bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Plasma Membrane Proteins from Ovarian Cancer Cells Using Mass Spectrometry
Authors: Springer David L. , Auberry Deanna L. , Ahram Mamoun , Adkins Joshua N. , Feldhaus Jane M. , Wahl Jon H. , Wunschel David S. , Rodland Karin D.
Published: June 2013

To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsin digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane‐associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.
Measurement Reproducibility in the Early Stages of Biomarker Development
Authors: Liggett Walter S. , Barker Peter E. , Semmes O. John , Cazares Lisa H.
Published: June 2013

Biomarker discovery and development requires measurement reproducibility studies in addition to case‐control studies. Parallel pursuit of reproducibility studies is especially important for emerging technologies such as protein biomarkers based on time‐of‐flight mass spectrometry, the case considered in this paper. For parallel studies, a way to improve reproducibility prior to identification of protein species is necessary. One approach is use of functional principal components analysis (PCA) as the basis for assessing measurement reproducibility. Reproducibility studies involve repeated measurement of a reference material such as a human serum standard. Measurement in our example is by SELDI‐TOF (surface‐enhanced laser desorption and ionization time‐of‐flight) mass spectrometry. Reproducibility is defined in reference to a source of variation, which in our example is associated with a type of commercially available protein biochip. We obtained spectra for 8 spots on each 11 chips. Two spectra are generally more alike when obtained from the same chip rather than different chips. Thus, our experiment indicates potential improvements from reducing variation in chip manufacture and chip handling during measurement. Our analysis involves careful registration of the spectra and characterization of the spectral differences. As shown by our example, a metrological analysis may enhance case‐control studies by guiding optimization of the measurements underlying the biomarker.
A Rational Approach for Discovering and Validating Cancer Markers in Very Small Samples Using Mass Spectrometry and ELISA Microarrays
Authors: Zangar Richard C. , Varnum Susan M. , Covington Chandice Y. , Smith Richard D.
Published: June 2013

Identifying useful markers of cancer can be problematic due to limited amounts of sample. Some samples such as nipple aspirate fluid (NAF) or early‐stage tumors are inherently small. Other samples such as serum are collected in larger volumes but archives of these samples are very valuable and only small amounts of each sample may be available for a single study. Also, given the diverse nature of cancer and the inherent variability in individual protein levels, it seems likely that the best approach to screen for cancer will be to determine the profile of a battery of proteins. As a result, a major challenge in identifying protein markers of disease is the ability to screen many proteins using very small amounts of sample. In this review, we outline some technological advances in proteomics that greatly advance this capability. Specifically, we propose a strategy for identifying markers of breast cancer in NAF that utilizes mass spectrometry (MS) to simultaneously screen hundreds or thousands of proteins in each sample. The best potential markers identified by the MS analysis can then be extensively characterized using an ELISA microarray assay. Because the microarray analysis is quantitative and large numbers of samples can be efficiently analyzed, this approach offers the ability to rapidly assess a battery of selected proteins in a manner that is directly relevant to traditional clinical assays.
SELDI‐TOF Serum Profiling for Prognostic and Diagnostic Classification of Breast Cancers
Authors: Laronga Christine , Becker Stephen , Watson Patrice , Gregory Betsy , Cazares Lisa , Lynch Henry , Perry Roger R. , Wright George L. , Drake Richard R. , Semmes O. John
Published: June 2013

Surface enhanced laser desorption/ionization (SELDI) time‐of‐flight mass spectrometry has emerged as a successful tool for serum based detection and differentiation of many cancer types, including breast cancers. In this study, we have applied the SELDI technology to evaluate three potential applications that could extend the effectiveness of established procedures and biomarkers used for prognostication of breast cancers. Paired serum samples obtained from women with breast cancers prior to surgery and post‐surgery (6–9 mos.) were examined. In 14/16 post‐treatment patients, serum protein profiles could be used to distinguish these samples from the pre‐treatment cancer samples. When compared to serum samples from normal healthy women, 11 of these post‐treatment samples retained global protein profiles not found in healthy women, including five low‐mass proteins that remained elevated in both pre‐treatment and post‐treatment serum groups. In another pilot study, serum profiles were compared for a group of 30 women who were known BRCA‐1 mutation carriers, half of whom subsequently developed breast cancer within three years of the sample procurement. SELDI protein profiling accurately classified 13/15 women with BRCA‐1 breast cancers from the 15 non‐cancer BRCA‐1 carriers. Additionally, the ability of SELDI to distinguish between the serum profiles from sentinel lymph node positive and sentinel lymph node negative patients was evaluated. In sentinel lymph node positive samples, 22/27 samples were correctly classified, in comparison to the correct classification of 55/71 sentinel lymph node negative samples. These initial results indicate the utility of protein profiling approaches for developing new diagnostic and prognostic assays for breast cancers.
Discovery of Ovarian Cancer Biomarkers in Serum Using NanoLC Electrospray Ionization TOF and FT‐ICR Mass Spectrometry
Authors: Bergen H. Robert , Vasmatzis George , Cliby William A. , Johnson Kenneth L. , Oberg Ann L. , Muddiman David C.
Published: June 2013

