Cathelicidin LL-37 Research Articles

Antimicrobial peptide cathelicidin LL-37 preserves intestinal barrier and organ function in rats with heat stroke

Global warming increases the incidence of heat stroke (HS) and HS causes the reduction of visceral blood flow during hyperthermia, leading to intestinal barrier disruption, microbial translocation, systemic inflammation and multiple organ failure. Cathelicidin LL-37 exhibits antimicrobial activities, helps innate immunity within the gut to maintain intestinal homeostasis, and augments intestinal wound healing and barrier function. Thus, we evaluated the effects and possible mechanisms of cathelicidin LL-37 on HS. Wistar rats were placed in a heating-chamber of 42 ̊C to induce HS. Changes in rectal temperature, hemodynamic parameters, and survival rate were measured during the experimental period. Blood samples and ilea were collected to analyze the effects of LL-37 on systemic inflammation, multiple organ dysfunction, and intestinal injury. Furthermore, LS174T and HT-29 cells were used to assess the underlying mechanisms. Our data showed cathelicidin LL-37 ameliorated the damage of intestinal cells induced by HS. Intestinal injury, systemic inflammation, and nitrosative stress (high nitric oxide level) caused by continuous hyperthermia were attenuated in HS rats treated with cathelicidin LL-37, and hence, improved multiple organ dysfunction, coagulopathy, and survival rate. These beneficial effects of cathelicidin LL-37 were attributed to the protection of intestinal goblet cells (by increasing transepithelial resistance, mucin-2 and Nrf2 expression) and the improvement of intestinal barrier function (less cyclooxygenase-2 expression and FITC-dextran translocation). Interestingly, high cathelicidin expression in the ileal samples of inflammatory bowel disease patients was associated with better clinical outcome. These results suggest that cathelicidin LL-37 could prevent heat stress-induced intestinal damage and heat-related illnesses.

Cardiolipin Strongly Inhibits the Leakage Activity of the Short Antimicrobial Peptide ATRA-1 in Comparison to LL-37, in Model Membranes Mimicking the Lipid Composition of Staphylococcus aureus

Cardiolipin is one of the main phospholipid components of Staphylococcus aureus membranes. This lipid is found at varying concentrations in the bilayer, depending on the growth stage of the bacteria, and as a response to environmental stress. Cardiolipin is an anionic phospholipid with four acyl chains, which modulates the bending properties of the membrane due to its inverted conical shape. It has been shown to inhibit the pore forming activity of several antimicrobial peptides, in general doubling the peptide concentration needed to induce leakage. Here we find that the short snake-derived antimicrobial peptide ATRA-1 is inhibited by several orders of magnitude in the presence of cardiolipin in saturated membranes (DMPG) compared to the human cathelicidin LL-37, which is only inhibited two-fold in its leakage-inducing concentration. The ATRA-1 is too short to span the membrane and its leakage activity is likely related to detergent-like alterations of bilayer structure. Fluorescence spectroscopy shows only a minor effect on ATRA-1 binding to DMPG membranes due to the presence of cardiolipin. However, FTIR spectroscopy shows that the acyl chain structure of DMPG membranes, containing cardiolipin, become more organized in the presence of ATRA-1, as reflected by an increase in the gel to liquid-crystalline phase transition temperature. Instead, a depression in the melting temperature is induced by ATRA-1 in DMPG in the absence of cardiolipin. In comparison, LL-37 induces a depression of the main phase transition of DMPG even in the presence of cardiolipin. These data suggest that cardiolipin inhibits the penetration of ATRA-1 into the membrane core, impeding its capacity to disrupt lipid packing.

Antibiofilm properties of cathelicidin LL-37: an in-depth review.

