ABSTRACT
Recombinant human growth hormone (rhGH) is a peptide comprising 191 amino acids, that is mainly used to promote the growth of children and plays an important antiaging role. In the present study, a simple and sensitive quantitation method for rhGH in rat plasma was established by LCMS/MS. After simple and rapid enzymatic digestion of the plasma sample, two suitable surrogate peptides (LFDNAMLR and FPTIPLSR) were selected for quantitative analysis. The results showed good linearity over calibration range 10-2000 ng/mL. The quality control (QC) accuracy ranged from -13.8 to 14.3%, and the accuracy of the lower limit of quantification (LLOQ) ranged from -12.9 to 19.0%. The intra-day and inter-day precision ranges for all QCs were 1.7-13.6% and 4.0-7.0%, respectively. The method was successfully applied to intravenous and subcutaneous pharmacokinetic studies in rats. In comparison with previously published methods, our method features simple sample preparation combined with a short sample processing time (3.5 h), wide linear range (10-2000 ng/mL), small plasma volume (35 µL), and LLOQ (10 ng/mL).
Subject(s)
Human Growth Hormone , Animals , Humans , Rats , Chromatography, Liquid/methods , Human Growth Hormone/analysis , Human Growth Hormone/blood , Quality Control , Reproducibility of Results , Tandem Mass Spectrometry/methods , Recombinant Proteins/analysis , Recombinant Proteins/bloodABSTRACT
In this work, we developed the method of preparative production of recombinant human cyclophilin A (rhCypA) in Escherichia coli. The full-length cDNA encoding the gene of human CypA (CYPA) was amplified by RT-PCR from the total RNA of human T cell lymphoma Jurkat. The nucleotide sequence of CYPA was optimized to provide highly effective translation in E. coli. Recombinant CYPA DNA was cloned into the pET22b(+) vector, and the resulted expression plasmid was used to transform E. coli strain BL21(DE3)Gold. The recombinant producer strain of E. coli produced soluble rhCypA in the bacterial cytoplasm. The synthesis efficiency of rhCypA was up to 50% of the total cell protein allowing to produce rhCypA in the amount of 1 g per liter of the culture. We also developed the method for rhCypA purification, consisting of a single-step tandem anion exchange chromatography on DEAE- and Q-Sepharose columns. The protein purity was 95% according to electrophoresis (SDS-PAGE), and its contamination with endotoxin did not exceed 0.05 ng per 1 mg of the protein that met the requirements of European pharmacopoeia for injectable preparations. The produced recombinant protein exhibited functional features of native CypA, i.e., isomerase activity and chemokine activity as assessed by stimulation of migration of mouse bone marrow hematopoietic stem cells in vivo. The generated producer strain of E. coli is a super-producer and could be used for large-scale experimental studies of rhCypA and in its preclinical and clinical trials as a drug.
Subject(s)
Cyclophilin A , Animals , Cyclophilin A/biosynthesis , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Humans , Mice , Plasmids , Recombinant Proteins/bloodABSTRACT
BACKGROUND: Coagulopathic bleeding is common during adult extracorporeal membrane oxygenation (ECMO), and acquired von Willebrand syndrome is a contributing factor. We compared ECMO patient blood samples that were treated in vitro with recombinant von Willebrand Factor concentrate and plasma-derived von Willebrand Factor concentrate. Our hypothesis was that recombinant von Willebrand Factor (vWF) would have greater efficacy in increasing vWF function. Secondarily, we hypothesized that recombinant vWF would have less impact on thrombin generation. METHODS: Thirty ECMO patients and 10 cardiac surgical controls were enrolled in the study. ECMO patient blood samples were treated in vitro with low- and high-dose recombinant vWFs and low- and high-dose plasma-derived vWFs. Whole blood ristocetin-induced platelet aggregation (RIPA), plasma ristocetin cofactor activity (RCo), and thrombin generation were compared between ECMO patient blood samples and control blood samples and between vWF-treated ECMO patient blood samples and nontreated samples. RESULTS: ECMO patient blood samples had severely reduced median RIPA compared to control samples 2 ohms (1-12 [25th-75th percentile]) vs 20 ohms (11-42) (P < .001). Treatment of ECMO patient blood samples with high-dose recombinant vWF significantly increased median RIPA to 10 ohms (2-15) (P < .001), while low-dose recombinant vWF and low- and high-dose plasma-derived vWFs did not significantly increase RIPA; 6 ohms (3-14), 4 ohms (1-13), and 6 ohms (2-10), respectively (P = .25, >.99, and >.99). Treatment with high-dose recombinant vWF and low- and high-dose plasma-derived vWFs significantly increased median plasma RCo to 4.7 international units (IU)/mL (3.7-5.9), 3.3 IU/mL (2.7-4.8), and 3.9 IU/mL (3.4-5.3), respectively, compared to controls 1.8 IU/mL (1.5-2.3) (all P < .001). Treatment with low- and high-dose plasma-derived vWFs significantly increased mean endogenous thrombin potential (6270.2 ± 2038.7 and 6313.1 ± 1913.3) compared to nontreated samples (5856.7 ± 1924.6) (P = .04 and .006), whereas treatment with low- and high-dose recombinant vWFs had no significant effect on mean endogenous thrombin potential (5776.1 ± 2087.3 and 5856.2 ± 1946.4) (P > .99 for both comparisons). CONCLUSIONS: In vitro treatment of ECMO patient blood samples with high-dose recombinant vWF was superior to low-dose recombinant vWF and plasma-derived vWF in terms of improving RIPA. In addition, recombinant vWF treatment did not increase endogenous thrombin potential, which may reduce overall thrombotic risk if it used to treat acquired von Willebrand syndrome in ECMO patients.
