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1.
AAPS PharmSciTech ; 25(5): 110, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740721

RESUMO

Antimicrobial peptide LL37 is a promising antibacterial candidate due to its potent antimicrobial activity with no known bacterial resistance. However, intrinsically LL37 is susceptible to degradation in wound fluids limits its effectiveness. Bacterial toxins which are released after cell lysis are found to hinder wound healing. To address these challenges, encapsulating LL37 in microspheres (MS) and loading the MS onto activated carbon (AC)-chitosan (CS) hydrogel. This advanced wound dressing not only protects LL37 from degradation but also targets bacterial toxins, aiding in the healing of chronic wound infections. First, LL37 MS and LL37-AC-CS hydrogel were prepared and characterised in terms of physicochemical properties, drug release, and peptide-polymer compatibility. Antibacterial and antibiofilm activity, bacterial toxin elimination, cell migration, and cell cytotoxicity activities were investigated. LL37-AC-CS hydrogel was effective against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. LL37-AC-CS hydrogel bound more endotoxin than AC with CS hydrogel alone. The hydrogel also induced cell migration after 72 h and showed no cytotoxicity towards NHDF after 72 h of treatment. In conclusion, the LL37-AC-CS hydrogel was shown to be a stable, non-toxic advanced wound dressing method with enhanced antimicrobial and antitoxin activity, and it can potentially be applied to chronic wound infections to accelerate wound healing.


Assuntos
Antibacterianos , Bandagens , Quitosana , Escherichia coli , Hidrogéis , Microesferas , Pseudomonas aeruginosa , Staphylococcus aureus , Quitosana/química , Antibacterianos/farmacologia , Antibacterianos/administração & dosagem , Antibacterianos/química , Hidrogéis/química , Hidrogéis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Humanos , Pseudomonas aeruginosa/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/prevenção & controle , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Catelicidinas , Testes de Sensibilidade Microbiana/métodos , Toxinas Bacterianas , Liberação Controlada de Fármacos , Movimento Celular/efeitos dos fármacos , Carbono/química , Biofilmes/efeitos dos fármacos
2.
Nutrients ; 16(9)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38732603

RESUMO

BACKGROUND: Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that a Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. METHODS: We used two cell culture models of THP-1 macrophages and Caco-2 monolayers to establish the effects of MAP infection on the vitamin D metabolic axis. We also tested the effects of Calcifediol, Calcitriol, and SB203580 treatments on the relative expression of the vitamin D metabolic genes, oxidative stress biomarkers, and inflammatory cytokines profile. RESULTS: In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 h post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78 ± 0.88 and 13.90 ± 3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93 ± 0.28, 1.86 ± 0.27, and 6.34 ± 0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. CONCLUSIONS: This study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase , Catelicidinas , Macrófagos , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Receptores de Calcitriol , Receptor 2 Toll-Like , Vitamina D , Proteínas Quinases p38 Ativadas por Mitógeno , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Humanos , Receptor 2 Toll-Like/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Vitamina D/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Receptores de Calcitriol/metabolismo , Células CACO-2 , Paratuberculose/microbiologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Células THP-1 , Sistema de Sinalização das MAP Quinases , Calcitriol/farmacologia , Transdução de Sinais
3.
J Immunol Methods ; 529: 113670, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38604530

RESUMO

Cytotoxicity studies determining hemolytic properties of antimicrobial peptides or other drugs are an important step in the development of novel therapeutics for clinical use. Hemolysis is an affordable, accessible, and rapid method for initial assessment of cellular toxicity for all drugs under development. However, variability in species of red blood cells and protocols used may result in significant differences in results. AMPs generally possess higher selectivity for bacterial cells but can have toxicity against host cells at high concentrations. Knowing the hemolytic activity of the peptides we are developing contributes to our understanding of their potential toxicity. Computational approaches for predicting hemolytic activity of AMPs exist and were tested head-to-head with our experimental results. RESULTS: Starting with an observation of high hemolytic activity of LL-37 peptide against human red blood cells that were collected in EDTA, we explored alternative approaches to develop a more robust, accurate and simple hemolysis assay using defibrinated human blood. We found significant differences between the sensitivity of defibrinated red blood cells and EDTA treated red blood cells. SIGNIFICANCE: Accurately determining the hemolytic activity using human red blood cells will allow for a more robust calculation of the therapeutic index of our potential antimicrobial compounds, a critical measure in their pre-clinical development. CONCLUSION: We introduce a standardized, more accurate protocol for assessing hemolytic activity using defibrinated human red blood cells. This approach, facilitated by the increased commercial availability of de-identified human blood and defibrination methods, offers a robust tool for evaluating toxicity of emerging drug compounds, especially AMPs.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Eritrócitos , Hemólise , Humanos , Hemólise/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Catelicidinas , Ácido Edético/farmacologia
4.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 36(1): 98-104, 2024 Mar 25.
Artigo em Chinês | MEDLINE | ID: mdl-38604693

