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Purpose: Magnetic separation of microbes can be an effective tool for pathogen identification and diagnostic applications to reduce the time needed for sample preparation. After peptide functionalization of superparamagnetic iron oxide nanoparticles (SPIONs) with an appropriate interface, they can be used for the separation of sepsis-associated yeasts like Candida albicans. Due to their magnetic properties, the magnetic extraction of the particles in the presence of an external magnetic field ensures the accumulation of the targeted yeast. Materials and Methods: In this study, we used SPIONs coated with 3-aminopropyltriethoxysilane (APTES) and functionalized with a peptide originating from GP340 (SPION-APTES-Pep). For the first time, we investigate whether this system is suitable for the separation and enrichment of Candida albicans, we investigated its physicochemical properties and by thermogravimetric analysis we determined the amount of peptide on the SPIONs. Further, the toxicological profile was evaluated by recording cell cycle and DNA degradation. The separation efficiency was investigated using Candida albicans in different experimental settings, and regrowth experiments were carried out to show the use of SPION-APTES-Pep as a sample preparation method for the identification of fungal infections. Conclusion: SPION-APTES-Pep can magnetically remove more than 80% of the microorganism and with a high selective host-pathogen distinction Candida albicans from water-based media and about 55% in blood after 8 minutes processing without compromising effects on the cell cycle of human blood cells. Moreover, the separated fungal cells could be regrown without any restrictions.

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Gram+ bacteria are very common in clinical medicine and responsible for a large number of infectious diseases. For example, Gram+ bacteria play a major role in causing bloodstream infections and sepsis. Therefore, the detection of Gram+ bacteria is of great importance for the diagnosis and treatment of infectious diseases. Furthermore, these bacteria are often present in biofilms that cover implants. Recent research work has mainly focused on the biologic activity and removal of Gram-negative bacteria or bacterial components such as lipopolysaccharides (LPS). In contrast, the effects of lipoteichoic acid (LTA) have been less well studied so the relevance of their removal from body fluids is possibly underestimated. To address this topic, we evaluated superparamagnetic iron oxide particles (SPION) carrying different peptides derived from the innate immune receptor (GP-340) for their ability to bind and remove Gram+ bacteria and LTA from different media. Our results show that, beyond S. aureus, effective agglutinating and removing of S. pneumoniae was possible. Furthermore, we were able to show for the first time that this was possible with LTA alone and that the magnetic removal of bacteria was also efficient under flow conditions. We also found that this method was able to capture Stapyhylococcus aureus from platelet concentrates, which can help to enhance the sensitivity of microbiological diagnostics, quality control measures, and blood product safety.

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Sepsis is a dysregulated host response of severe bloodstream infections, and given its frequency of occurrence and high mortality rate, therapeutic improvements are imperative. A reliable biomimetic strategy for the targeting and separation of bacterial pathogens in bloodstream infections involves the use of the broad-spectrum binding motif of human GP-340, a pattern-recognition receptor of the scavenger receptor cysteine rich (SRCR) superfamily that is expressed on epithelial surfaces but not found in blood. Here we show that these peptides, when conjugated to superparamagnetic iron oxide nanoparticles (SPIONs), can separate various bacterial endotoxins and intact microbes (E. coli, S. aureus, P. aeruginosa and S. marcescens) with high efficiency, especially at low and thus clinically relevant concentrations. This is accompanied by a subsequent strong depletion in cytokine release (TNF, IL-6, IL-1ß, Il-10 and IFN-γ), which could have a direct therapeutic impact since escalating immune responses complicates severe bloodstream infections and sepsis courses. SPIONs are coated with aminoalkylsilane and capture peptides are orthogonally ligated to this surface. The particles behave fully cyto- and hemocompatible and do not interfere with host structures. Thus, this approach additionally aims to dramatically reduce diagnostic times for patients with suspected bloodstream infections and accelerate targeted antibiotic therapy. STATEMENT OF SIGNIFICANCE: Sepsis is often associated with excessive release of cytokines. This aspect and slow diagnostic procedures are the major therapeutic obstacles. The use of magnetic particles conjugated with small peptides derived from the binding motif of a broad-spectrum mucosal pathogen recognition protein GP-340 provides a highly efficient scavenging platform. These peptides are not found in blood and therefore are not subject to inhibitory mechanisms like in other concepts (mannose binding lectine, aptamers, antibodies). In this work, data are shown on the broad bacterial binding spectrum, highly efficient toxin depletion, which directly reduces the release of cytokines. Host cells are not affected and antibiotics not adsorbed. The particle bound microbes can be recultured without restriction and thus be used directly for diagnostics.

