Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
Mais filtros

Bases de dados
Tipo de documento
Intervalo de ano de publicação
1.
Nat Immunol ; 23(8): 1246-1255, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35817845

RESUMO

Lymph nodes (LNs) comprise two main structural elements: fibroblastic reticular cells that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. Immunological challenge causes LNs to increase more than tenfold in size within a few days. Here, we characterized the biomechanics of LN swelling on the cellular and organ scale. We identified lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing fibroblastic reticular cells of the T-zone (TRCs) and their associated conduits to stretch. After an initial phase of relaxation, TRCs sensed the resulting strain through cell matrix adhesions, which coordinated local growth and remodeling of the stromal network. While the expanded TRC network readopted its typical configuration, a massive fibrotic reaction of the organ capsule set in and countered further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multitier fashion.


Assuntos
Linfonodos , Células Estromais , Animais , Fibroblastos , Linfócitos , Camundongos , Camundongos Endogâmicos C57BL
2.
Nat Immunol ; 22(5): 571-585, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33903764

RESUMO

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.


Assuntos
Células Dendríticas Foliculares/imunologia , Fibroblastos/imunologia , Linfonodos/imunologia , Células Estromais/imunologia , Idoso , Animais , Apoptose/genética , Apoptose/imunologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Dendríticas Foliculares/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Humanos , Imunidade Celular/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfonodos/citologia , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Análise de Célula Única , Células Estromais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
3.
Nature ; 618(7966): 827-833, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37258670

RESUMO

The immune phenotype of a tumour is a key predictor of its response to immunotherapy1-4. Patients who respond to checkpoint blockade generally present with immune-inflamed5-7 tumours that are highly infiltrated by T cells. However, not all inflamed tumours respond to therapy, and even lower response rates occur among tumours that lack T cells (immune desert) or that spatially exclude T cells to the periphery of the tumour lesion (immune excluded)8. Despite the importance of these tumour immune phenotypes in patients, little is known about their development, heterogeneity or dynamics owing to the technical difficulty of tracking these features in situ. Here we introduce skin tumour array by microporation (STAMP)-a preclinical approach that combines high-throughput time-lapse imaging with next-generation sequencing of tumour arrays. Using STAMP, we followed the development of thousands of arrayed tumours in vivo to show that tumour immune phenotypes and outcomes vary between adjacent tumours and are controlled by local factors within the tumour microenvironment. Particularly, the recruitment of T cells by fibroblasts and monocytes into the tumour core was supportive of T cell cytotoxic activity and tumour rejection. Tumour immune phenotypes were dynamic over time and an early conversion to an immune-inflamed phenotype was predictive of spontaneous or therapy-induced tumour rejection. Thus, STAMP captures the dynamic relationships of the spatial, cellular and molecular components of tumour rejection and has the potential to translate therapeutic concepts into successful clinical strategies.


Assuntos
Neoplasias , Linfócitos T , Microambiente Tumoral , Humanos , Imunoterapia , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Linfócitos T/imunologia , Fenótipo , Fibroblastos , Monócitos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico
4.
Nat Immunol ; 17(12): 1361-1372, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27798618

RESUMO

Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.


Assuntos
Infecções por Bactérias Gram-Negativas/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Heme/metabolismo , Hemólise/imunologia , Macrófagos/imunologia , Fagocitose , Sepse/imunologia , Animais , Antibacterianos/uso terapêutico , Citoesqueleto/metabolismo , Feminino , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Fatores de Troca do Nucleotídeo Guanina/genética , Heme Oxigenase-1/genética , Hemólise/efeitos dos fármacos , Humanos , Evasão da Resposta Imune , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Quinina/uso terapêutico , Células RAW 264.7 , Sepse/tratamento farmacológico , Proteína cdc42 de Ligação ao GTP/metabolismo
5.
Nature ; 595(7868): 578-584, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34135508

RESUMO

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis3-5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions1. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial-mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8+ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions.