RESUMO

Parasitic diseases caused by protozoan and helminth infections are still widespread across the world, notably in tropical and subtropical areas, which threaten the children and adult health. Long-term use of anti-parasitic drugs may result in reduced drug susceptibility and even drug resistance. Antimicrobial peptides have been demonstrated to inhibit parasite growth and development, which has potential antiparasitic values. LL-37, the only human antimicrobial peptide in the cathelicidin family, has been widely investigated. This paper reviews the progress of researches on the antiparasitic activity of LL-37, and discusses the prospects of LL-37 in the research of parasites.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Humanos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Catelicidinas/farmacologia
5.
Res Vet Sci ; 172: 105240, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38608347

RESUMO

Antimicrobial usage (AMU) could be reduced by differentiating the causative bacteria in cases of clinical mastitis (CM) as either Gram-positive or Gram-negative bacteria or identifying whether the case is culture-negative (no growth, NG) mastitis. Immunoassays for biomarker analysis and a Tandem Mass Tag (TMT) proteomic investigation were employed to identify differences between samples of milk from cows with CM caused by different bacteria. A total of 94 milk samples were collected from cows diagnosed with CM across seven farms in Scotland, categorized by severity as mild (score 1), moderate (score 2), or severe (score 3). Bovine haptoglobin (Hp), milk amyloid A (MAA), C-reactive protein (CRP), lactoferrin (LF), α-lactalbumin (LA) and cathelicidin (CATHL) were significantly higher in milk from cows with CM, regardless of culture results, than in milk from healthy cows (all P-values <0.001). Milk cathelicidin (CATHL) was evaluated using a novel ELISA technique that utilises an antibody to a peptide sequence of SSEANLYRLLELD (aa49-61) common to CATHL 1-7 isoforms. A classification tree was fitted on the six biomarkers to predict Gram-positive bacteria within mastitis severity scores 1 or 2, revealing that compared to the rest of the samples, Gram-positive samples were associated with CRP < 9.5 µg/ml and LF ≥ 325 µg/ml and MAA < 16 µg/ml. Sensitivity of the tree model was 64%, the specificity was 91%, and the overall misclassification rate was 18%. The area under the ROC curve for this tree model was 0.836 (95% bootstrap confidence interval: 0.742; 0.917). TMT proteomic analysis revealed little difference between the groups in protein abundance when the three groups (Gram-positive, Gram-negative and no growth) were compared, however when each group was compared against the entirety of the remaining samples, 28 differentially abundant protein were identified including ß-lactoglobulin and ribonuclease. Whilst further research is required to draw together and refine a suitable biomarker panel and diagnostic algorithm for differentiating Gram- positive/negative and NG CM, these results have highlighted a potential panel and diagnostic decision tree. Host-derived milk biomarkers offer significant potential to refine and reduce AMU and circumvent the many challenges associated with microbiological culture, both within the lab and on the farm, while providing the added benefit of reducing turnaround time from 14 to 16 h of microbiological culture to just 15 min with a lateral flow device (LFD).


Assuntos
Biomarcadores , Mastite Bovina , Leite , Animais , Bovinos , Feminino , Leite/química , Leite/microbiologia , Mastite Bovina/microbiologia , Mastite Bovina/diagnóstico , Biomarcadores/metabolismo , Proteoma , Proteínas do Leite/análise , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Catelicidinas
6.
Oral Health Prev Dent ; 22: 159-170, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38687028

RESUMO

PURPOSE: To study the therapeutic effect of hemagglutinin-2 and fimbrial (HA2-FimA) vaccine on experimental periodontitis in rats. MATERIALS AND METHODS: The first batch of rats was divided into two groups and immunised with pure water or pVAX1-HA2-FimA at the age of 6, 7, and 9 weeks. After sacrificing the animals, total RNA was extracted from the spleens for RNA high-throughput sequencing (RNA-Seq) analysis. The second batch of rats was divided into four groups (A, B, C, D), and an experimental periodontitis rat model was established by suturing silk thread around the maxillary second molars of rats in groups B, C, and D for 4 weeks. The rats were immunised with pure water, pVAX1-HA2-FimA vaccine, empty pVAX1 vector, and pure water at 10, 11, and 13 weeks of age, respectively. Secretory immunoglobulin A (SIgA) antibodies and cathelicidin antimicrobial peptide (CAMP) levels in saliva were measured by enzyme-linked immunosorbent assay (ELISA). All rats were euthanised at 17 weeks of age, and alveolar bone loss was examined using micro-computed tomography (Micro-CT). RESULTS: Through sequencing analysis, six key genes, including Camp, were identified. Compared with the other three groups, the rats in the periodontitis+pVAX1-HA2-FimA vaccine group showed higher levels of SIgA and CAMP (p < 0.05). Micro-CT results showed significantly less alveolar bone loss in the periodontitis+pVAX1-HA2-FimA vaccine group compared to the periodontitis+pVAX1 group and periodontitis+pure water group (p < 0.05). CONCLUSION: HA2-FimA DNA vaccine can increase the levels of SIgA and CAMP in the saliva of experimental periodontitis model rats and reduce alveolar bone loss.