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Streptococcus gordonii is a member of the viridans streptococci and is an early colonizer of the tooth surface. Adherence to the tooth surface is enabled by proteins present on the S. gordonii cell surface, among which SspB belongs to one of the most well studied cell-wall-anchored adhesin families: the antigen I/II (AgI/II) family. The C-terminal region of SspB consists of three tandemly connected individual domains that display the DEv-IgG fold. These C-terminal domains contain a conserved Ca2+-binding site and isopeptide bonds, and they adhere to glycoprotein 340 (Gp340; also known as salivary agglutinin, SAG). Here, the structural and functional characterization of the C123SspB domain at 2.7 Šresolution is reported. Although the individual C-terminal domains of Streptococcus mutans AgI/II and S. gordonii SspB show a high degree of both sequence and structural homology, superposition of these structures highlights substantial differences in their electrostatic surface plots, and this can be attributed to the relative orientation of the individual domains (C1, C2 and C3) with respect to each other and could reflect their specificity in binding to extracellular matrix molecules. Studies further confirmed that affinity for Gp340 or its scavenger receptor cysteine-rich (SRCR) domains requires two of the three domains of C123SspB, namely C12 or C23, which is different from AgI/II. Using protein-protein docking studies, models for this observed functional difference between C123SspB and C123AgI/II in their binding to SRCR1 are presented.

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BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

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The first months of life represent a crucial time period for an infant. Alongside establishing the early microbiome, the mucosal immunological homeostasis is being developed. Both processes may be perturbed in prematurely born infants. The glycoprotein SALSA plays a role in mucosal inflammation and microbial clearance. It is one of the most abundant molecules on the intestinal mucosal surfaces in early life. SALSA binds to many types of microbes and host defence molecules like IgA, C1q and collectin molecules. We here describe the development in faecal SALSA levels during the first three months of life. During these 90 days, the median SALSA level in full-term babies decreased from 1100 µg/mL (range 49-17 000 µg/mL) to 450 µg/mL (range 33-1000 µg/mL). Lower levels of SALSA were observed in prematurely born infants in the same time period. Our novel observation thus indicates an impact of prematurity on an important component of the infant intestinal immune system. Changes in SALSA in early life may have an effect on the early establishment of the human microbiome.

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Deleted in malignant brain tumors 1 (DMBT1) is part of the innate immune system and is expressed on mucosal surfaces in various tissues throughout the human body. However, to date, the localization of DMBT1 has not been investigated systematically and comprehensively in normal human tissues. In this study, we analyzed the mRNA expression of DMBT1 in human tissue by quantitative real-time PCR and examined its localization and distribution in the tissue by immunohistochemical staining using the monoclonal DMBT1 antibody HYB213-6. Anti-ovalbumin was used as an isotype control. The highest level of mRNA expression of DMBT1 was found in the small intestine, and the expression level was high throughout the luminal digestive tract. The expression of DMBT1 was especially high in the luminal digestive tract and salivary glands. The lowest expression level was found in the spleen. Immunohistochemical staining showed a high expression level of DMBT1 on mucosal surfaces throughout the body. There was a clear correlation between the mRNA expression and immunohistochemical expression of DMBT1 in the tissue. DMBT1 is strongly expressed on mucosal surfaces and in salivary glands.

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The Salivary Scavenger and Agglutinin (SALSA) protein is an innate immune protein with various alleged functions, including the regulation of inflammation and tissue remodeling. Transcriptomic studies of severe equine asthma (SEA) showed downregulation of the gene encoding SALSA in bronchial epithelium of asthmatic compared to non-asthmatic horses. This study aimed to characterize expression of SALSA in equine tissues by immunohistochemistry (IHC), corroborate potential differences in epithelial gene expression between asthmatic and non-asthmatic horses, and assess the structure of equine SALSA. An antibody against SALSA was validated through immunoprecipitation followed by mass spectrometry and Western blotting to recognize the equine protein. This antibody was applied to tissue microarrays (TMAs) containing 22 tissues each from four horses. A quantitative PCR assay was designed to compare gene expression for SALSA between six asthmatic and six non-asthmatic horses, before and after an asthmatic challenge, using cDNA from endoscopic bronchial biopsies as source material. The SALSA gene from bronchial cDNA samples of 10 horses, was amplified and sequenced, and translated to characterize the protein structure. Immunostaining for SALSA was detected in the mucosal surfaces of the trachea, bronchi, bronchioles, stomach, small intestine and bladder, in pancreatic and salivary gland ducts, and in uterine gland epithelium. Staining was strongest in the duodenum, and the intercalated ducts and Demilune cells of the salivary gland. SALSA was concentrated in the apical regions of the epithelial cell cytoplasm, suggestive of a secreted protein. Gene expression was significantly lower (p = 0.031) in asthmatic compared to non-asthmatic horses. Equine SALSA consisted of three to five scavenger receptor cysteine-rich (SRCR) domains, two CUB (C1r/C1s, uegf, bmp-1) domains and one Zona Pellucida domain. These domains mediate the binding of ligands involved in innate immunity. Varying numbers of SRCR domains were identified in different horses, indicating different isoforms. In summary, equine SALSA has a predilection for mucosal sites, has multiple isoforms, and has decreased expression in asthmatic horses, suggesting alterations in innate immunity in equine asthma.

