Carlo P.J.M. Brouwer

Synergistic Activity of the N-Terminal Peptide of Human Lactoferrin and Fluconazole against Candida Species

ABSTRACT In light of the need for new antifungal regimens, we report that at noncandidacidal concentrations, the lactoferrin-derived peptide hLF(1-11), which is highly active against fluconazole-resistant Candida albicans, acts synergistically with fluconazole against this yeast and a fluconazole-sensitive C. albicans strain as well as C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis. When these yeasts were exposed to hLF(1-11) for 5 min and then incubated with fluconazole, they were killed effectively, while no candidacidal activity was observed when they were incubated first with fluconazole and then exposed to the peptide, indicating that the candidacidal activity is initiated by the peptide while fluconazole is only required during the effector phase. Investigations of the effect of azide, which inhibits mitochondrial respiration, on the activity of combinations of hLF(1-11) and fluconazole against fluconazole-resistant C. albicans revealed that it inhibits this activity, even when added during the effector phase only. As expected, azide inhibited the accumulation of rhodamine 123 in mitochondria and the production and release of ATP by C. albicans that occurred upon exposure to the combination of hLF(1-11) and fluconazole. Accordingly, oxidized ATP (oATP), an antagonist of ATP receptors, completely blocked the candidacidal activity of the hLF(1-11)-fluconazole combination, whereas oATP did not block the activity when its presence was restricted to the effector phase. The candidacidal activity of combinations of hLF(1-11) and fluconazole, which is initiated by the peptide through the involvement of energized mitochondria, renders fluconazole-resistant C. albicans sensitive to this azole.

The Synthetic N-Terminal Peptide of Human Lactoferrin, hLF(1-11), Is Highly Effective against Experimental Infection Caused by Multidrug-Resistant Acinetobacter baumannii

ABSTRACT The lactoferrin-derived peptide hLF(1-11), but not its control peptide, was highly effective against five multidrug-resistant Acinetobacter baumannii strains in vitro (3 to 4 log reduction) and against four of these strains in an experimental infection in mice (2 to 3 log reduction). Therefore, this peptide is a promising candidate as a novel agent against infections with multidrug-resistant A. baumannii.

Discovery and development of a synthetic peptide derived from lactoferrin for clinical use.

There is an urgent need to develop new antimicrobial drugs especially for combating the rise of infections caused by multi-resistant pathogens such as MRSA and VRSA. The problem of antibiotic resistant micro-organisms is expected to increase disproportionally and controlling of infections is becoming difficult because of the rapid spread of those micro-organisms. Primary therapy with classical antibiotics is becoming more ineffective. Combinational therapy of antibiotics with antimicrobial peptides (AMPs) has been suggested as an alternative approach to improve treatment outcome. Their unique mechanism of action and safety profile makes AMPs appealing candidates for simultaneous or sequential use in different cases of infections. In this review, for antimicrobial treatment the application of synthetic antimicrobial peptide hLF(1-11), derived from the first 11 amino acids of human lactoferrin is evaluated in both pre-clinical and clinical settings. Present information indicates that this derivate from lactoferrin is well tolerated in pre-clinical tests and clinical trials and thus hLF(1-11) is an interesting candidate for further exploration in various clinical indications of obscure infections, including meningitis. Another approach of using AMPs is their use in prevention of infections e.g. as coating for dental or bone implants or in biosensing applications or useful as infection specific radiopharmaceutical.

Human Lactoferrin-Derived Peptide's Antifungal Activities against Disseminated Candida albicans Infection

BACKGROUND Because the human lactoferrin-derived peptide, hLF(1-11), exerts potent in vitro candidacidal activity, we investigated whether it displays antifungal activity against disseminated Candida albicans infections. METHODS Neutropenic mice were intravenously infected with C. albicans and, 24 h later, were injected with hLF(1-11); 18 h later, the number of viable yeasts in the kidneys was determined microbiologically, the size and number of infectious foci were determined histologically, and serum cytokine levels were determined by immunoassays. RESULTS hLF(1-11) was effective (maximum reduction, 1.5 logs) against disseminated C. albicans infections, and its antifungal activity leveled off at a concentration of 0.4 ng of hLF(1-11)/kg of body weight. The antifungal activity of hLF(1-11) was increased in mice injected with interleukin (IL)-10 neutralizing antibodies, which suggests that IL-10 reduces the antifungal activity of hLF(1-11). In agreement with this result was the finding that injection of high doses of hLF(1-11) into infected mice was accompanied by increased levels of IL-10 in serum. Microscopic analysis revealed that infectious foci in kidneys of hLF(1-11)-treated mice contained mainly blastoconidia, whereas filamentous forms were abundant in untreated mice. The peptide inhibited the in vitro morphological transition of C. albicans, in a dose-dependent manner. : hLF(1-11) is effective against disseminated C. albicans infections; and its effects on C. albicans viability and virulence and on host cells may explain this antifungal activity.

MycoBank gearing up for new horizons.

MycoBank, a registration system for fungi established in 2004 to capture all taxonomic novelties, acts as a coordination hub between repositories such as Index Fungorum and Fungal Names. Since January 2013, registration of fungal names is a mandatory requirement for valid publication under the International Code of Nomenclature for algae, fungi and plants (ICN). This review explains the database innovations that have been implemented over the past few years, and discusses new features such as advanced queries, registration of typification events (MBT numbers for lecto, epi- and neotypes), the multi-lingual database interface, the nomenclature discussion forum, annotation system, and web services with links to third parties. MycoBank has also introduced novel identification services, linking DNA sequence data to numerous related databases to enable intelligent search queries. Although MycoBank fills an important void for taxon registration, challenges for the future remain to improve links between taxonomic names and DNA data, and to also introduce a formal system for naming fungi known from DNA sequence data only. To further improve the quality of MycoBank data, remote access will now allow registered mycologists to act as MycoBank curators, using Citrix software.

