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1.
Mycoses ; 63(1): 21-29, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31610041

RESUMO

BACKGROUND: Despite the worldwide prevalence of dermatophyte infections, only a few genes are reported to be related to dermatophyte infections. In addition, the mechanism by which different ecological dermatophytes infection leads to varying intensity of inflammation remains unclear. OBJECTIVES: To investigate the mechanism of varying intensity of skin inflammation caused by different ecological dermatophytes infection. METHODS: We infected HaCaT cells with anthropophilic and geophilic dermatophytes to mimic various ecological dermatophyte infections. RNA-sequencing (RNA-seq) was employed to identify the change in the gene expression of HaCaT cells. To verify the expression of differentially expressed genes (DEGs), we selected 18 HaCaT cells genes to conduct qPCR experiments. In addition, immunoblotting was conducted to validate key genes from the MAPK signalling pathway. RESULTS: After HaCaT cells were infected with the anthropophilic Trichophyton rubrum (T rubrum) and the geophilic Microsporum gypseum (M gypseum), 118 and 619 differentially expressed genes were identified in HaCaT cells, respectively. These genes may provide a clue as to how keratinocytes respond to anthropophilic and geophilic dermatophytes. We also found that JUN may play a critical role in keratinocytes infected with M gypseum. CONCLUSIONS: Differential gene expression in HaCaT cells may account for the various clinical presentation caused by anthropophilic and geophilic dermatophytes infections. In addition, the intense inflammatory reaction of M gypseum infection may be triggered by activating the JNK-JUN signalling pathway.


Assuntos
Arthrodermataceae , Interações Hospedeiro-Patógeno/imunologia , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Arthrodermataceae/patogenicidade , Linhagem Celular , Dermatomicoses/genética , Dermatomicoses/imunologia , Dermatomicoses/microbiologia , Perfilação da Expressão Gênica , Humanos , Queratinócitos/imunologia , Microsporum/patogenicidade , Transdução de Sinais/genética , Trichophyton/patogenicidade
2.
J Appl Microbiol ; 128(2): 584-597, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31602730

RESUMO

AIMS: Probiotics have the ability to enhance the immune system, produce anti-inflammatory action and promote wound healing process. The first aim of the study was to isolate pathogenic micro-organisms from sites of chronic ulcerative lesion. The second aim was to evaluate probiotic efficacy of SYNBIO® (1:1 combination of Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502® ) in counteracting wound infections. METHODS AND RESULTS: Several bacterial pathogens were isolated from chronic ulcerative lesions and identified by morphological, biochemical and molecular techniques. SYNBIO® probiotic formulation was investigated for its antimicrobial activity, minimum inhibitory concentration, co-aggregation and adherence capacity against the isolated pathogens. Moreover, SYNBIO was also tested in combination with some medical devices, using an in vitro model, in order to simulate a real ulcerative wound infection. Probiotic formulation demonstrated an inhibitory action against all the tested pathogens and their mixture (MIX), with an increased ability of co-aggregation during time. In addition, the adhesion percentage of probiotic micro-organisms to human keratinocyte (HaCaT cells) and human fibroblasts (NHF), calculated by an in vitro model, was 19% and 17% respectively, highlighting the possibility to create a protective environment preventing pathogens' biofilm formation in order to contrast infections. CONCLUSIONS: SYNBIO® probiotics showed a very good antimicrobial capacity and adhesion percentage to HaCaT cells and fibroblasts, giving the opportunity to be successfully used as complement to conventional therapies in the treatment of chronic ulcerative lesions. SIGNIFICANCE AND IMPACT OF THE STUDY: A new therapeutic approach with probiotics (supplemented in topical applications, excluding side effects) able to eliminate pathogenic micro-organisms and improve healing of chronic ulcerative lesions.


