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From the Walter and Eliza Hall Institute of Medical Research: Hobbit and Blimp1 May Be Needed for Local Tissue Immune Response

Reporter: Stephen J Williams, PhD

 

Researchers uncover ‘local heroes’ of immune system

 

22 April 2016
Axel Kallies in the laboratory
Dr Axel Kallies and his team have identified genes that
control a molecular program to protect the body. 
Melbourne researchers have uncovered genes responsible for the way the body fights infection at the point of ‘invasion’ – whether it’s the skin, liver, lungs or the gut.

Research led by Dr Axel Kalliesand Dr Klaas van Gisbergen at the Walter and Eliza Hall Institute of Medical Research, and Dr Laura Mackay from the University of Melbourne at the Peter Doherty Institute for Infection and Immunity has identified the genes Hobit andBlimp1 and found that they control a universal molecular program responsible for placing immune cells at the ‘front lines’ of the body to fight infection and cancer.

The presence of these organ-residing cells, which differ strikingly from their counterparts circulating in the blood stream, is key to local protection against viruses and bacteria.

Walter and Eliza Hall Institute’s Dr Kallies said the human body was fighting disease-causing pathogens every minute of its life. Dr Kallies said identifying how immune cells remain in the part of the body where they are needed most was critical to developing better ways to protect us from infections such as malaria orHIV.

“Discovering these ‘local heroes’ and knowing how the localised immune response is established allows us to find ways to ensure the required cells are positioned where they are needed most,” Dr Kallies said.

“This research will help us understand how immune cells adapt, survive and respond within the organs they protect. This is critical to rid the body of pathogens even before they are established and may also have implications for understanding how the spread of cancer could be prevented.”

The Doherty Institute’s Dr Laura Mackay, who is also an associate investigator with the Australian Research Council Centre of Excellence in Advanced Molecular Imaging, said the factors that control the ‘tissue-residency’ of immune cells – their ability to locally reside in different organs of the body – was previously unknown.

“These results have major implications for developing strategies to induce immune cells in tissues that protect against infectious diseases,” Dr Mackay said.

“It’s a crucial discovery for future vaccine strategies – Hobit and Blimp1 would be key to placing immune cells in the tissues, which we know are really important for protection.”

The findings have just been published in the journal Science.

This research was supported by the Victorian State Government Operational Infrastructure Support and the Australian Government National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme.

Further information:

Ebru Yaman
Media and Publications Manager
M: 0428 034 089
E: yaman.e@wehi.edu.au

Immune cell ‘survival’ gene key to better myeloma treatments

4 February 2013
Dr Victor Peperzak, Dr Ingela Vikstrom and Associate Professor David Tarlinton in a laboratory
Dr Victor Peperzak (left), Dr Ingela Vikstrom
(centre) and Associate Professor Tarlinton led a
research team that identified a gene that is
essential for survival of antibody-producing cells.
Scientists have identified the gene essential for survival of antibody-producing cells, a finding that could lead to better treatments for diseases where these cells are out of control, such as myeloma and chronic immune disorders.

The discovery that a gene calledMcl1 is critical for keeping this vital immune cell population alive was made by researchers at Melbourne’s Walter and Eliza Hall Institute. Associate Professor David Tarlinton, Dr Victor Peperzak and Dr Ingela Vikstrom from the institute’s Immunology division led the research, which was published today in Nature Immunology.

Antibody-producing cells, also known as plasma cells, live in the bone marrow and make antibodies that provide a person with long-term protection from viruses and bacteria, Associate Professor Tarlinton said. “Plasma cells are produced after vaccination or infection and are responsible for the immune ‘memory’ that can persist in humans for 70 or 80 years. In this study, we found that plasma cells critically rely on Mcl1 for their continued survival and, without it, they die within two days,” he said.

Dr Peperzak said the team was surprised to find that plasma cells used this as a ‘failsafe’ mechanism in controlling their survival. “One of the interesting things we found is that because plasma cells rapidly destroy Mcl-1 proteins within the cell yet depend on it for their survival, they need continuous external signals to tell them to produce more Mcl-1 protein,” Dr Peperzak said. “This keeps the plasma cells under tight control, with Mcl-1 acting like a timer that constantly counts down and, if not reset, instructs the cell to die.”

Plasma cells are vital to the immune response, but can be dangerous if not properly controlled, Associate Professor Tarlinton said. “As with any immune cell, plasma cells are really quite dangerous in many respects and need to be tightly controlled,” he said. “When they are out of control they continue to make antibodies that can be very damaging if there are too many. This happens in conditions such as myeloma – a cancer of plasma cells – and various forms of autoimmunity, such as systemic lupus erythamatosus or rheumatoid arthritis, where there are excessive levels of antibodies.”

Myeloma is a blood cancer that affects more than 1200 Australians each year, and is more common in people over 60. It is caused by the uncontrolled production of abnormal plasma cells in the bone marrow and the build up of damaging antibodies in the blood. Rheumatoid arthritis and lupus are autoimmune diseases in which the antibodies produced by plasma cells attack and destroy the body’s own tissues.

Associate Professor Tarlinton said that his hope was that the discovery could be used to develop new treatments for these conditions. “Myeloma in particular has a very poor prognosis, and is generally considered incurable,” Associate Professor Tarlinton said. “Now that we know Mcl1 is the one essential gene needed to keep plasma cells alive, we have begun ‘working backwards’ to identify all the critical molecules and signals needed to switch on Mcl1 and keep the cells alive. Our hope is that we will identify some point in the internal cell signalling pathway, or a critical external molecule, that could be blocked to stop Mcl-1 being produced by the cell. This would be an important new platform for diseases that currently have no specific or effective treatment, such as myeloma, or offer new treatment options for people who don’t respond well to existing treatments for diseases such as lupus or rheumatoid arthritis.”

This research was supported by the Australian National Health and Medical Research Council, Multiple Myeloma Research Foundation, European Molecular Biology Organization and the Victorian Government.

Read the article in Nature ImmunologyMcl-1 is essential for the survival of plasma cells.

Further information:

Liz Williams
Media and Publications Manager
P: +61 3 9345 2928
M: +61 405 279 095
E: williams@wehi.edu.au

Australian researchers find immune ‘kill switch’

as reported in http://www.abc.net.au/am/content/2012/s3649437.htm

Martin Cuddihy reported this story on Friday, December 7, 2012 08:12:00

TONY EASTLEY: For want of a better description we all apparently have an immune system ‘kill switch’.

Melbourne researchers have discovered why the body reacts the way it does when under stress from a severe infection.

They’ve found that the immune system switch destroys blood stem cells, and they’ve also discovered how to turn it off.

The discovery could mean a faster recovery rates from blood infections and from bouts of chemotherapy.

Martin Cuddihy reports.

MARTIN CUDDIHY: Worldwide, sepsis or blood poisoning is one of the leading causes of death in the intensive care units of hospitals.

When someone develops the condition the body goes into shock and blood stem cells start dying.

SETH MASTERS: You can think about it like suicide. The cells know that they should die to try and get rid of the infection but if the infection is overwhelming as it is with sepsis, then we need them to stay alive to help fight any infection.

MARTIN CUDDIHY: Dr Seth Masters from the Walter and Eliza Hall Institute in Melbourne is part of a research team that’s discovered the kill switch that tells cells when they should die.

Normally that’s a good thing, except when there’s a massive infection.

SETH MASTERS: You have to repopulate those immune cells somehow and these come from progenitor cells in the bone marrow and we think that this cell death pathway is something we can block to try and help the new cells regenerate to fight the infections better.

MARTIN CUDDIHY: So what does this cell receptor normally do?

SETH MASTERS: We are not entirely clear about that. We think that when a progenitor cell gets infected it’d be really bad if it stayed alive for too long cause it would pass that infection along to all of its daughters and sons.

So instead of staying infected, it just commits suicide and dies via this new pathway.

MARTIN CUDDIHY: Dr Masters is part of an international research team that’s found blocking a certain cell receptor stops blood cells from dying.

The researchers hope the discovery could lead to a treatment for sepsis and a way to help boost the immune system of cancer patients undergoing chemotherapy.

SETH MASTERS: I think that probably the most likely avenue where it could be of use is in trying to help recovery from chemotherapy. That’s a period during which we really need as many cells to mobilise out of the bone marrow into the periphery as possible to try and fight any potential infections that might be coming along.

And so we think that this cell death pathway might be stopping that from happening quickly and if we can inhibit it, we can make it go faster.

MARTIN CUDDIHY: Does that mean then that someone could be subjected to a more intense round of chemotherapy if this was to work and therefore you could boost their immune system following that round of chemo?

SETH MASTERS: Yeah, that does seem like a relatively attractive proposal. It is not something we have actually validated just yet but that would have to be on the cards if we can do some more research down those lines.

MARTIN CUDDIHY: The findings are published today in the medical journal Immunity.

TONY EASTLEY: Martin Cuddihy.

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Colon cancer and organoids

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

 

Guts and Glory

An open mind and collaborative spirit have taken Hans Clevers on a journey from medicine to developmental biology, gastroenterology, cancer, and stem cells.

By Anna Azvolinsky    http://www.the-scientist.com/?articles.view/articleNo/45580/title/Guts-and-Glory

Ihave had to talk a lot about my science recently and it’s made me think about how science works,” says Hans Clevers. “Scientists are trained to think science is driven by hypotheses, but for [my lab], hypothesis-driven research has never worked. Instead, it has been about trying to be as open-minded as possible—which is not natural for our brains,” adds the Utrecht University molecular genetics professor. “The human mind is such that it tries to prove it’s right, so pursuing a hypothesis can result in disaster. My advice to my own team and others is to not preformulate an answer to a scientific question, but just observe and never be afraid of the unknown. What has worked well for us is to keep an open mind and do the experiments. And find a collaborator if it is outside our niche.”

“One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory.”

Clevers entered medical school at Utrecht University in The Netherlands in 1978 while simultaneously pursuing a master’s degree in biology. Drawn to working with people in the clinic, Clevers had a training position in pediatrics lined up after medical school, but then mentors persuaded him to spend an additional year converting the master’s degree to a PhD in immunology. “At the end of that year, looking back, I got more satisfaction from the research than from seeing patients.” Clevers also had an aptitude for benchwork, publishing four papers from his PhD year. “They were all projects I had made up myself. The department didn’t do the kind of research I was doing,” he says. “Now that I look back, it’s surprising that an inexperienced PhD student could come up with a project and publish independently.”

Clevers studied T- and B-cell signaling; he set up assays to visualize calcium ion flux and demonstrated that the ions act as messengers to activate human B cells, signaling through antibodies on the cell surface. “As soon as the experiment worked, I got T cells from the lab next door and did the same experiment. That was my strategy: as soon as something worked, I would apply it elsewhere and didn’t stop just because I was a B-cell biologist and not a T-cell biologist. What I learned then, that I have continued to benefit from, is that a lot of scientists tend to adhere to a niche. They cling to these niches and are not that flexible. You think scientists are, but really most are not.”

Here, Clevers talks about promoting a collaborative spirit in research, the art of doing a pilot experiment, and growing miniature organs in a dish.

Clevers Creates

Re-search? Clevers was born in Eindhoven, in the south of The Netherlands. The town was headquarters to Philips Electronics, where his father worked as a businessman, and his mother took care of Clevers and his three brothers. Clevers did well in school but his passion was sports, especially tennis and field hockey, “a big thing in Holland.” Then in 1975, at age 18, he moved to Utrecht University, where he entered an intensive, biology-focused program. “I knew I wanted to be a biology researcher since I was young. In Dutch, the word for research is ‘onderzoek’ and I knew the English word ‘research’ and had wondered why there was the ‘re’ in the word, because I wanted to search but I didn’t want to do re-search—to find what someone else had already found.”

Opportunity to travel. “I was very disappointed in my biology studies, which were old-fashioned and descriptive,” says Clevers. He thought medicine might be more interesting and enrolled in medical school while still pursuing a master’s degree in biology at Utrecht. For the master’s, Clevers had to do three rotations. He spent a year at the International Laboratory for Research on Animal Diseases (ILRAD) in Nairobi, Kenya, and six months in Bethesda, Maryland, at the National Institutes of Health. “Holland is really small, so everyone travels.” Clevers saw those two rotations more as travel explorations. In Nairobi, he went on safaris and explored the country in Land Rovers borrowed from the institute. While in Maryland in 1980, Clevers—with the consent of his advisor, who thought it was a good idea for him to get a feel for the U.S.—flew to Portland, Oregon, and drove back to Boston with a musician friend along the Canadian border. He met the fiancé of political activist and academic Angela Davis in New York City and even stayed in their empty apartment there.

Life and lab lessons. Back in Holland, Clevers joined Rudolf Eugène Ballieux’s lab at Utrecht University to pursue his PhD, for which he studied immune cell signaling. “I didn’t learn much science from him, but I learned that you always have to create trust and to trust people around you. This became a major theme in my own lab. We don’t distrust journals or reviewers or collaborators. We trust everyone and we share. There will be people who take advantage, but there have only been a few of those. So I learned from Ballieux to give everyone maximum trust and then change this strategy only if they fail that trust. We collaborate easily because we give out everything and we also easily get reagents and tools that we may need. It’s been valuable to me in my career. And it is fun!”

Clevers Concentrates

On a mission. “Once I decided to become a scientist, I knew I needed to train seriously. Up to that point, I was totally self-trained.” From an extensive reading of the immunology literature, Clevers became interested in how T cells recognize antigens, and headed off to spend a postdoc studying the problem in Cox Terhorst’s lab at Dana-Farber Cancer Institute in Boston. “Immunology was young, but it was very exciting and there was a lot to discover. I became a professional scientist there and experienced how tough science is.” In 1988, Clevers cloned and characterized the gene for a component of the T-cell receptor (TCR) called CD3-epsilon, which binds antigen and activates intracellular signaling pathways.

