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1.  Perchlorate Exposure is Associated with Oxidative Stress and Indicators of Serum Iron Homeostasis Among NHANES 2005–2008 Subjects 
Biomarker Insights  2015;10:9-19.
Perchlorate (ClO4−), an oxidizing agent, is a ubiquitous environmental pollutant. Several studies have investigated its thyroid hormone disrupting properties. Its associations with other biological measures are largely unknown. This study, combining 2005–2008 National Health and Nutrition Examination Surveys, investigated associations between urinary perchlorate and biomarkers of iron homeostasis, lipids, blood cell counts, and glucose metabolism. Healthy males (n = 3705), non-pregnant females (n = 2967), and pregnant females (n = 356), aged 12–59 years, were included in the linear regression models, which showed significant positive (+) and negative (−) associations for both males and non-pregnant females with serum uric acid (−), serum iron (−), RBC count (−), blood urea nitrogen (+), and lymphocyte count (+). Other significant associations were observed for either males or non-pregnant females. Among pregnant females, perchlorate was significantly associated with blood urea nitrogen (+) and serum iron (−). These associations may be indicators of perchlorate’s potential effect on several biological systems, which when considered in total, may implicate perturbation of iron homeostasis.
PMCID: PMC4310500
perchlorate; epidemiology; biomarkers; iron homeostasis
2.  Prednisolone-Induced Predisposition to Femoral Head Separation and the Accompanying Plasma Protein Changes in Chickens 
Biomarker Insights  2015;10:1-8.
Femoral head separation (FHS) is an idiopathic bone problem that causes lameness and production losses in commercial poultry. In a model of prednisolone-induced susceptibility to FHS, the changes in plasma proteins and peptides were analyzed to find possible biomarkers. Plasma samples from control and FHS-susceptible birds were depleted of their high abundance proteins by acetonitrile precipitation and were then subjected to cation exchange and reverse-phase (RP) fractionations. Analysis with matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) showed several differentially expressed peptides, two of which were isolated by RP-HPLC and identified as the fragments of apolipoprotein A-I. The acetonitrile fractionated plasma proteins were subjected to reduction/alkylation and trypsin digestion followed by liquid chromatography and tandem mass spectrometry, which showed the absence of protocadherin 15, vascular endothelial growth factor-C, and certain transcription and ubiquitin-mediated proteolytic factors in FHS-prone birds. It appears that prednisolone-induced dyslipidemia, vascular, and tissue adhesion problems may be consequential to FHS. Validity of these biomarkers in our model and the natural disease must be verified in future using traditional approaches.
Lameness because of femoral head separation (FHS) is a production and welfare problem in the poultry industry. Selection against FHS requires identification of the birds with subclinical disease with biomarkers from a source such as blood. Prednisolone can induce femoral head problems and predisposition to FHS. Using this experimental model, we analyzed the plasma peptides and proteins from normal and FHS-prone chickens by mass spectrometry to identify differentially expressed peptides and proteins. We found two peptides, both derived from apolipoprotein A-I, quantitatively elevated and two proteins, protocadherin 15 and VEGF-C, that were conspicuously absent in FHS-susceptible birds.
PMCID: PMC4295844  PMID: 25635167
femoral head separation; glucocorticoids; chickens; mass spectrometry; biomarker; proteomics
3.  Risk Factors Associated with Serum Levels of the Inflammatory Biomarker Soluble Urokinase Plasminogen Activator Receptor in a General Population 
Biomarker Insights  2014;9:91-100.
The soluble urokinase plasminogen activator receptor (suPAR) is a biomarker of mortality risk in various patient populations. However, little is known about the implications of lifestyle for suPAR levels in the general population. Lifestyle, demographic, and cardiovascular disease (CVD) risk factor data were collected from 5,538 participants in the Danish population-based Inter99 study. Their suPAR levels were measured using a sandwich enzyme-linked immunosorbent assay. In the final adjusted model, smoking and morbid obesity were strongly associated with higher suPAR levels (P < 0.001). An unhealthy diet and alcohol abstinence in men were also associated with higher suPAR levels. Physical activity in leisure time had a modest impact on suPAR levels in univariate analysis, but not in the final adjusted model. In conclusion, smoking and morbid obesity were strongly associated with higher serum suPAR levels in this general population. Diet and alcohol consumption also seemed to impact suPAR levels. Lifestyle changes are likely to affect suPAR since ex-smokers had suPAR levels comparable to those of never-smokers.
PMCID: PMC4269129  PMID: 25574132
biomarkers; epidemiologic studies; risk factors; urokinase plasminogen activator receptor
4.  Decreased Mitogen Inducible Gene 6 (MIG-6) Associated with Symptom Severity in Children with Autism 
Biomarker Insights  2014;9:85-89.
