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Understanding the diversity of gastrointestinal (GI) immune cells, especially in the muscularis propria, is crucial for understanding their role in the maintenance of enteric neurons and smooth muscle and their contribution to GI motility. Here, we present a detailed protocol for isolating single immune cells from the human gastric muscularis propria. We describe steps for tissue preservation, dissection, and dissociation of the muscularis propria. We then detail procedures for magnetic sorting of CD45+ cells and single-cell RNA sequencing (scRNA-seq) analysis. For complete details on the use and execution of this protocol, please refer to Chikkamenahalli et al.1.
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IMPORTANCE: We analyzed over 22,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes of patient samples tested at Mayo Clinic Laboratories during a 2-year period in the COVID-19 pandemic, which included Alpha, Delta, and Omicron variants of concern to examine the roles and relationships of Minnesota virus transmission. We found that Hennepin County, the most populous county, drove the transmission of SARS-CoV-2 viruses in the state after including the formation of earlier clades including 20A, 20C, and 20G, as well as variants of concern Alpha and Delta. We also found that Hennepin County was the source for most of the county-to-county introductions after an initial predicted introduction with the virus in early 2020 from an international source, while other counties acted as transmission "sinks." In addition, major policies, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, did not appear to have an impact on virus diversity across individual counties.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Minnesota/epidemiología , Pandemias , COVID-19/epidemiología , Control de Enfermedades Transmisibles , GenómicaRESUMEN
SARS-CoV-2 has had an unprecedented impact on human health and highlights the need for genomic epidemiology studies to increase our understanding of virus evolution and spread, and to inform policy decisions. We sequenced viral genomes from over 22,000 patient samples tested at Mayo Clinic Laboratories between 2020-2022 and use Bayesian phylodynamics to describe county and regional spread in Minnesota. The earliest introduction into Minnesota was to Hennepin County from a domestic source around January 22, 2020; six weeks before the first confirmed case in the state. This led to the virus spreading to Northern Minnesota, and eventually, the rest of the state. International introductions were most abundant in Hennepin (home to the Minneapolis/St. Paul International (MSP) airport) totaling 45 (out of 107) over the two-year period. Southern Minnesota counties were most common for domestic introductions with 19 (out of 64), potentially driven by bordering states such as Iowa and Wisconsin as well as Illinois which is nearby. Hennepin also was, by far, the most dominant source of in-state transmissions to other Minnesota locations (n=772) over the two-year period. We also analyzed the diversity of the location source of SARS-CoV-2 viruses in each county and noted the timing of state-wide policies as well as trends in clinical cases. Neither the number of clinical cases or major policy decisions, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, appeared to have impact on virus diversity across each individual county.
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BACKGROUND: Oral lichen planus confers a 1% risk of transformation to oral squamous cell carcinoma. While prior exome sequencing studies have identified multiple genetic mutations in oral squamous cell carcinoma, mutational analyses of lichen planus-derived OSCC are lacking. We sought to clarify genomic events associated with oral lichen planus transformation. METHODS: Using rigorous diagnostic criteria, we retrospectively identified patients with non-transforming oral lichen planus (i.e., known to be non-transforming with 5 years of clinical follow-up; n = 17), transforming oral lichen planus (tissue marginal to oral squamous cell carcinoma, n = 9), or oral squamous cell carcinoma arising in lichen planus (n = 17). Gene mutational profiles derived from whole-exome sequencing on fixed mucosal specimens were compared among the groups. RESULTS: The four most frequently mutated genes in transforming oral lichen planus and oral squamous cell carcinoma (TP53, CELSR1, CASP8, and KMT2D) identified 12/17 (71%) of oral squamous cell carcinomas and 5/9 (56%) of transforming oral lichen planus but were absent in non-transforming oral lichen planus. We identified other known oral squamous cell carcinoma mutations (TRRAP, OBSCN, and LRP2) but also previously unreported mutations (TENM3 and ASH1L) in lichen planus-associated oral squamous cell carcinomas. CONCLUSIONS: These findings suggest alterations in DNA damage response and apoptosis pathways underlie lichen planus-related oral squamous cell carcinoma transformation and are supported by mutational signatures indicative of DNA damage. This study characterized patterns of mutational events present in oral lichen planus associated with squamous cell carcinoma and in squamous cell carcinoma associated with oral lichen planus but not in non-transforming oral lichen planus.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Liquen Plano Oral , Liquen Plano , Neoplasias de la Boca , Apoptosis/genética , Carcinoma de Células Escamosas/diagnóstico , Daño del ADN/genética , Neoplasias de Cabeza y Cuello/complicaciones , Humanos , Liquen Plano/patología , Liquen Plano Oral/metabolismo , Proteínas de la Membrana , Neoplasias de la Boca/patología , Mutación , Proteínas del Tejido Nervioso , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello , Secuenciación del ExomaRESUMEN
BACKGROUND: Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3' mRNA-seq method sequences the 3' region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3' mRNA-Seq using Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation. RESULTS: The total mapped reads of QuantSeq 3' mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3' mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3' mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67). CONCLUSION: In this study, QuantSeq 3' mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3' mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.