Treatment of cancer patients is greatly facilitated by detection of the cancer prior to metastasis. One of the obstacles to early cancer detection is the lack of availability of biomarkers with sufficient specificity. With modern differential proteomic techniques, the potential exists to identify high specificity cancer biomarkers. We have delineated a set of protocols for the isolation and identification of serum biomarkers for ovarian cancer that exist in the low molecular weight serum fraction. After isolation of the low molecular weight fraction by ultrafiltration, the potential biomarkers are separated by reversed phase nano liquid chromatography. Detection via TOF or FT‐ICR yields a data set for each sample. We compared stage III/IV ovarian cancer serum with postmenopausal age‐matched controls. Using bioinformatics tools developed at Mayo, we normalized each sample for intensity and chromatographic alignment. Normalized data sets are subsequently compared and potential biomarkers identified. Several candidate biomarkers were found. One of these contains the sequence of fibrinopeptide‐A known to be elevated in many types of cancer including ovarian cancer. The protocols utilized will be examined and would be applicable to a wide variety of cancers or disease states.
APOA1‐75 G to A Substitution Associated with Severe Forms of CAD, Lower Levels of HDL and apoA‐I among Northern Indians
Authors: Chhabra S. , Narang R. , Lakshmy R. , Das N.
Published: June 2013

Apolipoprotein A‐I (APOA1 gene, apoA‐I protein) is the major protein for plasma high density lipoprotein (HDL). The relationship of APOA1‐75G/A polymorphism with lipid profile and coronary artery disease (CAD) is unclear. Out of 370 individuals initially recruited, 164 angiographically proven CAD patients (≥ 70% stenosis) and 36 individuals with normal coronaries or insignificant CAD (NCAD, ≤ 50% stenosis) from Delhi and adjoining areas were selected for analysis based on the set criteria. Polymorphism was determined by PCR followed by MspI restriction digestion. Lipid profile was estimated by enzymatic kit and apoA‐I levels by immunoturbidimetry. A highly significant increasing trend in ‘A’ allele frequency was observed with the rise in severity of CAD: NCAD (0.097) < SVD (single vessel disease) (0.117) < DVD (double vessel disease)
(0.223) < TVD (triple vessel disease) (0.291). In comparison to GG individuals, the OR of ‘A’ allele carriers to develop SVD,
DVD, TVD was 1.3, 2.8 and 4.2 respectively (ptrend = 0.007). Analysis of intergenotypic variations in the lipid profile revealed significantly lower levels of HDL and apoA‐I among ‘A’ allele carriers as compared to GG (patients). Our study, first of its kind from India, suggests that ‘A’ allele may contribute to severity of CAD and low levels of HDL & apoA‐I. However, an in depth study with a larger set of sample is necessary.
Daytime of Sampling, Tooth‐Brushing and Ascorbic Acid Influence Salivary Thiobarbituric Acid Reacting Substances – A Potential Clinical Marker of Gingival Status
Authors: Hodosy Július , Celec Peter
Published: June 2013

Background. Salivary thiobarbituric acid reacting substances (TBARS) have been previously shown to correlate with the impairment of gingival tissue. Although the details on the origin and the composition of this heterogeneous group of compounds in saliva are unknown, the potential clinical usefulness makes necessary the studies of factors influencing the salivary TBARS levels. Aim. To observe the effects of daily dynamics, tooth‐brushing and ascorbic acid administration on salivary TBARS levels. Subjects and methods. Self‐collected samples were obtained from 10 young healthy men collecting samples in the morning, in the afternoon and in the evening during 2 consecutive days. Ascorbic acid (250 mg) was administered orally after the last sampling on day 1 and before every sampling on day 2. Additional sampling was performed before and after tooth‐brushing. TBARS levels in saliva specimens were detected spectrofluorometrically. Sialic acid content was measured using a modified method of Warren. Results. Salivary TBARS levels vary significantly during a day (p < 0.001) with highest concentrations in the morning. Both,
tooth‐brushing (p < 0.05) and short‐term antioxidative treatment with ascorbic acid (p < 0.005) decrease salivary TBARS levels.
Sialic acid content of saliva is not influenced significantly by any of the investigated factors. Conclusion. TBARS levels in saliva are affected by daytime of sampling, tooth‐brushing and ascorbic acid pre‐treatment. These results must be considered in clinical research using salivary TBARS levels. Sialic acid seems not to be a major component of TBARS in saliva. Further studies should clarify the molecular compounds of salivary TBARS and uncover the role of oral microbial factors.
The Application of Plasma 1,5‐Anhydro‐D‐glucitol for Monitoring Type 2 Diabetic Patients
Authors: Dworacka Marzena , Winiarska Hanna
Published: June 2013

