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1.
Sci Rep ; 10(1): 2219, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32042039

RESUMO

Establishing clinically relevant single-cell (SC) transcriptomic workflows from cryopreserved tissue is essential to move this emerging immune monitoring technology from the bench to the bedside. Improper sample preparation leads to detrimental cascades, resulting in loss of precious time, money and finally compromised data. There is an urgent need to establish protocols specifically designed to overcome the inevitable variations in sample quality resulting from uncontrollable factors in a clinical setting. Here, we explore sample preparation techniques relevant to a range of clinically relevant scenarios, where SC gene expression and repertoire analysis are applied to a cryopreserved sample derived from a small amount of blood, with unknown or partially known preservation history. We compare a total of ten cell-counting, viability-improvement, and lymphocyte-enrichment methods to highlight a number of unexpected findings. Trypan blue-based automated counters, typically recommended for single-cell sample quantitation, consistently overestimate viability. Advanced sample clean-up procedures significantly impact total cell yield, while only modestly increasing viability. Finally, while pre-enrichment of B cells from whole peripheral blood mononuclear cells (PBMCs) results in the most reliable BCR repertoire data, comparable T-cell enrichment strategies distort the ratio of CD4+ and CD8+ cells. Furthermore, we provide high-resolution analysis of gene expression and clonotype repertoire of different B cell subtypes. Together these observations provide both qualitative and quantitative sample preparation guidelines that increase the chances of obtaining high-quality single-cell transcriptomic and repertoire data from human PBMCs in a variety of clinical settings.

2.
Exp Dermatol ; 29(1): 4-14, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30991456

RESUMO

Due to the clinical development of drugs such as secukinumab, ustekinumab and dupilumab, major changes have been achieved in the treatment of patients diagnosed with psoriasis and atopic dermatitis. In academia and the pharmaceutical industry, research is increasingly moving towards the development of bispecific antibodies and multi-specific nanobodies, as there is a compelling need for new treatment modalities for patients suffering from autoimmune or malignant disease. The purpose of this review is to discuss aspects of translational drug development with a particular emphasis on indications such as psoriasis and atopic dermatitis. The identification of biomarkers, the assessment of target organ pharmacokinetic and pharmacodynamics interactions and a wide range of in vitro, ex vivo and in vivo models should contribute to an appropriate prediction of a biological effect in the clinical setting. As human biology may not be perfectly reflected by approaches such as skin equivalents or animal models, novel approaches such as the use of human skin and dermal microperfusion assays in healthy volunteers and patients appear both reasonable and mandatory. These models may indeed generate highly translationally relevant data that have the potential to reduce the failure rate of drugs currently undergoing clinical development.

4.
Nat Commun ; 10(1): 4703, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619666

RESUMO

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.


Assuntos
Dermatite Atópica/genética , Interações entre Hospedeiro e Microrganismos/genética , Microbiota/genética , Psoríase/genética , Pele/metabolismo , Pele/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Dermatite Atópica/microbiologia , Disbiose/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Psoríase/microbiologia , RNA Ribossômico 16S , Adulto Jovem
5.
Cell ; 176(4): 757-774.e23, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30712866

RESUMO

ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.


Assuntos
Movimento Celular/fisiologia , Miosina Tipo II/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular/imunologia , Proteínas do Citoesqueleto , Feminino , Humanos , Interleucina-1alfa/metabolismo , Masculino , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Fosforilação , Proteômica , Receptor Cross-Talk/fisiologia , Transdução de Sinais , Microambiente Tumoral/imunologia
6.
Clin Cancer Res ; 24(20): 5098-5111, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30068707

RESUMO

Purpose: Highly aggressive triple-negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy.Experimental Design: We evaluated FRα expression in breast cancers by genomic (n = 3,414) and IHC (n = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the antitumor functions of an antibody recognizing FRα (MOv18-IgG1), in vitro, and in human TNBC xenograft models.Results: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in postneoadjuvant chemotherapy-residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1, and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death in vitro by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth.Conclusions: FRα is overexpressed in high-grade TNBC and postchemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as ADCs, or passive immunotherapy priming Fc-mediated antitumor immune cell responses. Clin Cancer Res; 24(20); 5098-111. ©2018 AACR.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Receptor 1 de Folato/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Modelos Biológicos , Terapia de Alvo Molecular , Neoplasia de Células Basais , Interferência de RNA , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Front Immunol ; 9: 493, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29628923

RESUMO

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.


