Wistar rats. Prospective study. Rats were bled to hemorrhagic hypovolemic shock and resuscitated with different resuscitation fluids. The mean arterial pressure and blood gas variables were measured. Hemorheology analysis was performed to evaluate the influence of resuscitation on RBCs and blood viscosity. The microvascular state was indirectly characterized in terms of monocyte chemotactic protein-1 and endothelial nitric oxide synthase that related to shear stress and vasodilation, respectively. The levels of inflammation-related factors and apoptosis-related proteins were used to evaluate the reperfusion injury in lungs. The results showed that PEGylated human serum albumin could improve the level of mean arterial pressure and blood gas variables more effectively at the end of resuscitation. poly(ethylene glycol) modification was able to increase the viscosity of human serum albumin to the level of effectively enhancing the expression of monocyte chemotactic protein-1 and endothelial nitric oxide synthase, which could promote microvascular perfusion. The hyperosmotic resuscitative agents including both 25% human serum albumin and PEGylated human serum albumins could greatly attenuate lung injury. No significant therapeutic advantages but some disadvantages were found for Y shaped poly(ethylene glycol) modification over linear poly(ethylene glycol) modification, such as causing the decrease of erythrocyte deformability. Linear high molecular weight site-specific PEGylated human serum albumin is recommended to be used as a hyperosmotic resuscitative agent.

Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis.

PubMed

Giménez, Estela; Balmaña, Meritxell; Figueras, Joan; Fort, Esther; de Bolós, Carme; Sanz-Nebot, Victòria; Peracaula, Rosa; Rizzi, Andreas

2015-03-25

In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α1-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

Relationship among serum taurine, serum adipokines, and body composition during 8-week human body weight control program.

PubMed

You, Jeong Soon; Park, Ji Yeon; Zhao, Xu; Jeong, Jin Seok; Choi, Mi Ja; Chang, Kyung Ja

2013-01-01

Human adipose tissue is not only a storage organ but also an active endocrine organ to release adipokines. This study was conducted to investigate the relationship among serum taurine and adipokine levels, and body composition during 8-week human body weight control program in obese female college students. The program consisted of diet therapy, exercise, and behavior modification. After the program, body weight, body fat mass, percent body fat, and body mass index (BMI) were significantly decreased. Serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels were significantly decreased. Also serum adiponectin level was significantly increased and serum leptin level was significantly decreased. There were no differences in serum taurine and homocysteine levels. The change of serum adiponectin level was positively correlated with change of body fat mass and percent body fat. These results may suggest that body fat loss by human body weight control program is associated with an increase in serum adiponectin in obese female college students. Therefore, further study such as taurine intervention study is needed to know more exact correlation between dietary taurine intake and serum adipokines or body composition.

Platelet-Rich Plasma (PRP) for Acute Muscle Injury: A Systematic Review

PubMed Central

A. Hamid, Mohamad Shariff; Yusof, Ashril; Mohamed Ali, Mohamed Razif

2014-01-01

Introduction Acute muscle injury is one of the commonest injuries that often result in loss of training and competition time. The best management for muscle injury has not been identified. Sports medicine practitioners used several approaches in attempt to accelerate time to recovery from muscle injury. More recently growing interest focussed on autologous blood product injection. Methods A literature search was conducted systematically using OvidMEDLINE, PubMed, EMBASE, SPORTDiscus and CINAHL databases to retrieve articles published until December 2012. Controlled trials and controlled laboratory studies comparing different strategies to promote early recovery of muscle injury were included. The methodological quality of studies was assessed. Results There are limited studies on the effects of PRP therapy for muscle injury. Three in vivo laboratory studies and one pilot human study were reviewed. The laboratory studies reported histological evidence on significant acceleration of muscle healing in animals treated with autologous conditioned serum (ACS), platelet-rich plasma (PRP) and platelet rich fibrin matrix (PRFM). A pilot human study found athletes treated with repeated ACS injection recovers significantly faster than retrospective controls. Conclusion Several in vivo laboratory studies suggest beneficial effects of ACS, PRP and PRFM in accelerating muscle recovery. Evidence to suggest similar effects on humans is however limited, as valuable information from robust human controlled trials is still not available at this moment. Hence, more studies of satisfactory methodological quality with platelet-rich plasma interventions on muscle injury are justified. PMID:24587389

Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry.

PubMed

Barnidge, David R; Dasari, Surendra; Ramirez-Alvarado, Marina; Fontan, Adrian; Willrich, Maria A V; Tschumper, Renee C; Jelinek, Diane F; Snyder, Melissa R; Dispenzieri, Angela; Katzmann, Jerry A; Murray, David L

2014-11-07

We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

Unexpected extent of immunochemical cross-reactions between rabbit and human serum proteins

PubMed Central

Johnson, P. K.; Yoder, J. M.

1970-01-01

Precipitin experiments indicated an unexpected extent of immunochemical cross-reactions between rabbit and human serum proteins. Commerical goat or horse antisera to human or rabbit serum were used. Two of the proteins involved in the cross-reactions were lipoproteins. Imagesp294-ap296-a PMID:4991121

[Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

PubMed

Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

2012-11-01

To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production.

PubMed

Uchida, Naoya; Demirci, Selami; Haro-Mora, Juan J; Fujita, Atsushi; Raines, Lydia N; Hsieh, Matthew M; Tisdale, John F

2018-06-15

In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro . To model therapeutic strategies, we transduced human CD34 + cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%-93%) in serum-free media than serum-containing media (20%-69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with β-globin lentiviral transduction in both CD34 + cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.

There Is a Method to the Madness: Strategies to Study Host Complement Evasion by Lyme Disease and Relapsing Fever Spirochetes.

PubMed

Marcinkiewicz, Ashley L; Kraiczy, Peter; Lin, Yi-Pin

2017-01-01

Lyme disease and relapsing fever are caused by various Borrelia species. Lyme disease borreliae , the most common vector-borne pathogens in both the U.S. and Europe, are transmitted by Ixodes ticks and disseminate from the site of tick bites to tissues leading to erythema migrans skin rash, arthritis, carditis, and neuroborreliosis. Relapsing fever borreliae , carried by ticks and lice, trigger reoccurring fever episodes. Following transmission, spirochetes survive in the blood to induce bacteremia at the early stages of infection, which is thought to promote evasion of the host complement system. The complement system acts as an important innate immune defense mechanism in humans and vertebrates. Upon activation, the cleaved complement components form complexes on the pathogen surface to eventually promote bacteriolysis. The complement system is negatively modulated by a number of functionally diverse regulators to avoid tissue damage. To evade and inhibit the complement system, spirochetes are capable of binding complement components and regulators. Complement inhibition results in bacterial survival in serum (serum resistance) and is thought to promote bloodstream survival, which facilitates spirochete dissemination and disease manifestations. In this review, we discuss current methodologies to elucidate the mechanisms of Borrelia spp. that promote serum resistance and bloodstream survival, as well as novel methods to study factors responsible for bloodstream survival of Lyme disease borreliae that can be applied to relapsing fever borreliae . Understanding the mechanisms these pathogens utilize to evade the complement system will ultimately aid in the development of novel therapeutic strategies and disease prevention to improve human health.

Assay of amoxicillin and clavulanic acid, the components of Augmentin, in biological fluids with high-performance liquid chromatography.

PubMed Central

Foulstone, M; Reading, C

1982-01-01

Augmentin is a new antibacterial formulation comprised of amoxicillin and the beta-lactamase inhibitor clavulanic acid. In the present paper, the use of high-performance liquid chromatography (HPLC) to provide a rapid assay of the components of Augmentin in body fluids is described. Clavulanic acid was assayed by reacting the sample with imidazole, which readily produces a derivative absorbing at 311 nm. This derivative chromatographs on reverse-phase HPLC columns clear of interfering components in both human serum and urine. Concentrations of clavulanic acid as low as 0.1 microgram/ml were readily detectable in human serum with this procedure. There was no interference from amoxicillin, amoxicillin penicilloic acid, or the acid and alkali degradation products of clavulanic acid when this assay system was used. Amoxicillin in body fluids was assayed directly by HPLC without derivatization. The same chromatographic conditions were employed for the assay of amoxicillin and the clavulanic acid derivative, simplifying the methodology. Amoxicillin, however, was determined of the antibiotic per ml. An alkali blanking procedure for amoxicillin and clavulanic acid is also described which allows the detection of any underlying peaks which may cochromatograph. The use of ultrafiltration to remove protein from serum samples before HPLC was successfully applied to the assay of clavulanic acid and amoxicillin. Ultrafiltration is not an essential procedure for these assays, but it prolongs column life and reduces interference in the amoxicillin assay. Results obtained by HPLC were compared with those obtained by using microbiological assays. PMID:7181486

Strain Background Modifies Phenotypes in the ATP8B1-Deficient Mouse

PubMed Central

Vargas, Julie C.; Xu, Hongmei; Groen, Annamiek; Paulusma, Coen C.; Grenert, James P.; Pawlikowska, Ludmila; Sen, Saunak; Elferink, Ronald P. J. Oude; Bull, Laura N.

2010-01-01

Background Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency. Methodology/Principal Findings We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains. Conclusions/Significance Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease. PMID:20126555

Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

PubMed

Kuchiike, Daisuke; Uto, Yoshihiro; Mukai, Hirotaka; Ishiyama, Noriko; Abe, Chiaki; Tanaka, Daichi; Kawai, Tomohito; Kubo, Kentaro; Mette, Martin; Inui, Toshio; Endo, Yoshio; Hori, Hitoshi

2013-07-01

The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

[Characterization of epithelial primary culture from human conjunctiva].

PubMed

Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

2014-01-01

To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.

PubMed

Kronewitter, Scott R; An, Hyun Joo; de Leoz, Maria Lorna; Lebrilla, Carlito B; Miyamoto, Suzanne; Leiserowitz, Gary S

2009-06-01

Annotation of the human serum N-linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state-transition networks. We find that at least 331 compositions are possible in the serum N-linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high-resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N-linked glycan mass library that was used for accurate high-throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan-based markers.

Interaction of benzo[a]pyrene diol epoxide isomers with human serum albumin: Site specific characterisation of adducts and associated kinetics

NASA Astrophysics Data System (ADS)

Motwani, Hitesh V.; Westberg, Emelie; Törnqvist, Margareta

2016-11-01

Carcinogenicity of benzo[a]pyrene {B[a]P, a polycyclic aromatic hydrocarbon (PAH)} involves DNA-modification by B[a]P diol epoxide (BPDE) metabolites. Adducts to serum albumin (SA) are not repaired, unlike DNA adducts, and therefore considered advantageous in assessment of in vivo dose of BPDEs. In the present work, kinetic experiments were performed in relation to the dose (i.e. concentration over time) of different BPDE isomers, where human SA (hSA) was incubated with respective BPDEs under physiological conditions. A liquid chromatography (LC) tandem mass spectrometry methodology was employed for characterising respective BPDE-adducts at histidine and lysine. This strategy allowed to structurally distinguish between the adducts from racemic anti- and syn-BPDE and between (+)- and (-)-anti-BPDE, which has not been attained earlier. The adduct levels quantified by LC-UV and the estimated rate of disappearance of BPDEs in presence of hSA gave an insight into the reactivity of the diol epoxides towards the N-sites on SA. The structure specific method and dosimetry described in this work could be used for accurate estimation of in vivo dose of the BPDEs following exposure to B[a]P, primarily in dose response studies of genotoxicity, e.g. in mice, to aid in quantitative risk assessment of PAHs.

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singaravelu, Ragunath; National Research Council of Canada, Ottawa, Ontario K1A 0R6; Lyn, Rodney K.

Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limitedmore » cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.« less

A novel culture method reveals unique neural stem/progenitors in mature porcine iris tissues that differentiate into neuronal and rod photoreceptor-like cells.

PubMed

Royall, Lars N; Lea, Daniel; Matsushita, Tamami; Takeda, Taka-Aki; Taketani, Shigeru; Araki, Masasuke

2017-11-15

Iris neural stem/progenitor cells from mature porcine eyes were investigated using a new protocol for tissue culture, which consists of dispase treatment and Matrigel embedding. We used a number of culture conditions and found an intense differentiation of neuronal cells from both the iris pigmented epithelial (IPE) cells and the stroma tissue cells. Rod photoreceptor-like cells were also observed but mostly in a later stage of culture. Neuronal differentiation does not require any additives such as fetal bovine serum or FGF2, although FGF2 and IGF2 appeared to promote neural differentiation in the IPE cultures. Furthermore, the stroma-derived cells were able to be maintained in vitro indefinitely. The evolutionary similarity between humans and domestic pigs highlight the potential for this methodology in the modeling of human diseases and characterizing human ocular stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Human umbilical cord blood serum promotes growth, proliferation, as well as differentiation of human bone marrow-derived progenitor cells.

PubMed

Phadnis, Smruti M; Joglekar, Mugdha V; Venkateshan, Vijayalakshmi; Ghaskadbi, Surendra M; Hardikar, Anandwardhan A; Bhonde, Ramesh R

2006-01-01

Fetal calf serum (FCS) is conventionally used for animal cell cultures due to its inherent growth-promoting activities. However animal welfare issues and stringent requirements for human transplantation studies demand a suitable alternative for FCS. With this view, we studied the effect of FCS, human AB serum (ABS), and human umbilical cord blood serum (UCBS) on murine islets of Langerhans and human bone marrow-derived mesenchymal-like cells (hBMCs). We found that there was no difference in morphology and functionality of mouse islets cultured in any of these three different serum supplements as indicated by insulin immunostaining. A comparative analysis of hBMCs maintained in each of these three different serum supplements demonstrated that UCBS supplemented media better supported proliferation of hBMCs. Moreover, a modification of adipogenic differentiation protocol using UCBS indicates that it can be used as a supplement to support differentiation of hBMCs into adipocytes. Our results demonstrate that UCBS not only is suitable for maintenance of murine pancreatic islets, but also supports attachment, propagation, and differentiation of hBMCs in vitro. We conclude that UCBS can serve as a better serum supplement for growth, maintenance, and differentiation of hBMCs, making it a more suitable supplement in cell systems that have therapeutic potential in human transplantation programs.

STUDY OF THE ADSORPTION ON AND DESORPTION FROM POLYSTYRENE-HUMAN SERUM ALBUMIN CONJUGATES OF RABBIT ANTI-HUMAN SERUM ALBUMIN ANTIBODIES HAVING DIFFERENT SPECIFICITIES

PubMed Central

Webb, Tom; Lapresle, Claude

1961-01-01

An insoluble specific adsorbent for anti-human serum albumin antibodies was prepared by coupling human serum albumin (H.S.A.) to polystyrene by azo bonds. Rabbit anti-H.S.A. immune serum was passed through a column of the adsorbent. It was shown that different volumes of the immune serum were required for the saturation of the different determinant groups of H.S.A. by their corresponding antibodies. The elution of the anti-H.S.A. antibodies adsorbed on the column was achieved by passing successively through the column an acetate buffer pH 3.0 and a solution of 0.1 N HCl in 0.15 M NaCl. The antibodies were eluted in three different fractions, each of which was composed of γ-globulins only. These three fractions contained different proportions of antibodies of different specificities. PMID:13783579

Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.

PubMed

Kunjithapatham, Rani; Geschwind, Jean-Francois; Devine, Lauren; Boronina, Tatiana N; O'Meally, Robert N; Cole, Robert N; Torbenson, Michael S; Ganapathy-Kanniappan, Shanmugasundaram

2015-04-03

Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

Sensitive determination of methadone in human serum and urine by dispersive liquid-liquid microextraction based on the solidification of a floating organic droplet followed by HPLC-UV.

PubMed

Taheri, Salman; Jalali, Fahimeh; Fattahi, Nazir; Jalili, Ronak; Bahrami, Gholamreza

2015-10-01

Dispersive liquid-liquid microextraction based on solidification of floating organic droplet was developed for the extraction of methadone and determination by high-performance liquid chromatography with UV detection. In this method, no microsyringe or fiber is required to support the organic microdrop due to the usage of an organic solvent with a low density and appropriate melting point. Furthermore, the extractant droplet can be collected easily by solidifying it at low temperature. 1-Undecanol and methanol were chosen as extraction and disperser solvents, respectively. Parameters that influence extraction efficiency, i.e. volumes of extracting and dispersing solvents, pH, and salt effect, were optimized by using response surface methodology. Under optimal conditions, enrichment factor for methadone was 134 and 160 in serum and urine samples, respectively. The limit of detection was 3.34 ng/mmL in serum and 1.67 ng/mL in urine samples. Compared with the traditional dispersive liquid-liquid microextraction, the proposed method obtained lower limit of detection. Moreover, the solidification of floating organic solvent facilitated the phase transfer. And most importantly, it avoided using high-density and toxic solvents of traditional dispersive liquid-liquid microextraction method. The proposed method was successfully applied to the determination of methadone in serum and urine samples of an addicted individual under methadone therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Serum osteopontin concentration is decreased by exercise-induced fat loss but is not correlated with body fat percentage in obese humans.

PubMed

You, Jeong Soon; Ji, Hye-In; Chang, Kyung Ja; Yoo, Myung Chul; Yang, Hyung-In; Jeong, In-Kyung; Kim, Kyoung Soo

2013-08-01

To evaluate the extent to which fat mass contributes to serum osteopontin (OPN) concentration, we investigated whether serum OPN levels are decreased by exercise-induced fat mass loss and whether they are associated with body fat percentage in obese humans. Twenty‑three female college students were recruited to participate in an 8‑week body weight control program. Body composition [body weight, soft lean mass, body fat mass, body fat percentage, waist-hip ratio and body mass index (BMI)] were assessed prior to and following the program. Serum lipid profiles and serum adiponectin, leptin and osteopontin levels were measured from serum collected prior to and following the program. To understand the effect of fat mass loss on the serum levels of adipokine, which is mainly produced in adipose tissue, the leptin and adiponectin levels were also measured prior to and following the program. Serum leptin levels (mean ± standard error of the mean) decreased significantly following the program (from 9.82±0.98 to 7.23±0.67 ng/ml) and were closely correlated with body fat percentage. In addition, serum adiponectin levels were negatively correlated with body fat percentage, while serum adiponectin levels were not significantly altered. By contrast, serum OPN levels decreased significantly following the program (from 16.03±2.34 to 10.65±1.22 ng/ml). However, serum OPN levels were not correlated with body fat percentage, suggesting that serum OPN levels are controlled by several other factors in humans. In conclusion, a high expression of OPN in adipose tissues may not be correlated with serum OPN levels in obese humans. Thus, tissues or physiological factors other than fat mass may have a greater contribution to the serum OPN levels.

Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma.

PubMed

Jeannot, Emmanuelle; Becette, Véronique; Campitelli, Maura; Calméjane, Marie-Ange; Lappartient, Emmanuelle; Ruff, Evelyne; Saada, Stéphanie; Holmes, Allyson; Bellet, Dominique; Sastre-Garau, Xavier

2016-10-01

Specific human papillomavirus genotypes are associated with most ano-genital carcinomas and a large subset of oro-pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus-associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus-16 or human papillomavirus-18-associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro-pharynx. As negative controls, 18 serum samples from women with human papillomavirus-16-associated high-grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at -80°C (27 cases) or at -20°C (43 cases). DNA was isolated from 200 µl of serum or plasma and droplet digital PCR was performed using human papillomavirus-16 E7 and human papillomavirus-18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at -80°C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 ± 85 copies/ml (stage I) to 1774 ± 3676 copies/ml (stage IV). Circulating human papillomavirus DNA is present in patients with human papillomavirus-associated invasive cancers even at sub-clinical stages and its level is related to tumour dynamics. Droplet digital PCR is a promising method for circulating human papillomavirus DNA detection and quantification. No positivity was found in patients with human papillomavirus-associated high grade cervical intraepithelial neoplasia.

Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus‐associated invasive carcinoma

PubMed Central

Jeannot, Emmanuelle; Becette, Véronique; Campitelli, Maura; Calméjane, Marie‐Ange; Lappartient, Emmanuelle; Ruff, Evelyne; Saada, Stéphanie; Holmes, Allyson; Bellet, Dominique

2016-01-01

Abstract Specific human papillomavirus genotypes are associated with most ano‐genital carcinomas and a large subset of oro‐pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus‐associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus‐16 or human papillomavirus‐18‐associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro‐pharynx. As negative controls, 18 serum samples from women with human papillomavirus‐16‐associated high‐grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at −80°C (27 cases) or at −20°C (43 cases). DNA was isolated from 200 µl of serum or plasma and droplet digital PCR was performed using human papillomavirus‐16 E7 and human papillomavirus‐18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at −80°C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 ± 85 copies/ml (stage I) to 1774 ± 3676 copies/ml (stage IV). Circulating human papillomavirus DNA is present in patients with human papillomavirus‐associated invasive cancers even at sub‐clinical stages and its level is related to tumour dynamics. Droplet digital PCR is a promising method for circulating human papillomavirus DNA detection and quantification. No positivity was found in patients with human papillomavirus‐associated high grade cervical intraepithelial neoplasia. PMID:27917295

Influence of Human Sera on the In Vitro Activity of the Echinocandin Caspofungin (MK-0991) against Aspergillus fumigatus

PubMed Central

Chiller, Tom; Farrokhshad, Kouros; Brummer, Elmer; Stevens, David A.

2000-01-01

There have been several reports that the activity of echinocandin antifungal agents is not affected or decreased in the presence of human sera. It is known that these drugs are bound >80% in animal and human sera. The activity of the echinocandin caspofungin (MK-0991), a 1,3-β-d-glucan synthase inhibitor, against Aspergillus fumigatus with and without human sera was studied. Conidia of A. fumigatus in microtest plate wells formed germlings after overnight culture in RPMI 1640. Caspofungin was then added with or without serum, and the germlings were incubated at 37°C for 24 h. Human serum (5%) in RPMI 1640 alone did not significantly inhibit the growth of A. fumigatus in vitro. Caspofungin in RPMI 1640 exhibited dose-dependent inhibition, with concentrations of 0.1 and 0.05 μg/ml inhibiting 24.9% +/− 10.4% and 11.7% +/− 3.6%, respectively (n = 10; P < 0.01). The addition of 5% human serum to caspofungin at 0.1 or 0.05 μg/ml increased the inhibition to 78.6% +/− 5.8% or 58.3% +/− 19.2%, respectively (n = 10; P < 0.01 versus controls and versus the drug without serum). Lower concentrations of serum also potentiated drug activity. The effect of human sera was further seen when using caspofungin that had lost activity (e.g., by storage) against A. fumigatus at 0.1 μg/ml. Inactive caspofungin alone demonstrated no significant inhibition of hyphal growth, whereas the addition of 5% human serum to the inactive drug showed 83% +/− 16.5% inhibition (n = 5; P < 0.01). The restoration of activity of caspofungin was seen at concentrations as low as 0.05% human serum. In contrast to prior reports, this study suggests that human serum acts synergistically with caspofungin to enhance its inhibitory activity in vitro against A. fumigatus. PMID:11083631

Capillary Electrophoresis of Mono- and Oligosaccharides.

PubMed

Toppazzini, Mila; Coslovi, Anna; Rossi, Marco; Flamigni, Anna; Baiutti, Edi; Campa, Cristiana

2016-01-01

This chapter reports an overview of the recent advances in the analysis of mono- and oligosaccharides by capillary electrophoresis (CE); furthermore, relevant reviews and research articles recently published in the field are tabulated. Additionally, pretreatments and procedures applied to uncharged and acidic carbohydrates (i.e., monosaccharides and lower oligosaccharides carrying carboxylate, sulfate, or phosphate groups) are described.Representative examples of such procedures are reported in detail, upon describing robust methodologies for the study of (1) neutral oligosaccharides derivatized by reductive amination and by formation of glycosylamines; (2) sialic acid derivatized with 2-aminoacridone, released from human serum immunoglobulin G; (3) anomeric couples of neutral glycosides separated using borate-based buffers; (4) unsaturated, underivatized oligosaccharides from lyase-treated alginate.

Plasma RNA integrity analysis: methodology and validation.

PubMed

Wong, Blenda C K; Lo, Y M Dennis

2006-09-01

The detection of cell-free RNA in plasma and serum of human subjects has found increasing applications in the field of medical diagnostics. However, many questions regarding the biology of circulating RNA remain to be addressed. One issue concerns the molecular nature of these circulating RNA species. We have recently developed a simple and quantitative method to investigate the integrity of plasma RNA. Our results have suggested that cell-free RNA in plasma is generally present as fragmented molecules instead of intact transcripts, with a predominance of 5' fragments. In this article, we summarize the basic principles in the experimental design for plasma RNA integrity analysis and highlight some of the important technical considerations for this type of investigation.

Lipid and Creatinine Adjustment to Evaluate Health Effects of Environmental Exposures.

PubMed

O'Brien, Katie M; Upson, Kristen; Buckley, Jessie P

2017-03-01

Urine- and serum-based biomarkers are useful for assessing individuals' exposure to environmental factors. However, variations in urinary creatinine (a measure of dilution) or serum lipid levels, if not adequately corrected for, can directly impact biomarker concentrations and bias exposure-disease association measures. Recent methodological literature has considered the complex relationships between creatinine or serum lipid levels, exposure biomarkers, outcomes, and other potentially relevant factors using directed acyclic graphs and simulation studies. The optimal measures of urinary dilution and serum lipids have also been investigated. Existing evidence supports the use of covariate-adjusted standardization plus creatinine adjustment for urinary biomarkers and standardization plus serum lipid adjustment for lipophilic, serum-based biomarkers. It is unclear which urinary dilution measure is best, but all serum lipid measures performed similarly. Future research should assess methods for pooled biomarkers and for studying diseases and exposures that affect creatinine or serum lipids directly.

Lipid and Creatinine Adjustment to Evaluate Health Effects of Environmental Exposures

PubMed Central

O’Brien, Katie M.; Upson, Kristen; Buckley, Jessie P.

2017-01-01

Purpose of review Urine- and serum-based biomarkers are useful for assessing individuals’ exposure to environmental factors. However, variations in urinary creatinine (a measure of dilution) or serum lipid levels, if not adequately corrected for, can directly impact biomarker concentrations and bias exposure-disease association measures. Recent findings Recent methodological literature has considered the complex relationships between creatinine or serum lipid levels, exposure biomarkers, outcomes, and other potentially relevant factors using directed acyclic graphs and simulation studies. The optimal measures of urinary dilution and serum lipids have also been investigated. Summary Existing evidence supports the use of covariate-adjusted standardization plus creatinine adjustment for urinary biomarkers and standardization plus serum lipid adjustment for lipophilic, serum-based biomarkers. It is unclear which urinary dilution measure is best, but all serum lipid measures performed similarly. Future research should assess methods for pooled biomarkers and for studying diseases and exposures that affect creatinine or serum lipids directly. PMID:28097619

Isomer-Specific Binding Affinity of Perfluorooctanesulfonate (PFOS) and Perfluorooctanoate (PFOA) to Serum Proteins.

PubMed

Beesoon, Sanjay; Martin, Jonathan W

2015-05-05

Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are among the most prominent contaminants in human serum, and these were historically manufactured as technical mixtures of linear and branched isomers. The isomers display unique pharmacokinetics in humans and in animal models, but molecular mechanisms underlying isomer-specific PFOS and PFOA disposition have not previously been studied. Here, ultrafiltration devices were used to examine (i) the dissociation constants (Kd) of individual PFOS and PFOA isomers with human serum albumin (HSA) and (ii) relative binding affinity of isomers in technical mixtures spiked to whole calf serum and human serum. Measurement of HSA Kd's demonstrated that linear PFOS (Kd=8(±4)×10(-8) M) was much more tightly bound than branched PFOS isomers (Kd range from 8(±1)×10(-5) M to 4(±2)×10(-4) M). Similarly, linear PFOA (Kd=1(±0.9)×10(-4) M) was more strongly bound to HSA compared to branched PFOA isomers (Kd range from 4(±2)×10(-4) M to 3(±2)×10(-4) M). The higher binding affinities of linear PFOS and PFOA to total serum protein were confirmed when both calf serum and human serum were spiked with technical mixtures. Overall, these data provide a mechanistic explanation for the longer biological half-life of PFOS in humans, compared to PFOA, and for the higher transplacental transfer efficiencies and renal clearance of branched PFOS and PFOA isomers, compared to the respective linear isomer.

Second-trimester maternal serum marker screening: maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, estriol, and their various combinations as predictors of pregnancy outcome.

PubMed

Yaron, Y; Cherry, M; Kramer, R L; O'Brien, J E; Hallak, M; Johnson, M P; Evans, M I

1999-10-01

We evaluated the value of all 3 common biochemical serum markers, maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, and unconjugated estriol, and combinations thereof as predictors of pregnancy outcome. A total of 60,040 patients underwent maternal serum screening. All patients had maternal serum alpha-fetoprotein measurements; beta-human chorionic gonadotropin was measured in 45,565 patients, and 24,504 patients had determination of all 3 markers, including unconjugated estriol. The incidences of various pregnancy outcomes were evaluated according to the serum marker levels by using clinically applied cutoff points. In confirmation of previous observations, increased maternal serum alpha-fetoprotein levels (>2.5 multiples of the median) were found to be significantly associated with pregnancy-induced hypertension, miscarriage, preterm delivery, intrauterine growth restriction, intrauterine fetal death, oligohydramnios, and abruptio placentae. Increased beta-human chorionic gonadotropin levels (>2.5 multiples of the median [MoM]) were significantly associated with pregnancy-induced hypertension, miscarriage, preterm delivery, and intrauterine fetal death. Finally, decreased unconjugated estriol levels (<0.5 MoM) were found to be significantly associated with pregnancy-induced hypertension, miscarriage, intrauterine growth restriction, and intrauterine fetal death. As with increased second-trimester maternal serum alpha-fetoprotein levels, increased serum beta-human chorionic gonadotropin and low unconjugated estriol levels are significantly associated with adverse pregnancy outcomes. These are most likely attributed to placental dysfunction. Multiple-marker screening can be used not only for the detection of fetal anomalies and aneu-ploidy but also for detection of high-risk pregnancies.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum*

PubMed Central

Wiedner, Susan D.; Burnum, Kristin E.; Pederson, LeeAnna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigné-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

2012-01-01

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli. PMID:22865858

Multiplexed activity-based protein profiling of the human pathogen Aspergillus fumigatus reveals large functional changes upon exposure to human serum.

PubMed

Wiedner, Susan D; Burnum, Kristin E; Pederson, LeeAnna M; Anderson, Lindsey N; Fortuin, Suereta; Chauvigné-Hines, Lacie M; Shukla, Anil K; Ansong, Charles; Panisko, Ellen A; Smith, Richard D; Wright, Aaron T

2012-09-28

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

PubMed

Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

2013-12-01

Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Physiological serum copper concentrations found in malignancies cause unfolding induced aggregation of human serum albumin in vitro.

PubMed

Rizvi, Asim; Furkan, Mohd; Naseem, Imrana

2017-12-15

Malignancies are characterized by several drastic metabolic changes, one of which is a progressive rise in the levels of serum copper. This rise in serum copper is documented across all malignancies and across malignancies in several species. This study aims to explore in vitro the effect of increased copper levels on the structure of the blood protein human serum albumin. Exposure of human serum albumin to physiologically relevant copper concentrations for 21 days resulted in structural modifications in the protein which were evident by changes in the intrinsic florescence. A loss of the predominantly alpha helical structure of human serum albumin was recorded along with a tendency to form protein aggregates. This aggregation was characterized by Thioflavin T and Congo Red assays. Rayleigh light scattering and turbidity assays confirmed aggregation. The aggregates were visually confirmed using transmission electron microscopy. This is the first report implicating increased copper levels as a cause of aggregation of blood proteins in malignancies. The physiological and biochemical implications of this phenomenon are discussed. Copyright © 2017. Published by Elsevier Inc.

Atomic structure and chemistry of human serum albumin

NASA Technical Reports Server (NTRS)

He, Xiao M.; Carter, Daniel C.

1992-01-01

The three-dimensional structure of human serum albumin has been determined crystallographically to a resolution of 2.8 A. It comprises three homologous domains that assemble to form a heart-shaped molecule. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and ILIA, which exhibit similar chemistry. The structure explains numerous physical phenomena and should provide insight into future pharmacokinetic and genetically engineered therapeutic applications of serum albumin.

[Lethal anaphylactic shock model induced by human mixed serum in guinea pigs].

PubMed

Ren, Guang-Mu; Bai, Ji-Wei; Gao, Cai-Rong

2005-08-01

To establish an anaphylactic shock model induced by human mixed serum in guinea pigs. Eighteen guinea pigs were divided into two groups: sensitized and control, The sensitized group were immunized intracutaneously with human mixed serum and then induced by endocardiac injection after 3 weeks. Symptoms of anaphylactic shock appeared in the sensitized group. The level of serum IgE were increased in the sensitized group significantly. An animal model of anaphylactic shock wer established successfully. It provide a tool for both forensic study and anaphylactic shock therapy.

Atomic structure and chemistry of human serum albumin

NASA Astrophysics Data System (ADS)

He, Xiao Min; Carter, Daniel C.

1992-07-01

The three-dimensional structure of human serum albumin has been determined crystallographically to a resolution of 2.8 Å. It comprises three homologous domains that assemble to form a heart-shaped molecule. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chemistry. The structure explains numerous physical phenomena and should provide insight into future pharmacokinetic and genetically engineered therapeutic applications of serum albumin.

The species origin of the serum in the culture medium influences the in vitro toxicity of silica nanoparticles to HepG2 cells.

PubMed

Pisani, Cédric; Rascol, Estelle; Dorandeu, Christophe; Gaillard, Jean-Charles; Charnay, Clarence; Guari, Yannick; Chopineau, Joël; Armengaud, Jean; Devoisselle, Jean-Marie; Prat, Odette

2017-01-01

The formation of a protein corona around nanoparticles can influence their toxicity, triggering cellular responses that may be totally different from those elicited by pristine nanoparticles. The main objective of this study was to investigate whether the species origin of the serum proteins forming the corona influences the in vitro toxicity assessment of silica nanoparticles. Coronas were preformed around nanoparticles before cell exposures by incubation in fetal bovine (FBS) or human (HS) serum. The compositions of these protein coronas were assessed by nano-LC MS/MS. The effects of these protein-coated nanoparticles on HepG2 cells were monitored using real-time cell impedance technology. The nanoparticle coronas formed in human or fetal bovine serum comprised many homologous proteins. Using human compared with fetal bovine serum, nanoparticle toxicity in HepG2 cells decreased by 4-fold and 1.5-fold, when used at 50 and 10μg/mL, respectively. It is likely that "markers of self" are present in the serum and are recognized by human cell receptors. Preforming a corona with human serum seems to be more appropriate for in vitro toxicity testing of potential nanocarriers using human cells. In vitro cytotoxicity assays must reflect in vivo conditions as closely as possible to provide solid and useful results.

Generation of immunosuppressive mesenchymal stem cells in allogeneic human serum.

PubMed

Le Blanc, Katarina; Samuelsson, Håkan; Lönnies, Lena; Sundin, Mikael; Ringdén, Olle

2007-10-27

Mesenchymal stem cells (MSC) may be used to treat acute graft-versus-host disease and for tissue repair. In vitro expansion of MSC has been achieved in the presence of fetal calf serum (FCS). For safety and regulatory reasons, we explored if FCS could be replaced by human blood group AB serum. Proliferation and fold increase of MSC was higher in the presence of AB-serum, compared to FCS. Similar to cells generated in FCS media, MSC from AB-serum media were more than 95% positive for CD90, CD105 and human leukocyte antigen (HLA) class I, and negative for hematopoietic and endothelial markers CD14, CD31, CD34, CD45, and CD80. HLA class II expression was higher in MSC generated in AB-serum, but decreased with higher passage numbers. MSC generated in AB-serum suppressed lymphocyte proliferation in mixed lymphocyte cultures and after stimulation with phytohemagglutinin. MSC expanded in AB-serum and FCS have similar in vitro properties.

Proteolysis inhibition by hibernating bear serum leads to increased protein content in human muscle cells.

PubMed

Chanon, Stéphanie; Chazarin, Blandine; Toubhans, Benoit; Durand, Christine; Chery, Isabelle; Robert, Maud; Vieille-Marchiset, Aurélie; Swenson, Jon E; Zedrosser, Andreas; Evans, Alina L; Brunberg, Sven; Arnemo, Jon M; Gauquelin-Koch, Guillemette; Storey, Kenneth B; Simon, Chantal; Blanc, Stéphane; Bertile, Fabrice; Lefai, Etienne

2018-04-03

Muscle atrophy is one of the main characteristics of human ageing and physical inactivity, with resulting adverse health outcomes. To date, there are still no efficient therapeutic strategies for its prevention and/or treatment. However, during hibernation, bears exhibit a unique ability for preserving muscle in conditions where muscle atrophy would be expected in humans. Therefore, our objective was to determine whether there are components of bear serum which can control protein balance in human muscles. In this study, we exposed cultured human differentiated muscle cells to bear serum collected during winter and summer periods, and measured the impact on cell protein content and turnover. In addition, we explored the signalling pathways that control rates of protein synthesis and degradation. We show that the protein turnover of human myotubes is reduced when incubated with winter bear serum, with a dramatic inhibition of proteolysis involving both proteasomal and lysosomal systems, and resulting in an increase in muscle cell protein content. By modulating intracellular signalling pathways and inducing a protein sparing phenotype in human muscle cells, winter bear serum therefore holds potential for developing new tools to fight human muscle atrophy and related metabolic disorders.

Analysis of human serum proteins by liquid phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry.

PubMed

Wang, Michael Z; Howard, Brandon; Campa, Michael J; Patz, Edward F; Fitzgerald, Michael C

2003-09-01

Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol.

Certified reference materials (GBW09170 and 09171) of creatinine in human serum.

PubMed

Dai, Xinhua; Fang, Xiang; Shao, Mingwu; Li, Ming; Huang, Zejian; Li, Hongmei; Jiang, You; Song, Dewei; He, Yajuan

2011-02-15

Creatinine is the most widely used clinical marker for assessing renal function. Concentrations of creatinine in human serum need to be carefully checked in order to ensure accurate diagnosis of renal function. Therefore, development of certified reference materials (CRMs) of creatinine in serum is of increasing importance. In this study, two new CRMs (Nos. GBW09170 and 09171) for creatinine in human serum have been developed. They were prepared with mixtures of several dozens of healthy people's and kidney disease patient's serum, respectively. The certified values of 8.10, 34.1 mg/kg for these two CRMs have been assigned by liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) method which was validated by using standard reference material (SRM) of SRM909b (a reference material obtained from National Institute of Standards and Technology, NIST). The expanded uncertainties of certified values for low and high concentrations were estimated to be 1.2 and 1.1%, respectively. The certified values were further confirmed by an international intercomparison for the determination of creatinine in human serum (Consultative Committee for Amount of Substance, CCQM) of K80 (CCQM-K80). These new CRMs of creatinine in human serum pool are totally native without additional creatinine spiked for enrichment. These new CRMs are capable of validating routine clinical methods for ensuring accuracy, reliability and comparability of analytical results from different clinical laboratories. They can also be used for instrument validation, development of secondary reference materials, and evaluating the accuracy of high order clinical methods for the determination of creatinine in human serum. Copyright © 2011 Elsevier B.V. All rights reserved.

Probing thyroglobulin in undiluted human serum based on pattern recognition and competitive adsorption of proteins

NASA Astrophysics Data System (ADS)

Wang, Ran; Huang, Shuai; Li, Jing; Chae, Junseok

2014-10-01

Thyroglobulin (Tg) is a sensitive indicator of persistent or recurrent differentiated thyroid cancer of follicular cell origin. Detection of Tg in human serum is challenging as bio-receptors, such as anti-Tg, used in immunoassay have relatively weak binding affinity. We engineer sensing surfaces using the competitive adsorption of proteins, termed the Vroman Effect. Coupled with Surface Plasmon Resonance, the "cross-responsive" interactions of Tg on the engineered surfaces produce uniquely distinguishable multiple signature patterns, which are discriminated using Linear Discriminant Analysis. Tg-spiked samples, down to 2 ng/ml Tg in undiluted human serum, are sensitively and selectively discriminated from the control (undiluted human serum).

The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

PubMed

Mirshafiee, Vahid; Kim, Raehyun; Mahmoudi, Morteza; Kraft, Mary L

2016-06-01

Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology.

PubMed

Knöspel, Fanny; Schindler, Rudolf K; Lübberstedt, Marc; Petzolt, Stephanie; Gerlach, Jörg C; Zeilinger, Katrin

2010-12-01

The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett-Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and L: -cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

Automation of a spectrophotometric method for measuring L -carnitine in human blood serum.

PubMed

Galan, A; Padros, A; Arambarri, M; Martin, S

1998-01-01

A spectrometric method for the determination of L-carnitine has been developed based on the reaction of the 5,5' dithiobis-(2-nitrobenzoic) acid (DTNB) and adapted to a Technicon RA-2000 automatic analyser Química Farmacéutica Bayer, S.A.). The detection limit of the method is 13.2 mumol/l, with a measurement interval ranging from 30 to 320 mumoll1. Imprecision and accuracy are good even at levels close to the detection limit (coeffcient of variation of 5.4% for within-run imprecision for a concentration of 35 mumol/l). A good correlation was observed between the method studied and the radiometric method. The method evaluated has suffcient analytical sensitivity to diagnose carnitine deficiencies. The short time period required for sample processing (30 samples in 40min), the simple methodology and apparatus, the ease of personnel training and the low cost of the reagents make this method a good alternative to the classical radiometric method for evaluating serum L-carnitine in clinical laboratories without radioactive installations.

Automation of a spectrophotometric method for measuring L -carnitine in human blood serum

PubMed Central

Galan, Amparo; Padros, Anna; Arambarri, Marta; Martin, Silvia

1998-01-01

A spectrometric method for the determination of L-carnitine has been developed based on the reaction of the 5, 5 ′ dithiobis-(2-nitrobenzoic) acid (DTNB) and adapted to a Technicon RA-2000 automatic analyser Química Farmacéutica Bayer, S.A.). The detection limit of the method is 13.2 μmol/l, with a measurement interval ranging from 30 to 320 μmoll1. Imprecision and accuracy are good even at levels close to the detection limit (coeffcient of variation of 5.4% for within-run imprecision for a concentration of 35 μmol/l). A good correlation was observed between the method studied and the radiometric method. The method evaluated has suffcient analytical sensitivity to diagnose carnitine deficiencies. The short time period required for sample processing (30 samples in 40min), the simple methodology and apparatus, the ease of personnel training and the low cost of the reagents make this method a good alternative to the classical radiometric method for evaluating serum L-carnitine in clinical laboratories without radioactive installations. PMID:18924818

Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

PubMed

Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey

2014-04-01

B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients. © 2013 APMIS. Published by John Wiley & Sons Ltd.

Aqueous Humor Rapidly Stimulates Myocilin Secretion from Human Trabecular Meshwork Cells

PubMed Central

Resch, Zachary T.; Hann, Cheryl R.; Cook, Kimberly A.; Fautsch, Michael P.

2010-01-01

Myocilin, a protein associated with the development of glaucoma, is expressed in most eye tissues with highest expression observed in trabecular meshwork cells. In culture, primary human trabecular meshwork cells incubated in 10% fetal bovine serum have reduced myocilin expression compared to in vivo, but incubation in human aqueous humor, their normal in vivo nutrient source, restores myocilin expression to near in vivo levels. To investigate the mechanism by which human aqueous humor stimulates myocilin accumulation in conditioned media from normal human trabecular meshwork cells, three independent trabecular meshwork cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing various supplements: fetal bovine serum (10%), human serum (0.2%), porcine aqueous humor (50%), bovine serum albumin (0.1%), dexamethasone (10−7 M), human aqueous humor (50%) or heat-inactivated human aqueous humor (50%). Conditioned media from cultured primary trabecular meshwork cells following incubation in human aqueous humor showed significant accumulation of myocilin in a time- (15 minutes) and dose-dependent manner (half maximal effective concentration ~ 30%) while intracellular myocilin levels decreased. Minimal myocilin accumulation was observed in conditioned media isolated from trabecular meshwork cells cultured in DMEM containing fetal bovine or human serum, bovine serum albumin, porcine aqueous humor, dexamethasone or DMEM alone. Heat inactivation of human aqueous humor nearly eliminated human aqueous humor-stimulated myocilin secretion. Inhibitors of new protein synthesis, gene transcription, the endoplasmic reticulum/Golgi system and endocytic/exocytic secretory pathways failed to inhibit human aqueous humor-stimulated myocilin secretion. Using immunolabeling and transmission electron microscopy, myocilin was found associated with 70–90 nm vesicle-like structures within the cytoplasm of human aqueous humor treated trabecular meshwork cells. These studies suggest that myocilin secretion from trabecular meshwork cells occurs in a Golgi-independent manner following human aqueous humor treatment. Heat-labile factors in human aqueous humor are responsible for the time- and dose-dependent release of myocilin from vesicle-like structures within the cytoplasm of trabecular meshwork cells. PMID:20932969

Molecular epidemiology of malaria in Cameroon. XV. Experimental studies on serum substitutes and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolates of Plasmodium falciparum.

PubMed

Basco, Leonardo K

2003-08-01

In vitro drug sensitivity assay is an important tool for various on-going studies aiming to establish the correlation between candidate molecular markers for drug resistance and drug response in laboratory-adapted strains and field isolates of Plasmodium falciparum. A widespread use of this technique in the field would require a suitable substitute that can replace human serum. In this study, several alternative sources of serum substitutes and supplements were evaluated for their capacity to sustain parasite growth for a single life cycle and their compatibility with in vitro assays for clinical isolates that have not been adapted to in vitro culture. Albumax, a commercial preparation of lipid-enriched bovine albumin, did not support parasite growth as much as human serum and fetal calf serum in several isolates. Other serum supplements (AmnioMax and Ultroser) supported parasite growth relatively well. The 50% inhibitory concentrations (IC50s) of chloroquine and antifolates determined with 0.05% Albumax were generally two or three times higher than with human serum. With 10% fetal calf serum, IC50s for chloroquine and antifolates were approximately two times higher and three times lower than with human serum, respectively. The use of AmnioMax and OptiMAb resulted in a greater than two-fold increase in IC50s and several uninterpretable assays. Despite possible batch-to-batch differences, fetal calf serum may be a suitable substitute for in vitro drug assays while awaiting the results of further studies on other serum substitutes and supplements.

In vitro cytotoxicity of superparamagnetic iron oxide nanoparticles on neuronal and glial cells. Evaluation of nanoparticle interference with viability tests.

PubMed

Costa, Carla; Brandão, Fátima; Bessa, Maria João; Costa, Solange; Valdiglesias, Vanessa; Kiliç, Gözde; Fernández-Bertólez, Natalia; Quaresma, Pedro; Pereira, Eulália; Pásaro, Eduardo; Laffon, Blanca; Teixeira, João Paulo

2016-03-01

Superparamagnetic iron oxide nanoparticles (ION) have attracted great interest for use in several biomedical fields. In general, they are considered biocompatible, but little is known of their effects on the human nervous system. The main objective of this work was to evaluate the cytotoxicity of two ION (magnetite), coated with silica and oleic acid, previously determining the possible interference of the ION with the methodological procedures to assure the reliability of the results obtained. Human neuroblastoma SHSY5Y and glioblastoma A172 cells were exposed to different concentrations of ION (5-300 µg ml(-1)), prepared in complete and serum-free cell culture medium for three exposure times (3, 6 and 24 h). Cytotoxicity was evaluated by means of the MTT, neutral red uptake and alamar blue assays. Characterization of the main physical-chemical properties of the ION tested was also performed. Results demonstrated that both ION could significantly alter absorbance readings. To reduce these interferences, protocols were modified by introducing additional washing steps and cell-free systems. Significant decreases in cell viability were observed for both cell lines in specific conditions by all assays. In general, oleic acid-coated ION were less cytotoxic than silica-coated ION; besides, a serum-protective effect was observed for both ION studied and cell lines. These results contribute to increase the knowledge of the potential harmful effects of ION on the human nervous system. Understanding these effects is essential to establish satisfactory regulatory policies on the safe use of magnetite nanoparticles in biomedical applications. Copyright © 2015 John Wiley & Sons, Ltd.

Isolation and recovery of selected polybrominated diphenyl ethers from human serum and sheep serum: coupling reversed-phase solid-phase disk extraction and liquid-liquid extraction techniques with a capillary gas chromatographic electron capture negative ion mass spectrometric determinative technique.

PubMed

Loconto, Paul R; Isenga, David; O'Keefe, Michael; Knottnerus, Mark

2008-01-01

Polybrominated diphenyl ethers (PBDEs) are isolated and recovered with acceptable percent recoveries from human serum via liquid-liquid extraction and column chromatographic cleanup and fractionation with quantitation using capillary gas chromatography-mass spectrometry with electron capture negative ion and selected ion monitoring. PBDEs are found in unspiked serum. An alternative sample preparation approach is developed using sheep serum that utilizes a formic acid pre-treatment followed by reversed-phase solid-phase disk extraction and normal-phase solid-phase cleanup using acidified silica gel that yields>50% recoveries. When these percent recoveries are combined with a minimized phase ratio for human serum and very low instrument detection limits, method detection limits below 500 parts-per-trillion are realized.

New methodology for the obtainment of antibothropic factors from the South American opossum (Didelphis marsupialis) and jararaca snake (Bothrops jararaca).

PubMed

Neves-Ferreira, A G; Valente, R H; Sá, P G; Rocha, S L; Moussatché, H; Domont, G B; Perales, J

1999-10-01

The antibothropic factor (ABF) from D. marsupialis was collected from perforated hollow plastic golf balls which were surgically implanted subcutaneously in anesthetized opossums, a technique originally described for the production of polyclonal antibodies. Two months after the implantation of the balls, approximately 15 ml of seromatous fluid from D. marsupialis (SFDm-50 mg total protein/ml) could be recovered monthly. Opossum serum as well as SFDm showed similar SDS-PAGE profiles and antihemorrhagic potencies against Bothrops jararaca snake venom (Bjv). The presence of ABF in SFDm was confirmed by immunoblotting, using rabbit polyclonal antibodies raised against ABF isolated from opossum serum. ABF isolated from SFDm or from serum by ion-exchange chromatography showed identical chromatographic and electrophoretic profiles. ABF fromboth sources displayed very similar antihemorrhagic and anticaseinolytic activities against Bjv. In the case of B. jararaca, polyethylene perforated tubes were inserted in the abdominal cavity and two months after implantation, approximately 4 ml of seromatous fluid from B. jararaca (SFBj-23 mg total protein/ml) were recovered. B.jararaca serum and SFBj showed the same native and SDS-PAGE band pattern. Both serum and SFBj inhibited Bjv hemorrhagic activity. We conclude that this new methodology is very suitable for continuously obtaining opossum ABF and SFBj, in large scale and in an easier way, avoiding animal suffering and eventual sacrifice.

Analysis of human serum phosphopeptidome by a focused database searching strategy.

PubMed

Zhu, Jun; Wang, Fangjun; Cheng, Kai; Song, Chunxia; Qin, Hongqiang; Hu, Lianghai; Figeys, Daniel; Ye, Mingliang; Zou, Hanfa

2013-01-14

As human serum is an important source for early diagnosis of many serious diseases, analysis of serum proteome and peptidome has been extensively performed. However, the serum phosphopeptidome was less explored probably because the effective method for database searching is lacking. Conventional database searching strategy always uses the whole proteome database, which is very time-consuming for phosphopeptidome search due to the huge searching space resulted from the high redundancy of the database and the setting of dynamic modifications during searching. In this work, a focused database searching strategy using an in-house collected human serum pro-peptidome target/decoy database (HuSPep) was established. It was found that the searching time was significantly decreased without compromising the identification sensitivity. By combining size-selective Ti (IV)-MCM-41 enrichment, RP-RP off-line separation, and complementary CID and ETD fragmentation with the new searching strategy, 143 unique endogenous phosphopeptides and 133 phosphorylation sites (109 novel sites) were identified from human serum with high reliability. Copyright © 2012 Elsevier B.V. All rights reserved.

Methods for the extraction and RNA profiling of exosomes

PubMed Central

Zeringer, Emily; Li, Mu; Barta, Tim; Schageman, Jeoffrey; Pedersen, Ketil Winther; Neurauter, Axl; Magdaleno, Susan; Setterquist, Robert; Vlassov, Alexander V

2013-01-01

AIM: To develop protocols for isolation of exosomes and characterization of their RNA content. METHODS: Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight® analysis, electron microscopy, and Western blots for CD63 marker. Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit. Finally, RNA was profiled using Bioanalyzer and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methodology. RESULTS: Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum, with subsequent isolation and analysis of RNA residing within these vesicles. The isolation procedure is completed in a fraction of the time, compared to the current standard protocols utilizing ultracentrifugation, and allows to recover fully intact exosomes in higher yields. Exosomes were found to contain a very diverse RNA cargo, primarily short sequences 20-200 nt (such as miRNA and fragments of mRNA), however longer RNA species were detected as well, including full-length 18S and 28S rRNA. CONCLUSION: We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes, followed by isolation of RNA and its analysis by qRT-PCR and other techniques. PMID:25237619

Carboxyl-rich plasma polymer surfaces in surface plasmon resonance immunosensing

NASA Astrophysics Data System (ADS)

Makhneva, Ekaterina; Obrusník, Adam; Farka, Zdeněk; Skládal, Petr; Vandenbossche, Marianne; Hegemann, Dirk; Zajíčková, Lenka

2018-01-01

Stable carboxyl-rich plasma polymers (PPs) were deposited onto the gold surface of surface plasmon resonance (SPR) chips under conditions that were chosen based on lumped kinetic model results. Carboxyl-rich films are of high interest for bio-applications thanks to their high reactivity, allowing the formation of covalent linkages between biomolecules and a surface. Accordingly, the monoclonal antibody, specific to human serum albumin (HSA), was immobilized and the performance of SPR immunosensors was evaluated by the immunoassay flow test. The developed sensors performed high level of stability and provided selective and high response to the HSA antigen solutions. The achieved results confirmed that the presented methodologies for the grafting of biomolecules on the gold surfaces have great potential for biosensing applications.

Aggregation of Human Eyelid Adipose-derived Stem Cells by Human Body Fluids

PubMed Central

Song, Yeonhwa; Yun, Sujin; Yang, Hye Jin; Yoon, A Young; Kim, Haekwon

2012-01-01

Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was 15.1±0.2×104 as the least for the low density group, and 29.3±2.8×104 as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids. PMID:25949109

Rapid killing of Capnocytophaga canimorsus and Capnocytophaga cynodegmi by human whole blood and serum is mediated via the complement system.

PubMed

Zangenah, Salah; Bergman, Peter

2015-01-01

Capnocytophaga canimorsus (Cani) and Capnocytophaga cynodegmi (Cyno) are found in the oral cavities of dogs and cats. They can be transmitted to humans via licks or bites and cause wound infections as well as severe systemic infections. Cani is considered to be more pathogenic than Cyno, but the pathophysiological mechanisms are not elucidated. Cani has been suggested to be resistant to serum bactericidal effects. Thus, we hypothesized that the more invasive Cani would exhibit a higher degree of serum-resistance than the less pathogenic Cyno. Whole blood and serum bactericidal assays were performed against Cani- (n = 8) and Cyno-strains (n = 15) isolated from blood and wound-specimens, respectively. Analysis of complement-function was performed by heat-inactivation, EGTA-treatment and by using C1q-depleted serum. Serum and whole blood were collected from healthy individuals and from patients (n = 3) with a history of sepsis caused by Cani. Both Cani and Cyno were equally susceptible to human whole blood and serum. Cani was preferentially killed by the classical pathway of the complement-system whereas Cyno was killed by a partly different mechanism. Serum from 2/3 Cani-infected patients were deficient in MBL-activity but still exhibited the same killing effect as control sera. Both Cani and Cyno were readily killed by human whole blood and serum in a complement-dependent way. Thus, it is not likely that serum bactericidal capacity is the key determinant for the clinical outcome in Cani or Cyno-infections.

Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

PubMed

Adams, Ralph; Griffin, Laura; Compson, Joanne E; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair Dg; Humphreys, David P; Heywood, Sam

2016-10-01

We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

Review of calcium methodologies.

PubMed

Zak, B; Epstein, E; Baginski, E S

1975-01-01

A review of calcium methodologies for serum has been described. The analytical systems developed over the past century have been classified as to type beginning with gravimetry and extending to isotope dilution-mass spectrometry by covering all of the commonly used technics which have evolved during that period. Screening and referee procedures are discussed along with comparative sensitivities encountered between atomic absorption spectrophotometry and molecular absorption spectrophotometry. A procedure involving a simple direct reaction for serum calcium using cresolphthalein complexone is recommended in which high blanks are minimized by repressing the ionization of the color reagent on lowering the dielectric constant characteristics of the mixture with dimethylsulfoxide. Reaction characteristics, errors which can be encountered, normal ranges and an interpretative resume are included in its discussion.

Determination of hydroxylated polychlorinated biphenyls (HO-PCBs) in blood plasma by high-performance liquid chromatography-electrospray ionization-tandem quadrupole mass spectrometry.

PubMed

Letcher, R J; Li, H X; Chu, S G

2005-01-01

Hydroxylated metabolites of polychlorinated biphenyls (HO-PCBs) and pentachlorophenol (PCP) are halogenated phenolic compounds, and they are increasingly common as environmental contaminants mainly in the blood of wildlife and humans. A methodology based on high-performance liquid chromatography (reversed-phase)-electrospray (negative) ionization-tandem quadrupole mass spectrometry (LC-ESI(-)-MS-MS) in the select ion monitoring or multiple reaction monitoring modes was developed for HO-PCB and PCP determination in blood plasma and serum. Among 11 environmentally relevant HO-PCB congeners and PCP spiked to fetal calf serum, quantitative assessments, including matrix effects on ESI(-) suppression/ enhancement, showed process (recovery) efficiencies of 73% to 89% without internal standard (IS) correction, and 88% to 103% with IS correction, and method limits of quantification ranging from 1 to 50 pg/g (wet weight). Using the developed LC-ESI(-)-MS methodology in comparison with GC-MS and GC-ECD based approaches, similar results were found for HO-PCB identification and quantification in the plasma of polar bear (Ursus maritimus) from the Canadian arctic. LC-ESI(-)-MS identified four HO-PCB congeners [4'-HO-2,2',4,6,6'-pentachlorobiphenyl (4'-HO-CB104), 4-HO-2,3,3',4',5-pentachlorobiphenyl (4-HO-CB107), 4-HO-2,3,3',5,5',6-hexachlorobiphenyl (4-HO-CB165) and 3'-HO-2,2',3,4,4',5,5'-heptachlorobiphenyl (3'-HO-CB180)], and 14 additional tetra- to hepta-chlorinated HO-PCBs isomers in the polar bear plasma.

Identification of fipronil metabolites by time-of-flight mass spectrometry for application in a human exposure study

PubMed Central

McMahen, Rebecca L.; Strynar, Mark J.; Dagnino, Sonia; Herr, David W.; Moser, Virginia C.; Garantziotis, Stavros; Andersen, Erik M.; Freeborn, Danielle L.; McMillan, Larry; Lindstrom, Andrew B.

2016-01-01

Fipronil is a phenylpyrazole insecticide commonly used in residential and agricultural applications. To understand more about the potential risks for human exposure associated with fipronil, urine and serum from dosed Long Evans adult rats (5 and 10 mg/kg bw) were analyzed to identify metabolites as potential biomarkers for use in human biomonitoring studies. Urine from treated rats was found to contain seven unique metabolites, two of which had not been previously reported—M4 and M7 which were putatively identified as a nitroso compound and an imine, respectively. Fipronil sulfone was confirmed to be the primary metabolite in rat serum. The fipronil metabolites identified in the respective matrices were then evaluated in matched human urine (n = 84) and serum (n = 96) samples from volunteers with no known pesticide exposures. Although no fipronil or metabolites were detected in human urine, fipronil sulfone was present in the serum of approximately 25% of the individuals at concentrations ranging from 0.1 to 4 ng/mL. These results indicate that many fipronil metabolites are produced following exposures in rats and that fipronil sulfone is a useful biomarker in human serum. Furthermore, human exposure to fipronil may occur regularly and require more extensive characterization. PMID:25687022

Identification of fipronil metabolites by time-of-flight mass spectrometry for application in a human exposure study.