On the fast track in Holland. Clevers returned to Utrecht University in 1989 as a professor of immunology. Within one month of setting up his lab, he had two graduate students and a technician, and the lab had cloned the first T cell–specific transcription factor, which they called TCF-1, in human T cells. When his former thesis advisor retired, Clevers was asked, at age 33, to become head of the immunology department. While the appointment was high-risk for him and for the department, Clevers says, he was chosen because he was good at multitasking and because he got along well with everyone.

Problem-solving strategy. “My strategy in research has always been opportunistic. One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory. I think there is an art to doing pilot experiments. So we have always just set up systems in which something happens and then you try and try things until a pattern appears and maybe you formulate a small hypothesis. But as soon as it turns out not to be exactly right, you abandon it. It’s a very open-minded type of research where you question whether what you are seeing is a real phenomenon without spending a year on doing all of the proper controls.”

Trial and error. Clevers’s lab found that while TCF-1 bound to DNA, it did not alter gene expression, despite the researchers’ tinkering with promoter and enhancer assays. “For about five years this was a problem. My first PhD students were leaving and they thought the whole TCF project was a failure,” says Clevers. His lab meanwhile cloned TCF homologs from several model organisms and made many reagents including antibodies against these homologs. To try to figure out the function of TCF-1, the lab performed a two-hybrid screen and identified components of the Wnt signaling pathway as binding partners of TCF-1. “We started to read about Wnt and realized that you study Wnt not in T cells but in frogs and flies, so we rapidly transformed into a developmental biology lab. We showed that we held the key for a major issue in developmental biology, the final protein in the Wnt cascade: TCF-1 binds b-catenin when b-catenin becomes available and activates transcription.” In 1996, Clevers published the mechanism of how the TCF-1 homolog in Xenopus embryos, called XTcf-3, is integrated into the Wnt signaling pathway.

Clevers Catapults

COURTESY OF HANS CLEVERS AND JEROEN HUIJBEN, NYMUS

3DCrypt building and colon cancer.

Clevers next collaborated with Bert Vogelstein’s lab at Johns Hopkins, linking TCF to Wnt signaling in colon cancer. In colon cancer cell lines with mutated forms of the tumor suppressor gene APC, the APC protein can’t rein in b-catenin, which accumulates in the cytoplasm, forms a complex with TCF-4 (later renamed TCF7L2) in the nucleus, and caninitiate colon cancer by changing gene expression. Then, the lab showed that Wnt signaling is necessary for self-renewal of adult stem cells, as mice missing TCF-4 do not have intestinal crypts, the site in the gut where stem cells reside. “This was the first time Wnt was shown to play a role in adults, not just during development, and to be crucial for adult stem cell maintenance,” says Clevers. “Then, when I started thinking about studying the gut, I realized it was by far the best way to study stem cells. And I also realized that almost no one in the world was studying the healthy gut. Almost everyone who researched the gut was studying a disease.” The main advantages of the murine model are rapid cell turnover and the presence of millions of stereotypic crypts throughout the entire intestine.

Against the grain. In 2007, Nick Barker, a senior scientist in the Clevers lab, identified the Wnt target gene Lgr5 as a unique marker of adult stem cells in several epithelial organs, including the intestine, hair follicle, and stomach. In the intestine, the gene codes for a plasma membrane protein on crypt stem cells that enable the intestinal epithelium to self-renew, but can also give rise to adenomas of the gut. Upon making mice with adult stem cell populations tagged with a fluorescent Lgr5-binding marker, the lab helped to overturn assumptions that “stem cells are rare, impossible to find, quiescent, and divide asymmetrically.”

On to organoids. Once the lab could identify adult stem cells within the crypts of the gut, postdoc Toshiro Sato discovered that a single stem cell, in the presence of Matrigel and just three growth factors, could generate a miniature crypt structure—what is now called an organoid. “Toshi is very Japanese and doesn’t always talk much,” says Clevers. “One day I had asked him, while he was at the microscope, if the gut stem cells were growing, and he said, ‘Yes.’ Then I looked under the microscope and saw the beautiful structures and said, ‘Why didn’t you tell me?’ and he said, ‘You didn’t ask.’ For three months he had been growing them!” The lab has since also grown mini-pancreases, -livers, -stomachs, and many other mini-organs.

Tumor Organoids. Clevers showed that organoids can be grown from diseased patients’ samples, a technique that could be used in the future to screen drugs. The lab is also building biobanks of organoidsderived from tumor samples and adjacent normal tissue, which could be especially useful for monitoring responses to chemotherapies. “It’s a similar approach to getting a bacterium cultured to identify which antibiotic to take. The most basic goal is not to give a toxic chemotherapy to a patient who will not respond anyway,” says Clevers. “Tumor organoids grow slower than healthy organoids, which seems counterintuitive, but with cancer cells, often they try to divide and often things go wrong because they don’t have normal numbers of chromosomes and [have] lots of mutations. So, I am not yet convinced that this approach will work for every patient. Sometimes, the tumor organoids may just grow too slowly.”

Selective memory. “When I received the Breakthrough Prize in 2013, I invited everyone who has ever worked with me to Amsterdam, about 100 people, and the lab organized a symposium where many of the researchers gave an account of what they had done in the lab,” says Clevers. “In my experience, my lab has been a straight line from cloning TCF-1 to where we are now. But when you hear them talk it was ‘Hans told me to try this and stop this’ and ‘Half of our knockout mice were never published,’ and I realized that the lab is an endless list of failures,” Clevers recalls. “The one thing we did well is that we would start something and, as soon as it didn’t look very good, we would stop it and try something else. And the few times when we seemed to hit gold, I would regroup my entire lab. We just tried a lot of things, and the 10 percent of what worked, those are the things I remember.”

Greatest Hits

  • Cloned the first T cell–specific transcription factor, TCF-1, and identified homologous genes in model organisms including the fruit fly, frog, and worm
  • Found that transcriptional activation by the abundant β-catenin/TCF-4 [TCF7L2] complex drives cancer initiation in colon cells missing the tumor suppressor protein APC
  • First to extend the role of Wnt signaling from developmental biology to adult stem cells by showing that the two Wnt pathway transcription factors, TCF-1 and TCF-4, are necessary for maintaining the stem cell compartments in the thymus and in the crypt structures of the small intestine, respectively
  • Identified Lgr5 as an adult stem cell marker of many epithelial stem cells including those of the colon, small intestine, hair follicle, and stomach, and found that Lgr5-expressing crypt cells in the small intestine divide constantly and symmetrically, disproving the common belief that stem cell division is asymmetrical and uncommon
  • Established a three-dimensional, stable model, the “organoid,” grown from adult stem cells, to study diseased patients’ tissues from the gut, stomach, liver, and prostate
 Regenerative Medicine Comes of Age   
“Anti-Aging Medicine” Sounds Vaguely Disreputable, So Serious Scientists Prefer to Speak of “Regenerative Medicine”
  • Induced pluripotent stem cells (iPSCs) and genome-editing techniques have facilitated manipulation of living organisms in innumerable ways at the cellular and genetic levels, respectively, and will underpin many aspects of regenerative medicine as it continues to evolve.

    An attitudinal change is also occurring. Experts in regenerative medicine have increasingly begun to embrace the view that comprehensively repairing the damage of aging is a practical and feasible goal.

    A notable proponent of this view is Aubrey de Grey, Ph.D., a biomedical gerontologist who has pioneered an regenerative medicine approach called Strategies for Engineered Negligible Senescence (SENS). He works to “develop, promote, and ensure widespread access to regenerative medicine solutions to the disabilities and diseases of aging” as CSO and co-founder of the SENS Research Foundation. He is also the editor-in-chief of Rejuvenation Research, published by Mary Ann Liebert.

    Dr. de Grey points out that stem cell treatments for age-related conditions such as Parkinson’s are already in clinical trials, and immune therapies to remove molecular waste products in the extracellular space, such as amyloid in Alzheimer’s, have succeeded in such trials. Recently, there has been progress in animal models in removing toxic cells that the body is failing to kill. The most encouraging work is in cancer immunotherapy, which is rapidly advancing after decades in the doldrums.

    Many damage-repair strategies are at an  early stage of research. Although these strategies look promising, they are handicapped by a lack of funding. If that does not change soon, the scientific community is at risk of failing to capitalize on the relevant technological advances.

    Regenerative medicine has moved beyond boutique applications. In degenerative disease, cells lose their function or suffer elimination because they harbor genetic defects. iPSC therapies have the potential to be curative, replacing the defective cells and eliminating symptoms in their entirety. One of the biggest hurdles to commercialization of iPSC therapies is manufacturing.

  • Building Stem Cell Factories

    Cellular Dynamics International (CDI) has been developing clinically compatible induced pluripotent stem cells (iPSCs) and iPSC-derived human retinal pigment epithelial (RPE) cells. CDI’s MyCell Retinal Pigment Epithelial Cells are part of a possible therapy for macular degeneration. They can be grown on bioengineered, nanofibrous scaffolds, and then the RPE cell–enriched scaffolds can be transplanted into patients’ eyes. In this pseudo-colored image, RPE cells are shown growing over the nanofibers. Each cell has thousands of “tongue” and “rod” protrusions that could naturally support rod and cone cells in the eye.

    “Now that an infrastructure is being developed to make unlimited cells for the tools business, new opportunities are being created. These cells can be employed in a therapeutic context, and they can be used to understand the efficacy and safety of drugs,” asserts Chris Parker, executive vice president and CBO, Cellular Dynamics International (CDI). “CDI has the capability to make a lot of cells from a single iPSC line that represents one person (a capability termed scale-up) as well as the capability to do it in parallel for multiple individuals (a capability termed scale-out).”

    Minimally manipulated adult stem cells have progressed relatively quickly to the clinic. In this scenario, cells are taken out of the body, expanded unchanged, then reintroduced. More preclinical rigor applies to potential iPSC therapy. In this case, hematopoietic blood cells are used to make stem cells, which are manufactured into the cell type of interest before reintroduction. Preclinical tests must demonstrate that iPSC-derived cells perform as intended, are safe, and possess little or no off-target activity.

    For example, CDI developed a Parkinsonian model in which iPSC-derived dopaminergic neurons were introduced to primates. The model showed engraftment and enervation, and it appeared to be free of proliferative stem cells.

    • “You will see iPSCs first used in clinical trials as a surrogate to understand efficacy and safety,” notes Mr. Parker. “In an ongoing drug-repurposing trial with GlaxoSmithKline and Harvard University, iPSC-derived motor neurons will be produced from patients with amyotrophic lateral sclerosis and tested in parallel with the drug.” CDI has three cell-therapy programs in their commercialization pipeline focusing on macular degeneration, Parkinson’s disease, and postmyocardial infarction.

    • Keeping an Eye on Aging Eyes

      The California Project to Cure Blindness is evaluating a stem cell–based treatment strategy for age-related macular degeneration. The strategy involves growing retinal pigment epithelium (RPE) cells on a biostable, synthetic scaffold, then implanting the RPE cell–enriched scaffold to replace RPE cells that are dying or dysfunctional. One of the project’s directors, Dennis Clegg, Ph.D., a researcher at the University of California, Santa Barbara, provided this image, which shows stem cell–derived RPE cells. Cell borders are green, and nuclei are red.

      The eye has multiple advantages over other organ systems for regenerative medicine. Advanced surgical methods can access the back of the eye, noninvasive imaging methods can follow the transplanted cells, good outcome parameters exist, and relatively few cells are needed.

      These advantages have attracted many groups to tackle ocular disease, in particular age-related macular degeneration, the leading cause of blindness in the elderly in the United States. Most cases of age-related macular degeneration are thought to be due to the death or dysfunction of cells in the retinal pigment epithelium (RPE). RPE cells are crucial support cells for the rods, cones, and photoreceptors. When RPE cells stop working or die, the photoreceptors die and a vision deficit results.

      A regenerated and restored RPE might prevent the irreversible loss of photoreceptors, possibly via the the transplantation of functionally polarized RPE monolayers derived from human embryonic stem cells. This approach is being explored by the California Project to Cure Blindness, a collaborative effort involving the University of Southern California (USC), the University of California, Santa Barbara (UCSB), the California Institute of Technology, City of Hope, and Regenerative Patch Technologies.

      The project, which is funded by the California Institute of Regenerative Medicine (CIRM), started in 2010, and an IND was filed early 2015. Clinical trial recruitment has begun.

      One of the project’s leaders is Dennis Clegg, Ph.D., Wilcox Family Chair in BioMedicine, UCSB. His laboratory developed the protocol to turn undifferentiated H9 embryonic stem cells into a homogenous population of RPE cells.

      “These are not easy experiments,” remarks Dr. Clegg. “Figuring out the biology and how to make the cell of interest is a challenge that everyone in regenerative medicine faces. About 100,000 RPE cells will be grown as a sheet on a 3 × 5 mm biostable, synthetic scaffold, and then implanted in the patients to replace the cells that are dying or dysfunctional. The idea is to preserve the photoreceptors and to halt disease progression.”

      Moving therapies such as this RPE treatment from concept to clinic is a huge team effort and requires various kinds of expertise. Besides benefitting from Dr. Clegg’s contribution, the RPE project incorporates the work of Mark Humayun, M.D., Ph.D., co-director of the USC Eye Institute and director of the USC Institute for Biomedical Therapeutics and recipient of the National Medal of Technology and Innovation, and David Hinton, Ph.D., a researcher at USC who has studied how actvated RPE cells can alter the local retinal microenvironment.

