In vivo TCR Signaling in CD4(+) T Cells Imprints a Cell-Intrinsic, Transient Low-Motility Pattern Independent of Chemokine Receptor Expression Levels, or Microtubular Network, Integrin, and Protein Kinase C Activity

Intravital imaging has revealed that T cells change their migratory behavior during physiological activation inside lymphoid tissue. Yet, it remains less well investigated how the intrinsic migratory capacity of activated T cells is regulated by chemokine receptor levels or other regulatory elements. Here, we used an adjuvant-driven inflammation model to examine how motility patterns corresponded with CCR7, CXCR4, and CXCR5 expression levels on ovalbumin-specific DO11.10 CD4(+) T cells in draining lymph nodes. We found that while CCR7 and CXCR4 surface levels remained essentially unaltered during the first 48-72 h after activation of CD4(+) T cells, their in vitro chemokinetic and directed migratory capacity to the respective ligands, CCL19, CCL21, and CXCL12, was substantially reduced during this time window. Activated T cells recovered from this temporary decrease in motility on day 6 post immunization, coinciding with increased migration to the CXCR5 ligand CXCL13. The transiently impaired CD4(+) T cell motility pattern correlated with increased LFA-1 expression and augmented phosphorylation of the microtubule regulator Stathmin on day 3 post immunization, yet neither microtubule destabilization nor integrin blocking could reverse TCR-imprinted unresponsiveness. Furthermore, protein kinase C (PKC) inhibition did not restore chemotactic activity, ruling out PKC-mediated receptor desensitization as mechanism for reduced migration in activated T cells. Thus, we identify a cell-intrinsic, chemokine receptor level-uncoupled decrease in motility in CD4(+) T cells shortly after activation, coinciding with clonal expansion. The transiently reduced ability to react to chemokinetic and chemotactic stimuli may contribute to the sequestering of activated CD4(+) T cells in reactive peripheral lymph nodes, allowing for integration of costimulatory signals required for full activation.

Standardized pelvic floor exercises improve stress urinary incontinence in women with intrinsic sphincter deficiency.

OBJECTIVES Pelvic floor rehabilitation is the conservative therapy of choice for women with stress urinary incontinence (SUI). The success rate of surgical procedures in SUI patients with intrinsic sphincter deficiency (ISD) is low. The aim of this study was to analyse the effect of a standardized physiotherapy on patients with SUI and normotonic urethra and ISD. METHODS In this study, 64 patients with ISD and 69 patients with normotonic urethra were enrolled. Maximum urethral pressure (MUCP) >20 cm H2 O was considered as normotonic urethral pressure. Before and after physiotherapy MUCP was measured and cough testing was performed. Additionally, patient reported outcome was assessed using the King's Health Questionnaire (KHQ). For statistical analyses Excel 2010 (Microsoft Inc; Redmond, Washington) and SPSS 20 (SPSS Inc; Chicago, Illinois) for Windows were used. Power calculation was based on the primary endpoint incontinence impact and general health. For power calculation, GraphPad Statmate version 2.00 for Windows was used. RESULTS Sixty-four patients with ISD and 69 patients with normotonic urethra were included in the study. In SUI patients with normotonic and hypotonic urethra KHQ-scores regarding the primary endpoins "general health" and "incontinence impact" significantly improved following standardized physiotherapy. In both groups MUCP increased after physiotherapy. In SUI patients with ISD standardized physiotherapy resulted in a decreased incidence of a positive cough test. CONCLUSIONS Standardized physiotherapy should be offered to patients with SUI and ISD. Long-term results are subject to future studies. Neurourol. Urodynam. © 2015 Wiley Periodicals, Inc.

A Synthetic Factor XIIa Inhibitor Blocks Selectively Intrinsic Coagulation Initiation.

Coagulation factor XII (FXII) inhibitors are of interest for the study of the protease in the intrinsic coagulation pathway, for the suppression of contact activation in blood coagulation assays, and they have potential application in antithrombotic therapy. However, synthetic FXII inhibitors developed to date have weak binding affinity and/or poor selectivity. Herein, we developed a peptide macrocycle that inhibits activated FXII (FXIIa) with an inhibitory constant Ki of 22 nM and a selectivity of >2000-fold over other proteases. Sequence and structure analysis revealed that one of the two macrocyclic rings of the in vitro evolved peptide mimics the combining loop of corn trypsin inhibitor, a natural protein-based inhibitor of FXIIa. The synthetic inhibitor blocked intrinsic coagulation initiation without affecting extrinsic coagulation. Furthermore, the peptide macrocycle efficiently suppressed plasma coagulation triggered by contact of blood with sample tubes and allowed specific investigation of tissue factor initiated coagulation.

