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1.
J Eur Acad Dermatol Venereol ; 34(6): 1248-1256, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31954077

RESUMO

BACKGROUND: Psoriatic arthritis (PsA) develops in ~30% of patients with psoriasis. The diagnosis of PsA is challenging, and there are no reliable molecular markers in clinical use. MicroRNAs are short non-coding regulatory RNAs, which can be actively packaged into extracellular vesicles (EVs) and secreted to the circulation. OBJECTIVES: To explore whether plasma-derived EV microRNAs may serve as biomarkers for PsA in patients with psoriasis. METHODS: Plasma samples were obtained from patients with cutaneous-only psoriasis (PsC) and patients with psoriasis and PsA. Plasma EVs were isolated using miRCURY™ Exosome Isolation Kit. RNA sequencing was used to identify differentially expressed EV miRNAs in the discovery phase (PsC, n = 15; PsA, n = 14). In the validation phase (PsC, n = 29; PsA, n = 28), 41 selected miRNAs were analysed in plasma EVs by qPCR. The association of the identified miRNAs with PsA was assessed by logistic regression analysis. RESULTS: RNA sequencing identified 19 plasma EV miRNAs with significantly different levels between PsA and PsC in the discovery cohort. Significantly lower levels of plasma EV let-7b-5p and miR-30e-5p in PsA vs. PsC were confirmed in the validation cohort, and their decreased levels were found to be associated with the presence of PsA. ROC analysis revealed an AUC of 0.68 (95% CI 0.53-0.83) for let-7b-5p and 0.69 (95% CI 0.55-0.84) for miR-30e-5p. CONCLUSIONS: Circulating EV microRNA levels are altered in patients with PsA as compared with PsC. Findings of this exploratory study suggest that circulating EV microRNAs may serve as biomarkers for arthritis in psoriasis patients.


Assuntos
Artrite Psoriásica , MicroRNA Circulante , Vesículas Extracelulares , MicroRNAs , Psoríase , Artrite Psoriásica/diagnóstico , Artrite Psoriásica/genética , Biomarcadores , Humanos , Psoríase/genética
2.
Br J Dermatol ; 169(3): 563-70, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23600954

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are endogenous, nonprotein-coding, regulatory RNAs with important roles in health and disease. miRNAs are present in the circulation in a stable form and their levels are altered in diseases. OBJECTIVES: To determine whether antitumour necrosis factor (TNF)-α therapy affects serum miRNA levels in patients with psoriasis. METHODS: Serum samples were obtained from healthy donors and from patients with chronic plaque psoriasis before and 12 weeks after the initiation of treatment with the TNF-inhibitor etanercept or methotrexate. miRNA expression profiling was utilized to identify miRNAs with altered serum level in psoriasis, as well as anti-TNF-α-regulated miRNAs in patients' sera. The expression of five miRNAs regulated by etanercept was measured by quantitative polymerase chain reaction (qPCR) in sera from patients and controls. RESULTS: Etanercept significantly suppressed a panel of 38 miRNAs, which were found to be predominantly immune-cell derived and which have been implicated in inflammation and autoimmunity. Validation by qPCR showed that serum levels of miR-106b, miR-26b, miR-142-3p, miR-223 and miR-126 were significantly downregulated by etanercept in responders (Psoriasis Area and Severity Index change > 50%). By contrast, methotrexate did not significantly affect the levels of these miRNAs. Serum levels of these miRNAs were not upregulated in patients with psoriasis compared with healthy controls. The level of four circulating miRNAs was significantly different (increased: miR-128a; decreased: let-7d, miR-142-3p, miR-181a) in psoriasis and healthy serum. CONCLUSIONS: The level of circulating miRNAs is altered in psoriasis. Anti-TNF-α therapy has a profound effect on the serum level of miRNAs; however, these are not related to disease severity. Our results suggest that changes in the miRNA level may reflect a previously unknown effect of anti-TNF-α therapy. Our results suggest the involvement of miRNAs in pathways affected by anti-TNF-α therapy and warrant further investigation of serum miRNAs as potential biomarkers for therapy response in psoriasis.


