Gene Therapy Laboratory

AUTORES: Daniel J Klionsky1745,1749*, Kotb Abdelmohsen840, Akihisa Abe1237, Md Joynal Abedin1762, Hagai Abeliovich425,
\nAbraham Acevedo Arozena789, Hiroaki Adachi1800, Christopher M Adams1669, Peter D Adams57, Khosrow Adeli1981,
\nPeter J Adhihetty1625, Sharon G Adler700, Galila Agam67, Rajesh Agarwal1587, Manish K Aghi1537, Maria Agnello1826,
\nPatrizia Agostinis664, Patricia V Aguilar1960, Julio Aguirre-Ghiso784,786, Edoardo M Airoldi89,422, Slimane Ait-Si-Ali1376,
\nTakahiko Akematsu2010, Emmanuel T Akporiaye1097, Mohamed Al-Rubeai1394, Guillermo M Albaiceta1294,
\nChris Albanese363, Diego Albani561, Matthew L Albert517, Jesus Aldudo128, Hana Alg€ul1164, Mehrdad Alirezaei1198,
\nIraide Alloza642,888, Alexandru Almasan206, Maylin Almonte-Beceril524, Emad S Alnemri1212, Covadonga Alonso544,
\nNihal Altan-Bonnet848, Dario C Altieri1205, Silvia Alvarez1497, Lydia Alvarez-Erviti1395, Sandro Alves107,
\nGiuseppina Amadoro860, Atsuo Amano930, Consuelo Amantini1554, Santiago Ambrosio1458, Ivano Amelio756,
\nAmal O Amer918, Mohamed Amessou2089, Angelika Amon726, Zhenyi An1538, Frank A Anania291, Stig U Andersen6,
\nUsha P Andley2079, Catherine K Andreadi1690, Nathalie Andrieu-Abadie502, Alberto Anel2027, David K Ann58,
\nShailendra Anoopkumar-Dukie388, Manuela Antonioli832,858, Hiroshi Aoki1791, Nadezda Apostolova2007,
\nSaveria Aquila1500, Katia Aquilano1876, Koichi Araki292, Eli Arama2098, Agustin Aranda456, Jun Araya591,
\nAlexandre Arcaro1472, Esperanza Arias26, Hirokazu Arimoto1225, Aileen R Ariosa1749, Jane L Armstrong1930,
\nThierry Arnould1773, Ivica Arsov2120, Katsuhiko Asanuma675, Valerie Askanas1924, Eric Asselin1867, Ryuichiro Atarashi794,
\nSally S Atherton369, Julie D Atkin713, Laura D Attardi1131, Patrick Auberger1787, Georg Auburger379, Laure Aurelian1727,
\nRiccardo Autelli1992, Laura Avagliano1029,1755, Maria Laura Avantaggiati364, Limor Avrahami1166, Suresh Awale1986,
\nNeelam Azad404, Tiziana Bachetti568, Jonathan M Backer28, Dong-Hun Bae1933, Jae-sung Bae677, Ok-Nam Bae409,
\nSoo Han Bae2117, Eric H Baehrecke1729, Seung-Hoon Baek17, Stephen Baghdiguian1368,
\nAgnieszka Bagniewska-Zadworna2, Hua Bai90, Jie Bai667, Xue-Yuan Bai1133, Yannick Bailly884,
\nKithiganahalli Narayanaswamy Balaji473, Walter Balduini2002, Andrea Ballabio316, Rena Balzan1711, Rajkumar Banerjee239,
\nG abor B anhegyi1052, Haijun Bao2109, Benoit Barbeau1363, Maria D Barrachina2007, Esther Barreiro467, Bonnie Bartel997,
\nAlberto Bartolom e222, Diane C Bassham550, Maria Teresa Bassi1046, Robert C Bast Jr1273, Alakananda Basu1798,
\nMaria Teresa Batista1578, Henri Batoko1336, Maurizio Battino970, Kyle Bauckman2085, Bradley L Baumgarner1909,
\nK Ulrich Bayer1594, Rupert Beale1553, Jean-Fran¸cois Beaulieu1360, George R. Beck Jr48,294, Christoph Becker336,
\nJ David Beckham1595, Pierre-Andr e B edard749, Patrick J Bednarski301, Thomas J Begley1135, Christian Behl1419,
\nChristian Behrends757, Georg MN Behrens406, Kevin E Behrns1627, Eloy Bejarano26, Amine Belaid490,
\nFrancesca Belleudi1041, Giovanni B enard497, Guy Berchem706, Daniele Bergamaschi983, Matteo Bergami1401,
\nBen Berkhout1441, Laura Berliocchi714, Am elie Bernard1749, Monique Bernard1354, Francesca Bernassola1880,