Intrinsic characteristics of the proton pump in the luminal membrane of a tight urinary epithelium. The relation between transport rate and delta mu H.

A number of tight urinary epithelia, as exemplified by the turtle bladder, acidify the luminal solution by active transport of H+ across the luminal cell membrane. The rate of active H+ transport (JH) decreases as the electrochemical potential difference for H+ [delta mu H = mu H(lumen) - mu H(serosa)] across the epithelium is increased. The luminal cell membrane has a low permeability for H+ equivalents and a high electrical resistance compared with the basolateral cell membrane. Changes in JH thus reflect changes in active H+ transport across the luminal membrane. To examine the control of JH by delta mu H in the turtle bladder, transepithelial electrical potential differences (delta psi) were imposed at constant acid-base conditions or the luminal pH was varied at delta psi = 0 and constant serosal PCO2 and pH. When the luminal compartment was acidified from pH 7 to 4 or was made electrically positive, JH decreased as a linear function of delta mu H as previously described. When the luminal compartment was made alkaline from pH 7 to 9 or was made electrically negative, JH reached a maximal value, which was the same whether the delta mu H was imposed as a delta pH or a delta psi. The nonlinear JH vs. delta mu H relation does not result from changes in the number of pumps in the luminal membrane or from changes in the intracellular pH, but is a characteristic of the H+ pumps themselves. We propose a general scheme, which, because of its structural features, can account for the nonlinearity of the JH vs. delta mu H relations and, more specifically, for the kinetic equivalence of the effects of the chemical and electrical components of delta mu H. According to this model, the pump complex consists of two components: a catalytic unit at the cytoplasmic side of the luminal membrane, which mediates the ATP-driven H+ translocation, and a transmembrane channel, which mediates the transfer of H+ from the catalytic unit to the luminal solution. These two components may be linked through a buffer compartment for H+ (an antechamber).

Intrinsic pH-Sensitivity of Cells in the Retrotrapezoid Nucleus: Possible Role of Glia in Respiratory Drive

An increase in carbon dioxide (CO2) and protons (H+) are the primary signals for breathing. Cells that sense changes in CO2/H+ levels and increase breathing accordingly are located in a region of the caudal medulla oblongata called the retrotrapezoid nucleus (RTN). Specifically, select RTN neurons are intrinsically pH sensitive and send excitatory projections to the respiratory rhythm generator to drive breathing. Glial cells in the RTN are thought to contribute to this respiratory drive, possibly by releasing ATP in response to increases in CO2/H+ levels. However, pH sensitivity of RTN glial cells has yet to be determined. Therefore, the goal of my thesis is to determine if acutely dissociated RTN cells can respond to changes in pH in isolation. To make this determination I used ratiometric fluorescent microscopy to measure intracellular calcium in dissociated RTN cells during changes in bath pH. I found that a small percentage of RTN cells (16%) respond to bath acidification from pH 7.3 to pH 6.9 with an increase in fluorescence indicating an increase in intracellular calcium. Preliminary electrophysiological findings suggest that responsive cells are unable to make action potentials, thus suggesting their identity to be glia. These results indicate that a subset of pH sensitive cells in the RTN are intrinsically pH sensitive and that glia cells may possibly play a role in central chemoreception.

Design, synthesis and cellular and molecular pharmacology of 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401): A novel non-cross-resistant anthracycline antibiotic with the intrinsic ability to circumvent P -glycoprotein-mediated drug resistance

