Novel human bioactive peptides identified in Apolipoprotein B: Evaluation of their therapeutic potential.

Host defence peptides (HDPs) are short, cationic amphipathic peptides that play a key role in the response to infection and inflammation in all complex life forms. It is increasingly emerging that HDPs generally have a modest direct activity against a broad range of microorganisms, and that their anti-infective properties are mainly due to their ability to modulate the immune response. Here, we report the recombinant production and characterization of two novel HDPs identified in human Apolipoprotein B (residues 887-922) by using a bioinformatics method recently developed by our group. We focused our attention on two variants of the identified HDP, here named r(P)ApoBL and r(P)ApoBS, 38- and 26-residue long, respectively. Both HDPs were found to be endowed with a broad-spectrum antimicrobial activity while they show neither toxic nor haemolytic effects towards eukaryotic cells. Interestingly, both HDPs were found to display a significant anti-biofilm activity, and to act in synergy with either commonly used antibiotics or EDTA. The latter was selected for its ability to affect bacterial outer membrane permeability, and to sensitize bacteria to several antibiotics. Circular dichroism analyses showed that SDS, TFE, and LPS significantly alter r(P)ApoBL conformation, whereas slighter or no significant effects were detected in the case of r(P)ApoBS peptide. Interestingly, both ApoB derived peptides were found to elicit anti-inflammatory effects, being able to mitigate the production of pro-inflammatory interleukin-6 and nitric oxide in LPS induced murine macrophages. It should also be emphasized that r(P)ApoBL peptide was found to play a role in human keratinocytes wound closure in vitro. Altogether, these findings open interesting perspectives on the therapeutic use of the herein identified HDPs.

Prophylactic administration of chicken cathelicidin-2 boosts zebrafish embryonic innate immunity.

Chicken cathelicidin-2 (CATH-2) is a host defense peptide that exhibits immunomodulatory and antibacterial properties. Here we examined effects of CATH-2 in zebrafish embryos in the absence and presence of infection. Yolk-injection of 0.2-1.5 h post-fertilized (hpf) zebrafish embryos with 2.6 ng/kg CATH-2 increased proliferation of phagocytic cells at 48 hpf by 30%. A lethal infection model was developed to test the prophylactic protective effect of CATH-2 peptide. Embryos (0.2-1.5 hpf) were injected with 2.6 ng/kg CATH-2, challenged with a lethal dose of fluorescently labeled Salmonella enteritidis pGMDs3 at 28 hpf and monitored for survival. Prophylactic treatment with CATH-2 was found to delay infection starting at 22 h post-infection (hpi). At 18-20 hpi, significantly lower (2-fold) fluorescence intensity and decreased bacterial loads were detected in peptide-treated embryos. Thus prophylactic administration of low CATH-2 concentrations confer partial protection in zebrafish embryos by boosting the innate immune system.

Antimicrobial and immunomodulatory activities of PR-39 derived peptides.

The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics.

Arginase release by primary hepatocytes and liver slices results in rapid conversion of arginine to urea in cell culture media.

Precision-cut liver slices and primary hepatocytes constitute suitable model systems for studying liver function. Frequently, urea cycle activity is used as a parameter to determine hepatocyte viability. Liver cells contain high levels of the urea cycle enzyme arginase, which converts arginine into urea and ornithine. Arginase can leak from the cells into the supernatants, converting arginine directly to urea and in this way circumventing the urea cycle. In this study, a hepatocellular cell line (HepG2 cells), a primary rat hepatocyte culture, and precision-cut rat liver slices were compared with respect to arginase leakage in the media by determining arginine conversion into urea. HepG2 cells did not show arginine conversion to urea during 24h incubations. In contrast, in both precision-cut liver slices and primary rat hepatocytes all arginine was converted to urea. Arginase activity was confirmed by showing that freshly added arginine to the cell-free supernatants again was converted to urea. In conclusion, when choosing urea production of primary hepatocytes cultures as a viability indicator, one has to take into account that arginase can leak from the cells into the supernatant. This can lead to an overestimation of the viability of the cells, since arginase converts arginine into urea without involvement of the urea cycle. We suggest using an extra incubation in an arginine-free buffer supplemented with ornithine and NH4Cl. In addition, arginase leakage can lead to depletion of the supernatant of arginine in primary hepatocytes cell cultures. This might have implications for studying cellular activities where arginine is involved, like, e.g. nitric oxide (NO) production.

In vitro and in vivo intrapulmonary distribution of fluorescently labeled surfactant.

OBJECTIVE: To determine the distribution of endotracheally administered surfactant at the alveolar level in an animal model of acute respiratory distress syndrome. DESIGN: Prospective, randomized animal study. SETTING: Research laboratory of a university hospital. SUBJECTS: Seventy-one male Sprague-Dawley rats, weighing 330-370 g. INTERVENTIONS: To measure surfactant distribution in vitro, a glass trough mimicking dichotomic lung anatomy was used to determine the spreading properties of bovine lung surfactant extract supplemented with fluorescent Bodipy-labeled surfactant protein B. To measure surfactant distribution in vivo, rats were anesthetized, and lipopolysaccharide was aerosolized (12 mg/kg body weight) to induce lung injury resembling acute respiratory distress syndrome; in control rats, buffered saline was aerosolized. Twenty-four hours later rats were anesthetized, tracheotomized, and mechanically ventilated (peak airway pressure = 20 mbar; positive end-expiratory pressure = 6 mbar; inspiration time = expiration time = 0.6 sec; Fio2 = 50%). Surfactant (bovine lung surfactant extract, supplemented with fluorescent Bodipy-labeled surfactant protein B; 50 mg/kg body weight) was applied as a bolus; in control rats, saline was administered as a bolus. Rats were ventilated for 5, 15, 30, or 60 mins (n = 8 or 9 for each group). Then, lungs were excised and sliced. Lung slices, divided into aerated (open), underinflated (dystelectatic), or collapsed (atelectatic) alveolar areas, were examined by both light and fluorescence microscopy. RESULTS: In vitro experiments revealed that surfactant spread independent of glass trough geometry and lowered the surface tension to equilibrium values (25 mN/m) within a few seconds. In vivo experiments showed that administered surfactant distributed preferentially into underinflated and aerated alveolar areas. Furthermore, surfactant distribution was not affected by length of mechanical ventilation. CONCLUSIONS: When conventional mechanical ventilation was used in lipopolysaccharide-induced lung injury, surfactant preferentially distributed into underinflated and aerated alveolar areas. Because surfactant rarely reached collapsed alveolar areas, methods aiding in alveolar recruitment (e.g., open lung concept or body positioning) should precede surfactant administration.