Human ß-defensins: The multi-functional natural peptide.

The increasing threat of antibiotic resistance among pathogenic microorganisms and the urgent demand for new antibiotics require immediate attention. Antimicrobial peptides exhibit effectiveness against microorganisms, fungi, viruses, and protozoa. The discovery of human ß-defensins represents a major milestone in biomedical research, opening new avenues for scientific investigation into the innate immune system and its resistance mechanisms against pathogenic microorganisms. Multiple defensins present a promising alternative in the context of antibiotic abuse. However, obstacles to the practical application of defensins as anti-infective therapies persist due to the unique properties of human ß-defensins themselves and serious pharmacological and technical challenges. To overcome these challenges, diverse delivery vehicles have been developed and progressively improved for the conjugation or encapsulation of human ß-defensins. This review briefly introduces the biology of human ß-defensins, focusing on their multistage structure and diverse functions. It also discusses several heterologous systems for producing human ß-defensins, various delivery systems created for these peptides, and patent applications related to their utilization, concluding with a summary of current challenges and potential solutions.

Immunotherapeutic effect of adenovirus encoding antimicrobial peptides in experimental pulmonary tuberculosis.

As components of the innate immune response, antimicrobial peptides (AMPs) efficiently contribute to infection control and maintenance of a latent state in pulmonary tuberculosis (TB). As a therapeutic strategy, the administration of recombinant AMPs could be limited by enzymatic degradation and high production costs. Likewise, strategies based on the induction of AMPs have generated controversial results. In this study, 2 recombinant type-5 adenoviruses (Ad) expressing the human ß-defensin 3 (HßD3) or cathelicidin (LL37) were assessed in a murine pulmonary TB model. Mice infected with either a high dose of a drug-sensitive (H37Rv) or a multidrug-resistant (MDR) strain of Mycobacterium tuberculosis (Mtb) were treated with a single administration of AdHßD3, AdLL37, AdGFP (control vector expressing a green fluorescent protein), or saline solution (SS). Lungs were obtained to determine the bacterial burden, histologic damage, and cytokine expression at different time points. Mice treated with AdHßD3 or AdLL37 showed significantly lower bacterial load and pneumonia, and higher proinflammatory cytokine expression than the control groups AdGFP and SS. A synergistic therapeutic effect could be observed when first- or second-line antibiotics (ABs) were administered with adenoviral therapy in animals infected with H37Rv or MDR strains, respectively. Adenovirus-delivered AMP's administration constitutes a promising adjuvant therapy for current anti-TB drugs by enhancing a protective immune response and potentially reducing current AB regimes' duration.

Effect of antimicrobial peptides HNP-1 and hBD-1 on Staphylococcus aureus strains in vitro and in vivo.

The aims of this study were: (i) To investigate the activity of recombinant AMPs HNP-1 and hBD-1 in combination with cefotaxime against Staphylococcus aureus strains (MSSA and MRSA) in vitro using checkerboard method; (ii) To investigate the activity of HNP-1 and hBD-1 encapsulated in silicon nanoparticles (niosomes) in the treatment of MRSA-infected wound in rats. For this S. aureus strains (MSSA and MRSA) were isolated from patients with diabetic foot infection. Cefotaxime, recombinant HNP-1 and hBD-1 (in all possible combinations with each other) were used for testing by the checkerboard method. Two niosomal topical gels with HNP-1/hBD-1 were prepared to treat MRSA-infected wounds in rats. Gels were administered once a day, the control group-without treatment. Wound healing rate was calculated on the 4th, 9th and 16th days of the experiment and compared using one-way ANOVA with Bonferroni correction. MIC of HNP-1 for MSSA and MRSA was the same-1 mg/L. MIC of hBD-1 for MSSA and MRSA was also the same-0.5 mg/L. Topical gels with niosomal HNP-1 (or hBD-1) showed a significantly faster wound healing in comparison with the control. The data obtained open up prospects for use of AMPs encapsulated in silica nanoparticles for the development of new antibiotics.

