Background: Microfibrillar-associated protein 4 (MFAP4) is a nnatricellular glycoprotein that colocalises with elastic fibres and is highly expressed in the lungs. The aim of this study was to test the hypothesis that plasma MFAP4 (pMFAP4) reflects clinical outcomes in chronic obstructive pulmonary disease (COPD).Methods: pMFAP4 was measured by an AlphaLISA immunoassay in stable COPD (n = 69) at baseline and at follow-up until 24 months after inclusion and in acute exacerbations of COPD (AECOPD) (n = 14) at baseline and until 6 months after inclusion.Results: The majority of patients (89%) were in GOLD II and III. Multiple linear regressions showed positive associations between pMFAP4 and the Global initiative for Obstructive Lung Disease (GOLD) grade (p = 0.01), modified Medical Research Council score (p < 0.0001) and BODE index (p = 0.04). Negative associations were found with 6-min walking distance (p = 0.04) and bronchodilator-induced reversibility (p = 0.02). The pMFAP4 levels varied less than 25% between the baseline and a 3 month follow-up in 83% of the patients. The pMFAP4 Levels appeared unaffected in the acute phase of severe AECOPD but rose to an increased stable level within one month after hospitalization.Conclusion: Increased pMFAP4 was associated to the severity in COPD and has the potential to serve as a stable disease biomarker. This observation warrants confirmation in a larger longitudinal COPD population. (C) 2014 Elsevier Ltd. All rights reserved.

Microfibrillar-associated protein 4 (MFAP4) is located in the extracellular matrix (ECM). We sought to identify tissues with high levels of MFAP4 mRNA and MFAP4 protein expression. Moreover, we aimed to evaluate the significance of MFAP4 as a marker of cardiovascular disease (CVD) and to correlate MFAP4 with other known ECM markers, such as fibulin-1, osteoprotegerin (OPG), and osteopontin (OPN). Quantitative real-time PCR demonstrated that MFAP4 mRNA was more highly expressed in the heart, lung, and intestine than in other elastic tissues. Immunohistochemical studies demonstrated high levels of MFAP4 protein mainly at sites rich in elastic fibers and within blood vessels in all tissues investigated. The AlphaLISA technique was used to determine serum MFAP4 levels in a clinical cohort of 172 patients consisting of 5 matched groups with varying degrees of CVD: 1: patients with ST elevation myocardial infarction (STEMI), 2: patients with non-STEMI, 3: patients destined for vascular surgery because of various atherosclerotic diseases (stable atherosclerotic disease), 4: apparently healthy individuals with documented coronary artery calcification (CAC-positive), and 5: apparently healthy individuals without signs of coronary artery calcification (CAC-negative). Serum MFAP4 levels were significantly lower in patients with stable atherosclerotic disease than CAC-negative individuals (p<0.05). Furthermore, lower serum MFAP4 levels were present in patients with stable atherosclerotic disease compared with STEMI and non-STEMI patients (p<0.05). In patients with stable atherosclerotic disease, positive correlations between MFAP4 and both fibulin-1 (ρ = 0.50; p = 0.0244) and OPG (ρ = 0.62; p = 0.0014) were found. Together, these results indicate that MFAP4 is mainly located in elastic fibers and is highly expressed in blood vessels. The present study suggests that serum MFAP4 varies in groups of patients with different cardiovascular conditions. Further studies are warranted to describe the role of serum MFAP4 as a biomarker of stable atherosclerotic disease.

Background: MFAP4 is a glycoprotein, co-localized with elastin and microfibrils in elastic fibres (Schlosser, Thomsen et al. (2006)). MFAP4 knock-out mice develop mild emphysema (not yet published data). We hypothesized that circulating MFAP4 reflects elastin degradation and can be used as a marker of emphysema severity. Methods: Plasma levels of MFAP4 (pMFAP4) were determined by Alphalisa® assay in 76 Danish COPD patients from the multicentre ECLIPSE study, (Vestbo, Anderson et al. 2008) 50 smokers and 54 non-smokers. Controls were healthy blooddonors. Associations between ln-transformed pMFAP4 levels and clinical outcomes were assessed by Pearson product moment correlation test and multiple regression analysis. Age, gender, packyears and carbonmonnoxide (as a indicator of current smoking) were included as covariates. Results: Levels of lnMFAP4 were significantly lower in smokers (mean:364ng/mL) than in non-smokers (471 ng/mL) and COPD patients (457 ng/mL). LnMFAP4 associated significantly with MMRC (Modified Medical Research Council score) in COPD patients (β: 0.09 p<0.05). There was a tendency of association between the composite BODE index (BMI, Obstruction index, Dyspnea score and Exercise capacity), it was borderline significant when adjusting for covariates (β:0.03 p:0.07). LnpMFAP4 did not associate significantly with FEV1 (β:-0.002 p=0.24) or obstruction index (β:-0.004 p=0.13). Conclusion: MFAP4 plasma levels are supressed by smoking, and are associated to dyspnea in COPD patients. References Schlosser, A., T. Thomsen, et al. (2006). "Microfibril-associated protein 4 binds to surfactant protein A (SP-A) and colocalizes with SP-A in the extracellular matrix of the lung." Scand J Immunol 64(2): 104-116. Vestbo J, W Anderson et al.(2008). "Evaluation of COPD longitudinally to identify predictive surrogate end-points (ECLIPSE)." Eur Respir J 31(4): 869-873

