The role of LL-37 in periodontal wound healing

Introduction: As a result of chronic inflammation during periodontal disease the junctional epithelium becomes micro-ulcerated. The inflammatory process is mediated by both bacterial and host cell products. Host defence peptides such as defensins, secretory leucocyte protease inhibitor (SLPI) and the sole human cathelicidin, LL-37, are secreted by both periodontal cells and neutrophils into gingival crevicular fluid (GCF). They have the ability to modulate the immune response in periodontitis and are thought to have a potential role in periodontal wound healing. Objectives: The aims of this study were to determine the role of LL-37 in the production of Interleukin (IL)-8, IL-6, hepatocyte growth factor (HGF) and basic-fibroblast growth factor (bFGF) by gingival fibroblasts. The role of LL-37 in modulating total matrix metalloproteinase (MMP) activity and expression of tissue inhibitors of metalloproteinase (TIMP)-1 and -2 by gingival fibroblasts was also investigated. Methods: Primary gingival fibroblasts were co-cultured with concentrations of LL-37 (1, 5 and 10µg/ml) for 24 hours and their supernatants tested for levels of IL-8 and IL-6, HGF, bFGF, TIMP-1 and TIMP-2 by ELISA. Rates of MMP turnover in the supernatants were tested by fluorogenic assay using fluorescence resonance energy transfer (FRET) peptide substrates. Cytotoxicity was measured by MTT assay. Statistical significance was measured using the independent t-test and p<0.05 was considered significant. Results: LL-37 significantly upregulated levels of IL-8, IL-6, HGF, bFGF and TIMP-1 (p<0.05) in a dose-dependent fashion. LL-37 significantly decreased the total MMP activity (p<0.05). None of the LL-37 concentrations tested were cytotoxic to gingival fibroblasts. Conclusion: These results indicate that LL-37 is involved in periodontal wound healing. LL-37 increased levels of proinflammatory cytokines and increased levels of growth factors involved in re-epithelialisation. LL-37 has the ability to regulate remodelling of the periodontium by controlling MMP overactivity both directly and by stimulating production of inhibitors by gingival fibroblasts.

LL-37 Degradation by GCF: A Role for Porphyromonas gingivalis Proteinases?

Background: LL-37, composed of 37 amino acid residues, is an innate host defence peptide of the cathelicidin family. It is expressed by neutrophils, monocytes and epithelial cells and exhibits both anti-bacterial and immunomodulatory properties. LL-37 is however prone to proteolytic degradation by proteinases, thus potentially limiting its inherent host defence properties in the inflammatory milieu. Objectives: The present study was designed to determine whether LL-37 was degraded by components of gingival crevicular fluid (GCF) from healthy subjects or those with periodontitis. In addition, we aimed to deduce whether degradation of the peptide was accelerated in GCF samples which were determined to be positive for the periodontopathic bacterium Porphyromonas gingivalis. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 individual sites in patients presenting with advanced periodontitis. In healthy subjects, GCF samples only were collected. Plaque samples were analysed by QPCR for the presence or absence of P. gingivalis. Pooled GCF samples from healthy sites; periodontitis sites which were P. gingivalis negative (Pg-); or periodontitis sites which were P. gingivalis positive (Pg+), were incubated with synthetic LL-37 for 0 – 180 min. The degradation products were then analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Results: LL-37 was not degraded when incubated with GCF from healthy subjects. In contrast, LL-37 was degraded after 30 min when incubated with Pg- GCF. However degradation of LL-37 was apparent after only 2 min incubation with Pg+ GCF and the parent molecule was almost completely degraded after 30 min. Conclusions: The rapid degradation of LL-37, particularly in Pg+ sites, highlights the limited role which this host defence peptide may play in the presence of biologically active proteinases. It also underscores a potent virulence mechanism of P. gingivalis used to circumvent innate host responses.

Identification of human neutrophil defensins (HNPs1-3) in biopsies of squamous cell carcinomas of the human tongue