Aim: Recent data have suggested that effective control of postprandial blood glucose can reduce the risk of macroangiopathic complications of diabetes, especially cardiovascular risk. 1,5‐Anhydro‐D‐glucitol (1,5‐AG) has been proposed as a marker of short‐term hyperglycaemic excursions. We aimed to evaluate its usefulness in patients with type 2 diabetes and have attempted to indicate when 1,5‐AG monitoring should be used in ordinary diabetes care settings. Methods: The study group consisted of 130 type 2 diabetic patients aged 36–69 years. 1,5‐AG plasma level, HbA1c concentrations and daily glucose profile were measured. Mean blood glucose (MBG), M‐value were calculated and maximal daily glycaemia (MxG) was established as indicators of short‐term hyperglycaemic episodes. Results: 1,5‐AG plasma level was negatively and HbA1c was positively correlated with fasting glycaemia (FG), MBG, M‐value and MxG. Multivariate regression analysis revealed that 1,5‐AG plasma level is determined by MxG only, while FG determined HbA1c concentration in blood. The analysis of 1,5‐AG level and HbA1c distributions in well and poorly controlled patients revealed that persons with low HbA1c values may have decreased 1,5‐AG plasma level. Conclusion: 1,5‐AG plasma level monitoring is the useful method to identify well controlled, exclusively based on HbA1c levels type 2 diabetic patients with transient hyperglycaemia, accordingly patients at high risk of macroangiopathic complications.
Over‐Expression of Dopamine D2 Receptor and Inwardly Rectifying Potassium Channel Genes in Drug‐Naive Schizophrenic Peripheral Blood Lymphocytes as Potential Diagnostic Markers
Authors: Zvara Ágnes , Szekeres György , Janka Zoltán , Kelemen János Z. , Cimmer Csongor , Sántha Miklós , Puskás László G.
Published: June 2013

Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL) express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3) was found to be over‐expressed in schizophrenic PBL and proposed to be a diagnostic and follow‐up marker for schizophrenia. In this study we screened PBL of 13 drug‐naive/drug‐free schizophrenic patients to identify additional markers of schizophrenia. One of the benefits of our study is the use of blood samples of non‐medicated, drug‐naive patients. This excludes the possibility that changes detected in gene expression levels might be attributed to the medication rather than to the disorder itself. Among others, genes for dopamine receptor D2 (DRD2) and the inwardly rectifying potassium channel (Kir2.3) were found to be over‐expressed in microarray analysis. Increased mRNA levels were confirmed by quantitative real‐time PCR (QRT‐PCR) using the SybrGreen method and dual labeled TaqMan probes. The use of both molecular markers allows a more rapid and precise prediction of schizophrenia and might help find the optimal medication for schizophrenic patients.
Patterns of Global DNA and Histone Methylation Appear to be Similar in Normal, Dysplastic and Neoplastic Oral Epithelium of Humans
Authors: Piyathilake Chandrika J. , Bell Walter C. , Jones Jennifer , Henao Olga L. , Heimburger Douglas C. , Niveleau Alain , Grizzle William E.
Published: June 2013

Although there is growing interest in the possibility that alterations in histone methylation may play a role in carcinogenesis, it has not been explored adequately in humans. Similarly, there are no reports of associations between this and a similar epigenetic event, DNA methylation. Using immunohistochemical staining, we compared the methylation of DNA and histones in histopathologically normal oral epithelium, dysplastic oral lesions, and squamous cell cancers (SCCs) from subjects with squamous cell cancer (n = 48) with those of normal oral epithelium from subjects without oral cancer (n = 93) who were matched on age and race. Monoclonal antibodies specific for 5 methyl cytosine (5‐mc), lysine 4 of histone H3 (H3‐Lys4), and lysine 9 of histone H3 (H3‐Lys9) were used in this study. The percentages of cells positive and a weighted average of the immunostaining intensity scores were calculated for each of these tissues, and Spearman correlation analyses were employed to study associations between DNA and histone methylation. Correlations between DNA and histone methylation, H3‐Lys4 and H3‐Lys9 were positive and statistically significant in all tissue types; they were strongest in normal oral epithelium from non‐cancer subjects (n = 0.63, p < 0.001 and r = 0.62, p < 0.001 respectively). Similarly, the positive correlations between
H3‐Lys4 and H3‐Lys9 were statistically significant in all tissue types and strongest in normal oral epithelium from non‐cancer subjects (r = 0.77, p < 0.001). Patterns of DNA and histone methylation are similar in tissues across the spectrum of oral carcinogenesis, and there is a significant positive association between these two epigenetic mechanisms.
RNA Profiling for Biomarker Discovery: Practical Considerations for Limiting Sample Sizes
Authors: Kelly Danny J. , Ghosh Sujoy
Published: June 2013

We have compared microarray data generated on Affymetrix™ chips from standard (8 micrograms) or low (100 nanograms) amounts of total RNA. We evaluated the gene signals and gene fold‐change estimates obtained from the two methods and validated a subset of the results by real time, polymerase chain reaction assays. The correlation of low RNA derived gene signals to gene signals obtained from standard RNA was poor for less to moderately abundant genes. Genes with high abundance showed better correlation in signals between the two methods. The signal correlation between the low RNA and standard RNA methods was improved by including a reference sample in the microarray analysis. In contrast, the fold‐change estimates for genes were better correlated between the two methods regardless of the magnitude of gene signals. A reference sample based method is suggested for studies that would end up comparing gene signal data from a combination of low and standard RNA templates; no such referencing appears to be necessary when comparing fold‐changes of gene expression between standard and low template reactions.
ɛ3ɛ4 Genotype as Risk Factor of Myocardial Infarction in Middle‐Aged People in Spain
Authors: Garcés Carmen , Maicas Carolina , Grande Rosario , Benavente Mercedes , Viturro Enrique , Cano Beatriz , López Dolores , de Oya Manuel
Published: June 2013