Assuntos
Antígenos de Neoplasias/química , Antineoplásicos Imunológicos/imunologia , Linfócitos B/imunologia , Citometria de Fluxo/métodos , Melanoma/imunologia , Receptor ErbB-2/imunologia , Animais , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/química , Linfócitos B/patologia , Feminino , Humanos , Masculino , Melanoma/patologia , Camundongos , Receptor ErbB-2/química
8.
Cell ; 169(6): 1119-1129.e11, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28552347

RESUMO

The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory T cells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of Tregs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs.


Assuntos
Folículo Piloso/citologia , Células-Tronco/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Células Epiteliais/metabolismo , Folículo Piloso/metabolismo , Humanos , Inflamação/metabolismo , Proteína Jagged-1/metabolismo , Camundongos
9.
Nat Commun ; 8: 13850, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-28054551

RESUMO

The diversity and activity of leukocytes is controlled by genetic and environmental influences to maintain balanced immune responses. However, the relative contribution of environmental compared with genetic factors that affect variations in immune traits is unknown. Here we analyse 23,394 immune phenotypes in 497 adult female twins. 76% of these traits show a predominantly heritable influence, whereas 24% are mostly influenced by environment. These data highlight the importance of shared childhood environmental influences such as diet, infections or microbes in shaping immune homeostasis for monocytes, B1 cells, γδ T cells and NKT cells, whereas dendritic cells, B2 cells, CD4+ T and CD8+ T cells are more influenced by genetics. Although leukocyte subsets are influenced by genetics and environment, adaptive immune traits are more affected by genetics, whereas innate immune traits are more affected by environment.


Assuntos
Adaptação Fisiológica/imunologia , Meio Ambiente , Imunidade Inata/genética , Idoso , Células Apresentadoras de Antígenos/imunologia , Estudos de Casos e Controles , Estudos de Coortes , Dieta , Feminino , Homeostase , Humanos , Imunofenotipagem , Microbiota , Pessoa de Meia-Idade , Monócitos/imunologia , Células T Matadoras Naturais/imunologia , Fenótipo , Medicina de Precisão , Linfócitos T/imunologia
10.
Cancer Res ; 77(5): 1127-1141, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28096174

RESUMO

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen-specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcε receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127-41. ©2017 AACR.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Receptor 1 de Folato/imunologia , Macrófagos/imunologia , Neoplasias Ovarianas/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Receptor 1 de Folato/antagonistas & inibidores , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Ratos , Ratos Wistar , Transdução de Sinais , Fator de Necrose Tumoral alfa/biossíntese
11.
Curr Opin Rheumatol ; 29(1): 71-78, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27875351

RESUMO

PURPOSE OF REVIEW: This review presents our current understanding of the pathogenesis and treatment of psoriasis with a particular focus on recent areas of research and emerging concepts. RECENT FINDINGS: Psoriasis arises in genetically predisposed individuals who have an abnormal innate and adaptive immune response to environmental factors. Recent studies have identified novel genetic, epigenetic and immunological factors that play a role in the disease pathogenesis. There is emerging evidence for the role of the skin microbiome in psoriasis. Studies have shown reduced diversity and altered composition of the skin microbiota in psoriasis. SUMMARY: Recent advances in our understanding of the complex immunopathogenesis of psoriasis have led to the identification of crucial cytokines and cell signalling pathways that are targeted by a range of immunotherapies.


