Antimicrobial Peptides and Their Use in Medicine - Springer Link

ISSN 00036838, Applied Biochemistry and Microbiology, 2010, Vol. 46, No. 9, pp. 803–814. © Pleiades Publishing, Inc., 2010. Original Russian Text © V.N. Lazarev,V.M. Govorun, 2010, published in Biotekhnologiya, 2010, No. 3, pp. 11–25.

Antimicrobial Peptides and Their Use in Medicine V. N. Lazarev and V. M. Govorun Federal State Research Institute of Physicochemical Medicine of the Federal MedicoBiological Agency, Moscow, 119435 Russia email: [email protected] Received April 23, 2010

Abstract—The review presents the current classification of antimicrobial peptides (AMP), which are the main component of innate immunity. The mechanism of their action and the molecular basis of the forma tion of resistance towards these peptides are described. Data on the use of AMP for the treatment of various infectious diseases, as well as the state of the art in genetic therapy using AMP, are given. Key words: antibiotic resistance, antimicrobial peptides, infectious diseases. DOI: 10.1134/S0003683810090012

Antimicrobial activity of human blood, leukocytes, and lymphoid tissue was discovered in the 1850s; in the 1920s–1950s, a multitude of antimicrobial substances was isolated from different human tissues. Their selec tive activity towards both grampositive and gram negative bacteria was demonstrated [1]. These sub stances included, for instance, basic proteins of high molecular weight, linear basic polypeptides, and the bacteriolytic substance from nasal mucosa which was later named lysozyme. The basic proteins of high molecular weight were found to be histone fractions and protamine: thus, the first step in the studies of humans’ innate resistance to microorganisms was made. James Hirsch was the first to obtain a purified antimicrobial substance from the extract of phagocyte granules [2]. Later, research groups led by Hans G. Boman, Michael Zasloff, and Robert I. Lehrer inde pendently isolated cecropins from insect cells [3], magainins from amphibians [4], and defensins from mammals [5]. It is presently known that already 2.6 million years ago, long before the appearance of adaptive immunity possessing the characteristics of specificity, memory, and complexity, a simple nonspecific system of innate immunity existed; even now this system is the main “protective barrier” and sometimes even an “attacking weapon” of all living organisms. The following char acteristics are essential for the innate immunity: quick response, “redundancy,” and multifunctionality. The antimicrobial function of the innate immunity usually relies on short, in most cases, positively charged pep tides (AMPs) active against both grampositive and gramnegative bacteria, fungi, and some viruses [6–8]. Abbreviations: AMPs—antimicrobial peptides, cAMPs—cat ionic AMPs, SIC—complement inhibitor.

More than 2000 antimicrobial peptides have been isolated from various sources and identified by now. These are peptides produced by various tissues and cell types of invertebrates, plants and animals [9–11], basic cytokines and chemokines [12, 13], some neu ropeptides and hormones [14, 15], and fragments of high molecular weight proteins [16, 17]. Databases of antimicrobial peptides are steadily expanded, espe cially due to the discovery of new fragments of high molecular weight proteins. The addresses of some of the databases of antimi crobial peptides sequences are given below: AMSDb—http://www.bbcm.univ.trieste.it/~tossi/ amsdb.html APD—http://www.aps.unmc.edu/AP/main.php RAPD—http://faculty.ist.unomaha.edu/chen/rapd/ index.php AMPer—http://www.cnbi2.com/cgibin/amp.pl Antimicrobial peptides receive growing attention as pharmaceuticals for the treatment of antibiotic resistant bacterial infections and septic shock [18, 19]. Besides, the potential of using these peptides as antivi ral [20] and antitumor drugs [21] is investigated. Further studies on the mechanism of action of antimicrobial peptides both in model membrane sys tems and in various microorganisms will allow for the determination of the parameters of optimal antimi crobial activity. STRUCTURAL CLASSIFICATION OF THE ANTIMICROBIAL PEPTIDES AMPs can be divided into several subtypes accord ing to their amino acid composition and structure. The vast majority of AMPs is formed from high molec ular weight precursors. Posttranslational modifica

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tions include proteolytic processing, glycosylation [22], carboxy terminal amidation, amino acid isomer isation [23], and halogenation [24]. Anionic antimicrobial peptides are small (720– 830 Da) peptides found in surfactant extracts, bron choalveolar lavage, and airway epithelium cells [25]. They are synthesized in millimolar concentrations, require zinc as the cofactor of antimicrobial activity, and suppress both grampositive and gramnegative bacteria. Their structure is similar to that of neutral zymogen propeptides of high molecular weight [26]. This family includes maximin H5 from amphibians [27]; small anionic peptides rich in glutamic and aspartic acid which were isolated from ovine, bovine, and human tissues [28]; and human dermcidin [29]. Cationic amphipathic αhelical peptides include approximately 300 short peptides containing less than 40 amino acid residues. Lack of cysteine residues in the molecule is characteristic of peptides of this group. These peptides have a disordered structure in aqueous solutions, but in the presence of trifluoroethanol, sodium dodecylsulfate, and liposomes, the whole molecule or a part of it is converted into an α – helix[30]. In the presence of 15 mM HC O 3 and 2–

S O 4 , the peptide molecules acquire an ordered heli cal structure [31]. The antibacterial activity of pep tides of this family was shown to grow concomitantly to the increase of structural order [32]. The family of cationic αhelical peptides includes, for instance, cecropin A, melittin from bee venom [33], magainins from amphibian tissues [4], and the human peptide LL37 [34]. Cationic peptides rich in amino acid residues of a certain type (approximately 45 members). This family includes bactenecins and the peptide PR39, which contains proline (33–49%) and arginine (13–33%) residues, porcine prophenin (57% proline residues and 19% phenylalanine residues), and bovine indoli cin rich in tryptophan residues [30]. Besides an increased content of amino acid residues of a certain type, the characteristic features of these peptides include a linear structure and the absence of cysteine residues. Cysteinecontaining anionic and cationic peptides forming disulfide bonds. This family contains several hundred peptides containing cysteine bonds capable of forming disulfide bonds, for example, protegrin iso lated from pig leucocytes (it contains 16 amino acid residues, including four cysteine residues forming two intramolecular disulfide bonds) [35], and the numer ous defensin family. More than 55 αdefensins have been isolated. The peptides from human neutrophils contain 29–35 amino acid residues, including six cys teine residues forming three intramolecular disulfide bonds [36]. Besides, this family includes 54 defensins isolated from insect tissues and 58 plant defensins. θ defensin from rhesus macaque is especially interesting

