The rockefeller university

The prevalence of asthma continues to increase in westernized countries, and optimal treatment remains a significant therapeutic challenge. Recently, CD1d-restricted invariant NKT (iNKT) cells were found to play a critical role in the induction of airway hyperreactivity (AHR) in animal models and are associated with asthma in humans. To test whether iNKT cell-targeted therapy could be used to treat allergen-induced airway disease, mice were sensitized with OVA and treated with di-palmitoyl-phosphatidyl-ethanolamine polyethylene glycol (DPPE-PEG), a CD1d-binding lipid antagonist. A single dose of DPPE-PEG prevented the development of AHR and pulmonary infiltration of lymphocytes upon OVA challenge, but had no effect on the development of OVA-specific Th2 responses. In addition, DPPE-PEG completely prevented the development of AHR after administration of alpha-galactosylceramide (alpha-GalCer) intranasally. Furthermore, we demonstrate that DPPE-PEG acts as antagonist to alpha-GalCer and competes with alpha-GalCer for binding to CD1d. Finally, we show that DPPE-PEG completely inhibits the alpha-GalCer-induced phosphorylation of ERK tyrosine kinase in iNKT cells, suggesting that DPPE-PEG specifically blocks TCR signaling and thus activation of iNKT cells. Because iNKT cells play a critical role in the development of AHR, the inhibition of iNKT activation by DPPE-PEG suggests a novel approach to treat iNKT cell-mediated diseases such as asthma. The Journal of Immunology, 2010,184: 2107-2115.

The zebrafish provides a useful experimental system for investigations of aural development. To permit the controlled expression of transgenes in developing hair cells, we isolated the genomic control regions of the parvalbumin 3a (pvalb3a) and parvalbumin 3b (pvalb3b) genes. Deletion analysis and somatic-cell transgenesis restricted the cis-acting control regions for hair cells to as little as 484 base pairs for pvalb3a and 650 base pairs for pvalb3b. Using both meganuclease-mediated and standard methods, we produced transgenic animals that transmit transgenes through their germ lines. These fish express GFP in hair cells in the inner ear and lateral line. Two stable transgenic lines express GFP prior to hair-bundle formation, so the associated promoter constructs are suitable for manipulating gene expression during bundle development. We additionally identified a transgenic line that offers variable labeling of supporting cells. (c) 2010 Elsevier B.V. All rights reserved.

Hepatitis C virus (HCV), which infects 2-3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation(1). The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.

BENCHMRK-1 and -2 are ongoing double-blind phase III studies of raltegravir in patients experiencing failure of antiretroviral therapy with triple-class drug-resistant human immunodeficiency virus infection. At week 96 (combined data), raltegravir (400 mg twice daily) plus optimized background therapy was generally well tolerated, with superior and durable antiretroviral and immunological efficacy, compared with optimized background therapy alone.

The experimental question is whether hypothalamic opioids, known to stimulate consummatory behavior, control a link to the nucleus accumbens (NAc). It was hypothesized that opioids injected in the hypothalamic paraventricular nucleus (PVN) alter the balance of dopamine (DA) and acetylcholine (ACh) in the NAc in a manner that fosters appetite for food or ethanol. Rats were implanted with two guide shafts, one in the NAc to measure extracellular DA and ACh by microdialysis and the other in the PVN for microinjection of opioid mu- and 8-agonists, an antagonist, or saline vehicle. The compounds tested were morphine, the mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin (DAMGO), the delta-receptor agonist D-Ala-Gly-Phe-Met-NH2 (DALA), and the opioid antagonist naloxone methiodide (m-naloxone). Morphine in the PVN increased the release of accumbens DA (+41%) and decreased ACh (-35%). Consistent with this, the opioid antagonist m-naloxone decreased DA (-24%) and increased ACh (+19%). In terms of receptor involvement, DAMGO dose-dependently increased DA to up to 209% of baseline. Simultaneously, ACh levels were markedly decreased to 55% of baseline. The agonist DALA produced a smaller but significant, 34% increase in DA, without affecting ACh. In contrast, control injections of saline had no significant effect. These results demonstrate that mu- and delta-opioids in the PVN contribute to the control of accumbens DA and ACh release and suggest that this circuit from the PVN to the NAc may be one of the mechanisms underlying opiate-induced ingestive behavior as well as naltrexone therapy for overeating and alcoholism. (C) 2009 Elsevier B.V. All rights reserved.

Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130 million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micropatterned cocultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence- and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics.