We designed and synthesized a novel daunorubicin (DNR) analogue that effectively circumvents P-glycoprotein (P-gp)-mediated drug resistance. The fully protected carbohydrate intermediate 1,2-dibromoacosamine was prepared from acosamine and effectively coupled to daunomycinone in high yield. Deprotection under alkaline conditions yielded 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401). The in vitro cytotoxicity and cellular and molecular pharmacology of WP401 were compared with those of DNR in a panel of wild-type cell lines (KB-3-1, P388S, and HL60S) and their multidrug-resistant (MDR) counterparts (KB-V1, P388/DOX, and HL60/DOX). Fluorescent spectrophotometry, flow cytometry, and confocal laser scanning microscopy were used to measure intracellular accumulation, retention, and subcellular distribution of these agents. All MDR cell lines exhibited reduced DNR uptake that was restored, upon incubation with either verapamil (VER) or cyclosporin A (CSA), to the level found in sensitive cell lines. In contrast, the uptake of WP401 was essentially the same in the absence or presence of VER or CSA in all tested cell lines. The in vitro cytotoxicity of WP401 was similar to that of DNR in the sensitive cell lines but significantly higher in resistant cell lines (resistance index (RI) of 2-6 for WP401 vs 75-85 for DNR). To ascertain whether drug-mediated cytotoxicity and retention were accompanied by DNA strand breaks, DNA single- and double-strand breaks were assessed by alkaline elution. High levels of such breaks were obtained using 0.1-2 $\mu$g/mL of WP401 in both sensitive and resistant cells. In contrast, DNR caused strand breaks only in sensitive cells and not much in resistant cells. We also compared drug-induced DNA fragmentation similar to that induced by DNR. However, in P-gp-positive cells, WP401 induced 2- to 5-fold more DNA fragmentation than DNR. This increased DNA strand breakage by WP401 was correlated with its increased uptake and cytotoxicity in these cell lines. Overall these results indicate that WP401 is more cytotoxic than DNR in MDR cells and that this phenomenon might be related to the reduced basicity of the amino group and increased lipophilicity of WP401. ^

Intrinsic oxidative stress in cancer cells: A biochemical basis for therapeutic selectivity using ROS -generating agents

A major goal of chemotherapy is to selectively kill cancer cells while minimizing toxicity to normal cells. Identifying biological differences between cancer and normal cells is essential in designing new strategies to improve therapeutic selectivity. Superoxide dismutases (SOD) are crucial antioxidant enzymes required for the elimination of superoxide (O2·− ), a free radical produced during normal cellular metabolism. Previous studies in our laboratory demonstrated that 2-methoxyestradiol (2-ME), an estradiol derivative, inhibits the function of SOD and selectively kills human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The present work was initiated to examine the biochemical basis for the selective anticancer activity of 2-ME. Investigations using two-parameter flow cytometric analyses and ROS scavengers established that O2·− is a primary and essential mediator of 2-ME-induced apoptosis in cancer cells. In addition, experiments using SOD overexpression vectors and SOD knockout cells found that SOD is a critical target of 2-ME. Importantly, the administration of 2-ME resulted in the selective accumulation of O 2·− and apoptosis in leukemia and ovarian cancer cells. The preferential activity of 2-ME was found to be due to increased intrinsic oxidative stress in these cancer cells versus their normal counterparts. This intrinsic oxidative stress was associated with the upregulation of the antioxidant enzymes SOD and catalase as a mechanism to cope with the increase in ROS. Furthermore, oxygen consumption experiments revealed that normal lymphocytes decrease their respiration rate in response to 2-ME-induced oxidative stress, while human leukemia cells seem to lack this regulatory mechanism. This leads to an uncontrolled production of O2·−, severe accumulation of ROS, and ultimately ROS-mediated apoptosis in leukemia cells treated with 2-ME. The biochemical differences between cancer and normal cells identified here provide a basis for the development of drug combination strategies using 2-ME with other ROS-generating agents to enhance anticancer activity. The effectiveness of such a combination strategy in killing cancer cells was demonstrated by the use of 2-ME with agents/modalities such as ionizing radiation and doxorubicin. Collectively, the data presented here strongly suggests that 2-ME may have important clinical implications for the selective killing of cancer cells. ^

Vínculos urbano-rurales y construcción de nuevas territorialidades en asentamientos de rango menor

La homogeneización productiva ha trastocado los procesos de construcción territorial induciendo cambios en la naturaleza, intensidad y direccionalidad de los vínculos urbano-rurales en región pampeana. La creación de nuevas articulaciones y/o la redefinición de las interacciones preexistentes involucran dimensiones funcionales, institucionales y espaciales, dando origen a formas específicas de resolución territorial. A partir de enfoques superadores de las aproximaciones sectoriales y dualistas intrínsecas al paradigma de la modernización, que han impregnado la interpretación de procesos de construcción territorial a partir de la oposición "campo-ciudad", se propone la indagación a partir de la convergencia de macroprocesos que trastocan las trayectorias territoriales, al tiempo que renuevan la naturaleza de los vínculos urbano-rurales (avance de la pluriactividad, emergencia de nuevos actores y usos del suelo en los espacios rurales, urbanización difusa). Dentro de esta perspectiva, los asentamientos de rango menor en región pampeana, constituyen ámbitos privilegiados para la exploración de las modalidades que asumen las articulaciones urbano-rurales, en tanto lugares alcanzados por el proceso de homogeneización productiva, mostrando cómo la emergencia de nuevos actores, lógicas y escalas de acción, interpela las aproximaciones tradicionales de análisis de los vínculos urbano-rurales y exige la necesidad de renovar enfoques, regulaciones e instrumentos solidarios con la gestión territorial sustentable