Assuntos
Carcinogênese , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Macrófagos/imunologia , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos/imunologia , Transição Epitelial-Mesenquimal , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Linfócitos T Reguladores/imunologia
6.
Int J Mol Sci ; 23(10)2022 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-35628310

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is a detrimental premature aging disease caused by a point mutation in the human LMNA gene. This mutation results in the abnormal accumulation of a truncated pre-lamin A protein called progerin. Among the drastically accelerated signs of aging in HGPS patients, severe skin phenotypes such as alopecia and sclerotic skins always develop with the disease progression. Here, we studied the HGPS molecular mechanisms focusing on early skin development by differentiating patient-derived induced pluripotent stem cells (iPSCs) to a keratinocyte lineage. Interestingly, HGPS iPSCs showed an accelerated commitment to the keratinocyte lineage than the normal control. To study potential signaling pathways that accelerated skin development in HGPS, we investigated the WNT pathway components during HGPS iPSCs-keratinocytes induction. Surprisingly, despite the unaffected ß-catenin activity, the expression of a critical WNT transcription factor LEF1 was diminished from an early stage in HGPS iPSCs-keratinocytes differentiation. A chromatin immunoprecipitation (ChIP) experiment further revealed strong bindings of LEF1 to the early-stage epithelial developmental markers K8 and K18 and that the LEF1 silencing by siRNA down-regulates the K8/K18 transcription. During the iPSCs-keratinocytes differentiation, correction of HGPS mutation by Adenine base editing (ABE), while in a partial level, rescued the phenotypes for accelerated keratinocyte lineage-commitment. ABE also reduced the cell death in HGPS iPSCs-derived keratinocytes. These findings brought new insight into the molecular basis and therapeutic application for the skin abnormalities in HGPS.


Assuntos
Células-Tronco Pluripotentes Induzidas , Fator 1 de Ligação ao Facilitador Linfoide , Progéria , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Progéria/genética , Progéria/metabolismo , Via de Sinalização Wnt
7.
Proc Natl Acad Sci U S A ; 114(45): 11832-11837, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29078370

RESUMO

The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes.


Assuntos
Algoritmos , Reprogramação Celular/genética , Biologia Computacional/métodos , Fibroblastos/citologia , Fatores de Transcrição/genética , Sítios de Ligação/genética , Ciclo Celular/genética , Diferenciação Celular , Células Cultivadas , Reprogramação Celular/fisiologia , Perfilação da Expressão Gênica , Genoma Humano/genética , Humanos , Modelos Genéticos
8.
J Immunol ; 199(2): 531-546, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28607115

RESUMO

The lysosome-associated membrane protein (LAMP) family includes the dendritic cell endocytic receptors DC-LAMP and CD68, as well as LAMP-1 and LAMP-2. In this study we identify LAMP-1 (CD107a) and LAMP-2 (CD107b) on the surface of human monocyte-derived dendritic cells (MoDC) and show only LAMP-2 is internalized after ligation by specific Abs, including H4B4, and traffics rapidly but transiently to the MHC class II loading compartment, as does Ag conjugated to H4B4. However, pulsing MoDC with conjugates of primary (keyhole limpet hemocyanin; KLH) and recall (Bet v 1) Ags (H4B4*KLH and H4B4*Bet v 1) induced significantly less CD4 cell proliferation than pulsing with native Ag or Ag conjugated to control mAb (ISO*KLH and ISO*Bet v 1). In H4B4*KLH-pulsed MoDC, the duration of KLH residence in MHC class II loading compartments was significantly reduced, as were surface HLA-DR and DR-bound KLH-derived peptides. Paradoxically, MoDC pulsed with H4B4*KLH, but not the other KLH preparations, induced robust proliferation of CD4 cells separated from them by a transwell membrane, indicating factors in the supernatant were responsible. Furthermore, extracellular vesicles from supernatants of H4B4*KLH-pulsed MoDC contained significantly more HLA-DR and KLH than those purified from control MoDC, and KLH was concentrated specifically in exosomes that were a uniquely effective source of Ag in standard T cell proliferation assays. In summary, we identify LAMP-2 as an endocytic receptor on human MoDC that routes cargo into unusual Ag processing pathways, which reduces surface expression of Ag-derived peptides while selectively enriching Ag within immunogenic exosomes. This novel pathway has implications for the initiation of immune responses both locally and at distant sites.