Chemokines are small secreted proteins that act as chemoattractants, and their role as neuromodulators in the brain has recently been appreciated. CXCL12 is one of the few chemokines found in neurons and expressed constitutively in the central nervous system. Previous data from our laboratory demonstrate the ability of CXCL12 to modulate the behavioral effects of cocaine, and this modulation is dependent on the central site of administration of CXCL12. The present study used single-staining immunohistochemical and dual-staining immunofluorescent methods to deter-mine the localization of the CXCL12 receptor, CXCR4, in the caudate putamen and nucleus accumbens of the adult rat brain. Results demonstrated that individual neurons in both the caudate putamen and lateral shell of the nucleus accumbens express both CXCR4 and D1 dopamine receptors. Immunofluorescent studies showed that CXCR4 was co-expressed with ChAT, a marker for cholinergic neurons, and with GAD C38, a marker for GABAergic neurons, in the caudate putamen and lateral shell of the accumbens. No evidence of CXCR4 was found in the medial shell or core regions of the nucleus accumbens. These data demonstrate that CXCR4 is expressed by subpopulations of cholinergic and GABAergic neurons in the striatum and suggest that CXCR4 is well-positioned to modulate striatal function. (C) 2009 Elsevier B.V. All rights reserved.

Skin, the biggest organ in mammals, protects the body from environmental hazards and prevents dehydration. Embryonic skin morphogenesis and homeostasis of adult skin require an accurately controlled gene expression in a spatiotemporally specific manner. Recently, the identification of microRNAs (miRNAs) in skin has added a new dimension in the regulatory network and attracted significant interest in this novel layer of gene regulation. Mammalian skin with its easy accessibility, well-defined lineages and established genetic tools offers an ideal system to unravel the functions of miRNAs in mammalian development and stem cells. In the past few years, significant progress has been made in determining the expression patterns of miRNAs, exploring their functions in skin morphogenesis and differentiation, as well as probing their functions in human skin diseases, for example, skin cancer. In this review, we summarized current progress in the study of miRNA in mammalian skin, provided insights gained from recent studies and offered our views for remaining challenges. Cell Death and Differentiation (2010) 17, 229-235; doi:10.1038/cdd.2009.92; published online 17 July 2009

Introduction. We examined two genetic markers established early in colorectal tumor development, microsatellite instability (MSI) and mutation of the KRAS proto-oncogene, to see if these genetic changes influence metastatic disease progression and survival. Patients and methods. MSI and KRAS mutation status were assessed in 532 primary adenocarcinomas (stage I-IV) from patients treated by colon resection. Median follow-up was 4.1 years (range 0-13.3 years) overall, 5.4 years for survivors. Results. MSI and KRAS mutation were detected in 12 and 36% of cases, respectively. MSI was more common in early-stage disease (I, 15%; II, 21%; III, 10%; IV, 2%; P = 0.0001). Prevalence of KRAS mutation did not vary with stage (I, 36%; II, 34%; III, 35%; IV, 40%; P = ns). Disease-specific survival was far superior for MSI tumors than for microsatellite stability (MSS) tumors (5-year survival 92 vs. 59%, P < 0.0001). KRAS mutation was a marker of poor survival (5-year survival 55 vs. 68%, P = 0.0002). Using Cox regression analysis MSI, KRAS mutation, and stage were strong independent predictors of survival in the entire patient population. A high-mortality group with MSS/KRAS-mutant tumors was identified within the stage I and II cohort. Conclusions. MSI and KRAS mutation provide fundamental genetic signatures influencing tumor behavior across patient subsets and stages of tumor development.

Measurements of inclusive charged-hadron transverse-momentum and pseudorapidity distributions are presented for proton-proton collisions at root s = 0.9 and 2.36 TeV. The data were collected with the CMS detector during the LHC commissioning in December 2009. For non-single-diffractive interactions, the average charged-hadron transverse momentum is measured to be 0.46 +/- 0.01 (stat.) +/- 0.01 (syst.) GeV/c at 0.9 TeV and 0.50 +/- 0.01 (stat.) +/- 0.01 (syst.) GeV/c at 2.36 TeV, for pseudorapidities between -2.4 and +2.4. At these energies, the measured pseudorapidity densities in the central region, dN(ch)/d eta vertical bar(vertical bar eta vertical bar<0.5), are 3.48 +/- 0.02 (stat.) +/- 0.13 (syst.) and 4.47 +/- 0.04 (stat.) +/- 0.16 (syst.), respectively. The results at 0.9 TeV are in agreement with previous measurements and confirm the expectation of near equal hadron production in p<(p)over bar> and pp collisions. The results at 2.36 TeV represent the highest-energy measurements at a particle collider to date.