It has previously been reported that the a-defensins, found in the granules of polymorphonuclear leukocytes (neutrophils/ PMNs), are cytolytic for human tumour cells in vitro. Objective: To identify and quantify the a- defensins, HNP-1, HNP-2 and HNP-3 in healthy and tumour tissue from patients with oral squamous cell carcinoma using HPLC, mass spectrometry and amino acid sequencing. Methods: All patients (n=5) were diagnosed with oral squamous cell carcinoma of the tongue.Biopsy tissue from the site of the tumour (n=5) and a non-affected region of the tongue (n=5) was snap frozen and subsequently stored at -70 ºC until analysed. Peptides were extracted from the 10 tissue biopsies using acidified ethanol. Peptide extracts were separated by reverse-phase HPLC . All tumour and control tissue samples were individually analysed under identical conditions with a flow rate of l ml/min, ambient column temperature and absorbance detection at 214 and 280 nm. Fractions (1ml) were collected automatically. HPLC fractions were analysed by MALDI-MS using a linear time-of-flight Voyager DE-mass spectrometer (PerSeptive Biosystems, UK). Using this system the detection limit was 10 fmol. Peptides with molecular masses corresponding to those reported for the a-defensins were deemed of interest and were further subject to complete structural analysis by automated Edman degradation using an Applied Biosystems 491 Procise microsequencer. Results: MALDI-MS revealed a triad of peptides of molecular masses 3442 Da, 3371 Da and 3486 Da in both healthy and tumour tissue. Full length sequence data were obtained for the three a-defensins, unequivocally identifying their presence in both tumour and healthy tissue. Analysis of the MALDI-MS and sequence data indicated that the a-defensins were overexpressed (up to 12 fold) in tumour tissue. Conclusion: This study demonstrates the feasibility of screening tumour tissue for novel peptides/proteins using HPLC and MALDI-MS.The role of a-defensins in oral squamous cell carcinoma of the tongue requires further investigation.

Doxorubicin induces the cleavage of Poly (ADP-ribose) polymerase, a marker of programmed cell death (apoptosis) in mouse cardiomyocytes

Cancer is one of the leading causes of death in the world. Despite this, a growing number of people are surviving the disease due to medical advancements and the development of numerous new therapies. Doxorubicin, a chemotherapeutic agent, is a widely-used and successful first-line anti-tumour treatment. However, the established toxic and deleterious effects of the drug on the cardiovascular system confer increased risk of congestive heart failure, thereby necessitating the use of reduced doxorubicin doses. In order to investigate how these events are initiated, mouse cardiomyocytes (HL-1) were treated in vitro with varying concentrations of doxorubicin (0.5-4.0 µmol/L). Following treatment (24h), a marked level of cell death was observed in comparison to untreated cardiomyocytes; the level of death appeared to correlate with the concentration of the drug used. Western blotting revealed the cleavage of full length Poly (ADP-ribose) polymerase (PARP) into 89 and 24kDa fragments, a process which is instrumental in triggering programmed cell death/apoptosis. Importantly, results suggested that this event may be independent of caspase 3 cleavage and thus activation. A number of previous studies have reported a functional role for both Mitofusin-2 (Mfn2) and NADPH oxidase 2 (Nox2) in the cardiotoxic response. Given that PARP cleavage is a validated indicator of cellular apoptosis, these results clearly indicate that this marker could be used in future studies when determining if depletion of the above proteins would cause a reduction in or eradicate the pro-apoptotic action of this agent on cardiomyocytes. Such investigations may lead to significant developments in ensuring that doxorubicin can achieve its full therapeutic anti-tumour potential without causing the subsequent deleterious effects on the cardiovascular system.

Cigarette smokers have exaggerated alveolar barrier disruption in response to lipopolysaccharide inhalation

RATIONALE: Cigarette smoke exposure is associated with an increased risk of the acute respiratory distress syndrome (ARDS); however, the mechanisms underlying this relationship remain largely unknown.

OBJECTIVE: To assess pathways of lung injury and inflammation in smokers and non-smokers with and without lipopolysaccharide (LPS) inhalation using established biomarkers.

METHODS: We measured plasma and bronchoalveolar lavage (BAL) biomarkers of inflammation and lung injury in smokers and non-smokers in two distinct cohorts of healthy volunteers, one unstimulated (n=20) and one undergoing 50 μg LPS inhalation (n=30).

MEASUREMENTS AND MAIN RESULTS: After LPS inhalation, cigarette smokers had increased alveolar-capillary membrane permeability as measured by BAL total protein, compared with non-smokers (median 274 vs 208 μg/mL, p=0.04). Smokers had exaggerated inflammation compared with non-smokers, with increased BAL interleukin-1β (p=0.002), neutrophils (p=0.02), plasma interleukin-8 (p=0.003), and plasma matrix metalloproteinase-8 (p=0.006). Alveolar epithelial injury after LPS was more severe in smokers than non-smokers, with increased plasma (p=0.04) and decreased BAL (p=0.02) surfactant protein D. Finally, smokers had decreased BAL vascular endothelial growth factor (VEGF) (p<0.0001) with increased soluble VEGF receptor-1 (p=0.0001).

CONCLUSIONS: Cigarette smoke exposure may predispose to ARDS through an abnormal response to a 'second hit,' with increased alveolar-capillary membrane permeability, exaggerated inflammation, increased epithelial injury and endothelial dysfunction. LPS inhalation may serve as a useful experimental model for evaluation of the acute pulmonary effects of existing and new tobacco products.