Assuntos
Apresentação de Antígeno , Células Dendríticas/imunologia , Exossomos/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Animais , Formação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Antígenos HLA/imunologia , Hemocianinas/imunologia , Humanos , Ativação Linfocitária , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Camundongos , Monócitos/imunologia , Peptídeos/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo
9.
Chromosoma ; 126(5): 655-667, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28343235

RESUMO

Human chromosomes occupy distinct territories in the interphase nucleus. Such chromosome territories (CTs) are positioned according to gene density. Gene-rich CTs are generally located in the center of the nucleus, while gene-poor CTs are positioned more towards the nuclear periphery. However, the association between gene expression levels and the radial positioning of genes within the CT is still under debate. In the present study, we performed three-dimensional fluorescence in situ hybridization experiments in the colorectal cancer cell lines DLD-1 and LoVo using whole chromosome painting probes for chromosomes 8 and 11 and BAC clones targeting four genes with different expression levels assessed by gene expression arrays and RT-PCR. Our results confirmed that the two over-expressed genes, MYC on chromosome 8 and CCND1 on chromosome 11, are located significantly further away from the center of the CT compared to under-expressed genes on the same chromosomes, i.e., DLC1 and SCN3B. When CCND1 expression was reduced after silencing the major transcription factor of the WNT/ß-catenin signaling pathway, TCF7L2, the gene was repositioned and mostly detected in the interior of the CT. Thus, we suggest a non-random distribution in which over-expressed genes are located more towards the periphery of the respective CTs.


Assuntos
Núcleo Celular/metabolismo , Cromossomos Humanos/metabolismo , Interfase , Transdução de Sinais , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Cromossomos Humanos/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente
10.
FASEB J ; 28(8): 3423-34, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24732130

RESUMO

Nuclear lamin B1 (LMNB1) constitutes one of the major structural proteins in the lamina mesh. We silenced the expression of LMNB1 by RNA interference in the colon cancer cell line DLD-1 and showed a dramatic redistribution of H3K27me3 from the periphery to a more homogeneous nuclear dispersion. In addition, we observed telomere attrition and an increased frequency of micronuclei and nuclear blebs. By 3D-FISH analyses, we demonstrated that the volume and surface of chromosome territories were significantly larger in LMNB1-depleted cells, suggesting that LMNB1 is required to maintain chromatin condensation in interphase nuclei. These changes led to a prolonged S phase due to activation of Chk1. Finally, silencing of LMNB1 resulted in extensive changes in alternative splicing of multiple genes and in a higher number of enlarged nuclear speckles. Taken together, our results suggest a mechanistic role of the nuclear lamina in the organization of chromosome territories, maintenance of genome integrity and proper gene splicing.


Assuntos
Lamina Tipo B/fisiologia , Fase S/fisiologia , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Núcleo Celular/ultraestrutura , Cromossomos Humanos Par 18/ultraestrutura , Cromossomos Humanos Par 19/ultraestrutura , Neoplasias do Colo/patologia , Heterocromatina/fisiologia , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Interfase , Lamina Tipo B/deficiência , Metilação , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Fase S/efeitos dos fármacos , Encurtamento do Telômero/efeitos dos fármacos
11.
J Am Soc Nephrol ; 25(3): 455-63, 2014 03.
Artigo em Inglês | MEDLINE | ID: mdl-24203998

