Four novel Loci (19q13, 6q24, 12q24, and 5q14) influence the microcirculation in vivo.

There is increasing evidence that the microcirculation plays an important role in the pathogenesis of cardiovascular diseases. Changes in retinal vascular caliber reflect early microvascular disease and predict incident cardiovascular events. We performed a genome-wide association study to identify genetic variants associated with retinal vascular caliber. We analyzed data from four population-based discovery cohorts with 15,358 unrelated Caucasian individuals, who are members of the Cohort for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and replicated findings in four independent Caucasian cohorts (n  =  6,652). All participants had retinal photography and retinal arteriolar and venular caliber measured from computer software. In the discovery cohorts, 179 single nucleotide polymorphisms (SNP) spread across five loci were significantly associated (p<5.0×10(-8)) with retinal venular caliber, but none showed association with arteriolar caliber. Collectively, these five loci explain 1.0%-3.2% of the variation in retinal venular caliber. Four out of these five loci were confirmed in independent replication samples. In the combined analyses, the top SNPs at each locus were: rs2287921 (19q13; p  =  1.61×10(-25), within the RASIP1 locus), rs225717 (6q24; p = 1.25×10(-16), adjacent to the VTA1 and NMBR loci), rs10774625 (12q24; p  =  2.15×10(-13), in the region of ATXN2,SH2B3 and PTPN11 loci), and rs17421627 (5q14; p = 7.32×10(-16), adjacent to the MEF2C locus). In two independent samples, locus 12q24 was also associated with coronary heart disease and hypertension. Our population-based genome-wide association study demonstrates four novel loci associated with retinal venular caliber, an endophenotype of the microcirculation associated with clinical cardiovascular disease. These data provide further insights into the contribution and biological mechanisms of microcirculatory changes that underlie cardiovascular disease.

In vivo time-resolved spectroscopy of the human bronchial early cancer autofluorescence.

Time-resolved measurements of tissue autofluorescence (AF) excited at 405 nm were carried out with an optical-fiber-based spectrometer in the bronchi of 11 patients. The objectives consisted of assessing the lifetime as a new tumor/normal (T/N) tissue contrast parameter and trying to explain the origin of the contrasts observed when using AF-based cancer detection imaging systems. No significant change in the AF lifetimes was found. AF bronchoscopy performed in parallel with an imaging device revealed both intensity and spectral contrasts. Our results suggest that the spectral contrast might be due to an enhanced blood concentration just below the epithelial layers of the lesion. The intensity contrast probably results from the thickening of the epithelium in the lesions. The absence of T/N lifetime contrast indicates that the quenching is not at the origin of the fluorescence intensity and spectral contrasts. These lifetimes (6.9 ns, 2.0 ns, and 0.2 ns) were consistent for all the examined sites. The fact that these lifetimes are the same for different emission domains ranging between 430 and 680 nm indicates that there is probably only one dominant fluorophore involved. The measured lifetimes suggest that this fluorophore is elastin.

MHC Class II Transactivator Is an In Vivo Regulator of Osteoclast Differentiation and Bone Homeostasis Co-opted From Adaptive Immunity.

The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo. © 2014 American Society for Bone and Mineral Research.

The histone-interacting domain of nuclear factor I activates simian virus 40 DNA replication in vivo.

Efficient initiation of SV40 DNA replication requires transcription factors that bind auxiliary sequences flanking the minimally required origin. To evaluate the possibility that transcription factors may activate SV40 replication by acting on the chromatin structure of the origin, we used an in vivo replication system in which we targeted GAL4 fusion proteins to the minimally required origin. We found that the proline-rich transcriptional activation domain of nuclear factor I (NF-I), which has been previously shown to interact with histone H3, specifically activates replication. Evaluation of a series of deletion and point mutants of NF-I indicates that the H3-binding domain and the replication activity coincide perfectly. Assays with other transcription factors, such as Sp1, confirmed the correlation between the interaction with H3 and the activation of replication. These findings imply that transcription factors such as NF-I can activate SV40 replication via direct interaction with chromatin components, thereby contributing to the relief of nucleosomal repression at the SV40 origin.

