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Chronic rhinosinusitis (CRS) is a chronic infection of the nasal cavity and paranasal sinuses associated with the presence of a microbial biofilm. Extracellular DNA (eDNA) is an important component of the biofilm matrix. Antimicrobial peptides (AMPs) are natural peptides with the ability to kill microorganisms. D-LL-31 is a synthetic variant of the AMP cathelicidin with increased resistance to proteolytic breakdown. In this study it is shown for 3 clinical CRS isolates that treatment of 24 h biofilms with DNase I enhanced the antimicrobial activity of D-LL-31. Conversely, co-incubation of D-LL-31 at the IC50 value with exogenous DNA resulted in reduced antimicrobial activity. DNase I alone did not show antimicrobial activity against the isolates tested but caused dispersal of an established biofilm. Hence, the presence of eDNA in the biofilm matrix reduced AMP-mediated killing. These results suggest that combination therapy with proteolysis resistant AMP D-LL-31 and DNase could be considered for effective treatment of CRS.

In this study, we discovered a panel of discriminative microRNAs in salivary gland tumors by application of statistical machine learning methods. We modelled multi-component interactions of salivary microRNAs to detect group-based associations among the features, enabling the distinction of malignant from benign tumors with a high predictive performance utilizing only seven microRNAs. Several of the identified microRNAs are separately known to be involved in cell cycle regulation. Integrated biological interpretation of identified microRNAs can provide potential new insights into the biology of salivary gland tumors and supports the development of non-invasive diagnostic tests to discriminate salivary gland tumor subtypes.

Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis-, adipose-, and gingiva-derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin-derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation.

Antimicrobial peptides have been found throughout living nature, yet antimicrobial sequences may still lie hidden within a wide variety of proteins. A rational strategy was developed to select interesting domains, based on the presumed common features of antimicrobial peptides, and to release these from accessible and safe proteins. In silico proteolysis simulations of bovine lactoferrin (bLF) with selected endoproteinases predicted the liberation of peptides that encompasses a cationic amphipathic alpha-helix. Three predicted peptides were synthesized and tested for their biological activity, demonstrating that one single enzyme was sufficient to obtain an antimicrobial peptide. The proof of principle demonstrated that a 32-mer fragment isolated from the endoproteinase AspN digestion of bLF possessed strong antimicrobial activity. Moreover, desalted crude digest had improved activity over native bLF. Hence, selective digestion of bLF increases its antimicrobial activity by release of antimicrobial stretches.

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections.

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-1β (IL-1β), 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-1β 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 P2X7 receptor) and the neutralizing antibody against P2X7 blocked IL-8 up-regulation in a dose-dependent manner, consistent with expression of the P2X7 receptor in HGFs.Conclusion: These findings indicate that LL-37 induces IL-8 expression via the P2X7 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.

De mond heeft het zwaar te verduren; bacteriën, mechanische stress door het kauwen van voedsel, en schadelijke stoffen uit drank, sigarettenrook en voeding kunnen het mondweefsel beschadigen. Speeksel beschermt de mond op een aantal manieren: als smeermiddel tegen schade door mechanische stress, als een barrière tegen bacteriële toxinen en schadelijke stoffen, als een buffer om de pH in de mond constant te houden en het bevat antimicrobiële peptiden die micro-organismen tegengaan. Histatine 1 (Hst1) is een speekselpeptide met vele eigenschappen, waaronder het binden van tannines (uit o.a. wijn en thee), bescherming van het tandglazuur en een mogelijke rol in wondgenezing. Maar de exacte werking van Hst1 op de weefsels in de mond waar het mee in aanraking komt was nog onbekend, en dat is dan ook het onderwerp van dit proefschrift. Wij hebben ontdekt dat Hst1 celhechting bevordert op substraten (dat kan van alles zijn, het hangt ervan af waarop de cellen zijn uitgezaaid, bijvoorbeeld glas of kweekplastic) en ook aan andere cellen. Dat gold zowel voor celtypen die voorkomen in de mond (zoals epitheelcellen en fibroblasten), als ook voor darmepitheel en zelfs endotheelcellen. Het effect van Hst1 op cel-substraat hechting heeft een mogelijke toepassing in de implantologie, aangezien cellen moeten hechten aan het implantaat om het stevig vast te zetten. Hst1 versterkte daarnaast de barrièrefunctie. De mond en de darmen bevatten een grote hoeveelheid bacteriën en de barrièrefunctie in deze organen is daarom erg belangrijk. We vonden bovendien dat cellen behandeld met Hst1 beter waren bestand tegen bacteriële translocatie.