from the structural point of view: it is a circular mole cule of 18 amino acid residues with three disulfide bonds [37]. Other AMPs are fragments of high molecular weight proteins. They have antimicrobial activity and the structure analogous to that of peptides belonging to the families described above; however, their function in the defense of the organism has not been completely elucidated yet. This group includes lactoferricin (a frag ment of lactoferrin), caseicidin I from human casein, and the antimicrobial domains of human hemoglobin, bovine αlactalbumin, and ovalbumin. Beside the structural classification, other AMP classifications exist. Antimicrobial peptides can be classified according to their origin, biosynthesis mechanism, localization, biological function, mecha nism of action, activity, and specificity. New AMPs, either isolated from natural sources or recombinant, chimeric, or synthetic, appear in databases each month. Accordingly, AMP classification may be mod ified; therefore, this is a dynamic classification. THE MECHANISM OF AMP ACTION A wide range of methods and instruments is used to study the mechanism of AMP action. However, a uni fied technique for the adequate determination and formulation of the mechanism of activity of various AMPs is currently not available. It is beyond doubt that the selective destruction of the cell membrane and the amphipathic structure of these peptides play an important role in the realization of this mechanism; however, the number of studies demonstrating an alternative, noncytolytic mechanism of AMPO action is increasing. The presence of polar phospholipid heads on the cell membrane and the charge distribution in the pep tide are the key factors in the peptide–membrane interaction [38]. A constantly increasing number of facts show that strongly hydrophobic AMPs recognize the anionic lipids exposed on the outer surface of the bacterial membrane [39]. In the eukaryotic cells, these lipids line the cytoplasmatic side of the membrane. This fact may account for a higher cytolytic activity of AMPs towards bacterial cells than towards eukaryotic cells. Generally speaking, the death of a bacterium due to the formation of pores in the bacterial membrane involves three processes: the binding of AMPs to the bacterial membrane, their aggregation inside the membrane, and the formation of pores causing the leakage of cell contents and the death of the cell. At the first stage, AMPs must pass through the negatively charged outer membrane of gramnegative bacteria which contains lipopolysaccharides or the outer mem brane of grampositive bacteria which contains acidic polysaccharides [40]. The metabolic activity of the target bacteria is often an important factor of pore for mation [41].

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ANTIMICROBIAL PEPTIDES AND THEIR USE IN MEDICINE

(a)

(b)

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(c)

Fig. 1. The models of the formation of pores in the bacterial membrane due to the action of AMPs: a—“barrelstave,” b—tor oidal pore formation, and c—“carpet” formation. The hydrophilic part of the AMP is shown in red, the hydrophobic part is shown in blue ([45]).

Several models explaining the increase in mem brane permeability due to AMP action exist currently. The three most popular models are the “barrel–stave” model, the model of toroidal pore formation and the “carpet” model. The “barrel–stave” model. According to this model, pore formation involves the formation of AMP dimers and multimers after the binding of AMP to the negatively charged bacterial membrane (Fig. 1a). Pep tide assembly is a critical step of pore formation: AMP multimers penetrate the membrane with their hydro phobic part facing the bilayer and their hydrophilic part forming internal lumen pores. The assembled peptide molecules inside the pore have a barrellike structure. The addition of monomeric molecule causes an increase in pore size leading to the leakage of cell contents and cell death. The mechanism of action of alamethicin from the peptaibol family is described by this model. This peptide forms an open barrellike pore consisting of 3–11 helical rods [42]. The Model of Toroidal Pore Formation. The way of toroidal pore formation is similar to that of the mech anism of pore formation in the “barrelstave” model (Fig. 1b); a distinctive feature of this model is the link ing (fixation) of the inner and outer lipid layers by the peptides included into the lipid bilayer. Currently this model is applied to the majority of AMPs, for exam ple, to melittin [43]. The “carpet” model. In this model (Fig. 1c) the peptides cover the surface of the outer membrane in a carpetlike fashion at the first step and then act as detergents, destroying the bilayer with concomitant APPLIED BIOCHEMISTRY AND MICROBIOLOGY

pore formation after the threshold concentration is reached. The pores are filled with “micellelike units” consisting of shell formed by AMP molecules and membrane fragments inside this shell. This model was first used to describe the action of dermaseptins (AMPs from amphibian skin) [44]. Other models of AMP action include socalled “molecular electroporation.” Some peptides can gen erate an electrostatic potential sufficient for the genera tion of pores according to the electroporation mecha nism. Pore formation requires a considerably high charge density which is achieved due to a high content of cationic amino acid residues in the peptide [46]. The “sinking float” model is also worth mentioning. The equilibrium in the lipid bilayer is destroyed after AMP penetration into it. Such peptides can form temporary pores which are still lethal for the bacterium [47]. Notwithstanding the multitude of models, the mechanism of action of many AMPs still remains unknown. Besides, some peptides can suppress micro bial growth according to several different mechanisms. Regardless of the model, the action of AMPs results in cell death. As already mentioned above, the bactericidal prop erties of most AMPs are due to the formation of pores in the lipid bilayer; however, alternative mechanisms of AMP action were put forward recently [45]. For example, defensins and cathelicidins can inactivate bacterial lipopolysaccharides by binding to a certain part of this molecule [48]. Many peptides act inside the microorganism’s cell by inhibiting certain intrac ellular processes. The model of “canal joining” postu

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LAZAREV, GOVORUN (a)

(b)

SIC

cAMP

protease

cAMP

(c)

(d)

cAMP

MtrCDE

cAMP

Fig. 2. Mechanisms of internal resistance of bacterial pathogens to cationic antimicrobial peptides (cAMP): a— linear αhelical cAMPs (for example, peptide LL37) are sensitive to proteolysis and targeted by many microbial proteases, b—the contact of cAMPs with the cell mem brane can be blocked by secreted bacterial proteins, such as complement inhibitor proteins from streptococci or Staphylococcus aureus staphylokinase, c—some cAMPs can be actively transported from the bacterial cell by extru sion, mediated, for example, by the multidrug resistance protein MtrCDE in Neisseria gonorrhoeae and Neisseria meningitides, and d—bacteria can also decrease the total negative charge on the cell wall, decreasing its affinity to AMPs [57].

lates the transport of peptides through the lipid bilayer without the formation of stable pores. Some peptides inhibit DNA synthesis [49], or protein biosynthesis [50], or both processes [51]. For example, histatin tar gets the mitochondria of fungal pathogens [52]. On the other hand, some authors state that AMPs can interfere with the pathogen’s metabolism, in addition to their direct cytolytic action. Sometimes they can trigger the synthesis of virulence factors, for example, hyaluronic acid and capsular polysaccharide [53]. Members of different classes of AMPs are listed in Table 1. MECHANISM OF FORMATION OF MICROORGANISM RESISTANCE TOWARDS AMPs It is currently believed that a transformation of a bacterial strain previously resistant to AMPs into a resistant strain is difficult, if possible at all. Innate

