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1.
EJNMMI Res ; 10(1): 151, 2020 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-33296043

RESUMEN

INTRODUCTION: Trialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an 18F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET. METHODS: A one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide (60 min synthesis time, > 98% radiochemical purity) has been developed, which was used to label the single cysteine residue in C2Am within 30 min at room temperature. Binding of 18F-C2Am to apoptotic and necrotic tumor cells was assessed in vitro, and also in vivo, by dynamic PET and biodistribution measurements in mice bearing human tumor xenografts treated with a TRAILR2 agonist or with conventional chemotherapy. C2Am detection of tumor cell death was validated by correlation of probe binding with histological markers of cell death in tumor sections obtained immediately after imaging. RESULTS: 18F-C2Am showed a favorable biodistribution profile, with predominantly renal clearance and minimal retention in spleen, liver, small intestine, bone and kidney, at 2 h following probe administration. 18F-C2Am generated tumor-to-muscle (T/m) ratios of 6.1 ± 2.1 and 10.7 ± 2.4 within 2 h of probe administration in colorectal and breast tumor models, respectively, following treatment with the TRAILR2 agonist. The levels of cell death (CC3 positivity) following treatment were 12.9-58.8% and 11.3-79.7% in the breast and colorectal xenografts, respectively. Overall, a 20% increase in CC3 positivity generated a one unit increase in the post/pre-treatment tumor contrast. Significant correlations were found between tracer uptake post-treatment, at 2 h post-probe administration, and histological markers of cell death (CC3: Pearson R = 0.733, P = 0.0005; TUNEL: Pearson R = 0.532, P = 0.023). CONCLUSION: The rapid clearance of 18F-C2Am from the blood pool and low kidney retention allowed the spatial distribution of cell death in a tumor to be imaged during the course of therapy, providing a rapid assessment of tumor treatment response. 18F-C2Am has the potential to be used in the clinic to assess early treatment response in tumors.

2.
Theranostics ; 8(14): 3991-4002, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30083276

RESUMEN

The extent of surgical resection is significantly correlated with outcome in glioma; however, current intraoperative navigational tools are useful only in a subset of patients. We show here that a new optical intraoperative technique, Cerenkov luminescence imaging (CLI) following intravenous injection of O­(2-[18F]fluoroethyl)-L-tyrosine (FET), can be used to accurately delineate glioma margins, performing better than the current standard of fluorescence imaging with 5-aminolevulinic acid (5-ALA). Methods: Rats implanted orthotopically with U87, F98 and C6 glioblastoma cells were injected with FET and 5-aminolevulinic acid (5-ALA). Positive and negative tumor regions on histopathology were compared with CL and fluorescence images. The capability of FET CLI and 5-ALA fluorescence imaging to detect tumor was assessed using receptor operator characteristic curves and optimal thresholds (CLIOptROC and 5-ALAOptROC) separating tumor from healthy brain tissue were determined. These thresholds were used to guide prospective tumor resections, where the presence of tumor cells in the resected material and in the remaining brain were assessed by Ki-67 staining. Results: FET CLI signal was correlated with signal in preoperative PET images (y = 1.06x - 0.01; p < 0.0001) and with expression of the amino acid transporter SLC7A5 (LAT1). FET CLI (AUC = 97%) discriminated between glioblastoma and normal brain in human and rat orthografts more accurately than 5-ALA fluorescence (AUC = 91%), with a sensitivity >92% and specificity >91%, and resulted in a more complete tumor resection. Conclusion: FET CLI can be used to accurately delineate glioblastoma tumor margins, performing better than the current standard of fluorescence imaging following 5-ALA administration, and is therefore a promising technique for clinical translation.


