Search

To comprehensively assess recent data on the effects of orthodontic forces on the dental pulp and to critically evaluate, whether any of the changes are permanent.Articles published between 2/2009 and 2/2022 were searched electronically on the PubMed, EMBASE and SCOPUS databases. The initial search retrieved 780 publications and, applying the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, 33 relevant articles were identified. Twenty articles fulfilled the requirements for high (Orthodontic forces appeared to cause a reduction in pulpal blood flow and a reduction in tooth sensibility, as indicated by increased response thresholds and increased amounts of negative responses to tooth sensibility tests. In addition, there were increases in the expression or activity levels of enzymes and neuropeptides associated with hypoxia and inflammation. Fibrotic tissue formation in the pulp was also reported.Except for some histological and morphological alterations, the observed pulpal changes were in most cases only temporary, appearing within days of initiating the treatment and usually lasting for weeks. There were no clear signs of permanent damage.

The aim of this study was to evaluate oral bacteria- and interleukin (IL)-1β-induced protein and mRNA expression profiles of monocyte chemoattractant protein-1-induced protein (MCPIP)-1 and mucosa-associated lymphoid tissue lymphoma translocation protein (MALT)-1 in human gingival keratinocyte monolayers and organotypic oral mucosal models.Human gingival keratinocyte (HMK) monolayers were incubated with Porphyromonas gingivalis, Fusobacterium nucleatum, P. gingivalis lipopolysaccharide (LPS) and IL-1β. The protein levels of MCPIP-1 and MALT-1 were examined by immunoblots and mRNA levels by qPCR. MCPIP-1 and MALT-1 protein expression levels were also analyzed immunohistochemically using an organotypic oral mucosal model. One-way analysis of variance followed by Tukey correction was used in statistical analyses.In keratinocyte monolayers, MCPIP-1 protein expression was suppressed by F. nucleatum and MALT-1 protein expression was suppressed by F. nucleatum, P. gingivalis LPS and IL-1β. P. gingivalis seemed to degrade MCPIP-1 and MALT-1 at all tested time points and degradation was inhibited when P. gingivalis was heat-killed. MCPIP-1 mRNA levels were increased by P. gingivalis, F. nucleatum, and IL-1β, however, no changes were observed in MALT-1 mRNA levels.Gingival keratinocyte MCPIP-1 and MALT-1 mRNA and protein expression responses are regulated by infection and inflammatory mediators. These findings suggest that periodontitis-associated bacteria-induced modifications in MCPIP-1 and MALT-1 responses can be a part of periodontal disease pathogenesis.

ObjectiveTo monitor salivary B-cell activating factor (BAFF), tumor necrosis factor-like weak inducer of apoptosis (TWEAK), and soluble (s)CD163 levels and arginase activity in periodontitis patients following nonsurgical periodontal treatment. BackgroundBAFF, TWEAK, and sCD163 and arginase are associated with activities of B cells and macrophages, which are important regulators of periodontal immune-inflammatory response and healing following treatment. Increased salivary BAFF and sCD163 levels and arginase activity in periodontitis have been demonstrated, but their changes following treatment have not been evaluated before. Materials and MethodsForty-four Stage III/IV periodontitis patients and 35 periodontally healthy controls were included in the study. Full-mouth periodontal measurements were recorded and unstimulated saliva was obtained from all participants at baseline. Sample collection and measurements were repeated in periodontitis patients at 2, 6, 12, and 24 weeks following full-mouth scaling and root debridement, whereas controls were only seen at baseline. BAFF, TWEAK, and sCD163 levels were analyzed with bead-based multiplexed immunoassay. Arginase activity was measured with Chinard's method. ResultsBAFF (p < .001) and sCD163 (p = .003) levels and arginase activity (p < .015) were higher in periodontitis patients compared to healthy controls. BAFF levels (p < .001) and arginase activity (p < .001) of periodontitis patients were reduced at 2 weeks posttreatment and continued to decrease up to 6 (p = .038) and 12 weeks (p = .024), respectively. The reduction of sCD163 levels became significant (p = .003) at 24 weeks posttreatment. ConclusionsThe decrease in salivary BAFF levels 2 weeks after periodontal treatment indicates a change in cell signaling toward limited B-cell activation. Decreasing arginase activity similarly reflects a significant reduction in inflammatory response. The reduction in sCD163 levels that are observed at 24 weeks may reflect a longstanding anti-inflammatory macrophage activation, given their multiple functions in immune response, inflammation, and healing.

Streptococcus pyogenes, also called group A streptococcus (GAS), is a human pathogen causing a wide range of infections ranging from mild tonsillitis to severe, life threatening conditions such as bacteraemia, necrotizing fasciitis, and streptococcal toxic shock syndrome. GAS may also colonise the oropharynx without causing any signs of disease which is known as asymptomatic carriage. This study aims to investigate IgA responses against GAS and oral streptococci from saliva samples collected from healthy Finnish adults. In addition, asymptomatic throat GAS carriage was studied. The study participants consisted of healthy adult volunteers who provided one saliva sample, a throat swab, and a background questionnaire. Total salivary IgA, and GAS specific IgA were analysed from the saliva samples using enzyme-linked immunosorbent assays (ELISA) and the results were compared to oral streptococci specific IgA levels. Asymptomatic GAS throat carriers were identified by bacterial culture, and the isolates were emm typed. Samples from a total of 182 individuals were analysed. The median salivary IgA concentration was 62.9 µg/ml (range 17.3–649.9 µg/ml), and median GAS and oral streptococcal specific IgA concentrations 2.7 and 3.3 arbitrary units (AU, range 1.4–7.4 AU and 1.6–12.0 AU), respectively. Three individuals with asymptomatic GAS throat carriage were identified.