Simulação e análise econômica da produção in vivo e in vitro de embriões em bovinos

O objetivo deste trabalho foi avaliar o efeito das variáveis número de receptoras, protocolo de sincronização, indicadores de eficiência reprodutiva e custo da prenhez na eficiência econômica da produção in vivo e in vitro de embriões, em bovinos. Elaborou-se um aplicativo de simulação que permite ao usuário inserir os parâmetros das variáveis de entrada. Um cenário base, originado a partir de taxas tradicionais de eficiência das técnicas de produção in vivo (TE) e in vitro (PIVE) de embriões bovinos, foi introduzido no aplicativo de simulação como critério para comparação dos resultados. O aplicativo desenvolvido foi capaz de reproduzir os cenários de TE e PIVE. A produção de embriões foi simulada com o uso de simulação estocástica. Posteriormente determinou-se o número ótimo de receptoras por análise de sensibilidade. O valor presente líquido e o custo por prenhez foram utilizados como parâmetros de decisão. A sincronização para inovulação em tempo fixo reduziu a ociosidade de receptoras e, consequentemente, o custo final da prenhez em comparação à metodologia tradicional. A sexagem fetal deve estar associada à produção in vivo de embriões bovinos. Além disso, o número ótimo de receptoras por doadora é variável e dependente dos parâmetros de entrada do sistema.

Plac8 is required for white adipocyte differentiation in vitro and cell number control in vivo.

Plac8 belongs to an evolutionary conserved family of proteins, mostly abundant in plants where they control fruit weight through regulation of cell number. In mice, Plac8 is expressed both in white and brown adipose tissues and we previously showed that Plac8(-/-) mice develop late-onset obesity, with abnormal brown fat differentiation and reduced thermogenic capacity. We also showed that in brown adipocytes, Plac8 is an upstream regulator of C/EBPβ expression. Here, we first assessed the role of Plac8 in white adipogenesis in vitro. We show that Plac8 is induced early after induction of 3T3-L1 adipocytes differentiation, a process that is prevented by Plac8 knockdown; similarly, embryonic fibroblasts obtained from Plac8 knockout mice failed to form adipocytes upon stimulation of differentiation. Knockdown of Plac8 in 3T3-L1 was associated with reduced expression of C/EBPβ, Krox20, and Klf4, early regulators of the white adipogenic program, and we show that Plac8 could transactivate the C/EBPβ promoter. In vivo, we show that absence of Plac8 led to increased white fat mass with enlarged adipocytes but reduced total number of adipocytes. Finally, even though Plac8(-/-) mice showed impaired thermogenesis due to brown fat dysfunction, this was not associated with changes in glycemia or plasma free fatty acid and triglyceride levels. Collectively, these data indicate that Plac8 is an upstream regulator of C/EBPβ required for adipogenesis in vitro. However, in vivo, Plac8 is dispensable for the differentiation of white adipocytes with preserved fat storage capacity but is required for normal fat cell number regulation.

Recent advances in tumour antigen-specific therapy: in vivo veritas.

Modern cancer therapies should strive not only to eliminate malignant tissues but also to preserve healthy tissues and the patient's quality of life. Antigen-specific immunotherapy approaches are promising for either aspect since they are designed to only act against tissues expressing 1 or more specified tumour antigens. In order to develop successful vaccine and adoptive transfer protocols, longitudinal monitoring of cancer patients taking part in clinical trials is mandatory. Here, in vivo expansion of antigen-specific cells, as well as their ex vivo functional status represent important parameters to be analysed. To obtain results that most closely reflect the cells' in vivo status, functional assays must be carried out with as little in vitro culturing as possible. The present minireview discusses recent advances in these domains.

CSP-A Model for In Vivo Presentation of Plasmodium berghei Sporozoite Antigens by Hepatocytes.

One target of protective immunity against the Plasmodium liver stage in BALB/c mice is represented by the circumsporozoite protein (CSP), and mainly involves its recognition by IFN-γ producing specific CD8+T-cells. In a previous in vitro study we showed that primary hepatocytes from BALB/c mice process Plasmodium berghei (Pb) CSP (PbCSP) and present CSP-derived peptides to specific H-2k(d) restricted CD8+T-cells with subsequent killing of the presenting cells. We now extend these observations to an in vivo infection model in which infected hepatocytes and antigen specific T-cell clones are transferred into recipient mice inducing protection from sporozoite (SPZ) challenge. In addition, using a similar protocol, we suggest the capacity of hepatocytes in priming of naïve T-cells to provide protection, as further confirmed by induction of protection after depletion of cross-presenting dendritic cells (DCs) by cytochrome c (cyt c) treatment or using traversal deficient parasites. Our results clearly show that hepatocytes present Plasmodium CSP to specific-primed CD8+T-cells, and could also prime naïve T-cells, leading to protection from infection. These results could contribute to a better understanding of liver stage immune response and design of malaria vaccines.