Assuntos
Imunoterapia/métodos , Psoríase/etiologia , Psoríase/terapia , Imunidade Adaptativa , Biomarcadores/sangue , Citocinas/imunologia , Epigenômica , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Humanos , Fatores Imunológicos/uso terapêutico , Microbiota , Psoríase/imunologia , Pele/microbiologia
12.
J Immunol ; 197(11): 4403-4412, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27798153

RESUMO

IL-17A has been strongly associated with epidermal hyperplasia in many cutaneous disorders. However, because IL-17A is mainly produced by αß and γδT cells in response to IL-23, the role of T cells and IL-23 has overshadowed any IL-17A-independent actions. In this article, we report that IL-17A gene transfer induces epidermal hyperplasia in Il23r-/-Rag1-/-- and Tcrδ-deficient mice, which can be prevented by neutrophil depletion. Moreover, adoptive transfer of CD11b+Gr-1hi cells, after IL-17A gene transfer, was sufficient to phenocopy the disease. We further show that the IL-17A-induced pathology was prevented in transgenic mice with impaired neutrophil extracellular trap formation and/or neutrophils with conditional deletion of the master regulator of selective autophagy, Wdfy3. Our data demonstrate a novel T cell-independent mechanism that is associated with neutrophil extracellular trap formation and selective autophagy in IL-17A-mediated epidermal hyperplasia.


Assuntos
Autofagia/imunologia , Epitélio/imunologia , Armadilhas Extracelulares/imunologia , Interleucina-17/imunologia , Linfócitos T/imunologia , Animais , Autofagia/genética , Epitélio/patologia , Armadilhas Extracelulares/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Hiperplasia/genética , Hiperplasia/imunologia , Hiperplasia/patologia , Interleucina-17/genética , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Linfócitos T/patologia
13.
Oncotarget ; 7(43): 70881-70897, 2016 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-27765920

RESUMO

Cancer cells are thought to use actin rich invadopodia to facilitate matrix degradation. Formation and maturation of invadopodia requires the co-ordained activity of Rho-GTPases, however the molecular mechanisms that underlie the invadopodia lifecycle are not fully elucidated. Previous work has suggested a formation and disassembly role for Rho family effector p-21 activated kinase 1 (PAK1) however, related family member PAK4 has not been explored. Systematic analysis of isoform specific depletion using in vitro and in vivo invasion assays revealed there are differential invadopodia-associated functions. We consolidated a role for PAK1 in the invadopodia formation phase and identified PAK4 as a novel invadopodia protein that is required for successful maturation. Furthermore, we find that PAK4 (but not PAK1) mediates invadopodia maturation likely via inhibition of PDZ-RhoGEF. Our work points to an essential role for both PAKs during melanoma invasion but provides a significant advance in our understanding of differential PAK function.


Assuntos
Melanoma/patologia , Podossomos/patologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Neoplasias Cutâneas/patologia , Quinases Ativadas por p21/metabolismo , Actinas , Animais , Linhagem Celular Tumoral , Imunofluorescência , Células HEK293 , Humanos , Invasividade Neoplásica/patologia , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Peixe-Zebra , Quinases Ativadas por p21/genética
14.
Sci Rep ; 6: 29736, 2016 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-27411958

RESUMO

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.


Assuntos
Linfócitos B/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Melanoma/imunologia , Neoplasias Cutâneas/imunologia , Linfócitos B/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Melanoma/genética , Melanoma/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Pele/imunologia , Pele/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo
15.
Curr Allergy Asthma Rep ; 16(1): 7, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26742760

