Episcopic 3D Imaging Methods: Tools for Researching Gene Function

This work aims at describing episcopic 3D imaging methods and at discussing how these methods can contribute to researching the genetic mechanisms driving embryogenesis and tissue remodelling, and the genesis of pathologies. Several episcopic 3D imaging methods exist. The most advanced are capable of generating high-resolution volume data (voxel sizes from 0.5x0.5x1 µm upwards) of small to large embryos of model organisms and tissue samples. Beside anatomy and tissue architecture, gene expression and gene product patterns can be three dimensionally analyzed in their precise anatomical and histological context with the aid of whole mount in situ hybridization or whole mount immunohistochemical staining techniques. Episcopic 3D imaging techniques were and are employed for analyzing the precise morphological phenotype of experimentally malformed, randomly produced, or genetically engineered embryos of biomedical model organisms. It has been shown that episcopic 3D imaging also fits for describing the spatial distribution of genes and gene products during embryogenesis, and that it can be used for analyzing tissue samples of adult model animals and humans. The latter offers the possibility to use episcopic 3D imaging techniques for researching the causality and treatment of pathologies or for staging cancer. Such applications, however, are not yet routine and currently only preliminary results are available. We conclude that, although episcopic 3D imaging is in its very beginnings, it represents an upcoming methodology, which in short terms will become an indispensable tool for researching the genetic regulation of embryo development as well as the genesis of malformations and diseases

Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium

Our objective was to investigate the role of phosphodiesterase (PDE)3 and PDE4 and cGMP in the control of cAMP metabolism and of phosphorylation of troponin I (TnI) and phospholamban (PLB) when 5-HT4 receptors are activated in pig left atrium. Electrically paced porcine left atrial muscles, mounted in organ baths, received stimulators of particulate guanylyl cyclase (pGC) or soluble guanylyl cyclase (sGC) and/or specific PDE inhibitors followed by 5-HT or the 5-HT4 receptor agonist prucalopride. Muscles were freeze-clamped at different moments of exposure to measure phosphorylation of the cAMP/protein kinase A targets TnI and PLB by immunoblotting and cAMP levels by enzyme immunoassay. Corresponding with the functional results, 5-HT only transiently increased cAMP content, but caused a less quickly declining phosphorylation of PLB and did not significantly change TnI phosphorylation. Under combined PDE3 and PDE4 inhibition, the 5-HT-induced increase in cAMP levels and PLB phosphorylation was enhanced and sustained, and TnI phosphorylation was now also increased. Responses to prucalopride per se and the influence thereupon of PDE3 and PDE4 inhibition were similar except that responses were generally smaller. Stimulation of pGC together with PDE4 inhibition increased 5-HT-induced PLB phosphorylation compared to 5-HT alone, consistent with functional responses. sGC stimulation hastened the fade of inotropic responses to 5-HT, while cAMP levels were not altered. PDE3 and PDE4 control the cAMP response to 5-HT4 receptor activation, causing a dampening of downstream signalling. Stimulation of pGC is able to enhance inotropic responses to 5-HT by increasing cAMP levels, while sGC stimulation decreases contraction to 5-HT cAMP independently

Two-component radiation model of the sonoluminescing bubble

Based on the experimental data from Weninger, Putterman & Barber, Phys. Rev. (E), 54, R2205 (1996), we offer an alternative interpretation of their experimetal results. A model of sonoluminescing bubble which proposes that the electromagnetic radiation originates from two sources: the isotropic black body or bramsstrahlung emitting core and dipole radiation-emitting shell of accelerated electrons driven by the liquid-bubble interface is outlined.Comment: 5 pages Revtex, submitted to Phys. Rev.

Generalized Lévy walks and the role of chemokines in migration of effector CD8+ T cells.

Chemokines have a central role in regulating processes essential to the immune function of T cells, such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of CD8+ T cells to control the pathogen Toxoplasma gondii in the brains of chronically infected mice. This chemokine boosts T-cell function in two different ways: it maintains the effector T-cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Notably, these statistics are not Brownian; rather, CD8+ T-cell motility in the brain is well described by a generalized Lévy walk. According to our model, this unexpected feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, CD8+ T-cell behaviour is similar to Lévy strategies reported in organisms ranging from mussels to marine predators and monkeys, and CXCL10 aids T cells in shortening the average time taken to find rare targets

Differences between blood and cerebrospinal fluid glial fibrillary Acidic protein levels: The effect of sample stability

