Robert Hinderer

Identification of 14-3-3θ as an antigen that induces a humoral response in lung cancer

We have implemented a strategy to identify tumor antigens that induce a humoral immune response in lung cancer based on the analysis of tumor cell proteins. Chromatographically fractionated protein extracts from three lung cancer cell lines were subjected to Western blotting and hybridization with individual sera to determine serum antibody binding. Two sets of sera were initially investigated. One set consisted of sera from 19 newly diagnosed subjects with lung adenocarcinoma and 19 matched controls. A second independent set consisted of sera from 26 newly diagnosed subjects with lung adenocarcinoma and 24 controls matched for age, gender, and smoking history. One protein that exhibited significant reactivity with both sets of cancer sera ( P = 0.0008) was confidently identified by mass spectrometry as 14-3-3𝛉. Remarkably, significant autoantibody reactivity against 14-3-3𝛉 was also observed in an analysis of a third set consisting of 18 prediagnostic lung cancer sera collected as part of the Beta-Carotene and Retinol Efficacy Trial cohort study, relative to 19 matched controls ( P = 0.0042). A receiver operating characteristic curve constructed with a panel of three proteins consisting of 14-3-3𝛉 identified in this study, plus annexin 1 and protein gene product 9.5 proteins previously identified as associated with autoantibodies in lung cancer, gave a sensitivity of 55% at 95% specificity (area under the curve, 0.838) in discriminating lung cancer at the preclinical stage from matched controls. [Cancer Res 2007;67(24):12000–6]

Stimulatory and Inhibitory Killer Ig-Like Receptor Molecules Are Expressed and Functional on Lupus T Cells

T cells from lupus patients have hypomethylated DNA and overexpress genes normally suppressed by DNA methylation that contribute to disease pathogenesis. We found that stimulatory and inhibitory killer cell Ig-like receptor (KIR) genes are aberrantly overexpressed on experimentally demethylated T cells. We therefore asked if lupus T cells also overexpress KIR, and if the proteins are functional. T cells from lupus patients were found to overexpress KIR genes, and expression was proportional to disease activity. Abs to the stimulatory molecule KIR2DL4 triggered IFN-γ release by lupus T cells, and production was proportional to disease activity. Similarly, cross-linking the inhibitory molecule KIR3DL1 prevented the autoreactive macrophage killing that characterizes lupus T cells. These results indicate that aberrant T cell KIR expression may contribute to IFN overproduction and macrophage killing in human lupus, and they suggest that Abs to inhibitory KIR may be a treatment for this disease.

Environmental Exposure, Estrogen and Two X Chromosomes are Required for Disease Development in an Epigenetic Model of Lupus

Systemic lupus erythematosus (SLE) is an autoimmune disease primarily afflicting women. The reason for the gender bias is unclear, but genetic susceptibility, estrogen and environmental agents appear to play significant roles in SLE pathogenesis. Environmental agents can contribute to lupus susceptibility through epigenetic mechanisms. We used (C57BL/6xSJL)F1 mice transgenic for a dominant-negative MEK (dnMEK) that was previously shown to be inducibly and selectively expressed in T cells. In this model, induction of the dnMEK by doxycycline treatment suppresses T cell ERK signaling, decreasing DNA-methyltransferase expression and resulting in DNA demethylation, overexpression of immune genes Itgal (CD11a) and Tnfsf7 (CD70), and anti-dsDNA antibody. To examine the role of gender and estrogen in this model, male and female transgenic mice were neutered and implanted with time-release pellets delivering placebo or estrogen. Doxycycline induced IgG anti-dsDNA antibodies in intact and neutered, placebo-treated control female but not male transgenic mice. Glomerular IgG deposits were also found in the kidneys of female but not male transgenic mice, and not in the absence of doxycycline. Estrogen enhanced anti-dsDNA IgG antibodies only in transgenic, ERK-impaired female mice. Decreased ERK activation also resulted in overexpression and demethylation of the X-linked methylation-sensitive gene CD40lg in female but not male mice, consistent with demethylation of the second X chromosome in the females. The results show that both estrogen and female gender contribute to the female predisposition in lupus susceptibility through hormonal and epigenetic X-chromosome effects and through suppression of ERK signaling by environmental agents.

Transforming properties of a Q18→E mutation of the microtubule regulator Op18

We have identified a somatic mutation in Op18 in a human esophageal adenocarcinoma. The mutant form of Op18 (M-Op18) was cloned and sequenced, revealing a substitution of a G for C at nucleotide 155, which results in a Q18-->E substitution in the protein. M-Op18 cDNA was expressed in NIH/3T3 cells, which resulted in foci formation and tumor growth in immunodeficient mice. Cell cycle analysis of M-Op18-expressing cells revealed a doubling in the percentage of cells in G2/M relative to cells overexpressing wild-type Op18, a decrease in M-Op18-specific phosphorylation, and alterations in tubulin ultrastructure in M-Op18-expressing cells. These results suggest that the somatic mutation identified in Op18 has profound effects on cell homeostasis that may lead to tumorigenicity.

Identification of proteins from two-dimensional gel electrophoresis of human erythroleukemia cells using capillary high performance liquid chromatography/electrospray-ion trap-reflectron time-of-flight mass spectrometry with two-dimensional topographic map analysis of in-gel tryptic digest products.

Protein spots from two-dimensional (2-D) gel electrophoresis of a human erythroleukemia cell line have been identified by analysis of the in-gel tryptic digests using capillary high performance liquid chromatography (HPLC) separation with on-line detection using electrospray ionization mass spectrometry (ESI-MS). This is performed using an electrospray/ion trap storage/reflectron time-of-flight mass spectrometer system (ESI-IT-reTOFMS). A 2-D topographic mapping display developed to process the on-line data acquired with this TOF system has been used to obtain mass identification of each peptide, even though the capillary HPLC only provides limited separation capability of the tryptic peptide mixtures studied herein. Using this method, a substantial fraction of the protein sequence can be covered and identified using the tryptic map. It is demonstrated that by entering the cell species, the approximate MW and pI range as determined by 2-D gel electrophoresis, and the tryptic peptide map into the database a unique match for identification of the protein generally results. It is also demonstrated that a much improved coverage of the protein sequence is obtained by this method relative to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

Identification of a specific Vimentin Isoform that induces an antibody response in pancreatic cancer

