RESUMO
Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential.
Assuntos
Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Membrana Celular/metabolismo , Transdução de Sinais , Glicólise , Glicoesfingolipídeos/metabolismoRESUMO
KRAS is mutated in 90% of human pancreatic ductal adenocarcinomas (PDACs). To function, KRAS must localize to the plasma membrane (PM) via a C-terminal membrane anchor that specifically engages phosphatidylserine (PtdSer). This anchor-binding specificity renders KRAS-PM localization and signaling capacity critically dependent on PM PtdSer content. We now show that the PtdSer lipid transport proteins, ORP5 and ORP8, which are essential for maintaining PM PtdSer levels and hence KRAS PM localization, are required for KRAS oncogenesis. Knockdown of either protein, separately or simultaneously, abrogated growth of KRAS-mutant but not KRAS-wild-type pancreatic cancer cell xenografts. ORP5 or ORP8 knockout also abrogated tumor growth in an immune-competent orthotopic pancreatic cancer mouse model. Analysis of human datasets revealed that all components of this PtdSer transport mechanism, including the PM-localized EFR3A-PI4KIIIα complex that generates phosphatidylinositol-4-phosphate (PI4P), and endoplasmic reticulum (ER)-localized SAC1 phosphatase that hydrolyzes counter transported PI4P, are significantly up-regulated in pancreatic tumors compared to normal tissue. Taken together, these results support targeting PI4KIIIα in KRAS-mutant cancers to deplete the PM-to-ER PI4P gradient, reducing PM PtdSer content. We therefore repurposed the US Food and Drug Administration-approved hepatitis C antiviral agent, simeprevir, as a PI4KIIIα inhibitor In a PDAC setting. Simeprevir potently mislocalized KRAS from the PM, reduced the clonogenic potential of pancreatic cancer cell lines in vitro, and abrogated the growth of KRAS-dependent tumors in vivo with enhanced efficacy when combined with MAPK and PI3K inhibitors. We conclude that the cellular ER-to-PM PtdSer transport mechanism is essential for KRAS PM localization and oncogenesis and is accessible to therapeutic intervention.
Assuntos
Antineoplásicos/farmacologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Receptores de Esteroides/metabolismo , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , Inibidores de Proteases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores de Esteroides/genética , Simeprevir/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
KRAS plays an essential role in regulating cell proliferation, differentiation, migration and survival. Mutated KRAS is a major driver of malignant transformation in multiple human cancers. We showed previously that fendiline (6) is an effective inhibitor of KRAS plasma membrane (PM) localization and function. In this study, we designed, synthesized and evaluated a series of new fendiline analogs to optimize its drug properties. Systemic structure-activity relationship studies by scaffold repurposing led to the discovery of several more active KRAS PM localization inhibitors such as compounds 12f (NY0244), 12h (NY0331) and 22 (NY0335) which exhibit nanomolar potencies. These compounds inhibited oncogenic KRAS-driven cancer cell proliferation at single-digit micromolar concentrations in vitro. In vivo studies in a xenograft model of pancreatic cancer revealed that 12h and 22 suppressed oncogenic KRAS-expressing MiaPaCa-2 tumor growth at a low dose range of 1-5 mg/kg with no vasodilatory effects, indicating their potential as chemical probes and anticancer therapeutics.
Assuntos
Antineoplásicos/farmacologia , Membrana Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fendilina/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Fendilina/análogos & derivados , Fendilina/química , Humanos , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Relação Estrutura-AtividadeRESUMO
K-Ras must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and K-RasG12V from the PM, resulting in abrogation of K-RasG12V signaling and potent, selective growth inhibition of mutant K-Ras-transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic K-Ras-expressing MiaPaCa-2 tumors but had no effect on the growth of the wild-type K-Ras-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a K-Ras ortholog) signaling in Caenorhabditis elegans, as evidenced by suppression of the induced multivulva phenotype. Using RNA interference against C. elegans genes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multivulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused K-RasG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic K-Ras-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for K-Ras function, which may present therapeutic options for the treatment of K-Ras-driven cancers.
