Assays evaluating the cytotoxic activity of the peptides against fresh mouse red blood cells (mRBCs) 1% Triton X-100 (triangle) served as positive control (100% hemolysis). (A) The release of hemoglobin was measured at 540 nm. In assays evaluating the cytotoxic activity of the different peptides and antibiotics against RAW 264.7 monocytes (B) L929 (C) and HEK-293 (D) cells were incubated for 24 h. cell viability was assessed by MTT assay. Values within the table correspond to the concentration (given in μM) of the Clavanin A (star), clavanin-MO (square) and LL-37 (asterisk). Data represent the mean of three experiments performed in triplicate. Data are expressed as mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. Statistical analysis was performed using a Student’s t test with Bonferroni correction for multiple testing.

In vitro assays. Effect of clavanin A and clavanin-MO on cytokine release (pg.mL⁻¹) from RAW264.7 macrophages. RAW264.7 cells were stimulated with LPS (10 ng.mL⁻¹) and treated with 2 μM of peptides. Cathelicidin LL-37 (1 μM) was used as control. All experiments were performed in triplicate. Pro-inflammatory cytokine levels for (A) IL-10, (B) IL-12p70 and (C) TNF-α were analyzed for samples collected 6, 12, 24 and 48 hours after treatment. Data are expressed as mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. Statistical analysis was performed using Bonferroni correction, where was compared NT group with the other groups. In vivo assays. Clavanin A and clavanin-MO induced migration of leukocytes into the peritoneal cavity of mice. Leukocyte migration was evaluated into the peritoneal cavity of uninfected C57BL/6 mice 30 min, 1, 2, 3, 4, 6 and 24 hours after treatment with 10 mg.kg⁻¹ of peptides (D). Thioglycolate (TGA) 3% was used as a positive control for leukocyte migration. Leukoyte recruitment to the peritoneal cavity was also assessed in mice infected with E. coli ATCC 8739 (E) or S. aureus ATCC 29213 (F). Leukocytes were counted 3 and 24 hours after infection and treatment with 10 mg.kg⁻¹ of peptide, 10 mg.kg^-1 of imipenem or PBS control. Data were expressed as mean ± standard deviation. Statistical analysis was performed using Fisher’s exact test. *p < 0.05; **p < 0.01; ***p < 0.001. Effects of clavanin A and clavanin-MO on the release of cytokines into the peritoneal cavity of mice infected with E. coli ATCC 8739 or S. aureus ATCC 29213. Cytokine release into the peritoneal cavity of C57BL/6 mice was measured by ELISA after 3 hours of peptide administration for mice infected with E. coli ATCC 8739 and treated with 10 mg.kg⁻¹ clavanin A or clavanin-MO (G). A separate group of mice were infected with S. aureus ATCC 29213, treated with 10 mg.kg⁻¹ of clavanin A or clavanin-MO, and peritoneal cytokine profiles were measured after 3 hours of administering peptides (H). PBS served as a negative control for these experiments. Data are expressed as mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. Statistical analysis was performed using Fisher’s exact test.

Mice were infected with ~2 × 10⁷ CFU of E. coli ATCC 8739 (A,E) or E. coli KPC-positive ID N°.1812446 (B F) or ~2 × 10⁹ CFU of S. aureus ATCC 29213 (C,G) or S. aureus MRSA ATCC 33591 (D,H). Mice were treated i.p. with a single dose of clavanin A , clavanin-MO , gentamicin , imipenem (10 mg.kg⁻¹) and PBS 3 h after infection. Survival of mice was evaluated over time during an 8-day period. Evaluation of the protective activity of the peptides in murine models of infection with ~2 × 10⁷ CFU of E. coli ATCC 8739 (I) or E. coli KPC-positive ID N°1812446 (J) or~2 × 10⁹ CFU of S. aureus ATCC 29213 (K) or S. aureus (MRSA) ATCC 33591 (L). Mice were treated i.p. with a single dose of peptide (10 mg.kg⁻¹) 3 hours after infection. The bacterial load was measured by counting bacteria in the peritoneal lavage 24 hours after infection. Data are expressed as mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. Statistical significance of differences between experimental groups of animals was determined using the Fisher’s exact test.