Elevated serum immunoglobulin (Ig) antibody levels are observed in Crohn’s disease patients. The aim of this study was to evaluate the salivary IgA and IgG antibody levels against Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia in Crohn’s disease patients. Eighty-eight participants (47 Crohn’s disease patients and 41 systemically healthy age- and gender-matched controls) were included in the study. Oral and medical health statuses were recorded and salivary samples were collected. Salivary P. gingivalis, T. forsythia, A. actinomycetemcomitans, and P. intermedia carriage were analyzed with DNA sequencing technique, salivary levels of IgG1, IgG2, IgG3, IgG4, and IgM were measured with the Luminex® xMAP™ technique, and salivary IgA and IgG antibody levels against P. gingivalis, T. forsythia, A. actinomycetemcomitans, and P. intermedia were detected by ELISA. As result, higher salivary IgG2 (p = 0.011) and IgG3 (p = 0.006), P. gingivalis IgA (p < 0.001), A. actinomycetemcomitans IgG (p = 0.001), and P. intermedia IgG (p < 0.001) antibody levels were detected in the Crohn’s disease group compared to the controls. Salivary P. gingivalis carriage was lower in the Crohn’s disease group in comparison to the controls (p = 0.024). In conclusion, salivary IgA antibody responses against P. gingivalis and IgG antibody responses against P. intermedia have independent associations with Crohn’s disease.

Background: Hinokitiol (β-thujaplicin), isolated from the wood of Chamaecyparis taiwanensis, has a wide variety of biological properties including anti-inflammatory, anti-microbial, and anti-tumor effects. Therefore, hinokitiol has become a frequent additive in oral and other healthcare products. Objectives: Our goal was to determine the anti-tumor activity of hinokitiol on human papillomavirus (HPV) positive (n = 3) and negative (n = 2) cell lines derived from cervical or head and neck squamous cell carcinoma (HNSCC) and keratinocyte cell lines (n = 3) transformed spontaneously or with HPV16E6 and E7 oncogenes. Methods: The cell-lines were exposed to hinokitiol at different concentrations (0–200 µM) for 24 h. Cell metabolism, proliferation, and the cell cycle distribution were assessed by MTT- and 3H-thymidine incorporation and flow cytometry. Expressions of p21 and on HPV16E6 and E7 oncogenes were assessed by qPCR. Results: In all carcinoma cell lines, hinokitiol treatment declined the metabolic activity irrespective of the HPV status. This decline was statistically significant, however, only in HPV-positive cell lines CaSki and UD-SCC-2 when exposed to hinokitiol concentrations at 100 and 200 µM, respectively (p < 0.05). Immortalized cell lines, HMK and HPV-positive IHGK, were more sensitive as a similar metabolic effect was achieved at lower hinokitiol concentrations of 3.1, 6.25, and 50 µM, respectively. Hinokitiol blocked DNA synthesis of all carcinoma cell lines without evident association with HPV status. G1 cell cycle arrest and p21 upregulation was found in all cell lines after hinokitiol treatment at higher concentration. However, when the p21 results of all HPV-positive cells were pooled together, the increase in p21 expression was statistically significantly higher in HPV-positive than in HPV-negative cell lines (p = 0.03), but only at the highest hinokitiol concentration (200 µM). In HPV-positive cell lines hinokitiol declined the expression of HPV16E7 and E6 along the increase of p21 expression. The dose-dependent inverse correlation between p21 and E7 was statistically significant in SiHa cells (r = −0.975, p-value = 0.03) and borderline in UD-SCC-2 cells (r = −0.944, p-value = 0.06), in which p21 and E6 were also inversely correlated (r = −0.989). Conclusions: Our results indicate that hinokitiol might have potential in preventing the progress of immortalized cells toward malignancy and the growth of malignant lesions. Hinokitiol can also influence on the progression of HPV-associated lesions by downregulating the E6 and E7 expression.

The scavenger receptor cysteine-rich (SRCR) family of proteins comprise more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ~110 amino acid SRCR domain, this class of proteins have widespread functions as anti-microbial molecules, scavenger- and signalling-receptors. Despite the high level of structural conservation of SRCR domains, no molecular basis for ligand interaction has been described. The SRCR protein SALSA, also known as dmbt1/gp340, is a key player in mucosal immunology. Based on detailed structures of the SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand binding mechanism for SALSA ligands. The binding interface incorporates a dual cation binding site, which is highly conserved across the SRCR super family. Along with the well-described cation dependency on most SRCR domain-ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for ligand recognition by the SRCR class of proteins.

Galectin-3-binding protein (Gal-3BP) is a ubiquitous and multifunctional secreted glycoprotein originally identified and mainly studied in the context of neoplastic transformation and cancer progression. However, Gal-3BP expression is induced in viral infection and by a multitude of molecules that either mimic or are characteristic for an ongoing inflammation and microbial infection, such as IFN-α, IFN-β, IFN-γ, TNF-α, poly(I:C), dsRNA, and dsDNA. Furthermore, Gal-3BP belongs to the scavenger receptor cysteine-rich (SRCR) domain-containing protein family, by virtue of its N-terminal SRCR domain. The SRCR domain is found in soluble or membrane-associated innate immunity-related proteins and is implicated in self-nonself discrimination. This review summarizes the current knowledge of structural features of Gal-3BP and its proposed intracellular and extracellular innate immunity functions with special emphasis on viral and bacterial infections.

