The Potential of Surface-Immobilized Antimicrobial Peptides for the Enhancement of Orthopaedic Medical Devices: A Review.

Due to the well-known phenomenon of antibiotic resistance, there is a constant need for antibiotics with novel mechanisms and different targets respect to those currently in use. In this regard, the antimicrobial peptides (AMPs) seem very promising by virtue of their bactericidal action, based on membrane permeabilization of susceptible microbes. Thanks to this feature, AMPs have a broad activity spectrum, including antibiotic-resistant strains, and microbial biofilms. Additionally, several AMPs display properties that can help tissue regeneration. A possible interesting field of application for AMPs is the development of antimicrobial coatings for implantable medical devices (e.g., orthopaedic prostheses) to prevent device-related infection. In this review, we will take note of the state of the art of AMP-based coatings for orthopaedic prostheses. We will review the most recent studies by focusing on covalently linked AMPs to titanium, their antimicrobial efficacy and plausible mode of action, and cytocompatibility. We will try to extrapolate some general rules for structure-activity (orientation, density) relationships, in order to identify the most suitable physical and chemical features of peptide candidates, and to optimize the coupling strategies to obtain antimicrobial surfaces with improved biological performance.

Membrane perturbation, altered morphology and killing of Staphylococcus epidermidis upon contact with a cytocompatible peptide-based antibacterial surface.

One possibility to prevent prosthetic infections is to produce biomaterials resistant to bacterial colonization by anchoring membrane active antimicrobial peptides (AMPs) onto the implant surface. In this perspective, a deeper understanding of the mode of action of the immobilized peptides should improve the development of AMP-inspired infection-resistant biomaterials. The aim of the present study was to characterize the bactericidal mechanism against Staphylococcus epidermidis of the AMP BMAP27(1-18), immobilized on titanium disks and on a model resin support, by applying viability counts, Field Emission Scanning Electron Microscopy (FE-SEM), and a fluorescence microplate assay with a membrane potential-sensitive dye. The cytocompatibility to osteoblast-like MG-63 cells was investigated in monoculture and in co-culture with bacteria. The impact of peptide orientation was explored by using N- and C- anchored analogues. On titanium, the ∼50 % drop in bacteria viability and dramatically affected morphology indicate a contact-killing action exerted by the N- and C-immobilized peptides to the same extent. As further shown by the fluorescence assay with the resin-anchored peptides, the bactericidal effect was mediated by rapid membrane perturbation, similar to free peptides. However, at peptide MBC resin equivalents the C-oriented analogue proved more effective with more than 99 % killing and maximum fluorescence increase, compared to half-maximum fluorescence with more than 90 % killing produced by the N-orientation. Confocal microscopy analyses revealed 4-5 times better MG-63 cell adhesion on peptide-functionalized titanium both in monoculture and in co-culture with bacteria, regardless of peptide orientation, thus stimulating further studies on the effects of the immobilized BMAP27(1-18) on osteoblast cells.

A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria.

BACKGROUND: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole Staphylococcus aureus and Staphylococcus epidermidis. METHODS: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide (diSC3(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. RESULTS: The ion channel-forming gramicidin D induced a rapid increase of diSC3(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. CONCLUSIONS: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs.

Covalent grafting of titanium with a cathelicidin peptide produces an osteoblast compatible surface with antistaphylococcal activity.

