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Vivo Observation (vivo + observation)
Selected AbstractsIn vivo observation of the locomotion of microglial cells in the retinaGLIA, Issue 14 2010Michel Paques Abstract Microglial cells (MCs) are active sensors and reactive phagocytes of neural tissues. They are known to migrate and accumulate in areas of neuronal damage. Thus, microglial locomotion is an essential feature of the inflammatory reaction in neural tissue. Yet, to our knowledge there has been no report of direct in vivo observation of the migration of MCs. Here, we show that intravitreally injected cyanine dyes (DiO, DiI, and indocyanine green) are sequestrated in MCs during several months, and subsequently in vivo images of these fluorescent MCs can be obtained by confocal scanning laser ophthalmoscopy. This enabled noninvasive, time-lapse observation of the migrating behavior of MCs, both in the basal state and following laser damage. In the basal state, a slow, intermittent, random-like locomotion was observed. Following focal laser damage, MCs promptly (i.e., within 1 h) initiated centripetal, convergent migration. MCs up to 400 ,m away migrated into the scar at velocities up to 7 ,m/min. This early phase of centripetal migration was followed by a more prolonged phase of nontargeted locomotion around and within injured sites during at least 24 h. Cyanine-positive cells persisted within the scar during several weeks. To our knowledge, this is the first in vivo observation of the locomotion of individual MCs. Our results show that the locomotion of MCs is not limited to translocation to acutely damaged area, but may also be observed in the basal state and after completion of the recruitment of MCs into scars. © 2010 Wiley-Liss, Inc. [source] Cellular iron status influences the functional relationship between microglia and oligodendrocytesGLIA, Issue 8 2006X. Zhang Abstract Previously, we have reported that there is a spatiotemporal relationship between iron accumulation in microglia and oligodendrocytes during normal development and in remyelination following injury. This in vivo observation has prompted us to develop a cell culture model to test the relationship between iron status of microglia and survival of oligodendrocytes. We found that conditioned media from iron-loaded microglia increases the survival of oligodendrocytes; but conditioned media from iron loaded activated microglia is toxic to oligodendrocytes. In the trophic condition, one of the proteins released by iron-loaded microglia is H-ferritin, and transfecting the microglia with siRNA for H-ferritin blocks the trophic response on oligodendrocytes. Lipopolysaccharide (LPS) activation decreases the amount of H-ferritin that is released from microglia and increases the release of the proinflammatory cytokines tumor necrosis factor-, and interleukin-1. LPS activation of iron-enriched microglia results in the activation of NF-kB and greater release of cytokines when compared with that of control microglia; whereas treating microglia with an iron chelator is associated with less NF-kB activation and less release of cytokines. These results indicate that microglia play an important role in iron homoeostasis and that their iron status can influence how microglia influence growth and survival of oligodendrocytes. The results further indicate that ferritin, released by microglia, is a significant source of iron for oligodendrocytes. © 2006 Wiley-Liss, Inc. [source] Diagnosis of Helicobacter pylori InfectionHELICOBACTER, Issue 2006Katarzyna Dzier, anowska-Fangrat Abstract A growing interest in non-invasive tests for the detection of Helicobacter pylori has been observed recently, reflecting a large number of studies published this year. New tests have been validated, and the old ones have been used in different clinical situations or for different purposes. Stool antigen tests have been extensively evaluated in pre- and post-treatment settings both in adults and children, and the urea breath test has been studied as a predictor of bacterial load, severity of gastric inflammation, and response to eradication treatment. Several studies have also explored the usefulness of some serologic markers as indicators of the gastric mucosa status. With regard to invasive tests, molecular methods are being used more and more, but the breakthrough this year was the direct in vivo observation of H. pylori during endoscopy. [source] Resting state sensorimotor functional connectivity in multiple sclerosis inversely correlates with transcallosal motor pathway transverse diffusivityHUMAN BRAIN MAPPING, Issue 7 2008Mark J. Lowe Abstract Recent studies