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Killing of Trypanosomatid Parasites by a Modified Bovine Host Defense Peptide, BMAP-18

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      Abstract

      BackgroundTropical diseases caused by parasites continue to cause socioeconomic devastation that reverberates worldwide. There is a growing need for new control measures for many of these diseases due to increasing drug resistance exhibited by the parasites and problems with drug toxicity. One new approach is to apply host defense peptides (HDP; formerly called antimicrobial peptides) to disease control, either to treat infected hosts, or to prevent disease transmission by interfering with parasites in their insect vectors. A potent anti-parasite effector is bovine myeloid antimicrobial peptide-27 (BMAP-27), a member of the cathelicidin family. Although BMAP-27 is a potent inhibitor of microbial growth, at higher concentrations it also exhibits cytotoxicity to mammalian cells. We tested the anti-parasite activity of BMAP-18, a truncated peptide that lacks the hydrophobic C-terminal sequence of the BMAP-27 parent molecule, an alteration that confers reduced toxicity to mammalian cells.Methodology/Principal FindingsBMAP-18 showed strong growth inhibitory activity against several species and life cycle stages of African trypanosomes, fish trypanosomes and Leishmania parasites in vitro. When compared to native BMAP-27, the truncated BMAP-18 peptide showed reduced cytotoxicity on a wide variety of mammalian and insect cells and on Sodalis glossindius, a bacterial symbiont of the tsetse vector. The fluorescent stain rhodamine 123 was used in immunofluorescence microscopy and flow cytometry experiments to show that BMAP-18 at low concentrations rapidly disrupted mitochondrial potential without obvious alteration of parasite plasma membranes, thus inducing death by apoptosis. Scanning electron microscopy revealed that higher concentrations of BMAP-18 induced membrane lesions in the parasites as early as 15 minutes after exposure, thus killing them by necrosis. In addition to direct killing of parasites, BMAP-18 was shown to inhibit LPS-induced secretion of tumour necrosis factor alpha (TNF-α), a cytokine that is associated with inflammation and cachexia (wasting) in sleeping sickness patients. As a prelude to in vivo applications, high affinity antibodies to BMAP-18 were produced in rabbits and used in immuno-mass spectrometry assays to detect the intact peptide in human blood and plasma.Conclusions/SignificanceBMAP-18, a truncated form of the potent antimicrobial BMAP-27, showed low toxicity to mammalian cells, insect cells and the tsetse bacterial symbiont Sodalis glossinidius while retaining an ability to kill a variety of species and life cycle stages of pathogenic kinetoplastid parasites in vitro. BMAP-18 also inhibited secretion of TNF-α, an inflammatory cytokine that plays a role in the cachexia associated with African sleeping sickness. These findings support the idea that BMAP-18 should be explored as a candidate for therapy of economically important trypanosome-infected hosts, such as cattle, fish and humans, and for paratransgenic expression in Sodalis glossinidius, a bacterial symbiont in the tsetse vector, as a strategy for interference with trypanosome transmission.

      Author Summary

      Protozoan parasites cause serious diseases in large areas of the tropics. Control of these diseases depends to a great extent on the use of therapeutic drugs, many of which are highly toxic. In addition, parasite resistance to several of the front-line drugs is increasing. Host defense peptides (HDP; formerly called antimicrobial peptides) have recently received attention as potential anti-parasite effector molecules. We earlier reported that one such peptide, bovine myeloid antimicrobial peptide (BMAP-27), is a potent inhibitor of the growth of trypanosomes and Leishmania in vitro. Here we report our studies on BMAP-18, a truncated form of BMAP-27, which showed reduced toxicity to mammalian and insect cells and yet retained its direct toxicity to parasites in vitro. BMAP-18 also strongly inhibited LPS-induced release of tumour-necrosis factor alpha (TNF-α) from human leukocytes, and thus has immunomodulatory activity. These findings suggest that BMAP-18 has potential as a therapeutic agent for treatment of infected animals or as an inhibitor of parasite transmission by their insect vectors. In anticipation of using BMAP-18 in vivo, we have also developed high affinity antibodies to BMAP-18 and have shown that these can be used, in conjunction with mass spectrometry, to detect the peptide in whole blood or plasma.

