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1.
Br J Cancer ; 123(6): 942-954, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32601464

RESUMO

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.


Assuntos
Quimiocina CCL20/biossíntese , Receptores ErbB/fisiologia , Neoplasias/imunologia , Microambiente Tumoral , Proteínas ras/fisiologia , Animais , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estadiamento de Neoplasias , Neoplasias/tratamento farmacológico , Neovascularização Patológica/etiologia , Receptores CCR6/fisiologia , Transdução de Sinais/fisiologia
2.
J Immunol ; 201(12): 3669-3676, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30464051

RESUMO

We have described a novel cytokine encoded by a gene called Meteorin-like (Metrnl). Metrnl is a small (∼28 kDa) secreted protein expressed by activated macrophages and barrier tissues (mucosa and skin). Metrnl production by bone marrow macrophages is induced by several cytokines including TNF-α, IL-17α, IL-12, and IL-4 and inhibited by IFN-γ and TGF-ß. Metrnl expression in macrophages is also induced by LPS, and its levels in circulation are associated with inflammatory responses in vivo. Furthermore, Metrnl regulates the production of several cytokines and chemokines in macrophages. We have produced a Metrnl-/- mouse, which is viable and shows normal development. However, it exhibits dysregulated cytokine production, alterations in IgG production, and is highly susceptible to LPS in a sepsis model. Furthermore, older Metrnl-/- mice develop inflammatory lesions, suggesting that Metrnl participates in the control of inflammatory responses. Taken together, these observations indicate that Metrnl encodes a novel immunoregulatory cytokine associated with inflammatory responses that we have designated Meteorin-ß.


Assuntos
Inflamação/imunologia , Macrófagos/fisiologia , Mucosa/metabolismo , Fatores de Crescimento Neural/metabolismo , Sepse/imunologia , Pele/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Imunomodulação , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Crescimento Neural/genética
3.
J Immunol ; 199(9): 3326-3335, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28978694

RESUMO

We describe a novel B cell-associated cytokine, encoded by an uncharacterized gene (C17orf99; chromosome 17 open reading frame 99), that is expressed in bone marrow and fetal liver and whose expression is also induced in peripheral B cells upon activation. C17orf99 is only present in mammalian genomes, and it encodes a small (∼27-kDa) secreted protein unrelated to other cytokine families, suggesting a function in mammalian immune responses. Accordingly, C17orf99 expression is induced in the mammary gland upon the onset of lactation, and a C17orf99-/- mouse exhibits reduced levels of IgA in the serum, gut, feces, and lactating mammary gland. C17orf99-/- mice have smaller and fewer Peyer's patches and lower numbers of IgA-secreting cells. The microbiome of C17orf99-/- mice exhibits altered composition, likely a consequence of the reduced levels of IgA in the gut. Although naive B cells can express C17orf99 upon activation, their production increases following culture with various cytokines, including IL-4 and TGF-ß1, suggesting that differentiation can result in the expansion of C17orf99-producing B cells during some immune responses. Taken together, these observations indicate that C17orf99 encodes a novel B cell-associated cytokine, which we have called IL-40, that plays an important role in humoral immune responses and may also play a role in B cell development. Importantly, IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observations suggest that it may play a role in the pathogenesis of certain human diseases.


Assuntos
Linfócitos B/imunologia , Regulação da Expressão Gênica/imunologia , Interleucinas/imunologia , Nódulos Linfáticos Agregados/imunologia , Animais , Humanos , Imunoglobulina A/imunologia , Interleucinas/genética , Células Jurkat , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Camundongos , Camundongos Knockout
4.
J Immunol ; 194(1): 29-33, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25411203

RESUMO

Chemokines are chemotactic cytokines that direct the traffic of leukocytes and other cells in the body. Chemokines bind to G protein-coupled receptors expressed on target cells to initiate signaling cascades and induce chemotaxis. Although the cognate receptors of most chemokines have been identified, the receptor for the mucosal chemokine CXCL17 is undefined. In this article, we show that GPR35 is the receptor of CXCL17. GPR35 is expressed in mucosal tissues, in CXCL17-responsive monocytes, and in the THP-1 monocytoid cell line. Transfection of GPR35 into Ba/F3 cells rendered them responsive to CXCL17, as measured by calcium-mobilization assays. Furthermore, GPR35 expression is downregulated in the lungs of Cxcl17(-/-) mice, which exhibit defects in macrophage recruitment to the lungs. We conclude that GPR35 is a novel chemokine receptor and suggest that it should be named CXCR8.


