LFchimera: a synthetic mimic of the two antimicrobial domains of bovine lactoferrin.

Saliva is essential for the maintenance of oral health. When salivary flow is impaired, the risk of various oral diseases such as caries and candidiasis increases drastically. Under healthy conditions, saliva provides effective protection against microbial colonization by the collaborative action of numerous host-defense molecules. This review describes how saliva has been the guideline for the design and characterization of a heterodimeric antimicrobial construct called LFchimera. This construct mimics the helical parts of two antimicrobial domains in the crystal structure of bovine lactoferrin. It shows high antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, fungi, and parasites including biowarfare agents such as Bacillus anthracis, Burkholderia pseudomallei, and Yersinia pestis. Further, sublethal concentrations of LFchimera inhibited biofilm formation, the invasiveness of HeLa cells by Yersinia spp., and prevented haemolysis of enteropathogenic Escherichia coli, demonstrating the versatility of these peptides.

Histatin 1 Enhances Cell Adhesion to Titanium in an Implant Integration Model.

Cellular adhesion is essential for successful integration of dental implants. Rapid soft tissue integration is important to create a seal around the implant and prevent infections, which commonly cause implant failure and can result in bone loss. In addition, soft tissue management is important to obtain good dental aesthetics. We previously demonstrated that the salivary peptide histatin 1 (Hst1) causes a more than 2-fold increase in the ability of human adherent cells to attach and spread on a glass surface. Cells treated with Hst1 attached more rapidly and firmly to the substrate and to each other. In the current study, we examine the potential application of Hst1 for promotion of dental implant integration. Our results show that Hst1 enhances the attachment and spreading of soft tissue cell types (oral epithelial cells and fibroblasts) to titanium (Ti) and hydroxyapatite (HAP), biomaterials that have found wide applications as implant material in dentistry and orthopedics. For improved visualization of cell adhesion to Ti, we developed a novel technique that uses sputtering to deposit a thin, transparent layer of Ti onto glass slides. This approach allows detailed, high-resolution analysis of cell adherence to Ti in real time. Furthermore, our results suggest that Hst1 has no negative effects on cell survival. Given its natural occurrence in the oral cavity, Hst1 could be an attractive agent for clinical application. Importantly, even though Hst1 is specific for saliva of humans and higher primates, it stimulated the attachment and spreading of canine cells, paving the way for preclinical studies in canine models.

CNS myelin induces regulatory functions of DC-SIGN-expressing, antigen-presenting cells via cognate interaction with MOG.

Myelin oligodendrocyte glycoprotein (MOG), a constituent of central nervous system myelin, is an important autoantigen in the neuroinflammatory disease multiple sclerosis (MS). However, its function remains unknown. Here, we show that, in healthy human myelin, MOG is decorated with fucosylated N-glycans that support recognition by the C-type lectin receptor (CLR) DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) on microglia and DCs. The interaction of MOG with DC-SIGN in the context of simultaneous TLR4 activation resulted in enhanced IL-10 secretion and decreased T cell proliferation in a DC-SIGN-, glycosylation-, and Raf1-dependent manner. Exposure of oligodendrocytes to proinflammatory factors resulted in the down-regulation of fucosyltransferase expression, reflected by altered glycosylation at the MS lesion site. Indeed, removal of fucose on myelin reduced DC-SIGN-dependent homeostatic control, and resulted in inflammasome activation, increased T cell proliferation, and differentiation toward a Th17-prone phenotype. These data demonstrate a new role for myelin glycosylation in the control of immune homeostasis in the healthy human brain through the MOG-DC-SIGN homeostatic regulatory axis, which is comprised by inflammatory insults that affect glycosylation. This phenomenon should be considered as a basis to restore immune tolerance in MS.

Involvement of P2X(7) purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: The antimicrobial peptide LL-37, derived from human neutrophils, can directly chemoattract leukocytes and up-regulate the expression of several immune-related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL-37 on interleukin-8 (IL-8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL-8 induction. MATERIAL AND METHODS: Cultured fibroblasts were treated with different concentrations of LL-37 or interleukin-1ß (IL-1ß), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT-PCR and real-time PCR were conducted to analyze the expression of IL-8 mRNA, and the IL-8 levels in cell-free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL-37. RESULTS: Nontoxic concentrations of LL-37 (up to 10 µm) and IL-1ß significantly up-regulated the expression of IL-8 mRNA in a dose-dependent manner (p < 0.05). The IL-8 protein levels were consistently significantly elevated in conditioned media of LL-37-treated HGFs (p < 0.05). IL-8 up-regulation by LL-37 was completely abrogated by 20 µm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X(7) receptor) and the neutralizing antibody against P2X(7) blocked IL-8 up-regulation in a dose-dependent manner, consistent with expression of the P2X(7) receptor in HGFs. CONCLUSION: These findings indicate that LL-37 induces IL-8 expression via the P2X(7) receptor and the MEK1/2-dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL-37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues.