Novel, comprehensive approaches for biomarker discovery and validation are urgently needed. One particular area of methodologic need is for discovery of novel genetic biomarkers in complex diseases and traits. Here, we review recent successes in the use of genome wide association (GWA) approaches to identify genetic biomarkers in common human diseases and traits. Such studies are yielding initial insights into the allelic architecture of complex traits. In general, it appears that complex diseases are associated with many common polymorphisms, implying profound genetic heterogeneity between affected individuals.We have compiled from literature and other sources a list of 1261 proteins believed to be differentially expressed in human cancer. These proteins, only some of which have been detected in plasma to date, represent a population of candidate plasma biomarkers that could be useful in early cancer detection and monitoring given sufficiently sensitive specific assays. We have begun to prioritize these markers for future validation by frequency of literature citations, both total and as a function of time. The candidates include proteins involved in oncogenesis, angiogenesis, development, differentiation, proliferation, apoptosis, hematopoiesis, immune and hormonal responses, cell signaling, nucleotide function, hydrolysis, cellular homing, cell cycle and structure, the acute phase response and hormonal control. Many have been detected in studies of tissue or nuclear components; nevertheless we hypothesize that most if not all should be present in plasma at some level. Of the 1261 candidates only 9 have been approved as “tumor associated antigens” by the FDA. We propose that systematic collection and large-scale validation of candidate biomarkers would fill the gap currently existing between basic research and clinical use of advanced diagnostics.Stratification of cardiac patients arriving at the emergency department is now being made according to the levels of acute cardiac biomarkers (i.e. cardiac troponin (cTn) or creatine kinase myocardial band (CK-MB)). Ongoing efforts are undertaken in an attempt to identify and validate additional cardiac biomarkers, for example, interleukin-6, soluble CD40L, and C-reactive protein, in order to further risk stratify patients with acute coronary syndrome. Several studies have also now shown an association of platelet transcriptome and genomic single nucleotide polymorphisms with myocardial infarction by using advanced genomic tools. A number of markers, such as myeloid-related protein 14 (MRP-14), cyclooxygenase-1 (COX-1), 5-lipoxygenase activating protein (FLAP), leukotriene A4 hydrolase (LTA4H) and myocyte enhancing factor 2A (MEF2A), have been linked to acute coronary syndromes, including myocardial infarction. In the future, these novel markers may pave the way toward personalized disease-prevention programs based on a person’s genomic, thrombotic and cardiovascular profiles. Current and future biomarkers and bioassays for identifying at-risk patients will be discussed in this review.Summary A number of “interesting” risk markers have been proposed as providing prognostic informations in acute coronary syndromes (ACS). Elevation in plasma inflammatory and necrosis biomarkers have been related to future cardiovascular events in individuals with or without prior myocardial infarction. Recently BNP and pro-BNP are entered in clinical practice to recognize patients at major risk, providing incremental information respect to the traditional markers. Together with these laboratory indexes, a few of promising laboratory markers once easily available, could become usefull in identification of patients at high risk. Several studies evaluated many markers of platelet aggregation, endothelial dysfunction and vascular thrombosis, but it is not yet clear whether each of the proposed markers may provide incremental predictive information. We describe, following the most studies reported in literature, the laboratory markers with potential clinical and prognostic power that could early help physicians in the identification of patients with impaired coronary disease and more narrowed coronary arteries.Efficient phagocytosis of cells undergoing apoptosis by macrophages is important to prevent immunological responses and development of chronic inflammatory disorders such as systemic lupus erythematosus, cystic fibrosis and atherosclerosis. To study phagocytosis of apoptotic cells (AC) by macrophages in tissue, we validated different apoptosis markers (DNA fragmentation, caspase-3 activation and cleavage of its substrate poly(ADP-ribose)polymerase-1) in combination with macrophage immunostaining. Human tonsils were used as a model because they show a high apoptosis frequency under physiological conditions as well as efficient phagocytosis of AC by macrophages. On the other hand, advanced human atherosclerotic plaques were examined since plaques show severely impaired phagocytosis of AC. Our results demonstrate that the presence of non-phagocytized terminal deoxynucleotidyl transferase end labelling (TUNEL)-positive AC represents a suitable marker of poor phagocytosis by macrophages in situ. Other markers for apoptosis, such as cleavage of caspase-3 or PARP-1, should not be used to assess phagocytosis efficiency, because activation of the caspase cascade and cleavage of their substrates can occur in AC when they have not yet been phagocytized by macrophages.Cervical cancer, a potentially preventable disease, remains the second most common malignancy in women worldwide. Human papillomavirus (HPV) is the single most important etiological agent in cervical cancer, contributing to neoplastic progression through the action of viral oncoproteins, mainly E6 and E7. Cervical screening programs using Pap smear testing have dramatically improved cervical cancer incidence and reduced deaths, but cervical cancer still remains a global health burden. The biomarker discovery for accurate detection and diagnosis of cervical carcinoma and its malignant precursors (collectively referred to as high-grade cervical disease) represents one of the current challenges in clinical medicine and cytopathology.Background Expressions of various biomarkers in non-small cell lung cancer (NSCLC) have been linked with the prognosis and involvement of mediastinal lymph nodes. Methods In this study, we utilized recursive partitioning analysis (RPA) by using P53, c-erb-B2, and P-glycoprotein (PGP) expressions evaluated by immunohistochemistry to estimate retrospectively the likelihood of the occult N2 mediastinal lymph node involvement in patients with operable NSCLC. Results In univariate tests, immunohistochemical staining of the primary tumor for these 3 markers in 61 patients undergoing surgery revealed no direct relationship with the N2 involvement. However, RPA demonstrated in patients aged <75 and with ≥4 mediastinal lymph nodes removed that, high PGP expression frequency (≥20%) predicted an increased likelihood of the N2 involvement (46.7%, R2 = 0.25). Univariate nominal logistic regression analysis revealed that RPA group affiliation, and the number of mediastinal lymph nodes resected (logarithmic transformation) were associated with the metastasis to N2 lymph nodes (χ2 = 17.59, p = 0.0005, and χ2 = 2.40, p = 0.0654, respectively). Multivariate analysis confirmed that only RPA group affiliation predicted the N2 involvement (χ2 = 14.63, p = 0.0022). Conclusion This study shows for the first time that PGP expression of the primary tumor may help to predict the occult N2 mediastinal lymph node involvement in NSCLC. Thus, further research is required to understand whether PGP expression may aid in the decision process for preoperative mediastinoscopy.Angiogenesis is essential to the survival, growth, invasion, and metastasis of various human solid tumors. We compared the microvessel density (MVD) and clinicopathologic features of two different groups of hepatocellular carcinoma (HCC), namely HCC with cirrhosis (HCC-C) and without cirrhosis (HCC-NC). A tissue microarray composed of 20 normal livers, 20 cirrhotic livers, tumor and adjacent background non-neoplastic liver tissues from 20 HCC-C and 20 HCC-NC were constructed and stained immunohistochemically with antibodies against the antigen CD34. The MVD was determined by the measurement of the area and density of CD34 positive sinusoidal endothelial cells using the Image Pro Plus software. There was a trend of increased MVD in cirrhotic liver compared to normal liver and in cirrhotic background non-neoplastic liver adjacent to the tumor compared to the non-cirrhotic background non-neoplastic liver. Tumor tissue of HCC-C and HCC-NC both showed significantly higher MVD than their adjacent background non-neoplastic liver tissue. There was no statistical difference in MVD between HCC-C and HCC-NC. A higher value of MVD was seen in tumors of intermediate size (5–10 cm), high histologic grade, the presence of lymphvascular space invasion, and the underlying etiology of hepatitis C and alcoholic steatohepatitis. This data indicates that MVD may play an important role in liver carcinogenesis and neoplastic progression. The difference in clinical behavior between HCC-C and HCC-NC does not seem to be associated with differences in tumor MVD. Objective measurement of MVD using standardized computer software could potentially be used as a clinical marker to predict patients’ prognosis.BRCA1 is a tumor suppressor which plays a crucial role in the repair of DNA double-strand breaks, and its abnormality is responsible for hereditary ovarian cancer syndrome. It has recently been reported that reduced expression of BRCA1 is also common in sporadic ovarian carcinoma via its promoter hypermethylation, and that ovarian carcinoma patients negative for BRCA1 expression showed favorable prognosis. To address if BRCA1 expression plays a role in