Assuntos
Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Caenorhabditis elegans , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Humanos , Camundongos , Camundongos Nus , Transdução de Sinais , Esfingomielinas/genética , Esfingomielinas/metabolismo , Proteínas ras/metabolismoRESUMO
Roquin is an E3 ligase that regulates mRNA stability. Mice with a mutation in the Rc3h1 gene and Roquin protein, referred to as Roquinsan/san or sanroque mice, develop broad-spectrum chronic inflammatory conditions and autoimmune pathologies. Our laboratory recently reported that sanroque mice also develop extensive inflammation that is localized in the small intestine but is rare in the colon. Here, we demonstrate that small intestinal intraepithelial lymphocytes (IELs) are present in the epithelium of sanroque mice but that cell recoverability is low using standard extraction techniques even though lamina propria lymphocytes (LPLs) can be recovered in normal numbers. In studies aimed at characterizing T cell costimulatory markers and activation molecules on LPLs in sanroque mice, we identified Ly6C and 4-1BB (CD137) as being expressed at elevated levels on sanroque small intestinal LPLs, and we show that both of those subsets, in conjunction with cells expressing the KLRG1 T cell activation molecule, are sources of IL-17A, IFN-γ, and TNFα. TNFα was primarily produced by 4-1BB+, KLRG1-cells, but was also made by some 4-1BB-, KLRG1-cells, and 4-1BB-, KLRG1+ cells. These findings collectively suggest that the small intestinal inflammatory response in sanroque mice is driven, at least in part, by LPL activation through Ly6C and 4-1BB signaling, and they provide further evidence in support of using the sanroque mouse as an animal model of chronic small intestinal inflammation.
Assuntos
Antígenos Ly/fisiologia , Linfócitos/metabolismo , Mucosa/metabolismo , Receptores Imunológicos/fisiologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/fisiologia , Animais , Doença de Crohn/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Heterozigoto , Inflamação , Interferon gama/metabolismo , Interleucina-17/metabolismo , Intestino Delgado/metabolismo , Lectinas Tipo C , Camundongos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The thyroid stimulating hormone beta-subunit (TSHß) with TSHα form a glycoprotein hormone that is produced by the anterior pituitary in the hypothalamus-pituitary-thyroid (HPT) axis. Although TSHß has been known for many years to be made by cells of the immune system, the role of immune system TSH has remained unclear. Recent studies demonstrated that cells of the immune system produce a novel splice variant isoform of TSHß (TSHßv), but little if any native TSHß. Here, we show that within three days of systemic infection of mice with Listeria monocytogenes, splenic leukocytes synthesized elevated levels of TSHßv. This was accompanied by an influx of CD14+, Ly6C+, Ly6G+ cells into the thyroid of infected mice, and increased levels of intrathyroidal TSHßv gene expression. Adoptive transfer of carboxyfluorescein succinimidyl ester (CFSE)-labeled splenic leukocytes from infected mice into non-infected mice migrated into the thyroid as early as forty-eight hours post-cell transfer, whereas CFSE-labeled cells from non-infected mice failed to traffic to the thyroid. These findings demonstrate for the first time that during bacterial infection peripheral leukocytes produce elevated levels of TSHßv, and that spleen cells traffic to the thyroid where they produce TSHßv intrathyroidally.
Assuntos
Leucócitos/citologia , Listeria monocytogenes , Listeriose/metabolismo , Isoformas de Proteínas/metabolismo , Baço/citologia , Glândula Tireoide/metabolismo , Tireotropina Subunidade beta/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Roquin-1, a RING finger E3 ubiquitin ligase, functions as a modulator of inflammation; however, nothing is known about how Rc3h1 expression is regulated. Here, we describe an opposing relationship between Roquin-1 and the IL-17 proinflammatory cytokine by demonstrating that enforced expression of Rc3h1 restricts Il17a expression, and that exposure of T cells to IL-10, a cytokine with immunosuppressive activity, increases Rc3h1 expression. Luciferase reporter assays conducted using eight transcription factor plasmids (STAT1, STAT3, STAT5, GATA2, c-Rel, IKZF1, IKZF2, and IKZF3) demonstrated that STAT1, STAT3, GATA2, and c-Rel increased Rc3h1 promoter activity, whereas IKZF2 decreased activity. Gene expression of those five transcription factors increased in T cells exposed to IL-10. Transcription factor-specific siRNAs suppressed the IL-10 effect on Rc3h1 transcription. These findings identify a role for IL-10 in regulating Rc3h1 transcription, and they have implications for understanding how Roquin-1 controls the immune response.