\nAnne Bertolotti791, Amanda S Bess272, S ebastien Besteiro1351, Saverio Bettuzzi1828, Savita Bhalla913,
\nShalmoli Bhattacharyya973, Sujit K Bhutia838, Caroline Biagosch1159, Michele Wolfe Bianchi520,1378,1381,
\nMartine Biard-Piechaczyk210, Viktor Billes298, Claudia Bincoletto1314, Baris Bingol350, Sara W Bird1128, Marc Bitoun1112,
\nIvana Bjedov1258, Craig Blackstone843, Lionel Blanc1183, Guillermo A Blanco1496, Heidi Kiil Blomhoff1812,
\nEmilio Boada-Romero1297, Stefan B€ockler1464, Marianne Boes1423, Kathleen Boesze-Battaglia1835, Lawrence H Boise286,287,
\nAlessandra Bolino2063, Andrea Boman693, Paolo Bonaldo1823, Matteo Bordi897, J€urgen Bosch608, Luis M Botana1308,
\nJoelle Botti1375, German Bou1405, Marina Bouch e1038, Marion Bouchecareilh1331, Marie-Jos ee Boucher1901,
\nMichael E Boulton481, Sebastien G Bouret1926, Patricia Boya133, Micha€el Boyer-Guittaut1345, Peter V Bozhkov1141,
\nNathan Brady374, Vania MM Braga469, Claudio Brancolini1997, Gerhard H Braus353, Jos e M Bravo-San Pedro299,393,508,1374,
\nLisa A Brennan322, Emery H Bresnick2022, Patrick Brest490, Dave Bridges1939, Marie-Agn es Bringer124, Marisa Brini1822,
\nGlauber C Brito1311, Bertha Brodin631, Paul S Brookes1872, Eric J Brown352, Karen Brown1690, Hal E Broxmeyer480,
\nAlain Bruhat486,1339, Patricia Chakur Brum1893, John H Brumell446, Nicola Brunetti-Pierri315,1171,
\nRobert J Bryson-Richardson781, Shilpa Buch1777, Alastair M Buchan1819, Hikmet Budak1022, Dmitry V Bulavin118,505,1789,
\nScott J Bultman1792, Geert Bultynck665, Vladimir Bumbasirevic1470, Yan Burelle1356, Robert E Burke216,217,
\nMargit Burmeister1750, Peter B€utikofer1473, Laura Caberlotto1987, Ken Cadwell896, Monika Cahova112, Dongsheng Cai24,
\nJingjing Cai2099, Qian Cai1018, Sara Calatayud2007, Nadine Camougrand1343, Michelangelo Campanella1700,
\nGrant R Campbell1525, Matthew Campbell1249, Silvia Campello556,1876, Robin Candau1769, Isabella Caniggia1983,
\nLavinia Cantoni560, Lizhi Cao116, Allan B Caplan1656, Michele Caraglia1051, Claudio Cardinali1043, Sandra Morais Cardoso1579, Jennifer S Carew208, Laura A Carleton874, Cathleen R Carlin101, Silvia Carloni2002,
\nSven R Carlsson1267, Didac Carmona-Gutierrez1643, Leticia AM Carneiro312, Oliana Carnevali971, Serena Carra1318,
\nAlice Carrier120, Bernadette Carroll900, Caty Casas1324, Josefina Casas1116, Giuliana Cassinelli324, Perrine Castets1462,
\nSusana Castro-Obregon214, Gabriella Cavallini1841, Isabella Ceccherini568, Francesco Cecconi253,555,1884,
\nArthur I Cederbaum459, Valent ın Ce~na199,1281, Simone Cenci1323,2064, Claudia Cerella444, Davide Cervia1996,
\nSilvia Cetrullo1478, Hassan Chaachouay2028, Han-Jung Chae187, Andrei S Chagin634, Chee-Yin Chai626,628,
\nGopal Chakrabarti1502, Georgios Chamilos1601, Edmond YW Chan1142, Matthew TV Chan181, Dhyan Chandra1003,
\nPallavi Chandra548, Chih-Peng Chang818, Raymond Chuen-Chung Chang1653, Ta Yuan Chang345, John C Chatham1434,
\nSaurabh Chatterjee1910, Santosh Chauhan527, Yongsheng Che62, Michael E Cheetham1263, Rajkumar Cheluvappa1783,
\nChun-Jung Chen1153, Gang Chen598,1676, Guang-Chao Chen9, Guoqiang Chen1078, Hongzhuan Chen1077, Jeff W Chen1514,
\nJian-Kang Chen370,371, Min Chen249, Mingzhou Chen2104, Peiwen Chen1823, Qi Chen1674, Quan Chen172,
