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1.
Traffic ; 22(1-2): 6-22, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33225555

RESUMO

In eukaryotic cells, clathrin-mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin-coated vesicles (CCVs) is AP-2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP-2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal-lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti-cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP-2 knockdown attenuated the global endocytic capacity, but clathrin-independent entry pathways were still operating, as indicated by persistent internalization of specific membrane-spanning and GPI-anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP-2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention.


Assuntos
Endocitose , Proteoma , Membrana Celular , Clatrina , Vesículas Revestidas por Clatrina
2.
Cancer Immunol Immunother ; 70(4): 893-907, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33006650

RESUMO

Thermal ablative therapies are standard treatments for localized hepatocellular carcinoma (HCC). In addition to local tumor destruction, ablation leads to abscopal effects in distant lesions most likely mediated by an anti-tumor immune response. Although microwave ablation (MWA) is increasingly substituting other ablative techniques, its systemic immunostimulatory effects are poorly studied. We analyzed tumor-specific immune responses in peripheral blood of HCC patients after thermal ablation with regard to T cell responses and disease outcome. While comprehensive flow cytometric analyses in sequential samples of a prospective patient cohort (n = 23) demonstrated only moderate effects of MWA on circulating immune cell subsets, fluorospot analyses of specific T cell responses against seven tumor-associated antigens (TTAs) revealed de-novo or enhanced tumor-specific immune responses in 30% of patients. This anti-tumor immune response was related to tumor control as Interferon-y and Interleukin-5 T cell responses against TAAs were more frequent in patients with a long-time remission (> 1 year) after MWA (7/16) compared to patients suffering from an early relapse (0/13 patients) and presence of tumor-specific T cell response (IFN-y and/or IL-5) was associated to longer progression-free survival (27.5 vs. 10.0 months). Digital image analysis of immunohistochemically stained archival HCC samples (n = 18) of patients receiving combined MWA and resection revealed a superior disease-free survival of patients with high T cell abundance at the time of thermal ablation (37.4 vs. 13.1 months). Our data demonstrates remarkable immune-related effects of MWA in HCC patients and provides additional evidence for a combination of local ablation and immunotherapy in this challenging disease.


Assuntos
Carcinoma Hepatocelular/imunologia , Ablação por Cateter/métodos , Imunidade/imunologia , Neoplasias Hepáticas/imunologia , Micro-Ondas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Feminino , Seguimentos , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Taxa de Sobrevida
3.
J Am Soc Nephrol ; 31(3): 532-542, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31924670

RESUMO

BACKGROUND: Inhibition of angiotensin II (AngII) signaling, a therapeutic mainstay of glomerular kidney diseases, is thought to act primarily through regulating glomerular blood flow and reducing filtration pressure. Although extravascular actions of AngII have been suggested, a direct effect of AngII on podocytes has not been demonstrated in vivo. METHODS: To study the effects of AngII on podocyte calcium levels in vivo, we used intravital microscopy of the kidney in mice expressing the calcium indicator protein GCaMP3. RESULTS: In healthy animals, podocytes displayed limited responsiveness to AngII stimulation. In contrast, in animals subjected to either adriamycin-induced acute chemical injury or genetic deletion of the podocin-encoding gene Nphs2, the consequent podocyte damage and proteinuria rendered the cells responsive to AngII and resulted in AngII-induced calcium transients in significantly more podocytes. The angiotensin type 1 receptor blocker losartan could fully inhibit this response. Also, responsiveness to AngII was at least partly mediated through the transient receptor potential channel 6, which has been implicated in podocyte calcium handling. Interestingly, loss of a single Nphs2 allele also increased podocytes' responsiveness to AngII signaling. This direct effect of AngII on injured podocytes results in increased calcium transients, which can further aggravate the underlying kidney disease. CONCLUSIONS: Our discovery that podocytes become sensitized to AngII-induced calcium signaling upon injury might explain results from large, randomized, controlled trials in which improved renal outcomes occur only in the subgroup of patients with proteinuria, indicating podocyte damage. Our findings also emphasize the need to treat every patient with a glomerular disease with either an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Losartan/farmacologia , Proteínas de Membrana/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Glomerulonefrite/metabolismo , Glomerulonefrite/fisiopatologia , Humanos , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Masculino , Camundongos , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Distribuição Aleatória , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Valores de Referência
4.
Biol Reprod ; 103(6): 1260-1274, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32915209

