Local anesthetics worsen renal function after ischemia-reperfusion injury in rats

. Local anesthetics worsen renal function after ischemia-reperfusion injury in rats. Am J Physiol Renal Physiol 286: F111-F119, 2004. First published September 30, 2003 10.1152 10. /ajprenal.00108.2003ics are widely used during the perioperative period, even in patients with preexisting renal disease. However, local anesthestics have been shown to cause cell death in multiple cell lines, including human kidney proximal tubule cells. We questioned whether local anesthetics potentiate renal dysfunction after ischemia-reperfusion (I/R) injury in vivo. Rats were implanted with subcutaneous miniosmotic pumps that continuously delivered lidocaine (2 mg⅐kg Ϫ1 ⅐h Ϫ1 ), bupivacaine (0.4 mg⅐kg Ϫ1 ⅐h Ϫ1 ), tetracaine (1 mg⅐kg Ϫ1 ⅐h Ϫ1 ), or saline vehicle, and 6 h later the rats were subjected to 30 min of renal ischemia or to sham operation. Renal function was assessed by measurement of plasma creatinine at 24 and 48 h after renal I/R injury in the presence or absence of chronic infusions of local anesthetics and correlated to histological changes indicative of necrosis. The degree of renal apoptosis was assessed by three methods: 1) DNA fragmentation detected by terminal deoxynucleotidyl transferase biotin-dUTP nickend labeling staining, 2) DNA laddering detected after agarose gel electrophoresis, and 3) morphological identification of apoptotic tubules at the corticomedullary junction. We also measured the expression of the proinflammatory markers ICAM-1 and TNF-␣. Continuous local anesthetic infusion with renal I/R injury resulted in an increased magnitude and duration of renal dysfunction compared with the saline-infused I/R group. Additionally, both apoptotic and necrotic renal cell death as well as inflammatory changes were significantly potentiated in local anesthetic-treated rat kidneys. Local anesthetic infusion alone without I/R injury had no effect on renal function. We conclude that local anesthetics potentiated renal injury after I/R by increasing necrosis, apoptosis, and inflammation. acute renal failure; apoptosis; bupivacaine; inflammation; lidocaine; necrosis; tetracaine ACUTE RENAL FAILURE (ARF) secondary to ischemia-reperfusion (I/R) injury continues to be a significant clinical problem Patients with impaired preoperative renal function undergoing aortovascular surgery are at greatest risk for developing perioperative ARF (26). Local anesthetics are widely used in clinical practice, even in patients with impaired preoperative renal function. Epidural infusions of local anesthetic are routinely used for intraoperative and postoperative analgesia (frequently lasting several days) in patients undergoing major abdominal and vascular procedures. During induction of general anesthesia for endotracheal intubation, intravenous lidocaine is given routinely to blunt the sympathetic reflex to direct laryngoscopy. Local anesthetics are used to provide surgical anesthesia and analgesia in peripheral and central nervous system nerve blocks (spinal and epidural anesthesia). In the intensive care unit, lidocaine is frequently used as an antiarrythmic agent. Several in vitro studies found that local anesthetics increase cell death via apoptosis in neuronal, lymphocytic, and osteoblastic cell lines MATERIALS AND METHODS Implantation of Miniosmotic Pumps and Renal I/R Injury All protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. Adult male Sprague-Dawley rats (225-275 g, Harlan Sprague-Dawley, Indianapolis, IN) were used. They had free access to rodent chow and water. Rats were anesthetized with intraperitoneal (ip) pentobarbital sodium (50 mg/kg or to effect) and implanted with subcutaneous miniosmotic pumps (model 2ML1, Alzet) that continuously delivered 10 l/h of 5% lidocaine (2 mg⅐kg Ϫ1 ⅐h Ϫ1 ), 1% bupivacaine (0.4 mg⅐kg Ϫ1 ⅐h Ϫ1 ), 2.5% tetracaine (1 mg⅐kg Ϫ1 ⅐h Ϫ1 ), or saline vehicle. The doses of local anesthetics delivered mimicked clinically administered doses for continuous epidural infusion for a 70-kg person during and after abdominal and vascular surgical procedures. Some rats were infused with 0.5% bupivacaine instead of 1% bupivacaine. Six hours later (the time required for osmotic pump priming), rats were reanesthetized with pentobarbital sodium. After 500 U of heparin were given ip, rats were placed on an electric heating pad under a warming light. Body temperature was monitored with a rectal probe and maintained at 37°C. They were allowed to spontaneously breath room air. After a laparotomy, rats were subjected to 30-min left renal ischemia after right nephrectomy. The duration of ischemia was shown in pilot studies to produce reversible and moderate renal dysfunction in rats. Some rats were subjected to only sham operation (anesthesia, laparotomy, and right nephrectomy) and received vehicle (saline) infusion, and others received a sham operation plus local anestheti

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Preclinical Development of Oncolytic Immunovirotherapy for Treatment of HPVPOS Cancers

Immunotherapy for HPVPOS malignancies is attractive because well-defined, viral, non-self tumor antigens exist as targets. Several approaches to vaccinate therapeutically against HPV E6 and E7 antigens have been adopted, including viral platforms such as VSV. A major advantage of VSV expressing these antigens is that VSV also acts as an oncolytic virus, leading to direct tumor cell killing and induction of effective anti-E6 and anti-E7 T cell responses. We have also shown that addition of immune adjuvant genes, such as IFNβ, further enhances safety and/or efficacy of VSV-based oncolytic immunovirotherapies. However, multiple designs of the viral vector are possible—with respect to levels of immunogen expression and method of virus attenuation—and optimal designs have not previously been tested head-to-head. Here, we tested three different VSV engineered to express a non-oncogenic HPV16 E7/6 fusion protein for their immunotherapeutic and oncolytic properties. We assessed their profiles of efficacy and toxicity against HPVPOS and HPVNEG murine tumor models and determined the optimal route of administration. Our data show that VSV is an excellent platform for the oncolytic immunovirotherapy of tumors expressing HPV target antigens, combining a balance of efficacy and safety suitable for evaluation in a first-in-human clinical trial. Keywords: VSV immunovirotherapy, HPV positive cancer, preclinica

Fibril diameters in the KO reticular dermis are smaller and more uniform than WT fibrils.