Assuntos
Bactérias/isolamento & purificação , Colite Ulcerativa/tratamento farmacológico , Probióticos/administração & dosagem , Bactérias/classificação , Bactérias/genética , Aderência Bacteriana , Fenômenos Fisiológicos Bacterianos , Doença Crônica/terapia , Colite Ulcerativa/microbiologia , Fibroblastos/microbiologia , Humanos , Queratinócitos/microbiologia , Lactobacillus paracasei/fisiologia , Lactobacillus rhamnosus/fisiologia
3.
Future Microbiol ; 14: 1133-1146, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31512521

RESUMO

Aim: This study aimed to evaluate the differences of biosurfactants produced by two Lactobacillus helveticus strains against the biofilm formation of Staphylococcus aureus in vitro and in vivo. Materials & methods: Scanning electron microscopy, Real time-quantitative PCR (RT-qPCR) and cell assay were used to analyze the inhibiting effect of biosurfactants against biofilm formation. Results & conclusion: Results showed that the biosurfactants have anti-adhesive and inhibiting effects on biofilm formation in vivo and in vitro. The biofilm-formative genes and autoinducer-2 signaling regulated these characteristics, and the biosurfactant L. helveticus 27170 is better than that of 27058. Host cell adhesion and invasion results indicated that the biosurfactants L. helveticus prevented the S. aureus invading the host cell, which may be a new strategy to eliminate biofilms.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Lactobacillus helveticus/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Tensoativos/farmacologia , Animais , Antibacterianos/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Células Cultivadas , Humanos , Queratinócitos/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Tensoativos/metabolismo
4.
Mycopathologia ; 184(5): 597-605, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31376042

RESUMO

Foot hyperkeratosis is common. They often coincide with fungal infections, are difficult to cure and relapse rates are high. In this case study, longstanding and intractable plantar hyperkeratotic lesions were investigated for potential causative agents by histological examinations, by using human cell culture medium to grow the infected skin tissue, by sequencing ribosomal DNA and whole genome. Aspergillus sydowii was identified as the pathogen in the hyperkeratotic lesions. A peculiars intracellular infection of the fungus appeared to merge with anucleated epithelial cells of the skin, in which not fungal cells but basophilic nucleus-like bodies and abundant fungal proteins were seen in the cells. The composite fungal-human zombie-like cells were found to grow in the culture and in hyperkeratotic lesions, and some were readily transformed to natural fungus. Such zombie cells might play roles in the pathogenesis and recurrences of plantar hyperkeratotic lesions, resistance to antifungal drugs and relapses of the fungal infections.


Assuntos
Aspergillus/isolamento & purificação , Queratinócitos/microbiologia , Queratinócitos/patologia , Ceratodermia Palmar e Plantar/microbiologia , Ceratodermia Palmar e Plantar/patologia , Aspergillus/classificação , Aspergillus/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
5.
Mycoses ; 62(10): 932-936, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31278884

RESUMO

The immediate immune response developed by the keratinocytes against Malassezia yeasts has been addressed yielding conflicting results. This study aims the assessment of cytokines and antimicrobial peptides gene expression elicited by M. sympodialis and M. furfur once in contact with a reconstructed human epidermis. A yeast suspension was prepared in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with Tween 60 and oleic acid to obtain approximately 1 × 106 cells in a volume of 100 µL. Clinical isolates of M. sympodialis (from pityriasis versicolor) and M. furfur (from seborrhoeic dermatitis) were inoculated, separately, onto a reconstructed human epidermis. A distinct expression pattern was found between the two tested species, with a tendency for overexpression of pro-inflammatory cytokines very soon after infection, whereas no significant expression or gene downregulation was often noticed following 24 and 48 h of incubation. A possible Malassezia species-dependent immune response pattern is highlighted.


Assuntos
Epiderme/imunologia , Epiderme/microbiologia , Interações Hospedeiro-Patógeno , Queratinócitos/imunologia , Queratinócitos/microbiologia , Malassezia/crescimento & desenvolvimento , Malassezia/imunologia , Peptídeos Catiônicos Antimicrobianos/análise , Citocinas/análise , Dermatomicoses/microbiologia , Dermatomicoses/patologia , Humanos , Modelos Teóricos
6.
PLoS Negl Trop Dis ; 13(6): e0007339, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31233498