\nShang-Der Chen138, Si Chen325, Steve S-L Chen10, Wei Chen2125, Wei-Jung Chen829, Wen Qiang Chen979, Wenli Chen1113,
\nXiangmei Chen1133, Yau-Hung Chen1157, Ye-Guang Chen1250, Yin Chen1447, Yingyu Chen953,955, Yongshun Chen2135,
\nYu-Jen Chen712, Yue-Qin Chen1145, Yujie Chen1208, Zhen Chen339, Zhong Chen2123, Alan Cheng1702,
\nChristopher HK Cheng184, Hua Cheng1728, Heesun Cheong814, Sara Cherry1836, Jason Chesney1703,
\nChun Hei Antonio Cheung817, Eric Chevet1359, Hsiang Cheng Chi140, Sung-Gil Chi656, Fulvio Chiacchiera308,
\nHui-Ling Chiang958, Roberto Chiarelli1826, Mario Chiariello235,567,577, Marcello Chieppa835, Lih-Shen Chin290,
\nMario Chiong1285, Gigi NC Chiu878, Dong-Hyung Cho676, Ssang-Goo Cho650, William C Cho982, Yong-Yeon Cho105,
\nYoung-Seok Cho1064, Augustine MK Choi2095, Eui-Ju Choi656, Eun-Kyoung Choi387,400,685, Jayoung Choi1563,
\nMary E Choi2093, Seung-Il Choi2116, Tsui-Fen Chou412, Salem Chouaib395, Divaker Choubey1574, Vinay Choubey1936,
\nKuan-Chih Chow822, Kamal Chowdhury730, Charleen T Chu1856, Tsung-Hsien Chuang827, Taehoon Chun657,
\nHyewon Chung652, Taijoon Chung978, Yuen-Li Chung1194, Yong-Joon Chwae18, Valentina Cianfanelli254,
\nRoberto Ciarcia1775, Iwona A Ciechomska886, Maria Rosa Ciriolo1876, Mara Cirone1042, Sofie Claerhout1694,
\nMichael J Clague1698, Joan Cl aria1457, Peter GH Clarke1687, Robert Clarke361, Emilio Clementi1045,1398, C edric Cleyrat1781,
\nMiriam Cnop1366, Eliana M Coccia574, Tiziana Cocco1459, Patrice Codogno1375, J€orn Coers271, Ezra EW Cohen1533,
\nDavid Colecchia235,567,577, Luisa Coletto25, N uria S Coll123, Emma Colucci-Guyon516, Sergio Comincini1829,
\nMaria Condello578, Katherine L Cook2073, Graham H Coombs1929, Cynthia D Cooper2076, J Mark Cooper1395,
\nIsabelle Coppens601, Maria Tiziana Corasaniti1387, Marco Corazzari485,1884, Ramon Corbalan1566,
\nElisabeth Corcelle-Termeau251, Mario D Cordero1899, Cristina Corral-Ramos1289, Olga Corti507,1109, Andrea Cossarizza1767,
\nPaola Costelli1993, Safia Costes1518, Susan L Cotman721, Ana Coto-Montes946, Sandra Cottet566,1688, Eduardo Couve1301,
\nLori R Covey1015, L Ashley Cowart762, Jeffery S Cox1536, Fraser P Coxon1427, Carolyn B Coyne1846, Mark S Cragg1919,
\nRolf J Craven1679, Tiziana Crepaldi1995, Jose L Crespo1300, Alfredo Criollo1285, Valeria Crippa558, Maria Teresa Cruz1576,
\nAna Maria Cuervo26, Jose M Cuezva1277, Taixing Cui1907, Pedro R Cutillas987, Mark J Czaja27, Maria F Czyzyk-Krzeska1572,
\nRuben K Dagda2068, Uta Dahmen1404, Chunsun Dai800, Wenjie Dai1187, Yun Dai2059, Kevin N Dalby1940,
\nLuisa Dalla Valle1822, Guillaume Dalmasso1340, Marcello D’Amelio557, Markus Damme188, Arlette Darfeuille-Michaud1340,
\nCatherine Dargemont950, Victor M Darley-Usmar1433, Srinivasan Dasarathy205, Biplab Dasgupta202, Srikanta Dash1254,
\nCrispin R Dass242, Hazel Marie Davey8, Lester M Davids1560, David D avila227, Roger J Davis1731, Ted M Dawson604,
\nValina L Dawson606, Paula Daza1898, Jackie de Belleroche470, Paul de Figueiredo1180,1182,
\nRegina Celia Bressan Queiroz de Figueiredo135, Jos e de la Fuente1023, Luisa De Martino1775,
\nAntonella De Matteis1171, Guido RY De Meyer1443, Angelo De Milito631, Mauro De Santi2002,