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Perfilación de la Expresión Génica , ARN , Formaldehído , Humanos , Adhesión en Parafina , ARN Mensajero/genética , Análisis de Secuencia de ARN , Fijación del TejidoRESUMEN
INTRODUCTION: The etiology of acute liver failure (ALF) remains an important prognostic factor. The Acute Liver Failure Study Group recently reported that 150 of 2,718 adult patients with ALF (5.5%) had an indeterminate etiology. Our aim was to use whole exome sequencing to identify genetic variants associated with phenotypic, biochemical, and histologic features among patients with indeterminate ALF. METHODS: This effort has defined a cohort of well-pedigreed patients with indeterminate ALF; DNA samples extracted from whole blood samples were obtained from 26 respective patients with indeterminate ALF. These samples were kept at the Acute Liver Failure Study Group repository at the NIDDK, Bethesda. Whole exome sequencing and bioinformatics analysis were performed at the Mayo Clinic Center of Individualized Medicine in Rochester, MN. RESULTS: Of the 26 patients, 8 survived spontaneously, 6 died, and 12 underwent a liver transplantation; all those transplanted were alive at 21 days after enrollment in the study. Twenty-two of the 26 patients presented as ALF. We found 12 variants associated with 11 genes. The most common variant was rs4940595 in the SERPINB11 gene which was found in 23 of the 26 patients. This variant had a stop codon; no reports of disorders have been associated with this variant. The next most commonly found variant was rs1135840 in the CYP2D6 gene; this mutation is a missense_variant and has been reported to be associated with hepatotoxicity of antituberculous therapy. None of our patients were receiving this therapy. We also found a significant asymmetric distribution of rs1800754 of the CYP2D7 gene and rs1135840 of the CYP2D6 gene between patients who survived spontaneously (75%) and those who died or underwent liver transplantation (30.5% and 25%, respectively). DISCUSSION: We found 12 variants of 11 genes significantly associated with ALF among adults with indeterminate etiology. We also found a significant asymmetric distribution of 2 variants belonging to the CYP2D7 and CYP2D6 genes, respectively, between those who survived spontaneously and those who died or underwent liver transplantation. The 2 most common variants, rs4940595 and rs1135840, of the SERPINB11 and CYP2D6 genes, respectively, found in our patients with ALF have been described as potentially important in the adaptive response combating the emergence of infectious diseases and associated with hepatotoxicity of antituberculous therapy, respectively. Our findings need to be expanded to include more patients with indeterminate ALF as well as viral, drug toxicity, and autoimmune etiologies to determine whether our findings are associated with the specific etiology, indeterminate, or with the overall ALF syndrome itself.
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Predisposición Genética a la Enfermedad , Fallo Hepático Agudo/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Biología Computacional , Citocromo P-450 CYP2D6/genética , Sistema Enzimático del Citocromo P-450/genética , Análisis Mutacional de ADN , Femenino , Humanos , Hígado/patología , Fallo Hepático Agudo/sangre , Fallo Hepático Agudo/mortalidad , Fallo Hepático Agudo/cirugía , Masculino , Persona de Mediana Edad , Mutación Missense , Proyectos Piloto , Polimorfismo de Nucleótido Simple , Pronóstico , Serpinas/genética , Secuenciación del Exoma , Adulto JovenRESUMEN
BACKGROUND AND OBJECTIVES: New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumour-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesise that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC-exosomes as the mediator of this lipolysis. DESIGN: Exosomes from patient-derived cell lines and from plasma of patients with PC and non-PC controls were isolated and characterised. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink Assay was used to study AM and adrenomedullin receptor (ADMR) interaction in adipocytes treated with exosomes. RESULTS: In murine and human adipocytes, we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by ADMR blockade. AM interacted with its receptor on the adipocytes, activated p38 and extracellular signal-regulated (ERK1/2) mitogen-activated protein kinases and promoted lipolysis by phosphorylating hormone-sensitive lipase. PKH67-labelled PC-exosomes were readily internalised into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. CONCLUSIONS: PC-secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal AM is a candidate mediator of this effect.