Notwithstanding ceaseless endeavors toward developing effective antibiofilm chemotherapeutics, biofilm-associated infections continue to be one of the most perplexing challenges confronting medicine today. Endogenous host defense peptides, such as the human cathelicidin LL-37, are being propounded as promising options for treating such infectious diseases. Over the past decennium, LL-37 has duly received tremendous research attention by virtue of its broad-spectrum antimicrobial activity and immunomodulatory properties. No attempt has hitherto been made, as far as we are aware, to comprehensively review the antibiofilm effects of LL-37. Accordingly, the intent in this paper is to provide a fairly all-embracing review of the literature available on the subject. Accumulating evidence suggests that LL-37 is able to prevent biofilm establishment by different bacterial pathogens such as Acinetobacter baumannii, Aggregatibacter actinomycetemcomitans, Bacteroides fragilis, Burkholderia thailandensis, Cutibacterium acnes, Escherichia coli, Francisella tularensis, Helicobacter pylori, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes. Inhibition of bacterial adhesion, downregulation of biofilm-associated genes, suppression of quorum-sensing pathways, degradation of biofilm matrix, and eradication of biofilm-residing cells are the major mechanisms responsible for antibiofilm properties of LL-37. In terms of its efficacy and safety in vivo, there are still many questions to be answered. Undoubtedly, LL-37 can open up new windows of opportunity to prevent and treat obstinate biofilm-mediated infections.

Cathelicidin LL-37 promotes EMT, migration and metastasis of hepatocellular carcinoma cells in vitro and mouse model

ABSTRACT The effect of cathelicidin hCAP18/LL-37 in hepatocellular carcinoma (HCC) metastasis remains unclear. Here, we confirmed that LL-37 expression enhanced endothelial-mesenchymal transition (EMT), migration and invasion in HCC cells. And the HER2/EGFR-MAPK/ERK signal participated in the process above. More frequent lung metastases were observed in an LL-37-overexpressing hematogenous metastasis model. Interestingly, 1,25(OH)2D3 together with si-LL-37 significantly enhanced 1,25(OH)2D3-induced inhibition of migration and invasion in PLC/PRF-5 cells, and also enhanced reversion of the EMT process. Therefore, LL-37 is involved in HCC metastases, and may act as an important factor to attenuate the inhibitory activity of 1,25(OH)2D3 on HCC metastasis. Targeting hCAP18/LL-37 may offer a potential strategy to improve the anticancer activity of 1,25(OH)2D3 in HCC therapy.

Effects of acrylic dentures on oral mucosa and salivary antimicrobial peptide content in denture wearers

Relevance. Aging of the body leads to the development of an imbalance in the secretory immunity of the oral mucosa, with the loss of natural teeth leading to an even more rapid decline in the host’s oral defense reactions. However, studies on salivary antimicrobial peptides in denture users and their potential role in protecting against the development of inflammation are still scarce at this time.Purpose. To evaluate the effects of full removable acrylic dentures on the oral mucosa and analyze the content of antimicrobial peptides in the saliva of wearers of such dentures.Materials and methods. Sixty-seven (21 men and 46 women) elderly patients (61 to 74 years old) with complete tooth loss were examined and divided into 3 study groups. In the 1st control group the patients did not use any dentures. Patients in groups 2 and 3 were made full removable acrylic dentures for the upper and lower jaws, and in group 2 from the first day of the adaptation period was used domestic denture fixation cream ASEPTA PARODONTAL. Assessment of the denture bed on the hard palate was performed visually using the Schiller – Pisarev test. Microbiological examination for Candida albicans was performed by PCR-diagnostics. The content of antimicrobial peptides in saliva was assessed using ELISA kits by Hycult Biotech.Results. The use of the adhesive cream was found to improve the condition of the palatal mucosa by 25% on the 15th day and by 50% on the 30th day of the adaptation period. Candida albicans was detected in oral material in more than 55% of patients in all groups during their initial examination. In the group of patients using denture fixation cream, a significant decrease in Candida albicans carriage was noted, while an increase in Candida albicans detection of up to 70% was noted in those not using the cream. There was also an increase in the level of cathelicidin LL-37 in the saliva of patients without the use of the cream and with a marked increase in inflammation. One month after dentures in patients without cream use the content of alpha-defensins (HNP 1-3) was significantly lower than that of the group with cream and the control group.Conclusion. On the basis of the carried out clinical and laboratory study, the domestic ACEPTA adhesive cream should be recommended for optimization of the adaptation period to removable acrylic dentures as well as for their permanent use.