VP22 light controlled delivery of oligonucleotides to ocular cells in vitro and in vivo.

PURPOSE: To study VP22 light controlled delivery of antisense oligonucleotide (ODN) to ocular cells in vitro and in vivo. METHODS: The C-terminal half of VP22 was expressed in Escherichia coli, purified and mixed with 20 mer phosphorothioate oligonucleotides (ODNs) to form light sensitive complex particles (vectosomes). Uptake of vectosomes and light induced redistribution of ODNs in human choroid melanoma cells (OCM-1) and in human retinal pigment epithelial cells (ARPE-19) were studied by confocal and electron microscopy. The effect of vectosomes formed with an antisense ODN corresponding to the 3'-untranslated region of the human c-raf kinase gene on the viability and the proliferation of OCM-1 cells was assessed before and after illumination. Cells incubated with vectosomes formed with a mismatched ODN, a free antisense ODN or a free mismatched ODN served as controls. White light transscleral illumination was carried out 24 h after the intravitreal injection of vectosomes in rat eyes. The distribution of fluorescent vectosomes and free fluorescent ODN was evaluated on cryosections by fluorescence microscopy before, and 1 h after illumination. RESULTS: Overnight incubation of human OCM-1 and ARPE-19 cells with vectosomes lead to intracellular internalization of the vectosomes. When not illuminated, internalized vectosomes remained stable within the cell cytoplasm. Disruption of vectosomes and release of the complexed ODN was induced by illumination of the cultures with a cold white light or a laser beam. In vitro, up to 60% inhibition of OCM-1 cell proliferation was observed in illuminated cultures incubated with vectosomes formed with antisense c-raf ODN. No inhibitory effect on the OCM-1 cell proliferation was observed in the absence of illumination or when the cells are incubated with a free antisense c-raf ODN and illuminated. In vivo, 24 h after intravitreal injection, vectosomes were observed within the various retinal layers accumulating in the cytoplasm of RPE cells. Transscleral illumination of the injected eyes with a cold white light induced disruption of the vectosomes and a preferential localization of the "released" ODNs within the cell nuclei of the ganglion cell layer, the inner nuclear layer and the RPE cells. CONCLUSIONS: In vitro, VP22 light controlled delivery of ODNs to ocular cells nuclei was feasible using white light or laser illumination. In vivo, a single intravitreal injection of vectosomes, followed by transscleral illumination allowed for the delivery of free ODNs to retinal and RPE cells.

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo.

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

Atividade antioxidante in vitro e in vivo de café bebida mole

O objetivo deste trabalho foi determinar a atividade antioxidante do café, bebida mole, in vivo e in vitro, antes e após a torração. Para a análise da atividade antioxidante in vitro, foram utilizados os métodos de sequestro de radicais livres (DPPH) e de atividade quelante de íons Fe2+. Foram utilizados, para o ensaio in vivo, ratos Zucker diabéticos, portadores de síndrome metabólica, e ratos Zucker controle. Os animais receberam doses diárias das bebidas de café, por gavagem, por 30 dias. Após o tratamento, foi realizada a avaliação de peroxidação lipídica. As amostras torradas apresentaram a maior percentagem de sequestro de radicais livres. As concentrações nas amostras de café verde e torrado foram similares às do padrão Trolox. Das amostras torradas, a torração média se destacou com maior atividade quelante de íons Fe2+. Os cafés verdes mostraram maior poder quelante do que os torrados. Compostos presentes no extrato diminuíram a lipoperoxidação hepática e renal que é comum em casos de diabetes e síndrome metabólica. O café apresenta atividade antioxidante e protege o fígado e os rins dos animais contra a lipoperoxidação comumente presente em quadros de diabetes mellitus tipo 2 e síndrome metabólica.

Nonactivated versus thrombin-activated platelets on wound healing and fibroblast-to-myofibroblast differentiation in vivo and in vitro.