Subject(s)
Extracorporeal Membrane Oxygenation/methods , Factor VIII/administration & dosage , von Willebrand Diseases/blood , von Willebrand Diseases/therapy , von Willebrand Factor/administration & dosage , Adult , Aged , Aged, 80 and over , Cohort Studies , Factor VIII/metabolism , Female , Humans , Male , Middle Aged , Plasma , Recombinant Proteins/administration & dosage , Recombinant Proteins/blood , Treatment Outcome , von Willebrand Factor/metabolismABSTRACT
Athletes abuse recombinant human erythropoietin (rhEPO) and erythropoiesis stimulating agents to increase hemoglobin mass and improve performance. To evade detection, athletes have developed sophisticated blood doping regimens, which often include rhEPO micro-dosing. Detection of these methods requires biomarkers with increased sensitivity and a sample matrix that is more amenable to frequent testing in the field. We have developed a method to measure two immature reticulocyte proteins, CD71 and ferrochelatase (FECH), and one total erythrocyte protein, Band 3, in dried blood spots (DBS). This method was tested in response to rhEPO administration after low doses, 40 IU/kg, micro-doses, 900 IU, or saline injection in 20 healthy subjects. During administration of low-dose rhEPO, the mean CD71/Band 3 and FECH/Band 3 ratio increased by 412 ± 197% and 250 ± 44%, respectively. The mean response for the current biomarker, RET%, increased by 195 ± 35%. During administration of rhEPO micro-doses, CD71/Band 3 increased to 127 ± 25% on day 35 and 139 ± 36% on day 39, while no increase was observed in RET%. After rhEPO administration, during the washout phase, mean values decreased to a minimum of 64 ± 4% and 64 ± 11% for CD71/Band 3 and RET%, respectively. However, CD71/Band 3 remained below 75% of baseline for at least 4 weeks after rhEPO injection, while RET% returned to baseline levels. The results demonstrate that immature reticulocyte proteins have a larger response to rhEPO administration than the current biomarker, RET%, and can be monitored in the DBS matrix.
Subject(s)
Dried Blood Spot Testing/methods , Erythropoietin/blood , Reticulocytes/chemistry , Substance Abuse Detection/methods , Adolescent , Adult , Cell Tracking/methods , Erythropoietin/administration & dosage , Erythropoietin/analysis , Humans , Male , Middle Aged , Placebo Effect , Recombinant Proteins/administration & dosage , Recombinant Proteins/analysis , Recombinant Proteins/blood , Reticulocytes/cytology , Young AdultABSTRACT
Pegvaliase (Palynziq®) is an enzyme substitution therapy using PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL) to reduce blood phenylalanine (Phe) levels in adults with phenylketonuria (PKU). In Phase 3 clinical studies, all subjects treated with pegvaliase developed anti-drug antibodies. To specifically evaluate pegvaliase-neutralizing antibodies (NAbs) and assess impact on pegvaliase efficacy, a novel hybrid ligand-binding/tandem mass spectrometry NAb assay was developed. Analysis of Phase 3 study samples revealed that pegvaliase NAb titers developed during early treatment (≤6 months after treatment initiation), and then plateaued and persisted in the majority of subjects during late treatment (>6 months). Subjects with the lowest/undetectable NAb titers had relatively high plasma pegvaliase concentrations and experienced the most rapid decline in blood Phe concentrations at relatively low pegvaliase dose concentrations. In contrast, subjects with higher NAb titers generally had lower plasma pegvaliase concentrations on similar low doses, with little change in blood Phe concentrations. However, with additional time on treatment and individualized dose titration, the majority of subjects achieved substantial and sustained blood Phe reduction, including those with higher NAb titers. Moreover, after maturation of the anti-pegvaliase immune response, NAb titers were stable over time and did not rise in response to dose increases; thus, subjects did not require additional dose increases to maintain reduction in blood Phe.