Bacterial infection of orthopaedic implants, often caused by Staphylococcus species, may ultimately lead to implant failure. The development of infection-resistant, osteoblast-compatible biomaterials could represent an effective strategy to prevent bacterial colonization of implants, reducing the need for antibiotics. In this study, the widely used biomaterial titanium was functionalized with BMAP27(1-18), an α-helical cathelicidin antimicrobial peptide that retains potent staphylocidal activity when immobilized on agarose beads. A derivative bearing a short spacer with a free thiol at the N-terminus was coupled to silanized titanium disks via thiol-maleimide chemistry. Tethering was successful, as assessed by Contact angle, Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), and X-ray Photoelectron Spectroscopy (XPS), with an average surface mass density of 456 ng/cm2 and a layer thickness of 3 nm. The functionalized titanium displayed antimicrobial properties against a reference strain of Staphylococcus epidermidis with well-known biofilm forming capability. Reduction of bacterial counts and morphological alterations of adhering bacteria, upon 2 h incubation, indicate a rapid contact-killing effect. The immobilized peptide was not toxic to osteoblasts, which adhered and spread better on functionalized titanium when co-cultured with bacteria, compared to non-coated surfaces. Results suggest that functionalization of titanium with BMAP27(1-18) could be promising for prevention of bacterial colonization in bone graft applications.

Evaluation of free or anchored antimicrobial peptides as candidates for the prevention of orthopaedic device-related infections.

The prevention of implant-associated infection, one the most feared complications in orthopaedic surgery, remains a major clinical challenge and urges development of effective methods to prevent bacterial colonization of implanted devices. Alpha-helical antimicrobial peptides (AMPs) may be promising candidates in this respect due to their potent and broad-spectrum antimicrobial activity, their low tendency to elicit resistance and possible retention of efficacy in the immobilized state. The aim of this study was to evaluate the potential of five different helical AMPs, the cathelicidins BMAP-27 and BMAP-28, their (1-18) fragments and the rationally designed, artificial P19(9/G7) peptide, for the prevention of orthopaedic implant infections. Peptides were effective at micromolar concentrations against 22 Staphylococcus and Streptococcus isolates from orthopaedic infections, while only BMAP-28 and to a lesser extent BMAP-27 were active against Enterococcus faecalis. Peptides in solution showed activities comparable to those of cefazolin and linezolid, on a molar basis, and also a variable capacity to neutralize bacterial lipopolysaccharide, while devoid of adverse effects on MG-63 osteoblast cells at concentrations corresponding to the MIC. The (1-18) BMAP fragments and P19(9/G7) were selected for further examination, based on better selectivity indices, and showed effectiveness in the presence of hyaluronic acid and in synovial fluid, while human serum affected their activity to variable extents, with BMAP-27(1-18) best retaining activity. This peptide was immobilized on streptavidin-resin beads and retained activity against reference Staphylococcus epidermidis and Staphylococcus aureus strains, with negligible toxicity towards osteoblasts, underlining its potential for the development of infection-resistant biomaterials for orthopaedic application. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Antimicrobial and host cell-directed activities of Gly/Ser-rich peptides from salmonid cathelicidins.

Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24-40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1-37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the immunostimulant ß-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture.

Antifungal activity of cathelicidin peptides against planktonic and biofilm cultures of Candida species isolated from vaginal infections.

