Human ß-Defensin 2 Mediated Immune Modulation as Treatment for Experimental Colitis.

Defensins represents an integral part of the innate immune system serving to ward off potential pathogens and to protect the intestinal barrier from microbial encroachment. In addition to their antimicrobial activities, defensins in general, and human ß-defensin 2 (hBD2) in particular, also exhibit immunomodulatory capabilities. In this report, we assessed the therapeutic efficacy of systemically administered recombinant hBD2 to ameliorate intestinal inflammation in three distinct animal models of inflammatory bowel disease; i.e., chemically induced mucosal injury (DSS), loss of mucosal tolerance (TNBS), and T-cell transfer into immunodeficient recipient mice. Treatment efficacy was confirmed in all tested models, where systemically administered hBD2 mitigated inflammation, improved disease activity index, and hindered colitis-induced body weight loss on par with anti-TNF-α and steroids. Treatment of lipopolysaccharide (LPS)-activated human peripheral blood mononuclear cells with rhBD2 confirmed the immunomodulatory capacity in the circulatory compartment. Subsequent analyzes revealed dendritic cells (DCs) as the main target population. Suppression of LPS-induced inflammation was dependent on chemokine receptor 2 (CCR2) expression. Mechanistically, hBD2 engaged with CCR2 on its DC target cell to decrease NF-κB, and increase CREB phosphorylation, hence curbing inflammation. To our knowledge, this is the first study showing in vivo efficacy of a systemically administered defensin in experimental disease.

Effect of Bacillus subtilis Strains on Intestinal Barrier Function and Inflammatory Response.

Strong tight junctions and curtailed inflammatory responses under stressful conditions are key for optimal digestive health. Bacillus-based probiotics are increasingly being used to maintain broilers' health, but their mode of action is often not well-defined. In the present study we used Caco-2 cells as a model for intestinal epithelia and assessed the effect of three Bacillus-based probiotics on intestinal barrier function and intestinal inflammation. Experimental results showed that one of the three tested strains, Bs 29784, significantly reinforced intestinal barrier integrity under basal conditions through an up-regulation of the expression of tight junction's proteins, whereas the others had no or detrimental effects. When Caco-2 cells were pre-treated with Bacillus subtilis strains, the subsequent IL-8 release to various pro-inflammatory signals (IL-1ß, deoxynivalenol, or flagellin) was blunted compared to cells that had not been pretreated, but to a different extent depending on the strain of Bacillus used. Bs 29784, was able to significantly decrease IL-8 production in all stressed conditions tested. Mechanistically, Bs 29784 appeared to limit nuclear translocation of NF-κB during IL-1ß exposure by preventing IκB degradation. The effects of Bs 29784 were observed independently with supernatant and cells but in a lesser extent than with the combination, indicating that they can thus likely be attributed to both secreted metabolites and cell-associated compounds. Moreover, under inflammatory conditions, Bs 29784 significantly reduced the upregulation of iNOS protein levels further underlining its intestinal anti-inflammatory potential. Our data show that Bacillus-based probiotics may indeed improve digestive health by strengthening intestinal barrier and limiting inflammatory responses and that these properties are strain-dependent.

Controlled Release of Plectasin NZ2114 from a Hybrid Silicone-Hydrogel Material for Inhibition of Staphylococcus aureus Biofilm.

Staphylococcus aureus is a major human pathogen in catheter-related infections. Modifying catheter material with interpenetrating polymer networks is a novel material technology that allows for impregnation with drugs and subsequent controlled release. Here, we evaluated the potential for combining this system with plectasin derivate NZ2114 in an attempt to design an S. aureus biofilm-resistant catheter. The material demonstrated promising antibiofilm properties, including properties against methicillin-resistant S. aureus, thus suggesting a novel application of this antimicrobial peptide.

Antimicrobial Peptide Novicidin Synergizes with Rifampin, Ceftriaxone, and Ceftazidime against Antibiotic-Resistant Enterobacteriaceae In Vitro.

The spread of antibiotic resistance among Gram-negative bacteria is a serious clinical threat, and infections with these organisms are a leading cause of mortality worldwide. Traditional novel drug development inevitably leads to the emergence of new resistant strains, rendering the new drugs ineffective. Therefore, reviving the therapeutic potentials of existing antibiotics represents an attractive novel strategy. Novicidin, a novel cationic antimicrobial peptide, is effective against Gram-negative bacteria. Here, we investigated novicidin as a possible antibiotic enhancer. The actions of novicidin in combination with rifampin, ceftriaxone, or ceftazidime were investigated against 94 antibiotic-resistant clinical Gram-negative isolates and 7 strains expressing New Delhi metallo-ß-lactamase-1. Using the checkerboard method, novicidin combined with rifampin showed synergy with >70% of the strains, reducing the MICs significantly. The combination of novicidin with ceftriaxone or ceftazidime was synergistic against 89.7% of the ceftriaxone-resistant strains and 94.1% of the ceftazidime-resistant strains. Synergistic interactions were confirmed using time-kill studies with multiple strains. Furthermore, novicidin increased the postantibiotic effect when combined with rifampin or ceftriaxone. Membrane depolarization assays revealed that novicidin alters the cytoplasmic membrane potential of Gram-negative bacteria. In vitro toxicology tests showed novicidin to have low hemolytic activity and no detrimental effect on cell cultures. We demonstrated that novicidin strongly rejuvenates the therapeutic potencies of ceftriaxone or ceftazidime against resistant Gram-negative bacteria in vitro. In addition, novicidin boosted the activity of rifampin. This strategy can have major clinical implications in our fight against antibiotic-resistant bacterial infections.

