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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.

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.

Due to the increasing use of automated milking systems, automated detection of clinical mastitis is becoming more important. Various in- or on-line diagnostic tests are in use, but generally suffer from false mastitis alerts. In this study, we explored a new diagnostic approach based on measurement of protease activity using fluorogenic protease substrates, which can be performed on site, at high speed, and at low costs. Samples from cows with clinical mastitis submitted for bacteriological culture at the University Farm Animal Practice were collected during several months and kept at -20°C until protease activity measurement. A reference set of milk samples from clinically healthy cows were collected on 9 different farms and were tested for protease activity directly and after freezing at -20°C to allow for comparison with the samples from clinical cases. The protease activity in mastitic milk samples was significantly higher than in samples from healthy animals. Based on 71 clinical mastitis samples and 180 milk samples from clinically healthy quarters, the area under the receiver-operating characteristic curve was estimated to be between 0.88 and 0.90, and at a threshold of 38 fluorescence per minute the test had a specificity of 0.99 at a sensitivity of 0.58. Protease activity measured in fresh milk from clinically healthy cows was significantly associated with somatic cell count and parity, but not with electrical conductivity, whereas protease activity in milk that had been frozen was statistically significantly associated with all 3 parameters. This study indicates that protease activity measurement as a stand-alone test can be used for detecting mastitis samples, using milk samples that have been frozen. Because protease activity acts in part on a different biological mechanism than somatic cell count or electrical conductivity, this test may increase the accuracy of mastitis diagnosis in combination with currently available in- or on-line tests in automated milking systems.

Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone. J. Cell. Physiol. 229: 210-225, 2014. (c) 2014 Wiley Periodicals, Inc.

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.

Anti-Microbial Peptides (AMPs) are part of the innate immune defense system and considered as promising lead compounds for the development of novel anti-bacterial agents. In general, AMPs are simple, short peptides with broad-spectrum activity against Gram-negative and Gram-positive bacteria, fungi, viruses, and parasites. AMPs are, inherently structured, to target and interact with bio-membranes. The AMPs in this study are derived from Lactoferrin. Lactoferrin is one of the antimicrobial components of the immune system of the body. In our laboratory, the antibacterial activity, cytotoxicity, the effects on host cell attachment and cell invasion of Lactoferrin and derived AMPs: lactoferricin, lactoferrampin and LFchimera (a chimera of lactoferricin and lactoferrampin) are studied against a broad range of bacteria including Bacillus antracis, Yersinia pestis and Burkholderia pseudomallei.

Bovine lactoferrin harbors 2 antimicrobial sequences (LFcin and LFampin), situated in close proximity in the N1-domain. To mimic their semi parallel configuration we have synthesized a chimeric peptide (LFchimera) in which these sequences are linked in a head-to-head fashion to the α- and ε-amino group, respectively, of a single lysine. In line with previously described bactericidal effects, this peptide was also a stronger candidacidal agent than the antimicrobial peptides LFcin17-30 and LFampin265-284, or a combination of these 2. Conditions that strongly reduced the candidacidal activities of LFcin17-30 and LFampin265-284, such as high ionic strength and energy depletion, had little influence on the activity of LFchimera. Freeze-fracture electron microscopy showed that LFchimera severely affected the membrane morphology, resulting in disintegration of the membrane bilayer and in an efflux of small and high molecular weight molecules such as ATP and proteins. The differential effects displayed by the chimeric peptide and a mixture of its constituent peptides clearly demonstrate the synergistic effect of linking these peptides in a fashion that allows a similar spatial arrangement as in the parent protein, suggesting that in bovine lactoferrrin the corresponding fragments act in concert in its candidacidal activity.

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.

Concentrations and output of lactoferrin and of low-M-r mucin MG2 were determined in saliva of subjects suffering from Actinobacillus actinomycetem-comitans-associated periodontal disease and healthy subjects. Periodontal patients were clinically examined and a microbiological sample was taken from the deepest bleeding pockets in each quadrant. The number of viable A. actinomycetemcomitans was determined in the sampled sites of each patient. The MG2 output in the diseased subjects (13.6 mu g protein/min) was decreased at least by a factor three compared to periodontal healthy subjects (44.3 mu g protein/min). On the other hand, output of lactoferrin was not significantly different in healthy (9.5 mu g/min) and diseased subjects (7.6 mu g/min). Western analyses demonstrated a higher iron-saturation of lactoferrin in diseased subjects in comparison with the healthy subjects. Lactoferrin degrading enzymes, probably derived from microbial sources, could be detected in saliva of the periodontally diseased subjects, but not in saliva of healthy subjects. The combination of iron-saturation and degradation of lactoferrin suggests that anti-microbial properties of lactoferrin are diminished in periodontitis patients. Moreover, the low concentration of mucin MG2 suggests a decline in mucin defence and consequently a higher susceptibility for oral infection. A negative correlation (r = -0.4, p