Fluid Protein Research Articles

PERFIL PROTEICO DO LÍQUIDO FOLICULAR COLETADO DE OVÁRIOS EM DIFERENTES FASES DO CICLO ESTRAL DE BOVINOS

The female gamete’s microenvironment is the follicular fluid. The proteins in the follicular fluid must interfere in the oocyte quality. The objective of this study was to verify the protein profile in the follicular fluid of different phases of bovine estrous cycle. The follicular fluid was taken from follicles of bovine ovaries obtained at slaughterhouse. The ovaries were classified into five different phases of estrous cycle, according to presence or absence of corpus luteum (CL) and the morphologic characteristics. Pools of follicular fluid were collected from follicles ranging from 2 to 7mm in diameter from ovaries in five different phases of estrous cycle (1=initial CL, hemorrhagic; 2= developing CL; 3= mature CL; 4=regressing CL and 5=absence CL). The protein profile was evaluated by poliacrilamide eletrophoresis and determined by percentages of samples from each phase. The polypeptides with molecular weigh of 35, 32, 30 and 18 KDa changed considerably in samples from different phases of estrous cycle. The polypeptidewith the same molecularweight ofIGFBP-2 is more observedin samples from follicular fluid in the end of estrous cycle. The most polypeptides were observed insmallfollicles from ovaries with regressing CL

309 PROTEOMIC ANALYSIS OF THE BLASTOCOEL FLUID AND REMAINING CELLS OF BOVINE BLASTOCYSTS

Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions such as Parkinson’s disease, diabetes, and spinal cord injury. One of today’s challenges in regenerative medicine is to define proper culture conditions for hESC. The natural milieu in the blastocyst may provide clues on how to improve culture conditions, and the aim of the present study was to determine the proteome of the blastocoel fluid and the remaining cells of bovine blastocysts. Bovine blastocysts were produced by in vitro fertilization of oocytes retrieved from slaughterhouse ovaries. The blastocoel from 195 blastocysts (1–8 nL per blastocyst) were isolated by micromanipulation and analysed by nano-HPLC tandem mass spectrometry along with the remaining cells of the blastocyst. Searching the mass spectrometry data against a combined bovine database (SwissProt/TrEMBL), we identified 263 proteins in the blastocoel fluid and 1606 proteins in the cellular compartment of the blastocyst. A Venn diagram showed 124 proteins in overlap between the two compartments of the blastocyst. Several heat shock proteins and specific antioxidants were identified in both the blastocoel and cell material. A selection of proteins identified in the blastocoel fluid is to be tested on hESC in cell culture experiments, with proliferation of undifferentiated cells as the primary endpoint. The results from this study provide new knowledge about early mammalian preimplantation development, and the data can be used in the continued pursue of improving culture conditions for hESC, which further facilitates the clinical application of these cells.

95 ALTERED PROTEIN AND AMINO-ACID METABOLISM IN PREIMPLANTATION EMBRYOS FROM DIABETIC RABBITS

During pregnancy the preimplantation period is a critical ontogenetic stage in embryo development. As the embryo is highly sensitive to its surrounding milieu and vulnerable to dysregulations by external stimuli, we investigated the influence of a maternal diabetes mellitus type 1 on blastocyst metabolism and protein modifications. Here we report on protein and amino acid metabolism in preimplantation embryos. A diabetes mellitus type 1 rabbit model was used to measure the protein and amino acid concentrations in blastocyst cavity fluid at Day 6 postcoitum. The protein concentration was enhanced in embryos from diabetic rabbits. The level of the various proteinogenic amino acids was unchanged except for the branched-chain amino acids (BCAAs; l-leucine, l-isoleucine, l-valine). The concentration of l-leucine [153.9 µM ± 60.14] and l-valine [231.95 µM ± 75.67] increased 2-fold and of l-isoleucine [99.85 µM ± 42.92] increased 3-fold. Due to the altered BCAA levels, we assumed a disturbed biodegradation. The expression of the BCAA oxidising enzymes (Bcat2, Bckdha, Dbt, and Dld) and BCAA transporters was determined by real-time PCR. Embryos grown in diabetic rabbits revealed a decreased expression of BCAA oxidizing enzymes and of the BCAA transporter LAT-2. The BCAA transporter LAT-2 was mainly localized in the embryoblast. The quality of proteins in blastocysts from diabetic rabbits was evaluated by analyzing protein glycation (advanced glycation end products, AGEs). Two specific AGE modifications, namely arg-pyrimidine and pentosidine, were detected in Day-6 blastocysts by Western Blot analysis. In blastocyst cavity fluid the AGE-specific fluorescence at 440ex/535em nm was significantly enhanced. Our findings show that not just the quantity of proteins but also the quality is affected in rabbit embryos grown in diabetic mothers. There is evidence to suggest that vital protein interactions and signalling pathways are misprogrammed with likely negative consequences for further life. Supported by EU FP 7 EpiHealth (N°278418).