Apolipoprotein E (apoE) plays an important role in lipid metabolism. Its ɛ4 allele has been consistently associated with lipoprotein disorders but its connection to myocardial infarction (MI) is controversial. Because ɛ4 frequency decreases with age we thought that the contradictory results in different studies could be due to the wide age range of the subjects included. To test our hypothesis, ApoE genotyping was performed in 474 MI cases and an analysis was performed by percentiles of age. The frequencies of ɛ3ɛ4 genotype and ɛ4 allele in the MI group as a whole (subjects aged 31 to 92) were not significantly different from those in our area general population. However, significant differences were observed when comparing by group of age. The frequencies decreased as age increased. The ɛ3ɛ4 and ɛ4 frequencies were significantly higher in MI subjects aged 31 to 56 than in subjects over 74. The ɛ3ɛ4 genotype prevalence in an age and sex matched control group of subjects aged 31 to 56 was significantly lower than in the 31–56 year‐old MI group. In conclusion, our data shows different ɛ3ɛ4 and ɛ4 frequencies depending on the age range of the subjects with MI, being significantly higher in the middle‐aged group. This finding may help explain the discrepancies between studies analyzing association between apoE genotype and MI, and emphasizes the idea of considering apoE genotype for prevention at early age.
Dietary Antioxidants Decrease Serum Soluble Adhesion Molecule (sVCAM‐1, sICAM‐1) but not Chemokine (JE/MCP‐1, KC) Concentrations, and Reduce Atherosclerosis in C57BL but Not ApoE*3 Leiden Mice Fed an Atherogenic Diet
Authors: Murphy Nuala , Grimsditch David C , Vidgeon-Hart Martin , Groot Pieter H.E. , Overend Philip , Benson G. Martin , Graham Annette
Published: June 2013

Dietary antioxidants are reported to suppress cellular expression of chemokines and adhesion molecules that recruit monocytes to the artery wall during atherosclerosis. In the present study we measured the effect of feeding apoE*3 Leiden mice or their non‐transgenic (C57BL) littermates with atherogenic diets either deficient in, or supplemented with, dietary antioxidants (vitamin E, vitamin C and β‐carotene) for 12 weeks, on serum levels of CC (JE/MCP‐1) and CXC (KC) chemokines and soluble adhesion molecules (sVCAM‐1, sICAM‐1) and atherosclerotic lesion size. ApoE*3 Leiden mice developed gross hypercholesterolaemia, and markedly accelerated (10–20 fold; P < 0.0001) atherogenesis, compared with non‐transgenic animals. Antioxidant consumption reduced lesion area in non‐transgenic, but not apoE*3 Leiden, mice. Serum sVCAM‐1 and sICAM‐1 levels were significantly (P < 0.0001) increased (sVCAM‐1 up to 3.9 fold; sICAM‐1 up to 2.4 fold) by 4—8 weeks in all groups, and then declined. The initial increase in the concentration of adhesion molecules was reduced by 38%— 61% (P < 0.05) by antioxidant consumption, particularly in non‐transgenic mice. By contrast, serum chemokine levels tended to increase more rapidly from baseline in apoE*3 Leiden mice, compared with non‐transgenic animals, but were unaffected by dietary antioxidants. We conclude that dietary antioxidants reduce circulating soluble adhesion molecules and atherosclerosis in C57BL mice.
Endostatin Polymorphism 4349G/A(D104N) is not Associated with Aggressiveness of Disease in Postate Cancer
Authors: Li He Cheng , Cai Qiu Yin , Shinohara Eric T. , Cai Hui , Cao Carolyn , Fei Wang Zuo , Teng Ming , Zheng Wei , Lu Bo
Published: June 2013

Endostatin is an important inhibitory molecule which mediates the sequential steps involved in angiogenesis. Lower level or impaired function of endostatin is associated with a higher risk of developing malignant solid tumors and with a worse prognosis of the disease. The endostatin N104 polymorphism might be associated with an impaired ability to inhibit angiogenesis. We analyzed the tissues from 98 Caucasian prostate cancer patients for the presence of D104N polymorphism. The frequencies of homozygous 4349G/G(104D/D), and heterozygous 4349G/A(104D/N) were 83.67%(82/98) and 16.33%(16/98), respectively; no individuals were homozygous 4349A/A(104N/N). With the Fisher’s exact test we found the genotype of D104N was not significantly related to age, tumor grade, PSA and clinical stage (P > 0.05). There was no difference in relapse free survival(RFS) or overall survival(OS) between patients with 104D/N and those with 104D/D (P = 0.8283, 0.3713 respectively). We concluded that endostatin polymorphism was not associated with the aggressiveness of prostate cancer in Caucasian patients.