PubMed

McMahen, Rebecca L; Strynar, Mark J; Dagnino, Sonia; Herr, David W; Moser, Virginia C; Garantziotis, Stavros; Andersen, Erik M; Freeborn, Danielle L; McMillan, Larry; Lindstrom, Andrew B

2015-05-01

Fipronil is a phenylpyrazole insecticide commonly used in residential and agricultural applications. To understand more about the potential risks for human exposure associated with fipronil, urine and serum from dosed Long Evans adult rats (5 and 10mg/kg bw) were analyzed to identify metabolites as potential biomarkers for use in human biomonitoring studies. Urine from treated rats was found to contain seven unique metabolites, two of which had not been previously reported-M4 and M7 which were putatively identified as a nitroso compound and an imine, respectively. Fipronil sulfone was confirmed to be the primary metabolite in rat serum. The fipronil metabolites identified in the respective matrices were then evaluated in matched human urine (n=84) and serum (n=96) samples from volunteers with no known pesticide exposures. Although no fipronil or metabolites were detected in human urine, fipronil sulfone was present in the serum of approximately 25% of the individuals at concentrations ranging from 0.1 to 4ng/mL. These results indicate that many fipronil metabolites are produced following exposures in rats and that fipronil sulfone is a useful biomarker in human serum. Furthermore, human exposure to fipronil may occur regularly and require more extensive characterization. Published by Elsevier Ltd.

ELISA measurement of stachylysin in serum to quantify human exposures to the indoor mold Stachybotrys chartarum.

PubMed

Van Emon, Jeanette M; Reed, Allan W; Yike, Iwona; Vesper, Stephen J

2003-06-01

The goal of this research was to develop a measurable indicator of human exposure to Stachyborys chartarum. Antibodies were produced against the hemolytic agent stachylysin obtained from the mold S. chartarum. These antibodies were used to develop two enzyme-linked immunosorbent assay methods for the analysis of stachylysin in human and rat sera and environmental samples. Stachylysin was measured in rat pups that received nasal instillations of S. chartarum conidia but not in control rat serum. Stachylysin in the serum of five human adults exposed to S. chartarum in water-damaged environments was 371 ng/mL but none was detected in the control serum. Stachylysin was also quantified in spore, wallboard, mycelial, and dust samples. The measurement of stachylysin may be a useful indicator in assessing human exposure to S. chartarum and in determining the presence of this indoor mold.

The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Szkudlarek-Haśnik, A.; Zubik-Skupień, I.; Góra, A.; Dubas, M.; Korzonek-Szlacheta, I.; Wielkoszyński, T.; Żurawiński, W.; Sosada, K.

2012-04-01

Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

The human serum metabolome of vitamin B-12 deficiency and repletion, and associations with neurological function

USDA-ARS?s Scientific Manuscript database

We characterize the human serum metabolome in sub-clinical vitamin B-12 (B-12) deficiency and repletion. A pre-post treatment study provided one injection of 10 mg B-12 to 27 community-dwelling elderly Chileans with B-12 deficiency evaluated with serum B-12, plasma homocysteine, methylmalonic acid a...

Silver vanadate nanoribbons: A label-free bioindicator in the conversion between human serum transferrin and apotransferrin via surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Zhou, Qing; Shao, Mingwang; Que, Ronghui; Cheng, Liang; Zhuo, Shujuan; Tong, Yanhua; Lee, Shuit-Tong

2011-05-01

Silver vanadate nanoribbons were synthesized via a hydrothermal process, which exhibited surface-enhanced Raman scattering effect. This surface-enhanced substrate was stable and reproducible for identifying human serum transferrin and human serum apotransferrin in the concentration of 1×10-5 M, which further exhibited significant sensitivity in monitoring the conversion of these two proteins in turn. This result showed that the silver vanadate nanoribbon might be employed as biomonitor in such systems.

Radioimmunoassay for glyburide in human serum. [Sulfonylurea; /sup 14/C and /sup 125/I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Royer, M.W.; Ko, H.; Evans, J.S.

1976-01-01

A radioimmunoassay (RIA) has been developed to measure nanogram amounts of drug-related materials in human serum, after oral administration of 1.25, 2.5, 3.75 or 5.0 mg glyburide, G, Micronase, a sulfonylurea. Of the compounds tested, only the known hydroxy metabolites of G cross-reacted significantly. Recoveries were quantitative (100.6 percent). In normal human volunteers, peak serum drug concentrations were observed at 4.3 +- 1.4 hrs (S.D., M = 32).

Sequences Of Amino Acids For Human Serum Albumin

NASA Technical Reports Server (NTRS)

Carter, Daniel C.

1992-01-01

Sequences of amino acids defined for use in making polypeptides one-third to one-sixth as large as parent human serum albumin molecule. Smaller, chemically stable peptides have diverse applications including service as artificial human serum and as active components of biosensors and chromatographic matrices. In applications involving production of artificial sera from new sequences, little or no concern about viral contaminants. Smaller genetically engineered polypeptides more easily expressed and produced in large quantities, making commercial isolation and production more feasible and profitable.

Probing Cocaine-Antibody Interactions in Buffer and Human Serum

PubMed Central

Ramakrishnan, Muthu; Alves De Melo, Fernando; Kinsey, Berma M.; Ladbury, John E.; Kosten, Thomas R.; Orson, Frank M.

2012-01-01

Background Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) we have evaluated the affinity properties of a representative mouse monoclonal (mAb08) as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum. Results MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20–50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC). This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies. Conclusions High sensitivity calorimetric determination of antibody binding to cocaine and its metabolites provide valuable information for characterization of their interactions and thermodynamic properties. In addition MST measurements of antibody affinity in the presence of biological fluids will provide a better opportunity to make reliable decisions and facilitate the design of cocaine vaccines and immunization conditions. The methods should be more widely adopted in characterization of antibody complexes. PMID:22859949

Detection of α-fetoprotein in human serum using carbon nanotube transistor

NASA Astrophysics Data System (ADS)

So, Hye-Mi; Park, Dong-Won; Lee, Seong-Kyu; Kim, Beom Soo; Chang, Hyunju; Lee, Jeong-O.

2009-03-01

We have fabricated antibody-coated carbon nanotube field effect transistor (CNT-FET) sensor for the detection of α-fetoprotein (AFP), single chain glycoprotein of 70 kDa that is normally expressed in the fetal liver, in human serum. The AFP-specific antibodies were immobilized on CNT with linker molecule such as pyrenebutyric acid N-hydroxysuccinimide ester. To prevent nonspecific adsorption of antigen, we performed blocking procedure using bovine serum albumin (BSA). Antibody-antigen binding was determined by measuring electrical conductance change of FET and took an average of thereshold voltage change before and after binding. Also we checked concentration-dependent conductance change in human serum using both p-type SWNT-FETs and n-type SWNT-FETs.

Brain cDNA clone for human cholinesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

McTiernan, C.; Adkins, S.; Chatonnet, A.

1987-10-01

A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum.more » The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase.« less

Application of silver films with different roughness parameter for septic human serum albumin detection by Surface Enhanced Raman Spectroscopy

NASA Astrophysics Data System (ADS)

Zyubin, A. Y.; Konstantinova, E. I.; Matveeva, K. I.; Slezhkin, V. A.; Samusev, I. G.; Demin, M. V.; Bryukhanov, V. V.

2018-01-01

In this paper, the rough silver films parameters investigation, used as media for surface enhancement Raman spectroscopy for health and septic human serum albumin (HSA) study results have been presented. The detection of small concentrations of HSA isolated from blood serum and it main vibrational groups identification has been done.

Enrichment of peptides in serum by C(8)-functionalized magnetic nanoparticles for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis.

PubMed

Yao, Ning; Chen, Hemei; Lin, Huaqing; Deng, Chunhui; Zhang, Xiangmin

2008-03-21

Human serum contains a complex array of proteolytically derived peptides (serum peptidome), which contain biomarkers of preclinical screening and disease diagnosis. Recently, commercial C(8)-functionalized magnetic beads (1-10 microm) were widely applied to the separation and enrichment of peptides in human serum, prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. In this work, laboratory-prepared C(8)-functionalized magnetic nanoparticles (about 50 nm) were prepared and applied to the fast separation and the enrichment of peptides from serum. At first, the C(8)-magnetic nanoparticles were synthesized by modifying amine-functionalized magnetic nanoparticles with chlorodimethyloctylsilane. These synthesized C(8)-amine-functionalized magnetic particles have excellent magnetic responsibility, high dispersibility and large surface area. Finally, the C(8)-magnetic nanoparticles were successfully applied to fast and efficient enrichment of low-abundance peptides from protein tryptic digestion and human serum followed by MALDI-TOF-MS analysis.

Isolation and partial characterisation of human riboflavin carrier protein and the estimation of its levels during human pregnancy.

PubMed

Natraj, U; George, S; Kadam, P

1988-06-01

Human cord serum contains protein(s) capable of binding to [14C]-riboflavin. Riboflavin-bound protein cross-reacts with anti-serum to chicken riboflavin carrier protein (cRCP). The carrier protein was isolated using affinity chromatography on a riboflavin AH Sepharose column. Bulk isolation and purification was also attempted by a combination of ion exchange, gel filtration and gel permeation chromatography on HPLC. The protein so isolated had a molecular weight of 36,000 +/- 2,000 daltons with an isoelectric point of 4.1. The levels of RCP in maternal serum during pregnancy were monitored using a sensitive heterologous radioimmunoassay system, using cRCP as standard and anti serum to cRCP. The levels of the protein increased after 4 months and remained significantly elevated up to 8 months. Although the level of the protein in the maternal serum remained low until 4 months, its level in amniotic fluid was elevated 2-3-fold as compared to that in serum.

Effect of the conditions of isolation on the physicochemical properties of human serum albumin in the norm and with pathology

NASA Astrophysics Data System (ADS)

Ivanov, A. I.; Zhbankov, R. G.; Korolenko, E. A.; Korolik, E. V.; Meleshchenko, L. A.; Sarnatskaya, V. V.; Nikolaev, V. G.; Nikolaichik, V. V.; Yushko, L. A.

1997-01-01

Differential scanning calorimetry and IR spectrosocopy were used to investigate the effect of the procedure of isolation of human serum albumin on its physicochemical characteristics. It is shown that fractionation of blood plasma with ethylene glycol followed by ion exchange chromatography can be used to obtain albumin of normal donors that is similar to the albumin in the nonfractionated plasma according to melting thermograms. Endotherms of human serum albumin samples that were obtained by affinity chromatography and preparative electrophoresis are bimodal, unlike the monophasic for albumin obtained by polyethylene glycol precipitation. These changes result from a higher content of nonetherified fatty acids in the albumin samples obtained by affinity chromatography and from modification of the secondary protein structure in the samples obtained by electrophoresis. Analysis of melting thermograms of serum albumin from patients with uremia, chronic hepatitis, and peritonitis shows that fractionation of blood with polyethylene glycol preserves the thermodynamic characteristics of the various pathological serum albumins to the greatest extent. The present results demonstrate the advantage of polyethylene glycol fractionation for isolation of native preparations of normal and “pathological” human serum albumin.

Identification of the trypanocidal factor in normal human serum: high density lipoprotein.

PubMed Central

Rifkin, M R

1978-01-01

The differentiation of Trypanosoma brucei from T. rhodesiense, the causative agent of human sleeping sickness, depends on their relative sensitivities to the cytotoxic effects of normal human serum. The molecule responsible for the specific lysis of T. brucei has now been isolated. Serum lipoproteins were fractionated and purified by ultracentrifugal flotation and chromatography on Bio-Gel A-5m. Trypanocidal activity was recovered in the high density lipoprotein fraction (density, 1.063-1.216 g/ml). Contamination by other serum proteins was checked by crossed immunoelectrophoresis and sodium dodecyl sulfate/acrylamide gel electrophoresis. Only a trace of beta-lipoprotein was found. The trypanocidal activity of pure human high density lipoprotein was identical to that of unfractionated serum when the following were tested: (i) time course of in vitro lysis of T. bruceli; (ii) in vivo destruction of T. brucei; (iii) relative resistance of T. rhodesiense to lysis. Rat or rabbit high density lipoprotein had no trypanocidal activity. Identification of the trypanocidal factor as high density lipoprotein was confirmed by the finding that serum from patients with Tangier disease, an autosomal recessive disorder characterized by a severe deficiency of high density lipoprotein, had no trypanocidal activity. Images PMID:210461

Single chain variable fragment displaying M13 phage library functionalized magnetic microsphere-based protein equalizer for human serum protein analysis.

PubMed

Zhu, Guijie; Zhao, Peng; Deng, Nan; Tao, Dingyin; Sun, Liangliang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2012-09-18

Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.

Beyond the Debye length in high ionic strength solution: direct protein detection with field-effect transistors (FETs) in human serum.

PubMed

Chu, Chia-Ho; Sarangadharan, Indu; Regmi, Abiral; Chen, Yen-Wen; Hsu, Chen-Pin; Chang, Wen-Hsin; Lee, Geng-Yen; Chyi, Jen-Inn; Chen, Chih-Chen; Shiesh, Shu-Chu; Lee, Gwo-Bin; Wang, Yu-Lin

2017-07-12

In this study, a new type of field-effect transistor (FET)-based biosensor is demonstrated to be able to overcome the problem of severe charge-screening effect caused by high ionic strength in solution and detect proteins in physiological environment. Antibody or aptamer-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) are used to directly detect proteins, including HIV-1 RT, CEA, NT-proBNP and CRP, in 1X PBS (with 1%BSA) or human sera. The samples do not need any dilution or washing process to reduce the ionic strength. The sensor shows high sensitivity and the detection takes only 5 minutes. The designs of the sensor, the methodology of the measurement, and the working mechanism of the sensor are discussed and investigated. A theoretical model is proposed based on the finding of the experiments. This sensor is promising for point-of-care, home healthcare, and mobile diagnostic device.

Chitosan-based ultrathin films as antifouling, anticoagulant and antibacterial protective coatings.

PubMed

Bulwan, Maria; Wójcik, Kinga; Zapotoczny, Szczepan; Nowakowska, Maria

2012-01-01

Ultrathin antifouling and antibacterial protective nanocoatings were prepared from ionic derivatives of chitosan using layer-by-layer deposition methodology. The surfaces of silicon, and glass protected by these nanocoatings were resistant to non-specific adsorption of proteins disregarding their net charges at physiological conditions (positively charged TGF-β1 growth factor and negatively charged bovine serum albumin) as well as human plasma components. The coatings also preserved surfaces from the formation of bacterial (Staphylococcus aureus) biofilm as shown using microscopic studies (SEM, AFM) and the MTT viability test. Moreover, the chitosan-based films adsorbed onto glass surface demonstrated the anticoagulant activity towards the human blood. The antifouling and antibacterial actions of the coatings were correlated with their physicochemical properties. The studied biologically relevant properties were also found to be dependent on the thickness of those nanocoatings. These materials are promising for biomedical applications, e.g., as protective coatings for medical devices, anticoagulant coatings and protective layers in membranes.

Lipidomics of human umbilical cord serum: identification of unique sterol sulfates.

PubMed

Wood, Paul L; Siljander, Heli; Knip, Mikael

2017-08-01

There are currently limited lipidomics data for human umbilical cord blood. Therefore, the lipidomes of cord sera from six newborns and sera from six nonpregnant females were compared. Sera lipidomics analyses were conducted using a high-resolution mass spectrometry analytical platform. Cord serum contained a diverse array of glycerophospholipids, albeit generally at lower concentrations than monitored in adult serum. The unexpected observations were that cord serum contained several neurosteroid sulfates and bile acid sulfates that were not detectable in adult serum. Our data are the first to demonstrate that cord serum contains bile acid sulfates that are synthesized early in the hydroxylase, neutral and acidic pathways of primary bile acid biosynthesis and support previous publications of cord blood perfluoralkyl toxins in newborns.

DIRECT AND INDIRECT FLUORESCENT-ANTIBODY TECHNIQUES FOR THE PSITTACOSIS-LYMPHOGRANULOMA VENEREUM-TRACHOMA GROUP OF AGENTS1

PubMed Central

Ross, Martin R.; Borman, Earle K.

1963-01-01

Ross, Martin R. (Connecticut State Department of Health, Hartford) and Earle K. Borman. Direct and indirect fluorescent-antibody techniques for the psittacosis-lymphogranuloma venereum-trachoma group of agents. J. Bacteriol. 85:851–858. 1963.—Direct and indirect fluorescent-antibody (FA) techniques were developed for the detection of group antigen in infected tissue cultures and the titration of group antibody in human antiserum. The growth of the agent of meningopneumonitis (MP) in mouse embryo lung cell monolayers was followed by infectivity and complement-fixing (CF) antigen titrations, and cytological examination of FA stained cultures. Although infectivity and CF antigen reached a peak at 2 days and remained constant for an additional 3 days, only cells tested 2 to 3 days after infection were suitable for FA staining with labeled anti-MP serum because of excessive artifacts in the older cultures. Fluorescein isothiocyanate-labeled rooster and guinea pig anti-MP serums and human antipsittacosis serums were titrated in direct FA and hemagglutination-inhibition (HI) tests. The rooster conjugate showed brighter staining and higher antibody titers than the guinea pig or human conjugates and was more effective in detecting minimal amounts of virus antigen. FA staining reactions with 1 and 2 units of labeled rooster serum were inhibited by unlabeled rooster serum but clear-cut inhibition with human antipsittacosis serum could not be demonstrated. The indirect FA technique was successfully used for the titration of group antibody in human serum. A comparison of the indirect FA, HI, and CF tests showed the indirect FA technique to be intermediate in sensitivity between the HI and CF tests. None of the three tests showed significant cross reactions with human serums reactive for influenza A and B; parainfluenza 1, 2, and 3; respiratory syncytial virus; Q fever; or the primary atypical pneumonia agent. PMID:14044954

Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

PubMed

Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

2010-02-15

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

Validation of the analytical method for the simultaneous determination of selected polybrominated diphenyl ethers, polychlorinated biphenyls and organochlorine pesticides in human blood serum by gas chromatography with microelectron capture detector.

PubMed

Matuszak, Małgorzata; Minorczyk, Maria; Góralczyk, Katarzyna; Hernik, Agnieszka; Struciński, Paweł; Liszewska, Monika; Czaja, Katarzyna; Korcz, Wojciech; Łyczewska, Monika; Ludwicki, Jan K

2016-01-01

Polybrominated diphenyl ethers (PBDEs) as other persistent organic pollutants like polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) pose a significant hazard to human health, mainly due to interference with the endocrine system and carcinogenetic effects. Humans are exposed to these substances mainly through a food of animal origin. These pollutants are globally detected in human matrices which requires to dispose reliable and simple analytical method that would enable further studies to assess the exposure of specific human populations to these compounds. The purpose of this study was to modify and validate of the analytical procedure for the simultaneous determination of selected PBDEs, PCBs and OCPs in human blood serum samples. The analytical measurement was performed by GC-µECD following preparation of serum samples (denaturation, multiple extraction, lipid removal). Identity of the compounds was confirmed by GC-MS. The method was characterised by the appropriate linearity, good repeatability (CV below 20%). The recoveries ranged from 52.9 to 125.0% depending on compound and level of fortification. The limit of quantification was set at 0.03 ng mL(-1) of serum. The modified analytical method proved to be suitable for the simultaneous determination of selected PBDEs, PCBs and OCPs in human blood serum by GC-µECD with good precision.

99M-technetium labeled macroaggregated human serum albumin pharmaceutical

DOEpatents

Winchell, Harry S.; Barak, Morton; Van Fleet, III, Parmer

1977-05-17

A reagent comprising macroaggregated human serum albumin having dispersed therein particles of stannous tin and a method for instantly making a labeled pharmaceutical therefrom, are disclosed. The labeled pharmaceutical is utilized in organ imaging.

[Presence of west Nile virus in northeast Mexico].

PubMed

Fernández-Salas, Ildefonso; de Lourdes Garza-Rodríguez, María; Beaty, Barry J; Jiménez, Javier Ramos; Rivas-Estilla, Ana María

2007-01-01

To investigate the presence of WNV in birds, horses and humans in northeast Mexico. Serum samples from 33 birds, 24 horses and 237 humans were screened by ELISA for Anti-WNV antibodies. Human serum samples were also screened for WNV RNA using an RT-PCR assay. Positive sera were found in three birds and 15 horses. Forty percent of the human serum samples were positive for IgG antibodies and 0% for IgM antibodies and viral RNA. The results of this study show that WNV is present in northeast Mexico and it is a new emergent infectious agent that represents a challenge for research and prevention programs in Mexico.

Human Milk and Matched Serum Demonstrate Concentration of Select miRNAs.

PubMed

Qin, Wenyi; Dasgupta, Santanu; Corradi, John; Sauter, Edward R

Pregnancy-associated breast cancers (PABCs), especially those diagnosed after childbirth, are often aggressive, with a poor prognosis. Factors influencing PABC are largely unknown. Micro(mi)RNAs are present in many human body fluids and shown to influence cancer development and/or growth. In six nursing mothers, we determined if breast cancer-associated miRNAs were (1) detectable in human breast milk and (2) if detectable, their relative expression in milk fractions compared to matched serum. We evaluated by quantitative PCR the expression of 11 cancer-associated miRNAs (10a-5p, 16, 21, 100, 140, 145, 155, 181, 199, 205, 212) in breast milk cells, fat and supernatant (skim milk), and matched serum. miRNA expression was detectable in all samples. For 10/11 miRNAs, mean relative expression compared to control (ΔCt) values was lowest in milk cells, the exception being miR205. Relative concentration was highest in the skim fraction of milk for all miRNAs. Expression was higher in skim milk than matched serum for 7/11 miRNAs and in serum for 4/11 miRNAs. miR205 expression was higher in all milk fractions than in matched serum. In conclusion, the expression of breast cancer-associated miRNAs is detectable in human breast milk and serum samples. The concentration is highest in skim milk, but is also detectable in milk fat and milk cells.

Is sonic Hedgehog involved in human fracture healing? --a prospective study on local and systemic concentrations of SHH.

PubMed

Eipeldauer, Stefan; Thomas, Anita; Hoechtl-Lee, Leonard; Kecht, Mathias; Binder, Harald; Koettstorfer, Julia; Gregori, Markus; Sarahrudi, Kambiz

2014-01-01

Sonic Hedgehog (SHH) is a new signalling pathway in bone repair. Evidence exist that SHH pathway plays a significant role in vasculogenesis and limb development during embryogenesis. Some in vitro and animal studies has already proven its potential for bone regeneration. However, no data on the role of SHH in the human fracture healing have been published so far. Seventy-five patients with long bone fractures were included into the study and divided in 2 groups. First group contained 69 patients with normal fracture healing. Four patients with impaired fracture healing formed the second group. 34 volunteers donated blood samples as control. Serum samples were collected over a period of 1 year following a standardized time schedule. In addition, SHH levels were measured in fracture haematoma and serum of 16 patients with bone fractures. Fracture haematoma and patients serum both contained lower SHH concentrations compared to control serum. The comparison between the patients' serum SHH level and the control serum revealed lower levels for the patients at all measurement time points. Significantly lower concentrations were observed at weeks 1 and 2 after fracture. SHH levels were slightly decreased in patients with impaired fracture healing without statistical significance. This is the first study to report local and systemic concentration of SHH in human fracture healing and SHH serum levels in healthy adults. A significant reduction of the SHH levels during the inflammatory phase of fracture healing was found. SHH concentrations in fracture haematoma and serum were lower than the concentration in control serum for the rest of the healing period. Our findings indicate that there is no relevant involvement of SHH in human fracture healing. Fracture repair process seem to reduce the SHH level in human. Further studies are definitely needed to clarify the underlying mechanisms.

pH Sensing Properties of Flexible, Bias-Free Graphene Microelectrodes in Complex Fluids: From Phosphate Buffer Solution to Human Serum.

PubMed

Ping, Jinglei; Blum, Jacquelyn E; Vishnubhotla, Ramya; Vrudhula, Amey; Naylor, Carl H; Gao, Zhaoli; Saven, Jeffery G; Johnson, Alan T Charlie

2017-08-01

Advances in techniques for monitoring pH in complex fluids can have a significant impact on analytical and biomedical applications. This study develops flexible graphene microelectrodes (GEs) for rapid (<5 s), very-low-power (femtowatt) detection of the pH of complex biofluids by measuring real-time Faradaic charge transfer between the GE and a solution at zero electrical bias. For an idealized sample of phosphate buffer solution (PBS), the Faradaic current is varied monotonically and systematically with the pH, with a resolution of ≈0.2 pH unit. The current-pH dependence is well described by a hybrid analytical-computational model, where the electric double layer derives from an intrinsic, pH-independent (positive) charge associated with the graphene-water interface and ionizable (negative) charged groups. For ferritin solution, the relative Faradaic current, defined as the difference between the measured current response and a baseline response due to PBS, shows a strong signal associated with ferritin disassembly and the release of ferric ions at pH ≈2.0. For samples of human serum, the Faradaic current shows a reproducible rapid (<20 s) response to pH. By combining the Faradaic current and real-time current variation, the methodology is potentially suitable for use to detect tumor-induced changes in extracellular pH. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Magnetic immunoassay coupled with inductively coupled plasma mass spectrometry for simultaneous quantification of alpha-fetoprotein and carcinoembryonic antigen in human serum

NASA Astrophysics Data System (ADS)

Zhang, Xing; Chen, Beibei; He, Man; Zhang, Yiwen; Xiao, Guangyang; Hu, Bin

2015-04-01

The absolute quantification of glycoproteins in complex biological samples is a challenge and of great significance. Herein, 4-mercaptophenylboronic acid functionalized magnetic beads were prepared to selectively capture glycoproteins, while antibody conjugated gold and silver nanoparticles were synthesized as element tags to label two different glycoproteins. Based on that, a new approach of magnetic immunoassay-inductively coupled plasma mass spectrometry (ICP-MS) was established for simultaneous quantitative analysis of glycoproteins. Taking biomarkers of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as two model glycoproteins, experimental parameters involved in the immunoassay procedure were carefully optimized and analytical performance of the proposed method was evaluated. The limits of detection (LODs) for AFP and CEA were 0.086 μg L- 1 and 0.054 μg L- 1 with the relative standard deviations (RSDs, n = 7, c = 5 μg L- 1) of 6.5% and 6.2% for AFP and CEA, respectively. Linear range for both AFP and CEA was 0.2-50 μg L- 1. To validate the applicability of the proposed method, human serum samples were analyzed, and the obtained results were in good agreement with that obtained by the clinical chemiluminescence immunoassay. The developed method exhibited good selectivity and sensitivity for the simultaneous determination of AFP and CEA, and extended the applicability of metal nanoparticle tags based on ICP-MS methodology in multiple glycoprotein quantifications.

The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

PubMed

Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

2009-12-01

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

Identification of a sulfate metabolite of PCB 11 in human serum

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; Koh, Wen Xin; DeWall, Jeanne; Teesch, Lynn M.; Hornbuckle, Keri C.; Thorne, Peter S.; Robertson, Larry W.; Duffel, Michael W.

2016-01-01

Despite increasing evidence for a major role for sulfation in the metabolism of lower-chlorinated polychlorinated biphenyls in vitro and in vivo, and initial evidence for potential bioactivities of the resulting sulfate ester metabolites, the formation of PCB sulfates in PCB exposed human populations had not been explored. The primary goal of this study was to determine if PCB sulfates, and potentially other conjugated PCB derivatives, are relevant classes of PCB metabolites in the serum of humans with known exposures to PCBs. In order to detect and quantify dichlorinated PCB sulfates in serum samples of 46 PCB-exposed individuals from either rural or urban communities, we developed a high-resolution mass spectrometry-based protocol using 4-PCB 11 sulfate as a model compound. The method also allowed the preliminary analysis of these 46 human serum extracts for the presence of other metabolites, such as glucuronic acid conjugates and hydroxylated PCBs. Sulfate ester metabolites derived from dichlorinated PCBs were detectable and quantifiable in more than 20 % of analyzed serum samples. Moreover, we were able to utilize this method to detect PCB glucuronides and hydroxylated PCBs, albeit at lower frequencies than PCB sulfates. Altogether, our results provide initial evidence for the presence of PCB sulfates in human serum. Considering the inability of previously employed analytical protocols for PCBs to extract these sulfate ester metabolites and the concentrations of these metabolites observed in our current study, our data support the hypothesis that total serum levels of PCB metabolites in exposed individuals may have been underestimated in the past. PMID:27816204

Identification of a sulfate metabolite of PCB 11 in human serum.

PubMed

Grimm, Fabian A; Lehmler, Hans-Joachim; Koh, Wen Xin; DeWall, Jeanne; Teesch, Lynn M; Hornbuckle, Keri C; Thorne, Peter S; Robertson, Larry W; Duffel, Michael W

2017-01-01

Despite increasing evidence for a major role for sulfation in the metabolism of lower-chlorinated polychlorinated biphenyls in vitro and in vivo, and initial evidence for potential bioactivities of the resulting sulfate ester metabolites, the formation of PCB sulfates in PCB exposed human populations had not been explored. The primary goal of this study was to determine if PCB sulfates, and potentially other conjugated PCB derivatives, are relevant classes of PCB metabolites in the serum of humans with known exposures to PCBs. In order to detect and quantify dichlorinated PCB sulfates in serum samples of 46 PCB-exposed individuals from either rural or urban communities, we developed a high-resolution mass spectrometry-based protocol using 4-PCB 11 sulfate as a model compound. The method also allowed the preliminary analysis of these 46 human serum extracts for the presence of other metabolites, such as glucuronic acid conjugates and hydroxylated PCBs. Sulfate ester metabolites derived from dichlorinated PCBs were detectable and quantifiable in more than 20% of analyzed serum samples. Moreover, we were able to utilize this method to detect PCB glucuronides and hydroxylated PCBs, albeit at lower frequencies than PCB sulfates. Altogether, our results provide initial evidence for the presence of PCB sulfates in human serum. Considering the inability of previously employed analytical protocols for PCBs to extract these sulfate ester metabolites and the concentrations of these metabolites observed in our current study, our data support the hypothesis that total serum levels of PCB metabolites in exposed individuals may have been underestimated in the past. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantification of Anti-Aggregation Activity of Chaperones: A Test-System Based on Dithiothreitol-Induced Aggregation of Bovine Serum Albumin

PubMed Central

Borzova, Vera A.; Markossian, Kira A.; Kara, Dmitriy A.; Chebotareva, Natalia A.; Makeeva, Valentina F.; Poliansky, Nikolay B.; Muranov, Konstantin O.; Kurganov, Boris I.

2013-01-01

The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin–target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5 = 53 and 58 mM, respectively). PMID:24058554

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET

PubMed Central

Madeira, Catarina; Loura, Luís MS; Prieto, Manuel; Fedorov, Aleksander; Aires-Barros, M Raquel

2008-01-01

Background Serum and high ionic strength solutions constitute important barriers to cationic lipid-mediated intravenous gene transfer. Preparation or incubation of lipoplexes in these media results in alteration of their biophysical properties, generally leading to a decrease in transfection efficiency. Accurate quantification of these changes is of paramount importance for the success of lipoplex-mediated gene transfer in vivo. Results In this work, a novel time-resolved fluorescence resonance energy transfer (FRET) methodology was used to monitor lipoplex structural changes in the presence of phosphate-buffered saline solution (PBS) and fetal bovine serum. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA lipoplexes, prepared in high and low ionic strength solutions, are compared in terms of complexation efficiency. Lipoplexes prepared in PBS show lower complexation efficiencies when compared to lipoplexes prepared in low ionic strength buffer followed by addition of PBS. Moreover, when serum is added to the referred formulation no significant effect on the complexation efficiency was observed. In physiological saline solutions and serum, a multilamellar arrangement of the lipoplexes is maintained, with reduced spacing distances between the FRET probes, relative to those in low ionic strength medium. Conclusion The time-resolved FRET methodology described in this work allowed us to monitor stability and characterize quantitatively the structural changes (variations in interchromophore spacing distances and complexation efficiencies) undergone by DOTAP/DNA complexes in high ionic strength solutions and in presence of serum, as well as to determine the minimum amount of potentially cytotoxic cationic lipid necessary for complete coverage of DNA. This constitutes essential information regarding thoughtful design of future in vivo applications. PMID:18302788

Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

PubMed

Ikeda, Mayumi; Ishima, Yu; Shibata, Akitomo; Chuang, Victor T G; Sawa, Tomohiro; Ihara, Hideshi; Watanabe, Hiroshi; Xian, Ming; Ouchi, Yuya; Shimizu, Taro; Ando, Hidenori; Ukawa, Masami; Ishida, Tatsuhiro; Akaike, Takaaki; Otagiri, Masaki; Maruyama, Toru

2017-05-29

Hydrogen sulfide (H 2 S) signaling involves polysulfide (RSS n SR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α 1 -anti-trypsin, α 1 -acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 μM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species. Copyright © 2017 Elsevier B.V. All rights reserved.

Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry.

PubMed

Zhang, Yong; Xie, Xinfang; Zhao, Xinyuan; Tian, Fang; Lv, Jicheng; Ying, Wantao; Qian, Xiaohong

2018-01-06

Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation. In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took advantage of the inherent properties of the new fragmentation method called EThcD, which provides more complete fragmentation information about O-glycosylated peptides and a more confident site localization of O-glycans than collision-induced dissociation (HCD). We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD was not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Finally, we got a largest data set of site-specific native O-glycoproteome from human serum samples. Furthermore, quantitative analysis of intact O-glycopeptides from the serum samples of IgA nephropathy (IgAN) patients and healthy donors was performed, and the results showed the potential of the strategy to discover O-glycosylation biomarkers. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and lead to exciting opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. Copyright © 2017. Published by Elsevier B.V.

Directed self-assembly of proteins into discrete radial patterns

PubMed Central

Thakur, Garima; Prashanthi, Kovur; Thundat, Thomas

2013-01-01

Unlike physical patterning of materials at nanometer scale, manipulating soft matter such as biomolecules into patterns is still in its infancy. Self-assembled monolayer (SAM) with surface density gradient has the capability to drive biomolecules in specific directions to create hierarchical and discrete structures. Here, we report on a two-step process of self-assembly of the human serum albumin (HSA) protein into discrete ring structures based on density gradient of SAM. The methodology involves first creating a 2-dimensional (2D) polyethylene glycol (PEG) islands with responsive carboxyl functionalities. Incubation of proteins on such pre-patterned surfaces results in direct self-assembly of protein molecules around PEG islands. Immobilization and adsorption of protein on such structures over time evolve into the self-assembled patterns. PMID:23719678

Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

NASA Astrophysics Data System (ADS)

Tran, Quang Huy; Hanh Nguyen, Thi Hong; Mai, Anh Tuan; Thuy Nguyen, Thi; Khue Vu, Quang; Nga Phan, Thi

2012-03-01

This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml-1 to 1 μg ml-1, and the limit of detection was about 10 ng ml-1. This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks.

The Human Serum Metabolome

PubMed Central

Psychogios, Nikolaos; Hau, David D.; Peng, Jun; Guo, An Chi; Mandal, Rupasri; Bouatra, Souhaila; Sinelnikov, Igor; Krishnamurthy, Ramanarayan; Eisner, Roman; Gautam, Bijaya; Young, Nelson; Xia, Jianguo; Knox, Craig; Dong, Edison; Huang, Paul; Hollander, Zsuzsanna; Pedersen, Theresa L.; Smith, Steven R.; Bamforth, Fiona; Greiner, Russ; McManus, Bruce; Newman, John W.; Goodfriend, Theodore; Wishart, David S.

2011-01-01

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca. PMID:21359215

Spectroscopic and molecular modeling studies of the interaction between cytidine and human serum albumin and its analytical application.

PubMed

Cui, Fengling; Wang, Junli; Yao, Xiaojun; Wang, Li; Zhang, Qiangzhai; Qu, Guirong

In this study, the interaction between cytidine and human serum albumin (HSA) was investigated for the first time by fluorescence spectroscopy in combination with UV absorption spectrum and molecular modeling under simulative physiological conditions. Experimental results indicated that cytidine had a strong ability to quench the intrinsic fluorescence of human serum albumin. The binding constants (K) at different temperatures, thermodynamic parameter enthalpy changes (DeltaH) and entropy changes (DeltaS) of HSA-cytidine had been calculated according to the relevant fluorescence data, which indicated that the hydrophobic and electrostatic interactions played a major role, which was in agreement with the results of molecular modeling study. In addition, the effects of other ions on the binding constants were also studied. Furthermore, synchronous fluorescence technology was successfully applied to the determination of human serum albumin added into the cytidine solution.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiedner, Susan D.; Burnum, Kristin E.; Pederson, Leeanna M.

2012-08-03

Environmental and metabolic adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised lung. We employed an activity-based protein profiling (ABPP) approach utilizing a new aryl vinyl sulfonate probe and a serine hydrolase probe combined with quantitative LC-MS based accurate mass and time (AMT) tag proteomics for the identification of functional pathway adaptation of A. fumigatus to environmental variability relevant to pulmonary Invasive Aspergillosis. When the fungal pathogen was grown with human serum, metabolism and energy processes were markedly decreased compared to no serum culture. Additionally, functional pathways associated with amino acid and protein biosynthesismore » were limited as the fungus scavenged from the serum to obtain essential nutrients. Our approach revealed significant metabolic adaptation by A. fumigatus, and provides direct insight into this pathogen’s ability to survive and proliferate.« less

Centrifugal ultrafiltration of human serum for improving immunoglobulin A quantification using attenuated total reflectance infrared spectroscopy.

PubMed

Elsohaby, Ibrahim; McClure, J Trenton; Riley, Christopher B; Bryanton, Janet; Bigsby, Kathryn; Shaw, R Anthony

2018-02-20

Attenuated total reflectance infrared (ATR-IR) spectroscopy is a simple, rapid and cost-effective method for the analysis of serum. However, the complex nature of serum remains a limiting factor to the reliability of this method. We investigated the benefits of coupling the centrifugal ultrafiltration with ATR-IR spectroscopy for quantification of human serum IgA concentration. Human serum samples (n = 196) were analyzed for IgA using an immunoturbidimetric assay. ATR-IR spectra were acquired for whole serum samples and for the retentate (residue) reconstituted with saline following 300 kDa centrifugal ultrafiltration. IR-based analytical methods were developed for each of the two spectroscopic datasets, and the accuracy of each of the two methods compared. Analytical methods were based upon partial least squares regression (PLSR) calibration models - one with 5-PLS factors (for whole serum) and the second with 9-PLS factors (for the reconstituted retentate). Comparison of the two sets of IR-based analytical results to reference IgA values revealed improvements in the Pearson correlation coefficient (from 0.66 to 0.76), and the root mean squared error of prediction in IR-based IgA concentrations (from 102 to 79 mg/dL) for the ultrafiltration retentate-based method as compared to the method built upon whole serum spectra. Depleting human serum low molecular weight proteins using a 300 kDa centrifugal filter thus enhances the accuracy IgA quantification by ATR-IR spectroscopy. Further evaluation and optimization of this general approach may ultimately lead to routine analysis of a range of high molecular-weight analytical targets that are otherwise unsuitable for IR-based analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Square Wave Voltammetric Determination of Diclofenac in Pharmaceutical Preparations and Human Serum

PubMed Central

Ciltas, Ulvihan; Yilmaz, Bilal; Kaban, Selcuk; Akcay, Bilge Kaan; Nazik, Gulsah

2015-01-01

In this study, a simple and reliable square wave voltammetric (SWV) method was developed and validated for determination of diclofenac in pharmaceutical preparations and human serum. The proposed method was based on electrooxidation of diclofenac at platinum electrode in 0.1 M TBAClO4/acetonitrile solution. The well-defined two oxidation peaks were observed at 0.87 and 1.27 V, respectively. Calibration curves that were obtained by using current values measured for second peak were linear over the concentration range of 1.5-17.5 μg mL-1 and 2-20 μg mL-1 in supporting electrolyte and serum, respectively. Precision and accuracy were also checked in all media. Intra- and inter-day precision values for diclofenac were less than 3.64, and accuracy (relative error) was better than 2.49%. Developed method in this study is accurate, precise and can be easily applied to Diclomec, Dicloflam and Voltaren tablets as pharmaceutical preparation. Also, the proposed technique was successfully applied to spiked human serum samples. No electroactive interferences from the endogenous substances were found in human serum. PMID:26330859

Square Wave Voltammetric Determination of Diclofenac in Pharmaceutical Preparations and Human Serum.

PubMed

Ciltas, Ulvihan; Yilmaz, Bilal; Kaban, Selcuk; Akcay, Bilge Kaan; Nazik, Gulsah

2015-01-01

In this study, a simple and reliable square wave voltammetric (SWV) method was developed and validated for determination of diclofenac in pharmaceutical preparations and human serum. The proposed method was based on electrooxidation of diclofenac at platinum electrode in 0.1 M TBAClO4/acetonitrile solution. The well-defined two oxidation peaks were observed at 0.87 and 1.27 V, respectively. Calibration curves that were obtained by using current values measured for second peak were linear over the concentration range of 1.5-17.5 μg mL(-1) and 2-20 μg mL(-1) in supporting electrolyte and serum, respectively. Precision and accuracy were also checked in all media. Intra- and inter-day precision values for diclofenac were less than 3.64, and accuracy (relative error) was better than 2.49%. Developed method in this study is accurate, precise and can be easily applied to Diclomec, Dicloflam and Voltaren tablets as pharmaceutical preparation. Also, the proposed technique was successfully applied to spiked human serum samples. No electroactive interferences from the endogenous substances were found in human serum.

24-hour human urine and serum profiles of bisphenol A: Evidence against sublingual absorption following ingestion in soup

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teeguarden, Justin G., E-mail: jt@pnl.gov; Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 93771; Twaddle, Nathan C., E-mail: nathan.twaddle@fda.hhs.gov

Extensive first-pass metabolism of ingested bisphenol A (BPA) in the gastro-intestinal tract and liver restricts blood concentrations of bioactive BPA to < 1% of total BPA in humans and non-human primates. Absorption of ingested BPA through non-metabolizing tissues of the oral cavity, recently demonstrated in dogs, could lead to the higher serum BPA concentrations reported in some human biomonitoring studies. We hypothesized that the extensive interaction with the oral mucosa by a liquid matrix, like soup, relative to solid food or capsules, might enhance absorption through non-metabolizing oral cavity tissues in humans, producing higher bioavailability and higher serum BPA concentrations.more » Concurrent serum and urine concentrations of d6-BPA, and its glucuronide and sulfate conjugates, were measured over a 24 hour period in 10 adult male volunteers following ingestion of 30 μg d6-BPA/kg body weight in soup. Absorption of d6-BPA was rapid (t{sub 1/2} = 0.45 h) and elimination of the administered dose was complete 24 h post-ingestion, evidence against any tissue depot for BPA. The maximum serum d6-BPA concentration was 0.43 nM at 1.6 h after administration and represented < 0.3% of total d6-BPA. Pharmacokinetic parameters, pharmacokinetic model simulations, and the significantly faster appearance half-life of d6-BPA-glucuronide compared to d6-BPA (0.29 h vs 0.45 h) were evidence against meaningful absorption of BPA in humans through any non-metabolizing tissue (< 1%). This study confirms that typical exposure to BPA in food produces picomolar to subpicomolar serum BPA concentrations in humans, not nM concentrations reported in some biomonitoring studies.« less

Spatial distribution of bisphenol S in surface water and human serum from Yangtze River watershed, China: Implications for exposure through drinking water.

PubMed

Wan, Yanjian; Xia, Wei; Yang, Shunyi; Pan, Xinyun; He, Zhenyu; Kannan, Kurunthachalam

2018-05-01

Bisphenol S (BPS) is an emerging environmental contaminant. The occurrence of this compound in humans and the environment is not well described. In this study, 120 surface water samples and 240 human serum samples were collected along the Yangtze River in 2015 for the determination of the occurrence of BPS. Surface water and human serum samples were extracted by solid phase extraction and liquid-liquid extraction, respectively, and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). BPS was detected in all river water samples at concentrations that ranged from 0.18 to 14.9 ng/L (median: 0.98 ng/L), with higher concentrations in spring than summer. The median estimated daily intake (EDI) of BPS through water ingestion by infants in spring and summer was 0.12 and 0.06 ng/kg body weight (bw)/day, respectively. BPS was detected in human serum with the highest concentrations in samples from Nanjing (median: 0.65 ng/mL, maximum: 169 ng/mL) among the four cities studied. No significant gender related difference in BPS concentrations was observed in human sera, while higher concentrations were found in younger individuals than elderly. The EDI of BPS calculated based on serum concentrations of adults in Nanjing was 22.8 ng/kg bw/day. Ingestion of water accounted for <1% of the total BPS intake by the Chinese population. This is the first report of the occurrence of BPS in water from the Yangtze River and human serum from several cities located along this river in China. Copyright © 2018 Elsevier Ltd. All rights reserved.

Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

PubMed

Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

2015-02-01

One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered. Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.

24-hour human urine and serum profiles of bisphenol A: Evidence against sublingual absorption following ingestion in soup.

PubMed

Teeguarden, Justin G; Twaddle, Nathan C; Churchwell, Mona I; Yang, Xiaoxia; Fisher, Jeffrey W; Seryak, Liesel M; Doerge, Daniel R

2015-10-15

Extensive first-pass metabolism of ingested bisphenol A (BPA) in the gastro-intestinal tract and liver restricts blood concentrations of bioactive BPA to <1% of total BPA in humans and non-human primates. Absorption of ingested BPA through non-metabolizing tissues of the oral cavity, recently demonstrated in dogs, could lead to the higher serum BPA concentrations reported in some human biomonitoring studies. We hypothesized that the extensive interaction with the oral mucosa by a liquid matrix, like soup, relative to solid food or capsules, might enhance absorption through non-metabolizing oral cavity tissues in humans, producing higher bioavailability and higher serum BPA concentrations. Concurrent serum and urine concentrations of d6-BPA, and its glucuronide and sulfate conjugates, were measured over a 24hour period in 10 adult male volunteers following ingestion of 30μg d6-BPA/kg body weight in soup. Absorption of d6-BPA was rapid (t1/2=0.45h) and elimination of the administered dose was complete 24h post-ingestion, evidence against any tissue depot for BPA. The maximum serum d6-BPA concentration was 0.43nM at 1.6h after administration and represented <0.3% of total d6-BPA. Pharmacokinetic parameters, pharmacokinetic model simulations, and the significantly faster appearance half-life of d6-BPA-glucuronide compared to d6-BPA (0.29h vs 0.45h) were evidence against meaningful absorption of BPA in humans through any non-metabolizing tissue (<1%). This study confirms that typical exposure to BPA in food produces picomolar to subpicomolar serum BPA concentrations in humans, not nM concentrations reported in some biomonitoring studies. Published by Elsevier Inc.

Comparison of methods used to diagnose generalized inflammatory disease in manatees (Trichechus manatus latirostris)

USGS Publications Warehouse

Harr, K.E.; Harvey, J.W.; Bonde, R.K.; Murphy, D.; Lowe, Mark; Menchaca, M.; Haubold, E.M.; Francis-Floyd, R.

2006-01-01

Manatees (Trichechus manatus latirostris) are afflicted with inflammatory and infectious disease secondary to human interaction, such as boat strike and entanglement, as well as “cold stress syndrome” and pneumonia. White-blood-cell count and fever, primary indicators of systemic inflammation in most species, are insensitive in diagnosing inflammatory disease in manatees. Acute phase-response proteins, such as haptoglobin and serum amyloid A, have proven to be sensitive measures of inflammation/infection in domestic large animal species. This study assessed diagnosis of generalized inflammatory disease by different methods including total white-blood-cell count, albumin: globulin ratio, gel electrophoresis analysis, C-reactive protein, alpha1 acid glycoprotein, haptoglobin, fibrinogen, and serum amyloid A. Samples were collected from 71 apparently healthy and 27 diseased animals during diagnostic medical examination. Serum amyloid A, measured by ELISA, followed by albumin:globulin ratio, measured by plasma gel electrophoresis, were most sensitive in diagnosing inflammatory disease, with diagnostic sensitivity and specificity of approximately 90%. The reference interval for serum amyloid A is <10–50 μg/ml with an equivocal interval of 51–70 μg/ml. The reference interval for albumin:globulin ratio by plasma gel electrophoresis is 0.7–1.1. Albumin: globulin ratio, calculated using biochemical techniques, was not accurate due to overestimation of albumin by bromcresol green dye-binding methodology. Albumin:globulin ratio, measured by serum gel electrophoresis, has a low sensitivity of 15% due to the lack of fibrinogen in the sample. Haptoglobin, measured by hemoglobin titration, had a reference interval of 0.4–2.4 mg/ml, a diagnostic sensitivity of 60%, and a diagnostic specificity of 93%. The haptoglobin assay is significantly affected by hemolysis. Fibrinogen, measured by heat precipitation, has a reference interval of 100–400 mg/dl, a diagnostic sensitivity of 40%, and a diagnostic specificity of 95%.

Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin.

PubMed Central

Stamler, J S; Jaraki, O; Osborne, J; Simon, D I; Keaney, J; Vita, J; Singel, D; Valeri, C R; Loscalzo, J

1992-01-01

We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endothelium-derived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for approximately 0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S--NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosothiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains approximately 7 microM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-serum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at approximately 1 microM; 60 min after administration of NG-monomethyl-L-arginine at 50 mg/ml, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by approximately 40% (greater than 95% of which were accounted for by S-nitrosoproteins, and approximately 80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be regulated for the maintenance of vascular tone. PMID:1502182

Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin.

PubMed

Stamler, J S; Jaraki, O; Osborne, J; Simon, D I; Keaney, J; Vita, J; Singel, D; Valeri, C R; Loscalzo, J

1992-08-15

We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endothelium-derived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for approximately 0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S--NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosothiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains approximately 7 microM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-serum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at approximately 1 microM; 60 min after administration of NG-monomethyl-L-arginine at 50 mg/ml, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by approximately 40% (greater than 95% of which were accounted for by S-nitrosoproteins, and approximately 80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be regulated for the maintenance of vascular tone.

Comparison of methods used to diagnose generalized inflammatory disease in manatees (Trichechus manatus latirostris).

PubMed

Harr, Kendal; Harvey, John; Bonde, Robert; Murphy, David; Lowe, Mark; Menchaca, Maya; Haubold, Elsa; Francis-Floyd, Ruth

2006-06-01

Manatees (Trichechus manatus latirostris) are afflicted with inflammatory and infectious disease secondary to human interaction, such as boat strike and entanglement, as well as "cold stress syndrome" and pneumonia. White-blood-cell count and fever, primary indicators of systemic inflammation in most species, are insensitive in diagnosing inflammatory disease in manatees. Acute phase-response proteins, such as haptoglobin and serum amyloid A, have proven to be sensitive measures of inflammation/infection in domestic large animal species. This study assessed diagnosis of generalized inflammatory disease by different methods including total white-blood-cell count, albumin: globulin ratio, gel electrophoresis analysis, C-reactive protein, alpha, acid glycoprotein, haptoglobin, fibrinogen, and serum amyloid A. Samples were collected from 71 apparently healthy and 27 diseased animals during diagnostic medical examination. Serum amyloid A, measured by ELISA, followed by albumin:globulin ratio, measured by plasma gel electrophoresis, were most sensitive in diagnosing inflammatory disease, with diagnostic sensitivity and specificity of approximately 90%. The reference interval for serum amyloid A is <10-50 microg/ml with an equivocal interval of 51-70 microg/ml. The reference interval for albumin:globulin ratio by plasma gel electrophoresis is 0.7-1.1. Albumin: globulin ratio, calculated using biochemical techniques, was not accurate due to overestimation of albumin by bromcresol green dye-binding methodology. Albumin:globulin ratio, measured by serum gel electrophoresis, has a low sensitivity of 15% due to the lack of fibrinogen in the sample. Haptoglobin, measured by hemoglobin titration, had a reference interval of 0.4-2.4 mg/ml, a diagnostic sensitivity of 60%, and a diagnostic specificity of 93%. The haptoglobin assay is significantly affected by hemolysis. Fibrinogen, measured by heat precipitation, has a reference interval of 100-400 mg/dl, a diagnostic sensitivity of 40%, and a diagnostic specificity of 95%.

Direct determination of trace refractory elements in human serum by ETV-ICP-MS with in-situ matrix removal.

PubMed

Li, Shengqing; Hu, Bin; Jiang, Zucheng; Chen, Rui

2004-08-01

A method for in-situ removal of matrix is proposed for direct determination of trace refractory elements in human serum by ETV-ICP-MS with the use of poly(tetrafluoroethylene) (PTFE) as fluorinating reagent. Attention has been paid to investigating the vaporization behavior both of refractory elements of interest and of matrix elements (Na, K, Ca, Mg, Cl, S, and P) in a graphite furnace with the PTFE modifier present or not. It was shown that potential interferences from the organic and inorganic matrices in the serum sample could be eliminated or reduced to a negligible level by appropriate dilution of the serum and deliberate optimization of the ETV temperature program. The proposed method has been applied to the direct simultaneous determination of V, Cr, Mo, Ba, La, Ce, and W in human serum. The limits of detection for fivefold diluted serum were 0.18 (V), 0.229 (Cr), 0.050 (Mo), 0.328 (Ba), 0.031 (La), 0.038 (Ce), and 0.019 ng mL(-1) (W), respectively, and the relative standard deviations of the method were in the range 4-15% (2 ng mL(-1) in serum, n=3).

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells.

PubMed

Zhang, Zhongqiang; Gao, Bingsi; Zhao, Chengjiang; Long, Cassandra; Qi, Haizhi; Ezzelarab, Mohamed; Cooper, David Kc; Hara, Hidetaka

2017-07-01

The results of the assay for measuring anti-non-Gal antibodies (which affect pig xenograft survival) in recipients are important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti-non-Gal antibody binding to porcine aortic endothelial cells (pAECs). Pooled human, naive, and sensitized baboon sera were incubated with wild-type, α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, and 3 hour) and concentrations (5, 10, 20, and 40 μL) was determined by flow cytometry. An increase in incubation time from 30 minutes to 2 hour was associated with increases in anti-non-Gal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human, naive and sensitized baboon sera (P<.05). Pooled human serum showed a significant increase in anti-non-Gal IgM (1.5 times) and a minimal increase in anti-non-Gal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2-hour incubation was 1.5 times and 2 times greater than after 30-minutes incubation, respectively, whereas naïve baboon sera showed minimal (non-significant) increase in anti-non-Gal IgM/IgG antibody binding. With 2-hour incubation, increasing the serum concentration from 5 μL to 20 μL significantly increased antibody binding to non-Gal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Increasing the serum incubation time contributed to improve the sensitivity of detecting anti-non-Gal antibodies, without affecting cell viability in vitro. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Development of certified reference materials for electrolytes in human serum (GBW09124-09126).

PubMed

Feng, Liuxing; Wang, Jun; Cui, Yanjie; Shi, Naijie; Li, Haifeng; Li, Hongmei

2017-05-01

Three reference materials, at relatively low, middle, and high concentrations, were developed for analysis of the mass fractions of electrolytes (K, Ca, Na, Mg, Cl, and Li) in human serum. The reference materials were prepared by adding high purity chloride salts to normal human serum. The concentration range of the three levels is within ±20% of normal human serum. It was shown that 14 units with duplicate analysis is enough to demonstrate the homogeneity of these candidate reference materials. The statistical results also showed no significant trends in both short-term stability test for 1 week at 40 °C and long-term stability test for 14 months. The certification methods of the six elements include isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS), inductively coupled plasma optical emission spectroscopy (ICP-OES), atomic absorption spectroscopy (AAS), ion chromatography (IC), and ion-selective electrode (ISE). The certification methods were validated by international comparisons among a number of national metrology institutes (NMIs). The combined relative standard uncertainties of the property values were estimated by considering the uncertainties of the analytical methods, homogeneity, and stability. The range of the expanded uncertainties of all the elements is from 2.2% to 3.9%. The certified reference materials (CRMs) are primarily intended for use in the calibration and validation of procedures in clinical analysis for the determination of electrolytes in human serum or plasma. Graphical Abstract Certified reference materials for K, Ca, Mg, Na, Cl and Li in human serum (GBW09124-09126).

Further studies on rat mast cell degranulation by IgE—anti-IgE and the inhibitory effect of drugs related to cAMP

PubMed Central

Kimura, Y.; Inoue, Yoshie; Honda, H.

1974-01-01

With a modified rat mast cell degranulation (RMCD) technique developed by Korotzer, Haddad and Lopapa (1971), the mechanism of mast cell degranulation by IgE—anti-IgE reaction and the inhibitory effect of cAMP-related compounds upon IgE-mediated mast cell degranulation were studied. Degranulations of 90 per cent or more were decreased to 13–16 per cent when the mast cells were pretreated with human IgE or normal human serum. However, if rat mast cells were pretreated with anti-human IgE rabbit serum or normal rabbit serum, the degranulation per cent in these cells by IgE—anti-IgE reaction was the same as in the nontreated cells. These results suggest the presence of receptors in rat mast cells for human IgE or normal human serum, and the lack of receptors in these cells for anti-human IgE rabbit serum or normal rabbit serum. Treatment of isolated rat mast cells with adenyl cyclase stimulating agents (isoprenaline, adrenaline, prostaglandin E1 and E2) and theophylline or aminophylline, which inhibit the enzymatic degradation of cAMP, also inhibited the morphological degranulation of the mast cells. Cromoglycate or chlorophenes in derivatives, which might have a stabilizing effect of the cell membrane, also inhibited the degranulation of the rat mast cells mediated by IgE—anti-IgE reaction. These results support the attractive hypothesis that cAMP occupies a central modulatory role in the in vitro mast cell degranulation by IgE—anti-IgE reaction. PMID:4368738

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor.

PubMed

Candal, F J; George, V G; Ades, E W

1991-07-01

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vaccine production and reduce costs.

Fast transmethylation of serum lipids using microwave irradiation.

PubMed

Lin, Yu Hong; Loewke, James D; Hyun, Duk Y; Leazer, Jay; Hibbeln, Joseph R

2012-11-01

Microwave irradiation as the energy source for one-step direct transesterification of fatty acids in human serum lipids was examined in a solvent system of methanol: hexane: acetyl chloride based on a Lepage & Roy assay. Innovative and explosion proof single-mode or multimode microwave accelerated reaction system was employed. Recoveries were calculated as the percentage of fatty acid concentrations measured by microwave assay to those by the reference method of the Lepage & Roy assay that utilized conductive heating at 100 °C for 60 min. Under conditions of 100 °C for 1 min in Single-mode (S4-100 × 1), or 125 °C for 5 min in Multimode (M5-125 × 5), the recoveries were 100-103 % for the total fatty acids and 96-106 % for each categorized fatty acid, including saturates, monounsaturates, n-6 PUFA, and n-3 PUFA. For individual PUFA, the mean recoveries were 102-105 % for 18:2n-6 and 18:3n-3; 99, 109, and 95 % for 20:4n-6, 20:5n-3, and 22:6n-3, respectively. Thus, fatty acid concentrations determined by microwave fatty acid assay were accurate to those results by the reference method, when the microwave conditions were optimal. In summary, the microwave irradiation could replace conductive heating in one-step direct transesterification, and reduce the duration from 60 min to 5 min or less. This methodology may be applied in both the absolute and relative quantification of serum total fatty acids.

Fast Transmethylation of Serum Lipids using Microwave Irradiation

PubMed Central

Lin, Yu Hong; Loewke, James D.; Hyun, Duk Y.; Leazer, Jay; Hibbeln, Joseph R.