A Monoclonal Antibody Against Bovine Adiponectin

Adiponectin (AdipoQ) is an adipokine mainly secreted by white fatty tissue, playing a major role in energy homeostasis and insulin sensitivity. For cattle, AdipoQ data are largely limited to mRNA expression; to our knowledge, valid information about the AdipoQ protein in bovine tissues and body fluids is not available. Therefore, we have developed a monoclonal antibody against bovine AdipoQ. This study describes the preparation, application, and characterization of a monoclonal antibody for use in ELISA, Western blot, and histology. The antibody was developed by PEG fusion of the SP2/0 cell line with splenic B cells from AdipoQ immunized C57Bl/6 mice. Antibody-producing cells were identified by ELISA and specified by immunoblotting and immunostaining of bovine retroperitoneal adipose tissue. The novel antibody detects AdipoQ in histological samples, ELISA, and Western blots.

Evaluation of the pulmonary effects of short-term nose-only cigarette smoke exposure in mice

Much is known about the chronic effects of cigarette smoke (CS) on lung function and inflammation and development of chronic obstructive pulmonary disease. However, the underlying pathophysiological mechanisms related to the short-term exposure to CS are not fully understood. Here, we assessed the effect of CS generated by nine consecutive cigarettes per day for four days in a nose-only exposure system on airway resistance measured using forced oscillation technique, lung inflammation and oxidative stress in BALB/c mice. Control mice were exposed to air. Mice exposed to CS showed a significant increase of neutrophils and lymphocytes numbers in bronchoalveolar lavage (BAL). The total protein and endothelin levels in BAL fluid were significantly augmented suggesting an increase of alveolar-capillary barrier permeability. Similarly, airway resistance was significantly increased in the CS group compared with controls. Furthermore, reactive oxygen species and lipid peroxidation levels in lung tissue were significantly increased. The antioxidant activities of reduced glutathione, glutathione S transferase and superoxide dismutase were all significantly increased following CS exposure, indicating that CS could trigger adaptive responses that counterbalance the potentially damaging activity of oxygen radicals induced by CS exposure. In conclusion, our data indicate that short-term nose-only exposure to CS causes lung inflammation and increase of airway resistance mediated at least partly through the oxidative stress.

Neuroproteomics: an insight into ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown aetiology. Diagnosis is made through physical examination, electrophysiological findings, and by excluding other conditions. There is not a single biomarker that concludes the diagnosis. The aim of this study was to investigate differentially expressed proteins in cerebrospinal fluid (CSF) of ALS patients compared to control subjects, with the purpose to identify a panel of possible biomarkers for the disease. The differentially expressed spots/proteins were submitted to two-dimensional (2D) electrophoresis and recognized with matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry. Parkin-like and many iron and zinc binding were some of the proteins found in ALS CSF. Parkin is a ligase involved in ubiquitin–proteasome pathway and mutations in the parkin gene are the most common cause of recessive familial Parkinson’s disease. Iron and zinc are involved with many important metabolic processes and are related to neurodegenerative disease. Common features of ALS comprise failure of the ubiquitin–proteasome system and increased levels of metal ions in the brain. Therefore, the identification of these proteins can be a significant step in ALS research. These and other identified proteins are discussed in this study.

Abstract IA01: New assays, old specimens: Multiplexing for plasma and urine.