RESUMO

Although Mycobacterium leprae (M.leprae) is usually found in macrophages and nerves of the dermis of patients with multibacillary leprosy, it is also present in all layers of the epidermis, basal, suprabasal, prickle cells, and keratin layers. However, the mechanism by which M.leprae invades the dermis remains unknown, whereas the underlying mechanism by which M.leprae invades peripheral nerves, especially Schwann cells, is well defined. M. leprae binds to the α-dystroglycan (DG) of Schwann cells via the interaction of α-DG and laminin (LN) -α2 in the basal lamina, thus permitting it to become attached to and invade peripheral nerves. In the current study, we investigated the issue of how M.leprae is phagocytosed by human epidermal keratinocytes, neonatal (HEKn). LN-5 is the predominant form of laminin in the epidermis and allows the epidermis to be stably attached to the dermis via its interaction with α/ß-DG as well as integrins that are produced by keratinocytes. We therefore focused on the role of LN-5 when M. leprae is internalized by HEKn cells. Our results show that M.leprae preferentially binds to LN-5-coated slides and this binding to LN-5 enhances its binding to HEKn cells. The findings also show that pre-treatment with an antibody against α-DG, integrin-ß1, or -ß4 inhibited the binding of LN-5-coated M.leprae to HEKn cells. These results suggest that M. leprae binds to keratinocytes by taking advantage of the interaction of LN-5 in the basal lamina of the epidermis and a surface receptor of keratinocytes, such as α-DG, integrin-ß1, or -ß4.


Assuntos
Aderência Bacteriana , Moléculas de Adesão Celular/metabolismo , Distroglicanas/metabolismo , Integrina beta1/metabolismo , Integrina beta4/metabolismo , Queratinócitos/microbiologia , Mycobacterium leprae/fisiologia , Células Cultivadas , Humanos , Fagocitose , Ligação Proteica
7.
Nat Commun ; 10(1): 2730, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227691

RESUMO

Recently our groups discovered lugdunin, a new cyclic peptide antibiotic that inhibits Staphylococcus aureus epithelial colonization in humans and rodents. In this work, we analyzed its immuno-modulatory and antimicrobial potential as a single agent or in combination with other microbiota- or host-derived factors. We show that pretreatment of primary human keratinocytes or mouse skin with lugdunin in combination with microbiota-derived factors results in a significant reduction of S. aureus colonization. Moreover, lugdunin increases expression and release of LL-37 and CXCL8/MIP-2 in human keratinocytes and mouse skin, and results in the recruitment of monocytes and neutrophils in vivo, both by a TLR/MyD88-dependent mechanism. Interestingly, S. aureus elimination by lugdunin is additionally achieved by synergistic antimicrobial activity with LL-37 and dermcidin-derived peptides. In summary, our results indicate that lugdunin provides multi-level protection against S. aureus and may thus become a promising treatment option for S. aureus skin infections in the future.


Assuntos
Antibacterianos/farmacologia , Imunidade Inata/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Tiazolidinas/farmacologia , Animais , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Queratinócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota/efeitos dos fármacos , Microbiota/imunologia , Peptídeos/imunologia , Peptídeos Cíclicos/uso terapêutico , Cultura Primária de Células , Pele/efeitos dos fármacos , Pele/imunologia , Pele/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Tiazolidinas/uso terapêutico
8.
Microb Pathog ; 134: 103603, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31226290

RESUMO

Staphylococcus aureus extracellular vesicles (EVs) deliver effector molecules to host cells and induce host cell pathology. This study investigated the disruption of S. aureus EVs by thymol along with its inhibitory effects on the cytotoxicity and inflammatory responses induced by EVs derived from two different S. aureus strains in cultured keratinocytes. Membrane disruption of the S. aureus EVs treated with thymol was determined using transmission electron microscopy. Human keratinocyte HaCaT cells were incubated with either intact or thymol-treated S. aureus EVs and then analyzed for cytotoxicity and pro-inflammatory cytokine gene expression. Thymol inhibited the growth of S. aureus strains and disrupted the membranes of the S. aureus EVs. The cytotoxicity and the expression levels of the pro-inflammatory cytokine genes towards HaCaT cells differed between the EVs derived from two S. aureus strains. Thymol-treated S. aureus EVs inhibited the cytotoxicity and the expression of the pro-inflammatory cytokine genes when compared to intact S. aureus EVs. Thymol-treated S. aureus EVs delivered lesser amounts of the EV component to host cells than intact EVs. Our results suggest that the thymol-induced disruption of the S. aureus EVs inhibits the delivery of effector molecules to host cells, resulting in the suppression of cytotoxicity and inflammatory responses in keratinocytes. Thymol may attenuate the host cell pathology induced by an S. aureus infection via both the antimicrobial activity against the bacteria and the disruption of the secreted EVs.