The Antimicrobial Peptide Cathelicidin Exerts Immunomodulatory Effects via Scavenger Receptors

An active form of cathelicidin antimicrobial peptide, LL-37, has immunomodulatory and stimulatory effects, though the specific pathways are not clear. The purpose of this study was to identify the cellular pathways by which LL-37 amplifies the inflammation induced by damage-associated molecular patterns (DAMPs). We performed DNA microarray, reverse transcription polymerase chain reaction, immunoblotting, and proximity ligation assays using cultured keratinocytes treated with LL-37 and/or the DAMP poly(I:C), a synthetic double-stranded RNA. In contrast to the combination of LL-37 and poly(I:C), LL-37 alone induced genes related to biological metabolic processes such as VEGFA and PTGS2 (COX-2). Inhibition of FPR2, a known receptor for cathelicidin, partially suppressed the induction of VEGFA and PTGS2. Importantly, VEGFA and PTGS2 induced by LL-37 alone were diminished by the knockdown of scavenger receptors including SCARB1 (SR-B1), OLR1 (SR-E1), and AGER (SR-J1). Moreover, LL-37 alone, as well as the combination of LL-37 and poly(I:C), showed proximity to the scavenger receptors, indicating that LL-37 acts via scavenger receptors and intermediates between them and poly(I:C). These results showed that the broad function of cathelicidin is generally dependent on scavenger receptors. Therefore, inhibitors of scavenger receptors or non-functional mock cathelicidin peptides may serve as new anti-inflammatory and immunosuppressive agents.

ETIOLOGICAL FACTORS IN DISEASES OF HARD TISSUES AND PERIODONTIUM IN PREGNANT WOMEN

Pregnant women have one of the highest risks of dental disease [3]. According to a number of authors, during the physiological course of pregnancy, the prevalence of dental caries is 91.4%, periodontal tissue diseases occur in 90% of cases, lesions of previously intact teeth with a predominantly acute course of the carious process - in 38% of pregnant patients [5 ]. Secondary caries, progression of the carious process, enamel hyperesthesia occurs in 79% of pregnant women [11]. At the same time, the intensity of the increase in dental caries in terms of the absolute increase in the index of caries-filling-removal during the gestational period is 0.83 [2]. In 50% of pregnant women and women in labor, the so-called gingivitis of pregnant women is observed during the normal course of the gestational period already at 2-3 months of pregnancy [12]. From the second half of pregnancy, the pathological process becomes more pronounced and more often proceeds as a generalized catarrhal or hypertrophic gingivitis, and pyogenic granuloma often develops. As pregnancy progresses, periodontal disease progresses continuously, and only in the postpartum period does the clinical picture improve [4]. In the long term, gingivitis that occurs during pregnancy becomes chronic [9, 10]. In a pregnant woman, against the background of altered reactivity and reduced body resistance, latent odontogenic foci of infection can lead to serious complications as a result of exacerbation of the inflammatory process [7, 8]. The greatest severity of inflammatory phenomena in periodontal tissues occurs in the second trimester of pregnancy, and the critical increase in the cariogenic situation in the oral cavity occurs in the third trimester [4, 2], which not only determines the optimal timing of dental examinations during pregnancy and the postpartum period, but also the differentiation of the approach to programs for the prevention and treatment of the most significant diseases of the oral cavity for this period of pregnancy. The decisive role in the development of caries and periodontitis in pregnant women belongs to hormonal substances (somatomammotropin, progesterone, gonadotropin) produced by the placenta, changes in mineral and protein metabolism, immunological status, oral microflora [1, 5]. The timing of the increased risk of the onset and progression of periodontal diseases, as well as the increase in the intensity of dental caries in pregnant women, is not enough to state; tires. This does not negate the significance of already established factors affecting the occurrence and development of dental caries, periodontal disease during pregnancy, but is aimed at competently combining diagnostically important known and newly identified criteria into a system of practical recommendations for identifying risk groups among pregnant women. on dental health and special monitoring of them. Immunoprotective peptides of biological media, including oral fluid, are markers of the intensity of local inflammation and are responsible for the implementation of innate antimicrobial immunity [6, 12]. In the oral fluid, a whole complex of immunoprotective peptides is isolated, among which lactoferrin, cathelicidin LL-37, and α-defensin are distinguished [14]. The inclusion of immunoprotective peptides in algorithms and models for ranking the risk of progression of dental caries during pregnancy will expand the boundaries of existing recommendations in this direction. When studying systemic immunity in pregnant women with periodontal diseases, oral fluid and peripheral blood are used as biological media [8]. At the same time, retroplacental blood and umbilical cord blood containing fetal and maternal blood, despite the easy method of selection after the birth of a child and placenta, not associated with invasive manipulations, is not used as a biological medium. Meanwhile, the determination of the spectrum of inflammatory mediators, the mineral composition of retroplacental and umbilical cord blood will allow answering the questions whether inflammation of the periodontal tissues of varying severity was accompanied by the "mother-placenta-fetus" system, whether it is possible to predict from the stage of childbirth in the future the appearance of caries of milk teeth? The purpose of this study is to optimize the system of providing dental care to pregnant women using clinical and laboratory indicators.

