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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

The role of cathelicidins in the innate host defenses of mammals.

The cathelicidin peptides comprise one of several families of antimicrobial peptides that are found in neutrophils and epithelia as components of the early host defenses of mammals against infection. All cathelicidin family members are synthesized and stored in cells as two-domain proteins. These are split on demand to produce a cathelin protein and an antimicrobial peptide.

The antimicrobial peptide BMAP-28 reduces lethality in mouse models of staphylococcal sepsis.

Objective: A mouse model of staphylococcal sepsis was used to compare the efficacy of the bovine antimicrobial peptide BMAP-28, a compound of the cathelicidin family, with that of conventional antibiotics.

Design: Prospective, randomized, controlled animal study.

Setting: Research laboratory in a university hospital.

Genome-wide transcriptional profiling of the Escherichia coli response to a proline-rich antimicrobial peptide.

Most antimicrobial peptides (AMPs) impair the viability of target bacteria by permeabilizing bacterial membranes. However, the proline-rich AMPs have been shown to kill susceptible organisms without causing significant membrane perturbation and may act by inhibiting the activity of bacterial targets. To gain initial insight into the events that follow interaction of a proline-rich peptide with bacterial cells, we used DNA macroarray technology to monitor transcriptional alterations of Escherichia coli in response to challenge with a subinhibitory concentration of the proline-rich Bac7(1-35).

In vitro activity of protegrin-1 and beta-defensin-1, alone and in combination with isoniazid, against Mycobacterium tuberculosis.

The antimicrobial peptide protegrin-1 (PG-1) inhibited the growth in vitro of drug-susceptible and multidrug-resistant Mycobacterium tuberculosis; a lower activity was shown by human beta-defensin-1 (HBD-1) against both strains. The combination of PG-1 or HBD-1 with isoniazid significantly reduced M. tuberculosis growth in comparison with the peptides or isoniazid alone.

In vitro and in vivo antimicrobial activity of two alpha-helical cathelicidin peptides and of their synthetic analogs.

Two alpha-helical antimicrobial peptides (BMAP-27 and -28) and four synthetic analogs were compared for in vitro and in vivo antimicrobial efficacy. All peptides proved active in vitro at micromolar concentrations against a range of clinical isolates, including antibiotic-resistant strains. BMAP-27 and two analogs were more effective towards Gram-negative, and BMAP-28 towards Gram-positive organisms.

Cathelicidin peptide sheep myeloid antimicrobial peptide-29 prevents endotoxin-induced mortality in rat models of septic shock.

The present study was designed to investigate the antiendotoxin activity and therapeutic efficacy of sheep myeloid antimicrobial peptide (SMAP)-29, a cathelicidin-derived peptide. The in vitro ability of SMAP-29 to bind LPS from Escherichia coli 0111:B4 was determined using a sensitive limulus chromogenic assay. Two rat models of septic shock were performed: (1) rats were injected intraperitoneally with 1 mg E.

Cathelicidins, multifunctional peptides of the innate immunity.

Cathelicidins comprise a family of mammalian proteins containing a C-terminal cationic antimicrobial domain that becomes active after being freed from the N-terminal cathelin portion of the holoprotein. Many other members of this family have been identified since the first cathelicidin sequences were reported 10 years ago. The mature peptides generally show a wide spectrum of antimicrobial activity and, more recently, some of them have also been found to exert other biological activities.

Antimicrobial activity of SMAP-29 against the Bacteroides fragilis group and clostridia.

Objectives: The cathelicidin-derived peptide SMAP-29 exerts rapid and broad-spectrum antimicrobial activity against aerobic bacteria and fungi. In this study, the effects of the peptide against the Bacteroides fragilis group, including antibiotic-resistant isolates, Clostridium perfringens and Clostridium difficile reference and clinical isolates, were investigated.

Methods: The microbicidal activity of SMAP-29 against eight reference and 100 clinical anaerobic strains from a national collection was assessed using a microdilution susceptibility assay, and by determining the killing kinetics on selected strains.

Neutralization of endotoxin in vitro and in vivo by Bac7(1-35), a proline-rich antibacterial peptide.

Lipopolysaccharides (LPS), or endotoxins, are structural components of gram-negative bacteria implicated in the pathogenesis of septic shock. In this study the antiendotoxin activity of Bac7(1-35), a synthetic peptide based on the sequence of a proline-rich antibacterial peptide from bovine neutrophils, was investigated in vitro and in an experimental rat model of gram-negative septic shock. The ability of Bac7(1-35) to bind LPS from Escherichia coli O111:B4 was determined using a sensitive Limulus chromogenic assay.

In vitro effect on Cryptosporidium parvum of short-term exposure to cathelicidin peptides.

Two laboratory methods, a cell culture system and double fluorogenic staining, were used to study the viability and infective ability of Cryptosporidium parvum sporozoites and oocysts after short-term exposure to four cathelicidin peptides. The compounds, SMAP-29, BMAP-28, PG-1 and Bac7(1-35), exerted a strong cytotoxic effect on sporozoites, but did not affect the viability and function of oocysts consistently. Overall, in the sporozoite series, a percentage of the viable population decreased rapidly to less than detectable levels after 15 and 60 min exposure to the peptides at concentrations of 100 and 10 micro g/mL, respectively.

Inducible expression of an antimicrobial peptide of the innate immunity in polymorphonuclear leukocytes.

Epithelia- and leukocyte-associated antimicrobial peptides provide immediate protection against microbial infections by rapidly inactivating potential pathogens. Bac5 is a member of the cathelicidin family of antimicrobial peptides and is stored in the cytoplasmic granules of bovine neutrophils. We investigated the expression of this gene in airway and intestine, and although the gene was not found to be locally expressed in these tissues, a strong Bac5 induction signal was detected by in situ hybridization in neutrophils infiltrating infected lung, consistent with expression of this gene in activated neutrophils.

Structural and functional characterization of hBD-1(Ser35), a peptide deduced from a DEFB1 polymorphism.

beta-Defensins are mammalian antimicrobial peptides that share a unique disulfide-bonding motif of six conserved cysteines. An intragenic polymorphism of the DEFB1 gene that changes a highly conserved Cys to Ser in the peptide coding region has recently been described. The deduced peptide cannot form three disulfide bonds, as one of the cysteines is unpaired.