RESUMO
In addition to comprising monomers of nucleic acids, nucleotides have signaling functions and act as second messengers in both prokaryotic and eukaryotic cells. The most common example is cyclic AMP (cAMP). Nucleotide signaling is a focus of great interest in bacteria. Cyclic di-AMP (c-di-AMP), cAMP, and cyclic di-GMP (c-di-GMP) participate in biological events such as bacterial growth, biofilm formation, sporulation, cell differentiation, motility, and virulence. Moreover, the cyclic-di-nucleotides (c-di-nucleotides) produced in pathogenic intracellular bacteria can affect eukaryotic host cells to allow for infection. On the other hand, non-cyclic nucleotide molecules pppGpp and ppGpp are alarmones involved in regulating the bacterial response to nutritional stress; they are also considered second messengers. These second messengers can potentially be used as therapeutic agents because of their immunological functions on eukaryotic cells. In this review, the role of c-di-nucleotides and cAMP as second messengers in different bacterial processes is addressed.
Assuntos
GMP Cíclico , Sistemas do Segundo Mensageiro , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia , Bactérias , AMP Cíclico , Nucleotídeos Cíclicos , Proteínas de BactériasRESUMO
Extracellular vesicles (EVs) are evaginations of the cytoplasmic membrane, containing nucleic acids, proteins, lipids, enzymes, and toxins. EVs participate in various bacterial physiological processes. Staphylococcus epidermidis interacts and communicates with the host skin. S. epidermidis' EVs may have an essential role in this communication mechanism, modulating the immunological environment. This work aimed to evaluate if S. epidermidis' EVs can modulate cytokine production by keratinocytes in vitro and in vivo using the imiquimod-induced psoriasis murine model. S. epidermidis' EVs were obtained from a commensal strain (ATC12228EVs) and a clinical isolated strain (983EVs). EVs from both origins induced IL-6 expression in HaCaT keratinocyte cultures; nevertheless, 983EVs promoted a higher expression of the pro-inflammatory cytokines VEGF-A, LL37, IL-8, and IL-17F than ATCC12228EVs. Moreover, in vivo imiquimod-induced psoriatic skin treated with ATCC12228EVs reduced the characteristic psoriatic skin features, such as acanthosis and cellular infiltrate, as well as VEGF-A, IL-6, KC, IL-23, IL-17F, IL-36γ, and IL-36R expression in a more efficient manner than 983EVs; however, in contrast, Foxp3 expression did not significantly change, and IL-36 receptor antagonist (IL-36Ra) was found to be increased. Our findings showed a distinctive immunological profile induction that is dependent on the clinical or commensal EV origin in a mice model of skin-like psoriasis. Characteristically, proteomics analysis showed differences in the EVs protein content, dependent on origin of the isolated EVs. Specifically, in ATCC12228EVs, we found the proteins glutamate dehydrogenase, ornithine carbamoyltransferase, arginine deiminase, carbamate kinase, catalase, superoxide dismutase, phenol-soluble ß1/ß2 modulin, and polyglycerol phosphate α-glucosyltransferase, which could be involved in the reduction of lesions in the murine imiquimod-induced psoriasis skin. Our results show that the commensal ATCC12228EVs have a greater protective/attenuating effect on the murine imiquimod-induced psoriasis by inducing IL-36Ra expression in comparison with EVs from a clinical isolate of S. epidermidis.