BackgroundMicrofibrillar-associated protein 4 (MFAP4) is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM), and variation in systemic MFAP4 (sMFAP4) has the potential to reflect diverse diseases with increased ECM turnover. Here, we aimed to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of sMFAP4 with an emphasis on the robustness of the assay. Moreover, we aimed to determine confounders influencing the basal sMFAP4 variability and the genetic contribution to the basal variation. MethodsThe sandwich ELISA was based on two monoclonal anti-MFAP4 antibodies and was optimized and calibrated with a standard of recombinant MFAP4. The importance of pre-analytical sample handling was evaluated regarding sample tube type, time, and temperature conditions. The mean value structure and variance structure was determined in a twin cohort including 1,417 Danish twins (age 18-67 years) by mixed-effect linear regression modeling. ResultsThe practical working range of the sandwich ELISA was estimated to be 4-75 U/ml. The maximum intra- and inter-assay variation was estimated to be 8.7% and 6.6%, respectively. Sample handling and processing appeared to influence MFAP4 measurements only marginally. The average concentration of sMFAP4 in the serum was 18.9 ± 8.4 (SD) U/ml in the twin cohort (95% CI: 18.5-19.4, median sMFAP4 17.3 U/ml). The mean structure model was demonstrated to include waist-hip ratio, age, and cigarette smoking status in interactions with gender. A relatively low heritability of h2 = 0.24 was found after applying a model including additive genetic factors and shared and non-shared environmental factors. ConclusionsThe described ELISA provides robust measures of the liver fibrosis marker sMFAP4. The low heritability and the relatively limited basal variation suggest that increased sMFAP4 reflects disease-induced processes.

The protein deleted in malignant brain tumors (DMBT1) and the trefoil factor (TFF) proteins have all been proposed to have roles in epithelial cell growth and cell differentiation and shown to be up regulated in inflammatory bowel diseases. A panel of monoclonal antibodies was raised against human DMBT1(gp340). Analysis of lung washings and colon tissue extracts by Western blotting in the unreduced state, two antibodies (Hyb213-1 and Hyb213-6) reacted with a double band of 290 kDa in lung lavage. Hyb213-6, in addition, reacted against a double band of 270 kDa in colon extract while Hyb213-1 showed no reaction. Hyb213-6 showed strong cytoplasmic staining in epithelial cells of both the small and large intestine whereas no staining was seen with Hyb213-1. The number of DMBT1(gp340) positive epithelial cells, stained with Hyb213-6, was significantly up regulated in inflammatory colon tissue sections from patients with ulcerative colitis (p<0.0001) and Crohn's disease (p = 0.006) compared to normal colon tissue. Immunohistochemical analysis of trefoil factor TFF1, 2 and 3 showed that TFF1 and 3 localized to goblet cells in both normal colon tissue and in tissue from patients with ulcerative colitis or Crohn's disease. No staining for TFF2 was seen in goblet cells in normal colon tissue whereas the majority of tissue sections in ulcerative colitis and Crohn's disease showed sparse and scattered TFF2 positive goblet cells. DMBT1 and TFF proteins did therefore not co-localize in the same cells but localized in adjacent cells in the colon. The interaction between DMBT1(gp340) and trefoil TFFs proteins was investigated using an ELISA assay. DMBT1(gp340) bound to solid-phase bound recombinant dimeric TFF3 in a calcium dependent manner (p<0.0001) but did not bind to recombinant forms of monomeric TFF3, TFF2 or glycosylated TFF2. This implies a role for DMBT1 and TFF3 together in inflammatory bowel disease.