Abstract A key concept in medical science is that by comparing DNA, RNA, or proteins from healthy and diseased individuals, insight can be gained into the etiology of disease leading to better diagnostics and therapeutics. In this context, biorepositories have long played a key role by allowing the accumulation of biological fluids and tissues from large numbers of individuals for longitudinal or cross-sectional comparisons. Approaches to understanding human disease are based on the central dogma that information flow in living systems is from DNA to RNA to protein. At all levels, aberrations could indicate either the presence of disease or suggest underlying mechanisms of disease. However, historically DNA and RNA have been more useful for identifying risk of disease than the presence of disease, except in surgical specimens. Proteins in biological fluids [blood, urine, aspirates, etc.] have played a more prominent role in diagnostics. Indeed given that proteins are in essence the effectors of gene expression a long held goal has been to associate specific proteins in peripheral circulation that indicate the presence of disease, e.g. biomarkers of disease, especially early disease before the appearance of symptoms or other clinical indicators. This presentation will focus on issues associated with the use of biorepository fluids in this context. Three issues are fundamental: 1) specimen volume is limited and precious, 2) specimen processing requirements are not known a'priori, and 3) analytical targets are often not precisely defined. Limited specimen volume is an inherent problem because each blood draw represents a discrete end point. Unlike nucleic acids, proteins are subject to numerous post-translational biological modifications and to extensive, often rapid, degradation post specimen collection. These issues drive method development and validation, ultimately determining the amount and quality of data that can be obtained from biorepository specimens. Protein analytical methods will be reviewed from this perspective with emphasis on recent developments in multi-analyte systems and newer, highly sensitive methods. Citation Format: Patrick M. Sluss. New assays, old specimens: Multiplexing for plasma and urine. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr IA01.

296 GPCR STIMULATED HYPOTHALAMIC PVN GαI2 PROTEIN-GATED PATHWAYS – THE KEY TO RENAL SYMPATHETIC NERVE MEDIATED SODIUM HOMEOSTASIS AND THE LONG-TERM CONTROL OF BLOOD PRESSURE

Aim: We have demonstrated CNS Gαi2 proteins, which are highly expressed in the hypothalamic PVN, mediate the renal sympathoinhibition and natriuresis evoked by α2-adrenoceptor activation and volume expansion in-vivo. Therefore, we examined the role(s) of PVN Gαi2 proteins in fluid & electrolyte homeostasis and MAP regulation following elevated dietary salt-intake. Methods: Intact or bilateral renal denervated (RDNX) Sprague-Dawley rats received a bilateral PVN or posterior hypothalamic (PH) infusion of a scrambled (SCR) or Gαi2 oligodeoxynucleotide (ODN-300ng/side/day) and a normal 0.4% (NS) or high 8% NaCl (HS) diet for 7-days. On day-7 MAP, 24 h metabolic balance, plasma norepinephrine, (NE) and PVN Gαi2 protein levels were determined (N = 5/group). Results: HS-intake evoked a significant 3-fold site-specific increase in PVN Gαi2 proteins. ODN-mediated PVN Gαi2 down-regulation, but not PH miss-injection or PVN SCR ODN pre-treatment, caused renal nerve-dependent sodium retention (24 h Na+ balance [meq] PH Gαi2 + HS 0.5 ± 0.2, PVN Gαi2 + HS 2.3 ± 0.4*, PVN Gαi2 RDNX + HS 0.8 ± 0.3τ), global sympathoexcitation (plasma norepinephrine [nmol/L] PH Gαi2 + HS 17 ± 4, PVN Gαi2 + HS 75 ± 9*, PVN Gαi2 RDNX + HS 24 ± 6τ) and hypertension (MAP [mmHg] PH Gαi2 + HS 128 ± 3, PVN Gαi2 + HS 140 ± 2*, PVN Gαi2 RDNX + HS 131 ± 3τ) *p<0.05 vs. PH Gαi2 + HS, τp < 0.05 vs. PVN Gαi2 + HS. Conclusion: Renal nerve dependent dysregulation of sympathetically driven sodium retaining mechanisms occurred following PVN Gαi2-protein down-regulation evoking salt-sensitive hypertension. We conclude PVN Gαi2 protein-gated pathways regulate renal nerve-dependent sodium excretion to facilitate sodium homeostasis and maintenance of a salt-resistant phenotype – R01HL107330.

