Human organoid biofilm model for assessing antibiofilm activity of novel agents.

Bacterial biofilms cause 65% of all human infections and are highly resistant to antibiotic therapy but lack specific treatments. To provide a human organoid model for studying host-microbe interplay and enabling screening for novel antibiofilm agents, a human epidermis organoid model with robust methicillin-resistant Staphylococcus aureus (MRSA) USA300 and Pseudomonas aeruginosa PAO1 biofilm was developed. Treatment of 1-day and 3-day MRSA and PAO1 biofilms with antibiofilm peptide DJK-5 significantly and substantially reduced the bacterial burden. This model enabled the screening of synthetic host defense peptides, revealing their superior antibiofilm activity against MRSA compared to the antibiotic mupirocin. The model was extended to evaluate thermally wounded skin infected with MRSA biofilms resulting in increased bacterial load, cytotoxicity, and pro-inflammatory cytokine levels that were all reduced upon treatment with DJK-5. Combination treatment of DJK-5 with an anti-inflammatory peptide, 1002, further reduced cytotoxicity and skin inflammation.

Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections.

One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies.IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.

Cyclic Derivative of Host-Defense Peptide IDR-1018 Improves Proteolytic Stability, Suppresses Inflammation, and Enhances In Vivo Activity.

Host-defense peptides have drawn significant attention as new drugs or drug adjuvants to combat multidrug-resistant bacteria. In this study, we report the development of cyclic derivatives of the immunomodulatory and antibiofilm innate defense regulator peptide (IDR)-1018 based on three different synthetic strategies including head-to-tail cyclization (C1), side-chain-to-tail cyclization (C2), and a disulfide bond cross-linkage (C3). The generated mimetics showed enhanced proteolytic stability and reduced aggregation in vitro and in vivo. The C2 derivative exhibited exceptional ability to suppress inflammation and significantly reduce bacterial loads in a high-density Staphylococcus aureus murine skin infection model. The findings describe different routes to the creation of enzymatically stable mimetics of IDR-1018 and identify a promising new cyclic analogue against bacterial infections.

Retinoic acid elicits a coordinated expression of gut homing markers on T lymphocytes of Zambian men receiving oral Vivotif, but not Rotarix, Dukoral or OPVERO vaccines.

All-trans retinoic acid (ATRA) up-regulates, in laboratory animals, the expression of the gut homing markers α4ß7 integrin and CCR9 on lymphocytes, increasing their gut tropism. Here, we show that, in healthy adult volunteers, ATRA induced an increase of these gut homing markers on T cells in vivo in a time dependent manner. The coordinated increase of α4ß7 and CCR9 by ATRA was seen in 57% (12/21) of volunteers and only when given together with an oral Vivotif vaccine. When this coordinated response to ATRA and Vivotif vaccine was present, it was strongly correlated with the gut immunoglobulin A (IgA) specific response to vaccine LPS (ρ = 0.82; P = 0.02). Using RNA-Seq analysis of whole blood transcription, patients receiving ATRA and Vivotif in conjunction showed transcriptomic changes in immune-related pathways, particularly including interferon α/ß signaling pathway, membrane-ECM interactions and immune hubs. These results suggest that exogenous ATRA can be used to manipulate responses to a subclass of oral vaccines, so far limited to a live attenuated Vivotif vaccine.

In silico optimization of a guava antimicrobial peptide enables combinatorial exploration for peptide design.

Plants are extensively used in traditional medicine, and several plant antimicrobial peptides have been described as potential alternatives to conventional antibiotics. However, after more than four decades of research no plant antimicrobial peptide is currently used for treating bacterial infections, due to their length, post-translational modifications or  high dose requirement for a therapeutic effect . Here we report the design of antimicrobial peptides derived from a guava glycine-rich peptide using a genetic algorithm. This approach yields guavanin peptides, arginine-rich α-helical peptides that possess an unusual hydrophobic counterpart mainly composed of tyrosine residues. Guavanin 2 is characterized as a prototype peptide in terms of structure and activity. Nuclear magnetic resonance analysis indicates that the peptide adopts an α-helical structure in hydrophobic environments. Guavanin 2 is bactericidal at low concentrations, causing membrane disruption and triggering hyperpolarization. This computational approach for the exploration of natural products could be used to design effective peptide antibiotics.

An Immunomodulatory Peptide Confers Protection in an Experimental Candidemia Murine Model.