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Adipocitos/metabolismo , Adrenomedulina/metabolismo , Exosomas/metabolismo , Lipólisis , Sistema de Señalización de MAP Quinasas , Neoplasias Pancreáticas/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Endocitosis/fisiología , Glicerol/metabolismo , Humanos , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptores de Adrenomedulina/antagonistas & inhibidores , Receptores de Adrenomedulina/metabolismo , Grasa Subcutánea/citología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
PURPOSE: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic ß-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches ß cells remote from the cancer to induce ß-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to ß cells and impair insulin secretion. EXPERIMENTAL METHODS: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 ß cells and human islets, and demonstrated the mechanism of exosome internalization into ß cells. We studied the interaction between ß-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in ß cells was shown. RESULTS: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered ß cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on ß cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. ß cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. CONCLUSIONS: Pancreatic cancer causes paraneoplastic ß-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response.
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Adrenomedulina/metabolismo , Diabetes Mellitus/etiología , Exosomas/metabolismo , Células Secretoras de Insulina/metabolismo , Neoplasias Pancreáticas/complicaciones , Western Blotting , Antígeno CA-19-9/metabolismo , Humanos , Microscopía Confocal , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Síndromes Paraneoplásicos/etiología , Síndromes Paraneoplásicos/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Autofagia , Exosomas/metabolismo , Neoplasias Pancreáticas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 7 Relacionada con la Autofagia , Beclina-1 , Transporte Biológico , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Expresión Génica , Glucosa/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Redes y Vías Metabólicas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/patología , Estrés Fisiológico , Enzimas Activadoras de Ubiquitina/metabolismo , GemcitabinaRESUMEN
Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.
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Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Oxigenasas de Función Mixta/fisiología , Proteínas Represoras/fisiología , Transcripción Genética , Animales , Neoplasias Encefálicas/genética , Hipoxia de la Célula , Línea Celular Tumoral , Proteína p300 Asociada a E1A/metabolismo , Glioblastoma/genética , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Although the importance of RGS-GAIP-interacting protein (GIPC) in the biology of malignant cells is well known, the molecular mechanism of GIPC in the inhibition of tumor progression has not been identified. This study focused on elucidating the molecular role of GIPC in breast cancer progression. By using a human breast tumor specimen, an in vivo mouse model, and breast cancer cell lines, we showed for the first time that GIPC is involved in breast cancer progression through regulation of breast cancer cell proliferation, survival, and invasion. Furthermore, we found that the Akt/Mdm2/p53 axis, insulin-like growth factor-1 receptor, matrix metalloproteinase-9, and Cdc42 were downstream of GIPC signaling in breast cancer cells. Moreover, we showed that wild-type p53 reduced GIPC-induced breast cancer cell survival, whereas mutant p53 inhibited GIPC-induced cell invasion. Finally, we demonstrated that an N-myristoylated GIPC peptide (CR1023, N-myristoyl-PSQSSSEA) capable of blocking the PDZ domain of GIPC successfully inhibited MDA-MB-231 cell proliferation, survival, and further in vivo tumor growth. Taken together, these findings demonstrate the importance of GIPC in breast tumor progression, which has a potentially significant impact on the development of therapies against many common cancers expressing GIPC, including breast and renal cancer.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Neoplasias de la Mama/genética , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Dominios PDZ , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Receptores de Somatomedina/metabolismo , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
This article describes one nurse's views about certification and its importance to patient care. The need to educate the public about the value of certification is also discussed.