2012-01-01

Microwave irradiation as the energy source for one–step direct transesterification of fatty acids in human serum lipids was examined in solvent system of methanol: hexane: acetyl chloride based on Lepage & Roy assay. Innovative and explosion proof single–mode or multimode microwave accelerate reaction system was employed. Recoveries were calculated as the percentage of fatty acid concentrations measured by microwave assay to those by reference method Lepage & Roy assay that utilized conductive heating at 100 °C for 60 min. At conditions of 100 °C for 1 min in Single–mode (S4–100×1), or 125 °C for 5 min in Multimode (M5–125×5), the recoveries were 100–103% for the total fatty acids and 96–106% for each categorized fatty acid, including saturates, monounsaturates, n-6 PUFA, and n-3 PUFA. For individual PUFA, the mean recoveries were 102–105% for 18:2n-6 and 18:3n-3; 99, 109, and 95% for 20:4n-6, 20:5n-3, and 22:6n-3, respectively. Thus, fatty acid concentrations determined by microwave fatty acid assay were accurate to those results by the reference method, when the microwave conditions were optimal. In summary, the microwave irradiation could replace conductive heating in one–step direct transesterification, and reduce duration from 60 min to 5 min or less. This methodology may be applied in both the absolute and relative quantification of serum total fatty acids. PMID:23015312

Thymosin α1 Interacts with Exposed Phosphatidylserine in Membrane Models and in Cells and Uses Serum Albumin as a Carrier.

PubMed

Mandaliti, Walter; Nepravishta, Ridvan; Sinibaldi Vallebona, Paola; Pica, Francesca; Garaci, Enrico; Paci, Maurizio

2016-03-15

Thymosin α1 is a peptidic hormone with pleiotropic activity and is used in the therapy of several diseases. It is unstructured in water solution and interacts with negative regions of vesicles by assuming two tracts of helical conformation with a structural break between them. This study reports on Thymosin α1's interaction with mixed phospholipids phosphatidylcholine and phosphatidylserine, the negative component of the membranes, by ¹H and natural abundance ¹⁵N nuclear magnetic resonance (NMR). The results indicate that interaction occurs when the membrane is negatively charged by exposing phosphatidylserine. Moreover, the direct interaction of Thymosin α1 with K562 cells with an overexposure of phosphatidylserine as a consequence of resveratrol-induced apoptosis was conducted. Thymosin α1's interaction with human serum albumin was also investigated by NMR spectroscopy. Steady-state saturation transfer, transfer nuclear Overhauser effect spectroscopy, and diffusion-ordered spectroscopy methodologies all reveal that the C-terminal region of Thymosin α1 is involved in the interaction with serum albumin. These results may shed more light on Thymosin α1's mechanism of action by its insertion in negative regions of membranes due to the exposure of phosphatidylserine. Once Thymosin α1's N-terminus has been inserted into the membrane, the rest may interact with nearby proteins and/or receptors acting as effectors and causing a biological signaling cascade, thus exerting thymosin α1's pleiotropy.

Preoperative nutrition status and postoperative outcome in elderly general surgery patients: a systematic review.

PubMed

van Stijn, Mireille F M; Korkic-Halilovic, Ines; Bakker, Marjan S M; van der Ploeg, Tjeerd; van Leeuwen, Paul A M; Houdijk, Alexander P J

2013-01-01

Poor nutrition status is considered a risk factor for postoperative complications in the adult population. In elderly patients, who often have a poor nutrition status, this relationship has not been substantiated. Thus, the aim of this systematic review was to assess the merit of preoperative nutrition parameters used to predict postoperative outcome in elderly patients undergoing general surgery. A systematic literature search of 10 consecutive years, 1998-2008, in PubMed, EMBASE, and Cochrane databases was performed. Search terms used were nutrition status, preoperative assessment, postoperative outcome, and surgery (hip or general), including their synonyms and MeSH terms. Limits used in the search were human studies, published in English, and age (65 years or older). Articles were screened using inclusion and exclusion criteria. All selected articles were checked on methodology and graded. Of 463 articles found, 15 were included. They showed profound heterogeneity in the parameters used for preoperative nutrition status and postoperative outcome. The only significant preoperative predictors of postoperative outcome in elderly general surgery patients were serum albumin and ≥ 10% weight loss in the previous 6 months. This systematic review revealed only 2 preoperative parameters to predict postoperative outcome in elderly general surgery patients: weight loss and serum albumin. Both are open to discussion in their use as a preoperative nutrition parameter. Nonetheless, serum albumin seems a reliable preoperative parameter to identify a patient at risk for nutrition deterioration and related complicated postoperative course.

Age and sex variation in serum albumin concentration: an observational study.

PubMed

Weaving, Gary; Batstone, Gifford F; Jones, Richard G

2016-01-01

In the UK, a common reference interval for serum albumin is widely used irrespective of age or sex. Implicit in this is that laboratories produce analytically similar results. This paper challenges the validity of this approach. A three-week collection of results sent to all primary care centres in England has been analysed by age, sex and laboratory. In all, 1,079,193 serum albumin reports were included in this analysis. The mean population serum albumin concentration increases to peak at around age 20 years and then decreases with increasing age. Values in females decrease more rapidly but become close to male values at 60 years. The variation between laboratories was large and potentially clinically significant. Reference intervals for serum albumin should be stratified by age and sex. Until there is greater methodological standardization, laboratories should determine their own reference intervals and not accept a single consensus reference interval. © The Author(s) 2015.

MicroRNA Expression Profiling of the Armed Forces Health Surveillance Branch Cohort for Identification of "Enviro-miRs" Associated With Deployment-Based Environmental Exposure.

PubMed

Dalgard, Clifton L; Polston, Keith F; Sukumar, Gauthaman; Mallon, Col Timothy M; Wilkerson, Matthew D; Pollard, Harvey B

2016-08-01

The aim of this study was to identify serum microRNA (miRNA) biomarkers that indicate deployment-associated exposures in service members at military installations with open burn pits. Another objective was to determine detection rates of miRNAs in Department of Defense Serum Repository (DoDSR) samples with a high-throughput methodology. Low-volume serum samples (n = 800) were profiled by miRNA-capture isolation, pre-amplification, and measurement by a quantitative PCR-based OpenArray platform. Normalized quantitative cycle values were used for differential expression analysis between groups. Assay specificity, dynamic range, reproducibility, and detection rates by OpenArray passed target desired specifications. Serum abundant miRNAs were consistently measured in study specimens. Four miRNAs were differentially expressed in the case deployment group subjects. miRNAs are suitable RNA species for biomarker discovery in the DoDSR serum specimens. Serum miRNAs are candidate biomarkers for deployment and environmental exposure in military service members.

Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

PubMed

Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

2016-01-01

The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Determination of element levels in human serum: Total reflection X-ray fluorescence applications

NASA Astrophysics Data System (ADS)

Majewska, U.; Łyżwa, P.; Łyżwa, K.; Banaś, D.; Kubala-Kukuś, A.; Wudarczyk-Moćko, J.; Stabrawa, I.; Braziewicz, J.; Pajek, M.; Antczak, G.; Borkowska, B.; Góźdź, S.

2016-08-01

Deficiency or excess of elements could disrupt proper functioning of the human body and could lead to several disorders. Determination of their concentrations in different biological human fluids and tissues should become a routine practice in medical treatment. Therefore the knowledge about appropriate element concentrations in human organism is required. The purpose of this study was to determine the concentration of several elements (P, S, Cl, K, Ca, Cr, Fe, Cu, Zn, Se, Br, Rb, Pb) in human serum and to define the reference values of element concentration. Samples of serum were obtained from 105 normal presumably healthy volunteers (66 women aged between 15 and 78 years old; 39 men aged between 15 and 77 years old). Analysis has been done for the whole studied population and for subgroups by sex and age. It is probably first so a wide study of elemental composition of serum performed in the case of Świętokrzyskie region. Total reflection X-ray fluorescence (TXRF) method was used to perform the elemental analysis. Spectrometer S2 Picofox (Bruker AXS Microanalysis GmbH) was used to identify and measure elemental composition of serum samples. Finally, 1st and 3rd quartiles were accepted as minimum and maximum values of concentration reference range.

Metrics for Systems Thinking in the Human Dimension

DTIC Science & Technology

2016-11-01

corpora of documents. 2 Methodology Overview We present a human-in-the- loop methodology that assists researchers and analysts by characterizing...supervised learning methods. Building on this foundation, we present an unsupervised, human-in-the- loop methodology that utilizes topic models to...the definition of strong systems thinking and in the interpretation of topics, but this is what makes the human-in-the- loop methodology so effective

Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

PubMed Central

Shi, Tujin; Sun, Xuefei; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y.; Smith, Richard D.; Tang, Keqi; Camp, David G.; Qian, Wei-Jun

2013-01-01

We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50–100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ~12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successful quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies. PMID:23763644

Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.

PubMed

Lübberstedt, Marc; Müller-Vieira, Ursula; Biemel, Klaus M; Darnell, Malin; Hoffmann, Stefan A; Knöspel, Fanny; Wönne, Eva C; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B; Zeilinger, Katrin

2015-09-01

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions. Copyright © 2012 John Wiley & Sons, Ltd.

Evaluation of a handheld point-of-care analyser for measurement of creatinine in cats.

PubMed

Reeve, Jenny; Warman, Sheena; Lewis, Daniel; Watson, Natalie; Papasouliotis, Kostas

2017-02-01

Objectives The aim of the study was to evaluate whether a handheld creatinine analyser (StatSensor Xpress; SSXp), available for human patients, can be used to measure creatinine reliably in cats. Methods Analytical performance was evaluated by determining within- and between-run coefficient of variation (CV, %), total error observed (TE obs , %) and sigma metrics. Fifty client-owned cats presenting for investigation of clinical disease had creatinine measured simultaneously, using SSXp (whole blood and plasma) and a reference instrument (Konelab, serum); 48 paired samples were included in the study. Creatinine correlation between methodologies (SSXp vs Konelab) and sample types (SSXp whole blood vs SSXp plasma ) was assessed by Spearman's correlation coefficient and agreement was determined using Bland-Altman difference plots. Each creatinine value was assigned an IRIS stage (1-4); correlation and agreement between Konelab and SSXp IRIS stages were evaluated. Results Within-run CV (4.23-8.85%), between-run CV (8.95-11.72%), TE obs (22.15-34.92%) and sigma metrics (⩽3) did not meet desired analytical requirements. Correlation between sample types was high (SSXp whole blood vs SSXp plasma ; r = 0.89), and between instruments was high (SSXp whole blood vs Konelab serum ; r = 0.85) to very high (SSXp plasma vs Konelab serum ; r = 0.91). Konelab and SSXp whole blood IRIS scores exhibited high correlation ( r = 0.76). Packed cell volume did not significantly affect SSXp determination of creatinine. Bland-Altman difference plots identified a positive bias for the SSXp (7.13 μmol/l SSXp whole blood ; 20.23 μmol/l SSXp plasma ) compared with the Konelab. Outliers (1/48 whole blood; 2/48 plasma) occurred exclusively at very high creatinine concentrations. The SSXp failed to identify 2/21 azotaemic cats. Conclusions and relevance Analytical performance of the SSXp in feline patients is not considered acceptable. The SSXp exhibited a high to very high correlation compared with the reference methodology but the two instruments cannot be used interchangeably. Improvements in the SSXp analytical performance are needed before its use can be recommended in feline clinical practice.

Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

PubMed Central

Hanga, Mariana P.; Heathman, Thomas R. J.; Coopman, Karen; Nienow, Alvin W.; Williams, David J.; Hewitt, Christopher J.

2017-01-01

ABSTRACT Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high‐throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum‐based medium was applied to a serum‐free process in the ambr15, resulting in >250% increase in yield compared to the serum‐based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum‐free and automated processing improved the reproducibility more than 10‐fold compared to the serum‐based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum‐free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253–2266. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:28627713

Mangifera indica L. (Vimang) protection against serum oxidative stress in elderly humans.

PubMed

Pardo-Andreu, Gilberto L; Philip, Sarah J; Riaño, Annia; Sánchez, Carlos; Viada, Carmen; Núñez-Sellés, Alberto J; Delgado, René

2006-01-01

We searched for the protective effect of a natural extract from stem bark of Mangifera indica L. extract (Vimang) on age-related oxidative stress. Healthy subjects were classified in two groups, elderly (>65 years) and young group (<26 years). The elderly group received a daily dose of 900 mg of extract (three coated Vimang tablets, 300 mg each, before meals) for 60 days. Serum concentration of lipid peroxides, serum peroxidation potential, extracellular superoxide dismutase activity (EC-SOD), glutathione status (GSH, GSSG, GSSG/GSH ratio)) and total antioxidant status (TAS) were determined before (both experimental groups) and 15, 30, and 60 days after treatment (only elderly group). We confirmed the existence of an age-associated oxidative stress in human serum as documented by an age-related increase in serum lipoperoxides and GSSG and a decrease in serum antioxidant capacity and EC-SOD activity. Vimang tablet supplementation increased EC-SOD activity (p <0.01) and serum TAS (p <0.01). It also decreased serum thiobarbituric reactive substances (p <0.01) and GSSG levels (p <0.05). We suggested that the antioxidant components of the extract could have been utilized by the cells (especially blood and endothelial cells), sparing the intra- and extracellular antioxidant system and increasing serum peroxil scavenging capacity, thus preventing age-associated increase in GSH oxidation and lipoperoxidation. Vimang tablets prevent age-associated oxidative stress in elderly humans, which could retard the onset of age-associated disease, improving the quality of life for elderly persons.

Assessment of serum HE4 levels throughout the normal menstrual cycle.

PubMed

Moore, Richard G; Plante, Beth; Hartnett, Erin; Mitchel, Jessica; Raker, Christine A; Vitek, Wendy; Eklund, Elizabeth; Lambert-Messerlian, Geralyn

2017-07-01

Human epididymis protein 4 is a serum biomarker to aid in differentiating benign and malignant disease in women with a pelvic mass. Interpretation of human epididymis protein 4 results relies on robust normative data. The purpose of this study was to evaluate whether human epididymis protein 4 levels are variable in women during the normal menstrual cycle. Healthy women, 18-45 years old, with regular menstrual cycles were recruited from community gynecologic practices in Rhode Island. Women consented to enroll and to participate by the donation of blood and urine samples at 5 specific times over the course of each cycle. Levels of reproductive hormones and human epididymis protein 4 were determined. Data were analyzed with the use of linear regression after log transformation. Among 74 enrolled cycles, 53 women had confirmed ovulation during the menstrual cycle and completed all 5 sample collections. Levels of estradiol, progesterone, and luteinizing hormone displayed the expected menstrual cycle patterns. Levels of human epididymis protein 4 in serum were relatively stable across the menstrual cycle, except for a small ovulatory (median, 37.0 pM) increase. Levels of human epididymis protein 4 in urine, after correction for creatinine, displayed the same pattern of secretion observed in serum. Serum human epididymis protein 4 levels are relatively stable across the menstrual cycle of reproductive-aged women and can be determined on any day to evaluate risk of ovarian malignancy. A slight increase is expected at ovulation; but even with this higher human epididymis protein 4 level, results are well within the healthy reference range for women (<120 pM). Levels of human epididymis protein 4 in urine warrant further investigation for use in clinical practice as a simple and convenient sample. Copyright © 2017 Elsevier Inc. All rights reserved.

Biological exposure indices of pyrrole adducts in serum and urine for hazard assessment of n-hexane exposure.

PubMed

Yin, Hongyin; Zhang, Chunling; Guo, Ying; Shao, Xiaoying; Zeng, Tao; Zhao, Xiulan; Xie, Keqin

2014-01-01

Pyrrole adducts might be used as a biomarker for monitoring occupational exposure to n-hexane, but the Biological Exposure Indices of pyrrole adducts in serum and urine are still unknown. The current study was designed to investigate the biological exposure limit of pyrrole adducts for hazard assessment of n-hexane. Male Wistar rats were given daily dose of 500, 1000, 1500, 2000, 4000 mg/kg bw n-hexane by gavage for 24 weeks. The levels of pyrrole adducts in serum and urine were determined at 8, 24 hours postdose once a week. The Biological Exposure Indices was evaluated by neurological evaluation and the levels of pyrrole adducts. The difference in pyrrole adducts formation between humans and rats were estimated by using in vitro test. Dose-dependent effects were observed between the doses of n-hexane and pyrrole adducts in serum and urine, and the levels of pyrrole adduct in serum and urine approached a plateau at week 4. There was a significantly negative correlation between the time to paralysis and the level of pyrrole adducts in serum and urine, while a positive correlation between gait score and levels of pyrrole adducts in serum and urine was observed. In vitro, pyrrole adducts formed in human serum was about two times more than those in rat serum at the same level of 2,5-HD. It was concluded that the BEIs of pyrrole adducts in humans were 23.1 ± 5.91 nmol/ml in serum 8 h postdose, 11.7 ± 2.64 nmol/ml in serum 24 h postdose, 253.8 ± 36.3 nmol/ml in urine 8 h postdose and 54.6 ± 15.42 nmol/ml in urine 24 h postdose.

Biological Exposure Indices of Pyrrole Adducts in Serum and Urine for Hazard Assessment of n-Hexane Exposure

PubMed Central

Yin, Hongyin; Zhang, Chunling; Guo, Ying; Shao, Xiaoying; Zeng, Tao; Zhao, Xiulan; Xie, Keqin

2014-01-01

Background Pyrrole adducts might be used as a biomarker for monitoring occupational exposure to n-hexane, but the Biological Exposure Indices of pyrrole adducts in serum and urine are still unknown. The current study was designed to investigate the biological exposure limit of pyrrole adducts for hazard assessment of n-hexane. Methods Male Wistar rats were given daily dose of 500, 1000, 1500, 2000, 4000 mg/kg bw n-hexane by gavage for 24 weeks. The levels of pyrrole adducts in serum and urine were determined at 8, 24 hours postdose once a week. The Biological Exposure Indices was evaluated by neurological evaluation and the levels of pyrrole adducts. The difference in pyrrole adducts formation between humans and rats were estimated by using in vitro test. Results Dose-dependent effects were observed between the doses of n-hexane and pyrrole adducts in serum and urine, and the levels of pyrrole adduct in serum and urine approached a plateau at week 4. There was a significantly negative correlation between the time to paralysis and the level of pyrrole adducts in serum and urine, while a positive correlation between gait score and levels of pyrrole adducts in serum and urine was observed. In vitro, pyrrole adducts formed in human serum was about two times more than those in rat serum at the same level of 2,5-HD. Conclusion It was concluded that the BEIs of pyrrole adducts in humans were 23.1±5.91 nmol/ml in serum 8 h postdose, 11.7±2.64 nmol/ml in serum 24 h postdose, 253.8±36.3 nmol/ml in urine 8 h postdose and 54.6±15.42 nmol/ml in urine 24 h postdose. PMID:24465904

Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum using an immobilized trypsin.

PubMed

Shibata, Kaito; Naito, Takafumi; Okamura, Jun; Hosokawa, Seiji; Mineta, Hiroyuki; Kawakami, Junichi

2017-11-30

Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) without an immunopurification technique have not been applied to the determination of serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-alkylated serum cetuximab without immunopurification was digested for 20minutes using immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the determination of serum samples in head and neck cancer patients treated with cetuximab. The chromatographic run time was 10minutes and no peaks interfering with surrogate peptides in serum digestion products were observed. The calibration curve of absolute cetuximab in serum was linear over the concentration range of 4-200μg/mL. The lower limit of quantification of cetuximab in human serum was 4μg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and 88.0-100.7%, respectively. The serum concentration range of cetuximab was 19-140μg/mL in patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r=0.899, P <0.01) and the mean bias was 1.5% in cancer patients. In conclusion, the present simple and rapid method with acceptable analytical performance can be helpful for evaluating the absolute concentration of serum cetuximab in clinical settings. Copyright © 2017 Elsevier B.V. All rights reserved.

Tocopherol isomer pattern in serum and stool of human following consumption of black currant seed press residue administered in whole grain bread.

PubMed

Helbig, Dorit; Wagner, Andreas; Schubert, Rainer; Jahreis, Gerhard

2009-12-01

Serum gamma-tocopherol is thought to be associated with human health. The dietary influence of tocopherol and fibre-rich black currant seed press residue on serum and stool tocopherol concentration was investigated in a controlled human intervention study. Thirty-six women consumed bread enriched with black currant press residue (4 weeks). The resultant faecal and serum tocopherol concentrations were compared with those after a period consuming control bread without press residue and a normal baseline diet. Fibre intake and excretion, antioxidant capacity (TEAC), and vitamin C concentrations in serum and urine were also determined. Samples were obtained with a 5-day standardised diet at the end of each period. The press residue bread lead to significantly increased beta-, gamma-, delta- and total tocopherol intake, serum alpha-, beta-, gamma- and total tocopherol concentration (with and without lipid adjustment), fibre intake and urinary vitamin C concentration compared to control bread (P<0.05). Faecal excretion of total tocopherols and fibre increased compared to baseline (P<0.05). Fibre intake and excretion influence total tocopherol concentration in lipid-adjusted serum and in stool. The outstandingly high increase of serum gamma-tocopherol concentration through seed press residue consumption could be due to a presumed interruption of the enzymatic tocopherol degradation mechanism by bread constituents.

Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

PubMed Central

Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha

2011-01-01

Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature. PMID:21779348

Scabies Mite Peritrophins Are Potential Targets of Human Host Innate Immunity

PubMed Central

Holt, Deborah C.; Kemp, Dave J.; Fischer, Katja

2011-01-01

Background Pruritic scabies lesions caused by Sarcoptes scabiei burrowing in the stratum corneum of human skin facilitate opportunistic bacterial infections. Emerging resistance to current therapeutics emphasizes the need to identify novel targets for protective intervention. We have characterized several protein families located in the mite gut as crucial factors for host-parasite interactions. Among these multiple proteins inhibit human complement, presumably to avoid complement-mediated damage of gut epithelial cells. Peritrophins are major components of the peritrophic matrix often found in the gut of arthropods. We hypothesized that a peritrophin, if abundant in the scabies mite gut, could be an activator of complement. Methodology/Principal Findings A novel full length scabies mite peritrophin (SsPTP1) was identified in a cDNA library from scabies mites. The amino acid sequence revealed four putative chitin binding domains (CBD). Recombinant expression of one CBD of the highly repetitive SsPTP1 sequence as TSP-hexaHis-fusion protein resulted in soluble protein, which demonstrated chitin binding activity in affinity chromatography assays. Antibodies against a recombinant SsPTP1 fragment were used to immunohistochemically localize native SsPTP1 in the mite gut and in fecal pellets within the upper epidermis, co-localizing with serum components such as host IgG and complement. Enzymatic deglycosylation confirmed strong N- and O-glycosylation of the native peritrophin. Serum incubation followed by immunoblotting with a monoclonal antibody against mannan binding lectin (MBL), the recognition molecule of the lectin pathway of human complement activation, indicated that MBL may specifically bind to glycosylated SsPTP1. Conclusions/Significance This study adds a new aspect to the accumulating evidence that complement plays a major role in scabies mite biology. It identifies a novel peritrophin localized in the mite gut as a potential target of the lectin pathway of the complement cascade. These initial findings indicate a novel role of scabies mite peritrophins in triggering a host innate immune response within the mite gut. PMID:21980545

Webinar Presentation: Suspect Screening of Environmental Organic Acids in Human Serum Using High-resolution Mass Spectrometry (HRMS)

EPA Pesticide Factsheets

This presentation, Suspect Screening of Environmental Organic Acids in Human Serum Using High-resolution Mass Spectrometry (HRMS), was given at the NIEHS/EPA Children's Centers 2016 Webinar Series: Exposome held on May 11, 2016.

Cytochrome P450 inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine-induced inflammation: the role of cytokines.

PubMed

Bleau, A M; Levitchi, M C; Maurice, H; du Souich, P

2000-08-01

Serum from humans with an acute upper respiratory viral infection and from rabbits with turpentine-induced inflammation reduce the catalytic activity of hepatic cytochrome P450 (P450). The aim of this study was to identify the serum mediators responsible for the decrease in P450 activity. Rabbit and human sera were fractionated by size exclusion chromatography and the fractions tested for their ability to reduce the activity and amount of P450 after 4 h of incubation with hepatocytes from turpentine-treated rabbits (H(INF)). Rabbit and human sera decreased P450 activity by around 40% without any change in the amount of CYP1A1 and 1A2 apoproteins. In rabbit serum, the fraction containing proteins of M(r) 23-15 kDa decreased P450 content by 41%, but did not alter the amount of the apoproteins. Anti-IL-6 antibody added to the M(r) 23-15 kDa fraction restored P450 content to 97% of control values, while anti-IL-1beta, TNF-alpha and IFN-gamma antibodies had no effect. Supporting the role of IL-6, incubation of H(INF) in the presence of IL-6 for 4 h reduced P450 content by 40%. In human serum, the fraction containing proteins of M(r) >95 kDa lowered P450 content by 43% without modifying the amounts of CYP1A1/2. Neutralization experiments showed that IFN-gamma, IL-6, and IL-1beta contributed to the decrease in P450 content. In conclusion, the present results demonstrate that IL-6, and IFN-gamma, IL-6 and IL-1beta are the serum mediators released in vivo by a turpentine-induced inflammatory reaction in the rabbit and an upper respiratory viral infection in humans, respectively, inactivating hepatic P450.

Cytochrome P450 inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine-induced inflammation: the role of cytokines

PubMed Central

Bleau, Anne-Marie; Levitchi, Mihaela C; Maurice, Hélène; du Souich, Patrick

2000-01-01

Serum from humans with an acute upper respiratory viral infection and from rabbits with turpentine-induced inflammation reduce the catalytic activity of hepatic cytochrome P450 (P450). The aim of this study was to identify the serum mediators responsible for the decrease in P450 activity.Rabbit and human sera were fractionated by size exclusion chromatography and the fractions tested for their ability to reduce the activity and amount of P450 after 4 h of incubation with hepatocytes from turpentine-treated rabbits (HINF). Rabbit and human sera decreased P450 activity by around 40% without any change in the amount of CYP1A1 and 1A2 apoproteins.In rabbit serum, the fraction containing proteins of Mr 23–15 kDa decreased P450 content by 41%, but did not alter the amount of the apoproteins. Anti-IL-6 antibody added to the Mr 23–15 kDa fraction restored P450 content to 97% of control values, while anti-IL-1β, TNF-α and IFN-γ antibodies had no effect. Supporting the role of IL-6, incubation of HINF in the presence of IL-6 for 4 h reduced P450 content by 40%.In human serum, the fraction containing proteins of Mr >95 kDa lowered P450 content by 43% without modifying the amounts of CYP1A1/2. Neutralization experiments showed that IFN-γ, IL-6, and IL-1β contributed to the decrease in P450 content.In conclusion, the present results demonstrate that IL-6, and IFN-γ, IL-6 and IL-1β are the serum mediators released in vivo by a turpentine-induced inflammatory reaction in the rabbit and an upper respiratory viral infection in humans, respectively, inactivating hepatic P450. PMID:10952665

Molecularly imprinted composite cryogel for albumin depletion from human serum.

PubMed

Andaç, Müge; Baydemir, Gözde; Yavuz, Handan; Denizli, Adil

2012-11-01

A new composite protein-imprinted macroporous cryogel was prepared for depletion of albumin from human serum prior to use in proteom applications. Polyhydroxyethyl-methacylate-based molecularly imprinted polymer (MIP) composite cryogel was prepared with high gel fraction yields up to 83%, and its morphology and porosity were characterized by Fourier transform infrared, scanning electron microscopy, swelling studies, flow dynamics, and surface area measurements. Selective binding experiments were performed in the presence of competitive proteins human transferrin (HTR) and myoglobin (MYB). MIP composite cryogel exhibited a high binding capacity and selectivity for human serum albumin (HSA) in the presence of HTR and MYB. The competitive adsorption amount for HSA in MIP composite cryogel is 722.1 mg/dL in the presence of competitive proteins (HTR and MYB). MIP composite cryogel column was successfully applied in the fast protein liquid chromatography system for selective depletion of albumin in human serum. The depletion ratio was highly increased by embedding beads into cryogel (85%). Finally, MIP composite cryogel can be reused many times with no apparent decrease in HSA adsorption capacity. Copyright © 2012 John Wiley & Sons, Ltd.

Binding of human serum proteins to titanium dioxide particles in vitro.

PubMed

Zaqout, Mazen S K; Sumizawa, Tomoyuki; Igisu, Hideki; Higashi, Toshiaki; Myojo, Toshihiko

2011-01-01

To determine the capacity of human serum proteins to bind to titanium dioxide (TiO(2)) particles of different polymorphs and sizes. TiO(2) particles were mixed with diluted human serum, purified human serum albumin (HSA) or purified human serum gamma-globulin (HGG) solutions. After incubation at 37°C for 1 h, the particles were sedimented by centrifugation, and proteins in the supernatant, as well as those bound to the particles, were analyzed. The total protein concentration in the supernatant was lowered by TiO(2), whereas the albumin/globulin ratio was elevated by the particles. Incubation with TiO(2) also lowered the immunoglobulin, pre-albumin, beta2-microglobulin, ceruloplasmin and retinol-binding protein levels, but not ferritin levels, in the supernatant. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins in the supernatant, especially HGG, were observed to decrease, while those released from the particles (after adding 1% SDS and heating) increased, depending on the dose of TiO(2). Purified HGG and HSA were also bound to TiO(2), although the former appeared to have a higher affinity. All the proteins tested showed the highest binding potency to the amorphous particles (<50 nm) and the lowest to the rutile particles (<5,000 nm), while binding to anatase particles was intermediate. The affinity to the larger anatase was higher than that to smaller anatase particles in most cases. Human serum proteins, including the two major components, HSA and HGG, are bound by TiO(2) particles. The polymorph of the particles seems to be important for determining the binding capacity of the particles and it may affect distribution of the particles in the body.

Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shibata, Akitomo; Ishima, Yu; Ikeda, Mayumi

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na{sub 2}S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximatelymore » 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases. - Highlights: • Hydropersulfide can behave as potent antioxidants. • We firstly detected human serum albumin hydropersulfide in healthy subjects. • Human serum albumin hydropersulfide in human subjects were reduced in chronic kidney disease but restored by hemodialysis.« less

Novel cell-based assay reveals associations of circulating serum AhR-ligands with metabolic syndrome and mitochondrial dysfunction.

PubMed

Park, Wook-Ha; Jun, Dae Won; Kim, Jin Taek; Jeong, Jae Hoon; Park, Hyokeun; Chang, Yoon-Seok; Park, Kyong Soo; Lee, Hong Kyu; Pak, Youngmi Kim

2013-01-01

Serum concentrations of environmental pollutants have been positively correlated with diabetes and metabolic syndrome in epidemiologic studies. In turn, abnormal mitochondrial function has been associated with the diseases. The relationships between these variables, however, have not been studied. We developed novel cell-based aryl hydrocarbon receptor (AhR) agonist bioassay system without solvent extraction process and analyzed whether low-dose circulating AhR ligands in human serum are associated with parameters of metabolic syndrome and mitochondrial function. Serum AhR ligand activities were measured as serum 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent (sTCDDeq) in pM using 10 μL human sera from 97 Korean participants (47 with glucose intolerance and 50 matched controls, average age of 46.6 ± 9.9 years, 53 male and 45 female). sTCDDeq were higher in participants with glucose intolerance than normal controls and were positively associated (P < 0.01) with obesity, blood pressure, serum triglyceride, and fasting glucose, but not with HDL-cholesterol. Body mass index was in a positive linear relationship with serum AhR ligands in healthy participants. When myoblast cells were incubated with human sera, ATP generating power of mitochondria became impaired in an AhR ligand concentration-dependent manner. Our results support that circulating AhR ligands may directly reduce mitochondrial function in tissues, leading to weight gain, glucose intolerance, and metabolic syndrome. Our rapid cell-based assay using minute volume of human serum may provide one of the best monitoring systems for circulating AhR ligands, good clinical biomarkers for the progress of disease and therapeutic efficacy. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Glycoblotting method allows for rapid and efficient glycome profiling of human Alzheimer's disease brain, serum and cerebrospinal fluid towards potential biomarker discovery.

PubMed

Gizaw, Solomon T; Ohashi, Tetsu; Tanaka, Masakazu; Hinou, Hiroshi; Nishimura, Shin-Ichiro

2016-08-01

Understanding of the significance of posttranslational glycosylation in Alzheimer's disease (AD) is of growing importance for the investigation of the pathogenesis of AD as well as discovery research of the disease-specific serum biomarkers. We designed a standard protocol for the glycoblotting combined with MALDI-TOFMS to perform rapid and quantitative profiling of the glycan parts of glycoproteins (N-glycans) and glycosphingolipids (GSLs) using human AD's post-mortem samples such as brain tissues (dissected cerebral cortices such as frontal, parietal, occipital, and temporal domains), serum and cerebrospinal fluid (CSF). The structural profiles of the major N-glycans released from glycoproteins and the total expression levels of the glycans were found to be mostly similar between the brain tissues of the AD patients and those of the normal control group. In contrast, the expression levels of the serum and CSF protein N-glycans such as bisect-type and multiply branched glycoforms were increased significantly in AD patient group. In addition, the levels of some gangliosides such as GM1, GM2 and GM3 appeared to alter in the AD patient brain and serum samples when compared with the normal control groups. Alteration of the expression levels of major N- and GSL-glycans in human brain tissues, serum and CSF of AD patients can be monitored quantitatively by means of the glycoblotting-based standard protocols. The changes in the expression levels of the glycans derived from the human post-mortem samples uncovered by the standardized glycoblotting method provides potential serum biomarkers in central nervous system disorders and can contribute to the insight into the molecular mechanisms in the pathogenesis of neurodegenerative diseases and future drug discovery. Most importantly, the present preliminary trials using human post-mortem samples of AD patients suggest that large-scale serum glycomics cohort by means of various-types of human AD patients as well as the normal control sera can facilitate the discovery research of highly sensitive and reliable serum biomarkers for an early diagnosis of AD. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. Copyright © 2016 Elsevier B.V. All rights reserved.

Development, validation and application of an ICP-MS/MS method to quantify minerals and (ultra-)trace elements in human serum.

PubMed

Meyer, Sören; Markova, Mariya; Pohl, Gabriele; Marschall, Talke A; Pivovarova, Olga; Pfeiffer, Andreas F H; Schwerdtle, Tanja

2018-09-01

Multi-element determination in human samples is very challenging. Especially in human intervention studies sample volumes are often limited to a few microliters and due to the high number of samples a high-throughput is indispensable. Here, we present a state-of-the-art ICP-MS/MS-based method for the analysis of essential (trace) elements, namely Mg, Ca, Fe, Cu, Zn, Mo, Se and I, as well as food-relevant toxic elements such as As and Cd. The developed method was validated regarding linearity of the calibration curves, method LODs and LOQs, selectivity and trueness as well as precision. The established reliable method was applied to quantify the element serum concentrations of participants of a human intervention study (LeguAN). The participants received isocaloric diets, either rich in plant protein or in animal protein. While the serum concentrations of Mg and Mo increased in participants receiving the plant protein-based diet (above all legumes), the Se concentration in serum decreased. In contrast, the animal protein-based diet, rich in meat and dairy products, resulted in an increased Se concentration in serum. Copyright © 2018 Elsevier GmbH. All rights reserved.

Binding affinity of aluminium to human serum transferrin and effects of carbohydrate chain modification as studied by HPLC/high-resolution ICP-MS--speciation of aluminium in human serum.

PubMed

Nagaoka, Megumi Hamano; Maitani, Tamio

2005-09-01

Aluminium (Al) in the blood is bound to transferrin (Tf), a glycoprotein of about 80kDa that is characterized by its need for a synergistic anion. In this focused review, the binding affinity of Al to Tf is surveyed in the context of our recent studies using on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry (HPLC/HR-ICP-MS). Al in human serum without any in vitro Al-spikes was present in a form bound to the N-lobe site of Tf. The influences of sialic acid in the carbohydrate chain of human serum Tf (hTf) were studied using asialo-hTf, obtained by treatment with sialidase. The binding affinity of Fe was similar between asialo-hTf and native-hTf, while that of Al for asialo-hTf was larger than that for native-hTf, especially in the presence of oxalate, a synergistic anion. The above findings are discussed in relation to diseases in which the serum concentrations of carbohydrate-deficient Tf and oxalate are augmented.

Detection of teratogens in human serum using rat embryo culture: cancer and epilepsy treatments. [Detecting teratogenicity of anticonvulsant and antineoplastic drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chatot, C. L.

1979-01-01

Growth (protein and DNA contents) of headfold stage rat embryos cultured for 48 hrs on human serum was enhanced by glucose supplementation. Embryo growth varied with the source of the serum. Sera from 3 of the 19 control subjects produced abnormal embryos. Sera from 5 subjects undergoing cancer chemotherapy and 6 subjects receiving anticonvulsants were either lethal or teratogenic to cultured rat embryos.

Ultrasensitive dual-channel detection of matrix metalloproteinase-2 in human serum using gold-quantum dot core-satellite nanoprobes.

PubMed

Zheng, Tingting; Zhang, Rui; Zhang, Qingfeng; Tan, Tingting; Zhang, Kui; Zhu, Jun-Jie; Wang, Hui

2013-09-18

We have developed a robust enzymatic peptide cleavage-based assay for the ultrasensitive dual-channel detection of matrix metalloproteinase-2 (MMP-2) in human serum using gold-quantum dot (Au-QD) core-satellite nanoprobes.

Fabrication of enzyme-degradable and size-controlled protein nanowires using single particle nano-fabrication technique

PubMed Central

Omichi, Masaaki; Asano, Atsushi; Tsukuda, Satoshi; Takano, Katsuyoshi; Sugimoto, Masaki; Saeki, Akinori; Sakamaki, Daisuke; Onoda, Akira; Hayashi, Takashi; Seki, Shu

2014-01-01

Protein nanowires exhibiting specific biological activities hold promise for interacting with living cells and controlling and predicting biological responses such as apoptosis, endocytosis and cell adhesion. Here we report the result of the interaction of a single high-energy charged particle with protein molecules, giving size-controlled protein nanowires with an ultra-high aspect ratio of over 1,000. Degradation of the human serum albumin nanowires was examined using trypsin. The biotinylated human serum albumin nanowires bound avidin, demonstrating the high affinity of the nanowires. Human serum albumin–avidin hybrid nanowires were also fabricated from a solid state mixture and exhibited good mechanical strength in phosphate-buffered saline. The biotinylated human serum albumin nanowires can be transformed into nanowires exhibiting a biological function such as avidin–biotinyl interactions and peroxidase activity. The present technique is a versatile platform for functionalizing the surface of any protein molecule with an extremely large surface area. PMID:24770668

Thermometric enzyme linked immunosorbent assay in continuous flow system: optimization and evaluation using human serum albumin as a model system.

PubMed

Borrebaeck, C; Börjeson, J; Mattiasson, B

1978-06-15

Thermometric enzyme-linked immunosorbent assay (TELISA) is described. After the procedure of optimization, human serum albumin was assayed using anti-human serum albumin bound to Sepharose CL 4-B in the enzyme thermistor unit and catalase as label on the free antigen. The model system was used for assays down to 10(-13)M and the preparation of immobilized antibodies was used repeatedly up to 100 times. Comparative studies of the TELISA technique with bromocresol green, immunoturbidimetric and rocket immunoelectrophoretic methods were carried out and showed that TELISA could be used as an alternative method.

Serum microRNAs are early indicators of survival after radiation-induced hematopoietic injury

PubMed Central

Acharya, Sanket S.; Fendler, Wojciech; Watson, Jacqueline; Hamilton, Abigail; Pan, Yunfeng; Gaudiano, Emily; Moskwa, Patryk; Bhanja, Payel; Saha, Subhrajit; Guha, Chandan; Parmar, Kalindi; Chowdhury, Dipanjan

2015-01-01

Accidental radiation exposure is a threat to human health that necessitates effective clinical planning and diagnosis. Minimally invasive biomarkers that can predict long-term radiation injury are urgently needed for optimal management after a radiation accident. We have identified serum microRNA (miRNA) signatures that indicate long-term impact of total body irradiation (TBI) in mice when measured within 24 hours of exposure. Impact of TBI on the hematopoietic system was systematically assessed to determine a correlation of residual hematopoietic stem cells (HSCs) with increasing doses of radiation. Serum miRNA signatures distinguished untreated mice from animals exposed to radiation and correlated with the impact of radiation on HSCs. Mice exposed to sublethal (6.5 Gy) and lethal (8 Gy) doses of radiation were indistinguishable for 3 to 4 weeks after exposure. A serum miRNA signature detectable 24 hours after radiation exposure consistently segregated these two cohorts. Furthermore, using either a radioprotective agent before, or radiation mitigation after, lethal radiation, we determined that the serum miRNA signature correlated with the impact of radiation on animal health rather than the radiation dose. Last, using humanized mice that had been engrafted with human CD34+ HSCs, we determined that the serum miRNA signature indicated radiation-induced injury to the human bone marrow cells. Our data suggest that serum miRNAs can serve as functional dosimeters of radiation, representing a potential breakthrough in early assessment of radiation-induced hematopoietic damage and timely use of medical countermeasures to mitigate the long-term impact of radiation. PMID:25972001

Asymmetric cellular responses in primary human myoblasts using sera of different origin and specification

PubMed Central

Rullman, Eric; Lilja, Mats; Mandić, Mirko; Melin, Michael; Olsson, Karl; Gustafsson, Thomas

2018-01-01

For successful growth and maintenance of primary myogenic cells in vitro, culture medium and addition of sera are the most important factors. At present it is not established as to what extent sera of different origin and composition, supplemented in media or serum-free media conditions influence myoblast function and responses to different stimuli. By assessing markers of proliferation, differentiation/fusion, quiescence, apoptosis and protein synthesis the aim of the current study was to elucidate how primary human myoblasts and myotubes are modulated by different commonly used serum using FCS (foetal calf serum), (CS-FCS charcoal-stripped FCS, a manufacturing process to remove hormones and growth factors from sera), HS (horse serum) as well as in serum free conditions (DMEM). To characterise the biological impact of the different serum, myoblasts were stimulated with Insulin (100 nM) and Vitamin D (100 nM; 1α,25(OH)2D3, 1α,25-Dihydroxycholecalciferol, Calcitriol), two factors with characterised effects on promoting fusion and protein synthesis or quiescence, respectively in human myoblasts/myotubes. We demonstrate that sera of different origin/formulation differentially affect myoblast proliferation and myotube protein synthesis. Importantly, we showed that quantifying the extent to which Insulin effects myoblasts in vitro is highly dependent upon serum addition and which type is present in the media. Upregulation of mRNA markers for myogenic fusion, Myogenin, with Insulin stimulation, relative to DMEM, appeared dampened at varying degrees with serum addition and effects on p70S6K phosphorylation as a marker of protein synthesis could not be identified unless serum was removed from media. We propose that these asymmetric molecular and biochemical responses in human myoblasts reflect the variable composition of mitogenic and anabolic factors in each of the sera. The results have implications for both the reproducibility and interpretation of results from experimental models in myoblast cells/myotubes. PMID:29401478

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line

PubMed Central

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-01-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines. PMID:28810654

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line.

PubMed

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-08-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines.

The ACVD task force on canine atopic dermatitis (XVI): laboratory evaluation of dogs with atopic dermatitis with serum-based "allergy" tests.

PubMed

DeBoer, D J; Hillier, A

2001-09-20

Serum-based in vitro "allergy tests" are commercially available to veterinarians, and are widely used in diagnostic evaluation of a canine atopic patient. Following initial clinical diagnosis, panels of allergen-specific IgE measurements may be performed in an attempt to identify to which allergens the atopic dog is hypersensitive. Methodology for these tests varies by laboratory; few critical studies have evaluated performance of these tests, and current inter-laboratory standardization and quality control measures are inadequate. Other areas where information is critically limited include the usefulness of these tests in diagnosis of food allergy, the effect of extrinsic factors such as season of the year on results, and the influence of corticosteroid treatment on test results. Allergen-specific IgE serological tests are never completely sensitive, nor completely specific. There is only partial correlation between the serum tests and intradermal testing; however, the significance of discrepant results is unknown and unstudied. Variation in test methodologies along with the absence of universal standardization and reporting procedures have created confusion, varying study results, and an inability to compare between studies performed by different investigators.

The robust bio-immobilization based on pulsed plasma polymerization of cyclopropylamine and glutaraldehyde coupling chemistry

NASA Astrophysics Data System (ADS)

Manakhov, Anton; Makhneva, Ekaterina; Skládal, Petr; Nečas, David; Čechal, Jan; Kalina, Lukáš; Eliáš, Marek; Zajíčková, Lenka

2016-01-01

The performance of immunosensing devices crucially depends on the methodology of antibody or antigen immobilization on the sensor surface. Hence, the stable intermediate layers capable of specific and reproducible binding of antibodies are required. Herein, we introduce the amine rich (NHx concentration of 6 at.%) layers prepared by pulsed plasma polymerization of cyclopropylamine (CPA) for functionalization of the quartz crystal microbalance (QCM) surface by the antibody specific to human serum albumin. In these layers the amine groups serve as anchor for the antibody binding. The sensitivity of QCM sensors prepared in this way surpasses the one for the previously reported sensors functionalized by the thiol-based self-assembled monolayers by the factor of 2. Our results thus show that CPA plasma polymers have a significant potential for further development of the active layers for biosensing applications.

Influence of human body composition on serum peak thyrotropin (TSH) after recombinant human TSH administration in patients with differentiated thyroid carcinoma.

PubMed

Castagna, Maria Grazia; Pinchera, Aldo; Marsili, Alessandro; Giannetti, Monica; Molinaro, Eleonora; Fierabracci, Paola; Grasso, Lucia; Pacini, Furio; Santini, Ferruccio; Elisei, Rossella

2005-07-01

In this study, we evaluated the influence of height, weight, body mass index (BMI), body surface area, and body composition [total lean body mass (LBM) and fat body mass] on serum peak TSH levels obtained after recombinant human (rh)TSH. Furthermore, to verify whether the serum peak TSH influenced the efficacy of radioiodine ((131)I), we compared the rate of thyroid remnant ablation according to the patients' BMI. We studied 105 patients with differentiated thyroid carcinoma who underwent rhTSH stimulation test. Serum TSH measurements were performed before and 24, 48, and 72 h after rhTSH administration. We also compared the rate of thyroid remnant ablation among 70 differentiated thyroid carcinoma patients with different BMI. The serum peak TSH after rhTSH was significantly lower in overweight and obese subjects compared with normal-weight subjects (92.1 +/- 41.8, 82.4 +/- 24.2, and 112.7 +/- 46.3 microU/ml, respectively; P = 0.01) and in males compared with females (74.6 +/- 22.3 and 105.0 +/- 43.0 microU/ml, respectively; P = 0.0002). By univariate analysis, serum peak TSH was negatively related to weight, height, body surface area, BMI, LBM, and fat body mass, but only LBM was independently associated with serum peak TSH levels. Although it was confirmed that overweight and obese patients had a lower serum peak TSH, the rate of ablation did not differ among normal-weight, overweight, and obese patients. With this study we demonstrated that LBM is the only parameter independently associated with serum peak TSH after rhTSH administration. However, the serum peak TSH does not influence the rate of (131)I remnant ablation.

OBSERVATIONS ON THE PRESENCE OF A TOXIC SUBSTANCE IN THE BLOOD AND URINE OF PATIENTS WITH SCARLET FEVER

PubMed Central

Trask, James D.; Blake, Francis G.

1924-01-01

A series of observations on the blood of patients acutely ill with scarlet fever has shown that a toxic substance can be demonstrated in the serum by means of intracutaneous injections of the serum in persons who have not had scarlet fever and whose serums fail to blanch the rash in scarlet fever. The reaction caused by this substance consists of a bright red local erythema, varying from 20 to 70 mm. in diameter, of 1 to 4 days duration. The severer reactions are moderately indurated and tender, and are followed bypigmentation and desquamation. Control injections in persons whose serums blanch the rash in scarlet fever cause no reaction. The toxic substance is not neutralized by mixture with a human serum which gives a negative blanching test but is readily neutralized by a human serum which gives a positive blanching test. It is not neutralized by normal horse serum, but is completely neutralized by Dochez's scarlatinal antistreptococcic serum. In a limited number of observations on the urine of patients with scarlet fever a similar toxic substance has been found in two out of five cases studied. Since the toxic substance described appears to resemble the toxic substance found in the filtrates of scarlatinal hemolytic streptococcus cultures by Dick and Dick and since it is neutralized not only by a blanching human serum but also by Dochez's scarlatinal antistreptococcic horse serum, the experiments reported support the conception that scarlet fever is a local infection of the throat by a particular type of Streptococcus hæmolyticus capable of producing a toxin which is absorbed and is the cause of the general manifestations of the disease. PMID:19868926

Creatinine elevation associated with nitromethane exposure: a marker of potential methanol toxicity.

PubMed

Cook, Matthew D; Clark, Richard F

2007-10-01

Nitromethane, methanol, and oil are the common components of radio-controlled (R/C) vehicle fuels. Nitromethane can cause a false elevation of serum creatinine concentration as measured by the widely used Jaffe colorimetric method. We gathered data from our poison control system and from previously published case reports to see if a correlation exists between serum methanol concentrations and spuriously elevated serum creatinine concentrations after human exposures to R/C fuel. The California Poison Control System (CPCS) computerized database was queried for all cases of human exposure to R/C vehicle fuel reported between December 1, 2002 and December 1, 2004. Serum creatinine and methanol concentrations were recorded when available, as was the method used to determine serum creatinine. A MEDLINE search was used to obtain previously published cases of human nitromethane exposure associated with falsely elevated creatinine concentrations. During the 2-year period, serum creatinine concentrations were recorded in 7 of 26 R/C fuel exposures (all ingestions), and 6 of these were abnormal (range of 1.9-11.5 mg/dL). In this series, the higher the serum creatinine concentration measured by Jaffe method, the higher the serum methanol concentration. The MEDLINE search yielded data from six previously published case reports on this topic. The data from these case reports seem to follow the trend seen in our case series. These data suggest that a spuriously elevated serum creatinine (by Jaffe method) may have value as an early surrogate marker of methanol poisoning in those who ingest R/C fuel. Also, the degree to which the serum creatinine is elevated may indicate the severity of methanol poisoning.

Serum starvation of ARPE-19 changes the cellular distribution of cholesterol and Fibulin3 in patterns reminiscent of age-related macular degeneration.

PubMed

Rajapakse, Dinusha; Peterson, Katherine; Mishra, Sanghamitra; Wistow, Graeme

2017-12-15

Retinal pigment epithelium (RPE) has been implicated as key source of cholesterol-rich deposits at Bruch's membrane (BrM) and in drusen in aging human eye. We have shown that serum-deprivation of confluent RPE cells is associated with upregulation of cholesterol synthesis and accumulation of unesterified cholesterol (UC). Here we investigate the cellular processes involved in this response. We compared the distribution and localization of UC and esterified cholesterol (EC); the age-related macular degeneration (AMD) associated EFEMP1/Fibulin3 (Fib3); and levels of acyl-coenzyme A (CoA): cholesterol acyltransferases (ACAT) ACAT1, ACAT2 and Apolipoprotein B (ApoB) in ARPE-19 cells cultured in serum-supplemented and serum-free media. The results were compared with distributions of these lipids and proteins in human donor eyes with AMD. Serum deprivation of ARPE-19 was associated with increased formation of FM dye-positive membrane vesicles, many of which co-labeled for UC. Additionally, UC colocalized with Fib3 in distinct granules. By day 5, serum-deprived cells grown on transwells secreted Fib3 basally into the matrix. While mRNA and protein levels of ACTA1 were constant over several days of serum-deprivation, ACAT2 levels increased significantly after serum-deprivation, suggesting increased formation of EC. The lower levels of intracellular EC observed under serum-deprivation were associated with increased formation and secretion of ApoB. The responses to serum-deprivation in RPE-derived cells: accumulation and secretion of lipids, lipoproteins, and Fib3 are very similar to patterns seen in human donor eyes with AMD and suggest that this model mimics processes relevant to disease progression. Published by Elsevier Inc.

Serum from pregnant women with gestational diabetes mellitus increases the expression of FABP4 mRNA in primary subcutaneous human pre-adipocytes

PubMed Central

Li, Lan; Lee, Se Jin; Kook, Song Yi; Ahn, Tae Gyu; Lee, Ji Yeon

2017-01-01

Objective Gestational diabetes mellitus (GDM) is defined as glucose intolerance first detected during pregnancy. It can result in pregnancy complications such as birth injury, stillbirth. Fatty acid-binding protein 4 (FABP4), found in adipose tissue, is associated with insulin resistance, and type 2 diabetes. The aim of this study was to investigate whether FABP4 in the placenta and decidua of pregnant women with GDM is higher than that in normal pregnant women, and whether serum from pregnant women with GDM may cause adipocytes to secrete more FABP4 than does serum from a normal pregnant group. Methods We obtained placentas, deciduas, and serum from 12 pregnant women with GDM and 12 normal pregnant women and performed enzyme-linked immunosorbent assay, real time quantitative-polymerase chain reaction. We cultured human pre-adipocytes for 17 days with GDM and non-GDM serum and performed western blot, real time quantitative-polymerase chain reaction, and oil red O staining. Results Expression of FABP4 in serum, placenta and decidua of pregnant women with GDM was significantly higher than that in normal pregnant women. Serum from pregnant women with GDM increased the expression of FABP4 mRNA and decreased the expression of adiponectin mRNA in human pre-adipocytes significantly. Adipocyte cultured in GDM serum showed significantly greater lipid accumulation than those cultured in normal serum. Conclusion Our results suggest that FABP4 is higher in placenta and decidua from pregnant women with GDM. Increased circulating FABP4 in maternal serum from pregnant women with GDM may originate from adipocytes and the placenta. Circulating FABP4 can induce increased insulin resistance and decreased insulin sensitivity. PMID:28534013

Serum from pregnant women with gestational diabetes mellitus increases the expression of FABP4 mRNA in primary subcutaneous human pre-adipocytes.

PubMed

Li, Lan; Lee, Se Jin; Kook, Song Yi; Ahn, Tae Gyu; Lee, Ji Yeon; Hwang, Jong Yun

2017-05-01

Gestational diabetes mellitus (GDM) is defined as glucose intolerance first detected during pregnancy. It can result in pregnancy complications such as birth injury, stillbirth. Fatty acid-binding protein 4 (FABP4), found in adipose tissue, is associated with insulin resistance, and type 2 diabetes. The aim of this study was to investigate whether FABP4 in the placenta and decidua of pregnant women with GDM is higher than that in normal pregnant women, and whether serum from pregnant women with GDM may cause adipocytes to secrete more FABP4 than does serum from a normal pregnant group. We obtained placentas, deciduas, and serum from 12 pregnant women with GDM and 12 normal pregnant women and performed enzyme-linked immunosorbent assay, real time quantitative-polymerase chain reaction. We cultured human pre-adipocytes for 17 days with GDM and non-GDM serum and performed western blot, real time quantitative-polymerase chain reaction, and oil red O staining. Expression of FABP4 in serum, placenta and decidua of pregnant women with GDM was significantly higher than that in normal pregnant women. Serum from pregnant women with GDM increased the expression of FABP4 mRNA and decreased the expression of adiponectin mRNA in human pre-adipocytes significantly. Adipocyte cultured in GDM serum showed significantly greater lipid accumulation than those cultured in normal serum. Our results suggest that FABP4 is higher in placenta and decidua from pregnant women with GDM. Increased circulating FABP4 in maternal serum from pregnant women with GDM may originate from adipocytes and the placenta. Circulating FABP4 can induce increased insulin resistance and decreased insulin sensitivity.

Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crow, J. Allen; Borazjani, Abdolsamad; Potter, Philip M.

2007-05-15

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrinmore » are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are {approx} 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts ({approx} 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected. Together, these results demonstrate that extrahepatic esterolytic metabolism of specific pyrethroids may be significant. Moreover, hepatic cytosolic and microsomal hydrolytic metabolism should each be considered during the development of pharmacokinetic models that predict the disposition of pyrethroids and other esterified compounds.« less

Correlation of human S100A12 (EN-RAGE) and high-sensitivity C-reactive protein as gingival crevicular fluid and serum markers of inflammation in chronic periodontitis and type 2 diabetes.

PubMed

Pradeep, A R; Martande, Santosh S; Singh, Sonender Pal; Suke, Deepak Kumar; Raju, Arjun P; Naik, Savitha B

2014-04-01

The aim of the present study was to evaluate the levels and correlation of human S100A12 and high-sensitivity C-reactive protein (hs-CRP) in gingival crevicular fluid (GCF) and serum in chronic periodontitis (CP) subjects with and without type 2 diabetes mellitus (DM). A total of 44 subjects were divided into three groups: group 1 had 10 periodontally healthy subjects, group 2 consisted of 17 CP subjects and group 3 had 17 type 2 DM subjects with CP. GCF and serum levels of human S100A12 and hs-CRP were quantified using enzyme-linked immunosorbent assay and immunoturbidimetric analysis, respectively. The clinical outcomes evaluated were gingival index, probing depth and clinical attachment level and the correlations of the two inflammatory mediators with clinical parameters were evaluated. Both human S100A12 and hs-CRP levels increased from group 1 to group 2 to group 3. The GCF and serum values of both these inflammatory mediators correlated positively with each other and with the periodontal parameters evaluated (p < 0.05). Human S100A12 and hs-CRP can be considered as possible GCF and serum markers of inflammatory activity in CP and DM.

Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface

PubMed Central

Marín, Elvira; Parra-Giraldo, Claudia M.; Hernández-Haro, Carolina; Hernáez, María L.; Nombela, César; Monteoliva, Lucía; Gil, Concha

2015-01-01

Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in NS than in HIS, as validated by immunofluorescence. PMID:26696967

ACTIVE IMMUNIZATION AGAINST POLIOMYELITIS IN MONKEYS.

PubMed

Brodie, M; Goldbloom, A

1931-05-31

1. A combination of poliomyelitis virus and specific human serum is effective for the production of active immunity. 2. For each gram of active virus given intradermally as an emulsion, 6 cc. of human immune serum, injected subcutaneously, was required in our experiments to protect a monkey from paralysis. Some degree of active immunity was induced. 3. Immunity, without symptom of the disease, was secured when the serum was given at the time of inoculation, or within 3 days preceding or following inoculation of the virus. 4. For the production of immunity, virus, preceded by serum administration, is probably less effective than when it is given simultaneously with, or before, the injection of serum. 5. The virus neutralization test is more sensitive than the direct intracerebral test for determining the production of immunity.

Simple method for determining human serum 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity - possible application in clinical studies on dietary antioxidants.

PubMed

Chrzczanowicz, Jacek; Gawron, Anna; Zwolinska, Anna; de Graft-Johnson, Jeffrey; Krajewski, Wojciech; Krol, Maciej; Markowski, Jaroslaw; Kostka, Tomasz; Nowak, Dariusz

2008-01-01

2,2-Diphenyl-1-picryl-hydrazyl (DPPH) radical decomposition in alcohol solution is widely used, characterizing plant antioxidants that can rise in serum after fruit and vegetable intake. However, this test failed reproducible results with serum due to protein precipitation. We describe the application of serum deproteinization with acetonitrile relating to the DPPH test. Assay sensitivity, linearity, repeatability and storage effect were determined in serum samples deproteinized with an equal volume of acetonitrile. Associations between the DPPH test and the ferric reducing ability of serum (FRAP) method, measuring total antioxidant potential, were evaluated in sera from 78 healthy non-smoking men. The effect of a single ingestion of 1 L of cloudy apple juice on the serum DPPH radical scavenging activity in healthy volunteers was also investigated. Assay linearity was within 5-25 microL (r=0.99, p<0.01). With 25 microL-deproteinized serum, coefficient of variation was 4.2% and detection limit was 0.5% of the initial amount of decomposed DPPH radical over 30 min incubation. There was no sera activity decrease over 14 days storage at -20 degrees C. Mean values of DPPH radical scavenging activity and FRAP obtained in human serum were 11.2+/-3.3% and 382.0+/-88.1 micromol/L, respectively. A positive significant linear correlation was observed between these two methods (r=0.42, p<0.01). Serum supplementation with 50 micromol/L of catechin, gallic acid, ascorbic acid or uric acid enhanced DPPH test results. One brisk serving of 1 L of apple juice caused a significant increment of serum DPPH radical scavenging activity (1.9+/-1.9%, p<0.01) in 12 healthy subjects 1 h after juice ingestion. Applicability of the DPPH test to deproteinized serum with acetonitrile revealed numerous advantages, validating its practicability, simplicity and cost effectiveness as a tool in the estimation of antioxidant status in humans.