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Retroviruses and Immunity

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Viral Remnants Help Regulate Human Immunity

Endogenous retroviruses in the human genome can regulate genes involved in innate immune responses.

By Jyoti Madhusoodanan | March 3, 2016
http://www.the-scientist.com//?articles.view/articleNo/45503/title/Viral-Remnants-Help-Regulate-Human-Immunity/

http://www.the-scientist.com/images/News/March2016/310ERVs.png

Dendrogram of various classes of endogenous retroviruses WIKIMEDIA, FGRAMMEN

Remnants of retroviruses that entered the human genome millions of years ago can regulate some innate immune responses. These viral sequences have previously been linked to controlling early mammalian development and formation of the placenta, among other things. A study published today (March 3) in Science establishes that one such endogenous retrovirus in human cells can also regulate the interferon response, which helps organisms quickly respond to infections. The work is one of the first reports to show that human cells could have adopted retroviral sequences to regulate their genes.

“Before we started this project . . . we knew our genomes were full of these elements and many of them are activated during normal development in cells,” said study coauthor Edward Chuong, a postdoc at the University of Utah in Salt Lake City. “Our motivation was: How can we take the next step and figure out their potential biological consequences?”

Chuong and his University of Utah mentors Nels Eldeand Cédric Feschotte began by scanning the sequences around interferon-induced genes, finding at least 27 transposable elements that likely originated from the long repeats at the ends of retroviral sequences. One such element, known as MER41, comes from a virus that invaded the genome approximately 45 million to 60 million years ago; the team found that its sequence in present-day human cells contained interferon-inducible binding sites.

The group then focused on a MER41 sequence that occurs 220 base pairs upstream of an interferon-induced gene called AIM2, which activates an inflammatory response in cells. When the researchers deleted this MER41 element in a cell line using CRISPR/Cas9 gene editing, interferon treatment could not trigger the AIM2 gene. Without the interferon-mediated response, these cells were more susceptible to viral infections, the team found.

“This is a really strong paper,” said Dixie Mager of the University of British Columbia who was not involved with the study. Although previous studies have considered the regulatory functions of endogenous retroviruses, most have been genome-wide correlations, Mager added. “[Here] they go in and delete the specific endogenous retroviruses and show an effect. That’s one of the things that sets this study apart.”

In addition to AIM2, the group found MER41 elements helped regulate at least three other interferon-inducible genes involved in human immunity. Looking across the genomes of other mammals, the researchers also found MER41-like regulatory elements in lemurs, bats, and other species.

The work is “simple and elegant,” said Todd Macfarlan of the Eunice Kennedy Shriver National Institute of Child Health and Human Development who was not involved with the study. “The novelty here is that it extends this idea that retroviruses are continually being coopted for things—not just for placental or early development, but also for other types of gene regulatory pathways. In the future the question might be: Are there any pathways where retroviruses don’t play a role?”

Whether host cells coopted the viral sequences for their regulatory needs or if ancient viruses used their regulatory abilities to control host immunity during invasion is still unknown, according to Feschotte. “We can only speculate why ancient viruses might have carried these regulatory switches to begin with, but data suggest they had these systems built into their sequence already,” he told The Scientist.

Endogenous retroviral elements make up about 8 percent of the human genome, and similar regulatory effects might be found on other mammalian gene functions, said Mager. “What’s cool about endogenous retroviruses is that their ends, known as LTRs, are optimized to have all these regulatory sequences in just 300 to 400 base pairs of DNA,” she said. “These units are powerhouses of regulatory potential.”

Future studies are needed to establish that these regulatory mechanisms are functional in animals, said Macfarlan. In subsequent work, Feschotte and his colleagues aim to extend their studies to a mouse model and immune cell lines.

To Feschotte’s mind, understanding how these sequences regulate human genes could shed light on previously unknown mechanisms of many diseases. While studies of cancer, autoimmune diseases, and other conditions have reported that endogenous retroviruses are reactivated in disease, the reasons for reactivation— and its consequences—are still unclear.

“What has plagued this field is that we don’t the consequences or molecular mechanisms by which these endogenous retroviruses contribute to disease,” he said.

E.B. Chuong et al., “Regulatory evolution of innate immunity through co-option of endogenous retroviruses,” Science, doi:10.1126/science.aad5497, 2016.

 

Regulatory evolution of innate immunity through co-option of endogenous retroviruses

 

Researchers Trace Spread of Ancient Viruses

Wed, 03/09/2016    Greg Watry, Digital Reporter    http://www.dddmag.com/articles/2016/03/researchers-trace-spread-ancient-viruses

Viruses have been present for billions of years, affecting the gamut of life from single celled to multicellular organisms. But these diminutive infectious agents don’t leave behind fossils. Therefore, understanding their origin and evolution has proven difficult.

However, researchers from Boston College have traced the spread of an ancient group of retroviruses—known as ERV-Fc—that affected 28 of 50 studied mammalian ancestors between 15 and 30 million years ago.

“Over the course of millions of years, genetic sequences from the viruses accumulate in the DNA genomes of living organisms (including humans),” the researchers wrote in their paper appearing in eLife. “These sequences can serve as molecular ‘fossils’ for exploring the natural history of viruses and their hosts.”

Retroviruses affect various populations, and included in that group are immunodeficiency viruses, such as HIV-1 and HIV-2, and T-cell leukemia viruses.

The ancient viruses studied “affected a diverse range of hosts, including carnivores, rodents and primates,” the researchers wrote. “The distribution of ERV-Fc among different mammals indicates that the viruses spread to every continent except Antarctica and Australia, and that they jumped between species more than 20 times.”

The ERV-Fc virus was traced to the beginning of the Oligocene Epoch, which was marked by the first appearance of elephants with trunks, early horses, and extensive grasslands, according to the Univ. of California Museum of Paleontology.

In order to trace the virus group, the researchers searched mammalian genome sequence databases for ERV-Fc loci, and then “reconstructed the sequences of proteins representing the virus that colonized the ancestors of that particular species,” according to eLife.

The researchers also followed the changing patterns in the ERV-Fc viruses’ genes as it adapted to various hosts.

“As part of this process, the viruses often exchanged genes with each other and with other types of viruses,” the researchers wrote. “Such genetic recombination is likely to have played a significant role in the evolutionary success of the ERV-Fc viruses.”

According to study co-author William E. Diehl, the research may help humanity predict the long-term effects of viral infections, and the future evolution of such organisms.

 

 

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Obesity Issues

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

The Changing Face of Obesity

Science tells us obesity is a chronic disease. Why does the outmoded and injurious notion that it is a problem of willpower persist?

By Joseph Proietto | November 1, 2015   http://www.the-scientist.com//?articles.view/articleNo/44288/title/The-Changing-Face-of-Obesity/

In Dante Alighieri’s Divine Comedy the narrator meets a man named Ciacco who had been sent to Hell for the “Damning sin of Gluttony.” According to Catholic theology, in order to end up in Hell one must willfully commit a serious sin. So Dante believed that fat people chose to be fat. This antiquated view of the cause of obesity is still widespread, even among medical professionals. The consequences of this misconception are significant, because it forms the basis for the discrimination suffered by the obese; for the wasting of scarce resources in attempts to change lifestyle habits by public education; and for the limited availability of subsidized obesity treatments.

http://www.the-scientist.com/November2015/critic1.jpg

While obesity is often labeled a lifestyle disease, poor lifestyle choices alone account for only a 6 to 8 kg weight gain. The body has a powerful negative feedback system to prevent excessive weight gain. The strongest inhibitor of hunger, the hormone leptin, is made by fat cells. A period of increased energy intake will result in fat deposition, which will increase leptin production. Leptin suppresses hunger and increases energy expenditure. This slows down weight gain. To become obese, it may be necessary to harbor a genetic difference that makes the individual resistant to the action of leptin.

Evidence from twin and adoption studies suggests that obesity has a genetic basis, and over the past two decades a number of genes associated with obesity have been described. The most common genetic defect in European populations leading to severe obesity is due to mutations in the gene coding for the melanocortin 4 receptor (MCR4). Still, this defect can explain severe obesity in only approximately 6 percent to 7 percent of cases (J Clin Invest, 106:271-79, 2000). Other genes have been discovered that can cause milder increases in weight; for example, variants of just one gene (FTO) can explain up to 3 kg of weight variation between individuals (Science, 316:889-94, 2007).

Genes do not directly cause weight gain. Rather, genes influence the desire for food and the feeling of satiety. In an environment with either poor access to food or access to only low-calorie food, obesity may not develop even in persons with a genetic predisposition. When there is an abundance of food and a sedentary lifestyle, however, an obesity-prone person will experience greater hunger and reduced satiety, increasing caloric intake and weight gain.

Since the 1980s, there has been a rapid rise in the prevalence of obesity worldwide, a trend that likely results from a variety of complex causes. There is increasing evidence, for example, that the development of obesity on individual or familial levels may be influenced by environmental experiences that occur in early life. For example, if a mother is malnourished during early pregnancy, this results in epigenetic changes to genes involved in the set points for hunger and satiety in the developing child. These changes may then become fixed, resulting in a tendency towards obesity in the offspring.

The biological basis of obesity is further highlighted by the vigorous defense of weight following weight loss. There are at least 10 circulating hormones that modulate hunger. Of these, only one has been confirmed as a hunger-inducing hormone (ghrelin), and it is made and released by the stomach. In contrast, nine hormones suppress hunger, including CCK, PYY, GLP-1, oxyntomodulin, and uroguanylin from the small bowel; leptin from fat cells; and insulin, amylin, and pancreatic polypeptide from the pancreas.

 

After weight loss, regardless of the diet employed, there are changes in circulating hormones involved in the regulation of body weight. Ghrelin levels tend to increase and levels of multiple appetite-suppressing hormones decrease. There is also a subjective increase in appetite. Researchers have shown that even after three years, these hormonal changes persist (NEJM, 365:1597-604, 2011; Lancet Diabetes and Endocrinology, 2:954-62, 2014). This explains why there is a high rate of weight regain after diet-induced weight loss.

Given that the physiological responses to weight loss predispose people to regain that weight, obesity must be considered a chronic disease. Data show that those who successfully maintain their weight after weight loss do so by remaining vigilant and constantly applying techniques to oppose weight regain. These techniques may involve strict diet and exercise practices and/or pharmacotherapy.

It is imperative for society to move away from a view that obesity is simply a lifestyle issue and to accept that it is a chronic disease. Such a change would not only relieve the stigma of obesity but would also empower politicians, scientists and clinicians to tackle the problem more effectively.

Joseph Proietto was the inaugural Sir Edward Dunlop Medical Research Foundation Professor of Medicine in the Department of Medicine, Austin Health at the University of Melbourne in Australia. He is a researcher and clinician investigating and treating obesity and type 2 diabetes.

 

 

A Weighty Anomaly

Why do some obese people actually experience health benefits?

By Jyoti Madhusoodanan | November 1, 2015     http://www.the-scientist.com//?articles.view/articleNo/44304/title/A-Weighty-Anomaly/

http://www.the-scientist.com/November2015/notebook4.jpg

THE ENDOCRINE THEORY: Some researchers have posited that fat cells may secrete molecules that affect glucose homeostasis in muscle or liver tissue.COURTESY OF MITCHELL LAZAR

In the early 19th century, Belgian mathematician Adolphe Quetelet was obsessed with a shape: the bell curve. While helping with a population census, Quetelet proposed that the spread of human traits such as height and weight followed this trend, also known as a Gaussian or normal distribution. On a quest to define a “normal man,” he showed that human height and weight data fell along his beloved bell curves, and in 1823 devised the “Quetelet Index”—more familiar to us today as the BMI, or body mass index, a ratio of weight to height.

Nearly two centuries later, clinicians, researchers, and fitness instructors continue to rely on this metric to pigeonhole people into categories: underweight, healthy, overweight, or obese. But Quetelet never intended the metric to serve as a way to define obesity. And now, a growing body of evidence suggests these categories fail to accurately reflect the health risks—or benefits—of being overweight.

Although there is considerable debate surrounding the prevalence of metabolically healthy obesity, when obesity is defined in terms of BMI (a BMI of 30 or higher), estimates suggest that about 10 percent of adults in the U.S. are obese yet metabolically healthy, while as many as 80 percent of those with a normal BMI may be metabolically unhealthy, with signs of insulin resistance and poor circulating lipid levels, even if they suffer no obvious ill effects. “If all we know about a person is that they have a certain body weight at a certain height, that’s not enough information to know their health risks from obesity,” says health-science researcher Paul McAuley of Winston-Salem State University. “We need better indicators of metabolic health.”

The dangers of being overweight, such as a higher risk of heart disease, type 2 diabetes, and other complications, are well known. But some obese individuals—dubbed the “fat fit”—appear to fare better on many measures of health when they’re heavier. Studies have found lower mortality rates, better response to hemodialysis in chronic kidney disease, and lower incidence of dementia in such people. Mortality, it’s been found, correlates with obesity in a U-shaped curve (J Sports Sci, 29:773-82, 2011). So does extra heft help or hurt?

To answer that question, researchers are trying to elucidate the metabolic reasons for this obesity paradox.

In a recent study, Harvard University epidemiologist Goodarz Danaei and his colleagues analyzed data from nine studies involving a total of more than 58,000 participants to tease apart how obesity and other well-known metabolic risk factors influence the risk of coronary heart disease. Controlling these other risk factors, such as hypertension or high cholesterol, with medication is simpler than curbing obesity itself, Danaei explains. “If you control a person’s obesity you get rid of some health risks, but if you control hypertension or diabetes, that also reduces health risks, and you can do the latter much more easily right now.”