the chemotherapeutic response, we analyzed the effect of BRCA1 suppression on the sensitivity to cisplatin and paclitaxel in ovarian cancer cells. Specific siRNA for BRCA1 gene was transfected into 3 ovarian cancer cell lines with various p53 status. Reduced expression of BRCA1 by transfection of BRCA1-siRNA resulted in a 5.3-fold increase in sensitivity to cisplatin in p53-wild A2780 cells, but not in p53-mutated A2780/CDDP and p53-deleted SKOV3 cells. Regarding the sensitivity to paclitaxel, BRCA1 suppression caused no significant changes in all the 3 cell lines. For ionizing radiation sensitivity, BRCA1 suppression also showed a significant higher sensitivity in A2780 cells. Growth curve and cell cycle analyses showed no significant differences between BRCA1-siRNA-transfected A2780 cells and control cells. However, cisplatin treatment under suppression of BRCA1 showed a significantly increased apoptosis along with up-regulation of p53 and p21 in A2780 cells. Accordingly, reduced expression of BRCA1 enhances the cisplatin sensitivity and apoptosis via up-regulation of p53 and p21, but does not affect the paclitaxel sensitivity. Expression of BRCA1 might be an important biomarker for cisplatin resistance in ovarian carcinoma.Rheumatoid arthritis (RA) is a common rheumatic disease in Caucasians and in other ethnic groups. Diagnosis is mainly based on clinical features. Before 1998, the only serological laboratory test that could contribute to the diagnosis was that for rheumatoid factor (RF). The disease activity markers for the evaluation of clinical symptoms or treatment outcome were the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). As a matter of fact, the diagnosis of early RA is quite impossible, as the clinical criteria are insufficient at the beginning stage of the disease. In 1998, Schelleken reported that a high percentage of RA patients had a specific antibody that could interact with a synthetic peptide which contained the amino acid citrulline. The high specificity (98%) for RA of this new serological marker, anti-cyclic citrullinated antibody (anti-CCP antibody), can be detected early in RA, before the typical clinical features appear. The presence or absence of this antibody can easily distinguish other rheumatic diseases from RA. Additionally, the titer of anti-CCP can be used to predict the prognosis and treatment outcome after DMARDs or biological therapy. Therefore, with improvement of sensitivity, the anti-CCP antibody will be widely used as a routine laboratory test in the clinical practice for RA.Sixteen longSAGE libraries from four different clinical stages of cervical intraepithelial neoplasia have enabled us to identify novel cell-surface biomarkers indicative of CIN stage. By comparing gene expression profiles of cervical tissue at early and advanced stages of CIN, several genes are identified to be novel genetic markers. We present fifty-six cell-surface gene products differentially expressed during progression of CIN. These cell surface proteins are being examined to establish their capacity for optical contrast agent binding. Contrast agent visualization will allow real-time assessment of the physiological state of the disease process bringing vast benefit to cancer care. The data discussed in this publication have been submitted to NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE6252.Expression of CCR2, CXCR3 and CCR4 on CD4+ T or CD8+ T cells in blood and cerebrospinal fluid (CSF) for multiple sclerosis (MS) was measured by 3-color flow cytometry, and compared to blood from healthy controls and CSF from patients with other inflammatory neurological diseases (INDs). CD4+CXCR3+/CD4+CCR4+ ratio (representing Th1/Th2 balance) was higher in both CSF and blood of MS patients than those of IND patients or healthy controls. Percentage of CCR2-positive T cells was significantly higher in CSF from MS patients. Increased CCR2 expression on T cells in CSF and Th1/Th2 imbalance may reflect the pathological processes involved in MS.Municipal effluents are complex mixtures of compounds such as heavy metals, aromatic and aliphatic hydrocarbons, and micro-organisms and are released in aquatic ecosystems. The purpose of this study was to verify whether changes in metallothioneins (MT) were associated with the accumulation of labile metals in tissue of freshwater mussels exposed to the dispersion plume of a major municipal effluent. Mussels were placed in experimental cages deployed at sites 1.5 km upstream, 8 km downstream and 12 km downstream of the outfall of a major, primary-treated municipal effluent in the St. Lawrence River (Québec, Canada). Mussels were analysed for MT and labile zinc levels in their gonads, gills and digestive glands. Lipogenic enzyme (isocitrate and glucose-6-phosphate dehydrogenase) and arachidonic acid cyclooxygenase (COX) activities were also measured in gonad and gill tissues. Although MT was induced in all the tissues examined, the results showed that labile zinc levels were significantly reduced in gill and gonad tissues, with an increase observed only at the 12 km downstream site in the digestive gland. COX activity was readily induced in gills and gonads. Glucose-6-phosphate dehydrogenase activity was reduced at both downstream sites, but isocitrate dehydrogenase activity was significantly induced at the farthest (12 km) site. Analysis of covariance revealed that MT levels in gills were more influenced by COX activity than with distance in the dispersion plume and was negatively correlated with labile zinc levels. In conclusion, MT induction was inversely related to the levels of labile zinc but positively so with the inflammation biomarker COX. Hence, the induction of MT in mussels exposed to the municipal effluent of a large city appears to be associated with either inflammatory processes or as compensation for the loss of labile essential metals. We propose that the simple and complimentary parameters of labile zinc and COX evaluations be used to link MT induction with divalent heavy metal exposure in environmental studies dealing with various type of contaminants in such complex contaminant mixture effluents.Human Glyco_18 domain-containing proteins constitute a family of chitinases and chitinase-like proteins. Chitotriosidase and AMCase are true enzymes which hydrolyse chitin and have a C-terminal chitin-binding domain. YKL-40, YKL-39, SI-CLP and murine YM1/2 proteins possess solely Glyco_18 domain and do not have the hydrolytic activity. The major sources of Glyco_18 containing proteins are macrophages, neutrophils, epithelial cells, chondrocytes, synovial cells, and cancer cells. Both macrophages and neutrophils use the regulated secretory mechanism for the release of Glyco_18 containing proteins. Glyco_18 containing proteins are established biomarkers for human diseases. Chitotriosidase is overproduced by lipid-laden macrophages and is a major marker for the inherited lysosomal storage Gaucher disease. AMCase and murine lectin YM1 are upregulated in Th2-environment, and enzymatic activity of AMCase contributes to asthma pathogenesis. YKL proteins act as soluble mediators for the cell proliferation and migration, and are also involved in rheumatoid arthritis, inflammatory bowel disease, hepatic fibrosis and cirrhosis. Chitotriosidase and YKL-40 reflect the macrophage activation in atherosclerotic plaques. Serum level of YKL-40 is a diagnostic and prognostic marker for numerous types of solid tumors. YKL-39 is a marker for the activation of chondrocytes and the progression of the osteoarthritis in human. Recently identified SI-CLP is upregulated by Th2 cytokine IL-4 as well as by glucocorticoids. This unique feature of SI-CLP makes it an attractive candidate for the examination of individual sensitivity of patients to glucocorticoid treatment and prediction of side effects of glucocorticoid therapy. Human chitinases and chitinase-like proteins are found in tissues and circulation, and can be detected by non-invasive technologies.In mammalian development, primordial germ cells (PGCs) represent the initial population of cells that are committed to the germ cell lineage. PGCs segregate early in development, triggered by signals from the extra-embryonic ectoderm. They are distinguished from surrounding cells by their unique gene expression patterns. Some of the more common genes used to identify them are Blimp1, Oct3/4, Fragilis, Stella, c-Kit, Mvh, Dazl and Gcna1. These genes are involved in regulating their migration and differentiation, and in maintaining the pluripotency of these cells. Recent research has demonstrated the possibility of obtaining PGCs, and subsequently, mature germ cells from a starting population of embryonic stem cells (ESCs) in culture. This phenomenon has been investigated using a variety of methods, and ESC lines of both mouse and human origin. Embryonic stem cells can differentiate into germ cells of both the male and female phenotype and in one case has resulted in the birth of live pups from the fertilization of oocytes with ESC derived sperm. This finding leads to the prospect of using ESC derived germ cells as a treatment for sterility. This review outlines the evolvement of germ cells from ESCs in vitro in relation to in vivo events.Free circulating DNA, which is thought to be derived from the primary tumour, can be detected in the blood of patients with cancer. Detection of genetic and epigenetic alteration in this tumour DNA offers a potential source of development of prognostic and predictive biomarkers for cancer. One such change is DNA methylation of the promotor region of tumour suppressor genes. This causes down regulation of tumour suppressor gene expression, a frequent event in carcinogenesis. Hypermethylation of the promotor region of a number of genes has been detected in many tumour types and more recently these changes have been detected in circulating tumour