Use of egg yolk antibody (IgY) as an immunoanalytical tool in the detection of Indian cobra (Naja naja naja) venom in biological samples of forensic origin.

PubMed

Brunda, G; Sashidhar, R B; Sarin, R K

2006-08-01

An immunoglobulin Y (IgY) based indirect double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed for the detection of Indian cobra (Naja naja naja) venom in the biological samples of forensic origin. Polyclonal antibodies were raised and purified from chick egg yolk and rabbit serum. The cobra venom was sandwiched between immobilized affinity purified IgY and the rabbit IgG. The detection concentration of cobra venom was in the range of 0.1 to 300ng. The calibration plot was based on linear regression analysis (y=0.2581x+0.4375, r(2)=0.9886). The limit of detection of the assay was found to be 0.1ng. The coefficient of variation (CV) of different concentrations of working range in inter (n=6) and intra-assay (n=6) was observed to be less than 10%. The recovery of venom was found to be in the range of 80-99%, when different concentrations (0.002, 0.1, 0.2, 1, and 2microg) of cobra venom were spiked to pooled normal human serum (ml(-1)). No cross reactivity was observed with krait and viper venom in the immunoassay system in the concentration range of 0.1-1000ng. The method was initially, validated by analyzing specimens (autopsy) of experimental rats injected with cobra venom (1.2mgkg(-1) body mass). Further, human specimens (autopsy and biopsy) of snake bite victims of forensic origin were also analyzed. The methodology developed may find diagnostic application in forensic laboratories.

Combination of human acetylcholinesterase and serum albumin sensing surfaces as highly informative analytical tool for inhibitor screening.

PubMed

Fabini, Edoardo; Tramarin, Anna; Bartolini, Manuela

2018-06-05

In the continuous research for potential drug lead candidates, the availability of highly informative screening methodologies may constitute a decisive element in the selection of best-in-class compounds. In the present study, a surface plasmon resonance (SPR)-based assay was developed and employed to investigate interactions between human recombinant AChE (hAChE) and four known ligands: galantamine, tacrine, donepezil and edrophonium. To this aim, a sensor chip was functionalized with hAChE using mild immobilization conditions to best preserve enzyme integrity. Binding affinities and, for the first time, kinetic rate constants for all drug-hAChE complexes formation/disruption were determined. Inhibitors were classified in two groups: slow-reversible and fast-reversible binders according to respective target residence time. Combining data obtained on drug-target residence time with data obtained on serum albumin binding levels, a good correlation with potency, plasma protein binding in vivo, and administration regimen was found. The outcomes of this work demonstrated that the developed SPR-based assay is suitable for the screening, the binding affinity ranking and the kinetic evaluation of hAChE inhibitors. The method proposed ensures a simpler and cost-effective assay to quantify kinetic rate constants for inhibitor-hAChE interaction as compared with other proposed and published methods. Eventually, the determination of residence time in combination with preliminary ADME studies might constitute a better tool to predict in vivo behaviour, a key information for the research of new potential drug candidates. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of the structure and dynamics of human serum albumin.

PubMed

Guizado, T R Cuya

2014-10-01

Human serum albumin (HSA) is a biologically relevant protein that binds a variety of drugs and other small molecules. No less than 50 structures are deposited in the RCSB Protein Data Bank (PDB). Based on these structures, we first performed a clustering analysis. Despite the diversity of ligands, only two well defined conformations are detected, with a deviation of 0.46 nm between the average structures of the two clusters, while deviations within each cluster are smaller than 0.08 nm. Those two conformations are representative of the apoprotein and the HSA-myristate complex already identified in previous literature. Considering the structures within each cluster as a representative sample of the dynamical states of the corresponding conformation, we scrutinize the structural and dynamical differences between both conformations. Analysis of the fluctuations within each cluster set reveals that domain II is the most rigid one and better matches both structures. Then, taking this domain as reference, we show that the structural difference between both conformations can be expressed in terms of twist and hinge motions of domains I and III, respectively. We also characterize the dynamical difference between conformations by computing correlations and principal components for each set of dynamical states. The two conformations display different collective motions. The results are compared with those obtained from the trajectories of short molecular dynamics simulations, giving consistent outcomes. Let us remark that, beyond the relevance of the results for the structural and dynamical characterization of HAS conformations, the present methodology could be extended to other proteins in the PDB archive.

Lack of Serologic Evidence of Neospora caninum in Humans, England

PubMed Central

McCann, Catherine M.; Vyse, Andrew J.; Salmon, Roland L; Thomas, Daniel; Williams, Diana J.L.; McGarry, John W.; Pebody, Richard

2008-01-01

Retrospective testing of 3,232 serum samples from the general population and 518 serum samples from a high-risk group showed no evidence of human exposure to Neospora caninum in England. Results were obtained by using immunofluorescence antibody testing and ELISA to analyze frequency distribution. PMID:18507920

Keratinocytes propagated in serum-free, feeder-free culture conditions fail to form stratified epidermis in a reconstituted skin model.

PubMed

Lamb, Rebecca; Ambler, Carrie A

2013-01-01

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model

PubMed Central

Lamb, Rebecca; Ambler, Carrie A.

2013-01-01

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification. PMID:23326335

Effect of resistant and digestible rice starches on human cytokine and lactate metabolic networks in serum.

PubMed

Wang, Huisong; Pang, Guangchang

2017-05-01

Resistant starch generated after treating ordinary starch is of great significance to human health in the countries with overnutrition. However, its functional evaluation in the human body has been rarely reported. By determining the lactate metabolic flux, 12 serum enzymes expression level and 38 serum cytokines in healthy volunteers, the variation in cytokine network and lactate metabolic network in serum were investigated to compare the mechanism of the physiological effects between the two starches. The results indicated that compared with digestible starch, resistant starch had anti-inflammatory effects, increased anabolism, and decreased catabolism. Further, the intercellular communication networks including cytokine and lactate metabolic networks were mapped out. The relationship suggested that resistant starch might affect and control the secretion of cytokines to regulate lactate metabolic network in the body, promoting the development of immunometabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biomonitoring of selenoprotein P in human serum by fast affinity chromatography coupled to ICP-MS.

PubMed

Heitland, Peter; Köster, Helmut D

2018-04-01

Most of the Se in human serum is bound to selenoprotein P (SEPP1) in which Se is present in form of selenocysteine. The SEPP1 is a new possible biomarker for the Se status and for this reason we developed a fast, simple and reliable method for the quantitative determination of SEPP1 in serum by affinity chromatography coupled to ICP-MS. It is possible to separate SEPP1 from other selenoproteins in serum in only 5 min, which allows high sample throughput in clinical laboratories. Measured and certified concentrations of total Se and Se(SEPP1) are in good agreement for the reference material SRM 1950. The SEPP1 concentration was stable in serum samples of 3 persons for a minimum of 2 weeks. Further results of method validation were described including internal and external quality assurance. The analytical method was applied for a biomonitoring study of the SEPP1 and total Se concentration in human serum of 50 occupationally non-exposed persons living in northern Germany. Concentration ranges and mean concentrations for Se(SEPP1) are 31.1-59.7 and 46.2 μg/L, respectively. The corresponding values for total Se are 62-120 and 83.5 μg/L. The mean percentage of total Se in serum present as SEPP1 is 58%. Copyright © 2018 Elsevier GmbH. All rights reserved.

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

PubMed Central

Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation. PMID:28465691

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions.

PubMed

Patrikoski, Mimmi; Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

Host plasma proteins on the surface of pathogenic Trichomonas vaginalis.

PubMed

Peterson, K M; Alderete, J F

1982-08-01

Sodium dodecyl sulfate-gel electrophoresis and fluorography and fluorography technology revealed that pathogenic Trichomonas vaginalis was able to acquire numerous loosely associated plasma proteins during incubation in normal human plasma. These proteins were readily removed by repeated washing of the parasite in phosphate-buffered saline. Plasma proteins avidly bound to the surface of T. vaginalis were also detected using a highly sensitive and specific agglutination assay with protein A-bearing Staphylococcus aureus pretreated with monospecific antiserum directed against individual human serum proteins. These avidly associated plasma proteins could not be removed by repeated washing in phosphate-buffered saline or by treatment of washed, live organisms with surface-modifying reagents such as trypsin and periodate. A combined radioimmunoprecipitation-gel electrophoresis-fluorography methodology indicated that parasite biosynthesis of hostlike macromolecules was not responsible for the observed agglutination and reinforced the idea of trichosomal acquisition of plasma components. Finally, incubation of trichomonads with plasma in various buffers at different pH values did not alter the agglutination patterns. These and other data suggest that specific membrane sites trichomonal binding of host proteins. The biological significance of our results is discussed.

THE INACTIVATION OF PENICILLINS F, G, K, AND X BY HUMAN AND RABBIT SERUM

PubMed Central

Eagle, Harry

1947-01-01

1. Penicillins F, G, K, and X were all inactivated by human and rabbit serum, but two qualitatively distinct mechanisms were apparently involved. 2. One was a slow inactivation of all four penicillins by a relatively thermostable serum component which was not demonstrably affected by heating for 60 minutes at 56°C. (a) In both human and rabbit serum this general inactivation of penicillin behaved like a pseudo first order reaction, with a velocity constant of 0.05–0.07 for penicillin X, and 0.09–0.11 for penicillins F and G. (b) The percentage of penicillins F, G, and X inactivated per hour was independent of their concentration over the range 0.4 to 50 micrograms per cc., averaging 9.5, 10, and 6.5 per cent, respectively, in human serum, and 9,8.5, and 5 per cent in rabbit serum. (c) The rate of inactivation varied linearly with the concentration of the serum factor. (d) Penicillin X was consistently and significantly less susceptible to inactivation than any of the other penicillins. Although minor differences were observed between F and G, these were not consistent, and are of questionable significance. 3. Superimposed on this slow inactivation of penicillins F, G, K, and X by a thermostable serum component was a much faster inactivation observed only with penicillin K. (a) In both rabbit and human serum, the serum factor responsible for this inactivation was highly thermolabile, and was almost completely destroyed within 5 minutes at 56°C., leaving only a thermostable component, not affected by further heating. (b) The inactivation of K by this thermolabile component was not a first order reaction, but varied with the concentration of both serum and penicillin. At high concentrations of K, the rate of inactivation due to the thermolabile factor was negligible, and penicillin K was destroyed no more rapidly than F, G, or X. The rate of inactivation increased as the concentration of penicillin was reduced. At penicillin K concentrations of 50, 10, 2, and 0.4 micrograms per cc., the hourly destruction in rabbit serum averaged 10, 16, 21, and 54 per cent. The corresponding figures in human serum were 10, 11, 14, and 54 per cent. The reservations entailed by the large serum error at the lower concentrations of penicillin are discussed in the text. 4. The temperature coefficient for the inactivation of penicillin K by fresh human or rabbit serum was 2.5 for each 10°C. No significant inactivation was observed in 24 hours at 20°C.; and this was true also of penicillins F, G, and X. 5. Heparinized plasma was just as active as serum, washed red blood cells had no effect, and the activity of whole blood was referable to its plasma content. 6. The nature of the serum factors responsible for these two types of penicillin inactivation are under present study. 7. The urinary excretion of penicillin is so rapid that the slow destruction of penicillins F, G, and X in the circulating blood as here described is of secondary significance therapeutically. It nevertheless must contribute to their rapid disappearance from the blood; and the fact that X is inactivated more slowly than either F or G could be reflected in higher and more sustained blood levels than are afforded by the latter two species. There are some reports that such is the case (15–17), and the following paper provides further evidence for the superiority of penicillin X in this respect over the other species so far studied. The serum inactivation of penicillin K, at a rate which increases as its concentration falls, should be reflected in significantly lower and more evanescent blood levels than are observed with penicillins F, G, or X. As will be discussed in the following paper, this has been found to be the case, and provides a simple explanation for its paradoxically low therapeutic activity in vivo (8–11). PMID:19871604

Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils

PubMed Central

Kobayashi, Scott D.; Porter, Adeline R.; Freedman, Brett; Pandey, Ruchi; Chen, Liang; Kreiswirth, Barry N.

2018-01-01

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae is a problem worldwide. A carbapenem-resistant K. pneumoniae lineage classified as multilocus sequence type 258 (ST258) is prominent in the health care setting in many regions of the world, including the United States. ST258 strains can be resistant to virtually all clinically useful antibiotics; treatment of infections caused by these organisms is difficult, and mortality is high. As a step toward promoting development of new therapeutics for ST258 infections, we tested the ability of rabbit antibodies specific for ST258 capsule polysaccharide to enhance human serum bactericidal activity and promote phagocytosis and killing of these bacteria by human neutrophils. We first demonstrated that an isogenic wzy deletion strain is significantly more susceptible to killing by human heparinized blood, serum, and neutrophils than a wild-type ST258 strain. Consistent with the importance of capsule as an immune evasion molecule, rabbit immune serum and purified IgG specific for ST258 capsule polysaccharide type 2 (CPS2) enhanced killing by human blood and serum in vitro. Moreover, antibodies specific for CPS2 promoted phagocytosis and killing of ST258 by human neutrophils. Collectively, our findings suggest that ST258 CPS2 is a viable target for immunoprophylactics and/or therapeutics. PMID:29535199

Thrombopoietin has a differentiative effect on late-stage human erythropoiesis.

PubMed

Liu, W; Wang, M; Tang, D C; Ding, I; Rodgers, G P

1999-05-01

To further explore the mechanism of the effect of thrombopoietin (TPO) on erythropoiesis, we used a two-phase culture system to investigate the effect of TPO on late-stage human erythroid lineage differentiation. In serum-free suspension and semisolid cultures of human peripheral blood derived erythroid progenitors, TPO alone did not produce benzidine-positive cells. However, in serum-containing culture, TPO alone stimulated erythroid cell proliferation and differentiation, demonstrated by erythroid colony formation, production of benzidine-positive cells and haemoglobin (Hb) synthesis. Monoclonal anti-human erythropoietin antibody and anti-human erythropoietin receptor antibody completely abrogated the erythroid differentiative ability of TPO in the serum-containing systems. This implied that binding of EPO and EPO-R was essential for erythropoiesis and the resultant signal transduction may be augmented by the signals emanating from TPO-c-Mpl interaction. Experiment of withdrawal of TPO further demonstrated the involvement of TPO in late-stage erythropoiesis. RT-PCR results showed that there was EPO-R but not c-Mpl expression on developing erythroblasts induced by TPO in serum-containing system. Our results establish that TPO affects not only the proliferation of erythroid progenitors but also the differentiation of erythroid progenitors to mature erythroid cells.

Alteration of human serum albumin tertiary structure induced by glycation. Spectroscopic study

NASA Astrophysics Data System (ADS)

Szkudlarek, A.; Maciążek-Jurczyk, M.; Chudzik, M.; Równicka-Zubik, J.; Sułkowska, A.

2016-01-01

The modification of human serum albumin (HSA) structure by non-enzymatic glycation is one of the underlying factors that contribute to the development of complications of diabetes and neurodegenerative diseases. The aim of the present work was to estimate how glycation of HSA altered its tertiary structure. Changes of albumin conformation were investigated by comparison of glycated (gHSA) and non-glycated human serum albumin (HSA) absorption spectra, red edge excitation shift (REES) and synchronous spectra. Effect of glycation on human serum albumin tertiary structure was also investigated by 1H NMR spectroscopy. Formation of gHSA Advanced Glycation End-products (AGEs) caused absorption of UV-VIS light between 310 nm and 400 nm while for non-glycated HSA in this region no absorbance has been registered. Analysis of red edge excitation shift effect allowed for observation of structural changes of gHSA in the hydrophobic pocket containing the tryptophanyl residue. Moreover changes in the microenvironment of tryptophanyl and tyrosyl residues brought about AGEs on the basis of synchronous fluorescence spectroscopy have been confirmed. The influence of glycation process on serum albumin binding to 5-dimethylaminonaphthalene-1-sulfonamide (DNSA), 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS), has been studied. Fluorescence analysis showed that environment of both binding site I and II is modified by galactose glycation.

Current pesticide profiles in blood serum of adults in Jiangsu Province of China and a comparison with other countries.

PubMed

Chang, Chunxin; Chen, Minjian; Gao, Jiawei; Luo, Jia; Wu, Keqin; Dong, Tianyu; Zhou, Kun; He, Xiaowei; Hu, Weiyue; Wu, Wei; Lu, Chuncheng; Hang, Bo; Meeker, John D; Wang, Xinru; Xia, Yankai

2017-05-01

Although various pesticides were used globally, the pesticides profiles in human blood serum remain largely unknown. We determined pesticide exposure profiles using solid-phase extraction and gas chromatography tandem with triple quadrupole mass spectrometry in 200 human blood serum samples from the adult population in Jiangsu Province, China. A systematic and comprehensive literature review was carried out to identify the articles investigating pesticide exposure and compare exposure data. Of the 88 pesticides, 76 were found in the blood serum of the population in Jiangsu Province. To the best of our knowledge, 58 pesticides were reported in human blood serum for the first time, and among these pesticides, parathion-methyl, pyrimethanil, fluacrypyrim, simazine, cloquintocet-mexyl and barban were debatable in more than half of the samples. By statistical comparison of the blood serum levels of pesticides between this study and other countries, we found the levels of several organochlorine pesticides were significantly higher in the female population of Jiangsu Province. Health risks related to the pesticide profiling were then revealed, which identified higher carcinogenic toxicity and teratogenic toxicity risk in the female adults of Jiangsu Province caused by organochlorine pesticide exposure. This study not only provides a high-throughput pesticide screening method for future studies of the exposome, but also presents the first human data on exposure to a number of pesticides. It may provide a knowledge database for the risk assessment and management of the pesticides. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human antirabies gamma globulin*

PubMed Central

Hosty, Thomas S.; Kissling, R. E.; Schaeffer, M.; Wallace, Gordon A.; Dibble, E. H.

1959-01-01

To obviate the foreign protein reactions experienced with the use of hyperimmune serum in rabies-exposed individuals, an attempt was made to produce a rabies antiserum of human origin. Five doses of an inactivated rabies virus duck-egg vaccine were administered to 34 volunteers at 4-day intervals (i.e., on days 0, 4, 8, 12 and 16). An additional dose of chick-embryo attenuated virus vaccine—Flury HEP (high egg passage)—was given on the 46th day, followed by a final booster dose of duck-egg vaccine on the 288th day. Twenty-four days later, i.e., on the 312th day after the first dose, the participants were bled and the serum pooled and converted to gamma globulin. These volunteers, having no initial antibody, responded with variable titres, the pooled serum having a titre of 1: 100 against 50 LD50 of rabies virus in neutralization tests and the gamma globulin prepared from this pool a titre of 1: 300. In five individuals inoculated with the antirabies gamma globulin, blood samples tested at intervals for residual antibody showed significant titres through 21 days. While the passive antibody levels resulting from the administration of a more potent immune horse serum were much higher than those achieved by the weaker human antirabies gamma globulin used, the decrease in titre was more gradual with the human globulin. With more booster inoculations in a larger group of human volunteers, it is believed that a human rabies immune gamma globulin could be produced which would be equal in effect to immune horse serum. The advantages of a human source of antibody in rabies prophylaxis are discussed. PMID:14403320

Headspace Solid Phase Micro Extraction Gas Chromatographic Determination of Fenthion in Human Serum

PubMed Central

Kasiotis, Konstantinos M.; Souki, Helen; Tsakirakis, Angelos N.; Carageorgiou, Haris; Theotokatos, Spiridon A.; Haroutounian, Serkos A.; Machera, Kyriaki

2008-01-01

A simple and effective analytical procedure was developed for the determination of fenthion residues in human serum samples. The sample treatment was performed using the headspace solid-phase micro extraction with polyacrylate fiber, which has the advantage to require low amount of serum (1 mL) without tedious pre-treatment. The quantification of fenthion was carried out by gas chromatography-mass spectrometry and the recoveries ranged from 79 to 104% at two spiking levels for 6 replicates. Detection and quantification limits were calculated as 1.51 and 4.54 ng/mL of serum respectively. Two fenthion metabolites fenoxon and fenthion–sulfoxide were also identified. PMID:19325792

Simultaneous quantitation of multiple contraceptive hormones in human serum by LC-MS/MS.

PubMed

Blue, Steven W; Winchell, Andrea J; Kaucher, Amy V; Lieberman, Rachel A; Gilles, Christopher T; Pyra, Maria N; Heffron, Renee; Hou, Xuanlin; Coombs, Robert W; Nanda, Kavita; Davis, Nicole L; Kourtis, Athena P; Herbeck, Joshua T; Baeten, Jared M; Lingappa, Jairam R; Erikson, David W

2018-04-01

The objective was to develop a method to simultaneously quantify five commonly used hormonal contraceptives (HCs) and two endogenous sex steroids by liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS/MS) and apply this method to human serum samples. We developed a method to simultaneously analyze ethinyl estradiol (EE2), etonogestrel (ENG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) and norethisterone (NET), along with estradiol (E2) and progesterone (P4), in human serum for a Shimadzu Nexera-LCMS-8050 LC-MS/MS platform. We analyzed serum collected from women self-reporting use of oral contraceptives, contraceptive implants or injectable contraceptives (n=14) and normally cycling women using no HC (n=15) as well as pooled samples from women administered various HCs (ENG, n=6; LNG, n=14; MPA, n=7; NET, n=5). Limits of quantitation were 0.010ng/mL for E2, EE2 and P4; 0.020ng/mL for ENG, LNG and MPA; and 0.040ng/mL for NET. Precisions for all assays, as indicated by coefficient of variation, were less than or equal to 12.1%. Accuracies for all assays were in the range of 95%-108%. Endogenous hormone values obtained from analysis of human serum samples are in agreement with levels previously reported in the literature for normally cycling women as well as for women taking the appropriate HC. We have developed a robust, accurate and sensitive method for simultaneously analyzing commonly used contraceptive steroids and endogenous sex steroids in human serum. This analytical method can be used for quantitating contraceptive steroid levels in women for monitoring systemic exposure to determine drug interactions, nonadherence, misreporting and proper dosing. Copyright © 2018 Elsevier Inc. All rights reserved.

Quantification of a biomarker of acetaminophen protein adducts in human serum by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: clinical and animal model applications.

PubMed

Cook, Sarah F; King, Amber D; Chang, Yan; Murray, Gordon J; Norris, Hye-Ryun K; Dart, Richard C; Green, Jody L; Curry, Steven C; Rollins, Douglas E; Wilkins, Diana G

2015-03-15

The aims of this study were to develop, validate, and apply a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantification of protein-derived 3-(cystein-S-yl)-acetaminophen (APAP-Cys) in human serum. Formation of acetaminophen (APAP) protein adducts is thought to be a critical, early event in the development of APAP-induced hepatotoxicity, and quantification of these protein adducts in human serum represents a valuable tool for assessment of APAP exposure, metabolism, and toxicity. In the reported procedure, serum samples were first dialyzed or passed through gel filtration columns to remove APAP-Cys not covalently bound to proteins. Serum eluates were then subjected to enzymatic protease digestion to liberate protein-bound APAP-Cys. Norbuprenorphine-D3 was utilized as an internal standard (IS). APAP-Cys and IS were recovered from digested serum by protein precipitation with acetonitrile, and sample extracts were analyzed by HPLC-ESI-MS/MS. The method was validated by assessment of intra- and inter-assay accuracy and imprecision on two different analytical instrument platforms. APAP-Cys could be accurately quantified from 0.010 to 10μM, and intra- and inter-assay imprecision were <15% on both analytical instruments. APAP-Cys was stable in human serum for three freeze-thaw cycles and for 24h at ambient temperature. Extracted samples were stable when stored in refrigerated autosamplers for the typical duration of analysis or when stored at -20°C for six days. Results from process efficiency and matrix effect experiments indicated adequate recovery from human serum and insignificant ion suppression or enhancement. The utility and sensitivity of the reported procedure were illustrated by analysis of clinical samples collected from subjects taking chronic, therapeutic doses of APAP. Applicability to other biological matrices was also demonstrated by measurement of protein-derived APAP-Cys in plasma collected from APAP-treated mice, a common animal model of APAP-induced hepatotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

Similarity of Bisphenol A Pharmacokinetics in Rhesus Monkeys and Mice: Relevance for Human Exposure

PubMed Central

Taylor, Julia A.; vom Saal, Frederick S.; Welshons, Wade V.; Drury, Bertram; Rottinghaus, George; Hunt, Patricia A.; Toutain, Pierre-Louis; Laffont, Céline M.; VandeVoort, Catherine A.

2011-01-01

Objective Daily adult human exposure to bisphenol A (BPA) has been estimated at < 1 μg/kg, with virtually complete first-pass conjugation in the liver in primates but not in mice. We measured unconjugated and conjugated BPA levels in serum from adult female rhesus monkeys and adult female mice after oral administration of BPA and compared findings in mice and monkeys with prior published data in women. Methods Eleven adult female rhesus macaques were fed 400 μg/kg deuterated BPA (dBPA) daily for 7 days. Levels of serum dBPA were analyzed by isotope-dilution liquid chromatography–mass spectrometry (0.2 ng/mL limit of quantitation) over 24 hr on day 1 and on day 7. The same dose of BPA was fed to adult female CD-1 mice; other female mice were administered 3H-BPA at doses ranging from 2 to 100,000 μg/kg. Results In monkeys, the maximum unconjugated serum dBPA concentration of 4 ng/mL was reached 1 hr after feeding and declined to low levels by 24 hr, with no significant bioaccumulation after seven daily doses. Mice and monkeys cleared unconjugated serum BPA at virtually identical rates. We observed a linear (proportional) relationship between administered dose and serum BPA in mice. Conclusions BPA pharmacokinetics in women, female monkeys, and mice is very similar. By comparison with approximately 2 ng/mL unconjugated serum BPA reported in multiple human studies, the average 24-hr unconjugated serum BPA concentration of 0.5 ng/mL in both monkeys and mice after a 400 μg/kg oral dose suggests that total daily human exposure is via multiple routes and is much higher than previously assumed. PMID:20855240

Development and validation of a rapid, selective, and sensitive LC-MS/MS method for simultaneous determination of D- and L-amino acids in human serum: application to the study of hepatocellular carcinoma.

PubMed

Han, Minlu; Xie, Mengyu; Han, Jun; Yuan, Daoyi; Yang, Tian; Xie, Ying

2018-04-01

A validated liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of D- and L-amino acids in human serum. Under the optimum conditions, except for DL-proline, L-glutamine, and D-lysine, the enantioseparation of the other 19 enantiomeric pairs of proteinogenic amino acids and nonchiral glycine was achieved with a CROWNPAK CR-I(+) chiral column within 13 min. The lower limits of quantitation for L-amino acids (including glycine) and D-amino acids were 5-56.25 μM and 0.625-500 nM, respectively, in human serum. The intraday precision and interday precision for all the analytes were less than 15%, and the accuracy ranged from -12.84% to 12.37% at three quality control levels. The proposed method, exhibiting high rapidity, enantioresolution, and sensitivity, was successfully applied to the quantification of D- and L-amino acid levels in serum from hepatocellular carcinoma patients and healthy individuals. The serum concentrations of L-arginine, L-isoleucine, L-aspartate, L-tryptophan, L-alanine, L-methionine, L-serine, glycine, L-valine, L-leucine, L-phenylalanine, L-threonine, D-isoleucine, D-alanine, D-glutamate, D-glutamine, D-methionine, and D-threonine were significantly reduced in the hepatocellular carcinoma patients compared with the healthy individuals (P < 0.01). D-Glutamate and D-glutamine were identified as the most downregulated serum markers (fold change greater than 1.5), which deserves further attention in hepatocellular carcinoma research. Graphical abstract Simultaneous determination of D- and L-amino acids in human serum from hepatocellular carcinoma patients and healthy individuals. AA amino acid, HCC hepatocellular carcinoma, LC liquid chromatography, MS/MS tandem mass spectrometry, NC normal control, TIC total ion chromatogram.

Stability of cytotoxic luteinizing hormone-releasing hormone conjugate (AN-152) containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro: implications for the design of preclinical studies.

PubMed

Nagy, A; Plonowski, A; Schally, A V

2000-01-18

Recently, we developed a series of cytotoxic peptide conjugates containing 14-O-glutaryl esters of doxorubicin (DOX) or 2-pyrrolino-DOX (AN-201). Serum carboxylesterase enzymes (CE) can partially hydrolyze these conjugates in the circulation, releasing the cytotoxic radical, before the targeting is complete. CE activity in serum of nude mice is about 10 times higher than in human serum. Thus, we found that the t(1/2) of AN-152, an analog of luteinizing hormone-releasing hormone (LH-RH) containing DOX, at 0.3 mg/ml is 19. 49 +/- 0.74 min in mouse serum and 126.06 +/- 3.03 min in human serum in vitro. The addition of a CE inhibitor, diisopropyl fluorophosphate (DFP), to mouse serum in vitro significantly (P < 0. 01) prolongs the t(1/2) of AN-152 to 69.63 +/- 4.44 min. When DFP is used in vivo, 400 nmol/kg cytotoxic somatostatin analog AN-238 containing AN-201 is well tolerated by mice, whereas all animals die after the same dose without DFP. In contrast, DFP has no effect on the tolerance of AN-201. A better tolerance to AN-238 after DFP treatment is due to the selective uptake of AN-238 by somatostatin receptor-positive tissues. Our results demonstrate that the suppression of the CE activity in nude mice greatly decreases the toxicity of cytotoxic hybrids containing 2-pyrrolino-DOX 14-O-hemiglutarate and brings this animal model closer to the conditions that exist in humans. The use of DFP together with these peptide conjugates in nude mice permits a better understanding of their mechanism of action and improves the clinical predictability of the oncological and toxicological results.

Direct and simultaneous analysis of loxoprofen and its diastereometric alcohol metabolites in human serum by on-line column switching liquid chromatography and its application to a pharmacokinetic study.

PubMed

Cho, Hea-Young; Park, Chan-Ho; Lee, Yong-Bok

2006-05-01

A simple, rapid, and accurate column-switching liquid chromatography method was developed and validated for direct and simultaneous analysis of loxoprofen and its metabolites (trans- and cis-alcohol metabolites) in human serum. After direct serum injection into the system, deproteinization and trace enrichment occurred on a Shim-pack MAYI-ODS pretreatment column (10 mm x 4.6 mm i.d.) by an eluent consisting of 20 mM phosphate buffer (pH 6.9)/acetonitrile (95/5, v/v) and 0.1% formic acid. The drug trapped by the pretreatment column was introduced to the Shim-pack VP-ODS analytical column (150 mm x 4.6 mm i.d.) using acetonitrile/water (45/55, v/v) containing 0.1% formic acid when the 6-port valve status was switched. Ketoprofen was used as the internal standard. The analysis was monitored on a UV detector at 225 nm. The chromatograms showed good resolution, sensitivity, and no interference by human serum. Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 15 and over 95%, respectively, in the concentration range of 0.1-20 microg/ml. With UV detection, the limit of quantitation was 0.1 microg/ml, and good linearity (r = 0.999) was observed for all the compounds with 50 microl serum samples. The mean absolute recoveries of loxoprofen, trans- and cis-alcohol for human serum were 89.6 +/- 3.9, 93.5 +/- 3.2, and 93.7 +/- 4.3%, respectively. Stability studies showed that loxoprofen and its metabolites in human serum were stable during storage and the assay procedure. This analytical method showed excellent sensitivity with small sample volume (50 microl), good precision, accuracy, and speed (total analytical time 18 min), without any loss in chromatographic efficiency. This method was successfully applied to the pharmacokinetic study of loxoprofen in human volunteers following a single oral administration of loxoprofen sodium (60 mg, anhydrate) tablet.

Electrochemical Detection of Anti-Breast-Cancer Agents in Human Serum by Cytochrome P450-Coated Carbon Nanotubes

PubMed Central

Baj-Rossi, Camilla; De Micheli, Giovanni; Carrara, Sandro

2012-01-01

We report on the electrochemical detection of anti-cancer drugs in human serum with sensitivity values in the range of 8–925 nA/μM. Multi-walled carbon nanotubes were functionalized with three different cytochrome P450 isoforms (CYP1A2, CYP2B6, and CYP3A4). A model used to effectively describe the cytochrome P450 deposition onto carbon nanotubes was confirmed by Monte Carlo simulations. Voltammetric measurements were performed in phosphate buffer saline (PBS) as well as in human serum, giving well-defined current responses upon addition of increasing concentrations of anti-cancer drugs. The results assert the capability to measure concentration of drugs in the pharmacological ranges in human serum. Another important result is the possibility to detect pairs of drugs present in the same sample, which is highly required in case of therapies with high side-effects risk and in anti-cancer pharmacological treatments based on mixtures of different drugs. Our technology holds potentials for inexpensive multi-panel drug-monitoring in personalized therapy. PMID:22778656

Circular dichroism study of the interaction between mutagens and bilirubin bound to different binding sites of serum albumins

NASA Astrophysics Data System (ADS)

Orlov, Sergey; Goncharova, Iryna; Urbanová, Marie

Although recent investigations have shown that bilirubin not only has a negative role in the organism but also exhibits significant antimutagenic properties, the mechanisms of interactions between bilirubin and mutagens are not clear. In this study, interaction between bilirubin bound to different binding sites of mammalian serum albumins with structural analogues of the mutagens 2-aminofluorene, 2,7-diaminofluorene and mutagen 2,4,7-trinitrofluorenone were investigated by circular dichroism and absorption spectroscopy. Homological human and bovine serum albumins were used as chiral matrices, which preferentially bind different conformers of bilirubin in the primary binding sites and make it observable by circular dichroism. These molecular systems approximated a real system for the study of mutagens in blood serum. Differences between the interaction of bilirubin bound to primary and to secondary binding sites of serum albumins with mutagens were shown. For bilirubin bound to secondary binding sites with low affinity, partial displacement and the formation of self-associates were observed in all studied mutagens. The associates of bilirubin bound to primary binding sites of serum albumins are formed with 2-aminofluorene and 2,4,7-trinitrofluorenone. It was proposed that 2,7-diaminofluorene does not interact with bilirubin bound to primary sites of human and bovine serum albumins due to the spatial hindrance of the albumins binding domains. The spatial arrangement of the bilirubin bound to serum albumin along with the studied mutagens was modelled using ligand docking, which revealed a possibility of an arrangement of the both bilirubin and 2-aminofluorene and 2,4,7-trinitrofluorenone in the primary binding site of human serum albumin.

Generation of therapeutic protein variants with the human serum albumin binding capacity via site-specific fatty acid conjugation.

PubMed

Cho, Jinhwan; Lim, Sung In; Yang, Byung Seop; Hahn, Young S; Kwon, Inchan

2017-12-21

Extension of the serum half-life is an important issue in developing new therapeutic proteins and expanding applications of existing therapeutic proteins. Conjugation of fatty acid, a natural human serum albumin ligand, to a therapeutic protein/peptide was developed as a technique to extend the serum half-life in vivo by taking advantages of unusually long serum half-life of human serum albumin (HSA). However, for broad applications of fatty acid-conjugation, several issues should be addressed, including a poor solubility of fatty acid and a substantial loss in the therapeutic activity. Therefore, herein we systematically investigate the conditions and components in conjugation of fatty acid to a therapeutic protein resulting in the HSA binding capacity without compromising therapeutic activities. By examining the crystal structure and performing dye conjugation assay, two sites (W160 and D112) of urate oxidase (Uox), a model therapeutic protein, were selected as sites for fatty acid-conjugation. Combination of site-specific incorporation of a clickable p-azido-L-phenylalanine to Uox and strain-promoted azide-alkyne cycloaddition allowed the conjugation of fatty acid (palmitic acid analog) to Uox with the HSA binding capacity and retained enzyme activity. Deoxycholic acid, a strong detergent, greatly enhanced the conjugation yield likely due to the enhanced solubility of palmitic acid analog.

Changes in lung ultrastructure following heterologous and homologous serum albumin infusion in the treatment of hemorrhagic shock.

PubMed Central

Moss, G S; Das Gupta, T K; Brinkman, R; Sehgal, L; Newsom, B

1979-01-01

The object of this study was to compare the ultrastructure pulmonary effects of the infusion of homologous and heterologous serum albumin solution in the treatment of hemorrhagic shock in baboons. Adult baboons subjected to hemorrhagic shock were resuscitated with either baboon serum albumin, human serum albumin, or Ringer's lactate solution. The lungs were fixed in vivo with potassium pyroantimony, a solution which produces electron dense interstitial precipitation of sodium. The lungs from animals resuscitated with baboon serum albumin showed evidence of interstitial edema, including dispersion of collagen fibers, interstitial smudging and increased interstital sodium concentrations. Similar changes were seen following human serum albumin infusions. Lung tissue from animals treated with Ringer's lactate solution showed minimal changes from normal. These results suggest that interstitial pulmonary edema develops after either homologous or heterologous serum albumin infusion in the treatment of hemorrhagic shock in baboons. Images Figs. 2a and b. Figs. 3a and b. Figs. 4a and b. Figs. 5a and b. Figs. 6a and b. PMID:106780

Detecting tau in serum of transgenic animal models after tau immunotherapy treatment.

PubMed

d'Abramo, Cristina; Acker, Christopher M; Schachter, Joel B; Terracina, Giuseppe; Wang, Xiaohai; Forest, Stefanie K; Davies, Peter

2016-01-01

In the attempt to elucidate if the "peripheral sink hypothesis" could be a potential mechanism of action for tau removal in passive immunotherapy experiments, we have examined tau levels in serum of chronically injected JNPL3 and Tg4510 transgenic animals. Measurement of tau in serum of mice treated with tau antibodies is challenging because of the antibody interference in sandwich enzyme-linked immunosorbent assays. To address this issue, we have developed a heat-treatment protocol at acidic pH to remove interfering molecules from serum, with excellent recovery of tau. The present data show that pan-tau and conformational antibodies do increase tau in mouse sera. However, these concentrations in serum do not consistently correlate with reductions of tau pathology in brain, suggesting that large elevations of tau species measured in serum are not predictive of efficacy. Here, we describe a reliable method to detect tau in serum of transgenic animals that have undergone tau immunotherapy. Levels of tau in human serum are less than the sensitivity of current assays, although artifactual signals are common. The method may be useful in similarly treated humans, a situation in which false positive signals are likely. Copyright © 2016 Elsevier Inc. All rights reserved.

Experimental Germ Tube Induction in Candida albicans: An Evaluation of the Effect of Sodium Bicarbonate on Morphogenesis and Comparison with Pooled Human Serum.

PubMed

Matare, Tapiwa; Nziramasanga, Pasipanodya; Gwanzura, Lovemore; Robertson, Valerie

2017-01-01

The potential of NaHCO 3 versus human serum to induce germ tube formation in Candida albicans was investigated. A total of 100 isolates were obtained from oral swabs of patients presenting with thrush. Approval for the study was granted by the Joint Research Ethics Committee (JREC/23/08). Confirmed C. albicans isolates by routine methods were tested for germ tube induction using 5 different concentrations of Tris-maleate buffered NaHCO 3 and Tris-maleate buffer control. Standard control strains included were C. albicans (ATCC 10231) and C. krusei (ATCC 6258). Microculture was done in 20  μ L inoculums on microscope slides for 3 hours at 37°C. The rate of germ tube formation at 10-minute intervals was determined on 100 isolates using the optimum 20 mM Tris-maleate buffered NaHCO 3 concentration. Parallel germ tube formation using human serum was done in test tubes. The optimum concentration of NaHCO 3 in Tris-maleate buffer for germ tube induction was 20 mM for 67% of isolates. Only 21% of isolates formed germ tubes in Tris-maleate buffer control. There was no significant difference in induction between human serum and Tris-maleate buffered NaHCO 3 . Tris-maleate buffered NaHCO 3 induced germ tube formation in C. albicans isolates at rates similar to human serum.

A spectroscopic and molecular docking approach on the binding of tinzaparin sodium with human serum albumin

NASA Astrophysics Data System (ADS)

Abdullah, Saleh M. S.; Fatma, Sana; Rabbani, Gulam; Ashraf, Jalaluddin M.

2017-01-01

Protein bound toxins are poorly removed by conventional extracorporeal therapies. Venous thromboembolism (VTE) is a major cause of morbidity and mortality in patients with cancer. The interaction between tinzaparin, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by tinzaparin (TP). The binding constants and binding stoichiometry can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG° reveals that the binding process is a spontaneous process. Thermodynamic analysis shows that the HSA-TP complex formation occurs via hydrogen bonds, hydrophobic interactions and undergoes slight structural changes as evident by far-UV CD. It indicated that the hydrophobic interactions play a main role in the binding of TP to human serum albumin. In addition, the distance between TP (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 2.21 nm according to the Förster's resonance energy transfer theory. For the deeper understanding of the interaction, thermodynamic, and molecular docking studies were performed as well. Our docking results suggest that TP forms stable complex with HSA (Kb ∼ 104) and its primary binding site is located in subdomain IIA (Sudlow Site I). The results obtained herein will be of biological significance in pharmacology and clinical medicine.

Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

2016-01-01

The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

Serum factors and clinical characteristics associated with serum E-Screen activity

PubMed Central

Wang, Jue; Trentham-Dietz, Amy; Hemming, Jocelyn D. C.; Hedman, Curtis J.; Sprague, Brian L.

2013-01-01

Background The E-Screen bioassay can measure the mitogenicity of human serum and thus may be useful as a biomarker in epidemiologic studies of breast cancer. While the assay’s MCF-7 cells are known to proliferate in response to estrogen, the specific determinants of variation in E-Screen activity in human serum samples are poorly understood. We sought to identify serum molecules and patient characteristics associated with serum E-Screen activity among postmenopausal women. Methods Postmenopausal women (N=219) aged 55–70 with no history of postmenopausal hormone use or breast cancer completed a questionnaire and provided a blood sample. Serum was analyzed for E-Screen activity and a variety of molecules including sex hormones, growth factors, and environmental chemicals. Stepwise selection procedures were used to identify correlates of E-Screen activity. Results Serum samples from all women had detectable E-Screen activity, with a median estradiol equivalents value of 0.027 ng/mL and interquartile range of 0.018–0.036 ng/mL. In the final multivariable-adjusted model, serum E-Screen activity was positively associated with serum estradiol, estrone, IGFBP-3, and testosterone levels (p<0.05), as well as body mass index (p=0.03). Serum E-Screen activity was lower among women with higher SHBG (p<0.0001) and progesterone levels (p=0.03). Conclusion Serum E-Screen activity varies according to levels of endogenous estrogens and other serum molecules. Obesity appears to confer additional serum mitogenicity beyond its impact on the measured hormones and growth factors. Impact By capturing mitogenicity due to a variety of patient and serum factors, the E-Screen may provide advantages for use as a biomarker in breast cancer studies. PMID:23588007

Alteration of human serum albumin binding properties induced by modifications: A review

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, Małgorzata; Szkudlarek, Agnieszka; Chudzik, Mariola; Pożycka, Jadwiga; Sułkowska, Anna

2018-01-01

Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

Resistance to serum bactericidal activity distinguishes Brazilian purpuric fever (BPF) case strains of Haemophilus influenzae biogroup aegyptius (H. aegyptius) from non-BPF strains. Brazilian Purpuric Fever Study Group.

PubMed

Porto, M H; Noel, G J; Edelson, P J

1989-04-01

We studied the ability of normal human serum to lyse H. influenzae biogroup aegyptius (H. aegyptius) isolates recovered from patients with Brazilian purpuric fever (BPF clone) or non-BPF clone strains. BPF clone isolates, although similar to non-BPF clone isolates with regard to the ability to fix C3 to their surfaces, could be distinguished from non-BPF clone strains by their resistance to lysis in vitro following incubation with normal adult human serum.

Imaging of experimental amyloidosis with /sup 131/I-labeled serum amyloid P component

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caspi, D.; Zalzman, S.; Baratz, M.

1987-11-01

/sup 131/I-labeled human serum amyloid P component, which was injected into mice with experimentally induced systemic AA amyloidosis and into controls, became specifically localized and was retained in amyloidotic organs. In comparison, it was rapidly and completely eliminated from unaffected tissues and from control animals. Distinctive images of this amyloid-specific deposition of labeled serum amyloid P component were derived from whole body scanning, in vivo, of amyloidotic mice. These findings suggest that such imaging may have applications for the diagnosis and quantitation of amyloid deposits in humans.

Resonance Raman spectroscopy and the preterm infant carotenoid status.

PubMed

Chan, Gary M; Chan, Melissa M; Gellermann, Werner; Ermakov, Igor; Ermakova, Maia; Bhosale, Prakash; Bernstein, Paul; Rau, Carrie

2013-05-01

The aim of the study was to validate the noninvasive resonance Raman spectroscopy (RRS) method in infants in comparison with the high-performance liquid chromatography (HPLC) method, and to evaluate the carotenoid status in preterm infants fed with mother's milk or formula. In the first phase of the study, resonance Raman measurements were made on male term infants' skin and correlated with tissue harvested at the time of circumcision. Each baby's foreskin was weighed, enzymatically digested, and the total carotenoids were extracted and quantitated by the HPLC. Next, to evaluate the carotenoid status of preterm infants (BW <1500 g), the skin and serum carotenoids in infants fed with either human milk or preterm formula were studied from the start of feedings and every 2 weeks until hospital discharge. Skin carotenoids were measured by RRS and the serum total carotenoids by HPLC. Foreskin carotenoid levels measured by RRS correlated with HPLC measurements of total serum carotenoids (R = 0.52, P < 0.01, n = 16). Forty preterm infants were studied for their carotenoid status. Thirty-two infants were fed mother's milk, whereas 8 were fed a preterm infant formula that was not enriched with carotenoids. The gestation and birth weight of the 2 feeding groups were similar. The infants fed human milk had a higher serum total carotenoid concentration and skin Raman counts than formula-fed infants. The skin Raman counts and total serum carotenoid correlated (R = 0.44, P = 0.01). The human milk-fed infants' serum total carotenoid concentrations and Raman values did not change during the study period; however, the formula-fed group's total serum and skin carotenoid decreased significantly during the study. RRS of infant's skin reliably assesses total carotenoid status noninvasively. Human milk-fed preterm infants have higher serum and skin carotenoids than formula-fed infants suggesting that formula-fed infants may benefit from carotenoid supplementation.

SERUM BIOMARKERS OF AGING IN THE BROWN NORWAY RAT

EPA Science Inventory

Serum biomarkers to identify susceptibility to disease in aged humans are well researched. On the other hand, our understanding of biomarkers in animal models of aging is limited. Hence, we applied a commercially available panel of 58 serum analytes to screen for possible biomark...

Comparative analyses of industrial-scale human platelet lysate preparations.

PubMed

Pierce, Jan; Benedetti, Eric; Preslar, Amber; Jacobson, Pam; Jin, Ping; Stroncek, David F; Reems, Jo-Anna

2017-12-01

Efforts are underway to eliminate fetal bovine serum from mammalian cell cultures for clinical use. An emerging, viable replacement option for fetal bovine serum is human platelet lysate (PL) as either a plasma-based or serum-based product. Nine industrial-scale, serum-based PL manufacturing runs (i.e., lots) were performed, consisting of an average ± standard deviation volume of 24.6 ± 2.2 liters of pooled, platelet-rich plasma units that were obtained from apheresis donors. Manufactured lots were compared by evaluating various biochemical and functional test results. Comprehensive cytokine profiles of PL lots and product stability tests were performed. Global gene expression profiles of mesenchymal stromal cells (MSCs) cultured with plasma-based or serum-based PL were compared to MSCs cultured with fetal bovine serum. Electrolyte and protein levels were relatively consistent among all serum-based PL lots, with only slight variations in glucose and calcium levels. All nine lots were as good as or better than fetal bovine serum in expanding MSCs. Serum-based PL stored at -80°C remained stable over 2 years. Quantitative cytokine arrays showed similarities as well as dissimilarities in the proteins present in serum-based PL. Greater differences in MSC gene expression profiles were attributable to the starting cell source rather than with the use of either PL or fetal bovine serum as a culture supplement. Using a large-scale, standardized method, lot-to-lot variations were noted for industrial-scale preparations of serum-based PL products. However, all lots performed as well as or better than fetal bovine serum in supporting MSC growth. Together, these data indicate that off-the-shelf PL is a feasible substitute for fetal bovine serum in MSC cultures. © 2017 AABB.

Investigation on predominant Leptospira serovars and its distribution in humans and livestock in Thailand, 2010-2015

PubMed Central

Chadsuthi, Sudarat; Bicout, Dominique J.; Wiratsudakul, Anuwat; Suwancharoen, Duangjai; Petkanchanapong, Wimol; Modchang, Charin; Triampo, Wannapong; Ratanakorn, Parntep; Chalvet-Monfray, Karine

2017-01-01

Background Leptospirosis is a worldwide zoonotic bacterial disease caused by infection with leptospires. Leptospirosis in humans and livestock is an endemic and epidemic disease in Thailand. Livestock may act as reservoirs for leptospires and source for human infection. Methodology/Principal findings Data on leptospirosis infection in humans and livestock (Buffaloes, Cattle, and Pigs) species during 2010 to 2015 were analyzed. Serum samples were examined using Microscopic Agglutination Test (MAT) to identify antibodies against Leptospira serovars using a cut-off titer ≥ 1:100. The seroprevalence was 23.7% in humans, 24.8% in buffaloes, 28.1% in cattle, and 11.3% in pigs. Region specific prevalence among humans and livestock was found in a wide range. The most predominant serovars were Shermani, followed by Bratislava, Panama, and Sejroe in human, Shermani, Ranarum, and Tarassovi in buffaloes, and Shermani and Ranarum in cattle and pigs. Equally highest MAT titers against multiple serovars per one sample were found mainly in buffaloes and cattle showing equally titers against Ranarum and Shermani. The correlations of distribution of serovars across Thailand’s regions were found to be similar in pattern for cattle but not for buffaloes. In humans, the serovar distribution in the south differed from other regions. By logistic regression, the results indicated that livestock is more susceptible to infection by serovar Shermani when compared to humans. Conclusions/Significance This study gives a detailed picture of the predominance of Leptospira serovars in relation to region, humans and typical livestock. The broad spatial distribution of seroprevalence was analyzed across and within species as well as regions in Thailand. Our finding may guide public health policy makers to implement appropriate control measures and help to reduce the impact of leptospirosis in Thailand. PMID:28182662

In vitro removal of human IgG autoantibodies by affinity filtration using immobilized L-histidine onto PEVA hollow fiber membranes.

PubMed

Ventura, R C; Zollner, R L; Legallais, C; Vijayalakshmi, M; Bueno, S M

2001-01-01

Histidine was immobilized onto PEVA membrane to obtain an affinity support for human IgG removal from serum with a view to clinical apheresis for the treatment of autoimmune diseases. These membranes were able to remove in vitro several autoantibodies from the serum of SLE patients.

Spectral Fluorescence Properties of an Anionic Oxacarbocyanine Dye in Complexes with Human Serum Albumin

NASA Astrophysics Data System (ADS)

Pronkin, P. G.; Tatikolov, A. S.

2015-07-01

The spectral fluorescence properties of the anionic oxacarbocyanine dye 3,3'-di-(γ-sulfopropyl)-5,5'-diphenyl-9-ethyloxacarbocyanine betaine (OCC) were studied in solutions and in complexes with human serum albumin (HSA). Interaction with HSA leads to a significant increase in the fluorescence of the dye. We studied quenching of the fluorescence of OCC in a complex with HSA by ibuprofen and warfarin. Data on quenching of fluorescence by ibuprofen indicate binding of the dye to binding site II of subdomain IIIA in the HSA molecule. Synchronous fluorescence spectra of human serum albumin in the presence of OCC showed that complexation with OCC does not lead to appreciable rearrangement of the protein molecule at the binding site.

Superparamagnetic nanoparticle-based viscosity test

NASA Astrophysics Data System (ADS)

Wu, Kai; Liu, Jinming; Wang, Yi; Ye, Clark; Feng, Yinglong; Wang, Jian-Ping

2015-08-01

Hyperviscosity syndrome is triggered by high blood viscosity in the human body. This syndrome can result in retinopathy, vertigo, coma, and other unanticipated complications. Serum viscosity is one of the important factors affecting whole blood viscosity, which is regarded as an indicator of general health. In this letter, we propose and demonstrate a Brownian relaxation-based mixing frequency method to test human serum viscosity. This method uses excitatory and detection coils and Brownian relaxation-dominated superparamagnetic nanoparticles, which are sensitive to variables of the liquid environment such as viscosity and temperature. We collect the harmonic signals produced by magnetic nanoparticles and estimate the viscosity of unknown solutions by comparison to the calibration curves. An in vitro human serum viscosity test is performed in less than 1.5 min.

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

PubMed Central

Muraglia, Anita; Nguyen, Van Thi; Nardini, Marta; Mogni, Massimo; Coviello, Domenico; Dozin, Beatrice; Strada, Paolo; Baldelli, Ilaria; Formica, Matteo; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-01-01

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype. PMID:29209609

Serum from dengue virus-infected patients with and without plasma leakage differentially affects endothelial cells barrier function in vitro

PubMed Central

Baimukanova, Gyulnar; Lanteri, Marion Christine; Keating, Sheila Marie; Moraes Ferreira, Frederico; Heitman, John; Pannuti, Cláudio Sérgio; Pati, Shibani; Romano, Camila Malta; Cerdeira Sabino, Ester

2017-01-01

Background Although most of cases of dengue infections are asymptomatic or mild symptomatic some individuals present warning signs progressing to severe dengue in which plasma leakage is a hallmark. Methodology/Principal findings The present study used Electric Cell-substrate Impedance Sensing (ECIS®) which allows for electrical monitoring of cellular barrier function measuring changes in Transendothelial Electric Resistance (TEER) to investigate the parameters associated with dengue induced leakage. Three groups of individuals were tested: dengue-positives with plasma leakage (leakage), dengue-positives without plasma leakage (no leakage), and dengue-negatives (control). Data show that TEER values of human umbilical vein endothelial cells (HUVECs) was significantly lower after incubation with serum from subjects of the leakage group in comparison to the no leakage or control groups. The serum levels of CXCL1, EGF, eotaxin, IFN-γ, sCD40L, and platelets were significantly decreased in the leakage group, while IL-10, IL-6, and IP-10 levels were significantly increased. We also found a strong correlation between TEER values and augmented levels of IP-10, GM-CSF, IL-1α, and IL-8, as well as decreased levels of CXCL1 and platelets. Conclusions/Significance The present work shows that the magnitude of the immune response contributes to the adverse plasma leakage outcomes in patients and that serum components are important mediators of changes in endothelial homeostasis during dengue infections. In particular, the increased levels of IP-10 and the decreased levels of CXCL1 and platelets seem to play a significant role in the disruption of vascular endothelium associated with leakage outcomes after DENV infection. These findings may have important implications for both diagnostic and therapeutic approaches to predict and mitigate vascular permeabilization in those experiencing the most severe clinical disease outcomes after dengue infection. PMID:28586397

Commutability of control materials for external quality assessment of serum apolipoprotein A-I measurement.

PubMed

Zeng, Jie; Qi, Tianqi; Wang, Shu; Zhang, Tianjiao; Zhou, Weiyan; Zhao, Haijian; Ma, Rong; Zhang, Jiangtao; Yan, Ying; Dong, Jun; Zhang, Chuanbao; Chen, Wenxiang

2018-04-25

The aim of the current study was to evaluate the commutability of commercial control materials and human serum pools and to investigate the suitability of the materials for the external quality assessment (EQA) of serum apolipoprotein A-I (apo A-I) measurement. The Clinical and Laboratory Standards Institute (CLSI) EP14-A3 protocol was used for the commutability study. Apo A-I concentrations in two levels of commercial control materials used in EQA program, two fresh-frozen human serum pools (FSPs) and two frozen human serum pools prepared from residual clinical specimens (RSPs) were measured along with 50 individual samples using nine commercial assays. Measurement results of the 50 individual samples obtained with different assays were pairwise analyzed by Deming regression, and 95% prediction intervals (PIs) were calculated. The commutability of the processed materials was evaluated by comparing the measurement results of the materials with the limits of the PIs. The FSP-1 was commutable for all the 36 assay pairs, and FSP-2 was commutable for 30 pairs; RSP-1 and RSP-2 showed commutability for 27/36 and 22/36 assay pairs, respectively, whereas the two EQA materials were commutable only for 4/36 and 5/36 assay pairs, respectively. Non-commutability of the tested EQA materials has been observed among current apo A-I assays. EQA programs need either to take into account the commutability-related biases in the interpretation of the EQA results or to use more commutable materials. Frozen human serum pools were commutable for most of the assays.

Potential ex vivo immunomodulatory effects of San-Huang-Xie-Xin-Tang and its component herbs on mice and humans.

PubMed

Li, Chia-Yang; Hou, Yu-Chi; Lee Chao, Pei-Dawn; Shia, Chi-Sheng; Hsu, Ian C; Fang, Shih-Hua

2010-02-03

San-Huang-Xie-Xin-Tang (SHXXT), an important Chinese medicine formula, contains Rhei Rhizoma (RR), Scutellariae Radix (SR) and Coptidis Rhizoma (CR). RR and SR are abundant in anthraquinone and flavonoid polyphenols. Pharmacokinetic study of SHXXT indicated that glucuronides were the predominant forms of polyphenols in rats. As an extension of pharmacokinetic study, the serum metabolites of SHXXT, RR, SR and CR were prepared from rats and quantitated, then the immunomodulation effects were examined by culturing these serum metabolites with murine and human immune cells. The results indicated that the inhibitions on nitric oxide (NO) and cytokine production from mitogen-activated peritoneal macrophages by the serum metabolites of SHXXT, RR, SR and CR were through reducing the protein expression of inducible NO synthase (iNOS) and the IC(50) were 0.8%, 1.5%, 3.0% and 0.8% of their blood concentrations, respectively. In addition, the serum metabolites of SHXXT, RR, SR and CR significantly decreased the ratios of interferon-gamma (IFN-gamma) to interleukin (IL)-4 in mitogen-stimulated mice spleen cells and human peripheral blood mononuclear cells (PBMCs). Moreover, the serum metabolites of SHXXT and SR significantly arrested the mitogen-stimulated mice spleen cells at G2/M stage. In conclusion, the serum metabolites of SHXXT and the component herbs exerted promising modulation activities on the immune functions and the cell cycle distribution of mice and human immune cells. We suggest that SHXXT is a promising remedy for immunomodulation through Th1/Th2 regulation. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Increased EPA levels in serum phospholipids of humans after four weeks daily ingestion of one portion chicken fed linseed and rapeseed oil.

PubMed

Haug, Anna; Nyquist, Nicole F; Mosti, Therese J; Andersen, Malin; Høstmark, Arne T

2012-08-22

Since the amounts of arachidonic acid (AA) and EPA in food may have implications for human health, we investigated whether a small change in chicken feed influenced the blood lipid concentration in humans ingesting the chicken. Forty-six young healthy volunteers (age 20-29) were randomly allocated into two groups in a double-blind dietary intervention trial, involving ingestion of about 160 g chicken meat per day for 4 weeks. The ingested meat was either from chickens given a feed concentrate resembling the commercial chicken feed, containing 4% soybean oil (SO), or the meat was from chickens given a feed where the soybean oil had been replaced by 2% rapeseed oil plus 2% linseed oil (RLO).Serum total cholesterol, LDL and HDL cholesterol, triacylglycerols, serum phospholipid fatty acid concentration, blood pressure, body weight and C-reactive protein were determined at baseline and post-intervention. In subjects consuming chicken meat from the RLO group there was a significantly (p < 0.001) increased concentration of EPA in serum phospholipids, and a reduced ratio between AA and EPA. The participants that had a low% of EPA + DHA in serum phospholipids (less than 4.6%), all increased their% of EPA + DHA after the four week intervention period when consuming the RLO chicken. No significant response differences in cholesterol, triacylglycerol, C-reactive protein, body weight or blood pressure were observed between the groups. This trial demonstrates that a simple change in chicken feed can have beneficial effects on amount of EPA and the AA/EPA ratio in human serum phospholipids.

