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1.
STAR Protoc ; 3(2): 101444, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35677615

RESUMO

Here, we present a protocol for flow cytometry analysis of endothelial cells (ECs) and CD8+ T cells in murine tumor models, at baseline and after cancer immunotherapy with anti-PD-1/anti-CTLA-4 antibodies. We provide gating strategies for identification of specific cell subsets including ECs from tumor-associated high endothelial venules (TA-HEVs), stem-like, and terminally exhausted CD8+ T cells. This protocol represents a valuable tool for the analysis of rare subsets of tumor ECs and CD8+ T cells with critical roles in antitumor immunity. For complete details on the use and execution of this protocol, please refer to Asrir et al. (2022).


Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Linfócitos T CD8-Positivos , Células Endoteliais , Citometria de Fluxo , Imunoterapia/métodos , Camundongos , Neoplasias/terapia
2.
Lipids ; 57(3): 183-195, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35318678

RESUMO

1-O-Acylceramides (1-OACs) have a fatty acid esterified to the 1-hydroxyl of the sphingosine head group of the ceramide, and recently we identified these lipids as natural components of human and mouse epidermis. Here we show epidermal 1-OACs arise shortly before birth during the establishment of the water permeability barrier in mice. Fractionation of human epidermis indicates 1-OACs concentrate in the stratum corneum. During in vitro maturation into reconstructed human epidermis, human keratinocytes dramatically increase 1-OAC levels indicating they are one source of epidermal 1-OACs. In search of potential enzymes responsible for 1-OAC synthesis in vivo, we analyzed mutant mice with deficiencies of ceramide synthases (Cers2, Cers3, or Cers4), diacylglycerol acyltransferases (Dgat1 or Dgat2), elongase of very long fatty acids 3 (Elovl3), lecithin cholesterol acyltransferase (Lcat), stearoyl-CoA desaturase 1 (Scd1), or acidic ceramidase (Asah1). Overall levels of 1-OACs did not decrease in any mouse model. In Cers3 and Dgat2-deficient epidermis they even increased in correlation with deficient skin barrier function. Dagt2 deficiency reshapes 1-OAC synthesis with an increase in 1-OACs with N-linked non-hydroxylated fatty acids and a 60% decrease compared to control in levels of 1-OACs with N-linked hydroxylated palmitate. As none of the single enzyme deficiencies we examined resulted in a lack of 1-OACs, we conclude that either there is functional redundancy in forming 1-OAC and more than one enzyme is involved, and/or an unknown acyltransferase of the epidermis performs the final step of 1-OAC synthesis, the implications of which are discussed.


Assuntos
Epiderme , Água , Animais , Ceramidas , Ácidos Graxos , Queratinócitos , Camundongos , Permeabilidade , Esfingosina N-Aciltransferase
3.
Cancer Res Commun ; 2(11): 1504-1519, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36970050

RESUMO

The receptor tyrosine kinase VEGFR-3 plays a crucial role in cancer-induced angiogenesis and lymphangiogenesis, promoting tumor development and metastasis. Here, we report the novel VEGFR-3 inhibitor EVT801 that presents a more selective and less toxic profile than two major inhibitors of VEGFRs (i.e., sorafenib and pazopanib). As monotherapy, EVT801 showed a potent antitumor effect in VEGFR-3-positive tumors, and in tumors with VEGFR-3-positive microenvironments. EVT801 suppressed VEGF-C-induced human endothelial cell proliferation in vitro and tumor (lymph)angiogenesis in different tumor mouse models. In addition to reduced tumor growth, EVT801 decreased tumor hypoxia, favored sustained tumor blood vessel homogenization (i.e., leaving fewer and overall larger vessels), and reduced important immunosuppressive cytokines (CCL4, CCL5) and myeloid-derived suppressor cells (MDSC) in circulation. Furthermore, in carcinoma mouse models, the combination of EVT801 with immune checkpoint therapy (ICT) yielded superior outcomes to either single treatment. Moreover, tumor growth inhibition was inversely correlated with levels of CCL4, CCL5, and MDSCs after treatment with EVT801, either alone or combined with ICT. Taken together, EVT801 represents a promising anti(lymph)angiogenic drug for improving ICT response rates in patients with VEGFR-3 positive tumors. Significance: The VEGFR-3 inhibitor EVT801 demonstrates superior selectivity and toxicity profile than other VEGFR-3 tyrosine kinase inhibitors. EVT801 showed potent antitumor effects in VEGFR-3-positive tumors, and tumors with VEGFR-3-positive microenvironments through blood vessel homogenization, and reduction of tumor hypoxia and limited immunosuppression. EVT801 increases immune checkpoint inhibitors' antitumor effects.