DNA. This review will summarise the literature detailing DNA methylation in circulating tumour DNA and discuss some of the current controversies and technical challenges facing its use as a potential biomarker for cancer.Receptor for advanced glycation end-products (RAGE) is known to be involved in both micro- and macro-vascular complications in diabetes. Among numerous truncated forms of RAGE recently described, the C-terminally truncated form of RAGE has received much attention. This form of RAGE, carrying all of the extracellular domains but devoid of the transmembrane and intracytoplasmic domains, is released outside from cells, binds ligands including AGEs, and is capable of neutralizing RAGE signaling on endothelial cells in culture. This form of RAGE is generated as a splice variant and is named endogenous secretory RAGE (esRAGE). Adenoviral overexpression of esRAGE reverses diabetic impairment of vascular dysfunction, suggesting that esRAGE may be an important inhibitor of RAGE signaling in vivo and potentially be useful for prevention of diabetic vascular complications. An ELISA system to measure plasma esRAGE was recently developed, and the pathophysiological roles of esRAGE have begun to be unveiled clinically. Plasma esRAGE levels are decreased in patients with several metabolic diseases including type 1 and type 2 diabetes, metabolic syndrome and hypertension. In cross-sectional analysis, plasma esRAGE levels are inversely correlated with carotid or femoral atherosclerosis. In an observational cohort of patients with end-stage renal disease, cumulative incidence of cardiovascular death was significantly higher in subjects with lower plasma esRAGE levels. These findings suggest that plasma esRAGE may act as a protective factor against and a novel biomarker for the occurrence of metabolic syndrome and cardiovascular diseases.We designed the present case-control study in order to examine the validity of apolipoprotein (apo) A-I, B, apoB/apoA-I ratio and Lp(a) as alternative markers of cardiovascular morbidity in end-stage renal disease (ESRD) patients undergoing chronic hemodialysis (HD). Twenty-five HD patients (18 males, mean age 63, range 52–69 years) comprised the group with prevalent cardiovascular disease (CVD) and 50 HD patients (35 males, mean age 62, range 40–77 years) with non evident cardiovascular disease history constituted the second study group. Patients with CVD had significantly higher concentrations of serum apoB, apoB/apoA-I ratio and Lp(a), and lower levels of apoA-I compared to patients without incident CVD. All three parameters studied were correlated with cardiovascular morbidity, i.e. apoA-I negatively and apoB and apoB/apoA-I ratio positively (r = −0.6, P < 0.05; r = 0.659, P < 0.01; and r = 0.614, P < 0.01, respectively). Furthermore, logCRP exhibited as well a significant positive correlation with cardiovascular morbidity (r = 0.704, P < 0.001), not this being the case for Lp(a) which was not found to exhibit such a correlation (r = 0.05, P = NS). Among them, apoB and apoB/apoA-I ratio exhibited the characteristics most coherent to CVD. The age- and sex-adjusted OR for the presence of CVD was 2.3 and 2.0, respectively, which remained independent of any confounding effect of inflammation. In conclusion, serum apoB levels and apoB/apoA-I ratio exhibit characteristics of credible independent markers of in HD patients.The clinical utility of a biomarker depends on its ability to identify high-risk individuals to optimally manage the patient. A new biomarker would be of clinical value if it is accurate and reliable, provides good sensitivity and specificity, and is available for widespread application. Data are accumulating on the potential clinical utility of integrating imaging technologies and circulating biomarkers for the identification of vulnerable (high-risk) cardiovascular patients. A multi-biomarker strategy consisting of markers of inflammation, hemostasis and thrombosis, proteolysis and oxidative stress, combined with new imaging modalities (optical coherence tomography, virtual histology plus IVUS, PET) can increase our ability to identify such thombosis-prone patients. In an ideal scenario, cardiovascular biomarkers and imaging combined will provide a better diagnostic tool to identify high-risk individuals and also more efficient methods for effective therapies to reduce such cardiovascular risk. However, additional studies are required in order to show that this approach can contribute to improved diagnostic and therapeutic of atherosclerotic disease.Metallothionein (MT) is a highly conserved, low-molecular-weight, cysteine-rich protein that occurs in 4 isoforms (MT-I to MT-IV), of which MT-I+II are the major and best characterized proteins. This review will focus on mammalian MT-I+II and their functional impact upon cellular survival and death, as seen in two rather contrasting pathological conditions: Neurodegeneration and neoplasms. MT-I+II have analogous functions including: 1) Antioxidant scavenging of reactive oxygen species (ROS); 2) Cytoprotection against degeneration and apoptosis; 3) Stimulation of cell growth and repair including angiogenesis/revascularization, activation of stem/progenitor cells, and neuroregeneration. Thereby, MT-I+II mediate neuroprotection, CNS restoration and clinical recovery during neurodegenerative disorders. Due to the promotion of cell survival, increased MT-I+II levels have been associated with poor tumor prognosis, although the data are less clear and direct causative roles of MT-I+II in oncogenesis remain to be identified. The MT-I+II molecular mechanisms of actions are not fully elucidated. However, their role in metal ion homeostasis might be fundamental in controlling Zn-dependent transcription factors, protein synthesis, cellular energy levels/metabolism and cell redox state. Here, the neuroprotective and regenerative functions of MT-I+II are reviewed, and the presumed link to oncogenesis is critically perused.Biomarkers are increasingly used to provide decision making data early in phase I by showing Proof of Mechanism or Proof of Concept (PoM/PoC). For antihypertensive agents, the administration of multiple doses (md) to hypertensive patients is assumed to be necessary for an early go/no-go decision. We compared the effects of an Angiotensin II receptor antagonist (ARA) on Plasma Renin and blood pressure (BP) following an oral single dose (sd) and once daily md for seven days to healthy volunteers and patients with essential hypertension (diastolic BP 95 mmHg to 114 mmHg; systolic BP 130 mmHg to 200 mmHg). Methods: 5–12 healthy male subjects/dose received 10 mg to 300 mg ARA sd and 50 to 300 mg md for 7 days; patients (9–10/dose) received 20 mg–400 mg ARA for 7 days. The studies were designed as randomized, single-blind, placebo-controlled, group comparison or crossover dose-escalation studies. Plasma Renin and BP were monitored up to 24 hours after dosing. Results: Plasma Renin showed a high interindividual variability in both healthy volunteers and patients. Healthy subjects showed a dose- and time-related increase in plasma Renin after sd from 40 mg to 300 mg and md of 50 mg to 300 mg (p < 0.05 for doses of 200 mg and 300 mg). In patients, increases in plasma Renin occurred at 8 hours and beyond starting at sd of 100 mg and md of 50 mg (p < 0.05 for the dose of 400 mg). While healthy volunteers showed no relevant decrease in BP, in hypertensive patients a reduction in BP in doses of 100 mg to 400 mg occurred (p < 0.05); effects were more pronounced after md compared to sd. Conclusion: Early PoM for an antihypertensive agent can be shown by use of laboratory biomarkers following sd to healthy subjects. PoC can be achieved after sd in hypertensive patients. Administration of sd to healthy volunteers is sufficient for an early go/no-decision.It has become very clear that a single molecular event is inadequate to accurately predict the biology (or pathophysiology) of cancer. Furthermore, using any single molecular event as a biomarker for the early detection of malignancy may not comprehensively identify the majority of individuals with that disease. Therefore, the fact that technologies have arisen that can simultaneously detect several, possibly hundreds, of biomarkers has propelled the field towards the development of multianalyte-based in vitro diagnostic early detection tests for cancer using body fluids such as serum, plasma, sputum, saliva, or urine. These multianalyte tests may be based on the detection of serum autoantibodies to tumor antigens, the presence of cancer-related proteins in serum, or the presence of tumor-specific genomic changes that appear in plasma as free DNA. The implementation of non-invasive diagnostic approaches to detect early stage cancer may provide the physician with evidence of cancer, but the question arises as to how the information will affect the pathway of clinical intervention. The confirmation of a positive result from an in vitro diagnostic cancer test may involve relatively invasive procedures to establish a true cancer diagnosis. If in vitro diagnostic tests are proven to be both specific, i.e. rarely produce false positive results due to unrelated conditions, and sufficiently sensitive, i.e. rarely produce false negative results, then such screening tests offer the potential for early detection and personalized therapeutics using multiple disease-related targets with convenient and non-invasive means. Here we discuss the technical and regulatory barriers inherent in development of clinical multianalyte biomarker assays.Disc degeneration plays a major role in this country’s medical, social and economic structure. The life-time prevalence of low back pain, which has disc degeneration as its cause, is about 80% in the general population. It is a primary cause of disability and estimated costs related to low back disorders exceed