<p><b>A.</b> Representative micrographs of skin in deep dermal areas near subcutaneous fat. (Scale bar = 0.5 μm) <b>B.</b> Histogram of the frequency of collagen fibrils with a given diameter range from WT (white bars) and KO (black bars). At least 200 fibrils from 3 WT and 3 KO animals at 5 months of age were used for analysis. P<0.05.</p

Stiff surfaces and TGFβ induce MRTF-A nuclear accumulation.

<p>SSc and control dermal fibroblast lines (both N = 3) were cultured on 6 well plates with collagen type I coated soft substrates (5 kPa Softwell), or hard substrate (50 kPa). <b>B. Induction of collagen transcription on hard surfaces requires MRTF-A.</b> Mouse fibroblasts from wild type (WT) and loss-of-function (KO) mice with collagen promoter driving GFP were cultured on fibronectin coated soft and hard surfaces. Pictures taken 24 hours after plating. Fluorescence was quantified using ImageJ. The numbers of cells in each image was counted (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126015#pone.0126015.s004" target="_blank">S4 Fig</a>) to determine the luminance per cell. <b>C.&D.</b> Normal control fibroblasts and SSc fibroblasts were cultured without serum for 16 hours then treated with TGFβ (4 ng/ml) for 0, 8 and 24 hours or treated with either saline or TGFβ (4 ng/ml) with or without CCG1423 (10μM) or NSC2376 (50 μM). Proteins (20 μg) from cytoplasm and nuclei were extracted using NE-PER Nuclear Protein Extraction Kit and separated on 4–12% gradient gel and visualized using MRTF-A antibody.</p

Increased expression of MRTF-A in the SSc epidermis and at the epidermal dermal junction in established SSc correlates with increased intracellular procollagen, and SMA.

<p>The expression of MRTF-A, procollagen type I, and SMA was detected by immunohistochemical staining in the epidermis and papillary dermis of early and established SSc patients and controls healthy control patients. Arrows point to staining in vascular cells (V), epidermis (e), and fibroblast (F).</p

Nuclear MRTF-A expression is more prominent in SSc skin then healthy control.

<p><b>A.</b> Representative pictures of healthy control and SSc sections of biopsy. Histological samples of human skin were stained with MRTF-A antibody (1:2000). Higher magnification of epithelium and small vessels (20X) in papillary dermis. Brown arrows = MRTF-A nuclei, Blue arrow = hematoxylin stained nuclei without MRTF-A. Graphical representations of % nuclei in epidermis, vasculature, and interstitial cells in papillary dermis. Histological samples of 5 healthy control and 9 scleroderma human skin were evaluated for nuclear staining. B. Total cells with MRTF-A nuclear localization in the epidermis, vasculature, and interstitial papillary dermal layers were counted and compared with the total amount of nuclei in the epidermal/papillary dermal layer. White bars = healthy controls, Black bars = SSc (* = p<0.01 using nonparametric Mann-Whitney U, two-tailed).</p

Mouse KO skin and lung are less stiff than WT skin and lung.

<p>A 3x1x1 mm strip of dorsal skin or lung tissue was placed longitudinally into a computer-controlled dual-mode lever arm force transducer system and stretched intermittently. <b>A.</b> Young’s module plotted at each strain. The Young’s module is the slope of the stress-strain curve. <b>B.</b> Dynamic stretch storage modulus describes the ability of the material to store elastic energy during the loading phase of a cyclic stretch <b>C</b>. Dynamic stretch loss modulus—The amount of energy lost (usually as heat) during a cycle. Vertical brackets denote the overall group differences using 2-way repeated measure ANOVA (*: <i>p</i><0.05, **: <i>p</i><0.01 and ***: <i>p</i><0.001) whereas horizontal brackets show Tukey’s post hoc differences between WT and KO at the same strain (Panel A) or frequency (Panels B and C). For the Young’s and loss moduli of lung tissue, there are also significant interactions between strain and group (panel A) as well as frequency and group (panel C, p<0.05). For the skin, there is a significant interaction between frequency and group only for the loss modulus (p<0.05).</p

Enhanced type I collagen and CCN2 expression as well as collagen gel contraction in scleroderma fibroblasts is dependent on MRTF-A pathway.

<p><b>A. The MRTF-A inhibitor, CCG-1423, blocks collagen and CCN2 synthesis.</b> Healthy control fibroblasts (Control) and scleroderma fibroblasts (SSc) from 3 independent isolates were cultured with or without the MRTF-A inhibitor CCG-1423 (10 μM). Basal CCN2 and type I collagen was increased in scleroderma cells and inhibited by CCG-1423. <b>B. Knockdown of MRTF-A by siRNA blocks collagen and CCN2 synthesis in SSc fibroblasts.</b> Control and SSc fibroblasts were treated with MRTF-A siRNA, (siMRTFA), a non-target siRNA (siNT), or vehicle (Cont). <b>C. Loss or inhibition of MRTF-A blocks contraction of collagen floating gels.</b> Knockdown (siMRTFA) and inhibition (CCG-1423) of MRTF-A partially blocks collagen floating gel contraction by SSc fibroblasts. * = p<0.05.</p