P1‐027: Astrocyte‐derived proteins in the cerebrospinal fluid as biomarkers for the pathobiological staging of Alzheimer's disease

P1‐027: Astrocyte‐derived proteins in the cerebrospinal fluid as biomarkers for the pathobiological staging of Alzheimer's disease

P2‐027: Development of a gold nanoparticle ELISA to detect low‐abundant proteins in cerebrospinal fluid for diagnosis of Alzheimer's disease

P2‐027: Development of a gold nanoparticle ELISA to detect low‐abundant proteins in cerebrospinal fluid for diagnosis of Alzheimer's disease

Evidence for an involvement of regulatory T-lymphocytes (Treg cells) in tolerance of pregnancy in mares

treatment-associated endometrial changes were seen at Day 19 than at Day 20. Cloprostenol treatment in pregnant mares markedly increased (10-100x) expression of many endometrial genes at Day 20. Of these, several genes involved in signaling (IL6, GABRP, IGFBP-1, IL1RN, CHGA, PENK), lipid metabolism (SPLA2, SCGB1A1, SAA1, EquC1), extracellular matrix modification (MMP3, HYAL4) and lipid transport (SAA1) were most increased in treated pregnant mares compared with control pregnant mares. The changes in expression of secretory phospholipase A2 (SPLA2), uteroglobin (SCGB1A1), and a lipocalin (EquC1) were also higher in non-pregnant mares than in control pregnant mares so these changes could partly reflect transition to the non-pregnant state after luteolysis. Endometrial expression of ST6GALNAC1, PLA2G6, PLA2G4, NCAM1 and GZMB were most reduced in pregnant mares in response to cloprostenol. By comparison, gene expression in Day 19 or 20 trophoblasts from control or treated mares were similar with only 14 significant differences in the 2-5 fold range observed. These included increases in serpins B2 and B10 after cloprostenol. Major alterations in proteins in uterine flush fluids and yolk-sac fluids were assessed in a separate series in which cloprostenol was administered 3 or 4 days before collection on Days 15-21. Identified proteins were ranked in relative amounts from trypsin digested peptides identified by LC/MS/MS and Scaffold 2 software performed at the Advanced Protein Technology Centre, Toronto. Proteins corresponding to SERPINA14, ANXA1, MUC6, SPLA2, and B2Mweremost increased in uterine flush fluids after cloprostenol treatment, with SERPINA14 and SPLA2 also being increased in yolk-sac fluid. By comparison, VNN2, VNN2, GZMB, CD109 and A2Mwere consistently reduced in uterine flush fluid from treated mares. These studies reveal several alterations in the endometrium and conceptus that might beharmful effectors, or biomarkers of adverse uterine conditions, useful for clarifying the mechanisms of pregnancy establishment and pregnancy failure in mares.

Life's Simple Measures: Unlocking the Proteome

Using modified nucleotides and selecting for slow off-rates in the SELEX procedure, we have evolved a special class of aptamers, called SOMAmers (slow off-rate modified aptamers), which bind tightly and specifically to proteins in body fluids. We use these in a novel assay that yields 1:1 complexes of the SOMAmers with their cognate proteins in body fluids. Measuring the SOMAmer concentrations of the resultant complexes reflects the concentration of the proteins in the fluids. This is simply done by hybridization to complementary sequences on solid supports, but it can also be done by any other DNA quantification technology (including NexGen sequencing). We use measurements of over 1000 proteins in under 100 μL of serum or plasma to answer important medical questions, two of which are reviewed here. A number of bioinformatics methods have guided our discoveries, including principal component analysis. We use various methods to evaluate sample handling procedures in our clinical samples and can identify many parameters that corrupt proteomics analysis.

A fatal course of neonatal meningo-encephalitis

Case presentationA pregnant woman (36 weeks gestation) underwent cesareansection and laparotomy because of fever and peritoneal signs. Anormal appendix and no signs of intra-abdominal infection wereseen. A girl was born in good condition.On the fourth day of life, the neonate became lethargic withcyanotic spells, requiring admission to the neonatal intensive careunit and antibiotic therapy was started. Cerebrospinal fluid (CSF)cell count, protein and glucose level and urine microscopy werenormal.Five days after birth, respiratory and circulatory failuredeveloped. CSF showed 26×10

