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1.
PLoS Pathog ; 8(2): e1002517, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22319449

RESUMEN

The early host response to viral infections involves transient activation of pattern recognition receptors leading to an induction of inflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα). Subsequent activation of cytokine receptors in an autocrine and paracrine manner results in an inflammatory cascade. The precise mechanisms by which viruses avert an inflammatory cascade are incompletely understood. Nuclear factor (NF)-κB is a central regulator of the inflammatory signaling cascade that is controlled by inhibitor of NF-κB (IκB) proteins and the IκB kinase (IKK) complex. In this study we show that murine cytomegalovirus inhibits the inflammatory cascade by blocking Toll-like receptor (TLR) and IL-1 receptor-dependent NF-κB activation. Inhibition occurs through an interaction of the viral M45 protein with the NF-κB essential modulator (NEMO), the regulatory subunit of the IKK complex. M45 induces proteasome-independent degradation of NEMO by targeting NEMO to autophagosomes for subsequent degradation in lysosomes. We propose that the selective and irreversible degradation of a central regulatory protein by autophagy represents a new viral strategy to dampen the inflammatory response.


Asunto(s)
Quinasa I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Muromegalovirus/metabolismo , FN-kappa B/antagonistas & inhibidores , Fagosomas/inmunología , Animales , Autofagia , Inflamación , Interleucina-1beta/metabolismo , Ratones , FN-kappa B/metabolismo , Células 3T3 NIH , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores de Interleucina-1/inmunología , Receptores de Interleucina-1/metabolismo , Transducción de Señal/inmunología , Receptores Toll-Like/antagonistas & inhibidores , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
2.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 2): 308-12, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23385466

RESUMEN

Successful cryogenic X-ray structure determination from a single high-pressure-frozen bovine enterovirus 2 crystal is reported. The presented high-pressure-freezing procedure is based on a commercially available device and allows the cryocooling of macromolecular crystals directly in their mother liquor without the time- and crystal-consuming search for optimal cryoconditions. The method is generally applicable and will allow cryogenic data collection from all types of macromolecular crystals.


Asunto(s)
Enterovirus Bovino/química , Congelación , Presión , Animales , Bovinos , Microscopía por Crioelectrón/métodos , Crioprotectores/farmacología , Cristalización , Cristalografía por Rayos X/métodos , Difusión , Enterovirus Bovino/efectos de la radiación , Congelación/efectos adversos
3.
J Hepatol ; 58(1): 112-8, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22989568

RESUMEN

BACKGROUND & AIMS: The liver can mitigate the inflammatory activity of infiltrating T cells by mechanisms that are not entirely clear. Here we investigated the role of liver sinusoidal endothelial cells (LSECs) in regulating the activity of inflammatory CD4 T cells. METHODS: Interactions between T helper (Th) 1 or Th17 cells and LSEC were studied by intravital microscopy and by in vitro stimulation assays. RESULTS: Circulating CD4 T cells established lasting and repeated interactions with liver endothelium in vivo. Stimulation of Th1 and Th17 cells by LSEC greatly inhibited their capacity to secrete interferon-γ or interleukin-17 in vitro; in contrast, stimulation by dendritic cells (DCs) resulted in considerable secretion of both cytokines. Cytokine release by Th1 or Th17 cells seemed to be actively suppressed by LSEC, as indicated by the inhibition of cytokine secretion even in the presence of Th1- and Th17-promoting DC. This inhibition of CD4 T cell effector function seemed to depend on the dominance of inhibitory over activating co-stimulatory signals on LSEC, since (1) cytokine secretion could be restored by increased CD28 co-activation; (2) LSEC from interleukin-10(-/-) mice, which manifest increased activating signals, such as MHC II, and decreased inhibitory signals, such as PD-L1, failed to suppress cytokine secretion; and (3) cytokine secretion by Th1 or Th17 cells that lacked PD-1, the ligand for inhibitory PD-L1, could not be suppressed by LSEC. CONCLUSIONS: LSEC inhibit inflammatory cytokine secretion of Th1 and Th17 effector CD4 T cells in dependence of interleukin-10 and PD-1.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Comunicación Celular/inmunología , Células Endoteliales/inmunología , Hígado/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Endoteliales/citología , Femenino , Tolerancia Inmunológica/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Hígado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal/inmunología , Células TH1/citología , Células TH1/inmunología , Células Th17/citología , Células Th17/inmunología
4.
J Virol ; 86(5): 2826-36, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22205742

