**4.2 Anti-inflammatory (Anti-endotoxin) roles of host defense peptides**

Bacterial lipopolysaccharides (LPS), also known as endotoxins, are major structural components of the outer membrane of Gram-negative bacteria that serve as a barrier and protective shield between them and their surrounding environment. LPS is considered to be a major virulence factor as it strongly stimulates the secretion of pro-inflammatory cytokines which mediate the host immune response and culminating in septic shock.

Early experiments determined that a number of host defense peptides from various sources bound to LPS from diverse Gram-negative bacteria and reduced LPS-induced release of proinflammatory cytokines (e.g. TNF-α, IL-1, IL-6) and nitric oxide from monocyte or macrophages and protected mice from LPS lethality (Larrick et al., 1994; Larrick et al., 1995; VanderMeer et al., 1995; Kirikae et al., 1998). Initial studies focused on the unprocessed form of cathelicidin, hCAP- 18 (Kirikae et al., 1998); however, it was later found that the LPSbinding properties of the peptide were contained within the processed 37-amino acid Cterminal domain, LL-37 (Turner et al., 1998). It has been proposed that the anti-endotoxic properties of these peptides are the result of the inhibition of binding of LPS to CD14 (Nagaoka et al., 2001) and lipopolysaccharide binding protein (LBP) (Scott et al., 2000), and/or indirect effects on cells (Scott et al., 2002). LL-37 has been shown to block a number of LPS-induced inflammatory responses, including contractility and (nitric oxide) NO release in aortic rings (Ciornei et al., 2003), pro-inflammatory cytokine production in a macrophage cell line and in animal models (Scott et al., 2000; Ohgami et al., 2003), suppression of leukocyte infiltration in a model of endotoxin-induced uveitis (Ohgami et al., 2003) and lethality in animal models of sepsis (Scott et al., 2002). These effects occur at concentrations in the physiological range for LL-37 (1–5 μg/ml) and may reflect a natural role for LL-37 in the body (e.g. balancing of the potential stimulus by endotoxin from commensals). This anti-endotoxin activity appears to correlate with an ability to dampen the pro-inflammatory effects of the Gram-positive surface molecule lipoteichoic acid (Scott et al., 2002; Gutsmann et al., 2010) designed a new class of peptides synthetic anti-LPS peptides (SALPs). SALPs were originally based on the LPS-binding domain of the *Limulus* anti-LPS factor (LALF) but were substantially changed in length and primary sequence for optimal binding to the lipid A portion of LPS. They observed that these peptides are highly efficient in neutralization of LPS and blockage of its immunopathological consequences *in vitro* and *in vivo*. SALPs combine excellent selectivity for LPS, with high neutralizing activity *in vitro*  and potent protection to septic shock using the murine model *in vivo*. They also demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the *Limulus* anti-LPS factor for systemic application. Efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock *in vivo* was analyzed, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. It seems that the lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure. These observations suggest a novel therapeutic role of AMPs.

#### **4.3 Anti-viral activity**

62 Antimicrobial Agents

MALDI-TOF MS, 28 peptides with a molecular mass below 15 kDa and belonging to different structural families were identified and could be classified into two groups. The first group contains the AMPs and their different isoforms. DIMs belonging to this group are likely to be effectors molecules of the immune response through their antimicrobial activity. The second group contains molecules for which the lack of similarity to any peptide prevents the proposition of any precise function. These peptides are suspected to serve as chemokines during the *Drosophila* immune response but the different approaches for investigating their role have so far been unsuccessful (Levy et al., 2004). On the other hand, our group has analyzed the peptides in the hemolymph of mosquitoes *An. albimanus* infected with malaria parasites. We found a complex pattern of peptides, including cecropin, which are released into the hemolymph. Similarly, gambicin, cecropin, and defensin are over-expressed in the intestinal epithelium and fat body of mosquitoes infected with *Plasmodium*. However, it is unknown whether these peptides participate in the elimination of the parasite. Cecropin has been consider an important AMP against Plasmodium, but in vitro assays with synthetic cecropin did not affect *Plasmodium* viability (unpublished results), but this peptide is over-expressed in mosquitoes infected with the parasite (Herrera-Ortiz, A. et al., 2010.). It would be interesting to analyze the peptides released into the hemolymph of these insects and their role in regulating the immune

**4.2 Anti-inflammatory (Anti-endotoxin) roles of host defense peptides** 

which mediate the host immune response and culminating in septic shock.