Subject(s)
Antibodies, Neutralizing/blood , Phenylalanine Ammonia-Lyase/blood , Phenylalanine Ammonia-Lyase/therapeutic use , Adult , Antibodies, Neutralizing/immunology , Humans , Phenylalanine/blood , Phenylalanine Ammonia-Lyase/adverse effects , Phenylalanine Ammonia-Lyase/immunology , Phenylketonurias/drug therapy , Recombinant Proteins/adverse effects , Recombinant Proteins/blood , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic useSubject(s)
Cerebral Intraventricular Hemorrhage/blood , Cerebral Intraventricular Hemorrhage/drug therapy , Erythropoietin/administration & dosage , Erythropoietin/blood , Infant, Premature/blood , Follow-Up Studies , Humans , Infant, Newborn , Randomized Controlled Trials as Topic/methods , Recombinant Proteins/administration & dosage , Recombinant Proteins/blood , Treatment OutcomeSubject(s)
Cerebral Intraventricular Hemorrhage/blood , Cerebral Intraventricular Hemorrhage/drug therapy , Erythropoietin/administration & dosage , Erythropoietin/blood , Infant, Premature/blood , Follow-Up Studies , Humans , Infant, Newborn , Randomized Controlled Trials as Topic/methods , Recombinant Proteins/administration & dosage , Recombinant Proteins/blood , Treatment OutcomeABSTRACT
A sensitive method for determination of PEG-IFN-α-2b in human serum was developed using ultra performance liquid chromatography aligned with tandem mass spectrometric detection. A two-treatment, two-period, cross over study was conducted to establish bioequivalence between a test and reference formulation and the method was successfully applied to the quantification of PEG-IFN-α-2b in serum samples of this clinical study. The sample concentrations obtained from LC-MS/MS technique were compared with the concentrations obtained from ELISA technique. PEG-IFN-α-2b was isolated from serum using protein precipitation technique with isopropyl alcohol followed by overnight tryptic digestion. The signature peptide formed as result of tryptic digestion was separated on a chromatograph and detected using a mass detector. The mass transition ion-pair of m/z 741.3â¯ââ¯1047.1 for PEG-IFN-α-2b and m/z 387.4â¯ââ¯205.2 for internal standard were used for MS/MS detection. The sample extraction involves a simple protein precipitation method followed by tryptic digestion of the supernatant and further sample cleanup was not needed. The method has been validated over a linear range of 1.028-3200â¯ng/mL with a correlation coefficientâ¯≥â¯0.99. The precision (%RSD) was 5.52 to 7.90 and accuracy (%RE) was within -1.80 to 1.68. The total run time was 22.0â¯min. The sensitivity of LC-MS/MS method was 1.0â¯ng/ml which was found to be more sensitive than ELISA and resulted in improving the overall study data by being able to quantify all the samples without any below LOQ results helping to further improve the pharmacokinetic modeling. This improved method is a promising anti-body free LC-MS/MS based methodology for estimation of PEG-IFN-α-2b in human serum and may be applied for other such pegylated molecules.
Subject(s)
Chromatography, High Pressure Liquid/methods , Interferon alpha-2/blood , Interferon-alpha/blood , Peptide Fragments/blood , Tandem Mass Spectrometry/methods , Cross-Over Studies , Humans , Interferon alpha-2/pharmacokinetics , Interferon-alpha/pharmacokinetics , Limit of Detection , Linear Models , Peptide Fragments/metabolism , Polyethylene Glycols/pharmacokinetics , Recombinant Proteins/blood , Recombinant Proteins/pharmacokinetics , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization/methods , Therapeutic Equivalency , Trypsin/metabolismABSTRACT
BACKGROUND: Lung cancer is one of the most common malignancies, and there is a trend of increasing incidence in young patients. The preoperative diagnosis of pulmonary nodules is mainly based on the combination of imaging and tumor markers. There is no relevant report on the diagnostic value of tumor markers in young pulmonary nodules. Our study was designed to explore the value of five tumor markers in young patients with pulmonary nodules. METHODS: We reviewed the medical records of 390 young patients (age ≤45 years) with pulmonary nodules treated at two separate centers from January 1, 2015, to January 1, 2021. Malignant pulmonary nodules were confirmed in 318 patients, and the other 72 patients were diagnosed with benign pulmonary nodules. The gold standard for diagnosis of pulmonary nodules was surgical biopsy. The conventional serum biomarkers included cytokeratin 19 (CYFRA21-1), pro-gastrin-releasing-peptide (ProGRP), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and squamous cell carcinoma-associated antigen (SCCA). The diagnostic values of five tumor markers were analyzed by receiver operating characteristic (ROC) curves. RESULTS: There were no significant differences in the expression of five tumor markers between the groups (p > 0.05). Single tumor marker (CYFRA21-1, ProGRP, CEA, NSE, and SCCA) showed a limited value in the diagnosis of malignant pulmonary nodules, with the AUC of 0.506, 0.503 0.532, 0.548, and 0.562, respectively. The AUC of the combined examination was only 0.502~0.596, which did not improve the diagnostic value. CONCLUSIONS: Five conventional tumor markers had a limited diagnostic value in young patients with pulmonary nodules.