Erratum of “Endostatin Polymorphism 4349G/A(D104N) Is Not Associated with Aggressiveness of Disease in Postate Cancer” dx.doi.org/10.1155/2005/318394
Clinical Application of Rapid Assay of Interleukin‐6 in Influenza‐Associated Encephalopathy
Authors: Nakamura Mikage , Yamanaka Gaku , Kawashima Hisashi , Watanabe Yoshiaki , Ioi Hiroaki , Kashiwagi Yasuyo , Takekuma Kouji , Hoshika Akinori , Hayakawa Mizuho , Suzuki Shigeru
Published: June 2013

The characteristics of influenza‐associated encephalopathy is the high mortality and nimble progress with coma which appears in general cases within 48 hours. Most of patients show no abnormalities in the standard blood checks on admission or in early stage. In this study we investigated if a rapid assay of interleukin (IL)‐6 is useful in influenza‐associated encephalopathy in early stages. The levels of IL‐6 in patients with influenza‐associated encephalopathy did not show any significant difference compared with those in patients with febrile convulsion and rotavirus‐associated convulsion. However the levels of IL‐6 in severe cases were significantly higher than those of mild cases with influenza‐associated encephalopathy. Consequently the rapid assay of serum IL‐6 is useful to evaluate and decide the therapies.
Serum Levels of Anti‐Myelin Antibodies in Relapsing‐Remitting Multiple Sclerosis Patients during Different Phases of Disease Activity and Immunomodulatory Therapy
Authors: Angelucci Francesco , Mirabella Massimiliano , Frisullo Giovanni , Caggiula Marcella , Tonali Pietro Attilio , Batocchi Anna Paola
Published: June 2013

Antibodies against myelin oligodendrocyte antigens have been found in the immunoreactive brain lesions of Multiple Sclerosis (MS) patients. Recently it has been proposed that these antibodies can be used as a prognostic marker in the course of disease. However, the serum levels of these autoantibodies during different phases of disease activity or after an immunomodulatory therapy have been poorly investigated. In this study the serum levels of anti‐myelin oligodendrocyte glycoprotein (MOG) (directed against the epitopes 1–26 and 15–40) and anti‐myelin basic protein (MBP) antibodies were sequentially measured in the same MS patient either in relapse or remission phases. We found that MS patients in the relapse phase had higher serum anti‐MOG (peptides 1–26 and 15–40) and anti‐MBP antibody levels than controls. In addition, the levels of anti‐MOG 1–26 were also elevated during the relapse as compared with the remission phase but no significant changes were found in the levels of anti‐MOG 15–40 of anti‐MBP antibodies. We also evaluated the effect of interferon‐beta (β) therapy on anti‐myelin antibodies. 1‐year of interferon‐β treatment did not induce any changes in the levels of anti‐MOG and anti‐MBP antibodies. In conclusion, these data indicate that the use of peripheral levels of autoantibodies against MOG and MBP as marker of multiple sclerosis might be complicated by the phase of disease activity and by the epitope of the MOG protein used.
The Use of Proteomics in Biomarker Discovery in Neurodegenerative Diseases
Authors: Davidsson Pia , Sjögren Magnus
Published: June 2013

Biomarkers for neurodegenerative diseases should reflect the central pathogenic processes of the diseases. The field of clinical proteomics is especially well suited for discovery of biomarkers in cerebrospinal fluid (CSF), which reflects the proteins in the brain under healthy conditions as well as in several neurodegenerative diseases. Known proteins involved in the pathology of neurodegenerative diseases are, respectively, normal tau protein, β‐amyloid (1‐42), synaptic proteins, amyloid precursor protein (APP), apolipoprotein E (apoE), which previously have been studied by protein immunoassays. The objective of this paper was to summarize results from proteomic studies of differential protein patterns in neurodegenerative diseases with focus on Alzheimer′s disease (AD). Today, discrimination of AD from controls and from other neurological diseases has been improved by simultaneous analysis of both β‐amyloid (1‐42), total‐tau, and phosphorylated tau, where a combination of low levels of CSF‐β‐amyloid 1‐42 and high levels of CSF‐tau and CSF‐phospho‐tau is associated with an AD diagnosis. Detection of new biomarkers will further strengthen diagnosis and provide useful information in drug trials. The combination of immunoassays and proteomic methods show that the CSF proteins express differential protein patterns in AD, FTD, and PD patients, which reflect divergent underlying pathophysiological mechanisms and neuropathological changes in these diseases.
Identification of Disease Markers in Human Cerebrospinal Fluid Using Lipidomic and Proteomic Methods
Authors: Fonteh Alfred N. , Harrington Robert J. , Huhmer Andreas F. , Biringer Roger G. , Riggins James N. , Harrington Michael G.
Published: June 2013

Lipids comprise the bulk of the dry mass of the brain. In addition to providing structural integrity to membranes, insulation to cells and acting as a source of energy, lipids can be rapidly converted to mediators of inflammation or to signaling molecules that control molecular and cellular events in the brain. The advent of soft ionization procedures such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) have made it possible for compositional studies of the diverse lipid structures that are present in brain. These include phospholipids, ceramides, sphingomyelin, cerebrosides, cholesterol and their oxidized derivatives. Lipid analyses have delineated metabolic defects in disease conditions including mental retardation, Parkinson′s Disease (PD), schizophrenia, Alzheimer′s Disease (AD), depression, brain development, and ischemic stroke. In this review, we examine the structure of the major lipid classes in the brain, describe methods used for their characterization, and evaluate their role in neurological diseases. The potential utility of characterizing lipid markers in the brain, with specific emphasis on disease mechanisms, will be discussed. Additionally, we describe several proteomic strategies for characterizing lipid‐metabolizing proteins in human cerebrospinal fluid (CSF). These proteins may be potential therapeutic targets since they transport lipids required for neuronal growth or convert lipids into molecules that control brain physiology. Combining lipidomics and proteomics will enhance existing knowledge of disease pathology and increase the likelihood of discovering specific markers and biochemical mechanisms of brain diseases.
Central Neurochemical Ultradian Variability in Depression
Authors: Salomon Ronald M. , Johnson Benjamin W. , Schmidt Dennis E.
Published: June 2013