We describe Bioconductor infrastructure for representing and computing on annotated genomic ranges and integrating genomic data with the statistical computing features of R and its extensions. At the core of the infrastructure are three packages: IRanges, GenomicRanges, and GenomicFeatures. These packages provide scalable data structures for representing annotated ranges on the genome, with special support for transcript structures, read alignments and coverage vectors. Computational facilities include efficient algorithms for overlap and nearest neighbor detection, coverage calculation and other range operations. This infrastructure directly supports more than 80 other Bioconductor packages, including those for sequence analysis, differential expression analysis and visualization.

Severe combined immunodeficiency–X1 (SCID-X1) is an X-linked inherited disorder characterized by an early block in T and natural killer (NK) lymphocyte differentiation. This block is caused by mutations of the gene encoding the γc cytokine receptor subunit of interleukin-2, -4, -7, -9, and -15 receptors, which participates in the delivery of growth, survival, and differentiation signals to early lymphoid progenitors. After preclinical studies, a gene therapy trial for SCID-X1 was initiated, based on the use of complementary DNA containing a defective γc Moloney retrovirus–derived vector and ex vivo infection of CD34 + cells. After a 10-month follow-up period, γc transgene–expressing T and NK cells were detected in two patients. T, B, and NK cell counts and function, including antigen-specific responses, were comparable to those of age-matched controls. Thus, gene therapy was able to provide full correction of disease phenotype and, hence, clinical benefit.

Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34(-)/low, c-Kit(+), Sca-1(+)) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and alpha-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Similar gender disparity is seen in mice given a chemical carcinogen, diethylnitrosamine (DEN). DEN administration caused greater increases in serum interleukin-6 (IL-6) concentration in males than it did in females. Furthermore, ablation of IL-6 abolished the gender differences in hepatocarcinogenesis in mice. DEN exposure promoted production of IL-6 in Kupffer cells (KCs) in a manner dependent on the Toll-like receptor adaptor protein MyD88, ablation of which also protected male mice from DEN-induced hepatocarcinogenesis. Estrogen inhibited secretion of IL-6 from KCs exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice. We propose that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females, and these findings may be used to prevent HCC in males.

Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis associated with tumors and other pathological conditions, including proliferative diabetic retinopathy and age-related macular degeneration. The murine anti-human VEGF monoclonal antibody (muMAb VEGF) A.4.6.1 has been shown to potently suppress angiogenesis and growth in a variety of human tumor cells lines transplanted in nude mice and also to inhibit neovascularization in a primate model of ischemic retinal disease. In this report, we describe the humanization of muMAb VEGF A.4.6.1. by site-directed mutagenesis of a human framework. Not only the residues involved in the six complementarity-determining regions but also several framework residues were changed from human to murine. Humanized anti-VEGF F(ab) and IgG1 variants bind VEGF with affinity very similar to that of the original murine antibody. Furthermore, recombinant humanized MAb VEGF inhibits VEGF-induced proliferation of endothelial cells in vitro and tumor growth in vivo with potency and efficacy very similar to those of muMAb VEGF A.4.6.1. Therefore, recombinant humanized MAb VEGF is suitable to test the hypothesis that inhibition of VEGF-induced angiogenesis is a valid strategy for the treatment of solid tumors and other disorders in humans.