RESUMEN

In macrophages, HIV-1 accumulates in intracellular vesicles designated virus-containing compartments (VCCs). These might play an important role in the constitution of macrophages as viral reservoirs and allow HIV-1 to evade the immune system by sequestration in an internal niche, which is difficult to access from the exterior. However, until now, evidence of whether internal virus accumulations are protected from the host's humoral immune response is still lacking. In order to be able to study the formation and antibody accessibility of VCCs, we generated HIV-1 with green fluorescent protein (GFP)-tagged Gag replicating in primary macrophages. Live-cell observations revealed faint initial cytosolic Gag expression and subsequent large intracellular Gag accumulations which stayed stable over days. Taking advantage of the opportunity to study the accessibility of intracellular VCCs via the cell surface, we demonstrate that macrophage internal HIV-1-containing compartments cannot be targeted by neutralizing antibodies. Furthermore, HIV-1 was efficiently transferred from antibody-treated macrophages to T cells. Three-dimensional reconstruction of electron microscopic slices revealed that Gag accumulations correspond to viral particles within enclosed compartments and convoluted membranes. Thus, although some VCCs were connected to the plasma membrane, the complex membrane architecture of the HIV-1-containing compartment might shield viral particles from neutralizing antibodies. In sum, our study provides evidence that HIV-1 is sequestered into a macrophage internal membranous web, posing an obstacle for the elimination of this viral reservoir.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Macrófagos/inmunología , Línea Celular , Regulación Viral de la Expresión Génica , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Evasión Inmune , Macrófagos/virología , Replicación Viral
5.
Cell Microbiol ; 14(5): 774-89, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22289149

RESUMEN

The mechanism of budding of influenza A virus revealed important deviation from the consensus mechanism of budding of retroviruses and of a growing number of negative-strand RNA viruses. This study is focused on the role of the influenza A virus matrix protein M1 in virus release. We found that a mutation of the proline residue at position 16 of the matrix protein induces inhibition of virus detachment from cells. Depletion of the M1-binding protein RACK1 also impairs virus release and RACK1 binding requires the proline residue at position 16 of M1. The impaired M1-RACK1 interaction does not affect the plasma membrane binding of M1; in contrast, RACK1 is recruited to detergent-resistant membranes in a M1-proline-16-dependent manner. The proline-16 mutation in M1 and depletion of RACK1 impairs the pinching-off of the budding virus particles. These findings reveal the active role of the viral matrix protein in the release of influenza A virus particles that involves a cross-talk with a RACK1-mediated pathway.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Interacciones Huésped-Patógeno , Subtipo H1N1 del Virus de la Influenza A/fisiología , Proteínas de Neoplasias/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus , Línea Celular , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Prolina/genética , Prolina/metabolismo , Mapeo de Interacción de Proteínas , Receptores de Cinasa C Activada , Proteínas de la Matriz Viral/genética
6.
J Infect Dis ; 205(11): 1654-64, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22457281

RESUMEN

Many enveloped viruses, including herpes viruses, hepatitis B virus (HBV), and hepatitis C virus (HCV), and human immunodeficiency virus (HIV), are among the most important human pathogens and are often responsible for coinfections involving ≥2 types of viruses. However, therapies that are effective against multiple virus classes are rare. Here we present a new class of synthetic anti-lipopolysaccharide peptides (SALPs) that bind to heparan sulfate moieties on the cell surface and inhibit infection with a variety of enveloped viruses. We demonstrate that SALPs inhibit entry of human immunodeficiency virus type 1 (HIV-1), herpes simplex virus (HSV) 1 and 2, HBV, and HCV to their respective host cells. Despite their high antiviral efficiency, SALPs were well tolerated, and neither toxicity nor measurable inhibitor-induced adverse effects were observed. Since these broad-spectrum antiviral peptides target a host cell rather than a viral component, they may also be useful for suppression of viruses that are resistant to antiviral drugs.