RESUMO

Pauci-immune focal necrotizing GN (piFNGN) is usually associated with ANCAs that are thought to be pathogenic. However, 10%-15% of patients are ANCA negative and the cause of their injury is unknown. We previously reported a high frequency of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in ANCA-associated piFNGN, and have now investigated whether the same is true in ANCA-negative patients. Of 11 patients, 8 (73%) had anti-hLAMP-2 antibodies detected by ELISA and confirmed by immunoblotting and indirect immunofluorescence. The autoantibodies from all 8 patients bound to native LAMP-2 purified from human glomeruli and recombinant hLAMP-2 expressed in ldlD cells, both with molecular masses of 110 kD. However, in contrast to anti-LAMP-2 antibodies from ANCA-positive patients, these antibodies from ANCA-negative patients failed to bind the more complexly glycosylated native neutrophil hLAMP-2 (190 kD). Treatment with the deglycosylating enzyme, endo-ß-galactosidase, reduced the mass of neutrophil hLAMP-2 to 110 kD and enabled autoantibody binding. Similarly, pretreating neutrophils with endo-ß-galactosidase or neuraminidase converted ANCA assay results from negative to positive. Finally, IgG from LAMP-2-positive ANCA-negative patients bound specifically to normal human kidney sections and to human glomerular endothelial cells in culture. In conclusion, in patients with ANCA-negative piFNGN, we have identified autoantibodies to hLAMP-2 that bind native glomerular but not neutrophil hLAMP-2, suggesting a role in pathogenesis.


Assuntos
Glomerulonefrite/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Adolescente , Adulto , Idoso , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Autoanticorpos/sangue , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
12.
PNAS Nexus ; 3(9): pgae374, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39262854

RESUMO

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic Escherichia coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse precolonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC(ΦStx2a). This study suggests that commensal E. coli engineered to deliver payloads that block essential virulence determinants can be developed as a new means to prevent and potentially treat infections including those due to antibiotic resistant microbes.

13.
bioRxiv ; 2024 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-39131305

RESUMO

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC(ΦStx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality.

14.
Cancers (Basel) ; 14(3)2022 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-35159051

RESUMO

Colon cancer tumorigenesis occurs incrementally. The process involves the acquisition of mutations which typically follow an established pattern: activation of WNT signaling, activation of RAS signaling, and inhibition of TGF-ß signaling. This arrangement recapitulates, to some degree, the stem cell niche of the intestinal epithelium, which maintains WNT and EGF activity while suppressing TGF-ß. The resemblance between the intestinal stem cell environment and colon cancer suggests that the concerted activity of these pathways generates and maintains a potent growth-inducing stimulus. However, each pathway has a myriad of downstream targets, making it difficult to identify which aspects of these pathways are drivers. To address this, we utilize the cell cycle, the ultimate regulator of cell proliferation, as a foundation for cross-pathway integration. We attempt to generate an overview of colon cancer signaling patterns by integrating the major colon cancer signaling pathways in the context of cell replication, specifically, the entrance from G1 into S-phase.

15.
Neoplasia ; 23(2): 257-269, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33422939

RESUMO

Canonical Wnt signaling is crucial for intestinal homeostasis as TCF4, the major Wnt signaling effector in the intestines, is required for stem cell maintenance. The capability of TCF4 to maintain the stem cell phenotype is contingent upon ß-catenin, a potent transcriptional activator, which interacts with histone acetyltransferases and chromatin remodeling complexes. We used RNAi to explore the influence of TCF4 on chromatin structure (Hi-C) and gene expression (RNA sequencing) across a 72-hour time series in colon cancer. We found that TCF4 reduction results in a disproportionate up-regulation of gene expression, including a powerful induction of SOX2. Integration of RNA sequencing and Hi-C data revealed a TAD boundary loss, which occurred concomitantly with the over-expression of a cluster of CEACAM genes on chromosome 19. We identified EMT and E2F as the 2 most deregulated pathways upon TCF4 depletion and LUM, TMPO, and AURKA as highly influential genes in these networks using measures of centrality. Results from gene expression, chromatin structure, and centrality analyses were integrated to generate a list of candidate transcription factors crucial for colon cancer cell homeostasis. The top ranked factor was c-JUN, an oncoprotein known to interact with TCF4 and ß-catenin, confirming the usefulness of this approach.