Assuntos
Quimiocinas/metabolismo , Quimiotaxia de Leucócito/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Quimiocinas/genética , Quimiocinas CXC/genética , Quimiocinas CXC/imunologia , Humanos , Pulmão/citologia , Pulmão/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Monócitos/metabolismo , Mucosa/metabolismo , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genética , Alinhamento de Sequência , Transfecção
5.
J Allergy Clin Immunol ; 138(2): 500-508.e24, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27212086

RESUMO

BACKGROUND: Pruritus is a cardinal symptom of atopic dermatitis, and an increased cutaneous sensory network is thought to contribute to pruritus. Although the immune cell-IL-31-neuron axis has been implicated in severe pruritus during atopic skin inflammation, IL-31's neuropoietic potential remains elusive. OBJECTIVE: We sought to analyze the IL-31-related transcriptome in sensory neurons and to investigate whether IL-31 promotes sensory nerve fiber outgrowth. METHODS: In vitro primary sensory neuron culture systems were subjected to whole-transcriptome sequencing, ingenuity pathway analysis, immunofluorescence, and nerve elongation, as well as branching assays after IL-31 stimulation. In vivo we investigated the cutaneous sensory neuronal network in wild-type, Il31-transgenic, and IL-31 pump-equipped mice. RESULTS: Transgenic Il31 overexpression and subcutaneously delivered IL-31 induced an increase in the cutaneous nerve fiber density in lesional skin in vivo. Transcriptional profiling of IL-31-activated dorsal root ganglia neurons revealed enrichment for genes promoting nervous system development and neuronal outgrowth and negatively regulating cell death. Moreover, the growth cones of primary small-diameter dorsal root ganglia neurons showed abundant IL-31 receptor α expression. Indeed, IL-31 selectively promoted nerve fiber extension only in small-diameter neurons. Signal transducer and activator of transcription 3 phosphorylation mediated IL-31-induced neuronal outgrowth, and pharmacologic inhibition of signal transducer and activator of transcription 3 completely abolished this effect. In contrast, transient receptor potential cation channel vanilloid subtype 1 channels were dispensable for IL-31-induced neuronal sprouting. CONCLUSIONS: The pruritus- and TH2-associated novel cytokine IL-31 induces a distinct transcriptional program in sensory neurons, leading to nerve elongation and branching both in vitro and in vivo. This finding might help us understand the clinical observation that patients with atopic dermatitis experience increased sensitivity to minimal stimuli inducing sustained itch.


Assuntos
Interleucinas/metabolismo , Prurido/imunologia , Prurido/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Animais , Análise por Conglomerados , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Interleucinas/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fibras Nervosas/metabolismo , Fosforilação , Prurido/genética , Fator de Transcrição STAT3/metabolismo , Pele/imunologia , Pele/inervação , Pele/metabolismo
7.
J Immunol ; 193(3): 1468-74, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24973458

RESUMO

Chemokines are a superfamily of chemotactic cytokines that direct the movement of cells throughout the body under homeostatic and inflammatory conditions. The mucosal chemokine CXCL17 was the last ligand of this superfamily to be characterized. Several recent studies have provided greater insight into the basic biology of this chemokine and have implicated CXCL17 in several human diseases. We sought to better characterize CXCL17's activity in vivo. To this end, we analyzed its chemoattractant properties in vivo and characterized a Cxcl17 (-/-) mouse. This mouse has a significantly reduced number of macrophages in its lungs compared with wild-type mice. In addition, we observed a concurrent increase in a new population of macrophage-like cells that are F4/80(+)CDllc(mid). These results indicate that CXCL17 is a novel macrophage chemoattractant that operates in mucosal tissues. Given the importance of macrophages in inflammation, these observations strongly suggest that CXCL17 is a major regulator of mucosal inflammatory responses.