RESUMO

Evidence suggests that maternal obesity (MO) can aggravate placental function causing severe pathologies during the perinatal window. However, molecular changes and mechanisms of placental dysfunction remain largely unknown. This work aimed to decipher structural and molecular alterations of the placental transfer zone associated with MO. To this end, mice were fed a high fat diet (HFD) to induce obesity before mating, and pregnant dams were sacrificed at E15.5 to receive placentas for molecular, histological, and ultrastructural analysis and to assess unidirectional materno-fetal transfer capacity. Laser-capture microdissection was used to collect specifically placental cells of the labyrinth zone for proteomics profiling. Using BeWo cells, fatty acid-mediated mechanisms of adherens junction stability, cell layer permeability, and lipid accumulation were deciphered. Proteomics profiling revealed downregulation of cell adhesion markers in the labyrinth zone of obese dams, and disturbed syncytial fusion and detachment of the basement membrane (BM) within this zone was observed, next to an increase in materno-fetal transfer in vivo across the placenta. We found that fetuses of obese dams develop a growth restriction and in those placentas, labyrinth zone volume-fraction was significantly reduced. Linoleic acid was shown to mediate beta-catenin level and increase cell layer permeability in vitro. Thus, MO causes fetal growth restriction, molecular and structural changes in the transfer zone leading to impaired trophoblast differentiation, BM disruption, and placental dysfunction despite increased materno-fetal transfer capacity. These adverse effects are probably mediated by fatty acids found in HFD demonstrating the need for obesity treatment to mitigate placental dysfunction and prevent offspring pathologies.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Obesidade/induzido quimicamente , Placenta/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos , Animais , Biomarcadores , Adesão Celular , Diferenciação Celular , Feminino , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Placenta/fisiologia , Placenta/ultraestrutura , Gravidez , Proteômica , Distribuição Aleatória , Transcriptoma
5.
Biol Chem ; 394(11): 1475-83, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23893688

RESUMO

Prion diseases are fatal neurodegenerative diseases which occur as sporadic, genetic, and transmissible disorders. A molecular hallmark of prion diseases is the conformational conversion of the host-encoded cellular form of the prion protein (PrPC) into its misfolded pathogenic isoform (PrPSc). PrPSc is the main component of the pathological and infectious prion agent. The study of the conversion mechanism from PrPC to PrPSc is a major field in prion research. PrPC is glycosylated and attached to the plasma membrane via its glycosyl phosphatidyl inositol (GPI)-anchor. In this study we established and characterised the expression of fully posttranslationally modified mammalian Syrian golden hamster PrPC in the yeast Pichia pastoris using native PrPC-specific N- and C-terminal signal sequences. In vivo as well as in vitro-studies demonstrated that the signal sequences controlled posttranslational processing and trafficking of native PrPC, resulting in PrPC localised in the plasma membrane of P. pastoris. In addition, the glycosylation pattern of native PrPC could be confirmed.


Assuntos
Pichia/química , Pichia/genética , Proteínas PrPSc/química , Proteínas PrPSc/genética , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular Transformada , Cricetinae , Vetores Genéticos , Glicosilação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mesocricetus , Pichia/metabolismo , Proteínas PrPSc/metabolismo , Processamento de Proteína Pós-Traducional/genética , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , Transfecção
6.
Clin Cancer Res ; 28(8): 1712-1723, 2022 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-35191474

RESUMO

PURPOSE: An increased risk to develop cancer is one of the most challenging negative side effects of long-term immunosuppression in organ transplant recipients and impaired cancer immunosurveillance is assumed as underlying mechanism. This study aims to elucidate transplant-related changes in the tumor immune microenvironment (TME) of cancer. EXPERIMENTAL DESIGN: Data from 123 organ transplant recipients (kidney, heart, lung, and liver) were compared with historic data from non-immunosuppressed patients. Digital image analysis of whole-section slides was used to assess abundance and spatial distribution of T cells and tertiary lymphoid structures (TLS) in the TME of 117 tumor samples. Expression of programmed cell death 1 ligand 1 (PD-L1) and human-leucocyte-antigen class I (HLA-I) was assessed on tissue microarrays. RESULTS: We found a remarkably reduced immune infiltrate in the center tumor (CT) regions as well as the invasive margins (IM) of post-transplant cancers. These differences were more pronounced in the IM than in the CT and larger for CD8+ T cells than for CD3+ T cells. The Immune-score integrating results from CT and IM was also lower in transplant recipients. Density of TLS was lower in cancer samples of transplant recipients. The fraction of samples with PD-L1 expression was higher in controls whereas decreased expression of HLA-I was more common in transplant recipients. CONCLUSIONS: Our study demonstrates the impact of immunosuppression on the TME and supports impaired cancer immunosurveillance as important cause of post-transplant cancer. Modern immunosuppressive protocols and cancer therapies should consider the distinct immune microenvironment of post-transplant malignancies.