Recently, efficient and long-term in vivo gene transfer by recombinant adeno-associated virus type 2 (rAAV) vectors has been demonstrated in a variety of tissues. Further improvement in vector titer and purity will expedite this in vivo exploration and provide preclinical information required for use in human gene therapy. In an effort to obtain higher titers, we constructed a novel AAV helper plasmid which utilizes translational control of AAV Rep genes (J. Li et al., J. Virol. 71:5236-5243, 1997). To address the issue of purity, in this study we report the first rAAV production method which is completely free of adenovirus (Ad) helper virus. The new production system uses a plasmid construct which contains a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes. This construct is missing some of the early and most of the late Ad genes and is incapable of producing infectious Ad. Transfection of 293 cells with the new mini-Ad helper and AAV packaging plasmids results in high-titer rAAV vectors with yields greater than 1,000 transducing units, or 10(5) viral particles per cell. When rAAV vectors were produced by using this production scheme and compared to traditional heat-inactivated rAAV preparations in vitro and in vivo, we observed transduction equivalent to or better than normal levels. The complete removal of infectious Ad from AAV production should facilitate a better understanding of immune response to AAV vectors in vivo, eliminate the need for developing replication-competent Ad assays, and provide a more defined reagent for clinical use.

Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-kappaB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.

Humoral hypercalcemia of malignancy is a common complication of lung and certain other cancers. The hypercalcemia results from the actions of tumor factors on bone and kidney. We report here the isolation of full-length complementary DNA clones of a putative hypercalcemia factor, and the expression from the cloned DNA of the active protein in mammalian cells. The clones encode a prepro peptide of 36 amino acids and a mature protein of 141 amino acids that has significant homology with parathyroid hormone in the amino-terminal region. This previously unrecognized hormone may be important in normal as well as abnormal calcium metabolism.

Members of the Toll-like receptor (TLR) family mediate dorsoventral patterning and cellular adhesion in insects as well as immune responses to microbial products in both insects and mammals. TLRs are characterized by extracellular leucine-rich repeat domains and an intracellular signaling domain that shares homology with cytoplasmic sequences of the mammalian IL-1 receptor and plant disease resistance genes. Ten human TLRs have been cloned as well as RP105, a protein similar to TLR4 but lacking the intracellular signaling domain. However, only five TLRs have described functions as receptors for bacterial products (e.g., LPS, lipoproteins). To identify potential sites of action, we used quantitative real-time RT-PCR to examine systematically the expression of mRNAs encoding all known human TLRs, RP105, and several other proteins important in TLR functions (e.g., MD-1, MD-2, CD14, MyD88). Most tissues tested expressed at least one TLR, and several expressed all (spleen, peripheral blood leukocytes). Analysis of TLR expression in fractionated primary human leukocytes (CD4(+), CD8(+), CD19(+), monocytes, and granulocytes) indicates that professional phagocytes express the greatest variety of TLR mRNAs although several TLRs appear more restricted to B cells, suggesting additional roles for TLRs in adaptive immunity. Monocyte-like THP-1 cells regulate TLR mRNA levels in response to a variety of stimuli including phorbol esters, LPS, bacterial lipoproteins, live bacteria, and cytokines. Furthermore, addition of Escherichia coli to human blood ex vivo caused distinct changes in TLR expression, suggesting that important roles exist for these receptors in the establishment and resolution of infections and inflammation.

OBJECTIVE: To develop a new evidence-based, pharmacologic treatment guideline for rheumatoid arthritis (RA). METHODS: We conducted systematic reviews to synthesize the evidence for the benefits and harms of various treatment options. We used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology to rate the quality of evidence. We employed a group consensus process to grade the strength of recommendations (either strong or conditional). A strong recommendation indicates that clinicians are certain that the benefits of an intervention far outweigh the harms (or vice versa). A conditional recommendation denotes uncertainty over the balance of benefits and harms and/or more significant variability in patient values and preferences. RESULTS: The guideline covers the use of traditional disease-modifying antirheumatic drugs (DMARDs), biologic agents, tofacitinib, and glucocorticoids in early (<6 months) and established (≥6 months) RA. In addition, it provides recommendations on using a treat-to-target approach, tapering and discontinuing medications, and the use of biologic agents and DMARDs in patients with hepatitis, congestive heart failure, malignancy, and serious infections. The guideline addresses the use of vaccines in patients starting/receiving DMARDs or biologic agents, screening for tuberculosis in patients starting/receiving biologic agents or tofacitinib, and laboratory monitoring for traditional DMARDs. The guideline includes 74 recommendations: 23% are strong and 77% are conditional. CONCLUSION: This RA guideline should serve as a tool for clinicians and patients (our two target audiences) for pharmacologic treatment decisions in commonly encountered clinical situations. These recommendations are not prescriptive, and the treatment decisions should be made by physicians and patients through a shared decision-making process taking into account patients' values, preferences, and comorbidities. These recommendations should not be used to limit or deny access to therapies.