Assuntos
Quimiocinas CXC/fisiologia , Pulmão/imunologia , Pulmão/metabolismo , Macrófagos Alveolares/imunologia , Animais , Homeostase/imunologia , Imunofenotipagem , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/fisiologia , Pulmão/citologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Knockout , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia
8.
Support Care Cancer ; 24(10): 4241-8, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27165055

RESUMO

BACKGROUND: The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes. OBJECTIVE: The objective of the study was to evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs. PATIENTS AND METHODS: Patients during prolonged treatment with EGFRIs (>3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (<25 years old) vs. old (> 65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro. RESULTS: There were progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation, and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA ß-Gal activity. There was significantly decreased baseline expression in EGFR density in aged skin, when compared to young controls. CONCLUSIONS: EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents.


Assuntos
Receptores ErbB/genética , Cloridrato de Erlotinib/uso terapêutico , Envelhecimento da Pele/genética , Dermatopatias/genética , Idoso , Idoso de 80 Anos ou mais , Envelhecimento , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/administração & dosagem , Cloridrato de Erlotinib/farmacologia , Feminino , Humanos , Estudos Retrospectivos
9.
Clin Immunol ; 156(2): 119-27, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25486603

RESUMO

Cytokines are involved in many functions of the immune system including initiating, amplifying and resolving immune responses. Through bioinformatics analyses of a comprehensive database of gene expression (BIGE: Body Index of Gene Expression) we observed that a small secreted protein encoded by a poorly characterized gene called meteorin-like (METRNL), is highly expressed in mucosal tissues, skin and activated macrophages. Further studies indicate that Metrnl is produced by Alternatively Activated Macrophages (AAM) and M-CSF cultured bone marrow macrophages (M2-like macrophages). In the skin, METRNL is expressed by resting fibroblasts and IFNγ-treated keratinocytes. A screen of human skin-associated diseases showed significant over-expression of METRNL in psoriasis, prurigo nodularis, actinic keratosis and atopic dermatitis. METRNL is also up-regulated in synovial membranes of human rheumatoid arthritis. Taken together, these results indicate that Metrnl represents a novel cytokine, which is likely involved in both innate and acquired immune responses.


Assuntos
Citocinas/biossíntese , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Fatores de Crescimento Neural/imunologia , Pele/metabolismo , Animais , Artrite Reumatoide/metabolismo , Células da Medula Óssea/metabolismo , Células Cultivadas , Dermatite Atópica/metabolismo , Células Endoteliais/metabolismo , Humanos , Queratinócitos/metabolismo , Ceratose Actínica/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/genética , Prurigo/metabolismo , Psoríase/metabolismo , Pele/citologia , Membrana Sinovial/metabolismo , Regulação para Cima
10.
Biol Chem ; 395(6): 577-91, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24497224

RESUMO

The mouse represents a key model system for the study of the physiology and biochemistry of skin. Comparison of skin between mouse and human is critical for interpretation and application of data from mouse experiments to human disease. Here, we review the current knowledge on structure and immunology of mouse and human skin. Moreover, we present a systematic comparison of human and mouse skin transcriptomes. To this end, we have recently used a genome-wide database of human gene expression to identify genes highly expressed in skin, with no, or limited expression elsewhere - human skin-associated genes (hSAGs). Analysis of our set of hSAGs allowed us to generate a comprehensive molecular characterization of healthy human skin. Here, we used a similar database to generate a list of mouse skin-associated genes (mSAGs). A comparative analysis between the top human (n=666) and mouse (n=873) skin-associated genes (SAGs) revealed a total of only 30.2% identity between the two lists. The majority of shared genes encode proteins that participate in structural and barrier functions. Analysis of the top functional annotation terms revealed an overlap for morphogenesis, cell adhesion, structure, and signal transduction. The results of this analysis, discussed in the context of published data, illustrate the diversity between the molecular make up of skin of both species and grants a probable explanation, why results generated in murine in vivo models often fail to translate into the human.