The host defense peptide ß-defensin confers protection against Vibrio anguillarum in ayu, Plecoglossus altivelis.

ß-defensin is a cationic host defense peptide actively participating in host innate immune response against pathogens. In teleost fish, ß-defensin exhibits a diversity in genotypes and functions. Herein, a ß-defensin homolog (PaBD) was identified from ayu, Plecoglossus altivelis, showing multiple tissues' upregulation against Vibrio anguillarum challenge. In vivo experiments revealed that intraperitoneal injection of chemically synthesized mature PaBD (mPaBD) increased the survival rate of V. anguillarum-infected ayu, accompanied by reduced bacterial load and decreased tissue mRNA levels of tumor necrosis factor α (PaTNF-α) and interleukin 1ß (PaIL-1ß). However, in vitro, mPaBD showed weak bactericidal activity against V. anguillarum. Interestingly, mPaBD enhanced phagocytosis, intracellular bacterial killing, and respiratory burst of ayu monocytes/macrophages (MO/MΦ). Moreover, it inhibited mRNA levels of PaIL-1ß and PaTNF-α in MO/MФ upon V. anguillarum infection. In conclusion, PaBD protects ayu against V. anguillarum challenge not only through its direct antibacterial ability, but also through its immunomodulation in MO/MΦ.

Enediyne-activated, EGFR-targeted human ß-defensin 1 has therapeutic efficacy against non-small cell lung carcinoma.

Human ß-defensins contain an oncolytic motif that binds to tumor cell membranes and mediate permeabilization, rapid induction of cytolysis, and apoptosis. Previous studies have indicated that a fragment of the mature human ß-defensin-1 (HBD1) peptide (DF) has antitumor properties. While targeted drug treatments using fusion proteins have been shown to increase drug efficacy, this phenomenon has not been studied for this defensin. Thus, in this study, we designed and prepared a fusion protein containing this HBD1 fragment and an epidermal growth factor receptor (EGFR)-targeting oligopeptide (Ec) as well as lidamycin (LDM), an extremely potent cytotoxic antitumor antibiotic, which consists of an apoprotein (LDP) and a highly active enediyne (AE). The fusion protein (Ec-LDP-DF) and its enediyne-integrated fusion protein (Ec-LDP(AE)-DF) were then purified and used to treat lung carcinoma cells in culture as well as lung carcinoma xenograft mouse models. The multifunctional fusion protein Ec-LDP-DF was shown to effectively bind to EGFR-expressing tumor cells. Furthermore, the enediyne-energized Ec-LDP(AE)-DF analog exhibited extremely potent cytotoxicity in NSCLC cell lines and an IC50 less than 10-10 mol/L. Ec-LDP(AE)-DF also significantly inhibited the growth of human carcinoma A549 and H460 xenografts in athymic mice at well-tolerated doses. Treatment resulted in cell cycle arrest and apoptosis in a dose-dependent manner. EGF-stimulated EGFR phosphorylation was also abolished by Ec-LDP(AE)-DF. In summary, our understanding of the role of defensins in cancer development and progression is continually expanding, and Ec-LDP(AE)-DF is a promising cancer cell-targeting agent for NSCLC.

Multi-Center, Double-Blind, Vehicle-Controlled Clinical Trial of an Alpha and Beta Defensin-Containing Anti-Aging Skin Care Regimen With Clinical, Histopathologic, Immunohistochemical, Photographic, and Ultrasound Evaluation.

