**5. Biological activities and therapeutic applications of cationic antimicrobial peptides**

#### **5.1. Antibacterial**

of CAPs that exert their effects on different targets may exhibit synergy, and provide more

Pleurocidin usually causes membrane disruption at high concentrations whereas at low con‐ centrations, it can translocate into the cytoplasm without causing cell lysis and exert its ef‐ fects intracellularly, inhibiting DNA, RNA and protein synthesis (Patrzykat et al., 2002). The intracellular effect of pleurocidin NRC-03 has since been probed using zebrafish embryos, and shown to target the mitochondria and generate superoxide (Morash et al., 2011). TU‐ NEL staining indicates that the DNA of some cells becomes degraded, whereas other cells undergo rapid lysis and cell death without DNA fragmentation. Pleurocidin variants NRC-03 and NRC-07 cause mitochondrial membrane damage and production of reactive

In most cases, killing is by nonreceptor-mediated mechanisms since all D-amino acid enan‐ tiomers are generally as active as the natural L-amino acid peptides. However, stereospecific receptor-mediated translocation has been described (Nicolas, 2009) and some CAPs trans‐ duce their effects via signaling networks upon interaction with receptors. Quite a diversity of receptors have been described, possibly reflecting the diversity in peptide structures. Up‐ take of apidaecin into Gram negative bacteria is proposed to by an energy-dependent mech‐ anism involving a permease or transporter (Castle et al., 1999). The detection of peptide:receptor complexes is technically very difficult but some receptors have been identi‐ fied. For example, the outer membrane protein OprI from *P. aeruginosa* (Lin et al., 2010) and the outer membrane lipoprotein Lpp in Enterobacteriaceae (Chang et al., 2012) have been shown to serve as receptors for α-helical CAPs. Histatin 5 and some defensins bind Ssa1/2 proteins on the cell surface of *C. albicans* in order to exert their activity, and the potassium transporter TRK1 is also required for histatin5 fungicidal activity (Vylkova et al., 2007). In eukaryotic cells, formyl peptide receptor-like 1 (FPRL1) is used as a receptor for LL-37 to act as a chemoattractant (Yang et al., 2000) and induce angiogenesis (Koczulla et al., 2003). LL-37 has also been shown to mediate keratinocyte migration and cytokine release by trans‐ activation of the epidermal growth factor receptor (Tokumaru et al., 2005) and P2X7 recep‐ tor (Elssner et al., 2004), respectively. However, activation of receptors could be via CAPinduced changes in membrane fluidity, ion transport and/or receptor aggregation (Braff et

Activation of mast cells by pleurocidin is G protein-dependent and proposed to involve FPRL1 and G protein coupled receptor signaling pathway (Kulka, unpub). The observation that pleurocidin can bind and activate FPRL1 has some important implications for human dis‐ ease. The FPRL1 receptor subtype is also a receptor for the bacterially-derived peptide fMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) making it an important innate immune receptor (Selvatici et al., 2006). FPRL1 activates key components of the innate immune sys‐ tem and is responsible for chemotactic responses, superoxide anion production and degranu‐ lation by neutrophils, macrophages and mast cells. FPRL function has been shown to be important in chronic obstructive pulmonary disease (COPD) due to cigarette smoking (Car‐

oxygen species (ROS) in MDA-MB-231 breast cancer cells (Hilchie et al., 2011).

126 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

potent killing activity.

**4.3. Receptors and binding proteins**

al., 2005) rather than direct binding.

Because of their rapid, broad spectrum bactericidal action, potency, and low host cytotox‐ icity, CAPs have elicited much excitement as alternatives to current antibiotics, particular‐ ly in the fight against antibiotic-resistant pathogens (Nijnik and Hancock, 2009). CAPs can cause bacterial cell death by disrupting the bacterial membrane, by entering the cell and inhibiting intracellular targets, or by stimulating the immune system to eliminate an infec‐ tion. They can be used alone or together with other antibacterial agents, with which they frequently synergize.

Pleurocidins show broad-spectrum antimicrobial activity at micromolar concentrations and also show synergistic activity with several antibiotics (Cole et al., 2000; Douglas et al., 2003; Patrzykat et al., 2003). In contrast to many CAPs, pleurocidin is insensitive to NaCl concen‐ trations up to 150 mM, and may therefore have application in relatively high-salt bodily flu‐ ids and in treatment of lung infections in patients with cystic fibrosis, who have even higher NaCl content (Goldman et al., 1997).