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Glycoprotein 340 (Gp340) is an innate immune receptor with well-defined roles in mucosal tissues. It is a normal component of mucosal fluids such as tears, breast milk, and saliva, and it is expressed in tissues such as the vagina, gastrointestinal tract, oral cavity, lung alveoli, and pancreas. In the eye, it is expressed in the lacrimal gland, cornea, conjunctiva, and retina. Investigations of the protein in wet-surfaced epithelia of the body show that the effects of Gp340 can be beneficial or harmful depending on the conformation in which it exists. In a fluid phase, Gp340 appears to be protective against mucosal infection, while in a surface-associated form it appears to promote infection. On the ocular surface, it is dysregulated in dry eye disease and inhibits twitching motility of P. aeruginosa in tears. This review discusses what is known about Gp340 in wet-surfaced mucosal epithelia and highlights the potential roles of the protein in ocular surface immunity, inflammation, and infections.

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This study was aimed to evaluate the role of B-cell epitopes of Epstein-Barr virus (EBV) Early antigen protein D (EA), envelope glycoprotein GP340/membrane antigen (MA), latent membrane protein (LMP)-1, and LMP-2A in systemic lupus erythematosus (SLE). B-cell epitopes were predicted by analyzing secondary structure, transmembrane domains, surface properties, and homological comparison. 60 female mice were randomized equally into 12 groups: 1-10 groups were immunized by epitope peptides (EPs) 1-10, respectively, while 11 and 12 groups were PBS and Keyhole limpet hemocyanin (KLH) control groups. Immunoglobulin G (IgG) and autoantibody to nuclear antigen (ANA) concentrations in mice serum were determined at week 8. Indirect levels of EP1-10 were further detected by enzyme-linked immuno sorbent assay (ELISA) in 119 SLE patients and 64 age- and gender-matched health controls (HCs). 10 probable EBV EA, MA, LMP-1, and LMP-2A B-cell epitopes related to SLE self-antigens were predicted and corresponding EP1-10 were synthesized. IgG concentrations at week 8 were increased in EP1-10 and KLH groups compared with PBS group in mice; while ANA levels were elevated in only EP1-4, EP6-7, and EP10 groups compared to KLH group by ELISA, and ANA-positive rates were increased in only EP1, EP2, EP4, EP6, and EP10 groups by indirect immunofluorescence assay. EP1-4, EP6, and EP10 indirect levels were increased in SLE patients than HCs, while EP1, EP3, EP6, and EP9 were correlated with SLE disease activity index score. In conclusion, EBV EA, MA, LMP-1, and LMP-2A B-cell EPs increased SLE-related autoantibodies in mice, and their indirect levels might be served as potential biomarkers for SLE diagnosis and disease severity.

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Deleted in Malignant Brain Tumor 1 (DMBT1, alias SAG or gp340) is a pattern recognition receptor involved in immune defense, cell polarization, differentiation and regeneration. To investigate the role of the protein in physiological and pathological processes, the protein has often been isolated from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol. Our data suggest that our FPLC-SEC protocol yields DMBT1 in a more native conformation.

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Complement is present mainly in blood. However, following mechanical damage or inflammation, serous exudates enter the mucosal surfaces. Here, the complement proteins interact with other endogenous molecules to keep microbes from entering the parenteral tissues. One of the mucosal proteins known to interact with the early complement components of both the classical and the lectin pathway is the salivary scavenger and agglutinin (SALSA). SALSA is also known as deleted in malignant brain tumors 1 and gp340. It is found both attached to the epithelium and secreted into the surrounding fluids of most mucosal surfaces. SALSA has been shown to bind directly to C1q, mannose-binding lectin, and the ficolins. Through these interactions SALSA regulates activation of the complement system. In addition, SALSA interacts with surfactant proteins A and D, secretory IgA, and lactoferrin. Ulcerative colitis and Crohn's disease are examples of diseases, where complement activation in mucosal tissues may occur. This review describes the latest advances in our understanding of how the early complement components interact with the SALSA molecule. Furthermore, we discuss how these interactions may affect disease propagation on mucosal surfaces in immunological and inflammatory diseases.