Asunto(s)
Neoplasias Encefálicas/cirugía , Glioma/cirugía , Mediciones Luminiscentes/métodos , Cirugía Asistida por Computador/métodos , Tirosina/análogos & derivados , Administración Intravenosa , Animales , Neoplasias Encefálicas/patología , Modelos Animales de Enfermedad , Glioma/patología , Xenoinjertos , Histocitoquímica , Trasplante de Neoplasias , Ratas , Resultado del Tratamiento , Tirosina/administración & dosificación
3.
J Immunol ; 200(7): 2426-2438, 2018 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-29459405

RESUMEN

Atherosclerosis is a complex inflammatory process characterized by monocyte recruitment into the arterial wall, their differentiation into macrophages, and lipid accumulation. Because integrin αMß2 (CD11b/CD18) mediates multiple diverse functions of leukocytes, we examined its role in atherogenesis. αM-/-/ApoE-/- and ApoE-/- mice were fed a control or high fat diet for 3 or 16 wk to induce atherogenesis. Unexpectedly, αM deficiency accelerated development of atherosclerosis in female but not in male mice. The size of aortic root lesions was 3-4.5-fold larger in female αM-/-/ApoE-/- than in ApoE-/- mice. Monocyte and macrophage content within the lesions was increased 2.5-fold in female αM-/-/ApoE-/- mice due to enhanced proliferation. αMß2 elimination promoted gender-dependent foam cell formation due to enhanced uptake of cholesterol by αM-/-/ApoE-/- macrophages. This difference was attributed to enhanced expression of lipid uptake receptors, CD36 and scavenger receptor A1 (SR-A1), in female mice. Macrophages from female αM-/-/ApoE-/- mice showed dramatically reduced expression of FoxM1 transcription factor and estrogen receptors (ER) α and ß. As their antagonists inhibited the effect of 17ß-estradiol (E2), E2 decreased CD36, SR-A1, and foam cell formation in ApoE-/- macrophages in an ERα- and ERß-dependent manner. However, female αM-/-/ApoE-/- macrophages failed to respond to E2 and maintained elevated CD36, SR-A1, and lipid accumulation. FoxM1 inhibition in ApoE-/- macrophages reduced ERs and enhanced CD36 and SR-A1 expression, whereas FoxM1 overexpression in αM-/-/ApoE-/- macrophages reversed their proatherogenic phenotype. We demonstrate a new, surprising atheroprotective role of αMß2 in female ApoE-/- mice. αMß2 maintains ER expression in macrophages and E2-dependent inhibition of foam cell formation.


Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/patología , Estradiol/metabolismo , Receptor alfa de Estrógeno/biosíntesis , Receptor beta de Estrógeno/biosíntesis , Antígeno de Macrófago-1/inmunología , Macrófagos/inmunología , Animales , Aterosclerosis/inmunología , Antígenos CD36 , Colesterol/metabolismo , Femenino , Células Espumosas/citología , Proteína Forkhead Box M1/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Depuradores de Clase A/inmunología
4.
EJNMMI Res ; 7(1): 99, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-29247446

RESUMEN

CORRECTION: Unfortunately, the original version of Figs. 4, 5 and 6b in the article [1] contained errors in the n numbers as indicated on the columns. Please note that column heights and error bars in the original figures and data in the ESM tables are correct and statistical tests are valid. These corrections do not affect any results or conclusions in this article.

5.
J Physiol ; 595(20): 6443-6462, 2017 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-28799653

RESUMEN

KEY POINTS: A reduction in Kindlin-2 levels in endothelial cells compromises vascular barrier function. Kindlin-2 is a previously unrecognized component of endothelial adherens junctions. By interacting directly and simultaneously with ß- or γ-catenin and cortical actin filaments, Kindlin-2 stabilizes adherens junctions. The Kindlin-2 binding sites for ß- and γ-catenin reside within its F1 and F3 subdomains. Although Kindlin-2 does not associate directly with tight junctions, its downregulation also destabilizes these junctions. Thus, impairment of both adherens and tight junctions may contribute to enhanced leakiness of vasculature in Kindlin-2+/- mice. ABSTRACT: Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischaemia or sepsis and cause severe organ dysfunction. Kindlin-2, which is primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin-2 in regulating vascular integrity using several different approaches to decrease Kindlin-2 levels in EC. Reduced levels of Kindlin-2 in Kindlin-2+/- mice aortic endothelial cells (MAECs) from these mice, and human umbilical ECs (HUVEC) treated with Kindlin-2 siRNA showed enhanced basal and platelet-activating factor (PAF) or lipopolysaccharide-stimulated vascular leakage compared to wild-type (WT) counterparts. PAF preferentially disrupted the Kindlin-2+/- MAECs barrier to BSA and dextran and reduced transendothelial resistance compared to WT cells. Kindlin-2 co-localized and co-immunoprecipitated with vascular endothelial cadherin-based complexes, including ß- and γ-catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin-2 with ß- and γ-catenin and actin was demonstrated in co-immunoprecipitation and surface plasmon resonance experiments. In thrombin-stimulated HUVECs, Kindlin-2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The ß- and γ-catenin binding site resides within the F1 and F3 subdomains of Kindlin-2 but not the integrin binding site in F3. These results establish a previously unrecognized and vital role of Kindlin-2 with respect to maintaining the vascular barrier by linking Vascuar endothelial cadherin-based complexes to cortical actin and thereby stabilizing AJ.