Fungal Candida species are commensals present in the mammalian skin and mucous membranes. Candida spp. are capable of breaching the epithelial barrier of immunocompromised patients with neutrophil and cell-mediated immune dysfunctions and can also disseminate to multiple organs through the bloodstream. Here we examined the action of innate defense regulator 1018 (IDR-1018), a 12-amino-acid-residue peptide derived from bovine bactenecin (Bac2A): IDR-1018 showed weak antifungal and antibiofilm activity against a Candida albicans laboratory strain (ATCC 10231) and a clinical isolate (CI) (MICs of 32 and 64 µg · ml-1, respectively), while 8-fold lower concentrations led to dissolution of the fungal cells from preformed biofilms. IDR-1018 at 128 µg · ml-1 was not hemolytic when tested against murine red blood cells and also has not shown a cytotoxic effect on murine monocyte RAW 264.7 and primary murine macrophage cells at the tested concentrations. IDR-1018 modulated the cytokine profile during challenge of murine bone marrow-derived macrophages with heat-killed C. albicans (HKCA) antigens by increasing monocyte chemoattractant protein 1 (MCP-1) and interleukin-10 (IL-10) levels, while suppressing tumor necrosis factor alpha (TNF-α), IL-1ß, IL-6, and IL-12 levels. Mice treated with IDR-1018 at 10 mg · kg-1 of body weight had an increased survival rate in the candidemia model compared with phosphate-buffered saline (PBS)-treated mice, together with a diminished kidney fungal burden. Thus, IDR-1018 was able to protect against murine experimental candidemia and has the potential as an adjunctive therapy.

Alternative strategies for the study and treatment of clinical bacterial biofilms.

Biofilms represent an adaptive lifestyle where microbes grow as structured aggregates in many different environments, e.g. on body surfaces and medical devices. They are a profound threat in medical (and industrial) settings and cause two-thirds of all infections. Biofilm bacteria are especially recalcitrant to common antibiotic treatments, demonstrating adaptive multidrug resistance. For this reason, novel methods to eradicate or prevent biofilm infections are greatly needed. Recent advances have been made in exploring alternative strategies that affect biofilm lifestyle, inhibit biofilm formation, degrade biofilm components and/or cause dispersal. As such, naturally derived compounds, molecules that interfere with bacterial signaling systems, anti-biofilm peptides and phages show great promise. Their implementation as either stand-alone drugs or complementary therapies has the potential to eradicate resilient biofilm infections. Additionally, altering the surface properties of indwelling medical devices through bioengineering approaches has been examined as a method for preventing biofilm formation. There is also a need for improving current biofilm detection methods since in vitro methods often do not accurately measure live bacteria in biofilms or mimic in vivo conditions. We propose that the design and development of novel compounds will be enabled by the improvement and use of appropriate in vitro and in vivo models.

Bacterial Abscess Formation Is Controlled by the Stringent Stress Response and Can Be Targeted Therapeutically.

Cutaneous abscess infections are difficult to treat with current therapies and alternatives to conventional antibiotics are needed. Understanding the regulatory mechanisms that govern abscess pathology should reveal therapeutic interventions for these recalcitrant infections. Here we demonstrated that the stringent stress response employed by bacteria to cope and adapt to environmental stressors was essential for the formation of lesions, but not bacterial growth, in a methicillin resistant Staphylococcus aureus (MRSA) cutaneous abscess mouse model. To pharmacologically confirm the role of the stringent response in abscess formation, a cationic peptide that causes rapid degradation of the stringent response mediator, guanosine tetraphosphate (ppGpp), was employed. The therapeutic application of this peptide strongly inhibited lesion formation in mice infected with Gram-positive MRSA and Gram-negative Pseudomonas aeruginosa. Overall, we provide insights into the mechanisms governing abscess formation and a paradigm for treating multidrug resistant cutaneous abscesses.

High throughput screening methods for assessing antibiofilm and immunomodulatory activities of synthetic peptides.