PATIENT SATISFACTION AND DIETARY OUTCOMES FROM ATTENDING A MULTIDISCIPLINARY CYSTINURIA CLINIC

Cystinuria is a genetic disease that leads to frequent cystine stone formation. A reduced methionine (precursor to cystine) diet has been recommended for cystinuria patients, involving maintenance of healthy weight, limiting intake of animal protein and salt whilst increasing vegetable protein, fruit, vegetables and fluid intake. The ability of patients to adhere to these dietary regimes has been questioned. We evaluated the dietetic service and dietary changes within our multidisciplinary cystinuria clinic using food frequency questionnaires (ffqs), dietary change questionnaires (dcqs) and diet histories. Most patients were seen by the dietitian 6–12 monthly. 100% of patients felt they benefited from dietetic input. There was a move away from ffqs due to them being difficult and cumbersome to complete. Dcqs and diet histories revealed 23/28 increased fruit and vegetable intake, 20/28 decreased salt intake, 20/28 decreased animal protein intake, 14/28 increased vegetable protein intake and 9/28 increased fluid intake. There were a few discrepancies in reported change between dcqs and diet histories, supporting the use of multi-source feedback for diet analysis. Patients had varying degrees of success with making changes to each dietary parameter. Multi-source diet analysis allowed us to develop tailored consultations. All patients made some positive dietary changes which may help prevent stone formation. The areas of least change were fluid (due to changes made prior to clinic attendance) and vegetable protein. Our results support the need for continued dietetic input. These results should be re-audited to check that patients are maintaining the changes made.

Proteomics of the ascitic fluid for the differentiation of advanced ovarian cancer.

e15510 Background: Ovarian cancers are classified as primary, if it arises in the ovary and secondary or metastatic, if the origin is from other parts of the body. The clinical manifestations of these cancers in the advanced stage are very similar making it difficult to distinguish clinically, hiostopathologically and radiologicaly. The therapeutics and management of the primary and secondary malignancies are completely different. While, the advanced primary malignancies are treated by cytoreduction followed by chemotherapy, the metastatic tumors are treated mainly with palliative chemotherapy. The prognosis is better for primary than secondary ovarian cancer making their diagnosis very crucial for patient care. Methods: 1D and 2D-gel based proteomic approaches were used to study the differentially expressed proteins in the ascitic fluid of patients (10 primary and 4 secondary) with advanced ovarian cancer. The relative ratios of the protein expression were estimated by densitometric analysis. The bands/spots with more than three fold difference were subjected to in-gel trypsin digestion and identified by mass spectrometric analysis. The differential expression of one of the proteins was further validated by western blot experiments and ELISA. Results: Programmed Cell Death 1-Ligand 2 and apolipoprotein A1 were seen to be up regulated in the advanced primary ovarian cancer while apolipoprotein A4, and chain L, humanized version of the anti-human fas antibody Hfe7a were seen to be up regulated in the metastatic variant. Validation for the expression of apolipoprotein A1 shows that a 61.8ng/ml cut off is ideal to differentiate the primary and secondary advanced ovarian cancers. The assay has 100% sensitivity, 75% specificity, positive predictive value of 90.9%, negative predictive value of 100%, accuracy of 92.85% and a pre-test odds positive of 2.5. Conclusions: Proteomics of ascitic fluid is useful for the differentiation of advanced ovarian cancers. There are proteins which are differentially expressed in the ascitic fluid of patients with primary and secondary ovarian cancer. Apolipoprotein A1 is a potential biomarker that can be used to differentiate the closely mimicking clinical scenarios of advanced ovarian cancer.

Pseudotumor Cerebri in a Turkish Boy With Fanconi Anemia

Pseudotumor cerebri (PC) is a clinical syndrome characterized by increased intracranial pressure with a normal cerebrospinal fluid cell count and protein level in the absence of a space-occupying lesion or apparent obstruction to the cerebrospinal fluid pathway. Although PC is described in patients with various hematological diseases including iron-deficiency anemia, megaloblastic anemia, acquired aplastic anemia, hemolytic anemia, sickle cell disease, and paroxysmal nocturnal hemoglobinuria, there is no case of PC with Fanconi anemia in the English literature. Here, we report a first case of PC in an 11-year-old boy with a diagnosis of Fanconi anemia.