Human papilloma viruses (HPV) are a common cause of transient infections on mucosal surfaces, also in the oral cavity. Some infections remain persistent and can, especially with high risk HPV genotypes, lead to malignancies in the oral-oropharyngeal area. Our understanding of the natural course of oral HPV infections is limited, and the local host responses are poorly known. In this study we show that anti-HPV16L1 antibodies, the IgA response being most abundant, can be measured in saliva of asymptomatic males. HPV16L1 specific multiplex serology and commercial ELISA methods were compared and also the total salivary IgA levels measured. The total salivary IgA concentrations varied from 36 to 163 μg/ml. All the assays could detect anti-HPV16 IgA from saliva, but the correlation between assays varied from non-significant 0.22 to highly significant 0.81, p 0.01. Salivary antibody responses did not correlate with the antibody responses detected in serum (Spearman correlations between -0.12 and 0.16) not even after adjusting the specific responses to differences in total IgA in saliva. Only six of 34 individuals were HPV16 DNA positive at the time of the sampling, but interestingly, three out of four with oral HPV16 DNA had salivary anti-HPV16 IgA responses below average. In conclusion, our results show that anti-HPV16 antibodies can be measured from saliva and the salivary response differs from that of serum. Individual differences in total salivary antibody concentrations may affect also the amount of HPV16 specific antibodies in saliva. Furthermore, different assay methods showed different specificities; thus comparisons between studies must be done with care.

The structures of SALSA SRCR domains 1 and 8 reveal a cation-dependent mechanism for ligand recognition, contributing to important roles in the immune system and cellular signalling. The cation-binding sites are conserved across all SRCR domains, suggesting conserved functional mechanisms., The scavenger receptor cysteine-rich (SRCR) family of proteins comprises more than 20 membrane-associated and secreted molecules. Characterised by the presence of one or more copies of the ∼110 amino-acid SRCR domain, this class of proteins have widespread functions as antimicrobial molecules, scavenger receptors, and signalling receptors. Despite the high level of structural conservation of SRCR domains, no unifying mechanism for ligand interaction has been described. The SRCR protein SALSA, also known as DMBT1/gp340, is a key player in mucosal immunology. Based on detailed structural data of SALSA SRCR domains 1 and 8, we here reveal a novel universal ligand-binding mechanism for SALSA ligands. The binding interface incorporates a dual cation-binding site, which is highly conserved across the SRCR superfamily. Along with the well-described cation dependency on most SRCR domain–ligand interactions, our data suggest that the binding mechanism described for the SALSA SRCR domains is applicable to all SRCR domains. We thus propose to have identified in SALSA a conserved functional mechanism for the SRCR class of proteins.

Streptococcus suis is a Gram-positive bacterium, which causes sepsis and meningitis in pigs and humans. This review examines the role of known S. suis virulence factors in adhesion and S. suis carbohydrate-based adhesion mechanisms, as well as the inhibition of S. suis adhesion by anti-adhesion compounds in in vitro assays. Carbohydrate-binding specificities of S. suis have been identified, and these studies have shown that many strains recognize Galα1-4Gal-containing oligosaccharides present in host glycolipids. In the era of increasing antibiotic resistance, new means to treat infections are needed. Since microbial adhesion to carbohydrates is important to establish disease, compounds blocking adhesion could be an alternative to antibiotics. The use of oligosaccharides as drugs is generally hampered by their relatively low affinity (micromolar) to compete with multivalent binding to host receptors. However, screening of a library of chemically modified Galα1-4Gal derivatives has identified compounds that inhibit S. suis adhesion in nanomolar range. Also, design of multivalent Galα1-4Gal-containing dendrimers has resulted in a significant increase of the inhibitory potency of the disaccharide. The S. suis adhesin binding to Galα1-4Gal-oligosaccharides, Streptococcal adhesin P (SadP), was recently identified. It has a Galα1-4Gal-binding N-terminal domain and a C-terminal LPNTG-motif for cell wall anchoring. The carbohydrate-binding domain has no homology to E. coli P fimbrial adhesin, which suggests that these Gram-positive and Gram-negative bacterial adhesins recognizing the same receptor have evolved by convergent evolution. SadP adhesin may represent a promising target for the design of anti-adhesion ligands for the prevention and treatment of S. suis infections.

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.

Bacterial adhesion is often a prerequisite for infection, and host cell surface carbohydrates play a major role as adhesion receptors. Streptococci are a leading cause of infectious diseases. However, only few carbohydrate-specific streptococcal adhesins are known. Streptococcus suis is an important pig pathogen and a zoonotic agent causing meningitis in pigs and humans. In this study, we have identified an adhesin that mediates the binding of S. suis to galactosyl-α1–4-galactose (Galα1–4Gal)-containing host receptors. A functionally unknown S. suis cell wall protein (SSU0253), designated here as SadP (streptococcal adhesin P), was identified using a Galα1–4Gal-containing affinity matrix and LC-ESI mass spectrometry. Although the function of the protein was not previously known, it was recently identified as an immunogenic cell wall protein in a proteomic study. Insertional inactivation of the sadP gene abolished S. suis Galα1–4Gal-dependent binding. The adhesin gene sadP was cloned and expressed in Escherichia coli. Characterization of its binding specificity showed that SadP recognizes Galα1–4Gal-oligosaccharides and binds its natural glycolipid receptor, GbO3 (CD77). The N terminus of SadP was shown to contain a Galα1-Gal-binding site and not to have apparent sequence similarity to other bacterial adhesins, including the E. coli P fimbrial adhesins, or to E. coli verotoxin or Pseudomonas aeruginosa lectin I also recognizing the same Galα1–4Gal disaccharide. The SadP and E. coli P adhesins represent a unique example of convergent evolution toward binding to the same host receptor structure.