Vulvovaginal candidiasis (VVC) is a frequent gynecological condition caused by Candida albicans and a few non-albicans Candida spp. It has a significant impact on the quality of life of the affected women also due to a considerable incidence of recurrent infections that are difficult to treat. The formation of fungal biofilm may contribute to the problematic management of recurrent VVC due to the intrinsic resistance of sessile cells to the currently available antifungals. Thus, alternative approaches for the prevention and control of biofilm-related infections are urgently needed. In this regard, the cationic antimicrobial peptides (AMPs) of the innate immunity are potential candidates for the development of novel antimicrobials as many of them display activity against biofilm formed by various microbial species. In the present study, we investigated the in vitro antifungal activities of the cathelicidin peptides LL-37 and BMAP-28 against pathogenic Candida spp. also including C. albicans, isolated from vaginal infections, and against C. albicans SC5314 as a reference strain. The antimicrobial activity was evaluated against planktonic and biofilm-grown Candida cells by using microdilution susceptibility and XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assays and, in the case of established biofilms, also by CFU enumeration and fluorescence microscopy. BMAP-28 was effective against planktonically grown yeasts in standard medium (MIC range, 2-32µM), and against isolates of C. albicans and Candida krusei in synthetic vaginal simulated fluid (MIC range 8-32µM, depending on the pH of the medium). Established 48-h old biofilms formed by C. albicans SC5314 and C. albicans and C. krusei isolates were 70-90% inhibited within 24h incubation with 16µM BMAP-28. As shown by propidium dye uptake and CFU enumeration, BMAP-28 at 32µM killed sessile C. albicans SC5314 by membrane permeabilization with a faster killing kinetics compared to 32µM miconazole (80-85% reduced biofilm viability in 90min vs 48h). In addition, BMAP-28 at 16µM prevented Candida biofilm formation on polystyrene and medical grade silicone surfaces by causing a >90% reduction in the viability of planktonic cells in 30min. LL-37 was overall less effective than BMAP-28 against planktonic Candida spp. (MIC range 4-≥64µM), and was ineffective against established Candida biofilms. However, LL-37 at 64µM prevented Candida biofilm development by inhibiting cell adhesion to polystyrene and silicone surfaces. Finally, Candida adhesion was strongly inhibited when silicone was pre-coated with a layer of BMAP-28 or LL-37, encouraging further studies for the development of peptide-based antimicrobial coatings.

Modulation of cytokine gene expression by cathelicidin BMAP-28 in LPS-stimulated and -unstimulated macrophages.

Apart from direct bacterial killing, antimicrobial host defence peptides (HDPs) exert various other biological activities that also include modulation of immune responses to infection. The bovine cathelicidin BMAP-28 has been extensively studied with regard to its direct antibacterial activity while little is known about its effects on immune cell function. We have investigated its ability to affect inflammatory pathways and to influence the proinflammatory response induced by LPS in RAW 264.7 macrophages, in terms of modulation of TLR4 activation and cytokine gene induction. BMAP-28 on its own elicited ERK1/2, p38 and NF-κB activation leading to upregulation of IL-1ß gene expression in these cells, suggesting it has the capacity to activate selected cellular pathways through direct effects on macrophages. As expected based on its in vitro LPS-binding properties, BMAP-28 blocked LPS-induced cytokine gene expression when added to the cell culture in combination with LPS. However it enhanced the induction of IL-1ß and IL-6 genes and suppressed that of IFN-ß when added prior to or following LPS stimulation over a 30-60 min time interval, or when co-administered with taxol as another TLR4 stimulant. It did not inhibit the expression of IFN-ß induced by the TLR3 ligand poly(I:C). Overall these results, and the fact that BMAP-28 increased the LPS-stimulated activation of NF-κB while diminishing that of IRF-3, suggest that the peptide potentiates the early TLR4-mediated proinflammatory cytokine response while inhibiting the TLR4/TRAM/TRIF signaling pathway leading to IRF-3 activation and IFN-ß gene expression. Using a TLR4-specific antibody we also found that BMAP-28 decreased the LPS-induced internalization of surface TLR4 required for initiating the TRAM/TRIF signaling pathway, which provides a mechanism for the inhibitory effect of the peptide on the TLR4/TRAM/TRIF pathway.

Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

The yeast-like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP-28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP-28 sterilized Prototheca cultures within 30-60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3-6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70-90% killing, suggesting they act via non-lytic mechanisms. In circular dichroism studies, the conformation of BMAP-28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP-28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non-lytic mechanisms may be exploited for the development of target-selective drugs.

Broad-spectrum activity against bacterial mastitis pathogens and activation of mammary epithelial cells support a protective role of neutrophil cathelicidins in bovine mastitis.