Individuals with autism spectrum disorders (ASDs) demonstrate impairment in social interactions and problems in verbal and nonverbal communication. Autism spectrum disorders are thought to affect 1 in 88 children in the US. Recent research has shown that epidermal growth factor receptor (EGFR) activation is associated with nerve cell development and repair. Mitogen inducible gene 6 (MIG-6) is a 58-kDa non-kinase scaffolding adaptor protein consisting of 462 amino-acids, which has been shown to be a negative feedback regulator of EGFR and Met receptor tyrosine kinase (RTK) signaling.
In this study, we determined plasma levels of MIG-6, which suppresses the EGFR RTK pathway in autistic children, and compared MIG-6 levels with the EGFR ligand, epidermal growth factor (EGF), and the cMET ligand, hepatocyte growth factor (HGF). MIG-6 levels were also compared to the symptom severity of 19 different autistic behaviors.
Plasma MIG-6 concentration was measured in 40 autistic children and 39 neurotypical, age, and gender similar controls using an enzyme linked immunosorbent assay (ELISA). Plasma MIG-6 levels were compared to putative biomarkers known to be associated with EGFR and cMET and severity levels of 19 autism related symptoms [awareness, expressive language, receptive language, (conversational) pragmatic language, focus/attention, hyperactivity, impulsivity, perseveration, fine motor skills, gross motor skills, hypotonia (low muscle tone), tip toeing, rocking/pacing, stimming, obsessions/fixations, eye contact, sound sensitivity, light sensitivity, and tactile sensitivity].
In this study, we found that plasma MIG-6 levels in autistic children (182.41 ± 24.3 pg/ml) were significantly lower than neurotypical controls (1779.76 ± 352.5; P = 1.76E − 5). Decreased MIG-6 levels correlated with serotonin, dopamine, Tumor necrosis factor alpha (TNF-alpha), and urokinase receptor (uPAR) concentration, but not with other tested putative biomarkers. MIG-6 levels also correlated significantly with severity of expressive language, receptive language, tip toeing, rocking/pacing, and hand flapping/stimming.
These results suggest a relationship between decreased plasma MIG-6 levels, biomarkers associated with the EGFR pathway, and symptom severity in autism. A strong correlation between plasma MIG-6 and dopamine and serotonin levels suggest that decreased MIG-6 levels may be associated with abnormal neurotransmitter synthesis and/or action. A strong correlation between MIG-6 and uPAR and the inflammatory marker TNF-alpha suggests that low MIG-6 levels may be associated with the HGF/Met signaling pathway, as well as inflammation in autistic children.
PMCID: PMC4197901  PMID: 25342879
MIG-6; EGFR; EGF; dopamine; serotonin; uPAR; TNF-alpha; autism; symptom severity
5.  Increased Expression of Phosphorylated FADD in Anaplastic Large Cell and Other T-Cell Lymphomas 
Biomarker Insights  2014;9:77-84.
FAS-associated protein with death domain (FADD) is a major adaptor protein involved in extrinsic apoptosis, embryogenesis, and lymphocyte homeostasis. Although abnormalities of the FADD/death receptor apoptotic pathways have been established in tumorigenesis, fewer studies have analyzed the expression and role of phosphorylated FADD (pFADD). Our identification of FADD as a lymphoma-associated autoantigen in T-cell lymphoma patients raises the possibility that pFADD, with its correlation with cell cycle, may possess role(s) in human T-cell lymphoma development. This immunohistochemical study investigated pFADD protein expression in a range of normal tissues and lymphomas, particularly T-cell lymphomas that require improved therapies. Whereas pFADD was expressed only in scattered normal T cells, it was detected at high levels in T-cell lymphomas (eg, 84% anaplastic large cell lymphoma and 65% peripheral T cell lymphomas, not otherwise specified). The increased expression of pFADD supports further study of its clinical relevance and role in lymphomagenesis, highlighting phosphorylation of FADD as a potential therapeutic target.
PMCID: PMC4159367  PMID: 25232277
FADD; pFADD; lymphoma; autoantigen; ALCL; PTCL
6.  Identification of Gene Signatures Used to Recognize Biological Characteristics of Gastric Cancer Upon Gene Expression Data 
Biomarker Insights  2014;9:67-76.
High-throughput gene expression microarrays can be examined by machine-learning algorithms to identify gene signatures that recognize the biological characteristics of specific human diseases, including cancer, with high sensitivity and specificity. A previous study compared 20 gastric cancer (GC) samples against 20 normal tissue (NT) samples and identified 1,519 differentially expressed genes (DEGs). In this study, Classification Information Index (CII), Information Gain Index (IGI), and RELIEF algorithms are used to mine the previously reported gene expression profiling data. In all, 29 of these genes are identified by all three algorithms and are treated as GC candidate biomarkers. Three biomarkers, COL1A2, ATP4B, and HADHSC, are selected and further examined using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) staining in two independent sets of GC and normal adjacent tissue (NAT) samples. Our study shows that COL1A2 and HADHSC are the two best biomarkers from the microarray data, distinguishing all GC from the NT, whereas ATP4B is diagnostically significant in lab tests because of its wider range of fold-changes in expression. Herein, a data-mining model applicable for small sample sizes is presented and discussed. Our result suggested that this mining model may be useful in small sample-size studies to identify putative biomarkers and potential biological features of GC.
PMCID: PMC4149392  PMID: 25210421
gastric cancer; gene signature; microarray; machine-learning algorithm
7.  Hydroxyproline, a Serum Biomarker Candidate for Gastric Ulcer in Rats: A Comparison Study of Metabolic Analysis of Gastric Ulcer Models Induced by Ethanol, Stress, and Aspirin 
Biomarker Insights  2014;9:61-66.
Gastrointestinal symptoms are a common manifestation of adverse drug effects. Non-steroid anti-inflammatory drugs (NSAIDs) are widely prescribed drugs that induce the serious side effect of gastric mucosal ulceration. Biomarkers for these side effects have not been identified and ulcers are now only detectable by endoscopy. We previously identified five metabolites as biomarker candidates for NSAID-induced gastric ulcer using capillary electrophoresis–mass spectrometry (CE–MS)-based metabolomic analysis of serum and stomach from rats. Here, to clarify mechanism of changes and limitations of indications of biomarker candidates, we performed CE–MS-based metabolomic profiling in stomach and serum from rats with gastric ulcers induced by ethanol, stress, and aspirin. The results suggest that a decrease in hydroxyproline reflects the induction of gastric injury and may be useful in identifying gastric ulcer induced by multiple causes. While extrapolation to humans requires further study, hydroxyproline can be a new serum biomarker of gastric injury regardless of cause.
PMCID: PMC4125369  PMID: 25125970
metabolomics; capillary electrophoresis–mass spectrometry (CE–MS); gastric injury; diagnostic marker candidate
8.  DNA Methylation at the Novel CpG Sites in the Promoter of MED15/PCQAP Gene as a Biomarker for Head and Neck Cancers 
Biomarker Insights  2014;9:53-60.
Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch’s t-test returning P < 0.05 and Mann–Whitney test P < 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P < 0.01) and 0.63 (P < 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC.
PMCID: PMC4085102  PMID: 25057238
Saliva; DNA methylation; diagnostics; non-invasiveness
9.  Integrative Genomic Analysis for the Discovery of Biomarkers in Prostate Cancer 
Biomarker Insights  2014;9:39-51.
Genome-wide association studies (GWAS) have achieved great success in identifying single nucleotide polymorphisms (SNPs, herein called genetic variants) and genes associated with risk of developing prostate cancer. However, GWAS do not typically link the genetic variants to the disease state or inform the broader context in which the genetic variants operate. Here, we present a novel integrative genomics approach that combines GWAS information with gene expression data to infer the causal association between gene expression and the disease and to identify the network states and biological pathways enriched for genetic variants. We identified gene regulatory networks and biological pathways enriched for genetic variants, including the prostate cancer, IGF-1, JAK2, androgen, and prolactin signaling pathways. The integration of GWAS information with gene expression data provides insights about the broader context in which genetic variants associated with an increased risk of developing prostate cancer operate.
PMCID: PMC4085106  PMID: 25057237
GWAS; genetic variants; gene expression; prostate cancer
10.  Folate Receptor Alpha, Mesothelin and Megakaryocyte Potentiating Factor as Potential Serum Markers of Chronic Kidney Disease 
Biomarker Insights  2014;9:29-37.
Renal disease is the eighth leading cause of death in the United States. Early diagnosis is usually based on the detection of proteinuria or elevated serum creatinine, a relatively poor biomarker that does not accurately predict renal disease progression. As a result, more predictive biomarkers of renal function are sought. We present preliminary data on three protein biomarkers, folate receptor alpha (FRA), mesothelin (MSLN), and megakaryocyte potentiating factor (MPF), currently being pursued for applications in oncology diagnostics, and evaluate serum and urine levels in subjects with renal disease. Compared to healthy subjects, a significant (P < 0.0001) increase in all three biomarkers in both serum and urine of subjects with renal disease was demonstrated. Further, serum levels of these three protein biomarkers increased with increasing stage of disease suggesting their potential value in predicting progression in subjects with renal disease and raising caution in interpretation of data in oncology applications.
PMCID: PMC4051790  PMID: 24932099
renal disease; folate receptor alpha; mesothelin; megakaryocyte potentiating factor; predictive biomarkers
11.  HLA-DRB1*08:02 Is Associated with Bucillamine-Induced Proteinuria in Japanese Rheumatoid Arthritis Patients 
Biomarker Insights  2014;9:23-28.
Drug-induced proteinuria can occur in rheumatoid arthritis (RA) patients treated with d-penicillamine, gold salts, or bucillamine (Buc), and represents a drug hypersensitivity reaction. Striking associations of human leukocyte antigen (HLA) alleles with adverse reactions have recently been reported for many drugs.
We investigated the association of HLA class II with Buc-induced proteinuria (BI-Pro) in 485 Japanese RA patients treated with Buc, of whom 25 had developed BI-Pro.
This preliminary study showed a highly significant association of DRB1*08:02 with BI-Pro (P = 1.09 × 10−6, corrected P [Pc] = 1.96 × 10−5, odds ratio [OR] 25.17, 95% confidence interval [CI] 7.98–79.38). DQB1*04:02 was also significantly associated with increased risk of BI-Pro (P = 2.44 × 10−5, Pc = 2.69 × 10−4, OR 10.35, 95%CI 3.99–26.83). These findings provide useful information for promoting personalized medicine for RA.
PMCID: PMC4038630  PMID: 24899791
bucillamine; drug-induced proteinuria; HLA-DRB1*08:02; rheumatoid arthritis; disease-modifying anti-rheumatic drugs
12.  Prognostic Value of DNA and mRNA E6/E7 of Human Papillomavirus in the Evolution of Cervical Intraepithelial Neoplasia Grade 2 
Biomarker Insights  2014;9:15-22.
This study aimed at evaluating whether human papillomavirus (HPV) groups and E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 are prognostic of cervical intraepithelial neoplasia (CIN) 2 outcome in women with a cervical smear showing a low-grade squamous intraepithelial lesion (LSIL).
This cohort study included women with biopsy-confirmed CIN 2 who were followed up for 12 months, with cervical smear and colposcopy performed every three months.
Women with a negative or low-risk HPV status showed 100% CIN 2 regression. The CIN 2 regression rates at the 12-month follow-up were 69.4% for women with alpha-9 HPV versus 91.7% for other HPV species or HPV-negative status (P < 0.05). For women with HPV 16, the CIN 2 regression rate at the 12-month follow-up was 61.4% versus 89.5% for other HPV types or HPV-negative status (P < 0.05). The CIN 2 regression rate was 68.3% for women who tested positive for HPV E6/E7 mRNA versus 82.0% for the negative results, but this difference was not statistically significant.
The expectant management for women with biopsy-confirmed CIN 2 and previous cytological tests showing LSIL exhibited a very high rate of spontaneous regression. HPV 16 is associated with a higher CIN 2 progression rate than other HPV infections. HPV E6/E7 mRNA is not a prognostic marker of the CIN 2 clinical outcome, although this analysis cannot be considered conclusive. Given the small sample size, this study could be considered a pilot for future larger studies on the role of predictive markers of CIN 2 evolution.
PMCID: PMC3999821  PMID: 24812482
viral oncogene proteins; papillomavirus E7 proteins; E6 protein; human papillomavirus-16; cervical intraepithelial neoplasia; neoplasm regression; spontaneous; disease progression
13.  The Range of Uncertainty: a Tool for Efficiently Addressing Result Variability Around Clinical Decision Points for Hepatitis C Response-guided Therapy 
Biomarker Insights  2014;9:9-13.
Despite efforts to standardize molecular diagnostic tests, performance differences are not rare. Laboratories are challenged in situations where treatment rules have been established using a reference assay that is different from the assays being used in daily practice. Assessing the viral load status of patients with chronic hepatitis C under modern triple therapy is a recognized example. We demonstrate the use of the range of uncertainty as an easy and efficient tool that can provide information on the certainty of a test result’s interpretation in the context of making hepatitis C virus treatment decisions.
PMCID: PMC3999813  PMID: 24812481
in vitro diagnostic tests; standardization; precision; hepatitis C; viral load; response guided therapy
14.  Comment on “Validation of a Blood-Based Laboratory Test to Aid in the Confirmation of a Diagnosis of Schizophrenia” 
Biomarker Insights  2014;9:7.