The recent observation that certain cationic peptides possess potent antibiofilm activity demonstrated that small peptides could be used to treat biofilm-associated infections. Other so-called innate defense regulator peptides possess potent immunomodulatory properties such as leukocyte recruitment and suppression of harmful inflammation. A peptide that directly targets biofilm cells while favorably modulating the immune response would be particularly advantageous for treating serious skin infections caused by Staphylococcus aureus. In the present work, using SPOT-synthesized peptide arrays on cellulose membranes, we outline a strategy for systematically assessing the antibiofilm activity of hundreds of IDR-1002 (VQRWLIVWRIRK-NH2) and IDR-HH2 (VQLRIRVAVIRA-NH2) peptide variants against MRSA biofilms. In addition, the ability of these peptides to stimulate production of a monocyte chemoattractant protein (MCP-1) and suppress LPS-induced interleukin (IL)-1ß production in human peripheral blood mononuclear cells (PBMCs) was evaluated. These results informed the synthesis of second-generation peptides resulting in a new peptide, IDR-2009 (KWRLLIRWRIQK-NH2), with enhanced MCP-1 stimulatory activity, favorable IL-1ß suppression characteristics and strong antibiofilm activity against MRSA and Pseudomonas aeruginosa biofilms. This work provides a proof-of-concept that multiple peptide activities can be optimized simultaneously to generate novel sequences that possess a variety of biological properties.

Expression of an engineered heterologous antimicrobial peptide in potato alters plant development and mitigates normal abiotic and biotic responses.

Antimicrobial cationic peptides (AMPs) are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s) and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani) and abiotic stressors (dark-induced senescence, wounding and temperature stress). msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR) and reactive oxygen species (ROS) responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s) of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield.

Synthetic analogues of bovine bactenecin dodecapeptide reduce herpes simplex virus type 2 infectivity in mice.

We have evaluated the potential of four synthetic peptides (denoted HH-2, 1002, 1006, 1018) with a distant relationship to the host defense peptide bovine bactenecin dodecapeptide for their ability to prevent genital infections with herpes simplex virus type 2 (HSV-2) in mice. All four peptides showed antiviral properties in vitro and reduced HSV-2 infection of Vero cells in a dose-dependent manner. Detailed analysis showed that the peptides were able to interfere with both viral attachment and entry, but not with replication post-entry, and were effective antivirals also when HSV-2 was introduced in human semen. Two of the peptides proved especially effective in reducing HSV-2 infection also in vivo. When admixed with virus prior to inoculation, both HH-2 and 1018 reduced viral replication and disease development in a genital model of HSV-2 infection in mice, and also when using very high infectious doses of HSV-2. These data show that peptides HH-2 and 1018 have antiviral properties and can be used to prevent genital herpes infection in mice.

Multilayered coating on titanium for controlled release of antimicrobial peptides for the prevention of implant-associated infections.

Prevention of bacterial colonization and formation of a bacterial biofilm on implant surfaces has been a challenge in orthopaedic surgery. The treatment of implant-associated infections with conventional antibiotics has become more complicated by the emergence of multi-drug resistant bacteria. Antimicrobial eluting coatings on implants is one of the most promising strategies that have been attempted. This study reports a controlled release of an antimicrobial peptide (AMP) from titanium surface through a non-cytotoxic multilayered coating. Three layers of vertically oriented TiO2 nanotubes, a thin layer of calcium phosphate coating and a phospholipid (POPC) film were impregnated with a potent broad-spectrum AMP (HHC-36). The coating with controlled and sustained release of AMP was highly effective against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. No cytotoxicity to osteoblast-like cells (MG-63) was observed. Moderate platelet activation and adhesion on the implant surface with no observable activation in solution, and very low red blood cell lysis was observed on the implant. This multi-layer assembly can be a potential approach to locally deliver AMPs to prevent peri-implant infection in orthopaedics without being toxic to host cells.

Innate defense regulator peptide 1018 in wound healing and wound infection.

Innate defense regulators (IDRs) are synthetic immunomodulatory versions of natural host defense peptides (HDP). IDRs mediate protection against bacterial challenge in the absence of direct antimicrobial activity, representing a novel approach to anti-infective and anti-inflammatory therapy. Previously, we reported that IDR-1018 selectively induced chemokine responses and suppressed pro-inflammatory responses. As there has been an increasing appreciation for the ability of HDPs to modulate complex immune processes, including wound healing, we characterized the wound healing activities of IDR-1018 in vitro. Further, we investigated the efficacy of IDR-1018 in diabetic and non-diabetic wound healing models. In all experiments, IDR-1018 was compared to the human HDP LL-37 and HDP-derived wound healing peptide HB-107. IDR-1018 was significantly less cytotoxic in vitro as compared to either LL-37 or HB-107. Furthermore, administration of IDR-1018 resulted in a dose-dependent increase in fibroblast cellular respiration. In vivo, IDR-1018 demonstrated significantly accelerated wound healing in S. aureus infected porcine and non-diabetic but not in diabetic murine wounds. However, no significant differences in bacterial colonization were observed. Our investigation demonstrates that in addition to previously reported immunomodulatory activities IDR-1018 promotes wound healing independent of direct antibacterial activity. Interestingly, these effects were not observed in diabetic wounds. It is anticipated that the wound healing activities of IDR-1018 can be attributed to modulation of host immune pathways that are suppressed in diabetic wounds and provide further evidence of the multiple immunomodulatory activities of IDR-1018.