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Actitud Frente a la Salud , Certificación , Comunicación , Cuidados Críticos , Opinión Pública , Humanos , Relaciones Enfermero-Paciente , Competencia Profesional , Especialidades de Enfermería/educación , Especialidades de Enfermería/organización & administraciónRESUMEN
Pancreatic adenocarcinoma (PCA) is an almost invariably fatal disease. Recently, it has been shown by several groups as well as ours that insulin-like growth factor-I receptor (IGF-IR) overexpression is related to higher proliferation, survival, angiogenesis, and highly invasive pancreatic tumors. Several studies have been carried out to understand the pathways that lead to growth factor-mediated signaling, but the molecular mechanism of receptor overexpression remains mostly unknown. Treatment with neutralizing antibodies or a specific kinase inhibitor against IGF-IR could block the receptor expression in PCA cells. Furthermore, we also showed that insulin receptor substrate (IRS)-2, but not IRS-1, is involved in regulation of IGF-IR expression, which is most likely not transcriptional control. By blocking mammalian target of rapamycin (mTOR) pathway with rapamycin as well as other biochemical analysis, we defined a unique regulation of IGF-IR expression mediated by protein kinase Cdelta (PKCdelta) and mTOR pathway. Moreover, we showed that the down-regulation of IGF-IR expression due to IRS-2 small interfering RNA can be compensated by overexpression of dominant-active mutant of PKCdelta, suggesting that PKCdelta is downstream of IGF-IR/IRS-2 axis. Overall, these findings suggest a novel regulatory role of IRS-2 on the expression of IGF-IR through PKCdelta and mTOR in pancreatic cancer cells.
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Adenocarcinoma/genética , Proteínas Sustrato del Receptor de Insulina/fisiología , Neoplasias Pancreáticas/genética , Proteína Quinasa C-delta/genética , Receptor IGF Tipo 1/genética , Adenocarcinoma/enzimología , Adenocarcinoma/fisiopatología , División Celular , Línea Celular Tumoral , Cartilla de ADN , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica , Neovascularización Patológica , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/fisiopatología , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Proteína Quinasa C-delta/metabolismo , Proteínas Quinasas/fisiología , ARN Neoplásico/genética , ARN Interferente Pequeño/genética , Serina-Treonina Quinasas TORRESUMEN
One of the key challenges in anticancer therapy is the toxicity and poor bioavailability of the anticancer drugs. Nanotechnology can play a pivotal role by delivering drugs in a targeted fashion to the malignant cells that will reduce the systemic toxicity of the anticancer drug. In this report, we show a stepwise development of a nanoparticle-based targeted delivery system for in vitro and in vivo therapeutic application in pancreatic cancer. In the first part of the study, we have shown the fabrication and characterization of the delivery system containing gold nanoparticle as a delivery vehicle, cetuximab as a targeting agent, and gemcitabine as an anticancer drug for in vitro application. Nanoconjugate was first characterized physico-chemically. In vitro targeting efficacy, tested against three pancreatic cancer cell lines (PANC-1, AsPC-1, and MIA Paca2) with variable epidermal growth factor receptor (EGFR) expression, showed that gold uptake correlated with EGFR expression. In the second part, we showed the in vivo therapeutic efficacy of the targeted delivery system. Administration of this targeted delivery system resulted in significant inhibition of pancreatic tumor cell proliferation in vitro and orthotopic pancreatic tumor growth in vivo. Tumor progression was monitored noninvasively by measuring bioluminescence of the implanted tumor cells. Pharmacokinetic experiments along with the quantitation of gold both in vitro and in vivo further confirmed that the inhibition of tumor growth was due to targeted delivery. This strategy could be used as a generalized approach for the treatment of a variety of cancers characterized by overexpression of EGFR.