Assuntos
Periodontite , Vacinas de DNA , Animais , Periodontite/prevenção & controle , Periodontite/imunologia , Ratos , Modelos Animais de Doenças , Imunoglobulina A Secretora/análise , Proteínas de Fímbrias/imunologia , Perda do Osso Alveolar/prevenção & controle , Catelicidinas , Ratos Sprague-Dawley , Ensaio de Imunoadsorção Enzimática , Saliva/imunologia , Hemaglutininas/imunologia , Microtomografia por Raio-X , Masculino
7.
Biochem Biophys Res Commun ; 712-713: 149962, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38642493

RESUMO

The human cathelicidin LL-37 shows activity against microorganisms, but it is also cytotoxic to host cells. The CAMP gene codes for the LL-37 precursor hCAP18 which is processed extracellularly to active LL-37. It has previously been shown that vitamin D stimulates CAMP gene activity, but less information is available demonstrating that vitamin D also can increase hCAP18/LL-37 protein production. Here, we show with RT-qPCR that a physiological concentration of vitamin D (50 nM) enhances CAMP mRNA levels by about 170 times in human THP-1 monocyte cells. Stimulation with 50 nM vitamin D increases hCAP18/LL-37 protein contents 3-4 times in THP-1 cell lysates demonstrated by both dot blot analysis and ELISA applying two different hCAP18/LL-37 antibodies. Treatment with the proteasome inhibitor MG132 enhances hCAP18/LL-37 levels, suggesting that turnover of hCAP18/LL-37 protein is regulated by the proteasome. The hCAP18/LL-37 concentration in vitamin D-stimulated THP-1 cells corresponds to 1.04 µM LL-37. Interestingly, synthetic LL-37, at this concentration, reduces viability of human osteoblast-like MG63 cells, whereas the THP-1 cells are less sensitive as demonstrated by the MTT assay. In summary, we show that vitamin D enhances hCAP18/LL-37 production, and that this effect can be of physiological/pathophysiological relevance for LL-37-induced human osteoblast toxicity.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Catelicidinas , Osteoblastos , Vitamina D , Humanos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Vitamina D/farmacologia , Vitamina D/metabolismo , Vitamina D/análogos & derivados , Células THP-1 , Complexo de Endopeptidases do Proteassoma/metabolismo , Sobrevivência Celular/efeitos dos fármacos
8.
Infect Immun ; 92(4): e0048323, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38501672

RESUMO

Aspergillus fumigatus (A. fumigatus) is one of the common pathogens of fungal keratitis. Fungal growth and invasion cause excessive inflammation and corneal damage, leading to severe vision loss. Neutrophils are the primary infiltrating cells critical for fungal clearance. Cathelicidin [LL-37 in humans and cathelicidin-related antimicrobial peptide (CRAMP) in mice], a natural antimicrobial peptide, can directly inhibit the growth of many pathogens and regulate immune responses. However, the role of cathelicidin and its effect on neutrophils in A. fumigatus keratitis remain unclear. By establishing A. fumigatus keratitis mouse models, we found that cathelicidin was increased in A. fumigatus keratitis. It could reduce fungal loads, lower clinical scores, and improve corneal transparency. Restriction of CRAMP on fungal proliferation was largely counteracted in CD18-/- mice, in which neutrophils cannot migrate into infected sites. When WT neutrophils were transferred into CD18-/- mice, corneal fungal loads were distinctly reduced, indicating that neutrophils are vital for CRAMP-mediated resistance. Furthermore, cathelicidin promoted neutrophils to phagocytose and degrade conidia both in vitro and in vivo. CXC chemokine receptor 2 (CXCR2) was reported to be a functional receptor of LL-37 on neutrophils. CXCR2 antagonist SB225002 or phospholipase C (PLC) inhibitor U73122 weakened LL-37-induced phagocytosis. Meanwhile, LL-37 induced PLC γ phosphorylation, which was attenuated by SB225002. SB225002 or the autophagy inhibitors Bafilomycin-A1 and 3-Methyladenine weakened LL-37-induced degradation of conidia. Transmission electron microscopy (TEM) observed that LL-37 increased autophagosomes in Aspergillus-infected neutrophils. Consistently, LL-37 elevated autophagy-associated protein expressions (Beclin-1 and LC3-II), but this effect was weakened by SB225002. Collectively, cathelicidin reduces fungal loads and improves the prognosis of A. fumigatus keratitis. Both in vitro and in vivo, cathelicidin promotes neutrophils to phagocytose and degrade conidia. LL-37/CXCR2 activates PLC γ to amplify neutrophils' phagocytosis and induces autophagy to eliminate intracellular conidia.