Asunto(s)
Uniones Adherentes/fisiología , Proteínas del Citoesqueleto/fisiología , Células Endoteliales/fisiología , Proteínas Musculares/fisiología , Animales , Aorta/citología , Sitios de Unión , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Células Endoteliales/metabolismo , Femenino , Células HEK293 , Humanos , Pulmón/irrigación sanguínea , Pulmón/fisiología , Masculino , Ratones Transgénicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Dominios Proteicos , Piel/irrigación sanguínea , Fenómenos Fisiológicos de la Piel , Tráquea/irrigación sanguínea , Tráquea/fisiología , Venas Umbilicales/citología , beta Catenina/metabolismo
7.
Nat Commun ; 8: 15559, 2017 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-28555620

RESUMEN

Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMß2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1-/-) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1-/- mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.


Asunto(s)
Plaquetas/inmunología , Leucocitos/metabolismo , Antígeno de Macrófago-1/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Trombosis/inmunología , Animales , Sitios de Unión , Tiempo de Sangría , Coagulación Sanguínea , Plaquetas/citología , Plaquetas/metabolismo , Arterias Carótidas/patología , Glucosamina/química , Hemostasis , Inflamación , Leucocitos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microcirculación , Células 3T3 NIH , Tiempo de Tromboplastina Parcial , Fagocitosis , Activación Plaquetaria , Recuento de Plaquetas , Unión Proteica , Dominios Proteicos , Transducción de Señal , Trombina/metabolismo
8.
J Nucl Med ; 58(6): 881-887, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28209913

RESUMEN

Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of synaptotagmin-I. Methods: The capability of near-infrared fluorophore-labeled and 99mTc- and 111In-labeled derivatives of C2Am for imaging tumor cell death, using planar near-infrared fluorescence imaging and SPECT, respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft. Results: The fluorophore-labeled C2Am derivative showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumor cell death (2%-5%). There was a significant correlation (R > 0.9, P < 0.05) between fluorescently labeled C2Am binding and histologic markers of cell death, including cleaved caspase-3, whereas there was no such correlation with a site-directed mutant of C2Am (iC2Am) that does not bind phosphatidylserine. 99mTc-C2Am and 111In-C2Am also showed favorable biodistribution profiles, with predominantly renal clearance and low nonspecific retention in the liver and spleen at 24 h after probe administration. 99mTc-C2Am and 111In-C2Am generated tumor-to-muscle ratios in drug-treated tumors of 4.3× and 2.2×, respectively, at 2 h and 7.3× and 4.1×, respectively, at 24 h after administration. Conclusion: Given the favorable biodistribution profile of 99mTc- and 111In-labeled C2Am, and their ability to produce rapid and cell death-specific image contrast, these agents have potential for clinical translation.


Asunto(s)
Apoptosis , Imagen Molecular/métodos , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Tomografía de Emisión de Positrones/métodos , Sinaptotagmina I/farmacocinética , Animales , Biomarcadores/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias Experimentales/diagnóstico por imagen , Dominios Proteicos , Radiofármacos/farmacocinética , Sinaptotagmina I/química , Distribución Tisular
9.
Theranostics ; 7(1): 40-50, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28042315