BACKGROUND/OBJECTIVES: Anti-aging strategies utilizing stem cells are in the forefront. Alpha and beta defensins are natural immune peptides that have been shown to activate an LGR6-positive stem cell locus in the hair follicle, identified as the source of most new epidermal cells during acute wound healing. We investigated the ability of biomimetic alpha and beta defensin molecules, supplemented with supportive cosmetic ingredients, formulated into three skin care products, at improving the structure and function of aging skin. METHODS: A participant- and investigator -blinded, placebo-controlled, multi-center trial was performed in outpatient settings. Forty-four healthy female subjects, aged 41-71 years, skin types I-V, completed the study with 2/3 receiving full formula and 1/3 receiving the placebo formula. A skin care regimen of 3 products (serum, cream, and mask) containing alpha-defensin 5 and beta-defensin 3, and other cosmetic ingredients, was applied to the face, post-auricular, and neck skin two times per day for 12 weeks in those receiving full formula, whereas the placebo group received the identically packaged regimen without the active ingredients. Methods of evaluation included histopathology and immunohistochemistry (7 subjects), clinical evaluation of pores, superficial and deep wrinkles based on Griffiths scale, and high-resolution photography (all subjects). In addition, a subset of 15 patients were evaluated with the QuantifiCare system (3-dimensional imaging and skin care scores for evenness, pores, oiliness) and Cortex measurements (high-resolution skin ultrasound, TEWL, elasticity, color, and hydration). Data points for evaluation included baseline, 6 weeks, and 12 weeks. All patients used the same sunscreen and cleanser, which was provided to them. RESULTS: The full formula regimen caused a significantly (P equals 0.027) increased thickness of the epidermis as seen in histology, not seen in the placebo group, with no signs of inflammation. No excessive cell proliferation was detected in either group as measured by Ki67-immunohistochemistry. Reduction in visible pores, superficial wrinkles, oiliness, pigmentation, and improvement of skin evenness, were statistically significant. A trend for improvement was also observed in skin elasticity, TEWL, and hydration; these did not achieve statistical significance. Ultrasound and histopathology demonstrated increases in dermal thickness in individual patients, without statistical significance. Comprehensive improvement in all 5 parameters, including visible pores, hyperpigmentation, superficial and deep wrinkles, and epidermal thickness, was statistically significant when the subset of participants assigned for histology in full formula group was compared with the placebo group participants. CONCLUSIONS: A 3-product skin care regimen containing alpha and beta defensins globally improves the visual appearance and structure of aging skin without irritation, dryness, or inflammation. Specifically, this regimen increases epidermal thickness, reduces appearance of pores, reduces wrinkles, and reduces melanin. This skin care regimen stimulates rejuvenation without evidence of increase of a marker of carcinogenic stimulation. This data is consistent with the hypothesis that a defensin-containing skin care regimen activates the body's own dormant stem cells to generate healthy new epidermal cells.

J Drugs Dermatol. 2018;17(4):426-441.

Novel synthetic analogues of avian ß-defensin-12: the role of charge, hydrophobicity, and disulfide bridges in biological functions.

BACKGROUND: Avian ß-defensins (AvBD) possess broad-spectrum antimicrobial, LPS neutralizing and chemotactic properties. AvBD-12 is a chemoattractant for avian immune cells and mammalian dendritic cells (JAWSII) - a unique feature that is relevant to the applications of AvBDs as chemotherapeutic agents in mammalian hosts. To identify the structural components essential to various biological functions, we have designed and evaluated seven AvBD analogues. RESULTS: In the first group of analogues, the three conserved disulfide bridges were eliminated by replacing cysteines with alanine and serine residues, peptide hydrophobicity and charge were increased by changing negatively charged amino acid residues to hydrophobic (AvBD-12A1) or positively charged residues (AvBD-12A2 and AvBD-12A3). All three analogues in this group showed improved antimicrobial activity, though AvBD-12A3, with a net positive charge of +9, hydrophobicity of 40% and a predicted CCR2 binding domain, was the most potent antimicrobial peptide. AvBD-12A3 also retained more than 50% of wild type chemotactic activity. In the second group of analogues (AvBD-12A4 to AvBD-12A6), one to three disulfide bridges were removed via substitution of cysteines with isosteric amino acids. Their antimicrobial activity was compromised and chemotactic activity abolished. The third type of analogue was a hybrid that had the backbone of AvBD-12 and positively charged amino acid residues AvBD-6. The antimicrobial and chemotactic activities of the hybrid resembled that of AvBD-6 and AvBD-12, respectively. CONCLUSIONS: While the net positive charge and charge distribution have a dominating effect on the antimicrobial potency of AvBDs, the three conserved disulfide bridges are essential to the chemotactic property and the maximum antimicrobial activity. Analogue AvBD-12A3 with a high net positive charge, a moderate degree of hydrophobicity and a CCR2-binding domain can serve as a template for the design of novel antimicrobial peptides with chemotactic property and salt resistance.