Altered Prion Protein Expression Pattern in CSF as a Biomarker for Creutzfeldt-Jakob Disease

Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are not fully understood, however neuronal loss followed by cell lysis is assumed to cause the increase in 14-3-3 levels, which also occurs in conditions such as brain ischemia. Here we investigated the relation between the levels of 14-3-3 protein, Lactate dehydrogenase (LDH) activity and expression of the prion protein (PrP) in CSF of sporadic and familial CJD cases. Unexpectedly, we found normal levels of LDH activity in CJD cases with moderate levels of 14-3-3 protein. Increased LDH activity was only observed in a percentage of the CSF samples that also exhibited high 14-3-3 levels. Analysis of the PrP expression pattern in CSF revealed a reduction in PrP levels in all CJD cases, as well as marked changes in its glycosylation pattern. PrP present in CSF of CJD cases was sensitive to proteases. The alterations in PrP expression observed in CJD cases were not detected in other pathologies affecting the nervous system, including cases of dementia and tropical spastic paraparesis/HTLV-1 associated myelopathy (HAM/TSP). Time course analysis in several CJD patients revealed that 14-3-3 levels in CSF are dynamic and show a high degree of variability during the end stage of the disease. Post-mortem analysis of brain tissue also indicated that 14-3-3 protein is upregulated in neuronal cells, suggesting that its expression is modulated during the course of the disease. These results suggest that a combined analysis of 14-3-3 and PrP expression pattern in CSF is a reliable biomarker to confirm the clinical diagnosis of CJD patients and follow disease progression.

Evaluation of high sensitivity C-reactive protein assay in cerebrospinal fluid on the Dimension RxL analyzer

Introduction: Low sensitivity and specificity in traditional laboratory tests became insufficient for accurate diagnostics and initiation of proper treatment of patients infected with bacterial meningitis. High sensitivity C reactive protein (hsCRP) may be an appropriate supplement for rapid diagnosis of bacterial meningitis. The subject of our investigation was the determination of C- reactive protein in cerebrospinal fluid (CSF) duringacute bacterial meningitis.Methods: HsCRP was analysed by a sensitive immunoturbidimetric assay using the Dimension RxL analyser (Siemens). Cerebrospinal fluid concentrations of C-reactive protein have been measured in 20 patients(age range,1 to 50 years) presenting with acute bacterial meningitis and also in a non-infected, non-inflamed control group (n=25).Results: The accuracy and precision of the method proved to be satisfactory. Repeatability of serial sampling for hsCRP described by coefficient of variation were CV=2.1-4.5%. This assay hsCRP in cerebrospinal fluid demonstrates adequate performance characteristics for routine clinical use. Elevated levels of CRP were found in 95% patients with bacterial meningitis. The mean CRP value in 25 uninfected control group was 0.25 mg/L (range 0.10-0.55). The mean CRP for patients with bacterial meningitis was 21.4 mg/L (range 0.40-100).Conclusions: A sensitive assay for CRP in CSF would be an useful adjunct to conventional investigation of acute infective meningitis.

Abstract 4555: Protein and glycan biomarkers of malignant potential in pancreatic cyst fluid

Abstract The early detection of a portion of pancreatic cancer precursors_cystic neoplasms of the pancreas_is possible through high-resolution abdominal imaging, providing an opportunity to successfully treat these lesions before they become invasive. The increased use of abdominal imaging has led to a higher rate of identifying pancreatic cysts, but currently it is not possible to accurately determine which cysts have high malignant potential and should be removed. Our previous work showed that the glycosylation and abundance of specific proteins in the cyst fluid was significantly different between potentially malignant cysts and benign cysts, providing leads for biomarkers to guide patient management. In this work we tested the hypothesis that a panel of specific protein glycoforms forms an accurate biomarker for pancreatic cysts with high malignant potential. Using antibody-lectin sandwich arrays, screens of nearly 100 different candidate markers and multiple glycoforms of each marker confirmed previous results and also showed that two distinct glycan structures are present on several proteins at high levels in the cyst fluid of cancer precursors (intraductal pancreatic mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN)) but not in benign cysts (serous cystadenomas (SC) and pseudocysts (PC)). The glycans, N-acetyl glucosamine and the blood group H structure, previously have not been recognized as cancer biomarkers. A six-marker panel comprising the measurement of one of these glycans on MUC5AC, MUC16, endorepellin, and fibronectin correctly detected 37 of 39 (95%) potential cancer precursors and 16 of 18 (89%) of the benign cysts. This performance significantly surpasses current diagnostic accuracy. The validation of this biomarker panel could lead to more accurate treatment decisions for patients with potentially malignant cysts while sparing patients with benign cysts from unnecessary procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4555. doi:1538-7445.AM2012-4555

Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.