Lyme borreliosis (LB) is a tickborne infectious disease caused by Borrelia burgdorferi sensu lato (Bbsl) which includes B. garinii (Bg), B. afzelii (Ba), and B. burgdorferi sensu stricto (Bbss) [1]. Late manifestations of LB are associated with different tissue tropism of borrelia genospecies. Bg has the tendency to cause neuroborreliosis, whereas Ba is mostly associated with chronic skin disorders, such as acrodermatitis chronica atrophicans and Bbss with Lyme arthritis. The molecular mechanisms behind this phenomenon are unknown. Bbsl has several surface proteins that mediate attachment to different tissues and molecules of the host. It can bind to host plasminogen and plasminogen activators [2] and to various extracellular matrix and cell surface molecules [3]. Several adhesins have been identified in Bbsl, which mediate adherence to decorin [4], glycosaminoglycans (GAGs) [5], integrins [6, 7], fibronectin [8–10], laminin [9, 11, 12], and collagen [9, 13]. Decorin-binding proteins (Dbp) A and B are 2 well-characterized adhesins of Bbss [4, 14]. DbpA and B are expressed during mammalian infection [15], and they mediate bacterial attachment to the proteoglycan decorin, which is associated with collagen fibers in the extracellular matrix [16]. The decorin binding site of DbpA has been mapped to a conserved peptide motif (EAKVRA) [17]. Decorin consists of a collagen-binding core protein and a single GAG side chain that can be either dermatan sulphate or chondroitin-6-sulphate [18]. The binding target of Dbps is the GAG side chain, especially dermatan sulphate, in decorin [19]. Decorin is widely expressed throughout the body. High concentrations are detected in the skin, joints, and endothelium [20]. Decorin binding activity has an important role in borrelial dissemination, and it is also suggested that tissues rich in decorin might serve as a protective niche for Bbss [21]. In mice, for example, mutant strains lacking either one or both of the proteins are infective, but their dissemination to distant organs is less efficient than is the dissemination of the wild-type strain [22]. Thus far, all studies concerning the biological functions of Dbps have been performed with Bbss, and little attention has been paid to the potential differences in the decorin binding activity among the 3 genospecies. In amino acid sequence comparison, only 40–60% similarity was seen among the Dbps of the genospecies [23]. Variation in decorin binding activity may be a factor that in part determines the different tissue tropism of the 3 genospecies. It may also have a role in immune evasion and persistence of LB. The aim of this study was to characterize and compare the biological activities of the Dbps of Bg, Ba, and Bbss. To accomplish that, we took 2 approaches: (1) we recombinantly expressed individual Dbps and characterized their decorin binding properties and (2) generated recombinant borrelia strains in a surface protein–deficient background strain (B313) [24] to study the role of Dbps in the adhesion of live borrelia to decorin and decorin-expressing cells.

Antiadhesion therapy is a promising approach to the fight against pathogens. Antibiotic resistance and the lack of effective vaccines have increased the search for new methods to prevent infectious diseases. Previous studies have shown the antiadhesion activity of juice from cultivated cranberries (Vaccinium macrocarpon Ait.) against bacteria, especially E. coli. In this study, the binding of two streptococcal strains, Streptococcus pneumoniae and Streptococcus agalactiae, to molecular size fractions (FI, FII and FIII,10 kDa, 10-100 kDa, and100 kDa, respectively) of berries and berry and fruit juices from 12 plant species were studied using a microtiter well assay. For Streptococcus suis a hemagglutination inhibition assay was used. In general, binding activity was detected especially to wild cranberry (Vaccinium oxycoccos L.) and to other Vaccinium species. S. pneumoniae cells bound most to cranberry juice fraction FI and S. agalactiae cells to cranberry fraction FIII. Hemagglutination induced by S. suis was most effectively inhibited by cranberry fraction FII. NMR spectra of some characteristic active and non-active fractions were also measured. They indicate that fractions FII and FIII contained proanthocyanidins and/or other phenolic compounds. The results suggest Vaccinium berries as possible sources of antiadhesives against bacterial infections.

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340.

Introduction: Most probiotic products are consumed orally and hence it is feasible that the bacteria in these products may also attach to oral surfaces; however, the effects of these bacteria on the oral ecosystem are mostly unknown. Our aim was to evaluate the oral colonization potential of different probiotic, dairy, and fecal Lactobacillus and Bifidobacterium strains in vitro. Methods: The binding of 17 Lactobacillus and seven Bifidobacterium strains to hydroxyapatite and microtitre wells coated with human saliva was tested. Binding of selected strains to human buccal epithelial cells and co-adherence with Fusobacterium nucleatum were also investigated. In addition, the survival in sterilized human whole saliva was examined. Results: There was a large variation in binding to saliva-coated surfaces and buccal epithelial cells but all strains survived in saliva. The binding pattern of the probiotics did not differ from the binding of the fecal strains. F. nucleatum altered the binding of both the low-binding bifidobacteria and the high-binding lactobacilli. Conclusion: The differences in binding in vitro may indicate that there are also differences in the persistence of the different probiotic strains in the oral cavity in vivo.

Human whole saliva contains two peroxidases, salivary peroxidase (hSPO) and myeloperoxidase (hMPO), which are part of the innate host defence in oral cavity. Both hSPO as well as human milk lactoperoxidase (hLPO) are coded by the same gene, but to what extent the different producing glands, salivary and mammary glands, affect the final conformation of the enzymes is not known. In human saliva the major function of hSPO and hMPO is to catalyze the oxidation of thiocyanate (SCN−) in the presence of hydrogen peroxide (H2O2) resulting in end products of wide antimicrobial potential. In addition cytotoxic H2O2 is degraded. Similar peroxidation reactions inactivate some mutagenic and carcinogenic compounds, which suggests another protective mechanism of peroxidases in human saliva. Although being target of an active antimicrobial research, the structure–function relationships of hSPO are poorly known. However, recently published method for recombinant hSPO production offers new tools for those investigations.