Recent studies have shown that pleurocidin possesses considerable activity against oral mi‐ croorganisms growing both planktonically and as a biofilm, even in the presence of saliva (Tao et al., 2011). Incorporation of a targeting moiety to pleurocidin NRC-04 has significant‐ ly increased its specificity towards *S. mutans*, the main cause of dental caries, relative to oth‐ er oral bacteria (Mai et al., 2011). Addition of 5 mM EDTA or 10 ppm sodium fluoride, two compounds commonly used in oral treatments, resulted in increased killing of *S. mutans*. Re‐ placement of Ser14 by His improved the activity of the targeted peptide at low pH similar to that found in the oral cavity, showing that this targeted pleurocidin has good potential as an anticaries agent.

Foodborne infections, especially those of seafood are a major health problem, yet only one CAP, nisin, has been approved by the FDA for use as a food preservative and it has only limited activity against Gram-negative bacteria or fungi (Burrowes et al., 2004).

Pleurocidin was tested against Gram-positive and Gram-negative bacteria of interest in food safety and shown to be highly effective against 17 of 18 strains, particularly the fish spoilage bacterium *Vibrio alginolyticus* (Burrowes et al., 2004). In addition, it was active against the yeasts *S. cerevisiae* and *P. pastoris*, and the mold *P. expansum*, but was not detrimental to hu‐ man red blood cells or intestinal epithelial cells, indicating that it would be safe to use as a food preservative. Furthermore, since pleurocidin is produced naturally by an edible fish, there are fewer concerns over its use in food preservation.

### **5.2. Antifungal**

The antifungal properties of CAPs have been recognized for some time (De Lucca and Walsh, 1999); however, it is only recently that the mechanism of action has been elucidated. While some CAPs such as LL-37 kill fungal cells by lysing the cell membrane, others such as hista‐ tin and defensins 2 and 3 kill in an energy-dependent and salt-sensitive fashion without cell lysis (den Hertog et al., 2005; Vylkova et al., 2007). Magainin 2 and dermaseptin S3(1-16) also cross the cell membrane and interfere with DNA integrity (Morton et al., 2007a; Morton et al., 2007b). Histatin 5, lactoferrin, arenicin-1 and several other CAPs inhibit mitochondrial respi‐ ration and induce the formation of ROS and subsequent apoptosis in *C. albicans* (Andres et al., 2008; Cho and Lee, 2011b; Helmerhorst et al., 2001; Hwang et al., 2011a; Hwang et al., 2011b).

Attempts to improve antifungal activity of CAPs have resulted in structural analogs with in‐ creased selectivity towards fungi and less host cytotoxicity. Hydrophobicity was shown to be a crucial factor in the antifungal activity of a series of CAP analogs against zygomycetes vs ascomycetes (Jiang et al., 2008). Synthetic, non-peptidic analogs of naturally-occurring CAPs have shown potent activity as anti-*Candida* agents against biofilms in the oral cavity, and reduced mammalian cytotoxicity (Hua et al., 2010).

Pleurocidin also possesses activity against *C. albicans* (Cole et al., 2000; Douglas et al., 2003; Patrzykat et al., 2003) and various derivatives truncated at the amino or carboxy-termini show reduced hemolytic activity although they are generally less potent (Lee and Lee, 2010). Mod‐ ification of pleurocidin to decrease hydrophobicity and α-helicity also resulted in active peptides with reduced hemolytic activity (Sung and Lee, 2008). A synthetic all-D amino acid entantiom‐ er of pleurocidin has also been designed that showed resistance to trypsin, plasmin and carboxypeptidase B proteases *in vitro* with no loss in antifungal activity (Jung et al., 2007). Pleurocidin induces ROS, oxidative stress, mitochondrial depolarization, caspase activation, and apoptosis in *C. albicans* with concomitant membrane phosphatidylserine externaliza‐ tion, cell shrinkage, DNA fragmentation and nuclear condensation (Cho and Lee, 2011a).