Cathelicidins are peptide components of the innate immune system of mammals. Apart from exerting a direct antibiotic activity, they can also trigger specific defense responses in the host. Their roles in various pathophysiological conditions have been studied, but there is a lack of published information on their expression and activities in the context of mastitis. The aims of this study were to investigate the expression of the bovine cathelicidins BMAP-27, BMAP-28, Bac5, and indolicidin in healthy and infected mammary tissue and in lipopolysaccharide (LPS)-treated cells, to determine their activities against bacteria isolated from bovine mastitis, and to examine their potentials to trigger defense responses in bovine mammary cells. The genes were found to be upregulated in LPS-stimulated neutrophils, but not in infected quarters or epithelial cells. All peptides showed a variably broad spectrum of activity against 28 bacterial isolates from bovine mastitis (MIC values, 0.5 to 32 microM), some of which were antibiotic resistant. The activity of each peptide was significantly enhanced when it was pairwise tested with the other peptides, reaching the synergy threshold when indolicidin was present. The bactericidal activity was sensitive to milk components; BMAP-27 and -28 were highly effective in mastitic bovine milk and inhibited in milk from healthy cows. Both peptides were also active in whey and in blood serum and triggered the expression of tumor necrosis factor alpha (TNF-alpha) in bovine mammary epithelial cells. Our results indicate multiple roles for the bovine cathelicidins in mastitis, with complementary and mutually enhanced antimicrobial activities against causative pathogens and the capacity to activate host cells.

Role of Cathelicidin Peptides in Bovine Host Defense and Healing.

The in vitro antimicrobial activities and biological effects on host cells were compared for the bovine cathelicidins BMAP-28, an alpha-helical AMP, and Bac5 and Bac7, proline-rich AMPs. Our results confirm that the broad-spectrum activity of BMAP-28 correlates with a high capacity to interact with and permeabilize bacterial membranes, whereas the proline-rich AMPs selectively internalize into the cytoplasm of susceptible Gram-negative bacteria with a non-lytic mechanism. All peptides efficiently translocated into mammalian fibroblastic cells, but while Bac5 and Bac7(1-35) localized to nuclear structures and induced cellular proliferation, BMAP-28 associated with mitochondria and did not induce proliferation. Moreover, BMAP-28 was considerably more cytotoxic than the proline-rich peptides due to cytolytic and pro-apoptotic effects. Our results highlight important functional differences among the bovine cathelicidins and suggest that they contribute to an integrated response of the host to infection, with distinct but complementary activities.

Structure dependence of biological activities for primate cathelicidins.

We have analysed the effects of variations in orang-utan (ppy), rhesus macaque (mmu) and leaf eater (pob) monkey orthologues of the human cathelicidin LL-37, on a range of relevant biological activities. These host defence peptides range in cationicity from +4 to +10, and while the more cationic pob and mmuRL-37 are in a monomeric and unstructured form in bulk solution (F-form), the human and ppyLL-37 are in an aggregated/helical form (A-form). The in vitro antibacterial activity depended strongly on both the structural form and the charge. F-form peptides were more potent against Gram-positive and -negative bacteria and less salt, medium or serum sensitive than A-form ones. CD studies suggested that A- and F-form peptides interact with LPS in different manners, but the ability to detoxify it did not correlate directly with either the charge or structure. Toxicity towards eukaryotic cells also showed a varied dependence on the peptides' physical characteristics. Haemolytic activity was similar for all the tested peptides while other cytotoxicity assays revealed the highly cationic, F-form pobRL-37 as the most toxic, followed by the A-form human LL-37. As shown with the human peptide, toxicity depended markedly on the nature and metabolic state of the target cell. Our results suggest that different evolutionary trajectories for each orthologue lead to distinct sets of physical characteristics, which significantly differentiates their biological activities.

The human cathelicidin LL-37 modulates the activities of the P2X7 receptor in a structure-dependent manner.