Asunto(s)
Antivirales/farmacología , Péptidos/farmacología , Acoplamiento Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Virus/efectos de los fármacos , Antivirales/toxicidad , Línea Celular , Supervivencia Celular , Heparitina Sulfato/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Péptidos/toxicidad , Unión Proteica
7.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 68(Pt 4): 495-500, 2012 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-22505429

RESUMEN

High-pressure freezing (HPF) is a method which allows sample vitrification without cryoprotectants. In the present work, protein crystals were cooled to cryogenic temperatures at a pressure of 210 MPa. In contrast to other HPF methods published to date in the field of cryocrystallography, this protocol involves rapid sample cooling using a standard HPF device. The fast cooling rates allow HPF of protein crystals directly in their mother liquor without the need for cryoprotectants or external reagents. HPF was first attempted with hen egg-white lysozyme and cubic insulin crystals, yielding good to excellent diffraction quality. Non-cryoprotected crystals of the membrane protein photosystem II have been successfully cryocooled for the first time. This indicates that the presented HPF method is well suited to the vitrification of challenging systems with large unit cells and weak crystal contacts.


Asunto(s)
Cristalografía por Rayos X/métodos , Insulina/análisis , Muramidasa/análisis , Animales , Pollos , Cristalografía por Rayos X/instrumentación , Congelación , Presión , Factores de Tiempo
8.
AIDS Res Ther ; 9(1): 2, 2012 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-22260499

RESUMEN

Recently, it has been shown that human ejaculate enhances human immunodeficiency virus 1 (HIV-1) infectivity. Enhancement of infectivity is conceived to be mediated by amyloid filaments from peptides that are proteolytically released from prostatic acid phosphatase (PAP), termed Semen-derived Enhancer of Virus Infection (SEVI). The aim of this study was to test the range of HIV-1 infectivity enhancing properties of a large number of individual semen samples (n = 47) in a TZM-bl reporter cell HIV infection system. We find that semen overall increased infectivity to 156% of the control experiment without semen, albeit with great inter- and intraindividual variability (range -53%-363%). Using transmission electron microscopy, we provide evidence for SEVI fibrils in fresh human semen for the first time. Moreover, we confirm that the infectivity enhancing property can be inhibited by the major green tea ingredient epigallocatechin-3-gallate (EGCG) at non-toxic concentrations. The median inhibition of infection by treatment with 0.4 mM EGCG was 70.6% (p < 0.0001) in our cohort. Yet, there were substantial variations of inhibition and in a minority of samples, infectivity enhancement was not inhibited by EGCG treatment at all. Thus, topical application of EGCG may be a feasible additional measure to prevent the sexual transmission of HIV. However, the reasons for the variability in the efficacy of the abrogation of semen-mediated enhancement of HIV-1 infectivity and EGCG efficacy have to be elucidated before therapeutic trials can be conducted.

9.
Proc Natl Acad Sci U S A ; 106(22): 9033-8, 2009 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-19451623

RESUMEN

Peptide fragments, derived from prostatic acidic phosphatase, are secreted in large amounts into human semen and form amyloid fibrils. These fibrillar structures, termed semen-derived enhancer of virus infection (SEVI), capture HIV virions and direct them to target cells. Thus, SEVI appears to be an important infectivity factor of HIV during sexual transmission. Here, we are able to demonstrate that epigallocatechin-3-gallate (EGCG), the major active constituent of green tea, targets SEVI for degradation. Furthermore, it is shown that EGCG inhibits SEVI activity and abrogates semen-mediated enhancement of HIV-1 infection in the absence of cellular toxicity. Therefore, EGCG appears to be a promising supplement to antiretroviral microbicides to reduce sexual transmission of HIV-1.


