Genetic Engineering & Biotechnology News

MAY1 2015

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14 | MAY 1, 2015 | GENengnews.com | Genetic Engineering & Biotechnology News Cell Signaling Continued from page 13 demonstrate that MHC class I-defcient tu- mor cells can escape immune surveillance by functionally inactivating NK cells. They further suggest that specifc cytokine thera- pies may be highly benefcial as a means of enhancing NK cell functions in cancer pa- tients, but mainly in patients where NK cells have been inactivated by tumor cells. Super-Resolution Marvels In 1873, the German physicist Ernst Abbe determined that the resolution of op- tical imaging instruments, such as micro- scopes and telescopes, was fundamentally limited by the diffraction of light. New ad- vances in microscopy have overcome this previously insurmountable barrier, and it is now possible to peer into the inner life of cells and their organelles. One of these advances garnered a Nobel Prize in 2014. The award went to three sci- entists (Eric Betzig, Stefan Hell, and William Moerner) who developed super-resolution microscopy. This technique exploits fuores- cent molecules to reveal the nanoscale fea- tures of cell membranes, intracellular organ- elles, and protein superstructures. Super-resolution imaging is already being used to pursue subjects of immunological interest, such as NK cell activation and the interaction of these white blood cells with tumor cells. Investigators visualizing these mechanisms include Daniel M. Davis, Ph.D., a professor of immunology at the University of Manchester. (Dr. Davis is perhaps more widely known as the author of The Compat- ibility Gene, a popular science book.) "There are many reasons for utilizing su- per-resolution microscopy, but the basic one is just that you can watch cells with unprece- dented resolution while they integrate signals and decide whether or not to respond," says Dr. Davis. "For example, by observing the interaction of an NK cell with proteins that are normally found only on tumor cells, one can see how the NK cell responds, and even observe unexpected phenomena, without a preformed hypothesis." In one study, Dr. Davis and colleagues wanted to determine how immune cells expressing germline receptors for viral pro- tein are able to differentiate between virus- infected cells and viral particles. A different immune response must be directed against virally infected cells rather than the par- ticles. NK cells secrete lytic granules that kill virus-infected or transformed cells, and they secrete cytokines to communicate with other cells. Integration of signals between activat- ing and inhibitory receptors on NK cells are driven by reactions to such target cells. This occurs across a structured interface called the immune synapse, which Dr. Davis co- discovered in the late 1990s. "We used super-resolution microscopy to follow the movements of F-actin, lytic gran- ules, and interferon-γ in primary human NK cells under different conditions," states Dr. Davis. "We found that NK cells can recog- nize infuenza virus particles but that the particles were not suffcient stimulus to open up the actin mesh. This happened only if the integrin LFA-1 was co-ligated. "These results suggest that integrin rec- ognition can help NK cells differentiate be- tween free pathogens and pathogen-infected cells. But the opening up of the actin mesh- work could be detected only with super-res- olution microscopy." Dr. Davis' studies demonstrate how cracking the limits of diffraction can open a whole new world of visualizing cellular events—even those that occur in confnes smaller than 100 nanometers. "The human body is one of the greatest wonders of the universe," observes Dr. Da- vis. "Its complexity, delicacy, and elegance are particularly illustrated in how our im- mune system works." Innate Sensing Pathways For more than a century, the immune stimulatory effects of DNA have been recog- nized. "Detection of foreign DNA, such as from an invading microbe, is a fundamental mechanism of host defense," notes Zhijian "James" Chen, Ph.D., a professor of molecu- lar biology at the University of Texas South- western Medical Center, and an investigator of the Howard Hughes Medical Institute. "We are studying the mechanisms under- lying detection of these danger signals and how they trigger the host innate immune response such as expression of type I inter- ferons." Dr. Chen and colleagues recently identi- fed cyclic guanosine monophosphate-ade- nosine monophosphate (cGAMP) synthase (cGAS) as a sensor of cytosolic DNA that triggers the production of infammatory cy- tokines and type I interferons. "cGAS binds generically to any DNA, and it catalyzes the synthesis of a specifc cGAMP isomer that functions as a second messenger for the binding and activation of the adapter protein STING," asserts Dr. Chen. "Once the pathway is engaged, STING activates two protein kinases (IκB kinase and TANK- binding kinase 1) that lead to induction of interferons and cytokines." To better understand the function of cGAS in vivo, Dr. Chen's team generated a cGas knockout mouse strain. They found that cells from cGAS-defcient mice, includ- ing fbroblasts, macrophages, and dendritic cells, could not produce type I interferons and other cytokines following DNA virus infection or DNA transfection. The knockout mice could not defend against infection by herpes simplex virus 1 as compared to wild-type mice. "Thus, cGAS is a general DNA sensor that activates the STING pathway," says Dr. Chen. Another fnding by Dr. Chen's group re- lated to cGAS-activated synthesis of a specifc cGAMP isomer, termed 2′3′cGAMP, the spe- cifc messenger that binds to STING. They de- termined that cGAMP is an effective adjuvant that can boost production of antigen-specifc antibodies and T-cell responses in mice. Dr. Chen states that although bacterial second messengers cyclic di-GMP and cyclic di-AMP are being studied for their potential use as vaccine adjuvants, 2′3′cGAMP is a much more potent ligand of STING: "It is possible that 2′3′cGAMP could be developed as an adjuvant for next-generation vaccines to treat infections and cancer." Infammasomes as Microbial Sensors The innate immune system provides the frst line of defense against invading patho- gens. Host pattern-recognition receptors (PRRs) recognize unique microbial signa- tures and engage signal transduction path- ways to combat infection. One of the major activation pathways is the infammasome pathway. The infamma- some, a multicomponent protein complex, activates the caspase-1 cascade, which leads to maturation of the proinfammatory cy- tokines interleukin 1β (IL-1β) and IL-18. It also induces pyroptosis, a form of caspase- dependent programmed cell death in which an infected immune cell produces cytokines, swells, and bursts, attracting still more cells to repeat the infammatory cycle. Gabriel Núñez, M.D., a professor of pa- thology at the University of Michigan Medi- cal School, is interested in understanding the mechanisms of PRRs including Nod-like re- ceptors (NLRs). "Of the four identifed infammasomes, three (NLRP1, NLRP3, and NLRP4) are activated by members of the NLR family," details Dr. Núñez. "The NLRs include an amino-terminal caspase-recruitment domain (CARD), pyrin domain, acidic transactivat- ing domain, a central nucleotide-binding-and- oligomerization domain (NOD) that mediates self-oligomerization, and a carboxy-terminal leucine-rich repeat believed to sense different microbes and endogenous damage." After pathogens infect a cell, NLR pro- teins sense the presence of released microbial ligands in the cytoplasm. For example, the DRUG DISCOVERY Activation and assembly of the infammasomes via Nod-like receptors (NLRs) in response to microbial and endogenous stimuli. This image, provided by Naohiro Inohara, Ph.D., of the University of Michigan, shows how oligomerization of NLRs induces the proximity and activation of caspase-1. ˝ 2015 Catalent Pharma Solutions. All rights reserved. *Titers may vary for specific antibodies. rapid development of high- performance cell lines biologics Proven GPEx ‰ gene expression platform: • 5 commercial products • Demonstrated titers to over 7 g/L* • Unmatched cell line stability Fully integrated drug substance and drug product manufacturing. Industry-leading global provider of advanced delivery technologies and development solutions. Discover more. + 1 888 SOLUTION solutions @ catalent.com catalent.com/biologics

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