Assuntos
Vesículas Extracelulares/metabolismo , Psoríase/terapia , Staphylococcus epidermidis/metabolismo , Animais , Antígenos Ly/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Vesículas Extracelulares/química , Vesículas Extracelulares/transplante , Humanos , Imiquimode/toxicidade , Interleucina-1/antagonistas & inibidores , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Infiltração de Neutrófilos , Psoríase/induzido quimicamente , Psoríase/patologia , Pele/metabolismo , Pele/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
During the progression of psoriatic lesions, abundant cellular infiltration of myeloid cells, such as macrophages and activated dendritic cells, occurs in the skin and the infiltrating cells interact with naive lymphoid cells to generate a T helper (Th)1 and Th17 environment. Therapies to treat psoriasis include phototherapy, nonsteroidal and steroidal drugs, as well as antibodies to block tumor necrosis factorα, interleukin (IL)17A and IL12/IL23, which all focus on decreasing the proinflammatory hallmark of psoriasis. The present study obtained the heptapeptide HP3 derived from phage display technology that blocks mononuclear cell adhesion to endothelial cells and inhibits transendothelial migration in vitro. The activity of the heptapeptide in a murine model of psoriasis was also assessed, which indicated that early administration inhibited the development of psoriatic lesions. Therefore, the results suggested that HP3 may serve as a potential therapeutic target for psoriasis.
Assuntos
Células Endoteliais/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Oligopeptídeos/uso terapêutico , Psoríase/tratamento farmacológico , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/patologia , Feminino , Humanos , Imiquimode , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Psoríase/induzido quimicamente , Psoríase/patologiaRESUMO
IL-36 cytokines (the agonists IL-36α, IL-36ß, IL-36γ, and the antagonist IL-36Ra) are expressed in the mouse uterus and associated with maternal immune response during pregnancy. Here, we characterize the expression of IL-36 members in human primary trophoblast cells (PTC) and trophoblastic cell lines (HTR-8/SVneo and JEG-3) and upon treatment with bacterial and viral components. Effects of recombinant IL-36 on the migration capacity of trophoblastic cells, their ability to interact with endothelial cells and the induction of angiogenic factors and miRNAs (angiomiRNAs) were examined. Constitutive protein expression of IL-36 (α, ß, and γ) and their receptor (IL-36R) was found in all cell types. In PTC, transcripts for all IL-36 subtypes were found, whereas in trophoblastic cell lines only for IL36G and IL36RN. A synthetic analog of double-stranded RNA (poly I:C) and lipopolysaccharide (LPS) induced the expression of IL-36 members in a cell-specific and time-dependent manner. In HTR-8/SVneo cells, IL-36 cytokines increased cell migration and their capacity to interact with endothelial cells. VEGFA and PGF mRNA and protein, as well as the angiomiRNAs miR-146a-3p and miR-141-5p were upregulated as IL-36 response in PTC and HTR-8/SVneo cells. In conclusion, IL-36 cytokines are modulated by microbial components and regulate trophoblast migration and interaction with endothelial cells. Therefore, a fundamental role of these cytokines in the placentation process and in response to infections may be expected.
Assuntos
Regulação da Expressão Gênica/genética , Interleucina-1/genética , Neovascularização Fisiológica/genética , Trofoblastos/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Neovascularização Fisiológica/fisiologia , Poli I-C/farmacologia , Prostaglandinas F/genética , Prostaglandinas F/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trofoblastos/citologia , Trofoblastos/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Staphylococci produce a large number of extracellular proteases, some of which are considered as potential virulence factors. Staphylococcus epidermidis is a causative agent of nosocomial infections in medical devices by the formation of biofilms. It has been proposed that proteases contribute to the different stages of biofilm formation. S. epidermidis secretes a small number of extracellular proteases, such as serine protease Esp, cysteine protease EcpA, and metalloprotease SepA that have a relatively low substrate specificity. Recent findings indicate a significant contribution of extracellular proteases in biofilm formation through the proteolytic inactivation of adhesion molecules. The objective of this work is to provide an overview of the current knowledge of S. epidermidis' extracellular proteases during pathogenicity, especially in the different stages of biofilm formation.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Cisteína Proteases/metabolismo , Metaloendopeptidases/metabolismo , Serina Proteases/metabolismo , Staphylococcus epidermidis/enzimologia , Moléculas de Adesão Celular/metabolismo , Infecção Hospitalar/microbiologia , Infecção Hospitalar/patologia , Humanos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus epidermidis/metabolismo , Staphylococcus epidermidis/patogenicidade , Fatores de Virulência/metabolismoRESUMO
PURPOSE: Staphylococcus epidermidis ATCC12228 lipoteichoic acid (LTA) inhibits TNFα production from keratinocytes that are activated with poly I:C. However, this effect has not been proven in clinical or commensal isolates. METHODOLOGY: The <10 kDa fractions of S. epidermidis isolates from ocular infections (n=56), healthy skin (n=35) and healthy conjunctiva (n=32) were obtained. TNFα production was determined by elisa in HaCaT keratinocytes stimulated with poly I:C and with the <10 kDa fractions. LTA in the cytoplasmic membrane and in the <10 kDa fractions of the isolates was determined during bacterial growth by flow cytometry, Western blot and electrospray ionization mass spectrometry. The expression levels of ugtP, ltaA and ltaS were evaluated. RESULTS: Two populations of isolates were found: a population that inhibited TNFα production (TNFα-inhibitor isolates) and a population that did not inhibit it (TNFα non-inhibitor isolates). The cells from the TNFα-inhibitor isolates had less LTA in the cytoplasmic membrane compared to the cells from the TNFα non-inhibitor isolates (P<0.05). Similarly, LTA was detected in the supernatants of TNFα-inhibitor isolates, and it was absent in TNFα non-inhibitor isolates. High expression levels of the ugtP and ltaA genes in the 1850I (TNFα-inhibitor isolate) and 37HS (TNFα non-inhibitor isolate) isolates were found during bacterial growth. However, the ltaS gene had a low expression level (P<0.05) in the 37HS isolate. CONCLUSION: The TNFα-inhibitor isolates release LTA due to high expression of the LTA synthesis genes. By contrast, TNFα non-inhibitor isolates do not release LTA due to low expression level of the ltaS gene.
Assuntos
Exocitose , Expressão Gênica , Genes Bacterianos , Lipopolissacarídeos/metabolismo , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/metabolismo , Ácidos Teicoicos/metabolismo , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Queratinócitos/imunologia , Queratinócitos/microbiologia , Espectrometria de Massas , Staphylococcus epidermidis/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 µg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR 95ΔG mutation. Isolates carrying the 95ΔG mutation had higher levels of norA expression compared with those that did not. To corroborate that the 95ΔG mutation is involved in HEA, a strain adapted to EtBr was obtained in vitro. This strain also presented the 95ΔG mutation and had a high level of norA expression and EF, indicating that the 95ΔG mutation is important for the HEA phenotype. The 95ΔG mutation produces a different structure in the Shine-Dalgarno region, which may promote better translation of norA mRNA. To our knowledge, this is the first report to demonstrate the participation of the 5'UTR 95ΔG mutation of the norA gene in the HEA phenotype of S. epidermidis isolates. Here, we propose that the efflux of EtBr is caused by an increment in the transcription and/or translation of the norA gene.
Assuntos
Regiões 5' não Traduzidas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Deleção de Sequência , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/metabolismo , Antibacterianos/farmacologia , Biofilmes , Resistencia a Medicamentos Antineoplásicos , Expressão Gênica , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/patogenicidadeRESUMO
The dynamic regulation of NF-κB activity in the uterus maintains a favorable environment of cytokines necessary to prepare for pregnancy throughout the estrous cycle. Recently, the mechanisms that directly regulate the NF-κB transcriptional activity in different tissues are of growing interest. IκBNS and BCL-3 are negative nuclear regulators of NF-κB activity that regulate IL-6 and TNF-α transcription, respectively. Both cytokines have been described as important factors in the remodeling of uterus for blastocyst implantation. In this work we analyzed in ICR mice the mRNA expression and protein production profile of IL-6, TNF-α, and their correspondent negative transcription regulators IκBNS or BCL-3 using real-time PCR, western blot and immunochemistry. We found that the expression of TNF-α and IL-6 was oscillatory along the estrous cycle, and its low expression coincided with the presence of BCL-3 and IκBNS, and vice versa, when the presence of the regulators was subtle, the expression of TNF-α and IL-6 was exacerbated. When we compared the production of TNF-α and IL-6 in the different estrous stages relating with diestrus we found that at estrus there is an important increase of the cytokines (p<0.05) decreasing at metestrus to reach the basal expression at diestrus. In the immunochemistry analysis we found that at diestrus BCL-3 is distributed all over the tissue with a barely detected TNF-α, but on the contrary, at estrus the expression of BCL-3 is not detected with TNF-α clearly observable along the tissue; the same phenomenon occur in the analysis of IκBNS and IL-6. With that evidence we suggest that the expression of TNF-α and IL-6 might be regulated through NF-κB nuclear regulators BCL-3 and IκBNS in the uterus of mice as has been demonstrated in other systems.
Assuntos
Interleucina-6/imunologia , NF-kappa B/imunologia , Proteínas/imunologia , Proteínas Proto-Oncogênicas/imunologia , Fatores de Transcrição/imunologia , Fator de Necrose Tumoral alfa/imunologia , Útero/metabolismo , Animais , Proteína 3 do Linfoma de Células B , Ciclo Estral/genética , Ciclo Estral/imunologia , Feminino , Regulação da Expressão Gênica , Interleucina-6/genética , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/genética , Gravidez , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica , Fator de Necrose Tumoral alfa/genética , Útero/imunologiaRESUMO
Psoriasis is a chronic inflammatory skin disease where the altered regulation in angiogenesis, inflammation, and proliferation of keratinocytes are the possible causes of the disease, and the transcription factor "hypoxia-inducible factor 1-alpha" (HIF-1α) is involved in the homeostasis of these three biological phenomena. In this review, the role of HIF-1α in the cross talk between the cytokines and cells of the immunological system involved in the pathogenesis of psoriasis is discussed.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Psoríase/etiologia , Animais , Polaridade Celular , Proliferação de Células , Citocinas/fisiologia , Modelos Animais de Doenças , Humanos , Interleucina-1/fisiologia , Camundongos , Neovascularização Fisiológica , Psoríase/imunologia , Células Th17/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Pregnancy is a complex process where several physiological pathways interact. The down-regulated inflammatory response and the abundance of anti-inflammatory molecules during gestation may explain the acceptance of the fetus and the lack of immune response against it, even though it is a foreign tissue for the mother. NF-κB is a key regulator of the transcription of inflammatory genes, such as IL-8, IL-1ß, TNF-α, or IL-6. Increased NF-κB activity that leads to the production of proinflammatory cytokines may induce obstetric disorders, such as preterm birth or abortion. Low activity of this transcription factor is associated with the beneficial anti-inflammatory environment during fetus development until delivery. Galectin-1 (Gal-1) is a lectin-type glycan-binding protein that is able to down-regulate inflammation. It has been shown that Gal-1 is abundantly expressed at the feto-maternal interface in humans, where it promotes maternal immune tolerance to the fetal semi-allograft. Gal-1 tolerance-promoting mechanisms have been established for adaptive immune cells, such as T cells and dendritic cells. However, the role of this lectin has not been established in non-immune cells at the feto-maternal interface. Here, we determined that Gal-1 is able to block the stimulating effect of LPS on IL-6 in human decidua cells. Our results show that Gal-1 acts by inhibiting the stimulation of the LPS-induced IκBζ expression, an NF-κB regulator involved in IL-6 gene transcription.
Assuntos
Decídua/imunologia , Galectina 1/imunologia , Proteínas I-kappa B/imunologia , Interleucina-6/imunologia , Lipopolissacarídeos/toxicidade , Placenta/imunologia , Proteínas Proto-Oncogênicas/imunologia , Adulto , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Gravidez , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologiaRESUMO
BACKGROUND: Keratitis caused by Staphylococcus aureus often leads to Vascular Endothelial Growth Factor (VEGF)-dependent neovascularization, but contribution of peptidoglycan (PGN), muramyl dipeptide (MDP) and lipoteichoic acid (LTA) from S. aureus to VEGF-dependent neovascularization has not been well-studied. This work was focused on the analysis of S. aureus cell wall components in the production of VEGF family members (VEGF-A, VEGF-B, VEGF-C and VEGF-D) in ocular limbal fibroblasts. METHODS: Primary culture of human limbal fibroblasts (PCHLFs) were stimulated with PGN, MDP, and LTA, and VEGF family; toll-like receptor 2 (TLR2), nucleotide-binding oligomerization domain 1 (NOD1), and NOD2 expression were determined by RT-PCR. Anti-TLR2 antibody, epidermal growth factor receptor (EGFR) signaling inhibitors (AG1478 and PD98059), and NFκB activation were used to analyze VEGF-A by ELISA. TLR2 and NOD1 expression were analyzed by flow cytometry. RESULTS: The stimulation of PCHLFs with PGN and MDP increased the levels of VEGF-A expression (mRNA and protein) in a time-dependent and dose-dependent manner. VEGF-B, VEGF-C and VEGF-D were expressed constitutively, and no further induction was observed in stimulated PCHLFs. LTA did not increase the expression levels of the VEGF family. TLR2 mRNA and protein were increased only when PCHLFs were stimulated with PGN. Treatment with an anti-TLR2 antibody blocked the interaction of PGN with the receptor, inhibiting VEGF-A over-expression; the presence of anti-TLR2 antibodies did not affect the over-production of VEGF-A after MDP treatment. PCHLFs stimulated with PGN and MDP, but not with LTA, activated NFκB. MDP stimulated the production of NOD1 and NOD2 mRNAs in a time-dependent and dose-dependent manner, and NOD2 protein was only increased by MDP. Treatment of PCHLFs with AG1478 and PD98059 inhibitors prior to stimulation with MDP resulted in the inhibition of VEGF-A over-production, compared with PCHLFs stimulated with MDP alone. CONCLUSIONS: Taken together, these results suggest that limbal fibroblasts produce VEGF-A through PGN-TLR2-NFκB and MDP-NOD2-EGFR.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Proteínas do Olho/metabolismo , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Peptidoglicano/farmacologia , Staphylococcus aureus/química , Fator A de Crescimento do Endotélio Vascular/genética , Acetilmuramil-Alanil-Isoglutamina/química , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Limbo da Córnea/citologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Peptidoglicano/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Receptor 2 Toll-Like/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: In hypoxic tumoral tissues, vascular endothelial growth factor (VEGF) expression is positively regulated by histone deacetylase 1 (HDAC1) and negatively regulated by the tumour suppressor protein von Hippel-Lindau (VHL) via transforming growth factor-alpha (HIF-1alpha). It has been reported that VEGF, HDAC1 and LL-37, but not VHL, are over-expressed in psoriatic skin. Although HIF-1alpha is constitutively expressed in normal keratinocytes, it is not known if HDAC1 and VHL can regulate VEGF production in these cells. METHODS: The participation of HDAC1 and VHL in the regulation of VEGF expression in HDAC-, VHL- and LL-37-transfected HaCaT cells, and in HaCaT cells treated with HDAC1 inhibitors, was studied. RESULTS: The production of VEGF was increased in HDAC1- and LL-37-transfected HaCaT cells and maintained in VHL-transfected cells under hypoxic conditions; meanwhile, VEGF production decreased in HaCaT cells treated with TSA, in cells transfected with HDAC1-siRNA, in cells co-transfected with HIF-1alpha-siRNA and pHDAC-1 and in VHL-transfected HaCaT cells. The levels of cytoplasmic HIF-1alpha were high in pLL37-transfected cells and low in pVHL- and pHDAC1-transfected cells; however, HIF-1alpha was detected in the nucleus of the HDAC1-transfected cells. The expression of VEGF was high in cells co-transfected with pHDAC1- and pLL-37, and the expression decreased when pVHL was present. CONCLUSIONS: These data demonstrate that HDAC1, LL-37 and VHL can modulate the production of VEGF via HIF-1alpha in HaCaT cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Peptídeos Catiônicos Antimicrobianos , Catelicidinas/biossíntese , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Hipóxia , Queratinócitos/citologia , Microscopia de Fluorescência/métodos , Psoríase/metabolismo , Pele/metabolismo , TransfecçãoRESUMO
Conventional microbiological culture techniques are frequently insufficient to confirm endophthalmitis clinical cases which could require urgent medical attention because it could lead to permanent vision loss. We are proposing PCR-DGGE and 16S rRNA gene libraries as an alternative to improve the detection and identification rate of bacterial species from endophthalmitis cases.
Assuntos
Humanos , Técnicas e Procedimentos Diagnósticos , Endoftalmite , Técnicas In Vitro , Infecções Estafilocócicas , Staphylococcus epidermidis/isolamento & purificação , Uveíte Supurativa , Diagnóstico , Incidência , Métodos , PacientesRESUMO
Lipopolysaccharide induces TLR-1-8 mRNAs over-expression in corneal fibroblast. Analyzing if other TLR-ligands can do the same, we found that peptidoglycan does, but not muramyldipeptide, lipoteichoic acid and polyI:C. This suggests that the recognition of lipopolysaccharide and peptidoglycan is enough to alert these cells against microorganisms through the over-expression of the majority TLRs.
Assuntos
Humanos , Córnea , Fibroblastos , Lipopolissacarídeos/análise , Peptidoglicano/análise , Infecções Estafilocócicas , Staphylococcus aureus , Métodos , MétodosRESUMO
Heat shock protein 60kDa (HSP60) could be involved in glaucoma because its expression in optical nerve, and autoantibodies to HSP60 have been found in glaucomatous patients; however, HSP60 antigenic sites recognized by glaucomatous patients are unknown. Therefore, in this work, the antibody levels in the sera of glaucoma patients, and HSP60 antigenic sites were determined. Analysis by the Hopp and Woods method with GENE 2.2 software identified the possible antigenic sites in the amino acid regions 200-213, 226-237, 278-290, 357-368 and 418-427 of Streptococcus pyogenes HSP60 (HSP60Sp). According to these, polypeptides were generated truncating the hsp60Sp gene by PCR. Antibody levels to HSP60Sp and the generated polypeptides were measured by ELISA in serum of glaucomatous patients and in healthy subjects. Glaucomatous patients showed higher antibody levels to rHSP60Sp (p=0.002) than healthy subjects. We found that the high anti-rHSP60Sp antibody levels are associated with the disease (OR=5.2; IC95% of 1.6-16.7; p<0.05). Here we report that amino acid regions 357-368 and 418-427 of HSP60Sp are the main antigenic sites, because when they are absent the level of anti-HSP60 antibodies decreased around 80%. These results suggest that the regions 357-368 and 418-427 of HSP60Sp could be associated with glaucoma.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Glaucoma/imunologia , Streptococcus pyogenes/imunologia , Sequência de Aminoácidos , Autoanticorpos/sangue , Sequência de Bases , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , Imunidade Humoral , Imunoglobulina G/sangue , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Análise de Sequência de ProteínaRESUMO
BACKGROUND: Vascular endothelial growth factor (VEGF)-transgenic mice develop psoriasiform plaques that resemble human psoriasis, demonstrating that VEGF is an important factor in the development of psoriasis. In human keratinocytes, VEGF is regulated by human cathelicidin LL37, and the expression of this peptide, as well as interleukin-13 receptor-alpha1 (IL-13Ralpha1) and VEGF, is increased in psoriatic skin. Peptidoglycan (PGN) stimulates innate immunity, inducing cytokines and antimicrobial peptides (cathelicidins), but PGN can also activate psoriatic T-helper-1 (Th1) lymphocytes. As neither the bacterial component that induces LL37 and VEGF expression nor the function of IL-13 in keratinocytes has been clarified, the role of PGN in the induction of these molecules was evaluated in this work. METHODS: HaCaT keratinocytes were treated with PGN from Staphylococcus aureus, recombinant VEGF (rVEGF), and recombinant IL-13 (rIL-13). The mRNA expression of LL37, VEGF, and IL-13 was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR), and VEGF and IL-13 production was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Keratinocytes treated with PGN show increased expression of LL37 at 24 h, VEGF at 36 h, and IL-13 at 72 h post-treatment. Anti-Toll-like receptor 2 (anti-TLR2) antibody blocked the production of these molecules. LL37 and VEGF expression in keratinocytes treated with PGN was dose dependent. rVEGF induced IL-13 production and rIL-13 induced VEGF production at 36 h. The anti-Flt-1 antibody blocked IL-13 expression. CONCLUSIONS: These results suggest that PGN from S. aureus can induce LL37 and VEGF expression in keratinocytes, and that this VEGF production can be amplified by subsequent IL-13 overproduction.
Assuntos
Interleucina-13/genética , Queratinócitos/fisiologia , Peptidoglicano/farmacologia , Staphylococcus aureus/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções Bacterianas/complicações , Infecções Bacterianas/imunologia , Catelicidinas , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Interleucina-13/metabolismo , Interleucina-13/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Psoríase/complicações , Psoríase/imunologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
PURPOSE: HLA class II, p-36 protein, heat shock protein and retinal antigens have been associated with pars planitis (PP), but their participation in the development of the disease are unknown. A search for new molecules related to PP is necessary. This work focused on the identification of peptides recognized by PP patient sera using the phage display method. METHODS: Sera of PP patients were used to isolate peptides fused to M13-phage pIII protein. The response of PP and healthy sera to peptides was determined by ELISA. PCR amplification and sequencing of peptide-encoding fragments from clones with high recognition by PP sera were used to characterize displayed peptides. RESULTS: One hundred clones were randomly selected from a phage display library after three panning rounds using serum proteins from a PP patient. The immunologic response level of 100 clones selected were determined with a major number of patients, it was found that one clone was recognized stronger in PP patients sera than in healthy sera (PP vs. healthy; P < 0.05). The peptide-encoding region of this clone was sequenced and translated. The peptide sequence corresponded to HSEAETGPP. An identical amino acid sequence to HSEAETGPP is found in the human proline-rich transmembrane protein 2 which has not been related with eye diseases. CONCLUSION: These results suggest that the peptide HSEAETGPP is associated with PP.
Assuntos
Pars Planite/sangue , Pars Planite/imunologia , Peptídeos/química , Peptídeos/imunologia , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Humanos , Biblioteca de Peptídeos , Peptídeos/genética , Reação em Cadeia da PolimeraseRESUMO
In the inflammosome complex, NALP3 or NALP1 binds to ASC and activates caspase-1 which induces IL-1beta. In murine LPS-induced ocular inflammation, the production of IL-1beta is increased. We suggest that NALP3- or NALP1-inflammasome complex can be participating in the LPS-induced ocular inflammation. In this work, eye, brain, testis, heart, spleen, and lung were obtained from C3H/HeN mice treated with LPS for 3 to 48 hours, and the expression of NALP1b, NALP3, ASC, caspase-1, IL-1beta, and IL-18 was determined. Infiltrated leukocytes producing IL-1beta in the anterior chamber were found at 12-hour posttreatment. A high upregulated expression of NALP3, ASC, caspase-1, IL-1beta, and IL-18 was found at the same time when infiltrated leukocytes were observed. NALP1b was not detected in the eye of treated mice. NALP3 was also overexpressed in heart and lung. These results suggest that NALP3-, but not NALP1-inflammosome complex, is participating in the murine LPS-induced ocular inflammation.
Assuntos
Proteínas de Transporte/imunologia , Olho/efeitos dos fármacos , Inflamação/induzido quimicamente , Lipopolissacarídeos/farmacologia , Animais , Antígenos/genética , Antígenos/imunologia , Proteínas de Transporte/genética , Caspase 1/metabolismo , Proteínas do Ovo/genética , Proteínas do Ovo/imunologia , Olho/imunologia , Olho/patologia , Imunidade Inata/fisiologia , Inflamação/imunologia , Interleucina-18/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Leucócitos/citologia , Leucócitos/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Proteína 3 que Contém Domínio de Pirina da Família NLR , Distribuição Tecidual , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: A feature of psoriasis is the rapid proliferation of keratinocytes, during which apoptosis is blocked and angiogenesis starts. It is known that tumor hypoxic cells produce histone deacetylase-1 (HDAC-1), which up-regulates hypoxia-inducible factor-1alpha (HIF-1alpha) and down-regulates von Hippel-Lindau (VHL) protein by up-regulating vascular endothelial growth factor (VEGF) expression. It has been reported recently that the porcine peptide PR39 (homologous to human LL-37) has angiogenic and antiapoptotic activity. Thus, LL-37, induced by insulin-like growth factor-1 (IGF-1), could help in the production of VEGF. PR39 also induces the expression of inhibitor of apoptosis protein-2 (IAP-2), which blocks apoptosis. The purpose of this work was to analyze whether these genes and their proteins are expressed in psoriatic biopsies. METHODS: Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) messenger RNA (mRNA) expression and immunohistochemical staining, we studied VHL, IAP-2, and related genes in skin biopsies from psoriatic patients and healthy subjects. RESULTS: An over-expression of the mRNA for HDAC-1, HIF-1alpha, LL-37, and IGF-1 in psoriatic skin, in comparison with skin from healthy subjects, was found. The antiangiogenic VHL mRNA and protein were under-expressed in psoriatic skin and highly expressed in healthy skin. The antiapoptotic IAP-2 was over-expressed in dermal endothelial cells from psoriatic skin. The pro-apoptotic Bax, Fas, and FasL mRNAs were expressed. CONCLUSIONS: These findings suggest that there could be an association of HDAC-1, HIF-1alpha, LL-37, VHL, and IAP-2 with angiogenic and apoptotic mechanisms in psoriasis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Histona Desacetilases/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Proteínas Inibidoras de Apoptose/biossíntese , Psoríase/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/biossíntese , Adulto , Idoso , Peptídeos Catiônicos Antimicrobianos/análise , Biópsia , Catelicidinas , Regulação da Expressão Gênica , Histona Desacetilase 1 , Histona Desacetilases/análise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Proteínas Inibidoras de Apoptose/análise , Pessoa de Meia-Idade , Psoríase/genética , Psoríase/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor Von Hippel-Lindau/análiseRESUMO
Corneal neovascularization can be induced by a severe ocular infection, injury or immunological diseases. The vascular endothelial growth factor (VEGF) is the main cytokine involved in this phenomenon, inducing angiogenesis from the vascularized ocular tissues. As the limbal tissue is located between conjunctival and corneal tissues, we suggest that the limbal cells are participating in the production of VEGF induced by bacterial components as LPS. In this work, RT-PCRs and immunoblots were used to investigate the expression of VEGF and other pro-angiogenic genes in primary cultures of human limbal fibroblasts (PCHLF) treated with lipopolysaccharide (LPS) from Escherichia coli. We found that the expression of VEGF was initiated at 6 h and reaches its highest expression at 72 h after stimulation with LPS. Up-regulation of toll-like receptor 4 (TLR4) after 3 h of treatment was also observed. LPS-induced the expression of VEGF in a dose-dependent manner, and the blocking of TLR4 with an anti-TLR4 antibody prevented VEGF expression. We also analyzed the molecules that modulate VEGF expression. LPS did not induce the up-regulation of LL-37 nor the hypoxia induced factor 1 alpha (HIF-1alpha) mRNA expression, however, an up-regulation of interleukin 13 receptor alpha 1 (IL-13Ralpha1) and interleukin 4 receptor alpha (IL-4Ralpha) were observed after 3 and 12 h of stimulation, respectively. The expression of interleukin 13 did not change throughout the treatment. These results suggest that TLR4, IL-13Ralpha1 and IL-4Ralpha induced by LPS in PCHLF could be playing an important role in the corneal neovascularization.