Bacterial lipopolysaccharides (LPS), also known as endotoxins, are major structural components of the outer membrane of Gram-negative bacteria that serve as a barrier and protective shield between them and their surrounding environment. LPS is considered to be a major virulence factor as it strongly stimulates the secretion of pro-inflammatory cytokines

Early experiments determined that a number of host defense peptides from various sources bound to LPS from diverse Gram-negative bacteria and reduced LPS-induced release of proinflammatory cytokines (e.g. TNF-α, IL-1, IL-6) and nitric oxide from monocyte or macrophages and protected mice from LPS lethality (Larrick et al., 1994; Larrick et al., 1995; VanderMeer et al., 1995; Kirikae et al., 1998). Initial studies focused on the unprocessed form of cathelicidin, hCAP- 18 (Kirikae et al., 1998); however, it was later found that the LPSbinding properties of the peptide were contained within the processed 37-amino acid Cterminal domain, LL-37 (Turner et al., 1998). It has been proposed that the anti-endotoxic properties of these peptides are the result of the inhibition of binding of LPS to CD14 (Nagaoka et al., 2001) and lipopolysaccharide binding protein (LBP) (Scott et al., 2000), and/or indirect effects on cells (Scott et al., 2002). LL-37 has been shown to block a number of LPS-induced inflammatory responses, including contractility and (nitric oxide) NO release in aortic rings (Ciornei et al., 2003), pro-inflammatory cytokine production in a macrophage cell line and in animal models (Scott et al., 2000; Ohgami et al., 2003), suppression of leukocyte infiltration in a model of endotoxin-induced uveitis (Ohgami et al., 2003) and lethality in animal models of sepsis (Scott et al., 2002). These effects occur at concentrations in the physiological range for LL-37 (1–5 μg/ml) and may reflect a natural role for LL-37 in the body (e.g. balancing of the potential stimulus by endotoxin from commensals). This anti-endotoxin activity appears to correlate with an ability to dampen the

response.

Apart from the antibacterial activity, AMPs also possess antiviral activity. For example, the α-defensins target the human immunodeficiency virus (HIV) activity by directly inactivating viral particles and affecting the ability of the virus to replicate within CD4 cells. Human α-defensins HNP-1 to -3 and HD-5 have been shown to block papillomavirus infection. Retrocyclin 2, a synthetic θ-defensin peptide that humans do not synthesize due to a mutation in the corresponding human gene, has the capacity to block influenza virus infection. Human β-defensins can also block HIV-1 replication, and interestingly, a single nucleotide polymorphism in a β-defensin gene has been associated with clinical manifestation of HIV-1 infection, suggesting that the human β-defensins play an important role in host defense against HIV. Cathelicidins, in contrast, have an inhibitory effect on lentiviral replication in vitro, and LL-37 appears capable of interfering with vaccinia virus replication in vitro and in mice. Dermaseptin S4, a 28-residue AMP isolated from frog skin, attenuates HIV infection in vitro. Other AMPs from frog skin including caerin 1.1, caerin 1.9, and maculatin 1.1 have also demonstrated inhibition of HIV in vitro(Albiol Matanic and Castilla, 2004).(Daher et al., 1986; Sinha et al., 2003; Yasin et al., 2004). Our group has worked with the peptide named scorpine from the venom of *Pandinus imperator* scorpion, where we observed a very interesting anti-virus dengue and anti-plasmodium activity (Carballar-Lejarazu et al., 2008). Scorpine is an antimicrobial peptide whose structure resembles a hybrid between a defensin and a cecropin. It exhibits antibacterial activity and inhibits the sporogonic development of parasites responsible for murine malaria. The recombinant expressed scorpine (RScp) in *Anopheles gambie* cells showed antibacterial activity against *Bacillus subtilis* and *Klebsiella pneumoniae*, at 5 and 10 µM, respectively. It also produced 98%mortality in sexual stages of *Plasmodium berghei* at 15 µM and 100% reduction in *Plasmodium falciparum* parasitemia at 5 µM. RScp also inhibited virus dengue-2 replication in C6/36 mosquito cells. In addition, we generated viable and fertile transgenic *Drosophila* that over-expresses and correctly secretes RScp into the insect hemolymph, suggesting that the generation of transgenic mosquitoes resistant to different pathogens may be viable. However, there is no knowledge of their mechanics, action. It is necessary to extend these studies with other peptides during infection induced with virus dengue and other pathogens.

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