Danaei’s team assessed BMI and metabolic markers such as systolic blood pressure, total serum cholesterol, and fasting blood glucose. The three metabolic markers only explained half of the increased risk of heart disease across all study participants. In obese individuals, the other half appeared to be mediated by fat itself, perhaps via inflammatory markers or other indirect mechanisms (Epidemiology, 26:153-62, 2015). While Danaei’s study was aimed at understanding how obesity hurts health, the results also uncovered unknown mechanisms by which excess adipose tissue might exert its effects. This particular study revealed obesity’s negative effects, but might these unknown mechanisms hold clues that explain the obesity paradox?

Other researchers have suggested additional possibilities—for example, that inflammatory markers such as TNF-α help combat conditions such as chronic kidney disease, or that obesity makes a body more capable of making changes to, and tolerating changes in, blood flow depending on systemic needs (Am J Clin Nutr, 81:543-54, 2005).

According to endocrinologist Mitchell Lazar at the University of Pennsylvania, the key to explaining the obesity paradox may be two nonexclusive ways fat tissue is hypothesized to function. One mechanism, termed the endocrine theory, suggests that fat cells secrete, or don’t secrete enough of, certain molecules that influence glucose homeostasis in other tissues, such as muscle or liver. The first such hormone to be discovered was leptin; later studies reported several other adipocyte-secreted factors, including adiponectin, resistin, and various cytokines.

The other hypothesis, dubbed the spillover theory, suggests that storing lipids in fat cells has some pluses. Adipose tissue might sequester fat-soluble endotoxins, and produce lipoproteins that can bind to and clear harmful lipids from circulation. When fat cells fill up, however, these endotoxins are stashed in the liver, pancreas, or other organs—and that’s when trouble begins. In “fat fit” people, problems typically linked to obesity such as high cholesterol or diabetes may be avoided simply because their adipocytes mop up more endotoxins.

“In this model, one could imagine that if you could store even more fat in fat cells, you could be even more obese, but you might be protected from problems [associated with] obesity because you’re protecting the other tissues from filling up with lipids that cause problems,” says Lazar. “This may be the most popular current model to explain the fat fit.”

Although obesity greatly increases the risk of type 2 diabetes—up to 93-fold in postmenopausal women, for example—not all obese people suffer from the condition. Similarly, a certain subtype of individuals with “normal” BMIs are at greater risk of developing insulin resistance and type 2 diabetes than others with BMIs in the same range. Precisely what distinguishes these two cohorts is still unclear. “Just as important as explaining why some obese people don’t get diabetes is to explain why other subgroups—normal-weight people or those with lipodystrophy—sometimes get it,” Lazar says. “If there are multiple subtypes of obesity and diabetes, can we figure out genetic aspects or biomarkers that cause one of these phenotypes and not the other?”

To Lazar, McAuley, and other researchers, it’s increasingly evident that BMI may not be that metric. Finding better ways to assess a healthy weight, however, has proven challenging. Researchers have tested measures, such as the body shape index (ABSI) or the waist-hip ratio, which attempt to gauge visceral fat—considered to be more metabolically harmful than fat in other body locations. However, these metrics have yet to be implemented widely in clinics, and few are as simple to understand as the BMI (Science, 341:856-58, 2013).

Independent of metrics, however, the health message regarding weight is still unanimous: exercise and healthy dietary choices benefit everyone. “At a certain point, despite all the so-called fit-fat people, the demographics say that there’s a huge risk of diabetes and heart disease at very high BMI,” notes Lazar. “We can’t assume we’ll be one of the lucky ones who will have a BMI in the obese category but will still be protected from heart disease.”

Correction (November 2): The original version of this article misattributed the pull quote above. The attribution for this quote has been corrected, and The Scientist regrets the error.

 

 

THE HEALTH RISK OF OBESITY—BETTER METRICS IMPERATIVE

 Science 23 Aug 2013;  341(6148): 856858     DOI: http://dx.doi.org:/10.1126/science.1241244
Obesity paradoxes.
In this review, we examine the original obesity paradox phenomenon (i.e. in cardiovascular disease populations, obese patients survive better), as well as three other related paradoxes (pre-obesity, “fat but fit” theory, and “healthy” obesity). An obesity paradox has been reported in a range of cardiovascular and non-cardiovascular conditions. Pre-obesity (defined as a body mass index of 25.0-29.9 kg · m⁻²) presents another paradox. Whereas “overweight” implies increased risk, it is in fact associated with decreased mortality risk compared with normal weight. Another paradox concerns the observation than when fitness is taken into account, the mortality risk associated with obesity is offset. The final paradox under consideration is the presence of a sizeable subset of obese individuals who are otherwise healthy. Consequently, a large segment of the overweight and obese population is not at increased risk for premature death. It appears therefore that low cardiorespiratory fitness and inactivity are a greater health threat than obesity, suggesting that more emphasis should be placed on increasing leisure time physical activity and cardiorespiratory fitness as the main strategy for reducing mortality risk in the broad population of overweight and obese adults.
Obesity, insulin resistance, and cardiovascular disease.
Recent Prog Horm Res. 2004;59:207-23.
The ability of insulin to stimulate glucose disposal varies more than six-fold in apparently healthy individuals. The one third of the population that is most insulin resistant is at greatly increased risk to develop cardiovascular disease (CVD), type 2 diabetes, hypertension, stroke, nonalcoholic fatty liver disease, polycystic ovary disease, and certain forms of cancer. Between 25-35% of the variability in insulin action is related to being overweight. The importance of the adverse effects of excess adiposity is apparent in light of the evidence that more than half of the adult population in the United States is classified as being overweight/obese, as defined by a body mass index greater than 25.0 kg/m(2). The current epidemic of overweight/obesity is most-likely related to a combination of increased caloric intake and decreased energy expenditure. In either instance, the fact that CVD risk is increased as individuals gain weight emphasizes the gravity of the health care dilemma posed by the explosive increase in the prevalence of overweight/obesity in the population at large. Given the enormity of the problem, it is necessary to differentiate between the CVD risk related to obesity per se, as distinct from the fact that the prevalence of insulin resistance and compensatory hyperinsulinemia are increased in overweight/obese individuals. Although the majority of individuals in the general population that can be considered insulin resistant are also overweight/obese, not all overweight/obese persons are insulin resistant. Furthermore, the cluster of abnormalities associated with insulin resistance – namely, glucose intolerance, hyperinsulinemia, dyslipidemia, and elevated plasma C-reactive protein concentrations — is limited to the subset of overweight/obese individuals that are also insulin resistant. Of greater clinical relevance is the fact that significant improvement in these metabolic abnormalities following weight loss is seen only in the subset of overweight/obese individuals that are also insulin resistant. In view of the large number of overweight/obese subjects at potential risk to be insulin resistant/hyperinsulinemic (and at increased CVD risk), and the difficulty in achieving weight loss, it seems essential to identify those overweight/obese individuals who are also insulin resistant and will benefit the most from weight loss, then target this population for the most-intensive efforts to bring about weight loss.
Long-Term Persistence of Hormonal Adaptations to Weight Loss

Priya Sumithran, Luke A. Prendergast, Elizabeth Delbridge, Katrina Purcell, Arthur Shulkes, Adamandia Kriketos, and Joseph Proietto

N Engl J Med 2011; 365:1597-1604   October 27, 2011http://dx.doi.org:/10.1056/NEJMoa1105816

After weight loss, changes in the circulating levels of several peripheral hormones involved in the homeostatic regulation of body weight occur. Whether these changes are transient or persist over time may be important for an understanding of the reasons behind the high rate of weight regain after diet-induced weight loss.

Weight loss (mean [±SE], 13.5±0.5 kg) led to significant reductions in levels of leptin, peptide YY, cholecystokinin, insulin (P<0.001 for all comparisons), and amylin (P=0.002) and to increases in levels of ghrelin (P<0.001), gastric inhibitory polypeptide (P=0.004), and pancreatic polypeptide (P=0.008). There was also a significant increase in subjective appetite (P<0.001). One year after the initial weight loss, there were still significant differences from baseline in the mean levels of leptin (P<0.001), peptide YY (P<0.001), cholecystokinin (P=0.04), insulin (P=0.01), ghrelin (P<0.001), gastric inhibitory polypeptide (P<0.001), and pancreatic polypeptide (P=0.002), as well as hunger (P<0.001).

What’s new in endocrinology and diabetes mellitus

Large genome wide association studies have demonstrated that variants in the FTO gene have the strongest association with obesity risk in the general population, but the mechanism of the association has been unclear. However, a nonocoding causal variant in FTO has now been identified that changes the function of adipocytes from energy utilization (beige fat) to energy storage (white fat) with a fivefold decrease in mitochondrial thermogenesis [17]. When the effect of the variant was blocked in genetically engineered mice, thermogenesis increased and weight gain did not occur, despite eating a high-fat diet. Blocking the gene’s effect in human adipocytes also increased energy utilization. This observation has important implications for potential new anti-obesity drugs. (See “Pathogenesis of obesity”, section on ‘FTO variants’.)

Liraglutide for the treatment of obesity (July 2015)

Along with diet, exercise, and behavior modification, drug therapy may be a helpful component of treatment for select patients who are overweight or obese. Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist, used for the treatment of type 2 diabetes, and can promote weight loss in patients with diabetes, as well as those without diabetes.

In a randomized trial in nondiabetic patients who had a body mass index (BMI) of ≥30 kg/m2 or ≥27 kg/m2 with dyslipidemia and/or hypertension, liraglutide 3 mg once daily, compared with placebo, resulted in greater mean weight loss (-8.0 versus -2.6 kg with placebo) [18]. In addition, cardiometabolic risk factors, glycated hemoglobin (A1C), and quality of life improved modestly. Gastrointestinal side effects transiently affected at least 40 percent of the liraglutide group and were the most common reason for withdrawal (6.4 percent). Liraglutide is an option for select overweight or obese patients, although gastrointestinal side effects (nausea, vomiting) and the need for a daily injection may limit the use of this drug. (See “Obesity in adults: Drug therapy”, section on ‘Liraglutide’.)

In a trial designed specifically to evaluate the effect of liraglutide on weight loss in overweight or obese patients with type 2 diabetes (mean weight 106 kg), liraglutide, compared with placebo, resulted in greater mean weight loss (-6.4 kg and -5.0 kg for liraglutide 3 mg and 1.8 mg, respectively, versus -2.2 kg for placebo) [19]. Treatment with liraglutide was associated with better glycemic control, a reduction in the use of oral hypoglycemic agents, and a reduction in systolic blood pressure. Although liraglutide is not considered as initial therapy for the majority of patients with type 2 diabetes, it is an option for select overweight or obese patients with type 2 diabetes who fail initial therapy with lifestyle intervention and metformin.  (See “Glucagon-like peptide-1 receptor agonists for the treatment of type 2 diabetes mellitus”, section on ‘Weight loss’.)

The Skinny on Fat Cells

Bruce Spiegelman has spent his career at the forefront of adipocyte differentiation and metabolism.

By Anna Azvolinsky | November 1, 2015

http://www.the-scientist.com//?articles.view/articleNo/44312/title/The-Skinny-on-Fat-Cells/

Bruce Spiegelman
Stanley J. Korsmeyer Professor of Cell Biology
and Medicine
Harvard Medical School
Director, Center for Energy Metabolism
and Chronic
Disease, Dana-Farber Cancer Institute, Boston

It’s hard to know whether you have the right stuff to be a scientist, but I had a passion for the research,” says Bruce Spiegelman, professor of cell biology at Harvard Medical School and the Dana-Farber Cancer Institute. After receiving his PhD in biochemistry from Princeton University in 1978, Spiegelman sent an application to do postdoctoral research to just one lab. “I wasn’t thinking I should apply to five different labs. I just marched forward more or less in a straight line,” he says. Spiegelman did know that he had no financial backup and depended on research fellowships throughout the early phase of his science career. “I thought it was fantastic, and still think so, that a PhD in science is supported by the government. I certainly appreciated that, because many of my friends in the humanities had to support themselves by cobbling together fellowships and teaching every semester, whereas we didn’t face similar challenges in the sciences.”

Since his graduate student days, Spiegelman has realized his potential, pioneering the study of adipose tissue biology and metabolism. He was introduced to the field in Howard Green’s laboratory, then at MIT, where Spiegelman began his one and only postdoc in 1978. Green had recently developed a system for culturing adipose cells and asked Spiegelman if he wanted to study fat cell differentiation. “I knew nothing about adipose tissue, but I was really interested in any model of how one cell switches to another. Whether skin or fat didn’t matter too much to me, because I was not coming at this from the perspective of physiology but from the perspective of how do these switches work at a molecular level?”

Spiegelman has stuck with studying the biology and differentiation of fat cells for more than 30 years. While looking for the master transcriptional regulator of fat development—which his laboratory found in 1994—Spiegelman’s group also discovered one of the first examples of a nuclear oncogene that functions as a transcription factor, and, more recently, the team found that brown fat and white fat come from completely different origins and that brown and beige fat are distinct cell types. Spiegelman was also the first to provide evidence for the connection between inflammation, insulin resistance, and fat tissue.

Here, Spiegelman talks about his strong affinity for the East Coast, his laboratory’s search for molecules that can crank up brown fat production and activity, and the culture of his laboratory’s weekly meeting.