Design, synthesis, and characterization of non-hemolytic antimicrobial peptides related to human cathelicidin LL-37.

We designed and synthesised the N-terminally labeled cationic and hydrophobic peptides, i.e., FFKKSKEKIGKEFKKIVQKI (P1) and FRRSRERIGREFRRIVQRI (P2) related to the human cathelicidin LL-37 peptide. The integrity and molecular weight of the peptides were confirmed by mass spectrometry. The purity and homogeneity of peptides P1 and P2 were determined by comparing LCMS or analytical HPLC chromatograms. The circular dichroism spectroscopy reveals the conformational transitions upon interaction with membranes. Predictably, peptides P1 and P2 showed a random coil structure in the buffer and formed α-helix secondary structure in TFE and SDS micelles. This assessment was further confirmed by 2D NMR spectroscopic methods. The analytical HPLC binding assay measurements revealed that peptides P1 and P2 display preferential interactions with the anionic lipid bilayer (POPC:POPG) moderately than zwitterionic (POPC). The efficacies of the peptides were tested against Gram-positive and Gram-negative bacteria. It is imperative to note here that the arginine-rich P2 exerted higher activity against all the test organisms as compared with that shown by the lysine-rich peptide P1. To test the toxicity of these peptides, a hemolytic assay was performed. P1 and P2 showed very little to no toxicity for a hemolytic assay, which is significant for P1 and P2 to be used as potential therapeutic agents in practical applications. Both peptides P1 and P2 were non-hemolytic and appeared to be more promising as they demonstrated wide-spectrum antimicrobial activity.

2350. LiaF is required for LiaX mediated protection against Daptomycin and LL-37 in Enterococcus faecalis OG1RF

Abstract Background Daptomycin (DAP) resistance in enterococci is regulated by the LiaFSR three-component regulatory system. Mutations in liaF have been linked to activation of this system, with increased expression of the protein LiaX, and resistance to DAP in clinical isolates. LiaX functions by recognizing DAP in the extracellular medium, and serves as a signal transduction molecule. However, the role of LiaF in signaling and its relationship with LiaX are unknown. Methods We generated a liaF null mutant in E. faecalis OG117 by adding four stop codons at amino acid positions 11–14 (OG117liaF*11-14) using a CRISPR-Cas9 system. The mutant was complemented by restoring wild-type liaF in its chromosomal location (OG117liaF*11-14::liaF). Mutants were confirmed with whole genome sequencing (WGS). Anionic phospholipid (AP) microdomain distribution was assessed on microscopy using 10-N-nonyl-acridine-orange (NAO). LiaFSR activation was assessed by surface expression of LiaX via ELISA. DAP MICs were performed by broth microdilution in the presence/absence of exogenous LiaX (eLiaX). LL37 (50µg/ml) and DAP (1.5µg/ml) killing assays with eLiaX were also performed in triplicate. Results Truncation of liaF did not have any effect on DAP MICs, AP microdomain distribution or LiaX surface expression compared to OG117. No additional mutations were observed by WGS in the mutants. In the presence eLiaX, DAP MIC increased 8-fold in wild-type OG117 but remained the same (1 µg/mL) in OG117liaF*11-14. Complementation of liaF restored the increase in DAP MIC in the presence of eLiaX. In the LL37 killing assay, survival of OG117 was significantly increased on the addition of eLiaX (2.9% vs 11.8%, respectively, P < .002). Addition of eLiaX was unable to rescue OG117liaF*11-14 in the presence of LL-37 (2.4% vs 2.5% respectively, P=0.51), but was able to increase survival in the complemented strain (2.8% vs 7.6%, respectively, P < .002). A similar trend was seen in the DAP killing assay, with increased survival for OG117 (8.7% vs 20%) and OG117liaF*11-14::liaF (11.6% vs 17%), but not OG117liaF*11-14 (0.38% vs 1%). Conclusion LiaF is required for full tolerance to the cathelicidin LL37 and DAP in E. faecalis OG1RF. These results suggest that LiaF may interact with LiaX as a part of the LiaFSR stress response. Disclosures William R. Miller, MD, Entasis: Grant/Research Support|UpToDate: Honoraria Cesar A. Arias, MD, PhD, Entasis: Grant/Research Support|MeMed Diagnostics Ltd: Grant/Research Support|Merck: Grant/Research Support.