Oncoprotein 18 levels and phosphorylation mediate megakaryocyte polyploidization in human erythroleukemia cells

100 billion per year in the U.S. alone. Biomarkers are becoming increasingly important as indicators of the presence of disease, and in evaluating outcomes during clinical treatment. Cell-based biologic therapies which are currently being developed to treat disc degeneration are going to be most efficacious when applied to the early stages of disc disease. In this article we ask: 1) Whether there are existing biomarkers which could play a role in detecting early stages of disc degeneration, and 2) Highlight exciting potentials in future biomarker screening for disc degeneration.Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na+/H+ exchange and HC3−/Cl− anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore damaged proteins, it is equipped with high activity of protective enzymes. Redox changes in the cell initiate various signalling pathways. Plasma membrane oxido-reductases (PMORs) are transmembrane electron transport systems that have been found in the membranes of all cells and have been extensively characterized in the human erythrocyte. Erythrocyte PMORs transfer reducing equivalents from intracellular reductants to extracellular oxidants, thus their most important role seems to be to enable the cell respond to changes in intra- and extra-cellular redox environments. So far the activity of erythrocyte PMORs in disease states has not been systematically investigated. This review summarizes present knowledge on erythrocyte electron transfer activity in humans (health, type 1 diabetes, diabetic nephropathy, and chronic uremia) and hypothesizes an integrated model of the functional organization of erythrocyte plasma membrane where electron pathways work in parallel with transport metabolons to maintain redox homeostasis.Purpose Telomere shortening is an important event during carcinogenesis. Although studies suggest role of multiple proteins in telomere length regulation, there is dearth of reports in oral cancer which is a leading malignancy in Asian countries especially in India. Thus the present study was carried out to study these mechanisms and explore the pathways involved in telomere—telomerase regulation and identify possible prognostic markers to understand the biology of oral tumors for better treatment approaches. Methods Telomere length was determined by Southern Hybridisation method, telomeric repeat binding factor (TRF) 1 and 2 expression was detected by Western blot method and telomerase activation by telomeric repeat amplification protocol. Statistical analysis was done using SPSS (Version 10) software. Results Significant shortening of telomeres was seen in the tumor tissues as compared to normal tissues. Poor prognosis was observed in the patients with higher telomere length in malignant tissue, higher tumor to normal telomere length ratio (T/N TRF LR). Expression of TRF-2 but not TRF-1 protein was significantly higher in the malignant tissues. We also observed telomerase activation in 75 malignant tissues. Conclusions Our results reveal significant clinical usefulness of telomere length, T/N TRF LR and telomerase activation in the prognosis of oral cancer patients. TRF-2 overexpression in malignant tissues appears to play an important role in telomere length shortening in oral cancer.In many subjects who are genetically susceptible to asthma, exposure to environmental stimuli may exacerbate their condition. However, it is unknown how the expression and function of a family of pattern-recognition receptors called toll-like receptors (TLR) are affected by exposure to particulate pollution. TLRs serve a critical function in alerting the immune system of tissue damage or infection—the so-called “danger signals”. We are interested in the role that TLRs play in directing appropriate responses by innate immunity, particularly dendritic cells (DC), after exposing them to particulate pollution. Dendritic cells serve a pivotal role in directing host immunity. Thus, we hypothesized that alterations in TLR expression could be further explored as potential biomarkers of effect related to DC exposure to particulate pollution. We show some preliminary data that indicates that inhaled particulate pollution acts directly on DC by down-regulating TLR expression and altering the activation state of DC. While further studies are warranted, we suggest that alterations in TLR2 and TLR4 expression should be explored as potential biomarkers of DC exposure to environmental particulate pollution.Parathyroid hormone (PTH) changes morphology of osteoclasts within minutes after its systemic administration. The aim of our study was to test in healthy men whether both exogenous and endogenous PTH could change acutely (minutes to hours) the serum cross-linked C-telopeptide of type I collagen (beta CTX), which is released during osteoclastic resorption of bone. Twelve healthy men (age range 24–34 yr) were each studied during 180 min on a control period, after a single subcutaneous injection of teriparatide, and after 30 min EDTA infusion to stimulate endogenous PTH secretion. The tests were started after overnight fast, 3 h after a standard calcium load. The EDTA infusion induced a significant decrease in serum ionized calcium (by 8.5% at 33 min) and a significant increase in plasma PTH (by 305% at 33 min). Both the EDTA and teriparatide resulted in a significant increase in beta CTX (p < 0.001) with maximum increases of 64% and 80%, respectively. A mild, but significant decrease in beta CTX was observed during the control test period. In conclusion, single-dose teriparatide injection as well as a stimulation of endogenous PTH in healthy men results in an acute increase of the bone resorption marker.Leukemia associated antigens (LAAs) are being increasingly identified by methods such as cytotoxic T-lymphocyte (CTL) cloning, serological analysis of recombinant cDNA expression libraries (SEREX) and mass spectrometry (MS). In additional, large scale screening techniques such as microarray, single nucleotide polymorphisms (SNPs), serial analysis of gene expression (SAGE) and 2-dimensional gel electrophoresis (2-DE) have expanded our understanding of the role that tumor antigens play in the biological processes which are perturbed in acute myeloid leukemia (AML). It has become increasingly apparent that these antigens play a dual role, not only as targets for immunotherapy, but also as biomarkers of disease state, stage, response to treatment and survival. We need biomarkers to enable the identification of the patients who are most likely to benefit from specific treatments (conventional and/or novel) and to help clinicians and scientists improve clinical end points and treatment design. Here we describe the LAAs identified in AML, to date, which have already been shown to play a dual role as biomarkers of AML disease.The objective was primarily to describe short term intra-individual variation in serum levels of soluble adhesion molecules (sCAMs: E-selectin, P-selectin, intercellular adhesion molecule-1(sICAM-1) and vascular cellular adhesion molecule-1(sVCAM-1)) in healthy subjects. Secondly, sCAMs were correlated to brachial artery flow mediated vasodilation (FMD). Forty healthy subjects aged 24–66 years had sCAMs measured twice with 4 week intervals and short-term intra-individual variation was estimated as variation in the paired measurements after correcting for the analytical precision of the used method. At baseline, brachial FMD was measured. No difference was observed in mean sCAMs in the whole study group. Estimated intra-subject variations in sCAMs were 7.6–11.3%. In a regression analysis, significant negative association was found between sE-selectin and FMD after controlling for possible confounders (p < 0.04) while no significant correlation could be demonstrated between the other sCAMs and FMD. In conclusion, short term intra-individual variations in sCAMs were 7.6–11.3% in healthy subjects. We also found a significant negative association between sE-selectin and FMD, indicating an possible association between inflammation and dysfunction of the vascular endothelium; however further studies are required to confirm this preliminary finding.YKL-39 is a Glyco_18 domain containing chitinase-like protein which is currently recognized as a biomarker for the activation of chondrocytes and the progress of the osteoarthritis in human. YKL-39 was identified as an abundantly secreted protein in primary culture of human articular chondrocytes. Two biological activities of YKL-39 might contribute to the disease progression. One is the induction of autoimmune response and second is the participation in tissue remodeling. Other mammalian chitinase-like proteins including chitotriosidase, SI-CLP, YKL-40 and YM1 are expressed by macrophages in various pathological conditions. In contrast, YKL-39 was never reported to be produced by macrophages. We used in vitro model of human monocyte-derived macrophage differentiation to analyse regulation of YKL-39 expression. Expression of YKL-39 was examined by real-time RT-PCR. CD14+ MACS sorted human monocytes differentiated for 6 days under different stimulations including IFNγ, IL-4, dexamethasone and TGF-β. We found that both IL-4 and TGF-β have weak stimulatory effect on YKL-39 expression in all donors tested (3.2 ± 1.7 fold, p = 0.006 and 6.3 ± 3.1 fold, p = 0.014 respectively). However the combination of IL-4 and TGF-β had strong stimulatory effect on the expression of YKL-39 in all analysed individual macrophage cultures (34 ± 36 fold, p = 0.05). IFN-γ did not show statistically significant effect of YKL-39 mRNA expression. Presence of dexamethasone almost completely abolished the stimulatory effects of IL-4 and TGF-β. In summary, we show here for the first time, that human cells of monocyte origin are able to produce YKL-39. Maturation of monocyte derived macrophages in the presence of Th2 cytokine IL-4 and TGF-β leads to the strong activation of YKL-39 expression. Thus elevated levels of YKL-39 observed during chronic inflammations can not be attributed solely to the activity of chondrocytes. In perspective, YKL-39 might serve as a useful biomarker to detect macrophage-specific response in pathologies like tumour, atherosclerosis and Alzheimer disease.Background Efficacy of thrombolysis in acute ischemic stroke is strongly related to physician’s ability to make an accurate diagnosis and to intervene within 3–6 h after event onset. In this context, the discovery and validation of very early blood