RESUMEN

The positron emission tomography (PET) tracer 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) has been proposed to measure cell proliferation non-invasively in vivo. Hence, it should provide valuable information for response assessment to tumor therapies. To date, [18F]FLT uptake has found limited use as a response biomarker in clinical trials in part because a better understanding is needed of the determinants of [18F]FLT uptake and therapy-induced changes of its retention in the tumor. In this systematic review of preclinical [18F]FLT studies, comprising 174 reports, we identify the factors governing [18F]FLT uptake in tumors, among which thymidine kinase 1 plays a primary role. The majority of publications (83 %) report that decreased [18F]FLT uptake reflects the effects of anticancer therapies. 144 times [18F]FLT uptake was related to changes in proliferation as determined by ex vivo analyses. Of these approaches, 77 % describe a positive relation, implying a good concordance of tracer accumulation and tumor biology. These preclinical data indicate that [18F]FLT uptake holds promise as an imaging biomarker for response assessment in clinical studies. Understanding of the parameters which influence cellular [18F]FLT uptake and retention as well as the mechanism of changes induced by therapy is essential for successful implementation of this PET tracer. Hence, our systematic review provides the background for the use of [18F]FLT in future clinical studies.


Asunto(s)
Didesoxinucleósidos/administración & dosificación , Monitoreo de Drogas/métodos , Neoplasias/diagnóstico por imagen , Neoplasias/terapia , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Animales , Evaluación Preclínica de Medicamentos
10.
Infect Immun ; 85(1)2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27799334

RESUMEN

Integrins αMß2 and αXß2 are homologous adhesive receptors that are expressed on many of the same leukocyte populations and bind many of the same ligands. Although αMß2 was extensively characterized and implicated in leukocyte inflammatory and immune functions, the roles of αXß2 remain largely obscure. Here, we tested the ability of mice deficient in integrin αMß2 or αXß2 to deal with opportunistic infections and the capacity of cells derived from these animals to execute inflammatory functions. The absence of αMß2 affected the recruitment of polymorphonuclear neutrophils (PMN) to bacterial and fungal pathogens as well as to model inflammatory stimuli, and αMß2-deficient PMN displayed defective inflammatory functions. In contrast, deficiency of αXß2 abrogated intraperitoneal recruitment and adhesive functions of monocytes and macrophages (Mϕ) and the ability of these cells to kill/phagocytose Candida albicans or Escherichia coli cells both ex vivo and in vivo During systemic candidiasis, the absence of αXß2 resulted in the loss of antifungal activity by tissue Mϕ and inhibited the production of tumor necrosis factor alpha (TNF-α)/interleukin-6 (IL-6) in infected kidneys. Deficiency of αMß2 suppressed Mϕ egress from the peritoneal cavity, decreased the production of anti-inflammatory IL-10, and stimulated the secretion of IL-6. The absence of αXß2, but not of αMß2, increased survival against a septic challenge with lipopolysaccharide (LPS) by 2-fold. Together, these results suggest that αMß2 plays a primary role in PMN inflammatory functions and regulates the anti-inflammatory functions of Mϕ, whereas αXß2 is central in the regulation of inflammatory functions of recruited and tissue-resident Mϕ.


Asunto(s)
Antiinfecciosos/metabolismo , Inflamación/metabolismo , Integrina alfaXbeta2/metabolismo , Leucocitos/metabolismo , Antígeno de Macrófago-1/metabolismo , Animales , Candida albicans/metabolismo , Candidiasis/metabolismo , Candidiasis/microbiología , Adhesión Celular/fisiología , Escherichia coli/metabolismo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Inflamación/microbiología , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Leucocitos/microbiología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Neutrófilos/microbiología , Fagocitosis/fisiología , Factor de Necrosis Tumoral alfa/metabolismo
11.
EJNMMI Res ; 6(1): 63, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27515446