Asunto(s)
Fármacos Anti-VIH/farmacología , Catequina/análogos & derivados , Infecciones por VIH/prevención & control , VIH-1 , Semen/efectos de los fármacos , Amiloide/antagonistas & inhibidores , Amiloide/metabolismo , Camellia sinensis/química , Catequina/farmacología , Células Cultivadas , Infecciones por VIH/transmisión , Humanos , Masculino , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Semen/virología
10.
Circ Res ; 105(3): 239-48, 2009 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-19590044

RESUMEN

RATIONALE: Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein (cMyBP)-C are frequent causes of hypertrophic cardiomyopathy, but the mechanisms leading from mutations to disease remain elusive. OBJECTIVE: The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations. METHODS AND RESULTS: We developed a cMyBP-C knock-in mouse carrying a point mutation. The level of total cMyBP-C mRNAs was 50% and 80% lower in heterozygotes and homozygotes, respectively. Surprisingly, the single G>A transition on the last nucleotide of exon 6 resulted in 3 different mutant mRNAs: missense (exchange of G for A), nonsense (exon skipping, frameshift, and premature stop codon) and deletion/insertion (as nonsense but with additional partial retention of downstream intron, restoring of the reading frame, and almost full-length protein). Inhibition of nonsense-mediated mRNA decay in cultured cardiac myocytes or in vivo with emetine or cycloheximide increased the level of nonsense mRNAs severalfold but not of the other mRNAs. By using sequential protein fractionation and a new antibody directed against novel amino acids produced by the frameshift, we showed that inhibition of the proteasome with epoxomicin via osmotic minipumps increased the level of (near) full-length mutants but not of truncated proteins. Homozygotes exhibited myocyte and left ventricular hypertrophy, reduced fractional shortening, and interstitial fibrosis; heterozygotes had no major phenotype. CONCLUSIONS: These data reveal (1) an unanticipated complexity of the expression of a single point mutation in the whole animal and (2) the involvement of both nonsense-mediated mRNA decay and the ubiquitin-proteasome system in lowering the level of mutant proteins.


Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Codón sin Sentido/genética , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad del ARN/genética , Ubiquitina/metabolismo , Animales , Células Cultivadas , Cicloheximida/farmacología , Modelos Animales de Enfermedad , Emetina/farmacología , Exones/genética , Técnicas de Sustitución del Gen , Homocigoto , Ratones , Ratones Mutantes , Ratones Transgénicos , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Células Musculares/patología , Mutación Puntual/genética , Inhibidores de la Síntesis de la Proteína/farmacología
11.
J Immunol ; 183(9): 5847-60, 2009 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-19812190

RESUMEN

Autophagy is a central lysosomal degradation process that is essential for the maintenance of cellular homeostasis. Autophagy has furthermore emerged as integral part of the host immune response. Autophagic processes promote the separation and degradation of intracellular microorganisms which contributes to the development of innate and adaptive immunity. Some pathogenic microbes have therefore evolved mechanisms to evade or impede autophagy. We analyzed the effects of the enteropathogenic bacterium Yersinia enterocolitica on autophagy in macrophages. Yersiniae use a number of defined adhesins and secreted proteins to manipulate host immune responses. Our results showed that Y. enterocolitica defective in type III protein secretion efficiently activated autophagy in macrophages. Autophagy was mediated by the Yersinia adhesins invasin and YadA and particularly depended on the engagement of beta(1) integrin receptors. Several autophagy-related events followed beta(1) integrin-mediated engulfment of the bacteria including the formation of autophagosomes, processing of the marker protein LC3, redistribution of GFP-LC3 to bacteria-containing vacuoles, and the segregation of intracellular bacteria by autophagosomal compartments. These results provide direct evidence for the linkage of beta(1) integrin-mediated phagocytosis and autophagy induction. Multiple microbes signal through integrin receptors, and our results suggest a general principle by which the sensing of an extracellular microbe triggers autophagy. Owing to the importance of autophagy as host defense response, wild-type Y. enterocolitica suppressed autophagy by mobilizing type III protein secretion. The subversion of autophagy may be part of the Y. enterocolitica virulence strategy that supports bacterial survival when beta(1) integrin-dependent internalization and autophagy activation by macrophages are deleterious for the pathogen.