Assuntos
Interleucina-10/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Roquin, an E3 ligase, is involved in curtailing autoimmune pathology as seen from studies using mice with mutated (Rc3h1(san/san)) or disrupted (Rc3h1(gt/gt)) Rc3h1 gene. The extent to which intestinal immunopathology is caused by insufficient Roquin expression in the immune system, or by Roquin impairment in non-hematopoietic cells, has not been determined. Using bone marrow cells from Rc3h1(gt/gt) mice transferred into irradiated normal mice (Rc3h1(gt/gt) â NL chimeras), we show that inflammation developed in the small intestine, kidney, lung, liver, and spleen. Proinflammatory cytokine levels were elevated in lamina propria lymphocytes (LPLs). Inflammation in the liver was accompanied by areas of hepatocyte apoptosis. Lung inflammation consisted of an influx of both T cells and B cells. Small intestinal LPLs had increased numbers of CD44(hi), CD62L(lo), KLRG1(+), ICOS(+) short-lived effector cells, indicating an influx of activated T cells. Following oral infection with L. monocytogenes, Rc3h1(gt/gt) â NL chimeras had more liver pathology and greater numbers of bacteria in the Peyer's patches than NL â NL chimeras. These findings demonstrate that small intestinal inflammation in Rc3h1(san/san) and Rc3h1(gt/gt) mice is due to a failure of Roquin expression in the immune system and not to insufficient systemic Roquin expression.
Assuntos
Enterite/sangue , Enterite/genética , Expressão Gênica , Hematopoese/genética , Ubiquitina-Proteína Ligases/genética , Animais , Antígenos de Superfície/metabolismo , Caspase 3 , Citocinas/metabolismo , Modelos Animais de Doenças , Enterite/imunologia , Enterite/metabolismo , Enterite/patologia , Memória Imunológica , Imunofenotipagem , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Knockout , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Ubiquitina-Proteína Ligases/deficiênciaRESUMO
BACKGROUND: A major cause of chronic inflammatory periodontal disease is Porphyromonas gingivalis, a non-motile, Gram-negative, rod-shaped, anaerobic bacterium. Within gingival tissue, both macrophages and fibroblasts participate in the immune response to foreign entities by releasing cytokines and expressing molecules to recruit and activate lymphocytes. However, the contribution of gingival macrophages and fibroblasts to the immune response to P. gingivalis infection is not fully known. METHODS: The AMJ2-C8 cell line (AM cells), a mouse alveolar macrophage cell line, and ESK-1 cells, a mouse gingival fibroblast cell line made in our laboratory, were treated with lipopolysaccharide (LPS) from either P. gingivalis or Escherichia coli. The expression of immune response molecules was quantified by real-time polymerase chain reaction and enzyme-linked immunoassay. RESULTS: AM and ESK-1 cells responded differently to P. gingivalis and E. coli LPS stimulation. The ESK-1 gingival fibroblast cell line was more responsive to E. coli LPS stimulation as seen by elevated levels of interleukin (IL)-6, inducible nitric oxide, and monocyte chemotactic protein-1 expression relative to stimulation by P. gingivalis LPS. Conversely, the AM macrophage cell line was more responsive to P. gingivalis LPS stimulation, particularly for interleukin IL-1ß, IL-6, and monocyte chemotactic protein-1, relative to stimulation by E. coli LPS. CONCLUSION: These findings demonstrate that E. coli LPS induces a stronger cytokine and chemokine response in gingival fibroblasts, whereas P. gingivalis LPS induces a stronger response in macrophages.