Assuntos
Vesículas Extracelulares/efeitos dos fármacos , Queratinócitos/imunologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Timol/farmacologia , Antibacterianos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Humanos , Queratinócitos/microbiologia , Queratinócitos/patologia , Microscopia Eletrônica de Transmissão , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/crescimento & desenvolvimento
9.
Appl Microbiol Biotechnol ; 103(12): 4767-4778, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31065753

RESUMO

Natural rubber latex (NRL) is a natural polymer which has arisen large interest in the biomedical field, mostly, due to its ability to facilitate angiogenesis and therefore, tissue repair. Moxifloxacin (MXF) is a broad-spectrum antibiotic orally administrated. Considering the biological properties of the NRL and its ability to deliver a wide range of compounds, the present study aimed to develop a novel device for infected chronic wound treatment. MXF-loaded NRL was obtained by a casting method. The results demonstrated that the incorporation of MXF in NRL did not promote any molecular interaction, preserving the integrity of the compounds. The mechanical properties of the biomaterial did not show any significant change, indicating enough elasticity for dermal application. The microbiological assays confirmed the ability of the polymer to deliver the drug without influencing its pharmacological properties. Moreover, it has expressed activity against major bacterial strains presented in wound infections. Finally, the biomaterial shown biocompatibility from the in vitro study. Thus, the present work has shown that MXF-loaded NRL membrane is a promising biomaterial to infected wound treatment.


Assuntos
Bandagens , Sistemas de Liberação de Medicamentos/instrumentação , Moxifloxacina/farmacologia , Polímeros/química , Infecção dos Ferimentos/terapia , Animais , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Linhagem Celular , Escherichia coli/efeitos dos fármacos , Fibroblastos/microbiologia , Humanos , Queratinócitos/microbiologia , Látex/química , Camundongos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Borracha/química , Cicatrização
10.
Nanoscale ; 11(21): 10472-10485, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31112150

RESUMO

Multidrug-resistant pathogens are prevalent in chronic wounds. There is an urgent need to develop novel antimicrobials and formulation strategies that can overcome antibiotic resistance and provide a safe alternative to traditional antibiotics. This work aimed to develop a novel nanocarrier for two cationic antibiotics, tetracycline hydrochloride and lincomycin hydrochloride which can potentially overcome antibiotic resistance. In this study, we report the use of surface functionalised polyacrylic copolymer nanogels as carriers for cationic antibiotics. These nanogels can encapsulate small cationic antimicrobial molecules and act as a drug delivery system. They were further functionalised with a biocompatible cationic polyelectrolyte, bPEI, to increase their affinity towards the negatively charged bacterial cell walls. These bPEI-coated nanocarrier-encapsulated antibiotics were assessed against a range of wound isolated pathogens, which had been shown through antimicrobial susceptibility testing (AST) to be resistant to tetracycline and lincomycin. Our data reveal that bPEI-coated nanogels with encapsulated tetracycline or lincomycin displayed increased antimicrobial performance against selected wound-derived bacteria, including strains highly resistant to the free antibiotic in solution. Additionally, our nanocarrier-based antibiotics showed no detectable cytotoxic effect against human keratinocytes. We attribute the increase in the antimicrobial activity of the cationically functionalised antibiotic-loaded nanogel carriers to specific electrostatic adhesion to the microbial cell wall delivering a higher local antibiotic concentration, confirmed by scanning electron microscopy. Such a nanotechnology based approach may enhance the effectiveness of a wide variety of existing antibiotics, offering a potentially new mechanism to overcome antibiotic resistance.


Assuntos
Antibacterianos , Portadores de Fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Lincomicina , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Tetraciclina , Antibacterianos/química , Antibacterianos/farmacologia , Linhagem Celular , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Humanos , Queratinócitos/microbiologia , Lincomicina/química , Lincomicina/farmacologia , Testes de Sensibilidade Microbiana , Tetraciclina/química , Tetraciclina/farmacologia , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia
11.
Microbiologyopen ; 8(9): e00841, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30950214