Immune responses to in ovo vaccine formulations containing inactivated fowl adenovirus 8b with poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) and avian beta defensin as adjuvants in chickens.

Inclusion body hepatitis (IBH) is one of the major viral infections causing substantial economic loss to the global poultry industry. The disease is characterized by a sudden onset of mortality (2-30%) and high morbidity (60-70%). IBH is caused by a number of serotypes of fowl adenovirus with substantially low levels of serotype cross protection. Thus far, there is no effective and safe vaccine commercially available in the North America for the control of IBH in chickens. Poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) is a high molecular weight, biodegradable water soluble polymer that has been well characterized as a safe and effective adjuvant for a number of experimental veterinary vaccines. Similarly, host defence peptides, including ß-defensins, have also been shown to exhibit strong adjuvant potential. In this study, we evaluated the adjuvant activity of PCEP and avian beta defensin (ABD) in a vaccine formulation containing inactivated fowl adenovirus (FAdV) serotype 8b administered in ovo. Our data showed that a combination of PCEP and inactivated virus is capable of inducing a robust and long lasting antibody response. Moreover, significant enhancement of IFN-γ, IFN-α, IL-12(p40) and IL-6 gene expression under the influence of PCEP suggests that as an in ovo adjuvant PCEP has the ability to activate a substantial balanced immune response in chickens. To our knowledge, these are the first studies in which PCEP and ABD have been characterized as adjuvants for the development of an in ovo poultry vaccine. It is expected that these preliminary studies will be helpful in the development of safer and more effective in ovo vaccine against IBH and other infectious diseases affecting chickens.

Targeting exogenous ß-Defensin to the endolysosomal compartment via a vehicle guided system.

A number of pathogens for which there are no effective treatments infect the cells via endocytosis. Once in the endosomes, the pathogens complete their life cycle by overriding normal lysosomal functions. Recently, our laboratory identified the lysosomal targeting signal of prosaposin, which is recognized by the sorting receptor "sortilin". Based on this evidence, we tested whether the antimicrobial peptide ß-Defensin linked to the targeting sequence of prosaposin (ßD-PSAP) could be redirected from its secretory pathway to the endolysosomal compartment. To this effect, ßD-PSAP was transfected into COS-7 cells. The sub-cellular distribution of ßD-PSAP was analyzed by confocal microscopy and differential centrifugation. Confocal microscopy demonstrated that ßD-PSAP overlaid with the lysosomal marker LAMP1, indicating that the construct reached endosomes and lysosomes. Differential centrifugation also showed that ßD-PSAP was in the lysosomal fractions. In addition, our binding inhibition assay demonstrated that ßD-PSAP bound specifically to sortilin. Similarly, the delivery of ßD-PSAP was abolished after overexpressing a truncated sortilin. These results indicate that the prosaposin C-terminus and D/C-domain (prosaposin targeting sequence) was an effective "guidance system" to redirect ßD-PSAP to the endolysosomal compartment. In the future, this and other fusion proteins with antimicrobial properties will be assembled to our "biotic vehicle" to target pathogens growing within these compartments.

Human ß-defensin 3 suppresses Porphyromonas gingivalis lipopolysaccharide-induced inflammation in RAW 264.7 cells and aortas of ApoE-deficient mice.