RESUMO

IgG4 is the least abundant subclass of IgG in normal human serum, but elevated IgG4 levels are triggered in response to a chronic antigenic stimulus and inflammation. Since the immune system is exposed to tumor-associated antigens over a relatively long period of time, and tumors notoriously promote inflammation, it is unsurprising that IgG4 has been implicated in certain tumor types. Despite differing from other IgG subclasses by only a few amino acids, IgG4 possesses unique structural characteristics that may be responsible for its poor effector function potency and immunomodulatory properties. We describe the unique attributes of IgG4 that may be responsible for these regulatory functions, particularly in the cancer context. We discuss the inflammatory conditions in tumors that support IgG4, the emerging and proposed mechanisms by which IgG4 may contribute to tumor-associated escape from immune surveillance and implications for cancer immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Imunoglobulina G/imunologia , Neoplasias/imunologia , Evasão Tumoral/imunologia , Humanos
17.
Curr Biol ; 25(22): 2899-914, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26526369

RESUMO

Cell migration underlies metastatic dissemination of cancer cells, and fast "amoeboid" migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility. How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood. Transforming growth factor ß (TGF-ß) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development. Surprisingly, we find that in melanoma, TGF-ß promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion. Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-ß-driven signature. We observe that downstream of TGF-ß, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces. Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis. CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients. Its expression is also enriched in the invasive fronts of lesions. Functionally, we show how the TGF-ß-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth. We propose a novel mechanism by which TGF-ß-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.


Assuntos
Movimento Celular/fisiologia , Melanoma/genética , Melanoma/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Actomiosina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Forma Celular/fisiologia , Transição Epitelial-Mesenquimal , Humanos , Queratinócitos/metabolismo , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/metabolismo , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Proteína Smad2/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima
19.
Clin Exp Rheumatol ; 33(5 Suppl 93): S2-6, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26472336

RESUMO

Psoriasis is a common, chronic inflammatory skin disease associated with multi-system manifestations including arthritis and obesity. Our knowledge of the aetiology of the condition, including the key genomic, immune and environmental factors, has led to the development of targeted, precision therapies that alleviate patient morbidity. This article reviews the key pathophysiological pathways and therapeutic targets and highlights future areas of interest in psoriasis research.


Assuntos
Anti-Inflamatórios/uso terapêutico , Terapia de Alvo Molecular , Medicina de Precisão , Psoríase/tratamento farmacológico , Psoríase/etiologia , Animais , Marcadores Genéticos , Predisposição Genética para Doença , Humanos , Seleção de Pacientes , Fenótipo , Psoríase/diagnóstico , Psoríase/genética , Psoríase/imunologia , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos
20.
Oncoimmunology ; 4(11): e1032492, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26451312

RESUMO

Emerging evidence suggests pathological and immunoregulatory functions for IgG4 antibodies and IgG4+ B cells in inflammatory diseases and malignancies. We previously reported that IgG4 antibodies restrict activation of immune effector cell functions and impair humoral responses in melanoma. Here, we investigate IgG4 as a predictor of risk for disease progression in a study of human sera (n = 271: 167 melanoma patients; 104 healthy volunteers) and peripheral blood B cells (n = 71: 47 melanoma patients; 24 healthy volunteers). IgG4 (IgG4/IgGtotal) serum levels were elevated in melanoma. High relative IgG4 levels negatively correlated with progression-free survival (PFS) and overall survival. In early stage (I-II) disease, serum IgG4 was independently negatively prognostic for progression-free survival, as was elevation of IgG4+ circulating B cells (CD45+CD22+CD19+CD3-CD14-). In human tissues (n = 256; 108 cutaneous melanomas; 56 involved lymph nodes; 60 distant metastases; 32 normal skin samples) IgG4+ cell infiltrates were found in 42.6% of melanomas, 21.4% of involved lymph nodes and 30% of metastases, suggesting inflammatory conditions that favor IgG4 at the peripheral and local levels. Consistent with emerging evidence for an immunosuppressive role for IgG4, these findings indicate association of elevated IgG4 with disease progression and less favorable clinical outcomes. Characterizing immunoglobulin and other humoral immune profiles in melanoma might identify valuable prognostic tools for patient stratification and in the future lead to more effective treatments less prone to tumor-induced blockade mechanisms.

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