RESUMO

Cutibacterium acnes, former Proprionibacterium acnes, is a heterogeneous species including acneic bacteria such as the RT4 strain, and commensal bacteria such as the RT6 strain. These strains have been characterized by metagenomic analysis but their physiology was not investigated until now. Bacteria were grown in different media, brain heart infusion medium (BHI), reinforced clostridial medium (RCM), and in sebum like medium (SLM) specifically designed to reproduce the lipid rich environment of the sebaceous gland. Whereas the RT4 acneic strain showed maximal growth in SLM and lower growth in RCM and BHI, the RT6 non acneic strain was growing preferentially in RCM and marginally in SLM. These differences were correlated with the lipophilic surface of the RT4 strain and to the more polar surface of the RT6 strain. Both strains also showed marked differences in biofilm formation activity which was maximal for the RT4 strain in BHI and for the RT6 strain in SLM. However, cytotoxicity of both strains on HaCaT keratinocytes remained identical and limited. The RT4 acneic strain showed higher inflammatory potential than the RT6 non acneic strain, but the growth medium was without significant influence. Both bacteria were also capable to stimulate ß-defensine 2 secretion by keratinocytes but no influence of the bacterial growth conditions was observed. Comparative proteomics analysis was performed by nano LC-MS/MS and revealed that whereas the RT4 strain only expressed triacylglycerol lipase, the principal C. acnes virulence factor, when it was grown in SLM, the RT6 strain expressed another virulence factor, the CAMP factor, exclusively when it was grown in BHI and RCM. This study demonstrates the key influence of growth conditions on virulence expression by C. acnesand suggest that acneic and non acneic strains are related to different environmental niches.


Assuntos
Adaptação Fisiológica , Propionibacterium acnes/crescimento & desenvolvimento , Propionibacterium acnes/metabolismo , Sebo/microbiologia , Proteínas de Bactérias/análise , Linhagem Celular , Meios de Cultura/química , Humanos , Queratinócitos/imunologia , Queratinócitos/microbiologia , Propionibacterium acnes/química , Proteoma/análise , Fatores de Virulência/análise
12.
Mycoses ; 62(7): 588-596, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30908750

RESUMO

The prevalence of atopic dermatitis (AD) has been increasing. Whereas AD symptoms are obvious and easy to recognise, the etiopathogenesis remains not fully elucidated. Recently, the role of microorganisms and their impact on the immunology of AD have been discussed. In this review, we summarise a possible role of Malassezia in the development and persistence of eczema in patients with atopic eczema/dermatitis syndrome. A high proportion of AD patients present with a positive reaction to Malassezia allergens. Several possible pathogenic mechanisms enable Malassezia to trigger the development of AD. Malassezia spp. may release more allergens in a less acidic (pH <6), typical for AD, environment. The similarity between fungal thioredoxin and human proteins causes T-cell cross-reactivity. TLR-mediated mechanisms are involved in host response against Malassezia spp. An interaction between Malassezia spp. and keratinocytes alters the profile of cytokine release, and what is more, yeast cells can survive when absorbed by keratinocytes. Dendritic cells of AD patients induced by Malassezia are less susceptible to lysis mediated by NK cells which exerts a pro-inflammatory effect. Despite the evidence that Malassezia spp. contribute to the development of AD, the pathogenetic mechanisms and relationship between Malassezia and immune defense remain partly unexplained and require further research.


Assuntos
Dermatite Atópica/patologia , Dermatite Atópica/fisiopatologia , Dermatomicoses/complicações , Dermatomicoses/patologia , Interações Hospedeiro-Patógeno , Malassezia/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Dermatite Atópica/epidemiologia , Dermatomicoses/epidemiologia , Humanos , Queratinócitos/imunologia , Queratinócitos/microbiologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/microbiologia , Prevalência
13.
Med Mycol J ; 60(1): 5-10, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30814468

RESUMO

Candida species are opportunistic fungal pathogens that cause superficial or invasive infections. Recently, the incidence of infection by non-Candida albicans species, especially Candida glabrata, has increased. In this study, we analyzed the adhesion and cytotoxicity of various Candida spp. that are part of the normal human microbiota. C. albicans adheres well to cell culture plates and to cultured cells. C. glabrata selectively adheres to epithelial cells rather than to cell culture plates. Candida parapsilosis insufficiently adheres to confluent monolayers of human lung epithelial A549 and keratinocyte HaCaT cells. We then analyzed the cytotoxicity of C. albicans and C. glabrata, which adhered well to epithelial cells. C. glabrata has been found to cause more damage to A549 cells than to HaCaT cells, suggesting that resident Candida spp. have distinct cytotoxic effects in different tissues. It is important to clarify the properties of Candida spp. as there is evidence that normal microbiota can cause infections. Our data suggest that it is necessary to use appropriate cell lines for characterizing the adherence and cytotoxicity of Candida spp.