Subject(s)
Biomarkers, Tumor/blood , Lung Neoplasms/diagnosis , Multiple Pulmonary Nodules/diagnosis , Solitary Pulmonary Nodule/diagnosis , Adult , Antigens, Neoplasm/blood , Carcinoembryonic Antigen/blood , Diagnosis, Differential , Female , Follow-Up Studies , GPI-Linked Proteins/blood , Humans , Keratin-19/blood , Lung Neoplasms/blood , Lung Neoplasms/surgery , Male , Middle Aged , Multiple Pulmonary Nodules/blood , Multiple Pulmonary Nodules/surgery , Peptide Fragments/blood , Phosphopyruvate Hydratase/blood , Prognosis , ROC Curve , Recombinant Proteins/blood , Retrospective Studies , Serpins/blood , Solitary Pulmonary Nodule/blood , Solitary Pulmonary Nodule/surgeryABSTRACT
Reduced activity of paraoxonase 1 (PON1), a high-density lipoprotein (HDL)-associated enzyme, has been implicated in the development of atherosclerosis. Post-translational modifications of PON1 may represent important mechanisms leading to reduced PON1 activity. Under atherosclerotic conditions, myeloperoxidase (MPO) is known to associate with HDL. MPO generates the oxidants hypochlorous acid and nitrogen dioxide, which can lead to post-translational modification of PON1, including tyrosine modifications that inhibit PON1 activity. Nitrogen dioxide also drives lipid peroxidation, leading to the formation of reactive lipid dicarbonyls such as malondialdehyde and isolevuglandins, which modify HDL and could inhibit PON1 activity. Because isolevuglandins are more reactive than malondialdehyde, we used in vitro models containing HDL, PON1, and MPO to test the hypothesis that IsoLG formation by MPO and its subsequent modification of HDL contributes to MPO-mediated reductions in PON1 activity. Incubation of MPO with HDL led to modification of HDL proteins, including PON1, by IsoLG. Incubation of HDL with IsoLG reduced PON1 lactonase and antiperoxidation activities. IsoLG modification of recombinant PON1 markedly inhibited its activity, while irreversible IsoLG modification of HDL before adding recombinant PON1 only slightly inhibited the ability of HDL to enhance the catalytic activity of recombinant PON1. Together, these studies support the notion that association of MPO with HDL leads to lower PON1 activity in part via IsoLG-mediated modification of PON1, so that IsoLG modification of PON1 could contribute to increased risk for atherosclerosis, and blocking this modification might prove beneficial to reduce atherosclerosis.
Subject(s)
Aryldialkylphosphatase/antagonists & inhibitors , Lipids/chemistry , Lipoproteins, HDL/metabolism , Peroxidase/metabolism , Aryldialkylphosphatase/blood , Humans , Lipid Peroxidation/drug effects , Lipids/pharmacology , Recombinant Proteins/blood , Recombinant Proteins/metabolismABSTRACT
The extent of liver fibrosis predicts prognosis and is important for determining treatment strategies for chronic hepatitis. During the fibrosis progression, serum levels of Mac2 binding protein (M2BP) increase and the N-glycan structure changes to enable binding to Wisteria floribunda agglutinin (WFA) lectin. As a novel diagnostic marker, glycosylation isomer of M2BP (M2BPGi) has been developed. However, its glycan structures recognized by WFA are unclear. In this study, we analyzed site-specific N-glycan structures of serum M2BP using Glyco-RIDGE (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile) method. We evaluated five sample types: (1) M2BP immunoprecipitated from normal healthy sera (NHS-IP(+)), (2) M2BP immunoprecipitated from sera of patients with liver cirrhosis (stage 4; F4-IP(+)), (3) M2BP captured with WFA from serum of patients with liver cirrhosis (stage 4; F4-WFA(+)), (4) recombinant M2BP produced by HEK293 cells (rM2BP) and (5) WFA-captured rM2BP (rM2BP-WFA(+)). In NHS-IP(+) M2BP, bi-antennary N-glycan was the main structure, and LacNAc extended to its branches. In F4-IP(+) M2BP, many branched structures, including tri-antennary and tetra-antennary N-glycans, were found. F4-WFA(+) showed a remarkable increase in branched structures relative to the quantity before enrichment. In recombinant M2BP, both no sialylated-LacdiNAc and -branched LacNAc structures were emerged. The LacdiNAc structure was not found in serum M2BP. Glycosidase-assisted HISCL assays suggest that reactivity with WFA of both serum and recombinant M2BP depends on unsialylated and branched LacNAc and in part of recombinant depends on LacdiNAc. On M2BPGi, the highly branched LacNAc, probably dense cluster of LacNAc, would be recognized by WFA.
