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1.
Nat Immunol ; 20(10): 1299-1310, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31534238

RESUMO

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

3.
Theranostics ; 8(20): 5645-5659, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30555570

RESUMO

Given the strong clinical evidence that copper levels are significantly elevated in a wide spectrum of tumors, copper homeostasis is considered as an emerging target for anticancer drug design. Monitoring copper levels in vivo is therefore of paramount importance when assessing the efficacy of copper-targeting drugs. Herein, we investigated the activity of the copper-targeting compound Dextran-Catechin by developing a [64Cu]CuCl2 PET imaging protocol to monitor its effect on copper homeostasis in tumors. Methods: Protein expression of copper transporter 1 (CTR1) in tissue microarrays representing 90 neuroblastoma patient tumors was assessed by immunohistochemistry. Western blotting analysis was used to study the effect of Dextran-Catechin on the expression of CTR1 in neuroblastoma cell lines and in tumors. A preclinical human neuroblastoma xenograft model was used to study anticancer activity of Dextran-Catechin in vivo and its effect on tumor copper homeostasis. PET imaging with [64Cu]CuCl2 was performed in such preclinical neuroblastoma model to monitor alteration of copper levels in tumors during treatment. Results: CTR1 protein was found to be highly expressed in patient neuroblastoma tumors by immunohistochemistry. Treatment of neuroblastoma cell lines with Dextran-Catechin resulted in decreased levels of glutathione and in downregulation of CTR1 expression, which caused a significant decrease of intracellular copper. No changes in CTR1 expression was observed in normal human astrocytes after Dextran-Catechin treatment. In vivo studies and PET imaging analysis using the neuroblastoma preclinical model revealed elevated [64Cu]CuCl2 retention in the tumor mass. Following treatment with Dextran-Catechin, there was a significant reduction in radioactive uptake, as well as reduced tumor growth. Ex vivo analysis of tumors collected from Dextran-Catechin treated mice confirmed the reduced levels of CTR1. Interestingly, copper levels in blood were not affected by treatment, demonstrating potential tumor specificity of Dextran-Catechin activity. Conclusion: Dextran-Catechin mediates its activity by lowering CTR1 and intracellular copper levels in tumors. This finding further reveals a potential therapeutic strategy for targeting copper-dependent cancers and presents a novel PET imaging method to assess patient response to copper-targeting anticancer treatments.


Assuntos
Tomografia por Emissão de Pósitrons/métodos , Animais , Catequina , Proteínas de Transporte de Cátions , Linhagem Celular Tumoral , Cobre , Dextranos , Feminino , Homeostase , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Imagem Molecular , Neuroblastoma , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Análise Serial de Tecidos
4.
J Exp Med ; 215(8): 2073-2095, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30018075

RESUMO

Gain-of-function (GOF) mutations in PIK3CD, encoding the p110δ subunit of phosphatidylinositide 3-kinase (PI3K), cause a primary immunodeficiency. Affected individuals display impaired humoral immune responses following infection or immunization. To establish mechanisms underlying these immune defects, we studied a large cohort of patients with PIK3CD GOF mutations and established a novel mouse model using CRISPR/Cas9-mediated gene editing to introduce a common pathogenic mutation in Pik3cd In both species, hyperactive PI3K severely affected B cell development and differentiation in the bone marrow and the periphery. Furthermore, PI3K GOF B cells exhibited intrinsic defects in class-switch recombination (CSR) due to impaired induction of activation-induced cytidine deaminase (AID) and failure to acquire a plasmablast gene signature and phenotype. Importantly, defects in CSR, AID expression, and Ig secretion were restored by leniolisib, a specific p110δ inhibitor. Our findings reveal key roles for balanced PI3K signaling in B cell development and long-lived humoral immunity and memory and establish the validity of treating affected individuals with p110δ inhibitors.


Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação em Linhagem Germinativa/genética , Fosfatidilinositol 3-Quinases/genética , Animais , Afinidade de Anticorpos/imunologia , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Criança , Mutação com Ganho de Função/genética , Humanos , Switching de Imunoglobulina , Imunoglobulinas/metabolismo , Interleucinas/farmacologia , Camundongos , Modelos Animais , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Plasmócitos/metabolismo , Transdução de Sinais
5.
PLoS One ; 13(1): e0190927, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29324879

RESUMO

The development of shark vertebrae and the possible drivers of inter- and intra-specific differences in vertebral structure are poorly understood. Shark vertebrae are used to examine life-history traits related to trophic ecology, movement patterns, and the management of fisheries; a better understanding of their development would be beneficial to many fields of research that rely on these calcified structures. This study used Scanning X-ray Fluorescence Microscopy to observe zinc distribution within vertebrae of ten shark species from five different orders. Zinc was mostly localised within the intermedialis and was generally detected at levels an order of magnitude lower in the corpus calcareum. In most species, zinc concentrations were higher pre-birth mark, indicating a high rate of pre-natal zinc deposition. These results suggest there are inter-specific differences in elemental deposition within vertebrae. Since the deposition of zinc is physiologically-driven, these differences suggest that the processes of growth and deposition are potentially different in the intermedialis and corpus calcareum, and that caution should be taken when extrapolating information such as annual growth bands from one structure to the other. Together these results suggest that the high inter-specific variation in vertebral zinc deposition and associated physiologies may explain the varying effectiveness of ageing methodologies applied to elasmobranch vertebrae.


Assuntos
Envelhecimento/metabolismo , Tubarões/crescimento & desenvolvimento , Tubarões/metabolismo , Coluna Vertebral/crescimento & desenvolvimento , Coluna Vertebral/metabolismo , Zinco/metabolismo , Envelhecimento/patologia , Animais , Cálcio/metabolismo , Modelos Lineares , Microscopia de Fluorescência , Tubarões/anatomia & histologia , Especificidade da Espécie , Coluna Vertebral/diagnóstico por imagem
6.
Sci Rep ; 6: 29698, 2016 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-27424768

RESUMO

Determining the age of sharks using vertebral banding is a vital component of management, but the causes of banding are not fully understood. Traditional shark ageing is based on fish otolith ageing methods where growth bands are assumed to result from varied seasonal calcification rates. Here we investigate these assumptions by mapping elemental distribution within the growth bands of vertebrae from six species of sharks representing four different taxonomic orders using scanning x-ray fluorescence microscopy. Traditional visual growth bands, determined with light microscopy, were more closely correlated to strontium than calcium in all species tested. Elemental distributions suggest that vertebral strontium bands may be related to environmental variations in salinity. These results highlight the requirement for a better understanding of shark movements, and their influence on vertebral development, if confidence in age estimates is to be improved. Analysis of shark vertebrae using similar strontium-focused elemental techniques, once validated for a given species, may allow more successful estimations of age on individuals with few or no visible vertebral bands.


Assuntos
Tubarões/metabolismo , Coluna Vertebral/metabolismo , Estrôncio/metabolismo , Determinação da Idade pelo Esqueleto , Animais , Calcificação Fisiológica , Cálcio/metabolismo , Feminino , Masculino , Tubarões/crescimento & desenvolvimento , Espectrometria por Raios X , Coluna Vertebral/crescimento & desenvolvimento
7.
Phys Med Biol ; 61(3): 1371-88, 2016 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-26797268

RESUMO

In PET imaging, research groups have recently proposed different experimental set ups allowing multiple animals to be simultaneously imaged in a scanner in order to reduce the costs and increase the throughput. In those studies, the technical feasibility was demonstrated and the signal degradation caused by additional mice in the FOV characterized, however, the impact of the signal degradation on the outcome of a PET study has not yet been studied. Here we thoroughly investigated, using Monte Carlo simulated [18F]FDG and [11C]Raclopride PET studies, different experimental designs for whole-body and brain acquisitions of two mice and assessed the actual impact on the detection of biological variations as compared to a single-mouse setting. First, we extended the validation of the PET-SORTEO Monte Carlo simulation platform for the simultaneous simulation of two animals. Then, we designed [18F]FDG and [11C]Raclopride input mouse models for the simulation of realistic whole-body and brain PET studies. Simulated studies allowed us to accurately estimate the differences in detection between single- and dual-mode acquisition settings that are purely the result of having two animals in the FOV. Validation results showed that PET-SORTEO accurately reproduced the spatial resolution and noise degradations that were observed with actual dual phantom experiments. The simulated [18F]FDG whole-body study showed that the resolution loss due to the off-center positioning of the mice was the biggest contributing factor in signal degradation at the pixel level and a minimal inter-animal distance as well as the use of reconstruction methods with resolution modeling should be preferred. Dual mode acquisition did not have a major impact on ROI-based analysis except in situations where uptake values in organs from the same subject were compared. The simulated [11C]Raclopride study however showed that dual-mice imaging strongly reduced the sensitivity to variations when mice were positioned side-by-side while no sensitivity reduction was observed when they were facing each other. This is the first study showing the impact of different experimental designs for whole-body and brain acquisitions of two mice on the quality of the results using Monte Carlo simulated [18F]FDG and [11C]Raclopride PET studies.


