Cathelicidin LL-37 Research Articles

Chlamydial-Secreted Protease Chlamydia High Temperature Requirement Protein A (cHtrA) Degrades Human Cathelicidin LL-37 and Suppresses Its Anti-Chlamydial Activity.

BackgroundChlamydia trachomatis is an obligate intracellular pathogen that can cause severe reproductive tract complications while ascending infection occurs. When spreading from cell to cell in a host, C. trachomatis utilizes various survival strategies to offset host defense mechanisms. One such strategy is to degrade host antimicrobial defense proteins before they can attack the invading C. trachomatis cells.Material/MethodsWe expressed and purified recombinant chlamydia high temperature requirement protein A (cHtrA) including 2 cHtrA mutants (MT-H143A and MT-S247A), and also extracted endogenous cHtrA. Proteins were identified and their purity evaluated by SDS-PAGE and Western blot. The anti-chlamydial activity and degradation of 5 antimicrobial peptides (cathelicidin LL-37, α-defensin-1 and -3, and β-defensin-2 and -4) by cHtrA and 2 cHtrA mutants (MT-H143A and MT-S247A) were tested by immunoassay and Western blot.ResultsOf the 5 antimicrobial peptides (cathelicidin LL-37, α-defensin-1 and -3, and β-defensin-2 and -4) tested, cathelicidin LL-37 showed the strongest anti-chlamydial activity. Interestingly, cHtrA effectively and specifically degraded LL-37, suppressing its anti-chlamydial activity. The 2 cHtrA mutants (MT-H143A and MT-S247A) were unable to degrade LL-37. Comparison of cHtrA activity from C. trachomatis D, L2, and MoPn strains on LL-37 showed similar responses.ConclusionscHtrA may contribute to C. trachomatis pathogenicity by clearing the passage of invasion by specific LL-37 degradation.

THE USE OF MOLECULAR AND BIOCHEMICAL MARKERS OF BONE METABOLISM FOR SCREENING OF DENTAL HEALTH OF THE POPULATION

At present, the research interests of both dentists and clinical laboratory diagnosticians lie in the field of using oral fluid as an object of diagnostic estimation of molecular and biochemical markers in oral cavity diseases. It is commonly known that oral fluid has both organic and non-organic components. Non-organic components of saliva are represented by macro- and microelements that can be parts of various compounds or stay in the ionized form. Organic components are represented by proteins, carbohydrates, lipids, essential nitrogenous compounds (urea and uric acid), vitamins, hormones, organic and nucleic acids.
 Compared with the traditional method of blood testing, the identification of markers in the oral fluid has several advantages. They are non-invasiveness, atraumatic character for the patient, the absence of stress typical of venipuncture; simpler conditions of storage and transportation due to the liquid state (unlike whole blood, which tends to coagulate); being able to take biological material an unlimited number of times; no need for qualified personnel and special equipment when taking oral fluid.
 The introduction of biological markers into the complex of dental checkups is one of the topical tasks of modern dentistry and clinical laboratory diagnostics. This is particularly important for those dental patients whose living or working conditions are accompanied by harmful chemical factors.
 The authors have performed complex research of lactoferrin, cathelicidin, and MMP8 present in the oral fluid of 50 patients who worked under harmful conditions (group 1), and of other 50 patients whose work is not associated with the harmful influence of chemical factors (group 2). The oral fluid was collected on an empty stomach in the morning by spitting into a sterile glass tube. The biomass was centrifuged and stored at 300C, and the amount of lactoferrin (Hycto Biotech, the Netherlands), cathelicidin (Hycult Biotech, the Netherlands), and MMP8 (Matrix Metalloproteinase-8) was determined in the test samples. The analysis was performed by enzyme-linked immunosorbent assay based on the use of a "sandwich" variant of solid-phase enzyme-linked immunosorbent assay. The procedure was performed on the enzyme-linked immunosorbent assay ImmunoChem-2100 (USA). The analysis was performed in 96-well microplates, the bottom of the wells was covered with monoclonal antibodies to the corresponding molecular marker.
 The analysis of the oral cavity condition in patients of both groups demonstrated the presence of caries (100 % of group 1 patients, 80% of group 2 patients) and partial adentia (75% of group 1 patients, 60% of group 2 patients). Secondary to the above-mentioned abnormalities, group 1 and 2 patients had periodontal diseases. Such abnormalities were registered in 87% of group 1 patients; in group 2 patients, such changes were less explicit and were present in 67% of the patients. On the contrary, it was established that the patients of both groups presented with a statistically significant increase of lactoferrin in the oral fluid (on average, 81% and 40% higher, respectively, in groups 1 and 2) and MMP8 (64% and 24% higher), as well as a decrease in cathelicidin concentration (87% and 42% lower) in comparison with the patients of group 3.
 The established pathological biochemical changes indicate pathological processes in the oral cavities of the research groups of patients.
 In modern practice, Lf is used as an organ-specific marker of the activation of a pathological process to diagnose and predict the course of mucosal and periodontal diseases. At the same time, the decrease in oral cathelicidin LL37 reflects the suppression of local immunity in the oral cavity and is regarded as a pathogenic chain in the progression of diseases of the mucosa, periodontium, including dental caries in patients. Similar dynamics of cathelicidin in group 1 patients, in our opinion, explains the intensity of oral disease development in this category of patients. Thus, neutrophils and macrophages die under the action of aggressive chemical factors, as a result of which the pool of antimicrobial peptides, including cathelicidin, decreases.
 The present markers determine the topicality and great potential of further research of the given molecular markers for both fundamental investigations and understanding the pathogenesis of oral cavity diseases in this category of patients. Besides, these indices can be used as independent markers for diagnosis, screening, and effective treatment of oral cavity diseases.