#### **5.3. Antiviral**

Enveloped viruses are often susceptible to attack by CAPs. Four different linear CAPs were shown to inactivate vaccinia virus by removal of the outer membrane, thereby rendering the inner membrane susceptible to neutralizing antibody against the exposed antigens (Dean et al., 2010). Novel HIV-1-inhibitory peptides have recently been identified by screening the APD Antimicrobial Peptide Database for CAPs with promising antiviral properties. This un‐ covered 30 candidate CAPs which, when synthesized and tested, resulted in 11 peptides with EC50<10 µM against HIV-1 (Wang et al., 2010). Some CAPs are able to exert antiviral effects by blocking cell surface receptors used for viral entry. For example, the polyphemu‐ sin analog T22 binds CXCR4 on T cells and prevents T cell line-tropic HIV-1 entry (Muraka‐ mi et al., 1997).

The antiviral activity of pleurocidin variants has been tested against vaccinia virus grown in HeLa cells and four lead candidates were identified based on viral inhibition without HeLa cytotoxicity at both high and low multiplicity of infection (MOI) (Johnston, pers. comm.). At the more biologically relevant low MOI, two peptides were inhibitory with an IC50< 1 µg/ml. Further studies are required to determine the mechanism by which pleurocidin exerts its an‐ tiviral effect.

#### **5.4. Anti-parasitic**

**5.2. Antifungal**

**5.3. Antiviral**

mi et al., 1997).

The antifungal properties of CAPs have been recognized for some time (De Lucca and Walsh, 1999); however, it is only recently that the mechanism of action has been elucidated. While some CAPs such as LL-37 kill fungal cells by lysing the cell membrane, others such as hista‐ tin and defensins 2 and 3 kill in an energy-dependent and salt-sensitive fashion without cell lysis (den Hertog et al., 2005; Vylkova et al., 2007). Magainin 2 and dermaseptin S3(1-16) also cross the cell membrane and interfere with DNA integrity (Morton et al., 2007a; Morton et al., 2007b). Histatin 5, lactoferrin, arenicin-1 and several other CAPs inhibit mitochondrial respi‐ ration and induce the formation of ROS and subsequent apoptosis in *C. albicans* (Andres et al., 2008; Cho and Lee, 2011b; Helmerhorst et al., 2001; Hwang et al., 2011a; Hwang et al., 2011b).

Attempts to improve antifungal activity of CAPs have resulted in structural analogs with in‐ creased selectivity towards fungi and less host cytotoxicity. Hydrophobicity was shown to be a crucial factor in the antifungal activity of a series of CAP analogs against zygomycetes vs ascomycetes (Jiang et al., 2008). Synthetic, non-peptidic analogs of naturally-occurring CAPs have shown potent activity as anti-*Candida* agents against biofilms in the oral cavity,

Pleurocidin also possesses activity against *C. albicans* (Cole et al., 2000; Douglas et al., 2003; Patrzykat et al., 2003) and various derivatives truncated at the amino or carboxy-termini show reduced hemolytic activity although they are generally less potent (Lee and Lee, 2010). Mod‐ ification of pleurocidin to decrease hydrophobicity and α-helicity also resulted in active peptides with reduced hemolytic activity (Sung and Lee, 2008). A synthetic all-D amino acid entantiom‐ er of pleurocidin has also been designed that showed resistance to trypsin, plasmin and carboxypeptidase B proteases *in vitro* with no loss in antifungal activity (Jung et al., 2007). Pleurocidin induces ROS, oxidative stress, mitochondrial depolarization, caspase activation, and apoptosis in *C. albicans* with concomitant membrane phosphatidylserine externaliza‐ tion, cell shrinkage, DNA fragmentation and nuclear condensation (Cho and Lee, 2011a).

Enveloped viruses are often susceptible to attack by CAPs. Four different linear CAPs were shown to inactivate vaccinia virus by removal of the outer membrane, thereby rendering the inner membrane susceptible to neutralizing antibody against the exposed antigens (Dean et al., 2010). Novel HIV-1-inhibitory peptides have recently been identified by screening the APD Antimicrobial Peptide Database for CAPs with promising antiviral properties. This un‐ covered 30 candidate CAPs which, when synthesized and tested, resulted in 11 peptides with EC50<10 µM against HIV-1 (Wang et al., 2010). Some CAPs are able to exert antiviral effects by blocking cell surface receptors used for viral entry. For example, the polyphemu‐ sin analog T22 binds CXCR4 on T cells and prevents T cell line-tropic HIV-1 entry (Muraka‐

The antiviral activity of pleurocidin variants has been tested against vaccinia virus grown in HeLa cells and four lead candidates were identified based on viral inhibition without HeLa cytotoxicity at both high and low multiplicity of infection (MOI) (Johnston, pers. comm.). At

and reduced mammalian cytotoxicity (Hua et al., 2010).

