Micro-pharmacokinetics: quantifying local drug concentration at live cell membranes

Fundamental equations for determining pharmacological parameters, such as the binding afnity of a ligand for its target receptor, assume a homogeneous distribution of ligand, with concentrations in the immediate vicinity of the receptor being the same as those in the bulk aqueous phase. It is, however, known that drugs are able to interact directly with the plasma membrane, potentially increasing local ligand concentrations around the receptor. We have previously reported an infuence of ligand-phospholipid interactions on ligand binding kinetics at the β2-adrenoceptor, which resulted in distinct “micro-pharmacokinetic” ligand profles. Here, we directly quantifed the local concentration of BODIPY630/650-PEG8-S-propranolol (BY-propranolol), a fuorescent derivative of the classical β-blocker propranolol, at various distances above membranes of single living cells using fuorescence correlation spectroscopy. We show for the frst time a signifcantly increased ligand concentration immediatel adjacent to the cell membrane compared to the bulk aqueous phase. We further show a clear role of both the cell membrane and the β2-adrenoceptor in determining high local BY-propranolol concentrations at the cell surface. These data suggest that the true binding afnity of BY-propranolol for the β2-adrenoceptor is likely far lower than previously reported and highlights the critical importance of understanding the “micro-pharmacokinetic” profles of ligands for membrane-associated proteins

New insights into the distribution and conservation status of the Golden-White Tassel-Ear Marmoset Mico chrysoleucos (Primates, Callitrichidae)

Among the 13 Mico species recognized by the IUCN Red List of Threatened Species, six are listed as "Data Deficient". The geographic range of most of the Mico species has been estimated from only a few records. We report new localities and the geographic extension of Mico chrysoleucos. In addition, we confirmed the presence of the species in two distinct protected areas. We modeled the habitat suitability of M. chrysoleucos using the maximum entropy method and including new records obtained by the authors in the state of Amazonas, Brazil. From the total area of occurrence calculated for the species, 22.8% is covered by protected areas and indigenous lands. The annual mean deforestation rate estimated between 2000 and 2015 was 2.95%, and the total area deforested by 2015 was 3354 km2 or 8.6% of the total distribution limits of the species. The habitat lost between 2000 and 2015 was 3.2% (1131 km2 ) of the total potential distribution, while the habitat loss area legally protected was 31 km2, and the habitat loss in settlements was equal to 691 km2. Our results extend the geographic distribution of the species about 100 km farther south, with the Maracanã River being a possible geographic barrier for the species. The significantly low rate of habitat loss inside protected areas and indigenous land, when compared to unprotected areas, points out the importance of these areas to M. chrysoleucos conservation. The species is relatively wide-ranging, legally protected, and resilient to regional anthropic threats. However, the hydroelectric schemes and the improvement of the road system in southern Amazonia pose an imminent threat to the species

Irradiation-Induced Up-Regulation of HLA-E on Macrovascular Endothelial Cells Confers Protection against Killing by Activated Natural Killer Cells

BACKGROUND: Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), endoglin (CD105) and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs) differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK) cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary macrovascular human umbilical vein endothelial cells (HUVECs) only express UL16 binding protein 2 (ULBP2) and the major histocompatibility complex (MHC) class I chain-related protein MIC-A (MIC-A) as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70), intercellular adhesion molecule ICAM-1 (CD54) and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD) plus IL-2 (TKD/IL-2)-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54) and HLA-E expression on the former which drops to the initial low levels (below 5%) when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected. CONCLUSION/SIGNIFICANCE: These data emphasize that an irradiation-induced, transient up-regulation of HLA-E on macrovascular ECs might confer protection against NK cell-mediated vascular injury

Sequential morphological characteristics of murine fetal liver hematopoietic microenvironment in Swiss Webster mice

Embryonic hematopoiesis occurs via dynamic development with cells migrating into various organs. Fetal liver is the main hematopoietic organ responsible for hematopoietic cell expansion during embryologic development. We describe the morphological sequential characteristics of murine fetal liver niches that favor the settlement and migration of hematopoietic cells from 12 days post-coitum (dpc) to 0 day post-partum. Liver sections were stained with hematoxylin and eosin, Lennert’s Giemsa, Sirius Red pH 10.2, Gomori’s Reticulin, and Periodic Acid Schiff/Alcian Blue pH 1.0 and pH 2.5 and were analyzed by bright-field microscopy. Indirect imunohistochemistry for fibronectin, matrix metalloproteinase-1 (MMP-1), and MMP-9 and histochemistry for naphthol AS-D chloroacetate esterase (NCAE) were analyzed by confocal microscopy. The results showed that fibronectin was related to the promotion of hepatocyte and trabecular differentiation; reticular fibers did not appear to participate in fetal hematopoiesis but contributed to the physical support of the liver after 18 dpc. During the immature phase, hepatocytes acted as the fundamental stroma for the erythroid lineage. The appearance of myeloid cells in the liver was related to perivascular and subcapsular collagen, and NCAE preceded MMP-1 expression in neutrophils, an occurrence that appeared to contribute to their liver evasion. Thus, the murine fetal liver during ontogenesis shows two different phases: one immature and mainly endodermic (<14 dpc) and the other more developed (endodermic-mesenchymal; >15 dpc) with the maturation of hepatocytes, a better definition of trabecular pattern, and an increase in the connective tissue in the capsule, portal spaces, and liver parenchyma. The decrease of hepatic hematopoiesis (migration) coincides with hepatic maturation