Assuntos
Citocinas/imunologia , Escherichia coli/imunologia , Fibroblastos/imunologia , Gengiva/imunologia , Lipopolissacarídeos/imunologia , Macrófagos Alveolares/imunologia , Porphyromonas gingivalis/imunologia , Animais , Linhagem Celular Transformada , Quimiocina CCL2/análise , Quimiocina CCL2/imunologia , Quimiocina CCL3/análise , Quimiocina CCL3/imunologia , Quimiocina CCL4/análise , Quimiocina CCL4/imunologia , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/microbiologia , Gengiva/citologia , Gengiva/microbiologia , Interleucina-1beta/análise , Interleucina-1beta/imunologia , Interleucina-23/análise , Interleucina-23/imunologia , Interleucina-6/análise , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The small intestine has been shown to be an extra-pituitary site of thyroid stimulating hormone (TSH) production, and previous in vivo studies have shown that TSH synthesis localizes within areas of enteric virus infection within the small intestine; however, the cellular source of intestinal TSH has not been adequately determined. In the present study, we have used the murine MODE-K small intestinal epithelial cell line to demonstrate both at the transcriptional level and as a secreted hormone, as measured in a TSHbeta-specific enzyme-linked assay, that epithelial cells in fact respond to infection with reovirus serotype 3 Dearing strain by upregulating TSH synthesis. Moreover, sequence analysis of a PCR-amplified TSHbeta product from MODE-K cells revealed homology to mouse pituitary TSHbeta. These findings have direct functional implications for understanding a TSH immune-endocrine circuit in the small intestine.
Assuntos
Células Epiteliais/metabolismo , Intestinos/citologia , Orthoreovirus Mamífero 3/fisiologia , Tireotropina Subunidade beta/biossíntese , Animais , Linhagem Celular , Camundongos , Análise de Sequência , Tireotropina Subunidade beta/genética , Tireotropina Subunidade beta/metabolismo , Regulação para CimaRESUMO
IL-10-deficient mice develop enterocolitis due to a failure of cytokine regulation; however, the full scope of that response remains poorly defined. Using multiplex analysis to quantify the activity of 23 regulatory and effector cytokines produced by colonic leukocytes, we demonstrate a vast dysregulation process of 18 cytokines in IL-10-/- mice from 7 to 27 weeks of age. Of those, IL-12p40, IL-6, granulocyte macrophage colony-stimulating factor, IFN-gamma, IL-13 and monocyte chemoattractant protein-1 (MCP-1) had the highest single correlations with pathology (r = 0.7766-0.7016). Importantly, there were strong associations (r = 0.7071-0.9074) between those cytokines and as many as 10 additional cytokines, indicating a high degree of cytokine complexity as disease progressed. IL-17 was notable in that it was produced at high levels by colonic leukocytes from IL-10-/- mice with pathology ranging from mild to severe, though it was not produced by healthy IL-10-/- mice lacking pathology. Tumor necrosis factor alpha (TNFalpha) by itself displayed only a modest association with pathology (r = 0.6340), ranking sixth lowest, though it cross-correlated strongly with the synthesis of 12 other cytokines, implying that the destructive effects associated with TNFalpha may be due to interactions of multiple cytokine activities. IL-23 expression did not correlate with pathology, possibly suggesting that IL-23 is involved in the initiation but not the perpetuation of inflammation. Four cytokines (IL-2, IL-3, IL-4 and IL-5) remained negative in IL-10-/- mice, demonstrating that cytokine dysregulation was not universal. These findings emphasize the need to better understand cytokine networks in chronic inflammation and they provide a rationale for combining immunotherapies in the treatment of intestinal inflammation.
Assuntos
Colite/imunologia , Colite/fisiopatologia , Colo/patologia , Citocinas/metabolismo , Células Epiteliais/patologia , Interleucina-10/deficiência , Leucócitos/patologia , Animais , Colite/patologia , Colo/citologia , Colo/imunologia , Citocinas/genética , Citocinas/imunologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Feminino , Interleucina-10/genética , Leucócitos/citologia , Leucócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The vast majority of peripheral T cells exist as resting lymphocytes until a signal for activation has been received. In response to antigen, this activation involves ligation of the T-cell receptor (TCR) and signal transmission through the CD3 complex, which then initiates a cascade of intracellular events that lead to the expression of genes used in T-cell activation. T-cell activation also requires soluble mediators in the form of cytokines and chemokines that regulate the process in both positive and negative ways, and costimulatory signals received in conjunction with TCR/CD3 signaling are important in the activation of T cells. Unlike T cells in other peripheral immune compartments, small and large intestinal intraepithelial lymphocytes (IELs) bear some but not all properties of activated T cells, suggesting that they constitute a large population of 'partially activated' effector cells. Thus, regulation of the IEL activation process must be held in tight check, yet it must be ready to respond to foreign antigen rapidly and effectively. We discuss how costimulatory molecules may hold the key to controlling IEL activation through a multiphase process beginning with cells that have already entered into the early stage of activation.