Assuntos
Neoplasias , Estruturas Linfoides Terciárias , Antígeno B7-H1 , Antígenos de Histocompatibilidade Classe I , Humanos , Linfócitos do Interstício Tumoral , Monitorização Imunológica , Neoplasias/etiologia , Neoplasias/metabolismo , Microambiente Tumoral
7.
Invest Ophthalmol Vis Sci ; 61(12): 14, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33057669

RESUMO

Purpose: The purpose of this study was to gain insights on the pathogenesis of chronic progressive external ophthalmoplegia, thus we investigated the vulnerability of five extra ocular muscles (EOMs) fiber types to pathogenic mitochondrial DNA deletions in a mouse model expressing a mutated mitochondrial helicase TWINKLE. Methods: Consecutive pairs of EOM sections were analyzed by cytochrome C oxidase (COX)/succinate dehydrogenase (SDH) assay and fiber type specific immunohistochemistry (type I, IIA, IIB, embryonic, and EOM-specific staining). Results: The mean average of COX deficient fibers (COX-) in the recti muscles of mutant mice was 1.04 ± 0.52% at 12 months and increased with age (7.01 ± 1.53% at 24 months). A significant proportion of these COX- fibers were of the fast-twitch, glycolytic type IIB (> 50% and > 35% total COX- fibers at 12 and 24 months, respectively), whereas embryonic myosin heavy chain-expressing fibers were almost completely spared. Furthermore, the proportion of COX- fibers in the type IIB-rich retractor bulbi muscle was > 2-fold higher compared to the M. recti at both 12 (2.6 ± 0.78%) and 24 months (20.85 ± 2.69%). Collectively, these results demonstrate a selective vulnerability of type IIB fibers to mitochondrial DNA (mtDNA) deletions in EOMs and retractor bulbi muscle. We also show that EOMs of mutant mice display histopathological abnormalities, including altered fiber type composition, increased fibrosis, ragged red fibers, and infiltration of mononucleated nonmuscle cells. Conclusions: Our results point to the existence of fiber type IIB-intrinsic factors and/or molecular mechanisms that predispose them to increased generation, clonal expansion, and detrimental effects of mtDNA deletions.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias Musculares/patologia , Doenças Mitocondriais/patologia , Fibras Musculares de Contração Rápida/patologia , Músculos Oculomotores/patologia , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Musculares/enzimologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Cadeias Pesadas de Miosina/metabolismo , Músculos Oculomotores/enzimologia , Oftalmoplegia Externa Progressiva Crônica/etiologia , Reação em Cadeia da Polimerase em Tempo Real , Succinato Desidrogenase/metabolismo
8.
Life Sci Alliance ; 3(6)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32321733

RESUMO

Lipid droplets (LDs) are metabolic organelles that store neutral lipids and dynamically respond to changes in energy availability by accumulating or mobilizing triacylglycerols (TAGs). How the plastic behavior of LDs is regulated is poorly understood. Hereditary spastic paraplegia is a central motor axonopathy predominantly caused by mutations in SPAST, encoding the microtubule-severing protein spastin. The spastin-M1 isoform localizes to nascent LDs in mammalian cells; however, the mechanistic significance of this targeting is not fully explained. Here, we show that tightly controlled levels of spastin-M1 are required to inhibit LD biogenesis and TAG accumulation. Spastin-M1 maintains the morphogenesis of the ER when TAG synthesis is prevented, independent from microtubule binding. Moreover, spastin plays a microtubule-dependent role in mediating the dispersion of LDs from the ER upon glucose starvation. Our results reveal a dual role of spastin to shape ER tubules and to regulate LD movement along microtubules, opening new perspectives for the pathogenesis of hereditary spastic paraplegia.


