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BACKGROUND: We report a proof-of-mechanism study of RG7112, a small-molecule MDM2 antagonist, in patients with chemotherapy-naive primary or relapsed well-differentiated or dedifferentiated MDM2-amplified liposarcoma who were eligible for resection. METHODS: Patients with well-differentiated or dedifferentiated liposarcoma were enrolled at four centres in France. Patients received up to three 28-day neoadjuvant treatment cycles of RG7112 1440 mg/m(2) per day for 10 days. If a patient progressed at any point after the first cycle, the lesion was resected or, if unresectable, an end-of-study biopsy was done. The primary endpoint was to assess markers of RG7112-dependent MDM2 inhibition and P53 pathway activation (P53, P21, MDM2, Ki-67, macrophage inhibitory cytokine-1 [MIC-1], and apoptosis). All analyses were per protocol. This trial is registered with EudraCT, number 2009-015522-10. RESULTS: Between June 3, and Dec 14, 2010, 20 patients were enrolled and completed pretreatment and day 8 biopsies. 18 of 20 patients had TP53 wild-type tumours and two carried missense TP53 mutations. 14 of 17 assessed patients had MDM2 gene amplification. Compared with baseline, P53 and P21 concentrations, assessed by immunohistochemistry, had increased by a median of 4·86 times (IQR 4·38-7·97; p=0·0001) and 3·48 times (2·05-4·09; p=0·0001), respectively, at day 8 (give or take 2 days). At the same timepoint, relative MDM2 mRNA expression had increased by a median of 3·03 times (1·23-4·93; p=0·003) that at baseline. The median change from baseline for Ki-67-positive tumour cells was -5·05% (IQR -12·55 to 0·05; p=0·01). Drug exposure correlated with blood concentrations of MIC-1 (p<0·0001) and haematological toxicity. One patient had a confirmed partial response and 14 had stable disease. All patients experienced at least one adverse event, mostly nausea (14 patients), vomiting (11 patients), asthenia (nine patients), diarrhoea (nine patients), and thrombocytopenia (eight patients). There were 12 serious adverse events in eight patients, the most common of which were neutropenia (six patients) and thrombocytopenia (three patients). DISCUSSION: MDM2 inhibition activates the P53 pathway and decreases cell proliferation in MDM2-amplified liposarcoma. This study suggests that it is feasible to undertake neoadjuvant biopsy-driven biomarker studies in liposarcoma. FUNDING: F Hoffmann-La Roche.
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Antineoplásicos , Liposarcoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor , Adulto , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Apoptosis , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Supervivencia sin Enfermedad , Femenino , Factor 15 de Diferenciación de Crecimiento/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Adulto JovenRESUMEN
Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. A rapidly expanding use of this technology is the acquisition of information about the posttranslational modifications of proteins reflecting the activity state of signal pathways and networks, and is now employed for the analysis of biopsy samples in clinical trial research.