Assuntos
Aspergillus fumigatus , Ceratite , Compostos de Fenilureia , Humanos , Animais , Camundongos , Neutrófilos , Antifúngicos/metabolismo , Catelicidinas , Fosfolipase C gama/metabolismo , Ceratite/microbiologia , Prognóstico , Camundongos Endogâmicos C57BL
9.
J Cutan Med Surg ; 28(2): 178-189, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38450615

RESUMO

Rosacea is a chronic inflammatory condition of which there is no cure. The pathogenesis of rosacea is likely multifactorial, involving genetic and environmental contributions. Current understanding suggests that pro-inflammatory pathways involving cathelicidins and inflammasome complexes are central to rosacea pathogenesis. Common rosacea triggers modulate these pathways in a complex manner, which may contribute to the varying severity and clinical presentations of rosacea. Established and emerging rosacea treatments may owe their efficacy to their ability to target different players in these pro-inflammatory pathways. Improving our molecular understanding of rosacea will guide the development of new therapies and the use of combination therapies.


Assuntos
Rosácea , Humanos , Rosácea/terapia , Rosácea/tratamento farmacológico , Catelicidinas/uso terapêutico
10.
Sci Rep ; 14(1): 6750, 2024 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-38514730

RESUMO

Signals for the maintenance of epithelial homeostasis are provided in part by commensal bacteria metabolites, that promote tissue homeostasis in the gut and remote organs as microbiota metabolites enter the bloodstream. In our study, we investigated the effects of bile acid metabolites, 3-oxolithocholic acid (3-oxoLCA), alloisolithocholic acid (AILCA) and isolithocholic acid (ILCA) produced from lithocholic acid (LCA) by microbiota, on the regulation of innate immune responses connected to the expression of host defense peptide cathelicidin in lung epithelial cells. The bile acid metabolites enhanced expression of cathelicidin at low concentrations in human bronchial epithelial cell line BCi-NS1.1 and primary bronchial/tracheal cells (HBEpC), indicating physiological relevance for modulation of innate immunity in airway epithelium by bile acid metabolites. Our study concentrated on deciphering signaling pathways regulating expression of human cathelicidin, revealing that LCA and 3-oxoLCA activate the surface G protein-coupled bile acid receptor 1 (TGR5, Takeda-G-protein-receptor-5)-extracellular signal-regulated kinase (ERK1/2) cascade, rather than the nuclear receptors, aryl hydrocarbon receptor, farnesoid X receptor and vitamin D3 receptor in bronchial epithelium. Overall, our study provides new insights into the modulation of innate immune responses by microbiota bile acid metabolites in the gut-lung axis, highlighting the differences in epithelial responses between different tissues.


Assuntos
Ácidos e Sais Biliares , Catelicidinas , Humanos , Ácidos e Sais Biliares/metabolismo , Catelicidinas/metabolismo , Sistema de Sinalização das MAP Quinases , Receptores Acoplados a Proteínas G/metabolismo , Epitélio/metabolismo , Ácido Litocólico/farmacologia , Ácido Litocólico/metabolismo
11.
Antiviral Res ; 225: 105855, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38460762

RESUMO

Zika virus (ZIKV) is an enveloped, single-stranded and positive-stranded RNA virus of the genus Flavivirus in the family Flaviviridae. ZIKV can cross the placental barrier and infect the fetus, causing microcephaly, congenital ZIKV syndrome, and even fetal death. ZIKV infection can also lead to testicular damage and male sterility. But no effective drugs and vaccines are available up to now. Previous studies have shown that the cathelicidin antimicrobial peptide LL-37 can protect against ZIKV infection. However, LL-37 is a secreted peptide, which can be easily degraded in vivo. We herein constructed exosome-loaded LL-37 (named LL-37-TM-exo and TM-LL-37-exo) using the transmembrane protein TM to load LL-37 onto the membrane of exosome. We found that exosome-loaded LL-37 could significantly inhibit ZIKV infection in vitro and in vivo, and LL-37-TM-exo had stronger antiviral activity than that of TM-LL-37-exo, which could significantly reduce ZIKV-induced testicular injury and sperm injury, and had broad-spectrum antiviral effect. Compared to free LL-37, exosome-loaded LL-37 showed a better serum stability, higher efficiency to cross the placental barrier, and stronger antiviral activity. The mechanism of exosome-loaded LL-37 against ZIKV infection was consistent with that of free LL-37, which could directly inactivate viral particles, reduce the susceptibility of host cells, and act on viral replication stage. Our study provides a novel strategy for the development of LL-37 against viral infection.