HBD-2 variants and SARS-CoV-2: New insights into inter-individual susceptibility.

A deep understanding of the causes of liability to SARS-CoV-2 is essential to develop new diagnostic tests and therapeutics against this serious virus in order to overcome this pandemic completely. In the light of the discovered role of antimicrobial peptides [such as human b-defensin-2 (hBD-2) and cathelicidin LL-37] in the defense against SARS-CoV-2, it became important to identify the damaging missense mutations in the genes of these molecules and study their role in the pathogenesis of COVID-19. We conducted a comprehensive analysis with multiple in silico approaches to identify the damaging missense SNPs for hBD-2 and LL-37; moreover, we applied docking methods and molecular dynamics analysis to study the impact of the filtered mutations. The comprehensive analysis reveals the presence of three damaging SNPs in hBD-2; these SNPs were predicted to decrease the stability of hBD-2 with a damaging impact on hBD-2 structure as well. G51D and C53G mutations were located in highly conserved positions and were associated with differences in the secondary structures of hBD-2. Docking-coupled molecular dynamics simulation analysis revealed compromised binding affinity for hBD-2 SNPs towards the SARS-CoV-2 spike domain. Different protein-protein binding profiles for hBD-2 SNPs, in relation to their native form, were guided through residue-wise levels and differential adopted conformation/orientation. The presented model paves the way for identifying patients prone to COVID-19 in a way that would guide the personalization of both the diagnostic and management protocols for this serious disease.

Cathelicidin LL-37 Activates Human Keratinocyte Autophagy through the P2X₇, Mechanistic Target of Rapamycin, and MAPK Pathways

Human cathelicidin LL-37 is a multifunctional antimicrobial peptide that exhibits antimicrobial and immunomodulatory activities. LL-37 regulates skin barrier function and was recently reported to activate autophagy in macrophages. Because autophagy deficiency is associated with skin diseases characterized by a dysfunctional epidermal barrier, we hypothesized that LL-37 might regulate the skin barrier through autophagy modulation. We showed that LL-37 activated autophagy in human keratinocytes and three-dimensional skin equivalent models as indicated by increases in LC3 puncta formation, decreases in p62, and autophagosome and autolysosome formation. LL-37‒induced autophagy was suppressed by P2X7 receptor, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 inhibitors, suggesting that the P2X7, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 pathways are involved. Moreover, LL-37 enhanced the phosphorylation of adenosine monophosphate‒activated protein kinase and unc-51-like kinase 1. In addition, LL-37‒mediated autophagy involves the mechanistic target of rapamycin and MAPK pathways. Interestingly, the LL-37‒induced distribution of tight junction proteins and improvement in the tight junction barrier were inhibited in autophagy-deficient keratinocytes and keratinocytes and skin models treated with autophagy inhibitors, indicating that the LL-37‒mediated tight junction barrier is associated with autophagy activation. Collectively, these findings suggest that LL-37 is a potential therapeutic target for skin diseases characterized by dysfunctional autophagy and skin barriers.

Novel Alligator Cathelicidin As-CATH8 Demonstrates Anti-Infective Activity against Clinically Relevant and Crocodylian Bacterial Pathogens

Host defense peptides (HDPs) represent an alternative way to address the emergence of antibiotic resistance. Crocodylians are interesting species for the study of these molecules because of their potent immune system, which confers high resistance to infection. Profile hidden Markov models were used to screen the genomes of four crocodylian species for encoded cathelicidins and eighteen novel sequences were identified. Synthetic cathelicidins showed broad spectrum antimicrobial and antibiofilm activity against several clinically important antibiotic-resistant bacteria. In particular, the As-CATH8 cathelicidin showed potent in vitro activity profiles similar to the last-resort antibiotics vancomycin and polymyxin B. In addition, As-CATH8 demonstrated rapid killing of planktonic and biofilm cells, which correlated with its ability to cause cytoplasmic membrane depolarization and permeabilization as well as binding to DNA. As-CATH8 displayed greater antibiofilm activity than the human cathelicidin LL-37 against methicillin-resistant Staphylococcus aureus in a human organoid model of biofilm skin infection. Furthermore, As-CATH8 demonstrated strong antibacterial effects in a murine abscess model of high-density bacterial infections against clinical isolates of S. aureus and Acinetobacter baumannii, two of the most common bacterial species causing skin infections globally. Overall, this work expands the repertoire of cathelicidin peptides known in crocodylians, including one with considerable therapeutic promise for treating common skin infections.