Human beta-defensin 3 (hBD3) is an antimicrobial peptide showing immunomodulatory effect on both innate and acquired immune response. Atherosclerosis is an inflammatory disease characterized by accumulation of lipids in the vascular wall. In this study, we evaluated whether hBD3 could attenuate the atherosclerosis development accelerated by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) with apolipoprotein E-deficient (ApoE(-/-)) mice. We observed that, in vivo, hBD3 inhibited serum MCP-1, sICAM-1 levels of ApoE-deficient mice exposed to Pg-LPS in a chronic inflammation model. Serum levels of total cholesterol (TC) and low-density lipoprotein (LDL) were also markedly reduced with hBD3 intervention. In addition, thinned vascular walls, less macrophage infiltration and the formation of atherosclerotic lesions were observed in the hBD3-treated group. Furthermore, in vitro, hBD3 profoundly suppressed the production of TNF-α and IL-6 in RAW 264.7 cells induced by Pg-LPS in a dose-dependent manner. Moreover, hBD3 attenuated the phosphorylation of p38 and ERK1/2 in the mitogen-activated protein kinase (MAPK) pathway. Taken together, our work has revealed that hBD3 exhibits potent anti-inflammatory properties both in vitro and in vivo, and this effect might be correlated with inhibition of MAPK pathway.

Use of recombinant porcine ß-defensin 2 as a medicated feed additive for weaned piglets.

Post-weaning diarrhoea (PWD) in piglets is associated with colonization of the intestine with bacterial pathogens. In this study, we evaluated the use of recombinant porcine ß-defensin 2 (rpBD2) as a medicated feed additive for weaned piglets. The crude extract from the culture supernatant of rpBD2-expressing Pichia pastoris was used as a medicated feed additive for weaned piglets. Dietary treatments included a positive control (basal diet + antibiotics, designated PC) and three different rpBD2 treatments without antibiotics (basal diet supplemented with 1, 5, or 15 g of crude rpBD2/kg basal diet, designated 1PD, 5PD, and 15PD, respectively). Of all the treatments, 5PD had the greatest impact on the weaned piglets. It increased their body weight, average daily weight gain, average daily feed intake, and intestinal villus height in the duodenum and jejunum, and reduced the incidence of PWD. The diversity of the cecal digesta and mucosa microflora was compared between the weaned piglets in the PC and 5PD groups. Piglets treated with 5PD had lower diversity indices and fewer bacterial pathogens in their cecal digesta and mucosa than the PC group. Our results demonstrate that crude rpBD2 could provide an alternative to the traditional antibiotic feed additives given to weaned piglets.

Oral administration of synthetic porcine beta-defensin-2 improves growth performance and cecal microbial flora and down-regulates the expression of intestinal toll-like receptor-4 and inflammatory cytokines in weaned piglets challenged with enterotoxigenic Escherichia coli.

Synthetic porcine beta-defensin-2 (pBD-2) was tested as an alternative to antimicrobial growth-promoters in pig production. Thirty 21-day weaned piglets were challenged with enterotoxigenic Escherichia coli, and orally dosed with either sterile water (CON), pBD-2 (BD) or neomycin sulphate (NS) twice daily for 21 days. pBD-2 and NS led to higher growth performance, jejunum villus height and increased expression of insulin-like growth factor-I compared with the CON group (P < 0.05). Hemolytic E. coli scores from rectal swabs, and copy numbers of E. coli, Bacteroides fragilis and Streptococcus in the cecal digesta of the BD- or NS-treated piglets were lower than those in the CON group (P < 0.05). Messenger RNA levels of toll-like receptor 4, tumor necrosis factor-α, interleukin (IL)-1ß, and IL-8 in the jejunum mucosa of the BD and NS groups were lower than those in the CON group (P < 0.05). Copy numbers of Lactobacilli and Bifidobacteria in the cecal digesta of the BD group were higher than those of the CON and NS groups (P < 0.05). Therefore, pBD-2 has antimicrobial activity in piglets, and it can improve growth performance, reduce inflammatory cytokine expression and affect intestinal morphological indices in the same way as probiotics. © 2015 Japanese Society of Animal Science.

Administration of Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) and Avian Beta Defensin as Adjuvants in Inactivated Inclusion Body Hepatitis Virus and its Hexon Protein-Based Experimental Vaccine Formulations in Chickens.