Spiegelman Sets Out

First publication. Spiegelman grew up in Massapequa, New York, a town on Long Island. “Birds, insects, fish, and animals were fascinating to me. As a kid, I imagined I would be a wildlife ranger,” he says. Spiegelman and his brother were the first in their family to attend college; Spiegelman entered the College of William and Mary in 1970 thinking he would major in psychology. But before taking his first psychology course, he had to take a biology course, really loved it, and switched his major. For his senior thesis, he chose one of the few labs that did biochemistry-related research. He studied cultures of the filamentous fungus Aspergillus ornatus in which he induced the upregulation of a metabolic enzyme. Spiegelman applied a calculus transformation that related the age of the culture to the age of individual cells, something that had not been previously done. The work earned him his first first-author publication in 1975. “It was not a great breakthrough, but I think it showed that I was maybe applying myself more than the typical undergraduate.”

Full steam ahead. “My interest in laboratory research was intense. Even though it was not particularly inspired work, the first-author publication in a college where not many of the professors published a lot gave me a lot of confidence. It was probably out of proportion to the quality of the actual work.” That confidence and Spiegelman’s interest in the chemistry of living things led him to pursue a PhD in biochemistry at Princeton University. “Very early on, I felt that I couldn’t understand biology if it didn’t go to the molecular level. To me, just describing how an animal lived without understanding how it worked was very unsatisfying. I think it was one of the best decisions that I made in my life, to do a PhD in biochemistry,” he says, “because if you really want to understand living systems, you are very limited in how you can understand them without having a strong background in biochemistry because these are, essentially, chemical systems.”

Embracing molecular biology. Spiegelman initially joined Arthur Pardee’s laboratory, but switched when Pardee left Princeton for Harvard University in 1975. Because he was already collaborating with Marc Kirschner, a cell biologist and biochemist who studies the regulation of the cell cycle and how the cytoskeleton works, it was an easy transition to transfer to the new laboratory. In Kirschner’s group, Spiegelman became the cell biologist among many protein biochemists working on microtubule assembly in vitro. Rather than understanding how the proteins fit together to form the filamentous structures, Spiegelman wanted to understand what controlled their assembly inside cells. Working in mammalian cells, Spiegelman published three consecutive Cell papers on how microtubule assembly occurs in vivo. The firstpaper, from 1977, demonstrated that a nucleotide functions to stabilize the tubulin molecule rather than to regulate tubulin assembly in vivo.

Spiegelman Simmers

A new tool. For his next move, Spiegelman wanted to marry his background in biochemistry and molecular biology with a good cellular model system. He became interested in differentiation at the end of his PhD, while studying how the cytoskeleton is reorganized during neural differentiation, and settled on Green’s MIT laboratory for his postdoc. Green had developed a way to study both skin and fat cell differentiation. Again, Spiegelman was the odd man out, working on the molecular biology of fat cell differentiation while most of the graduate students and postdocs focused on the cellular biology of skin cell differentiation. While there, Spiegelman learned how to clone cDNA—a new method that some researchers thought was just another new fad, he says. “I thought it was pretty obvious that this was a tool that would be a game changer. I could see how I could clone some of the cDNAs and genes that were regulated in the fat cell lineage and then try to understand the regulation of these genes.”

Setting the stage. Spiegelman demonstrated that cAMP regulates the synthesis of certain enzymes in fat cells during differentiation. But while this was the most influential paper from his postdoc, says Spiegelman, it was his demonstration of cloning mRNAs from adipocytes, published in 1983, that set the stage for cloning fat-selective genes. The work, mostly done when Spiegelman was already a new faculty member at the Dana-Farber Cancer Institute, stemmed from his learning molecular cloning in Phillip Sharp’s lab at MIT and Bryan Roberts’s lab at Harvard. “This was the raw material from which we eventually cloned PPARγ and showed it to be the master regulator of fat [cell] development.”

Roots. Spiegelman became an assistant professor at the Harvard Medical School in 1982, when he was not yet 30. Although he had entertained the idea of moving to the West Coast with his wife, whom he had met at Princeton where she obtained a PhD in French literature, Spiegelman says he is really an East Coaster at heart. “My wife and I came to love Boston and were very comfortable there. Our families were both in New York, which was close, but not too close, and we really enjoyed the culture and pace of Boston; it was more ‘us.’ We really liked to visit California but didn’t particularly want to move there. We’re both real Northeastern people.”

Relating to Sisyphus. The transition from doing a postdoc to setting up his own laboratory was “very exciting and terribly stressful,” says Spiegelman. “When I think back, I always tried to be professional with my laboratory, but I was so stressed at suddenly being on my own with no management training.” The people resources he had encountered in his graduate and postdoctoral training labs were also not there yet, and he says his first publication as a principal investigator was like pushing a rock up a hill. But eventually, Spiegelman’s lab built a reputation and reached a critical mass of talented people who advanced the science. Again in 1983, Spiegelman produced a publication showing that morphological manipulation can affect gene expression and adipose differentiation.

End goal. Spiegelman’s goal was to find a master molecule that  orchestrates the conversion of adipocyte precursor cells into bona fide fat cells. Piece by piece, his lab identified the enhancers, promoters, and other regulatory elements involved in adipocyte differentiation. In 1994, graduate student Peter Tontonoz finallyfound that the PPARγ gene, inserted via a retroviral vector into fibroblasts, could induce the cells to become adipose cells. “It took 10 years,” Spiegelman says. Along the way, the laboratory found that c-fos, the product of a famous nuclear oncogene, bound to the promoters of fat-specific genes and worked as a transcription factor. “It was not really known how nuclear oncogenes worked. This was one of the first papers showing that these oncogenes bound to gene promoters and were transcription factors.”

A wider scope. In 1993, graduate student Gökhan Hotamisligil found that tumor necrosis factor-alpha(TNF-α), is induced in the fat tissue of rodent models of obesity and diabetes. The paper sparked the formation of the field of immunometabolism and resulted in the expansion of Spiegelman’s lab into the physiology arena, partly thanks to the guidance of C. Ronald Kahn and Jeff Flier, who both study metabolism and diabetes. But the work initially encountered pushback, says Spiegelman, partly because it was the merging of two fields.

Spiegelman Scales Up

Fat color palette. Brown fat tissue, abundant in infants but scarce in adults, is a metabolically active form of fat that is chock full of mitochondria and is found in pockets in the body distinct from white fat tissue.Pere Puigserver, then a postdoc in Spiegelman’s lab, found that the coactivator PCG-1, binding to PPARγ and other nuclear receptors, could stimulate mitochondrial biogenesis. The PCG-1 gene is turned on by stimuli such as exercise or a cold environment. Later, postdoc Patrick Seale, Spiegelman, and their colleagues showed brown fat cells derive from the same lineage that gives rise to skeletal muscle. “This was a big surprise, maybe the biggest surprise we ever uncovered in the lab,” says Spiegelman.

A paler shade of brown. More recently, in 2012, Spiegelman’s laboratory showed that within adult white adipose tissue, there are pockets of a yet another type of fat tissue that he called beige fat. “I think the evidence is very good from rodents that if you activate brown and beige fat, you get metabolic benefit both in obesity and diabetes. So the question now is: Can that be done in humans in a way that’s beneficial and not toxic?”  The lab is now looking to identify molecules that can either ramp up the activity of brown and beige fat or increase the production of both cell types as possible therapeutics for metabolic disorders or even cancer-associated cachexia. “Anyone who says that either approach will work better is being foolish. We just don’t know enough to go after just one or the other.”

On the irisin controversy. After reporting in 2012 that a muscle-related hormone called irisin could switch white fat to metabolically active brown fat, Spiegelman became embroiled in a media-covered debate about whether the molecule really exists; he was also the victim of a potential fraud plot. Most recently, Spiegelman provided thorough evidence that irisin does in fact exist. On the controversy, he says it’s a fine line between defending his scientific integrity and not adding more fuel to the fire or engaging with his harassers. “We have a long track record of doing credible and reproducible science and it was not that complicated to address the paper that claimed irisin was ‘a myth.’ That study used very outmoded scientific approaches.”

Raw talent. Many of Spiegelman’s trainees have gone on to become very successful scientists, including Tontonoz, Hotamisligil, Evan Rosen, and Randy Johnson. “It’s a quantum change in the experience of doing science when you get people who have their own visions. I would have thought that interacting with smart people would mainly help me get my scientific vision accomplished. And that was partly true, but also it changed my vision. When you have people challenging you on a day-to-day basis, you learn from them through the questions they ask and the way they challenge you in a constructive way. They made me a much better scientist.”

Rigorous mentorship.  “I feel very passionately that a major part of my job is to prepare the next generation of scientists. Everyone who comes through my lab will tell you that I take that very seriously. We make sure my students give a lot of talks and get critical assessments of their presentations to our lab group. I am very hands-on both scientifically and in developing the way students project their vision. I had a very good mentor, Marc Kirschner, and I’d like to think that I learned how to be a mentor from him. I want to make sure that when people walk out of my lab they are prepared to run independent research programs.”

Greatest Hits

  • Identified the master regulator of adipogenesis, the nuclear receptor PPARγ
  • Was the first to show that a nuclear oncogene, c-fos, codes for a transcription factor that binds to the promoters of genes
  • Demonstrated that adipose tissue synthesizes tumor necrosis factor-alpha (TNF-α), providing the first direct link between obesity, inflammation, insulin resistance, and fat tissue.
  • Showed that brown fat cells are not developmentally related to white fat
  • Identified beige fat as a distinct cell type, different from either white or brown fat

 

Fanning the Flames

Obesity triggers a fatty acid synthesis pathway, which in turn helps drive T cell differentiation and inflammation.

By Kate Yandell | November 1, 2015

http://www.the-scientist.com//?articles.view/articleNo/44306/title/Fanning-the-Flames/

EDITOR’S CHOICE IN IMMUNOLOGY

The paper
Y. Endo et al., “Obesity drives Th17 cell differentiation by inducing the lipid metabolic kinase, ACC1,” Cell Reports, 12:1042-55, 2015.

Cell Rep. 2015 Aug 11;12(6):1042-55.   http://dx.doi.org:/10.1016/j.celrep.2015.07.014. Epub 2015 Jul 30.
Obesity Drives Th17 Cell Differentiation by Inducing the Lipid Metabolic Kinase, ACC1.
  • A high-fat diet augments Th17 cell development and the expression of Acaca
  • ACC1 controls Th17 cell development in vitro and Th17 cell pathogenicity in vivo
  • ACC1 modulates RORγt function in developing Th17 cells
  • Obesity in humans induces ACACA and IL-17A expression in CD4 T cells

Chronic inflammation due to obesity contributes to the development of metabolic diseases, autoimmune diseases, and cancer. Reciprocal interactions between metabolic systems and immune cells have pivotal roles in the pathogenesis of obesity-associated diseases, although the mechanisms regulating obesity-associated inflammatory diseases are still unclear. In the present study, we performed transcriptional profiling of memory phenotype CD4 T cells in high-fat-fed mice and identified acetyl-CoA carboxylase 1 (ACC1, the gene product of Acaca) as an essential regulator of Th17 cell differentiation in vitro and of the pathogenicity of Th17 cells in vivo. ACC1 modulates the DNA binding of RORγt to target genes in differentiating Th17 cells. In addition, we found a strong correlation between IL-17A-producing CD45RO(+)CD4 T cells and the expression of ACACA in obese subjects. Thus, ACC1 confers the appropriate function of RORγt through fatty acid synthesis and regulates the obesity-related pathology of Th17 cells.

Figure thumbnail fx1

http://www.cell.com/cms/attachment/2035221719/2050630604/fx1.jpg

 

 

http://www.the-scientist.com/November2015/NovMediLit_310px.jpg

FEEDING INFLAMMATION: When mice eat a diet high in fat, their CD4 T cells show increased expression of the fatty acid biosynthesis gene Acaca, which encodes the enzyme ACC1 (1). Products of the ACC1 fatty acid synthesis pathway encourage the transcription factor RORγt to bind near the gene encoding the cytokine IL-17A (2). There, RORγt recruits an enzyme called p300 to modify the genome epigenetically and turn on IL-17A. The memory T cells then differentiate into inflammatory T helper 17 cells.
See full infographic: PDF
© STEVE GRAEPEL

Obesity often comes with a side of chronic inflammation, causing inflammatory chemicals and immune cells to flood adipose tissue, the hypothalamus, the liver, and other areas of the body. Inflammation is a big part of what makes obesity such an unhealthy condition, contributing to Type 2 diabetes, heart disease, cancers, autoimmune disorders, and possibly even neurodegenerative diseases.

To better understand the relationship between obesity and inflammation, Toshinori Nakayama, Yusuke Endo, and their colleagues at Chiba University in Japan started with what often leads to obesity: a high-fat diet. They fed mice rich meals for a couple of months and looked at how gene expression in the animals’ T cells compared to gene expression in the T cells of mice fed a normal diet. Most notably, they found increased expression ofAcaca, a gene that codes for a fatty acid synthesis enzyme called acetyl coA carboxylase 1 (ACC1). They went on to show that the resulting increase in fatty acid levels pushed CD4 T cells to differentiate into inflammatory T helper 17 (Th17) cells.

Th17 cells help fight off invading fungi and some bacteria. But these immune cells can also spin out of control in autoimmune diseases such as multiple sclerosis. Nakayama’s team showed that either blocking ACC1 activity with a drug called TOFA or deleting a key portion of Acaca in mouse CD4 T cells reduced the generation of pathologic Th17 cells. Overexpressing Acaca increased Th17-cell generation.

The researchers also demonstrated that mice fed a high-fat diet had elevated susceptibility to a multiple sclerosis–like disease, and that TOFA reduced the symptoms.