Assuntos
Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Microtúbulos/metabolismo , Transdução de Sinais/genética , Paraplegia Espástica Hereditária/metabolismo , Espastina/deficiência , Animais , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Isoenzimas , Camundongos , Neurônios Motores/metabolismo , Mutação , Paraplegia Espástica Hereditária/genética , Espastina/genética , Transfecção , Triglicerídeos/metabolismo
9.
Nutrients ; 12(2)2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31979004

RESUMO

Obesity during pregnancy is a known health risk for mother and child. Since obesity is associated with increased inflammatory markers, our objectives were to determine interleukin-6 (IL-6) levels in obese mice and to examine the effect of IL-6 on placental endothelial cells. Placentas, blood, and adipose tissue of C57BL/6N mice, kept on high fat diet before and during pregnancy, were harvested at E15.5. Serum IL-6 levels were determined and endothelial cell markers and IL-6 expression were measured by qRT-PCR and western blot. Immunostaining was used to determine surface and length densities of fetal capillary profiles and placental endothelial cell homeostasis. Human placental vein endothelial cells were cultured and subjected to proliferation, apoptosis, senescence, and tube formation assays after stimulation with hyperIL-6. Placental endothelial cell markers were downregulated and the percentage of senescent endothelial cells was higher in the placental exchange zone of obese dams and placental vascularization was strongly reduced. Additionally, maternal IL-6 serum levels and IL-6 protein levels in adipose tissue were increased. Stimulation with hyperIL-6 provoked a dose dependent increase of senescence in cultured endothelial cells without any effects on proliferation or apoptosis. Diet-induced maternal obesity led to an IUGR phenotype accompanied by increased maternal IL-6 serum levels. In the placenta of obese dams, this may result in a disturbed endothelial cell homeostasis and impaired fetal vasculature. Cell culture experiments confirmed that IL-6 is capable of inducing endothelial cell senescence.


Assuntos
Células Endoteliais/metabolismo , Interleucina-6/metabolismo , Obesidade Materna/metabolismo , Placenta/metabolismo , Tecido Adiposo/metabolismo , Animais , Técnicas de Cultura de Células , Senescência Celular , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Feto/irrigação sanguínea , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Obesidade Materna/etiologia , Gravidez
10.
Cell Stem Cell ; 24(5): 736-752.e12, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982769

RESUMO

The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivo due to differences in gene dosage sensitivity between mice and humans. This is illustrated by branchiooculofacial syndrome (BOFS), a rare congenital disorder caused by heterozygous mutations within TFAP2A, a neural crest regulator for which humans, but not mice, are haploinsufficient. Here, we present a BOFS patient carrying a heterozygous inversion with one breakpoint located within a topologically associating domain (TAD) containing enhancers essential for TFAP2A expression in human neural crest cells (hNCCs). Using patient-specific hiPSCs, we show that, although the inversion shuffles the TFAP2A hNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption. Instead, the inversion disconnects one TFAP2A allele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2A expression in patient hNCCs. Our work illustrates the power of hiPSC differentiation to unveil long-range pathomechanisms.


Assuntos
Síndrome Brânquio-Otorrenal/genética , Variação Estrutural do Genoma/genética , Mutação/genética , Crista Neural/fisiologia , Fator de Transcrição AP-2/metabolismo , Adolescente , Alelos , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Elementos Facilitadores Genéticos/genética , Haploinsuficiência , Humanos , Masculino , Camundongos , Análise de Célula Única , Fator de Transcrição AP-2/genética
11.
Nat Commun ; 9(1): 3622, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30190464

RESUMO

Increasing brown adipose tissue (BAT) thermogenesis in mice and humans improves metabolic health and understanding BAT function is of interest for novel approaches to counteract obesity. The role of long noncoding RNAs (lncRNAs) in these processes remains elusive. We observed maternally expressed, imprinted lncRNA H19 increased upon cold-activation and decreased in obesity in BAT. Inverse correlations of H19 with BMI were also observed in humans. H19 overexpression promoted, while silencing of H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown but not white adipocytes. In vivo, H19 overexpression protected against DIO, improved insulin sensitivity and mitochondrial biogenesis, whereas fat H19 loss sensitized towards HFD weight gains. Strikingly, paternally expressed genes (PEG) were largely absent from BAT and we demonstrated that H19 recruits PEG-inactivating H19-MBD1 complexes and acts as BAT-selective PEG gatekeeper. This has implications for our understanding how monoallelic gene expression affects metabolism in rodents and, potentially, humans.