Local delivery of antimicrobial peptides using self-organized TiO2 nanotube arrays for peri-implant infections.

Peri-implant infections have been reported as one of the major complications that lead to the failure of orthopedic implants. An ideal solution to the peri-implant infection is to locally deliver antimicrobial agents through the implant surface. The rising problem of infections caused by multiple antibiotic-resistant bacteria makes traditional antibiotics less desirable for the prevention of peri-implant infections. One of the promising alternatives is the family of antimicrobial peptides (AMPs). In this study, we report the local delivery of AMPs through the nanotubular structure processed on titanium surface. Self-organized and vertically oriented TiO2 nanotubes, about 80 nm in diameter and 7 µm thick, were prepared by the anodization technique. HHC-36 (KRWWKWWRR), one of the most potent broad-spectrum AMPs, was loaded onto the TiO2 nanotubes via a simple vacuum-assisted physical adsorption method. Antimicrobial activity testing against Gram-positive bacterium, Staphylococcus aureus, demonstrated that this AMP-loaded nanotubular surface could effectively kill the bacteria (≈ 99.9% killing) and reduce the total bacterial number adhered to the surface after 4 h of culture. In vitro AMP elution from the nanotubes was investigated using liquid chromatography-mass spectrometry (LC-MS). The release profiles strongly depended on the crystallinity of the TiO2 nanotubes. Anatase TiO2 nanotubes released significantly higher amounts of AMP than amorphous nanotubes during the initial burst release stage. Both followed almost the same slow release profile from 4 h up to 7 days. Despite the differences in release kinetics, no significant difference was observed between these two groups in bactericidal efficiency.

Antimicrobial peptides on calcium phosphate-coated titanium for the prevention of implant-associated infections.

Prevention of implant-associated infections has been one of the main challenges in orthopaedic surgery. This challenge is further complicated by the concern over the development of antibiotic resistance as a result of using traditional antibiotics for infection prophylaxis. The objective of this study was to develop a technique that enables the loading and local delivery of a unique group of cationic antimicrobial peptides (AMP) through implant surfaces. A thin layer of micro-porous calcium phosphate (CaP) coating was processed by electrolytic deposition onto the surface of titanium as the drug carrier. The broad spectrum AMP Tet213 (KRWWKWWRRC) was selected and loaded onto the CaP coating. SEM, XRD and FTIR analyses confirmed the CaP coating to be micro-porous octacalcium phosphate. By using a luminescence spectrometer technique, it was demonstrated that a 7 µm thick porous CaP coating could load up to 9 µg of AMP/cm² using a simple soaking technique. The drug-loaded CaP coating (CaP-Tet213) was not cytotoxic for MG-63 osteoblast-like cells. The CaP-Tet213 implants had antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria with 106-fold reductions of both bacterial strains within 30 min as assessed by measuring colony-forming units (CFU). Repeated CFU assays on the same CaP-Tet213 specimen demonstrated retention of antimicrobial activity by the CaP-Tet213 surfaces through four test cycles. The susceptibility of bacteria to the CaP-Tet213 surfaces was also evaluated by assessing the inhibition of luminescence of P. aeruginosa containing a luxCDABE cassette at 4 h and 24 h with ∼92% and ∼77% inhibition of luminescence, respectively. It was demonstrated that CaP-Tet213 was a more efficient antimicrobial coating than CaP-MX226, CaP-hLF1-11 or CaP-tobramycin following incubation of CaP implants with equimolar concentrations of Tet213, the commercially developed antimicrobial peptide MX-226, hLF1-11 or tobramycin. A device coated with CaP-Tet213 could be a potential solution for the prevention of the peri-implant infection in orthopaedics.