Surfactant protein D (SP‐D) is part of the innate immune system involved in lung homeostasis. SP‐D knockout mice show accumulations of foamy alveolar macrophages, alveolar lipoproteinosis and pulmonary emphysema. Three single nucleotide polymorphisms (SNPs) have been described in the coding sequence of the human SP‐D gene SFTPD. Clinical studies showed that the SNP SFTPD with a nucleotide change from A to C resulting in a Met to Thr substitution at position 11 in the protein (Met(11)Thr), is relevant. This study set out to create a humanised mouse model of the Met(11)Thr SNP. Transgenic mice lines expressing either Met(11) or Thr(11) SP‐D under the control of the ubiquitously expressed pROSA26 promoter in C57Bl/6 SP‐D deficient mice (DKO) was created. Both Met(11) (142 ± 52 ng mL−1) and Thr(11) (228 ± 76 ng mL−1) mice lines expressed human SP‐D at almost similar levels. According to the literature this was within the range of SP‐D levels found in wildtype (WT) mice (253 ± 22 ng mL−1). Met(11) or Thr(11) SP‐D in serum from transgenic mice bound maltose in a calcium‐dependent manner, and binding was inhibited in the presence of EDTA or maltose. Bronchoalveolar lavage showed for both transgenic mice lines complementation of the DKO phenotype by restoring cell counts, phospholipid levels and protein content back to WT levels. Cytospins of BAL pellet cells showed a resemblance to WT but both mice lines showed some foamy alveolar macrophages. The stereological analysis showed for none of the mice lines a complete abrogation of emphysematous alterations. However, both Met(11) and Thr(11) mice lines were partially reverted back to a WT phenotype when compared with DKO mice, indicating important effects on surfactant metabolism in vivo.

Time 8:30 Glycobiology of Human Pluripotent Stem Cells; Steve Dalton, University of Georgia #1 9:00 Siglec-Sialoglycan Binding Regulates Cell-Cell Interactions; Ronald Schnaar, Johns Hopkins School of Medicine 9:30 Stem Cell Therapies in Epidermolysis Bullosa; Angela Christiano, Columbia University Medical Center 10:00 – 10:30 am Coffee Break (Grande Foyer and Grande Ballroom A) 10:30 am – 12:00 pm Plenary II: Biomaterials and Matrix Engineering (Grande Ballroom B & C) Chair: Adam Engler, University of California, San Diego 10:30 Hydrogels as Synthetic Extracellular Matrices; Kristi Anseth, University of Colorado at Boulder 11:00 Mechanical Regulation of Cell Adhesion and Function; Christopher Chen, University of Pennsylvania Joint Meeting of the Society for Glycobiology & American Society for Matrix Biology Conference Program by A rm en Peosyan on N ovem er 2, 2012 http://glycfordjournals.org/ D ow nladed from

We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm A. marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass of 27 and 31 kDa in the reduced state. The N-terminal amino acid sequences were identical in these two bands. In the unreduced state, a complex band pattern was observed with bands from 35 kDa to more than 200 kDa. Two different full-length clones encoding polypeptides of 241 and 243 amino acids, respectively, were isolated from a coelomocyte cDNA library. The two clones, designated AML-1a and AML-1b, were 92% identical at the protein level and represent a novel type of protein sequence family. Purified AML-1 induced agglutination of rabbit erythrocytes, which could be inhibited by N-acetylated saccharides. Recombinant AML-1b showed the same band pattern as the native protein, whereas recombinant AML-1a in the reduced state lacked a 27 kDa band. AML-1b bound GlcNAc-derivatized columns and chitin, whereas AML-1a did not bind to these matrices. Immunohistochemical analysis revealed that AML-1 is expressed by coelomocytes in the nephridium and in round cells in the epidermis and in eggs. Moreover, AML-1 expression was up-regulated in response to a parasitic infection. We conclude that AML-1 purified from coelomic fluid is encoded by AML-1b and represents a novel type of protein family that binds acetylated components.

CD163-L1 belongs to the group B scavenger receptor cysteine-rich family of proteins, where the CD163-L1 gene arose by duplication of the gene encoding the hemoglobin scavenger receptor CD163 in late evolution. The current data demonstrate that CD163-L1 is highly expressed and colocalizes with CD163 on large subsets of macrophages, but in contrast to CD163 the expression is low or absent in monocytes and in alveolar macrophages, glia, and Kupffer cells. The expression of CD163-L1 increases when cultured monocytes are M-CSF stimulated to macrophages, and the expression is further increased by the acute-phase mediator IL-6 and the anti-inflammatory mediator IL-10 but is suppressed by the proinflammatory mediators IL-4, IL-13, TNF-α, and LPS/IFN-γ. Furthermore, we show that CD163-L1 is an endocytic receptor, which internalizes independently of cross-linking through a clathrin-mediated pathway. Two cytoplasmic splice variants of CD163-L1 are differentially expressed and have different subcellular distribution patterns. Despite its many similarities to CD163, CD163-L1 does not possess measurable affinity for CD163 ligands such as the haptoglobin-hemoglobin complex or various bacteria. In conclusion, CD163-L1 exhibits similarity to CD163 in terms of structure and regulated expression in cultured monocytes but shows clear differences compared with the known CD163 ligand preferences and expression pattern in the pool of tissue macrophages. We postulate that CD163-L1 functions as a scavenger receptor for one or several ligands that might have a role in resolution of inflammation.