Assuntos
Tecido Adiposo Marrom/fisiologia , Impressão Genômica , Obesidade/genética , RNA Longo não Codificante/genética , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Obesidade/etiologia
12.
J Cell Biol ; 216(3): 675-693, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28188211

RESUMO

Mitochondria are essential organelles that host crucial metabolic pathways and produce adenosine triphosphate. The mitochondrial proteome is heterogeneous among tissues and can dynamically change in response to different metabolic conditions. Although the transcriptional programs that govern mitochondrial biogenesis and respiratory function are well known, posttranscriptional regulatory mechanisms remain unclear. In this study, we show that the cytosolic RNA-binding protein clustered mitochondria homologue (CLUH) regulates the expression of a mitochondrial protein network supporting key metabolic programs required under nutrient deprivation. CLUH exerts its function by controlling the stability and translation of target messenger RNAs. In the absence of Cluh, mitochondria are severely depleted of crucial enzymes involved in catabolic energy-converting pathways. CLUH preserves oxidative mitochondrial function and glucose homeostasis, thus preventing death at the fetal-neonatal transition. In the adult liver, CLUH ensures maximal respiration capacity and the metabolic response to starvation. Our results shed new light on the posttranscriptional mechanisms controlling the expression of mitochondrial proteins and suggest novel strategies to tailor mitochondrial function to physiological and pathological conditions.


Assuntos
Mitocôndrias/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Citosol/metabolismo , Citosol/fisiologia , Metabolismo Energético/fisiologia , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Metabolismo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Interferência de RNA/fisiologia
13.
Oncotarget ; 8(27): 44418-44433, 2017 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-28574843

RESUMO

The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors (n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa (n = 7) of 34 treatment-naïve HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA-/CCR7-). Naïve T cells (CD45RA+/CCR7+) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4+/CD25+/CD127low and CD4+/CD39+) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the 'Immunoscore' (CD3+ and CD8+ cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance.


Assuntos
Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Imunomodulação , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Biomarcadores , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Carcinoma de Células Escamosas/patologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imunofenotipagem , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Fenótipo , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Microambiente Tumoral/imunologia
14.
Biomaterials ; 32(8): 2070-6, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21167596

RESUMO

Substrate-bound gradients expressed in numerous spatio-temporal patterns play a crucial role during the development of complex neural circuits. A deeper understanding of the axon guidance mechanism is provided by studying the effect of a defined substrate-bound cue on a confined neural network. In this study, we constructed a discontinuous substrate-bound gradient to control neuronal cell position, the path of neurite growth, and axon directionality. A variety of gradient patterns, with slight changes in slope, width, and length were designed and fabricated by microcontact printing using laminin/poly-l-lysine (PLL) or PLL alone. The gradients were tested for neurite growth and their impact on axon guidance of embryonic rat cortical neurons. The neurite length was determined and the axon was evaluated by Tau-1 immunostaining. We found that the microgradients of laminin/PLL and PLL directed neurons' adhesion, differentially controlled the neurite growth, and guided up to 84% of the axons. The effect of the protein micropattern on axon guidance and neurite growth depended on the protein and geometric parameters used. Our approach proved to be very successful in guiding axons of single multipolar neurons with very high efficiency. It could thereby be useful to engineer defined neural networks for analyzing signal processing of functional circuits, as well as to unravel fundamental questions of the axon guidance mechanism.


Assuntos
Axônios/metabolismo , Movimento Celular/fisiologia , Córtex Cerebral/citologia , Neurônios/citologia , Neurônios/fisiologia , Animais , Axônios/ultraestrutura , Técnicas de Cultura de Células , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Laminina/metabolismo , Teste de Materiais , Neuritos/metabolismo , Neuritos/ultraestrutura , Polilisina/metabolismo , Impressão , Ratos , Ratos Wistar , Propriedades de Superfície
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