Bile acids induce arrhythmias in human atrial myocardium--implications for altered serum bile acid composition in patients with atrial fibrillation.

PubMed

Rainer, Peter P; Primessnig, Uwe; Harenkamp, Sandra; Doleschal, Bernhard; Wallner, Markus; Fauler, Guenter; Stojakovic, Tatjana; Wachter, Rolf; Yates, Ameli; Groschner, Klaus; Trauner, Michael; Pieske, Burkert M; von Lewinski, Dirk

2013-11-01

High bile acid serum concentrations have been implicated in cardiac disease, particularly in arrhythmias. Most data originate from in vitro studies and animal models. We tested the hypotheses that (1) high bile acid concentrations are arrhythmogenic in adult human myocardium, (2) serum bile acid concentrations and composition are altered in patients with atrial fibrillation (AF) and (3) the therapeutically used ursodeoxycholic acid has different effects than other potentially toxic bile acids. Multicellular human atrial preparations ('trabeculae') were exposed to primary bile acids and the incidence of arrhythmic events was assessed. Bile acid concentrations were measured in serum samples from 250 patients and their association with AF and ECG parameters analysed. Additionally, we conducted electrophysiological studies in murine myocytes. Taurocholic acid (TCA) concentration-dependently induced arrhythmias in atrial trabeculae (14/28 at 300 µM TCA, p<0.01) while ursodeoxycholic acid did not. Patients with AF had significantly decreased serum levels of ursodeoxycholic acid conjugates and increased levels of non-ursodeoxycholic bile acids. In isolated myocytes, TCA depolarised the resting membrane potential, enhanced Na(+)/Ca(2+) exchanger (NCX) tail current density and induced afterdepolarisations. Inhibition of NCX prevented arrhythmias in atrial trabeculae. High TCA concentrations induce arrhythmias in adult human atria while ursodeoxycholic acid does not. AF is associated with higher serum levels of non-ursodeoxycholic bile acid conjugates and low levels of ursodeoxycholic acid conjugates. These data suggest that higher levels of toxic (arrhythmogenic) and low levels of protective bile acids create a milieu with a decreased arrhythmic threshold and thus may facilitate arrhythmic events.

Immunosignature Differentiation of Non-Infectious Meningoencephalomyelitis and Intracranial Neoplasia in Dogs.

PubMed

Lake, Bathilda B; Rossmeisl, John Henry; Cecere, Julie; Stafford, Phillip; Zimmerman, Kurt L

2018-01-01

A variety of inflammatory conditions of unknown cause (meningoencephalomyelitis of unknown etiology-MUE) and neoplastic diseases can affect the central nervous system (CNS) of dogs. MUE can mimic intracranial neoplasia both clinically, radiologically and even in some cases, histologically. Serum immunosignature protein microarray assays have been used in humans to identify CNS diseases such as Alzheimer's and neoplasia, and in dogs, to detect lymphoma and its progression. This study evaluated the effectiveness of immunosignature profiles for distinguishing between three cohorts of dogs: healthy, intracranial neoplasia, and MUE. Using the learned peptide patterns for these three cohorts, classification prediction was evaluated for the same groups using a 10-fold cross validation methodology. Accuracy for classification was 100%, as well as 100% specific and 100% sensitive. This pilot study demonstrates that immunosignature profiles may help serve as a minimally invasive tool to distinguish between MUE and intracranial neoplasia in dogs.

Ozone Exposure Increases Circulating Stress Hormones and Lipid Metabolites in Humans

PubMed Central

Miller, Desinia B.; Ghio, Andrew J.; Karoly, Edward D.; Bell, Lauren N.; Snow, Samantha J.; Madden, Michael C.; Soukup, Joleen; Cascio, Wayne E.; Gilmour, M. Ian

2016-01-01

Rationale: Air pollution has been associated with increased prevalence of type 2 diabetes; however, the mechanisms remain unknown. We have shown that acute ozone exposure in rats induces release of stress hormones, hyperglycemia, leptinemia, and glucose intolerance that are associated with global changes in peripheral glucose, lipid, and amino acid metabolism. Objectives: To examine ozone-induced metabolic derangement in humans using serum metabolomic assessment, establish human-to-rodent coherence, and identify novel nonprotein biomarkers. Methods: Serum samples were obtained from a crossover clinical study that included two clinic visits (n = 24 each) where each subject was blindly exposed in the morning to either filtered air or 0.3 parts per million ozone for 2 hours during 15-minute on-off exercise. Serum samples collected within 1 hour after exposure were assessed for changes in metabolites using a metabolomic approach. Measurements and Main Results: Metabolomic analysis revealed that ozone exposure markedly increased serum cortisol and corticosterone together with increases in monoacylglycerol, glycerol, and medium- and long-chain free fatty acids, reflective of lipid mobilization and catabolism. Additionally, ozone exposure increased serum lysolipids, potentially originating from membrane lipid breakdown. Ozone exposure also increased circulating mitochondrial β-oxidation–derived metabolites, such as acylcarnitines, together with increases in the ketone body 3-hydroxybutyrate. These changes suggested saturation of β-oxidation by ozone in exercising humans. Conclusions: As in rodents, acute ozone exposure increased stress hormones and globally altered peripheral lipid metabolism in humans, likely through activation of a neurohormonally mediated stress response pathway. The metabolomic assessment revealed new biomarkers and allowed for establishment of rodent-to-human coherence. Clinical trial registered with www.clinicaltrials.gov (NCT 01492517). PMID:26745856

Simultaneous LC-MS/MS determination of JWH-210, RCS-4, ∆(9)-tetrahydrocannabinol, and their main metabolites in pig and human serum, whole blood, and urine for comparing pharmacokinetic data.

PubMed

Schaefer, Nadine; Kettner, Mattias; Laschke, Matthias W; Schlote, Julia; Peters, Benjamin; Bregel, Dietmar; Menger, Michael D; Maurer, Hans H; Ewald, Andreas H; Schmidt, Peter H

2015-05-01

A series of new synthetic cannabinoids (SC) has been consumed without any toxicological testing. For example, pharmacokinetic data have to be collected from forensic toxicological case work and/or animal studies. To develop a corresponding model for assessing such data, samples of controlled pig studies with two selected SC (JWH-210, RCS-4) and, as reference, ∆(9)-tetrahydrocannabinol (THC) should be analyzed as well as those of human cases. Therefore, a method for determination of JWH-210, RCS-4, THC, and their main metabolites in pig and human serum, whole blood, and urine samples is presented. Specimens were analyzed by liquid-chromatography tandem mass spectrometry and multiple-reaction monitoring with three transitions per compound. Full validation was carried out for the pig specimens and cross-validation for the human specimens concerning precision and bias. For the pig studies, the limits of detection were between 0.05 and 0.50 ng/mL in serum and whole blood and between 0.05 and 1.0 ng/mL in urine, the lower limits of quantification between 0.25 and 1.0 ng/mL in serum and 0.50 and 2.0 ng/mL in whole blood and urine, and the intra- and interday precision values lower than 15% and bias values within ±15%. The applicability was tested with samples taken from a pharmacokinetic pilot study with pigs following intravenous administration of a mixture of 200 μg/kg body mass dose each of JWH-210, RCS-4, and THC. The cross-validation data for human serum, whole blood, and urine showed that this approach should also be suitable for human specimens, e.g., of clinical or forensic cases.

Determination of perfluorinated alkyl acid concentrations in human serum and milk standard reference materials.

PubMed

Keller, Jennifer M; Calafat, Antonia M; Kato, Kayoko; Ellefson, Mark E; Reagen, William K; Strynar, Mark; O'Connell, Steven; Butt, Craig M; Mabury, Scott A; Small, Jeff; Muir, Derek C G; Leigh, Stefan D; Schantz, Michele M

2010-05-01

Standard Reference Materials (SRMs) are certified reference materials produced by the National Institute of Standards and Technology that are homogeneous materials well characterized with values for specified properties, such as environmental contaminant concentrations. They can be used to validate measurement methods and are critical in improving data quality. Disagreements in perfluorinated alkyl acid (PFAA) concentrations measured in environmental matrices during past interlaboratory comparisons emphasized the need for SRMs with values assigned for PFAAs. We performed a new interlaboratory comparison among six laboratories and provided, for the first time, value assignment of PFAAs in SRMs. Concentrations for perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and other PFAAs in two human serum and two human milk SRMs are reported. PFAA concentration measurements agreed for serum SRM 1957 using different analytical methods in six laboratories and for milk SRM 1954 in three laboratories. The interlaboratory relative standard deviation for PFOS in SRM 1957 was 7%, which is an improvement over past interlaboratory studies. Matrix interferences are discussed, as well as temporal trends and the percentage of branched vs. linear isomers. The concentrations in these SRMs are similar to the present-day average concentrations measured in human serum and milk, resulting in representative and useful control materials for PFAA human monitoring studies.

Human SAP is a novel peptidoglycan recognition protein that induces complement- independent phagocytosis of Staphylococcus aureus

PubMed Central

An, Jang-Hyun; Kurokawa, Kenji; Jung, Dong-Jun; Kim, Min-Jung; Kim, Chan-Hee; Fujimoto, Yukari; Fukase, Koichi; Coggeshall, K. Mark; Lee, Bok Luel

2014-01-01

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin (MBL) binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer, a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an Fcγ receptor-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain (NOD)-like receptors. PMID:23966633

Enoxacin penetration into human prostatic tissue.

PubMed Central

Bergeron, M G; Roy, R; Lessard, C; Foucault, P

1988-01-01

Concurrent enoxacin concentrations in serum and prostatic tissue were determined in 14 patients. The mean ratios of enoxacin concentration in tissue over concentration in serum were 1.4 +/- 0.2 (standard error of the mean). The levels in serum and prostatic tissue were above the MICs for most urinary pathogens. PMID:3196004

Direct extraction of lead (II) from untreated human blood serum using restricted access carbon nanotubes and its determination by atomic absorption spectrometry.

PubMed

Barbosa, Valéria Maria Pereira; Barbosa, Adriano Francisco; Bettini, Jefferson; Luccas, Pedro Orival; Figueiredo, Eduardo Costa

2016-01-15

Oxidized carbon nanotubes were covered with layers of bovine serum albumin to result in so-called restricted-access carbon nanotubes (RACNTs). This material can extract Pb(2+) ions directly from untreated human blood serum while excluding all the serum proteins. The RACNTs have a protein exclusion capacity of almost 100% and a maximum Pb(2+) adsorption capacity of 34.5mg g(-1). High resolution transmission electron microscopy, scanning transmission electron microscopy and energy dispersive spectroscopy were used to confirm the BSA layer and Pb(2+) adsorption sites. A mini-column filled with RACNTs was used in an on-line solid phase extraction system coupled to a thermospray flame furnace atomic absorption spectrometry. At optimized experimental conditions, the method has a detection limit as low as 2.1µg L(-1), an enrichment factor of 5.5, and inter- and intra-day precisions (expressed as relative standard deviation) of <8.1%. Recoveries of the Pb(2+) spiked samples ranged from 89.4% to 107.3% for the extraction from untreated human blood serum. Copyright © 2015 Elsevier B.V. All rights reserved.

The Use of Breast Magnetic Resonance Imaging Parameters to Identify Possible Signaling Pathways of a Serum Biomarker, HE4.

PubMed

Durur-Karakaya, Afak; Durur-Subasi, Irmak; Karaman, Adem; Akcay, Mufide Nuran; Palabiyik, Saziye Sezin; Erdemci, Burak; Alper, Fatih; Acemoglu, Hamit

2016-01-01

This study aimed to investigate the relationship between breast magnetic resonance imaging (MRI) parameters; clinical features such as age, tumor diameter, N, T, and TNM stages; and serum human epididymis protein 4 (HE4) levels in patients with breast carcinoma and use this as a means of estimating possible signaling pathways of the biomarker, HE4. Thirty-seven patients with breast cancer were evaluated by breast MRI and serum HE4 levels before therapy. Correlations between parameters including age, tumor diameter T and N, dynamic curve type, enhancement ratio (ER), slope washin (S-WI), time to peak (TTP), slope washout (S-WO), and the serum level of HE4 were investigated statistically. Human epididymis protein 4 levels of early and advanced stage of disease were also compared statistically. Breast MRI parameters showed correlation to serum HE4 levels and correlations were statistically significant. Of these MRI parameters, S-WI had higher correlation coefficient than the others. Human epididymis protein 4 levels were not statistically different in early and advanced stage of disease. High correlation with MRI parameters related to neoangiogenesis may indicate signaling pathway of HE4.

Determination of miloxacin and metabolites in human serum and urine by high-pressure liquid chromatography.

PubMed Central

Yoshitake, A; Kawahara, K; Shono, F; Umeda, I; Izawa, A; Komatsu, T

1980-01-01

A sensitive and reliable high-pressure liquid chromatography (HPLC) assay for miloxacin and its two principal metabolites, 5,8-dihydro-8-oxo-2H-1,3-dioxolo[4,5-g]quinoline-7-carboxylic acid (M-1) and 1,4-dihydro-1,6-dimethoxy-7-hydroxy-4-oxoquinoline-3-carboxylic acid (M-2), in human serum and urine was developed. A strong anion-exchange Zipax SAX column using a mobile phase of 0.01 M citric acid solution containing 0.03 M sodium nitrate with pH 5.0 was used to achieve separation of the three compounds. The retention times of miloxacin, M-1, and M-2 were 3.8, 9.3, and 5.9 min, respectively. Serum and urine concentrations of these compounds as low as 10 ng/ml were measured. When results from the HPLC assay were compared with those from the microbiological assay of serum and urine samples from human subjects receiving miloxacin orally, the correlation coefficients were 0.94 for the serum and 0.99 for the urine. The HPLC assay method presents an alternative to the microbiological assay and permits future pharmacokinetic investigations of miloxacin. PMID:7416751

Occurrence of West Nile virus antibodies in wild birds, horses, and humans in Poland.

PubMed

Niczyporuk, Jowita Samanta; Samorek-Salamonowicz, Elżbieta; Lecollinet, Sylvie; Pancewicz, Sławomir Andrzej; Kozdruń, Wojciech; Czekaj, Hanna

2015-01-01

Serum samples of 474 wild birds, 378 horses, and 42 humans with meningitis and lymphocytic meningitis were collected between 2010 and 2014 from different areas of Poland. West Nile virus (WNV) antibodies were detected using competition enzyme linked immunosorbent assays: ELISA-1 ID Screen West Nile Competition, IDvet, ELISA-2 ID Screen West Nile IgM Capture, and ELISA-3 Ingezim West Nile Compac. The antibodies were found in 63 (13.29%) out of 474 wild bird serum samples and in one (0.26%) out of 378 horse serum samples. Fourteen (33.33%) out of 42 sera from patients were positive against WNV antigen and one serum was doubtful. Positive samples obtained in birds were next retested with virus microneutralisation test to confirm positive results and cross-reactions with other antigens of the Japanese encephalitis complex. We suspect that positive serological results in humans, birds, and horses indicate that WNV can be somehow closely related with the ecosystem in Poland.

Occurrence of West Nile Virus Antibodies in Wild Birds, Horses, and Humans in Poland

PubMed Central

Niczyporuk, Jowita Samanta; Samorek-Salamonowicz, Elżbieta; Lecollinet, Sylvie; Pancewicz, Sławomir Andrzej; Kozdruń, Wojciech; Czekaj, Hanna

2015-01-01

Serum samples of 474 wild birds, 378 horses, and 42 humans with meningitis and lymphocytic meningitis were collected between 2010 and 2014 from different areas of Poland. West Nile virus (WNV) antibodies were detected using competition enzyme linked immunosorbent assays: ELISA-1 ID Screen West Nile Competition, IDvet, ELISA-2 ID Screen West Nile IgM Capture, and ELISA-3 Ingezim West Nile Compac. The antibodies were found in 63 (13.29%) out of 474 wild bird serum samples and in one (0.26%) out of 378 horse serum samples. Fourteen (33.33%) out of 42 sera from patients were positive against WNV antigen and one serum was doubtful. Positive samples obtained in birds were next retested with virus microneutralisation test to confirm positive results and cross-reactions with other antigens of the Japanese encephalitis complex. We suspect that positive serological results in humans, birds, and horses indicate that WNV can be somehow closely related with the ecosystem in Poland. PMID:25866767

Test of neural inertia in humans during general anaesthesia.

PubMed

Kuizenga, M H; Colin, P J; Reyntjens, K M E M; Touw, D J; Nalbat, H; Knotnerus, F H; Vereecke, H E M; Struys, M M R F

2018-03-01

Neural inertia is defined as the tendency of the central nervous system to resist transitions between arousal states. This phenomenon has been observed in mice and Drosophila anaesthetized with volatile anaesthetics: the effect-site concentration required to induce anaesthesia in 50% of the population (C 50 ) was significantly higher than the effect-site concentration for 50% of the population to recover from anaesthesia. We evaluated this phenomenon in humans using propofol or sevoflurane (both with or without remifentanil) as anaesthetic agents. Thirty-six healthy volunteers received four sessions of anaesthesia with different drug combinations in a step-up/step-down design. Propofol or sevoflurane was administered with or without remifentanil. Serum concentrations of propofol and remifentanil were measured from arterial blood samples. Loss and return of responsiveness (LOR-ROR), response to pain (PAIN), Patient State Index (PSI) and spectral edge frequency (SEF) were modeled with NONMEM®. For propofol, the C 50 for induction and recovery of anaesthesia was not significantly different across the different endpoints. For sevoflurane, for all endpoints except SEF, significant differences were found. For some endpoints (LOR and PAIN) the difference was significant only when sevoflurane was combined with remifentanil. Our results nuance earlier findings with volatile anaesthetics in mice and Drosophila. Methodological aspects of the study, such as the measured endpoint, influence the detection of neural inertia. A more thorough definition of neural inertia, with a robust methodological framework for clinical studies is required to advance our knowledge of this phenomenon. NCT 02043938. Copyright © 2017 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.

Effect of Platelet Lysate on Human Cells Involved in Different Phases of Wound Healing

PubMed Central

Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

2013-01-01

Background Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Methodology/Principal Findings Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control), comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. Conclusion/Significance These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing. PMID:24386412

The cytotoxic effect of spiroflavanone derivatives, their binding ability to human serum albumin (HSA) and a DFT study on the mechanism of their synthesis

NASA Astrophysics Data System (ADS)

Budzisz, Elzbieta; Paneth, Piotr; Geromino, Inacrist; Muzioł, Tadeusz; Rozalski, Marek; Krajewska, Urszula; Pipiak, Paulina; Ponczek, Michał B.; Małecka, Magdalena; Kupcewicz, Bogumiła

2017-06-01

This paper examines the cytotoxic effect of nine compounds with spiropyrazoline structures, and determines the reaction mechanism between diazomethane and selected benzylideneflavanones, their lipophilicity, and their binding ability to human serum albumin. The cytotoxic effect was determined on two human leukaemia cell lines (HL-60 and NALM-6) and melanoma WM-115 cells, as well as on normal human umbilical vein endothelial cells (HUVEC). The highest cytotoxicity was exhibited by compound B7: it was found to have an IC50 of less than 10 μM for all three cancer cell lines, with five to 12-fold lower sensitivity against normal cells (HUVEC). All the compounds exhibit comparable affinity energy in human serum albumin binding (from -8.1 to -8.6 kcal mol-1) but vary in their binding sites depending on the substituent. X-ray crystallography of two derivatives confirmed their synthetic pathway, and their structures were carefully examined.

Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woolbright, Benjamin L.; Dorko, Kenneth; Antoine, Daniel J.

Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with,more » or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury compared to rodents. • Primary human hepatocytes largely undergo necrosis in response to BA toxicity. • Cholestatic liver injury in vivo is predominantly necrotic with minor apoptosis. • Rodent models of bile acid toxicity may not recapitulate the injury in man.« less

The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer.

PubMed

Fu, Xiaoling; Liu, Yangyang; Qiu, Ruiyun; Foda, Mohamed F; Zhang, Yong; Wang, Tao; Li, Jinshan

2018-03-01

The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA) was presented for the clinical determination and analysis of human epididymis protein 4 (HE4) in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0-1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA), with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum.

Detection of endogenous cortisol in equine tears and blood at rest and after simulated stress.

PubMed

Monk, Caroline S; Hart, Kelsey A; Berghaus, Roy D; Norton, Natalie A; Moore, Phillip A; Myrna, Kathern E

2014-07-01

To determine whether cortisol is present in equine tears at rest and during simulated stress and compare tear cortisol to serum free and total cortisol. Fourteen healthy adult horses were included. Paired tear total cortisol and serum total and free cortisol concentrations were measured with ELISA, chemiluminescent immunoassay, and ultrafiltration methodology, respectively, in 10 horses at rest once daily for five consecutive days. In an additional four horses, paired tear and serum samples were collected for cortisol measurement before and after adrenocorticotropic hormone (ACTH) stimulation (cosyntropin, 1 μg/kg IV). Cortisol was detectable in equine tears at rest. Following ACTH stimulation, tear cortisol increased significantly from baseline at 60-120 min (P ≤ 0.001). Serum total and free cortisol also increased significantly at 30-180 min after ACTH stimulation (P ≤ 0.001). Both serum and tear cortisol returned to baseline concentrations by 360 min. Changes in tear cortisol were similarly associated with changes in serum total and free cortisol, although high tear cortisol concentrations suggest a portion of tear cortisol may be protein-bound. Cortisol is present in equine tears and increases in concert with serum cortisol following ACTH stimulation. Further study is needed to determine whether endogenous cortisol in tears contributes to ocular pathology. © 2013 American College of Veterinary Ophthalmologists.

Serum creatinine levels are significantly influenced by renal size in the normal pediatric population.

PubMed

Di Zazzo, Giacomo; Stringini, Gilda; Matteucci, Maria Chiara; Muraca, Maurizio; Malena, Saverio; Emma, Francesco

2011-01-01

Clinical and experimental data have shown that differences in nephron endowment result in differences in renal mass and predisposition to chronic renal failure, hypertension, and proteinuria. We hypothesized that a significant proportion of the variance in GFR, as estimated by serum creatinine, is attributable to differences in renal size in normal children. A total of 1748 normal renal ultrasounds that were performed in children older than 6 months were reviewed. For each ultrasound, serum creatinine, serum blood urea nitrogen, and systolic and diastolic office BP were recorded. Renal size was evaluated as a function of renal length and thickness. All data were normalized for height, weight, age, and gender. When expressed as SD scores, a significant correlation was found between kidney size and serum creatinine (P < 0.0001) and between kidney size and serum blood urea nitrogen (P < 0.002). When dividing kidney size data per quintiles, a difference of 0.51 SD score in serum creatinine was observed between the lowest and highest quintile. No significant correlation was found with office BP measurements. These data show that, even in the normal pediatric population, differences in renal function are significantly explained by differences in renal mass. Methodologic limitations of this study are likely to underestimate this relationship.

Increased Serum Oxidative Stress Markers in Women with Uterine Leiomyoma

PubMed Central

Santulli, Pietro; Borghese, Bruno; Lemaréchal, Herve; Leconte, Mahaut; Millischer, Anne-Elodie; Batteux, Frédéric

2013-01-01

Background Uterine leiomyomas (fibroids) are the most common gynaecological benign tumors in premenopausal women. Evidences support the role of oxidative stress in the development of uterine leiomyoma. We have analysed oxidative stress markers (thiols, advanced oxidized protein products (AOPP), protein carbonyls and nitrates/nitrites) in preoperative sera from women with histologically proven uterine leiomyoma. Methodology/Principal Findings We conducted a laboratory study in a tertiary-care university hospital. Fifty-nine women with histologically proven uterine leiomyoma and ninety-two leiomyoma-free control women have been enrolled in this study. Complete surgical exploration of the abdominopelvic cavity was performed in each patient. Preoperative serum samples were obtained from all study participants to assay serum thiols, AOPP, protein carbonyls and nitrates/nitrites. Concentrations of serum protein carbonyl groups and AOPP were higher in leiomyoma patients than in the control group (p=0.005 and p<0.001, respectively). By contrast, serum thiol levels were lower in leiomyoma patients (p<0.001). We found positive correlations between serum AOPP concentrations and total fibroids weight (r=0.339; p=0.028), serum AOPP and serum protein carbonyls with duration of infertility (r=0.762; p=0.006 and r=0.683; p=0.021, respectively). Conclusions/Significance This study, for the first time, reveals a significant increase of protein oxidative stress status and reduced antioxidant capacity in sera from women with uterine leiomyoma. PMID:23951284

Use of transfer learning to detect diffuse degenerative hepatic diseases from ultrasound images in dogs: A methodological study.

PubMed

Banzato, T; Bonsembiante, F; Aresu, L; Gelain, M E; Burti, S; Zotti, A

2018-03-01

The aim of this methodological study was to develop a deep convolutional neural network (DNN) to detect degenerative hepatic disease from ultrasound images of the liver in dogs and to compare the diagnostic accuracy of the newly developed DNN with that of serum biochemistry and cytology on the same samples, using histopathology as a standard. Dogs with suspected hepatic disease that had no prior history of neoplastic disease, no hepatic nodular pathology, no ascites and ultrasonography performed 24h prior to death were included in the study (n=52). Ultrasonography and serum biochemistry were performed as part of the routine clinical evaluation. On the basis of histopathology, dogs were categorised as 'normal' (n=8), or having 'vascular abnormalities'(n=8), or 'inflammatory'(n=0), 'neoplastic' (n=4) or 'degenerative'(n=32) disease; dogs with 'neoplastic' disease were excluded from further analysis. On cytological evaluation, dogs were categorised as 'normal' (n=11), or having 'inflammatory' (n=0), 'neoplastic' (n=4) or 'degenerative' (n=37) disease. Dogs were categorised as having 'degenerative' (n=32) or 'non-degenerative' (n=16) liver disease for analysis due to the limited sample size. The DNN was developed using a transfer learning methodology on a pre-trained neural network that was retrained and fine-tuned to our data set. The resultant DNN had a high diagnostic accuracy for degenerative liver disease (area under the curve 0.91; sensitivity 100%; specificity 82.8%). Cytology and serum biochemical markers (alanine transaminase and aspartate transaminase) had poor diagnostic accuracy in the detection of degenerative liver disease. The DNN outperformed all the other non-invasive diagnostic tests in the detection of degenerative liver disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Doxorubicin-loaded glycyrrhetinic acid modified recombinant human serum albumin nanoparticles for targeting liver tumor chemotherapy.

PubMed

Qi, Wen-Wen; Yu, Hai-Yan; Guo, Hui; Lou, Jun; Wang, Zhi-Ming; Liu, Peng; Sapin-Minet, Anne; Maincent, Philippe; Hong, Xue-Chuan; Hu, Xian-Ming; Xiao, Yu-Ling

2015-03-02

Due to overexpression of glycyrrhetinic acid (GA) receptor in liver cancer cells, glycyrrhetinic acid modified recombinant human serum albumin (rHSA) nanoparticles for targeting liver tumor cells may result in increased therapeutic efficacy and decreased adverse effects of cancer therapy. In this study, doxorubicin (DOX) loaded and glycyrrhetinic acid modified recombinant human serum albumin nanoparticles (DOX/GA-rHSA NPs) were prepared for targeting therapy for liver cancer. GA was covalently coupled to recombinant human serum albumin nanoparticles, which could efficiently deliver DOX into liver cancer cells. The resultant GA-rHSA NPs exhibited uniform spherical shape and high stability in plasma with fixed negative charge (∼-25 mV) and a size about 170 nm. DOX was loaded into GA-rHSA NPs with a maximal encapsulation efficiency of 75.8%. Moreover, the targeted NPs (DOX/GA-rHSA NPs) showed increased cytotoxic activity in liver tumor cells compared to the nontargeted NPs (DOX/rHSA NPs, DOX loaded recombinant human serum albumin nanoparticles without GA conjugating). The targeted NPs exhibited higher cellular uptake in a GA receptor-positive liver cancer cell line than nontargeted NPs as measured by both flow cytometry and confocal laser scanning microscopy. Biodistribution experiments showed that DOX/GA-rHSA NPs exhibited a much higher level of tumor accumulation than nontargeted NPs at 1 h after injection in hepatoma-bearing Balb/c mice. Therefore, the DOX/GA-rHSA NPs could be considered as an efficient nanoplatform for targeting drug delivery system for liver cancer.

Lymphocyte-dependent antibody-mediated cytotoxicity in Hashimoto thyroiditis

PubMed Central

Calder, Elizabeth A.; Penhale, W. J.; McLeman, Dena; Barnes, E. W.; Irvine, W. J.

1973-01-01

In the presence of normal human lymphocytes, decomplemented sera from twentynine out of thirty-nine patients with Hashimoto thyroiditis caused significant lysis of thyroglobulin-coated chicken red blood cells, as estimated by the release of 51Cr; the mean% specific 51Cr release being 14·1 ± 1·9 (SEM). Serum from twenty-one control subjects studied concurrently caused no significant lysis of thyroglobulin-coated chicken red blood cells; the mean% specific 51Cr release being −1·6±0·7 (SEM). The degree of cytotoxicity correlated with the titre of thyroglobulin antibodies in the serum, determined by tanned red cell haemagglutination. The active component in the Hashimoto serum was localized in the 19S fraction, was unaffected by pre-absorption with anti-human IgM serum, but was neutralized by pre-absorption with anti-human IgG serum. These findings suggest that the cytotoxic activity of serum from patients with Hashimoto thyroiditis is due to the presence of thyroglobulin antibody of the IgG class in the form of complexes, either alone or with antigen. It is postulated that non-specific lymphocytes may play an important role in the pathogenesis of Hashimoto thyroiditis, being activated by the presence in the gland of thyroglobulin antibody, either alone or in the form of complexes attached to thyroid cells. PMID:4740445

Thermometric enzyme linked immunosorbent assay: TELISA.

PubMed

Mattiasson, B; Borrebaeck, C; Sanfridson, B; Mosbach, K

1977-08-11

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

PubMed

Wei, M; Chen, Y; Xi, J; Ru, S; Ji, M; Zhang, D; Fang, Q; Tang, B

Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

Resistance to serum bactericidal activity distinguishes Brazilian purpuric fever (BPF) case strains of Haemophilus influenzae biogroup aegyptius (H. aegyptius) from non-BPF strains. Brazilian Purpuric Fever Study Group.

PubMed Central

Porto, M H; Noel, G J; Edelson, P J

1989-01-01

We studied the ability of normal human serum to lyse H. influenzae biogroup aegyptius (H. aegyptius) isolates recovered from patients with Brazilian purpuric fever (BPF clone) or non-BPF clone strains. BPF clone isolates, although similar to non-BPF clone isolates with regard to the ability to fix C3 to their surfaces, could be distinguished from non-BPF clone strains by their resistance to lysis in vitro following incubation with normal adult human serum. PMID:2786003

Molecular Structure-Affinity Relationship of Flavonoids in Lotus Leaf (Nelumbo nucifera Gaertn.) on Binding to Human Serum Albumin and Bovine Serum Albumin by Spectroscopic Method.

PubMed

Tang, Xiaosheng; Tang, Ping; Liu, Liangliang

2017-06-23

Lotus leaf has gained growing popularity as an ingredient in herbal formulations due to its various activities. As main functional components of lotus leaf, the difference in structure of flavonoids affected their binding properties and activities. In this paper, the existence of 11 flavonoids in lotus leaf extract was confirmed by High Performance Liquid Chromatography (HPLC) analysis and 11 flavonoids showed various contents in lotus leaf. The interactions between lotus leaf extract and two kinds of serum albumins (human serum albumin (HSA) and bovine serum albumin (BSA)) were investigated by spectroscopic methods. Based on the fluorescence quenching, the interactions between these flavonoids and serum albumins were further checked in detail. The relationship between the molecular properties of flavonoids and their affinities for serum albumins were analyzed and compared. The hydroxylation on 3 and 3' position increased the affinities for serum albumins. Moreover, both of the methylation on 3' position of quercetin and the C₂=C₃ double bond of apigenin and quercetin decreased the affinities for HSA and BSA. The glycosylation lowered the affinities for HSA and BSA depending on the type of sugar moiety. It revealed that the hydrogen bond force played an important role in binding flavonoids to HSA and BSA.

Commercial radioimmunoassay for beta subunit of human chorionic gonadotropin: falsely positive determinations due to elevated serum luteinizing hormone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, J.E. Jr.; Platoff, G.E.; Kubrock, C.A.

1982-01-01

Among 17 men who had received seemingly curative treatment for unilateral non-seminomatous germ cell tumors for the testis and who had consistently normal serum human chorionic gonadotropin (HCG) levels at a reference laboratory, 7 (41%) had at least one falsely positive commercial serum HCG determination. To investigate the cause of these falsely positive determinations the authors measured the cross reactivity of luteinizing hormone (LH) and follicle stimulating hormone (FSH) standards in the commercial HCG assay, and studied the relationships between commercial HCG levels and serum LH levels, serum FSH levels and gonadal status in men with and without normal gonadalmore » function. The falsely positive HCG determinations appeared to be due to elevated serum LH levels and cross reactivity of LH in the commercial HCG assay because: 1) there was substantial cross reactivity of the LH standards in the commercial assay, 2) the serum LH was elevated in four of six men with solitary testes, 3) there was a striking correlation between elevated serum LH levels and falsely elevated commercial HCG levels in ten men with solitary or absent testes, and 4) there were no falsely positive HCG determinations in 13 normal men but there were falsely positive HCG determinations in seven of ten anorchid men.« less

Ghrelin in the pilosebaceous unit: alteration of ghrelin in patients with acne vulgaris.

PubMed

Cicek, Demet; Demir, Betul; Erder, Ilker; Kuloglu, Tuncay; Ucer, Ozlem; Aydin, Suleyman; Ucak, Haydar; Dertlioglu, Selma; Kalayci, Mehmet

2015-01-01

Ghrelin in the pilosebaceous tissues of human skin and ghrelin levels in patients with acne vulgaris have not yet been investigated. The purpose of this study was to screen ghrelin immunoreactivity by immunohistochemistry in human pilosebaceous tissues of human skin and also to determine the quantities of ghrelin in the serum of the patients with acne vulgaris. 30 patients presenting with acne vulgaris and 30 control subjects participated in this study. Ghrelin levels were determined by enzyme linked immunosorbent assay (ELISA). Human hair follicles and sebaceous glands were immunohistochemically examined. Immunohistochemistry results showed that there is a strong ghrelin immunoreactivity in the hair follicles and sebaceous glands in sections of human skin. The mean serum ghrelin levels (27.58 ・} 15.44 pg/mL) in patients with acne vulgaris was significantly lower than those of controls (35.62・}20.46 pg/mL). Ghrelin produced in hair follicles and sebaceous glands of the skin might participate in the pathogenesis of acne vulgaris and also acne vulgaris in humans might be associated with decreased serum ghrelin.

Inactivation of human interferon by body fluids

NASA Technical Reports Server (NTRS)

Cesario, T. C.; Mandell, A.; Tilles, J. G.

1973-01-01

Description of the effects of human feces, bile, saliva, serum, and cerebrospinal fluid on interferon activity. It is shown that crude interferon is inactivated by at least 50% more than with the control medium used, when incubated for 4 hr in vitro in the presence of serum, saliva, or cerebrospinal liquid, and by close to 100% when incubated with stool extract or bile.

Human Ozone (O3) Exposure Alters Serum Profile of Lipid Metabolites

EPA Science Inventory

HUMAN OZONE (O3) EXPOSURE ALTERS SERUM PROFILE OF LIPID METABOLITES Miller, D B.1; Kodavanti, U P.2 Karoly, E D.3; Cascio W.E2, Ghio, A J. 21. UNC-Chapel Hill, Chapel Hill, N.C., United States. 2. NHEERL, U.S. EPA, RTP, N.C., United States. 3. METABOLON INC., Durham, N.C., United...

Investigation of the interaction of deltamethrin (DM) with human serum albumin by multi-spectroscopic method

NASA Astrophysics Data System (ADS)

Wang, Jiaman; Ma, Liang; Zhang, Yuhao; Jiang, Tao

2017-02-01

The interaction of Deltamethrin (DM) with human serum albumin (HSA) under the condition of simulating human blood pH environment (pH = 7.4) was investigated by fluorescence, UV-Vis absorbance and circular dichroism (CD) spectroscopy. It was shown that DM was a static quencher of HSA. The binding constants (Ka) are 3.598 × 104 L mol-1 (25 °C); the thermodynamic parameters (ΔH = -3.269 × 104 kJ mol-1, ΔS = -22.81 kJ mol-1 k-1, ΔG = -25889.8 kJ mol-1) obtained with the thermodynamic equation. The hydrogen bond and Vander Waals were the main driving force. The effect of DM on the conformation of HSA was observed by three-dimensional (3D) fluorescence and circular dichroism spectra, indicating that the interaction between DM and HSA was achieved through the binding of DM with the tryptophan and tyrosine residues of HSA. The study on the interaction of DM and Bovine Serum Albumin (BSA) was researched and compared. Difference exists in the interactions of with each of the serum albumins. We will verify and supplement that DM residue in animals and human metabolism, toxicology and other mechanisms are different.

Role of vaspin in human eating behaviour.

PubMed

Breitfeld, Jana; Tönjes, Anke; Gast, Marie-Therese; Schleinitz, Dorit; Blüher, Matthias; Stumvoll, Michael; Kovacs, Peter; Böttcher, Yvonne

2013-01-01

The adipokine vaspin (visceral adipose tissue derived serine protease inhibitor, serpinA12) follows a meal-related diurnal variation in humans and intracerebroventricular vaspin administration leads to acutely reduced food intake in db/db mice. We therefore hypothesized that vaspin may play a role in human eating behaviour. We measured serum vaspin concentrations in 548 subjects from a self-contained population of Sorbs (Germany) who underwent detailed metabolic testing including eating behaviour assessments using the three-factor eating questionnaire. In addition, genetic variation within vaspin was assessed by genotyping 28 single nucleotide polymorphisms (SNPs) in all study subjects. Serum vaspin concentrations correlated positively with restraint, disinhibition and hunger (all P<0.05), although the correlations did not withstand further adjustments for age, gender and BMI (all P>0.05). Independent of observed correlations, genetic variants in vaspin were associated with serum vaspin levels but showed no significant association with any of the eating behaviour phenotypes after accounting for multiple testing (P≥0.05 after adjusting for age, gender and BMI). Our data suggest that serum vaspin concentrations might modulate human eating behaviour, which does not seem to be affected by common genetic variation in vaspin.

Non-covalent binding analysis of sulfamethoxazole to human serum albumin: Fluorescence spectroscopy, UV-vis, FT-IR, voltammetric and molecular modeling.

PubMed

Naik, Praveen N; Nandibewoor, Sharanappa T; Chimatadar, Shivamurthi A

2015-06-01

This study was designed to examine the interaction of sulfamethoxazole (SMZ) with human serum albumin(HSA). Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of human serum albumin by SMZ was static mechanism. The binding constant values for the SMZ-HSA system were obtained to be 22,500 L/mol at 288 K, 15,600 L/mol at 298 K, and 8500 L/mol at 308 K. The distance r between donor and acceptor was evaluated according to the theory of Föster energy transfer. The results of spectroscopic analysis and molecular modeling techniques showed that the conformation of human serum albumin had been changed in the presence of SMZ. The thermodynamic parameters, namely enthalpy change (∆ H 0 ) -36.0 kJ/mol, entropy change (∆ S 0 ) -41.3 J/mol K and free energy change (∆ G 0 ) -23.7 kJ/mol, were calculated by using van׳t Hoff equation. The effect of common ions on the binding of SMZ to HSA was tested.

HPLC-MS/MS method optimisation for matrix metalloproteinase 3 and matrix metalloproteinase 9 determination in human blood serum using target analysis.

PubMed

Kotnik, Petra; Krajnc, Metka Koren; Pahor, Artur; Finšgar, Matjaž; Knez, Željko

2018-02-20

A quantitative analysis of zinc endopeptidases matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 3 (MMP3) from human blood serum are presented. Both matrix metalloproteinases (MMP) are present in human blood serum and can be used as biomarkers for different diseases. The analysis was performed using LC-MS/MS with a triple quadrupole mass spectrometer, based on two specific peptides of each MMP in comparison with an enzyme-linked immunosorbent assay (ELISA). While the conditions for the LC-MS/MS analysis of MMP9 peptides were previously reported for bronchoalveolar lavage fluid, the analysis of MMP3 peptides was newly quantified for human blood serum herein for the first time. For MMP3, the linear behaviour was determined in the concentration range from 1.0-200.0ng/mL (R 2 =0.997) with an LLOD of 0.5ng/mL. For MMP9, linearity was determined in the concentration range from 6.5-65.0ng/mL (R 2 =0.995) with an LLOD of 2.0ng/mL. Copyright © 2017 Elsevier B.V. All rights reserved.

Buyang Huanwu Decoction Vigorously Rescues PC12 Cells Against 6-OHDA-Induced Neurotoxicity via Akt/GSK3β Pathway Based on Serum Pharmacology Methodology.

PubMed

Li, Zeyan; Wang, Hui; Wang, Qian; Sun, Jinhao

2016-12-01

Buyang Huanwu decoction (BYHWD), as a popular traditional Chinese medicine formula, was widely used for treating ischemic diseases. However, in the area of neurodegenerative diseases, the researches focused on BYHWD are rare but promising, and molecular mechanisms underlying are largely elusive. 6-Hydroxydopamine (6-OHDA), a dopaminergic-specific neurotoxin, is extensively used to establish neurotoxic model in vivo and in vitro. In our present study, we prepared drug-containing serum of BYHWD (Buyang Huanwu drug-containing serum [BYHWS]) based on serum pharmacology methodology. Neurotoxic model in vitro was established in PC12 cells, and innovative experimental grouping method was adopted to investigate neuroprotective effects of BYHWS on neurotoxicity induced by 6-OHDA exposure. Remarkably, BYHWS vigorously rescued PC12 cells from 6-OHDA-induced neurotoxicity even to surpass 100% in cell viability. Moreover, Hoechst/propidium iodide (PI) staining revealed that cell apoptotic rate was reduced significantly after incubation of BYHWS. Besides, BYHWS effectively restored the disruption of mitochondrial membrane potential and attenuated the elevation of intracellular reactive oxygen species level caused by 6-OHDA insult. Furthermore, BYHWS remarkably reversed the dephosphorylation of Akt (protein kinase B) and glycogen synthase kinase-3β (GSK3β) evoked by 6-OHDA. The above protective effects were attenuated by coculturing with Akt inhibitor LY294002. In summary, we concluded that the BYHWS vigorously blocked 6-OHDA-induced neurotoxicity via Akt/GSK3β pathway and provided a novel insight into roles of BYHWD in the clinical practices on neurodegenerative diseases.

Can serums be replaced by Mueller-Hinton agar in germ tube test?

PubMed

Atalay, M A; Koc, A N; Parkan, O M; Aydemir, G; Elmali, F; Sav, H

2017-01-01

The germ tube test (GTT) is inexpensive, easy, and well-defined test that differentiates Candida albicans (excluding Candida dubliniensis and Candida africana) from other species. The aim of this study was to evaluate various serums (i.e., human, rabbit, horse, and fetal bovine serum) used in the GTT and Mueller-Hinton agar (MHA). Fifty species isolated from various clinical samples that were defined as C. albicans by both conventional and DNA sequence analysis methods were included in the study. One to two colonies of C. albicans were mixed into 0.5-1 ml of fetal bovine serum, horse serum, rabbit serum, and human serum. Serums and MHA were incubated at 37°C for GTT. They were removed from the incubator and evaluated after 30 min, 1 h, 2 h, and 3 h of incubation. The GTT was accepted to be positive only if germ tube was 1/2 the width and 3 times the length of the parent yeast cell and with no constriction at the point of origin. When the use of serums and MHA for GTT was statistically evaluated, according to the positive scoring, the best results were obtained with MHA and with rabbit, horse, and fetal bovine serum, respectively. The best definition over time statistically was the third hour. It is suggested that inexpensive MHA is a fast, appropriate, and reliable medium for the probable diagnosis of GTT and C. albicans; however, additional studies are still needed to define other Candida species.

Human milk polyunsaturated long-chain fatty acids and secretory immunoglobulin A antibodies and early childhood allergy.

PubMed

Duchén, K; Casas, R; Fagerås-Böttcher, M; Yu, G; Björkstén, B

2000-02-01

The possible protective effect of breast milk against atopic manifestations in infancy, i.e. atopic eczema and food allergy, has been controversial for the last decades. Besides the methodological problems, differences in the composition of human milk could explain these controversies. The aim of this study was to investigate the composition of polyunsaturated fatty acids (PUFA) and secretory immunoglobulin A (S-IgA) levels to food proteins (ovalbumin and beta-lactoglobulin) and an inhalant allergen (cat) in milk from mothers of allergic and non-allergic children. Blood samples were obtained at birth and at 3 months from 120 children. Skin prick tests were performed at 6, 12 and 18 months, and the development of atopic diseases was assessed in the children. Breast milk samples were collected from their mothers at birth and monthly during the lactation period. Milk PUFA composition was measured by gas chromatography, and enzyme-linked immunosorbent assay (ELISA) was used to measure total S-IgA, anti-cat S-IgA, anti-ovalbumin S-IgA, and anti-beta-lactoglobulin S-IgA. Allergic disease developed in 44/120 children (22/63 children of allergic mothers and 22/57 children of non-allergic mothers). Lower levels of eicosapentaenoic acid, C20:5 n-3 (EPA), docosapentaenoic acid C22:5 n-3 (DPA), and docosatetraenoic acid C22:4 n-6 (DHA) (p < 0.05 for all) were found in mature milk from mothers of allergic as compared to milk from mothers of non-allergic children. The total n-6:total n-3 and the arachidonic acid, C20:4 n-6 (AA):EPA ratios were significantly lower in transitional and mature milk from mothers of allergic children, as compared to milk from mothers of non-allergic children. The PUFA levels in serum of allergic and non-allergic children were largely similar, except for higher levels of C22:4 n-6 and C22:5 n-6 (p < 0.05 for both) and a higher AA:EPA ratio in serum phospholipids in the former group (p < 0.05). Changes in the levels of milk PUFA were reflected in changes in PUFA serum phospholipids, particularly for the n-6 PUFA. The AA: EPA ratio in maternal milk was related, however, to the AA:EPA only in serum from non-allergic children, while this was not the case in allergic children. The levels of total S-IgA, anti-cat S-IgA, anti-ovalbumin S-IgA, and anti-beta-lactoglobulin S-IgA in milk from mothers of allergic, as compared to non-allergic, children were similar through the first 3 months of lactation. Low levels of n-3 PUFA in human milk, and particularly a high AA:EPA ratio in maternal milk and serum phospholipids in the infants, were related to the development of symptoms of allergic disease at 18 months of age. The milk PUFA composition influenced the composition of PUFA in serum phospholipids of the children. We also showed that the lower levels of colostral anti-ovalbumin S-IgA and lower total S-IgA in mature milk from atopic mothers did not influence the development of allergic disease in the children up to 18 months of age. The findings indicate that low alpha-linolenic acid, C18:3 n-3 (LNA) and n-3 long-chain polyunsaturated fatty acids (LCP) 20-22 carbon chains, but not the levels of S-IgA antibodies to allergens, are related to the development of atopy in children.

Constitutive secretion of soluble interleukin-2 receptor by human T cell lymphoma xenografted into SCID mice. Correlation of tumor volume with concentration of tumor-derived soluble interleukin-2 receptor in body fluids of the host mice.

PubMed Central

Wasik, M. A.; Sioutos, N.; Tuttle, M.; Butmarc, J. R.; Kaplan, W. D.; Kadin, M. E.

1994-01-01

Increased serum concentration of soluble alpha-chain receptor for interleukin-2 (sIL-2R) has been noted in patients with a variety of inflammatory conditions and lymphoid malignancies including T cell leukemia and lymphoma. Elevated sIL-2R serum levels seen in lymphoid malignancies appear to correlate with the clinical stage of disease. However, because sIL-2R is produced by normal activated lymphocytes, it has been uncertain whether serum sIL-2R in such conditions is derived from tumor cells or normal immune cells responding to the tumor. To address this question, we used a model of human (CD30+) anaplastic, large T cell lymphoma transplanted into immunodeficient SCID mice. Reverse transcription polymerase chain reaction of tumor RNA showed that the tumor, designated mJB6, contains mRNA for alpha-chain of human IL-2R. Furthermore, 15 to 25% of tumor cells stained with anti-human IL-2R alpha-chain mAb. Solid phase ELISA analysis of serum samples from mice bearing mJB6 lymphoma showed high concentrations of human sIL-2R. None of the control mice without lymphoma or with human nonlymphoid tumors (prostatic carcinoma, ovarian carcinoma, and glioblastoma multiforme) showed detectable human sIL-2R. The sIL-2R serum titers of mJB6-bearing mice correlated strongly with tumor volume (P < 0.0001). Tumors as small as 0.4 to 0.8 mm3 could be detected by this method. The sensitivity of sIL-2R ELISA exceeded at least 150 times the sensitivity of conventional radioisotopic tumor detection. Total resection of mJB6 tumors resulted in complete clearance of sIL-2R from the murine serum within 48 hours with a half-life of 6 hours. Accordingly, partial resection led to a significant decrease in sIL-2R followed by gradual increase with tumor regrowth. sIL-2R was also detected in the urine of mJB6-transplanted mice. As in serum, urine concentrations of sIL-2R were proportional to tumor mass (P < 0.02). Based on these findings we postulate that malignant cells are a major source of serum sIL-2R in patients with lymphoid tumors. In addition, our data further support monitoring sIL-2R concentration in body fluids as a sensitive method to detect change in tumor volume in such patients. Images Figure 1 Figure 2 PMID:8178932

RNA-mediated gene silencing in Candida albicans: inhibition of hyphae formation by use of RNAi technology.

PubMed

Moazeni, Maryam; Khoramizadeh, Mohammad Reza; Kordbacheh, Parivash; Sepehrizadeh, Zargham; Zeraati, Hojat; Noorbakhsh, Fatemeh; Teimoori-Toolabi, Ladan; Rezaie, Sassan

2012-09-01

The introduction of RNA silencing machinery in fungi has led to the promising application of RNAi methodology to knock down essential vital factor or virulence factor genes in the microorganisms. Efg1p is required for development of a true hyphal growth form which is known to be essential for interactions with human host cells and for the yeast's pathogenesis. In this paper, we describe the development of a system for presenting and studying the RNAi function on the EFG1 gene in C. albicans. The 19-nucleotide siRNA was designed on the basis of the cDNA sequence of the EFG1 gene in C. albicans and transfection was performed by use of a modified-PEG/LiAc method. To investigate EFG1 gene silencing in siRNA-treated cells, the yeasts were grown in human serum; to induce germ tubes a solid medium was used with the serum. Quantitative changes in expression of the EFG1 gene were analyzed by measuring the cognate EFG1 mRNA level by use of a quantitative real-time RT-PCR assay. Compared with the positive control, true hyphae formation was significantly reduced by siRNA at concentrations of 1 μM, 500 nM, and 100 nM (P < 0.05). In addition, siRNA at a concentration of 1 μM was revealed to inhibit expression of the EFG1 gene effectively (P < 0.05). On the basis of the potential of post-transcriptional gene silencing to control the expression of specific genes, these techniques may be regarded as promising means of drug discovery, with applications in biomedicine and functional genomics analysis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kapil, Sanjay; Oberst, R. D.; Bieker, Jill Marie

Chemical disinfection and inactivation of viruses is largely understudied, but is very important especially in the case of highly infectious viruses. The purpose of this LDRD was to determine the efficacy of the Sandia National Laboratories developed decontamination formulations against Bovine Coronavirus (BCV) as a surrogate for the coronavirus that causes Severe Acute Respiratory Syndrome (SARS) in humans. The outbreak of SARS in late 2002 resulted from a highly infectious virus that was able to survive and remain infectious for extended periods. For this study, preliminary testing with Escherichia coli MS-2 (MS-2) and Escherichia coli T4 (T4) bacteriophages was conductedmore » to develop virucidal methodology for verifying the inactivation after treatment with the test formulations following AOAC germicidal methodologies. After the determination of various experimental parameters (i.e. exposure, concentration) of the formulations, final testing was conducted on BCV. All experiments were conducted with various organic challenges (horse serum, bovine feces, compost) for results that more accurately represent field use condition. The MS-2 and T4 were slightly more resistant than BCV and required a 2 minute exposure while BCV was completely inactivated after a 1 minute exposure. These results were also consistent for the testing conducted in the presence of the various organic challenges indicating that the test formulations are highly effective for real world application.« less

The cholesterol-lowering effect of coconut flakes in humans with moderately raised serum cholesterol.

PubMed

Trinidad, Trinidad P; Loyola, Anacleta S; Mallillin, Aida C; Valdez, Divinagracia H; Askali, Faridah C; Castillo, Joan C; Resaba, Rosario L; Masa, Dina B

2004-01-01

This study investigated the effect of coconut flakes on serum cholesterol levels of humans with moderately raised serum cholesterol in 21 subjects. The serum total cholesterol of subjects differed and ranged from 259 to 283 mg/dL. The study was conducted in a double-blind randomized crossover design on a 14-week period, consisting of four 2-week experimental periods, with each experimental period separated by a 2-week washout period. The test foods were as follows: corn flakes as the control food, oat bran flakes as the reference food, and corn flakes with 15% and 25% dietary fiber from coconut flakes (made from coconut flour production). Results showed a significant percent reduction in serum total and low-density lipoprotein (LDL) cholesterol (in mg/dL) for all test foods, except for corn flakes, as follows: oat bran flakes, 8.4 +/- 1.4 and 8.8 +/- 6.0, respectively; 15% coconut flakes, 6.9 +/- 1.1 and 11.0 +/- 4.0, respectively; and 25% coconut flakes, 10.8 +/- 1.3 and 9.2 +/- 5.4, respectively. Serum triglycerides were significantly reduced for all test foods: corn flakes, 14.5 +/- 6.3%; oat bran flakes, 22.7 +/- 2.9%; 15% coconut flakes, 19.3 +/- 5.7%; and 25% coconut flakes, 21.8 +/- 6.0%. Only 60% of the subjects were considered for serum triglycerides reduction (serum triglycerides >170 mg/dL). In conclusion, both 15% and 25% coconut flakes reduced serum total and LDL cholesterol and serum triglycerides of humans with moderately raised serum cholesterol levels. Coconut flour is a good source of both soluble and insoluble dietary fiber, and both types of fiber may have significant role in the reduction of the above lipid biomarker. To our knowledge, this is the first study conducted to show a relationship between dietary fiber from a coconut by-product and a lipid biomarker. Results from this study serves as a good basis in the development of coconut flakes/flour as a functional food, justifying the increased production of coconut and coconut by-products.

Acoustic analysis of the composition of human blood serum

NASA Astrophysics Data System (ADS)

Gurbatov, S. N.; Demin, I. Yu.; Klemina, A. V.; Klemin, V. A.

2009-10-01

New acoustic methods of determining total protein, protein fractions, and lipid components of the human blood serum are presented. Acoustic methods are based on high-precision measurements of velocity and temperature dependences and frequency and temperature dependences of ultrasound absorption. Acoustic characteristics of the blood serum were measured using the method of a fixed length interferometer in acoustic cells ˜80 mcl in volume in the temperature range from 15 to 40°C and the 4-9 MHz frequency range with the acoustic analyzer developed by BIOM company. An error in measuring ultrasound velocity in the blood serum was 3 × 10-5; that of absorption, 2 × 10-2. The developed acoustic methods were clinically tested and recommended for application at clinical diagnostic laboratories with RF treatment-and-prophylactics establishments.

Macrophages Exhibit a Large Repertoire of Activation States via Multiple Mechanisms of Macrophage-activating Factors.

PubMed

Sumiya, Y U; Inoue, Takahiro; Ishikawa, Mami; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

2016-07-01

Macrophages are important components of human defense systems and consequently key to antitumor immunity. Human-serum macrophage activation factor (serum MAF) can activate macrophages, making it a promising reagent for anticancer therapy. We established four different macrophage subtypes through introduction of different culture conditions to THP-1- and U937-derived macrophages. We assessed phagocytic activity to understand subtype responses to typical macrophage activation factors (MAFs) and the activation mechanisms of serum MAF. All four macrophage subtypes differed in their response to all MAFs. Moreover, serum MAF had two different activation mechanisms: N-acetylgalactosamine (GalNAc)-dependent and GalNAc-independent. Macrophage activation states and mechanisms are heterogeneous. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum.

PubMed

Ramos, Inês I; Magalhães, Luís M; Barreiros, Luisa; Reis, Salette; Lima, José L F C; Segundo, Marcela A

2018-01-01

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (μ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL -1 in undiluted sample was found. The method allowed IgG determination in the range 100-300 μg mL -1 (corresponding to 5.0-60 mg mL -1 in undiluted samples), with a detection limit of 33 μg mL -1 (1.7 mg mL -1 for samples, dilution factor of 50). RSD values were < 9.4 and < 11.7%, for intra and inter-assay precision, respectively, while recovery values for human serum spiked with IgG at high pathological levels were 97.8-101.4%. Comparison to commercial ELISA kit showed no significant difference for tested samples (n = 8). Moreover, time-to-result decreased from several hours to < 5 min and analysis cost decreased 10 times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab-on-Valve (LOV) platforms using a miniaturised protein G bioaffinity separative approach. IgG are separated from serum matrix components upon quantification with low non-specific binding in less than 5 min.

EFFECTS OF PREDNISOLONE AND TRIAMCINOLONE ON CORTICOSTEROID LEVELS IN PAROTID FLUID, SERUM, AND URINE.

DTIC Science & Technology

A simultaneous determination was made of 17-OHCS in serum, urine, and parotid fluid, by the Porter-Silber reaction, after the oral administration of...prednisolone, triamcinolone, or placebo to 240 normal human subjects. The data clearly demonstrate the homology of serum and parotid fluid 17-OHCS

Thyroid stimulation with recombinant human thyrotropin in healthy cats, cats with non-thyroidal illness and in cats with low serum thyroxin and azotaemia after treatment of hyperthyroidism.

PubMed

van Hoek, Ingrid M; Vandermeulen, Eva; Peremans, Kathelijne; Daminet, Sylvie

2010-02-01

This study investigated the recombinant human thyrotropin (rhTSH) stimulation test in healthy cats (group 1), cats with non-thyroidal illness (group 2) and cats with low serum total T(4) (TT(4)) and azotaemia after (131)I treatment (group 3). Serum TT(4) responses and thyroidal pertechnetate uptake after administration of 25 microg rhTSH IV were assessed. Baseline serum TT(4) was significantly lower in group 3 compared with group 1, but not between other group pairs. Serum TT(4) increased significantly in groups 1 and 2 but not in group 3 after rhTSH administration. Post-rhTSH serum TT(4) concentrations differed significantly between groups 1 and 3 and groups 2 and 3, but not between groups 1 and 2. Thyroid/salivary gland uptake ratio (T/S uptake ratio) differed only significantly between groups 1 and 3. Stimulation with rhTSH is valuable to differentiate euthyroidism from iatrogenic hypothyroidism in cats. Copyright 2009 ESFM and AAFP. Published by Elsevier Ltd. All rights reserved.

[Determination of ferulic acid absorbed into human serum from oral decoction of rhizoma chuanxiong and pharmacokinetic study of ferulic acid].