Assuntos
Células A549/microbiologia , Células A549/patologia , Candida albicans/patogenicidade , Candida glabrata/patogenicidade , Queratinócitos/microbiologia , Queratinócitos/patologia , Células Cultivadas , Humanos
14.
J Periodontal Res ; 54(4): 396-404, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30793777

RESUMO

AIM: To explore the role of keratinocyte myeloid differentiation primary response 88 (MyD88) expression in the adhesion of Porphyromonas gingivalis to the cells and its subsequent invasion and intracellular survival. MATERIALS AND METHODS: Primary mouse keratinocytes from wild-type (WT) or Myd88-/- mice were infected with P gingivalis alone or co-infected with Fusobacterium nucleatum. Bacterial adhesion and invasion were measured using fluorescent microscopy and flow cytometry, and intracellular survival in keratinocytes was quantified by an antibiotic protection assay. Keratinocyte expression of antimicrobial peptides was measured by real-time PCR. RESULTS: In the absence of MyD88, P gingivalis adherence, invasion, and intracellular survival were enhanced compared with WT keratinocytes. The presence of F nucleatum during infection increased the adhesion of P gingivalis to WT keratinocytes but reduced the adhesion to Myd88-/- keratinocytes. Fusobacterium nucleatum improved mildly the invasion and survival of P gingivalis in both cell types. Baseline expression of beta-defensin 2, 3, 4 and RegIII-γ was elevated in Myd88-/- keratinocytes compared to WT cells; however, following infection beta-defensin expression was strongly induced in WT cells but decreased dramatically in the MyD88 deficient cells. CONCLUSION: In the absence of MyD88 expression, P gingivalis adhesion to keratinocytes is improved, and invasion and intracellular survival are increased. Furthermore, keratinocyte infection by P gingivalis induces antimicrobial peptide expression in a MyD88-dependent manner. Thus, MyD88 plays a key role in the interaction between P gingivalis and keratinocytes.


Assuntos
Infecções por Bacteroidaceae/imunologia , Queratinócitos/microbiologia , Fator 88 de Diferenciação Mieloide/imunologia , Porphyromonas gingivalis , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Aderência Bacteriana , Fusobacterium nucleatum , Queratinócitos/imunologia , Camundongos , Camundongos Knockout
15.
J Nanobiotechnology ; 17(1): 22, 2019 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-30711007

RESUMO

BACKGROUND: Human plasma gelsolin (pGSN) is a multifunctional actin-binding protein involved in a variety of biological processes, including neutralization of pro-inflammatory molecules such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and modulation of host inflammatory response. It was found that PBP10, a synthetic rhodamine B-conjugated peptide, based on the phosphoinositide-binding site of pGSN, exerts bactericidal activity against Gram-positive and Gram-negative bacteria, interacts specifically with LPS and LTA, and limits microbial-induced inflammatory effects. The therapeutic efficiency of PBP10 when immobilized on the surface of iron oxide-based magnetic nanoparticles was not evaluated, to date. RESULTS: Using the human keratinocyte cell line HaCaT stimulated by bacterially-derived LPS and LTA as an in vitro model of bacterial infection, we examined the anti-inflammatory effects of nanosystems consisting of iron oxide-based magnetic nanoparticles with aminosilane (MNP@NH2) or gold shells (MNP@Au) functionalized by a set of peptides, derived from the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding site of the human plasma protein gelsolin, which also binds LPS and LTA. Our results indicate that these nanosystems can kill both Gram-positive and Gram-negative bacteria and limit the production of inflammatory mediators, including nitric oxide (NO), reactive oxygen species (ROS), and interleukin-8 (IL-8) in the response to heat-killed microbes or extracted bacterial cell wall components. The nanoparticles possess the potential to improve therapeutic efficacy and are characterized by lower toxicity and improved hemocompatibility when compared to free peptides. Atomic force microscopy (AFM) showed that these PBP10-based nanosystems prevented changes in nanomechanical properties of cells that were otherwise stimulated by LPS. CONCLUSIONS: Neutralization of endotoxemia-mediated cellular effects by gelsolin-derived peptides and PBP10-containing nanosystems might be considered as potent therapeutic agents in the improved therapy of bacterial infections and microbial-induced inflammation.