Assuntos
Catelicidinas , Exossomos , Infecção por Zika virus , Zika virus , Feminino , Humanos , Masculino , Gravidez , Antivirais/uso terapêutico , Exossomos/metabolismo , Placenta , Replicação Viral , Zika virus/efeitos dos fármacos , Zika virus/fisiologia , Infecção por Zika virus/prevenção & controle , Infecção por Zika virus/virologia
12.
J Innate Immun ; 16(1): 203-215, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38471488

RESUMO

INTRODUCTION: TNFα-inducible matrix metalloproteinases play a critical role in the process of airway remodeling in respiratory inflammatory disease including asthma. The cationic host defense peptide LL-37 is elevated in the lungs during airway inflammation. However, the impact of LL-37 on TNFα-driven processes is not well understood. Here, we examined the effect of LL-37 on TNFα-mediated responses in human bronchial epithelial cells (HBECs). METHODS: We used a slow off-rate modified aptamer-based proteomics approach to define the HBEC proteome altered in response to TNFα. Abundance of selected protein candidates and signaling intermediates was examined using immunoassays, ELISA and Western blots, and mRNA abundance was examined by qRT-PCR. RESULTS: Proteomics analysis revealed that 124 proteins were significantly altered, 12 proteins were enhanced by ≥2-fold compared to unstimulated cells, in response to TNFα. MMP9 was the topmost increased protein in response to TNFα, enhanced by ∼10-fold, and MMP13 was increased by ∼3-fold, compared to unstimulated cells. Furthermore, we demonstrated that LL-37 significantly suppressed TNFα-mediated MMP9 and MMP13 in HBEC. Mechanistic data revealed that TNFα-mediated MMP9 and MMP13 production is controlled by SRC kinase and that LL-37 enhances related upstream negative regulators, namely, phospho-AKT (T308) and TNFα-mediated TNFAIP3 or A20. CONCLUSIONS: The findings of this study suggest that LL-37 may play a role in intervening in the process of airway remodeling in chronic inflammatory respiratory disease such as asthma.


Assuntos
Remodelação das Vias Aéreas , Peptídeos Catiônicos Antimicrobianos , Asma , Brônquios , Catelicidinas , Células Epiteliais , Metaloproteinase 13 da Matriz , Metaloproteinase 9 da Matriz , Fator de Necrose Tumoral alfa , Humanos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Asma/imunologia , Asma/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteômica , Mucosa Respiratória/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
13.
Biomolecules ; 14(3)2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38540740

RESUMO

Antimicrobial peptides (AMPs), as well as host defense peptides (HDPs), constitute the first line of defense as part of the innate immune system. Humans are known to express antimicrobial precursor proteins, which are further processed to generate AMPs, including several types of α/ß defensins, histatins, and cathelicidin-derived AMPs like LL37. The broad-spectrum activity of AMPs is crucial to defend against infections caused by pathogenic bacteria, viruses, fungi, and parasites. The emergence of multi-drug resistant pathogenic bacteria is of global concern for public health. The prospects of targeting antibiotic-resistant strains of bacteria with AMPs are of high significance for developing new generations of antimicrobial agents. The 37-residue long LL37, the only cathelicidin family of AMP in humans, has been the major focus for the past few decades of research. The host defense activity of LL37 is likely underscored by its expression throughout the body, spanning from the epithelial cells of various organs-testis, skin, respiratory tract, and gastrointestinal tract-to immune cells. Remarkably, apart from canonical direct killing of pathogenic organisms, LL37 exerts several other host defense activities, including inflammatory response modulation, chemo-attraction, and wound healing and closure at the infected sites. In addition, LL37 and its derived peptides are bestowed with anti-cancer and anti-amyloidogenic properties. In this review article, we aim to develop integrative, mechanistic insight into LL37 and its derived peptides, based on the known biophysical, structural, and functional studies in recent years. We believe that this review will pave the way for future research on the structures, biochemical and biophysical properties, and design of novel LL37-based molecules.