Assuntos
Imunoglobulina G/uso terapêutico , Fatores Imunológicos/uso terapêutico , MicroRNAs/metabolismo , Psoríase/tratamento farmacológico , Receptores do Fator de Necrose Tumoral/uso terapêutico , Doença Crônica , Etanercepte , Feminino , Humanos , Masculino , MicroRNAs/efeitos dos fármacos , Pessoa de Meia-Idade , Psoríase/sangue , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
3.
Oncogenesis ; 1: e3, 2012 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-23552555

RESUMO

Basal cell carcinoma (BCC) of the skin represents the most common malignancy in humans. MicroRNAs (miRNAs), small regulatory RNAs with pleiotropic function, are commonly misregulated in cancer. Here we identify miR-203, a miRNA abundantly and preferentially expressed in skin, to be downregulated in BCCs. We show that activation of the Hedgehog (HH) pathway, critically involved in the pathogenesis of BCCs, as well as the EGFR/MEK/ERK/c-JUN signaling pathway suppresses miR-203. We identify c-JUN, a key effector of the HH pathway, as a novel direct target for miR-203 in vivo. Further supporting the role of miR-203 as a tumor suppressor, in vivo delivery of miR-203 mimics in a BCC mouse model results in the reduction of tumor growth. Our results identify a regulatory circuit involving miR-203 and c-JUN, which provides functional control over basal cell proliferation and differentiation. We propose that miR-203 functions as a 'bona fide' tumor suppressor in BCC, whose suppressed expression contributes to oncogenic transformation via derepression of multiple stemness- and proliferation-related genes, and its overexpression could be of therapeutic value.

4.
J Cell Mol Med ; 13(1): 24-38, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19175698

RESUMO

Since their discovery in 1993 and the introduction of the term microRNA in 2001, it has become evident that microRNAs (miRNAs) involved in many biological processes, including development, differentiation, proliferation and apoptosis. The function of miRNA the control of protein production in cells by sequence-specific targeting of mRNAs for translational repression or mRNA degradati Interestingly, immune genes are apparently preferentially targeted by miRNAs compared to the average of the human genome, indicat the significance of miRNA-mediated regulation for normal immune responses. Here, we review what is known about the role of miRN in the pathogenesis of immune-related diseases such as chronic inflammatory skin diseases, autoimmunity and viral infections.


Assuntos
Imunidade , Inflamação , MicroRNAs , Animais , Autoimunidade/genética , Humanos , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/imunologia , Imunidade/genética , Imunidade/imunologia , Inflamação/imunologia , Inflamação/fisiopatologia , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Genéticos , Vírus/genética , Vírus/imunologia
5.
Clin Exp Dermatol ; 33(3): 312-5, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18419608

RESUMO

Compelling evidence indicates that microRNAs (miRNAs), short, non-protein coding RNAs, are critical for the development and survival of multicellular organisms. Recently, miRNAs were implicated in the pathogenesis of psoriasis and atopic eczema (AE), the two most common chronic inflammatory disorders in skin. In particular, miR-203, the first skin-specific miRNA, showing an intriguing expression profile being confined to skin epithelium, is specifically overexpressed in psoriasis. MiR-146a, another miRNA showing specific upregulation in psoriasis, is involved in the regulation of innate immune responses and the tumour necrosis factor (TNF)-alpha pathway. Interestingly, miR-125b, another miRNA involved in the TNF-alpha pathway, is also deregulated in psoriasis and AE. As skin inflammation may serve as a model for chronic inflammatory disorders, it is likely that miRNAs involved in skin inflammation will eventually emerge in other inflammatory or autoimmune disorders, and some of these may become disease markers and therapeutic targets. In this review we present an overview of what is currently known about the roles of miRNAs in chronic inflammatory skin disorders.