resistance of microorganisms to these peptides is also a very rare phenomenon. The resistance of several human pathogens towards AMPs is now regarded as one of the most important factors of their virulence. Bacteria resistant to AMPs are often very resistant to the standard antibiotics [54]. There are two mechanisms of resistance: internal (constitutive) and adaptive (inducible). Some of the constitutive resistance mechanisms are the following: loss of electrostatic affinity to AMPs, change of mem brane energetic, and electrostatic shielding. The main mechanisms of the formation of internal resistance of bacteria towards AMPs are described below (Fig. 2). The anionic molecules, such as peptidoglycan, teichoic acid, lipid A, or phospholipids, in the bacte rial cell wall are synthesized in the course of compli cated biological processes, and it is highly doubtful that bacteria can substitute them with molecules hav ing a lower affinity to AMPs. Nevertheless, many bac teria can modify the above mentioned compounds, causing a decrease of the total negative charge on the membrane and in the affinity of the bacterial cell wall to AMPs. For example, teichoic acid polymers found in the cell wall of grampositive bacteria carry a strong negative charge [55]. Staphylococcus aureus, Strepto coccus agalacticae, Listeria monocytogenes, and some other bacteria can, however, partially neutralize this negative charge by modifying the teichoic acid by D alanine residues carrying a positively charged amino group (this process is mediated by proteins coded by the genes of the dltABCD operon) [56]. Staphylococcus aureus uses a similar mechanism to change the negative charge of phosphatidylglycerine to a positive charge by modification with Llysine [58] (Fig. 2, model d). The gene coding for the protein MprF catalyzing this modification is present in many bacterial genomes and plays a key role in the putative strategy of the spreading of the resistance towards AMPs. A similar mechanism is used by Salmonella enteric and Pseudomonas aeruginosa when lipid A, a conservative integral membrane component of gram negative bacteria, is modified by positively charged aminoarabinose molecules [59]. Inactivation of the genes responsible for the modi fication of the cell wall by Dalanine, Llysine, or aminoarabinose makes the affected bacterial strains highly sensitive to AMP and other defense factors, such as lysozyme or secreted A2 phospholipase of the group IIa, and causes a complete attenuation of viru lence [60]. It is necessary to note that any bacterium with a cell wall thus modified remains sensitive to higher concentration of AMP because cell wall neu tralization is always incomplete. The ability of bacteria to reduce the negative surface charge is obviously lim ited. Many AMPs have been shown to act efficiently against dltABCD and mprFpositive strains of S. aureus [61]. This may contradict the belief that a strengthen


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RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLGRPFPa RRRPRPPYLRPRPPPFFPPRLPPRIPPGFPPRFPPRFPa ILPWKWPWWPWRRa GNNRPVYIPQPRPPHPRI VDKGSYLPRPTPPRPIYNRN DSHAKRHHGYKRKFHEKHHSHRGY

QKLC1QRPSGTWSGVC2GNNNAC3KNQC4IRLEKARHGSC2NYVFPAHC3IC4YFPC1 DC1SGRYKGPC2AVWDNETC3RRVC4KEEGRSSGHC2SPSLKC3WCjEGC4 DTHFPIC1IFC2C3GC4C1HRSKC2GMC3C4KT

DC1YC2RIPAC3IAGERRYGTC2IYQGRLWAFC3C1 NPVSC1VRNKGIC2VPIRC3PGSMKQIGTC2VGRAVKC1C3RKK GFC1RC2LC3RRGVC3RC2IC1TR ATC1DLLSGTGINHSAC2AAHC3LLRGNRGGYC2NGKAVC3VC1RN TTC1C2PSIVARSNFNVC3RIPGTPEAIC3ATYTGC2IIIPGATC1PGDYAN

RWC1FRVC2YRGIC2YRKC1 Ra RSVC1RQIKIC2RRGGC2YYKC1TNRPY RGGRLC1YC2RRRFC2VC1VGRa

RLCRJWIRVCR GSKKPVPIIYCNRRTGKCQRM VNPIILGVLPKVCLITKKC FLGGLIKIVPAMICAVTKKC SMLSVLKNLGKVGLGFVACKINKQC GIFSKLGRKKIKNLLISGLKNVGKEVGMDWRTGIDIAGCKIKGEC

KWKLFKKIEKVGQNIRDGIIKAGPAVAWGQATQIAKa GIGKFLHSAKKFGKAFVGEIMNS GIGKFLKKAKKFGKAFVKILKKa ALWKTMLKKLGTMALHAGKAALGAAADTISQGTO LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES TRSSRAGLQFPVGRVHRLLRK

Sequence

Cow Pig Cow Bee Insects Human

Radish Drosophila Human

Human Cow Monkey Insects Plants

Horseshoe crab Scorpio Pig

Cow Insects Rana frog Same '' ''

Silkworm Frog Synthetic Frog Human Vertebrates

Source

BC BC BC H H Saliva

Seeds, E H Liver

BC, E E, BC BC E, BC E

BC H BC

BC BC E E E E

E, BC H E E E, BC E

Tissue

Notes: Pairs of cysteine residues (C) are indicated with numbers; Cterminal amides are indicated with a; the terminal amino acid residues in q=defensin are joined with a peptide bond; BC—blood cels; H—hemolymph cells; E—epithelial cells.

Linear nonhelical Bac 5 PR39 Indolicidin Epidecin Pirrocoricin Histatin 5

Containing four disulfide bonds Defensin Drosomycin Hepcidin

Containing three disulfide bonds αDefensin (HNP3) βDefensin (TAP) θDefensin (HNP3) Sapecin A Thionin (crambin)

Containing two disulfide bonds Tachyplesin Androctonin Protegrin 1

Containing one disulfide bond Bactenecin 1 Tanatin Brevicin 1T Ranalexin Ranateurin 1 Esculentin 1

αHelical Cecropin A Magainin 2 Pexiganan Dermaseptin 1 LL37 Buforin 2

Peptides

Table 1. Peptides isolated from various natural sources (*)

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LAZAREV, GOVORUN

ing of cationic properties of the AMPs is a result of the evolution of these peptides targeting the bacteria which become resistant due to the neutralization of the surface charge. The human defensin hBD3 has a potential antimicrobial activity against S. aureus and is known to carry a higher negative charge (+10) than the other cAMPs. Paradoxically, a high quantity of positively charged amino acid residues causes a decrease in peptide hydrophobicity. In fact, the action of hBD3 on membranes is weaker than that of the other defensins, but it can also act according to a dif ferent mechanism, activating bacterial enzymes that cleave the cell wall during cell division (autolysins), by displacing them from the anchoring sites in the cell wall [62]. As mentioned above, another type of microorgan ism resistance towards AMPs is inducible. Although the structure of “AMP traps” and “AMP extruders” is still not completely elucidated, it is clear that they rec ognize certain sequences and structural motifs in the AMPs. Accordingly, any slight changes in the peptide sequence have distinct effects on the level of resis tance. For example, SIC protein of S. aureus provides for a moderate resistance towards human βdefensins 2 and 3 (hBD2 and hBD3) but not towards βdefensin 1 (hBD1) [63]. The transporter protein MtrCDE removes the peptide protegrin 1 from the cells but is inactive towards tachyplesin having a similar struc ture; neutrophil defensins also cannot serve as sub strates for MtrCDE [64]. Generally speaking, inducible resistance is based on the activation of factors necessary for microorgan ism survival in the presence of sublethal AMP doses. This mechanism is often based on the functioning of twocomponent systems, for example PhoP/PhoQ of gramnegative bacteria. The mechanism involves extracellular structural modification, protease activa tion, extrusion activation, and the modification of the extracellular targets of the AMPs [65]. AMPs can function as ligands of the bacterial sensory kinase PhoQ for the initiation of virulence and adaptive response. Consequently, the therapeutic use of AMPs can aggravate the infection process due to an increase of virulence and selection of resistant mutants. On the other hand, the understanding of the mechanisms of inducible resistance lays the foundation for the opti mization and selection of novel AMPs. For example, the recent investigation of the genemediated response of the nosocomial pathogen Staphylococcus epidermi dis to hBD3 allowed for the identification of a unique threecomponent AMPsensing system controlling resistance development in grampositive bacteria [66]. The components of this system are promising targets for novel antimicrobial preparations. It is necessary to note that only a small number of amino acid residues in most AMPs are necessary for the antimicrobial action, while other residues can be substituted without an effect on the functional activity