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Adenocarcinoma/tratamiento farmacológico , Anticuerpos Monoclonales/administración & dosificación , Antimetabolitos Antineoplásicos/administración & dosificación , Desoxicitidina/análogos & derivados , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas del Metal/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Adenocarcinoma/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/farmacocinética , Línea Celular Tumoral , Cetuximab , Desoxicitidina/administración & dosificación , Desoxicitidina/química , Desoxicitidina/farmacocinética , Receptores ErbB/biosíntesis , Receptores ErbB/inmunología , Oro/administración & dosificación , Oro/farmacocinética , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Nanopartículas del Metal/química , Neoplasias Pancreáticas/metabolismo , GemcitabinaRESUMEN
Under normoxia, FIH-1 (factor inhibiting HIF-1) inhibits the transcriptional activity of hypoxia-inducible factor (HIF); however, under such conditions, we observed a significant level of HIF activity in renal cell carcinoma (RCC). This phenomenon could be attributed to a decrease in the level of functional FIH that has been identified in our previous work. Nonetheless, the molecular mechanism of FIH regulation in cancer, in particular RCC, was unclear until now. In this communication, we have demonstrated that in RCC, the Cut-like homeodomain protein (CDP/Cut) is involved in FIH transcriptional regulation and is controlled by a specific signaling event involving protein kinase C (PKC) zeta. Furthermore, we have defined a unique CDP/Cut binding site on the FIH promoter. With chromatin immunoprecipitation assays, we show that CDP binds to the FIH-1 promoter in vivo and that this binding is PKC zeta dependent. Moreover, we have also defined a potential phosphorylation site in CDP (serine 987) that modulates FIH expression. CDP/Cut is a transcriptional repressor that decreases FIH-1 expression and subsequently leads to a decrease in the repressor activity of FIH-1. Without this repression, HIF activity increases, allowing for the increased transcription of the genes it regulates, such as the vascular endothelial growth factor and GLUT-1 genes. Both CDP and HIF levels are increased in several cancers and are responsible for the metastatic progression of the tumors. Taken together, our results suggest for the first time a potential connection between CDP and FIH that could lead to the development of future therapeutic interventions.
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Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Carcinoma de Células Renales/metabolismo , Línea Celular , Proteínas de Homeodominio/genética , Humanos , Neoplasias Renales/metabolismo , Oxigenasas de Función Mixta , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Regulator of G-protein signaling-GAIP-interacting protein COOH terminus (GIPC) is involved in protein trafficking, endocytosis, and receptor clustering and is associated with insulin-like growth factor I receptor (IGF-IR), a receptor important for proliferation and anchorage-independent growth. Here, we described GIPC expression in different human pancreatic adenocarcinoma (PCA) cell lines and we examined the role of GIPC in the regulation of IGF-IR protein levels in PCA. Interestingly, inhibition of GIPC expression by RNA interference led to reduced IGF-IR protein levels and a subsequent decrease in proliferation of PCA cells. We also determined that the PDZ domain of GIPC is essential for the post-translational regulation and the binding of IGF-IR. The importance of GIPC in pancreatic cancer development and progression is supported by tissue microarray data of 300 pancreatic cancer specimens where GIPC is highly expressed in PCA. Taken together, our data suggest that GIPC is a central molecule for the stability of IGF-IR and could be a target for future therapy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Adenocarcinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/química , Adenocarcinoma/patología , Línea Celular Tumoral , Proliferación Celular , Citoplasma/química , Humanos , Neoplasias Pancreáticas/patología , ARN Mensajero/análisis , Receptor IGF Tipo 1/análisis , Receptor IGF Tipo 1/genética , Análisis de Matrices TisularesRESUMEN
Dendritic cells (DC) are key regulators of immune responses. In the current study, we hypothesized that cigarette smoke-induced aberrance in DC function is an important mechanism by which smokers develop cancer, infection, and allergy--diseases common in smokers. We demonstrate that cigarette smoke extract (CSE) inhibits DC-mediated priming of T cells, specifically inhibiting the secretion of IFN-gamma whereas enhancing the production of IL-4 in the MLR. Conditioning with CSE did not effect cytokine (IL-10, IL-6, or IL-12) production from immature DCs, but significantly inhibited IL-12p70 release by LPS-matured DCs. In contrast, IL-10 secretion by LPS-activated CSE-conditioned DCs was enhanced when compared with control DCs. CSE also induced cyclooxygenase-2 protein levels in maturing DCs and significantly augmented endogenous PGE2 release. Conditioning of DCs with CSE also suppressed LPS-mediated induction of CD40, CD80, and CD86, and suppressed maturation-associated CCR7 expression. Although CSE has been reported to induce apoptosis of fibroblasts and epithelial cells, the immunomodulatory effects observed with CSE were not due to diminished DC viability. The effects of CSE on DC function were not exclusively mediated by nicotine, because equivalent, or even higher concentrations of nicotine than those found in CSE, failed to suppress DC-induced T cell priming. These data provide evidence that soluble components extracted from cigarette smoke suppress key DC functions and favor the development of Th-2 immunity.