Overexpression of a Minimal Domain of Calpastatin Suppresses IL-6 Production and Th17 Development via Reduced NF-κB and Increased STAT5 Signals

Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA

Doxorubicin and paclitaxel enhance the antitumor efficacy of vaccines directed against HER 2/neu in a murine mammary carcinoma model

INTRODUCTION: The purpose of the present study was to determine whether cytotoxic chemotherapeutic agents administered prior to immunotherapy with gene vaccines could augment the efficacy of the vaccines. METHODS: Mice were injected in the mammary fat pad with an aggressive breast tumor cell line that expresses HER2/neu. The mice were treated 3 days later with a noncurative dose of either doxorubicin or paclitaxel, and the following day with a gene vaccine to HER2/neu. Two more doses of vaccine were given 14 days apart. Two types of gene vaccines were tested: a plasmid vaccine encoding a self-replicating RNA (replicon) of Sindbis virus (SINCP), in which the viral structural proteins were replaced by the gene for neu; and a viral replicon particle derived from an attenuated strain of Venezuelan equine encephalitis virus, containing a replicon RNA in which the Venezuelan equine encephalitis virus structural proteins were replaced by the gene for neu. RESULTS: Neither vaccination alone nor chemotherapy alone significantly reduced the growth of the mammary carcinoma. In contrast, chemotherapy followed by vaccination reduced tumor growth by a small, but significant amount. Antigen-specific CD8(+ )T lymphocytes were induced by the combined treatment, indicating that the control of tumor growth was most probably due to an immunological mechanism. The results demonstrated that doxorubicin and paclitaxel, commonly used chemotherapeutic agents for the treatment of breast cancer, when used at immunomodulating doses augmented the antitumor efficacy of gene vaccines directed against HER2/neu. CONCLUSIONS: The combination of chemotherapeutic agents plus vaccine immunotherapy may induce a tumor-specific immune response that could be beneficial for the adjuvant treatment of patients with minimal residual disease. The regimen warrants further evaluation in a clinical setting

Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Search for the direct production of charginos and neutralinos in final states with tau leptons in √s=13 TeV collisions with the ATLAS detector

A search for the direct production of charginos and neutralinos in final states with at least two hadronically decaying tau leptons is presented. The analysis uses a dataset of pp collisions corresponding to an integrated luminosity of 36.1 fb−1, recorded with the ATLAS detector at the Large Hadron Collider at a centre-of-mass energy of 13TeV.Nosignificant deviation from the expected Standard Model background is observed. Limits are derived in scenarios of ˜χ+1 ˜χ−1 pair production and of ˜χ±1 ˜χ02 and ˜χ+1 ˜χ−1 production in simplified models where the neutralinos and charginos decay solely via intermediate left-handed staus and tau sneutrinos, and the mass of the ˜ τL state is set to be halfway between the masses of the ˜χ±1 and the ˜χ01. Chargino masses up to 630 GeV are excluded at 95% confidence level in the scenario of direct production of ˜χ+1 ˜χ−1 for a massless ˜χ01. Common ˜χ±1 and ˜χ02 masses up to 760 GeV are excluded in the case of production of ˜χ±1 ˜χ02 and ˜χ+1 ˜χ−1 assuming a massless ˜χ01. Exclusion limits for additional benchmark scenarios with large and small mass-splitting between the ˜χ±1 and the ˜χ01 are also studied by varying the ˜ τL mass between the masses of the ˜χ±1 and the ˜χ01

Measurement of the cross section for inclusive isolated-photon production in pp collisions at √s=13TeV using the ATLAS detector

Inclusive isolated-photon production in pp collisions at a centre-of-mass energy of 13TeVis studied with the ATLAS detector at the LHC using a data set with an integrated luminosity of 3.2fb−1. The cross section is measured as a function of the photon transverse energy above 125GeVin different regions of photon pseudorapidity. Next-to-leading-order perturbative QCD and Monte Carlo event-generator predictions are compared to the cross-section measurements and provide an adequate description of the data