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Neoplasias/genética , Análisis por Matrices de Proteínas/métodos , Transducción de Señal , Animales , Anticuerpos , Biotecnología , Humanos , Neoplasias/terapia , Proteoma , Sensibilidad y EspecificidadAsunto(s)
Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Imidazolinas/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Adulto , Anciano , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Imidazolinas/efectos adversos , Leucemia Mieloide Aguda/enzimología , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-mdm2/metabolismoRESUMEN
Little is known about the preanalytical fluctuations of phosphoproteins during tissue procurement for molecular profiling. This information is crucial to establish guidelines for the reliable measurement of these analytes. To develop phosphoprotein profiles of tissue subjected to the trauma of excision, we measured the fidelity of 53 signal pathway phosphoproteins over time in tissue specimens procured in a community clinical practice. This information provides strategies for potential surrogate markers of stability and the design of phosphoprotein preservative/fixation solutions. Eleven different specimen collection time course experiments revealed augmentation (+/-20% from the time 0 sample) of signal pathway phosphoprotein levels as well as decreases over time independent of tissue type, post-translational modification, and protein subcellular location (tissues included breast, colon, lung, ovary, and uterus (endometrium/myometrium) and metastatic melanoma). Comparison across tissue specimens showed an >20% decrease of protein kinase B (AKT) Ser-473 (p < 0.002) and myristoylated alanine-rich C-kinase substrate protein Ser-152/156 (p < 0.0001) within the first 90-min postexcision. Proteins in apoptotic (cleaved caspase-3 Asp-175 (p < 0.001)), proliferation/survival/hypoxia (IRS-1 Ser-612 (p < 0.0003), AMP-activated protein kinase beta Ser-108 (p < 0.005), ERK Thr-202/Tyr-204 (p < 0.003), and GSK3alphabeta Ser-21/9 (p < 0.01)), and transcription factor pathways (STAT1 Tyr-701 (p < 0.005) and cAMP response element-binding protein Ser-133 (p < 0.01)) showed >20% increases within 90-min postprocurement. Endothelial nitric-oxide synthase Ser-1177 did not change over the time period evaluated with breast or leiomyoma tissue. Treatment with phosphatase or kinase inhibitors alone revealed that tissue kinase pathways are active ex vivo. Combinations of kinase and phosphatase inhibitors appeared to stabilize proteins that exhibited increases in the presence of phosphatase inhibitors alone (ATF-2 Thr-71, SAPK/JNK Thr-183/Tyr-185, STAT1 Tyr-701, JAK1 Tyr-1022/1023, and PAK1/PAK2 Ser-199/204/192/197). This time course study 1) establishes the dynamic nature of specific phosphoproteins in excised tissue, 2) demonstrates augmented phosphorylation in the presence of phosphatase inhibitors, 3) shows that kinase inhibitors block the upsurge in phosphorylation of phosphoproteins, 4) provides a rational strategy for room temperature preservation of proteins, and 5) constitutes a foundation for developing evidence-based tissue procurement guidelines.
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Fosfoproteínas/metabolismo , Obtención de Tejidos y Órganos , Ambiente , Inhibidores Enzimáticos , Femenino , Hospitales , Humanos , Especificidad de Órganos , Fosforilación , Análisis por Matrices de Proteínas , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Transducción de Señal , Temperatura , Factores de Tiempo , Conservación de Tejido , Supervivencia TisularRESUMEN
A previously untapped bank of information resides within the low molecular weight proteomic fraction of blood. Intensive efforts are underway to harness this information so that it can be used for early diagnosis of diseases such as cancer. The physicochemical malleability and high surface areas of nanoparticle surfaces make them ideal candidates for developing biomarker harvesting platforms. Given the variety of engineering strategies afforded through nanoparticle technologies, a significant goal is to tailor nanoparticle surfaces to selectively bind a subset of biomarkers, sequestering them for later study using high sensitivity proteomic tests. To date, applications of nanoparticles have largely focused on imaging systems and drug delivery vectors. As such, biomarker harvesting is an underutilized application of nanoparticle technology and is an area of nanotechnology research that will likely undergo substantial growth.
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Biomarcadores/química , Nanoestructuras/química , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Humanos , Modelos Moleculares , Peso Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low-abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement.
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Biomarcadores/sangre , Proteínas Portadoras/química , Péptidos/sangre , Humanos , Espectrometría de Masas/métodos , Modelos Biológicos , Peso Molecular , Proteoma/análisisRESUMEN
Reverse phase protein microarrays (RPMA) enable high throughput screening of posttranslational modifications of important signaling proteins within diseased cells. One limitation of protein-based molecular profiling is the lack of a PCR-like intrinsic amplification system for proteins. Enhancement of protein microarray sensitivities is an important goal, especially because many molecular targets within patient tissues are of low abundance. The ideal array substrate will have a high protein-binding affinity and low intrinsic signal. To date, nitrocellulose-coated glass has provided an effective substrate for protein binding in the microarray format when using chromogenic detection systems. As fluorescent systems, such as quantum dots, are explored as potential reporter agents, the intrinsic fluorescent properties of nitrocellulose-coated glass slides limit the ability to image microarrays for extended periods of time where increases in net sensitivity can be attained. Silicon, with low intrinsic autofluorescence, is being explored as a potential microarray surface. Native silicon has low binding potential. Through titrated reactive ion etching (RIE), varying surface areas have been created on silicon in order to enhance protein binding. Further, via chemical modification, reactive groups have been added to the surfaces for comparison of relative protein binding. Using this combinatorial method of surface roughening and surface coating, 3-aminopropyltriethoxysilane (APTES) and mercaptopropyltrimethoxysilane (MPTMS) treatments were shown to transform native silicon into a protein-binding substrate comparable to nitrocellulose.