RESUMEN

BACKGROUND: Recent studies have shown that 3'-deoxy-3'-[(18)F] fluorothymidine ([(18)F]FLT)) uptake depends on endogenous tumour thymidine concentration. The purpose of this study was to investigate tumour thymidine concentrations and whether they correlated with [(18)F]FLT uptake across a broad spectrum of murine cancer models. A modified liquid chromatography-mass spectrometry (LC-MS/MS) method was used to determine endogenous thymidine concentrations in plasma and tissues of tumour-bearing and non-tumour bearing mice and rats. Thymidine concentrations were determined in 22 tumour models, including xenografts, syngeneic and spontaneous tumours, from six research centres, and a subset was compared for [(18)F]FLT uptake, described by the maximum and mean tumour-to-liver uptake ratio (TTL) and SUV. RESULTS: The LC-MS/MS method used to measure thymidine in plasma and tissue was modified to improve sensitivity and reproducibility. Thymidine concentrations determined in the plasma of 7 murine strains and one rat strain were between 0.61 ± 0.12 µM and 2.04 ± 0.64 µM, while the concentrations in 22 tumour models ranged from 0.54 ± 0.17 µM to 20.65 ± 3.65 µM. TTL at 60 min after [(18)F]FLT injection, determined in 14 of the 22 tumour models, ranged from 1.07 ± 0.16 to 5.22 ± 0.83 for the maximum and 0.67 ± 0.17 to 2.10 ± 0.18 for the mean uptake. TTL did not correlate with tumour thymidine concentrations. CONCLUSIONS: Endogenous tumour thymidine concentrations alone are not predictive of [(18)F]FLT uptake in murine cancer models.

12.
Am J Physiol Heart Circ Physiol ; 311(3): H725-34, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27288438

RESUMEN

Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains ß-1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of ß-1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 µmol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of αIIbß3 BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans GLc5 decreased ATP release and TGF-ß1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimulation and reduce platelet-neutrophil interaction.


Asunto(s)
Plaquetas/efectos de los fármacos , Glucósidos/farmacología , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , beta-Glucanos/farmacología , Adenosina Trifosfato/metabolismo , Plaquetas/metabolismo , Candida albicans , Fibrinógeno/efectos de los fármacos , Fibrinógeno/metabolismo , Hongos/química , Humanos , Neutrófilos , Selectina-P/efectos de los fármacos , Selectina-P/metabolismo , Fosforilación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Trombina/efectos de los fármacos , Trombina/metabolismo , Receptor Toll-Like 4/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba
13.
Cancer J ; 21(2): 129-36, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25815854

RESUMEN

Positron emission tomography (PET) is an extraordinarily sensitive clinical imaging modality for interrogating tumor metabolism. Radiolabeled PET substrates can be traced at subphysiological concentrations, allowing noninvasive imaging of metabolism and intratumoral heterogeneity in systems ranging from advanced cancer models to patients in the clinic. There are a wide range of novel and more established PET radiotracers, which can be used to investigate various aspects of the tumor, including carbohydrate, amino acid, and fatty acid metabolism. In this review, we briefly discuss the more established metabolic tracers and describe recent work on the development of new tracers. Some of the unanswered questions in tumor metabolism are considered alongside new technical developments, such as combined PET/magnetic resonance imaging scanners, which could provide new imaging solutions to some of the outstanding diagnostic challenges facing modern cancer medicine.


Asunto(s)
Metabolismo Energético , Neoplasias/diagnóstico , Neoplasias/metabolismo , Tomografía de Emisión de Positrones , Aminoácidos/metabolismo , Animales , Metabolismo de los Hidratos de Carbono , Diagnóstico por Imagen , Ácidos Grasos/metabolismo , Humanos , Redes y Vías Metabólicas , Tomografía de Emisión de Positrones/métodos , Radiofármacos
14.
J Immunol ; 193(9): 4712-21, 2014 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-25261488

RESUMEN

Polymorphonuclear neutrophils (PMNs) and macrophages are crucial contributors to neovascularization, serving as a source of chemokines, growth factors, and proteases. α(M)ß(2)(CD11b/CD18) and α(L)ß(2)(CD11a/CD18) are expressed prominently and have been implicated in various responses of these cell types. Thus, we investigated the role of these ß2 integrins in angiogenesis. Angiogenesis was analyzed in wild-type (WT), α(M)-knockout (α(M)(-/-)), and α(L)-deficient (α(L)(-/-)) mice using B16F10 melanoma, RM1 prostate cancer, and Matrigel implants. In all models, vascular area was decreased by 50-70% in α(M)(-/-) mice, resulting in stunted tumor growth as compared with WT mice. In contrast, α(L) deficiency did not impair angiogenesis and tumor growth. The neovessels in α(M)(-/-) mice were leaky and immature because they lacked smooth muscle cell and pericytes. Defective angiogenesis in the α(M)(-/-) mice was associated with attenuated PMN and macrophage recruitment into tumors. In contrast to WT or the α(L)(-/-) leukocytes, the α(M)(-/-) myeloid cells showed impaired plasmin (Plm)-dependent extracellular matrix invasion, resulting from 50-75% decrease in plasminogen (Plg) binding and pericellular Plm activity. Surface plasmon resonance verified direct interaction of the α(M)I-domain, the major ligand binding site in the ß(2) integrins, with Plg. However, the α(L)I-domain failed to bind Plg. In addition, endothelial cells failed to form tubes in the presence of conditioned medium collected from TNF-α-stimulated PMNs derived from the α(M)(-/-) mice because of severely impaired degranulation and secretion of VEGF. Thus, α(M)ß(2) plays a dual role in angiogenesis, supporting not only Plm-dependent recruitment of myeloid cells to angiogenic niches, but also secretion of VEGF by these cells.