Salivary scavenger receptor cysteine-rich protein gp340 aggregates streptococci and other bacteria as part of the host innate defense system at mucosal surfaces. In this article, we have investigated the properties of fluid-phase gp340 and hydroxylapatite surface-adsorbed gp340 in aggregation and adherence, respectively, of viridans group streptococci (e.g., Streptococcus gordonii and Streptococcus mutans) , non-viridans group streptococci (e.g., Streptococcus pyogenes and Streptococcus suis ), and oral Actinomyces . Fluid-phase gp340 and surface-phase gp340 bioforms were differentially recognized by streptococci, which formed three phenotypic groupings according to their modes of interaction with gp340. Group I streptococci were aggregated by and adhered to gp340, and group II streptococci preferentially adhered to surface-bound gp340, while group III streptococci were preferentially aggregated by gp340. Each species of Streptococcus tested was found to contain strains representative of at least two of these gp340 interaction groupings. The gp340 interaction modes I to III and sugar specificities of gp340 binding strains coincided for several species. Many gp340 interactions were sialidase sensitive, and each of the interaction modes (I to III) for S. gordonii was correlated with a variant of sialic acid specificity. Adherence of S. gordonii DL1 (Challis) to surface-bound gp340 was dependent upon expression of the sialic acid binding adhesin Hsa. However, aggregation of cells by fluid-phase gp340 was independent of Hsa and involved SspA and SspB (antigen I/II family) polypeptides. Conversely, both gp340-mediated aggregation and adherence of S. mutans NG8 involved antigen I/II polypeptide. Deletion of the mga virulence regulator gene in S. pyogenes resulted in increased cell aggregation by gp340. These results suggest that salivary gp340 recognizes different bacterial receptors according to whether gp340 is present in the fluid phase or surface bound. This phase-associated differential recognition by gp340 of streptococcal species of different levels of virulence and diverse origins may mediate alternative host responses to commensal or pathogenic bacterial phenotypes.

We have used a conceptually novel way to construct antibody mimics based on the binding of a noncatalytic enzyme to its substrate. Bacteriophage-derived endosialidase cleaves polysialic acid (polySia), an important oncofetal and bacterial antigen, which is poorly immunogenic. We fused to green fluorescent protein (GFP) a catalytically inactive endosialidase known to bind but not degrade polysialic acid. The fusion protein is a convenient single-step reagent in fluorescence microscopy, binding assays and immunoblots. It efficiently and specifically detected polysialic acid in developing brain, neuroblastoma cells and bacteria causing meningitis. Enzyme-substrate interactions represent an unexploited source of molecular recognition events. Some of these could be used in designing well-defined substitute antibodies for the study of target molecules which are difficult to purify, available in low quantities, are unstable or have poor immunogenity.

A series of mono-, di-, and tetravalent galabiose (Galα1−4Gal) compounds were synthesized in good yields by coupling of a general carboxylic acid-bearing sugar building block to dendritic scaffolds based on the 3,5-di-(2-aminoethoxy)benzoic acid branching unit. Furthermore, a poly(amidoamine)- (PAMAM-) based dendritic galabioside was synthesized containing eight galabiose units. All galabiosides were tested in a hemagglutination assay and a surface plasmon resonance (SPR) competition assay in order to establish their potency in the binding to the bacterial Gram-positive pathogen Streptococcus suis. A monovalent galabioside containing a short spacer was used as a reference compound in all the assays. Variations in the scaffold as well as in the spacer arms were introduced to determine their influence on the inhibition. The best inhibitor of hemagglutination was an octavalent galabioside with a minimal inhibitory concentration (MIC) of 0.3 nM, to the best of our knowledge the first example of inhibition of ...

Helicobacter pylori has frequently been isolated from human dental plaque, and oral spread via saliva is thought to be one of its principal modes of transmission. Among other innate defence systems human saliva contains peroxidase enzymes and lysozyme. The sensitivity of H. pylori to physiological concentrations of lactoperoxidase and its salivary substrate thiocyanate, and different amounts of hydrogen peroxide (H(2)O(2)) was investigated in buffer and in human whole saliva. The effect of lysozyme was also studied in saliva. All tested H. pylori strains, ATCC 43504(T) and five clinical isolates, were efficiently inhibited by the peroxidase system with high concentrations of H(2)O(2) in buffer. The inhibition was stronger at lower pH. However, in human saliva these high concentrations of H(2)O(2) generated less hypothiocyanite, the antibacterial product of the peroxidase system and the effects of the peroxidase system were weaker. Physiological concentration of lysozyme was not bacteriocidal against H. pylori, nor did it enhance the effect of the peroxidase system in saliva. Thus, further studies are needed to enhance the efficacy of peroxidase systems in human saliva to make it more beneficial not only against dental but also against gastric pathogens.

Passive local immunization against dental caries is a promising approach to its prevention, as clinical evidence of active oral or nasal immunization is still limited and controversial. By means of systemic immunization of pregnant cows with a multivalent vaccine, high titres of IgG antibodies against human cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus, were produced in bovine colostrum. The purified immune product (IP) of this preparation has a number of anticariogenic properties, such as inhibition of streptococcal adherence to saliva-coated hydroxyapatite and inhibition of glucosyltransferase enzymes. This study investigated whether IP antibodies remained active and functional when added to ultra-high temperature (UHT)-treated milk or to Lactobacillus rhamnosus GG (LGG)-fermented milk stored for an extended time. LGG was chosen because of its widely known health benefits in humans and animals. A commercial UHT toddler's milk was supplemented with IP and stored for 2 months at 5, 21 and 30 degrees C. The antistreptococcal titres in UHT milk did not decline at any temperature during storage, and UHT-IP inhibited the adherence of S. mutans for up to 2 months. This was not the case with UHT toddler's milk without IgG antibodies. Milk was fermented with live LGG cells in the presence or absence of 5% IP. The antistreptococcal titres declined to about 30% of the original titres after storage. Fresh milk alone slightly enhanced streptococcal adhesion but fresh milk with IP inhibited the adherence of S. mutans by over 50%. LGG-positive fermented milk without antibodies also inhibited (P < 0.05) the adhesion by about 40%. In both LGG-fermented and UHT immune milk, the activity of antibodies against cariogenic streptococci was maintained during the expected shelf-life of these products. From the anticariogenic point of view it may be beneficial to add bovine-specific antibodies against mutans streptococci to probiotic LGG-containing milk products.