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Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Silicio/química , Adsorción , Albúminas/química , Animales , Biotinilación , Colodión/química , Humanos , Iones , Compuestos de Organosilicio , Propilaminas , Proteínas/química , Silanos/química , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
Reverse-phase protein microarrays (RPPMAs) enable heterogeneous mixtures of proteins from cellular extracts to be directly spotted onto a substrate (such as a protein biochip) in minute volumes (nanoliter-to-picoliter volumes). The protein spots can then be probed with primary antibodies to detect important posttranslational modifications such as phosphorylations that are important for protein activation and the regulation of cellular signaling. Previously, we relied on chromogenic signals for detection. However, quantum dots (QDs) represent a more versatile detection system because the signals can be time averaged and the narrow-emission spectra enable multiple protein targets to be quantified within the same spot. We found that commercially available pegylated, streptavidin-conjugated QDs are effective detection agents, with low-background binding to heterogeneous protein mixtures. This type of test, the RPPMAs, is at the forefront of an exciting, clinically-oriented discipline that is emerging, namely tissue or clinical proteomics.
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Fluorescencia , Análisis por Matrices de Proteínas/métodos , Proteínas/análisis , Puntos Cuánticos , Nanotecnología , Proteínas/química , Proteínas/metabolismo , Proteómica/métodos , Estreptavidina/químicaRESUMEN
OBJECTIVE: The rapid diagnosis of acute graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (HCT) is important for optimizing the management of this life-threatening complication. Current diagnostic techniques are time-consuming and require invasive tissue sampling. We investigated serum protein pattern analysis using surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry as a tool to diagnose GVHD. PATIENTS AND METHODS: Eighty-eight serum samples were obtained from 34 patients undergoing HCT either pretransplant (n = 28 samples) or at various time points posttransplant (n = 60 samples), including 22 samples obtained on the day of onset of acute GVHD symptoms. Serum proteomic spectra generated from a "training set" of known samples were used to identify distinct proteomic patterns that best categorized a sample as either pretransplant, posttransplant non-GVHD, or GVHD; these distinct proteomic signatures were subsequently used to classify samples from a masked "test" sample set into the appropriate diagnostic category. RESULTS: Proteomic pattern analysis accurately distinguished GVHD samples from both posttransplant non-GVHD samples and pretransplant samples (100% specificity and 100% sensitivity in both cases). Furthermore, distinct serum proteomic signatures were identified that distinguished pretransplant from posttransplant non-GVHD samples (100% specificity and 94% sensitivity). CONCLUSION: These preliminary data suggest a potential application of SELDI-TOF-based proteomic analysis as a rapid and accurate method to diagnose acute GVHD.