Assuntos
Algoritmos , Tomografia por Emissão de Pósitrons/métodos , Animais , Fluordesoxiglucose F18/farmacocinética , Camundongos , Modelos Teóricos , Tomografia por Emissão de Pósitrons/instrumentação , Racloprida/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética
8.
Nat Commun ; 5: 5452, 2014 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-25406832

RESUMO

The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer's disease to anxiety. Here we show that global C57BL/6-Tspo(tm1GuWu(GuwiyangWurra))-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from (GuwiyangWurra)TSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of (GuwiyangWurra)TSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs.


Assuntos
Glândulas Suprarrenais/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Rim/diagnóstico por imagem , Microglia/metabolismo , Receptores de GABA/genética , Trifosfato de Adenosina/metabolismo , Animais , Comportamento Animal , Colesterol/metabolismo , Fertilidade/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tomografia por Emissão de Pósitrons , Pregnenolona/sangue , Protoporfirinas/metabolismo , Baço/diagnóstico por imagem , Testículo/diagnóstico por imagem , Imagem Corporal Total
9.
Exp Neurol ; 257: 162-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24825369

RESUMO

Evidence suggests that there is a link between the endocannabinoid system (ECS) and neuropsychiatric illnesses, including schizophrenia. Whilst the ECS has been shown to be involved in immune system regulation in various ways, it is known that infections during pregnancy can modulate the immune system of the mother and increase the risk for schizophrenia in offspring. In animal studies, maternal immune activation following administration of viral or bacterial mimics has been shown to reproduce many key structural, behavioural, and pharmacological abnormalities in offspring that resemble schizophrenia. In the present study, we used Positron Emission Tomography (PET) and [(18)F]MK-9470, a selective high-affinity inverse agonist radioligand for cannabinoid type 1 receptors (CB1R), to longitudinally assess CB1R expression in the progeny of female rats exposed to the viral mimic polyriboinosinic-polyribocytidilic acid (poly I:C) (4mg/kg i.v.) or vehicle at gestational day 15 (GD 15). PET scans were performed in offspring at postnatal days (PND) 32-42 (adolescence) and in the same animals again at PNDs 75-79 (adulthood). Sixteen regions of interest were assessed, encompassing the whole rat brain. At adolescence, offspring exposed prenatally to poly I:C had significantly lower CB1R relative Standard Uptake Values (rSUV) compared to controls in the globus pallidus (p=0.046). In adulthood, however, poly I:C exposed offspring had higher levels of CB1R rSUV in sensory cortex (p=0.034) and hypothalamus (p=0.032) compared to controls. Our results suggest that prenatal poly I:C leads to long term alterations in the integrity of the ECS that are age and region-specific. The increased CB1R expression in adulthood following poly I:C mirrors the increased CB1R observed in patients with schizophrenia in post-mortem and in vivo PET studies.


Assuntos
Encéfalo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Poli I-C/farmacologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Receptor CB1 de Canabinoide/efeitos dos fármacos , Fatores Etários , Animais , Peso Corporal/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Mapeamento Encefálico , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Imagem por Ressonância Magnética , Masculino , Tomografia por Emissão de Pósitrons , Gravidez , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacocinética , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismo
10.
J Nucl Med ; 54(10): 1833-40, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24009279