The salivary scavenger and agglutinin (SALSA), also known as gp340 and dmbt1, is an antimicrobial and inflammation-regulating molecule located at the mucosal surfaces. The present study revealed that SALSA was present in the amniotic fluid (AF) and exceptionally enriched in both meconium and feces of infants. Based on immunological and mass spectrometric analysis, SALSA was estimated to constitute up to 4–10% of the total protein amount in meconium, making it one of the most abundant proteins. SALSA proteins in the AF and intestinal samples were polymorphic and exhibited varying polypeptide compositions. In particular, a different abundance of peptides corresponding to functionally important structures was found in the AF and intestinal SALSA. The AF form of SALSA had a more intact structure and contained peptides from the zona pellucida domain, which is involved in cell differentiation and oligomerization. In contrast, the intestinal SALSA was more enriched with the scavenger receptor cysteine-rich domains. The AF, but not the meconium SALSA, bound to Streptococcus pyogenes, S. agalactiae, S. gordonii, and Escherichia coli. Furthermore, differential binding was observed also to known endogenous ligands C1q, mannose-binding lectin, and secretory IgA. Our results have thus identified mucosal body compartments, where SALSA is particularly abundant, and suggest that SALSA exhibits varying functions in the different mucosal locations. The high levels of SALSA in AF and the infant intestine suggest a robust and important function for SALSA during the fetal development and in the mucosal innate immune defense of infants.

Objective: The aim of our study was to evaluate the effects of acute alcohol consumption on saliva secretion rate and selected salivary parameters in healthy nonalcoholic volunteers. Study Design: Twenty-four volunteers (37.7 ± 9.6 years, mean ± SD) consumed 0.6 g or 0.7 g alcohol/kg of body weight (for women and men, respectively) in a soft drink. Saliva samples were collected, first (S0) before any alcohol was consumed, 45 minutes after consumption (S1) and, finally, 60 minutes after S1 (S2). Flow rates of both resting whole saliva and paraffin-stimulated (SWS) whole saliva were assessed. SWS was assessed for amylase, total protein, inorganic phosphate (PO43–), sodium (Na+), potassium (K+), and calcium (Ca2+) content. Results: SWS, but not resting whole saliva (in milliliters/minute), decreased significantly after consumption of alcohol. Amylase activity (P =.010) and the concentrations of Na+ (P =.000) and Ca2+ (P =.002) decreased significantly between S0 and S1. When SWS was analyzed for output, the total protein concentration (S0 to S1, P =.000; S0 to S2, P =.033) and amylase activity (S0 to S1, P =.000) decreased significantly. Further, the output of all the studied electrolytes decreased significantly as blood alcohol concentration increased. Conclusions: We conclude that acute alcohol consumption causes a decrease in SWS flow rate. The decrease in flow rate also results in impaired output of total protein and amylase, as well as in a decrease in the output of electrolytes.(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:292-8)

Previous studies have shown that the peroxidase system with iodide is particularly effective against Actinobacillus actinomycetemcomitans. In the present study, the effects of iodide, chloride and thiocyanate in combinations with lactoperoxidase (LP) and myeloperoxidase (MP) on the viability of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and S. rattus were analysed. Bacteria were incubated in buffer solution containing peroxidase, substrate(s) and H2O2 (all in oral physiological concentrations), and plated after 0, 0.5 and 1 h. The oxidation product of iodide was the most bactericidal against all the bacteria tested. The effect was significantly weaker on mutans streptococci. Physiological concentrations of thiocyanate abolished the effects of LP-H2O2-iodide and MP-H2O2-iodide/chloride combinations. Thiocyanate-peroxidase systems have already been used in oral hygiene products. The incorporation of iodide into these products could make them much more potent against periodontal pathogens, and also help to prevent transmission of these pathogens from person to person via saliva.

Passive immunisation, based on bovine colostral preparations, is an area of active research. Specific bovine antibodies inhibit the virulence factors of target pathogens but the interactions between whey preparations and human immune defence cells are not well known. Bovine colostrum inhibits the phagocytic activity of bovine leucocytes and this may reflect the biological activity of immunoglobulins in it. Therefore, this study aimed to examine the effects of bovine whey protein preparations from the colostrum of Streptococcus mutans/S. sobrinus-immunised and sham-immunised cows on binding, ingestion and killing of these bacteria by human leucocytes. Binding and ingestion of FITC-labelled bacteria were estimated by flow cytometry and leukocyte activation was measured as chemiluminescence. Killing rate was estimated by plate counting and by measuring bioluminescence from S. mutans- containing the insect luciferase gene. Colostral whey protein preparation from hyperimmunised cows activated human leucocytes by opsonising specific bacteria. Neutrophils, eosinophils and monocytes weakly phagocytosed non-opsonised bacteria and bacteria opsonised with control product. On the contrary, binding and ingestion were efficient in the presence of the preparation from immunised cows. Thus, these results show that bovine colostral whey proteins are able to support the activation of human phagocytes against pathogenic microbes and that this property is related to specific antibodies in whey preparations. These whey proteins may also be clinically useful, especially in preventing the colonisation of newly erupted teeth by mutans streptococci.

Colostral products from non-immunized cows (CP) and cows immunized with mutans streptococci (IP) were used as mouth rinses in a short-term human study. The acidogenic potential of the products was tested and found to be negligible in vivo before application to subsequent rinsing tests. At first, all the participants received a professional tooth cleaning, after which they rinsed with one of the solutions (IP; CP; water) three times per day for 3 d. After each rinsing period, the resting pH and decrease in plaque pH after sucrose challenge were determined, the amount of plaque was estimated, and all available plaque was collected. No significant differences were recorded in the composition or in the amounts of accumulated plaque. The resting pH values of plaques with low "innate" pH were increased after the IP rinsing period. Surprisingly, the lowest pH values after the sucrose challenge were recorded in IP plaques. The number of cultivable facultative flora or total streptococci were not affected by different rinsings, but the relative number of mutans streptococci significantly decreased after the IP rinsing period when compared to the CP period. Thus, the short term rinsing indicates favourable effects of bovine immune whey on human dental plaque.