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Proteínas Sanguíneas/análisis , Enfermedad Injerto contra Huésped/sangre , Proteómica , Adulto , Anciano , Biomarcadores/análisis , Femenino , Enfermedad Injerto contra Huésped/diagnóstico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/complicaciones , Neoplasias/terapia , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trasplante HomólogoRESUMEN
PURPOSE: The TWEAK-Fn14 pathway represents a novel anticancer target that is being actively investigated. Understanding the relationship between pharmacokinetics of anti-TWEAK therapeutics and tumor pharmacodynamics is critical. We investigated exposure-response relationships of RG7212, an anti-TWEAK mAb, in patients with Fn14-expressing tumors. EXPERIMENTAL DESIGN: Patients with Fn14-positive tumors (IHC ≥ 1+) treated in a phase I first-in-human study with ascending doses of RG7212 were the basis for this analysis. Pharmacokinetics of RG7212 and dynamics of TWEAK were determined, as were changes in tumor TWEAK-Fn14 signaling in paired pre- and posttreatment tumor biopsies. The objectives of the analysis were to define exposure-response relationships and the relationship between pretreatment tumor Fn14 expression and pharmacodynamic effect. Associations between changes in TWEAK-Fn14 signaling and clinical outcome were explored. RESULTS: Thirty-six patients were included in the analysis. RG7212 reduced plasma TWEAK to undetectable levels at all observed RG7212 exposures. In contrast, reductions in tumor Fn14 and TRAF1 protein expression were observed only at higher exposure (≥ 300 mg*h/mL). Significant reductions in tumor Ki-67 expression and early changes in serum concentrations of CCL-2 and MMP-9 were observed exclusively in patients with higher drug exposure who had high pretreatment tumor Fn14 expression. Pretreatment tumor Fn14 expression was not associated with outcome, but a trend toward longer time on study was observed with high versus low RG7212 exposure. CONCLUSIONS: RG7212 reduced tumor TWEAK-Fn14 signaling in a systemic exposure-dependent manner. In addition to higher exposure, relatively high Fn14 expression might be required for pharmacodynamic effect of anti-TWEAK monoclonal antibodies.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/sangre , Neoplasias Colorrectales/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral/sangre , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/toxicidad , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Quimiocina CCL2/sangre , Neoplasias Colorrectales/sangre , Citocina TWEAK , Femenino , Expresión Génica , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/sangre , Dosis Máxima Tolerada , Persona de Mediana Edad , Receptores del Factor de Necrosis Tumoral/genética , Factor 1 Asociado a Receptor de TNF/metabolismo , Receptor de TWEAK , Resultado del Tratamiento , Inhibidores del Factor de Necrosis Tumoral , Adulto JovenRESUMEN
Molecular diagnostics research within the field of cancer is increasingly focused on detecting low-abundance protein endpoints that can be used to define a patient's disease more completely. Protein microarrays represent an important Clinical Proteomics tool for directly measuring protein endpoints in samples extracted from patient tissues. By combining laser capture microdissection, arraying devices, validated isoform-specific antibodies and advanced reporter technology tools, Clinical Proteomics laboratories are currently generating molecular portraits of cancer cells harvested from patient biopsies.:
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PURPOSE: Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible molecule 14 (Fn14) are a ligand-receptor pair frequently overexpressed in solid tumors. TWEAK: Fn14 signaling regulates multiple oncogenic processes through MAPK, AKT, and NFκB pathway activation. A phase I study of RG7212, a humanized anti-TWEAK IgG1κ monoclonal antibody, was conducted in patients with advanced solid tumors expressing Fn14. EXPERIMENTAL DESIGN: Dose escalations, over a 200- to 7,200-mg range, were performed with patients enrolled in weekly (QW), bi-weekly (Q2W), or every-three-week (Q3W) schedules. Primary objectives included determination of dose and safety profile. Secondary endpoints included assessments related to inhibition of TWEAK: Fn14 signaling, tumor proliferation, tumor immune cell infiltration, and pharmacokinetics. RESULTS: In 192 treatment cycles administered to 54 patients, RG7212 was well-tolerated with no dose-limiting toxicities observed. More than 95% of related adverse events were limited to grade 1/2. Pharmacokinetics were dose proportional for all cohorts, with a t1/2 of 11 to 12 days. Pharmacodynamic changes included clearance of free and total TWEAK ligand and reductions in tumor Ki-67 and TRAF1. A patient with BRAF wild-type melanoma who received 36 weeks of RG7212 therapy had tumor regression and pharmacodynamic changes consistent with antitumor effects. Fifteen patients (28%) received 16 or more weeks of RG7212 treatment. CONCLUSION: RG7212 demonstrated excellent tolerability and favorable pharmacokinetics. Pharmacodynamic endpoints were consistent with reduced TWEAK: Fn14 signaling. Tumor regression was observed and prolonged stable disease was demonstrated in multiple heavily pretreated patients with solid tumors. These encouraging results support further study of RG7212. Clin Cancer Res; 21(2); 258-66. ©2014 AACR.