of the peptide. Many verterbrates synthesize multiple AMPs with a similar structure. The presence of five different hepcidins in flatfish [67] and only one such peptide in the human organism, as well as the presence of 26 αdefensins (cryptdins) in mouse cells and only two such peptides in human cells [69], are typical examples of the variations in cAMP number in differ ent species. The presence of multiple structurally sim ilar peptide variants is one of the obstacles towards the development of microorganism resistance to AMPs. THERAPEUTIC USE OF AMPs Antimicrobial peptides are currently among the most promising agents for the treatment of infections. The situation is paradoxical: after several decades of galloping development of antibiotic chemistry, we are returning to antimicrobial peptides—the most ancient biological substances. The motive for the search for new antimicrobial substances is certainly the widespread resistance of microorganisms to the currently used antibiotics [70]. Pharmaceutical com panies continuously modify the existing antibiotics and create new ones. Unfortunately, it is evident now that the rate of synthesis of the new antibiotics is lower than the rate of resistance and multiresistance devel opment in microorganisms. It is doubtless that antimicrobial peptides have sev eral advantages over antibiotics, namely: (1) Limited capacity for the development of resis tance against AMP due to the peculiarities of the mechanism of antimicrobial action. (2) Local application of the drug. (3) Wider spectrum of antimicrobial action. (4) Activity in the nanomolar concentration range. (5) Unlimited possibilities of chemical synthesis of AMP analogs with modified biological properties including decreased toxicity for the eukaryotic cell. Theoretically, antimicrobial peptides can be used for the therapy in several different ways [71]: as indi vidual antimicrobial preparations, in combination with conventional antibiotics in order to achieve an additive or synergistic effect, for the stimulation of the innate immune system, and for the neutralization of endotoxins in order to prevent complications, includ ing septic shock caused by bacterial virulence factors. The use of AMPs as individual preparations is cur rently considered the most promising strategy, though the use of these peptides as endotoxinneutralizing agents also receives a considerable attention. Chemically and structurally diverse antimicrobial peptides are currently considered an alternative to conventional antibiotics. As mentioned above, the probability of the development of resistance and/or side effects during the use of AMPs is much lower than in the case of antibiotics; therefore AMPs are cur rently used for the development of novel antibacterial, antifungal, and antiviral preparations [72–74], prepa


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rations for the treatment of infections caused by para sites [75], and antitumor drugs [76, 77]. The therapeu tic use of AMPs as antimicrobial drugs is considered below in more detail. INDUCTION OF AMP EXPRESSION Proinflammatory cytokines are known to induce the endogenous expression of AMP genes [78, 79]. Schlee and coauthors studied the stimulating effect of the probiotic Escherichia coli strain Nissle, 1917, on the expression of hBD2 and identified the bacterial factor responsible for the induction [80]. The stimula tion in vitro took place in the presence of flagellin. In an attempt to elucidate the currently unknown rela tionship between psychological stress and an increased sensitivity to infection, Alberg and coauthors showed that stress causes an increase in the concentration of two key AMPs (cathelinlike AMP and βdefensin) in mouse skin upon an increase in endogenous glucocor ticoid production [81]. These data confirm the hypothesis concerning the blockade of the normal antibiotic function of the skin by glucocorticoids upon psychological stress. Raqib and coauthors (see [82] recently suggested an alternative treatment method for acute shigellosis [82]. Their experiments on animals showed that oral use of sodium butyrate can have a therapeutic effect due to the induction of endogenous AMPs and their secretion into the colon and rectum. Data on the influence of the hormonally active form of the vitamin D3 (1,25(OH)2D3) on the expression of cathelicidins both in normal and fibrous bronchial cells were presented in [83]. Vitamin D also stimulates the expression of cathelicidins active against airway pathogens, such as Bordetella bronchiseptica and Pseudomonas aeruginosa. It is important to elucidate the mechanism of innate immunity induction before the start of clinical trials. AMPS IN THE THERAPY OF INFECTIOUS DISEASES Cathelicidins—antimicrobial compounds of a new class—occupy the leading positions in infection treat ment. The antibacterial effect of these peptides is due to an increase in membrane permeability and binding to the membrane polysaccharide. Due to these prop erties, many cathelicidins are capable of decreasing the lethality in a septic shock model after intravenous infusion in rats and in staphylococcal sepsis models in mice after parenteral infusion [84]. Cathelicidin LL 37 and its orthologue CRAMP are the key defense fac tors in E. coli infections of mouse urogenital tract [85]. The role of LL37 in the pathogenesis of different infections is now actively discussed. The protective effect of LL37 in the case of lethal sepsis caused by grampositive bacteria was demonstrated [86]. Cathe licidins efficiently contribute to the healing of stomach APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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ulcers in rats due to the stimulation of cell prolifera tion and angiogenesis [87]. Phase III of clinical studies of a synthetic peptide MBI226 as a preparation for the prevention of infec tious complications after blood vessel catheterization [88]. Unfortunately, the trials were unsuccessful. The study involving 1400 patients from 29 clinics in the United States did not give statistically significant proof of a higher efficiency of this AMP compared to the povidoneiodine preparation (control). Nevertheless, the use of MBI226 gave two statistically significant results: lower colonization of the catheter by microf lora (p = 0.002) and a decrease in the number of cases of local infection (p = 0.004). The data on clinical tests of AMPs and some of their properties are given in Table 2. Another variant of indolicidinlike peptides— MBI 594AN—was designed for the topical treatment of acne. The Propionibacterium acne bacteria are most often associated with this disease; the resistance of these bacteria to antibiotics is growing dramatically. Most isolates are constitutively resistant to erythromy cin, clindamycin, and other antibiotics [89]. Preclinical studies of MBI 594AN demonstrated an excellent effect against both sensitive and resistant P. acnes strains in vitro; the preparation applied topi cally was nontoxic for animals. Topical application of the cream containing 2.5% of MBI 594AN twice a day in the phase IIb of clinical studies involving 255 patients from nine centers caused a statistically signif icant decrease of inflammation (p < 0.004) relative to control after 6 weeks of treatment [71]. Antimicrobial activity of a peptide K4S4(1–15)a from frog skin towards oral cavity pathogens associ ated with caries and periodontitis was demonstrated in vitro. The results confirmed high activity of this pep tide against strains of Streptococcus mutans, Strepto coccus sobrinus, Lactobacillus paracasei, and Actyno myces viscosus resistant to the peptide LL37 [90]. Various (nonlinear αhelical) variants of histatin P113 (Table 2), cationic dodecapeptide derivatives of histatin 5 found in human saliva, are also worth men tioning [91]. P113 is highly active in vitro against Can dida albicans and many grampositive and gramneg ative bacteria. P113 was used in HIVinfected patients having oral candidosis as a component of a mouth wash in the phase I/II of clinical trials. The use of this peptide was shown to decrease the symptoms of gingi vitis and gum bleeding without any side effects. The possibility of using these peptides in mucoviscidosis patients for the treatment of lung infections caused by Pseudomonas aeruginosa is under consideration. Protegrin1, a member of the θdefensin family, may become a very promising antimicrobial agent in patients with mucoviscidosis complicated by lung infections [92]. The efficiency of an aerosol prepara tion containing a synthetic analog of protegrin was demonstrated in phase I of clinical trials in these