Synthetic peptides derived from human antimicrobial peptide ubiquicidin accumulate at sites of infections and eradicate (multi-drug resistant) Staphylococcus aureus in mice

The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely recognized as an evolutionary preserved part of the innate immune system. Based on evidence in animal models and humans, AMPs are now positioned as novel anti-infective agents. The current study aimed to evaluate the potential antimicrobial activity of ubiquicidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the alpha-helix or beta-sheet domains of the human cationic peptide ubiquicidin (UBI 1-59), allowed selection of AMPs for possible treatment of MRSA infections. The strongest antibacterial activity was observed for the entire peptide UBI 1-59 and for synthetic fragments comprising amino acids 31-38. The availability, chemical synthesis opportunities, and size of these small peptides, combined with their strong antimicrobial activity towards MRSA make these compounds promising candidates for antimicrobial therapy and detection of infections in man.

Histatin and lactoferrin derived peptides: antimicrobial properties and effects on mammalian cells

In order to analyze the clinical potential of two antimicrobial peptides, human lactoferrin 1-11 (hLF1-11) and synthetic histatin analogue Dhvar-5, we measured the killing effect on bacteria, and the potential toxicity on erythrocytes and bone cells. The antimicrobial activity was determined in a killing assay on six strains, including methicillin resistant Staphylococcus Aureus. The effect on human erythrocytes and MC3T3 mouse bone cells was measured with a hemolysis assay and a viability assay, respectively. Both hLF1-11 and Dhvar-5 dose-dependently killed all bacterial strains, starting at concentrations of 6 microg/mL. hLF1-11 had no effect on mammalian cells at concentrations up to 400 microg/mL, but Dhvar-5 induced significant hemolysis (37% at 200 microg/mL) and bone cell death (70% at 400 microg/mL). This indicates that both peptides are able to kill various resistant and non-resistant bacteria, but Dhvar-5 may exert a cytotoxic effect on host cells at higher concentrations.

Histatin-Derived Monomeric and Dimeric Synthetic Peptides Show Strong Bactericidal Activity towards Multidrug-Resistant Staphylococcus aureus In Vivo

ABSTRACT Homodimerization of histatin-derived peptides generally led to improved bactericidal activity against Staphylococcus aureus in vitro. In vivo, monomers and dimers were equally active in killing bacteria in mice with a soft tissue infection. Altogether, these peptides are promising compounds for the development of novel therapeutics against infections with drug-resistant bacteria.

Various routes of administration of 99mTc-labeled synthetic lactoferrin antimicrobial peptide hLF 1–11 enables monitoring and effective killing of multidrug-resistant Staphylococcus aureus infections in mice

The synthetic antimicrobial peptide representative of the first 11 N-terminal amino acids of human lactoferrin (hLF 1-11) kills multidrug-resistant Staphylococcus aureus (MRSA). This study displays antimicrobial activity of hLF 1-11, via various routes of administration, against MRSA infections in mice. Radiolabeling hLF 1-11 with technetium-99m ((99m)Tc-hLF 1-11) enables scintigraphic monitoring directly after administration. (99m)Tc-hLF 1-11 was taken up by the gall bladder, intestines, and kidneys. Most of the radioactivity was captured in the urinary bladder and about 1% of the injected dose accumulated into infected thigh muscles. At 2 or 24h after either intravenously, subcutaneously, intraperitoneally, or orally injected a single dose of 0.04 mg/kg hLF 1-11 in mice significantly reduced (20-60 times) the number of viable MRSA. In a dose-response setting in immunocompetent mice maximum bactericidal effects (10,000 times reduction) of intravenously injected (99m)Tc-hLF 1-11 was seen with 40 mg/kg whereas the same dose of orally administered (99m)Tc-hLF 1-11 induced about approximately 100 times reduction. In conclusion, intravenously and orally administrated (99m)Tc-hLF 1-11 accumulates in infected tissues and is highly effective against experimental infections with MRSA. Moreover, scintigraphy is an excellent tool to study the pharmacology of experimental compounds and to determine the uptake in infected tissues.

Current Status of Imaging Infections with Radiolabeled Anti-Infective Agents

Infection specific radiopharmaceuticals can be used for diagnosis, decision-making in therapy and treatment follow-up. Research has been ongoing to develop infection specific markers since clinically used tracers cannot discriminate between infection and inflammation. A specific radiopharmaceutical for infection imaging should satisfy the following criteria: high and specific uptake at the infection site, rapid infection detection and background clearance, minimal accumulation in non-target tissues, low toxicity, zero immune response and especially the ability to differentiate infection from sterile inflammation. Radiolabeled anti-infective agents can fulfill the majority of these requirements. Technetium-99m (99mTc) labeled antibiotics have potential to differentiate sterile inflammation from infection. There are numerous studies reporting the use of radiolabeled antibacterial and antifungal agents for infection detection. Other promising agents are antimicrobial peptides (AMPs) since they preferentially bind to bacteria membranes instead of those of mammalian cells thus distinguishing between infection and sterile inflammation. Synthetic AMPs derived from human natural peptides offers possibility for studying the effects of polymerization and substituting the amino acid sequence to design a specific micro-organism seeking tracer. 99mTc-labeled anti-infectives are ideal as infection-seeking agents because of its direct and fast accumulation. Clinical studies already undertaken and further evaluation with different pathogen types such as viruses, fungi, parasites and intracellular pathogens in humans will improve the potential of these compounds. Radiochemical techniques for labeling anti-infectives have been developed to optimize biodistribution and targeting properties of tracers. An important issue is the technetium-99m specific coordination site in the anti-infective molecule and its chemical and biochemical characterization.