PubMed

Sun, S; Huang, X; Zhang, L; Ren, P; Chang, Z

2001-10-01

To improve the HPLC method for determining ferulic acid(FA) in the human serum and to research FA clinical pharmacokinetics. Serum concentrations of FA were determined by HPLC using methanol-water-acetic acid (40:59.7:0.3, v/v) as the mobile phase and the column packed with ODS (150 mm x 4.6 mm, 5 microns) as a fixed phase and the flow rate was 1.0 ml/min. FA was detected at 320 nm wave length. The internal standard was coumarin. The serum samples were treated by the water-boiling method. Peak of FA in serum was recognized by photodiode-array detection. FA and internal standard were separated completely under the condition described as above. FA was linear in the range of (40.16-8032 ng/ml) (r = 0.9975). The CV was less than 10%, the average recovery was 100.3%, and the limit concentration in serum was 25.1 ng/ml. PK was in accordance with open bicameral model. This method can be applied to clinical pharmacokinetic study of FA.

Elevated serum level of human alkaline phosphatase in obesity.

PubMed

Khan, Abdul Rehman; Awan, Fazli Rabbi; Najam, Syeda Sadia; Islam, Mehboob; Siddique, Tehmina; Zain, Maryam

2015-11-01

To investigate a correlation between serum alkaline phosphatase level and body mass index in human subjects. The comparative cross-sectional study was carried out at the National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan, from April 2012 to June 2013. Blood serum alkaline phosphatase levels were estimated and the subjects were divided into three sub-groups on the basis of their body mass. normal weight (<25kg/m2), overweight (25-27kg/m2) and obese (>27kg/m2) subjects. The serum samples were used for the estimation of clinically important biochemical parameters, using commercial kits on clinical chemistry analyser. Of the 197 subjects, 97(49%) were obese and 100(51%) were non-obese. The serum alkaline phosphatase level increased in obese (214±6.4 IU/L) compared to the non-obese subjects (184.5±5 IU/L). Furthermore, a significant linear relationship (r=0.3;p-0.0001) was found between serum alkaline phosphatase and body mass index. Other biochemical variables were not correlated to the body mass index. Over activity and higher amounts of alkaline phosphatase were linked to the development of obesity.

Activation of lipoprotein lipase by lipoprotein fractions of human serum.

PubMed

Bier, D M; Havel, R J

1970-11-01

Triglycerides in fat emulsions are hydrolyzed by lipoprotein lipase only when they are "activated" by serum lipoproteins. The contribution of different lipoprotein fractions to hydrolysis of triglycerides in soybean oil emulsion was assessed by determining the quantity of lipoprotein fraction required to give half-maximal hydrolysis. Most of the activator property of whole serum from normolipidemic, postabsorptive subjects was in high density lipoproteins. Low density lipoproteins and serum from which all lipoprotein classes were removed had little or no activity. Also, little activator was present in guinea pig serum or in very low density poor serum from an individual with lecithin:cholesterol acyltransferase deficiency, both of which are deficient in high density lipoproteins. Human very low density lipoproteins are potent activators and are much more active than predicted from their content of high density lipoprotein-protein. Per unit weight of protein, very low density lipoproteins had 13 times the activity of high density lipoproteins. These observations suggest that one or more of the major apoproteins of very low density lipoproteins, present as a minor constituent of high density lipoproteins, may be required for the activation process.

Antigenic Variation of East/Central/South African and Asian Chikungunya Virus Genotypes in Neutralization by Immune Sera

PubMed Central

Chua, Chong-Long; Sam, I-Ching; Merits, Andres; Chan, Yoke-Fun

2016-01-01

Background Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus which causes epidemics of fever, severe joint pain and rash. Between 2005 and 2010, the East/Central/South African (ECSA) genotype was responsible for global explosive outbreaks across India, the Indian Ocean and Southeast Asia. From late 2013, Asian genotype CHIKV has caused outbreaks in the Americas. The characteristics of cross-antibody efficacy and epitopes are poorly understood. Methodology/Principal Findings We characterized human immune sera collected during two independent outbreaks in Malaysia of the Asian genotype in 2006 and the ECSA genotype in 2008–2010. Neutralizing capacity was analyzed against representative clinical isolates as well as viruses rescued from infectious clones of ECSA and Asian CHIKV. Using whole virus antigen and recombinant E1 and E2 envelope glycoproteins, we further investigated antibody binding sites, epitopes, and antibody titers. Both ECSA and Asian sera demonstrated stronger neutralizing capacity against the ECSA genotype, which corresponded to strong epitope-antibody interaction. ECSA serum targeted conformational epitope sites in the E1-E2 glycoprotein, and E1-E211K, E2-I2T, E2-H5N, E2-G118S and E2-S194G are key amino acids that enhance cross-neutralizing efficacy. As for Asian serum, the antibodies targeting E2 glycoprotein correlated with neutralizing efficacy, and I2T, H5N, G118S and S194G altered and improved the neutralization profile. Rabbit polyclonal antibody against the N-terminal linear neutralizing epitope from the ECSA sequence has reduced binding capacity and neutralization efficacy against Asian CHIKV. These findings imply that the choice of vaccine strain may impact cross-protection against different genotypes. Conclusion/Significance Immune serum from humans infected with CHIKV of either ECSA or Asian genotypes showed differences in binding and neutralization characteristics. These findings have implications for the continued outbreaks of co-circulating CHIKV genotypes and effective design of vaccines and diagnostic serological assays. PMID:27571254

Separation of Two Distinct O-Glycoforms of Human IgA1 by Serial Lectin Chromatography Followed by Mass Spectrometry O-Glycan Analysis.

PubMed

Lehoux, S; Ju, T

2017-01-01

Human immunoglobulin A1 (IgA1), which carries four to six mucin-type O-glycans (O-glycans) on its hinge region (HR), is the most abundant O-glycoprotein in plasma or serum. While normal O-glycans from hematopoietic-originated cells are core 1-based complex structures, many reports showed that the IgA1 from patients with IgA nephropathy (IgAN) carries undergalactosylated or truncated O-glycans such as the Tn antigen and its sialylated version the SialylTn (STn) antigen on the HR. Yet, there is still a debate whether Tn/STn on the HR of IgA1 is specific to the IgA1 from patients with IgAN since these antigens have also been seen in serum IgA1 of healthy individuals. An additional question is whether the O-glycans at all sites on the two HRs of one IgA1 molecule are homogeneous (either all normal or all Tn/STn) or heterogeneous (both normal and Tn/STn O-glycans). To address these questions, we conducted a systematic study on the O-glycans of plasma IgA1 from both IgAN patients and healthy controls using serial HPA and PNA lectin chromatography followed by western blotting and further analysis of O-glycans from HPA-bound and PNA-bound IgA1 fractions by mass spectrometry. Unexpectedly, we found that a variable minor fraction of IgA1 from both IgAN patients and healthy controls had Tn/STn antigens, and that the O-glycoprotein IgA1 molecules from most samples had only two distinct O-glycoforms: one major glycoform with homogeneous normal core 1-based O-glycans and one minor glycoform with homogeneous Tn/STn antigens. These results raised a serious question about the role of Tn/STn antigens on IgA1 in pathogenesis of IgAN, and there is a demand for a practical methodology that any laboratory can utilize to analyze the O-glycans of IgA1. Herein, we describe the methodology we developed in more detail. The method could also be applied to the analysis of any other O-glycosylated proteins. © 2017 Elsevier Inc. All rights reserved.

Chemical functionalization of bioceramics to enhance endothelial cells adhesion for tissue engineering.

PubMed

Borcard, Françoise; Staedler, Davide; Comas, Horacio; Juillerat, Franziska Krauss; Sturzenegger, Philip N; Heuberger, Roman; Gonzenbach, Urs T; Juillerat-Jeanneret, Lucienne; Gerber-Lemaire, Sandrine

2012-09-27

To control the selective adhesion of human endothelial cells and human serum proteins to bioceramics of different compositions, a multifunctional ligand containing a cyclic arginine-glycine-aspartate (RGD) peptide, a tetraethylene glycol spacer, and a gallate moiety was designed, synthesized, and characterized. The binding of this ligand to alumina-based, hydroxyapatite-based, and calcium phosphate-based bioceramics was demonstrated. The conjugation of this ligand to the bioceramics induced a decrease in the nonselective and integrin-selective binding of human serum proteins, whereas the binding and adhesion of human endothelial cells was enhanced, dependent on the particular bioceramics.

Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Leng, Jiapeng; Zhu, Dong; Wu, Duojiao; Zhu, Tongyu; Zhao, Ningwei; Guo, Yinlong

2012-11-15

Peptidomics analysis of human serum is challenging due to the low abundance of serum peptides and interference from the complex matrix. This study analyzed the differentially expressed (DE) low molecular weight peptides in human serum integrating a DMPITC-based N-terminal isotope labeling technique with nano-liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (nano-LC/MALDI-MS). The workflow introduced a [d(6)]-4,6-dimethoxypyrimidine-2-isothiocyanate (DMPITC)-labeled mixture of aliquots from test samples as the internal standard. The spiked [d(0)]-DMPITC-labeled samples were separated by nano-LC then spotted on the MALDI target. Both quantitative and qualitative studies for serum peptides were achieved based on the isotope-labeled peaks. The DMPITC labeling technique combined with nano-LC/MALDI-MS not only minimized the errors in peptide quantitation, but also allowed convenient recognition of the labeled peptides due to the 6 Da mass difference. The data showed that the entire research procedure as well as the subsequent data analysis method were effective, reproducible, and sensitive for the analysis of DE serum peptides. This study successfully established a research model for DE serum peptides using DMPITC-based N-terminal isotope labeling and nano-LC/MALDI-MS. Application of the DMPITC-based N-terminal labeling technique is expected to provide a promising tool for the investigation of peptides in vivo, especially for the analysis of DE peptides under different biological conditions. Copyright © 2012 John Wiley & Sons, Ltd.

Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

PubMed Central

Polci, Maria Letizia; Rossi, Stefania; Cordella, Martina; Carlucci, Giuseppe; Marchetti, Paolo; Antonini-Cappellini, Giancarlo; Facchiano, Antonio; D'Arcangelo, Daniela; Facchiano, Francesco

2013-01-01

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05). Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera. PMID:23533572

Differential regulation of membrane-associated mucins in the human ocular surface epithelium.

PubMed

Hori, Yuichi; Spurr-Michaud, Sandra; Russo, Cindy Leigh; Argüeso, Pablo; Gipson, Ilene K

2004-01-01

Membrane-associated mucins present in the apical cells of the ocular surface epithelium (MUC1, -4, and -16) are believed to contribute to the maintenance of a hydrated and wet-surfaced epithelial phenotype. Serum and retinoic acid (RA) have been used to treat drying ocular surface diseases. The goal of this study was to determine whether serum or RA regulates the production of membrane-associated mucins in human conjunctival epithelial cells. A telomerase-immortalized human conjunctival epithelial cell line (HCjE) was used. Cells were cultured in serum-free medium to confluence and then cultured with either 10% calf serum or with 100 nM RA for 0 to 72 hours. Conventional RT-PCR was used to determine the expression of retinoic acid receptors (RARs) and quantitative real-time PCR was used to investigate the mRNA expression of MUC1, -4, and -16. Protein levels were assayed by immunoblot analysis, using the antibodies HMFG-2, 1G8, or OC125, which are specific to MUC1, -4 and -16, respectively. To determine whether RA-associated MUC4 mRNA induction is a direct or indirect effect, HCjE cells were treated with RA and the protein synthesis inhibitor cycloheximide (1.0 microg/mL) for 12 hours. MUC1 and -16, but not -4, mRNAs were detectable in HCjE cells grown in serum-free medium. Real-time PCR revealed that MUC4 mRNA was significantly induced by serum 3 hours after its addition, and that MUC1 and MUC16 mRNA levels were significantly upregulated at 72 hours. Western blot analysis demonstrated that the MUC1, -4, and -16 proteins increased over time after addition of serum. Conventional RT-PCR analysis demonstrated that RAR-alpha and -gamma mRNA were expressed in native human conjunctival tissue as well as in the HCjE cells. Treatment with RA upregulated the expression of both MUC4 and -16 mRNA and protein, but MUC1 was unaffected. Because the protein synthesis inhibitor cycloheximide did not prevent the RA-associated induction of MUC4 mRNA, the action of RA on the MUC4 promoter may be direct. The membrane-associated mucins of the ocular surface epithelia, MUC1, -4, and -16, are differentially regulated by serum and RA in the telomerase-immortalized human conjunctival epithelial cell line. Serum derived from vessels in the conjunctiva may play an important role in mucin regulation in the ocular surface epithelia. These data also support the clinical efficacy of autologous serum and RA application in patients with ocular surface diseases. Furthermore, the data suggest that MUC4 and -16 are particularly important hydrophilic molecules involved in maintenance of a healthy ocular surface.

Activity of the de novo engineered antimicrobial peptide WLBU2 against Pseudomonas aeruginosa in human serum and whole blood: implications for systemic applications.

PubMed

Deslouches, Berthony; Islam, Kazi; Craigo, Jodi K; Paranjape, Shruti M; Montelaro, Ronald C; Mietzner, Timothy A

2005-08-01

Cationic amphipathic peptides have been extensively investigated as a potential source of new antimicrobials that can complement current antibiotic regimens in the face of emerging drug-resistant bacteria. However, the suppression of antimicrobial activity under certain biologically relevant conditions (e.g., serum and physiological salt concentrations) has hampered efforts to develop safe and effective antimicrobial peptides for clinical use. We have analyzed the activity and selectivity of the human peptide LL37 and the de novo engineered antimicrobial peptide WLBU2 in several biologically relevant conditions. The host-derived synthetic peptide LL37 displayed high activity against Pseudomonas aeruginosa but demonstrated staphylococcus-specific sensitivity to NaCl concentrations varying from 50 to 300 mM. Moreover, LL37 potency was variably suppressed in the presence of 1 to 6 mM Mg(2+) and Ca(2+) ions. In contrast, WLBU2 maintained its activity in NaCl and physiologic serum concentrations of Mg(2+) and Ca(2+). WLBU2 is able to kill P. aeruginosa (10(6) CFU/ml) in human serum, with a minimum bactericidal concentration of <9 microM. Conversely, LL37 is inactive in the presence of human serum. Bacterial killing kinetic assays in serum revealed that WLBU2 achieved complete bacterial killing in 20 min. Consistent with these results was the ability of WLBU2 (15 to 20 microM) to eradicate bacteria from ex vivo samples of whole blood. The selectivity of WLBU2 was further demonstrated by its ability to specifically eliminate P. aeruginosa in coculture with human monocytes or skin fibroblasts without detectable adverse effects to the host cells. Finally, WLBU2 displayed potent efficacy against P. aeruginosa in an intraperitoneal infection model using female Swiss Webster mice. These results establish a potential application of WLBU2 in the treatment of bacterial sepsis.

Activity of the De Novo Engineered Antimicrobial Peptide WLBU2 against Pseudomonas aeruginosa in Human Serum and Whole Blood: Implications for Systemic Applications

PubMed Central

Deslouches, Berthony; Islam, Kazi; Craigo, Jodi K.; Paranjape, Shruti M.; Montelaro, Ronald C.; Mietzner, Timothy A.

2005-01-01

Cationic amphipathic peptides have been extensively investigated as a potential source of new antimicrobials that can complement current antibiotic regimens in the face of emerging drug-resistant bacteria. However, the suppression of antimicrobial activity under certain biologically relevant conditions (e.g., serum and physiological salt concentrations) has hampered efforts to develop safe and effective antimicrobial peptides for clinical use. We have analyzed the activity and selectivity of the human peptide LL37 and the de novo engineered antimicrobial peptide WLBU2 in several biologically relevant conditions. The host-derived synthetic peptide LL37 displayed high activity against Pseudomonas aeruginosa but demonstrated staphylococcus-specific sensitivity to NaCl concentrations varying from 50 to 300 mM. Moreover, LL37 potency was variably suppressed in the presence of 1 to 6 mM Mg2+ and Ca2+ ions. In contrast, WLBU2 maintained its activity in NaCl and physiologic serum concentrations of Mg2+ and Ca2+. WLBU2 is able to kill P. aeruginosa (106 CFU/ml) in human serum, with a minimum bactericidal concentration of <9 μM. Conversely, LL37 is inactive in the presence of human serum. Bacterial killing kinetic assays in serum revealed that WLBU2 achieved complete bacterial killing in 20 min. Consistent with these results was the ability of WLBU2 (15 to 20 μM) to eradicate bacteria from ex vivo samples of whole blood. The selectivity of WLBU2 was further demonstrated by its ability to specifically eliminate P. aeruginosa in coculture with human monocytes or skin fibroblasts without detectable adverse effects to the host cells. Finally, WLBU2 displayed potent efficacy against P. aeruginosa in an intraperitoneal infection model using female Swiss Webster mice. These results establish a potential application of WLBU2 in the treatment of bacterial sepsis. PMID:16048927

The Lectin Pathway in Thrombotic Conditions-A Systematic Review.

PubMed

Larsen, Julie Brogaard; Hvas, Christine Lodberg; Hvas, Anne-Mette

2018-06-04

The lectin pathway of the complement system can activate the coagulation system in vitro, but the role of the lectin pathway in haemostatic activation and thrombosis in vivo is not clear. We performed a systematic review of the existing literature on associations between the lectin pathway and arterial and venous thrombosis, in accordance with the Assessing the Methodological Quality of Systematic Reviews guidelines. PubMed and Embase were searched from January 1990 to March 2017. We included original studies on human study populations investigating associations between the lectin pathway (protein serum levels, genotype or gene expression) and thrombotic conditions or laboratory coagulation markers. Exclusion criteria were case studies including fewer than five cases, conference abstracts or any other language than English. In total, 43 studies were included which investigated associations between the lectin pathway and cardiovascular thrombotic events (CVEs) ( n  = 22), ischaemic stroke ( n  = 9), CVE and stroke ( n  = 1) and other conditions (systemic lupus erythematosus [ n  = 6], sepsis-related coagulopathy [ n  = 3], pulmonary embolism [ n  = 1], asparaginase treatment [ n  = 1]). Studies on the lectin pathway and CVE risk reported discrepant results, as both high and low mannose-binding lectin (MBL) serum levels were found to correlate with increased CVE risk. In ischaemic stroke patients, occurrence of stroke as well as increased stroke severity and poor outcome were consistently associated with high serum MBL. For other thromboembolic conditions, only few studies were identified. In conclusion, lectin pathway activation may negatively influence outcome after ischaemic stroke and possibly contribute to CVE risk. Further research is warranted to elucidate the role of the lectin pathway in other thrombotic conditions. Schattauer GmbH Stuttgart.

Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS

PubMed Central

Lancrajan, Ioana; Schneider-Stock, Regine; Naschberger, Elisabeth; Schellerer, Vera S; Stürzl, Michael; Enz, Ralf

2015-01-01

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using 15N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts. PMID:25823874

Increased EPA levels in serum phospholipids of humans after four weeks daily ingestion of one portion chicken fed linseed and rapeseed oil

PubMed Central

2012-01-01

Since the amounts of arachidonic acid (AA) and EPA in food may have implications for human health, we investigated whether a small change in chicken feed influenced the blood lipid concentration in humans ingesting the chicken. Forty-six young healthy volunteers (age 20–29) were randomly allocated into two groups in a double-blind dietary intervention trial, involving ingestion of about 160 g chicken meat per day for 4 weeks. The ingested meat was either from chickens given a feed concentrate resembling the commercial chicken feed, containing 4% soybean oil (SO), or the meat was from chickens given a feed where the soybean oil had been replaced by 2% rapeseed oil plus 2% linseed oil (RLO). Serum total cholesterol, LDL and HDL cholesterol, triacylglycerols, serum phospholipid fatty acid concentration, blood pressure, body weight and C-reactive protein were determined at baseline and post-intervention. In subjects consuming chicken meat from the RLO group there was a significantly (p < 0.001) increased concentration of EPA in serum phospholipids, and a reduced ratio between AA and EPA. The participants that had a low% of EPA + DHA in serum phospholipids (less than 4.6%), all increased their% of EPA + DHA after the four week intervention period when consuming the RLO chicken. No significant response differences in cholesterol, triacylglycerol, C-reactive protein, body weight or blood pressure were observed between the groups. This trial demonstrates that a simple change in chicken feed can have beneficial effects on amount of EPA and the AA/EPA ratio in human serum phospholipids. PMID:22913248

Development of a physiologically based pharmacokinetic model for assessment of human exposure to bisphenol A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xiaoxia, E-mail: xiaoxia.yang@fda.hhs.gov; Doerge, Daniel R.; Teeguarden, Justin G.

A previously developed physiologically based pharmacokinetic (PBPK) model for bisphenol A (BPA) in adult rhesus monkeys was modified to characterize the pharmacokinetics of BPA and its phase II conjugates in adult humans following oral ingestion. Coupled with in vitro studies on BPA metabolism in the liver and the small intestine, the PBPK model was parameterized using oral pharmacokinetic data with deuterated-BPA (d{sub 6}-BPA) delivered in cookies to adult humans after overnight fasting. The availability of the serum concentration time course of unconjugated d{sub 6}-BPA offered direct empirical evidence for the calibration of BPA model parameters. The recalibrated PBPK adult humanmore » model for BPA was then evaluated against published human pharmacokinetic studies with BPA. A hypothesis of decreased oral uptake was needed to account for the reduced peak levels observed in adult humans, where d{sub 6}-BPA was delivered in soup and food was provided prior to BPA ingestion, suggesting the potential impact of dosing vehicles and/or fasting on BPA disposition. With the incorporation of Monte Carlo analysis, the recalibrated adult human model was used to address the inter-individual variability in the internal dose metrics of BPA for the U.S. general population. Model-predicted peak BPA serum levels were in the range of pM, with 95% of human variability falling within an order of magnitude. This recalibrated PBPK model for BPA in adult humans provides a scientific basis for assessing human exposure to BPA that can serve to minimize uncertainties incurred during extrapolations across doses and species. - Highlights: • A PBPK model predicts the kinetics of bisphenol A (BPA) in adult humans. • Serum concentrations of aglycone BPA are available for model calibration. • Model predicted peak BPA serum levels for adult humans were in the range of pM. • Model predicted 95% of human variability fell within an order of magnitude.« less

Treatment of obesity hypoventilation syndrome and serum leptin.

PubMed

Yee, Brendon J; Cheung, Jane; Phipps, Paul; Banerjee, Dev; Piper, Amanda J; Grunstein, Ronald R

2006-01-01

Leptin is a protein produced by adipose tissue that circulates to the brain and interacts with receptors in the hypothalamus to inhibit eating. In obese humans, serum leptin is up to four times higher than in lean subjects, indicating that human obesity is associated with a central resistance to the weight-lowering effects of leptin. Although the leptin-deficient mouse (ob/ob) develops obesity hypoventilation syndrome (OHS), in humans with OHS, serum leptin is a better predictor of awake hypercapnia in obesity than the body mass index (BMI). This suggests that central leptin resistance may promote the development of OHS in humans. We speculated that the reversal of OHS by regular non-invasive ventilation (NIV) therapy decreases leptin levels. The aim of this study was to investigate whether ventilatory treatment of OHS would alter circulating leptin concentrations. We measured fasting serum leptin levels, BMI, spirometry and arterial blood gases in 14 obese hypercapnic subjects undergoing a diagnostic sleep study. The average age of the subjects was (mean +/- SE) 62 +/- 13 years, BMI 40.9 +/- 2.2 kg/m(2), PaCO(2) 6.7 +/- 0.2 kPa, PaO(2 )8.9 +/- 0.4 kPa and total respiratory disturbance index 44 +/- 35 events/hour. Subjects were clinically reviewed after a median of 2.3 years (range 1.6-3) with repeat investigations. Nine patients were regular NIV users and 5 were non-users. NIV users had a significant reduction in serum leptin levels (p = 0.001), without a change in BMI. In these patients, there was a trend towards an improved daytime hypercapnia and hypoxemia, while in the 5 non-users, no changes in serum leptin, BMI or arterial blood gases occurred. Regular NIV use reduces serum leptin in OHS. Leptin may be a modulator of respiratory drive in patients with OHS.

Stability of cytotoxic luteinizing hormone-releasing hormone conjugate (AN-152) containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro: Implications for the design of preclinical studies

PubMed Central

Nagy, Attila; Plonowski, Artur; Schally, Andrew V.

2000-01-01

Recently, we developed a series of cytotoxic peptide conjugates containing 14-O-glutaryl esters of doxorubicin (DOX) or 2-pyrrolino-DOX (AN-201). Serum carboxylesterase enzymes (CE) can partially hydrolyze these conjugates in the circulation, releasing the cytotoxic radical, before the targeting is complete. CE activity in serum of nude mice is about 10 times higher than in human serum. Thus, we found that the t1/2 of AN-152, an analog of luteinizing hormone-releasing hormone (LH-RH) containing DOX, at 0.3 mg/ml is 19.49 ± 0.74 min in mouse serum and 126.06 ± 3.03 min in human serum in vitro. The addition of a CE inhibitor, diisopropyl fluorophosphate (DFP), to mouse serum in vitro significantly (P < 0.01) prolongs the t1/2 of AN-152 to 69.63 ± 4.44 min. When DFP is used in vivo, 400 nmol/kg cytotoxic somatostatin analog AN-238 containing AN-201 is well tolerated by mice, whereas all animals die after the same dose without DFP. In contrast, DFP has no effect on the tolerance of AN-201. A better tolerance to AN-238 after DFP treatment is due to the selective uptake of AN-238 by somatostatin receptor-positive tissues. Our results demonstrate that the suppression of the CE activity in nude mice greatly decreases the toxicity of cytotoxic hybrids containing 2-pyrrolino-DOX 14-O-hemiglutarate and brings this animal model closer to the conditions that exist in humans. The use of DFP together with these peptide conjugates in nude mice permits a better understanding of their mechanism of action and improves the clinical predictability of the oncological and toxicological results. PMID:10639165

Serum thioredoxin reductase is highly increased in mice with hepatocellular carcinoma and its activity is restrained by several mechanisms.

PubMed

Zhang, Le; Cheng, Qing; Zhang, Longjie; Wang, Yijun; Merrill, Gary F; Ilani, Tal; Fass, Deborah; Arnér, Elias S J; Zhang, Jinsong

2016-10-01

Increased thioredoxin reductase (TrxR) levels in serum were recently identified as possible prognostic markers for human prostate cancer or hepatocellular carcinoma. We had earlier shown that serum levels of TrxR protein are very low in healthy mice, but can in close correlation to alanine aminotransferase (ALT) increase more than 200-fold upon chemically induced liver damage. We also found that enzymatic TrxR activity in serum is counteracted by a yet unidentified oxidase activity in serum. In the present study we found that mice carrying H22 hepatocellular carcinoma tumors present highly increased levels of TrxR in serum, similarly to that reported in human patients. In this case ALT levels did not parallel those of TrxR. We also discovered here that the TrxR-antagonistic oxidase activity in serum is due to the presence of quiescin Q6 sulfhydryl oxidase 1 (QSOX1). We furthermore found that the chemotherapeutic agents cisplatin or auranofin, when given systemically to H22 tumor bearing mice, can further inhibit TrxR activities in serum. The TrxR serum activity was also inhibited by endogenous electrophilic inhibitors, found to increase in tumor-bearing mice and to include protoporphyrin IX (PpIX) and 4-hydroxynonenal (HNE). Thus, hepatocellular carcinoma triggers high levels of serum TrxR that are not paralleled by ALT, and TrxR enzyme activity in serum is counteracted by several different mechanisms. The physiological role of TrxR in serum, if any, as well as its potential value as a prognostic marker for tumor progression, needs to be studied further. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibitor(s) of the classical complement pathway in mouse serum limit the utility of mice as experimental models of neuromyelitis optica.

PubMed

Ratelade, Julien; Verkman, A S

2014-11-01

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which anti-aquaporin-4 (AQP4) autoantibodies (AQP4-IgG) cause damage to astrocytes by complement-dependent cytotoxicity (CDC). Various approaches have been attempted to produce NMO lesions in rodents, some involving genetically modified mice with altered immune cell function. Here, we found that mouse serum strongly inhibits complement from multiple species, preventing AQP4-IgG-dependent CDC. Effects of mouse serum on complement activation were tested in CDC assays in which AQP4-expressing cells were incubated with AQP4-IgG and complement from different species. Biochemical assays and mass spectrometry were used to characterize complement inhibitor(s) in mouse serum. Sera from different strains of mice produced almost no AQP4-IgG-dependent CDC compared with human, rat and guinea pig sera. Remarkably, addition of mouse serum prevented AQP4-IgG-dependent CDC caused by human, rat or guinea pig serum, with 50% inhibition at <5% mouse serum. Hemolysis assays indicated that the inhibitor(s) in mouse serum target the classical and not the alternative complement pathway. We found that the complement inhibitor(s) in mouse serum were contained in a serum fraction purified with protein-A resin; however, the inhibitor was not IgG as determined using serum from IgG-deficient mice. Mass spectrometry on the protein A-purified fraction produced several inhibitor candidates. The low intrinsic complement activity of mouse serum and the presence of complement inhibitor(s) limit the utility of mouse models to study disorders, such as NMO, involving the classical complement pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of proteomic profiles of serum, plasma, and modified media supplements used for cell culture and expansion

PubMed Central

Ayache, Saleh; Panelli, Monica C; Byrne, Karen M; Slezak, Stefanie; Leitman, Susan F; Marincola, Francesco M; Stroncek, David F

2006-01-01

Background The culture and expansion of human cells for clinical use requires the presence of human serum or plasma in culture media. Although these supplements have been extensively characterized in their chemical composition, only recently it has been possible to provide by high throughput protein analysis, a comprehensive profile of the soluble factors contributing to cell survival. This study analyzed and compared the presence of 100 proteins including chemokines, cytokines and soluble factors in six different types of media supplements: serum, plasma, recalcified plasma, heat inactivated serum, heat inactivated plasma and heat inactivated recalcified plasma. Methods Serum, plasma, recalcified plasma, and heat inactivated supplements were prepared from ten healthy subjects. The levels of 100 soluble factors were measured in each sample using a multiplexed ELISA assay and compared by Eisen hierarchical clustering analysis. Results A comparison of serum and plasma levels of soluble factors found that 2 were greater in plasma but 18 factors were greater in serum including 11 chemokines. The levels of only four factors differed between recalcified plasma and plasma. Heat inactivation had the greatest effect on soluble factors. Supervised Eisen hierarchical clustering indicated that the differences between heat inactivated supplements and those that were not were greater than the differences within these two groups. The levels of 36 factors differed between heat inactivated plasma and plasma. Thirty one of these factors had a lower concentration in heat inactivated plasma including 12 chemokines, 4 growth factors, 4 matrix metalloproteases, and 3 adhesion molecules. Heat inactivated decalcified plasma is often used in place of heat inactivated serum and the levels of 19 soluble factors differed between these two supplements. Conclusion Our report provides a comprehensive protein profile of serum, plasma recalcified plasma, and heat inactivated supplements. This profile represents a qualitative and quantitative database that can aid in the selection of the appropriate blood derived supplement for human cell cultures with special requirements. PMID:17020621

Micro-RNA Profiling in Human Serum Reveals Compartment-Specific Roles of miR-571 and miR-652 in Liver Cirrhosis

PubMed Central

Roderburg, Christoph; Mollnow, Tobias; Bongaerts, Brenda; Elfimova, Natalia; Vargas Cardenas, David; Berger, Katharina; Zimmermann, Henning; Koch, Alexander; Vucur, Mihael; Luedde, Mark; Hellerbrand, Claus; Odenthal, Margarete; Trautwein, Christian; Tacke, Frank; Luedde, Tom

2012-01-01

Background and Aims Micro-RNAs (miRNAs) have recently emerged as crucial modulators of molecular processes involved in chronic liver diseases. The few miRNAs with previously proposed roles in liver cirrhosis were identified in screening approaches on liver parenchyma, mostly in rodent models. Therefore, in the present study we performed a systematic screening approach in order to identify miRNAs with altered levels in the serum of patients with chronic liver disease and liver cirrhosis. Methods We performed a systematic, array-based miRNA expression analysis on serum samples from patients with liver cirrhosis. In functional experiments we evaluated the relationship between alterations of miRNA serum levels and their role in distinct cellular compartments involved in hepatic cirrhosis. Results The array analysis and the subsequent confirmation by qPCR in a larger patient cohort identified significant alterations in serum levels of miR-513-3p, miR-571 and miR-652, three previously uncharacterized miRNAs, in patients with alcoholic or hepatitis C induced liver cirrhosis. Of these, miR-571 serum levels closely correlated with disease stages, thus revealing potential as a novel biomarker for hepatic cirrhosis. Further analysis revealed that up-regulation of miR-571 in serum reflected a concordant regulation in cirrhotic liver tissue. In isolated primary human liver cells, miR-571 was up-regulated in human hepatocytes and hepatic stellate cells in response to the pro-fibrogenic cytokine TGF-β. In contrast, alterations in serum levels of miR-652 were stage-independent, reflecting a concordant down-regulation of this miRNA in circulating monocytes of patients with liver cirrhosis, which was inducible by proinflammatory stimuli like bacterial lipopolysaccharide. Conclusion Alterations of miR571 and miR-652 serum levels in patients with chronic liver disease reflect their putative roles in the mediation of fibrogenic and inflammatory processes in distinct cellular compartments involved in the pathogenesis of liver cirrhosis. PMID:22412969

Potent Innate Immune Response to Pathogenic Leptospira in Human Whole Blood

PubMed Central

Hartskeerl, Rudy A.; van Gorp, Eric C. M.; Schuller, Simone; Monahan, Avril M.; Nally, Jarlath E.; van der Poll, Tom; van 't Veer, Cornelis

2011-01-01

Background Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires. Methodology/Principal Findings We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires. Conclusions/Significance Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response. PMID:21483834

Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.

PubMed

He, Xiaoli; H'ng, Shiau-Chen; Leong, David T; Hutmacher, Dietmar W; Melendez, Alirio J

2010-08-01

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

Supplements in human islet culture: human serum albumin is inferior to fetal bovine serum.

PubMed

Avgoustiniatos, Efstathios S; Scott, William E; Suszynski, Thomas M; Schuurman, Henk-Jan; Nelson, Rebecca A; Rozak, Phillip R; Mueller, Kate R; Balamurugan, A N; Ansite, Jeffrey D; Fraga, Daniel W; Friberg, Andrew S; Wildey, Gina M; Tanaka, Tomohiro; Lyons, Connor A; Sutherland, David E R; Hering, Bernhard J; Papas, Klearchos K

2012-01-01

Culture of human islets before clinical transplantation or distribution for research purposes is standard practice. At the time the Edmonton protocol was introduced, clinical islet manufacturing did not include culture, and human serum albumin (HSA), instead of fetal bovine serum (FBS), was used during other steps of the process to avoid the introduction of xenogeneic material. When culture was subsequently introduced, HSA was also used for medium supplementation instead of FBS, which was typically used for research islet culture. The use of HSA as culture supplement was not evaluated before this implementation. We performed a retrospective analysis of 103 high-purity islet preparations (76 research preparations, all with FBS culture supplementation, and 27 clinical preparations, all with HSA supplementation) for oxygen consumption rate per DNA content (OCR/DNA; a measure of viability) and diabetes reversal rate in diabetic nude mice (a measure of potency). After 2-day culture, research preparations exhibited an average OCR/DNA 51% higher (p < 0.001) and an average diabetes reversal rate 54% higher (p < 0.05) than clinical preparations, despite 87% of the research islet preparations having been derived from research-grade pancreata that are considered of lower quality. In a prospective paired study on islets from eight research preparations, OCR/DNA was, on average, 27% higher with FBS supplementation than that with HSA supplementation (p < 0.05). We conclude that the quality of clinical islet preparations can be improved when culture is performed in media supplemented with serum instead of albumin.

Indirect electrochemical detection for total bile acids in human serum.

PubMed

Zhang, Xiaoqing; Zhu, Mingsong; Xu, Biao; Cui, Yue; Tian, Gang; Shi, Zhenghu; Ding, Min

2016-11-15

Bile acids level in serum is a useful index for screening and diagnosis of hepatobiliary diseases. As bile acids concentration is closely related to the degree of hepatobiliary diseases, detecting it is a vital factor to understand the stage of the diseases. The prevalent determination for bile acids is the enzymatic cycling method which has low sensitivity while reagent-consuming. It is desirable to develop a new method with lower cost and higher sensitivity. An indirect electrochemical detection (IED) for bile acids in human serum was established using the screen printed carbon electrode (SPCE). Since bile acids do not show electrochemical signals, they were converted to 3-ketosteroids by 3-α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of nicotinamide adenine dinucleotide (NAD(+)), which was reduced to NADH. NADH could then be oxidized on the surface of SPCE, generating a signal that was used to calculate the total bile acids (TBA) concentration. A good linear calibration for TBA was obtained at the concentration range from 5.00μM to 400μM in human serum. Both the precisions and recoveries were sufficient to be used in a clinical setting. The TBA concentrations in 35 human serum samples by our IED method didn't show significant difference with the result by enzymatic cycling method, using the paired t-test. Moreover, our IED method is reagent-saving, sensitive and cost-effective. Copyright © 2016. Published by Elsevier B.V.

The Application of SILAC Mouse in Human Body Fluid Proteomics Analysis Reveals Protein Patterns Associated with IgA Nephropathy.

PubMed

Zhao, Shilin; Li, Rongxia; Cai, Xiaofan; Chen, Wanjia; Li, Qingrun; Xing, Tao; Zhu, Wenjie; Chen, Y Eugene; Zeng, Rong; Deng, Yueyi

2013-01-01

Body fluid proteome is the most informative proteome from a medical viewpoint. But the lack of accurate quantitation method for complicated body fluid limited its application in disease research and biomarker discovery. To address this problem, we introduced a novel strategy, in which SILAC-labeled mouse serum was used as internal standard for human serum and urine proteome analysis. The SILAC-labeled mouse serum was mixed with human serum and urine, and multidimensional separation coupled with tandem mass spectrometry (IEF-LC-MS/MS) analysis was performed. The shared peptides between two species were quantified by their SILAC pairs, and the human-only peptides were quantified by mouse peptides with coelution. The comparison for the results from two replicate experiments indicated the high repeatability of our strategy. Then the urine from Immunoglobulin A nephropathy patients treated and untreated was compared by this quantitation strategy. Fifty-three peptides were found to be significantly changed between two groups, including both known diagnostic markers for IgAN and novel candidates, such as Complement C3, Albumin, VDBP, ApoA,1 and IGFBP7. In conclusion, we have developed a practical and accurate quantitation strategy for comparison of complicated human body fluid proteome. The results from such strategy could provide potential disease-related biomarkers for evaluation of treatment.

Apolipoprotein L1 Variant Associated with Increased Susceptibility to Trypanosome Infection

PubMed Central

Cuypers, Bart; Lecordier, Laurence; Meehan, Conor J.; Van den Broeck, Frederik; Imamura, Hideo; Büscher, Philippe; Dujardin, Jean-Claude; Laukens, Kris; Schnaufer, Achim; Dewar, Caroline; Lewis, Michael; Balmer, Oliver; Azurago, Thomas; Kyei-Faried, Sardick; Ohene, Sally-Ann; Duah, Boateng; Homiah, Prince; Mensah, Ebenezer Kofi; Anleah, Francis; Franco, Jose Ramon; Pays, Etienne

2016-01-01

ABSTRACT African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity. PMID:27073096

The Prevalence of Brucellosis in Cattle, Goats and Humans in Rural Uganda: A Comparative Study.

PubMed

Miller, R; Nakavuma, J L; Ssajjakambwe, P; Vudriko, P; Musisi, N; Kaneene, J B

2016-12-01

A cross-sectional study was conducted to determine the presence of brucellosis in cattle, goats and humans in farms from south-western Uganda and identify risk factors associated with brucellosis in these three host groups. Data and serum samples were collected from 768 cattle, 315 goats and 236 humans, with 635 samples of bovine milk, from 70 farms in two different study areas in south-western Uganda. Sera from livestock were tested with the Rose Bengal Plate test, using B. abortus and B. melitensis antigens, and human sera were tested with a commercial IgG/IgM lateral flow assay. Milk samples were tested using the OIE-approved milk ring test. Screening tests for brucellosis were positive in 14% of cattle serum, 29% of bovine milk, 17% of goat serum and 11% of human serum samples. There were significant differences in the test prevalence of brucellosis by study site, with levels higher in the study area near Lake Mburo National Park than in the study area near Queen Elizabeth National Park. Multivariable regression models identified risk factors associated with increasing test positivity at the individual and farm levels for cattle, goats and humans. Positive associations were seen between increasing seropositivity of brucellosis in goats, cattle and humans. Results of multivariable analyses suggest that improvements in farm biosecurity and hygiene may reduce the risk of brucellosis on the farm and suggest a role for ticks in bovine brucellosis. Although cattle are the focus of brucellosis control in Uganda, the significant associations between seropositivity in humans and seropositivity in goats suggest that brucellosis in goats may be an important contributor to the epidemiology of the disease on the farm. © 2015 Blackwell Verlag GmbH.

Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors

NASA Astrophysics Data System (ADS)

Huy, Tran Quang; Hanh, Nguyen Thi Hong; Van Chung, Pham; Anh, Dang Duc; Nga, Phan Thi; Tuan, Mai Anh

2011-06-01

In this paper, we describes different methods to immobilize Japanese encephalitis virus (JEV) antibodies in human serum onto the interdigitated surface of a microelectrode sensor for optimizing electrochemical detection: (1) direct covalent binding to the silanized surface, (2) binding to the silanized surface via a cross-linker of glutaraldehyde (GA), (3) binding to glutaraldehyde/silanized surface via goat anti-human IgG polyclonal antibody and (4) binding to glutaraldehyde/silanized surface via protein A (PrA). Field emission scanning electron microscopy, Fourier transform infrared spectrometry, and fluorescence microscopy are used to verify the characteristics of antibodies on the interdigitated surface after the serum antibodies immobilization. The analyzed results indicate that the use of protein A is an effective choice for immobilization and orientation of antibodies in serum for electrochemical biosensors. This study provides an advantageous immobilization method of serum containing antiviral antibodies to develop electrochemical biosensors for preliminary screening of viruses in clinical samples from outbreaks.

[An attempt for standardization of serum CA19-9 levels, in order to dissolve the gap between three different methods].

PubMed

Hayashi, Kuniki; Hoshino, Tadashi; Yanai, Mitsuru; Tsuchiya, Tatsuyuki; Kumasaka, Kazunari; Kawano, Kinya

2004-06-01

It is well known that serious method-related differences exist in results of serum CA19-9, and the necessity of standardization has been pointed out. In this study, differences of serum tumor marker CA19-9 levels obtained by various immunoassay kits (CLEIA, FEIA, LPIA and RIA) were evaluated in sixty-seven clinical samples and five calibrators and the possibility to improve the inter-methodological differences were observed not only for clinical samples but also for calibrators. We supposed an assumed standard material using by a calibrator. We calculated the serum levels of CA19-9 when using the assumed standard material for three different measurement methods. We approximate the CA19-9 values using by this method. It is suggested that the obtained CA19-9 values could be approximated by recalculation with the assumed standard material would be able to correct between-method and between-laboratory discrepancies in particular systematic errors.

The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

PubMed Central

Villarino Romero, Rodrigo; Bibova, Ilona; Cerny, Ondrej; Vecerek, Branislav; Wald, Tomas; Benada, Oldrich; Zavadilova, Jana; Sebo, Peter

2013-01-01

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice. PMID:23690400

Maternal biochemical serum screening for Down syndrome in pregnancy with human immunodeficiency virus infection.

PubMed

Le Meaux, Jean-Patrick; Tsatsaris, Vassilis; Schmitz, Thomas; Fulla, Yvonne; Launay, Odile; Goffinet, François; Azria, Elie

2008-08-01

To estimate the influence of human immunodeficiency virus (HIV) infection and antiretroviral therapy on maternal serum markers levels and the false-positive rate with biochemical maternal serum screening for Down syndrome. We performed a 1:1 matched case-control study comparing 132 HIV-infected women with single pregnancy to controls selected among non-HIV-infected women matched on geographical origin and fetal sex. Of HIV-infected women, 47.7% were receiving antiretroviral therapy. Groups did not differ in multiples of the median (MoM) levels of total human chorionic gonadotrophin. The MoM alpha fetoprotein level did not differ between total HIV-infected women and control women but was significantly lower for untreated HIV-positive women compared with control women (0.91 compared with 1.03 MoM, P<.01) and compared with treated HIV-positive women (0.91 compared with 1.18 MoM, P<.01). The false-positive rate of biochemical screening did not differ between groups. Untreated HIV infection is associated with lower maternal serum alpha fetoprotein levels. Nevertheless, the false-positive rate of double-marker second-trimester Down syndrome serum screening did not appear to be affected in our sample of HIV-infected women, whether women were receiving antiretroviral therapy at the time of the test or not.

[Silymarin Protects Umbilical Cord-Derived Mesenchymal Stem Cells against Apoptosis Induced by Serum-Deprivation].

PubMed

Wei, Xiao-Juan; Zhang, Hong-Chao; Guo, Zi-Kuan; Zheng, Hai-Bin; Yang, Lei-Lei; Liu, Chao-Zhong

2015-10-01

To investigate the protection of silymarin against the human mesenchymal stem cell (MSC) apoptosis induced by serum deprivation and its underlying mechanism. Human umbilical cord MSCs were cultured in the absence of serum, and the silymain of different concentration (1-10 µg/ml) was added into the medium. MTT test was performed to observe the cell proliferation status. After being cultured for 72 hours, the cells were collected, and flow cytometry with Annexin-V-PI double-staining was used to detect the apoptotic cells from the control and silymarin-treated groups. Furthermore, the intracellular contents of BAX and BCL-2 were detected by Western blot for exploring the potential mechanism. The silymarin promoted the proliferation of human UC-MSCs in a dose-dependent manner, reaching its maximal at a dose of 5 µg/ml. Moreover, silymarin could inhibit the serum deprivation-induced apoptosis of MSCs and, the inhibitory rate reached up to 30% when it was added at a concentration of 5 µg/ml. The content of intracellular BAX was obviously elevated after serum-deprivation treatment, and this increase could be blunted by the addition of silymarin. Meanwhile, the content of BCL-2 was not obviously changed. The silymarin can stimulate MSC growth and inhibit the apoptosis of MSCs probably by the mitochondria pathway.

The Effects of Fetal Gender on Serum Human Chorionic Gonadotropin and Testosterone in Normotensive and Preeclamptic Pregnancies

PubMed Central

Lorzadeh, Nahid; Kazemirad, Sirous

2012-01-01

Introduction. The aim of the present study was to evaluate the effects of fetal sex on serum human chorionic gonadotropin (hCG) and testosterone in normotensive and preeclamptic pregnancies. Materials and Methods. This is a cross-sectional study and 139 women with singleton pregnancies in the third trimester were studied. Seventy-one pregnancies were uncomplicated; among those were 35 male and 36 female fetuses. Sixty-eight pregnancies were complicated by preeclampsia; among those were 35 male and 33 female fetuses. Human chorionic gonadotropin and total testosterone were measured in maternal peripheral blood. Data analyzed by SPSS software. Results. In male-bearing pregnancies, maternal hCG and testosterone serum levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001 and P < 0.001, resp.) in female-bearing pregnancies testosterone levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001). Total testosterone levels were significantly higher in pregnancies with either gender and significantly higher in mlae-bearing than in female-bearing pregnancies. Conclusion. According to our results, there is a correlation between maternal serum hCG and testosterone levels and preeclampsia. Therefore these tests can be used as routine during 30–38 weeks of gestation. High maternal serum concentrations of these markers can predict preeclampsia. PMID:22518314

Identification and monitoring of metabolite markers of dry bean consumption in parallel human and mouse studies.

PubMed

Perera, Thushanthi; Young, Matthew R; Zhang, Zhiying; Murphy, Gwen; Colburn, Nancy H; Lanza, Elaine; Hartman, Terryl J; Cross, Amanda J; Bobe, Gerd

2015-04-01

Aim of the study was to identify and monitor metabolite markers of dry bean consumption in parallel human and mouse studies that each had shown chemopreventive effects of dry bean consumption on colorectal neoplasia risk. Using LC/mass spectroscopy ± ESI and GC/mass spectroscopy, serum metabolites of dry beans were measured in 46 men before and after a 4-week dry bean enriched diet (250 g/day) and 12 mice that received a standardized diet containing either 0 or 10% navy bean ethanol extract for 6 weeks; we also investigated fecal metabolites in the mice. The serum metabolites identified in these controlled feeding studies were then investigated in 212 polyp-free participants from the Polyp Prevention Trial who self-reported either increased (≥+31 g/day from baseline), high dry bean intake of ≥42 g/day in year 3 or low, unchanged dry bean consumption of <8 g/day; serum was analyzed from baseline and year 3. Serum pipecolic acid and S-methyl cysteine were elevated after dry bean consumption in human and mouse studies and reflected dry bean consumption in the Polyp Prevention Trial. Serum levels of pipecolic acid and S-methyl cysteine are useful biomarkers of dry bean consumption. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model.

PubMed

Krawczyk, Krzysztof M; Matak, Damian; Szymanski, Lukasz; Szczylik, Cezary; Porta, Camillo; Czarnecka, Anna M

2018-04-01

The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco's Modified Eagle's Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nie, Song; Shi, Tujin; Fillmore, Thomas L.

Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low ng/mL to sub-ng/mL level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundant but biologically important proteins (e.g., ≤100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging. To address this need, we have developed an antibody-independent Deep-Dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide enrichment combined withmore » precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ~5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue has been demonstrated to enable precise quantification of endogenous proteins at ~10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibody is not available.« less

Identification and monitoring of metabolite markers of dry bean consumption in parallel human and mouse studies

PubMed Central

Perera, Thushanthi; Young, Matthew R.; Zhang, Zhiying; Murphy, Gwen; Colburn, Nancy H.; Lanza, Elaine; Hartman, Terryl J.; Cross, Amanda J.; Bobe, Gerd

2015-01-01

Scope Aim of the study was to identify and monitor metabolite markers of dry bean consumption in parallel human and mouse studies that each had shown chemopreventive effects of dry bean consumption on colorectal neoplasia risk. Methods and Results Using liquid chromatography/mass spectroscopy +/− electrospray ionization and gas chromatography/mass spectroscopy, serum metabolites of dry beans were measured in 46 men before and after a four-week dry bean-enriched diet (250 g/d) and 12 mice that received a standardized diet containing either 0 or 10% navy bean ethanol extract for 6 weeks; we also investigated fecal metabolites in the mice. The serum metabolites identified in these controlled feeding studies were then investigated in 212 polyp-free participants from the Polyp Prevention Trial who self-reported either increased (≥+31 g/d from baseline), high dry bean intake of ≥42 g/d in year 3 or low, unchanged dry bean consumption of <8 g/d; serum was analyzed from baseline and year 3. Serum pipecolic acid and S-methyl-cysteine were elevated after dry bean consumption in human and mouse studies and reflected dry bean consumption in the Polyp Prevention Trial. Conclusions Serum levels of pipecolic acid and S-methyl-cysteine are useful biomarkers of dry bean consumption. PMID:25641932

Is DTPA a good competing chelating agent for Th(IV) in human serum and suitable in targeted alpha therapy?

PubMed

Le Du, Alicia; Sabatié-Gogova, Andrea; Morgenstern, Alfred; Montavon, Gilles

2012-04-01

The interaction between thorium and human serum components was studied using difference ultraviolet spectroscopy (DUS), ultrafiltration and high-pressure-anion exchange chromatography (HPAEC) with external inductively conducted plasma mass spectrometry (ICP-MS) analysis. Experimental data are compared with modelling results based on the law of mass action. Human serum transferrin (HSTF) interacts strongly with Th(IV), forming a ternary complex including two synergistic carbonate anions. This complex governs Th(IV) speciation under blood serum conditions. Considering the generally used Langmuir-type model, values of 10(33.5) and 10(32.5) were obtained for strong and weak sites, respectively. We showed that trace amounts of diethylene triamine pentaacetic acid (DTPA) cannot complex Th(IV) in the blood serum at equilibrium. Unexpectedly this effect is not related to the competition with HSTF but is due to the strong competition with major divalent metal ions for DTPA. However, Th-DTPA complex was shown to be stable for a few hours when it is formed before addition in the biological medium; this is related to the high kinetic stability of the complex. This makes DTPA a potential chelating agent for synthesis of (226)Th-labelled biomolecules for application in targeted alpha therapy. Copyright © 2011 Elsevier Inc. All rights reserved.

Lithium/aluminium alloys. A problem material for biological monitoring.

PubMed

Bencze, K; Pelikan, C; Bahemann-Hoffmeister, A; Kronseder, A

1991-01-01

Investigations on aqueous and nonaqueous media have shown that the dust particles of lithium/aluminium alloy are readily soluble in blood serum. Concentration of 10 mg A1 and 3 mg Li in 1 l serum constitute the saturation values in human blood serum at 20 degrees C. Further observations are intended to clarify the danger at the workplace. With a short-term exposure, the change in concentration of Li and Al was followed in the working environment and in the staff. Both elements (Li and Al) were determined in the working air, in the urine, the blood serum and on human skin with electrothermal AAS. On human skin, microerosions arise owing to hydrolytic destruction of the epidermis after long-term contact. The intensive irritant action of Li/Al dust is most relevant for preventive measures. It must also be considered that the Al values in the blood serum are raised by up to 50% in the presence of Li. The resorption and/or the bioavailability of Al is altered by Li. In the presence of Li up to a Li content of 2.5% in Al dust, a MAK (biological tolerance value, threshold limit value) of 1 mg Al/m3 room air (for the total dust) is recommended.

A methodology for the evaluation of the human-bioclimatic performance of open spaces

NASA Astrophysics Data System (ADS)

Charalampopoulos, Ioannis; Tsiros, Ioannis; Chronopoulou-Sereli, Aik.; Matzarakis, Andreas

2017-05-01

The purpose of this paper is to present a simple methodology to improve the evaluation of the human-biometeorological benefits of open spaces. It is based on two groups of new indices using as basis the well-known PET index. This simple methodology along with the accompanying indices allows a qualitative and quantitative evaluation of the climatic behavior of the selected sites. The proposed methodology was applied in a human-biometeorology research in the city of Athens, Greece. The results of this study are in line with the results of other related studies indicating the considerable influence of the sky view factor (SVF), the existence of the vegetation and the building material on human-biometeorological conditions. The proposed methodology may provide new insights in the decision-making process related to urban open spaces' best configuration.

Serum hepcidin is significantly associated with iron absorption from food and supplemental sources in healthy young woman

USDA-ARS?s Scientific Manuscript database

Hepcidin is a key regulator of iron homeostasis, but to date no studies have examined the effect of hepcidin on iron absorption in humans. Our objective was to assess relations between both serum hepcidin and serum prohepcidin with nonheme-iron absorption in the presence and absence of food with the...

Serum metabolome biomarkers associate low-level environmental perfluorinated compound exposure with oxidative /nitrosative stress in humans.

PubMed

Wang, Xiaofei; Liu, Liangpo; Zhang, Weibing; Zhang, Jie; Du, Xiaoyan; Huang, Qingyu; Tian, Meiping; Shen, Heqing

2017-10-01

Previous in vivo and in vitro studies have linked perfluorinated compound (PFC) exposure with metabolic interruption, but the inter-species difference and high treatment doses usually make the results difficult to be extrapolated to humans directly. The best strategy for identifying the metabolic interruption may be to establish the direct correlations between monitored PFCs data and metabolic data on human samples. In this study, serum metabolome data and PFC concentrations were acquired for a Chinese adult male cohort. The most abundant PFCs are PFOA and PFOS with concentration medians 7.56 and 12.78 nM, respectively; in together they count around 81.6% of the total PFCs. PFC concentration-related serum metabolic profile changes and the related metabolic biomarkers were explored by using partial least squares-discriminant analysis (PLS-DA). Respectively taking PFOS, PFOA and total PFC as the classifiers, serum metabolome can be differentiated between the lowest dose group (1st quartile PFCs) and the highest PFC dose group (4th quartile PFCs). Ten potential PFC biomarkers were identified, mainly involving in pollutant detoxification, antioxidation and nitric oxide (NO) signal pathways. These suggested that low-level environmental PFC exposure has significantly adverse impacts on glutathione (GSH) cycle, Krebs cycle, nitric oxide (NO) generation and purine oxidation in humans. To the best of our knowledge, this is the first report investigating the association of environmental PFC exposure with human serum metabolome alteration. Given the important biological functions of the identified biomarkers, we suggest that PFC could increase the metabolism syndromes risk including diabetes and cardiovascular diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

PubMed Central

Krayukhina, Elena; Noda, Masanori; Ishii, Kentaro; Maruno, Takahiro; Wakabayashi, Hirotsugu; Tada, Minoru; Suzuki, Takuo; Ishii-Watabe, Akiko; Kato, Masahiko; Uchiyama, Susumu

2017-01-01

ABSTRACT A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins. PMID:28387583

Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations.

PubMed

Springer, Jan; White, P Lewis; Hamilton, Shanna; Michel, Denise; Barnes, Rosemary A; Einsele, Hermann; Löffler, Juergen

2016-03-01

Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time to diagnosis may be enhanced by testing multiple contemporaneously obtained sample types. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Clinical peptidomic analysis by a one-step direct transfer technology: its potential utility for monitoring of pathophysiological status in female reproductive system disorders.

PubMed

Araki, Yoshihiko; Nonaka, Daisuke; Hamamura, Kensuke; Yanagida, Mitsuaki; Ishikawa, Hitoshi; Banzai, Michio; Maruyama, Mayuko; Endo, Shuichiro; Tajima, Atsushi; Lee, Lyang-Ja; Nojima, Michio; Takamori, Kenji; Yoshida, Koyo; Takeda, Satoru; Tanaka, Kenji

2013-10-01

To date, numerous studies have searched for candidate molecules or clinical examination methods as potential biomarkers for monitoring intractable diseases, such as carcinomas. Evidence accumulated over the past decade shows that many proteolytic peptides appear in human humoral fluids, including peripheral blood, in association with an individual's health condition. Although an analysis of the whole peptide (the 'peptidome') using mass spectrometry is thought to be one of the most powerful and promising experimental approaches, it has failed to identify biomarkers in the clinical blood samples, presumably due to the methodological limitations. In general, commonly used techniques for proteomic analysis of blood require the removal of large amounts of serum/plasma proteins prior to mass spectrometry analysis, and this step seems to have resulted in the overlooking of important biomarkers during the analytical process. Here, we provide a brief overview of a new quantitative peptidomic analysis by a one-step direct transfer technology without depletion of major blood proteins. Using this technology, we herein report experimental data on serum peptidomic analysis for patients with pregnancy-induced hypertension as a clinical model. In addition, we refer to the potential utility of this approach for the monitoring of pathophysiological status in female reproductive system disorders in general. © 2013 The Authors. Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology.

Relationship of serum levels of TNF-α, IL-6 and IL-18 and schizophrenia-like symptoms in chronic ketamine abusers

PubMed Central

Fan, Ni; Luo, Yayan; Xu, Ke; Zhang, Minling; Ke, Xiaoyin; Huang, Xini; Ding, Yi; Wang, Daping; Ning, Yuping; Deng, Xuefeng; He, Hongbo

2016-01-01

Objective Exposing to NMDAR receptor antagonists, such as ketamine, produces schizophrenia-like symptoms in humans and deteriorates symptoms in schizophrenia patients. Meanwhile, schizophrenia is associated with alterations of cytokines in the immune system. This study aims to examine the serum TNF-α, IL-6 and IL-18 levels in chronic human ketamine users as compared to healthy subjects. The correlations between the serum cytokines levels with the demographic, ketamine use characteristics and psychiatric symptoms were also assessed. Methods 155 subjects who fulfilled the criteria of ketamine dependence and 80 healthy control subjects were recruited. Serum TNF-α, IL-6 and IL-18 levels were measured using an enzyme-linked immunosorbent assay (ELISA). The psychiatric symptoms of the ketamine abusers were assessed using the Positive and Negative Syndrome Scale (PANSS). Results Serum IL-6 and IL-18 levels were significantly higher, while serum TNF-α level was significantly lower among ketamine users than among healthy controls (p < 0.05). Serum TNF-α levels showed a significant negative association with PANSS total score (r = −0.210, p < 0.01) and negative subscore (r = −0.300, p < 0.01). No significant association was found between PANSS score and serum levels of IL-6 and IL-18. Conclusions Serum levels of TNF-α, IL-6 and IL-18 were altered in chronic ketamine abusers which may play a role in schizophrenia-like symptoms in chronic ketamine abusers. PMID:26589393

Massive glutamine cyclization to pyroglutamic acid in human serum discovered using NMR spectroscopy.