Extracellular ATP, released at sites of inflammation or tissue damage, activates the P2X(7) receptor, which in turn triggers a range of responses also including cell proliferation. In this study the ability of the human cathelicidin LL-37 to stimulate fibroblast growth was inhibited by commonly used P2X(7) blockers. We investigated the structural requirements of the growth-promoting activity of LL-37 and found that it did not depend on helix sense (the all-d analog was active) but did require a strong helix-forming propensity in aqueous solution (a scrambled analog and primate LL-37 orthologs devoid of this property were inactive). The involvement of P2X(7) was analyzed using P2X(7)-expressing HEK293 cells. LL-37 induced proliferation of these cells, triggered Ca(2+) influx, promoted ethidium bromide uptake, and synergized with benzoyl ATP to enhance the pore and channel functions of P2X(7). The activity of LL-37 had an absolute requirement for P2X(7) expression as it was blocked by the P2X(7) inhibitor KN-62, was absent in cells lacking P2X(7), and was restored by P2X(7) transfection. Of particular interest, LL-37 led to pore-forming activity in cells expressing a truncated P2X(7) receptor unable to generate the non-selective pore typical of the full-length receptor. Our results indicate that P2X(7) is involved in the proliferative cell response to LL-37 and that the structural/aggregational properties of LL-37 determine its capacity to modulate the activation state of P2X(7).

Efficacy of LL-37 and granulocyte colony-stimulating factor in a neutropenic murine sepsis due to Pseudomonas aeruginosa.

A promising therapeutic strategy for the management of severe Pseudomonas infection in neutropenic patients may result from the coadministration of colony-stimulating factors (CSFs) that help maintain immune competence and antimicrobial peptides, a novel generation of adjunctive therapeutic agents with antimicrobial and anti-inflammatory properties. A promising peptide with these properties is LL-37, the only member of the cathelicidin family of antimicrobial peptides found in humans. BALB/c male mice were rendered neutropenic by intraperitoneal administration of cyclophosphamide on days -4 and -2 preinfection. Septic shock was induced at time 0 by intraperitoneal injection of 2x10 colony-forming units of P. aeruginosa American Type Culture Collection (ATCC) 27853. All animals were randomized to receive intravenously isotonic sodium chloride solution, 1 mg/kg of LL-37, 20 mg/kg of imipenem, 0.1 mg/kg of granulocyte CSF (G-CSF), 1 mg/kg of LL-37+0.1 mg/kg of G-CSF, or 20 mg/kg of imipenem+0.1 mg/kg of G-CSF. Lethality and bacterial growth in blood, peritoneum, spleen, liver, and kidney were evaluated. All regimens were significantly superior to controls at reducing the mouse lethality rate and bacterial burden in organs. Particularly, the combination between LL-37 and G-CSF was the most effective in protecting neutropenic mice from the onset of sepsis and in vitro significantly reduced the apoptosis of neutrophils. Combination therapy between LL-37 and G-CSF is a promising therapeutic strategy for the management of severe Pseudomonas infection complicated by neutropenia.

BMAP-28 improves the efficacy of vancomycin in rat models of gram-positive cocci ureteral stent infection.

An experimental study was performed to evaluate the efficacy of BMAP-28 alone and in combination with vancomycin in animal models ureteral stent infection due to Enterococcus faecalis and Staphylococcus aureus. Study included a control group without bacterial challenge to evaluate the sterility of surgical procedure, a challenged control group that did not receive any antibiotic prophylaxis and for each bacterial strain three challenged groups that received (a) 10 mg/kg vancomycin intraperitoneally, immediately after stent implantation, (b) BMAP-28-coated ureteral stents where 0.2-cm(2) sterile ureteral stents were incubated in 1mg/l BMAP-28 solution for 30 min immediately before implantation and (c) intraperitoneal vancomycin plus BMAP-28-coated ureteral stent at the above concentrations. Experiments were performed in duplicate. Ureteral stents were explanted at day 5 following implantation and biofilm bacteria enumerated. Our data showed that rats that received intraperitoneal vancomycin showed the lowest bacterial numbers. BMAP-28 combined with vancomycin showed efficacies higher than that of each single compound. These results highlight the potential usefulness of this combination in preventing ureteral stent-associated in gram-positive infections.

RNAIII-inhibiting peptide in combination with the cathelicidin BMAP-28 reduces lethality in mouse models of staphylococcal sepsis.