Depression is characterized by blunted behavior and neuroendocrine function that generally improve with antidepressant treatment. This study examined intrinsic variability in brain neurotransmitter function, since it may be a source of blunted behavior and neuroendocrine function in depression and a marker for the illness, and has not previously been analyzed using wavelet decomposition. To measure variability in monoamine metabolites, lumbar cerebrospinal fluid (CSF) was collected in serial samples in depressed patients before and after treatment. We hypothesized that changes in variability would be observed after treatment. Mechanisms that control such variability may be critical to the pathophysiology of depression. Method: Time series data was obtained from serial ten‐min sampling over a 24‐hr period (N = 144) from thirteen depressed patients, with a repeat collection after 5 weeks of antidepressant (sertraline or bupropion) treatment. Concentrations of tryptophan (TRP), the monoamine metabolites 5‐HIAA (metabolite of serotonin) and HVA (metabolite of dopamine), and the HVA:5HIAA ratio were transformed to examine power in slowly (160 min/cycle) to rapidly (20 min/cycle) occurring events. Power, the sum of the squares of the coefficients in each d (detail) wavelet, reflects variability within a limited frequency bandwidth for that wavelet. Pre‐treatment to post‐treatment comparisons were conducted with repeated measures ANOVA. Results: Antidepressant treatment was associated with increased power in the d2 wavelet from the HVA (p = 0.03) and the HVA:5‐HIAA ratio (p = 0.03) series. The d1 and d3 wavelets showed increased power following antidepressant treatment for the ratio series (d1, p = 0.01; d3, p = 0.05). Significant changes in power were not observed for the 5‐HIAA data series. Power differences among analytes suggest that the findings are specific to each system. Conclusion: The wavelet transform analysis shows changes in neurochemical signal variability following antidepressant treatment. Patterns or degrees of variability may be as important as, or possibly more important than, the mean levels of monoamine transmitters. Studies of variability observed in healthy individuals and a larger depressed sample will be needed to verify a relationship with mood and treatment response. Neurochemical measures of time‐variability may be a pivotal marker in depression.
Altered Glutathione Redox State in Schizophrenia
Authors: Yao Jeffrey K. , Leonard Sherry , Reddy Ravinder
Published: June 2013

Altered antioxidant status has been reported in schizophrenia. The glutathione (GSH) redox system is important for reducing oxidative stress. GSH, a radical scavenger, is converted to oxidized glutathione (GSSG) through glutathione peroxidase (GPx), and converted back to GSH by glutathione reductase (GR). Measurements of GSH, GSSG and its related enzymatic reactions are thus important for evaluating the redox and antioxidant status. In the present study, levels of GSH, GSSG, GPx and GR were assessed in the caudate region of postmortem brains from schizophrenic patients and control subjects (with and without other psychiatric disorders). Significantly lower levels of GSH, GPx, and GR were found in schizophrenic group than in control groups without any psychiatric disorders. Concomitantly, a decreased GSH:GSSG ratio was also found in schizophrenic group. Moreover, both GSSG and GR levels were significantly and inversely correlated to age of schizophrenic patients, but not control subjects. No significant differences were found in any GSH redox measures between control subjects and individuals with other types of psychiatric disorders. There were, however, positive correlations between GSH and GPx, GSH and GR, as well as GPx and GR levels in control subjects without psychiatric disorders. These positive correlations suggest a dynamic state is kept in check during the redox coupling under normal conditions. By contrast, lack of such correlations in schizophrenia point to a disturbance of redox coupling mechanisms in the antioxidant defense system, possibly resulting from a decreased level of GSH as well as age‐related decreases of GSSG and GR activities.
Microglia Activation in the Brain as Inflammatory Biomarker of Alzheimer’s Disease Neuropathology and Clinical Dementia
Authors: Xiang Zhongmin , Haroutunian Vahram , Ho Lap , Purohit Dushant , Pasinetti Giulio Maria
Published: June 2013