RESUMO

UNLABELLED: The Inveon small-animal SPECT system comes with several types of multipinhole collimator plates. We evaluate here the performance measurements of the Inveon SPECT system using 6 different collimators: 3 dedicated for mouse imaging and 3 for rat imaging. METHODS: The measured performance parameters include the sensitivity, the spatial resolution using line sources, the ultra-micro Derenzo phantom, the recovery coefficient and the noise measurements using the National Electrical Manufacturers Association NU-4 image quality phantom, obtained with the 2 reconstruction algorithms available with the Inveon Acquisition Workplace, version 1.5-the 3-dimensional ordered-subset expectation maximization (3DOSEM) and the 3-dimensional maximum a posteriori (3DMAP). Further, the overall performance of the system is illustrated by an animal experiment. RESULTS: The results show that the Inveon SPECT scanner offers a spatial resolution, measured at the center of the field of view, ranging from 0.6 to 1 mm with the collimator plates dedicated to mouse imaging and from 1.2 to less than 2 mm with rat collimator plates. The system sensitivity varies from 29 to 404 cps/MBq for mouse collimators and from 53 to 175 cps/MBq for rat collimators. The image quality study showed that 3DMAP allows better noise reduction while preserving the recovery coefficient, compared with other regularization strategies such as the premature termination of the 3DOSEM reconstruction or 3DOSEM followed by gaussian filtering. CONCLUSION: The acquisition parameters, such as the collimator set and the radius of rotation, offer a wide range of possibilities to apply to a large number of biologic studies. However, special care must be taken because this increase in sensitivity can be offset by image degradation, such as image artifacts caused by projection overlap and statistical noise due to a higher number of iterations required for convergence. 3DMAP allowed better noise reduction while maintaining relatively constant recovery coefficients, as compared with other reconstruction strategies.


Assuntos
Tomografia Computadorizada de Emissão de Fóton Único/instrumentação , Animais , Camundongos , Imagens de Fantasmas , Ratos , Razão Sinal-Ruído , Tecnécio Tc 99m Sestamibi
11.
J Nucl Med ; 54(2): 291-8, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23321458

RESUMO

UNLABELLED: Glial neuroinflammation is associated with the development and progression of multiple sclerosis. PET imaging offers a unique opportunity to evaluate neuroinflammatory processes longitudinally in a noninvasive and clinically translational manner. (18)F-PBR111 is a newly developed PET radiopharmaceutical with high affinity and selectivity for the translocator protein (TSPO), expressed on activated glia. This study aimed to investigate neuroinflammation at different phases of relapsing-remitting (RR) experimental autoimmune encephalomyelitis (EAE) in the brains of SJL/J mice by postmortem histologic analysis and in vivo by PET imaging with (18)F-PBR111. METHODS: RR EAE was induced by immunization with PLP(139-151) peptide in complete Freund's adjuvant. Naive female SJL/J mice and mice immunized with saline-complete Freund's adjuvant were used as controls. The biodistribution of (18)F-PBR111 was measured in 13 areas of the central nervous system and compared with PET imaging results during different phases of RR EAE. The extents of TSPO expression and glial activation were assessed with immunohistochemistry, immunofluorescence, and a real-time polymerase chain reaction. RESULTS: There was significant TSPO expression in all of the central nervous system areas studied at the peak of the first clinical episode and, importantly, at the preclinical stage. In contrast, only a few TSPO-positive cells were observed at the second episode. At the third episode, there was again an increase in TSPO expression. TSPO expression was associated with microglial cells or macrophages without obvious astrocyte labeling. The dynamics of (18)F-PBR111 uptake in the brain, as measured by in vivo PET imaging and biodistribution, followed the pattern of TSPO expression during RR EAE. CONCLUSION: PET imaging with the TSPO ligand (18)F-PBR111 clearly reflected the dynamics of microglial activation in the SJL/J mouse model of RR EAE. The results are the first to highlight the discrepancy between the clinical symptoms of EAE and TSPO expression in the brain, as measured by PET imaging at the peaks of various EAE episodes. The results suggest a significant role for PET imaging investigations of neuroinflammation in multiple sclerosis and allow for in vivo follow-up of antiinflammatory treatment strategies.


Assuntos
Sistema Nervoso Central/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Tomografia por Emissão de Pósitrons/métodos , Receptores de GABA/biossíntese , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Imuno-Histoquímica/métodos , Inflamação , Macrófagos/citologia , Camundongos , Microscopia de Fluorescência/métodos , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Transporte Proteico , Reação em Cadeia da Polimerase em Tempo Real/métodos
12.
Brain Res Bull ; 87(2-3): 172-9, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22155282