Asunto(s)
Autofagia/inmunología , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , Integrina beta1/fisiología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Yersinia enterocolitica/inmunología , Adhesinas Bacterianas/fisiología , Animales , Autofagia/genética , Proteínas Bacterianas/genética , Biomarcadores/metabolismo , Línea Celular , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Integrina beta1/biosíntesis , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/fisiología , Plásmidos/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Virulencia/genética , Virulencia/inmunología , Yersinia enterocolitica/genética , Yersinia enterocolitica/patogenicidad
12.
Mol Imaging ; 9(4): 211-22, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20643024

RESUMEN

The objective of this study was to quantify enzymatic activity on the surface of T cells by magnetic resonance imaging (MRI) using R2 and R2* relaxometry. Lymphoma cells expressing adenosine diphosphate (ADP)-ribosyltransferase 2 (ART2) were incubated with increasing doses of its substrate etheno-nicotinamide adenine dinucleotide (NAD), resulting in increasing amounts of surface protein ADP-ribosylation. Etheno-ADP-ribosylated proteins were detected with monoclonal antibody 1G4 and superparamagnetic iron oxide conjugated secondary antibodies (Ab-SPIO). Labeling efficiency was determined with R2 and R2* relaxometry on a clinical 3.0 T scanner. Parallel aliquots of cells were analyzed by flow cytometry. Cell-bound SPIO conjugates were detected by immunofluorescence and electron microscopy and quantified by atomic absorption spectroscopy. To mimic an inflammatory site in vivo, Ab-SPIO-labeled cells were injected subcutaneously in mice and analyzed by MRI. Immunofluorescence and electron microscopy confirmed cell-surface localization of Ab-SPIO. MRI of Ab-SPIO-labeled cells showed a corresponding signal reduction. Increases in R2 and R2* determined by magnetic resonance relaxometry correlated linearly with the expression level of ART2 and the concentration of the ART2 substrate etheno-NAD. R2 and R2* increases correlated linearly with the results from flow cytometry and atomic absorption spectroscopy analyses. Quantitative R2 and R2* mapping enable noninvasive determination of enzymatic activity on T cells and holds promise for characterization of inflammatory sites in vivo by MRI.


Asunto(s)
ADP Ribosa Transferasas/metabolismo , Membrana Celular/enzimología , Compuestos Férricos/metabolismo , Imagen por Resonancia Magnética/métodos , Linfocitos T/enzimología , Adenosina Difosfato Ribosa/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Compuestos Férricos/química , Citometría de Flujo , Modelos Lineales , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , NAD/metabolismo , NAD/farmacología , Reproducibilidad de los Resultados , Imagen de Cuerpo Entero
13.
J Am Chem Soc ; 130(23): 7315-20, 2008 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-18484723

RESUMEN

Magnetic nanoparticles have been assembled into the bilayer membrane of block copolymer vesicles. The nanoparticles decorate the hydrophobic/hydrophilic interface, which leads to bridging of adjacent bilayers and the formation of oligo-lamellar vesicles. The nanoparticle uptake of the vesicles is sufficiently high to become magnetophoretic in external magnetic fields as shown by video microscopy.


Asunto(s)
Hemiterpenos/química , Látex/química , Membrana Dobles de Lípidos/química , Magnetismo , Nanopartículas/química , Polietilenglicoles/química , Compuestos Férricos/química , Interacciones Hidrofóbicas e Hidrofílicas , Difracción de Neutrones , Dispersión del Ángulo Pequeño
14.
Contrast Media Mol Imaging ; 10(2): 153-62, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25078884

RESUMEN

Accurate determination of tissue concentration of ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) using T2 * MR relaxometry is still challenging. We present a reliable quantification method for local USPIO amount with the estimation of the liver specific relaxivity r2 * using monodisperse (59) Fe-core-labeled USPIO ((59) FeUSPIO). Dynamic and relaxometric in vivo characteristics of unlabeled monodisperse USPIO were determined in MRI at 3 T. The in vivo MR studies were performed for liver tissue with (59) FeUSPIO using iron dosages of 9 (n = 3), 18 (n = 2) and 27 (n = 3) µmol Fe kg(-1) body weight. The R2 * of the liver before and after USPIO injection (∆R2 *) was measured and correlated with (59) Fe activity measurements of excised organs by a whole body radioactivity counter (HAMCO) to define the dependency of ∆R2 * and (59) FeUSPIO liver concentration and calculate the r2 * of (59) FeUSPIO for the liver. Ultrastructural analysis of liver uptake was performed by histology and transmission electron microscopy. ∆R2 * of the liver revealed a dosage-dependent accumulation of (59) FeUSPIO with a percentage uptake of 70-88% of the injection dose. Hepatic ∆R2 * showed a dose-dependent linear correlation to (59) FeUSPIO activity measurements (r = 0.92) and an r2 * in the liver of 481 ± 74.9 mm(-1) s(-1) in comparison to an in vitro r2 * of 60.5 ± 3.3 mm(-1) s(-1) . Our results indicate that core-labeled (59) FeUSPIO can be used to quantify the local amount of USPIO and to estimate the liver-specific relaxivity r2 *.