A mouse model of staphylococcal sepsis was used to evaluate the efficacy of RNAIII-inhibiting peptide (RIP) combined with the cathelicidin BMAP-28. Preliminary in vitro studies showed that both peptides, alone or combined, were able to inhibit the lipoteichoic acid-induced production of tumor necrosis factor alpha and nitric oxide by RAW 264.7 cells. For in vivo experiments, the main outcome measures were lethality, quantitative blood cultures, and detection of tumor necrosis factor alpha and interleukin 6 plasma levels. BALB/c mice were injected i.v. with 2.0 x 10(6) colony-forming units of live Staphylococcus aureus ATCC 25923 or with 5.0 x 10(8) heat-killed cells of the same strain. All animals were randomized to receive i.v. isotonic sodium chloride solution, 10-mg/kg RIP, alone or in combination with 2-mg/kg BMAP-28, 7-mg/kg imipenem, or 7-mg/kg vancomycin, immediately and at 6 hours after bacterial challenge. In in vivo experiments performed with live bacteria, all compounds reduced lethality rates and bacteremia when compared with controls. In general, combined-treated groups had significantly lower bacteremia when compared with single-treated groups. Lowest lethality rates and bacteremia were obtained when RIP was administered in combination with BMAP-28 or vancomycin. In the experiments performed using heat-killed organisms, only BMAP-28 demonstrated significant efficacy on lethality rates and cytokines plasma levels when compared with controls. RIP combined with BMAP-28 exhibited the highest efficacy on all main outcome measurements. These data were observed on both immediate and delayed treatments. These results highlight the capacity of RIP and BMAP-28 to reduce the septic effects of bacterial cell components and exotoxins, and suggest their potential use in the treatment of severe staphylococcus-associated sepsis.

Effects of the antimicrobial peptide BMAP-27 in a mouse model of obstructive jaundice stimulated by lipopolysaccharide.

An experimental study was designed to investigate the efficacy of BMAP-27, a compound of the cathelicidin family, in neutralizing Escherichia coli 0111:B4 lipopolysaccharide (LPS) in bile duct-ligated mice. Main outcome measures were: endotoxin and TNF-alpha concentrations in plasma, evidence of bacterial translocation in blood and peritoneum, and lethality. Adult male BALB/c mice were injected intraperitoneally with 2 mg/kg E. coli 0111:B4 LPS 1 week after sham operation or bile duct ligation (BDL). Six groups were studied: sham with placebo, sham with 120 mg/kg tazobactam-piperacillin (TZP), sham with 1 mg/kg BMAP-27, BDL with placebo, BDL with 120 mg/kg TZP, and BDL with 1mg/kg BMAP-27. After LPS, TNF-alpha plasma levels were significantly higher in BDL mice compared to sham-operated animals. BMAP-27 achieved a significant reduction of plasma endotoxin and TNF-alpha concentration when compared with placebo- and TZP-treated groups. On the other hand, both TZP and BMAP-27 significantly reduced the bacterial growth compared with saline treatment. Finally, LPS induced 60% and 55% lethality in BDL placebo- and TZP-treated treated mice and no lethality in sham-operated mice, while only BMAP-27 significantly reduced the lethality to 10%. In light of its dual antimicrobial and anti-endotoxin properties, BMAP-27 could be an interesting compound to inhibit bacterial translocation and endotoxin release in obstructive jaundice.

Pre-treatment of central venous catheters with the cathelicidin BMAP-28 enhances the efficacy of antistaphylococcal agents in the treatment of experimental catheter-related infection.