Inclusion body hepatitis (IBH) is one of the major infectious diseases adversely affecting the poultry industry of the United States and Canada. Currently, no effective and safe vaccine is available for the control of IBH virus (IBHV) infection in chickens. However, based on the excellent safety and immunogenic profiles of experimental veterinary vaccines developed with the use of new generation adjuvants, we hypothesized that characterization of vaccine formulations containing inactivated IBHV or its capsid protein hexon as antigens, along with poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) and avian beta defensin 2 (ABD2) as vaccine adjuvants, will be helpful in development of an effective and safe vaccine formulation for IBH. Our data demonstrated that experimental administration of vaccine formulations containing inactivated IBHV and a mixture of PCEP with or without ABD2 as an adjuvant induced significantly higher antibody responses compared with other vaccine formulations, while hexon protein-based vaccine formulations showed relatively lower levels of antibody responses. Thus, a vaccine formulation containing inactivated IBHV with PCEP or a mixture of PCEP and ABD2 (with a reduced dosage of PCEP) as an adjuvant may serve as a potential vaccine candidate. However, in order to overcome the risks associated with whole virus inactivated vaccines, characterization of additional viral capsid proteins, including fiber protein and penton of IBHV along with hexon protein in combination with more new generation adjuvants, will be helpful in further improvements of vaccines against IBHV infection.

Toxicity assessment of (99m)technetium-labeled human beta-defensin-3 in CD1 mice.

OBJECTIVE: Human beta-defensin-3 (HBD-3) is an antimicrobial peptide which is up-regulated during inflammation. Based on the previously demonstrated capacity of technetium-99m ((99m)Tc) labelled HBD-3 of distinguishing infection from inflammation in rats, we have decided to collect information on the potential toxicity of the tracer in view of its possible use for imaging in humans. MATERIALS AND METHODS: Recombinant HBD-3 underwent labeling with (99m)Tc. The CD1 mice were selected as standard rodent species. Ten mice, 5 male and 5 female, were subjected to physical examination and housed in a dedicated room in 5 per cage. After 9 days pre-test period, all mice were weighted for dose adjustment and received intravenously 6mcg/mouse of (99m)Tc-HBD-3. Mortality was recorded daily, while body weight was registered once a week. Clinical observation of animals was performed daily for sickness symptoms due to the drug treatment. At day 19 a second dose of 6mcg/mouse (99m)Tc-HBD-3, was administered. Twenty-four hours after the second dose (day 20) the animals were euthanized. A piece of liver, kidneys, heart and lungs was collected for histopathological analysis. RESULTS: Our results showed that the labelled-HBD-3 dose did not induce significant toxicity in mice. Of course these parameters were not sufficient to authorize use in humans. This non-toxic dose of HBD-3 when translated from animals to humans resulted in an equivalent dose of approximately 25 times higher than that needed for imaging. CONCLUSION: Our non toxicity data of using (99m)Tc-beta-defensin-3 in mice offer a further indication in favour of the clinical use of this radiopharmaceutical in all cases where discrimination between infection and inflammation is needed.

Construction of Recombinant Pichia pastoris Carrying a Constitutive AvBD9 Gene and Analysis of Its Activity.