RESUMO

Cannabis use has been shown to alter brain metabolism in both rat models and humans although the observations between both species are conflicting. In the present study, we examined the short term effects of a single-dose injection of the synthetic cannabinoid agonist HU210 on glucose metabolism in the rat brain using small animal (18)F-2-fluoro-deoxyglucose (FDG) Positron Emission Tomography (PET) 15 min (Day 1) and 24h (Day 2) post-injection of the agonist in the same animal. Young adult male Wistar rats received an intra-peritoneal injection of HU210 (100 µg/kg, n=7) or vehicle (n=5) on Day 1. Approximately 1mCi of (18)F-FDG was injected intravenously into each animal at 15 min (Day 1) and 24h (Day 2) post-injection of HU210. A 5-min Computer Tomography (CT) scan followed by a 20-min PET scan was performed 40 min after each (18)F-FDG injection. Standardised Uptake Values (SUVs) were calculated for 10 brain regions of interest (ROIs). Global increased SUVs in the whole brain, hence global brain metabolism, were observed following HU210 treatment on Day 1 compared to the controls (21%, P<0.0001), but not in individual brain regions. On Day 2, however, no statistically significant differences were observed between the treated and control groups. At the 24h time point (Day 2), SUVs in the HU210 treated group returned to control levels (21-30% decrease compared to Day 1), in all ROIs investigated (P<0.0001). In the control group, SUVs did not differ between the two acquisition days in all brain regions. The present results suggest that high-dose HU210 increases brain glucose metabolism in the rat brain shortly after administration, in line with normalised human in vivo studies, an effect that was no longer apparent 24 h later.


Assuntos
Encéfalo , Dronabinol/análogos & derivados , Fluordesoxiglucose F18 , Fármacos Neuroprotetores/farmacologia , Tomografia por Emissão de Pósitrons , Análise de Variância , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Mapeamento Encefálico , Dronabinol/farmacologia , Masculino , Ratos , Ratos Wistar , Fatores de Tempo , Tomografia Computadorizada por Raios X
13.
Int J Mol Imaging ; 2011: 548123, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22187642

RESUMO

Despite the important role of cannabinoid CB(1) receptors (CB(1)R) in brain development, little is known about their status during adolescence, a critical period for both the development of psychosis and for initiation to substance abuse. In the present study, we assessed the ontogeny of CB(1)R in adolescent and adult rats in vivo using positron emission tomography with [(18)F]MK-9470. Analysis of covariance (ANCOVA) to control for body weight that would potentially influence [(18)F]MK-9470 values between the two groups revealed a main effect of age (F(1,109)=5.0, P = 0.02) on [(18)F]MK-9470 absolute binding (calculated as percentage of injected dose) with adult estimated marginal means being higher compared to adolescents amongst 11 brain regions. This finding was confirmed using in vitro autoradiography with [(3)H]CP55,940 (F(10,99)=140.1, P < 0.0001). This ontogenetic pattern, suggesting increase of CB(1)R during the transition from adolescence to adulthood, is the opposite of most other neuroreceptor systems undergoing pruning during this period.

14.
Comput Med Imaging Graph ; 34(4): 321-32, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20044235

RESUMO

3D shape context is a method to define matching points between similar shapes. It can be used as a pre-processing step in a non-rigid registration. The main limitation of the method is point mismatching, which includes long geodesic distance mismatch causing wrong topological structure, and neighbors crossing mismatch between two adjacent points. In this paper, we propose a topological structure verification method to correct the long geodesic distance mismatch and a correspondence field smoothing method to correct the neighbors crossing mismatch. A robust 3D shape context model is generated and further combined with thin-plate spline model for non-rigid registration. The method was tested on phantoms and applied to rat hind limb and mouse hind limb skeletons registration from micro-CT images. Errors between the registered surfaces were reduced by using the proposed method. The robustness of the method is demonstrated.


Assuntos
Osso e Ossos/diagnóstico por imagem , Membro Posterior/diagnóstico por imagem , Imagem Tridimensional/métodos , Reconhecimento Automatizado de Padrão/métodos , Interpretação de Imagem Radiográfica Assistida por Computador/métodos , Técnica de Subtração , Tomografia Computadorizada por Raios X/métodos , Algoritmos , Animais , Inteligência Artificial , Camundongos , Intensificação de Imagem Radiográfica/métodos , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
15.
J Biol Chem ; 285(10): 7633-44, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20044484