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Cream

Application

Planned clinical trials


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Neuprex (rBP121)

XMP.629

Histatin (human)

Xoma (US) Berkeley, (Cali Same fornia, USA)

Xoma (US) Berkeley, (Cali BPI (bactericidal fornia, USA) permeability increasing protein histatin (human)

P113P113D Demegen, Pittsburgh, PA Dow Pharmaceutical Sci ences, Patuloma (Califor nia, USA)

Same

Injections

BPIcationic, Cream 450 amino acid res idues

Nonlinear αheli Mouthwash cal peptide

Phase III of clinical trials (treatment Phase I/II of clinical trials for a of meningococcemia in children) was decrease in complications after unsuccessful heart surgery in children

Phase III of clinical trials (topical No treatment of acne) was unsuccessful

Phase II of clinical trials (mouthwash As an inhaled form for the in HIVinfected patients with candi treatment of pseudomonas dosis) was finished successfully infections in mucoviscidosis patients

Phase IIb of clinical trials demon Phase III of clinical trials strated the efficiency of the peptide in topical acne treatment

Indolicidin Nonlinear αheli Cream (cow erythrocytes) cal peptide

MBI 594AN Microbiologix Biotech Vav couver, BC, (Canada)

Possible repetition of phase III

Phase III of clinical trials (topical application for the prevention of bloodstream infections caused by catheterization) was unsuccessful

Synthetic analogue Nonlinear αheli Cream of indolicidin cal peptide

Phase III of clinical trials (mouth Phase IIa, as an aerosol for the wash in stomatitis, use as an aerosol in treatment of chronic respira pneumonia) was unsuccessful tory infections in mucoviscido sis patients

Phase III of clinical trials did not No demonstrate any advantages over conventional antibiotics for the treat ment of impetigo and diabetic ulcers. Not approved by FDA

Clinical trial results

Omiganan Microbiologix Biotech Vav (MBI226) couver, BC, (Canada)

Peptide containing Mouthwash two disulfide bonds

Intrabiotics Pharmaceuticals Protegrin Inc. Mountainview, (Cali (pig leucocytes) fornia, USA)

Structure

Iseganan (IB367)

AMP type (source

Genaera Plymouth Meeting, Magainin 2 αHelix PA (known previously as (Xenopus frog skin) Magainin Pharmaceutical Inc.)

Manufacturer

Pexiganan (MSI78)

Name

Table 2.

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patients [93]. It is necessary to note that a high con centration of salt ions on the mucosa surface in muco viscidosis [94] patients inhibits the action of saltsen sitive AMPs, and, therefore, it is necessary to design analogs of these peptides resistant to media of high ionic strength. GENE THERAPY OF INFECTIOUS DISEASES USING ANTIMICROBIAL PEPTIDES Notwithstanding the obvious advantages of antimi crobial peptides, the therapeutic use of these sub stances, either synthetic or isolated from natural sources, is currently limited. The main obstacles are high production cost, high toxicity towards human cells and low selectivity of AMPs. Recently, antimicrobial peptides were used as the agents of gene therapy due to a wide spectrum of bio logical activity. One of the first studies reported the intratracheal injection of a recombinant adenovirus carrying a gene coding for a peptide LL37 of the cathelicidin family into mice [95]. The survival rate of injected animals after an infection with Pseudomonas aeruginosa PAO1 or E. coli CP9 was higher than that of control animals. The results of this successful study were later con firmed by other studies [96, 97]. The expression of the gene coding for the peptide PR39 (belonging to the cathelicidin family from pig skin) or LL37 was shown to inhibit an infection caused by group A streptococci in mice. The antimicrobial peptides were delivered using lentivirus vectors. Successful delivery of a gene coding for the peptide LL37 into an isolated organ in mucoviscidosis was demonstrated as early as 1999 [98]. However, AMPs are known to cause side effects, influencing chemotaxis and angiogenesis in macroor ganisms [99, 100]. Therefore, the peptide cathelicidin was expressed as a precursor in this model. Another successful gene therapy scheme involved the delivery of the gene coding for the antimicrobial peptide elafin into the lungs of mice infected by S. aureus [101] using recombinant adenoviruses. Currently, an opinion about the advantages of use the genes of antimicrobial peptides over the use of chemically synthesized peptides is being formed. The study demonstrating a higher efficiency of the subcu taneous injection of the recombinant adenovirus expressing the gene of human cathelicidin hCAP 18/LL37 compared to the use of a synthetic peptide in the treatment of burn wounds [102] can serve as an example. In addition to the treatment of infectious diseases, AMPs are used in gene therapy of other pathologies, such as atopic dermatitis, Crohn’s disease, mucovisc idosis, and tumors [103–105]. It is necessary to note that most studies dealing with the gene therapy of infectious disease using AMP genes encounter the cytotoxic action of peptides on APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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macroorganism cells. Systems of precise regulation of the expression of AMP genes can be used to solve this problem. Such a system was used for the first time in a proce dure of gene therapy of mycoplasmoses and chlamydi oses in laboratory animals [106]. A plasmid vector expressing the gene of a linear αhelical cationic pep tide melittin controlled by a tetracyclinedependent promoter of human cytomegalovirus was intravagi nally introduced into mice. Chlamydia and myco plasms were eliminated from more than 2/3 of the infected animals. The same vector was successfully used for aerosol injection in broiler chickens infected with Mycoplasma gallisepticum [107]. An increase in pathogen resistance to antibiotics is observed in the whole world during recent years. The appearance of antibiotic resistance is a natural biolog ical response to the use of antibiotics which create a selective pressure promoting the selection, survival, and proliferation of resistant microorganism strains. Antibiotic resistance has a great social and economical impact and is regarded as a threat to national security by developed countries. Infections caused by resistant strains are characterized by prolonged duration, more often require hospitalization, increase the duration of inpatient treatment and worsen the prognosis. In case when specific antibiotics are inefficient, it is necessary to use drugs of second and third line which are often more expensive, less safe, and not always available. All this increases direct and indirect economical costs, as well as the risk of resistant strain spreading in the pop ulation. During the last 5 years, the pharmaceutical indus try spent more than 30 billion dollars for the develop ment of new antibiotics. If the resistance of microor ganisms to drugs develops quickly enough, most of these investments may prove to be useless (http:// www.who.int/topics/drug_resistance/en). Accord ingly, the development of alternative therapeutics, the resistance to which is lacking or limited, becomes a question of great importance. Antimicrobial pep tides—a unique and wonderfully diverse group of compounds constituting the main component of innate immunity—can be potentially used as such therapeutics.