Asunto(s)
Leucocitos/inmunología , Leucocitos/metabolismo , Antígeno de Macrófago-1/genética , Neovascularización Patológica/genética , Animales , Trasplante de Médula Ósea , Quimiotaxis/genética , Quimiotaxis/inmunología , Modelos Animales de Enfermedad , Antígeno de Macrófago-1/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Noqueados , Neovascularización Patológica/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Plasminógeno/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Carga Tumoral , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genética
15.
J Nucl Med ; 55(7): 1144-9, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24777291

RESUMEN

UNLABELLED: Tumors are often characterized by high levels of de novo fatty acid synthesis. The kinetics of acetate incorporation into tricarboxylic acid cycle intermediates and into lipids suggest that detection of tumors with [1-(11)C]acetate PET could be improved by imaging at later time points. METHODS: The uptake and metabolism of [1-(11)C], [1-(13)C], and [1-(14)C]acetate were measured in mouse prostate and lung cancer models to investigate the time course of (11)C label incorporation into tumor metabolites. RESULTS: Radioactivity in the lipid fraction, as compared with the aqueous fraction, in extracts of C4-2B human prostate xenografts peaked at 90 min after [1-(14)C]acetate injection, which coincided with peak (13)C label incorporation into the fatty acids palmitate and stearate. Contrast between the tumor and tissues, such as blood and muscle, increased in PET images acquired over a period of 120 min after [1-(11)C]acetate injection, and Patlak plots were linear from 17.5 min after injection. Similar results were obtained in a genetically engineered K-ras(G12D); p53(null) lung cancer model, in which the mean tumor-to-lung ratio at 90 min after [1-(14)C]acetate injection was 4.4-fold higher than at 15 min. CONCLUSION: These findings suggest that when imaging de novo fatty acid synthesis with [1-(11)C]acetate it is preferable to measure uptake at later time points, when the effects of perfusion and (11)C incorporation into tricarboxylic acid cycle intermediates and bicarbonate are declining. The data presented here suggest that future clinical PET scans of tumors should be acquired later than 30 min, when tracer accumulation due to de novo fatty acid synthesis prevails.


Asunto(s)
Acetatos , Ácidos Grasos/biosíntesis , Neoplasias Pulmonares/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Neoplasias de la Próstata/diagnóstico por imagen , Animales , Radioisótopos de Carbono , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Neoplasias de la Próstata/metabolismo , Factores de Tiempo , Tomografía Computarizada por Rayos X
16.
Proc Natl Acad Sci U S A ; 111(1): 415-20, 2014 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-24347640

RESUMEN

The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd(3+) ion for the radionuclide, (111)In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging.


Asunto(s)
Genes Reporteros , Imagen por Resonancia Magnética/métodos , Animales , Línea Celular Tumoral , Medios de Contraste/química , Femenino , Gadolinio/química , Gadolinio DTPA/química , Células HCT116 , Células HEK293 , Humanos , Aumento de la Imagen/métodos , Iones , Células MCF-7 , Ratones , Ratones SCID , Microscopía Fluorescente/métodos , Trasplante de Neoplasias , Transportadores de Anión Orgánico/metabolismo , Tomografía Computarizada de Emisión de Fotón Único/métodos
17.
Blood ; 122(14): 2491-9, 2013 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-23896409

RESUMEN

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2(+/-) mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2(+/-) mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2(+/-) mice, kindlin-2(+/-) endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5'-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5'-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73) were increased twofold to threefold on kindlin-2(+/-) ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2(+/-) mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.