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      Most cited references 60

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      Drug resistance in leishmaniasis.

      Leishmaniasis is a complex disease, with visceral and cutaneous manifestations, and is caused by over 15 different species of the protozoan parasite genus Leishmania. There are significant differences in the sensitivity of these species both to the standard drugs, for example, pentavalent antimonials and miltefosine, and those on clinical trial, for example, paromomycin. Over 60% of patients with visceral leishmaniasis in Bihar State, India, do not respond to treatment with pentavalent antimonials. This is now considered to be due to acquired resistance. Although this class of drugs has been used for over 60 years for leishmaniasis treatment, it is only in the past 2 years that the mechanisms of action and resistance have been identified, related to drug metabolism, thiol metabolism, and drug efflux. With the introduction of new therapies, including miltefosine in 2002 and paromomycin in 2005-2006, it is essential that there be a strategy to prevent the emergence of resistance to new drugs; combination therapy, monitoring of therapy, and improved diagnostics could play an essential role in this strategy.
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        QUANTITATIVE STUDIES OF THE GROWTH OF MOUSE EMBRYO CELLS IN CULTURE AND THEIR DEVELOPMENT INTO ESTABLISHED LINES

        Disaggregated mouse embryo cells, grown in monolayers, underwent a progressive decline in growth rate upon successive transfer, the rapidity of the decline depending, among other things, on the inoculation density. Nevertheless, nearly all cultures developed into established lines within 3 months of culture. The first sign of the emergence of an established line was the ability of the cells to maintain a constant or rising potential growth rate. This occurred while the cultures were morphologically unchanged. The growth rate continued to increase until it equaled or exceeded that of the original culture. The early established cells showed an increasing metabolic autonomy, as indicated by decreasing dependence on cell-to-cell feeding. It is suggested that the process of establishment involves an alteration in cell permeability properties. Chromosome studies indicated that the cells responsible for the upturn in growth rate were diploid, but later the population shifted to the tetraploid range, often very rapidly. Still later, marker chromosomes appeared. Different lines acquired different properties, depending on the culture conditions employed; one line developed which is extremely sensitive to contact inhibition.
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          Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication.

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            Author and article information

            Affiliations
            [1 ]Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada
            [2 ]Department of Biology, University of Victoria, Victoria, British Columbia, Canada
            [3 ]Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada
            National Institute of Allergy and Infectious Diseases, United States of America
            Author notes
            [¤]

            Current address: Liverpool School of Tropical Medicine, Liverpool, United Kingdom

            Conceived and designed the experiments: LRH TWP. Performed the experiments: LRH JMT AMJ BAE MR CNW BG. Analyzed the data: LRH JMT AMJ BAE MR TWP. Contributed reagents/materials/analysis tools: BG REWH TWP. Wrote the paper: LRH JMT REWH TWP.

            Contributors
            Role: Editor
            Journal
            PLoS Negl Trop Dis
            plos
            plosntds
            PLoS Neglected Tropical Diseases
            Public Library of Science (San Francisco, USA )
            1935-2727
            1935-2735
            February 2009
            3 February 2009
            : 3
            : 2
            2628741
            19190729
            08-PNTD-RA-0325R2
            10.1371/journal.pntd.0000373
            (Editor)
            Haines et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
            Counts
            Pages: 13
            Categories
            Research Article
            Biochemistry/Protein Chemistry
            Cell Biology/Cell Growth and Division
            Immunology/Immunity to Infections
            Infectious Diseases/Antimicrobials and Drug Resistance

            Infectious disease & Microbiology

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