The aim of this study was to examine the effect of bovine colostral whey proteins from cows immunized with Streptococcus mutans/Strep. sobrinus on the adherence and aggregation of caries-inducing bacteria, i.e. mutans streptococci. Both adherence and aggregation are important phenomena in the bacterial colonization of the human oral cavity. In all adherence experiments there was a significant difference between treatments by immune product (IP; from immunized cows) and a control product (CP; a similar product from non-immunized cows). The adherence of 35S-labelled Strep. mutans cells (serotype c) to parotid saliva-coated hydroxyapatite (SHA) was dose-dependently inhibited by both IP and CP if SHA was coated with either product before exposure to bacteria, but markedly lower concentrations of IP than CP were effective. When instead of SHA the bacterial cells were pretreated with IP or CP, only IP strongly and dose-dependently inhibited streptococcal adherence. When bacteria, IP or CP, and SHA were incubated simultaneously, a significant difference between IP and CP treatments was again found. Further, IP effectively aggregated both Strep. mutans and Strep. sobrinus cells, whereas hardly any effect was seen with CP. Both IP and CP aggregated the control bacterium Strep. sanguis, which affected the adherence of the pretreated bacteria.

The oral bacterium Streptococcus mutans was transformed by electroporation with a shuttle vector (pCSS945) containing insect luciferase gene from a click beetle ( Pyrophorus plagiophthalamus ) resulting in a bioluminescent phenotype. This S. mutans strain was used in experiments in which light emission was used as a rapid and, compared to conventional CFU counting, more convenient means of estimating the effects of various antimicrobial treatments. The basic parameters affecting in vivo light production by the strain were studied. It was found that pH 6.0 was optimal for incorporation of the substrate d -luciferin for the luciferase reaction. The optimum concentration of d -luciferin was approximately 150 μM at room temperature. Under optimum conditions the light emission in vivo increased rapidly to a constant level and thereafter had a decay of 0.6%/min when logarithmic-growth-phase cells were used. The light emission closely paralleled the numbers of CFU, giving a detectable signal from 30,000 cells and having a dynamic measurement range over 4 log CFU/relative light unit. The cells were treated with various antimicrobial agents, and the emitted bioluminescence was measured. With the bioluminescent measurements, the results were obtained within hours compared to the days required for agar plates, and also, the kinetics of the antibacterial actions could be followed. Thus, the light emission was found to be a reliable, sensitive, and real-time indicator of the bacteriostatic actions of the antimicrobial agents tested.

Due to potential side-effects of active immunization by cariogenic mutans streptococci, oral administration of passively-derived antibodies could be a more acceptable way to reduce colonization and virulence of these microorganisms in human dentition. The aim of this study was to produce antistreptococcal immunoglobulins into bovine colostrum and explore the possible antibacterial mechanisms of these immunoglobulins against mutans streptococci. Specific serum IgG antibodies to whole cell antigens of both Streptococcus mutans and Streptococcus sobrinus increased rapidly in cows during immunization and were high also in the final whey-product. Low concentration (0.5% w/v) of bovine immune preparation inhibited significantly the incorporation of [14C]glucose by both S. mutans and S. sobrinus. Higher concentration (> 1%) was needed to inhibit the glucosyltransferase or fructosyltransferase activities of these bacteria. No such inhibitory effects were observed with the control preparation from the non-immunized cows. Our results indicate that bovine immune colostrum has a significant inhibitory potential against mutans streptococci, apparently dependent on the presence of specific IgG antibodies against S. mutans and S. sobrinus.

Pathogenesis of many bacterially-induced inflammatory diseases is driven by toll- like receptor (TLR) mediated immune responses following recognition of bacterial factors by different TLRs. Periodontitis is a chronic inflammation of the tooth supporting apparatus often leading to tooth loss, and is caused by a Gram-negative bacterial consortium that includes Tannerella forsythia. This bacterium expresses a virulence factor, the BspA, which drives periodontal inflammation by activating TLR2. The N- terminal portion of the BspA protein comprises a leucine-rich repeat (LRR) domain previously shown to be involved in the binding and activation of TLR2. The objective of the current study was to identify specific epitopes in the LRR domain of BspA that interact with TLR2. Our results demonstrate that a sequence motif GC(S/T)GLXSIT is involved in mediating the interaction of BspA with TLR2. Thus, our study has identified a peptide motif that mediates the binding of a bacterial protein to TLR2 and highlights the promiscuous nature of TLR2 with respect to ligand binding. This work could provide a structural basis for designing peptidomimetics to modulate the activity of TLR2 in order to block bacterially-induced inflammation.

Because smoking negatively affects oral health, we investigated whether this could be due to adverse effects of smoking per se on human oral microflora. Faecal microflora was also analysed for comparison. Samples of whole saliva, dental plaque, pharynx and faeces were collected from 48 healthy young military conscripts, 24 smokers and 24 non-smokers, and studied by gas–liquid chromatography of bacterial cellular fatty acids. The method has proved to be fast and sensitive in detecting microfloral differences and their magnitudes. The results showed significant differences in microflorae between the different sample types (saliva, dental plaque, pharyngeal or faecal). However, there were no significant differences within any sample type between smokers and non-smokers. When oral health is good, smoking history short (mean 5.4 years) and groups otherwise uniform, moderate daily smoking does not seem to change human microbial florae.Keywords: smoking, gas–liquid chromatography, bacterial fatty acids, microbial flora.