PubMed

Nagana Gowda, G A; Gowda, Yashas N; Raftery, Daniel

2015-04-07

Glutamine is one of the most abundant metabolites in blood and is a precursor as well as end product central to numerous important metabolic pathways. A number of surprising and unexpected roles for glutamine, including cancer cell glutamine addiction discovered recently, stress the importance of accurate analysis of glutamine concentrations for understanding its role in health and numerous diseases. Utilizing a recently developed NMR approach that offers access to an unprecedented number of quantifiable blood metabolites, we have identified a surprising glutamine cyclization to pyroglutamic acid that occurs during protein removal. Intact, ultrafiltered and protein precipitated samples from the same pool of human serum were comprehensively investigated using (1)H NMR spectroscopy at 800 MHz to detect and quantitatively evaluate the phenomenon. Interestingly, although glutamine cyclization occurs in both ultrafiltered and protein precipitated serum, the cyclization was not detected in intact serum. Strikingly, due to cyclization, the apparent serum glutamine level drops by up to 75% and, concomitantly, the pyroglutamic acid level increases proportionately. Further, virtually under identical conditions, the magnitude of cyclization is vastly different for different portions of samples from the same pool of human serum. However, the sum of glutamine and pyroglutamic acid concentrations in each sample remains the same for all portions. These unexpected findings indicate the importance of considering the sum of apparent glutamine and pyroglutamic acid levels, obtained from the contemporary analytical methods, as the actual blood glutamine level for biomarker discovery and biological interpretations.

Simultaneous determination of neonicotinoid insecticides in human serum and urine using diatomaceous earth-assisted extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Yamamuro, Tadashi; Ohta, Hikoto; Aoyama, Mika; Watanabe, Daisuke

2014-10-15

A rapid and sensitive analytical method was developed for simultaneous determination of eight neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) and three specific metabolites of acetamiprid (N-desmethylacetamiprid, 5-(N-acetyl-N-methylaminomethyl)-2-chloropyridine and 5-(N-acetylaminomethyl)-2-chloropyridine) in human serum and urine. A diatomaceous earth-assisted extraction using Extrelut NT3 column with chloroform/2-propanol (3:1, v/v) as eluent was selected for the single step cleanup procedure for all the target compounds. Qualitative and quantitative analyses were conducted by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring mode. The limits of detection and the limits of quantification of eleven compounds were in the ranges of 0.1-0.2ng/mL and 0.5-10ng/mL for serum, 0.1-1ng/mL and 1-10ng/mL for urine, respectively. The extraction recoveries were between 80.9% and 101.8% for serum samples, 91.9% and 106% for urine samples. The intra-day RSDs and the inter-day RSDs were less than 11.5% and 13.2% for serum, less than 8.3% and 8.8% for urine. The proposed procedure will be suitable for forensic investigations of human poisoning cases with neonicotinoid insecticides. This is the first report of simultaneous determination of eight neonicotinoids in serum and urine samples. Copyright © 2014. Published by Elsevier B.V.

Serum S100B Represents a New Biomarker for Mood Disorders

PubMed Central

Schroeter, Matthias L.; Sacher, Julia; Steiner, Johann; Schoenknecht, Peter; Mueller, Karsten

2013-01-01

Recently, mood disorders have been discussed to be characterized by glial pathology. The protein S100B, a growth and differentiation factor, is located in, and may actively be released by astro- and oligodendrocytes. This protein is easily assessed in human serum and provides a useful parameter for glial activation or injury. Here, we review studies investigating the glial marker S100B in serum of patients with mood disorders. Studies consistently show that S100B is elevated in mood disorders; more strongly in major depressive than bipolar disorder. Consistent with the glial hypothesis of mood disorders, serum S100B levels interact with age with higher levels in elderly depressed subjects. Successful antidepressive treatment has been associated with serum S100B reduction in major depression, whereas there is no evidence of treatment effects in mania. In contrast to the glial marker S100B, the neuronal marker protein neuron-specific enolase is unaltered in mood disorders. Recently, serum S100B has been linked to specific imaging parameters in the human white matter suggesting a role for S100B as an oligodendrocytic marker protein. In sum, serum S100B can be regarded as a promising in vivo biomarker for mood disorders deepening the understanding of the pathogenesis and plasticity-changes in these disorders. Future longitudinal studies combining serum S100B with other cell-specific serum parameters and multimodal imaging are warranted to further explore this serum protein in the development, monitoring and treatment of mood disorders. PMID:23701298

Association between Nitrogen Stable Isotope Ratios in Human Hair and Serum Levels of Leptin.

PubMed

Ahn, Song Vogue; Koh, Sang-Baek; Lee, Kwang-Sik; Bong, Yeon-Sik; Park, Jong-Ku

2017-10-01

Stable isotope ratios have been reported to be potential biomarkers of dietary intake and nutritional status. High serum levels of leptin, a hormone which regulates energy metabolism and food intake, are associated with insulin resistance and metabolic syndrome. However, little is known about the association between stable isotope ratios and the metabolic risk in humans. We investigated whether the carbon and nitrogen stable isotope ratios in hair are associated with serum leptin levels. Hair samples were collected from 399 healthy adults (233 men and 166 women) aged 40 to 70 years of a community-based cohort in Korea and the bulk stable isotope ratios of carbon (δ 13 C) and nitrogen (δ 15 N) were measured for all hair samples. Serum leptin levels were analyzed by radioimmunoassay. δ 15 N showed positive correlations with serum leptin levels. In multivariate models, increasing δ 15 N were associated with elevated serum leptin levels (defined as ≥ the median values), whereas δ 13 C were not significantly associated with serum leptin levels. The odds ratio (95% confidence interval) per 1‰ increase in δ 15 N for an elevated serum leptin level was 1.58 (1.11-2.26). In participants with high body mass index, δ 15 N showed positive associations with serum leptin levels, whereas these associations were not seen in participants with low body mass index. The nitrogen stable isotopic ratio in hair is positively associated with serum leptin levels. The hair δ 15 N could be used as a clinical marker to estimate metabolic risk.

Serum antibodies to outer membrane proteins (OMPs) of Moraxella (Branhamella) catarrhalis in patients with bronchiectasis: identification of OMP B1 as an important antigen.

PubMed Central

Sethi, S; Hill, S L; Murphy, T F

1995-01-01

Moraxella (Branhamella) catarrhalis is a common cause of lower respiratory tract infections in adults and of otitis media in children. Little is known about the human immune response to this bacterium. In this study, immunoblot assays were performed to detect serum immunoglobulin G antibodies directed at purified outer membrane of M. catarrhalis. Twelve serum samples, two each from six patients with bronchiectasis who were persistently colonized with this organism, were tested with their homologous M. catarrhalis sputum isolates. In all the sera, the most prominent and consistent antibody response was to a minor 84-kDa outer membrane protein, OMP B1. Immunoblot adsorption assays show that these antibodies recognize surface exposed epitopes on OMP B1. Further analysis of human serum antibodies eluted from the surface of intact bacterial cells shows that these surface-exposed epitopes on OMP B1 are heterogeneous among strains of M. catarrhalis. OMP B1 is therefore an important OMP antigen on the surface of M. catarrhalis for the human immune response to infection by this bacterium. PMID:7890418

Strategies for Characterization of Low-Abundant Intact or Truncated Low-Molecular-Weight Proteins From Human Plasma.

PubMed

Cai, Tanxi; Yang, Fuquan

2017-01-01

Low-molecular-weight region (LMW, MW≤30kDa) of human serum/plasma proteins, including small intact proteins, truncated fragments of larger proteins, along with some other small components, has been associated with the ongoing physiological and pathological events, and thereby represent a treasure trove of diagnostic molecules. Great progress in the mining of novel biomarkers from this diagnostic treasure trove for disease diagnosis and health monitoring has been achieved based on serum samples from healthy individuals and patients and powerful new approaches in biochemistry and systems biology. However, cumulative evidence indicates that many potential LMW protein biomarkers might still have escaped from detection due to their low abundance, the dynamic complexity of serum/plasma, and the limited efficiency of characterization approaches. Here, we provide an overview of the current state of knowledge with respect to strategies for the characterization of low-abundant LMW proteins (small intact or truncated proteins) from human serum/plasma, involving prefractionation or enrichment methods to reduce dynamic range and mass spectrometry-based characterization of low-abundant LMW proteins. © 2017 Elsevier Inc. All rights reserved.

Role of the Protein Corona Derived from Human Plasma in Cellular Interactions between Nanoporous Human Serum Albumin Particles and Endothelial Cells.

PubMed

Zyuzin, Mikhail V; Yan, Yan; Hartmann, Raimo; Gause, Katelyn T; Nazarenus, Moritz; Cui, Jiwei; Caruso, Frank; Parak, Wolfgang J

2017-08-16

The presence of a protein corona on various synthetic nanomaterials has been shown to strongly influence how they interact with cells. However, it is unclear if the protein corona also exists on protein particles, and if so, its role in particle-cell interactions. In this study, pure human serum albumin (HSA) particles were fabricated via mesoporous silica particle templating. Our data reveal that various serum proteins adsorbed on the particles, when exposed to human blood plasma, forming a corona. In human umbilical vein endothelial cells (HUVECs), the corona was shown to decrease particle binding to the cell membrane, increase the residence time of particles in early endosomes, and reduce the amount of internalized particles within the first hours of exposure to particles. These findings reveal important information regarding the mechanisms used by vascular endothelial cells to internalize protein-based particulate materials exposed to blood plasma. The ability to control the cellular recognition of these organic particles is expected to aid the advancement of HSA-based materials for intravenous drug delivery.

Repertoire of BALB/c Mice Natural Anti-Carbohydrate Antibodies: Mice vs. Humans Difference, and Otherness of Individual Animals

PubMed Central

Bello-Gil, Daniel; Khasbiullina, Nailya; Shilova, Nadezhda; Bovin, Nicolai; Mañez, Rafael

2017-01-01

One of the most common genetic backgrounds for mice used as a model to investigate human diseases is the inbred BALB/c strain. This work is aimed to characterize the pattern of natural anti-carbohydrate antibodies present in the serum of 20 BALB/c mice by printed glycan array technology and to compare their binding specificities with that of human natural anti-carbohydrate antibodies. Natural antibodies (NAbs) from the serum of BALB/c mice interacted with 71 glycans from a library of 419 different carbohydrate structures. However, only seven of these glycans were recognized by the serum of all the animals studied, and other five glycans by at least 80% of mice. The pattern of the 12 glycans mostly recognized by the circulating antibodies of BALB/c mice differed significantly from that observed with natural anti-carbohydrate antibodies in humans. This lack of identical repertoires of natural anti-carbohydrate antibodies between individual inbred mice, and between mice and humans, should be taken into consideration when mouse models are intended to be used for investigation of NAbs in biomedical research. PMID:29163519

Phage display for identification of serum biomarkers of traumatic brain injury.

PubMed

Ghoshal, Sarbani; Bondada, Vimala; Saatman, Kathryn E; Guttmann, Rodney P; Geddes, James W

2016-10-15

The extent and severity of traumatic brain injuries (TBIs) can be difficult to determine with current diagnostic methods. To address this, there has been increased interest in developing biomarkers to assist in the diagnosis, determination of injury severity, evaluation of recovery and therapeutic efficacy, and prediction of outcomes. Several promising serum TBI biomarkers have been identified using hypothesis-driven approaches, largely examining proteins that are abundant in neurons and non-neural cells in the CNS. An unbiased approach, phage display, was used to identify serum TBI biomarkers. In this proof-of-concept study, mice received a TBI using the controlled cortical impact model of TBI (1mm injury depth, 3.5m/s velocity) and phage display was utilized to identify putative serum biomarkers at 6h postinjury. An engineered phage which preferentially bound to injured serum was sequenced to identify the 12-mer 'recognizer' peptide expressed on the coat protein. Following synthesis of the recognizer peptide, pull down, and mass spectrometry analysis, the target protein was identified as glial fibrillary acidic protein (GFAP). GFAP has previously been identified as a promising TBI biomarker. The results provide proof of concept regarding the ability of phage display to identify TBI serum biomarkers. This methodology is currently being applied to serum biomarkers of mild TBI. Copyright © 2016 Elsevier B.V. All rights reserved.

Serum Immunoglobulin G4 in Discriminating Autoimmune Pancreatitis From Pancreatic Cancer: A Diagnostic Meta-analysis.

PubMed

Dai, Cong; Cao, Qin; Jiang, Min; Sun, Ming-Jun

2018-03-01

Differentiation between autoimmune pancreatitis (AIP) and pancreatic cancer (PC) is a clinical challenge. Emerging published data on the accuracy of serum immunoglobulin G4 (IgG4) for the differential diagnosis between AIP and PC are inconsistent. The objective of our study was to perform a meta-analysis evaluating the clinical utility of serum IgG4 in the differential diagnosis between AIP and PC. We performed a systematic literature search of multiple electronic databases. The methodological quality of each study was assessed according to the Quality Assessment of Diagnostic Accuracy Studies checklist. Random-effects model was used to summarize the diagnostic odds ratio and other measures of accuracy. Eleven studies comprising 523 AIP patients and 771 PC patients were included in the meta-analysis. The summary estimates for serum IgG4 in distinguishing AIP from PC were as follows: diagnostic odds ratio, 57.30 (95% confidence interval [CI], 23.17-141.67); sensitivity, 0.72 (95% CI, 0.68-0.76); specificity, 0.93 (95% CI, 0.91-0.94). The area under the curve of serum IgG4 in distinguishing AIP from PC was 0.9200. Our meta-analysis found that serum IgG4 has high specificity and relatively low sensitivity in the differential diagnosis between AIP and PC. Therefore, serum IgG4 is useful in distinguishing AIP from PC.

Parathyroid hormone related protein concentration in human serum and CSF correlates with age.

PubMed

Kushnir, Mark M; Peterson, Lisa K; Strathmann, Frederick G

2018-02-01

Parathyroid Hormone-Related Protein (PTHrP) is involved in intracellular calcium (Ca) regulation, and has been demonstrated to participate in regulation of Ca in brain cells, activation of neurons, and modulation of pain. However, there are conflicting reports regarding the presence of PTHrP in CSF. PTHrP and Ca were quantified in paired CSF and serum samples using mass spectrometry-based methods. Associations between PTHrP and Ca concentrations with age, sex and concentrations of nine CSF diagnostic markers in a set of 140 paired serum and CSF patient samples were evaluated. The observed median PTHrP concentration in CSF was 51 times higher than in serum; the median concentration of Ca in CSF was 1.8 times lower than in serum. We observed positive correlation between concentrations of PTHrP in CSF and serum (p=0.013). Distribution of PTHrP concentrations in serum was associated with age (p=0.0068) and the concentrations were higher in women. In samples with serum calcium concentrations within the reference intervals (n=118), central 95% distribution of concentrations for Ca-CSF, PTHrP-serum and PTHrP-CSF were 5.4 (4.5-6.1) mg/dL, 1.2 (0.5-2.5) pmol/L, 62 (22-125) pmol/L, respectively. Our data demonstrate that PTHrP is a normal constituent of human CSF with median concentrations 51 fold higher than in serum. Elevated serum PTHrP concentrations were positively correlated with age and significantly higher in women. Our data suggest that CSF could be a significant source of circulating PTHrP. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Posaconazole in Human Serum: a Greater Pharmacodynamic Effect than Predicted by the Non-Protein-Bound Serum Concentration ▿

PubMed Central

Lignell, Anders; Löwdin, Elisabeth; Cars, Otto; Chryssanthou, Erja; Sjölin, Jan

2011-01-01

It is generally accepted that only the unbound fraction of a drug is pharmacologically active. Posaconazole is an antifungal agent with a protein binding of 98 to 99%. Taking into account the degree of protein binding, plasma levels in patients, and MIC levels of susceptible strains, it can be assumed that the free concentration of posaconazole sometimes will be too low to exert the expected antifungal effect. The aim was therefore to test the activity of posaconazole in serum in comparison with that of the calculated unbound concentrations in protein-free media. Significant differences (P < 0.05) from the serum control were found at serum concentrations of posaconazole of 1.0 and 0.10 mg/liter, with calculated free concentrations corresponding to 1× MIC and 0.1× MIC, respectively, against one Candida lusitaniae strain selected for proof of principle. In RPMI 1640, the corresponding calculated unbound concentration of 0.015 mg/liter resulted in a significant effect, whereas that of 0.0015 mg/liter did not. Also, against seven additional Candida strains tested, there was an effect of the low posaconazole concentration in serum, in contrast to the results in RPMI 1640. Fluconazole, a low-grade-protein-bound antifungal, was used for comparison at corresponding concentrations in serum and RPMI 1640. No effect was observed at the serum concentration, resulting in a calculated unbound concentration of 0.1× MIC. In summary, there was a substantially greater pharmacodynamic effect of posaconazole in human serum than could be predicted by the non-protein-bound serum concentration. A flux from serum protein-bound to fungal lanosterol 14α-demethylase-bound posaconazole is suggested. PMID:21502622

Human serum miR-34a as an indicator of exposure to ionizing radiation.

PubMed

Halimi, Mohammad; Shahabi, Ahmad; Moslemi, Dariush; Parsian, Hadi; Asghari, S Mohsen; Sariri, Reyhaneh; Yeganeh, Farshid; Zabihi, Ebrahim

2016-11-01

Radiation exposure in industrial accidents or nuclear device attacks is a major public health concern. There is an urgent need for markers that rapidly identify people exposed to ionizing radiation (IR). Finding a blood-based marker is advantageous because of the ease of sample collection. This study was designed to test the hypothesis that serum miR-34a could serve as an indicator of exposure to IR. Therefore, 44 women with breast cancer, where radiotherapy was part of their therapeutic protocol, were investigated in this study. After demonstrating the appropriateness of our microRNA (miRNA) extraction efficiency and miRNA assay in human serum, we analyzed the miR-34a level in paired serum samples before and after radiotherapy. Fifty Gy X-ray irradiation in daily dose fractions of 2 Gy, 5 days per week, was used in this study. We demonstrated that IR significantly increased serum level of miR-34a. By measuring miR-34a in serum, we could distinguish irradiated patients with sensitivity of 65 % and specificity of 75 %. According to this study, serum miR-34a has the potential to be used as an indicator of radiation exposure.

Improvement in skin wrinkles using a preparation containing human growth factors and hyaluronic acid serum.

PubMed

Lee, Do Hyun; Oh, In Young; Koo, Kyo Tan; Suk, Jang Mi; Jung, Sang Wook; Park, Jin Oh; Kim, Beom Joon; Choi, Yoo Mi

2015-02-01

Skin aging is accompanied by wrinkle formation. At some sites, such as the periorbital skin, this is a relatively early phenomenon. We evaluated the anti-wrinkle effect of a preparation containing human growth factor and hyaluronic acid serum on periorbital wrinkles (crow's feet). In total, 23 Korean women (age range: 39-59 years), who were not pregnant, nursing, or undergoing any concurrent therapy, were enrolled in this study. All the patients completed an 8-week trial of twice-daily application of human growth factor and hyaluronic acid serum on the entire face. Efficacy was based on a global photodamage score, photographs, and image analysis using replicas and visiometer analysis every 4 weeks. The standard wrinkle and roughness parameters used in assessing skin by visiometer were calculated and statistically analyzed. Periorbital wrinkles were significantly improved after treatment, with improvements noted both by physician's assessment and visiometer analysis. Topical application of human growth factor and hyaluronic acid was beneficial in reducing periorbital wrinkles.

CYTOPATHIC EFFECT OF THE ATYPICAL PNEUMONIA ORGANISM IN CULTURES OF HUMAN TISSUE

PubMed Central

Eaton, Monroe D.; Farnham, Ann E.; Levinthal, Jeana D.; Scala, Anthony R.

1962-01-01

Eaton, Monroe D. (Harvard Medical School, Boston, Mass.), Ann E. Farnham, Jeana D. Levinthal, and Anthony R. Scala. Cytopathic effect of the atypical pneumonia organism in cultures of human tissue. J. Bacteriol. 84:1330–1337. 1962.—Three strains of the atypical pneumonia agent were adapted to grow in continuous cell cultures of human amnion or human embryonic lung, with production of initial increased acidity followed by destruction of the cells. Evidence is presented that cytopathic effects of the organism were associated with intracellular growth and formation of microcolonies. Clumps of organisms stained specifically with fluorescein-labeled antibody, and showed distinctive tinctorial reactions with the May Grünwald-Giemsa stain. The cytopathic effect was prevented by fresh serum from a rabbit immunized with an egg-passage strain of the atypical pneumonia agent. Heating the immune serum to 56 C for 30 min abolished the neutralizing effect. The significance of heat-labile serum constituents in killing or inhibition of mycoplasma is discussed. Images PMID:16561984

Kinetic assay of antitrypsin in human serum by a surface acoustic wave(SAW)-impedance sensor.

PubMed

Cai, Q; Wei, W; Wang, R; Nie, L; Yao, S

1996-08-01

Antitrypsin in human serum was determined by using both the SAW-impedance sensor system and spectrophotometry, indicating that the mean value for women was significantly higher than the mean value for men; the value for acute pancreasis patients is about 2-folds of the normal values, and there is no significant difference between the acute pancreasis patients and the pancreatic cancer patients.

Self-Assembly of Human Serum Albumin: A Simplex Phenomenon

PubMed Central

Thakur, Garima; Prashanthi, Kovur; Jiang, Keren; Thundat, Thomas

2017-01-01

Spontaneous self-assemblies of biomolecules can generate geometrical patterns. Our findings provide an insight into the mechanism of self-assembled ring pattern generation by human serum albumin (HSA). The self-assembly is a process guided by kinetic and thermodynamic parameters. The generated protein ring patterns display a behavior which is geometrically related to a n-simplex model and is explained through thermodynamics and chemical kinetics. PMID:28930179

Effect of human serum albumin upon the permeabilizing activity of sticholysin II, a pore forming toxin from Stichodactyla heliantus.

PubMed

Celedón, Gloria; González, Gustavo; Gulppi, Felipe; Pazos, Fabiola; Lanio, María E; Alvarez, Carlos; Calderón, Cristian; Montecinos, Rodrigo; Lissi, Eduardo

2013-12-01

Sticholysin II (St II) is a haemolytic toxin isolated from the sea anemone Stichodactyla helianthus. The high haemolytic activity of this toxin is strongly dependent on the red cell status and the macromolecule conformation. In the present communication we evaluate the effect of human serum albumin on St II haemolytic activity and its capacity to form pores in the bilayer of synthetic liposomes. St II retains its pore forming capacity in the presence of large concentrations (up to 500 μM) of human serum albumin. This effect is observed both in its capacity to produce red blood cells haemolysis and to generate functional pores in liposomes. In particular, the capacity of the toxin to lyse red blood cells increases in the presence of human serum albumin (HSA). Regarding the rate of the pore forming process, it is moderately decreased in liposomes and in red blood cells, in spite of an almost total coverage of the interface by albumin. All the data obtained in red cells and model membranes show that St II remains lytically active even in the presence of high HSA concentrations. This stubbornness can explain why the toxin is able to exert its haemolytic activity on membranes immersed in complex plasma matrixes such as those present in living organisms.

Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens.

PubMed

Prendergast, Emily N; de Souza Fonseca, Marcos Abraão; Dezem, Felipe Segato; Lester, Jenny; Karlan, Beth Y; Noushmehr, Houtan; Lin, Xianzhi; Lawrenson, Kate

2018-01-01

Exosomes are endosome-derived membrane vesicles that contain proteins, lipids, and nucleic acids. The exosomal transcriptome mediates intercellular communication, and represents an understudied reservoir of novel biomarkers for human diseases. Next-generation sequencing enables complex quantitative characterization of exosomal RNAs from diverse sources. However, detailed protocols describing exosome purification for preparation of exosomal RNA-sequence (RNA-Seq) libraries are lacking. Here we compared methods for isolation of exosomes and extraction of exosomal RNA from human cell-free serum, as well as strategies for attaining equal representation of samples within pooled RNA-Seq libraries. We compared commercial precipitation with ultracentrifugation for exosome purification and confirmed the presence of exosomes via both transmission electron microscopy and immunoblotting. Exosomal RNA extraction was compared using four different RNA purification methods. We determined the minimal starting volume of serum required for exosome preparation and showed that high quality exosomal RNA can be isolated from sera stored for over a decade. Finally, RNA-Seq libraries were successfully prepared with exosomal RNAs extracted from human cell-free serum, cataloguing both coding and non-coding exosomal transcripts. This method provides researchers with strategic options to prepare RNA-Seq libraries and compare RNA-Seq data quantitatively from minimal volumes of fresh and archival human cell-free serum for disease biomarker discovery.

Affinity chromatography on monolithic supports for simultaneous and high-throughput isolation of immunoglobulins from human serum.

PubMed

Martinović, Tamara; Andjelković, Uroš; Klobučar, Marko; Černigoj, Urh; Vidič, Jana; Lučić, Marina; Pavelić, Krešimir; Josić, Djuro

2017-11-01

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Can nail, hair and urine be used for biomonitoring of human exposure to perfluorooctane sulfonate and perfluorooctanoic acid?

PubMed

Li, Jingguang; Guo, Feifei; Wang, Yuxin; Zhang, Jialing; Zhong, Yuxin; Zhao, Yunfeng; Wu, Yongning

2013-03-01

Because of the disadvantages of invasive sampling, it is desirable to explore non-invasive matrices for human biomonitoring of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA). The aim of this study was to evaluate the application of nail, hair and urine for human biomonitoring of PFOS and PFOA. The concentrations of PFOS and PFOA in matched nail, hair, urine and serum samples collected from 64 donors were measured. The chemicals of interest were detected with high detection frequency in these matrices (90%-100%) except for PFOA in urine samples (56%). Generally, the gender influences on the levels of PFOS and PFOA in these non-invasive matrices were in agreement with that in serum. For PFOS, the coefficients of Spearman correlation between serum samples and nail, hair and urine samples were 0.786 (p<0.001), 0.545 (p<0.001) and 0.302 (p<0.05), respectively. For PFOA, the correlation was only observed between nail samples and serum samples with a correlation coefficient of 0.299 (p<0.05). The results suggested that nail has more potential than hair and urine to be applied in human biomonitoring for PFOS and PFOA in general populations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Simple method to distinguish between primary and secondary C3 deficiencies.

PubMed

Pereira de Carvalho Florido, Marlene; Ferreira de Paula, Patrícia; Isaac, Lourdes

2003-03-01

Due to the increasing numbers of reported clinical cases of complement deficiency in medical centers, clinicians are now more aware of the role of the complement system in the protection against infections caused by microorganisms. Therefore, clinical laboratories are now prepared to perform a number of diagnostic tests of the complement system other than the standard 50% hemolytic component assay. Deficiencies of alternative complement pathway proteins are related to severe and recurrent infections; and the application of easy, reliable, and low-cost methods for their detection and distinction are always welcome, notably in developing countries. When activation of the alternative complement pathway is evaluated in hemolytic agarose plates, some but not all human sera cross-react to form a late linear lysis. Since the formation of this linear lysis is dependent on C3 and factor B, it is possible to use late linear lysis to routinely screen for the presence of deficiencies of alternative human complement pathway proteins such as factor B. Furthermore, since linear lysis is observed between normal human serum and primary C3-deficient serum but not between normal human serum and secondary C3-deficient serum caused by the lack of factor H or factor I, this assay may also be used to discriminate between primary and secondary C3 deficiencies.

Detection of Novel Sequences Related to African Swine Fever Virus in Human Serum and Sewage▿ †

PubMed Central

Loh, Joy; Zhao, Guoyan; Presti, Rachel M.; Holtz, Lori R.; Finkbeiner, Stacy R.; Droit, Lindsay; Villasana, Zoilmar; Todd, Collin; Pipas, James M.; Calgua, Byron; Girones, Rosina; Wang, David; Virgin, Herbert W.

2009-01-01

The family Asfarviridae contains only a single virus species, African swine fever virus (ASFV). ASFV is a viral agent with significant economic impact due to its devastating effects on populations of domesticated pigs during outbreaks but has not been reported to infect humans. We report here the discovery of novel viral sequences in human serum and sewage which are clearly related to the asfarvirus family but highly divergent from ASFV. Detection of these sequences suggests that greater genetic diversity may exist among asfarviruses than previously thought and raises the possibility that human infection by asfarviruses may occur. PMID:19812170

42 CFR 71.53 - Requirements for importers of nonhuman primates.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Trade in Endangered Species. ELISA means enzyme-linked immunosorbent assay, a type of laboratory test... serum for immunoglobulin G (IgG) antibodies to filovirus by using an ELISA methodology, or other method... for filovirus antigen by using the antigen-capture ELISA method must be submitted to a qualified...

42 CFR 71.53 - Requirements for importers of nonhuman primates.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Trade in Endangered Species. ELISA means enzyme-linked immunosorbent assay, a type of laboratory test... serum for immunoglobulin G (IgG) antibodies to filovirus by using an ELISA methodology, or other method... for filovirus antigen by using the antigen-capture ELISA method must be submitted to a qualified...

Milk and serum standard reference materials for monitoring organic contaminants in human samples.

PubMed

Schantz, Michele M; Eppe, Gauthier; Focant, Jean-François; Hamilton, Coreen; Heckert, N Alan; Heltsley, Rebecca M; Hoover, Dale; Keller, Jennifer M; Leigh, Stefan D; Patterson, Donald G; Pintar, Adam L; Sharpless, Katherine E; Sjödin, Andreas; Turner, Wayman E; Vander Pol, Stacy S; Wise, Stephen A

2013-02-01

Four new Standard Reference Materials (SRMs) have been developed to assist in the quality assurance of chemical contaminant measurements required for human biomonitoring studies, SRM 1953 Organic Contaminants in Non-Fortified Human Milk, SRM 1954 Organic Contaminants in Fortified Human Milk, SRM 1957 Organic Contaminants in Non-Fortified Human Serum, and SRM 1958 Organic Contaminants in Fortified Human Serum. These materials were developed as part of a collaboration between the National Institute of Standards and Technology (NIST) and the Centers for Disease Control and Prevention (CDC) with both agencies contributing data used in the certification of mass fraction values for a wide range of organic contaminants including polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, polybrominated diphenyl ether (PBDE) congeners, and polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners. The certified mass fractions of the organic contaminants in unfortified samples, SRM 1953 and SRM 1957, ranged from 12 ng/kg to 2200 ng/kg with the exception of 4,4'-DDE in SRM 1953 at 7400 ng/kg with expanded uncertainties generally <14 %. This agreement suggests that there were no significant biases existing among the multiple methods used for analysis.

Milk and serum standard reference materials for monitoring organic contaminants in human samples

PubMed Central

Eppe, Gauthier; Focant, Jean-François; Hamilton, Coreen; Heckert, N. Alan; Heltsley, Rebecca M.; Hoover, Dale; Keller, Jennifer M.; Leigh, Stefan D.; Patterson, Donald G.; Pintar, Adam L.; Sharpless, Katherine E.; Sjödin, Andreas; Turner, Wayman E.; Vander Pol, Stacy S.; Wise, Stephen A.

2016-01-01

Four new Standard Reference Materials (SRMs) have been developed to assist in the quality assurance of chemical contaminant measurements required for human biomonitoring studies, SRM 1953 Organic Contaminants in Non-Fortified Human Milk, SRM 1954 Organic Contaminants in Fortified Human Milk, SRM 1957 Organic Contaminants in Non-Fortified Human Serum, and SRM 1958 Organic Contaminants in Fortified Human Serum. These materials were developed as part of a collaboration between the National Institute of Standards and Technology (NIST) and the Centers for Disease Control and Prevention (CDC) with both agencies contributing data used in the certification of mass fraction values for a wide range of organic contaminants including polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, polybrominated diphenyl ether (PBDE) congeners, and polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners. The certified mass fractions of the organic contaminants in unfortified samples, SRM 1953 and SRM 1957, ranged from 12 ng/kg to 2200 ng/kg with the exception of 4,4′-DDE in SRM 1953 at 7400 ng/kg with expanded uncertainties generally <14 %. This agreement suggests that there were no significant biases existing among the multiple methods used for analysis. PMID:23132544

Pre-flight report on cultured human embryonic kidney cell handling and cell electrophoresis. Prepared prior to continuous-flow electrophoretic separation experiments aboard space shuttle flight STS-8

NASA Technical Reports Server (NTRS)

Todd, P. W.; Sarnoff, B. E.; Li, Z. K.

1985-01-01

Studies of the physical properties of continuous-flow zero-G electrophoretic separator (CFES) buffer, the electrokinetic properties of human erythrocytes in the CFES buffer, the electrokinetic properties of human embryonic kidney cells in the CFES buffer, and the viability and yield of human embryonc kidney cells subjected to flight handling procedures are discussed. In general, the procedure for cell handling and electrophoresis of HEK-8514 cells in 1st or 2nd passage on STS-8 is acceptable if executed properly. The CFES buffer has ionic strength that is barely compatible with cell viability and membrane stability, as seen in experiments with human erythrocytes and trypan-blue staining of human kidney cells. Cells suspended in 10% dialysed horse serum for 3 days in the cold appear to be more stable than freshly trypsinized cells. 10% horse serum appears to be superior to 5% horse serum for this purpose. The mean absolute raw mobility of HEK-8514 cells in CFES buffer at 6 degrees, conductivity 0.055 mmho/cm, is 1.1 to 1.4 um-cm/V-sec, with a range of nearly a whole mobility unit.

The hook effect in calcitonin immunoradiometric assay: a case report.

PubMed

Fangous, Marie-Sarah; Kerspern, Hélène; Moineau, Marie-Pierre; Kerlan, Véronique; Alavi, Zarrin; Carré, Jean-Luc

2012-12-01

The hook effect, which has long been detected and documented for immunoradiometric assays (IRMA) such as those measuring prolactin or thyroglobulin, occurs when the serum antigen level is extremely high, thus inducing a bias in the methodology of measurement. We report the case of an 80-year-old man with confirmed medullary thyroid carcinoma (MTC). In the case reported here, the clinical status of the patient contrasts with his tumor antigen, serum calcitonin (CT), concentrations. The measured increased CT concentrations revealed the presence of a hook effect. This phenomenon occurs due to an excess of antigen during the one-step IRMA where the signal antibodies, bound to the non-captured antigens, are washed out during the measurement, inducing the loss of signal. Aiming to prevent the "hook effect", successive dilutions of the same sample of serum were done. Previous studies have shown when one-step IRMA reveals high concentrations of a tumor serum antigen (i.e. prolactin or thyroglobulin), a two-step IRMA or a systematic 1:10 dilution of the serum sample prevents the formation of the "hook effect". In our case report, the CT "hook effect" formation was prevented by performing serial dilutions of the serum sample. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Active transport of cimetidine into human milk.

PubMed

Oo, C Y; Kuhn, R J; Desai, N; McNamara, P J

1995-11-01

Most xenobiotics are transferred from blood into breast milk by passive diffusion. However, an active transport mechanism has been speculated for cimetidine, and the purpose of this study was to characterize cimetidine transfer into human milk. Twelve healthy lactating volunteers received single oral doses of 100, 600, and 1200 mg cimetidine in a randomized, crossover design on 3 different days. Blood and milk specimens were collected and assayed for cimetidine. In vitro measurements, including skim to whole milk concentration ratio, milk pH, and free fractions in serum and milk were used for a diffusion model prediction of milk to serum concentration ratio of cimetidine; the mean milk/serum ratio (+/- SD) was 1.05 +/- 0.18. The observed milk/serum ratio (5.77 +/- 1.24) was 5.5 times higher than the milk/serum ratio predicted by diffusion. The observed milk/serum ratio for the three dosing regimens were not significantly different from one another. Time of peak concentration (tmax) in milk (3.3 +/- 0.7 hours) displayed a delay compared with serum tmax (1.7 +/- 0.6 hours). Oral clearance for 1200 mg cimetidine dose (0.47 +/- 0.11 L/hr/kg) was significantly lower compared with oral clearance values for 100 and 600 mg cimetidine doses (0.59 +/- 0.11 and 0.57 +/- 0.13 L/hr/kg, respectively). The maternal dose of cimetidine ingested by a suckling infant based on body weight was estimated to be 6.7%, which appears to be safe under normal conditions. This study provides the first definitive evidence of an active transport system for drug transfer into human milk, which may have broader consequences for the suckling infant.

High Consumption of Iron Exacerbates Hyperlipidemia, Atherosclerosis, and Female Sterility in Zebrafish via Acceleration of Glycation and Degradation of Serum Lipoproteins.

PubMed

Kim, So-Hee; Yadav, Dhananjay; Kim, Suk-Jeong; Kim, Jae-Ryong; Cho, Kyung-Hyun

2017-07-02

Elevated serum iron level is linked with an increased risk of diabetes and atherosclerosis. However, the pathological mechanism by which iron affects serum lipoprotein levels is unknown. To elucidate the mechanism, a high dose of ferrous ion was applied (final 60 µM, 120 µM) to human serum lipoproteins, macrophages, and human dermal fibroblast (HDF) cells. Iron-treated lipoproteins showed loss of antioxidant ability along with protein degradation and multimerization, especially co-treatment with fructose (final 10 mM). In the presence of fructose, HDF cells showed 3.5-fold more severe cellular senescence, as compared to the control, dependent on the dosage of fructose. In macrophages, phagocytosis of acetylated low-density lipoprotein (acLDL) was more accelerated by ferrous ion, occurring at a rate that was up to 1.8-fold higher, than acLDL alone. After 24 weeks supplementation with 0.05% and 0.1% ferrous ion in the diet (wt/wt), serum total cholesterol (TC) level was elevated 3.7- and 2.1-fold, respectively, under normal diet (ND). Serum triglyceride (TG) was elevated 1.4- and 1.7-fold, respectively, under ND upon 0.05% and 0.1% ferrous ion supplementation. Serum glucose level was elevated 2.4- and 1.2-fold under ND and high cholesterol diet (HCD), respectively. However, body weight was decreased by the Fe 2+ consumption. Iron consumption caused severe reduction of embryo laying and reproduction ability, especially in female zebrafish via impairment of follicular development. In conclusion, ferrous ion treatment caused more pro-atherogenic, and pro-senescence processes in human macrophages and dermal cells. High consumption of iron exacerbated hyperlipidemia and hyperglycemia as well as induced fatty liver changes and sterility along with reduction of female fertility.

Determination of nifedipine in serum of women in preterm labor by high-performance liquid chromatography with diode array detection.

PubMed

Kowalczuk, Dorota; Wawrzycka, Maria Bozena; Haratym Maj, Agnieszka

2006-01-01

Nifedipine (Nif) is widely used in treating cardiovascular disorders (especially hypertension) and for inhibiting preterm labor. A fully validated selective high-performance liquid chromatographic method with diode array detection, using solid-phase extraction, was developed for the determination of Nif in human serum. To assess specificity, Nif and its degradation products were separated on a Purospher RP-18 (5 microm, 125 x 4 mm) column plus a LiChrospher 100 RP-18 (5 microm, 4 x 4 mm) precolumn with a mobile phase of methanol-10 mM aqueous trifluoroacetic acid, pH 7.3 (57 + 43, v/v); chromatographic separation was followed by UV detection at 238 nm. For toxicological analysis, Nif in the presence of other calcium-channel antagonist drugs was identified under optimum chromatographic conditions. The calibration graph was constructed over the concentration range of 12.5-400 ng/mL in serum with good correlation (r = 0.9956). This method was not subject to interference by other plasma components and was successfully applied to the assay of Nif in spiked human serum and in serum of women in preterm labor after sublingual administration of 30 mg Nif per day divided into 3 equal doses. The mean recovery based on the ratio of the slopes of serum and mobile phase standard curves was 96.5%. The detection and quantification limits of the drug in spiked human serum were found to be 6 and 17.5 ng/mL, respectively. Validation of the method demonstrated good intraday and interday precision, which ranged from 2.18 to 6.67% and from 6.52 to 11.93%, respectively.

Radiation therapy induces circulating serum Hsp72 in patients with prostate cancer.

PubMed

Hurwitz, Mark D; Kaur, Punit; Nagaraja, Ganachari M; Bausero, Maria A; Manola, Judith; Asea, Alexzander

2010-06-01

Hsp72 found in the extracellular milieu has been shown to play an important role in immune regulation. The impact of common cancer therapies on extracellular release of Hsp72 however, has been to date undefined. Serum from 13 patients undergoing radiation therapy (XRT) for prostate cancer with or without hormonal therapy (ADT) was measured for levels of circulating serum Hsp72 and pro-inflammatory cytokines (IL-6 and TNF-alpha) using the classical sandwich ELISA technique and the relative expression of CD8(+) T lymphocytes and natural killer (NK) cells was measured using flow cytometry. Mouse orthotopic xenograft of human prostate cancer tumors (DU-145 and PC-3) were used to validate and further characterize the response noted in the clinical setting. The biological significance of tumor released Hsp72 was studied in human dendritic cells (DC) in vitro. Circulating serum Hsp72 levels increased an average of 3.5-fold (median per patient 4.8-fold) with XRT but not with ADT (p=0.0002). Increases in IL-6 (3.3-fold), TNF-alpha (1.8-fold), CD8(+) CTL (2.1-fold) and NK cells (3.2-fold) also occurred. Using PC-3 and DU-145 human prostate cancer xenograft models in mice, we confirmed that XRT induces Hsp72 release primarily from implanted tumors. In vitro studies using supernatant recovered from irradiated human prostate cancer cells point to exosomes containing Hsp72 as a possible stimulator of pro-inflammatory cytokine production and costimulatory molecules expression in human DC. The current study confirms for the first time in an actual clinical setting elevation of circulating serum Hsp72 with XRT. The accompanying studies in mice and in vitro identify the released exosomes containing Hsp72 as playing a pivotal role in stimulating pro-inflammatory immune responses. These findings, if validated, may lead to new treatment paradigms for common human malignancies. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Radiation therapy induces circulating serum Hsp72 in patients with prostate cancer

PubMed Central

Hurwitz, Mark D.; Kaur, Punit; Nagaraja, Ganachari M.; Bausero, Maria A.; Manola, Judith; Asea, Alexzander

2010-01-01

Background and purpose Hsp72 found in the extracellular milieu has been shown to play an important role in immune regulation. The impact of common cancer therapies on extracellular release of Hsp72 however, has been to date undefined. Materials and methods Serum from 13 patients undergoing radiation therapy (XRT) for prostate cancer with or without hormonal therapy (ADT) was measured for levels of circulating serum Hsp72 and pro-inflammatory cytokines (IL-6 and TNF-α) using the classical sandwich ELISA technique and the relative expression of CD8+ T lymphocytes and natural killer (NK) cells was measured using flow cytometry. Mouse orthotopic xenograft of human prostate cancer tumors (DU145 and PC3) were used to validate and further characterize the response noted in the clinical setting. The biological significance of tumor released Hsp72 was studied in human dendritic cells (DC) in vitro. Results Circulating serum Hsp72 levels increased an average of 3.5-fold (median per patient 4.8-fold) with XRT but not with ADT (p = 0.0002). Increases in IL-6 (3.3-fold), TNF-α (1.8-fold), CD8+ CTL (2.1-fold) and NK cells (3.2-fold) also occurred. Using PC3 and DU145 human prostate cancer xenograft models in mice, we confirmed that XRT induces Hsp72 release primarily from implanted tumors. In vitro studies using supernatant recovered from irradiated human prostate cancer cells point to exosomes containing Hsp72 as a possible stimulator of pro-inflammatory cytokine production and costimulatory molecules expression in human DC. Conclusions The current study confirms for the first time in an actual clinical setting elevation of circulating serum Hsp72 with XRT. The accompanying studies in mice and in vitro identify the released exosomes containing Hsp72 as playing a pivotal role in stimulating pro-inflammatory immune responses. These findings, if validated, may lead to new treatment paradigms for common human malignancies. PMID:20430459

Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

PubMed

Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

2014-11-01

Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides. Copyright © 2014 Elsevier Inc. All rights reserved.

Serum 25-hydroxyvitamin d levels and C-reactive protein in persons with human immunodeficiency virus infection.

PubMed

Poudel-Tandukar, Kalpana; Poudel, Krishna C; Jimba, Masamine; Kobayashi, Jun; Johnson, C Anderson; Palmer, Paula H

2013-03-01

Human immunodeficiency virus (HIV) infection has frequently been associated with vitamin D deficiency as well as chronic inflammatory response. We tested the hypothesis of an independent relationship between serum concentrations of 25-hydroxyvitamin D [25(OH)D] and high-sensitivity C-reactive protein (CRP) in a cohort of HIV-positive people. A cross-sectional survey was conducted among 316 HIV-positive people (181 men and 135 women) aged 16 to 60 years residing in the Kathmandu Valley, Nepal. Serum high-sensitivity CRP concentrations and serum 25(OH)D levels were measured by the latex agglutination nephelometry method and the competitive protein-binding assay, respectively. The relationship between serum CRP concentrations and 25(OH)D serum level was assessed using multiple logistic regression analysis with adjustment of potential cardiovascular and HIV-related factors. The proportions of participants with 25(OH)D serum levels <20 ng/ml, 20-30 ng/ml, and ≥30 ng/ml were 83.2%, 15.5%, and 1.3%, respectively. The mean 25(OH)D serum levels in men and women were 15.3 ng/ml and 14.4 ng/ml, respectively. Participants with a 25(OH)D serum level of <20 ng/ml had a 3.2-fold higher odds of high CRP (>3 mg/liter) compared to those with a 25(OH)D serum level of ≥20 ng/ml (p=0.005). Men and women with a 25(OH)D serum level of <20 ng/ml had 3.2- and 2.7-fold higher odds of high CRP (>3 mg/liter), respectively, compared to those with a 25(OH)D serum level of ≥20 ng/ml. The relationships remained significant only in men (p =0.02) but not in women (p=0.28). The risk of having a high level of inflammation (CRP>3 mg/liter) may be high among HIV-positive men and women with a 25(OH)D serum level of <20 ng/ml.

Novel Replication-Competent Circular DNA Molecules from Healthy Cattle Serum and Milk and Multiple Sclerosis-Affected Human Brain Tissue

PubMed Central

Whitley, Corinna; Gunst, Karin; Müller, Hermann; Funk, Mathis; zur Hausen, Harald

2014-01-01

Epidemiological data point to the involvement of a cow milk factor in the etiology of multiple sclerosis (MS). Eleven circular DNA molecules closely related to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 1.76 were isolated from healthy cattle serum, cow milk, and serum and brain tissue from MS patients. PMID:25169859

Polybrominated Diphenyl Ethers in Human Milk and Serum from the U.S. EPA MAMA Study: Modeled Predictions of Infant Exposure and Considerations for Risk Assessment

EPA Science Inventory

Background: Serum concentrations of polybrominated diphenyl ethers (PBDEs) in U.S. women are believed to be among the world’s highest; however, little information exists on the partitioning of PBDEs between serum and breast milk and how this may affect infant exposure. Obj...

Polybrominated Diphenyl Ethers in Human Milk and Serum from the U.S. EPA MAMA Study: Modeled Predictions of Infant Exposure and Considerations for Risk Assessment

EPA Science Inventory

Background: Serum concentrations of polybrominated diphenyl ethers (PBDEs) in U.S. women are believed to be among the world’s highest; however, little information exists on the partitioning of PBDEs between serum and breast milk and how this may affect infant exposure. Objecti...

Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum.

PubMed

Dussart, Philippe; Labeau, Bhety; Lagathu, Gisèle; Louis, Philippe; Nunes, Marcio R T; Rodrigues, Sueli G; Storck-Herrmann, Cécile; Cesaire, Raymond; Morvan, Jacques; Flamand, Marie; Baril, Laurence

2006-11-01

We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Regulation of leptin production in humans.

PubMed

Fried, S K; Ricci, M R; Russell, C D; Laferrère, B

2000-12-01

Serum levels of the adipocyte hormone leptin are increased in proportion to body fat stores as a result of increased production in enlarged fat cells from obese subjects. In vitro studies indicate that insulin and glucocorticoids work directly on adipose tissue to upregulate in a synergistic manner leptin mRNA levels and rates of leptin secretion in human adipose tissue over the long term. Thus, the increased leptin expression observed in obesity could result from the chronic hyperinsulinemia and increased cortisol turnover. Superimposed upon the long-term regulation, nutritional status can influence serum leptin over the short term, independent of adiposity. Fasting leads to a gradual decline in serum leptin that is probably attributable to the decline in insulin and the ability of catecholamines to decrease leptin expression, as observed in both in vivo and in vitro studies. In addition, increases in serum leptin occur approximately 4-7 h after meals. Increasing evidence indicates that insulin, in concert with permissive effects of cortisol, can increase serum leptin over this time frame and likely contributes to meal-induced increases in serum leptin. Further research is required to elucidate the cellular and molecular mechanisms underlying short- and long-term nutritional and hormonal regulation of leptin production and secretion.

The beneficial effects of honeybee-venom serum on facial wrinkles in humans

PubMed Central

Han, Sang Mi; Hong, In Phyo; Woo, Soon Ok; Chun, Sung Nam; Park, Kwan Kyu; Nicholls, Young Mee; Pak, Sok Cheon

2015-01-01

Facial wrinkles are an undesirable outcome caused by extrinsic photodamage and intrinsic aging processes. Currently, no effective strategies are known to prevent facial wrinkles. We assessed the beneficial effects of bee-venom serum on the clinical signs of aging skin. Our results show that bee-venom serum treatment clinically improved facial wrinkles by decreasing total wrinkle area, total wrinkle count, and average wrinkle depth. Therefore, bee-venom serum may be effective for the improvement of skin wrinkles. PMID:26491274

21 CFR 114.90 - Methodology.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 2 2013-04-01 2013-04-01 false Methodology. 114.90 Section 114.90 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION ACIDIFIED FOODS Production and Process Controls § 114.90 Methodology. Methods that may be used to...

21 CFR 114.90 - Methodology.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 2 2014-04-01 2014-04-01 false Methodology. 114.90 Section 114.90 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION ACIDIFIED FOODS Production and Process Controls § 114.90 Methodology. Methods that may be used to...

21 CFR 114.90 - Methodology.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 2 2012-04-01 2012-04-01 false Methodology. 114.90 Section 114.90 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION ACIDIFIED FOODS Production and Process Controls § 114.90 Methodology. Methods that may be used to...

Financial Support for the Humanities: A Special Methodological Report.

ERIC Educational Resources Information Center

Gomberg, Irene L.; Atelsek, Frank J.

Findings and methodological problems of a survey on financial support for humanities in higher education are discussed. Usable data were gathered from 351 of 671 Higher Education Panel member institutions. Two weighting methodologies were employed. The conventional method assumed that nonrespondents were similar to respondents, whereas a…

Anti-complement activities of human breast-milk.

PubMed

Ogundele, M O

1999-08-01

It has long been observed that the human milk possesses significant anti-inflammatory properties, while simultaneously protecting the infant against many intestinal and respiratory pathogens. There is, however, a paucity of information on the degree and extent of this anti-inflammatory activity. In the present study, the inhibitory effects of different fractions of human milk on serum complement activity were analysed. Colostrum and milk samples from healthy voluntary lactating donors at different postpartum ages were obtained and pooled normal human serum was used as source of complement in a modified CH50 assay. Inherent complement activity in human milk was also investigated by measuring the deposition of an activated C3 fragment on a serum-sensitive bacteria, and by haemolytic assays. Most whole- and defatted-milk samples consistently showed a dose-dependent inhibition of the serum complement activity. This inhibition was greater in mature milk compared to transitional milk samples. It was enhanced by inactivation of milk complement, and diminished by centrifugation of milk samples, which partly removed fat and larger protein components including casein micelles. Inherent complement activity in human milk was also demonstrated by haemolysis of sensitised sheep erythrocytes and deposition of C3 fragments on solid-phase bacteria. These activities were highest in the colostrum and gradually decreased as lactation proceeded. Several natural components abundant in the fluid phase of the human breast-milk have been shown to be inhibitors of complement activation in vitro. Their physiological significance probably reside in their ability to prevent inflammatory-induced tissue damage of the delicate immature gastrointestinal tract of the new-born as well as the mammary gland itself, which may arise from ongoing complement activation.

Determination of novel brominated flame retardants and polybrominated diphenyl ethers in serum using gas chromatography-mass spectrometry with two simplified sample preparation procedures.

PubMed

Gao, Le; Li, Jian; Wu, Yandan; Yu, Miaohao; Chen, Tian; Shi, Zhixiong; Zhou, Xianqing; Sun, Zhiwei

2016-11-01

Two simple and efficient pretreatment procedures have been developed for the simultaneous extraction and cleanup of six novel brominated flame retardants (NBFRs) and eight common polybrominated diphenyl ethers (PBDEs) in human serum. The first sample pretreatment procedure was a quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based approach. An acetone/hexane mixture was employed to isolate the lipid and analytes from the serum with a combination of MgSO 4 and NaCl, followed by a dispersive solid-phase extraction (d-SPE) step using C18 particles as a sorbent. The second sample pretreatment procedure was based on solid-phase extraction. The sample extraction and cleanup were conducted directly on an Oasis HLB SPE column using 5 % aqueous isopropanol, concentrated sulfuric acid, and 10 % aqueous methanol, followed by elution with dichloromethane. The NBFRs and PBDEs were then detected using gas chromatography-negative chemical ionization mass spectrometry (GC-NCI MS). The methods were assessed for repeatability, accuracy, selectivity, limits of detection (LODs), and linearity. The results of spike recovery experiments in fetal bovine serum showed that average recoveries ranged from 77.9 % to 128.8 % with relative standard deviations (RSDs) from 0.73 % to 12.37 % for most of the analytes. The LODs for the analytes in fetal bovine serum ranged from 0.3 to 50.8 pg/mL except for decabromodiphenyl ethane. The proposed method was successfully applied to the determination of the 14 brominated flame retardants in human serum. The two pretreatment procedures described here are simple, accurate, and precise, and are suitable for the routine analysis of human serum. Graphical Abstract Workflow of a QuEChERS-based approach (top) and an SPE-based approach (bottom) for the detection of PBDEs and NBFRs in serum.

Effects of 2-year calorie restriction on circulating levels of IGF-1, IGF-binding proteins and cortisol in nonobese men and women: a randomized clinical trial.

PubMed

Fontana, Luigi; Villareal, Dennis T; Das, Sai K; Smith, Steven R; Meydani, Simin N; Pittas, Anastassios G; Klein, Samuel; Bhapkar, Manjushri; Rochon, James; Ravussin, Eric; Holloszy, John O

2016-02-01

Young-onset calorie restriction (CR) in rodents decreases serum IGF-1 concentration and increases serum corticosterone levels, which have been hypothesized to play major roles in mediating its anticancer and anti-aging effects. However, little is known on the effects of CR on the IGF-1 system and cortisol in humans. To test the sustained effects of CR on these key hormonal adaptations, we performed a multicenter randomized trial of a 2-year 25% CR intervention in 218 nonobese (body mass index between 22 and 27.8 kg m(-2) ) young and middle-aged (20-50 years age range) men and women. Average CR during the first 6 months was 19.5 ± 0.8% and 9.1 ± 0.7% over the next 18 months of the study. Weight loss averaged 7.6 ± 0.3 kg over the 2-years period of which 71% was fat mass loss (P < 0.0001). Average CR during the CR caused a significant 21% increase in serum IGFBP-1 and a 42% reduction in IGF-1:IGFBP-1 ratio at 2 years (P < 0.008), but did not change IGF-1 and IGF-1:IGFBP-3 ratio levels. Serum cortisol concentrations were slightly but significantly increased by CR at 1 year only (P = 0.003). Calorie restriction had no effect on serum concentrations of PDGF-AB and TGFβ-1. We conclude, on the basis of the present and previous findings, that, in contrast to rodents, humans do not respond to CR with a decrease in serum IGF-1 concentration or with a sustained and biological relevant increase in serum cortisol. However, long-term CR in humans significantly and persistently increases serum IGFBP-1 concentration. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

PubMed

Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

2014-02-01

Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

NASA Astrophysics Data System (ADS)

He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

2017-05-01

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines.

PubMed

Chen, Di; Xin, Xiao-Xuan; Qian, Hao-Cheng; Yu, Zhang-Yin; Shen, Li-Rong

2016-06-01

Royal jelly (RJ) is a well-known bioactive substance. It contains large amounts of major royal jelly proteins (MRJPs), which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum (FBS) in different types of human cell culture, the proliferation of the complex serum with different ratios of MRJPs/FBS (M/F) was evaluated on five cell lines: 293T, HFL-I, 231, HCT116, and Changliver using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor (EGF) and insulin-transferrin-selenium (ITS), promoted proliferations of Changliver, 293T, HCT116, and HFL-I by 18.73%‒56.19% (P<0.01). Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.

The effect of intravenous lactated Ringer's solution versus 0.9% sodium chloride solution on serum osmolality in human volunteers.

PubMed

Williams, E L; Hildebrand, K L; McCormick, S A; Bedel, M J

1999-05-01

Animal studies have shown that large volumes of IV lactated Ringer's solution (LR) decrease serum osmolality, thereby increasing cerebral water. These studies have led to recommendations to limit LR to avoid cerebral edema in neurosurgical patients. Eighteen healthy human volunteers aged 20-48 yr received 50 mL/kg LR over 1 h on one occasion and 0.9% sodium chloride (NS) on another. Venous samples were taken at baseline (T1), at infusion end (T2), and 1 h after T2 (T3). Time until first urination was noted. With LR, serum osmolality decreased by 4+/-3 mOsm/kg from T1 to T2 and increased insignificantly with NS. At T3, osmolality returned almost to baseline in the LR group. Blood pH increased from T1 to T2 with LR by 0.04+/-0.04 and decreased with NS by 0.04+/-0.04. These pH changes persisted at T3. Subjective mental changes occurred only with NS. Abdominal discomfort was more common with NS. Time until first urination was longer with NS (106+/-11 min) than with LR (75+/-10 min) (P < 0.001). In healthy humans, an infusion of large volumes of LR, but not NS, transiently decreased serum osmolality, whereas acidosis associated with NS persisted and urinary output was slower with NS. Large volumes of lactated Ringer's solution administered to healthy humans produced small transient changes in serum osmolality. Large volumes of sodium chloride did not change osmolality but resulted in lower pH.

Isolation and culture of primary human pancreatic stellate cells that reflect the context of their tissue of origin.

PubMed

Strobel, Oliver; Dadabaeva, Nigora; Felix, Klaus; Hackert, Thilo; Giese, Nathalia A; Jesenofsky, Ralf; Werner, Jens

2016-02-01

Pancreatic stellate cells (PSCs) play a critical role in pancreatic ductal adenocarcinoma (PDAC). Activated PSCs are the main source of fibrosis in chronic pancreatitis and of desmoplasia in PDAC. The majority of studies on PSC are based on in vitro experiments relying on immortalized cell lines derived from diseased human pancreas or from animal models. These PSCs are usually activated and may not represent the biological context of their tissue of origin. (1) To isolate and culture primary human PSC from different disease contexts with minimal impact on their state of activation. (2) To perform a comparative analysis of phenotypes of PSC derived from different contexts. PSCs were isolated from normal pancreas, chronic pancreatitis, and PDAC using a hybrid method of digestion and outgrowth. To minimize activation by serum compounds, cells were cultured in a low-serum environment (2.5 % fetal bovine serum (FBS)). Expression patterns of commonly used markers for PSC phenotype and activity were compared between primary PSC lines derived from different contexts and correlated to expression in their original tissues. Isolation was successful from 14 of 17 tissues (82 %). Isolated PSC displayed stable viability and phenotype in low-serum environment. Expression profiles of isolated PSC and matched original tissues were closely correlated. PDAC-derived PSC tended to have a higher status of activation if compared to PSC derived from non-cancerous tissues. Primary human PSCs isolated from different contexts and cultured in a low-serum environment maintain a phenotype that reflects the stromal activity present in their tissue of origin.