GPR40 is a member of a subfamily of homologous G protein-coupled receptors that include GPR41 and GPR43 and that have no current function or ligand ascribed. Ligand fishing experiments in HEK293 cells expressing human GPR40 revealed that a range of saturated and unsaturated carboxylic acids with carbon chain lengths greater than six were able to induce an elevation of [Ca(2+)](i), measured using a fluorometric imaging plate reader. 5,8,11-Eicosatriynoic acid was the most potent fatty acid tested, with a pEC(50) of 5.7. G protein coupling of GPR40 was examined in Chinese hamster ovary cells expressing the G alpha(q/i)-responsive Gal4-Elk1 reporter system. Expression of human GPR40 led to a constitutive induction of luciferase activity, which was further increased by exposure of the cells to eicosatriynoic acid. Neither the constitutive nor ligand-mediated luciferase induction was inhibited by pertussis toxin treatment, suggesting that GPR40 was coupled to G alpha(q/11.) Expression analysis by quantitative reverse transcription-PCR showed that GPR40 was specifically expressed in brain and pancreas, with expression in rodent pancreas being localized to insulin-producing beta-cells. These data suggest that some of the physiological effects of fatty acids in pancreatic islets and brain may be mediated through a cell-surface receptor.

Exomes, transcriptomes and copy-number alterations in a sample of more than 70 primary human colonic tumours were analysed in an attempt to characterize the genomic landscape; in addition to finding alterations in genes associated with commonly mutated signalling pathways, recurrent gene fusions involving R-spondin family members were also found to occur in approximately 10% of colonic tumours, revealing a potential new therapeutic target. An analysis of exomes, transcriptomes and copy-number alterations in more than 70 primary human colon tumours and matched normal controls has identified more than 35,000 protein-altering somatic mutations, most of which have been validated. In addition to alterations in genes involved in the Wnt pathway, chromatin remodelling and receptor-tyrosine-kinase signalling, the authors identify recurrent gene fusions involving R-spondin family members that collectively occur in 10% of colon tumors; as such they may provide a potential therapeutic target. There is evidence to suggest that these fusions may have a role in the activation of Wnt signalling and tumorigenesis. Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics1. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.

Syndrome X, typified by obesity, insulin resistance (IR), dyslipidemia, and other metabolic abnormalities, is responsive to antidiabetic thiazolidinediones (TZDs). Peroxisome proliferator-activated receptor (PPAR) gamma, a target of TZDs, is expressed abundantly in adipocytes, suggesting an important role for this tissue in the etiology and treatment of IR. Targeted deletion of PPARgamma in adipose tissue resulted in marked adipocyte hypocellularity and hypertrophy, elevated levels of plasma free fatty acids and triglyceride, and decreased levels of plasma leptin and ACRP30. In addition, increased hepatic glucogenesis and IR were observed. Despite these defects, blood glucose, glucose and insulin tolerance, and insulin-stimulated muscle glucose uptake were all comparable to those of control mice. However, targeted mice were significantly more susceptible to high-fat diet-induced steatosis, hyperinsulinemia, and IR. Surprisingly, TZD treatment effectively reversed liver IR, whereas it failed to lower plasma free fatty acids. These results suggest that syndrome X may be comprised of separable PPARgamma-dependent components whose origins and therapeutic sites may reside in distinct tissues.

Inflammasomes are a set of intracellular protein complexes that enable autocatalytic activation of inflammatory caspases, which drive host and immune responses by releasing cytokines and alarmins into circulation and by inducing pyroptosis, a proinflammatory cell death mode. The inflammasome type mediating these responses varies with the microbial pathogen or stress factor that poses a threat to the organism. Since the discovery that polymorphisms in inflammasome genes are linked to common autoimmune diseases and less frequent periodic fever syndromes, inflammasome signaling has been dissected at the molecular level. In this review, we present recently gained insight on the distinct inflammasome types, their activation and effector mechanisms, and their modulation by microbial virulence factors. In addition, we discuss recently gained knowledge on the role of deregulated inflammasome activity in human autoinflammatory, autoimmune, and infectious diseases.