Asunto(s)
Medios de Contraste , Compuestos Férricos , Marcaje Isotópico/métodos , Hígado , Imagen por Resonancia Magnética/métodos , Nanopartículas/química , Animales , Medios de Contraste/química , Medios de Contraste/farmacología , Relación Dosis-Respuesta a Droga , Compuestos Férricos/química , Compuestos Férricos/farmacología , Isótopos de Hierro/química , Isótopos de Hierro/farmacología , Hígado/diagnóstico por imagen , Hígado/metabolismo , Ratones , Ratones SCID , Radiografía
15.
PLoS One ; 7(5): e36258, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22590529

RESUMEN

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via "wick-in-needle" technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antineoplásicos/farmacología , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/inmunología , Sitios de Unión , Células CACO-2 , Moléculas de Adhesión Celular/inmunología , Molécula de Adhesión Celular Epitelial , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones SCID , Invasividad Neoplásica , Proteínas de Neoplasias/inmunología , Trasplante de Neoplasias , Presión , Radiografía , Trasplante Heterólogo
16.
ACS Nano ; 6(8): 7318-25, 2012 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-22793497

RESUMEN

A simple, fast, efficient, and widely applicable method to radiolabel the cores of monodisperse superparamagnetic iron oxide nanoparticles (SPIOs) with (59)Fe was developed. These cores can be used as precursors for a variety of functionalized nanodevices. A quality control using filtration techniques, size-exclusion chromatography, chemical degradation methods, transmission electron microscopy, and magnetic resonance imaging showed that the nanoparticles were stably labeled with (59)Fe. Furthermore, the particle structure and the magnetic properties of the SPIOs were unchanged. In a second approach, monodisperse SPIOs stabilized with (14)C-oleic acid were synthesized, and the stability of this shell labeling was studied. In proof of principle experiments, the (59)Fe-SPIOs coated with different shells to make them water-soluble were used to evaluate and compare in vivo pharmacokinetic parameters such as blood half-life. It could also be shown that our radiolabeled SPIOs embedded in recombinant lipoproteins can be used to quantify physiological processes in closer detail than hitherto possible. In vitro and in vivo experiments showed that the (59)Fe label is stable enough to be applied in vivo, whereas the (14)C label is rapidly removed from the iron core and is not adequate for in vivo studies. To obtain meaningful results in in vivo experiments, only (59)Fe-labeled SPIOs should be used.


Asunto(s)
Dextranos/química , Radioisótopos de Hierro , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/química , Imagen de Cuerpo Entero/métodos , Animales , Medios de Contraste , Radioisótopos de Hierro/química , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Radiofármacos , Distribución Tisular
17.
PLoS One ; 6(12): e29112, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22216177

RESUMEN

Merkel Cell Polyomavirus (MCPyV) genomes are clonally integrated in tumor tissues of approximately 85% of all Merkel cell carcinoma (MCC) cases, a highly aggressive tumor of the skin which predominantly afflicts elderly and immunosuppressed patients. All integrated viral genomes recovered from MCC tissue or MCC cell lines harbor signature mutations in the early gene transcript encoding for the large T-Antigen (LT-Ag). These mutations selectively abrogate the ability of LT-Ag to support viral replication while still maintaining its Rb-binding activity, suggesting a continuous requirement for LT-Ag mediated cell cycle deregulation during MCC pathogenesis. To gain a better understanding of MCPyV biology, in vitro MCPyV replication systems are required. We have generated a synthetic MCPyV genomic clone (MCVSyn) based on the consensus sequence of MCC-derived sequences deposited in the NCBI database. Here, we demonstrate that transfection of recircularized MCVSyn DNA into some human cell lines recapitulates efficient replication of the viral genome, early and late gene expression together with virus particle formation. However, serial transmission of infectious virus was not observed. This in vitro culturing system allows the study of viral replication and will facilitate the molecular dissection of important aspects of the MCPyV lifecycle.