MODERN DIAGNOSTICS METHODS OF NON-SPECIFIC PROTECTION OF PERIODONTAL TISSUES IN RESIDENTS OF THE INDUSTRIAL REGION

The aim of the article is to study the peculiarities of antimicrobial immunity of the oral cavity of the industrial region’s inhabitants in order to optimize special preventive programs of major dental diseases.
 The object and research methods. 178 patients was examined from 21 to 50 years old, who turned to the University Dental Center of Zaporizhzhia State Medical University. The study group consisted of 126 patients with generalized periodontitis of the initial (8), I (32), II (68) and III (18) degree of chronic course, complicated by harmful factors of manufacturing. The comparison group consisted of 32 patients with periodontitis (5), I (10), II (11) and III (6) degrees who did not work in adverse conditions. The control group consisted of 20 relatively healthy individuals aged from 19 to 25 years without signs of generalized periodontitis.
 To study the factors of local immunity of the oral cavity, oral fluid was collected from each examined person, which was obtained without stimulation, spitting into sterile tubes. Then the oral fluid was centrifuged for 15 minutes at 8,000 rpm. The supernatant part of the oral liquid was poured into plastic tubes and stored at 30°C. The quantitative determination of markers in the oral fluid was performed by standard enzyme immunoassay kits according to the instructions of the "Lactoferrin-strip" ("VectorBest") manufacturers. The result was expressed in mµg / ml. The content of cathelicidin LL-37 was determined by enzyme-linked immunosorbent assay using NycultBiotechhuman LL-37 ELISA (Netherlands) firm reagent kit-manufacturers. The result was expressed in µg / ml. Statistica 13.0 licensed number JPZ804I382130ARCN10-J was used to process the results.
 The results of the study and their discussion. The hygienic condition in the examined patients of the study group was assessed as unsatisfactory: from 2.24 ± 0.5 to 2.99 ± 0.47 points depending on the age and length of service at the enterprise, but the PI and SBI values were increased.
 It was found, that the lactoferrin level in the oral fluid was higher in the all patients with periodontitis, than the same index in comparison with healthy control group and consistently increased with increasing severity of the process. In the patients’ study group, working in hazardous conditions of production, the lactoferrin content has increased in the oral fluid relative to the level of the healthy patients was more pronounced in comparison with the patients, suffering of periodontal tissue disease, but do not work in harmful manufacturing conditions. Increased lactoferrin in the oral fluid in the steelmaking workers can be considered as a means of compensation, that provides protection of the oral mucosa from colonization of microorganisms.
 In the steel industry workers, a statistically significant cathelicidin content decrease in the oral fluid was observed compared to healthy control patients’ group. In the clinical group of the patients without adverse factors of production, the cathelicidin’s concentration in the oral fluid was also reduced.
 The increasing level of lactoferrin in the oral fluid in parallel with the cathelicidin’s decrease in saliva are markers of the inflammatory phase, as well as the destructive phase of connective tissue.
 Conclusions. Thus, our observations have established a direct relationship between the lactoferrin’s concentration in the oral fluid and the severity of periodontal tissue diseases and the feedback between the cathelicidin’s content in the oral liquid and the activity of the pathological process.
 The determination of lactoferrin and cathelicidin in the oral fluid of the patients with harmful manufacturing factors allow us to identify the signs of pathological process in the oral cavity.

Cationic amphipathic peptide analogs of cathelicidin LL-37 as a probe in the development of antimicrobial/anticancer agents.

Cathelicidin LL-37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL-37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13-amino acid cathelicidin 17-29 amide segment F17 KRIV21 QR23 IK25 DF27 LR-NH2 were prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were performed on residues R23 and K25 in the hydrophilic side, V21 and F27 in the hydrophobic side of the interphase, and F17 that interacts with cell membranes. Specific motifs IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals were also inserted into the 17-29 fragment to investigate how they affect activity. Substitution of the amino-terminal positive charge by acetylation and replacement of lysine by the aliphatic leucine in the peptide analog Ac-FKRIVQRIL25 DFLR-NH2 resulted in significant cytotoxicity against A549 cancer cells with an IC50 value 3.90 μg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac-FKRIVQI23 IKK26 FLR-NH2 , which incorporates the IIKK motif and the peptides FKRIVQL23 L24 KK26 L27 LR-NH2 and Ac-FKRIVQL23 L24 KK26 L27 LR-NH2 , which incorporate the LLKKL motif, displayed potent antimicrobial activity against gram-negative bacteria (MIC 3-7.5 μg/mL) and substantial cytotoxicity against bronchial epithelial cancer cells, (IC50 12.9-9.8 μg/mL), with no cytotoxic activity for human erythrocytes. The helical conformation of the synthetic peptides was confirmed by circular dichroism. Our study shows that appropriate substitutions, mainly in positions of the interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin 17-29 segment, may lead to the preparation of effective biological compounds.

329 Cathelicidin LL-37 facilitates damage-associated molecular patterns to be bioactive substances by intermediating between scavenger receptors

Damage-associated molecular patterns (DAMPs) are group of bioactive molecules, which are generated by damaged host cells following injury or infection. DAMPs provoke intracellular signaling leading to chemokines and cytokines production. However, the fundamental mechanism how host cells recognize DAMPs on cellular surface has not been fully elucidated. Human cathelicidin LL-37, a biphasic and cationic peptide, is an essential component of innate immune responses and acts as an alarmin. Inappropriate expression of cathelicidin is observed in inflammatory skin diseases such as psoriasis and rosacea, and cathelicidin provokes and exacerbates innate immune type skin inflammation in these dermatoses.

Antiviral and Immunomodulatory Properties of Antimicrobial Peptides Produced by Human Keratinocytes.

Keratinocytes, the main cells of the epidermis, are the first site of replication as well as the first line of defense against many viruses such as arboviruses, enteroviruses, herpes viruses, human papillomaviruses, or vaccinia virus. During viral replication, these cells can sense virus associated molecular patterns leading to the initiation of an innate immune response composed of pro-inflammatory cytokines, chemokines, and antimicrobial peptides. Human keratinocytes produce and secrete at least nine antimicrobial peptides: human cathelicidin LL-37, types 1–4 human β-defensins, S100 peptides such as psoriasin (S100A7), calprotectin (S100A8/9) and koebnerisin (S100A15), and RNase 7. These peptides can exert direct antiviral effects on the viral particle or its replication cycle, and indirect antiviral activity, by modulating the host immune response. The purpose of this review is to summarize current knowledge of antiviral and immunomodulatory properties of human keratinocyte antimicrobial peptides.

RB137 and RB138 antibodies recognize human cathelicidin LL37 by ELISA

LL37 is a cationic antimicrobial peptide (AMP), which can undergo post-translational modifications (PTM), such as citrullination and carbamylation. We demonstrate here that recombinant antibodies RB137 and RB138 are specific for native LL37 and do not recognize modified LL37 (citrullinated and carbamylated). They thus represent tools to assess the presence of native, unmodified LL37 in body fluids by ELISA.

Generation of Monoclonal Antibodies Specific for Native LL37 and Citrullinated LL37 That Discriminate the Two LL37 Forms in the Skin and Circulation of Cutaneous/Systemic Lupus Erythematosus and Rheumatoid Arthritis Patients

Human cathelicidin LL37 is a cationic antimicrobial peptide active against bacteria and viruses and exerting immune modulatory functions. LL37 can be also a target of autoreactive B- and T-lymphocytes in autoimmune settings. Irreversible post-translational modifications, such as citrullination and carbamylation, mainly occurring at the level of cationic amino acids arginine and lysine, can affect the inflammatory properties and reduce antibacterial effects. Moreover, these modifications could be implicated in the rupture of immune tolerance to LL37 in chronic conditions such as psoriatic disease and cutaneous lupus (LE)/systemic lupus erythematosus (SLE). Here, we describe the generation and fine specificity of six recombinant antibodies (MRB137–MRB142), produced as a monovalent mouse antibody with the antigen-binding scFv portion fused to a mouse IgG2a Fc, and their ability to recognize either native or citrullinated LL37 (cit-LL37) and not cross-react to carbamylated LL37. By using these antibodies, we detected native LL37 or cit-LL37 in SLE and rheumatoid arthritis (RA) sera, and in LE skin, by ELISA and immunohistochemistry, respectively. Such antibodies represent previously unavailable and useful tools to address relationships between the presence of post-translational modified LL37 and the immune system status (in terms of innate/adaptive responses activation) and the clinical characteristics of patients affected by chronic immune-mediated diseases or infectious diseases.

Mutations in the critical domain of human cathelicidin LL-37 affect neutralization of IAV

Abstract Influenza A Virus (IAV) is the virus responsible for the flu and affects 3 to 5 million people around the world. Although vaccines exist for common strains of IAV, whenever pandemic strains of IAV arise humans are largely unprotected. Thus, new treatment strategies are required for pandemic strains of IAV as well as serious cases of IAV infection that can result in a bacterial superinfection. Treatments that reduce viral load in an infected host would be optimal to reduce virus spread as well as to potentially alleviate viral burden on the host. Mammals have many host defense peptides such defensins, lectins and cathelicidins. Cathelicidins are short cationic peptides that are antimicrobial in nature, and have been shown to have anti-IAV properties along with other antimicrobial functions. A critical domain of LL-37, the only human cathelicidin, has been shown to have a central helix with a hydrophobic surface. This critical domain is the minimum sequence of LL-37 required to have antimicrobial properties. There is no consensus on the mechanism LL-37 utilizes to neutralize bacteria and viruses. However, we hypothesize that by mutating single hydrophobic residues in the critical domain to a hydrophilic residue we can see changes in LL-37 neutralization of various IAV strains. Using fluorescent focus assays, we can quantify IAV titers after exposure to LL-37 and other cationic peptides. Here we show that reducing the hydrophobic residues within the critical domain of LL-37 reduces seasonal and pandemic IAV titers in vitro. These results lend credence to the theory of the hydrophobic surface of LL-37 is responsible for its anti-IAV effects.

The Human Host-Defense Peptide Cathelicidin LL-37 is a Nanomolar Inhibitor of Amyloid Self-Assembly of Islet Amyloid Polypeptide (IAPP).

Amyloid self‐assembly of islet amyloid polypeptide (IAPP) is linked to pancreatic inflammation, β‐cell degeneration, and the pathogenesis of type 2 diabetes (T2D). The multifunctional host‐defence peptides (HDPs) cathelicidins play crucial roles in inflammation. Here, we show that the antimicrobial and immunomodulatory polypeptide human cathelicidin LL‐37 binds IAPP with nanomolar affinity and effectively suppresses its amyloid self‐assembly and related pancreatic β‐cell damage in vitro. In addition, we identify key LL‐37 segments that mediate its interaction with IAPP. Our results suggest a possible protective role for LL‐37 in T2D pathogenesis and offer a molecular basis for the design of LL‐37‐derived peptides that combine antimicrobial, immunomodulatory, and T2D‐related anti‐amyloid functions as promising candidates for multifunctional drugs.

Resistome of Staphylococcus aureus in Response to Human Cathelicidin LL-37 and Its Engineered Antimicrobial Peptides.

Staphylococcus aureus is notoriously known for its rapid development of resistance to conventional antibiotics. S. aureus can alter its membrane composition to reduce the killing effect of antibiotics and antimicrobial peptides (AMPs). To obtain a more complete picture, this study identified the resistance genes of S. aureus in response to human cathelicidin LL-37 peptides by screening the Nebraska Transposon Mutant Library. In total, 24 resistant genes were identified. Among them, six mutants, including the one with the known membrane-modifying gene (mprF) disabled, became more membrane permeable to the LL-37 engineered peptide 17BIPHE2 than the wild type. Mass spectrometry analysis detected minimal lysyl-phosphatidylglycerol (lysylPG) from the mprF mutant of S. aureus JE2, confirming loss-of-function of this gene. Moreover, multiple mutants showed reduced surface adhesion and biofilm formation. In addition, four S. aureus mutants were unable to infect wax moth Galleria mellonella. There appears to be a connection between the ability of bacterial attachment/biofilm formation and infection. These results underscore the multiple functional roles of the identified peptide-response genes in bacterial growth, infection, and biofilm formation. Therefore, S. aureus utilizes a set of resistant genes to weave a complex molecular network to handle the danger posed by cationic LL-37. It appears that different genes are involved depending on the nature of antimicrobials. These resistant genes may offer a novel avenue to designing more potent antibiotics that target the Achilles heels of S. aureus USA300, a community-associated pathogen of great threat.

Osthole, a Natural Plant Derivative Inhibits MRGPRX2 Induced Mast Cell Responses.

Mast cells are tissue-resident innate immune cells known for their prominent role in mediating allergic reactions. MAS-related G-protein coupled receptor-X2 (MRGPRX2) is a promiscuous G-protein coupled receptor (GPCR) expressed on mast cells that is activated by several ligands that share cationic and amphipathic properties. Interestingly, MRGPRX2 ligands include certain FDA-approved drugs, antimicrobial peptides, and neuropeptides. Consequently, this receptor has been implicated in causing mast cell-dependent pseudo-allergic reactions to these drugs and chronic inflammation associated with asthma, urticaria and rosacea in humans. In the current study we examined the role of osthole, a natural plant coumarin, in regulating mast cell responses when activated by the MRGPRX2 ligands, including compound 48/80, the neuropeptide substance P, and the cathelicidin LL-37. We demonstrate that osthole attenuates both the early (Ca2+ mobilization and degranulation) and delayed events (chemokine/cytokine production) of mast cell activation via MRGPRX2 in vitro. Osthole also inhibits MrgprB2- (mouse ortholog of human MRGPRX2) dependent inflammation in in vivo mouse models of pseudo-allergy. Molecular docking analysis suggests that osthole does not compete with the MRGPRX2 ligands for interaction with the receptor, but rather regulates MRGPRX2 activation via allosteric modifications. Furthermore, flow cytometry and confocal microscopy experiments reveal that osthole reduces both surface and intracellular expression levels of MRGPRX2 in mast cells. Collectively, our data demonstrate that osthole inhibits MRGPRX2/MrgprB2-induced mast cell responses and provides a rationale for the use of this natural compound as a safer alternative treatment for pseudo-allergic reactions in humans.

MP07-06 CHRONIC LL-37 INDUCED CYSTITIS PRODUCES PAIN INDEPENDENT OF INFLAMMATION AND MODIFIED GLYCOSAMINOGLYCANS ARE POTENT NON-OPIOID ANALGESICS

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis (MP07)1 Apr 2020MP07-06 CHRONIC LL-37 INDUCED CYSTITIS PRODUCES PAIN INDEPENDENT OF INFLAMMATION AND MODIFIED GLYCOSAMINOGLYCANS ARE POTENT NON-OPIOID ANALGESICS Austin Schults*, Wanjian Jia, M. Martin Jensen, Glenn Prestwich, and Siam Oottamasathien Austin Schults*Austin Schults* More articles by this author , Wanjian JiaWanjian Jia More articles by this author , M. Martin JensenM. Martin Jensen More articles by this author , Glenn PrestwichGlenn Prestwich More articles by this author , and Siam OottamasathienSiam Oottamasathien More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000827.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Interstitial cystitis/painful bladder syndrome (IC/PBS) can be a debilitating condition that dramatically impacts the quality of life for patients. The primary goal of patients with IC/PBS is to provide symptomatic relief. The immunomodulatory human cathelicidin (antimicrobial peptide) LL-37 induces profound acute bladder inflammation and pain. Our objectives were to evaluate a chronic pain profile associated with an LL-37-induced murine model of IC/PBS, along with the analgesic effects of modified glycosaminoglycan ether compounds. We hypothesized that chronic pain could be produced that is independent of inflammation and that a semi-synthetic glycosaminoglycan ether (SAGE GM-0111) could be a potent non-opioid analgesic in the setting of chronic LL-37-induced cystitis. METHODS: Bladder instillation of 150μL of 80 µM LL-37 for 1-hr, biweekly for four weeks induced the chronic IC/PBS model. SAGE analgesic effects were tested by instilling SAGE GM-0111. Pain responses to suprapubic stimulation were assessed using von Frey filaments (0.04gm to 4.0gm) before LL-37 instillation and before sacrifice. Inflammation and fibrosis were evaluated using tissue myeloperoxidase (MPO) concentration, collagen content, gross inspection, and microscopic histologic examination. RESULTS: SAGE GM-0111 reduced pain in treated animals significantly (54 ± 32% response rate) in animals exposed to LL-37 insult (96 ± 7% response rate, p<0.001) at 1g of stimulation. Collagen content, gross anatomy, histology with H&E and Masson’s trichrome staining indicated that fibrosis did not occur over the duration of the study. At the time of sacrifice there was no significant variation in tissue inflammation between groups. CONCLUSIONS: LL-37-induced cystitis yielded pain to be independent of inflammation. These findings further substantiate the physiological applicability of the LL-37-induced cystitis model to mimic human IC/PBS pathophysiology. Finally, our SAGE compound demonstrated potent non-opioid analgesic effects within this surrogate model. Source of Funding: NIH NIDDK R01 DK100868 (SO), Primary Children’s Hospital Integrated Science Award (SO), NIH K12 UL1RR025764 (SO), National Science Foundation Graduate Research Fellowship 1256065 (MMJ) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e97-e97 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Austin Schults* More articles by this author Wanjian Jia More articles by this author M. Martin Jensen More articles by this author Glenn Prestwich More articles by this author Siam Oottamasathien More articles by this author Expand All Advertisement PDF downloadLoading ...

Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism.

BackgroundSmall vessel vasculitis (SVV) is a group of systemic autoimmune diseases that are mediated by neutrophil extracellular traps (NETs) in response to cathelicidin LL37, an aging molecular marker, which could be induced by telomere dysfunction. Therefore, in this study, we evaluated the hypothesis that telomere dysfunction in neutrophils may promote SVV via an LL37-NETs-dependent mechanism.MethodsWe contrasted the release of neutrophil NETs from mice with telomere dysfunction, mice with DNA damage and wide-type mice. Neutrophil telomere length, the expression of LL37, and the formation of NETs were measured in SVV patients and healthy controls (HCs). The co-expression of γH2AX, LL37, and NETs were detected in SVV patients to evaluate the association of the immune aging of neutrophils and pro-inflammatory conditions. LL37 inhibitor was used to verify its key role in NETs release in SVV patients and DNA damage mice.ResultsWe found that NETs were over-induced by telomere dysfunction and DNA damage in mice, which may be associated with a marked increase in LL37. For patients with SVV, telomeres in neutrophils were significantly shortened, which was also associated with higher levels of LL37 and NETs. Inhibition of LL37 reduced the NETs released from neutrophils.ConclusionsTaken together, the results of these studies suggest that dysfunction of telomeres may promote SVV through the mechanism of LL37-dependent NETs. Thus, targeting the LL37-NETs may be a novel therapy for SVV.

Short-Term versus Long-Term Culture of A549 Cells for Evaluating the Effects of Lipopolysaccharide on Oxidative Stress, Surfactant Proteins and Cathelicidin LL-37.

Alveolar epithelial type II (ATII) cells and their proper function are essential for maintaining lung integrity and homeostasis. However, they can be damaged by lipopolysaccharide (LPS) during Gram-negative bacterial infection. Thus, this study evaluated and compared the effects of LPS on short and long-term cultures of A549 cells by determining the cell viability, levels of oxidative stress and antimicrobial peptide cathelicidin LL-37 and changes in the expression of surfactant proteins (SPs). Moreover, we compared A549 cell response to LPS in the presence of different serum concentrations. Additionally, the effect of N-acetylcysteine (NAC) on LPS-induced oxidative stress as a possible treatment was determined. Our results indicate that A549 cells are relatively resistant to LPS and able to maintain integrity even at high LPS concentrations. Their response to endotoxin is partially dependent on serum concentration. NAC failed to lower LPS-induced oxidative stress in A549 cells. Finally, LPS modulates SP gene expression in A549 cells in a time dependent manner and differences between short and long-term cultures were present. Our results support the idea that long-term cultivation of A549 cells could promote a more ATII-like phenotype and thus could be a more suitable model for ATII cells, especially for in vitro studies dealing with surfactant production.

Citrullination Alters the Antiviral and Immunomodulatory Activities of the Human Cathelicidin LL-37 During Rhinovirus Infection

Human rhinoviruses (HRV) are the most common cause of viral respiratory tract infections. While normally mild and self-limiting in healthy adults, HRV infections are associated with bronchiolitis in infants, pneumonia in immunocompromised patients, and exacerbations of asthma and COPD. The human cathelicidin LL-37 is a host defense peptide (HDP) with broad immunomodulatory and antimicrobial activities that has direct antiviral effects against HRV. However, LL-37 is known to be susceptible to the enzymatic activity of peptidyl arginine deiminases (PAD), and exposure of the peptide to these enzymes results in the conversion of positively charged arginines to neutral citrullines (citrullination). Here, we demonstrate that citrullination of LL-37 reduced its direct antiviral activity against HRV. Furthermore, while the anti-rhinovirus activity of LL-37 results in dampened epithelial cell inflammatory responses, citrullination of the peptide, and a loss in antiviral activity, ameliorates this effect. This study also demonstrates that HRV infection upregulates PAD2 protein expression, and increases levels of protein citrullination, including histone H3, in human bronchial epithelial cells. Increased PADI gene expression and HDP citrullination during infection may represent a novel viral evasion mechanism, likely applicable to a wide range of pathogens, and should therefore be considered in the design of therapeutic peptide derivatives.

Значение уровня кателицидина LL-37 в сыворотке крови и содержимом трахеобронхиального дерева при респираторном дистресс-синдроме и врожденной пневмонии у недоношенных новорожденных

Значение уровня кателицидина LL-37 в сыворотке крови и содержимом трахеобронхиального дерева при респираторном дистресс-синдроме и врожденной пневмонии у недоношенных новорожденных

Linezolid and Rifampicin Combination to Combat cfr-Positive Multidrug-Resistant MRSA in Murine Models of Bacteremia and Skin and Skin Structure Infection.