Assuntos
Dermatopatias/patologia , Pele/anatomia & histologia , Animais , Didesoxinucleosídeos , Perfilação da Expressão Gênica , Humanos , Camundongos , Modelos Animais , Pele/metabolismo , Pele/patologia , Dermatopatias/metabolismo , Transcriptoma
11.
J Immunol ; 188(12): 6399-406, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22611239

RESUMO

The mucosal immune network is a crucial barrier preventing pathogens from entering the body. The network of immune cells that mediates the defensive mechanisms in the mucosa is likely shaped by chemokines, which attract a wide range of immune cells to specific sites of the body. Chemokines have been divided into homeostatic or inflammatory depending upon their expression patterns. Additionally, several chemokines mediate direct killing of invading pathogens, as exemplified by CCL28, a mucosa-associated chemokine that exhibits antimicrobial activity against a range of pathogens. CXCL17 was the last chemokine ligand to be described and is the 17th member of the CXC chemokine family. Its expression pattern in 105 human tissues and cells indicates that CXCL17 is a homeostatic, mucosa-associated chemokine. Its strategic expression in mucosal tissues suggests that it is involved in innate immunity and/or sterility of the mucosa. To test the latter hypothesis, we tested CXCL17 for possible antibacterial activity against a panel of pathogenic and opportunistic bacteria. Our results indicate that CXCL17 has potent antimicrobial activities and that its mechanism of antimicrobial action involves peptide-mediated bacterial membrane disruption. Because CXCL17 is strongly expressed in bronchi, we measured it in bronchoalveolar lavage fluids and observed that it is strongly upregulated in idiopathic pulmonary fibrosis. We conclude that CXCL17 is an antimicrobial mucosal chemokine that may play a role in the pathogenesis of interstitial lung diseases.


Assuntos
Antibacterianos/imunologia , Quimiocinas CXC/imunologia , Fibrose Pulmonar Idiopática/imunologia , Imunidade Inata/imunologia , Mucosa Respiratória/imunologia , Idoso , Antibacterianos/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocinas CXC/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Mucosa Respiratória/química , Mucosa Respiratória/metabolismo
12.
Clin Immunol ; 149(3): 388-99, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24211713

RESUMO

We have identified Tspan33 as a gene encoding a transmembrane protein exhibiting a restricted expression pattern including expression in activated B cells. TSPAN33 is a member of the tetraspanin family. TSPAN33 is not expressed in resting B cells, but is strongly induced in primary human B cells following activation. Human 2E2 cells, a Burkitt's lymphoma-derived B cell model of activation and differentiation, also upregulate TSPAN33 upon activation. TSPAN33 is expressed in several lymphomas including Hodgkin's and Diffuse large B cell lymphoma. TSPAN33 is also expressed in some autoimmune diseases where B cells participate in the pathology, including rheumatoid arthritis patients, systemic lupus erythematosus (SLE), and in spleen B cells from MRL/Fas(lpr/lpr) mice (a mouse model of SLE). We conclude that TSPAN33 may be used as a diagnostic biomarker or as a target for therapeutic antibodies for treatment of certain B cell lymphomas or autoimmune diseases.


Assuntos
Linfócitos B/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/imunologia , Tetraspaninas/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Lipopolissacarídeos/farmacologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Cultura Primária de Células , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Tetraspaninas/genética
13.
Lasers Surg Med ; 45(2): 67-75, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23440713

RESUMO

BACKGROUND AND OBJECTIVES: Neither the pathogenesis of port wine stain (PWS) birthmarks nor tissue effects of pulsed dye laser (PDL) treatment of these lesions is fully understood. There are few published reports utilizing gene expression analysis in human PWS skin. We aim to compare gene expression in PWS before and after PDL, using DNA microarrays that represent most, if not all, human genes to obtain comprehensive molecular profiles of PWS lesions and PDL-associated tissue effects. MATERIALS AND METHODS: Five human subjects had PDL treatment of their PWS. One week later, three biopsies were taken from each subject: normal skin (N); untreated PWS (PWS); PWS post-PDL (PWS + PDL). Samples included two lower extremity lesions, two facial lesions, and one facial nodule. High-quality total RNA isolated from skin biopsies was processed and applied to Affymetrix Human gene 1.0ST microarrays for gene expression analysis. We performed a 16 pair-wise comparison identifying either up- or down-regulated genes between N versus PWS and PWS versus PWS + PDL for four of the donor samples. The PWS nodule (nPWS) was analyzed separately. RESULTS: There was significant variation in gene expression profiles between individuals. By doing pair-wise comparisons between samples taken from the same donor, we were able to identify genes that may participate in the formation of PWS lesions and PDL tissue effects. Genes associated with immune, epidermal, and lipid metabolism were up-regulated in PWS skin. The nPWS exhibited more profound differences in gene expression than the rest of the samples, with significant differential expression of genes associated with angiogenesis, tumorigenesis, and inflammation. CONCLUSION: In summary, gene expression profiles from N, PWS, and PWS + PDL demonstrated significant variation within samples from the same donor and between donors. By doing pair-wise comparisons between samples taken from the same donor and comparing these results between donors, we were able to identify genes that may participate in formation of PWS and PDL effects. Our preliminary results indicate changes in gene expression of angiogenesis-related genes, suggesting that dysregulation of angiogenic signals and/or components may contribute to PWS pathology.