indicate that functional connectivity using low-frequency BOLD fluctuations (LFBFs) is reduced between the bilateral primary sensorimotor regions in multiple sclerosis. In addition, it has been shown that pathway-dependent measures of the transverse diffusivity of water in white matter correlate with related clinical measures of functional deficit in multiple sclerosis. Taken together, these methods suggest that MRI methods can be used to probe both functional connectivity and anatomic connectivity in subjects with known white matter impairment. We report the results of a study comparing anatomic connectivity of the transcallosal motor pathway, as measured with diffusion tensor imaging (DTI) and functional connectivity of the bilateral primary sensorimotor cortices (SMC), as measured with LFBFs in the resting state. High angular resolution diffusion imaging was combined with functional MRI to define the transcallosal white matter pathway connecting the bilateral primary SMC. Maps were generated from the probabilistic tracking employed and these maps were used to calculate the mean pathway diffusion measures fractional anisotropy ,FA,, mean diffusivity ,MD,, longitudinal diffusivity ,,1,, and transverse diffusivity ,,2,. These were compared with LFBF-based functional connectivity measures (Fc) obtained at rest in a cohort of 11 multiple sclerosis patients and ,10 age- and gender-matched control subjects. The correlation between ,FA, and Fc for MS patients was r = ,0.63, P < 0.04. The correlation between all subjects ,,2, and Fc was r = 0.42, P < 0.05. The correlation between all subjects ,,2, and Fc was r = ,0.50, P < 0.02. None of the control subject correlations were significant, nor were ,FA,, ,,1,, or ,MD, significantly correlated with Fc for MS patients. This constitutes the first in vivo observation of a correlation between measures of anatomic connectivity and functional connectivity using spontaneous LFBFs. Hum Brain Mapp, 2008. © 2008 Wiley-Liss, Inc. [source] Orthogonal polarization technique in the assessment of human skin microcirculationINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 5 2008Omar Lupi MD Background The "gold standard" for the study of the in vivo microcirculation is intravital microscopy. The recently developed method of orthogonal polarization of light [orthogonal polarization spectral (OPS) imaging] allows for the in vivo transcutaneous evaluation of the microcirculation without the need for invasive surgical procedures. Methods The application of polarized light originating from a 100 W halogen tungsten lamp is able to penetrate tissues at a depth of up to 3 mm, and generates reissued light from this depth. The evaluation of this depolarized light, from a deeper origin, may be carried out separately from the light reflected by the more superficial layers of the tissue under study because this light retains photon polarization, whereas the former light undergoes real depolarization. Results The process of validation of the OPS technique, when compared with intravital microscopy, the "gold standard" for the in vivo observation of the microcirculation, has shown that it is as effective and reliable as the gold standard, reaching the same resolution level in the visualization of blood vessels, but without the need for invasive surgical procedures. Conclusions The OPS technique is a very promising tool for dermatologists and researchers, especially in the study of vasculitis, chronic venous insufficiency, and skin tumors. [source] Ring Opening of the Cyclobutane in a Thymine Dimer Radical AnionCHEMISTRY - A EUROPEAN JOURNAL, Issue 32 2007Chryssostomos Chatgilialoglu Dr. Abstract The reactions of hydrated electrons (eaq,) with thymine dimer 2 and thymidine have been investigated by radiolytic methods coupled with product studies, and addressed computationally by means of BB1K-HMDFT calculations. Pulse radiolysis revealed that one-electron reduction of the thymine dimer 2 affords the radical anion of thymidine (5) with t1/2<35,ns. Indeed, the theoretical study suggests that radical anion 3, in which the spin density and charge distribution are located in both thymine rings, undergoes a fast partially ionic splitting of the cyclobutane with a half-life of a few ps. This model fits with the in vivo observation of thymine dimer repair in DNA by photolyase. ,-Radiolysis of thymine dimer 2 demonstrates that the one-electron reduction and the subsequent cleavage of the cyclobutane ring does not proceed by means of a radical chain mechanism, that is, in this model reaction the T,. is unable to transfer an electron to the thymine dimer 2. [source] The circulatory system in Chilopoda: functional morphology and phylogenetic aspectsACTA ZOOLOGICA, Issue 3 2002Christian S. Wirkner Abstract The circulatory organs of nine representative species of all five chilopod orders were examined by light microscopy and by in vivo observations of haemolymph flow. In Scutigera coleoptrata, the heart ultrastructure was studied. The circulatory system in Craterostigmomorpha is described for the first time. Further focus is placed on the Geophilomorpha since previous descriptions in this group have been only superficial. In all investigated species, the circulatory system consists of two longitudinal central vessels which are connected in the first body segment by the maxilliped arch. The posterior part of these vessels is contractile and thus haemolymph is pumped anteriorly in the heart, while it is pumped posteriorly in the supraneural vessel. From these central vessels numerous peripheral vessels branch off. Differences among the chilopod orders lie mainly in the distribution of the peripheral vessels. The circulatory system in Scutigeromorpha shows some striking morphological adaptations with regard to the functional coupling of circulatory and respiratory tasks. The most peculiar structures are the aortic diverticles which act as accessory pumps in the head. Phylogenetic analysis of the circulatory organ traits within Chilopoda supports the Pleurostigmophora hypothesis. Synapomorphies supporting the Myriapoda hypothesis or the Tracheata concept were not found. [source] MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay,HUMAN MUTATION, Issue 2 2010Sara Molatore Abstract MUTYH -associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh,/, mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1,8, 2009. © 2009 Wiley-Liss, Inc. [source] Defining the molecular action of HDAC inhibitors and synergism with androgen deprivation in ERG-positive prostate cancerINTERNATIONAL JOURNAL OF CANCER, Issue 12 2008Mari Björkman Abstract Gene fusions between prostate-specific, androgen responsive TMPRSS2 gene and oncogenic ETS factors, such as ERG, occur in up to 50% of all prostate cancers. We recently defined a gene signature that was characteristic to prostate cancers with ERG activation. This suggested epigenetic reprogramming, such as upregulation of histone deactylase 1 (HDAC1) gene and downregulation of its target genes. We then hypothesized that patients with ERG -positive prostate cancers may benefit from epigenetic therapy such as HDAC inhibition (HDACi), especially in combination with antiandrogens. Here, we exposed ERG -positive prostate cancer cell lines to HDAC inhibitors Trichostatin A (TSA), MS-275 and suberoylanilide hydroxamic acid (SAHA) with or without androgen deprivation. We explored the effects on cell phenotype, gene expression as well as ERG and androgen receptor (AR) signaling. When compared with 5 other prostate cell lines, ERG -positive VCaP and DuCap cells were extremely sensitive to HDACi, in particular TSA, showing synergy with concomitant androgen deprivation increasing apoptosis. Both of the HDAC inhibitors studied caused repression of the ERG -fusion gene, whereas the pan-HDAC inhibitor TSA prominently repressed the ERG -associated gene signature. Additionally, HDACi and flutamide caused retention of AR in the cytoplasm, indicating blockage of androgen signaling. Our results support the hypothesis that HDACi, especially in combination with androgen deprivation, is effective against TMPRSS2-ERG -fusion positive prostate cancer in vitro. Together with our previous in vivo observations of an "epigenetic reprogramming gene signature" in clinical ERG -positive prostate cancers, these studies provide mechanistic insights to ERG -associated tumorigenesis and suggest therapeutic paradigms to be tested in vivo. © 2008 Wiley-Liss, Inc. [source] Regulation of lipopolysaccharide-induced inflammatory response and endotoxemia by ,-arrestins,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Katie J. Porter ,-Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that ,-arrestin-1 (,-arr-1) and -2 knockout (KO) mice are protected from TLR4-mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild-type (WT) and ,-arr-1 and -2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS-induced inflammatory cytokine levels in the plasma were markedly decreased in both ,-arr-1 and -2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11b+ and CD11b, populations) from WT, ,-arr-1, and -2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS-induced inflammatory cytokines were significantly blocked in both splenocyte populations from the ,-arr-2 KO compared to the WT mice. This effect in the ,-arr-1 KO mice, however, was