Subject(s)
Antigens, Neoplasm/chemistry , Biomarkers, Tumor/chemistry , Liver Cirrhosis/blood , Plant Lectins/chemistry , Polysaccharides/chemistry , Receptors, N-Acetylglucosamine/chemistry , Antigens, Neoplasm/blood , Biomarkers, Tumor/blood , HEK293 Cells , Healthy Volunteers , Humans , Plant Lectins/blood , Polysaccharides/blood , Protein Array Analysis , Receptors, N-Acetylglucosamine/blood , Recombinant Proteins/blood , Recombinant Proteins/chemistryABSTRACT
Background MEDI6012 is recombinant human lecithin cholesterol acyltransferase, the rate-limiting enzyme in reverse cholesterol transport. Infusions of lecithin cholesterol acyltransferase have the potential to enhance reverse cholesterol transport and benefit patients with coronary heart disease. The purpose of this study was to test the safety, pharmacokinetic, and pharmacodynamic profile of MEDI6012. Methods and Results This phase 2a double-blind study randomized 48 subjects with stable coronary heart disease on a statin to a single dose of MEDI6012 or placebo (6:2) (NCT02601560) with ascending doses administered intravenously (24, 80, 240, and 800 mg) and subcutaneously (80 and 600 mg). MEDI6012 demonstrated rates of treatment-emergent adverse events that were similar to those of placebo. Dose-dependent increases in high-density lipoprotein cholesterol were observed with area under the concentration-time curves from 0 to 96 hours of 728, 1640, 3035, and 5318 should be: mg·h/mL in the intravenous dose groups and 422 and 2845 mg·h/mL in the subcutaneous dose groups. Peak mean high-density lipoprotein cholesterol percent change was 31.4%, 71.4%, 125%, and 177.8% in the intravenous dose groups and 18.3% and 111.2% in the subcutaneous dose groups, and was accompanied by increases in endogenous apoA1 (apolipoprotein A1) and non-ATP-binding cassette transporter A1 cholesterol efflux capacity. Decreases in apoB (apolipoprotein B) were observed across all dose levels and decreases in atherogenic small low-density lipoprotein particles by 41%, 88%, and 79% at the 80-, 240-, and 800-mg IV doses, respectively. Conclusions MEDI6012 demonstrated an acceptable safety profile and increased high-density lipoprotein cholesterol, endogenous apoA1, and non-ATP-binding cassette transporter A1 cholesterol efflux capacity while reducing the number of atherogenic low-density lipoprotein particles. These findings are supportive of enhanced reverse cholesterol transport and a functional high-density lipoprotein phenotype. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02601560.
Subject(s)
Coronary Disease/drug therapy , Lipoproteins, HDL/administration & dosage , Lipoproteins, LDL/administration & dosage , Phosphatidylcholine-Sterol O-Acyltransferase/administration & dosage , Recombinant Proteins/administration & dosage , Administration, Intravenous , Adult , Aged , Apolipoprotein A-I/blood , Dose-Response Relationship, Drug , Double-Blind Method , Female , Humans , Injections, Subcutaneous , Lipoproteins, HDL/adverse effects , Lipoproteins, HDL/blood , Lipoproteins, LDL/adverse effects , Lipoproteins, LDL/blood , Male , Middle Aged , Phosphatidylcholine-Sterol O-Acyltransferase/adverse effects , Phosphatidylcholine-Sterol O-Acyltransferase/blood , Recombinant Proteins/adverse effects , Recombinant Proteins/blood , Treatment OutcomeABSTRACT
Recombinant batroxobin (S3101) is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system. A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies. Therefore, a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101. In this study, a sensitive bioanalytical method was developed and validated, using a Quanterix single molecular array (Simoa) assay. Moreover, to thoroughly assess the platform, enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed, and their performance was compared with that of this novel technology platform. The assay was validated in compliance with the current guidelines. Measurements with the Simoa assay were precise and accurate, presenting a valid assay range from 6.55 to 4000 pg/mL. The intra- and inter-run accuracy and precision were within -19.3% to 15.3% and 5.5% to 17.0%, respectively. S3101 was stable in human serum for 280 days at -20°C and -70°C, for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature (22°C-28°C), respectively, and after five and two freeze-thaw cycles at -70°C and -20°C, respectively. The Simoa assay also demonstrated sufficient dilution linearity, assay sensitivity, and parallelism for quantifying S3101 in human serum. The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.
Subject(s)
Batroxobin/blood , Enzyme-Linked Immunosorbent Assay , Recombinant Proteins/blood , Batroxobin/isolation & purification , Batroxobin/pharmacokinetics , Female , Fibrinogen/metabolism , Humans , Male , Protein Binding/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacokineticsABSTRACT
BACKGROUND: Serum biomarkers have been widely adopted in clinical practice for assisting lung cancer diagnoses, therapeutic monitoring, and prognostication. The function of a well-performing tumor biomarker depends on a reliable reference interval (RI) with consideration of the study subjects' age, gender, and geographical location. This study aimed to establish a RI for each of 6 lung cancer biomarkers for use in the whole country of China on Mindray platform. METHODS: The levels of serum 6 lung cancer biomarkers-namely progastrin-releasing peptide (ProGRP), neuron-specific enolase (NSE), squamous cell carcinoma antigen (SCC), carcinoembryonic antigen (CEA), cytokeratin-19 fragment (CYFRA21-1), and human epididymis protein 4 (HE4)-were measured utilizing the chemiluminescence immunoassay on the Mindray CL-6000i platform following the laboratory standard operating procedures in apparently healthy Chinese individuals on large cohort, multicenter, and geographical consideration bases. The CLSI EP28-A3C guideline was followed for the enrollment of study subjects. RESULTS: The age-stratified, gender-specific RIs for ProGRP, NSE, SCC, CEA, CYFRA21-1, and HE4 lung cancer biomarkers in the Chinese population have been established as described in the results and discussion in this work. In addition, various levels of the six lung cancer biomarkers among nine geographical locations in China have been observed. CONCLUSIONS: The sample volume of study cohort, age, and geographical location should be considered upon establishing a reliable biomarker RI. A RI for each of six lung cancer biomarkers has been established. The results from this study would be helpful for clinical laboratories in interpreting the analytical results and for clinicians in patient management.
Subject(s)
Biomarkers, Tumor/blood , Immunoassay/methods , Lung Neoplasms/diagnosis , Adolescent , Adult , Age Factors , Antigens, Neoplasm/blood , Carcinoembryonic Antigen/blood , Carrier Proteins/blood , China , Cohort Studies , Female , GPI-Linked Proteins/blood , Geography , Humans , Keratin-19/blood , Lung Neoplasms/blood , Male , Middle Aged , Peptide Fragments/blood , Prognosis , Recombinant Proteins/blood , Reference Values , Serpins/blood , Sex Factors , WAP Four-Disulfide Core Domain Protein 2/analysis , Young AdultABSTRACT
OBJECTIVE: To improve the clinical understanding of anti-gamma-aminobutyric-acid B receptor encephalitis (anti-GABABR encephalitis) by analyzing 13 cases. METHODS: We retrospectively studied demographic and clinical features including clinical symptoms, serum/cerebrospinal fluid (CSF) laboratory findings (including antibody test), brain magnetic resonance imaging (MRI), electroencephalogram (EEG), treatment plan, and treatment effect for 13 patients with a definitive diagnosis of anti-GABABR encephalitis. RESULTS: Seven patients (53.8%, 7/13) were complicated with lung cancer. Epileptic seizures were the most common symptoms at onset in 11 patients (84.6%, 11/13). All patients had seizures in the course of the disease. Abnormalities in craniocerebral MRI examination, including hippocampus, occipital lobe, insular lobe, were found in six of nine tested patients, and EEG abnormalities were found in seven out of nine tested patients. Elevated pro-gastrin releasing peptide (ProGRP) levels were found in 70% of patients with a median value of 490.10 pg/ml; and CSF oligoclonal bands were positive for 4 of 10 tested cases. However, there were no significant differences in modified Rankin Scale (mRS) between the ProGRP or CSF oligoclonal band positive and negative groups at admission and follow-up (p > .05). The value between SCLC and non-SCLC subgroup was significantly different (p < .05). Ten patients received immunotherapy (three patients refused treatment). After immunotherapy, the frequency of seizures was significantly reduced. There was a significant difference in mRS between admission and after treatment (p < .05). The average survival time after onset was 27.7 months. CONCLUSIONS: Epilepsy is the most common clinical manifestation of Anti-GABABR encephalitis. The prognosis of anti-GABABR encephalitis is poor. Section of anti-GABABR encephalitis patients have higher level of serum ProGRP and positive GSF oligoclonal bands. Elevated ProGRP or positive CSF oligoclonal bands with classic clinical features can potentially help to improve early recognition of anti-GABABR encephalitis.
Subject(s)
Brain/diagnostic imaging , Encephalitis/diagnosis , Encephalitis/immunology , Peptide Fragments/cerebrospinal fluid , Receptors, GABA-B/immunology , Adult , Aged , Brain/physiopathology , China , Electroencephalography , Encephalitis/metabolism , Encephalitis/physiopathology , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Oligoclonal Bands , Peptide Fragments/blood , Recombinant Proteins/blood , Recombinant Proteins/cerebrospinal fluid , Retrospective StudiesABSTRACT
The dodecapeptide angiotensin-(1-12) [Ang-(1-12)] functions as an intracrine/paracrine substrate for local production of angiotensin II. We developed a reliable and specific radioimmunoassay (RIA) method for the measurement of Ang-(1-12) in human plasma and urine using an affinity purified antibody fraction directed towards the C-terminus of the human Ang-(1-12) sequence. The RIA method was applied to quantify the Ang-(1-12) in plasma and urine collected from thirty-four human subjects (29 treated with antihypertensive medicines and 5 untreated patients). Plasma Ang-(1-12) level was significantly higher (P < 0.05) in patients with systolic blood pressure ≥140 mm Hg (n = 10) compared to the group with systolic blood pressure <140 mm Hg (n = 24). No significant difference (P = 0.22) was found in spot urine between the groups. Our study also shows that the polyclonal antibody neutralizes the cleavage sites of the human Ang-(1-12) from recombinant human chymase (rhChymase) and serum angiotensin converting enzyme (ACE) mediated Ang II generating hydrolysis. Overall, this newly developed RIA method is reliable and applicable to accurately quantify the Ang-(1-12) level in clinical samples (plasma and urine). Further, our in vitro neutralization study suggests that the anti-Ang-(1-12)-antibody might be used as an in vivo therapeutic agent for preventing Ang-(1-12)/Ang II-mediated hypertension and organ damage.
Subject(s)
Angiotensinogen/blood , Angiotensinogen/urine , Hypertension/genetics , Peptide Fragments/blood , Peptide Fragments/urine , Radioimmunoassay/methods , Renin-Angiotensin System/genetics , Aged , Angiotensin II/blood , Angiotensin II/genetics , Angiotensin II/urine , Angiotensinogen/genetics , Antibodies/chemistry , Antibodies/isolation & purification , Antihypertensive Agents/therapeutic use , Blood Pressure/genetics , Case-Control Studies , Chymases/blood , Chymases/genetics , Female , Gene Expression Regulation , Humans , Hypertension/blood , Hypertension/drug therapy , Hypertension/urine , Limit of Detection , Male , Middle Aged , Peptide Fragments/genetics , Radioimmunoassay/standards , Recombinant Proteins/blood , Recombinant Proteins/genetics , Signal Transduction , Water-Electrolyte Balance/geneticsABSTRACT
BACKGROUND: Due to insufficient scientific evidence, panels of tumour markers (TMs) are currently not recommended for use in suspected cancer. However, recent well-designed studies have revealed a potential clinical value in lung cancer. We analysed the diagnostic accuracy of a panel of 11 circulating TMs with clinically controlled thresholds in the differentiation of cancer from nonmalignant diseases. METHODS: We prospectively recruited 4776 consecutive patients presenting with focal or nonspecific symptoms suggestive of cancer who underwent testing for 11 serum TMs before diagnosis was known. The study abided by 2015 STARD guidelines. Tumour markers included, among others, carbohydrate antigen 19-9, carcinoembryonic antigen, alpha-fetoprotein, squamous cell carcinoma-associated antigen, prostate-specific antigen (males), neuron-specific enolase, progastrin-releasing peptide and carbohydrate antigen 125. Thresholds were adjusted for the presence of kidney failure, liver disease, effusions and dermatological disorders. Results showing ≥1 TMs with concentrations above threshold were considered positive. RESULTS: Benign diseases were diagnosed in 3281 (68.7%) patients and cancer in 1495 (31.3%), with epithelial cancers in 1214 (77% at stage IV). When applying criteria for controlled thresholds, overall specificity was 98%. Overall sensitivity of the panel in epithelial cancers was 72.2%, positive predictive value 93% and negative predictive value 90.5%. The area under the receiver operating characteristic curve was 0.920 (95% confidence interval, 0.902-0.924). CONCLUSIONS: By using clinically controlled cut-offs, the combined panel demonstrated an excellent ability to discriminate epithelial cancers from nonmalignant diseases. However, its use in clinical practice would need formal validation through a multicentre controlled trial assessing a panel-guided strategy vs. standard diagnosis.
Subject(s)
Biomarkers, Tumor/blood , Neoplasms/blood , Abdominal Pain/physiopathology , Aged , Antigens, Neoplasm/blood , CA-125 Antigen/blood , CA-19-9 Antigen/blood , Carcinoembryonic Antigen/blood , Carcinoma/blood , Carcinoma/diagnosis , Case-Control Studies , Dyspnea/physiopathology , Female , Hematologic Neoplasms/blood , Hematologic Neoplasms/diagnosis , Humans , Keratin-19/blood , Lymphadenopathy/physiopathology , Lymphoma/blood , Lymphoma/diagnosis , Male , Melanoma/blood , Melanoma/diagnosis , Middle Aged , Mucin-1/blood , Neoplasms/diagnosis , Neoplasms/physiopathology , Nervous System Diseases/physiopathology , Pain/physiopathology , Peptide Fragments/blood , Phosphopyruvate Hydratase/blood , Prostate-Specific Antigen/blood , Recombinant Proteins/blood , Sarcoma/blood , Sarcoma/diagnosis , Sensitivity and Specificity , Serpins/blood , Weight Loss , alpha-Fetoproteins/metabolismABSTRACT
Hyperferritinemia is associated with poor outcomes in critically ill patients with sepsis, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndromes (MAS) and coronavirus disease 19 (COVID-19). Autopsies of hyperferritinemic patients that succumbed to either sepsis, HLH, MAS or COVID-19 have revealed disseminated microvascular thromboses with von Willebrand factor (VWF)-, platelets-, and/or fibrin-rich microthrombi. It is unknown whether high plasma ferritin concentration actively promotes microvascular thrombosis, or merely serves as a prognostic biomarker in these patients. Here, we show that secretion of VWF from human umbilical vein endothelial cells (HUVEC) is significantly enhanced by 100,000 ng/ml of recombinant ferritin heavy chain protein (FHC). Ferritin fraction that was isolated by size exclusion chromatography from the plasma of critically ill HLH patients promoted VWF secretion from HUVEC, compared to similar fraction from non-critically ill control plasma. Furthermore, recombinant FHC moderately suppressed the activity of VWF cleaving metalloprotease ADAMTS-13. These observations suggest that a state of marked hyperferritinemia could promote thrombosis and organ injury by inducing endothelial VWF secretion and reducing the ADAMTS-13 activity.