Asunto(s)
Replicación del ADN , Expresión Génica , Genoma Viral , Poliomavirus de Células de Merkel/genética , Virión , Replicación Viral , Línea Celular Tumoral , Humanos , Poliomavirus de Células de Merkel/fisiología , Mutación , Transcripción Genética
18.
PLoS One ; 6(12): e28030, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22162753

RESUMEN

Members of the carcinoembryonic antigen cell adhesion molecules (CEACAMs) family are the prototype of tumour markers. Classically they are used as serum markers, however, CEACAMs could serve as targets for molecular imaging as well.In order to test the anti CEACAM monoclonal antibody T84.1 for imaging purposes, CEACAM expression was analysed using this antibody. Twelve human cancer cell lines from different entities were screened for their CEACAM expression using qPCR, Western Blot and FACS analysis. In addition, CEACAM expression was analyzed in primary tumour xenografts of these cells. Nine of 12 tumour cell lines expressed CEACAM mRNA and protein when grown in vitro. Pancreatic and colon cancer cell lines showed the highest expression levels with good correlation of mRNA and protein level. However, when grown in vivo, the CEACAM expression was generally downregulated except for the melanoma cell lines. As the CEACAM expression showed pronounced expression in FemX-1 primary tumours, this model system was used for further experiments. As the accessibility of the antibody after i.v. application is critical for its use in molecular imaging, the binding of the T84.1 monoclonal antibody was assessed after i.v. injection into SCID mice harbouring a FemX-1 primary tumour. When applied i.v., the CEACAM specific T84.1 antibody bound to tumour cells in the vicinity of blood vessels. This binding pattern was particularly pronounced in the periphery of the tumour xenograft, however, some antibody binding was also observed in the central areas of the tumour around blood vessels. Still, a general penetration of the tumour by i.v. application of the anti CEACAM antibody could not be achieved despite homogenous CEACAM expression of all melanoma cells when analysed in tissue sections. This lack of penetration is probably due to the increased interstitial fluid pressure in tumours caused by the absence of functional lymphatic vessels.


Asunto(s)
Antígeno Carcinoembrionario/metabolismo , Moléculas de Adhesión Celular/metabolismo , Albúminas/metabolismo , Animales , Antígenos CD/biosíntesis , Biomarcadores/metabolismo , Células CACO-2 , Antígeno Carcinoembrionario/química , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/química , Línea Celular Tumoral , Azul de Evans/farmacología , Femenino , Humanos , Inmunohistoquímica/métodos , Vasos Linfáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Imagen Molecular/métodos , Metástasis de la Neoplasia , Trasplante de Neoplasias , ARN Mensajero/metabolismo
19.
PLoS One ; 5(2): e9174, 2010 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-20161802

RESUMEN

Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus-infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.


Asunto(s)
Citomegalovirus/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Antivirales/farmacología , Núcleo Celular/metabolismo , Células Cultivadas , Citomegalovirus/efectos de los fármacos , Citomegalovirus/genética , Evaluación Preclínica de Medicamentos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Ganciclovir/farmacología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Luminiscentes/genética , Masculino , Microscopía Fluorescente , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Espectrometría de Fluorescencia , Transactivadores/genética , Transactivadores/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/genética , Replicación Viral/efectos de los fármacos
20.
PLoS One ; 4(6): e6084, 2009 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-19562035

RESUMEN

Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner.


Asunto(s)
Citoplasma/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Filamentos Intermediarios/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Receptores de Glucocorticoides/metabolismo , Vimentina/biosíntesis , Animales , Ciclo Celular , Diferenciación Celular , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Glioma/metabolismo , Humanos , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/metabolismo , Nestina , ARN/metabolismo , Ratas , Vimentina/metabolismo
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