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Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacocinética , Neoplasias Colorrectales/diagnóstico por imagen , Femenino , Fluorodesoxiglucosa F18 , Humanos , Masculino , Dosis Máxima Tolerada , Melanoma/diagnóstico por imagen , Persona de Mediana Edad , Cintigrafía , Radiofármacos , Resultado del TratamientoRESUMEN
PURPOSE: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models. EXPERIMENTAL DESIGN: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized. RESULTS: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias/inmunología , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Inhibidores del Factor de Necrosis Tumoral , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocina TWEAK , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Neoplasias/genética , Neoplasias/patología , Carga Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Factores de Necrosis Tumoral/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: (a) molecular size sieving, (b) affinity capture of all solution-phase target molecules, and (c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis.
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Biomarcadores , Cromatografía de Afinidad/métodos , Cromatografía en Gel/métodos , Hidrogeles , HidrólisisRESUMEN
Determination of the expression and spatial distribution of molecular epitopes, or antigens, in patient tissue specimens has substantially improved the pathologist's ability to classify disease processes. Certain disease pathophysiologies are marked by characteristic increased or decreased expression of developmentally controlled antigens, defined as Cluster of Differentiation markers, that currently form the foundation for understanding lymphoid malignancies. While chromogens and organic fluorophores have been utilitized for some time in immunohistochemical analyses, developments in synthetic, inorganic fluorophore semiconductors, namely quantum dots, offer a versatile alternative reporter system. Quantum dots are stable fluorophores, are resistant to photobleaching, and are attributed with wide excitation ranges and narrow emission spectra. To date, routinely processed, formalin-fixed tissues have only been probed with two quantum dot reporters simultaneously. In the present study, streptavidin-conjugated quantum dots with distinct emission spectra were tested for their utility in identifying a variety of differentially expressed antigens (surface, cytoplasmic, and nuclear). Slides were analyzed using confocal laser scanning microscopy, which enabled with a single excitation wavelength (488 nm argon laser) the detection of up to seven signals (streptavidin-conjugated quantum dots 525, 565, 585, 605, 655, 705 and 805 nm) plus the detection of 4'6-DiAmidino-2-PhenylIndole with an infra-red laser tuned to 760 nm for two photon excitation. Each of these signals was specific for the intended morphologic immunohistochemical target. In addition, five of the seven streptavidin-conjugated quantum dots tested (not streptavidin-conjugated quantum dots 585 or 805 nm) were used on the same tissue section and could be analyzed simultaneously on routinely processed formalin-fixed, paraffin-embedded sections. Application of this multiplexing method will enable investigators to explore the clinically relevant multidimensional cellular interactions that underlie diseases, simultaneously.