markers have recently become an urgent, yet unmet, goal of stroke research. Ubiquitin fusion degradation protein 1 is increased in human postmortem CSF, a model of global brain insult, suggesting that its measurement in blood may prove useful as a biomarker of stroke. Methods Enzyme-linked immunosorbent assay (ELISA) was used to measure UFD1 in plasma and sera in three independent cohorts, European (Swiss and Spanish) and North-American retrospective analysis encompassing a total of 123 consecutive stroke and 90 control subjects. Results Highly significant increase of ubiquitin fusion degradation protein 1 (UFD1) was found in Swiss stroke patients with 71% sensitivity (95% CI, 52–85.8%), and 90% specificity (95% CI, 74.2–98%) (N = 31, p < 0.0001). Significantly elevated concentration of this marker was then validated in Spanish (N = 39, p < 0.0001, 95% sensitivity (95% CI, 82.7–99.4%)), 76% specificity (95% CI, 56.5–89.7%)) and North-American stroke patients (N = 53, 62% sensitivity (95% CI, 47.9–75.2%), 90% specificity (95% CI, 73.5–97.9%), p < 0.0001). Its concentration was increased within 3 h of stroke onset, on both the Swiss (p < 0.0001) and Spanish (p = 0.0004) cohorts. Conclusions UFD1 emerges as a reliable plasma biomarker for the early diagnosis of stroke, and in the future, might be used in conjunction with clinical assessments, neuroimaging and other blood markers.Accurate interpretation and correlation of tissue spectroscopy with pathological conditions requires disease-specific tissue metabolite databases; however, specimens for research are often kept in frozen storage for various lengths of time. Whether such frozen storage results in alterations to the measured metabolites is a critical but largely unknown issue. In this study, human prostate tissues from specimens that had been stored at −80 °C for 32 months were analyzed with high resolution magic angle spinning (HRMAS) magnetic resonance (MR) spectroscopy, and compared with the initial measurements of the adjacent specimens from the same cases when snap frozen in the operation room and kept frozen for less than 24 hours. Results of the current study indicate that that the storage-induced metabolite alterations are below the limits that tissue MR spectroscopy can discriminate. Furthermore, quantitative pathology evaluations suggest the observed alterations in metabolite profiles measured from the adjacent specimens of the same prostates may be accounted for by tissue pathological heterogeneities and are not a result of storage conditions. Hence, these results indicate that long-term frozen storage of prostate specimens can be quantitatively analyzed by HRMAS MR spectroscopy without concerns regarding significant metabolic degradation or alteration.Summary The urinary ratio of 6 β-hydroxycortisol/cortisol (6 β-OHC/C) as a biomarker of CYP3A4 metabolizing activity has been studied in Egyptian patients with chronic liver cirrhosis associated with previous hepatic Schistosomiasis infection to determine any possible alteration in enzyme activity. The ratio of 6-β OHC/C was determined in morning urine samples collected from 8:00 a.m. to 12:00 p.m. in healthy adults (n = 36) and patients with liver cirrhosis (n = 57). The median age for control was 27 years (range: 18–50 years) and 50 years (range: 27–75 years) for patients. 6 β-OHC was detected in urine by ELIZA kits (Stabiligen, France). Patients with liver cirrhosis were categorized according to Child Pugh Classification into Child B (n = 28) and Child C (n = 29) classes. Cholestasis was observed in 9/28 of Child B class and 8/29 of Child C class of patients. The control subjects showed gender-related difference in the urinary ratio of 6 β-OHC/C. A significant reduction (P < 0.001) in 6 β-OHC/C ratio was observed only in Child C patients in comparison with control subjects. Regression analysis showed a significant correlation (P < 0.05) between 6 β-OHC/C ratio and serum albumin. The influence of cholestasis on the urinary ratio of 6-β OHC/C was observed on cirrhotic patients of Child B class. In conclusion, patients with chronic liver cirrhosis might have a reduction of metabolizing activity of CYP3A4 enzymes which could be identified by measuring the urinary ratio of 6 β-OHC/C. This reduction is more apparent in severe liver injury (Child C class). Therefore, it is important to understand the metabolic fate of drugs metabolized by 3A4 enzymes in patients with liver cirrhosis to avoid drug accumulation that might lead to development of drug toxicity.A dysfunction in copper homeostasis seems to occur in Alzheimer’s disease (AD). We previously evidenced that an excess of non-ceruloplasmin-copper (NCC) correlated with the main functional, anatomical as well as cerebrospinal markers of the disease. Aim of our study was to investigate ceruloplasmin isoforms as potential actors in this AD copper dysfunction. Our data show that AD patients have ceruloplasmin fragments of low molecular weight (<50 kDa) both in their serum and brain, contrary to healthy controls. Ceruloplasmin isoforms of higher molecular weight (115 and 135 kDa in serum and 135 kDa in brain), as well as copper levels in the brain, instead, do not seem to mark a difference between AD and healthy subjects. These data suggest a ceruloplasmin fragmentation in the serum of AD patients. Some clues in this direction have been found also in the AD brain.Malignant mesothelioma (MM) is a malignant tumor derived from mesothelial cells, native cells of the body cavities. Exposure to asbestos is the most strongly established etiologic factor, predominantly for the most common disease form, pleural mesothelioma. The pathogenesis of MM involves the accumulation of extensive cytogenetic changes, as well as cancer-related phenotypic alterations that facilitate tumor cell survival, invasion and metastasis. This review presents current knowledge regarding the biological characteristics of this disease that are linked to the so-called hallmarks of cancer. In addition, data suggesting that the anatomic site (solid tumor vs. effusion) affects the expression of metastasis-associated and regulatory molecules in MM are presented. Finally, recent work in which high-throughput methodology has been applied to MM research is reviewed. The data obtained in the reviewed research may aid in defining new prognostic markers and therapeutic targets for this aggressive disease in the future.In 1981 de Bold et al. reported that the injection of atrial tissue extracts in rats induced natriuresis, a discovery that initiated a flurry of research leading to the discovery of a new class of cardiac hormones, the natriuretic peptides.1 These peptides represent the first line of response of the heart to defend the body against a number of processes, especially plasma volume expansion. Since circulating concentrations of natriuretic peptides are elevated in heart failure (HF) and given that clinical parameters for the evaluation and management of HF have a poor sensitivity and specificity, these peptides were a more than welcome addition to standard clinical evaluation, as a marker to identify HF. Nowadays, Brain Natriuretic Peptide (BNP) and its cleavage equivalent aminoterminal-proBNP (NT-proBNP) have proven its clinical diagnostic but also prognostic value in HF, and have shown to be superior as a diagnostic parameter for HF when compared to chest X-ray and parameters of physical examination.2–4 In this paper, we aim to discuss the background, the value, and of course the potential pitfalls encountered by clinicians using NT-proBNP testing in their daily clinical practice.The Gastric Cancer (Biomarkers) Knowledgebase (GCBKB) (http://biomarkers.bii.a-star.edu.sg/background/gastricCancerBiomarkersKb.php) is a curated and fully integrated knowledgebase that provides data relating to putative biomarkers that may be used in the diagnosis and prognosis of gastric cancer. It is freely available to all users. The data contained in the knowledgebase was derived from a large literature source and the putative biomarkers therein have been annotated with data from the public domain. The knowledgebase is maintained by a curation team who update the data from a defined source. As well as mining data from the literature, the knowledgebase will also be populated with unpublished experimental data from investigators working in the gastric cancer biomarker discovery field. Users can perform searches to identify potential markers defined by experiment type, tissue type and disease state. Search results may be saved, manipulated and retrieved at a later date. As far as the authors are aware this is the first open access database dedicated to the discovery and investigation of gastric cancer biomarkers.Biological markers for assessment of exposure to a variety of environmental carcinogens has been widely applied in both basic as well as clinical research. Exposure to tobacco smoke presents an ideal environment with which to develop, characterize, and refine biological markers, especially of those carcinogens found in tobacco. In the present study, a sensitive gas chromatography/mass spectrometry (GC/MS) method was developed to measure nitrosamine- hemoglobin adducts (HPB-Hb (4-Hydroxy-3-pyridinyl-1-butanone) at trace levels in red blood cells of both African-American and Caucasian smoking and nonsmoking mothers and their infants. Gas chromatographic and mass spectrometric methods with chemical ionization (CI) of methane reagent gas in both positive and negative ion mode as well as electron ionization (EI) were studied to determine differences in sensitivity of detection among the various ionization methods. Detection limits using both positive and negative chemical ionization modes were found to be 30 femtomoles of HPB, whereas detection using electron impact modes yielded a detection limit of 80 femtomoles of HBP. Comparative derivatization of HPB was performed using O-bis(Trimethylsilyl)-trifluoroacetamide (BSTFA) and 2, 3, 4, 5, 6-Pentafluorobenzoylchloride (PFBC). Both Negative CI and Positive CI modes of analysis were compared to the more widely accepted EI modes of mass spectrometric analysis.Transthyretin (TTR) which exists in various isoforms, is a valid marker for acute phase response and subclinical malnutrition. The aim of the study was to investigate the relationship between inflammation, oxidative stress and the occurrence of changes in microheterogeneity of TTR. A prospective, observational study at a level-I trauma center of a large urban medical university was performed. Patients were severely injured (n = 18; injury severity score (ISS): 34–66), and were observed within the first 24 hours of admittance and over the following days until day 20 after injury. 20 healthy subjects, matched by age and sex, were used as controls. TTR was enriched by immunoprecipitation. Microheterogeneity of TTR was determined by linear matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Four major mass signals were observed for TTR representing native, S-cysteinylated, S-cysteinglycinylated and S-glutathionylated TTR. In the course of their ICU stay, 14 of the 18 patients showed a transient change in microheterogeneity in favour of the S-cysteinglycinylated form of TTR (p < 0.05 vs. controls). The occurrence of this variant was not associated with the severity of trauma or the intensity of the acute-phase response, but was associated with oxidative stress as evidenced by Trolox. Our results demonstrate that changes in microheterogeneity of TTR occur in a substantial number of ICU trauma patients. The diagnostic values of these changes remains to be elucidated. It is speculated that TTR modification may well be the mechanism underlying the morphological manifestation of amyloidose or Alzheimer’s diseases in patients surviving multiple trauma.Background: Pesticides are of concern in Bolivia because of increasing use. Frequent intoxications have been demonstrated due to use of very toxic pesticides, insuffi cient control of distribution and sale and little knowledge among farmers of protective measures and hygienic procedures. Method: Questionnaires were applied and blood tests taken from 81 volunteers from La Paz County, of whom 48 were pesticide exposed farmers and 33 non-exposed controls. Sixty males and 21 females participated with a mean age of 37.3 years (range 17–76). Data of exposure and possible genetic damage were collected and evaluated by well known statistical methods, controlling for relevant confounders. To measure genetic damage chromosomal aberrations and the comet assay analysis were performed. Results: Pesticide exposed farmers had a higher degree of genetic damage compared to the control group. The number of chromosomal aberrations increased with the intensity of pesticide exposure. Females had a lower number of chromosomal aberrations than males, and people living at altitudes above 2500 metres seemed to exhibit more DNA damage measured by the comet assay. Conclusions: Bolivian farmers showed signs of genotoxic damage, probably related to exposure to pesticides. Due to the potentially negative long term health effects of genetic damage on reproduction and the development of cancer, preventive measures are recommended. Effective control with imports and sales, banning of the most toxic pesticides, education and information are possible measures, which could help preventing the negative effects of pesticides on human health and the environment.Primary and recurrent venous thromboembolic disease (VTE, deep venous thrombosis and pulmonary embolism) remain a significant source of morbidity and mortality in the hospitalized patient. Non-specific subjective complaints and lack of specific objective findings related to acute deep venous thrombosis (DVT) and pulmonary embolism (PE) complicate the diagnosis. There remains no single serum marker available to exclusively confirm the diagnosis of VTE. While D-dimer is highly sensitive and useful for diagnostic exclusion, it lacks the specificity necessary for diagnostic confirmation resulting in the need for a variety of additional studies (i.e.: duplex ultrasound, venography, V/Q scanning, helical thoracic and pelvic CT scans and pulmoary angiography). There is evolving research supporting the utility of various plasma markers as novel “biomarkers” for VTE including selectins, microparticles, interleukin-10 and other cytokines. This review attempts to examine recent literature assessing the utility of P-selectin, microparticles, D-dimer, E-selectin, thrombin, interleukins and fibrin monomers in the diagnosis and guidance of therapy for VTE.Bladder cancers are a mixture of heterogeneous cell populations, and numerous factors are likely to be involved in dictating their recurrence, progression and the patient’s survival. For any candidate prognostic marker to have considerable clinical relevance, it must add some predictive capacity beyond that offered by conventional clinical and pathologic parameters. Here, the current situation in bladder cancer research with respect to identification of suitable prognostic markers is reviewed. A number of individual molecular markers that might predict bladder cancer recurrence and progression have been identified but many are not sufficiently sensitive or specific for the whole spectrum of bladder cancer diseases seen in routine clinical practice. These limitations have led to interest in other molecular parameters that could enable more accurate prognosis for bladder cancer patients. Of particular interest is the epigenetic silencing of tumor suppressor genes. Since the methylation of these genes can correlate with a poor prognosis, the methylation profile may represent a new bio-marker that indicates the risk of transitional cell carcinoma development. In addition, bladder cancer research is likely to be revolutionized by high-throughput molecular technologies, which allow rapid and global gene expression analysis of thousands of tumor samples. Initial studies employing these technologies have considerably expanded our ability to classify bladder cancers with respect to their survivability. Future microarray analyses are likely to reveal particular gene expression signatures that predict the likelihood of bladder cancer progression and recurrence, as well as patient’s survival and responsiveness to different anti-cancer therapies, with great specificity and sensitivity.Protein Tyrosine Phosphatase gamma (PTPγ) is a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPγ is known to regulate haematopoietic differentiation in a murine embryonic stem cells model. We have recently demonstrated that PTPγ mRNA is expressed in monocytes, tissue-localized myeloid dendritic cells and in both myeloid and plasmacytoid dendritic cells in peripheral blood. We now developed a PTPγ specific antibody that recognizes the protein by flow cytometry. PTPγ expression was detected in monocytes and both myeloid and plasmacytoid dendritic cells, while PMN showed a low but consistent staining in contrast with previous mRNA data. B cells were found to express the phosphatase while T cells were negative. In keeping with RNA data, PTPγ was detected in monocyte-derived dendritic cells and its expression rose upon LPS stimulation. Finally, we discovered that CD34+ haematopoietic precursors express high PTPγ level that drops during in vitro expansion induced by IL-3 and SCF growth factors. We therefore propose PTPγ as a new functionally regulated leukocyte marker whose role in normal and pathological context deserve further investigation.Pancreatic cancer has a poor prognosis, in part due to lack of early detection. The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Proteins from a pancreatic adenocarcinoma cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for autoantibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis and 15 healthy subjects were analyzed. Autoantibodies were detected against a protein identified by mass spectrometry as vimentin, in sera from 16/36 patients with pancreatic cancer (44.4%). Only one of 18 chronic pancreatitis patients and none of the healthy controls exhibited reactivity against this vimentin isoform. Interestingly, none of several other isoforms of vimentin detectable in 2-D gels exhibited reactivity with patient sera. Vimentin protein expression levels were investigated by comparing the integrated intensity of spots visualized in 2-D PAGE gels of various cancers. Pancreatic tumor tissues showed greater than a 3-fold higher expression of total vimentin protein than did the lung, colon, and ovarian tumors that were analyzed. The specific antigenic isoform was found at 5–10 fold higher levels. The detection of autoantibodies to this specific isoform of vimentin may have utility for the early diagnosis of pancreatic cancer.High sensitivity serum C-reactive protein (hs-CRP) has come into clinical use as a marker of risk for cardiovascular disease (CVD). In addition to a role as a marker of disease, CRP has also been implicated in the pathogenesis of CVD. Specific small-molecule inhibitors of CRP have recently been developed with the intent of mitigating cardiac damage during acute myocardial infarction. However, the use of CRP, both as a risk marker and a disease target are controversial for several reasons. Serum hs-CRP concentrations can be elevated on the basis of genetics, female gender, and non-Caucasian ethnicity. It is not clear, in these contexts, that elevations of hs-CRP have any pathological significance. As a non-specific indicator of inflammation, CRP is also not a specific indicator of a single disease state such as cardiovascular disease but elevated concentrations can be seen in association with other comorbidities including obesity and pulmonary disease. In sharp contrast to the proposed inhibition of CRP for cardiovascular disease treatment, the infusion of CRP has been shown to have profound therapeutic benefits for autoimmune disease and septic shock. The balance between the risks and benefits of these competing views of the role of CRP in disease and disease therapy is reminiscent of the ongoing controversy regarding the use of non-steroidal anti-inflammatory drugs (NSAIDs) for musculoskeletal disease and their cardiovascular side effects. Soon, NSAIDs may not be the only agents about which Rheumatologists and Cardiologists may spar.Summary: The causes of many important diseases in animals are complex and multifactorial, which present unique challenges. Biomarkers indicate the presence or extent of a biological process, which is directly linked to the clinical manifestations and outcome of a particular disease. Identifying biomarkers or biomarker profiles will be an important step towards disease characterization and management of disease in animals. The emergence of post-genomic technologies has led to the development of strategies aimed at identifying specific and sensitive biomarkers from the thousands of molecules present in a tissue or biological fluid. This review will summarize the current developments in biomarker discovery and will focus on the role of transcriptomics, proteomics and metabolomics in biomarker discovery for animal health and disease.