Assuntos
Neoplasias , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Humanos , Camundongos , Animais , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Neoplasias/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Imunoterapia , Microambiente Tumoral
5.
Hum Mol Genet ; 26(10): 1787-1800, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28369476

RESUMO

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of monogenic genodermatoses that encompasses non-syndromic disorders of keratinization. The pathophysiology of ARCI has been linked to a disturbance in epidermal lipid metabolism that impaired the stratum corneum function, leading to permeability barrier defects. Functional characterization of some genes involved in ARCI contributed to the identification of molecular actors involved in epidermal lipid synthesis, transport or processing. Recently, PNPLA1 has been identified as a gene causing ARCI. While other members of PNPLA family are key elements in lipid metabolism, the function of PNPLA1 remained unclear. We identified 5 novel PNPLA1 mutations in ARCI patients, mainly localized in the putative active enzymatic domain of PNPLA1. To investigate Pnpla1 biological role, we analysed Pnpla1-deficient mice. KO mice died soon after birth from severe epidermal permeability defects. Pnpla1-deficient skin presented an important impairment in the composition and organization of the epidermal lipids. Quantification of epidermal ceramide species highlighted a blockade in the production of ω-O-acylceramides with a concomitant accumulation of their precursors in the KO. The virtually loss of ω-O-acylceramides in the stratum corneum was linked to a defective lipid coverage of the resistant pericellular shell encapsulating corneocytes, the so-called cornified envelope, and most probably disorganized the extracellular lipid matrix. Finally, these defects in ω-O-acylceramides synthesis and cornified envelope formation were also evidenced in the stratum corneum from PNPLA1-mutated patients. Overall, our data support that PNPLA1/Pnpla1 is a key player in the formation of ω-O-acylceramide, a crucial process for the epidermal permeability barrier function.


Assuntos
Ictiose Lamelar/genética , Lipase/genética , Lipase/metabolismo , Idoso , Animais , Ceramidas/metabolismo , Criança , Epiderme/metabolismo , Matriz Extracelular/metabolismo , Feminino , Genes Recessivos , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Permeabilidade , Pele/metabolismo
7.
Gut ; 62(12): 1714-23, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23172891

RESUMO

OBJECTIVE: Inflammatory bowel diseases (IBD) have been intrinsically linked to a deregulated cytokine network, but novel therapeutic principles are urgently needed. Here we identify the interleukin (IL)-33 and its receptor ST2 as key negative regulators of wound healing and permeability in the colon of mice. DESIGN: Expression of IL-33 and ST2 was determined by qRT-PCR, ELISA, immunohistochemistry and western-blot analysis. Wild-type and St2(-/-) mice were used in wound healing experiments and in two experimental models of IBD triggered by 2,4,6-trinitrobenzene sulphonic acid or dextran sodium sulphate (DSS). Neutralisation of ST2 was performed by using a specific blocking antibody. RESULTS: Nuclear localisation and enhanced expression of IL-33 in myofibroblasts and enterocytes was linked to disease involvement independently of inflammation, while the expression of ST2 was primarily restricted to the colonic epithelia. In two experimental models of IBD, genetic ablation of ST2 significantly improved signs of colitis, while a sustained epithelial expression of the cyto-protective factor connexin-43 was observed in DSS-treated St2-deficient mice. Unexpectedly, absence of ST2 in non-hematopoietic cells was sufficient to protect against colitis. Consistently, specific inhibition of endogenous ST2-mediated signalling by treatment with neutralising antibody improved DSS-induced colitis. In addition, IL-33 treatment impaired epithelial barrier permeability in vitro and in vivo, whereas absence of ST2 enhanced wound healing response upon acute mechanical injury in the colon. CONCLUSIONS: Our study unveiled a novel non-hematopoietic function of IL-33 in epithelial barrier function and wound healing. Therefore, blocking the IL-33/ST2 axis may represent an efficient therapy in IBD.


Assuntos
Colite Ulcerativa/etiologia , Interleucinas/fisiologia , Receptores de Interleucina/fisiologia , Animais , Western Blotting , Células CACO-2 , Colite Ulcerativa/fisiopatologia , Colite Ulcerativa/terapia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Reação em Cadeia da Polimerase em Tempo Real , Cicatrização/fisiologia
8.
J Immunol ; 188(7): 3488-95, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22371395

RESUMO

IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33-LacZ gene trap reporter strain (Il-33(Gt/Gt)) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti-IL-33 Abs, using Il-33(Gt/Gt) (Il-33-deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33-LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.


Assuntos
Encéfalo/metabolismo , Núcleo Celular/metabolismo , Células Epiteliais/metabolismo , Inflamação/metabolismo , Interleucinas/fisiologia , Tecido Linfoide/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Proteínas Fetais/biossíntese , Proteínas Fetais/genética , Genes Reporter , Humanos , Interleucina-33 , Interleucinas/biossíntese , Interleucinas/genética , Óperon Lac , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Eosinofilia Pulmonar/induzido quimicamente , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/metabolismo , Choque Séptico/imunologia , Choque Séptico/metabolismo , Especificidade da Espécie
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