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REFERENCES 1. Skarnes, R.C. and Watson, D.W., Bacteriol. Rev., 1957, vol. 21, pp. 273–294. 2. Hirsch, J.G., J. Exp. Med., 1956, vol. 103, pp. 589– 611. 3. Steiner, H., Hultmark, D,. Engstrom, A., Bennich, H., and Boman, H.G., Nature, 1981, vol. 292, pp. 246– 248. 4. Zasloff, M., Proc. Natl. Acad. Sci. USA, 1987, vol. 84, pp. 5449–5453. 5. Ganz, T., Selested, M.E., and Lehrer, R.I., Eur. J. Haematol., 1990, vol. 44, pp. 1–8. No. 9

2010

812

LAZAREV, GOVORUN

6. Auynet, C. and Rosenstein, Y., FEBS J, 2009, vol. 276, pp. 6497–6508. 7. Vaara, M., Curr. Opin. Pharmacol., 2009, vol. 9, pp. 571–576. 8. Diamond, G., Beckloff, N., Weinberg, A., and Kisich, K.O., Curr. Pharm. Des., 2009, vol. 15, pp. 23770–2392. 9. Ganz, T., Nature Rev. Immunol., 2003, vol. 3, pp. 710– 720. 10. Lehrer, R.I., Nature Rev. Microbiol, 2004, vol. 2, pp. 727–738. 11. Zasloff, M., Nature, 2002, vol. 415, pp. 389–395. 12. Cole, A.M., Ganz, T., Liese, A.M., Burdick, M.D., Liu, L., and Strieter, R.M., J. Immunol., 2001, vol. 167, pp. 623–627. 13. Yang, D., Chen, Q., Hoover, D.M., Staley, P., Tucker, K.D., Lubkowski, J., and Oppenheim, J.J., J. Leukoc. Biol., 2003, vol. 74, pp. 448–455. 14. Allaker, R.P. and Kapas, S., Regul. Pept., 2003, vol. 112, pp. 147–152. 15. Kowalska, K., Carr, D.B., and Lipkowski, A.W., Life Sci., 2002, vol. 71, pp. 747–750. 16. Kuwata, H., Yip, T.T., Yip, C.L., Tomita, M., and Hutchens, T.W., Biochem. Biophys. Res. Commun., 1998, vol. 245, pp. 764–773. 17. Liepke, C., Baxmann, S., Heine, C., Breithaupt, N., Standker, L., and Forsmann, W.G., J. Chromat. B. Analyt. Technol. Biomed. Life Sci., 2003, vol. 791, pp. 345–356. 18. Finlay, B.B. and Hancock, R.E., Nature Rev. Micro biol., 2004, vol. 2, pp. 497–504. 19. Hancock, R.E. and Patrzykat, A., Curr. Drug. Targets Infect. Disord., 2002, vol. 2, pp. 79–83. 20. Cole, A.M., Prot. Pept. Lett., 2005, vol. 12, pp. 41–47. 21. Ling, C.Q., Li, B., Zhang, C., Zhu, D.Z., Huang, X.Q., Gu, W., and Li, S.X., Ann. Oncol., 2005, vol. 16, pp. 109–115. 22. Bulet, P., Dimarcq, J.L., Hetru, C., Lageux, M., Charlet, M., Hegy, G., Van Dorsselaer, A., and Hoff mann, J.A., J. Biol. Chem., 1993, vol. 268, pp. 14893– 14897. 23. Simmaco, M., Mignogna, G., and Barra, D., Biopoly mers, 1998, vol. 47, pp. 435–450. 24. Shinnar, A.E., Uzzell, T., Rao, M.N., Spooner, E., Lane, W.S., and Zasloff, M.A., Proc. 14th Am. Peptide Symp., Kaumaya, P. and Hodges, R., Eds., Leiden: Mayflower Scientific, 1996, pp. 189–191. 25. Huttner, K.M., Infect. Immun., 1999, vol. 67, pp. 4256–4259. 26. Brogden, K.A., Ackermann, M., and Huttner, K.M., Antimicrob. Agents Chem., 1997, vol. 41, pp. 1615– 1617. 27. Lai, R., Ku, H., Hui Lee, W., and Zhang, Y., Biochem. Biophys. Res. Commun., 2002, vol. 295, pp. 796–799. 28. Brogden, K.A., De Lucca, A.J., Bland, J., and Elliott, S., Proc. Natl. Acad. Sci. USA, 1996, vol. 93, pp. 412–416. 29. Schittek, B., Hipfel, R., Sauer, B., Bauer, J., Kal bacher, H., Stevanovic, S., Schirle, M., Schroeder, K.,