Histatins enhance wound closure with oral and non-oral cells.

The role of human saliva in oral wound-healing has never been fully elucidated. We previously demonstrated that parotid-salivary histatins enhance in vitro wound closure. The question remains whether other salivary-gland secretions enhance wound closure, and also the effects of histatins on primary and non-oral cells. Since the presence of histatins is not limited to parotid saliva, we expected to observe wound-closure activity of other salivary-gland secretions. However, here we show that non-parotid saliva does not stimulate wound closure, most probably due to the presence of mucins, since the addition of MUC5B to parotid saliva abolished its effect. Furthermore, we found that histatins stimulated wound closure of (primary) cells of both oral and non-oral origin. This suggests that the cellular receptor of histatins is widely expressed and not confined to cells derived from the oral cavity. These findings encourage the future therapeutic application of histatins in the treatment of all kinds of wounds.

Salivary proteins: protective and diagnostic value in cariology?

Saliva is essential for a lifelong conservation of the dentition. Various functions of saliva are implicated in the maintenance of oral health and the protection of our teeth: (i) The tooth surface is continuously protected against wear by a film of salivary mucins and proline-rich glycoprotein. (ii) The early pellicle proteins, proline-rich proteins and statherin, promote remineralization of the enamel by attracting calcium ions. (iii) Demineralization is retarded by the pellicle proteins, in concert with calcium and phosphate ions in saliva and in the plaque fluid. (iv) Several salivary (glyco)proteins prevent the adherence of oral microorganisms to the enamel pellicle and inhibit their growth. (v) The salivary bicarbonate/carbonate buffer system is responsible for rapid neutralization of acids. An overview is presented on the major antimicrobial systems in human saliva. Not only the well-known major salivary glycoproteins, including mucins, proline-rich glycoprotein and immunoglobulins, but also a number of minor salivary (glyco)proteins, including agglutinin, lactoferrin, cystatins and lysozyme, are involved in the first line of defense in the oral cavity. Besides, small cationic antimicrobial peptides, e.g. defensins, cathelicidin and the histatins, have come into focus. These are potentially suited as templates for the design of a new generation of antibiotics, since they kill a broad spectrum of microorganisms, while hardly evoking resistance, in contrast to the classical antibiotics.

Distinct localization of MUC5B glycoforms in the human salivary glands.

Salivary mucins, encoded by the MUC5B gene, make up a heterogeneous family of molecules, which are secreted by several glands, including the submandibular, sublingual, and palatine glands. Previous studies have shown that heterogeneity in the salivary mucin population is related to its multiglandular origin. In the present study we address the question to what extent the mucin (MUC5B) population from a single human salivary gland is made up of different glycoforms. Using monoclonal antibodies to defined protein and sulfated carbohydrate epitopes specific to MUC5B, we conduct an immunohistochemical study of different salivary gland types, including submandibular, sublingual, and labial glands. In all tissues studied we found a mosaic expression pattern of sulfo-Lewis a antigen, recognized by mAb F2, which in salivary glands is exclusively present on MUC5B. On the other hand, mucous acini were uniformly labeled by mAb EU-MUC5Bb, evoked against a peptide-stretch of the tandem repeat region of MUC5B. Double staining with both antibodies confirmed the presence of MUC5B-positive/sulfo-Lewis a-positive cells, as well as MUC5B-positive/sulfo-Lewis a-negative cells within one glandular unit. These results indicate that one and the same salivary gland synthesizes different MUC5B glycoforms.

A rapid solid-phase fluorimetric assay for measuring bacterial adherence, using DNA-binding stains.

In this report, we describe the validation of a rapid, single-step, microtiter plate method for quantifying bacterial adherence, based on fluorescent labeling of microorganisms with cell-permeable fluorescent DNA-binding probes. We have tested the binding to saliva-coated microtiter plates of bacteria, including Helicobacter pylori and viridans streptococci (S. mitis, S. gordonii, S. sanguis), known to interact with salivary components. Furthermore, we tested the short-term and longer-term temporal stability of a saliva-mediated adherence of these bacteria in a healthy population (N=30). The assay exhibited excellent reliability statistics, yielding within-assay variability coefficients ranging from 4.9% to 11%. A range of approximately 5 x 10(4)-1 x 10(7) cells could be detected. This method may be generally applicable to study surface binding of virtually any microbial species, while obviating the need of radioactive materials or specific antibodies for quantification, thus providing a procedure that is useful to both basic and clinical research.