RESUMO

The complement anaphylatoxin C5a is a proinflammatory component of host defense that functions through two identified receptors, C5a receptor (C5aR) and C5L2. C5aR is a classical G protein-coupled receptor, whereas C5L2 is structurally homologous but deficient in G protein coupling. In human neutrophils, we show C5L2 is predominantly intracellular, whereas C5aR is expressed on the plasma membrane. Confocal analysis shows internalized C5aR following ligand binding is co-localized with both C5L2 and beta-arrestin. Antibody blockade of C5L2 results in a dramatic increase in C5a-mediated chemotaxis and ERK1/2 phosphorylation but does not alter C5a-mediated calcium mobilization, supporting its role in modulation of the beta-arrestin pathway. Association of C5L2 with beta-arrestin is confirmed by cellular co-immunoprecipitation assays. C5L2 blockade also has no effect on ligand uptake or C5aR endocytosis in human polymorphonuclear leukocytes, distinguishing its role from that of a rapid recycling or scavenging receptor in this cell type. This is thus the first example of a naturally occurring seven-transmembrane segment receptor that is both obligately uncoupled from G proteins and a negative modulator of signal transduction through the beta-arrestin pathway. Physiologically, these properties provide the possibility for additional fine-tuning of host defense.


Assuntos
Receptor da Anafilatoxina C5a/metabolismo , Receptores de Quimiocinas/metabolismo , Transdução de Sinais/fisiologia , Animais , Arrestinas/metabolismo , Linhagem Celular , Quimiotaxia de Leucócito/fisiologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neutrófilos/citologia , Neutrófilos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptor da Anafilatoxina C5a/química , Receptor da Anafilatoxina C5a/genética , Receptores de Quimiocinas/química , Receptores de Quimiocinas/genética , Distribuição Tecidual , beta-Arrestinas
16.
Nat Biotechnol ; 24(10): 1279-84, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16980974

RESUMO

Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of approximately 40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.


Assuntos
Anticorpos Monoclonais/farmacologia , Inflamação/tratamento farmacológico , Proteínas de Membrana/genética , Receptores de Complemento/genética , Animais , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Humanos , Inflamação/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/imunologia , Receptor da Anafilatoxina C5a , Receptores de Complemento/metabolismo
17.
J Rheumatol ; 29(4): 787-95, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11950023

RESUMO

OBJECTIVE: The antiinflammatory effects of glucocorticoids are mediated by several mechanisms, including inhibition of nuclear factor-kappaB (NF-kappaB) nuclear translocation and DNA binding. This mechanism is not evident in some cell types, including endothelial cells and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). We determined the effect of glucocorticoids and tumor necrosis factor (TNF) on nuclear localization and DNA binding of the transcription factor NF-kappaB in osteoarthritic (OA) synovial tissue. METHODS: Explants of synovial tissue from patients undergoing joint replacement surgery for arthritis were placed in culture and treated with dexamethasone 10(-6) M for 18 h and again at 30 min prior to stimulation with TNF for a further 30 min. NF-kappaB and AP-1 DNA binding activities were determined by electrophoretic mobility shift analysis of nuclear extracts prepared from 6 whole tissue explants. Nuclear localization of NF-kappaB was determined by quantitative immunohistochemistry for Rel-A(p65) in thin sections of 5 synovial tissue explants. RESULTS: TNF induced NF-kappaB nuclear translocation and DNA binding in all OA synovial tissue explants, although there were no consistent effects on AP-1 DNA binding. Dexamethasone reduced TNF stimulated nuclear translocation of RelA(p65) in all 5 OA synovial explants analyzed by immunohistochemistry. Dexamethasone partially decreased NF-kappaB DNA binding in 5 of 6 TNF stimulated explants and 4 of 6 unstimulated explants. In cultured rheumatoid arthritis and OA fibroblast-like synoviocytes and Mono Mac 6 cells the effects of dexamethasone on NF-kappaB DNA binding were not evident. CONCLUSION: Dexamethasone partially inhibits TNF induced NF-kappaB DNA binding in human synovial tissue. It is feasible to use explants of intact fresh human synovium as a substrate for the action of antirheumatic drugs targeting a transcription factor.


Assuntos
Anti-Inflamatórios/farmacologia , DNA/metabolismo , Dexametasona/farmacologia , NF-kappa B/biossíntese , Osteoartrite , Membrana Sinovial/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Combinação de Medicamentos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , NF-kappa B/antagonistas & inibidores , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteínas Recombinantes , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa/farmacologia
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