Human cathelicidin LL-37 exerts amelioration effects against EHEC O157:H7 infection regarding inflammation, enteric dysbacteriosis, and impairment of gut barrier function

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection impairs intestinal barrier function, causing intestinal inflammation and enteric dysbacteriosis. The human cathelicidin LL-37 can regulate excessive inflammatory responses, barrier function, and balance the intestinal microbial community; however, little is known about its effects on inflammation, intestinal barrier function, and microbiota disorders in EHEC O157:H7-infected mice. In this study, we investigated the protective effect of LL-37 against EHEC O157:H7 infection and elucidated the underlying mechanism using a mouse model. LL-37 treatment was found to inhibit body weight loss, restore edema and destruction of the intestinal villi, and significantly reduce epithelial apoptosis (P < 0.05) in EHEC O157:H7-infected mice. Furthermore, inflammatory infiltration of macrophages and neutrophils into the jejunum and colon was significantly decreased (P < 0.05). LL-37 significantly downregulated the production of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) (P < 0.05) and upregulated the anti-inflammatory cytokine (IL-10) during EHEC O157:H7 infection. LL-37 increased the expression of tight junction proteins (ZO-1, ZO-2, claudin-1, and occludin), which are associated with intestinal barrier function, and had a positive effect on EHEC O157:H7-induced microbial disorders, particularly in terms of the inflammation-related microbiota. LL-37 also significantly decreased the E. coli load in the liver and spleen (P < 0.01) and restored the structure of the liver and kidney. Taken together, LL-37 conferred protection in a EHEC O157:H7-induced mouse model by reducing intestinal inflammation, enhancing intestinal barrier function, and restoring the balance of the intestinal microbiota, which indicates the therapeutic potential of LL-37 against pathogen infection.

Nanofiber Aerogels with Precision Macrochannels and LL-37-Mimic Peptides Synergistically Promote Diabetic Wound Healing.

Fast healing of diabetic wounds remains a major clinical challenge. Herein, this work reports a strategy to combine nanofiber aerogels containing precision macrochannels and the LL-37-mimic peptide W379 for rapid diabetic wound healing. Nanofiber aerogels consisting of poly(glycolide-co-lactide) (PGLA 90:10)/gelatin and poly-p-dioxanone (PDO)/gelatin short electrospun fiber segments were prepared by partially anisotropic freeze-drying, crosslinking, and sacrificial templating with three-dimensional (3D)-printed meshes, exhibiting nanofibrous architecture and precision micro-/macrochannels. Like human cathelicidin LL-37, W379 peptide at a concentration of 3 μg/mL enhanced the migration and proliferation of keratinocytes and dermal fibroblasts in a cell scratch assay and a proliferation assay. In vivo studies show that nanofiber aerogels with precision macrochannels can greatly promote cell penetration compared to aerogels without macrochannels. Relative to control and aerogels with and without macrochannels, adding W379 peptides to aerogels with precision macrochannels shows the best efficacy in healing diabetic wounds in mice in terms of cell infiltration, neovascularization, and re-epithelialization. The fast re-epithelization could be due to upregulation of phospho-extracellular signal-regulated kinase (p38 MAPK) after treatment with W379. Together, the approach developed in this work could be promising for the treatment of diabetic wounds and other chronic wounds.