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Tejido Linfoide/química , Microquímica/métodos , Nanotecnología/métodos , Puntos Cuánticos , Espectrometría de Fluorescencia/métodos , Antígenos/análisis , Linfocitos B/química , Linfocitos B/patología , Biomarcadores/análisis , Compuestos Cromogénicos , Células Dendríticas Foliculares/química , Células Dendríticas Foliculares/patología , Diagnóstico por Imagen , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Humanos , Rayos Láser , Tejido Linfoide/patología , Microquímica/instrumentación , Microscopía Confocal , Nanotecnología/instrumentación , Semiconductores , Espectrometría de Fluorescencia/instrumentación , Estreptavidina/química , Linfocitos T/química , Linfocitos T/patologíaRESUMEN
BACKGROUND & AIMS: Hermansky-Pudlak syndrome (HPS), a rare autosomal recessive disorder characterized by oculocutaneous albinism and platelet dysfunction, results from mutations in 1 of at least 7 different genes. Some patients develop a fatal pulmonary fibrosis and others a disabling colitis. This study aimed to document the occurrence of colitis among HPS patients, characterize gastrointestinal tract involvement in HPS, and analyze the distribution of colitis among HPS genotypes. METHODS: Of the 122 HPS patients followed at the National Institutes of Health Clinical Center between 1993 and 2005, 24 were evaluated by endoscopy for gastrointestinal complaints. The histology of gastrointestinal biopsies was retrospectively examined to assess for inflammatory changes, granulomata, and pigmented macrophages. These data were compared with symptoms and HPS genetic subtypes. RESULTS: At colonoscopy, 7 of 23 patients (30%) had endoscopic mucosal abnormalities, including nodularity, erythema, petechiae, or erosions. Six of these 7 patients (86%) had findings of colitis on biopsy. Of the 16 patients with normal-appearing colonic mucosa, 2 patients (12%) had colitis on biopsy. Pigmented macrophages were also observed in the colonic lamina propria in 16 of the 23 patients (70%). Of the 8 patients with confirmed colitis, 7 had the HPS-1 subtype, and 1 had the HPS-4 subtype. CONCLUSIONS: There is an increased frequency of colitis in our population of 122 HPS patients (8/122, 7%) and in HPS patients referred specifically for symptom evaluation (8/24, 33%). Colitis was found in patients with HPS-1 and HPS-4 genotypes.
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Colitis/genética , Síndrome de Hermanski-Pudlak/genética , Adolescente , Adulto , Niño , Preescolar , Colitis/patología , Colonoscopía , Femenino , Genotipo , Síndrome de Hermanski-Pudlak/clasificación , Síndrome de Hermanski-Pudlak/fisiopatología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1-1.5 h.
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Rayos Láser , Microdisección/instrumentación , Microdisección/métodos , Separación Celular/instrumentación , Separación Celular/métodos , ADN , Rayos Infrarrojos , Proteínas , ARN , Coloración y Etiquetado , Rayos UltravioletaRESUMEN
Numerous previously uncharacterized molecules resident within the low molecular weight circulatory proteome may provide a picture of the ongoing pathophysiology of an organism. Recently, proteomic signatures composed of low molecular weight molecules have been identified using mass spectrometry combined with bioinformatic algorithms. Attempts to sequence and identify the molecules that underpin the fingerprints are currently underway. The finding that many of these low molecular weight molecules may exist bound to circulating carrier proteins affords a new opportunity for fractionation and separation techniques prior to mass spectrometry-based analysis. In this study we demonstrate a method whereby nanoporous substrates may be used for the facile and reproducible fractionation and selective binding of the serum-based biomarker material, including subcellular proteins found within the serum. Aminopropyl-coated nanoporous silicon, when exposed to serum, can deplete serum of proteins and yield a serum with a distinct, altered MS profile. Additionally, aminopropyl-coated, nanoporous controlled-pore glass beads are able to bind a subset of serum proteins and release them with stringent elution. The eluted proteins have distinct MS profiles, gel electrophoresis profiles, and differential peptide sequence identities, which vary based on the size of the nanopores. These material surfaces could be employed in strategies for the harvesting and preservation of labile and carrier-protein-bound molecules in the blood.
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Nanoestructuras/química , Suero/química , Electroforesis en Gel de Poliacrilamida , Vidrio , Humanos , Espectrometría de Masas , Microscopía Electrónica , Nanoestructuras/ultraestructura , Péptidos/química , Péptidos/metabolismo , Silicio/química , Especificidad por SustratoRESUMEN
Recent advances in understanding the complex biology of the microenvironment that underlies tumor invasion and migration have revealed novel and promising therapeutic targets. Pharmacological blockade of intra- and extracellular signaling events that regulate migration and survival of multiple cell types may disrupt the host-tumor conspiracy that allows escape from normal developmental regulation.