Assuntos
Anti-Infecciosos , Catelicidinas , Humanos , Peptídeos Catiônicos Antimicrobianos/química , Anti-Infecciosos/farmacologia , Cicatrização , Pele/metabolismo
14.
Int J Mol Sci ; 25(5)2024 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-38474156

RESUMO

Obesity represents a worldwide health challenge, and the condition is accompanied by elevated risk of cardiovascular diseases caused by metabolic dysfunction and proinflammatory adipokines. Among those, the immune-modulatory cathelicidin antimicrobial peptide (human: CAMP; murine: CRAMP) might contribute to the interaction of the innate immune system and metabolism in these settings. We investigated systemic CAMP/CRAMP levels in experimental murine models of atherosclerosis, myocardial infarction and cardiovascular patients. Atherosclerosis was induced in low-density lipoprotein receptor-deficient (Ldlr-/-) mice by high-fat diet (HFD). C57BL/6J wild-type mice were subjected to myocardial infarction by permanent or transient left anterior descending (LAD)-ligation. Cramp gene expression in murine organs and tissues was investigated via real-time PCR. Blood samples of 234 adult individuals with or without coronary artery disease (CAD) were collected. Human and murine CAMP/CRAMP serum levels were quantified by ELISA. Atherosclerotic mice exhibited significantly increased CRAMP serum levels and induced Cramp gene expression in the spleen and liver, whereas experimental myocardial infarction substantially decreased CRAMP serum levels. Human CAMP serum quantities were not significantly affected by CAD while being correlated with leukocytes and pro-inflammatory cytokines. Our data show an influence of cathelicidin in experimental atherosclerosis, myocardial infarction, as well as in patients with CAD. Further studies are needed to elucidate the pathophysiological mechanism.


Assuntos
Aterosclerose , Doença da Artéria Coronariana , Infarto do Miocárdio , Adulto , Animais , Humanos , Camundongos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Catelicidinas , Camundongos Endogâmicos C57BL , Camundongos Knockout
15.
ACS Infect Dis ; 10(3): 951-960, 2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38315114

RESUMO

The emergence of multidrug-resistant (MDR) bacteria presents a significant challenge to public health, increasing the risk of infections that are resistant to current antibiotic treatment. Antimicrobial peptides (AMPs) offer a promising alternative to conventional antibiotics in the prevention of MDR bacterial infections. In the present study, we identified a novel cathelicidin AMP from Gekko japonicus, which exhibited broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging from 2.34 to 4.69 µg/mL. To improve its potential therapeutic application, a series of peptides was synthesized based on the active region of the gecko-derived cathelicidin. The lead peptide (RH-16) showed an antimicrobial activity comparable to that of the parent peptide. Structural characterization revealed that RH-16 adopted an amphipathic α-helical conformation. Furthermore, RH-16 demonstrated neither hemolytic nor cytotoxic activity but effectively killed a wide range of clinically isolated, drug-resistant bacteria. The antimicrobial activity of RH-16 was attributed to the nonspecific targeting of bacterial membranes, leading to rapid bacterial membrane permeabilization and rupture. RH-16 also retained its antibacterial activity in plasma and exhibited mild toxicity in vivo. Notably, RH-16 offered robust protection against skin infection in a murine model. Therefore, this newly identified cathelicidin AMP may be a strong candidate for future pharmacological development targeting multidrug resistance. The use of a rational design approach for isolating the minimal antimicrobial unit may accelerate the transition of natural AMPs to clinically applicable antibacterial agents.


Assuntos
Anti-Infecciosos , Catelicidinas , Lagartos , Camundongos , Animais , Catelicidinas/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Antimicrobianos , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Bactérias
16.
Peptides ; 175: 171183, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38423213

RESUMO

Diabetic patients often experience impaired wound healing. Human cathelicidin LL-37 possesses various biological functions, such as anti-microbial, anti-inflammatory, and pro-wound healing activities. Autophagy has important effects on skin wound healing. However, little is known about whether LL-37 accelerates diabetic wound healing by regulating autophagy. In the study, we aimed to investigate the role of autophagy in LL-37-induced wound healing and uncover the underlying mechanisms involved. A full-thickness wound closure model was established in diabetic mice to evaluate the effects of LL-37 and an autophagy inhibitor (3-MA) on wound healing. The roles of LL-37 and 3-MA in regulating keratinocyte migration were assessed using transwell migration and wound healing assays. The activation of transcription factor EB (TFEB) was measured using western blotting and immunofluorescence (IF) assays of its nuclear translocation. The results showed that LL-37 treatment improved wound healing in diabetic mice, whereas these effects were reversed by 3-MA. In vitro, 3-MA decreased the effects of LL-37 on promoting HaCat keratinocyte migration in the presence of high glucose (HG). Mechanistically, LL-37 promoted TFEB activation and resulted in subsequent activation of autophagy, as evidenced by increased nuclear translocation of TFEB and increased expression of ATG5, ATG7, and beclin 1 (BECN1), whereas these changes were blocked by TFEB knockdown. As expected, TFEB knockdown damaged the effects of LL-37 on promoting keratinocyte migration. Collectively, these results suggest that LL-37 accelerates wound healing in diabetic mice by activating TFEB-dependent autophagy, providing new insights into the mechanism by which LL-37 promotes diabetic wound healing.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Catelicidinas , Diabetes Mellitus Experimental , Animais , Humanos , Camundongos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Catelicidinas/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Cicatrização
17.
Front Immunol ; 15: 1295168, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38384468