Asunto(s)
Plaquetas/metabolismo , Clatrina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células Endoteliales/metabolismo , Hemostasis/fisiología , Proteínas Musculares/metabolismo , 5'-Nucleotidasa/biosíntesis , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Antígenos CD/biosíntesis , Apirasa/biosíntesis , Membrana Celular/metabolismo , Femenino , Citometría de Flujo , Inmunoprecipitación , Masculino , Ratones , Ratones Noqueados , Agregación Plaquetaria/fisiología , Transporte de Proteínas/fisiología , Resonancia por Plasmón de Superficie
18.
J Immunol ; 189(5): 2468-77, 2012 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-22844116

RESUMEN

The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin α(X)ß2 in the pathogenesis of fungal infection was assessed. Both purified α(X)ß2 and α(X)ß2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by ß-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of α(M)ß2. Mice deficient in α(X)ß2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in α(X)ß2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of α(X)ß2-deficient mice within 9 d. Organs taken from α(X)ß2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that α(X)ß2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use α(X)ß2 to control fungal invasion.


Asunto(s)
Candida albicans/patogenicidad , Candidiasis/inmunología , Candidiasis/prevención & control , Integrina alfaXbeta2/metabolismo , Leucocitos/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Candidiasis/microbiología , Adhesión Celular/inmunología , Línea Celular , Movimiento Celular/inmunología , Citofagocitosis/inmunología , Células HEK293 , Humanos , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/fisiología , Leucocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores Inmunológicos/fisiología
19.
Eur J Cancer ; 48(4): 416-24, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22209266

RESUMEN

The paradigm of drug development is shifting towards early use of imaging biomarkers as surrogate end-points in clinical trials. Quantitative Imaging in Cancer: Connecting Cellular Processes (QuIC-ConCePT) is an initiative to qualify complementary imaging biomarkers (IB) of proliferation, cell death and tumour heterogeneity as possible tools in early phase clinical trials to help pharmaceutical developers in 'go, no-go' decisions early in the process of drug development. One of the IBs is [(18)F]3'-deoxy-3'-fluorothymidine with Positron Emission Tomography (FLT-PET). We review results of recent clinical trials using FLT-PET for monitoring tumour response to drug treatment and discuss the potential and the possible pitfalls of using this IB as a surrogate end-point in early phase clinical trials for assessing tumour response to drug treatment. From first human trial results it seems that the degree of FLT accumulation in tumours is governed not only by the tumour proliferation rate but also by other factors. Nevertheless FLT-PET could potentially be used as a negative predictor of tumour response to chemotherapy, and hence evaluation of this IB is granted in multi-centre clinical trials.


Asunto(s)
Proliferación Celular , Didesoxinucleósidos , Neoplasias/diagnóstico por imagen , Neoplasias/terapia , Biomarcadores Farmacológicos/análisis , Biomarcadores de Tumor/análisis , Diagnóstico por Imagen/métodos , Didesoxinucleósidos/análisis , Humanos , Neoplasias/patología , Tomografía de Emisión de Positrones/métodos , Pronóstico , Trazadores Radiactivos , Resultado del Tratamiento
20.
Circ Res ; 109(11): 1269-79, 2011 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-21998326

RESUMEN

RATIONALE: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. OBJECTIVE: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. METHODS AND RESULTS: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the ß-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr(-/-) mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. CONCLUSIONS: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.


Asunto(s)
Aterosclerosis/metabolismo , Ligando de CD40/metabolismo , Quimiotaxis de Leucocito/fisiología , Antígeno de Macrófago-1/metabolismo , Trombosis/etiología , Secuencias de Aminoácidos , Animales , Aterosclerosis/genética , Aterosclerosis/prevención & control , Tiempo de Sangría , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/fisiología , Células CHO , Células Cultivadas , Cricetinae , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Péptidos Cíclicos/farmacología , Peritonitis/sangre , Peritonitis/prevención & control , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Receptores de LDL/deficiencia , Proteínas Recombinantes de Fusión/fisiología , Resonancia por Plasmón de Superficie
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