Assuntos
Citocinas/metabolismo , Dermatite/metabolismo , Queratinócitos/metabolismo , MicroRNAs/metabolismo , Psoríase/metabolismo , Pele/metabolismo , Dermatite/etiologia , Regulação da Expressão Gênica , Humanos , Psoríase/etiologia , Regulação para Cima
6.
Int Immunopharmacol ; 6(3): 358-68, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16428071

RESUMO

Topical immunosuppressant therapy is widely used in the treatment of inflammatory skin diseases such as psoriasis and atopic dermatitis. Besides its beneficial therapeutic effects, application of topical anti-inflammatory drugs may render the epidermis more vulnerable to invading pathogens by suppressing innate immune responses in keratinocytes, such as cytokine production and Toll-like receptor (TLR) expression. In order to evaluate and compare the immunosuppressive effects of different immunosuppressant drugs on keratinocytes, we treated lipopolysaccharide (LPS)-stimulated and -unstimulated normal human keratinocytes with the synthetic corticosteroid budesonide and the macrolide tacrolimus. The expressions of the pattern recognition receptors (PRRs) TLR2 and TLR4 were measured by quantitative RT-PCR, pro-inflammatory cytokines IL-1alpha, IL-8 and TNF-alpha were monitored by quantitative RT-PCR and by ELISA, and alterations in TLR2 protein level were measured by flow cytometry. Budesonide had a suppressive effect on both constitutive and LPS-induced IL-8 gene expression. The amount of TNF-alpha mRNA was diminished in unstimulated keratinocytes, while TLR2 mRNA expression was markedly enhanced both in unstimulated and LPS-treated cells after incubation with budesonide. This increase in TLR2 mRNA expression was also detectable at the protein level in LPS-stimulated cells. Tacrolimus had no effect on any of the examined genes. Budesonide, but not tacrolimus, significantly inhibited the NF-kappaB-dependent luciferase reporter activity in HaCaT cells after induction with LPS or TNF-alpha. Although tacrolimus and budesonide are both effective treatments in some inflammatory skin diseases, the data provided here imply differences in local therapeutic and adverse effects of these two topical immunosuppressants.


Assuntos
Anti-Inflamatórios/farmacologia , Budesonida/farmacologia , Imunossupressores/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Tacrolimo/farmacologia , Linhagem Celular , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Interleucina-8/biossíntese , Interleucina-8/genética , Queratinócitos/metabolismo , Lipopolissacarídeos/imunologia , NF-kappa B/fisiologia , RNA Mensageiro/metabolismo , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética
7.
Acta Microbiol Immunol Hung ; 51(3): 303-10, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15571070

RESUMO

Human keratinocytes are known to kill living microbes. They express different pattern recognition receptors (PRRs) such as the Toll-like receptor 2 (TLR2), TLR4, the CD1d molecule and a keratinocyte mannose-binding receptor (KcMR). In response to challenge with microbes or microbial-derived substances the activation and nuclear translocation of NF-kappaB, the production of nitric oxide (NO) and inflammatory cytokines occur in keratinocytes, in a TLR-dependent manner. Blocking of NF-kappaB activation or NO production inhibit the Candida albicans-killing activity of keratinocytes. This Candida killing activity could be inhibited by blocking of KcMR. Recognition of invading pathogens in the epidermis triggers cytokine production in keratinocytes leading to elimination of pathogens and the activation of the adaptive immune system. These findings stress the importance of the role of keratinocytes in innate immunity.


Assuntos
Imunidade Inata , Queratinócitos/imunologia , Receptores de Superfície Celular/fisiologia , Pele/citologia , Humanos , Queratinócitos/citologia , Queratinócitos/microbiologia , Queratinócitos/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores Imunológicos/fisiologia , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like
8.
Dermatology ; 206(2): 96-105, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12592074

RESUMO

Acne is a multifactorial disease of the pilosebaceous follicle. The most significant pathogenetic factors of acne are: abnormal ductal keratinization, increased sebum secretion, abnormalities of the microbial flora and inflammation. The pilosebaceous unit is an immunocompetent organ. Keratinocytes and sebocytes may act as immune cells capable of pathogen recognition and abnormal lipid presentation, and they might have an important role in initiating and perpetuating the activation of both innate and adaptive immune responses. The elements of the skin immune system are involved in the development of both noninflammatory and inflammatory acne lesions.