RESUMO

Candida albicans remains the predominant cause of fungal infections, where adhered microbial cells form biofilms - densely packed communities. The central feature of C. albicans biofilms is the production of an extracellular matrix (ECM) consisting of polymers and extracellular nucleic acids (eDNA, eRNA), which significantly impedes the infiltration of host cells. Neutrophils, as crucial players in the innate host defense, employ several mechanisms to eradicate the fungal infection, including NETosis, endocytosis, or the release of granules containing, among others, antimicrobial peptides (AMPs). The main representative of these is the positively charged peptide LL-37 formed from an inactive precursor (hCAP18). In addition to its antimicrobial functions, this peptide possesses a propensity to interact with negatively charged molecules, including nucleic acids. Our in vitro studies have demonstrated that LL-37 contacting with C. albicans nucleic acids, isolated from biofilm, are complexed by the peptide and its shorter derivatives, as confirmed by electrophoretic mobility shift assays. We indicated that the generation of the complexes induces discernible alterations in the neutrophil response to fungal nucleic acids compared to the effects of unconjugated molecules. Our analyses involving fluorescence microscopy, flow cytometry, and Western blotting revealed that stimulation of neutrophils with DNA:LL-37 or RNA:LL-37 complexes hamper the activation of pro-apoptotic caspases 3 and 7 and fosters increased activation of anti-apoptotic pathways mediated by the Mcl-1 protein. Furthermore, the formation of complexes elicits a dual effect on neutrophil immune response. Firstly, they facilitate increased nucleic acid uptake, as evidenced by microscopic observations, and enhance the pro-inflammatory response, promoting IL-8 production. Secondly, the complexes detection suppresses the production of reactive oxygen species and attenuates NETosis activation. In conclusion, these findings may imply that the neutrophil immune response shifts toward mobilizing the immune system as a whole, rather than inactivating the pathogen locally. Our findings shed new light on the intricate interplay between the constituents of the C. albicans biofilm and the host's immune response and indicate possible reasons for the elimination of NETosis from the arsenal of the neutrophil response during contact with the fungal biofilm.


Assuntos
Candida albicans , Ácidos Nucleicos , Candida albicans/fisiologia , Neutrófilos , Catelicidinas/farmacologia , Ácidos Nucleicos/metabolismo , Biofilmes
18.
Sci Rep ; 14(1): 3991, 2024 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-38368484

RESUMO

The number of genome-level resources for non-model species continues to rapidly expand. However, frog species remain underrepresented, with up to 90% of frog genera having no genomic or transcriptomic data. Here, we assemble the first genomic and transcriptomic resources for the recently described southern stuttering frog (Mixophyes australis). The southern stuttering frog is ground-dwelling, inhabiting naturally vegetated riverbanks in south-eastern Australia. Using PacBio HiFi long-read sequencing and Hi-C scaffolding, we generated a high-quality genome assembly, with a scaffold N50 of 369.3 Mb and 95.1% of the genome contained in twelve scaffolds. Using this assembly, we identified the mitochondrial genome, and assembled six tissue-specific transcriptomes. We also bioinformatically characterised novel sequences of two families of antimicrobial peptides (AMPs) in the southern stuttering frog, the cathelicidins and ß-defensins. While traditional peptidomic approaches to peptide discovery have typically identified one or two AMPs in a frog species from skin secretions, our bioinformatic approach discovered 12 cathelicidins and two ß-defensins that were expressed in a range of tissues. We investigated the novelty of the peptides and found diverse predicted activities. Our bioinformatic approach highlights the benefits of multi-omics resources in peptide discovery and contributes valuable genomic resources in an under-represented taxon.