“This is a very intriguing finding, suggesting not only that obesity can directly induce Th17 differentiation but also indicating that pharmacologic targeting of fatty acid synthesis may help to interfere with obesity-associated inflammation,” Tim Sparwasser of the Twincore Center for Experimental and Clinical Infection Research in Hannover, Germany, says in an email. Sparwasser and his colleagues had previously shown that ACC1 is required for the differentiation of Th17 cells in mice and humans.

Nakayama explains that CD4 T cells must undergo profound metabolic changes as they mature and differentiate. “The intracellular metabolites, including fatty acids, are essential for cell proliferation and cell growth,” he says in an email. When fatty acid levels in T cells increase, the cells are activated and begin to proliferate.

“It’s a nice illustration of how, really, immune response is so highly connected to the metabolic state of the cell,” says Gökhan S. Hotamisligil of Harvard University’s T.H. Chan School of Public Health who was not involved in the study. “The immune system launches its responses commensurate with the sources of nutrients and energy from the environment,” he adds in an email.

There are still missing pieces in the path from high-fat diet to increased Acaca expression to ACC1’s influence on T-cell differentiation. It also remains to be seen how this plays out in obese humans, although Nakayama and colleagues did show that inhibiting ACC1 reduced pathologic Th17 generation in human immune cell cultures, and that the T cells of obese humans contain elevated levels of ACC1 and show signs of increased differentiation into Th17 cells.

 

The prevalence of obesity has been increasing worldwide, and obesity is now a major public health problem in most developed countries (Gregor and Hotamisligil, 2011, Ng et al., 2014). Obesity-induced inflammation contributes to the development of various chronic diseases, such as autoimmune diseases, metabolic diseases, and cancer (Kanneganti and Dixit, 2012, Kim et al., 2014,Osborn and Olefsky, 2012, Winer et al., 2009a). A number of studies have pointed out the importance of reciprocal interactions between metabolic systems and immune cells in the pathogenesis of obesity-associated diseases (Kaminski and Randall, 2010, Kanneganti and Dixit, 2012, Kim et al., 2014, Mauer et al., 2014, Stienstra et al., 2012, Winer et al., 2011).

Elucidating the molecular mechanisms by which naive CD4 T cells differentiate into effector T cells is crucial for understanding helper T (Th) cell-mediated immune pathogenicity. After antigen stimulation, naive CD4 T cells differentiate into at least four distinct Th cell subsets: Th1, Th2, Th17, and inducible regulatory T (iTreg) cells (O’Shea and Paul, 2010, Reiner, 2007). Several specific master transcription factors that regulate Th1/Th2/Th17/iTreg cell differentiation have been identified, including T-bet for Th1 (Szabo et al., 2000), GATA3 (Yamashita et al., 2004, Zheng and Flavell, 1997) for Th2, retinoic-acid-receptor-related orphan receptor γt (RORγt) for Th17 (Ivanov et al., 2006), and forkhead box protein 3 (Foxp3) for iTreg (Sakaguchi et al., 2008). The appropriate expression and function of these transcription factors is essential for proper immune regulation by each Th cell subset.

Among these Th cell subsets, Th17 cells contribute to the host defense against fungi and extracellular bacteria (Milner et al., 2008). However, the pathogenicity of IL-17-producing T cells has been recognized in various autoimmune diseases, including multiple sclerosis, psoriasis, inflammatory bowel diseases, and steroid-resistant asthma (Bettelli et al., 2006, Coccia et al., 2012, Ivanov et al., 2006,Leonardi et al., 2012, McGeachy and Cua, 2008, Nylander and Hafler, 2012,Stockinger et al., 2007, Sundrud et al., 2009).

An HFD Promotes Th17 Cell Differentiation and Affects the Expression of Fatty Acid Enzymes in Memory CD4 T Cells In Vivo

Inhibition of ACC1 Function Results in Decreased Th17 Cell Differentiation and Ameliorates the Development of Autoimmune Disease

ACC1 Controls the Differentiation of Th17 Cells Both In Vitro and In Vivo

ACC1 Controls the Function, but Not Expression, of RORγt in Differentiating Th17 Cells

Extrinsic Fatty Acid Supplementation Restored Acaca−/− Th17 Cell Differentiation through the Functional Improvement of RORγt

Obese Subjects Show Upregulation of ACACA and Increased Th17 Cells in CD45RO+ Memory CD4 T Cells

We herein identified a critical role that ACC1 plays in Th17 cell differentiation and the pathogenicity of Th17 cells through the control of the RORγt function under obese circumstances. High-fat-induced obesity augments Th17 cell differentiation and the expression of enzymes involved in fatty acid metabolism, including ACC1. Pharmacological inhibition or genetic deletion of ACC1 resulted in impaired Th17 cell differentiation in both mice and humans. In contrast, overexpression of Acaca induced Th17 cells in vivo, leaving the expression ofIfng and Il4 largely unchanged. ACC1 modulated the binding of RORγt to theIl17a gene and the subsequent p300 recruitment in differentiating Th17 cells. Memory CD4 T cells from peripheral blood mononuclear cells (PBMCs) of obese subjects showed increased IL-17A production and ACACA expression. Furthermore, a strong correlation was detected between the proportion of IL-17A-producing cells and the expression level of ACACA in memory CD4 T cells in obese subjects. Thus, our findings provide evidence of a mechanism wherein obesity can exacerbate IL-17-mediated pathology via the induction of ACC1.

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Demet Sag, PhD, CRA, GCP

 

Gene engineering and editing specifically are becoming more attractive. There are many applications derived from microbial origins to correct genomes in many organisms including human to find solutions in health.

There are four customizable DNA specific binding protein applications to edit the gene expression in translational genomics. The targeted DNA double-strand breaks (DSBs) could greatly stimulate genome editing through HR-mediated recombination events.  We can mainly name these site-specific DNA DSBs:

 

  1. meganucleases derived from microbial mobile genetic elements (Smith et al., 2006),
  2. zinc finger (ZF) nucleases based on eukaryotic transcription factors (Urnov et al., 2005;Miller et al., 2007),
  3. transcription activator-like effectors (TALEs) from Xanthomonasbacteria (Christian et al., 2010Miller et al., 2011Boch et al., 2009; Moscou and Bogdanove, 2009), and
  4. most recently the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (Cong et al., 2013;Mali et al., 2013a).

There is a new ground breaking study published in Science by Valentino Gantz and Ethan Bier of the University of California, San Diego, described an approach called mutagenic chain reaction (MCR).

This group developed a new technology for editing genes that can be transferable change to the next generation by combining microbial immune defense mechanism, CRISPR/Cas9 that is the latest ground breaking technology for translational genomics with gene therapy-like approach.

  • In short, this so-called “mutagenic chain reaction” (MCR) introduces a recessive mutation defined by CRISPR/Cas9 that lead into a high rate of transferable information to the next generation. They reported that when they crossed the female MCR offspring to wild type flies, the yellow phenotype observed more than 95 percent efficiency.

 

Development and Applications of CRISPR-Cas9 for Genome Engineeri

Structural and Metagenomic Diversity of Cas9 Orthologs

(A) Crystal structure of Streptococcus pyogenes Cas9 in complex with guide RNA and target DNA.

(B) Canonical CRISPR locus organization from type II CRISPR systems, which can be classified into IIA-IIC based on their cas gene clusters. Whereas type IIC CRISPR loci contain the minimal set of cas9, cas1, andcas2, IIA and IIB retain their signature csn2 and cas4 genes, respectively.

(C) Histogram displaying length distribution of known Cas9 orthologs as described in UniProt, HAMAP protein family profile MF_01480.

(D) Phylogenetic tree displaying the microbial origin of Cas9 nucleases from the type II CRISPR immune system. Taxonomic information was derived from greengenes 16S rRNA gene sequence alignment, and the tree was visualized using the Interactive Tree of Life tool (iTol).

(E) Four Cas9 orthologs from families IIA, IIB, and IIC were aligned by ClustalW (BLOSUM). Domain alignment is based on the Streptococcus pyogenes Cas9, whereas residues highlighted in red indicate highly conserved catalytic residues within the RuvC I and HNH nuclease domains.

(Cell. Author manuscript; available in PMC 2015 Feb 27.Published in final edited form as:

Cell. 2014 Jun 5; 157(6): 1262–1278.doi:  10.1016/j.cell.2014.05.010)

 

The uniqueness of this study comes from:

 

  • There is a big difference between the new type of mutation and traditional mutation is expressivity of the character since previously mutations were passive and non-transferable at 100% rate. However,  in classical Mendelian Genetics, only one fourth f the recessive traits can be presented in new generation. Yet, in this case this can be achieve about 97% plus transferred to new generation.

 

  • MCR alterations is active that is they convert matching sequences at the same target site so mutated sites took over the wild type character without degenerating by wild type alleles segregating independently during the breeding process

 

  • Therefore, the altered sequences routinely replace the wild type (original) sequences at that site. The data demonstrated that among 92 flies, only one female became wild type but remaining 41 females had yellow eyes yet all 50 males showed wild type eye coloring at the second generation.

 

  • The genetic engineering of the genome occurred in a single generation with high efficiency.

 

Their technique developed by Gantz and Bier had three basic parts:

 

  1. Both somatic and germline cells expressed a Cas9 gene,

 

  1. A guide RNA (gRNA) targeted to a genomic sequence of interest,

 

  1. The Cas9/gRNA cassettes have the flanking homolog arms that matches the two genomic sequences immediately adjacent to either side of the target cut site

 

There are many applications in translational genomics that requires multiple steps to make it perfect for complicated organisms, such as plants, mosquitoes and human diseases.

Short Walk from Past to the Future of CRISPR/Cas9

Development and Applications of CRISPR-Cas9 for Genome Engineeri

The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.

CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner.

The latest ground-breaking technology for genome editing is based on RNA-guided engineered nucleases, which already hold great promise due to their:

  • simplicity,
  • efficiency and
  • versality

Although CRISPR arrays were first identified in the Escherichia coli genome in 1987 (Ishino et al., 1987),

their biological function was not understood until 2005, when it was shown that the spacers were homologous to viral and plasmid sequences suggesting a role in adaptive immunity (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005).

Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007).

The mechanism of this immune system based on RNA-mediated DNA targeting was demonstrated shortly thereafter (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010; Marraffini and Sontheimer, 2008).

 

The most widely used system is the type II clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 (CRISPR-associated) system from Streptococcus pyogenes (Jinek et al., 2012).

Then, five independent groups demonstrated that the two-component system was functional in eukaryotes (human, mouse and zebrafish), indicating that the other functions of the CRISPR locus genes were supported by endogenous eukaryotic enzymes (Cho et al., 2013Cong et al., 2013Hwang et al., 2013Jinek et al., 2013 and Mali et al., 2013).

Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified colonial cell lines can be derived within 2-3 weeks

 

Development and Applications of CRISPR-Cas9 for Genome Engineeri

Genome editing with site-specific nucleases.

Double-strand breaks induced by a nuclease at a specific site can be repaired either by non-homologous end joining (NHEJ) or homologous recombination (HR).  In most cases, NHEJ causes random insertions or deletions (indels), which can result in frameshift mutations if they occur in the coding region of a gene, effectively creating a gene knockout.

Alternatively, when the DSB generates overhangs, NHEJ can mediate the targeted introduction of a double-stranded DNA template with compatible overhangs

Even though the generation of breaks in both DNA strands induces recombination at specific genomic loci, NHEJ is by far the most common DSB repair mechanism in most organisms, including higher plants, and the frequency of targeted integration by HR remains much lower than random integration.

  • Unlike its predecessors, the CRISPR/Cas9 system does not require any protein engineering steps, making it much more straightforward to test multiple gRNAs for each target gene

 

  • Unlike ZFNs and TALENs, the CRISPR/Cas9 system can cleave methylated DNA in human cells (Hsu et al., 2013), allowing genomic modifications that are beyond the reach of the other nucleases (Ding et al., 2013).

 

  • The main practical advantage of CRISPR/Cas9 compared to ZFNs and TALENs is the ease of multiplexing. The simultaneous introduction of DSBs at multiple sites can be used to edit several genes at the same time (Li et al., 2013; Mao et al., 2013) and can be particularly useful to knock out redundant genes or parallel pathways.

 

  • Finally, the open access policy of the CRISPR research community has promoted the widespread uptake and use of this technology in contrast, for example, to the proprietary nature of the ZFN platform.

The community provides access to plasmids (e.g., via the non-profit repository Addgene), web tools for selecting gRNA sequences and predicting specificity:

Downside:

One area that will likely need to be addressed when moving to more complex genomes, for instance, is off-target CRISPR/Cas9 activity since fruit fly has only four chromosomes and less likely to have off-target effects. However, this study provided proof of principle.

  • Yet, this critics is not new since one of the few criticisms of the CRISPR/Cas9 technology is the relatively high frequency of off-target mutations reported in some of the earlier studies (Cong et al., 2013; Fu et al., 2013; Hsu et al., 2013; Jiang et al., 2013a; Mali et al., 2013; Pattanayak et al., 2013).

 

Several strategies have been developed to reduce off-target genome editing, the most important of which is the considered design of the gRNA.

 

  • fusions of catalytically inactive Cas9 and FokI nuclease have been generated, and these show comparable efficiency to the nickases but substantially higher (N140-fold) specificity than the wild-type enzyme (Guilinger et al., 2014; Tsai et al., 2014)

 

  • Altering the length of the gRNA can also minimize non-target modifications. Guide RNAs with two additional guanidine residues at the 5′ end were able to avoid off-target sites more efficiently than normal gRNAs but were also slightly less active at on-target sites (Cho et al., 2014)

Development and Applications of CRISPR-Cas9 for Genome Engineeri

What more:

The CRISPR/Cas9 system can be used for several purposes in addition to genome editing:

  • The ectopic regulation of gene expression, which can provide useful information about gene functions and can also be used to engineer novel genetic regulatory circuits for synthetic biology applications.