Assuntos
Acne Vulgar/imunologia , Proteínas de Drosophila , Imunidade Inata , Acne Vulgar/microbiologia , Acne Vulgar/fisiopatologia , Antígenos CD1/imunologia , Antígenos CD1d , Humanos , Mediadores da Inflamação/imunologia , Queratinócitos/imunologia , Glicoproteínas de Membrana/imunologia , Propionibacterium acnes/imunologia , Receptores de Superfície Celular/imunologia , Pele/imunologia , Pele/microbiologia , Pele/fisiopatologia , Receptores Toll-Like
9.
J Invest Dermatol ; 117(2): 205-13, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11511295

RESUMO

Human keratinocytes are known to kill Candida albicans in vitro, but the mechanism of killing is not yet understood. Here, we demonstrate that spontaneous, ultraviolet-B-light-induced, alpha-melanocyte-stimulating-hormone-induced, and interleukin-8-induced Candida killing by keratinocytes can be inhibited with mannan and mannosylated bovine serum albumin (Man-BSA). A polyclonal goat serum raised against the human macrophage mannose receptor stained suprabasal keratinocytes, but no staining was observed on keratinocytes with a monoclonal antibody (mAb15) specific for the human macrophage mannose receptor. Mannose-affinity chromatography of keratinocyte extract isolated a 200 kDa protein, and on the Western blot the goat antiserum reacted with a 200 kDa protein. In radioligand binding studies, the binding of 125I-Man-BSA to human keratinocytes was inhibited by mannan in a concentration-dependent manner. Analysis of the binding revealed a single class keratinocyte mannose receptor with a KD of 1.4 x 10(-8) M and a Bmax of 1 x 10(4) binding sites per cell. The binding of 125I-Man- BSA to keratinocytes proved to be time-dependent, acid-precipitable, and Ca2+- and trypsin-sensitive. After trypsinization the receptors underwent a rapid recovery at 37 degrees C. These results demonstrate the presence of mannose receptor on human keratinocytes, and its active involvement in the killing of Candida albicans.


Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Lectinas Tipo C , Lectinas de Ligação a Manose , Receptores de Superfície Celular/biossíntese , Anticorpos Monoclonais , Western Blotting , Cálcio/metabolismo , Candidíase/metabolismo , Adesão Celular , Quelantes/farmacologia , Reações Cruzadas , Ácido Egtázico/farmacologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Radioisótopos do Iodo , Queratinócitos/citologia , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/microbiologia , Mananas/farmacologia , Manose/farmacocinética , Receptor de Manose , Ensaio Radioligante , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/imunologia , Albumina Sérica/farmacocinética , Pele/citologia , Pele/microbiologia
10.
Arch Dermatol Res ; 293(4): 206-13, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11380154

RESUMO

In the highly coordinated programme of gene expression during keratinocyte proliferation and differentiation, alpha5 integrin and keratins 1 and 10 (K1/K10) may play important regulatory roles. We were interested in seeing whether, in continuously growing, immortalized HaCaT keratinocytes, similar to normal keratinocytes, the expression of alpha5 integrin and K1/K10 was related to cell proliferation and differentiation. After release from cell quiescence the expression of alpha5 integrin, both at the mRNA and protein levels, was upregulated in the cells. At the same time, K1/K10 mRNA and protein expression decreased dramatically, while the mRNA for D1 cyclin became detectable, and the cells became highly proliferative. These findings indicate that alpha5 integrin and K1/K10 are involved in the regulation of HaCaT proliferation and differentiation, as in normal keratinocytes. However, HaCaT cells are different from normal keratinocytes in their ability to lose K1/K10 expression. There is no evidence that the expression of K1/K10 can be reversed in normal keratinocytes. This ability of dedifferentiation might be a unique feature of HaCaT cells and may be a key component of their immortalized nature. We also found that serum factors regulate mRNA expression of alpha5 integrin and K1, but not of K10, in HaCaT cells. This information could be relevant to the understanding of normal epidermal differentiation.