An in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms developed on 96-well polystyrene tissue culture plates was performed to elucidate the activity of the 27 residues cathelicidin peptide BMAP-28, quinupristin/dalfopristin (Q/D), linezolid, and vancomycin. Efficacy studies were performed in a rat model of staphylococcal CVC infection. Silastic catheters were implanted into the superior cava. Twenty-four hours after implantation the catheters were filled with BMAP-28. Thirty minutes later rats were challenged via the CVC with 1.0x10(6) CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC at a concentration equal to the MBC observed using adherent cells, or at a much higher concentration (1024 microg/mL) began 24 h later. The inhibition activities of all antibiotics against adherent bacteria were at least two-four-fold lower that against freely growing cells. When antibiotics were used in BMAP-28 pre-treated wells, they showed higher activities. The in vivo studies showed that when CVCs were pre-treated with BMAP-28 or with a high dose of antibiotics, biofilm bacterial load was reduced from 10(7) to 10(3) CFU/mL and bacteremia reduced from 10(3) to 10(1) CFU/mL. When CVCs were treated with both BMAP-28 and antibiotics, biofilm bacterial load was further decreased to 10(1) CFU/mL and bacteremia was not detected. These results suggest that CVC pre-treated with BMAP-28 represents an attractive choice for the treatment of device-related infections caused by staphylococci.

LL-37 protects rats against lethal sepsis caused by gram-negative bacteria.

We investigated the efficacy of LL-37, the C-terminal part of the only cathelicidin in humans identified to date (termed human cationic antimicrobial protein), in three experimental rat models of gram-negative sepsis. Adult male Wistar rats (i) were given an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS, (ii) were given 2 x 10(10) CFU of Escherichia coli ATCC 25922, or (iii) had intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intravenously isotonic sodium chloride solution, 1-mg/kg LL-37, 1-mg/kg polymyxin B, 20-mg/kg imipenem, or 60-mg/kg piperacillin. Lethality; growth of bacteria in blood, peritoneum, spleen, liver, and mesenteric lymph nodes; and endotoxin and tumor necrosis factor alpha (TNF-alpha) concentrations in plasma were evaluated. All compounds reduced lethality compared to levels in controls. Endotoxin and TNF-alpha plasma levels were significantly higher in conventional antibiotic-treated rats than in LL-37- and polymyxin B-treated animals. All drugs tested significantly reduced bacterial growth compared to saline treatment. No statistically significant differences between LL-37 and polymyxin B were noted for antimicrobial and antiendotoxin activities. LL-37 and imipenem proved to be the most effective treatments in reducing all variables measured. Due to its multifunctional properties, LL-37 may become an important future consideration for the treatment of sepsis.

Mechanistic and functional studies of the interaction of a proline-rich antimicrobial peptide with mammalian cells.

Mammalian antimicrobial peptides provide rapid defense against infection by inactivating pathogens and by influencing the functions of cells involved in defense responses. Although the direct antibacterial properties of these peptides have been widely characterized, their multiple effects on host cells are only beginning to surface. Here we investigated the mechanistic and functional aspects of the interaction of the proline-rich antimicrobial peptide Bac7(1-35) with mammalian cells, as compared with a truncated analog, Bac7(5-35), lacking four critical N-terminal residues (RRIR) of the Bac7(1-35) sequence. By using confocal microscopy and flow cytometry, we showed that although the truncated analog Bac7(5-35) remains on the cell surface, Bac7(1-35) is rapidly taken up into 3T3 and U937 cells through a nontoxic energy- and temperature-dependent process. Cell biology-based assays using selective endocytosis inhibitors and spectroscopic and surface plasmon resonance studies of the interaction of Bac7(1-35) with phosphatidylcholine/cholesterol model membranes collectively suggest the concurrent contribution of macropinocytosis and direct membrane translocation. Structural studies with model membranes indicated that membrane-bound Bac7(5-35) is significantly more aggregated than Bac7(1-35) due to the absence of the N-terminal cationic cluster, thus providing an explanation for hampered cellular internalization of the truncated form. Further investigations aimed to reveal functional implications of intracellular uptake of Bac7(1-35) demonstrated that it correlates with enhanced S phase entry of 3T3 cells, indicating a novel function for this proline-rich peptide.