The role of microglia‐mediated inflammation in the progression of Alzheimer’s disease (AD) neuropathology remains unclear. In this study, postmortem brain sections from AD and control cases were subjected to Human Leukocyte Antigen (HLA)‐DR immunohistochemistry to examine microglia activation in the progression of AD assessed by pre‐mortem clinical dementia rating (CDR) and postmortem pathological manifestations of neuritic plaque (NP) and neurofibrillary tangle (NT) according to the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). In both gray and white matter of the entorhinal cortex (EC) and HLA‐DR immunostaining increased with the progression of CDR or CERAD NP, and to a lesser degree with CERAD NT. Between CDR stages HLA‐DR significance was found in moderate (CDR 2) to severe dementia (CDR 5) where as between CERAD NP stages staining increased significantly from NP 0 (no plaque) to NP 1 (sparse plaques), suggesting increased microglia activation begins with amyloid NP deposition. In the hippocampus, a significant increase in microglia immunostaining was found in the pyramidal cell layer of CA1 as early as CDR 1, and in the upper molecular layer of the dentate gyrus in CDR 0.5. This increase continues with the progression of CDR and reaches maximum in CDR 5. When assessed by CERAD NP stages however, a significant increase in microglia immunostaining was found only in mid‐to‐late stages (NP 3) and reduced staining was seen in NP 5. These results suggest that microglia activation increases with the progression of AD, with the increase varying depending on the involved brain region.
Molecular Detection of Disseminated Tumor Cells in the Peripheral Blood of Patients with Gastric Cancer: Evaluation of Their Prognostic Significance
Authors: Wu C. H. , Lin S. R. , Hsieh J. S. , Chen F. M. , Lu C. Y. , Yu F. J. , Cheng T. L. , Huang T. J. , Huang S. Y. , Wang J. Y.
Published: June 2013

Early detection of disseminated tumor cells in the peripheral blood of patients with early stage gastric cancer could help to improve the outcome after tumor resection. The aim of this study is to evaluate the prognostic significance of tumor‐related mRNA for the detection of circulating tumor cells in gastric cancer patients by a reverse‐transcriptase polymerase chain reaction (RT‐PCR) method. We simultaneously analyzed human telomerase reverse transcriptase (hTERT), cytokeratin‐19 (CK‐19), cytokeratin‐20 (CK‐20) and carcinoembryonic antigen (CEA) mRNA (messenger RNA) expression in the peripheral blood of 42 gastric cancer patients and 30 healthy individuals. Additionally, analyses were carried out for the correlation of these four molecular markers with patients’ clinicopathologic features, as well as the occurrence of postoperative recurrence/metastasis. Among 42 gastric cancer patients, the prevalence of mRNA for hTERT, CK‐19, CK‐20, and CEA was 61.9% (26/42), 69% (29/42), 61.9% (26/42), and 78.6% (33/42), respectively. All 30 healthy individuals were negative for hTERT and CEA mRNA, while two were positive for either CK‐19 mRNA or CK‐20 mRNA. Positive CEA mRNA was significantly correlated with tumor size (p = 0.008), vessel invasion (p = 0.001), depth of tumor invasion (p = 0.007), lymph node metastasis (p < 0.001), and TNM stage (p < 0.001). In addition, the multivariate logistic regression demonstrated that CEA mRNA expression was an independent and significant predictor for postoperative recurrence/metastasis (p = 0.032). Our findings suggest that CEA mRNA may be a more reliable marker than hTERT, CK‐19 and CK‐20 for the detection of circulating cancer cells in gastric cancer patients′ peripheral blood. Patients with positive CEA mRNA expression in peripheral blood have a significantly higher risk of postoperative recurrence/metastasis.
Potential Biomarkers Found by Protein Profiling May Provide Insight for the Macrovascular Pathogenesis of Diabetes Mellitus
Authors: Cho William C. S. , Yip Tai-Tung , Chung Wai-Shing , Leung Albert W. N. , Cheng Christopher H. K. , Yue Kevin K. M.
Published: June 2013

Diabetes mellitus (DM) is an alarming threat to health of mankind, yet its pathogenesis is unclear. The purpose of this study was to find potential biomarkers to serve as indicators for the pathogenesis of DM in a time course manner. Based on our previous findings that oxidative stress occurred at week 8, aorta lysate and sera of 102 streptozotocin (STZ)‐induced diabetic and 85 control male Sprague‐Dawley rats were obtained at the 4th, 8th and 12th week after STZ injection. The protein profiles were studied employing surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry technology in attomole sensitivity range. In the aorta, a multiple biomarker panel was discovered at the 4th week. At the 8th week, 4 biomarkers were found, while at the 12th week, 3 biomarkers were identified. In the sera, a triplet of 3 peaks and 2 biomarkers were all discovered to have 100% classification accuracy rate to differentiate the DM and control groups at all time intervals. Besides, 2 biomarkers were also found to have high classification value at week 12. Comparing the aorta and sera from DM and non‐DM rats, a bundle of potential biomarkers with significant changes in peak intensities and high classification values were found. Two of the serum biomarkers matched with islet amyloid polypeptide and resistin in the SWISS‐PROT knowledgebase. Validation has been conducted using immunoassay kits. These potential biomarkers may provide valuable insight on the pathogenesis of DM and macrovascular complications.
Multiple Sclerosis Cerebrospinal Fluid Biomarkers
Authors: Giovannoni Gavin
Published: June 2013

