Antifungal activity of human antimicrobial peptides targeting apoptosis in Candida auris.

Introduction. Innovative antifungal therapies are of crucial importance to combat the potentially life-threatening infections linked to the multidrug-resistant fungal pathogen Candida auris. Induction of regulated cell death, apoptosis, could provide an outline for future therapeutics. Human antimicrobial peptides (AMPs), well-known antifungal compounds, have shown the ability to induce apoptosis in pathogenic fungi.Hypothesis/Gap Statement . Although it is known that AMPs possess antifungal activity against C. auris, their ability to induce apoptosis requires further investigations.Aim. This study evaluated the effects of AMPs on the induction of apoptosis in C. auris.Methods. Human neutrophil peptide-1 (HNP-1), human ß-Defensins-3 (hBD-3) and human salivary histatin 5 (His 5) were assessed against two clinical C. auris isolates. Apoptosis hallmarks were examined using FITC-Annexin V/PI double labelling assay and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick-end labelling (TUNEL) to detect phosphatidylserine externalization and DNA fragmentation, respectively. Then, several intracellular triggers were studied using JC-10 staining, spectrophotometric assay and 2',7'-dichlorofluorescin diacetate staining to measure the mitochondrial membrane potential, cytochrome-c release and reactive oxygen species (ROS) production, respectively.Results and conclusion. FITC-Annexin V/PI staining and TUNEL analysis revealed that exposure of C. auris cells to HNP-1 and hBD-3 triggered both early and late apoptosis, while His 5 caused significant necrosis. Furthermore, HNP-1 and hBD-3 induced significant mitochondrial membrane depolarization, which resulted in substantial cytochrome c release. In contrast to His 5, which showed minimal mitochondrial depolarization and no cytochrome c release. At last, all peptides significantly increased ROS production, which is related to both types of cell death. Therefore, these peptides represent promising and effective antifungal agents for treating invasive infections caused by multidrug-resistant C. auris.

FOXO inhibition rescues α-defensin expression in human intestinal organoids.

To mediate critical host-microbe interactions in the human small intestine, Paneth cells constitutively produce abundant levels of α-defensins and other antimicrobials. We report that the expression profile of these antimicrobials is dramatically askew in human small intestinal organoids (enteroids) as compared to that in paired tissue from which they are derived, with a reduction of α-defensins to nearly undetectable levels. Murine enteroids, however, recapitulate the expression profile of Paneth cell α-defensins seen in tissue. WNT/TCF signaling has been found to be instrumental in the regulation of α-defensins, yet in human enteroids exogenous stimulation of WNT signaling appears insufficient to rescue α-defensin expression. By stark contrast, forkhead box O (FOXO) inhibitor AS1842856 induced the expression of α-defensin mRNA in enteroids by >100,000-fold, restoring DEFA5 and DEFA6 to levels comparable to those found in primary human tissue. These results newly identify FOXO signaling as a pathway of biological and potentially therapeutic relevance for the regulation of human Paneth cell α-defensins in health and disease.

The Function and Modification of Human Defensin 5.

The antibacterial and antiviral functions of human defensin 5 lay the foundation for its role as a core host protective component. In addition, HD5 also has the function of inhibiting tumor proliferation and immune regulation. However, everything has two sides; cytotoxic and proinflammatory properties may exist, while HD5 performs physiological functions. Accordingly, the modification and engineering of HD5 are particularly important. Therefore, this review summarizes the role of HD5 in various aspects of host defense, as well as modification of HD5 to ameliorate the biological activity, with a view to promoting the clinical use of HD5.

Host Cationic Antimicrobial Molecules Inhibit S. aureus Exotoxin Production.