Measuring and Validating the Levels of Brain-Derived Neurotrophic Factor in Human Serum

PubMed Central

Naegelin, Yvonne; Dingsdale, Hayley; Säuberli, Katharina; Schädelin, Sabine; Kappos, Ludwig

2018-01-01

Brain-derived neurotrophic factor (BDNF) secreted by neurons is a significant component of synaptic plasticity. In humans, it is also present in blood platelets where it accumulates following its biosynthesis in megakaryocytes. BDNF levels are thus readily detectable in human serum and it has been abundantly speculated that they may somehow serve as an indicator of brain function. However, there is a great deal of uncertainty with regard to the range of BDNF levels that can be considered normal, how stable these values are over time and even whether BDNF levels can be reliably measured in serum. Using monoclonal antibodies and a sandwich ELISA, this study reports on BDNF levels in the serum of 259 volunteers with a mean value of 32.69 ± 8.33 ng/ml (SD). The mean value for the same cohort after 12 months was not significantly different (N = 226, 32.97 ± 8.36 ng/ml SD, p = 0.19). Power analysis of these values indicates that relatively large cohorts are necessary to identify significant differences, requiring a group size of 60 to detect a 20% change. The levels determined by ELISA could be validated by Western blot analyses using a BDNF monoclonal antibody. While no association was observed with gender, a weak, positive correlation was found with age. The overall conclusions are that BDNF levels can be reliably measured in human serum, that these levels are quite stable over one year, and that comparisons between two populations may only be meaningful if cohorts of sufficient sizes are assembled. PMID:29662942

Correlation between μCT imaging, histology and functional capacity of the osteoarthritic knee in the rat model of osteoarthritis.

PubMed

Bagi, Cedo M; Zakur, David E; Berryman, Edwin; Andresen, Catharine J; Wilkie, Dean

2015-08-25

To acquire the most meaningful understanding of human arthritis, it is essential to select the disease model and methodology translatable to human conditions. The primary objective of this study was to evaluate a number of analytic techniques and biomarkers for their ability to accurately gauge bone and cartilage morphology and metabolism in the medial meniscal tear (MMT) model of osteoarthritis (OA). MMT surgery was performed in rats to induce OA. A dynamic weight bearing system (DWB) system was deployed to evaluate the weight-bearing capacity of the front and hind legs in rats. At the end of a 10-week study cartilage pathology was evaluated by micro computed tomography (μCT), contrast enhanced μCT (EPIC μCT) imaging and traditional histology. Bone tissue was evaluated at the tibial metaphysis and epiphysis, including the subchondral bone. Histological techniques and dynamic histomorphometry were used to evaluate cartilage morphology and bone mineralization. The study results showed a negative impact of MMT surgery on the weight-bearing capacity of the operated limb. Surgery caused severe and extensive deterioration of the articular cartilage at the medial tibial plateau, as evidenced by elevated CTX-II in serum, EPIC μCT and histology. Bone analysis by μCT showed thickening of the subchondral bone beneath the damaged cartilage, loss of cancellous bone at the metaphysis and active osteophyte formation. The study emphasizes the need for using various methodologies that complement each other to provide a comprehensive understanding of the pathophysiology of OA at the organ, tissue and cellular levels. Results from this study suggest that use of histology, μCT and EPIC μCT, and functional DWB tests provide powerful combination to fully assess the key aspects of OA and enhance data interpretation.

Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis

NASA Astrophysics Data System (ADS)

Szkudlarek, A.; Sułkowska, A.; Maciążek-Jurczyk, M.; Chudzik, M.; Równicka-Zubik, J.

2016-01-01

Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors - glycation of HSA - occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSAGLC) with HSA glycated by fructose (gHSAFRC). We focused on presenting the differences between gHSAFRC and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335 nm and λem 420 nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSAFRC is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSAFRC becomes less accessible for the negatively charged quencher (I-), KSV value is smaller for gHSAFRC than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the unmodified serum albumin, while KSV values for gHSA-KP systems are only slightly lower than that obtained for HSA-KP. The affinity of PHB to the glycated HSA is stronger than to the non-glycated in the first class binding sites within subdomain IIA, in the vicinity of Trp-214. Ketoprofen bound to unmodified human serum albumin stronger than for glycated albumin and one class of binding sites is observed (Scatchard linear plots).

Homogeneous immunoglobulins in sera of Rhesus monkeys after lethal irradiation and bone marrow transplantation

PubMed Central

Rádl, J.; van den Berg, P.; Voormolen, M.; Hendriks, W. D. H.; Schaefer, U. W.

1974-01-01

The immunoglobulin pattern in the sera of lethally irradiated and bone marrow transplanted Rhesus monkeys was studied during the reconstitution of their immune system. All of the irradiated monkeys which survived longer than 30 days, and in which reconstitution of their immune system took place, also developed homogeneous immunoglobulins (HI) in their sera. These homogeneous, sometimes multiple, immunoglobulins were transient. However, they persisted frequently in the sera for several months. In two monkeys which were additionally immunized with a complex antigen (normal human serum), clear-cut M-components appeared in the serum about 10 days later. These HI of IgG class did not precipitate the antigen in immunodiffusion techniques; however, when passing the serum through an immunoadsorbent prepared from normal human serum, only the HI were specifically retained on the column and afterwards isolated by elution. ImagesFIG. 1FIG. 2 PMID:4143277

Increased (/sup 125/I)trypsin-binding in serum from cystic fibrosis patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cox, K.L.; Frates, R.C. Jr.; Sheikholislam, B.M.

1982-01-01

The capacities of normal and cystic fibrosis (CF) sera to bind to exogenous human (/sup 125/I)trypsin were compared. Sera from eight older CF patients bound significantly more exogenous human (/sup 125/I)trypsin than did sera from eight normal subjects (p less than 0.001). Disregarding the increased trypsin-binding (TB) of CF sera, serum immunoreactive trypsinogen (SIRT) levels were not detectable in these eight older CF patients. However, when SIRT levels were corrected for TB, four CF patients had normal SIRT concentrations and four had low but detectable SIRT levels. As compared to five normal newborns' sera, serum from a newborn with CFmore » had normal TB and the SIRT levels were very high. In conclusion, increased TB in CF serum lowers results of SIRT assays. Therefore, unless SIRT levels are corrected for TB, results obtained from currently available SIRT kits may be invalid.« less

Correlation of serum unconjugated oestriol to red cell 2,3-diphosphoglycerate levels in diabetic pregnancy.

PubMed

Madsen, H; Ditzel, J

1983-03-01

In order to evaluate the possible underlying factors for the increase in red cell 2,3-diphosphoglycerate content observed in late diabetic pregnancy, its relationship with serum unconjugated oestriol, human placental lactogen, haemoglobin and hydrogen ion concentrations was investigated in 42 pregnant diabetic women. A significant correlation was found between red cell 2,3-diphosphoglycerate and serum unconjugated oestriol (r = 0.54, p less than 0.001), whereas no correlation was present between 2,3-diphosphoglycerate and the following variables: arterial pH, haemoglobin concentration and human placental lactogen. The content of 2,3-diphosphoglycerate correlated significantly with haemoglobin-oxygen affinity expressed as P50 at pH 7.4 (r = 0.34, p less than 0.05). The results of this study indicate that serum unconjugated oestriol may participate in the regulation of red cell 2,3-diphosphoglycerate content and thereby of the maternal blood oxygen release to the fetus.

Ternary Complex Factors and Cofactors Are Essential for Human T-Cell Leukemia Virus Type 1 Tax Transactivation of the Serum Response Element

PubMed Central

Shuh, Maureen; Derse, David

2000-01-01

The human T-cell leukemia virus type 1 Tax protein activates the expression of cellular immediate early genes controlled by the serum response element (SRE), which contains both the serum response factor (SRF) binding element (CArG box) and the ternary complex factor (TCF) binding element (Ets box). We show that TCF binding is necessary for Tax activation of the SRE and that Tax directly interacts with TCFs in vitro. In addition, Tax interactions with CREB binding protein (CBP) and p300- and CBP-associated factor were found to be essential for Tax activation of SRF-mediated transcription. PMID:11070040

[Identification and characterization of proteins from human bronchial secretion (author's transl)].

PubMed

Laine, A; Hayem, A

1976-03-01

An analysis of bronchial mucus proteins was carried out by crossed immunoelectrophoresis. Before electrophoretic migration, sputum was treated with Ecteola-cellulose, which retains acid mucins. The proteins were then extracted by a phosphate/saline buffer pH 7.5. Crossed immunoelectrophoresis of the "bronchial extracts" was carried out with an anti-human serum: fifteen proteins were detected. Among them, IgA and protease inhibitiors play an important role in bronchial pathology. Bronchial extracts were also studied with immune serums against milk proteins, whole saliva and proteins of bronchial mucus. Bronchotransferrin, amylase and two esterases were characterized. Four other proteins were also detected with immune serums against bronchial mucus-proteins: their biological role is still unknown.

Effect of molecular parameters on the binding of phenoxyacetic acid derivatives to albumins.

PubMed

Cserháti, T; Forgács, E; Deyl, Z; Miksík, I

2001-03-25

The interaction of 12 phenoxyacetic acid derivatives with human and serum albumin as well as with egg albumin was studied by charge-transfer reversed-phase (RP) thin-layer chromatography (TLC) and the relative strength of interaction was calculated. Each phenoxyacetic acid derivative interacted with human and bovine serum albumins whereas no interaction was observed with egg albumin. Stepwise regression analysis proved that the lipophilicity of the derivatives exert a significant impact on their capacity to bind to serum albumins. This result supports the hypothesis that the binding of phenoxyacetic acid derivatives to albumins may involve hydrophobic forces occurring between the corresponding apolar substructures of these derivatives and the amino acid side chains.

Forster resonance energy transfer in the system of human serum albumin-xanthene dyes

NASA Astrophysics Data System (ADS)

Kochubey, V. I.; Pravdin, A. B.; Melnikov, A. G.; Konstantinova, I.; Alonova, I. V.

2016-04-01

The processes of interaction of fluorescent probes: eosin and erythrosine with human serum albumin (HSA) were studied by the methods of absorption and fluorescence spectroscopy. Extinction coefficients of probes were determined. Critical transfer radius and the energy transfer efficiency were defined by fluorescence quenching of HSA. Analysis of the excitation spectra of HSA revealed that the energy transfer process is carried out mainly between tryptophanyl and probes.

Determination of estradiol valerate in pharmaceutical preparations and human serum by flow injection chemiluminescence.

PubMed

Liu, Wenwen; Xie, Liangxiao; Liu, Hongshuang; Xu, Shichao; Hu, Bingcheng; Cao, Wei

2013-01-01

A novel method for the detection of trace estradiol valerate (EV) in pharmaceutical preparations and human serum was developed by inhibition of luminol chemiluminescence (CL) by estradiol valerate on the zinc deuteroporphyrin (ZnDP)-enhanced luminol-K3 Fe(CN)6 chemiluminescence system. Under optimized experimental conditions, CL intensity and concentration of estradiol valerate had a good linear relationship in the ranges of 8.0 × 10(-8) to 1.0 × 10(-5) g/mL. Detection limit (3σ) was estimated to be 3.5 × 10(-8) g/mL. The proposed method was applied successfully for the determination of estradiol valerate in pharmaceutical preparations and human serum and recoveries were 97.0-105.0% and 95.5-106.0%, respectively. The possible mechanism of the CL system is discussed. Copyright © 2012 John Wiley & Sons, Ltd.

Ex Vivo Expansion of Human Mesenchymal Stem Cells in Defined Serum-Free Media

PubMed Central

Jung, Sunghoon; Panchalingam, Krishna M.; Rosenberg, Lawrence; Behie, Leo A.

2012-01-01

Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSCs appear to be safe; however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies. PMID:22645619

Displacement of Drugs from Human Serum Albumin: From Molecular Interactions to Clinical Significance.

PubMed

Rimac, Hrvoje; Debeljak, Željko; Bojić, Mirza; Miller, Larisa

2017-01-01

Human serum albumin (HSA) is the most abundant protein in human serum. It has numerous functions, one of which is transport of small hydrophobic molecules, including drugs, toxins, nutrients, hormones and metabolites. HSA has the ability to interact with a wide variety of structurally different compounds. This promiscuous, nonspecific affinity can lead to sudden changes in concentrations caused by displacement, when two or more compounds compete for binding to the same molecular site. It is important to consider drug combinations and their binding to HSA when defining dosing regimens, as this can directly influence drug's free, active concentration in blood. In present paper we review drug interactions with potential for displacement from HSA, situations in which they are likely to occur and their clinical significance. We also offer guidelines in designing drugs with decreased binding to HSA. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Per- and polyfluoroalkyl substances in human serum and urine samples from a residentially exposed community.

PubMed

Worley, Rachel Rogers; Moore, Susan McAfee; Tierney, Bruce C; Ye, Xiaoyun; Calafat, Antonia M; Campbell, Sean; Woudneh, Million B; Fisher, Jeffrey

2017-09-01

Per- and polyfluoroalkyl substances (PFAS) are considered chemicals of emerging concern, in part due to their environmental and biological persistence and the potential for widespread human exposure. In 2007, a PFAS manufacturer near Decatur, Alabama notified the United States Environmental Protection Agency (EPA) it had discharged PFAS into a wastewater treatment plant, resulting in environmental contamination and potential exposures to the local community. To characterize PFAS exposure over time, the Agency for Toxic Substances and Disease Registry (ATSDR) collected blood and urine samples from local residents. Eight PFAS were measured in serum in 2010 (n=153). Eleven PFAS were measured in serum, and five PFAS were measured in urine (n=45) from some of the same residents in 2016. Serum concentrations were compared to nationally representative data and change in serum concentration over time was evaluated. Biological half-lives were estimated for perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and perfluorohexane sulfonic acid (PFHxS) using a one-compartment pharmacokinetic model. In 2010 and 2016, geometric mean PFOA and PFOS serum concentrations were elevated in participants compared to the general U.S. In 2016, the geometric mean PFHxS serum concentration was elevated compared to the general U.S. Geometric mean serum concentrations of PFOA, PFOS, and perfluorononanoic acid (PFNA) were significantly (p≤0.0001) lower (49%, 53%, and 58%, respectively) in 2016 compared to 2010. Half-lives for PFOA, PFOS, and PFHxS were estimated to be 3.9, 3.3, and 15.5years, respectively. Concentrations of PFOA in serum and urine were highly correlated (r=0.75) in males. Serum concentrations of some PFAS are decreasing in this residentially exposed community, but remain elevated compared to the U.S. general population. Published by Elsevier Ltd.

[The improvement of mixed human serum-induced anaphylactic reaction death model in guinea pigs].

PubMed

Chen, Jiong-Yuan; Lai, Yue; Li, Dang-Ri; Yue, Xia; Wang, Hui-Jun

2012-12-01

To increase the death rate of fatal anaphylaxis in guinea pigs and the detectahie level of the tryptase of mast cell in hlood serum. Seventy-four guinea pigs were randomly divided into five groups: original model group, original model control group, improved model group, improved model control group, improved model with non-anaphylaxis group. Using mixed human serum as the allergen, the way of injection, sensitization and induction were improved. ELISA was used to detect the serum mast cell tryptase and total IgE in guinea pigs of each group. The death rate of fatal anaphylaxis in original model group was 54.2% with the different degree of hemopericardium. The severe pericardial tamponade appeared in 9 guinea pigs in original model group and original model control group. The death rate of fatal anaphylaxis in improved model group was 75% without pericardial tamponade. The concentration of the serum total IgE showed no statistically difference hetween original model group and original model control group (P > 0.05), hut the serum mast cell tryptase level was higher in the original model group than that in the original model control group (P > 0.05). The concentration of the serum total IgE and the serum mast cell tryptase level were significantly higher in improved model group than that in the improved model control group (P < 0.05). The death rate of the improved model significantly increases, which can provide effective animal model for the study of serum total IgE and mast cell tryptase.

The determination of ultrafiltrable calcium and magnesium in serum.

PubMed

Danielson, B G; Pallin, E; Sohtell, M

1982-01-01

Ultrafiltrate of human serum was investigated in order to evaluate the serum content of calcium and magnesium. The acid and base concentrations and pH of the serum was altered through titration with HCl- or NaOH-solutions. The Pco2 was varied in the titrated serum using different carbon dioxide tensions. This was performed when serum was filtered in a recycling system. It is shown that the analysis of calcium and magnesium have to be done under anaerobic conditions or at standardized pH and Pco2 situations, as the concentrations vary with both pH and Pco2. The concentration ratio between ultrafiltrate and serum for calcium and magnesium was found to be 0.56 and 0.74 respectively at pH=7.41 and Pco2=40 mmHg.

Thyroid hormone induction of human cholesterol 7 alpha-hydroxylase (Cyp7a1) in vitro.

PubMed

Lammel Lindemann, Jan A; Angajala, Anusha; Engler, David A; Webb, Paul; Ayers, Stephen D

2014-05-05

Thyroid hormone (TH) modulates serum cholesterol by acting on TH receptor β1 (TRβ1) in liver to regulate metabolic gene sets. In rodents, one important TH regulated step involves induction of Cyp7a1, an enzyme in the cytochrome P450 family, which enhances cholesterol to bile acid conversion and plays a crucial role in regulation of serum cholesterol levels. Current models suggest, however, that Cyp7a1 has lost the capacity to respond to THs in humans. We were prompted to re-examine TH effects on cholesterol metabolic genes in human liver cells by a recent study of a synthetic TH mimetic which showed that serum cholesterol reductions were accompanied by increases in a marker for bile acid synthesis in humans. Here, we show that TH effects upon cholesterol metabolic genes are almost identical in mouse liver, mouse and human liver primary cells and human hepatocyte cell lines. Moreover, Cyp7a1 is a direct TR target gene that responds to physiologic TR levels through a set of distinct response elements in its promoter. These findings suggest that THs regulate cholesterol to bile acid conversion in similar ways in humans and rodent experimental models and that manipulation of hormone signaling pathways could provide a strategy to enhance Cyp7a1 activity in human patients. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

PubMed

Peng, Xian-E; Wu, Yun-Li; Zhu, Yi-Bing; Huang, Rong-Dong; Lu, Qing-Qing; Lin, Xu

2015-01-01

Liver fatty acid-binding protein (L-FABP), also known as fatty acid-binding protein 1 (FABP1), is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD) and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs) of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182) from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032).Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05). The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01). In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = -0.320, P = 0.003), while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014). Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans.

Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels

PubMed Central

Zhu, Yi-bing; Huang, Rong-dong; Lu, Qing-Qing; Lin, Xu

2015-01-01

Liver fatty acid-binding protein (L-FABP), also known as fatty acid-binding protein 1 (FABP1), is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD) and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs) of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182) from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032).Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05). The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01). In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = —0.320, P = 0.003), while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014). Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans. PMID:26439934

The serum vaspin levels are reduced in Japanese chronic hemodialysis patients.

PubMed

Inoue, Junko; Wada, Jun; Teshigawara, Sanae; Hida, Kazuyuki; Nakatsuka, Atsuko; Takatori, Yuji; Kojo, Shoichirou; Akagi, Shigeru; Nakao, Kazushi; Miyatake, Nobuyuki; McDonald, John F; Makino, Hirofumi

2012-12-03

Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified in genetically obese rats that correlates with insulin resistance and obesity in humans. Recently, we found that 7% of the Japanese population with the minor allele sequence (A) of rs77060950 exhibit higher levels of serum vaspin. We therefore evaluated the serum vaspin levels in Japanese chronic hemodialysis patients. Healthy Japanese control volunteers (control; n = 95, 49.9 ± 6.91 years) and Japanese patients undergoing hemodialysis therapy (HD; n = 138, 51.4 ± 10.5 years) were enrolled in this study, and serum samples were subjected to the human vaspin RIA system. The measurement of the serum vaspin levels demonstrated that a fraction of control subjects (n = 5) and HD patients (n = 11) exhibited much higher levels (> 10 ng/ml; Vaspin High group), while the rest of the population exhibited lower levels (< 3 ng/ml; Vaspin Low group). By comparing the patients in the Vaspin Low group, the serum vaspin levels were found to be significantly higher in the control subjects (0.87 ± 0.24 ng/ml) than in the HD patients (0.32 ± 0.15 ng/ml) (p < 0.0001). In the stepwise regression analyses, the serum creatinine and triglyceride levels were found to be independently and significantly associated with the vaspin concentrations in all subjects. The creatinine levels are negatively correlated with the serum vaspin levels and were significantly reduced in the Japanese HD patients in the Vaspin Low group.

The entrapment of corrosion products from CoCr implant alloys in the deposits of calcium phosphate: a comparison of serum, synovial fluid, albumin, EDTA, and water.

PubMed

Lewis, A C; Kilburn, M R; Heard, P J; Scott, T B; Hallam, K R; Allen, G C; Learmonth, I D

2006-08-01

Physical wear of orthopedic implants is inevitable. CoCr alloy samples, typically used in joint reconstruction, corrode rapidly after removal of the protective oxide layer. The behavior of CoCr pellets immersed in human serum, foetal bovine serum (FBS), synovial fluid, albumin in phosphate-buffered saline (PBS), EDTA in PBS, and water were studied using X-ray Photoelectron Spectroscopy (XPS) and Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS). The difference in the corrosive nature of human serum, water, albumin in PBS and synovial fluid after 5 days of immersion was highlighted by the oxide layer, which was respectively 15, 3.5, 1.5, and 1.5 nm thick. The thickness of an additional calcium phosphate deposit from human serum and synovial fluid was 40 and 2 nm, respectively. Co and Cr ions migrated from the bulk metal surface and were trapped in this deposit by the phosphate anion. This may account for the composition of wear debris from CoCr orthopedic implants, which is known to consist predominantly of hydroxy-phosphate compounds. Known components of synovial fluid including proteoglycans, pyrophosphates, phospholipids, lubricin, and superficial zone protein (SZP), have been identified as possible causes for the lack of significant calcium phosphate deposition in this environment. Circulation of these compounds around the whole implant may inhibit calcium phosphate deposition.

Serum podocalyxin is significantly increased in early-onset preeclampsia and may represent a novel marker of maternal endothelial cell dysfunction.

PubMed

Chen, Qi; Wang, Yao; Li, Ying; Zhao, Min; Nie, Guiying

2017-11-01

Podocalyxin is a glomerular podocyte protein and increased in urine of preeclampsia. However, podocalyxin is also expressed in endothelial cells of other organs. Here we investigated whether podocalyxin is detectable in pregnant serum and whether the levels are altered in preeclampsia. Podocalyxin was determined by ELISA in sera collected from normal pregnancy across gestation (n = 44) and from preeclamptic pregnancies at diagnosis (n = 34) with gestation-age-matched controls (n = 68). Immunohistochemistry examined podocalyxin in placentas and in 32 human tissues on a tissue array. Human umbilical vein endothelial cells (HUVECs) were treated with interleukin (IL)-6 and podocalyxin was analysed by ELISA and western blotting. Podocalyxin was detected in serum of normal pregnancy, with levels increasing progressively with advancing gestation. Podocalyxin serum levels were significantly elevated in preeclampsia, especially the early-onset subtype. Within the placenta, blood vessels but not trophoblasts expressed podocalyxin, and preeclampsia didn't differ from controls. Endothelial cells in all 32 human organs examined, as well as HUVECs, expressed podocalyxin. Its levels increased in the conditioned media but decreased in the lysates when HUVECs were treated with IL-6. Podocalyxin likely derived from maternal endothelial cells is present in pregnant serum and significantly increased in early-onset preeclampsia. Podocalyxin release was stimulated by IL-6 in HUVECs.

Human Growth Factor Cream and Hyaluronic Acid Serum in Conjunction with Micro Laser Peel

PubMed Central

Katz, Bruce E.; Cohen, Joel L.; Biron, Julie

2010-01-01

The present study investigated the use of a novel hyaluronic acid serum in combination with a cream comprising a mixture of human growth factors in conjunction with the micro laser peel procedure for skin rejuvenation. After preconditioning the face with the hyaluronic acid serum followed by the cream twice daily for one month, 15 female volunteers between 35 to 65 years of age with demonstrable facial wrinkling received a micro laser peel on the entire face using an erbium-doped yttrium aluminium garnet laser. Immediately following the laser procedure, the subjects applied the test products twice daily until the second laser peel one month later. Immediately following the second procedure, the subjects reapplied the test products for another month. In the large majority of subjects, erythema or edema, crusts or erosions, and transitory stinging or burning sensations after the micro laser peel were minimal or mild when the skin was treated with the serum followed by the cream. The micro laser peel in conjunction with the test products helped to significantly improve hyperpigmentation, wrinkles, and texture as compared to before treatment. This study with the micro laser peel device demonstrated that a novel hyaluronic acid serum combined with the human growth factor cream can be successfully used for skin rejuvenation in conjunction with light-to-medium invasive laser skin treatments. PMID:21203354

Human CRISP-3 binds serum alpha(1)B-glycoprotein across species.

PubMed

Udby, Lene; Johnsen, Anders H; Borregaard, Niels

2010-04-01

CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments. We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing. We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG. It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera. Copyright 2009 Elsevier B.V. All rights reserved.

Alemtuzumab: validation of a sensitive and simple enzyme-linked immunosorbent assay.

PubMed

Jilani, Iman; Keating, Michael; Giles, Francis J; O'Brien, Susan; Kantarjian, Hagop M; Albitar, Maher

2004-12-01

Alemtuzumab (MabCampath) is a humanized rat monoclonal antibody that targets the CD52 antigen. It has been approved for the treatment of patients with resistant chronic lymphocytic leukaemia (CLL). Measuring plasma/serum levels of alemtuzumab is important for optimizing the dosing and scheduling of therapy; however, current assays in serum or plasma, based on the capture of alemtuzumab using CD52, are complicated and difficult to adapt for high throughput testing. We developed a simple sandwich enzyme-linked immunosorbent assay (ELISA) to measure alemtuzumab that takes advantage of the remaining rat sequence in alemtuzumab. Using specific anti-rat immunoglobulin (Ig) antibodies (absorbed against human Ig), alemtuzumab levels were measured in the serum and plasma of patients treated with alemtuzumab. Levels were similar between plasma and serum samples, in fresh samples and samples stored at 4 degrees C for 24 h, but were significantly lower in samples stored at room temperature for 24h. The assay was successfully used to determine serum alemtuzumab pre- and post-treatment. This assay is simple and adaptable for high throughput testing, with a limit of detection of 0.05 microg/ml and a coefficient of variation of +/-12.5%. No false positivity was observed in >200 samples tested. This validated assay should help optimize the dosing and scheduling of alemtuzumab therapy. The underlying principles are also applicable to the measurement of other humanized antibodies using an appropriate anti-Ig.

The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

PubMed Central

Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; Rendina, Erino Angelo; Calogero, Antonella; Pierelli, Luca; Ibrahim, Mohsen; De Falco, Elena

2015-01-01

Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs. PMID:26495284

The potential of GMP-compliant platelet lysate to induce a permissive state for cardiovascular transdifferentiation in human mediastinal adipose tissue-derived mesenchymal stem cells.

PubMed

Siciliano, Camilla; Chimenti, Isotta; Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; Peruzzi, Mariangela; Rendina, Erino Angelo; Calogero, Antonella; Pierelli, Luca; Ibrahim, Mohsen; De Falco, Elena

2015-01-01

Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

The natural compound berberine positively affects macrophage functions involved in atherogenesis.

PubMed

Zimetti, F; Adorni, M P; Ronda, N; Gatti, R; Bernini, F; Favari, E

2015-02-01

We investigated the effect of berberine (BBR), an alkaloid showing antiatherogenic properties beyond the cholesterol lowering capacity, on macrophage cholesterol handling upon exposure to human serum and on macrophage responses to excess free cholesterol (FC) loading. Mouse and human macrophages were utilized as cellular models. Cholesterol content was measured by a fluorimetric assay; cholesterol efflux, cytotoxicity and membrane FC distribution were evaluated by radioisotopic assays. Monocyte chemotactic protein-1 (MCP-1) secretion was measured by ELISA; membrane ruffling and macropinocytosis were visualized by confocal microscopy. Exposure of cholesterol-enriched MPM to serum in the presence of 1 μM BBR resulted in a reduction of intracellular cholesterol content twice greater than exposure to serum alone (-52%; p < 0.01 and -21%; p < 0.05), an effect not mediated by an increase of cholesterol efflux, but rather by the inhibition of cholesterol uptake from serum. Consistently, BBR inhibited in a dose-dependent manner cholesterol accumulation in human macrophages exposed to hypercholesterolemic serum. Confocal microscope analysis revealed that BBR inhibited macropinocytosis, an independent-receptor process involved in LDL internalization. Macrophage FC-enrichment increased MCP-1 release by 1.5 folds, increased cytotoxicity by 2 fold, and induced membrane ruffling; all these responses were markedly inhibited by BBR. FC-enrichment led to an increase in plasma membrane cholesterol by 4.5 folds, an effect counteracted by BBR. We showed novel potentially atheroprotective activities of BBR in macrophages, consisting in the inhibition of serum-induced cholesterol accumulation, occurring at least in part through an impairment of macropinocytosis, and of FC-induced deleterious effects. Copyright © 2014 Elsevier B.V. All rights reserved.

Determination of phosphonoformate (foscarnet) in calf and human serum by automated solid-phase extraction and high-performance liquid chromatography with amperometric detection.

PubMed

Ba, B B; Corniot, A G; Ducint, D; Breilh, D; Grellet, J; Saux, M C

1999-03-05

An isocratic high-performance liquid chromatographic method with automated solid-phase extraction has been developed to determine foscarnet in calf and human serums. Extraction was performed with an anion exchanger, SAX, from which the analyte was eluted with a 50 mM potassium pyrophosphate buffer, pH 8.4. The mobile phase consisted of methanol-40 mM disodium hydrogenphosphate, pH 7.6 containing 0.25 mM tetrahexylammonium hydrogensulphate (25:75, v/v). The analyte was separated on a polyether ether ketone (PEEK) column 150x4.6 mm I.D. packed with Kromasil 100 C18, 5 microm. Amperometric detection allowed a quantification limit of 15 microM. The assay was linear from 15 to 240 microM. The recovery of foscarnet from calf serum ranged from 60.65+/-1.89% for 15 microM to 67.45+/-1.24% for 200 microM. The coefficient of variation was < or = 3.73% for intra-assay precision and < or =7.24% for inter-assay precision for calf serum concentrations ranged from 15 to 800 microM. For the same samples, the deviation from the nominal value ranged from -8.97% to +5.40% for same day accuracy and from -4.50% to +2.77% for day-to-day accuracy. Selectivity was satisfactory towards potential co-medications. Replacement of human serum by calf serum for calibration standards and quality control samples was validated. Automation brought more protection against biohazards and increase in productivity for routine monitoring and pharmacokinetic studies.

SEPARATION AND CHARACTERIZATION OF HUMAN SERUM CHYLOMICRONS

PubMed Central

Scanu, Angelo; Page, Irvine H.

1959-01-01

Chylomicrons were separated by low and high speed ultracentrifugation from lipemic sera of human subjects in the absorptive phase. The final chylomicron preparation was free from other serum components and contained a small constant amount of protein, approximately 2 per cent of the chylomicron fraction. Electrophoresis, immunochemical analysis, and absorption experiments identified the protein component as derived from a mixture of beta and alpha1 serum lipoproteins. Large aliquots of an emulsion of serum freed of chylomicrons and coconut oil were incubated at 37°C. for 2 hours and ultracentrifuged as in the preparation of chylomicrons. The fat particles now showed the presence of minute amounts of beta and alpha1 serum lipoproteins in almost the same proportion as found in chylomicrons. "Finger prints" of delipidized samples of chylomicrons and particles from serum-coconut oil emulsion gave similar, although not identical patterns. The data on "clearing factor" activity corroborated the finding that serum alpha1 lipoproteins are contained in chylomicrons and particles from serum-coconut oil emulsion. These two lipide particles, partially delipidized, were both able to activate a "clearing factor" system in vitro, a property exhibited only by intact or partially delipidized alpha1 serum lipoproteins. Clearing activity was satisfactorily determined by using an emulsion of coconut oil mixed in agar as a substrate to give an opaque gel, in which the diffusing enzyme showed its activity by areas of clearing. The results obtained by this technique were in agreement with those based on fall in optical density and non-esterified fatty acid production. Chemical analysis of serum chylomicrons showed a concentration of cholesterol and phospholipides higher than could be accounted for by the attached beta and alpha1 serum lipoproteins. On the basis of these results the assumption is made that in the blood stream small amounts of serum lipoproteins, by a process of adsorption, form a complex with the absorbed triglycerides, cholesterol, and phospholipides, to produce chylomicrons. PMID:13620852

Effect of transdermal magnesium cream on serum and urinary magnesium levels in humans: A pilot study.

PubMed

Kass, Lindsy; Rosanoff, Andrea; Tanner, Amy; Sullivan, Keith; McAuley, William; Plesset, Michael

2017-01-01

Oral magnesium supplementation is commonly used to support a low magnesium diet. This investigation set out to determine whether magnesium in a cream could be absorbed transdermally in humans to improve magnesium status. In this single blind, parallel designed pilot study, n = 25 participants (aged 34.3+/-14.8y, height 171.5+/-11cm, weight 75.9 +/-14 Kg) were randomly assigned to either a 56mg/day magnesium cream or placebo cream group for two weeks. Magnesium serum and 24hour urinary excretion were measured at baseline and at 14 days intervention. Food diaries were recorded for 8 days during this period. Mg test and placebo groups' serum and urinary Mg did not differ at baseline. After the Mg2+ cream intervention there was a clinically relevant increase in serum magnesium (0.82 to 0.89 mmol/l,p = 0.29) that was not seen in the placebo group (0.77 to 0.79 mmol/L), but was only statistically significant (p = 0.02)) in a subgroup of non-athletes. Magnesium urinary excretion increased from baseline slightly in the Mg2+ group but with no statistical significance (p = 0.48). The Mg2+ group showed an 8.54% increase in serum Mg2+ and a 9.1% increase in urinary Mg2+ while these figures for the placebo group were smaller, i.e. +2.6% for serum Mg2+ and -32% for urinary Mg2+. In the placebo group, both serum and urine concentrations showed no statistically significant change after the application of the placebo cream. No previous studies have looked at transdermal absorbency of Mg2+ in human subjects. In this pilot study, transdermal delivery of 56 mg Mg/day (a low dose compared with commercial transdermal Mg2+ products available) showed a larger percentage rise in both serum and urinary markers from pre to post intervention compared with subjects using the placebo cream, but statistical significance was achieved only for serum Mg2+ in a subgroup of non-athletes. Future studies should look at higher dosage of magnesium cream for longer durations. ISRCTN registry ID No. ISRTN15136969.

Determination of crenolanib in human serum and cerebrospinal fluid by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

PubMed

Roberts, Michael S; Turner, David C; Owens, Thandranese S; Ramachandran, Abhijit; Wetmore, Cynthia; Throm, Stacy L; Stewart, Clinton F

2013-06-15

A LC-ESI-MS/MS method for the determination of crenolanib (CP-868,596) in human serum was developed and validated employing d4-CP-868,596 as an internal standard (ISTD). In addition to human serum, the method was also partially validated for crenolanib determination in human cerebrospinal fluid (CSF) samples. Sample aliquots (50μl of serum or CSF) were prepared for analysis using liquid-liquid extraction (LLE) with tert-butyl methyl ether. Chromatography was performed using a phenomenex Gemini C18 column (3μm, 100mm×4.6mm I.D.) in a column heater set at 50°C and an isocratic mobile phase (methanol/water/formic acid at a volume ratio of 25/25/0.15, v/v/v). The flow rate was 0.45mL/min, and the retention time for both analyte and ISTD was less than 3.5min. Samples were analyzed with an API-5500 LC-MS/MS system (ESI) in positive ionization mode coupled to a Shimadzu HPLC system. The ion transitions monitored were m/z 444.4→373.1 and m/z 448.2→374.2 for crenolanib and ISTD, respectively. The method was linear over the range of 5-1000ng/mL for serum and 0.5-1000ng/mL for CSF. For human serum, both intra-day and inter-day precision were <4%, while intra-day and inter-day accuracy were within 8% of nominal values. Recovery was greater than 50% for both the analyte and ISTD. For CSF samples, both intra-day and inter-day precision were <9% except at the lower limit of quantification (LLOQ) which was <17%. The intra-day and inter-day accuracy were within 11% of the nominal CSF concentrations. After validation, this method was successfully applied to the analysis of serial pharmacokinetic samples obtained from a child treated with oral crenolanib. Copyright © 2013 Elsevier B.V. All rights reserved.

[Methodological aspects of evaluation of potential lipid capacity for peroxidation from the serum levels of TBA-active products during iron ion stimulation].

PubMed

Kulikova, A I; Tugusheva, F A; Zubina, I M; Shepilova, I N

2008-05-01

The authors propose a simple and reproducible procedure for using iron ions to stimulate serum lipid peroxidation, with TBA-active products being further determined. The procedure determines the reserve of lipids that can be oxidized during oxidative stress. A combination of direct studies and correlation analysis suggests that low-density lipoproteins and very low-density lipoproteins are the major substrates for lipid peroxidation while high-density lipoproteins show a high resistance to this process. The presented procedure may be used to monitor lipid peroxidation in various conditions and upon various exposures in common laboratory practice.

Antibody binding of circulating ergot alkaloids in cattle grazing tall fescue.

PubMed

Hill, N S; Thompson, F N; Dawe, D L; Stuedemann, J A

1994-03-01

Direct evidence linking alkaloids found in endophyte-infected tall fescue forage with the livestock disorder known as fescue toxicosis is lacking. Physiologic effects of fescue toxicosis include reduced serum prolactin concentration in cattle. A monoclonal antibody specific to the lysergic moiety of ergot alkaloids was developed in mice after creating an immunogen by linking lysergol to human serum albumin. The antibody was specific to the lysergic moiety and, therefore, it cross-reacted with ergot alkaloids, lysergic acid, and lysergol. The antibody did not cross-react with alkaloid derivatives that had bromated or hydrogenated lysergic ring moieties. Fescue toxicosis conditions were elicited in yearling Angus steers by permitting them to graze endophyte-infected tall fescue containing > 650 micrograms/kg of ergovaline for 60 days. Passive immunization of steers by infusion of the monoclonal antibody increased serum prolactin concentration by 7 ng/ml, beginning immediately after infusion. Control steers did not respond to treatment with bovine serum albumin. Active immunization of yearling Angus heifers with immunogens containing lysergol or ergonovine linked to human serum albumin resulted in an antibody response.

Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

PubMed

de Puig, Helena; Bosch, Irene; Carré-Camps, Marc; Hamad-Schifferli, Kimberly

2017-01-18

We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, K D eff . Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

Association of canine obesity with reduced serum levels of C-reactive protein.

PubMed

Veiga, Angela P M; Price, Christopher A; de Oliveira, Simone T; Dos Santos, Andréa P; Campos, Rómulo; Barbosa, Patricia R; González, Félix H D

2008-03-01

The prevalence of obesity is increasing in dogs as well as in humans. C-reactive protein (CRP) is an important tool for the detection of inflammation and/or early tissue damage and is linked to obesity in humans. The objective of the present study was to determine if serum CRP levels are altered in obese dogs. Fifteen lean (control group) and 16 overweight (obese group) dogs were examined. Blood samples were collected under fasted conditions for serum determination of CRP, glucose, insulin, cholesterol, triglyceride, and fructosamine. Results indicated that obese dogs were insulin resistant because serum insulin and insulin/glucose ratios were higher than in lean dogs (P < or = 0.05). Serum CRP concentrations were lower in obese dogs than in controls (P < or = 0.001). C-reactive protein was negatively correlated with insulin/glucose ratio (R = -0.42) and cholesterol (R = -0.39; P < or = 0.05). Furthermore, levels of cholesterol, triglycerides, and fructosamine were increased in the obese group compared with the control group. Based on these results, it can be postulated that CRP production is inhibited by obesity and insulin resistance in dogs.

Critical Evaluation of Gamma-Irradiated Serum Used as Feeder in the Culture and Demonstration of Putative Nanobacteria and Calcifying Nanoparticles

PubMed Central

Young, John D.

2010-01-01

The culture and demonstration of putative nanobacteria (NB) and calcifying nanoparticles (CNP) from human and animal tissues has relied primarily on the use of a culture supplement consisting of FBS that had been γ-irradiated at a dose of 30 kGy (γ-FBS). The use of γ-FBS is based on the assumption that this sterilized fluid has been rid entirely of any residual NB/CNP, while it continues to promote the slow growth in culture of NB/CNP from human/animal tissues. We show here that γ-irradiation (5–50 kGy) produces extensive dose-dependent serum protein breakdown as demonstrated through UV and visible light spectrophotometry, fluorometry, Fourier-transformed infrared spectroscopy, and gel electrophoresis. Yet, both γ-FBS and γ-irradiated human serum (γ-HS) produce NB/CNP in cell culture conditions that are morphologically and chemically indistinguishable from their normal serum counterparts. Contrary to earlier claims, γ-FBS does not enhance the formation of NB/CNP from several human body fluids (saliva, urine, ascites, and synovial fluid) tested. In the presence of additional precipitating ions, both γ-irradiated serum (FBS and HS) and γ-irradiated proteins (albumin and fetuin-A) retain the inherent dual NB inhibitory and seeding capabilities seen also with their untreated counterparts. By gel electrophoresis, the particles formed from both γ-FBS and γ-HS are seen to have assimilated into their scaffold the same smeared protein profiles found in the γ-irradiated sera. However, their protein compositions as identified by proteomics are virtually identical to those seen with particles formed from untreated serum. Moreover, particles derived from human fluids and cultured in the presence of γ-FBS contain proteins derived from both γ-FBS and the human fluid under investigation—a confusing and unprecedented scenario indicating that these particles harbor proteins from both the host tissue and the FBS used as feeder. Thus, the NB/CNP described in the literature clearly bear hybrid protein compositions belonging to different species. We conclude that there is no basis to justify the use of γ-FBS as a feeder for the growth and demonstration of NB/CNP or any NB-like particles in culture. Moreover, our results call into question the validity of the entire body of literature accumulated to date on NB and CNP. PMID:20436679

Mechanistically linked serum miRNAs distinguish between drug induced and fatty liver disease of different grades.

PubMed

Liu, Zhichao; Wang, Yuping; Borlak, Jürgen; Tong, Weida

2016-04-05

Hepatic steatosis is characterised by excessive triglyceride accumulation in the form of lipid droplets (LD); however, mechanisms differ in drug induced (DIS) and/or non-alcoholic fatty liver disease (NAFLD). Here we hypothesized distinct molecular circuits of microRNA/LD-associated target genes and searched for mechanistically linked serum and tissue biomarkers that would distinguish between DIS and human NAFLD of different grades. We analysed >800 rat hepatic whole genome data for 17 steatotic drugs and identified 157 distinct miRNAs targeting 77 DIS regulated genes. Subsequently, genomic data of N = 105 cases of human NAFLD and N = 32 healthy controls were compared to serum miRNA profiles of N = 167 NAFLD patients. This revealed N = 195 tissue-specific miRNAs being mechanistically linked to LD-coding genes and 24 and 9 miRNAs were commonly regulated in serum and tissue of advanced and mild NAFLD, respectively. The NASH serum regulated miRNAs informed on hepatic inflammation, adipocytokine and insulin signalling, ER-and caveolae associated activities and altered glycerolipid metabolism. Conversely, serum miRNAs associated with blunt steatosis specifically highlighted activity of FOXO1&HNF4α on CPT2, the lipid droplet and ER-lipid-raft associated PLIN3 and Erlin1. Altogether, serum miRNAs informed on the molecular pathophysiology of NAFLD and permitted differentiation between DIS and NAFLD of different grades.

Screening the low molecular weight fraction of human serum using ATR-IR spectroscopy.

PubMed

Bonnier, Franck; Brachet, Guillaume; Duong, Romain; Sojinrin, Tobiloba; Respaud, Renaud; Aubrey, Nicolas; Baker, Matthew J; Byrne, Hugh J; Chourpa, Igor

2016-10-01

Vibrational spectroscopic techniques can detect small variations in molecular content, linked with disease, showing promise for screening and early diagnosis. Biological fluids, particularly blood serum, are potentially valuable for diagnosis purposes. The so-called Low Molecular Weight Fraction (LMWF) contains the associated peptidome and metabolome and has been identified as potentially the most relevant molecular population for disease-associated biomarker research. Although vibrational spectroscopy can deliver a specific chemical fingerprint of the samples, the High Molecular Weight Fraction (HMWF), composed of the most abundant serum proteins, strongly dominates the response and ultimately makes the detection of minor spectral variations a challenging task. Spectroscopic detection of potential serum biomarkers present at relatively low concentrations can be improved using pre-analytical depletion of the HMWF. In the present study, human serum fractionation by centrifugal filtration was used prior to analysis by Attenuated Total Reflection infrared spectroscopy. Using a model sample based on glycine spiked serum, it is demonstrated that the screening of the LMWF can be applied to quantify blinded concentrations up to 50 times lower. Moreover, the approach is easily transferable to different bodily fluids which would support the development of more efficient and suitable clinical protocols exploring vibrational spectroscopy based ex-vivo diagnostic tools. Revealing serum LMWF for spectral serological diagnostic applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Effect of Trimethoprim on Serum Folate Levels in Humans: A Randomized, Double-Blind, Placebo-Controlled Trial.

PubMed

Meidahl Petersen, Kasper; Eplov, Kasper; Kjær Nielsen, Torben; Jimenez-Solem, Espen; Petersen, Morten; Broedbaek, Kasper; Daugaard Popik, Sara; Kallehave Hansen, Lina; Enghusen Poulsen, Henrik; Trærup Andersen, Jon

2016-01-01

Trimethoprim antagonize the actions of folate by inhibition of dihydrofolate reductase. This could diminish serum folate levels in humans and causes folate deficiency in some patients. We conducted a randomized, double-blind, placebo-controlled trial, to investigate the effect of trimethoprim on serum folate levels in healthy participants after a 7-day trial period. Thirty young, healthy males were randomly allocated to receive trimethoprim, 200 mg twice daily, and 30 were randomly allocated to placebo. Before trial initiation, participant numbers were given randomly generated treatment allocations within sealed opaque envelopes. Participants and all staff were kept blinded to treatment allocations during the trial. Serum folate was measured at baseline and at end of trial. In the 58 participants analyzed (30 in the trimethoprim group and 28 in the placebo group), 8 had folate deficiency at baseline. Within the trimethoprim group, serum folate was significantly decreased (P = 0.018) after the trial. We found a mean decrease in serum folate among trimethoprim exposed of 1.95 nmol/L, compared with a 0.21 nmol/L mean increase in the placebo group (P = 0.040). The proportion of folate-deficient participants increased significantly within the trimethoprim group (P = 0.034). No serious adverse events were observed. In conclusion, we found that a daily dose of 400 mg trimethoprim for 7 days significantly lowered serum folate levels in healthy study participants.

A novel, non-invasive transdermal fluid sampling methodology: IGF-I measurement following exercise

USDA-ARS?s Scientific Manuscript database

This study tested the hypothesis that transdermal fluid (TDF) provides a more sensitive and accurate measure of exercise-induced increases in insulin-like growth factor-I (IGF-I) than serum, and that these increases are detectable proximal, but not distal, to the exercising muscle. A novel, noninvas...

Lipid fatty acid profile analyses in liver and serum in rats with nonalcoholic steatohepatitis using improved gas chromatography-mass spectrometry methodology

USDA-ARS?s Scientific Manuscript database

Fatty acids (FA) are essential components of lipids and exhibit important biological functions. The analyses of FAs are routinely carried out by gas chromatography-mass spectrometry, after multi-step sample preparation. In this study, several key experimental factors were carefully examined, validat...

Development of high protein, high fiber smoothie as a grab-and-go breakfast option using response surface methodology.

PubMed

Mehta, Dipakkumar; Kumar, M H Sathish; Sabikhi, Latha

2017-11-01

The current work aimed to formulate smoothie by optimizing varying levels of soy protein isolate (1.5-2.5% w/w), sucralose (150-190 ppm) and pectin (0.3-0.5% w/w) along with milk, legume (chickpea), vegetable (carrot), fruit (mango), honey and trisodium citrate by response surface methodology on the basis of sensory (color and appearance, flavor, consistency, sweetness and overall acceptability) and physical (expressible serum and viscosity) responses. Soy protein isolate and pectin levels influenced color and appearance, flavor, consistency and overall acceptability significantly. Soy protein isolate and pectin showed a positive correlation with viscosity of smoothie with reduced expressible serum. Smoothie was optimized with 1.8% (w/w) soy protein isolate, 166.8 ppm sucralose, and 0.5% (w/w) pectin with acceptable quality. One serving (325 ml) of optimized smoothie provides approximately 23% protein, 27% dietary fiber of the recommended daily values and provides approximately 74 kcal per 100 ml of smoothie, which renders smoothie as a high protein, high fiber, grab-and-go breakfast option.

Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy.

PubMed

Yasutake, Junichi; Suzuki, Yusuke; Suzuki, Hitoshi; Hiura, Naoko; Yanagawa, Hiroyuki; Makita, Yuko; Kaneko, Etsuji; Tomino, Yasuhiko

2015-08-01

Galactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA). Rats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens. Measurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA. This novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA.

Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy

PubMed Central

Yasutake, Junichi; Suzuki, Yusuke; Suzuki, Hitoshi; Hiura, Naoko; Yanagawa, Hiroyuki; Makita, Yuko; Kaneko, Etsuji; Tomino, Yasuhiko

2015-01-01

Background Galactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA). Methods Rats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens. Results Measurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA. Conclusion This novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease. PMID:26109484

Calcium-independent haemolysis via the lectin pathway of complement activation in the guinea-pig and other *

PubMed Central

Zhang, Y; Suankratay, C; Zhang, X-H; Jones, D R; Lint, T F; Gewurz, H

1999-01-01

We previously reported that complement-dependent haemolysis of sheep erythrocytes (E) coated with mannan (M) and sensitized with human mannan-binding lectin (MBL) via the lectin pathway in man occurs in Mg-EGTA and requires alternative pathway amplification. Calcium was required for MBL binding to E-M, but once the E-M-MBL intermediate was formed, MBL was retained and haemolysis occurred in the absence of calcium. Comparable or greater lectin pathway haemolysis in the absence of calcium was observed upon incubation of E-M-MBL in guinea-pig, rat, dog and pig sera, and was further investigated in the guinea-pig, in which titres were much higher (∼14-fold) than in man, and in contrast to humans, greater than classical pathway haemolytic activity. As in human serum, no lysis was observed in C4- or C2-deficient guinea-pig serum until purified C4 or C2, respectively, were restored. However, lectin pathway haemolytic activity in the guinea-pig did not require the alternative pathway. Removal (>98%) of factor D activity by three sequential passages through Sephadex G-75, resulting in serum which retained a normal classical pathway but no alternative pathway haemolytic activity, did not reduce the ability of guinea-pig serum to mediate haemolysis via the lectin pathway. Further, the C3-convertase formed via the lectin pathway (E-M-MBL-C4,2) lysed in C2-deficient guinea-pig but not human serum chelated with EDTA, a condition which precludes alternative pathway amplification. Thus, lectin pathway haemolysis occurs efficiently in guinea-pig serum, in the absence of calcium and without requirement for alternative pathway amplification. The guinea-pig provides a model for studying the assembly and haemolytic function of a lectin pathway which contrasts with the lectin pathway of man, and allows for comparisons that may help clarify the role of this pathway in complement biology. PMID:10457224

Creatine metabolism and safety profiles after six-week oral guanidinoacetic acid administration in healthy humans.

PubMed

Ostojic, Sergej M; Niess, Barbara; Stojanovic, Marko; Obrenovic, Milos

2013-01-01

Guanidinoacetic acid (GAA) is a natural precursor of creatine, yet the potential use of GAA as a nutritional additive for restoring creatine availability in humans has been limited by unclear efficacy and safety after exogenous GAA administration. The present study evaluated the effects of orally administered GAA on serum and urinary GAA, creatine and creatinine concentration, and on the occurrence of adverse events in healthy humans. Twenty-four healthy volunteers were randomized in a double-blind design to receive either GAA (2.4 grams daily) or placebo (PLA) by oral administration for 6 weeks. www.clinicaltrials.gov, identification number NCT01133899. Serum creatine and creatinine increased significantly from before to after administration in GAA-supplemented participants (P < 0.05). The proportion of participants who reported minor side effects was 58.3% in the GAA group and 45.5% in the placebo group (P = 0.68). A few participants experienced serum creatine levels above 70 µmol/L. Exogenous GAA is metabolized to creatine, resulting in a significant increase of fasting serum creatine after intervention. GAA had an acceptable side-effects profile with a low incidence of biochemical abnormalities.

Creatine Metabolism and Safety Profiles after Six-Week Oral Guanidinoacetic Acid Administration in Healthy Humans

PubMed Central

Ostojic, Sergej M.; Niess, Barbara; Stojanovic, Marko; Obrenovic, Milos

2013-01-01

Objectives; Guanidinoacetic acid (GAA) is a natural precursor of creatine, yet the potential use of GAA as a nutritional additive for restoring creatine availability in humans has been limited by unclear efficacy and safety after exogenous GAA administration. The present study evaluated the effects of orally administered GAA on serum and urinary GAA, creatine and creatinine concentration, and on the occurrence of adverse events in healthy humans. Methods and Results; Twenty-four healthy volunteers were randomized in a double-blind design to receive either GAA (2.4 grams daily) or placebo (PLA) by oral administration for 6 weeks. Clinical trial registration: www.clinicaltrials.gov, identification number NCT01133899. Serum creatine and creatinine increased significantly from before to after administration in GAA-supplemented participants (P < 0.05). The proportion of participants who reported minor side effects was 58.3% in the GAA group and 45.5% in the placebo group (P = 0.68). A few participants experienced serum creatine levels above 70 µmol/L. Conclusion; Exogenous GAA is metabolized to creatine, resulting in a significant increase of fasting serum creatine after intervention. GAA had an acceptable side-effects profile with a low incidence of biochemical abnormalities. PMID:23329885

Evaluation of the binding effect of human serum albumin on the properties of granules.

PubMed

Kristó, Katalin; Bajdik, János; Eros, István; Pintye-Hódi, Klára

2008-11-01

The main objective of this study was the application of a solution of human serum albumin as a granulating fluid. The properties of the granules formed were evaluated and compared with those when a conventional binder was applied in the same concentration. The powder mixture contained a soluble (mannitol) and an insoluble component (different types of cellulose). The protein solution applied exerted an appropriate aggregating effect if the system contained microcrystalline celluloses. Powdered cellulose was not suitable for the granulation with human serum albumin solution. As compared with the same concentration of the conventionally applied cellulose ethers as binder, the prepared granules exhibited a larger particle size, a significantly better compressibility, a higher breaking hardness and a favourable deformation process. These findings mainly reflect the good adhesive properties of the protein. The best compressibility and mechanical behaviour were attained on the application of the microcrystalline cellulose Vivapur type 105. This favourable behaviour may be connected with the wettability of cellulose. These results suggest that the formulation of tablets may be easier from an active agent in the serum that binds to albumin (e.g. interferon) since the amount of additives (binder) can be reduced.

A facile route to glycated albumin detection.

PubMed

Bohli, Nadra; Meilhac, Olivier; Rondeau, Philippe; Gueffrache, Syrine; Mora, Laurence; Abdelghani, Adnane

2018-07-01

In this paper we propose an easy way to detect the glycated form of human serum albumin which is biomarker for several diseases such as diabetes and Alzheimer. The detection platform is a label free impedimetric immunosensor, in which we used a monoclonal human serum albumin antibody as a bioreceptor and electrochemical impedance as a transducing method. The antibody was deposited onto a gold surface by simple physisorption technique. Bovine serum albumin was used as a blocking agent for non-specific binding interactions. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the characterization of each layer. Human serum albumin was glycated at different levels with several concentrations of glucose ranging from 0 mM to 500 mM representing physiological, pathological (diabetic albumin) and suprapathological concentration of glucose. Through the calibration curves, we could clearly distinguish between two different areas related to physiological and pathological albumin glycation levels. The immunosensor displayed a linear range from 7.49% to 15.79% of glycated albumin to total albumin with a good sensitivity. Surface plasmon resonance imaging was also used to characterize the developed immunosensor. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of dried serum spot sampling techniques for the assessment of trace elements in serum samples by LA-ICP-MS.

PubMed

Chantada-Vázquez, María Pilar; Moreda-Piñeiro, Jorge; Cantarero-Roldán, Alicia; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

2018-08-15

A novel approach for serum analysis by dried matrix spot (DMS) technique is proposed. The methodology consists of sampling filter paper discs (2.7 mm in diameter) containing the large amount of serum retained after a single spotting. Several oxidizers (sodium chlorate, sodium azide, acetic acid, formic acid, 1-butyl-3-methylimidazoliumm chloride/bromide) were tested (oxidizers premixed with the sample before spotting, and papers previously soaked in concentrated additive/oxidizer solutions). Direct multi-element determination (Al, Be, Ca, Cu, Fe, K, Li, Mg, Mn, Mo, Na, P, Rb, Se, V, and Zn) in dried serum spots at very low levels was therefore assessed by laser ablation (LA) coupled with inductively coupled plasma - mass spectrometry (ICP-MS). Laser ablation was performed using a focused Nd: YAG laser beam in lineal scan mode (wavelength 213 nm, laser fluency 2.2 J cm -2 , repetition rate 20 Hz, laser spot diameter 90 µm, depth 0 µm, scanning speed 12 µm s -1 ). Matrix-matched calibration mode and 13 C as internal standard (for signal intensities normalization) was used throughout the work. Limits of quantification were found to be from 21 µg L -1 to 221 mg L -1 . Repeatability (seven ablations of the same dried serum spot) and reproducibility (two ablations of seven dried serum spot from the same material) offered RSDs below 12% for all analytes, which seems satisfactory for clinical purposes. The method was validated by analyzing several certified reference materials (Seronorm™ level I and II trace elements in serum), and it was applied to several DMS from serum samples from healthy adults. Copyright © 2018 Elsevier B.V. All rights reserved.

Online immunocapture ICP-MS for the determination of the metalloprotein ceruloplasmin in human serum.

PubMed

Bernevic, Bogdan; El-Khatib, Ahmed H; Jakubowski, Norbert; Weller, Michael G

2018-04-02

The human copper-protein ceruloplasmin (Cp) is the major copper-containing protein in the human body. The accurate determination of Cp is mandatory for the reliable diagnosis of several diseases. However, the analysis of Cp has proven to be difficult. The aim of our work was a proof of concept for the determination of a metalloprotein-based on online immunocapture ICP-MS. The immuno-affinity step is responsible for the enrichment and isolation of the analyte from serum, whereas the compound-independent quantitation with ICP-MS delivers the sensitivity, precision, and large dynamic range. Off-line ELISA (enzyme-linked immunosorbent assay) was used in parallel to confirm the elution profile of the analyte with a structure-selective method. The total protein elution was observed with the 32 S mass trace. The ICP-MS signals were normalized on a 59 Co signal. The human copper-protein Cp could be selectively determined. This was shown with pure Cp and with a sample of human serum. The good correlation with off-line ELISA shows that Cp could be captured and eluted selectively from the anti-Cp affinity column and subsequently determined by the copper signal of ICP-MS.

Correlation Between Iron and alpha and pi Glutathione-S-Transferase Levels in Humans

DTIC Science & Technology

2012-09-01

assays were performed as described in the Biotrin High Sensitivity Alpha GST EIA kit protocol. First, serum samples were diluted 1:10 with wash solution...immunosorbent assays were performed as described in the Biotrin Pi GST EIA kit protocol. First, plasma samples were diluted 1:5 with sample diluent...immunosorbent assays were performed as described in the AssayMax Human Transferrin ELISA kit protocol. First, serum samples were diluted 1:2000 with MIX