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IPF PharmaCeuticals, Hannover, Germany
3Correspondence: Institute for Anatomy III, JW Goethe University, Theodor-Stern-Kai-7, Frankfurt, D-60590, Germany. E-mail: e.maronde{at}em.uni-frankfurt.de
SPECIFIC AIMS
We sought to investigate the physiological properties of the novel human ßbeta;-defensin DEFB123 in comparison to the well-studied ßbeta;-defensin DEFB3.
PRINCIPAL FINDINGS
1. Similar to other members of the ßbeta;-defensin peptide family, also DEFB123 inhibits the growth of Gram-positive bacteria like Staphylococcus carnosus (MIC 18.8 µg/ml), Staphylococcus aureus or Streptococcus pneumoniae, and Gram-negative bacteria-like Klebsiella pneumoniae (MIC 75 µg/ml), Escherichia coli (37.5 µg/ml), or Pseudomonas aeruginosa. 2. Similar to the synthetic peptide MBI-28, DEFB123 binds to LPS according to a Limulus amoebocyte lysate assay (LDL), whereas DEFB3 does not. 3. When cells of the murine macrophage cell line RAW264.7 are treated with LPS they release TNF-
. However, if these cells were preincubated with DEFB123, it dose-dependently inhibited the TNF- release with an IC50 of 4.4 µM (Fig. 1
). 4. It is well established that in the murine macrophage cell line RAW264.7, LPS treatment induces phosphorylation of p42/44 mitogen-activated protein kinase (pMAPK). However, when LPS and DEFB123 were applied in combination, the LPS-induced pMAPK was markedly attenuated. The LPS-binding peptide MBI-28 also inhibited LPS-induced pMAPK elevation, whereas DEFB3 did not. 5. When D-galactosamine (D-GalN)-sensitized mice are injected with 100 ng LPS over 80% of the mice die within 24 h. However, both under nontherapeutic (preinjection of protective agent) and therapeutic (LPS injection before protective agent on contralateral side of the peritoneum) conditions, DEFB123, at 50mg/kg, and LL-37, a known LPS-protective agent at 500 µg/kg, lead to survival of 75% and 70%, respectively, of the treated mice. Ten-fold (DEFB123) and five-fold lower amounts did not protect the mice from LPS-induced death and were indistinguishable from the control mice treated with LPS alone. The complete data are shown in Table 1
.
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CONCLUSIONS AND SIGNIFICANCE
We show here that, like other ßbeta;-defensins, DEFB123 exerts antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative germs. In addition, the peptide shows LPS-binding activity in a Limulus amoebocyte lysate (LAL) assay. Moreover, DEFB123 prevented LPS-induced tumor necrosis factor (TNF)- secretion in a murine monocyte cell line, RAW264.7. Accordingly, DEFB123 abolished LPS-mediated pMAPK induction in these cells, suggesting that DEFB123 may also be protective against LPS-induced lethality in an in vivo model for sepsis. Our experiments show that synthetic ßbeta;-defensin, DEFB123, prevents LPS-induced mortality in C57BL/6 mice in a therapeutic approach. We therefore propose that the physiological role of ßbeta;-defensins may include the interference with LPS-action on macrophages, a function formerly thought to be restricted to the family of cathelicidins, an antimicrobial peptide group, which is structurally unrelated to the ßbeta;-defensin family.
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FOOTNOTES
1 Current address: Deutsches Primatenzentrum GmbH, Kellnerweg 4, Göttingen 37077, Germany. 
2 These authors contributed equally to this work.
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4970fje
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