Blin, N., Meier, F., Rassner, G., and Garbe, C., Nature Immunol., 2001, vol. 2, pp. 1133–1137. 30. Gennaro, R. and Zanetti, M., Biopolymers, 2000, vol. 55, pp. 31–49. 31. Johansson, J., Gudmundsson, G.H., Rottenberg, M.E., Bendt, K.D., and Agerberth, B., J. Biol. Chem., 1998, vol. 273, pp. 3718–3724. 32. Park, C.B., Yi, K.S., Matusuzaki, K., Kim, M.S., and Kim, S.C., Proc. Natl. Acad. Sci. USA, 2000, vol. 97, pp. 8245–8250. 33. Bechinger, B., J. Membr. Biol., 1997, vol. 156, pp. 197–211. 34. Travis, S.M., Anderson, N.N., Forsyth, W.R., Espir itu, C., Conway, B.D., Greenberg, E.P., McCray, P.B., Jr., Lehrer, R.I., Welsh, M.J., and Tack, B.F., Infect. Immun., 2000, vol. 68, pp. 2748–2755. 35. Ostberg, N. and Kaznessis, Y., Peptides, 2005, vol. 26, pp. 197–206. 36. Ganz, T., Science, 2002, vol. 298, pp. 977–979. 37. Tang, Y.Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C.J., Ouellettem, A.J., and Selsted, M.E., Sci ence, 1999, vol. 286, pp. 498–502. 38. Lee, J., Park, C., Park, S.C., Woo, E.R., Park, T., Hahm, K.S., Lee, D.G., J. Pept. Sci., 2009, vol. 16, pp. 103–109. 39. Tytler, E.M., Anantharamaiah, G.M., Walker, D.E., Mishra, V.K., Palgunachari, M.N., and Segrest, J.P., Biochemistry, 1995, vol. 34, pp. 4393–4401. 40. Hancock, R.E., J. Med. Microbiol., 1997, vol. 46, pp. 1–3. 41. Lehrer, R.I., Barton, A., Daher, K.A., Harwig, S.S., Ganz, T., and Selsted, M.E., J. Clin. Invest., 1989, vol. 84, pp. 553–561. 42. Kouzayaha, A., Nasir, M.N., Buchet, R., Wattraint, O., Sarazin, C., and Besson, F., J. Phys. Chem. B, 2009, vol. 113, pp. 7012–7019. 43. Yang, L., Harroun, T.A., Weiss, T.M., Ding, L., and Huang, H.W., Biophys. J., 2001, vol. 81, pp. 1475– 1485. 44. Dagan, A., Efron, K., Gaidukov, L., Mor, A., and Ginsburg, H., Antimicrob. Agents Chemother., 2002, vol. 46, pp. 1059–1066. 45. Brogden, K.A., Nat. Rev. Microbiol., 2005, vol. 3, pp. 238–250. 46. Chan, D.I., Prenner, E.J., and Vogel, H.J., Biochim. Biophys. Acta, 2006, vol. 1758, pp. 1184–1202. 47. Dawson, R.M. and Liu, C.Q., Crit. Rev. Microbiol., 2008, vol. 34, pp. 89–107. 48. Bals, R., Respir. Res., 2000, vol. 1, pp. 141–150. 49. Subbalakshmi, C. and Sitaram, N., FEMS Microbiol. Letts., 1998, vol. 160, pp. 91–96. 50. Gennaro, R., Zanetti, M., Benincasa, M., Podda, E., and Miani, M., Curr. Pharm. Des., 2002, vol. 8, pp. 763–778. 51. Boman, H.G., Agerberth, B., and Boman, A., Infect. Immun., 1993, vol. 61, pp. 2978–2984. 52. Tsai, H., and Bobek, L.A., Crit. Rev. Oral. Biol. Med., 1998, vol. 9, pp. 480–497. 53. Gryllos, I., TranWinkler, H.J., Cheng, M.F., Chung, H., Bolcome, R., 3rd, Lu, W., Lehrer, R.I.,


Vol. 46

No. 9

2010

ANTIMICROBIAL PEPTIDES AND THEIR USE IN MEDICINE and Wessels, M.R., Proc. Natl. Acad. Sci. USA, 2008, vol. 105, pp. 16755–16760. 54. Mantovani, H.C. and Russell, J.B., Appl. Environ. Microbiol., 2001, vol. 67, pp. 808–813. 55. Neuhaus, F.C. and Baddiley, J., Microbiol. Mol. Biol. Rev., 2003, vol. 67, pp. 686–723. 56. Kristian, S.A., Datta, V., Weidenmaier, C., Kansal, R., Fedtke, I., Peschel, A., Gallo, R.L., and Nizet, V., J. Bacteriol., 2005, vol. 187, pp. 6719–6725. 57. Peschel, A. and Sahl, H.G., Nat. Rev. Microbiol., 2006, vol. 4, pp. 529–536. 58. Peschel, A., Jack, R.W., Otto, M., Vollins, L.V., Staubitz, P., Nichlson, G., Kalbacher, H., Nieuwen huizen, W.F., Jung, G., Tarkowski, A., van Kessel, K.P., and van Strijp, J.A., J. Exp. Med., 2001, vol. 193, pp. 1067–1076. 59. Miller, S.I., Ernst, R.K., and Bader, M.W., Nat. Rev. Microbiol., 2005, vol. 3, pp. 36–46. 60. Weidenmaier, C., Peschel, A., Kempf, V.A., Lucindo, N., Yeaman, M.R., and Bayer, A.S., Infect. Immun., 2005, vol. 73, pp. 8033–8038. 61. Midorikawa, K., Iuhara, K., Komatsuzawa, H., Kawai, T., Yamada, S., Fujiara, T., Yamazaki, K., Sayama, K., Taubman, M.A., Kurihara, H., Hasgim oti, J., and Sugai, M., Infect. Immun., 2003, vol. 71, pp. 3730–3739. 62. Grenfell, B.T., Pybus, O.G., Gog, J.R., Wood, J.L., Daly, J.M., Mumford, J.A., and Holmes, E.C., Sci ence, 2004, vol. 303, pp. 327–332. 63. FernieKing, B.A., Seilly, D.J., and Lachmann, P.J., Immunology, 2004, vol. 111, pp. 444–452. 64. Shafer, W.M., Qu, X., Waring, A.J., and Lehrer, R.I., Proc. Natl. Acad. Sci. USA, 1998, vol. 95, pp. 1829– 1833. 65. Yeaman, M.R. and Yount, N.Y., Pharmacol. Rev., 2003, vol. 55, pp. 27–55. 66. Li, M., Lai, Y., Villaruz, A.E., Cha, D.J., Sturdevant, D.E., and Otto, M., Proc. Natl. Acad. Sci. USA, 2007, vol. 104, pp. 9469–9474. 67. Douglas, S.E., Gallant, J.W., Liebscher, R.S., Dacanay, A., and Tsoi, S.C., Dev. Comp. Immunol., 2003, vol. 27, pp. 589–601. 68. Park, C.H., Valore, E.V., Waring, A.J., and Ganz, T., J. Biol. Chem., 2001, vol. 276, pp. 7806–7810. 69. Quellette, A.J. and Selsted, M.E., FASEB J., 1996, vol. 10, pp. 1280–1289. 70. Verhoef, J., Adv. Exp. Med. Biol., 2003, vol. 531, pp. 301–313. 71. Gordon, Y.J., Romanowski, E.G., and McDermott, A.M., Curr. Eye Res., 2005, vol. 30, pp. 505–515. 72. Sit, C.S. and Mederas, J.C., Biocohem. Cell. Biol., 2008, vol. 86, pp. 116–123. 73. Lupett, A., Van Dissel, J.T., Brouwer, C.P., and Nib bering, P.H., Eur. J. Clin. Microbiol. Infect. Dis., 2008, vol. 27, pp. 1125–1129. 74. CarrielGomes, M.C., Kratz, J.M., Barracco, M.A., Bachere, E., Barardi, C.R., and Simoes, C.M., Mem. Inst. Oswaldo Cruz., 2007, vol. 102, pp. 469–472. 75. Moreira, C.K., Rodrigues, F.G., Ghosh, A., Varotti Fde, P., Miranda, A., Daffre, S., JacobsLorena, M., APPLIED BIOCHEMISTRY AND MICROBIOLOGY