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Group B Streptococcus (GBS) is a leading cause of neonatal sepsis, pneumonia and meningitis, and is responsible for a rising number of severe invasive infections in adults. For all disease manifestations, colonisation is a critical first step. GBS has frequently been isolated from the oropharynx of neonates and adults. However, little is understood about the mechanisms of GBS colonisation at this site. In this study it is shown that three GBS strains (COH1, NEM316, 515) have capacity to adhere to human salivary pellicle. Heterologous expression of GBS pilus island (PI) genes in Lactococcus lactis to form surface-expressed pili demonstrated that GBS PI-2a and PI-1 pili bound glycoprotein-340 (gp340), a component of salivary pellicle. By contrast, PI-2b pili did not interact with gp340. The variation was attributable to differences in capacities for backbone and ancillary protein subunits of each pilus to bind gp340. Furthermore, while GBS strains were aggregated by fluid-phase gp340, this mechanism was not mediated by pili, which displayed specificity for immobilised gp340. Thus pili may enable GBS to colonise the soft and hard tissues of the oropharynx, while evading an innate mucosal defence, with implications for risk of progression to severe diseases such as meningitis and sepsis.

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In the United States, composites accounted for nearly 70% of the 173.2 million composite and amalgam restorations placed in 2006 (Kingman et al., 2012), and it is likely that the use of composite will continue to increase as dentists phase out dental amalgam. This trend is not, however, without consequences. The failure rate of composite restorations is double that of amalgam (Ferracane, 2013). Composite restorations accumulate more biofilm, experience more secondary decay, and require more frequent replacement. In vivo biodegradation of the adhesive bond at the composite-tooth interface is a major contributor to the cascade of events leading to restoration failure. Binding by proteins, particularly gp340, from the salivary pellicle leads to biofilm attachment, which accelerates degradation of the interfacial bond and demineralization of the tooth by recruiting the pioneer bacterium Streptococcus mutans to the surface. Bacterial production of lactic acid lowers the pH of the oral microenvironment, erodes hydroxyapatite in enamel and dentin, and promotes hydrolysis of the adhesive. Secreted esterases further hydrolyze the adhesive polymer, exposing the soft underlying collagenous dentinal matrix and allowing further infiltration by the pathogenic biofilm. Manifold approaches are being pursued to increase the longevity of composite dental restorations based on the major contributing factors responsible for degradation. The key material and biological components and the interactions involved in the destructive processes, including recent advances in understanding the structural and molecular basis of biofilm recruitment, are described in this review. Innovative strategies to mitigate these pathogenic effects and slow deterioration are discussed.

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Glycoprotein 340 (gp340), an innate immunity molecule is secreted luminally by monolayered epithelia and associated glands within the human oral cavity. Gp340 contains 14 scavenger receptor cysteine rich (SRCR) domains, two CUB (C1r/C1s Uegf Bmp1) domains and one zona pellucida (ZP) domain. Oral streptococci are known to adhere to the tooth immobilized gp340 via its surface protein Antigen I/II (AgI/II), which is considered to be the critical first step in pathogenesis that eventually results in colonization and infection. In order to decipher the interactions between gp340's domains and oral streptococcal AgI/II domains, we undertook to express human gp340's first SRCR domain (SRCR1) and the first three tandem SRCR domains (SRCR123) in Drosophila S2 cells. While our initial attempts with human codons did not produce optimal results, codon-optimization for expression in Drosophila S2 cells and usage of inducible/secretory Drosophila expression system (DES) pMT/BiP/V5-HisA vector greatly enhanced the expression of the SRCR domains. Here we report the successful cloning, expression, and purification of the SRCR domains of gp340. Recognition of expressed SRCRs by the conformational dependent gp340 antibody indicate that these domains are appropriately folded and furthermore, surface plasmon resonance studies confirmed functional adherence of the SRCR domains to AgI/II.

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The salivary scavenger and agglutinin (SALSA), also known as gp340, salivary agglutinin and deleted in malignant brain tumor 1, is a 340-kDa glycoprotein expressed on mucosal surfaces and secreted into several body fluids. SALSA binds to a broad variety of microbes and endogenous ligands, such as complement factor C1q, surfactant proteins D and A, and IgA. Our search for novel ligands of SALSA by direct protein-interaction studies led to the identification of mannan-binding lectin (MBL) as a new binding partner. We observed that surface-associated SALSA activates complement via binding of MBL. On the other hand, soluble SALSA was found to inhibit Candida albicans-induced complement activation. Thus, SALSA has a dual complement activation modifying function. It activates the lectin pathway when bound to a surface and inhibits it when free in the fluid phase. These activities are mediated via a direct interaction with MBL. This suggests that SALSA could target the innate immune responses to certain microorganisms and simultaneously limit complement activation in the fluid phase.