128 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

The anti-parasitic properties of magainins and cecropins have been known for over twenty years and have subsequently been reported for many other CAPs (Harrington, 2011; Mor, 2009). CAPs are able to traverse the membranes of parasite-infected erythrocytes and are proposed to change the properties of the parasite cell membrane, cause membrane lysis, or become internalized and interfere with biological processes. BMAP-28, BMAP-27 and the less cytotoxic truncated derivative BMAP-18 have recently been shown to possess excellent *in vitro* activity against *Leishmania* and African trypanosomes growing both in mammalian cells and in cells of the insect vector (Haines et al., 2009; Lynn et al., 2011). Importantly, BMAP-18 was also able to inhibit release of leukocyte LPS-induced TNF-α associated with inflammation and cachexia in patients with sleeping sickness. Further *in vivo* studies of nat‐ urally-occurring and engineered CAPs are needed to demonstrate their potential in the treatment of serious parasite diseases of humans.

Pleurocidin inhibited the bloodstream form of *Trypanosoma brucei* at low concentration but was not effective against the procyclic culture form or the tsetse symbiont (Haines et al., 2003).

#### **5.5. Anticancer**

In contrast to conventional chemotherapy drugs that target all actively proliferating cells, CAPs can show selective cytotoxicity towards cancer cells including dormant, slow-grow‐ ing, and multidrug resistant cells (see Hoskin and Ramamoorthy, 2008)). Both apoptotic (Jin et al., 2010) and non-apoptotic (Ceron et al., 2010; Hilchie et al., 2011; Morash et al., 2011) mechanisms have been proposed. The α-helical CAP, temporin-1CEa, exhibits cytotoxicity towards all of 12 tested human carcinoma cell lines in a concentration-dependent manner, yet no significant cytotoxicity to normal human umbilical vein smooth muscle cells at con‐ centrations that showed potent antitumor activity (Wang et al., 2011). The basis for selectivi‐ ty is thought to be the presence of phosphatidylserine, which is exposed on non-apoptotic tumor cells including malignant metastatic cells and primary cell cultures. Susceptibility of metastatic cells suggests that CAPs may be useful in treating metastatic as well as primary cancers (Riedl et al., 2011).

In addition to direct killing of cancer cells, CAPs can affect T-cell dependent tumor regula‐ tion. Recently, intratumoral administration of a lactoferricin derivative into lymphomas es‐ tablished in mice resulted in tumor necrosis, infiltration of inflammatory cells, and regression of tumors. Transfer of spleen cells from treated mice provided long-term, specific T cell-dependent immunity against the lymphoma, suggesting therapeutic vaccination against cancer using CAPs may be possible (Berge et al., 2010). In contrast, LL-37 was able to induce apoptosis of T regulatory cells, thus inhibiting their immune suppressor activity and thereby enhancing the anti-tumor response (Mader et al., 2011).

Pleurocidins selectively killed human leukemia cells at low concentrations (<32 µg/mL) (Mo‐ rash et al., 2011) as well as multiple breast cancer cell lines (Hilchie et al., 2011) by a predom‐ inantly membranolytic mechanism. Disruption of the cell membrane also augmented the activity of the chemotherapeutic cisplatin, presumably by enhancing access to the nucleus. Furthermore, when administered intratumorally into breast cancer xenografts in mice, pleu‐ rocidin inhibited tumor growth and induced tumor necrosis, while not causing observable adverse effects on the mice (Hilchie et al., 2011). These advantageous properties indicate that pleurocidins should be pursued as novel anticancer agents.