Introduction: Recent evidence has shown that the marker of reactive astrogliosis, glial fibrillary acidic protein (GFAP), has a stronger relationship with cerebral amyloid beta (Aβ) pathology in blood than in cerebrospinal fluid (CSF). This study investigates if pre-analytical treatment of blood and CSF contribute to these unexpected findings. Methods: Paired CSF and serum samples from 49 individuals (Aβ-negative = 28; Aβ-positive = 21) underwent a series of seven freeze-thaw cycles (FTCs). All samples were analyzed for GFAP and neurofilament light (NfL) using single molecule array technology including a fresh unfrozen sample from each patient. Results: FTC significantly affected CSF GFAP concentration (−188.12 pg/ml per FTC) but not serum GFAP. In the same samples, NfL remained stable. Serum GFAP had a higher discrimination of Aβ burden than CSF GFAP, irrespective of FTC, which also included unfrozen samples. Discussion: This study demonstrates large stability differences of GFAP in CSF and serum. However, this disparity does not seem to fully explain the stronger association of serum GFAP with Aβ pathology. Further work should investigate mechanisms of GFAP release into the bloodstream under pathological conditions

A Mouse Model of Vitiligo with Focused Epidermal Depigmentation Requires IFN-γ for Autoreactive CD8+CD8^+ T Cell Accumulation in the Skin

Vitiligo is an autoimmune disease of the skin causing disfiguring patchy depigmentation of the epidermis and, less commonly, hair. Therapeutic options for vitiligo are limited, reflecting in part limited knowledge of disease pathogenesis. Existing mouse models of vitiligo consist of hair depigmentation but lack prominent epidermal involvement, which is the hallmark of human disease. They are thus unable to provide a platform to fully investigate disease mechanisms and treatment. $CD8^+$ T cells have been implicated in the pathogenesis of vitiligo and expression of interferon-gamma (IFN-γ) is increased in the lesional skin of patients, however it is currently unknown what role IFN-γ plays in disease. Here, we have developed an adoptive transfer mouse model of vitiligo using melanocyte-specific $CD8^+$ T cells, which recapitulates the human condition by inducing epidermal depigmentation while sparing the hair. Like active lesions in human vitiligo, histology of depigmenting skin reveals a patchy mononuclear infiltrate and single-cell infiltration of the epidermis. Depigmentation is accompanied by accumulation of autoreactive $CD8^+$ T cells in the skin, quantifiable loss of tyrosinase transcript, and local IFN-γ production. Neutralization of IFN-γ with antibody prevents $CD8^+$ T cell accumulation and depigmentation, suggesting a therapeutic potential for this approach

CXCL12 Mediates CCR7-independent Homing of Central Memory Cells, But Not Naive T Cells, in Peripheral Lymph Nodes

Central memory CD8+ T cells (TCM) confer superior protective immunity against infections compared with other T cell subsets. TCM recirculate mainly through secondary lymphoid organs, including peripheral lymph nodes (PLNs). Here, we report that TCM, unlike naive T cells, can home to PLNs in both a CCR7-dependent and -independent manner. Homing experiments in paucity of lymph node T cells (plt/plt) mice, which do not express CCR7 ligands in secondary lymphoid organs, revealed that TCM migrate to PLNs at ∼20% of wild-type (WT) levels, whereas homing of naive T cells was reduced by 95%. Accordingly, a large fraction of endogenous CD8+ T cells in plt/plt PLNs displayed a TCM phenotype. Intravital microscopy of plt/plt subiliac lymph nodes showed that TCM rolled and firmly adhered (sticking) in high endothelial venules (HEVs), whereas naive T cells were incapable of sticking. Sticking of TCM in plt/plt HEVs was pertussis toxin sensitive and was blocked by anti-CXCL12 (SDF-1α). Anti-CXCL12 also reduced homing of TCM to PLNs in WT animals by 20%, indicating a nonredundant role for this chemokine in the presence of physiologic CCR7 agonists. Together, these data distinguish naive T cells from TCM, whereby only the latter display greater migratory flexibility by virtue of their increased responsiveness to both CCR7 ligands and CXCL12 during homing to PLN

Visualizing Vertebrate Embryos with Episcopic 3D Imaging Techniques

The creation of highly detailed, three-dimensional (3D) computer models is essential in order to understand the evolution and development of vertebrate embryos, and the pathogenesis of hereditary diseases. A still-increasing number of methods allow for generating digital volume data sets as the basis of virtual 3D computer models. This work aims to provide a brief overview about modern volume data–generation techniques, focusing on episcopic 3D imaging methods. The technical principles, advantages, and problems of episcopic 3D imaging are described. The strengths and weaknesses in its ability to visualize embryo anatomy and labeled gene product patterns, specifically, are discussed