 

  • The external control of gene expression typically relies on the use of inducible or repressible promoters, requiring the introduction of a new promoter and a particular treatment (physical or chemical) for promoter activation or repression.

 

  • Disabled nucleases can be used to regulate gene expression because they can still bind to their target DNA sequence. This is the case with the catalytically inactive version of Cas9 which is known as dead Cas9 (dCas9).

 

  • Preparing the host for an immunotherapy is possible if it is combined with TLR mechanism:

On the other hand, the host mechanism needs to be review carefully for the design of an effective outcome.

The mechanism of microbial response and infectious tolerance are complex.

 

During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells.

 

Uniqueness of TLR comes from four major characteristics of each individual TLR :

 

  1. ligand specificity,
  2. signal transduction pathways,
  3. expression profiles and
  4. cellular localization.

 

Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

 

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression  levels Specific TLR stimulat ion links innate and acquired responses through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.

 

Some examples of ligand – TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2, double stranded (ds) RNAs through TLR3, lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5, single stranded RNAs through TLR7/8, synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9.

 

The specificity is established by correct pairing of a TLR with its proinflammatory cytokine(s), so that these permutations influence creation and maintenance of cell differentiat ion.

Development and Applications of CRISPR-Cas9 for Genome Engineeri

 

  • Immunotherapy: The immune cells can be used as a sensor to scavenger the circulating malformed cells in vivo diagnostics or attack and remember them, for instance, relapse of cancer, re-infection with a same or similar agent (bacteria or virus) etc.

Not only using unique microbial and other model organism properties but also using the human host defense mechanism during innate immune responses may bring a new combat to create a new method of precision medicine. This can be a new type of immunotherapy, immune cell mediated gene therapy or vaccine even a step for an in vivo diagnostics.

 

Molecular Genetics took a long road from discovery of restriction enzymes, developing PCR assays, cloning were the beginning. Now, having technology to sequence and compare the sequences between organisms also help to design more sophisticated methods.

Generating mutant lines in Drosophila with the classical genetics methods relies on P elements, a type of transposon and balancers after crossing selected flies with specific markers. This fly pushing is a very tedious work but powerful to identify primary pathways, mechanisms and gene interactions in system and translational  genomics.

 Thus, Microbial Immunomodulation is an important factor not only using the microorganisms or their mechanisms but also modulating the immune cells based on the host interaction may generate new types of diagnostics and targeted therapy tools.

 

Microbial immunomodulation. Microbes from the environment, and from the various microbiota, modulate the immune system. Some of this is due to direct effects of defined microbial products on elements of the immune system. But modulation of the immune system also secondarily alters the host–microbiota relationship and leads to changes in the composition of the microbiota, and so to further changes in immunoregulation (shown as indirect pathways). At the end of the day balance is the key for survival.

microbial immunomodulationGrahamnihms199923f2 A. W. Rook,*,1 Christopher A. Lowry,2 and Charles L. Raison3  Microbial ‘Old Friends’, immunoregulation and stress resilience  Evol Med Public Health. 2013; 2013(1): 46–64. Published online 2013 Apr 9. doi:  10.1093/emph/eot004 PMCID: PMC3868387

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881665/bin/nihms199923f2.jpg

 

CRISPR-Cas9 mediated NHEJ in transient transfection experiments.

Table 1.
Species Transformation method Cas9 codon optimization Promoters (Cas9,  gRNA) Target Mutation frequency Detection method Off-target (no. of sites analyzed) Detection method Multiplex (deletion) Reference
Arabidopsis thaliana PEG-protoplast transfection Arabidopsis (with intron) CaMV35SPDK, AtU6 PDS3<comma> FLS2 1.1–5.6% PCR + sequencing Li et al. (2013)
A. thaliana Leaf agroinfiltration Arabidopsis (with intron) CaMV35SPDK, AtU6 PDS3 2.70% PCR + sequencing Yes (48 bp) Li et al. (2013)
A. thaliana PEG-protoplast transfection Arabidopsis (with intron) CaMV35SPDK,  AtU6 RACK1b<comma> RACK1c 2.5–2.7% PCR + sequencing No (1 site) PCR + sequencing Li et al. (2013)
A. thaliana Leaf agroinfiltration C. reinhardtii CaMV35S, AtU6 Co-transfected GFP n.a. Pre-digested PCR + RE Jiang et al. 2013a and Jiang et al. 2013b
Nicotiana benthamiana PEG-protoplast transfection Arabidopsis (with intron) CaMV35SPDK, AtU6 PDS3 37.7–38.5% PCR + sequencing Li et al. (2013)
N. benthamiana Leaf agroinfiltration Arabidopsis (with intron) CaMV35SPDK,  AtU6 PDS3 4.80% PCR + sequencing Li et al. (2013)
N. benthamiana Leaf agroinfiltration Human CaMV35S,  AtU6 PDS 1.8–2.4% PCR + RE No (18 sites) PCR + RE Nekrasov et al. (2013)
N. benthamiana Leaf agroinfiltration C. reinhardtii CaMV35S, AtU6 Co-transfected GFP n.a. pre-digested PCR + RE Jiang et al. 2013a and Jiang et al. 2013b
N. benthamiana Leaf agroinfiltration Human CaMV35S, CaMV35S PDS 12.7–13.8% Upadhyay et al. (2013)
Nicotiana tabacum PEG-protoplast transfection Tobacco 2xCaMV35S, AtU6 PDS<comma> PDR6 16.27–20.3% PCR + RE Yes (1.8 kb) Gao et al. (2014)
Oryza sativa PEG-protoplast transfection Rice 2xCaMV35S, OsU3 PDS<comma> BADH2<comma> MPK2<comma> Os02g23823 14.5–38.0% PCR + RE Noa (3 sites) PCR + RE Shan et al. (2013)
O. sativa PEG-protoplast transfection Human CaMV35S,  OsU3 or OsU6 MPK5 3–8% RE + qPCR and T7E1 assay No (2 sites) Yes (1 site with a mismatch at position 12) RE + PCR Xie and Yang (2013)
O. sativa PEG-protoplast transfection Rice CaMV35S,  OsU6 SWEET14 n.a. pre-digested PCR + RE Jiang et al. 2013a and Jiang et al. 2013b
O. sativa PEG-protoplast transfection Rice ZmUbi,  OsU6 KO1 KOL5; CPS4 CYP99A2; CYP76M5 CYP76M6 n.a. PCR + sequencing Yes (115<comma> 170<comma> 245 kb) Zhou et al. (2014)
Triticum aestivum PEG-protoplast transfection Rice 2xCaMV35S, TaU6 MLO 28.50% PCR + RE Shan et al. (2013)
T. aestivum PEG-protoplast transfection Plant ZmUbi, TaU6 MLO-A1 36% T7E1 Wang et al. 2014a and Wang et al. 2014b
T. aestivum Agrotransfection of cells from immature embryos Human CaMV35S,  CaMV35S PDS<comma> INOX 18–22% PCR + sequencing Upadhyay et al. (2013)
T. aestivum Agrotransfection of cells from immature embryos Human CaMV35S,  CaMV35S INOX PCR + sequencing No* PCR + RE Yes (53 bp) Upadhyay et al. (2013)
Zea mays PEG-protoplast transfection Rice 2xCaMV35S,  ZmU3 IPK 16.4–19.1% PCR + RE Liang et al. (2014)
Citrus sinensis Leaf agroinfiltration Human CaMv35S,  CaMV35S PDS 3.2–3.9% PCR + RE No (8 sites) PCR + RE Jia et al. (2014)

 

 

 

References:

A brief overview of CRISPR-mediated immunity and explain how the emerging new properties of this defense system are being repurposed for genome engineering in bacteria, yeast, human cells, insects, fish, worms, plants, frogs, pigs, and rodents.

Also look at F1000Prime Rep. 2014; 6: 3. For the list of microorganisms use in CRISPR applications.

Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41:7429–37. doi: 10.1093/nar/gkt520.

Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013;31:233–9. doi: 10.1038/nbt.2508.

Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013;152:1173–83. doi: 10.1016/j.cell.2013.02.022

Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods.2013;10:1116–21. doi: 10.1038/nmeth.2681.

Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.Cell. 2013;154:442–51. doi: 10.1016/j.cell.2013.06.044.

DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 2013;41:4336–43. doi: 10.1093/nar/gkt135.

Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res.2013;23:1163–71. doi: 10.1038/cr.2013.122.

 Hou Z, Zhang Y, Propson NE, Howden SE, Chu L, Sontheimer EJ, Thomson JA. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA. 2013;110:15644–9. doi: 10.1073/pnas.1313587110.

Ran FA, Hsu PD, Lin C, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154:1380–9. doi: 10.1016/j.cell.2013.08.021.

Cho SW, Kim S, Kim JM, Kim J. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31:230–2. doi: 10.1038/nbt.2507.

Le Cong, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013;339:819–23. doi: 10.1126/science.1231143.

Cradick TJ, Fine EJ, Antico CJ, Bao G. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res.2013;41:9584–92. doi: 10.1093/nar/gkt714.

Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell. 2013;12:393–4. doi: 10.1016/j.stem.2013.03.006.

Ebina H, Misawa N, Kanemura Y, Koyanagi Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep. 2013;3:2510. doi: 10.1038/srep02510.

Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat Biotechnol. 2013;31:822–6. doi: 10.1038/nbt.2623.

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–32. doi: 10.1038/nbt.2647.

Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. Elife. 2013;2:e00471. doi: 10.7554/eLife.00471.

Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013;10:977–9. doi: 10.1038/nmeth.2598.

Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013;31:833–8. doi: 10.1038/nbt.2675.

Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6. doi: 10.1126/science.1232033.

Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013;31:839–43. doi: 10.1038/nbt.2673.

Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods. 2013;10:973–6. doi: 10.1038/nmeth.2600.

Yang L, Guell M, Byrne S, Yang JL, Los Angeles A de, Mali P, Aach J, Kim-Kiselak C, Briggs AW, Rios X, Huang P, Daley G, Church G. Optimization of scarless human stem cell genome editing. Nucleic Acids Res. 2013;41:9049–61. doi: 10.1093/nar/gkt555.

Bassett AR, Tibbit C, Ponting CP, Liu J. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 2013;4:220–8. doi: 10.1016/j.celrep.2013.06.020.

Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O’Connor-Giles KM. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics. 2013;194:1029–35. doi: 10.1534/genetics.113.152710.

Yu Z, Ren M, Wang Z, Zhang B, Rong YS, Jiao R, Gao G. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics.2013;195:289–91. doi: 10.1534/genetics.113.153825.

Kondo S, Ueda R. Highly improved gene targeting by germline-specific cas9 expression in Drosophila. Genetics. 2013;195:715–21. doi: 10.1534/genetics.113.156737.

Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong J, Xi JJ. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 2013;23:465–72. doi: 10.1038/cr.2013.45.

Hwang WY, Fu Y, Reyon D, Maeder ML, Kaini P, Sander JD, Joung JK, Peterson RT, Yeh JJ. Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS ONE. 2013;8:e68708. doi: 10.1371/journal.pone.0068708.

Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JJ, Joung JK. Efficient genome editing in zebrafish using a CRISPR-Cas system.Nat Biotechnol. 2013;31:227–9. doi: 10.1038/nbt.2501.

Jao L, Wente SR, Chen W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA. 2013;110:13904–9. doi: 10.1073/pnas.1308335110.

Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, Zu Y, Li W, Huang P, Tong X, Zhu Z, Lin S, Zhang B. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 2013;41:e141. doi: 10.1093/nar/gkt464.

Chen C, Fenk LA, Bono M de. Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination. Nucleic Acids Res.2013;41:e193. doi: 10.1093/nar/gkt805.

Chiu H, Schwartz HT, Antoshechkin I, Sternberg PW. Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas. Genetics. 2013;195:1167–71. doi: 10.1534/genetics.113.155879.

Cho SW, Lee J, Carroll D, Kim J, Lee J. Heritable Gene Knockout in Caenorhabditis elegans by Direct Injection of Cas9-sgRNA Ribonucleoproteins.Genetics. 2013;195:1177–80. doi: 10.1534/genetics.113.155853.

Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods.2013;10:741–3. doi: 10.1038/nmeth.2532.

Katic I, Großhans H. Targeted Heritable Mutation and Gene Conversion by Cas9-CRISPR in Caenorhabditis elegans. Genetics. 2013;195:1173–6. doi: 10.1534/genetics.113.155754.

Lo T, Pickle CS, Lin S, Ralston EJ, Gurling M, Schartner CM, Bian Q, Doudna JA, Meyer BJ. Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions.Genetics. 2013;195:331–48. doi: 10.1534/genetics.113.155382.

Tzur YB, Friedland AE, Nadarajan S, Church GM, Calarco JA, Colaiácovo MP. Heritable Custom Genomic Modifications in Caenorhabditis elegans via a CRISPR-Cas9 System. Genetics. 2013;195:1181–5. doi: 10.1534/genetics.113.156075.

Waaijers S, Portegijs V, Kerver J, Lemmens BBLG, Tijsterman M, van den Heuvel S, Boxem M. CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans. Genetics. 2013;195:1187–91. doi: 10.1534/genetics.113.156299.

Dickinson DJ, Ward JD, Reiner DJ, Goldstein B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.Nat Methods. 2013;10:1028–34. doi: 10.1038/nmeth.2641.

Feng Z, Zhang B, Ding W, Liu X, Yang D, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu J. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res.2013;23:1229–32. doi: 10.1038/cr.2013.114.