Vínculos urbano-rurales y construcción de nuevas territorialidades en asentamientos de rango menor

La homogeneización productiva ha trastocado los procesos de construcción territorial induciendo cambios en la naturaleza, intensidad y direccionalidad de los vínculos urbano-rurales en región pampeana. La creación de nuevas articulaciones y/o la redefinición de las interacciones preexistentes involucran dimensiones funcionales, institucionales y espaciales, dando origen a formas específicas de resolución territorial. A partir de enfoques superadores de las aproximaciones sectoriales y dualistas intrínsecas al paradigma de la modernización, que han impregnado la interpretación de procesos de construcción territorial a partir de la oposición "campo-ciudad", se propone la indagación a partir de la convergencia de macroprocesos que trastocan las trayectorias territoriales, al tiempo que renuevan la naturaleza de los vínculos urbano-rurales (avance de la pluriactividad, emergencia de nuevos actores y usos del suelo en los espacios rurales, urbanización difusa). Dentro de esta perspectiva, los asentamientos de rango menor en región pampeana, constituyen ámbitos privilegiados para la exploración de las modalidades que asumen las articulaciones urbano-rurales, en tanto lugares alcanzados por el proceso de homogeneización productiva, mostrando cómo la emergencia de nuevos actores, lógicas y escalas de acción, interpela las aproximaciones tradicionales de análisis de los vínculos urbano-rurales y exige la necesidad de renovar enfoques, regulaciones e instrumentos solidarios con la gestión territorial sustentable

Vínculos urbano-rurales y construcción de nuevas territorialidades en asentamientos de rango menor

La homogeneización productiva ha trastocado los procesos de construcción territorial induciendo cambios en la naturaleza, intensidad y direccionalidad de los vínculos urbano-rurales en región pampeana. La creación de nuevas articulaciones y/o la redefinición de las interacciones preexistentes involucran dimensiones funcionales, institucionales y espaciales, dando origen a formas específicas de resolución territorial. A partir de enfoques superadores de las aproximaciones sectoriales y dualistas intrínsecas al paradigma de la modernización, que han impregnado la interpretación de procesos de construcción territorial a partir de la oposición "campo-ciudad", se propone la indagación a partir de la convergencia de macroprocesos que trastocan las trayectorias territoriales, al tiempo que renuevan la naturaleza de los vínculos urbano-rurales (avance de la pluriactividad, emergencia de nuevos actores y usos del suelo en los espacios rurales, urbanización difusa). Dentro de esta perspectiva, los asentamientos de rango menor en región pampeana, constituyen ámbitos privilegiados para la exploración de las modalidades que asumen las articulaciones urbano-rurales, en tanto lugares alcanzados por el proceso de homogeneización productiva, mostrando cómo la emergencia de nuevos actores, lógicas y escalas de acción, interpela las aproximaciones tradicionales de análisis de los vínculos urbano-rurales y exige la necesidad de renovar enfoques, regulaciones e instrumentos solidarios con la gestión territorial sustentable

Planimetric ice-flow convergence and flow-orthonormal strain rate across the Antarctic Ice Sheet, with links to model result files

Fast-flowing ice streams discharge most of the ice from the interior of the Antarctic Ice Sheet coastward. Understanding how their tributary organisation is governed and evolves is essential for developing reliable models of the ice sheet's response to climate change. Despite much research on ice-stream mechanics, this problem is unsolved, because the complexity of flow within and across the tributary networks has hardly been interrogated. Here I present the first map of planimetric flow convergence across the ice sheet, calculated from satellite measurements of ice surface velocity, and use it to explore this complexity. The convergence map of Antarctica elucidates how ice-stream tributaries draw ice from the interior. It also reveals curvilinear zones of convergence along lateral shear margins of streaming, and abundant convergence ripples associated with nonlinear ice rheology and changes in bed topography and friction. Flow convergence on ice-stream tributaries and their feeding zones is markedly uneven, and interspersed with divergence at distances of the order of kilometres. For individual drainage basins as well as the ice sheet as a whole, the range of convergence and divergence decreases systematically with flow speed, implying that fast flow cannot converge or diverge as much as slow flow. I therefore deduce that flow in ice-stream networks is subject to mechanical regulation that limits flow-orthonormal strain rates. These properties and the gridded data of convergence and flow-orthonormal strain rate in this archive provide targets for ice- sheet simulations and motivate more research into the origin and dynamics of tributarization.