Linezolid resistance mediated by the cfr gene in MRSA represents a global concern. We investigated relevant phenotype differences between cfr-positive and -negative MRSA that contribute to pathogenesis, and the efficacy of linezolid-based combination therapies in murine models of bacteremia and skin and skin structure infection (SSSI). As a group, cfr-positive MRSA exhibited significantly reduced susceptibilities to the host defense peptides tPMPs, human neutrophil peptide-1 (hNP-1), and cathelicidin LL-37 (P < 0.01). In addition, increased binding to fibronectin (FN) and endothelial cells paralleled robust biofilm formation in cfr-positive vs. -negative MRSA. In vitro phenotypes of cfr-positive MRSA translated into poor outcomes of linezolid monotherapy in vivo in murine bacteremia and SSSI models. Importantly, rifampicin showed synergistic activity as a combinatorial partner with linezolid, and the EC50 of linezolid decreased 6-fold in the presence of rifampicin. Furthermore, this combination therapy displayed efficacy against cfr-positive MRSA at clinically relevant doses. Altogether, these data suggest that the use of linezolid in combination with rifampicin poses a viable therapeutic alternative for bacteremia and SSSI caused by cfr-positive multidrug resistant MRSA.

Dynamics of immune and biochemical features of oral fluid in persons with caries receiving vitamin D

The aim of the study was to evaluate the content of the vitamin D metabolite (25(OH)D3) in the blood serum, the level of some indicators of the immune and peroxidic status in the oral fluid in people with carious processes of different intensities while taking the native solution of vitamin D (international non-proprietary name Cholecalciferol). The study included 47 people aged 20 to 22 with caries and low levels of 25(OH)D3 in blood serum. The control group consisted of 13 people with DMFT index of 0.00 [0.00; 0.00] and a normal serum content of 25(OH)D3. The amount of antimicrobial peptide cathelicidin LL-37, secretory immunoglobulin A, lipopolysaccharide binding protein (LBP) was determined in the oral fluid using ELISA reagent kits, and the content of intermediate lipid peroxidation products such as malondialdehyde (TBA-active products) and the level of total antioxidant activity were spectrophotometrically evaluated. Reduced serum concentrations of 25(OH)D3 were recorded predominantly in individuals with a DMFT index of more than 9.0. In patients with caries, at a concentration of 25(OH)D3 in blood serum below 30 ng/ml, a decrease in the values of secretory immunoglobulin A, lipopolysaccharide-binding protein, cathelicidin, and the level of total antioxidant activity was observed, accompanied by an increase in the number of intermediate products of lyoperoxidation in the oral fluid. The course intake of the native AquaDtrim vitamin D solution (Cholecalciferol) led to normalization of the values of certain immune and biochemical parameters (cathelicidin LL-37, secretory immunoglobulin A, LBP of the oral fluid) and restoration of balance in the system «lipid peroxidation - antioxidants». The results indicate the advisability of assessing the level of 25(OH)D3 in the body for the diagnosis of pathological processes in the oral cavity and the use of vitamin D preparations in the prevention and complex therapy of carious process.

A Profound Membrane Reorganization Defines Susceptibility of &lt;i&gt;Plasmodium falciparum&lt;/i&gt; Infected Red Blood Cells to Lysis by Granulysin and Perforin

Malaria remains one of the most serious health problems in developing countries. The causative agent of malaria, Plasmodium spp., have a complex life cycle involving multiple developmental stages as well as different morphological, biochemical and metabolic requirements. We recently found that gd T cells control parasite growth using pore-forming proteins to deliver their cytotoxic proteases, the granzymes, into blood residing parasites.Here, we demonstrate that Plasmodium falciparum infection depletes the red blood cells of cholesterol, which renders the parasite surrounding membranes susceptible to lysis by prokaryotic membrane disrupting proteins, such as lymphocytic granulysin or the human cathelicidin LL-37. Interestingly, not the cholesterol depletion but rather the simultaneous exposure of phosphatidylserine, a negatively charged phospholipid, triggers resistance of late stage parasitized red blood cells towards the eukaryotic pore forming protein perforin. Overall, we establish here a pathogen-host interaction that involves host cell membrane remodeling that defines the susceptibility towards cytolytic molecules.