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunosupresores , Activación de Linfocitos/inmunología , Nicotina , Humo , Células Th2/inmunología , Células Th2/metabolismo , Adyuvantes Inmunológicos , Muerte Celular/inmunología , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/fisiología , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Células Dendríticas/citología , Dinoprostona/metabolismo , Regulación hacia Abajo/inmunología , Inhibidores de Crecimiento/farmacología , Humanos , Inmunofenotipificación , Interleucina-10/metabolismo , Interleucina-12/antagonistas & inhibidores , Interleucina-12/metabolismo , Lipopolisacáridos/inmunología , Activación de Linfocitos/efectos de los fármacos , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/fisiología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células Th2/citología , Regulación hacia Arriba/inmunologíaRESUMEN
Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that plays an important role in tumor growth and progression. Recent evidence suggests an alternate, albeit indirect, role of VEGF on host immune response to tumors. VEGF appears to diminish host immunity by altering the function of major antigen-presenting cells such as dendritic cells (DCs) [D.I. Gabrilovich, T. Ishida, S. Nadaf, J.E. Ohm, D.P. Carbone, Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function, Clin. Cancer Res. 5 (1999) 2963-2970, D. Gabrilovich, T. Ishida, T. Oyama, S. Ran, V. Kravtsov, S. Nadaf, D.P. Carbone, Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo, Blood 92 (1998) 4150-4166, T. Oyama, S. Ran, T. Ishida, S. Nadaf, L. Kerr, D.P. Carbone, D.I. Gabrilovich, Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-kappa B activation in hemopoietic progenitor cells, J. Immunol. 160 (1998) 1224-1232.]. DCs are prime initiators of host immunity as they are known to activate both primary as well as secondary immune responses [J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque, Y.J. Liu, B. Pulendran, K. Palucka, Immunobiology of dendritic cells, Ann. Rev. Immunol. 18 (2000) 767-811.]. However, the exact nature of how VEGF suppresses DC function is not fully clear. In this report, we show that DCs cultured in the presence of VEGF are less potent in stimulating antigen-specific T-cells. Furthermore, by using DCs derived from Id1(-/-) mice that are defective in Flt-1 signaling, we demonstrated that the inhibitory function of VEGF on DC function is most likely mediated by Flt-1. Thus, the role of VEGF in downregulating host immunity may highlight a unique role of VEGF in the pathogenesis of cancer.
Asunto(s)
Células Dendríticas/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Humanos , Proteína 1 Inhibidora de la Diferenciación , Ratones , Transducción de Señal/fisiologíaRESUMEN
Contemporary approaches for vaccination and immunotherapy are often capable of eliciting strong T-cell responses against tumor antigens. However, such responses are not parallel to clinical tumor regression. The development of evasion mechanisms within tumor microenvironment may be responsible for poor therapeutic responses. We report here that constitutive or inducible expression of B7-H1, a B7 family molecule widely expressed by cancers, confers resistance to therapeutic anti-CD137 antibody in mice with established tumors. The resistance is accompanied with failure of antigen-specific CD8+ CTLs to destroy tumor cells without impairment of CTL function. Blockade of B7-H1 or PD-1 by specific monoclonal antibodies could reverse this resistance and profoundly enhance therapeutic efficacy. Our findings support that B7-H1/PD-1 forms a molecular shield to prevent destruction by CTLs and implicate new approaches for immunotherapy of human cancers.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/inmunología , Antígeno B7-1/inmunología , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/inmunología , Neoplasias Experimentales/inmunología , Péptidos/antagonistas & inhibidores , Péptidos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Proteínas Reguladoras de la Apoptosis , Antígeno B7-1/biosíntesis , Antígeno B7-H1 , Anergia Clonal , Femenino , Inmunoterapia/métodos , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Desnudos , Neoplasias Experimentales/terapia , Receptor de Muerte Celular Programada 1 , Linfocitos T Citotóxicos/inmunologíaRESUMEN
B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.