Schwarz et al. Validation of a Blood-Based Laboratory Test to Aid in the Confirmation of a Diagnosis of Schizophrenia. Biomarker Insights. 2010:5 39–47 doi:10.4137/BMI.S4877.
PMCID: PMC3964184  PMID: 24678240
schizophrenia; biomarker; diagnosis; clinical test
15.  Utility of C-terminal Telopeptide in Evaluating Levothyroxine Replacement Therapy-Induced Bone Loss 
Biomarker Insights  2014;9:1-6.
Levothyroxine (LT4) therapy has shown to have effects on bone metabolism though its deleterious effect on bone remodeling is debatable. This study was aimed at assessing the diagnostic utility of the bone remodeling marker C-terminal telopeptide (CTx) in detecting early bone loss.
In this case–control study, 84 premenopausal women of 30–45 years of age were selected. Out of them, 28 were recently diagnosed of hypothyroidism (not on LT4), 28 were on LT4 replacement therapy (100–200 μg/day) for more than five years, and 28 had euthyroid. Plasma CTx levels were estimated. Bone mineral density (BMD) was measured by quantitative ultrasound (QUS) method. Pearson’s coefficient of correlation and ANOVA were used for statistical analysis.
CTx was most elevated in LT4-treated group (0.497 ± 0.209 ng/mL). It showed a significant negative correlation with T-score and Z-score of BMD values. In the treatment group of more than 150 μg/day, CTx showed significantly negative correlation with TSH (r = −0.462, P = 0.047).
LT4 therapy induces bone loss in hypothyroid patients. CTx levels can measure such bone loss along with BMD. Regular monitoring of CTx with adjustment in LT4 doses may help delay osteoporosis induced by prolonged LT4 replacement therapy.
PMCID: PMC3948734  PMID: 24634578
hypothyroidism; bone loss; C-terminal telopeptide; LT4 - levothyroxine therapy; Bone mineral density; bone remodeling; osteoporosis
16.  Quantification of Plasma miRNAs by Digital PCR for Cancer Diagnosis 
Biomarker Insights  2013;8:127-136.
Analysis of plasma microRNAs (miRNAs) by quantitative polymerase chain reaction (qPCR) provides a potential approach for cancer diagnosis. However, absolutely quantifying low abundant plasma miRNAs is challenging with qPCR. Digital PCR offers a unique means for assessment of nucleic acids presenting at low levels in plasma. This study aimed to evaluate the efficacy of digital PCR for quantification of plasma miRNAs and the potential utility of this technique for cancer diagnosis. We used digital PCR to quantify the copy number of plasma microRNA-21-5p (miR-21–5p) and microRNA-335–3p (miR-335–3p) in 36 lung cancer patients and 38 controls. Digital PCR showed a high degree of linearity and quantitative correlation with miRNAs in a dynamic range from 1 to 10,000 copies/μL of input, with high reproducibility. qPCR exhibited a dynamic range from 100 to 1×107 copies/μL of input. Digital PCR had a higher sensitivity to detect copy number of the miRNAs compared with qPCR. In plasma, digital PCR could detect copy number of both miR-21–5p and miR-335–3p, whereas qPCR was only able to assess miR-21–5p. Quantification of the plasma miRNAs by digital PCR provided 71.8% sensitivity and 80.6% specificity in distinguishing lung cancer patients from cancer-free subjects.
PMCID: PMC3836484  PMID: 24277982
digital PCR; miRNAs; plasma; diagnosis; lung cancer
17.  Perspectives on the Value of Biomarkers in Acute Cardiac Care and Implications for Strategic Management 
Biomarker Insights  2013;8:115-126.
Biomarkers in acute cardiac care are gaining increasing interest given their clinical benefits. This study is a review of the major conditions in acute cardiac care, with a focus on biomarkers for diagnostic and prognostic assessment. Through a PubMed search, 110 relevant articles were selected. The most commonly used cardiac biomarkers (cardiac troponin, natriuretic peptides, and C-reactive protein) are presented first, followed by a description of variable acute cardiac conditions with their relevant biomarkers. In addition to the conventional use of natriuretic peptides, cardiac troponin, and C-reactive protein, other biomarkers are outlined in variable critical conditions that may be related to acute cardiac illness. These include ST2 and chromogranin A in acute dyspnea and acute heart failure, matrix metalloproteinase in acute chest pain, heart-type fatty acid binding protein in acute coronary syndrome, CD40 ligand and interleukin-6 in acute myocardial infarction, blood ammonia and lactate in cardiac arrest, as well as tumor necrosis factor-alpha in atrial fibrillation. Endothelial dysfunction, oxidative stress and inflammation are involved in the physiopathology of most cardiac diseases, whether acute or chronic. In summary, natriuretic peptides, cardiac troponin, C-reactive protein are currently the most relevant biomarkers in acute cardiac care. Point-of-care testing and multi-markers use are essential for prompt diagnostic approach and tailored strategic management.
PMCID: PMC3771707  PMID: 24046510
biomarker; acute cardiac care; point-of-care; management; assays
18.  Decreased Hepatocyte Growth Factor (HGF) and Gamma Aminobutyric Acid (GABA) in Individuals with Obsessive-Compulsive Disorder (OCD) 
Biomarker Insights  2013;8:107-114.
There is support for the role of gamma aminobutyric acid (GABA) in the etiology of mood disorders. Recent research has shown that hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility. This study was designed to determine and correlate plasma levels of HGF and GABA as well as symptom severity in individuals with obsessive-compulsive disorder (OCD).
Subjects and methods
Plasma from 15 individuals with OCD (9 males, 6 females;, mean age 38.7 years) and 17 neurotypical controls (10 males, 7 females; mean age 35.2 years) was assessed for HGF, GABA, urokinase plasminogen activator (uPA), and urokinase plasminogen activator receptor (uPAR) concentration using enzyme-linked immunosorbest assays ELISAs. Symptom severity was assessed in these OCD individuals and compared with HGF and GABA concentrations.
In this preliminary study, individuals with OCD had significantly decreased HGF levels, decreased plasma levels of GABA and decreased uPA. We found that both uPA and uPAR levels correlate with HGF. Both low uPA and low uPAR levels correlate with high symptom severity in individuals with OCD. Low GABA levels in OCD individuals also correlate with high symptom severity.
These results demonstrate a preliminary association between HGF, GABA, uPA levels, and OCD and suggest that plasma GABA and uPA levels are related to symptom severity in individuals with OCD.
PMCID: PMC3762604  PMID: 24023510
GABA; HGF; obsessive-compulsive disorder; OCD; uPA; uPAR; symptom severity
19.  Markers that can Reflect Asthmatic Activity before and after Reduction of Inhaled Corticosteroids: A Pilot Study 
Biomarker Insights  2013;8:97-105.
Evaluation of airway inflammation is important in achieving adequate dosing of inhaled corticosteroids (ICS) for treating bronchial asthma. However, there is no evaluation tool that can be used in clinical settings. We examined biomarkers that can precisely reflect airway inflammation when ICS are decreased in stable asthmatic patients. This was a 12-week, single-arm, open-label clinical study performed at a single university hospital. Twenty-five patients (6 male and 19 female) with stable asthma were included in this study. We investigated whether the levels of nitrite and nitrate in exhaled breath condensate (EBC) increase after ICS reduction. We also investigated whether blood eosinophils, serum immunoglobulin E (IgE), high-sensitivity C reactive protein (hs-CRP), interleukin (IL)-13, IL-17, and periostin are different before and after ICS reduction. Peak expiratory flow (PEF), pulmonary function tests, asthma control test (ACT), and asthma quality of life questionnaire (AQLQ) were also examined. We considered an unscheduled hospital visit due to asthmatic symptoms and decline in average PEF over one week by more than 10% to indicate disease instability, and compared patients with stable and unstable disease for analysis. Unstable status was detected in 5 patients. Age, sex, asthma duration, ACT and AQLQ scores, and the level of serum IgE did not differ between stable and unstable groups. In the unstable group, the total concentration of nitrite and nitrate at the last visit was 9.84 (6.65–11.24) μM. Surprisingly, this was similar to the concentration at the first visit (5.58 (2.94–17.29) μM). Serum periostin before ICS reduction (141.9 [107.7–147.7] pg/mL) was higher in the unstable group than in the stable group (91.5 [78.75–103.5] pg/mL). The unstable group had a higher peripheral blood eosinophil count and wider diurnal variation of PEF at the first visit compared to the stable group. Higher eosinophils in peripheral blood and wider diurnal variation of PEF were predictive markers for unstable disease after ICS reduction. Serum periostin is another candidate for the predictive marker.
PMCID: PMC3738382  PMID: 23943655
bronchial asthma; inhaled corticosteroid; exhaled breath condensate; nitric oxides; periostin
20.  Identification of Potential Prognostic Markers for Knee Osteoarthritis by Serum Proteomic Analysis 
Biomarker Insights  2013;8:85-95.
As osteoarthritis (OA) is a highly heterogeneous disease in terms of progression, establishment of prognostic biomarkers would be highly beneficial for treatment. The present study was performed to identify novel biomarkers capable of predicting the progression of knee OA.
A total of 69 plasma samples (OA patients undergoing radiographic progression, n = 25; nonprogression, n = 33; healthy donors, n = 11) were analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), and ion peaks of interest were identified by liquid chromatography and matrix-assisted laser desorption/ionization (MALDI)-TOF MS. The identities of these proteins were further validated by immunoprecipitation combined with SELDI-TOF MS analysis.
SELDI-TOF MS analysis indicated that the intensities of 3 ion peaks differed significantly between progressors and nonprogressors. Subsequent analyses indicated that these peaks corresponded to apolipoprotein C-I, C-III, and an N-terminal truncated form of transthyretin, respectively. The identities of these proteins were confirmed by the loss of ion peaks in SELDI-TOF MS spectra by immunoprecipitation using specific antibodies for the respective proteins.
Three potential biomarkers were identified whose serum levels differed significantly between OA progressors and nonprogressors. These biomarkers are expected to be prognostic biomarkers for knee OA and to facilitate the development of novel disease-modifying treatments for OA.
PMCID: PMC3735238  PMID: 23935359
osteoarthritis; biomarker; proteomics; surface-enhanced laser desorption/ionization (SELDI)
21.  CIINE Reflects Collagenase-Specific CII Breakdown in Cartilage Explant and Whole Body of Canine 
Biomarker Insights  2013;8:77-83.
To evaluate collagenase inhibitors for the treatment of osteoarthritis and to correlate them with clinical pathology, canine cartilage explant and anterior cruciate ligament transection (ACLT) models were examined by quantifying the CII neoepitope (CIINE). This peptide is a putative marker for collagenase-specific type II collagen (CII) degradation, which is a critical step in osteoarthritis pathology.
The concentration of CIINE in supernatants of canine cartilage explants showed increase upon IL-1β—stimulation and collagenase inhibitors suppressed this elevation of CIINE. In the canine ACLT model, levels of CIINE in urine (uCIINE) increased as lesions of knee joint cartilage developed and decreased in response to collagenase inhibitors.
Our results suggest that CIINE reflects collagenase-specific CII degradation in canine explants and whole bodies. It is anticipated that these data will establish a tool for clarifying and bridging the efficacy and mechanism of collagenase inhibitors at the preclinical stage of drug discovery.
PMCID: PMC3694827  PMID: 23825438
CII neoepitope; collagenase; type II collagen; osteoarthritis; biomarker; matrix metalloproteinase
22.  Correlation Between Hepatocyte Growth Factor (HGF) and Gamma-Aminobutyric Acid (GABA) Plasma Levels in Autistic Children 
Biomarker Insights  2013;8:69-75.
There is much support for the role of Gamma-Aminobutyric acid (GABA) in the etiology of autism. Recent research has shown that hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility. This study was designed to determine and correlate plasma levels of HGF, GABA, as well as symptom severity, in autistic children and neurotypical controls. Plasma from 48 autistic children and 29 neurotypical controls was assessed for HGF and GABA concentration using ELISAs. Symptom severity was assessed in these autistic individuals and compared to HGF and GABA concentrations. We previously reported that autistic children had significantly decreased levels of HGF. In this study, the same autistic children had significantly increased plasma levels of GABA (P = 0.002) and decreased HGF levels correlated with these increased GABA levels (r = 0.3; P = 0.05). High GABA levels correlated with increasing hyperactivity (r = 0.6; P = 0.0007) and impulsivity severity (r = 0.5; P = 0.007), tip toeing severity (r = 0.35; P = 0.03), light sensitivity (r = 0.4; P = 0.02), and tactile sensitivity (r = 0.4; P = 0.01). HGF levels did not correlate significantly with any symptom severity. These results suggest an association between HGF and GABA levels and suggest that plasma GABA levels are related to symptom severity in autistic children.
PMCID: PMC3694825  PMID: 23825437
GABA; HGF; autism; symptom severity
23.  Biomarkers of Dose and Effect of Inhaled Ozone in Resting versus Exercising Human Subjects: Comparison with Resting Rats 
Biomarker Insights  2013;8:53-67.
To determine the influence of exercise on pulmonary dose of inhaled pollutants, we compared biomarkers of inhaled ozone (O3) dose and toxic effect between exercise levels in humans, and between humans and rats. Resting human subjects were exposed to labeled O3 (18O3, 0.4 ppm, for 2 hours) and alveolar O3 dose measured as the concentration of excess 18O in cells and extracellular material of nasal, bronchial, and bronchoalveolar lavage fluid (BALF). We related O3 dose to effects (changes in BALF protein, LDH, IL-6, and antioxidant substances) measurable in the BALF. A parallel study of resting subjects examined lung function (FEV1) changes following O3. Subjects exposed while resting had 18O concentrations in BALF cells that were 1/5th of those of exercising subjects and directly proportional to the amount of O3 breathed during exposure. Quantitative measures of alveolar O3 dose and toxicity that were observed previously in exercising subjects were greatly reduced or non-observable in O3 exposed resting subjects. Resting rats and resting humans were found to have a similar alveolar O3 dose.
PMCID: PMC3663491  PMID: 23761957
ozone; inhalation toxicology; exercise; animal human extrapolation
24.  Oxidative Stress and DNA Damage Induced by Chromium in Liver and Kidney of Goldfish, Carassius auratus 
Biomarker Insights  2013;8:43-51.
Chromium (Cr) is an abundant element in the Earth’s crust. It exhibits various oxidation states, from divalent to hexavalent forms. Cr has diverse applications in various industrial processes and inadequate treatment of the industrial effluents leads to the contamination of the surrounding water resources. Hexavalent chromium (Cr (VI)) is the most toxic form, and its toxicity has been associated with oxidative stress. The present study was designed to investigate the toxic potential of Cr (VI) in fish. In this research, we investigated the role of oxidative stress in chromium-induced genotoxicity in the liver and kidney cells of goldfish, Carassius auratus. Goldfish were acclimatized to the laboratory conditions and exposed them to 5% and 10% of 96 hr-LC50 (85.7 mg/L) of aqueous Cr (VI) in a continuous flow through system. Fish were sampled every 7 days for a period of 28 days to analyze the lipid hydroperoxides (LHP) levels and genotoxic potentials in the liver and kidney. LHP levels were analyzed by spectrophotometry while genotoxicity was assessed by single cell gel electrophoresis (comet) assay. LHP levels in the liver increased significantly at week 1, followed by a decrease. LHP levels in the kidney increased significantly at weeks 1, 2, and 3, and decreased at week 4 compared to the control. The percentage of DNA damage increased in both liver and kidney at both test concentrations. The results clearly indicate that Cr (VI) induces significant levels of DNA damage in liver and kidney cells of goldfish. The induced LHP levels in both organs were concentration-dependent and were directly correlated with the levels of DNA damage. The two tested Cr (VI) concentrations induced significant levels of oxidative stress in both organs, however the kidney appears to be more vulnerable and sensitive to Cr-induced toxicity than the liver.
PMCID: PMC3653851  PMID: 23700361
chromium; goldfish; sub-chronic exposure; oxidative stress; DNA damage
25.  Decreased Epidermal Growth Factor (EGF) Associated with HMGB1 and Increased Hyperactivity in Children with Autism 
Biomarker Insights  2013;8:35-41.
Autism spectrum disorders (ASDs), characterized by impaired social interactions and deficits in verbal and nonverbal communication, are thought to affect 1 in 88 children in the United States. There is much support for the role of growth factors in the etiology of autism. Recent research has shown that epithelial growth factor (EGF) is decreased in young autistic children (2–4 years of age). This study was designed to determine plasma levels of EGF in an older group of autistic children (mean age 10.6 years) and to correlate these EGF levels with putative biomarkers HGF, uPA, uPAR, GAD2, MPO GABA, and HMGB1, as well as symptom severity of 19 different symptoms.
Subjects and methods
Plasma from 38 autistic children, 11 children with pervasive developmental disorder (PDD-NOS) and 40 neurotypical, age and gender similar controls was assessed for EGF concentration using ELISAs. Severity of 19 symptoms (awareness, expressive language, receptive language, (conversational) pragmatic language, focus/attention, hyperactivity, impulsivity, perseveration, fine motor skills, gross motor skills, hypotonia (low muscle tone), tiptoeing, rocking/pacing, stimming, obsessions/fixations, eye contact, sound sensitivity, light sensitivity, and tactile sensitivity) was assessed and then compared to EGF concentrations.
In this study, we found EGF levels in autistic children and those with PDD-NOS to be significantly lower when compared with neurotypical controls. EGF levels correlated with HMGB1 levels but not the other tested putative biomarkers, and EGF correlated negatively with hyperactivity, gross motor skills, and tiptoeing but not other symptoms.
These results suggest an association between decreased plasma EGF levels and selected symptom severity. We also found a strong correlation between plasma EGF and HMGB1, suggesting inflammation is associated with decreased EGF.
PMCID: PMC3623607  PMID: 23645980
EGF; HMGB1; GABA; autism; PDD; symptom severity

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