Screening and characterization of surface-tethered cationic peptides for antimicrobial activity.

There is an urgent need to coat the surfaces of medical devices, including implants, with antimicrobial agents to reduce the risk of infection. A peptide array technology was modified to permit the screening of short peptides for antimicrobial activity while tethered to a surface. Cellulose-amino-hydroxypropyl ether (CAPE) linker chemistry was used to synthesize, on a cellulose support, peptides that remained covalently bound during biological assays. Among 122 tested sequences, the best surface-tethered 9-, 12-, and 13-mer peptides were found to be highly antimicrobial against bacteria and fungi, as confirmed using alternative surface materials and coupling strategies as well as coupling through the C and N termini of the peptides. Structure-activity modeling of the structural features determining the activity of tethered peptides indicated that the extent and positioning of positive charges and hydrophobic residues were influential in determining activity.

The roles of cathelicidin LL-37 in immune defences and novel clinical applications.

PURPOSE OF REVIEW: LL-37 is the only member of the cathelicidin family of host defence peptides expressed in humans. It is primarily produced by phagocytic leucocytes and epithelial cells, and mediates a wide range of biological responses: direct killing of microorganisms, chemotaxis and chemokine induction, regulation of inflammatory responses, as well as adjuvant, angiogenic and wound healing effects. In this review we will cover the recent advances in the understanding of LL-37 biology: its activities, the mechanisms of its induction and roles in immune defence. RECENT FINDINGS: Recent studies advanced our understanding of the mechanisms controlling LL-37 expression, demonstrating the key involvement of the vitamin D3 and the hypoxia response pathways, and the impacts of commensal and pathogenic microorganisms on its production. The synergistic and antagonistic interactions between LL-37 and other immune mediators have been further elucidated. Furthermore, studies in animal models and human patients further characterized the roles of cathelicidins in immunity, with roles in infectious and inflammatory conditions. The underlying properties of LL-37 have been exploited to create innate defence regulator peptides that represent a novel immunomodulatory approach to treating infections. SUMMARY: The understanding of the biological properties and functions of LL-37 and other host defence peptides advances our knowledge of innate immunity, the interactions of the host with pathogens and the microflora, as well as the pathology of infectious and inflammatory diseases, creating many strategies and opportunities for therapeutic intervention.

Using microarray gene signatures to elucidate mechanisms of antibiotic action and resistance.

Microarray analyses reveal global changes in gene expression in response to environmental changes and, thus, are well suited to providing a detailed picture of bacterial responses to antibiotic treatment. These responses are represented by patterns of gene expression, termed expression signatures, which provide insight into the mechanism of action of antibiotics as well as the general physiological responses of bacteria to antibiotic-related stresses. The complexity of such signatures is challenging the notion that antibiotics act on single targets and this is consistent with the concept that there are multiple targets coupled with common stress responses. A more detailed knowledge of how known antibiotics act should reveal new strategies for antimicrobial drug discovery.

Antimicrobial peptide therapeutics for cystic fibrosis.

Greater than 90% of lung infections in cystic fibrosis (CF) patients are caused by Pseudomonas aeruginosa, and the majority of these patients subsequently die from lung damage. Current therapies are either targeted at reducing obstruction, reducing inflammation, or reducing infection. To identify potential therapeutic agents for the CF lung, 150 antimicrobial peptides consisting of three distinct structural classes were screened against mucoid and multidrug-resistant clinical isolates of P. aeruginosa, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Staphylococcus aureus. Five peptides that retained potent antimicrobial activities in physiological salt and divalent cation environment were further characterized in vivo using a rat chronic lung infection model. All animals were inoculated intratracheally with 10(4) P. aeruginosa mucoid PAO1 cells in agar beads. Three days following inoculation treatment was initiated. Animals were treated daily for 3 days with 100 microl of peptide solution (1 mg/ml) in 10 mM sodium citrate, which was deposited via either intratracheal instillation or aerosolization. Control animals received daily exposure to vehicle alone. At the end of the treatment the lungs of the animals were removed for quantitative culture. Four peptides, HBCM2, HBCM3, HBCPalpha-2, and HB71, demonstrated significant reduction in Pseudomonas bioburden in the lung of rats. Further in vivo studies provided direct evidence that anti-inflammatory activity was associated with three of these peptides. Therefore, small bioactive peptides have the potential to attack two of the components responsible for the progression of lung damage in the CF disease: infection and inflammation.