813

and Moreira, L.A., Exp. Parasitol., 2007, vol. 116, pp. 346–353. 76. Ghavami, S., Asoodeh, A., Klonisch, T., Halayko, A.J., Kadkhoda, K., Kroczak, T.J., Gibson, S.B., Booy, E.P., NaderiManesh, H., and Los, M., J. Cell. Mol. Med., 2008, vol. 12, pp. 1005–1022. 77. Suttmann, H., Suttmann, H., Retz, M., Paulsen, F., Harder, J., Zwergel, U., Kamradt, J., Wullich, B., Unteregger, G., Stockle, M., and Lehmann, J., BMC Urol., 2008, vol. 8, p. 5. 78. Kolls, J.K., McCray, P.B., Jr., and Chan, Y.R., Nat. Rev. Immunol., 2008, vol. 8, pp. 829–835. 79. Han, S., Han, S., Bishop, B.M., and van Hoek, M.L., Biochem. Biphys. Res. Commun., 2008, vol. 371, pp. 670–674. 80. Schlee, M., Wehkamp, J., Altenhoefer, A., Oelschlae ger, T.A., Stange, E.F., and Fellermann, K., Infect. Immun., 2007, vol. 75, pp. 2399–2407. 81. Aberg, K.M., Radek, K.A., Choi, E.H., Kim, D.K., Demerjan, M., Hupe, M., Kerbleski, J., Gallo, R.L., Ganz, T., Nauro, T., Feingold, K.R., and Elias, P.M., J. Clin. Invest., 2007, vol. 117, pp. 3339–3349. 82. Raqib, R., Sarker, P., Bergman, P., Ara, G., Kindh, M., Sack, D.A., Nasirul Islam, K.M., Gud mundsson, G.H., Andersson, J., and Agerberth, B., Proc. Natl. Acad. Sci. USA, 2006, vol. 103, pp. 9178– 9183. 83. Yim, S., Dhawan, P., Ragunath, C., Christakos, S., and Diamond, G., J. Cyst. Fibros., 2007, vol. 6, pp. 403–410. 84. Giacometti, A., Cirioni, O., Ghiselli, R., Mocchegi ani, F., D’Amato, G., Circo, R., Orlando, F., Skerla vaj, B., Silvestri, C., Saba, V., Zanetti, M., and Scalise, G., Am. J. Respir. Crit. Care Med., 2004, vol. 169, pp. 187–194. 85. Chromek, M. Slamova, Z., Bergman, P., Kovacs, L., Podracka, L., Ehren, I., Hokfelt, T., Gudmundsson, G.H., Gallo, R.L., Agerberth, B., and Brauner, A., Nat. Med., 2006, vol. 12, pp. 636–641. 86. Cirioni, O., Giacometti, A., Ghiselli, R., Berghach, C., Orlando, F., Silvestri, C., Mocchegiani, F., Licci, A., Skerlavaj, B., Rocchi, M., Saba, V., Zanetti, M., and Scalise, G., Antimicrob. Agents Chemother., 2006, vol. 50, pp. 1672–1679. 87. Yang, Y.H., Wun, W.K., Tai, E.K., Wong, H.P., Lam, E.K., So, W.H., Shin, V.Y., and Cho, C.H., J. Pharmacol. Exp. Ther., 2006, vol. 318, pp. 547–554. 88. Isaacson, R.E., Curr. Opin. Investig. Drugs, 2003, vol. 4, pp. 999–1003. 89. Tan, H.H., Am. J. Clin. Dermatol., 2004, vol. 5, pp. 79–84. 90. Alman, H., Steingerg, D., Porat, Y., Mor, A., Frid man, D., Friedman, M., and Bachrach, G., J. Antimi crob. Chemother., 2006, vol. 58, pp. 198–201. 91. Kavanagh, K. and Dowd, S., J. Pharm. Pharmacol., 2004, vol. 56, pp. 285–289. 92. Cazzola, M., Sanduzzi, A., and Matera, M.G., Pulm. Pharmacol. Ther., 2003, vol. 16, pp. 131–145. 93. Toney, J.H., Curr. Opin. Investig. Drugs, 2002, vol. 3, pp. 225–228.

Vol. 46

No. 9

2010

814

LAZAREV, GOVORUN

94. Smith, J.J., Travis, S.M., Greenberg, E.P., and Welsh, M.J., Cell, 1996, vol. 85, pp. 229–236. 95. Bals, R., Weiner, D.J., Moscioni, A.D., Meegalla, R.L., and Wilson, J.M., Infect. Immun., 1999, vol. 67, pp. 6084–6089. 96. Lee, P.H., Ohtake, T., Zaiou, M., Murakami, M., Rid isill, J.A., Lin, K.Y., and Gallo, R.L., Proc. Natl. Acad. Sci. USA, 2005, vol. 102, pp. 3750–3755. 97. Braff, M.H., Zaiou, M., Fiere, J., Nizet, V., and Gallo, R.L., Infect. Immun., 2005, vol. 73, pp. 6771– 6781. 98. Bals, R. and Wilson, J.M., Neth. J. Med., 1999, vol. 54, pp. 10–S11. 99. Huang, H.J., Ross, C.R., and Blecha, F., J. Leukoc. Biol., 1997, vol. 61, pp. 624–629. 100. Li, J., Post, M., Volk, R., Gao, Y., Li, M., Metais, C., Sato, K., Tsai, J., Aird, W., Rosenberg, R.D., Hamp ton, T.G., Sellke, F., Carmeliet, P., and Simons, M., Nat. Med., 2000, vol. 6, pp. 49–55. 101. McMichael, J.W., Maxerll, A.I., Hayashi, K., Taylor, K., Wallace, W.A., Govan, J.R., Dorin, J.R.,

and Sallenave, J.M., Infect. Immun., 2005, vol. 73, pp. 3609–3617. 102. Jacobsen, F., Hirsch, T., Beller, J., Mittler, D., Sorkin, M., Pazdierny, G., Jacobsen, F., Eriksson, E., and Steinau, H.U., Gene Ther., 2005, vol. 12, pp. 1494–1502. 103. Samad, A., Sultana, Y., and Aqil, M., Curr. Drug. Deliv., 2007, vol. 4, pp. 297–305. 104. Meyer, J.E. and Harder, J., Curr. Pharm. Des., 2007, vol. 13, pp. 3119–3130. 105. Wehkamp, J., Koslowski, M., Wang, G., and Stange, E.F., Mucosal Immunol., 2008, vol. 1, pp. S67–S74. 106. Lazarev, V.M., Shkarupeta, M.M., Titova, G.A., Kos trjukova, E.S., Akopian, T.A., and Govorun, V.M., Biochem. Biophys. Res. Commun., 2005, vol. 338, pp. 946–950. 107. Lazarev, V.N., Stipkovits, L., Biro, J., Miklodi, D., Shkarupeta, M.M., Titova, G.A., Akppian, T.A., and Govorun, V.M., Microb. Infect., 2004, vol. 6, pp. 536– 541.


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