Assuntos
Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Ativação Linfocitária/imunologia , Modelos Imunológicos , Linfócitos T/imunologia , Animais , Células Epiteliais/citologia , Células Epiteliais/imunologia , Humanos , Mucosa Intestinal/citologiaRESUMO
Achondroplasia, the most common short-limbed dwarfism in humans, results from a single nucleotide substitution in the gene for fibroblast growth factor receptor 3 (FGFR3). FGFR3 regulates bone growth in part via the mitogen-activated protein kinase pathway (MAPK). To examine the role of this pathway in chondrocyte differentiation, a transgenic mouse was generated that expresses a constitutively active mutant of MEK1 in chondrocytes and exhibits dwarfing characteristics typical of human achondroplasia, i.e., shortened axial and appendicular skeletons, mid-facial hypoplasia, and dome-shaped cranium. In this study, cephalometrics of the MEK1 mutant skulls were assessed to determine if the MEK1 mice are a good model of achondroplasia. Skull length, arc of the cranial vault, and area, maximum and minimum diameters of the brain case were measured on digitized radiographs of skulls of MEK1 and control mice. Cranial base and nasal bone length and foramen magnum diameter were measured on midsagittal micro-CT sections. Data were normalized by dividing by the cube root of each animal's weight. Transgenic mice exhibited a domed skull, deficient midface, and (relatively) prognathic mandible and had a shorter cranial base and nasal bone than the wild-type. Skull length was significantly less in transgenic mice, but cranial arc was significantly greater. The brain case was larger and more circular and minimum diameter of the brain case was significantly greater in transgenic mice. The foramen magnum was displaced anteriorly but not narrowed. MEK1 mouse cephalometrics confirm these mice as a model for achondroplasia, demonstrating that the MAP kinase signaling pathway is involved in FGF signaling in skeletal development.
Assuntos
Acondroplasia/patologia , Modelos Animais de Doenças , Camundongos Transgênicos/anormalidades , Crânio/patologia , Acondroplasia/diagnóstico por imagem , Acondroplasia/genética , Animais , Cefalometria , Camundongos , Radiografia , Crânio/diagnóstico por imagemRESUMO
Murine intestinal intraepithelial lymphocytes (IELs) can be classified according to expression of a CD43 glycoform recognized by the S7 monoclonal antibody. In this study, we examined the response of S7+ and S7- IELs in mice during acute reovirus serotype 3 (Dearing strain) infection, which was confirmed by virus-specific real-time PCR. In vivo proliferation increased significantly for both S7- and S7+ IELs on day 4 post-infection as determined by BrdU incorporation; however, expression of the inducible costimulatory (ICOS) molecule, which peaked on day 7 post-infection, was upregulated on S7+ CD4+ T cells, most of which were CD4+8- IELs. In vitro ICOS stimulation by syngeneic peritoneal macrophages induced IFN-gamma secretion from IELs from day 7 infected mice, and was suppressed by treatment with anti-ICOS mAb. Additionally, IFN-gamma mRNA increased in CD4+ IELs on day 6 post-infection. These findings indicate that S7- and S7+ IELs are differentially mobilized during the immune response to reovirus infection; that the regulated expression of ICOS is associated with S7+ IELs; and that stimulation of IELs through ICOS enhances IFN-gamma synthesis during infection.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células Epiteliais/metabolismo , Intestino Delgado/citologia , Leucossialina/metabolismo , Infecções por Reoviridae/metabolismo , Regulação para Cima/genética , Animais , Anticorpos Monoclonais , Antígeno B7-1/metabolismo , Proliferação de Células , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Citometria de Fluxo , Ligante Coestimulador de Linfócitos T Induzíveis , Proteína Coestimuladora de Linfócitos T Induzíveis , Interferon gama/biossíntese , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
PURPOSE: Surprisingly little is known about the cellular composition of the temporomandibular joint (TMJ) disc, which is a crucial piece of the puzzle in tissue engineering efforts. Toward this end, cell types were identified and quantified regionally in the TMJ disc. MATERIALS AND METHODS: Porcine TMJ discs were examined by histology, electron microscopy, and immunohistochemistry. Histology consisted of hematoxylin and eosin staining to identify regional variation of cell type and cell numbers. Transmission electron microscopy was used to elucidate differences in organelle content and pericellular matrix between TMJ disc cells and chondrocytes from hyaline cartilage. Immunohistochemistry was used to assess the presence of smooth and skeletal muscle character in the TMJ disc. RESULTS: The overall ratio of fibroblasts to chondrocyte-like cells in the TMJ disc was approximately 2.35 to 1, with the highest relative number of chondrocyte-like cells in the intermediate zone. Electron microscopy revealed distinct differences between TMJ disc chondrocyte-like cells and chondrocytes from hyaline cartilage with respect to organelles and the pericellular region. Immunostaining identified smooth muscle in the form of vessels, which were most prominent in the anterior band. Skeletal muscle was not observed. CONCLUSION: The cells of the TMJ disc are distinctly different from cells of hyaline cartilage, and consequently should not be referred to as chondrocytes. TMJ disc cells are comprised of heterogeneously distributed subpopulations, with fibroblasts predominating over fibrochondrocytes.