Avian beta-defensin 9 (AvBD9) is a small cationic peptide consisting of 41 amino acids that plays a crucial rule in innate immunity and acquired immunity in chickens. Owing to its wide antibacterial spectrum, lack of a residue, and failure to induce bacterial drug resistance, AvBD9 is expected to become a substitute for conventional antibiotics in the livestock and poultry industries. Using the preferred codon of Pichia pastoris, the mature AvBD9 peptide was designed and synthesized, based on the sequence from GenBank. The P. pastoris constitutive expression vector pGHKα was used to construct a pGHKα-AvBD9 recombinant plasmid. Restriction enzyme digestion was performed using SacI and BglII to remove the ampicillin resistance gene, and the plasmid was electrotransformed into P. pastoris GS115. High-expression strains with G418 resistance were screened, and the culture supernatant was analyzed by Tricine-SDS-PAGE and western blot assay to identify target bands of about 6 kDa. A concentrate of the supernatant containing AvBD9 was used for determination of antimicrobial activity. The supernatant concentrate was effective against Escherichia coli, Salmonella paratyphi, Salmonella pullorum, Pseudomonas aeruginosa, Enterococcus faecalis, and Enterobacter cloacae. The fermentation product of P. pastoris carrying the recombinant AvBD9 plasmid was adjusted to 1.0 × 10(8) CFU/ml and added to the drinking water of white feather broilers at different concentrations. The daily average weight gain and immune organ indices in broilers older than 7 days were significantly improved by the AvBD9 treatment.

Ultrasound microbubbles enhance human ß-defensin 3 against biofilms.

BACKGROUND: The infection of orthopedic implantation devices with Staphylococcus has been a serious concern within the biomaterial community. Treatments are not always successful because of antibiotic-resistant bacteria biofilm infection. Recent studies have shown that combination of antibiotics with low-frequency ultrasound (US) can enhance the bactericidal activity effectively against the formation of biofilms in vitro pilot study. Meanwhile, microbubbles evolved as targeted drug-delivery agents can provide nuclei for inertial cavitation and lower the threshold for US-induced cavitation. Human ß-defensin 3 (HBD-3) is a cationic antimicrobial peptide considered particularly promising for future bactericidal employment and has effect on antibiotic-resistant Staphylococcus biofilms. But the effect has not been reported when combined with US-targeted microbubble destruction (UTMD) in vivo. METHODS: In this study, we evaluated the effect of HBD-3 combined with UTMD on two tested Staphylococcus by the spread plate method, crystal violet staining, confocal laser scanning microscopy, scanning electron microscopy, and real-time polymerase chain reaction. RESULTS: In the study, we found that the biofilm densities, the percentage of live cells, and the viable counts of two tested Staphylococcus that recovered from the biofilm on the titanium surface in mice were significantly decreased in the group of the HBD-3 combined with UTMD, compared with those of other groups. Furthermore, in the experiment, we found out that UTMD could enhance HBD-3 activity, which inhibits the biofilm-associated genes expression of icaAD and the methicillin-resistance genes expression of MecA by promoting the icaR expression simultaneously. CONCLUSIONS: The combination of HBD-3 with UTMD can play a significant role on the elimination of the antibiotic-resistant Staphylococcus biofilms in vivo.

Prophylactic potential of defensins and L-isoleucine in tuberculosis household contacts: an experimental model.

AIM: Patients with pulmonary tuberculosis (TB) are the most important source for TB infection, being the risk of infection determined by the source case infectiousness and the contact closeness. Currently, the administration of isoniazid is used to prevent the infection to some extent in household contacts. At experimental level, defensins are efficient molecules for the treatment of TB and other infectious diseases. MATERIALS & METHODS: In this work, we used a model of Mycobacterium tuberculosis transmission by long cohabitation of infected and noninfected mice, and treated the latter group with antimicrobial peptides in order to determine the potential capacity of defensins to prevent the infection. RESULTS: Our results showed that the intratracheal administration of human neutrophil peptide-1, human ß-defensin-2 alone or in combination and the use of L-isoleucine significantly prevents bacterial transmission, diminishing pulmonary lesions and bacterial loads. CONCLUSION: Data suggest the potential use of L-isoleucine as prophylactic for TB household contacts.

Utilization of plant-derived recombinant human ß-defensins (hBD-1 and hBD-2) for averting salmonellosis.

We describe the use of plant-made ß-defensins as effective antimicrobial substances for controlling salmonellosis, a deadly infection caused by Salmonella typhimurium (referred to further as S. typhi). Human ß-defensin-1 (hBD-1) and -2 (hBD-2) were expressed under the control of strong constitutive promoters in tobacco plants, and bio-active ß-defensins were successfully extracted. In the in vitro studies, enriched recombinant plant-derived human ß-defensin-1 (phBD-1) and -2 (phBD-2) obtained from both T1 and T2 transgenic plants showed significant antimicrobial activity against Escherichia coli and S. typhi when used individually and in various combinations. The 2:1 peptide combination of phBD-1:phBD-2 with peptides isolated from T1-and T2-generation plants reduced the growth of S. typhi by 96 and 85 %, respectively. In vivo studies employing the mouse model (Balb/c) of Salmonella infection clearly demonstrated that the administration of plant-derived defensins individually and in different combinations enhanced the mean survival time of Salmonella-infected animals. When treatment consisted of the 2:1 phBD-1:phBD-2 combination, approximately 50 % of the infected mice were still alive at 206 h post-inoculation; the lowest number of viable S. typhi was observed in the liver and spleen of infected animals. We conclude that plant-made recombinant ß-defensins (phBD-1 and phBD-2) are promising antimicrobial substances and have the potential to become additional tools against salmonellosis, particularly when used in combination.

Therapeutic potential of adenovirus-mediated delivery of ß-defensin 2 for experimental otitis media.

Otitis media (OM), one of the most prevalent diseases in young children, is clinically important owing to its high incidence in children and its potential impact on language development and motor coordination. OM is the most common reason for the prescription of antibiotics (accounting for 25% of prescriptions) due to its extremely high incidence. A recent increase in antibiotic resistance among OM pathogens is emerging as a major public health concern globally, which led us to consider non-antibiotic approaches for the management of OM. In this study, we evaluated gene transfer of an antimicrobial peptide, human ß-defensin 2 (DEFB4), using an adenoviral vector (Ad5 with deletions of E1/E3/E4) as a potential therapeutic approach. We demonstrated that the transduction of human ß-defensin 2 induces the production of human ß-defensin 2 and suppresses non-typeable Haemophilus influenzae (NTHi) adhesion to human middle ear epithelial cells. Moreover, intratympanic inoculation of Ad-DEFB4 was found to attenuate NTHi-induced middle ear effusions without eliciting a significant immune response. Most importantly, intratympanic inoculation of Ad-DEFB4 appeared to significantly augment clearance of NTHi from middle ear cavity. Collectively, our results suggest that intratympanic gene delivery of antimicrobial molecules may serve as an alternative/adjuvant approach for the management of OM.

Beta-defensin 2 enhances immunogenicity and protection of an adenovirus-based H5N1 influenza vaccine at an early time.

Reports of human infections with highly pathogenic H5N1 avian influenza viruses in many countries in Asia and Africa with varying case fatality rates highlight the pandemic potential of these viruses. In order to contain a rapidly spreading influenza virus in a pandemic scenario, a vaccine which can induce rapid and robust immune responses, preferably in a single dose, is necessary. Murine beta-defensin 2 (Mbd2), a small molecular weight protein expressed by epithelial cells, has been shown to enhance antigen-specific immune responses by recruiting and activating professional antigen presenting cells to the site of vaccination. This study assessed the potential of Mbd2 to enhance the immunogenicity and protective efficacy of a human adenovirus (HAd)-based vaccine expressing the hemagglutinin (HA) and nucleoprotein (NP) [HAd-HA-NP] of an H5N1 influenza virus. A single inoculation of mice with both HAd-HA-NP and a HAd vector expressing Murine ß-defensin 2 (HAd-Mbd2) resulted in significantly higher levels of both humoral and cell-mediated immune responses compared to the groups vaccinated only with HAd-HA-NP. These responses were evident even at day 7 post-immunization. Furthermore, the HAd-HA-NP+HAd-Mbd2-immunized group receiving the lowest vector dose (2 × 10(7)+1 × 10(7)) was completely protected against an rgH5N1 virus challenge on day 7 post-vaccination. These results highlight the potential of Mbd2 as a genetic adjuvant in inducing rapid and robust immune responses to a HAd-based vaccine.