Assuntos
Regulação da Expressão Gênica , Inativação Gênica , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Neoplasias do Colo/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Família Multigênica , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt
16.
Genome Med ; 13(1): 93, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34034815

RESUMO

BACKGROUND: Many carcinomas have recurrent chromosomal aneuploidies specific to the tissue of tumor origin. The reason for this specificity is not completely understood. METHODS: In this study, we looked at the frequency of chromosomal arm gains and losses in different cancer types from the The Cancer Genome Atlas (TCGA) and compared them to the mean gene expression of each chromosome arm in corresponding normal tissues of origin from the Genotype-Tissue Expression (GTEx) database, in addition to the distribution of tissue-specific oncogenes and tumor suppressors on different chromosome arms. RESULTS: This analysis revealed a complex picture of factors driving tumor karyotype evolution in which some recurrent chromosomal copy number reflect the chromosome arm-wide gene expression levels of the their normal tissue of tumor origin. CONCLUSIONS: We conclude that the cancer type-specific distribution of chromosomal arm gains and losses is potentially "hardwiring" gene expression levels characteristic of the normal tissue of tumor origin, in addition to broadly modulating the expression of tissue-specific tumor driver genes.


Assuntos
Aneuploidia , Biomarcadores Tumorais , Mapeamento Cromossômico , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Algoritmos , Análise por Conglomerados , Biologia Computacional/métodos , Metilação de DNA , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Mutação , Oncogenes , Especificidade de Órgãos/genética
17.
Clin Cancer Res ; 26(13): 3468-3480, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32253233

RESUMO

PURPOSE: The standard treatment of patients with locally advanced rectal cancer consists of preoperative chemoradiotherapy (CRT) followed by surgery. However, the response of individual tumors to CRT is extremely diverse, presenting a clinical dilemma. This broad variability in treatment response is likely attributable to intratumor heterogeneity (ITH). EXPERIMENTAL DESIGN: We addressed the impact of ITH on response to CRT by establishing single-cell-derived cell lines (SCDCL) from a treatment-naïve rectal cancer biopsy after xenografting. RESULTS: Individual SCDCLs derived from the same tumor responded profoundly different to CRT in vitro. Clonal reconstruction of the tumor and derived cell lines based on whole-exome sequencing revealed nine separate clusters with distinct proportions in the SCDCLs. Missense mutations in SV2A and ZWINT were clonal in the resistant SCDCL, but not detected in the sensitive SCDCL. Single-cell genetic analysis by multiplex FISH revealed the expansion of a clone with a loss of PIK3CA in the resistant SCDCL. Gene expression profiling by tRNA-sequencing identified the activation of the Wnt, Akt, and Hedgehog signaling pathways in the resistant SCDCLs. Wnt pathway activation in the resistant SCDCLs was confirmed using a reporter assay. CONCLUSIONS: Our model system of patient-derived SCDCLs provides evidence for the critical role of ITH for treatment response in patients with rectal cancer and shows that distinct genetic aberration profiles are associated with treatment response. We identified specific pathways as the molecular basis of treatment response of individual clones, which could be targeted in resistant subclones of a heterogenous tumor.