Assuntos
Regulação para Baixo , Perfilação da Expressão Gênica , Lasers de Corante/uso terapêutico , Análise de Sequência com Séries de Oligonucleotídeos , Mancha Vinho do Porto/cirurgia , Regulação para Cima , Adulto , Análise por Conglomerados , DNA , Feminino , Marcadores Genéticos , Humanos , Masculino , Neovascularização Fisiológica/genética , Mancha Vinho do Porto/genética , Análise de Componente Principal , Resultado do Tratamento
16.
BMC Neurosci ; 10: 20, 2009 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-19284629

RESUMO

BACKGROUND: Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. RESULTS: We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. CONCLUSION: SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.


Assuntos
Células Epiteliais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Sódio/metabolismo , Papilas Gustativas/metabolismo , Animais , Canais de Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.2 , Canal de Sódio Disparado por Voltagem NAV1.3 , Canal de Sódio Disparado por Voltagem NAV1.7 , Proteínas do Tecido Nervoso/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Sódio/genética , Canais de Cátion TRPM/metabolismo , Paladar/fisiologia , Língua/metabolismo
17.
J Neuroimmunol ; 171(1-2): 145-55, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16300832

RESUMO

We have previously reported the characterization of a novel immunoglobulin supergene family member, designated class-I MHC-restricted T cell associated molecule (CRTAM). Here we further characterize human CRTAM and find that it is highly expressed in the cerebellum, notably in Purkinje neurons. We identify CRTAM as a new member of the nectin-like (Necls) family and find significant expression of Necl-2 (IGSF4), a protein known to bind CRTAM and another member of the nectin superfamily, in the cerebellum. These findings suggest that CRTAM/Necl-2 binding may contribute to neuronal interactions. We also show that, in the immune system, CRTAM expression is restricted to activated class-I MHC-restricted T cells, including NKT and CD8 T cells. CRTAM represents one of the most highly expressed surface markers of activated human CD8 T cells and NKT cells, suggesting it may have diagnostic uses in various human viral and autoimmune diseases.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Cerebelo/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Imunoglobulinas/metabolismo , Ativação Linfocitária/fisiologia , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting/métodos , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Cerebelo/citologia , Relação Dose-Resposta a Droga , Ativação Enzimática/fisiologia , Feminino , Citometria de Fluxo/métodos , Humanos , Imuno-Histoquímica/métodos , Ionomicina/farmacologia , Leucócitos Mononucleares/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Filogenia , Células de Purkinje/metabolismo , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Alinhamento de Sequência/métodos , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismo
18.
FASEB J ; 19(10): 1356-8, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15955843

RESUMO

The Affymetrix GeneChip platform was used to build a gene expression database of the normal human body. Postmortem human tissues represent a valuable source of biological materials for this type of study, but their use entails some delays before harvesting such tissues. We first evaluated the RNA quality obtained from tissues obtained 3-5 h postmortem and found variations that were both tissue and donor-dependent. RNAs extracted from brain regions were of higher quality than those obtained from the gut, while the cause of death was a significant factor in donor-dependent differences. To avoid these variables, we used rat duodenum to determine the effects of RNA degradation on the analysis of gene expression. Surprisingly, even samples exhibiting significant RNA degradation yielded robust gene expression results, comparable to those obtained using intact samples at a certain signal intensity cutoff. We extended these findings to our human expression database and obtained similar results, indicating that the Affymetrix platform, which is biased to the 3' end of transcripts for detection, can tolerate significant RNA degradation, while still yielding high quality expression data. Our resulting body index expression database is a valuable research tool. As examples of potential uses, we report novel expression sites for four potential therapeutic targets--CCL27, GPR22, GPR113 and GPR128--and as well as a set of thymus-specific genes, including three not previously associated with the thymus.


Assuntos
Perfilação da Expressão Gênica , Estabilidade de RNA , Animais , Encéfalo/metabolismo , Quimiocina CCL27 , Quimiocinas CC/genética , Duodeno/química , Duodeno/metabolismo , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , RNA , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Timo/metabolismo
19.
J Exp Ther Oncol ; 5(4): 273-86, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17024968

RESUMO

Integrin alpha5beta1, the principal fibronectin receptor, is an important survival factor, playing a key role in angiogenesis. Angiogenesis is critical for tumor growth, and anti-angiogenic therapies have met clinical success. To validate the therapeutic potential of an anti-alpha5beta1 strategy, we generated volociximab (M200) a chimeric human IgG4 version of the alpha5beta1 function-blocking murine antibody IIA1; and F200, the Fab derivative. Volociximab, F200 and IIA1 showed similar activity by ELISA (EC50= 0.2nM), Biacore (Kd= 0.1-0.4nM) and inhibition of fibronectin binding (IC50= 2-3nM). The inhibitory potential of alpha5beta1 antibodies was compared to HuMV833, an anti-VEGF antibody. Both volociximab and HuMV833 inhibited HUVEC proliferation (IC50 of volociximab = 0.2-0.5nM; IC50 of HuMV833 = 45nM). However, IIA1, volociximab and F200 were also potent inhibitors of an in vitro model of angiogenesis (HUVEC tube formation assay), unlike HuMV833. Additionally, volociximab inhibited in vitro tube formation induced by VEGF and/or bFGF, suggesting a mechanism of action independent of growth factor stimulus. In fact, inhibition of alpha5beta1 function by volociximab induced apoptosis of actively proliferating, but not resting, endothelial cells. Volociximab does not cross-react with rodent alpha5beta1, therefore in vivo validation of an anti-alpha5beta1 approach was conducted in a cynomolgus model of choroidal revascularization. Volociximab and F200 were potent inhibitors of neovessel formation in this model. These data demonstrate that volociximab has therapeutic potential in diseases in which new vessel formation is a component of the pathology.


Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Integrina alfa5beta1/imunologia , Animais , Anticorpos Monoclonais Murinos , Células COS , Chlorocebus aethiops , Matriz Extracelular/metabolismo , Humanos , Concentração Inibidora 50 , Integrina alfa5beta1/química , Cinética , Macaca fascicularis , Degeneração Macular/tratamento farmacológico , Neovascularização Patológica , Rituximab
20.
Cancer Res ; 63(19): 6387-94, 2003 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-14559828

RESUMO

We have used the Eos Hu03 GeneChip array, which represents over 92% of the transcribed human genome, to measure gene expression in a panel of normal and diseased human tissues. This analysis revealed that E-selectin mRNA is selectively overexpressed in prostate cancer epithelium, a finding that correlated strongly with E-selectin protein expression as assessed by immunohistochemistry. Antibodies against E-selectin that blocked function failed to impede cancer cell growth, suggesting that overexpression of E-selectin was not essential for cell growth. However, a novel auristatin E-based antibody drug conjugate (ADC), E-selectin antibody valine-citrulline monomethyl-auristatin E, was a potent and selective agent against E-selectin-expressing cancer cell lines in vitro, with the degree of cytotoxicity varying with surface antigen density. Interestingly, sensitivity to the ADC differed among cell lines from different tissues expressing similar amounts of E-selectin and was found to correlate with sensitivity to free auristatin E. Furthermore, E-selectin-expressing tumors grown as xenografts in severe combined immunodeficient mice were responsive to treatment with E-selectin antibody valine-citrulline monomethyl-auristatin E in vivo, with more than 85% inhibition of tumor growth observed in treated mice. These findings demonstrate that an E-selectin-targeting ADC has potential as a prostate cancer therapy and validates a genomics-based paradigm for the identification of cancer-specific antigens suitable for targeted therapy.


Assuntos
Antineoplásicos/administração & dosagem , Selectina E/biossíntese , Imunotoxinas/metabolismo , Oligopeptídeos/administração & dosagem , Neoplasias da Próstata/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Selectina E/genética , Selectina E/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imunotoxinas/imunologia , Imunotoxinas/farmacologia , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
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