Innate immune molecules, including antimicrobial peptides (for example, defensins) and lysozyme, function to delay or prevent bacterial infections. These molecules are commonly found on mucosal and skin surfaces. Staphylococcus aureus is a common pathogen and causes millions of infections annually. It is well known that innate immune molecules, such as defensins and lysozyme, either poorly inhibit or do not inhibit the growth of S. aureus. Our current studies show that the α-defensin human neutrophil α-defensin-1 (HNP-1) and lysozyme inhibit exotoxin production, both hemolysins and superantigens, which are required for S. aureus infection. HNP-1 inhibited exotoxin production at concentrations as low as 0.001 µg/mL. Lysozyme inhibited exotoxin production at 0.05 to 0.5 µg/mL. Both HNP-1 and lysozyme functioned through at least one two-component system (SrrA/B). The ß-defensin human ß-defensin 1 (HBD-1) inhibited hemolysin but not superantigen production. The cation chelator S100A8/A9 (calprotectin), compared to EDTA, was tested for the ability to inhibit exotoxin production. EDTA at high concentrations inhibited exotoxin production; these were the same concentrations that interfered with staphylococcal growth. S100A8/A9 at the highest concentration tested (10 µg/mL) had no effect on S. aureus growth but enhanced exotoxin production. Lower concentrations had no effect on growth or exotoxin production. Lysostaphin is regularly used to lyse S. aureus. The lytic concentrations of lysostaphin were the only concentrations that also inhibited growth and exotoxin production. Our studies demonstrate that a major activity of innate defensin peptides and lysozyme is inhibition of staphylococcal exotoxin production but not inhibition of growth. IMPORTANCE Staphylococcus aureus causes large numbers of both relatively benign and serious human infections, which are mediated in large part by the organisms' secreted exotoxins. Since 1921, it has been known that lysozyme and, as shown later in the 1900s, other innate immune peptides, including human neutrophil α-defensin-1 (HNP-1) and human ß-defensin 1 (HBD-1), are either not antistaphylococcal or are only weakly inhibitory to growth. Our study confirms those findings but, importantly, shows that at subgrowth inhibitory concentrations, these positively charged innate immune peptides inhibit exotoxin production, including both hemolysins and the superantigen toxic shock syndrome toxin-1. The data show that the principal activity of innate immune peptides in the host is likely to be inhibition of exotoxin production required for staphylococcal mucosal or skin colonization rather than growth inhibition.

Distinct mode of membrane interaction and disintegration by diverse class of antimicrobial peptides.

The exploitation of conventional antibiotics in conjunction with the adeptness of microbes has led to the emergence of multi-drug-resistant pathogens. This has posed a severe threat to combating life-threatening infectious diseases. Antimicrobial peptides (AMP), which are considered to be the first line of defense in all living organisms, are being developed for therapeutic use. Herein, we determined the NMR solution structure of Rhesus macaque Myeloid Alpha Defensin-4 (RMAD4), a defensin AMP. Additionally, the distinct modes of membrane perturbation for two structurally dissimilar classes of AMPs was studied using biophysical methods namely, Solid-state 31P NMR, DSC and cryo-TEM. The cathelicidin - Bovine myeloid antimicrobial peptide (BMAP-28 (1-18)), which adopts a helical conformation, and the defensin RMAD4 peptide that natively folds to form ß-sheets appeared to engage differently with the bacterial membrane. The helical BMAP-28 (1-18) peptide initiates lipid segregation and membrane thinning followed by pore formation, while the ß-stranded RMAD4 peptide demonstrates fragmentation of the bilayer by the carpet or detergent-like mechanism of action. Molecular dynamics studies sufficiently corroborated these findings. The structure and mechanism of action of the AMPs studied using experimental and computational approaches are believed to help in providing a platform for the rational design of new competent and cost-effective antimicrobial peptides for therapeutic applications.

The capsule of Bacillus anthracis protects it from the bactericidal activity of human defensins and other cationic antimicrobial peptides.