Assuntos
Heterogeneidade Genética , Neoplasias Retais/etiologia , Neoplasias Retais/patologia , Análise de Célula Única , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Terapia Combinada , Hibridização Genômica Comparativa , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Neoplasias Retais/metabolismo , Neoplasias Retais/terapia , Transdução de Sinais , Resultado do Tratamento , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Neoplasia ; 21(7): 721-729, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174021

RESUMO

Chromosomal aneuploidy is a defining feature of epithelial cancers. The pattern of aneuploidies is cancer-type specific. For instance, the gain of chromosome 13 occurs almost exclusively in colorectal cancer. We used microcell-mediated chromosome transfer to generate gains of chromosome 13 in the diploid human colorectal cancer cell line DLD-1. Extra copies of chromosome 13 resulted in a significant and reproducible up-regulation of transcript levels of genes on chromosome 13 (P = .0004, FDR = 0.01) and a genome-wide transcriptional deregulation in all 8 independent clones generated. Genes contained in two clusters were particularly affected: the first cluster on cytoband 13q13 contained 7 highly up-regulated genes (NBEA, MAB21L1, DCLK1, SOHLH2, CCDC169, SPG20 and CCNA1, P = .0003) in all clones. A second cluster was located on 13q32.1 and contained five upregulated genes (ABCC4, CLDN10, DZIP1, DNAJC3 and UGGT2, P = .003). One gene, RASL11A, localized on chromosome band 13q12.2, escaped the copy number-induced overexpression and was reproducibly and significantly down-regulated on the mRNA and protein level (P = .0001, FDR = 0.002). RASL11A expression levels were also lower in primary colorectal tumors as compared to matched normal mucosa (P = .0001, FDR = 0.0001. Overexpression of RASL11A increases cell proliferation and anchorage independent growth while decreasing cell migration in +13 clones. In summary, we observed a strict correlation of genomic copy number and resident gene expression levels, and aneuploidy dependent consistent genome-wide transcriptional deregulation.


Assuntos
Cromossomos/genética , Neoplasias Colorretais/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Transcriptoma/genética , Aneuploidia , Neoplasias Colorretais/patologia , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , Ativação Transcricional/genética
19.
Clin Exp Metastasis ; 35(5-6): 443-454, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29796854

RESUMO

Breast cancer metastasizes through the lymphovascular system to the regional lymph nodes in the axilla and to both visceral and non-visceral sites. Renewed interest in the route by which tumor cells gain access to blood and lymphatic capillaries are the subject of research at mechanical, anatomic, pathologic, genetic, epidemiologic and molecular levels. Two papers presented at the 7th International Symposium on Cancer Metastasis in San Francisco showed tumor cells entering the systemic circulation through the sentinel lymph node. This information challenges the current paradigm where clinicians believe that access is gained through intra- and peri-tumoral blood vessels and that metastasis to axillary lymph nodes is an interesting epi-phenomenon. The sentinel lymph node era has changed the modern surgical approach to the axilla and the basis of this change is summarized in this paper. A new approach to the management of axillary metastases after systemic therapy relies on determining whether there is a complete pathologic response; if no tumor is found in the previously biopsied node, a complete axillary lymph node dissection may be avoided. African American women seem to inherit a trait from West African ancestors and tend to develop more lethal types of breast cancer. These tumors may have a molecular machinery that enhances their ability to metastasize to visceral sites and future research may unearth the mechanisms for this phenomenon.


Assuntos
Neoplasias da Mama/epidemiologia , Metástase Linfática/genética , Sistema Linfático/patologia , Biópsia de Linfonodo Sentinela , Neoplasias da Mama/genética , Feminino , Humanos , Linfonodos/patologia , Linfonodos/cirurgia , Metástase Linfática/patologia , Linfonodo Sentinela/patologia
20.
J Cell Biol ; 217(6): 2205-2221, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29650776

RESUMO

Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.


Assuntos
Movimento Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Exossomos/metabolismo , Vasos Linfáticos/metabolismo , Animais , Linhagem Celular Tumoral , Extensões da Superfície Celular/metabolismo , Microambiente Celular , Quimiocina CX3CL1/metabolismo , Colágeno/metabolismo , Sinais (Psicologia) , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Exossomos/ultraestrutura , Humanos , Inflamação/patologia , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteômica , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA