**3. AMP classical functions**

The classical function of AMP has been their role as major effectors of the innate immune system; AMPs complement the highly specific but relatively slow adaptive immune system. Unlike the acquired immune mechanisms, endogenous AMPs, which are constitutively expressed or induced, provide fast and effective means of defense. Most of these geneencoded peptides are mobilized shortly after microbial infection and act rapidly to

Natural Antimicrobial Peptides from Eukaryotic Organisms 61

al., 2000; Niyonsaba et al., 2002). Defensins can also activate effector cells that can work together with the complement system to destroy microbial invaders. The α-defensins HNP1~3 have been reported to increase the production of TNFa and IL-1 while decreasing the production of IL-10 by monocytes (De et al., 2000). Some α-defensins enhance expression of adhesion molecules including ICAM-1, CD11b, and CD11c by neutrophils and facilitate the recruitment and enhance the microbicidal activity (Van Wetering et al., 1997; Chaly et al., 2000; Di Nardo et al., 2003) (FÈger et al., 2002). β-defensins induce mast cell degranulation and release of histamine and prostaglandin D2 (Yamashita and Saito, 1989; Befus et al., 1999; Niyonsaba et al., 2001) increase the expression of CXCL8 and CXCL5 (Van Wetering et al., 1997; van Wetering et al., 2002). Furthermore, murine β-defensin 2 has been shown to act directly on immature DCs as an endogeneous ligand for Toll like receptor 4 (TLR-4), inducing up regulation of co-stimulatory molecules and DC maturation, triggering robust, Th1 polarized adaptive immune responses in vivo (Biragyn et al., 2002). However, the mechanisms that regulate these functions are not well studied. Defensins attract inflammatory cells as neutrophils, B lymphocytes and macrophages. All these cells release inflammatory mediators such as IL-8, IFNγ, IL-6, IL-10 and LTB4. It is interesting that defensins may also have anti-inflammatory activity by the induction of IL-10 or SLPI.(Durr and Peschel, 2002; Zasloff, 2002).The synthesis of β-defensins by epithelial cells and the recruitment of peripheral blood granulocytes α-defensin-rich site of inflammation generates a high concentration of them. Also have direct antimicrobial effects, defensins facilitate and amplify the subsequent immune response. Indeed, spleen cells stimulated with α-defensins increase the production of human cytokines and lymphocyte proliferation. This same type of defensins, when administered to mice, produces increased serum IgG1, IgG2 and IgG2b. In addition, small amounts of HNP extend the antibody response against a singenic tumor

These results indicate, without doubt, the AMPs have a role in the regulation of the immune response. On the other hand, recent studies have identified several structurally diverse endogenous mediators of innate immunity with certain features: firstly, they are rapidly released in response to infection or tissue injury; secondly, they have both chemotactic and activating effects on APCs; and thirdly, they exhibit particularly potent *in vivo* immunoenhancing activity and enhance DC differentiation from DC precursors. This subset of mediators alerts host defenses by augmenting innate and adaptive immune responses to tissue injury and/or infection. On the basis of their unique activities, they are called 'alarmins' (Oppenheim and Yang, 2005). Innate-immune mediators possessing alarmin activity include defensins, cathelicidin, eosinophil-derived neurotoxin (EDN), and high mobility group box protein 1 (HMGB1) (Oppenheim and Yang, 2005). The concept of alarmins is very interesting. This has only been observed in mammals, but is likely to exist in other groups of animals including insects. It has been observed overexpression of antimicrobial peptides during infection with various pathogens and even damage to the cuticle. Many groups of insects have been used to understand the basic characteristics of the innate immunity. However, surprisingly, the study of AMPs in insects has been limited study of their bactericidal or antiparasitic activity and virtually no information on the alternative role that could have the AMPs on the immune response

Differential analyses after bacterial or fungal challenge showed the regulation of more than a 100 molecules in adult *Drosophila* hemolymph (Levy et al., 2004). Using differential

(Tani et al., 2000).

in these organisms.

neutralize a broad range of microbes (bacteria, virus and protozoa). The ubiquitous nature of antimicrobial peptides suggests that their role in nature has been long standing and must have contributed to an organism's fitness. Many of these molecules exert mechanisms of action that appear to be unique and highly complex. However, AMPs exhibit varying, and in some cases, significant degrees of host cytotoxicity, reflecting non-selective cell targeting (Shin et al., 1999). It is likely that distinct antimicrobial peptides have evolved to function within specific physiologic and anatomic contexts to minimize their potential to concomitantly injuring the host cells. An intensive area of focus regarding antimicrobial peptide biochemistry relates to the precise mechanisms by which these molecules cause cell death. A long-held paradigm for microbicidal action has been that AMPs kill microorganisms by initiating multiple injuries in target microbial cell membranes. The principal theory suggest that peptides may create membrane pores in the organism, making a leakage of some metabolites, ensuing depolarization, loss of membrane-coupled respiration and biopolymer synthesis, and ultimately cell death. However, other authors suggest additional mechanisms, , where membrane permeabilization alone appears to be insufficient to cause cell death. These evidences come from studies documenting a clear dissociation between membrane perturbation and cell death. In these cases, cell killing may proceed in the absence of significant disruption in membrane architecture, due rather to disruptions in cellular function (Zhang et al., 2000). The functional integrity of the cytoplasmic membrane is crucial to essential functions of microbial pathogens, including gradient formation and selective permeability, cellular energetics, and synthesis of biomolecules (Yeaman et al., 1998).

The general membrane effects of AMP are the membrane perturbation however alone may be insufficient for microbicidal effects of certain peptides. Permeabilization alone does not invariably result in staphylococcal death due antimicrobial peptides. Different peptides with varying staphylocidal potencies exhibited disparate capacities of membrane permeabilization and cell killing (Koo et al., 2001). Similar studies showed that gramicidin S rapidly depolarizes *Pseudomonas aeruginosa*, but did not kill it, suggesting that the concept of membrane perturbation and eventual cell killing may be independent (Zhang et al., 2000). Bacterial membrane energetic also appears to be involved in AMP mechanisms of action (Yeaman et al., 1998). It is now widely recognized that the AMP concept could play a promising role in fighting the presently raging microbial resistance to conventional antibiotics.