Introduction: The use of probiotic bacteria is increasing worldwide and at least some of them can transiently colonize the oral cavity. Several studies have shown that probiotic bacteria, which are often thought of in relation only to intestinal health, can also affect the oral ecology, but the mechanisms for this are largely unknown. The aim of this study was to investigate in vitro if the probiotic bacteria used in commercial products affect the protein composition of the salivary pellicle and the adherence of other oral bacteria. Methods: Salivary pellicle on hydroxyapatite and the adhesion of two oral streptococci, Streptococcus mutans and Streptococcus gordonii, were used as a model. Results: Probiotic bacteria that bound to saliva-coated hydroxyapatite reduced the adhesion of S. mutans but the inhibitory effect on the adherence of S. gordonii was weaker. Salivary pellicle protein composition was modified by all the strains tested. The modifications in the pellicle affected the adherence of S. mutans but not of S. gordonii. Two of the proteins missing from the pellicles made of saliva-treated with the probiotic bacteria were identified as salivary agglutinin gp340 and salivary peroxidase. All bacterial strains bound salivary agglutinin gp340. The ability of the probiotic bacteria to degrade peroxidase was demonstrated with purified bovine lactoperoxidase and two of the probiotic strains. Conclusion: This in vitro study showed that probiotic strains used in commercial products may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of the salivary pellicle.

Uropathogenic P-fimbriated Escherichia coli adheres to host cells by specific adhesins recognizing galabiose (Galalpha1-4Gal)-containing structures on cell surfaces. In search of agents inhibiting this first step of infection, the inhibition potency of a set of synthetic mono- and multivalent galabiose compounds was evaluated. In order to mimic the flow conditions of natural infections, a live-bacteria application of surface plasmon resonance (SPR) was established.For the measurement of the binding of E. coli to a surface containing galabiose, live bacteria were injected over the flow cell, and the inhibition of adhesion caused by the galabiose inhibitors was recorded. Quantitative binding data were recorded in real-time for each inhibitor. The results were compared with those of conventional static haemagglutination and ELISA-based cell adhesion assays. Compared with the Gram-positive Streptococcus suis bacteria, which also bind to galabiose and whose binding inhibition is strongly dependent on the multivalency of the inhibitor, E. coli inhibition was only moderately affected by the valency. However, a novel octavalent compound was found to be the most effective inhibitor of E. coli PapG(J96) adhesion, with an IC50 value of 2 microM.Measurement of bacterial adhesion by SPR is an efficient way to characterize the adhesion of whole bacterial cells and allows the characterization of the inhibitory potency of adhesion inhibitors under dynamic flow conditions. Under these conditions, multivalency increases the anti-adhesion potency of galabiose-based inhibitors of P-fimbriated E. coli adhesion and provides a promising approach for the design of high-affinity anti-adhesion agents.

Salivary proline-rich proteins (PRPs) attach commensal Actinomyces and Streptococcus species to teeth. Here, gel filtration, mass spectrometry and Edman degradation were applied to show the release of a pentapeptide, RGRPQ, from PRP-1 upon proteolysis by Streptococcus gordonii . Moreover, synthetic RGRPQ and derivatives were used to investigate associated innate properties and responsible motifs. The RGRPQ peptide increased 2.5-fold the growth rate of S. gordonii via a Q-dependent sequence motif and selectively stimulated oral colonization of this organism in a rat model in vivo. In contrast, the growth of Streptococcus mutans , implicated in caries, was not affected. While the entire RGRPQ sequence was required to block sucrose-induced pH-decrease by S. gordonii and S. mutans , the N-terminal Arg residue mediated the pH increase (i.e., ammonia production) by S. gordonii alone (which exhibits Arg catabolism to ammonia). Strains of commensal viridans streptococci exhibited PRP degradation and Arg catabolism, whereas cariogenic species did not. The RGRPQ peptide mediated via a differential Q-dependent sequence motif, adhesion inhibition, and desorption of PRP-1-binding strains of A. naeslundii genospecies 2 (5 of 10 strains) but not of S. gordonii ( n = 5). The inhibitable A. naeslundii strains alone displayed the same binding profile as S. gordonii to hybrid peptides terminating in RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1. The present findings indicate the presence of a host-bacterium interaction in which a host peptide released by bacterial proteolysis affects key properties in biofilm formation.

Udgivelsesdato: 2007-Apr Glycoprotein gp-340 aggregates bacteria in saliva as part of innate defence at mucosal surfaces. We have detected size- and glycoforms of gp-340 between human saliva samples (n = 7) and lung gp-340 from a proteinosis patient using antibodies and lectins in Western blots and ELISA measurements. Western blots of saliva samples, and of gp-340 purified, from the seven donors using a gp-340 specific antibody distinguished four gp-340 size variants, designated I to IV (n = 2,2,2 and 1). While saliva gp-340 variants I to III had single bands of increasing sizes, variant IV and lung gp-340 had double bands. Purified I to IV proteins all revealed a N-terminal sequence TGGWIP upon Edman degradation. Moreover, purified gp-340 from the seven donors and lung gp-340 shared N-glycans, sialylated Galbeta1-3GalNAc and (poly)lactosamine structures. However, the larger size gp-340 grouping II/III (n = 4) and smaller size grouping I/IV correlated with a secretor, Se(+), and a non secretor, Se(-), dependent glycoform of gp-340, respectively (p = 0.03). The Se(+) glycoforms contained ABH, Le(b), Le(y) and polylactosamine structures, while the Se(-) glycoforms lacked ABH antigens but expressed Le(a), Le(x) and lactosamine structures. By contrast, lung gp-340 completely lacked ABH, Le(a/b), Le(x/y) or sLe(x) structures. Gp-340 and secretor typing of saliva from additional donors (n = 29) showed gp-340 glycoforms I to IV for 6, 16, 4 and 0 donors, respectively, and 3 non-typeable donors, and verified that gp-340 glycoforms I and II/III correlate with Se(-) and Se(+) phenotypes, respectively (p < 0.0001). The glycoforms of saliva and lung gp-340 mediated differential aggregation of Le(b)- (Helicobacter pylori), sialylpolylactosamine- (Streptococcus suis) or sialic acid- (Streptococcus mutans) binding bacteria. In conclusion, variant size- and glycoforms of gp-340 are expressed by different individuals and may modulate the biological properties of gp-340 pertinent to health and disease.