Induction of the autoantigen proliferating cell nuclear antigen in T lymphocytes by a mycobacterial antigen

Megakaryocytes undergo an unusual cell cycle during differentiation that results in polyploidy through largely unknown mechanism(s). It has been shown that serine phosphorylation of oncoprotein 18 (Op18) is required for cell cycle progression specifically at the G2/M transition. Moreover, mutant forms of Op18 that are defective in one or more of the four serine residues induce G2/M arrest and subsequent polyploidization. Op18 phosphorylation is rapidly induced with phorbol myristate acetate (PMA) treatment in a wide range of human cells. In this study, we investigated the role of Op18 in PMA induced polyploidization during megakaryocyte differentiation of the human erythroleukemia cell line. Crucial to the molecular analysis of megakaryocyte differentiation, is the ability to fractionate cell populations with different ploidy levels. We have utilized cell elutriation as a fractionation strategy to analyze Op18 expression in synchronized cell subpopulations in different phases of the cell cycle or with progressive megakaryocyte polyploidization. In the absence of PMA, increased phosphorylation of Op18 was observed in HEL cells during cell cycle progression, as for other proliferating cells. However, in contrast to Jurkat leukemia cells chosen as control, HEL cells exhibited a lack of Op18 phosphorylation in response to PMA, which was accompanied by polyploidization and differentiation along the megakaryocytic lineage. To further determine the role of Op18 in polyploidization, HEL cells were transfected with different Op18 expression constructs. Differences in cell survival and polyploidization were observed between high and low Op18 expressors. An increased Op18 level reduced cell survival during the early stage of PMA induced megakaryocyte differentiation, but enhanced polyploidization efficiency. Our findings suggest that maintenance of a high level of unphosphorylated Op18 is required for efficient polyploidization during the differentiation program of megakaryocytes.

Isoelectric focusing nonporous RP HPLC: A two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry

Mycobacteria have been implicated in the pathogenesis of autoimmunity. To determine the potential effect of mycobacterial antigens on peripheral blood mononuclear cells (PBMC), we analyzed PBMC incubated with the acetone-precipitable fraction of Mycobacterium tuberculosis (APMT) for changes in cellular protein expression. Two-dimensional gel analysis showed induction of a 36-kD polypeptide identified as proliferating cell nuclear antigen (PCNA), a known autoantigen, after incubation with AP-MT. PCNA plays a role in cell proliferation and is expressed as a late growth regulated factor. However, its synthesis in response to AP-MT was induced as an early event. The early induction of PCNA was regulated at a posttranscriptional level and was restricted to T cells. Treatment of PBMC with known T cell mitogens, namely PHA, anti-CD3 antibodies, and staphylococcal superantigens failed to induce an early PCNA increase. The distinct characteristics of the AP-MT effect on PCNA expression suggest a separate mechanism of induction in response to AP-MT, compared with the late increase observed in response to mitogens. The induction of PCNA in response to mycobacterial antigens may represent a pathogenically relevant mechanism in autoimmunity.