#### **5.6. Immunomodulatory**

CAPs show multiple activities associated with modulation of the immune system (Jenssen and Hancock, 2010; Yeung et al., 2011) and have been postulated to represent an evolution‐ ary link bridging innate and adaptive immune responses (Selsted and Ouellette, 2005). CAPs are directly chemotactic for immune cells and also stimulate chemokine and cytokine secretion (Lai and Gallo, 2009). Some CAPs are able to boost protein antigens and vaccines that have low immunogenicity by inducing proinflammatory cytokines such as TNF, IFN and IL-1β (Kindrachuk et al., 2009; Mutwiri et al., 2007; Tavano et al., 2011), suggesting that they would be good adjuvants (Huang et al., 2011; Li et al., 2008). CAPs play a critical role in wound healing (Steinstraesser et al., 2008), promoting keratinocyte migration (Aung et al., 2011a), re-epithelialization (Hirsch et al., 2009), deposition of extracellular matrix (Oono et al., 2002), and angiogenesis (Koczulla et al., 2003). Human β-defensins and LL-37 are able to stimulate mast cells, and recently the neuroendocrine CAP catestatin has been reported to induce migration and degranulation of mast cells, release of lipid mediators and production of cytokines and chemokines (Aung et al., 2011b). CAPs also show promise in counteracting sepsis, a major cause of morbidity and mortality in hospitalized patients. For example, intra‐ peritoneal injection of LPS-sensitized mice with S-thanatin reduced serum endotoxin and TNF-α levels, resulting in 100% survival (Wu et al., 2011).

Pleurocidin has a proinflammatory effect in fish cells *in vitro*, inducing expression of IL-1β and COX-2 (Chiou et al., 2006). In addition, pleurocidin variant NRC-08, which has no anti‐ bacterial activity, induced the expression of the co-stimulatory marker CD40 as well as IL-12p40, TNF, and TGF from mouse bone marrow-derived immature dendritic cells (Phil‐ lips, Lee & Douglas, pers. comm.). The potential immunomodulating effects of this particu‐ lar pleurocidin variant, which promotes the maturation of DCs, suggest that it could be further explored as a candidate adjuvant for a Th1 response to vaccine antigens.

Pleurocidins activate human mast cells to release granule contents such as histamine and proteases (Kulka, per. comm.) both of which are important in allergic inflammation and tis‐ sue hometostasis. Mast cells are critical regulators of the tissue microenvironment capable of responding to many different stimuli including allergens and releasing a huge variety of pro-inflammatory and immunomodulatory mediators. Interestingly, pleurocidin activation of mast cells is unique from that of allergens. Allergens bind to and crosslink surface high affinity immunoglobulin E receptors (FcεRI), which activates increases in intracellular calci‐ um and initiates several signaling pathways. Whereas FcεRI activation results in degranula‐ tion, arachidonic acid metabolite release and pro-inflammatory mediator production, pleurocidin activation of FPRL1 initiates degranulation and production of relatively small amounts of chemokines. Furthermore, whereas FcεRI engagement does not activate mast cell chemotaxis, pleurocidins modify adhesion via the fibronectin receptor (CD29) and pro‐ mote mast cell migration. This suggests that pleurocidins activate selective signaling path‐ ways in mast cells and may be useful tools in targeted mast cell-dependent therapy.

#### **5.7. Cytotoxic**

induce apoptosis of T regulatory cells, thus inhibiting their immune suppressor activity and

Pleurocidins selectively killed human leukemia cells at low concentrations (<32 µg/mL) (Mo‐ rash et al., 2011) as well as multiple breast cancer cell lines (Hilchie et al., 2011) by a predom‐ inantly membranolytic mechanism. Disruption of the cell membrane also augmented the activity of the chemotherapeutic cisplatin, presumably by enhancing access to the nucleus. Furthermore, when administered intratumorally into breast cancer xenografts in mice, pleu‐ rocidin inhibited tumor growth and induced tumor necrosis, while not causing observable adverse effects on the mice (Hilchie et al., 2011). These advantageous properties indicate that