Assuntos
Antibacterianos/farmacologia , Gelsolina/química , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Nanopartículas de Magnetita/química , Fragmentos de Peptídeos/química , Antibacterianos/química , Bactérias/efeitos dos fármacos , Sítios de Ligação , Gelsolina/farmacologia , Humanos , Mediadores da Inflamação/metabolismo , Queratinócitos/microbiologia , Lipopolissacarídeos/química , Lipopolissacarídeos/toxicidade , Fragmentos de Peptídeos/farmacologia , Peptídeos/química , Dermatopatias Bacterianas/imunologia , Dermatopatias Bacterianas/microbiologia , Ácidos Teicoicos/química , Ácidos Teicoicos/toxicidade
17.
Molecules ; 24(2)2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30634461

RESUMO

Acne is associated with hyperkeratosis, elevated levels of skin sebum and growth of Propionibacterium acnes (P. acnes) and Staphylococcus aureus (S. aureus). Furthermore, P. acnes promotes inflammation by inducing IL-6 production and oxidative stress. The aim of this study was to assess the antioxidant, anti-inflammatory and antibacterial potential of a hop-CO2-extract with 50% humulone and lupulone. The susceptibility of P. acnes and S. aureus to the hop extract was tested by using the broth microdilution technique. The minimal inhibitory concentrations (MIC) for P. acnes and S. aureus were 3.1 and 9.4 µg/mL, respectively. In addition, the hop extract showed an antioxidative effect with a half maximal inhibitory concentration (IC50) of 29.43 µg/mL as well as additional anti-inflammatory effects by reducing the IL-6 expression (IC50: 0.8 µg/mL). In addition, a gel formulation with 0.3% hop extract (w/w) had antibacterial activity against P. acnes and S. aureus (inhibition zone value: 5.5 mm and 3 mm, respectively) which was significantly superior to the placebo gel. The positive control (a gel with the antibiotic clindamycin) showed an inhibition zone of 9 mm. Due to its antioxidant, anti-inflammatory and antibacterial effects hop extract might be a treatment option for acne-prone skin.


Assuntos
Antibacterianos/farmacologia , Antioxidantes/farmacologia , Humulus/química , Extratos Vegetais/farmacologia , Propionibacterium acnes/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Acne Vulgar/tratamento farmacológico , Acne Vulgar/metabolismo , Acne Vulgar/microbiologia , Antibacterianos/química , Antioxidantes/química , Células Cultivadas , Humanos , Interleucina-6/metabolismo , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/microbiologia , Testes de Sensibilidade Microbiana , Extratos Vegetais/química , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/metabolismo
18.
J Antimicrob Chemother ; 74(2): 365-372, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388236

RESUMO

Background: Sodium hypochlorite (NaClO, SHC)/hypochlorous acid (HClO, HCA) wound irrigation solutions have experienced a renaissance in the prevention and treatment of low-level wound infections. They are attributed with lower cytotoxicity and have therefore gained increasing attention in daily clinical practice. Objectives: To determine the cytotoxicity and antimicrobial efficacy of six NaClO/HClO wound irrigation solutions. Methods: For cytotoxicity evaluation (based on DIN EN 10993-5), human keratinocytes (HaCaT) and human skin fibroblasts (BJ) were used. Staphylococcus aureus and Pseudomonas aeruginosa were used for antimicrobial efficacy evaluation (based on DIN EN 13727). Solutions were evaluated after 1, 5 and 15 min of exposure. Additionally, physicochemical properties (pH and oxidation-reduction potential values) were investigated. Results: Efficacy and cytotoxicity varied significantly between solutions. Generally, increasing antimicrobial activity was associated with decreasing cell viability. Furthermore, a concentration- and time-dependent impact on pathogens and cells was observed: cytotoxic and antimicrobial activity increased with rising NaClO/HClO solution concentrations and extended exposure times. Based on these in vitro evaluations, the following ranking (lowest to highest microbicidal effect and cytotoxic impact) was found: Microdacyn60® (SHC/HCA-M) < Granudacyn® (SHC/HCA-G) < Veriforte™ (SHC/HCA-V) < KerraSol™ (SHC-K) < Lavanox® (SHC-L) ≪ ActiMaris®forte (SHC/SM-A). Conclusions: The presented results indicate that microbicidal effects are almost always associated with certain negative side effects on cell proliferation. Efficacy and biocompatibility of NaClO/HClO solutions depend on their specific formulation and physicochemical properties. The investigations also underline the necessity for exact product- and application-specific efficacy profiles.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Desinfetantes/farmacologia , Ácido Hipocloroso/farmacologia , Hipoclorito de Sódio/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/microbiologia , Humanos , Concentração de Íons de Hidrogênio , Queratinócitos/efeitos dos fármacos , Queratinócitos/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Irrigação Terapêutica , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia
19.
FASEB J ; 33(2): 2144-2155, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30260708