Assuntos
Gagueira , beta-Defensinas , Animais , Peptídeos Antimicrobianos , beta-Defensinas/genética , Multiômica , Austrália , Catelicidinas/genética , Anuros/genética , Cromossomos
19.
Cancer Chemother Pharmacol ; 93(5): 455-469, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38280033

RESUMO

OBJECTIVE: Glioblastoma multiforme (GBM) is the most aggressive and fatal malignant primary brain tumor. The enhancement of the survival rate for glioma patients remains limited, even with the utilization of a combined treatment approach involving surgery, radiotherapy, and chemotherapy. This study was designed to assess the expression of IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX in glioblastoma tissue from 11 patients. We investigated the anticancer impact and combined effects of cathelicidin (LL-37), protegrin-1 (PG-1), with chemotherapy-temozolomide (TMZ), doxorubicin (DOX), carboplatin (CB), cisplatin (CPL), and etoposide (ETO) in primary GBM cells. In addition, we examined the effect of LL-37, PG-1 on normal human fibroblasts and in the C6/Wistar rat intracerebral glioma model. METHODS: For this study, 11 cases of glioblastoma were evaluated immunohistochemically for IDH1, TP53, EGFR, Ki-67, GFAP, H3K27M, MGMT, VEGF, NOS, CD99, and ATRX. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cells viability and to determine cytotoxic effects of LL-37, PG-1 and their combination with chemotherapy in primary GBM cells. Synergism or antagonism was determined using combination index (CI) method. Finally, we established C6 glioblastoma model in Wistar rats to investigate the antitumor activity. RESULTS: Peptides showed a strong cytotoxic effect on primary GBM cells in the MTT test (IC50 2-16 and 1-32 µM) compared to chemotherapy. The dual-drug combinations of LL-37 + DOX, LL-37 + CB (CI 0.46-0.75) and PG-1 + DOX, PG-1 + CB, PG-1 + TMZ (CI 0.11-0.77), demonstrated a synergism in primary GBM cells. In rat C6 intracerebral GBM model, survival of rats in experimental group (66.75 ± 12.6 days) was prolonged compared with that in control cohort (26.2 ± 2.66 days, p = 0.0008). After LL-37 treatment, experimental group rats showed significantly lower tumor volumes (31.00 ± 8.8 mm3) and weight (49.4 ± 13.3 mg) compared with control group rats (153.8 ± 43.53 mg, p = 0.038; 82.50 ± 7.60 mm3, respectively). CONCLUSIONS: The combination of antimicrobial peptides and chemical drugs enhances the cytotoxicity of chemotherapy and exerts synergistic antitumor effects in primary GBM cells. Moreover, in vivo study provided the first evidence that LL-37 could effectively inhibit brain tumor growth in rat C6 intracerebral GBM model. These results suggested a significant strategy for proposing a promising therapy for the treatment of GBM.


Assuntos
Neoplasias Encefálicas , Sinergismo Farmacológico , Glioblastoma , Ratos Wistar , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Animais , Ratos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Masculino , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Feminino , Pessoa de Meia-Idade , Linhagem Celular Tumoral , Biomarcadores Tumorais/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Idoso , Catelicidinas , Adulto , Temozolomida/farmacologia , Temozolomida/administração & dosagem
20.
J Cosmet Dermatol ; 23(5): 1654-1662, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38284129

RESUMO

BACKGROUND: Rosacea is a prevalent chronic dermatological condition marked by facial inflammation and erythema, significantly compromising the quality of life for affected individuals. Current treatment methods for rosacea are not considered ideal because of the complex etiology of the disease. Mussel adhesive protein (MAP) is a glycoprotein derived from the foot gland of mussels. The protein exhibits anti-inflammatory properties, relieves skin itching, and promotes wound healing. AIMS: We aimed to explore the feasibility of using MAP administered via microneedle delivery for treating rosacea and the potential molecular mechanism involved. MATERIALS AND METHODS: The therapeutic effect and mechanism of MAP microneedle delivery in an LL-37-induced rosacea-like mouse model were observed using morphological and histological methods. Twenty-seven patients with erythematotelangiectatic rosacea (ETR) underwent treatment once every 1 month, with three treatments constituting one treatment course. The therapeutic effect was evaluated by comparing the clinical images taken at baseline, after the first treatment course, and after the second treatment course. The red value, CEA, and GFSS score were also calculated. RESULTS: In response to the microneedle delivery of MAP, innate immunity, inflammatory infiltration, and abnormal neurovascular regulation improved significantly in rosacea-like mice. In the clinical experiments, the microneedle delivery of MAP significantly improved the symptoms of erythema, flushing, and telangiectasia in patients with ETR, and no obvious adverse reactions were observed. CONCLUSIONS: MAP delivered by microneedling is effective and safe for treating ETR.


Assuntos
Agulhas , Rosácea , Rosácea/terapia , Animais , Humanos , Feminino , Camundongos , Pessoa de Meia-Idade , Adulto , Agulhas/efeitos adversos , Masculino , Modelos Animais de Doenças , Proteínas/administração & dosagem , Resultado do Tratamento , Estudos de Viabilidade , Pele/patologia , Administração Cutânea , Eritema/etiologia , Eritema/terapia , Catelicidinas , Indução Percutânea de Colágeno
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