Manganous superoxide dismutase (MnSOD) scavenges potentially toxic superoxide radicals produced in the mitochondria. Tumor necrosis factor-alpha (TNF-alpha) was found to induce the messenger RNA for MnSOD, but not the mRNAs for other antioxidant or mitochondrial enzymes tested. The increase in MnSOD mRNA occurred rapidly and was blocked by actinomycin D, but not by cycloheximide. Induction of MnSOD mRNA was also observed with TNF-beta, interleukin-1 alpha (IL-1 alpha), and IL-1 beta but not with other cytokines or agents tested. TNF-alpha induced MnSOD mRNA in all cell lines and normal cells examined in vitro and in various organs of mice in vivo. These effects of TNF-alpha and IL-1 on target cells may contribute to their reported protective activity against radiation as well as their ability to induce resistance to cell killing induced by the combination of TNF-alpha and cycloheximide.

T cells are required for normal host defense against fungal infection, and individuals with T cell-deficiency syndromes are highly susceptible to fungal pathogens. Interleukin (IL)-17A is a proinflammatory cytokine that interconnects myeloid and lymphoid host defense. The role of murine (m) IL-17A/mIL-17A receptor (R) interactions was evaluated in a murine model of systemic candidiasis. In response to systemic challenge with Candida albicans, expression of mIL-17A was induced, and IL-17AR knockout (IL-17AR(-/-)) mice had dose-dependent, substantially reduced survival. Fungal burden in the kidneys of IL-17AR(-/-) mice was dramatically increased (25-fold at 96 h). In IL-17AR(-/-) mice, both mobilization of peripheral neutrophils and their influx to infected organs were significantly impaired and delayed. In vivo expression of mIL-17A protected normal mice from a lethal dose of C. albicans (100% at day 7 and 65% at day 42). The data suggest that the mIL-17A/mIL-17AR system is required for normal fungal host defense in vivo. IL-17A could have potential as a therapeutic cytokine for systemic C. albicans infections in immunocompromised patients with cancer or advanced acquired immunodeficiency syndrome.

Aberrant regulation of the Wnt signaling pathway is a prevalent theme in cancer biology. From the earliest observation that Wnt overexpression could lead to malignant transformation of mouse mammary tissue to the most recent genetic discoveries gleaned from tumor genome sequencing, the Wnt pathway continues to evolve as a central mechanism in cancer biology. This article summarizes the evidence supporting a role for Wnt signaling in human cancer. This includes a review of the genetic mutations affecting Wnt pathway components, as well as some of epigenetic mechanisms that alter expression of genes relevant to Wnt. I also highlight some research on the cooperativity of Wnt with other signaling pathways in cancer. Finally, some emphasis is placed on laboratory research that provides a proof of concept for the therapeutic inhibition of Wnt signaling in cancer.

IL-22 belongs to a family of cytokines structurally related to IL-10, including IL-19, IL-20, IL-24, and IL-26. In contrast to IL-10, IL-22 has proinflammatory activities. IL-22 signals through a class II cytokine receptor composed of an IL-22-binding chain, IL-22RA1, and the IL-10RB subunit, which is shared with the IL-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional IL-22R but no IL-10R. Accordingly, IL-22 but not IL-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that IL-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, IL-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that IL-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that IL-22 plays an important role in skin inflammatory processes and wound healing.

IGF-I is a pleiotropic hormone reported to affect linear growth, glucose metabolism, organ homeostasis, and the immune and neurologic systems. In contrast to IGF-II, IGF-I is expressed at low levels embryonically and has been thought to be more important for postnatal growth and development. To investigate the role of IGF-I in normal development we generated mice with an inactive IGF-I gene by homologous recombination in ES cells. Heterozygous mice are healthy and fertile, but they are 10-20% smaller than wild-type littermates and have lower than normal levels of IGF-I. The size reduction is attributable to a decrease in organs and muscle and bone mass. However, all tissues appear histologically normal. At birth homozygous mutant mice (IGF-I-/-) are < 60% body weight of wild type. Greater than 95% of IGF-I-/- pups die perinatally. Histopathology is characterized by underdevelopment of muscle tissue. Lungs of late embryonic and neonates also appeared less organized with ill-defined alveolae. IGF-I appears to be essential for correct embryonic development in mice.