In silico assessment of missense point mutations on human cathelicidin LL-37

Cathelicidin antimicrobial peptides are a diverse family of cationic amphipathic peptides with multiple activities. In humans, cathelicidin LL-37 is one of the main host defense peptides with a remarkable medical and biotechnological potential. Deregulation of LL-37 expression has been associated with inflammatory diseases. However the effects of point mutations driven by single nucleotide polymorphisms (SNPs) on LL-37 are unknown. Here we applied an array of computational tools to investigate the effects of such mutations on LL-37 structure and activity. Due to the fact that, on cathelicidins, the prodomain is more conserved than the mature peptide, the SNP effect predictions were biased and, overall, resulted in neutral effects; and due to the slight changes in physicochemical properties, the antimicrobial predictions indicated the maintenance of such activity. Nonetheless, R07P, R07W, R29Q, R29W mutations reduced the peptide net charge, which in turn could result in less active LL-37 variants. Molecular dynamics data indicated that R07Q and N30Y mutations altered the LL-37 structure, leading to potential deleterious effects. In addition, the helix dipole is altered in G03A, R07P, R07W and L31P mutations, which could also alter the antimicrobial activity. Our results indicated that despite the mutations did not alter the residues from LL-37 active core, they could influence the antimicrobial activity and consequently, could be involved in inflammatory diseases.

Cathelicidin LL-37 level in presence and absence of vitamin D in cultured macrophages isolated from elderly women

Introduction: Vitamin D deficiency and frequent infections are the two common worldwide phenomenon among elderly. Recent studies have demonstrated that vitamin D regulates the expression of specific endogenous antimicrobial peptides like cathelicidin LL-37 of macrophages and neutrophils, which is active against a broad spectrum of infectious agents. Therefore, the objective of the present study was to determine the level of cathelicidin LL-37 in macrophages of elderly women (classified according to serum 25(OH)D level) after exposure to Vibrio cholera infection and to find out the effect of 1,25(OH)2D added in vitro. Methods: This study was conducted among 40 randomly selected rural elderly women aged between 60 to 70 years of age. Their vitamin D status was assessed by the estimation of serum 25(OH)D and classified into three groups viz. sufficient (14 members), insufficient (13 members), and deficient (13 members). Later, their peripheral blood mononuclear cells (PBMC) were isolated and cultured from fresh blood. 1,25(OH)2D supplementation was given selectively at a dose of 10 ×10-8 M for 72 hours in the culture media; then exposed to infection and screened according to the objectives of this study. Results: Macrophages in all groups, except vitamin D deficient group, responded significantly in terms of LL-37 release during exposure to Vibrio cholera infection. Considering in vitro 1,25(OH)2D, supplementation responded significantly (p&lt;0.05) in all three groups. Conclusion: Vitamin D can be used as a prophylaxis to enhance cathelicidin LL-37 release for all three groups as in the present study.

In vitro Screening of Herbal Medicinal Products for Their Supportive Curing Potential in the Context of SARS-CoV-2.

COVID-19 herbal medicinal products may have the potential for symptom relief in nonsevere or moderate disease cases. In this in vitro study we screened the five herbal medicinal products Sinupret extract (SINx), Bronchipret thyme-ivy (BRO-TE), Bronchipret thyme-primula (BRO TP), Imupret (IMU), and Tonsipret (TOP) with regard to their potential to (i) interfere with the binding of the human angiotensin-converting enzyme 2 (ACE2) receptor with the SARS-CoV-2 spike S1 protein, (ii) modulate the release of the human defensin HBD1 and cathelicidin LL-37 from human A549 lung cells upon spike S1 protein stimulation, and (iii) modulate the release of IFN-γ from activated human peripheral blood mononuclear cells (PBMC). The effect of the extracts on the interaction of spike S1 protein and the human ACE2 receptor was measured by ELISA. The effects on the intracellular IFN-γ expression in stimulated human PBMC were measured by flow cytometry. Regulation of HBD1 and LL-37 expression and secretion was assessed in 25 d long-term cultured human lung A549 epithelial cells by RT-PCR and ELISA. IMU and BRO-TE concentration-dependently inhibited the interaction between spike S1 protein and the ACE2 receptor. SINx, TOP, and BRO-TE significantly upregulated the intracellular expression of anti-viral IFN-γ from stimulated PBMC. Cotreatment of A549 cells with IMU or BRO TP together with SARS-CoV-2 spike protein significantly upregulated mRNA expression (IMU) and release of HBD1 (IMU and BRO TP) and LL-37 (BRO TP). The in vitro screening results provide first evidence for an immune-activating potential of some of the tested herbal medicinal extracts in the context of SARS-CoV-2. Whether these could be supportive in symptom relief or curing from SARS-CoV-2 infection needs deeper understanding of the observations.