RESUMO

Decellularized matrices of biologic tissue have performed well as wound care dressings. Extracellular matrix-based dressings are subject to rapid degradation by excessive protease activity at the wound environment. Stabilized, acellular, equine pericardial collagen matrix (sPCM) wound care dressing is flexible cross-linked proteolytic enzyme degradation resistant. sPCM was structurally characterized utilizing scanning electron and atomic force microscopy. In murine excisional wounds, sPCM was effective in mounting an acute inflammatory response. Postwound inflammation resolved rapidly, as indicated by elevated levels of IL-10, arginase-1, and VEGF, and lowering of IL-1ß and TNF-α. sPCM induced antimicrobial proteins S100A9 and ß-defensin-1 in keratinocytes. Adherence of Pseudomonas aeruginosa and Staphylococcus aureus on sPCM pre-exposed to host immune cells in vivo was inhibited. Excisional wounds dressed with sPCM showed complete closure at d 14, while control wounds remained open. sPCM accelerated wound re-epithelialization. sPCM not only accelerated wound closure but also improved the quality of healing by increased collagen deposition and maturation. Thus, sPCM is capable of presenting scaffold functionality during the course of wound healing. In addition to inducing endogenous antimicrobial defense systems, the dressing itself has properties that minimize biofilm formation. It mounts robust inflammation, a process that rapidly resolves, making way for wound healing to advance.-El Masry, M. S., Chaffee, S., Das Ghatak, P., Mathew-Steiner, S. S., Das, A., Higuita-Castro, N., Roy, S., Anani, R. A., Sen, C. K. Stabilized collagen matrix dressing improves wound macrophage function and epithelialization.


Assuntos
Bandagens , Colágeno/farmacologia , Matriz Extracelular/metabolismo , Inflamação/prevenção & controle , Queratinócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Reepitelização , Cicatrização/efeitos dos fármacos , Animais , Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Células Cultivadas , Modelos Animais de Doenças , Cavalos , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
20.
Acta Derm Venereol ; 99(2): 181-187, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30328471

RESUMO

Staphylococcus epidermidis is an abundant skin commensal capable of activating cutaneous defense responses, such as induction of cytokines and antimicrobial peptides. To permanently colonize human skin and prevent inflammation S. epidermidis needs to control the induction of host defense mediators. We report here that S. epidermidis induces expression of the host regulator protein A20 in human keratinocytes, thereby controlling expression and release of interleukin-1 beta. siRNA-mediated knockdown of A20 expression strongly enhanced the induction of interleukin-1 beta gene expression and protein release in keratinocytes stimulated with S. epidermidis. Furthermore, siRNA-mediated knockdown of A20 resulted in enhanced gene expression and secretion of the antimicrobial peptide human beta-defensin-2 in keratinocytes facing S. epidermidis. Mechanistically, A20 negatively controlled S. epidermidis-induced activation of the transcription factor NF-kappaB. Together, these data indicate that S. epidermidis exploits A20 to attenuate cutaneous defense responses, which may help S. epidermidis to persist on human skin.


Assuntos
Interleucina-1beta/metabolismo , Queratinócitos/microbiologia , Pele/microbiologia , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus epidermidis/patogenicidade , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , beta-Defensinas/metabolismo , Células Cultivadas , Interações Hospedeiro-Patógeno , Humanos , Interleucina-1beta/genética , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Pele/metabolismo , Infecções Cutâneas Estafilocócicas/genética , Infecções Cutâneas Estafilocócicas/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Regulação para Cima , beta-Defensinas/genética
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