Background: Platelet preparations for tissue healing are usually preactivated before application to deliver concentrated growth factors. In this study, the authors investigated the differences between nonactivated and thrombin-activated platelets in wound healing.Methods: The healing effects (i.e., wound closure, myofibroblast formation, and angiogenesis) of nonactivated and thrombin-activated platelets were compared in experimental wounds in diabetic (db/db) animals. In vitro, fibroblast phenotype and function were tested in response to platelets and activated platelets. No treatment served as a negative control.Results: Wounds treated with platelets reached 90 percent closure after 15 days, faster than activated platelets (26 days), and with higher levels of myofibroblasts and angiogenesis. In vitro, platelets enhanced cell migration and induced twofold higher myofibroblast differentiation and contraction compared with activated platelets.Conclusions: Platelets stimulate wound healing more efficiently compared with activated platelets by enhancing fibroblast differentiation and contractile function. Similar levels of growth factors may induce different biological effects when delivered "on demand" rather than in an initial bolus. (Plast. Reconstr. Surg. 129: 46e, 2012.)

Long-term monitoring of post-stroke plasticity after transient cerebral ischemia in mice using in vivo and ex vivo diffusion tensor MRI.

WE USED A MURINE MODEL OF TRANSIENT FOCAL CEREBRAL ISCHEMIA TO STUDY: 1) in vivo DTI long-term temporal evolution of the apparent diffusion coefficient (ADC) and diffusion fractional anisotropy (FA) at days 4, 10, 15 and 21 after stroke 2) ex vivo distribution of a plasticity-related protein (GAP-43) and its relationship with the ex vivo DTI characteristics of the striato-thalamic pathway (21 days). All animals recovered motor function. In vivo ADC within the infarct was significantly increased after stroke. In the stroke group, GAP-43 expression and FA values were significantly higher in the ipsilateral (IL) striatum and contralateral (CL) hippocampus compared to the shams. DTI tractography showed fiber trajectories connecting the CL striatum to the stroke region, where increased GAP43 and FA were observed and fiber tracts from the CL striatum terminating in the IL hippocampus.Our data demonstrate that DTI changes parallel histological remodeling and recovery of function.

Mechanisms underlying functional recovery after in vivo focal cerebral ischemia : from preconditioning to diffusion MRI