Assuntos
Condrócitos , Fibroblastos , Disco da Articulação Temporomandibular/citologia , Animais , Contagem de Células , Condrócitos/ultraestrutura , Feminino , Fibroblastos/ultraestrutura , Cartilagem Hialina/citologia , Microscopia Eletrônica de Transmissão e Varredura , Suínos , Disco da Articulação Temporomandibular/ultraestruturaRESUMO
Methods for obtaining preparation of intestinal intraepithelial lymphocytes (IELs) present special challenges for immunologists due to difficulties in recovering IELs devoid of contaminating enterocytes. Although high-purity preparations can be achieved using techniques such as flow cytometric or magnetic-activated cell sorting, those methods may not be feasible on a routine basis and may result in low overall cell recoveries. Thus, most procedures today rely on density gradient centrifugation as a means of separating IEL and non-hematopoietic cells; however, the purity of IELs from those preparations can vary considerably. Here, we describe a modification of an IEL purification technique that uses two sequential Percoll gradients rather than one gradient in the purification scheme. This alteration consistently results in 80-85% IEL purity in cell preparations. Moreover, it requires no additional reagents, has no adverse effect on the phenotypic composition of recovered IELs or on the cell viability, and adds minimal additional time to the isolation protocol. It is expected that this procedure will have practical benefit as a means of isolating IELs with high purity on a routine basis that can be used for in vivo or in vitro studies of IEL function.
Assuntos
Separação Celular/métodos , Intestino Delgado/citologia , Intestino Delgado/imunologia , Linfócitos/citologia , Animais , Células Epiteliais/citologia , Células Epiteliais/imunologia , Feminino , Citometria de Fluxo/métodos , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/imunologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Povidona , Dióxido de SilícioRESUMO
PURPOSE: The purpose of this study was to test the hypothesis that spatial and temporal localization of growth factors FGF-2 and VEGF in a rabbit tooth extraction socket model correlate with the histologic events of healing. MATERIALS AND METHODS: Twenty-four male New Zealand white rabbits divided into 8 groups of 3 were used in the study. Incisor teeth were extracted from both jaws and the healing extraction socket with surrounding jaw bone was harvested at 48 hours, 4 days, 1, 2, 4, 8, 12, and 16 weeks. Tissues were fixed, decalcified, and processed for hematoxylin-eosin and immunohistochemical staining. The sections were stained to detect FGF-2 and VEGF. The stained sections were then imaged and an automated computer program was used to detect the brown diaminobenzidine stain that represented the growth factors of interest. Data was obtained in the form of percentage area and intensity of stain and analyzed using the analysis of variance (ANOVA - Tukey Kramer and Scheffe's post-test). RESULTS: Spatial and temporal differences in localization of FGF-2 and VEGF were observed across all time frames in both jaws. Statistically significant differences in percentage area and intensity of brown diaminobenzidine stain were seen temporally between FGF-2 and VEGF (P < .05). CONCLUSION: The results of this study showed positive correlation of histologic events to spatial and temporal localization of FGF-2 and VEGF in a rabbit tooth extraction model.