During infection, Bacillus anthracis bacilli encounter potent antimicrobial peptides (AMPs) such as defensins. We examined the role that B. anthracis capsule plays in protecting bacilli from defensins and other cationic AMPs by comparing their effects on a fully virulent encapsulated wild type (WT) strain and an isogenic capsule-deficient capA mutant strain. We identified several human defensins and non-human AMPs that were capable of killing B. anthracis. The human alpha defensins 1-6 (HNP-1-4, HD-5-6), the human beta defensins 1-4 (HBD-1-4), and the non-human AMPs, protegrin, gramicidin D, polymyxin B, nisin, and melittin were all capable of killing both encapsulated WT and non-encapsulated capA mutant B. anthracis. However, non-encapsulated capA mutant bacilli were significantly more susceptible than encapsulated WT bacilli to killing by nearly all of the AMPs tested. We demonstrated that purified capsule bound HBD-2, HBD-3, and HNP-1 in an electrophoretic mobility shift assay. Furthermore, we determined that the capsule layer enveloping WT bacilli bound and trapped HBD-3, substantially reducing the amount reaching the cell wall. To assess whether released capsule might also play a protective role, we pre-incubated HBD-2, HBD-3, or HNP-1 with purified capsule before their addition to non-encapsulated capA mutant bacilli. We found that free capsule completely rescued the capA mutant bacilli from killing by HBD-2 and -3 while killing by HNP-1 was reduced to the level observed with WT bacilli. Together, these results suggest an immune evasion mechanism by which the capsule, both that enveloping the bacilli and released fragments, contributes to virulence by binding to and inhibiting the antimicrobial activity of cationic AMPs.

Human α-Defensin-6 Neutralizes Clostridioides difficile Toxins TcdA and TcdB by Direct Binding.

Rising incidences and mortalities have drawn attention to Clostridioides difficile infections (CDIs) in recent years. The main virulence factors of this bacterium are the exotoxins TcdA and TcdB, which glucosylate Rho-GTPases and thereby inhibit Rho/actin-mediated processes in cells. This results in cell rounding, gut barrier disruption and characteristic clinical symptoms. So far, treatment of CDIs is limited and mainly restricted to some antibiotics, often leading to a vicious circle of antibiotic-induced disease recurrence. Here, we demonstrate the protective effect of the human antimicrobial peptide α-defensin-6 against TcdA, TcdB and the combination of both toxins in vitro and in vivo and unravel the underlying molecular mechanism. The defensin prevented toxin-mediated glucosylation of Rho-GTPases in cells and protected human cells, model epithelial barriers as well as zebrafish embryos from toxic effects. In vitro analyses revealed direct binding to TcdB in an SPR approach and the rapid formation of TcdB/α-defensin-6 complexes, as analyzed with fluorescent TcdB by time-lapse microscopy. In conclusion, the results imply that α-defensin-6 rapidly sequesters the toxin into complexes, which prevents its cytotoxic activity. These findings extend the understanding of how human peptides neutralize bacterial protein toxins and might be a starting point for the development of novel therapeutic options against CDIs.

Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain.

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a ß-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.

VP4 Is a Determinant of Alpha-Defensin Modulation of Rotaviral Infection.

Fecal-oral pathogens encounter constitutively expressed enteric alpha-defensins in the intestine during replication and transmission. Alpha-defensins can be potently antiviral and antibacterial; however, their primary sequences, the number of isoforms, and their activity against specific microorganisms often vary greatly between species, reflecting adaptation to species-specific pathogens. Therefore, alpha-defensins might influence not only microbial evolution and tissue tropism within a host but also species tropism and zoonotic potential. To investigate these concepts, we generated a panel of enteric and myeloid alpha-defensins from humans, rhesus macaques, and mice and tested their activity against group A rotaviruses, an important enteric viral pathogen of humans and animals. Rotaviral adaptation to the rhesus macaque correlated with resistance to rhesus enteric, but not myeloid, alpha-defensins and sensitivity to human alpha-defensins. While mouse rotaviral infection was increased in the presence of mouse enteric alpha-defensins, two prominent genotypes of human rotaviruses were differentially sensitive to human enteric alpha-defensins. Furthermore, the effects of cross-species alpha-defensins on human and mouse rotaviruses did not follow an obvious pattern. Thus, exposure to alpha-defensins may have shaped the evolution of some, but not all, rotaviruses. We then used a genetic approach to identify the viral attachment and penetration protein, VP4, as a determinant of alpha-defensin sensitivity. Our results provide a foundation for future studies of the VP4-dependent mechanism of defensin neutralization, highlight the species-specific activities of alpha-defensins, and focus future efforts on a broader range of rotaviruses that differ in VP4 to uncover the potential for enteric alpha-defensins to influence species tropism. IMPORTANCE Rotavirus is a leading cause of severe diarrhea in young children. Like other fecal-oral pathogens, rotaviruses encounter abundant, constitutively expressed defensins in the small intestine. These peptides are a vital part of the vertebrate innate immune system. By investigating the impact that defensins from multiple species have on the infectivity of different strains of rotavirus, we show that some rotaviral infections can be inhibited by defensins. We also found that some, but not all, rotaviruses may have evolved resistance to defensins in the intestine of their host species, and some even appropriate defensins to increase their infectivity. Because rotaviruses infect a broad range of animals and rotaviral infections are highly prevalent in children, identifying immune defenses against infection and how they vary across species and among viral genotypes is important for our understanding of the evolution, transmission, and zoonotic potential of these viruses as well as the improvement of vaccines.

Recombinant HNP-1 Produced by Escherichia coli Triggers Bacterial Apoptosis and Exhibits Antibacterial Activity against Drug-Resistant Bacteria.

Human neutrophil peptide-1 (HNP-1) is a promising antibiotic candidate, but its clinical applications have been hampered by challenges during mass production and an inadequate understanding of its bactericidal mechanisms. In this study, we demonstrated that Escherichia coli expressing full-length preproHNP-1 secretes a soluble form of HNP-1, which can be recovered from the total cell lysate after isopropyl thio-ß-d-galactoside (IPTG) induction and ultrafiltration. Label-free quantitative proteomics and co-immunoprecipitation experiments revealed that HNP-1 induces cell apoptosis in bacteria by causing DNA and membrane damage. Notably, we found that HNP-1 disrupts the DNA damage response pathway by interfering with the binding of RecA to single-stranded DNA (ssDNA). Further experiments demonstrated that HNP-1 encapsulated in liposomes inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) and meropenem-resistant Pseudomonas aeruginosa (MRPA). These results indicated that recombinant protein expression may be a simple and cost-effective solution to produce HNP-1 and that RecA inhibition via HNP-1 may serve as an alternative strategy to counteract antibiotic resistance. IMPORTANCE Human neutrophil peptide-1 (HNP-1) is a promising antibiotic candidate, but its clinical application has been hampered by the difficulty of mass production and an inadequate understanding of its bactericidal mechanisms. In this study, we demonstrated that recombinant protein expression combined with ultrafiltration may be a simple and cost-effective solution to HNP-1 production. We further found that HNP-1 induces bacterial apoptosis and prevents its SOS repair pathway from binding to the RecA protein, which may be a new antibacterial mechanism. In addition, we showed that HNP-1 encapsulated in liposomes inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) and meropenem-resistant Pseudomonas aeruginosa (MRPA). These results provide new insights into the production and antibacterial mechanism of HNP-1, both of which may promote its clinical application.

Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.

Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.

Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin.

Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB5-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates α-subunits of inhibitory G-proteins (Gαi) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by α-defensin-1 and -5, but not ß-defensin-1. The amount of ADP-ribosylated Gαi was significantly reduced in PT-treated cells, in the presence of α-defensin-1 and -5. Moreover, both α-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the Gαi-mediated signal transduction (iGIST) assay. Taken together, we identified the human peptides α-defensin-1 and -5 as inhibitors of PT activity, suggesting that these human peptides bear potential for developing novel therapeutic strategies against whooping cough.

Alpha-defensins: risk factor for thrombosis in COVID-19 infection.

The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions.

Inhibition and eradication activity of truncated α-defensin analogs against multidrug resistant uropathogenic Escherichia coli biofilm.

Today the development of antibiotic resistance, especially in the treatment of bacterial infections associated with biofilms, has led to increasing the importance of antimicrobial peptides (AMPs). In this work, antimicrobial and synergistic activity of three truncated HNP-1 analogs (2Abz14S29, 2Abz23S29, and HNP1ΔC18A) with ß-lactam (amoxicillin and cefixime) and fluoroquinolones (ciprofloxacin and norfloxacin) antibiotics against multidrug-resistant (MDR) uropathogenic E. coli clinical isolates were evaluated. The anti-biofilm potential of peptides at different stages was also investigated. All peptides exhibited additive activity just with ß-lactam antibiotics in a checkerboard synergy assay. Inhibition and eradication of MDR uropathogenic E. coli biofilm were shown by all test peptides at different concentrations. Thus, truncated HNP-1 analogs (2Abz14S29, 2Abz23S29, and HNP1ΔC18A) may have the potential for the treatment of urinary tract infections (UTIs) caused by biofilm-forming MDR uropathogenic E. coli.

Anticancer potential of human intestinal defensin 5 against 1, 2-dimethylhydrazine dihydrochloride induced colon cancer: A therapeutic approach.

The escalating predicament of multidrug resistant cancer cells and associated side effects of conventional chemotherapy necessitates the exploration of alternative anticancer therapies. The present study evaluated anticancer therapeutic potential of human defensin 5 (HD-5) against colon cancer. The in vivo anticancer efficacy of HD-5 against 1,2-dimethylhydrazine (DMH) induced colon cancer was elucidated in terms of tumor biostatistics, number of aberrant crypt foci (ACF), in situ apoptosis assay,changes in morphological as well as histological architecture of colon(s). The direct interaction of peptide was investigated by incubating peptide with normal and/or cancerous colonocytes followed by phase contrast, Hoechst 3342 and AO/PI staining as well as confocal microscopy. Changes in membrane dynamics were evaluated by MC 540 and N-NBD-PE staining. In vivo decrease(s) in tumor parameters, number of aberrant crypt foci along with marked increase in the rate of apoptosis was observed.H&E staining revealed neutrophils infiltration and restoration of normal architecture in treated colon(s) which was consistent with scanning electron microscopic observations. Furthermore, non-membranolytic mechanism was found to be acquired by peptide as it could traverse cell membrane gaining access to nucleus and cytoplasm thereby disintegrating cellular architecture. MC 540 and NBD-PE staining revealed that peptide could bind to cancerous cells by taking advantage of altered fluidity levels. Our results indicated that HD-5 exhibited strong cancer cell killing and does not affect normal host cells. The peptide can be exploited as promising option to combat developing menace of colon cancer and/or can at least be used as an adjunct to present day chemotherapies.

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.

RTD-1 therapeutically normalizes synovial gene signatures in rat autoimmune arthritis and suppresses proinflammatory mediators in RA synovial fibroblasts.

Rhesus theta defensin-1 (RTD-1), a macrocyclic immunomodulatory host defense peptide from Old World monkeys, is therapeutic in pristane-induced arthritis (PIA) in rats, a model of rheumatoid arthritis (RA). RNA-sequence (RNA-Seq) analysis was used to interrogate the changes in gene expression in PIA rats, which identified 617 differentially expressed genes (DEGs) in PIA synovial tissue of diseased rats. Upstream regulator analysis showed upregulation of gene expression pathways regulated by TNF, IL1B, IL6, proinflammatory cytokines, and matrix metalloproteases (MMPs) involved in RA. In contrast, ligand-dependent nuclear receptors like the liver X-receptors NR1H2 and NR1H3 and peroxisome proliferator-activated receptor gamma (PPARG) were downregulated in arthritic synovia. Daily RTD-1 treatment of PIA rats for 1-5 days following disease presentation modulated 340 of the 617 disease genes, and synovial gene expression in PIA rats treated 5 days with RTD-1 closely resembled the gene signature of naive synovium. Systemic RTD-1 inhibited proinflammatory upstream regulators such as TNF, IL1, and IL6 and activated antiarthritic ligand-dependent nuclear receptor pathways, including PPARG, NR1H2, and NR1H3, that were suppressed in untreated PIA rats. RTD-1 also inhibited proinflammatory responses in IL-1ß-stimulated human RA fibroblast-like synoviocytes (FLS) in vitro and diminished expression of human orthologs of disease genes that are induced in rat PIA synovium. Thus, the antiarthritic mechanisms of systemic RTD-1 include homeostatic regulation of arthritogenic gene networks in a manner that correlates temporally with clinical resolution of rat PIA.

Antimicrobial Peptide-Conjugated Hierarchical Antifouling Polymer Brushes for Functionalized Catheter Surfaces.

Catheter-related infection is a great challenge to modern medicine, which causes significant economic burden and increases patient morbidity. Hence, there is a great requirement for functionalized surfaces with inherently antibacterial properties and biocompatibility that prevent bacterial colonization and attachment of blood cells. Herein, we developed a strategy for constructing polymer brushes with hierarchical architecture on polyurethane (PU) via surface-initiated atom-transfer radical polymerization (SI-ATRP). Surface-functionalized PU (PU-DMH) was readily prepared, which comprised of poly(3-[dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]azaniumyl]propane-1-sulfonate) (PDMAPS) brushes as the lower layer and antimicrobial peptide-conjugated poly(methacrylic acid) (PMAA) brushes as the upper layer. The PU-DMH surface showed excellent bactericidal property against both Gram-positive and Gram-negative bacteria and could prevent accumulation of bacterial debris on surfaces. Simultaneously, the PU-DMH samples possessed good hemocompatibility and low cytotoxicity. Furthermore, the integrated antifouling and bactericidal properties of PU-DMH under hydrodynamic conditions were confirmed by an in vitro circulating model. The functionalized surface possessed persistent antifouling and bactericidal performances both under static and hydrodynamic conditions. The microbiological and histological results of animal experiments also verified the in vivo anti-infection performance. The present work might find promising clinical applications for preventing catheter-related infection.

A mutated rabbit defensin NP-1 produced by Chlorella ellipsoidea can improve the growth performance of broiler chickens.

The demand for alternatives to antibiotics to improve the growth performance of food animals is increasing. Defensins constitute the first line of defence against pathogens in the innate immune system of animals and humans. A transgenic Chlorella ellipsoidea strain producing mNP-1 (a mutated rabbit defensin NP-1) was previously obtained in our laboratory. In this study, a process for producing the transgenic strain on a large scale was developed, and the C. ellipsoidea strain producing mNP-1 was used as a feed additive to improve the health and growth performance of chickens. The volume of C. ellipsoidea producing mNP-1 can be scaled up to 10,000 L with approximately 100 g/L dry biomass, and the mNP-1 content of transgenic microalgal powder (TMP) was 90-105 mg/L. A TMP-to-regular feed ratio of 1‰, as the optimal effective dose, can promote the growth of broiler chickens by increasing weight by 9.27-12.95%. mNP-1 can improve duodenum morphology by promoting long and thin villi and affect the microbial community of the duodenum by increasing the diversity and abundance of beneficial microbes. These results suggested that transgenic Chlorella producing mNP-1 can be industrially produced and used as an effective feed additive and an alternative to antibiotics for improving the health and growth performance of broiler chickens or other types of food animals/poultry.

Exploiting chitosan and gold nanoparticles for antimycobacterial activity of in silico identified antimicrobial motif of human neutrophil peptide-1.

The upsurge of drug resistant tuberculosis is major health threat globally. To counteract, antimicrobial peptides are being explored as possible alternatives. However, certain limitations of peptide-based drugs such as potential toxicity, high cost and relatively low stability need to be addressed to enhance their clinical applicability. Use of computer predicted short active motifs of AMPs along with nanotechnology could not only overcome the limitations of AMPs but also potentiate their antimicrobial activity. Therefore, present study was proposed to in silico identify short antimicrobial motif (Pep-H) of human neutrophil peptide-1 (HNP-1) and explore its antimycobacterial activity in free form and using nanoparticles-based delivery systems. Based on colony forming unit analysis, motif Pep-H led to killing of more than 90% M. tb in vitro at 10 µg/ml, whereas, similar activity against intracellularly growing M. tb was observed at 5 µg/ml only. Thereafter, chitosan (244 nm) and gold nanoparticles (20 nm) were prepared for Pep-H with both the formulations showing minimal effects on the viability of human monocyte derived macrophages (MDMs) and RBC integrity. The antimycobacterial activity of Pep-H against intracellular mycobacteria was enhanced in both the nanoformulations as evident by significant reduction in CFU (>90%) at 5-10 times lower concentrations than that observed for free Pep-H. Thus, Pep-H is an effective antimycobacterial motif of HNP-1 and its activity is further enhanced by chitosan and gold nanoformulations.