CAPs show multiple activities associated with modulation of the immune system (Jenssen and Hancock, 2010; Yeung et al., 2011) and have been postulated to represent an evolution‐ ary link bridging innate and adaptive immune responses (Selsted and Ouellette, 2005). CAPs are directly chemotactic for immune cells and also stimulate chemokine and cytokine secretion (Lai and Gallo, 2009). Some CAPs are able to boost protein antigens and vaccines that have low immunogenicity by inducing proinflammatory cytokines such as TNF, IFN and IL-1β (Kindrachuk et al., 2009; Mutwiri et al., 2007; Tavano et al., 2011), suggesting that they would be good adjuvants (Huang et al., 2011; Li et al., 2008). CAPs play a critical role in wound healing (Steinstraesser et al., 2008), promoting keratinocyte migration (Aung et al., 2011a), re-epithelialization (Hirsch et al., 2009), deposition of extracellular matrix (Oono et al., 2002), and angiogenesis (Koczulla et al., 2003). Human β-defensins and LL-37 are able to stimulate mast cells, and recently the neuroendocrine CAP catestatin has been reported to induce migration and degranulation of mast cells, release of lipid mediators and production of cytokines and chemokines (Aung et al., 2011b). CAPs also show promise in counteracting sepsis, a major cause of morbidity and mortality in hospitalized patients. For example, intra‐ peritoneal injection of LPS-sensitized mice with S-thanatin reduced serum endotoxin and

Pleurocidin has a proinflammatory effect in fish cells *in vitro*, inducing expression of IL-1β and COX-2 (Chiou et al., 2006). In addition, pleurocidin variant NRC-08, which has no anti‐ bacterial activity, induced the expression of the co-stimulatory marker CD40 as well as IL-12p40, TNF, and TGF from mouse bone marrow-derived immature dendritic cells (Phil‐ lips, Lee & Douglas, pers. comm.). The potential immunomodulating effects of this particu‐ lar pleurocidin variant, which promotes the maturation of DCs, suggest that it could be

Pleurocidins activate human mast cells to release granule contents such as histamine and proteases (Kulka, per. comm.) both of which are important in allergic inflammation and tis‐ sue hometostasis. Mast cells are critical regulators of the tissue microenvironment capable of responding to many different stimuli including allergens and releasing a huge variety of pro-inflammatory and immunomodulatory mediators. Interestingly, pleurocidin activation of mast cells is unique from that of allergens. Allergens bind to and crosslink surface high

further explored as a candidate adjuvant for a Th1 response to vaccine antigens.

thereby enhancing the anti-tumor response (Mader et al., 2011).

130 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

pleurocidins should be pursued as novel anticancer agents.

TNF-α levels, resulting in 100% survival (Wu et al., 2011).

**5.6. Immunomodulatory**

#### *5.7.1. Hemolysis and host cell lysis*

Although most CAPs are not hemolytic or cytolytic at the concentrations used for killing mi‐ crobes or cancer cells, some do exhibit that characteristic, melittin being the most notorious (Tosteson et al., 1985). Magainin also forms pores in human cell membranes and enters the cell within minutes, accumulating in the nucleus and mitochondrion (Imura et al., 2008). Bo‐ vine myeloid antimicrobial peptides BMAP-27 and BMAP-28 at 10 times minimal inhibitory concentration (MIC) are toxic towards human erythrocytes and polymorphonuclear cells and induce apoptosis in transformed cell lines and *in vitro*-activated lymphocytes at concen‐ trations near MIC (Risso et al., 1998). A recent high-throughput method to assess cytotoxici‐ ty of melittin and structural variants differing in hydrophobicity, amphipathicity and helicity, showed it to be more sensitive than standard tests such as erythrocyte lysis and MTT assay and holds promise for identifying CAP variants with non-hemolytic properties (Walsh et al., 2011).

Hemolysis assays of 26 variants of pleurocidin showed that only one, a histidine-rich variant NRC-19, showed any ability to lyse red blood cells up to a concentration of 128 µg/mL, well above the concentrations required to kill bacterial and cancer cells (Morash et al., 2011). The selectivity of these peptides indicates that they would be excellent candidates as antibacteri‐ al and anti-cancer agents.

#### *5.7.2. Sepsis*

While many CAPs can reduce sepsis, some actually exacerbate the problem. This is because they are so effective at killing bacterial cells that they cause the release of endotoxin from lysed cells, eliciting an excessive inflammatory response (Risso et al., 1998; Steinstraesser et al., 2003).

Preliminary studies using a mouse cytokine array showed that pleurocidin variants NRC-03 and NRC-08 are able to repress LPS-induced TNF-α and IL-10 secretion from mouse RAW264.7 macrophages, indicating that they may be considered for the control of sepsis (Carroll, Patrzykat & Douglas, pers. comm.).