Assuntos
Regeneração Óssea/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Alvéolo Dental/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia , Análise de Variância , Animais , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/análise , Imuno-Histoquímica , Masculino , Modelos Animais , Coelhos , Estatísticas não Paramétricas , Distribuição Tecidual , Extração Dentária , Alvéolo Dental/química , Alvéolo Dental/fisiologia , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
T cells are present in large numbers in the epithelial lining of the small and large intestine of humans and mice. Those cells, referred to as intraepithelial lymphocytes (IELs), are critical for maintaining an effective mucosal immune response against the onslaught of enteric infectious agents and intestinal neoplasia. However, because intestinal immunity must by necessity occur rapidly and efficiently, it is concomitantly important that the local intestinal immune response be curtailed so as not to result in conditions that lead to a destructive inflammatory environment as occurs in inflammatory bowel disease (IBD). Although many aspects of the IEL activation process remain to be understood, emerging evidence indicates that costimulatory molecules on IELs are critical for activation and that they hold the key to regulating intestinal immunity across many levels. In this article, the involvement of three IEL costimulatory molecules (CD43, OX40, and Ly-6C) - working independently or in collaboration-will be discussed in the context of immunity and disease in the human and mouse intestine, and the involvement of those in sustaining the IELs in a uniquely precarious but effective state of activation readiness will be explored.
RESUMO
Cartilage oligomeric matrix protein (COMP) is a large extracellular matrix protein expressed in cartilage, ligament and tendon. Mutations in the COMP gene cause two dominantly inherited skeletal dysplasias, pseudoachondroplasia (PSACH) and Multiple Epiphyseal Dysplasia (MED/EDM1). We report on a novel point mutation D511Y in the seventh calcium-binding repeat of the COMP gene and the resulting iliac crest growth plate pathology. The PSACH iliac crest growth plate is comprised of a large region of resting chondrocytes above a narrow region composed of clusters of disorganized proliferative and hypertrophic chondrocytes. Chondrocytes in all zones show massive intracellular retention of COMP and the surrounding extracellular matrix is deficient in COMP. Moreover, the 511Y COMP mutation selectively affects type IX collagen as little is found in the growth plate matrix whereas type II collagen and aggrecan are abundant in the matrix. Chondrocyte remnants are observed in the chondrocyte clusters and dead cells are found throughout the growth plate. Apoptosis studies demonstrate an unusual pattern of TUNEL staining in the PSACH chondrocytes compared to the control growth plate. These in vivo findings support our previous observation that retention of COMP leads to chondrocyte death. These results also add to the increasing evidence that PSACH and EDM1 are rER storage diseases and that impaired linear growth and joint erosion are caused by the disruptive effect of massive amounts of COMP within the chondrocytes.
Assuntos
Acondroplasia/patologia , Apoptose , Condrócitos/patologia , Colágeno Tipo X/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Lâmina de Crescimento/metabolismo , Acondroplasia/genética , Acondroplasia/metabolismo , Agrecanas , Proteína de Matriz Oligomérica de Cartilagem , Criança , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Colágeno Tipo X/genética , Análise Mutacional de DNA , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Glicoproteínas/genética , Lâmina de Crescimento/patologia , Humanos , Ílio , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Lectinas Tipo C , Proteínas Matrilinas , Mutação Puntual , Proteoglicanas/metabolismoRESUMO
Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by inappropriate chondrocyte proliferation and bone growth arising at the juxtaepiphyseal region of the long bones. HME is caused by mutations in the EXT 1 and EXT 2 genes, which have glycosyltransferase activity. These genes are responsible for synthesis of heparan sulfate (HS) chains, which are important signaling molecules in chondrocyte differentiation. HME chondrocytes in monolayer culture have been shown by transmission electron and deconvolution microscopy to contain enormous bundles of actin, cross-linked with muscle specific alpha-actinin. Here additional ultrastructural anomalies in HME chondrocytes are reported, including lobulated nuclei, shortened channels of rER, large numbers of cell processes and podosomes, nontypical junctions, elongated, bulbous-ended mitochondria, and reduced extracellular matrix. Microfilaments are present throughout the cytoplasm, compartmentalizing it, and isolating organelles. The excess microfilaments, attributed to increased cell adhesiveness, are likely to interfere with secretion and cytokinesis, and sterically hinder intracellular organelle differentiation. The observed surface modifications and cytoskeletal abnormalities are proposed to play a role in development of the mutant phenotype, via changes in cell adhesiveness and/or binding of signals to receptors, which results in loss of the unidirectionality of growth in the epiphyseal plate.