Assuntos
Antígenos CD/genética , Regulação da Expressão Gênica/fisiologia , Queratinócitos/metabolismo , Queratinas/genética , Fenômenos Fisiológicos Sanguíneos , Divisão Celular/fisiologia , Linhagem Celular Transformada , Meio Ambiente , Humanos , Integrina alfa5 , Queratina-10 , Queratinócitos/citologia , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Fatores de Tempo
11.
Inflamm Res ; 50(1): 44-9, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11235021

RESUMO

OBJECTIVE: Dithranol is highly effective in the treatment of psoriasis, however its mode of action is still not well known. Since interleukin-8 and interleukin-10 are involved in the pathogenesis of psoriasis, the aim of our study was to investigate the effect of dithranol on interleukin-8, interleukin-10 mRNA production and interleukin-10 receptor expression of the HaCaT keratinocyte cell line which is commonly used in experiments examining the effects of therapeutic drugs on keratinocytes. MATERIALS AND METHODS: Cultured HaCaT cells were treated with 0.1-0.5 microg/ml dithranol for 30 minutes. After 2 and 4 h total cellular RNA isolated from HaCaT cells was reverse transcribed (RT) to cDNA which was subjected to polymerase chain reaction (PCR) with specific primer pairs for interleukin-8, interleukin-10 and interleukin-10 receptor. For immunohistochemistry cultured HaCaT cells were stained with a monoclonal antibody against the human interleukin-10 receptor. RESULTS: Our results showed that dithranol treatment did not change the highly elevated level of interleukin-8 mRNA of HaCaT cells. Interleukin-10 mRNA signal with RT-PCR could not be detected in HaCaT cells. Depending on the concentration dithranol increased the mRNA production of interleukin-10 receptors in HaCaT cells. This dithranol induced dose dependent upregulation of IL-10 receptors in HaCaT cells was also observed on the protein level using immunohistochemistry. CONCLUSIONS: Since the interleukin-10 receptor expression of keratinocytes in psoriatic lesional skin is downregulated, the dithranol induced upregulation of the receptor in our model system might help to reveal the therapeutic action of the drug.


Assuntos
Antralina/farmacologia , Anti-Inflamatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Queratinócitos/metabolismo , Receptores de Interleucina/genética , Actinas/genética , Administração Tópica , Antralina/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Anticorpos Monoclonais/farmacologia , Linhagem Celular , Linhagem Celular Transformada , Células Cultivadas , Humanos , Imuno-Histoquímica , Interleucina-10/genética , Interleucina-8/genética , RNA Mensageiro/análise , Receptores de Interleucina/imunologia , Receptores de Interleucina-10 , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
J Invest Dermatol ; 115(3): 345-52, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10951267

RESUMO

Histamine has been implicated as one of the mediators involved in regulation of proliferation in both normal and neoplastic tissues. Histidine decarboxylase, the only enzyme that catalyzes the formation of histamine from L-histidine, is an essential regulator of histamine levels. In this study, we investigated the gene and protein expression of histidine decarboxylase in melanoma. Reverse transcriptase polymerase chain reaction and in situ hybridization studies of WM-35, WM-983/B, HT-168, and M1 human melanoma cell lines both resulted in positive signals for histidine decarboxylase messenger RNA. A polyclonal chicken antibody was developed against human histidine decarboxylase and protein expression was confirmed by western blot analysis of the cell lysates, revealing a predominant immunoreactive band at approximately 54 kDa corresponding to monomeric histidine decarboxylase. Protein expression of histidine decarboxylase was also shown by flow cytometric analysis and strong punctate cytoplasmic staining of melanoma cell lines. Moreover, both primary and metastatic human melanoma tissues were brightly stained for histidine decarboxylase. When compared with the very weak or no reactions on cultivated human melanocytes both western blot and immunohistochemical studies showed much stronger histidine decarboxylase expression in melanoma cells. These findings suggest that expression of histidine decarboxylase is elevated in human melanoma.


Assuntos
Histidina Descarboxilase/genética , Western Blotting , Citometria de Fluxo , Expressão Gênica , Histidina Descarboxilase/imunologia , Humanos , Melanoma/secundário , Sondas Moleculares/análise , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
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