Cerebrospinal fluid (CSF) is the body fluid closest to the pathology of multiple sclerosis (MS). For many candidate biomarkers CSF is the only fluid that can be investigated. Several factors need to be standardized when sampling CSF for biomarker research: time/volume of CSF collection, sample processing/storage, and the temporal relationship of sampling to clinical or MRI markers of disease activity. Assays used for biomarker detection must be validated so as to optimize the power of the studies. A formal method for establishing whether or not a particular biomarker can be used as a surrogate end‐point needs to be adopted. This process is similar to that used in clinical trials, where the reporting of studies has to be done in a standardized way with sufficient detail to permit a critical review of the study and to enable others to reproduce the study design. A commitment must be made to report negative studies so as to prevent publication bias. Pre‐defined consensus criteria need to be developed for MS‐related prognostic biomarkers. Currently no candidate biomarker is suitable as a surrogate end‐point. Bulk biomarkers of the neurodegenerative process such as glial fibrillary acidic protein (GFAP) and neurofilaments (NF) have advantages over intermittent inflammatory markers.
Biomarkers in Multiple Sclerosis: Role of Antibodies
Authors: Berger Thomas , Reindl Markus
Published: June 2013

The first international workshop on “Biomarkers in Multiple Sclerosis” was organized by B. Bielekova, R. Hohlfeld, R. Martin and U. Utz from April 14–16, 2004, in Washington, DC. The workshop intended to discuss the current status and potential applicability of biological markers for the understanding of the pathogenesis, diagnosis, and therapy of multiple sclerosis. The present review summarizes the presentation on the potential role of antibodies as biomarkers for diagnosis, disease activity, classification and prediction of clinical courses in multiple sclerosis.
Comprehensive Phenotyping in Multiple Sclerosis: Discovery Based Proteomics and the Current Understanding of Putative Biomarkers
Authors: O’Connor Kevin C. , Roy Sushmita Mimi , Becker Christopher H. , Hafler David A. , Kantor Aaron B.
Published: June 2013

Currently, there is no single test for multiple sclerosis (MS). Diagnosis is confirmed through clinical evaluation, abnormalities revealed by magnetic resonance imaging (MRI), and analysis of cerebrospinal fluid (CSF) chemistry. The early and accurate diagnosis of the disease, monitoring of progression, and gauging of therapeutic intervention are important but elusive elements of patient care. Moreover, a deeper understanding of the disease pathology is needed, including discovery of accurate biomarkers for MS. Herein we review putative biomarkers of MS relating to neurodegeneration and contributions to neuropathology, with particular focus on autoimmunity. In addition, novel assessments of biomarkers not driven by hypotheses are discussed, featuring our application of advanced proteomics and metabolomics for comprehensive phenotyping of CSF and blood. This strategy allows comparison of component expression levels in CSF and serum between MS and control groups. Examination of these preliminary data suggests that several CSF proteins in MS are differentially expressed, and thus, represent putative biomarkers deserving of further evaluation.
Chemokine Receptors as Biomarkers in Multiple Sclerosis
Authors: Fox Robert J. , Kivisäkk Pia , Lee Jar-Chi , Tucky Barbara , Lucchinetti Claudia , Rudick Richard A. , Ransohoff Richard M.
Published: June 2013

Leukocyte infiltrates characterize tissue inflammation and are thought to be integral in the pathogenesis of multiple sclerosis (MS). This attribute underlines the importance of understanding mechanisms of leukocyte migration. Chemokines are secreted proteins which govern leukocyte trafficking into targeted organs. Chemokine receptors (CKR) are differentially expressed on leukocytes and their modulation is a potential target for MS disease modifying therapies. Chemokines and their receptors are also potential biomarkers of both disease activity and response to treatment. We describe the fluctuations in CKR expression on peripheral leukocytes in a group of MS patients followed longitudinally for up to 36 months. We observed little fluctuation in CKR expression within each patient over time, despite considerable variability in CKR expression between patients. These observations suggest that individual patients have a CKR set point, and this set point varies from one patient to another. Evaluation of chemokines or chemokine receptors as biomarkers in MS will need to account for this individual variability in CKR expression.
Biomarkers that Discriminate Multiple Myeloma Patients with or without Skeletal Involvement Detected Using SELDI‐TOF Mass Spectrometry and Statistical and Machine Learning Tools
Authors: Bhattacharyya Sudeepa , Epstein Joshua , Suva Larry J.
Published: June 2013

Multiple Myeloma (MM) is a severely debilitating neoplastic disease of B cell origin, with the primary source of morbidity and mortality associated with unrestrained bone destruction. Surface enhanced laser desorption/ionization time‐of‐flight mass spectrometry (SELDI‐TOF MS) was used to screen for potential biomarkers indicative of skeletal involvement in patients with MM. Serum samples from 48 MM patients, 24 with more than three bone lesions and 24 with no evidence of bone lesions were fractionated and analyzed in duplicate using copper ion loaded immobilized metal affinity SELDI chip arrays. The spectra obtained were compiled, normalized, and mass peaks with mass‐to‐charge ratios (m/z) between 2000 and 20,000 Da identified. Peak information from all fractions was combined together and analyzed using univariate statistics, as well as a linear, partial least squares discriminant analysis (PLS‐DA), and a non‐linear, random forest (RF), classification algorithm. The PLS‐DA model resulted in prediction accuracy between 96–100%, while the RF model was able to achieve a specificity and sensitivity of 87.5% each. Both models as well as multiple comparison adjusted univariate analysis identified a set of four peaks that were the most discriminating between the two groups of patients and hold promise as potential biomarkers for future diagnostic and/or therapeutic purposes.