Intracellular activity of the peptide antibiotic NZ2114: studies with Staphylococcus aureus and human THP-1 monocytes, and comparison with daptomycin and vancomycin.

OBJECTIVES: Staphylococcus aureus survives inside eukaryotic cells. Our objective was to assess the activity of NZ2114, a novel peptidic antibiotic, against intracellular S. aureus in comparison with established antistaphylococcal agents acting on the bacterial envelope with a distinct mechanism. METHODS: The extracellular (broth) and intracellular (THP-1 monocytes) activities of NZ2114 were compared with those of vancomycin and daptomycin against methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). RESULTS: All three compounds showed an extracellular bactericidal effect (>3 log(10) kill) against MSSA and MRSA. Daptomycin and NZ2114 also exhibited bactericidal activity against VRSA. The extracellular killing was concentration dependent for all three compounds within the range of drug concentrations tested. The intracellular experiments demonstrated a maximal intracellular effect of NZ2114 after 24 h as a 5 log(10) cfu reduction against MSSA (ATCC 25923), while the activity was a 0.9 log(10) cfu reduction against MRSA and a 0.2 log(10) cfu reduction against VRSA. For comparison, the intracellular activity of daptomycin was a 1.0 log(10) cfu reduction against MSSA, a 0.8 log(10) cfu reduction against MRSA and a 0.3 log(10) cfu reduction against VRSA. Vancomycin showed activity against both MSSA and MRSA (0.6 log(10) cfu reduction), whereas VRSA was resistant to vancomycin. CONCLUSIONS: NZ2114 displayed similar extracellular and intracellular activities as daptomycin, and was more effective than vancomycin against the intracellular forms of susceptible bacteria. However, the study also showed that the intracellular activities of NZ2114 and daptomycin are weaker than their extracellular activities.

Plectasin shows intracellular activity against Staphylococcus aureus in human THP-1 monocytes and in a mouse peritonitis model.

Antimicrobial therapy of infections with Staphylococcus aureus can pose a challenge due to slow response to therapy and recurrence of infection. These treatment difficulties can partly be explained by intracellular survival of staphylococci, which is why the intracellular activity of antistaphylococcal compounds has received increased attention within recent years. The intracellular activity of plectasin, an antimicrobial peptide, against S. aureus was determined both in vitro and in vivo. In vitro studies using THP-1 monocytes showed that some intracellular antibacterial activity of plectasin was maintained (maximal relative efficacy [E(max)], 1.0- to 1.3-log reduction in CFU) even though efficacy was inferior to that of extracellular killing (E(max), >4.5-log CFU reduction). Animal studies included a novel use of the mouse peritonitis model, exploiting extra- and intracellular differentiation assays, and assessment of the correlations between activity and pharmacokinetic (PK) parameters. The intracellular activity of plectasin was in accordance with the in vitro studies, with an E(max) of a 1.1-log CFU reduction. The parameter most important for activity was fC(peak)/MIC, where fC(peak) is the free peak concentration. These findings stress the importance of performing studies of extra- and intracellular activity since these features cannot be predicted from traditional MIC and killing kinetic studies. Application of both the THP-1 and the mouse peritonitis models showed that the in vitro results were similar to findings in the in vivo model with respect to demonstration of intracellular activity. Therefore the in vitro model was a good screening model for intracellular activity. However, animal models should be applied if further information on activity, PK/pharmacodynamic parameters, and optimal dosing regimens is required.

Influence of antidrug antibodies on plectasin efficacy and pharmacokinetics.

Plectasin is a 4.4-kDa antimicrobial peptide with the potential to be a treatment of infections caused by gram-positive bacteria. Since plectasin is a large molecule compared to conventional antibiotics, the development of antidrug antibodies (ADAs) could be anticipated. The immunogenic properties of plectasin were assessed through immunization studies. In mice treated for 5 days with one to two daily subcutaneous doses of plectasin, no antibody response was observed. If the animals were immunized again, after a rest period, low levels of antibodies developed in approximately half the animals. Additionally, mice were immunized with plectasin in Freund's incomplete adjuvant (FIA). Ninety-two percent of these mice developed ADAs after repeated immunizations, with two-thirds having high levels of antibodies. An agar diffusion bioassay showed that sera from animals immunized with plectasin did not inhibit the efficacy of the drug, while hyperimmune sera from animals in which an immune response was provoked by immunization with plectasin in FIA reduced the efficacy of plectasin at the lowest concentration tested. Studies in the murine peritonitis model showed an excellent efficacy of plectasin for the treatment of Streptococcus pneumoniae infections both in naïve animals and in animals with ADAs. No difference in bacterial counts was seen when the animals were treated with plectasin at 2.5 mg/kg of body weight, a dose below the expected therapeutic level. When animals were treated with plectasin at 0.625 mg/kg, the effect was reduced but not neutralized in animals with high levels of ADAs. No animals showed signs of hypersensitivity or injection site reactions toward plectasin, and the half-life of the compound did not vary between animals with and without antibodies.