Subject(s)
ADAMTS13 Protein/metabolism , COVID-19/blood , COVID-19/complications , Ferritins/metabolism , Hyperferritinemia/blood , Hyperferritinemia/complications , von Willebrand Factor/metabolism , ADAMTS13 Protein/antagonists & inhibitors , COVID-19/immunology , Critical Illness , Ferritins/blood , Human Umbilical Vein Endothelial Cells , Humans , Lymphohistiocytosis, Hemophagocytic/blood , Lymphohistiocytosis, Hemophagocytic/complications , Oxidoreductases/blood , Oxidoreductases/metabolism , Recombinant Proteins/blood , Recombinant Proteins/metabolism , SARS-CoV-2 , Thrombosis/blood , Thrombosis/etiologyABSTRACT
Hepatitis B virus (HBV) infection is a major public health problem worldwide. The study aimed to evaluate the efficacy of pegylated interferon (Peg-IFN) alfa-2a treatment for seroclearance of HBs antigen (HBsAg) in HBe antigen (HBeAg)-negative chronic hepatitis B (CHB) patients. This retrospective study investigated 16 HBeAg-negative CHB patients who received Peg-IFN alfa-2a weekly for 48 weeks. Thereafter, the patients were followed-up for 48 weeks after the end of therapy. The following criteria were also used for inclusion: HBV-DNA < 5.0 log copies/mL and without nucleot(s)ide analogs. Four HBsAg-positive cases became HBsAg negative. The HBsAg levels of the 4 patients who achieved HBsAg seroclearance were lower significantly than that of the non-seroclearance group (p = 0.007). The mean HBsAg levels in these 4 cases were 68 IU/mL, while the mean HBsAg levels in the non-seroclearance group were 2,114 IU/mL. The mean HBV-DNA levels in the 4 HBsAg seroclearance cases were 2.8 log copies/mL as compared to 3.6 log copies/mL in HBsAg-non-seroclearance cases (p = 0.01). Cases that are HBeAg negative, with HBV-DNA levels < 5 log copies/mL, and HBsAg titers < 120 IU/mL cases may achieve HBsAg clearance with Peg-IFN therapy.
Subject(s)
Hepatitis B e Antigens/analysis , Hepatitis B/blood , Interferon-alpha/analysis , Polyethylene Glycols/analysis , Time Factors , Adult , Aftercare/methods , Chi-Square Distribution , Female , Hepatitis B/physiopathology , Hepatitis B e Antigens/blood , Humans , Interferon-alpha/blood , Male , Middle Aged , Recombinant Proteins/analysis , Recombinant Proteins/blood , Seroconversion , Treatment OutcomeABSTRACT
This study evaluated whether recombinant human erythropoietin (rhEpo) treatment combined with chronic hypoxia provided an additive erythropoietic response and whether the athlete biological passport (ABP) sensitivity improved with hypoxia. Two interventions were completed, each containing 4 weeks baseline, 4 weeks exposure at sea level or 2,320 m of altitude, and 4 weeks follow-up. Participants were randomly assigned to 20 IU·kg bw-1 rhEpo or placebo injections every second day for 3 weeks during the exposure period at sea level (rhEpo n = 25, placebo n = 9) or at altitude (rhEpo n = 12, placebo n = 27). Venous blood was analyzed weekly. Combining rhEpo and hypoxia induced larger changes compared with rhEpo or hypoxia alone for [Hb] (p < 0.001 and p > 0.05, respectively), reticulocyte percentage (p < 0.001), and OFF-hr score (p < 0.01 and p < 0.001, respectively). The most pronounced effect was observed for reticulocyte percentage with up to ~35% (p < 0.001) and ~45% (p < 0.001) higher levels compared with rhEpo or hypoxia only, respectively. The ABP sensitivity for the combined treatment was 54 and 35 percentage points higher for [Hb] (p < 0.05) and reticulocyte percentage (p < 0.05), respectively, but similar for OFF-hr score, compared with rhEpo at sea level. Across any time point, [Hb] and OFF-hr score combined identified 14 unique true-positive participants (56%) at sea level and 12 unique true-positive participants (100%) at altitude. However, a concurrent reduction in specificity existed at altitude. In conclusion, rhEpo treatment combined with hypoxic exposure provided an additive erythropoietic response compared with rhEpo or hypoxic exposure alone. Correspondingly, ABP was more sensitive to rhEpo at altitude than at sea level, but a compromised specificity existed with hypoxic exposure.