Antimicrobial Peptides in Early-Life Host Defense, Perinatal Infections, and Necrotizing Enterocolitis-An Update.

Host defense against early-life infections such as chorioamnionitis, neonatal sepsis, or necrotizing enterocolitis (NEC) relies primarily on innate immunity, in which antimicrobial peptides (AMPs) play a major role. AMPs that are important for the fetus and neonate include α and β defensins, cathelicidin LL-37, antiproteases (elafin, SLPI), and hepcidin. They can be produced by the fetus or neonate, the placenta, chorioamniotic membranes, recruited neutrophils, and milk-protein ingestion or proteolysis. They possess antimicrobial, immunomodulating, inflammation-regulating, and tissue-repairing properties. AMPs are expressed as early as the 13th week and increase progressively through gestation. Limited studies are available on AMP expression and levels in the fetus and neonate. Nevertheless, existing evidence supports the role of AMPs in pathogenesis of chorioamnionitis, neonatal sepsis, and NEC, and their association with disease severity. This suggests a potential role of AMPs in diagnosis, prevention, prognosis, and treatment of sepsis and NEC. Herein, we present an overview of the antimicrobial and immunomodulating properties of human AMPs, their sources in the intrauterine environment, fetus, and neonate, and their changes during pre- and post-natal infections and NEC. We also discuss emerging data regarding the potential utility of AMPs in early-life infections, as diagnostic or predictive biomarkers and as therapeutic alternatives or adjuncts to antibiotic therapy considering the increase of antibiotic resistance in neonatal intensive care units.

Bicarbonate Effects on Antibacterial Immunity and Mucus Glycobiology in the Cystic Fibrosis Lung: A Review With Selected Experimental Observations.

The primary defect in cystic fibrosis (CF) is abnormal chloride and bicarbonate transport in the cystic fibrosis transmembrane conductance regulator (CFTR) epithelial ion channel. The apical surface of the respiratory tract is lined by an airway surface liquid layer (ASL) composed of mucin comprising mainly MUC5A and MUC5B glycoproteins. ASL homeostasis depends on sodium bicarbonate secretion into the airways and secretion deficits alter mucus properties leading to airway obstruction, inflammation, and infections. Downstream effects of abnormal ion transport in the lungs include altered intrinsic immune defenses. We observed that neutrophils killed Pseudomonas aeruginosa more efficiently when it had been exposed to sodium bicarbonate, and formation of neutrophil extracellular traps (NETs) by neutrophils was augmented in the presence of increasing bicarbonate concentrations. Physiological levels of bicarbonate sensitized P. aeruginosa to the antimicrobial peptide cathelicidin LL-37, which is present in both lung ASL and in NETs. Sodium bicarbonate has various uses in clinical medicine and in the care of CF patients, and could be further explored as a therapeutic adjunct against Pseudomonas infections.

A Scoping Analysis of Cathelicidin in Response to Organic Dust Exposure and Related Chronic Lung Illnesses.

Over two billion people worldwide are exposed to organic dust, which can cause respiratory disorders. The discovery of the cathelicidin peptide provides novel insights into the lung’s response to organic dust; however, its role in the lung’s response to organic dust exposure and chronic lung diseases remains limited. We conducted a scoping review to map the current evidence on the role of cathelicidin LL-37/CRAMP in response to organic dust exposure and related chronic lung diseases: hypersensitivity pneumonitis (HP), chronic obstructive pulmonary disease (COPD) and asthma. We included a total of n = 53 peer-reviewed articles in this review, following the process of (i) a preliminary screening; (ii) a systematic MEDLINE/PubMed database search; (iii) title, abstract and full-text screening; (iv) data extraction and charting. Cathelicidin levels were shown to be altered in all clinical settings investigated; its pleiotropic function was confirmed. It was found that cathelicidin contributes to maintaining homeostasis and participates in lung injury response and repair, in addition to exerting a positive effect against microbial load and infections. In addition, LL-37 was found to sustain continuous inflammation, increase mucus formation and inhibit microorganisms and corticosteroids. In addition, studies investigated cathelicidin as a treatment modality, such as cathelicidin inhalation in experimental HP, which had positive effects. However, the primary focus of the included articles was on LL-37’s antibacterial effect, leading to the conclusion that the beneficial LL-37 activity has not been adequately examined and that further research is required.