restricted to the CD11b, splenocytes. Our studies further indicate that regulation of cytokine production by ,-arrestins is likely independent of MAPK and I,B,-NF,B pathways. Our results, however, suggest that LPS-induced chromatin modification is dependent on ,-arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by ,-arrestins in vivo. Taken together, these results indicate that ,-arr-1 and -2 mediate LPS-induced cytokine secretion in a cell-type specific manner and that both ,-arrestins have overlapping but non-redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406,416, 2010. © 2010 Wiley-Liss, Inc. [source] Trophic factors attenuate nitric oxide mediated neuronal and axonal injury in vitro: roles and interactions of mitogen-activated protein kinase signalling pathwaysJOURNAL OF NEUROCHEMISTRY, Issue 6 2005Alastair Wilkins Abstract Inflammation in the central nervous system occurs in diseases such as multiple sclerosis and leads to axon dysfunction and destruction. Both in vitro and in vivo observations have suggested an important role for nitric oxide (NO) in mediating inflammatory axonopathy. The purposes of this study were to model inflammatory axonopathy in vitro and identify modulators of the process. Rat cortical neurones were cultured and exposed to an NO-donor plus potential protective factors. Cultures were then assessed for neuronal survival, axon survival and markers of intracellular signalling pathways. The NO-donor produced dose-dependent neuronal loss and a large degree of axon destruction. Oligodendrocyte conditioned medium (OCM) and insulin-like growth factor type-1 (IGF-1), but not glial cell line-derived neurotrophic factor (GDNF), improved survival of neurones exposed to NO donors. In addition p38 MAP kinase was activated by NO exposure and inhibition of p38 signalling led to neuronal and axonal survival effects. OCM and IGF-1 (but not GDNF) reduced p38 activation in NO-exposed cortical neurones. OCM, IGF-1 and GDNF improved axon survival in cultures exposed to NO, a process dependent on mitogen-activated protein kinase/extracellular signal-related kinase signalling. This study emphasizes that different mechanisms may underlie neuronal/axonal destructive processes, and suggests that trophic factors may modulate NO-mediated neurone/axon destruction via specific pathways. [source] Mechanistic comparison of blood undergoing laser photocoagulation at 532 and 1,064 nmLASERS IN SURGERY AND MEDICINE, Issue 2 2005John F. Black PhD Abstract Background and Objectives We seek to compare and contrast the mechanisms of blood photocoagulation under 532 and 1,064 nm laser irradiation in vitro in order to better understand the in vivo observations. We also seek to validate a finite element model (FEM) developed to study the thermodynamics of coagulation. Study Design/Materials and Methods We study the photocoagulation of whole blood in vitro at 532 and 1,064 nm using time-domain spectroscopic and optical coherence tomography (OCT)-based imaging techniques. We model the coagulation using an FEM program that includes the latent heat of vaporization (LHV) of water, consideration of the pulse shape of the laser, and the bathochromic shift in the hemoglobin absorption spectrum. Results We find significant similarities in the spectroscopic, chemical, and structural changes occurring in hemoglobin and in the blood matrix during photocoagulation despite the very large difference in the absorption coefficients. The more uniform temperature profile developed by the deeper-penetrating 1,064 nm laser allows us to resolve the structural phase transition in the red blood cells (going from biconcave disc to spherocyte) and the chemical transition creating met-hemoglobin. We find that the RBC morphology transition happens first, and that the met-Hb transition happens at a much higher temperature (,>,90°C) than is found in slow bath heating. The FEM analysis with the LHV constraint and bathochromic shift predicts accurately the imaging results in both cases, and can be used to show that at 1,064 nm there is the potential for a runaway increase in absorption during the laser pulse. Conclusions Photothermally mediated processes dominate the in vitro coagulation dynamics in both regimes despite the difference in absorption coefficients. There is a significant risk under 1,064 nm irradiation of vascular lesions in vivo that the dynamic optical properties of blood will cause runaway absorption and heating. This may in turn explain some recent results at this wavelength where full-thickness burns resulted from laser treatment. Lasers Surg. Med. 36:155,165, 2005. © 2005 Wiley-Liss, Inc. [source] | |||||||||||||