Version abregée L'ischémie cérébrale est la troisième cause de mort dans les pays développés, et la maladie responsable des plus sérieux handicaps neurologiques. La compréhension des bases moléculaires et anatomiques de la récupération fonctionnelle après l'ischémie cérébrale est donc extrêmement importante et représente un domaine d'intérêt crucial pour la recherche fondamentale et clinique. Durant les deux dernières décennies, les chercheurs ont tenté de combattre les effets nocifs de l'ischémie cérébrale à l'aide de substances exogènes qui, bien que testées avec succès dans le domaine expérimental, ont montré un effet contradictoire dans l'application clinique. Une approche différente mais complémentaire est de stimuler des mécanismes intrinsèques de neuroprotection en utilisant le «modèle de préconditionnement» : une brève insulte protège contre des épisodes d'ischémie plus sévères à travers la stimulation de voies de signalisation endogènes qui augmentent la résistance à l'ischémie. Cette approche peut offrir des éléments importants pour clarifier les mécanismes endogènes de neuroprotection et fournir de nouvelles stratégies pour rendre les neurones et la glie plus résistants à l'attaque ischémique cérébrale. Dans un premier temps, nous avons donc étudié les mécanismes de neuroprotection intrinsèques stimulés par la thrombine, un neuroprotecteur «préconditionnant» dont on a montré, à l'aide de modèles expérimentaux in vitro et in vivo, qu'il réduit la mort neuronale. En appliquant une technique de microchirurgie pour induire une ischémie cérébrale transitoire chez la souris, nous avons montré que la thrombine peut stimuler les voies de signalisation intracellulaire médiées par MAPK et JNK par une approche moléculaire et l'analyse in vivo d'un inhibiteur spécifique de JNK (L JNK) .Nous avons également étudié l'impact de la thrombine sur la récupération fonctionnelle après une attaque et avons pu démontrer que ces mécanismes moléculaires peuvent améliorer la récupération motrice. La deuxième partie de cette étude des mécanismes de récupération après ischémie cérébrale est basée sur l'investigation des bases anatomiques de la plasticité des connections cérébrales, soit dans le modèle animal d'ischémie transitoire, soit chez l'homme. Selon des résultats précédemment publiés par divers groupes ,nous savons que des mécanismes de plasticité aboutissant à des degrés divers de récupération fonctionnelle sont mis enjeu après une lésion ischémique. Le résultat de cette réorganisation est une nouvelle architecture fonctionnelle et structurelle, qui varie individuellement selon l'anatomie de la lésion, l'âge du sujet et la chronicité de la lésion. Le succès de toute intervention thérapeutique dépendra donc de son interaction avec la nouvelle architecture anatomique. Pour cette raison, nous avons appliqué deux techniques de diffusion en résonance magnétique qui permettent de détecter les changements de microstructure cérébrale et de connexions anatomiques suite à une attaque : IRM par tenseur de diffusion (DT-IR1V) et IRM par spectre de diffusion (DSIRM). Grâce à la DT-IRM hautement sophistiquée, nous avons pu effectuer une étude de follow-up à long terme chez des souris ayant subi une ischémie cérébrale transitoire, qui a mis en évidence que les changements microstructurels dans l'infarctus ainsi que la modification des voies anatomiques sont corrélés à la récupération fonctionnelle. De plus, nous avons observé une réorganisation axonale dans des aires où l'on détecte une augmentation d'expression d'une protéine de plasticité exprimée dans le cône de croissance des axones (GAP-43). En appliquant la même technique, nous avons également effectué deux études, rétrospective et prospective, qui ont montré comment des paramètres obtenus avec DT-IRM peuvent monitorer la rapidité de récupération et mettre en évidence un changement structurel dans les voies impliquées dans les manifestations cliniques. Dans la dernière partie de ce travail, nous avons décrit la manière dont la DS-IRM peut être appliquée dans le domaine expérimental et clinique pour étudier la plasticité cérébrale après ischémie. Abstract Ischemic stroke is the third leading cause of death in developed countries and the disease responsible for the most serious long-term neurological disability. Understanding molecular and anatomical basis of stroke recovery is, therefore, extremely important and represents a major field of interest for basic and clinical research. Over the past 2 decades, much attention has focused on counteracting noxious effect of the ischemic insult with exogenous substances (oxygen radical scavengers, AMPA and NMDA receptor antagonists, MMP inhibitors etc) which were successfully tested in the experimental field -but which turned out to have controversial effects in clinical trials. A different but complementary approach to address ischemia pathophysiology and treatment options is to stimulate and investigate intrinsic mechanisms of neuroprotection using the "preconditioning effect": applying a brief insult protects against subsequent prolonged and detrimental ischemic episodes, by up-regulating powerful endogenous pathways that increase resistance to injury. We believe that this approach might offer an important insight into the molecular mechanisms responsible for endogenous neuroprotection. In addition, results from preconditioning model experiment may provide new strategies for making brain cells "naturally" more resistant to ischemic injury and accelerate their rate of functional recovery. In the first part of this work, we investigated down-stream mechanisms of neuroprotection induced by thrombin, a well known neuroprotectant which has been demonstrated to reduce stroke-induced cell death in vitro and in vivo experimental models. Using microsurgery to induce transient brain ischemia in mice, we showed that thrombin can stimulate both MAPK and JNK intracellular pathways through a molecular biology approach and an in vivo analysis of a specific kinase inhibitor (L JNK1). We also studied thrombin's impact on functional recovery demonstrating that these molecular mechanisms could enhance post-stroke motor outcome. The second part of this study is based on investigating the anatomical basis underlying connectivity remodeling, leading to functional improvement after stroke. To do this, we used both a mouse model of experimental ischemia and human subjects with stroke. It is known from previous data published in literature, that the brain adapts to damage in a way that attempts to preserve motor function. The result of this reorganization is a new functional and structural architecture, which will vary from patient to patient depending on the anatomy of the damage, the biological age of the patient and the chronicity of the lesion. The success of any given therapeutic intervention will depend on how well it interacts with this new architecture. For this reason, we applied diffusion magnetic resonance techniques able to detect micro-structural and connectivity changes following an ischemic lesion: diffusion tensor MRI (DT-MRI) and diffusion spectrum MRI (DS-MRI). Using DT-MRI, we performed along-term follow up study of stroke mice which showed how diffusion changes in the stroke region and fiber tract remodeling is correlating with stroke recovery. In addition, axonal reorganization is shown in areas of increased plasticity related protein expression (GAP 43, growth axonal cone related protein). Applying the same technique, we then performed a retrospective and a prospective study in humans demonstrating how specific DTI parameters could help to monitor the speed of recovery and show longitudinal changes in damaged tracts involved in clinical symptoms. Finally, in the last part of this study we showed how DS-MRI could be applied both to experimental and human stroke and which perspectives it can open to further investigate post stroke plasticity.