Surfactant protein D (SP-D) is an oligomeric lung-derived lectin with important roles in innate host defence. Previous studies have suggested serum SP-D as a reliable biomarker for inflammatory lung diseases. Our objective was to validate for the first time in the Lebanese population, SP-D as a clinical biomarker for asthma, COPD and chronic bronchitis. In addition, we studied the correlation between SP-D level and the disease severity. A case-control study is being conducted in the Lebanese population. To date, 46 cases of asthma, 24 cases of COPD, 23 cases of chronic bronchitis patients and 46 controls have been analysed. Lung function measurements were performed by spirometry, a standardized questionnaire was filled and serum levels of SP-D, CRP and plasma fibrinogen levels were measured. The mean level of SP-D was significantly elevated in asthmatics (1394.26 ng.ml-1; p = 0.023), in COPD patients (1507.24 ng.ml-1; p = 0.013) and in chronic bronchitis patients (1477.27 ng.ml-1; p = 0.007) when compared to controls (980.1 ng.ml-1). Serum SP-D levels correlated significantly with the disease severity only in COPD (p = 0.007). Conversely, levels of CRP and fibrinogen did not correlate with disease severity. We conclude that SP-D could be used as a biomarker for asthma, COPD and chronic bronchitis and seems to be more specific than general systemic markers of inflammation, CRP and fibrinogen. The ongoing study should provide further information on the cut off values and/or absolute values of serum Sp-D reflecting the degree of inflammation in the investigated diseases.

Surfactant protein D (SP-D) is an oligomeric calcium-dependent lectin with important roles in innate host defence against infectious microorganisms. Several studies have shown that patients with inflammatory lung disease have elevated levels of circulating SP-D, and serum SP-D has been suggested to be used as a biomarker for disease e.g. in COPD. We aimed to investigate the variation of circulating SP-D in healthy individuals in and between days for 6 months. In addition, we studied the SP-D response to a standardized physical exercise programme. SP-D was measured in serum using a 5-layered ELISA technique. We found that circulating SP-D remained constant over a 6-month period. However, during the course of one day SP-D varied significantly. Median SP-D peaked at 10 a.m. at 1009 ng/ml (95% CI: 803-1497), subsequently decreasing to nadir at 10 p.m. at 867 ng/ml (95% CI: 650-1148)(P<0.00005). Median pre-exercise level of SP-D was 746 ng/ml (95% CI: 384-2035), and immediately after cessation of physical activity the median SP-D level was 767 ng/ml (95% CI: 367-1885) (P=0.248). Our findings underscore the importance of standardized blood sampling conditions in future studies on the potential role of SP-D as a biomarker. Importantly, stable measures of systemic SP-D over a prolonged period support that SP-D is suitable for biomarker studies.

Bovine conglutinin is a serum protein involved in innate immunity. It binds calcium dependently to iC3b, a product of the complement component C3 deposited on cell surfaces, immune complexes or artificial surfaces after complement activation. We here present a simple and efficient two-step procedure for the purification of conglutinin. In the first step, bovine serum is incubated with non-coupled chromatographic TSK beads at 37°C to allow complement activation and iC3b deposition on the beads and subsequent binding of conglutinin to iC3b. Conglutinin is then eluted from the beads by EDTA. In the second step, conglutinin is separated from iC3b and IgM by ion-exchange chromatography. This purification procedure yielded 81 μg of conglutinin per ml of serum with a recovery of 61.2%. Surface plasmon resonance analysis showed that the purified conglutinin had a high affinity for mannan (K(d)=2.3-3.2 nM). SDS-PAGE and time-resolved immunofluorometric assays showed that the conglutinin was not contaminated with other serum collectins such as collectin-43 or mannan-binding lectin.

Meyer HE1, Molleken2 C, Sitek B1, Henkel C1, Poschmann G1, Sipos B3, Wiese S1, Warscheid B1, Broelsch C4, Reiser M2, Friedman SL5, Tornoe I6, Schlosser A6, Kloppel G3, Schmiegel W2,7, Holmskov U6, und Stuhler K1 1Medizinisches Proteom-Center, Ruhr-Universitat Bochum, Germany; 2Department of Internal Medicine, Bergmannsheil, Ruhr-Universitat Bochum, Germany; 3Department of Pathology, Christian Albrechts University, Kiel, Germany, 4Department of General Surgery and Transplantation, University Hospital, Essen, Germany; 5Division of Liver Diseases, Mount Sinai School of Medicine, New York, USA; 6Department of Medical Biology, University of Southern Denmark, Odense, Denmark; 7Department of Internal Medicine, Knappschaftskrankenhaus, Ruhr-Universitat Bochum, Germany