Li J, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013;31:688–91. doi: 10.1038/nbt.2654.

Nekrasov V, Staskawicz B, Weigel D, Jones JDG, Kamoun S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31:691–3. doi: 10.1038/nbt.2655.

Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu J, Gao C. Targeted genome modification of crop plants using a CRISPR-Cas system.Nat Biotechnol. 2013;31:686–8. doi: 10.1038/nbt.2650.

 Xie K, Yang Y. RNA-Guided Genome Editing in Plants Using a CRISPR-Cas System. Mol Plant. 2013;6:1975–83. doi: 10.1093/mp/sst119.

Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu L. Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res. 2013;23:1233–6. doi: 10.1038/cr.2013.123.

Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 2013;41:e188. doi: 10.1093/nar/gkt780.

Upadhyay SK, Kumar J, Alok A, Tuli R. RNA Guided Genome Editing for Target Gene Mutations in Wheat. G3 (Bethesda) 2013

Nakayama T, Fish MB, Fisher M, Oomen-Hajagos J, Thomsen GH, Grainger RM. Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. Genesis. 2013 doi: 10.1002/dvg.22720.

Tan W, Carlson DF, Lancto CA, Garbe JR, Webster DA, Hackett PB, Fahrenkrug SC. Efficient nonmeiotic allele introgression in livestock using custom endonucleases. Proc Natl Acad Sci USA. 2013;110:16526–31. doi: 10.1073/pnas.1310478110.

Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, Zhao Y, Liu M. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system.Nat Biotechnol. 2013;31:681–3. doi: 10.1038/nbt.2661.

Li W, Teng F, Li T, Zhou Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol.2013;31:684–6. doi: 10.1038/nbt.2652.

Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 2013;23:720–3. doi: 10.1038/cr.2013.46.

Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153:910–8. doi: 10.1016/j.cell.2013.04.025.

 

Previously Published at Leaders in Pharmaceutical Intelligence:

 

CRISPR/Cas9: Contributions on Endoribonuclease Structure and Function, Role in Immunity and Applications in Genome Engineering larryhbern 2015/03/27
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CRISPR-CAS editing brings cloning of woolly mammoth one step closer to reality 2012pharmaceutical 2015/03/26
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GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases 2012pharmaceutical 2014/12/22
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The Patents for CRISPR, the DNA editing technology as the Biggest Biotech Discovery of the Century 2012pharmaceutical 2014/12/05
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CRISPR: Applications for Autoimmune Diseases @UCSF 2012pharmaceutical 2014/11/04
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“Gene Editing at CRISPR Speed”: Services and Tools 2012pharmaceutical 2014/10/29
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Licensing CRISPR-Cas9 Technology from Broad Institute: Clontech, Horizon Discovery, Sage Labs 2012pharmaceutical 2014/10/28
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CRISPR-Cas9 Discovery and Development of Programmable Genome Engineering – Gabbay Award Lectures in Biotechnology and Medicine – Hosted by Rosenstiel Basic Medical Sciences Research Center, 10/27/14 3:30PM Brandeis University, Gerstenzang 121 2012pharmaceutical 2014/10/26
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Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells using a bacterial CRISPR/Cas 2012pharmaceutical 2014/10/24
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CRISPR-Cas9 Foundational Technology originated at UC, Berkeley & UCSF, Broad Institute is developing Biotech Applications — Intellectual Property emerging as Legal Potential Dispute 2012pharmaceutical 2014/06/18
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2:15 – 2:45, 6/13/2014, Jennifer Doudna “The biology of CRISPRs: from genome defense to genetic engineering” 2012pharmaceutical 2014/06/13
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CRISPR @MIT – Genome Surgery 2012pharmaceutical 2014/04/21
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Gene Therapy and the Genetic Study of Disease: @Berkeley and @UCSF – New DNA-editing technology spawns bold UC initiative as Crispr Goes Global 2012pharmaceutical 2014/03/27
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Evaluate your Cas9 Gene Editing Vectors: CRISPR/Cas Mediated Genome Engineering – Is your CRISPR gRNA optimized for your cell lines? 2012pharmaceutical 2014/03/25
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CRISPR-Cas: A powerful new tool for precise genetic engineering 2012pharmaceutical 2013/11/29
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A NEW ERA OF GENETIC MANIPULATION   Demet Sag, Ph.D., CRA, GCP 31 mins ago
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Manipulate Signaling Pathways [7.6] larryhbern 2015/04/08
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RNAi – On Transcription and Metabolic Control larryhbern 2015/03/26
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Real Time Conference Coverage for Scientific and Business Media: Unique Twitter Hashtags and Handles per Conference Presentation/Session 2012pharmaceutical 2015/03/24
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Advances in Gene Editing Technology: New Gene Therapy Options in Personalized Medicine 2012pharmaceutical 2015/03/16
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Annual Margaret Pittman Lecture, honors the NIH’s first female lab chief, March 11, 2015, 3:00:00 PM by Jennifer Doudna, Ph.D., University of California, Berkeley 2012pharmaceutical 2015/03/11
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About the author:

Dr Sag has a Bachelor’s degree in Basic and Industrial Microbiology as a Sum cum Laude among 450 graduating class of Science faculty,  an MSc in Microbial Engineering and Biotechnology (Bioprocessing improvement) and PhD in Molecular and Developmental Genetics (Functional Genome and Stem Cell Biology).

She is an translational functional genomic scientist to develop diagnostics and targeted therapies by non-invasive methods for personalized medicine from bench to bedside and engineering tools through clinical trials and regulatory affairs.

You may contact with her at 858-729-4942 or by demet.sag@gmail.com if you have questions.

 

 

Read Full Post »


Upcoming Meetings on Cancer Immunogenetics

 

Curator: Stephen J. Williams, Ph.D.

Below is a curation of upcoming 2014-15 Cancer Immunogenetics symposia. Some listed have CME credits.

August 2014

Target Discovery for T Cell Therapy Symposium
Next Step to Advance Immunotherapies
August 14, 2014 | Part of ImVacS – The Immunotherapies and Vaccine Summit
Learn more | View Agenda PDF | Register by July 18 & SAVE up to $200

 

Q&A with Dr. Adrian Bot of Kite Pharma

 

SITC 2014 Meetings

The Society for Immunotherapy of Cancer (SITC) is a 501 (c)(3) non-profit society of medical professionals. Recent advances in immunology and biology have opened up new horizons in the field of cancer therapy, with an upsurge in the integration of new biologic agents into clinical practice. With several high-caliber scientific meetings with a focus on clinical and translational aspects of biologic approaches to cancer treatment and numerous networking opportunities unique to this organization, the Society for Immunotherapy of Cancer (SITC) has developed into the premier destination for interaction and innovation in the cancer biologics community.

Upcoming SITC Meetings and Activities

sitc banner

Advances in Cancer Immunotherapy™ (ACI™) Regional CME-Certified Programs

  • La Jolla, CA – Friday, August 22, 2014
  • Portland, OR – Friday, October 3, 2014
    Charlotte, NC – Friday, October 3, 2014
  • Tampa, FL – Friday, December 5, 2014

 ACI

September 2014

 

 aacrmeetinghemoto2014

  Hematologic Malignancies: Translating Discoveries to Novel Therapies
    September 20-23, 2014 • Sheraton Philadelphia Downtown • Philadelphia, PA

The AACR is proud to announce our conference focused on the blood-based cancers and associated disorders categorized as hematologic malignancies. Sessions will include presentations on leukemia, lymphoma, myeloma, myelodysplastic syndrome, and myeloproliferative neoplasms.

 

Advances in Melanoma: From Biology to Therapy

Loews Philadelphia • Philadelphia, PA • September 20-23, 2014

With so many recent advances in treating metastatic melanoma, including approaches like immunotherapies, targeted therapies, and combination therapies, melanoma research is at a critical point where it is extremely important for the field to have a continuous exchange of information. Despite the success of various “targeted” inhibitors, therapeutic responses in melanoma patients are often short-lived due to rapidly acquired drug resistance. Therefore, it is essential that melanoma researchers translate the novel understanding of melanoma biology to decipher the mechanisms of innate and acquired drug resistance for the development of improved therapeutic options. To bridge the gap between scientists and clinician-scientists’ professional practice, this conference will provide a platform for discussion and potential collaborations for the discovery of new therapeutic targets.

 

 proimmunegif

The 4th Mastering Immunogenicity Summit

September 15-16, 2014

British Consulate-General, Boston MA, USA

Join leaders in the immunogenicity field for a two day conference to learn what constitutes a successful strategy for managing immunogenicity risk, and explore the business case for introducing immunogenicity assessment into your program.

  • Learn about the latest strategies and exciting new technologies
  • Discuss current and developing challenges and exchange new ideas
  • Improve the outcome of your R&D programs

Our 4th Mastering Immunogenicity Conference will continue to have a strong focus on immunogenicity sciences, particularly on what basic research needs to be carried out to improve our understanding of immune regulation to biotherapeutics. We will review progress made in correlating data from pre-clinical predictive tools to clinical outcomes, as well as continuing our discussions surrounding the benefits that Quality by Design has on reduced immunogenicity, considering subsequent patient benefits as well as competitive advantage. Presentations by experts will provide an overview of the wide range of technologies currently used for immunogenicity risk management and how they can be incorporated for a ‘quality by design’ approach.

 

Immunogenomics 2014

September 29 – October 1, 2014

HudsonAlpha Biotechnology Campus
Huntsville, Alabama, USA

The HudsonAlpha-Science Conference on Immunogenomics will bring together preeminent leaders and thinkers at the intersection of genomics and immunology.

October 2014

canerrersinstlogo

Cancer Immunotherapy: Out of the Gate

October 06, 2014 Grand Hyatt New York Hotel at Grand Central, New York, NY

The Cancer Research Institute (CRI) will host its 22nd Annual International Cancer Immunotherapy Symposium October 6-8, 2014 at The Grand Hyatt in New York City. Attracting clinicians, laboratory scientists, postdoctoral fellows, and graduate students, the symposium will feature plenary presentations from leaders in immunology and cancer immunotherapy, a poster session, and numerous networking opportunities.

This year’s CRI symposium, entitled Cancer Immunotherapy: Out of the Gate, will harness the excitement and enthusiasm generated by recent clinical successes to explore new and emerging areas of basic, translational, and clinical research. Topics such as the use of genomic methods to catalogue cancer heterogeneity, mechanistic studies of checkpoint blockage antibodies, new views on immunosurveillance and immunoregulation, and emerging therapies that are altering the landscape of cancer treatment will be discussed.

– See more at: http://www.cancerresearch.org/grants-programs/conferences-meetings/annual-international-cancer-immunotherapy-symposia/2014-symposium#sthash.PnY56e5E.dpuf

Cytokines 2014

October 26–29, Melbourne, Australia

EMBO Conference: Innate Lymphoid Cells
September 29–October 1, Paris, France

Recommended reading

Laurie Dempsey

 

November 2014

SITC 2014 – November 6-9, 2014

  • Gaylord National Hotel & Convention Center, National Harbor, MD
  • SITC 29th Annual Meeting
  • SITC Workshop on Combination Immunotherapy: Where Do We Go From Here?
  • SITC Primer on Tumor Immunology and Cancer Immunotherapy™
  • SITC Hot Topic Symposium – including two topics explored concurrently:
    • Accelerating Tumor Immunity with Agonist Antibodies
    • Engineered T Cell Toxicities
  • Professional Development Session: A Roadmap for Thriving in Your Career

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The Delicate Connection:  IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

Author and Curator: Demet Sag, PhD, CRA, GCP      

Table of Contents:

  1. Abstract
  2. Dual role for IDO
  3. Immune System and IDO
  4. Autoimmune disorders and IDO
  5. Cancer and Ido
  6. Clinical Interventions
  7. Clinical Trials
  8. Future Actions for Molecular Dx and Targeted Therapies:
  9. Conclusion
  10. References

TABLE 1- IDO Clinical Trials

TABLE 2- Kyn induced Genes

TABLE 3 Possible biomarkers and molecular diagnostics targets

TABLE 4: Current Interventions ______________________________________________________________________________________________________________

ABSTRACT:

Overall purpose is to find a method to manipulate IDO for clinical applications, mainly the focus of this review is is cancer prevention and treatment.  The first study proving the connection between IDO and immune response came from, a very natural event, a protection of pregnancy in human. This led to discover that high IDO expression is a common factor in cancer tumors. Thus, attention promoted investigations on IDO’s role in various disease states, immune disorders, transplantation, inflammation, women health, mood disorders.
Many approaches, vaccines and adjuvants are underway to find new immunotherapies by combining the power of DCs in immune response regulation and specific direction of siRNA.  As a result, with this unique qualities of IDO, DCs and siRNA, we orchestrated a novel intervention for immunomodulation of IDO by inhibiting with small interference RNA, called siRNA-IDO-DCvax.  Proven that our DCvax created a delay and regression of tumor growth without changing the natural structure and characterization of DCs in melanoma and breast cancers in vivo. (** The shRNA IDO- DCvax is developed by Regen BioPhrama, San Diego, CA ,  Thomas Ichim, Ph.D, CSO. and David Koos, CEO)

______________________________________________________________________________________________________________

Double-Edged Sword of IDO: The Good and The Bad for Clinical intervention and Developments

IDO almost has a dual role. There is a positive side of high expression of IDO during pregnancy (29; 28; 114), transplants (115; 116; 117; 118; 119), infectious diseases (96) and but this tolerance is negative during autoimmune-disorders (120; 121; 122), tumors of cancer (123; 124; 117; 121; 125; 126; 127) (127), and mood disorders (46). The increased IDO expression has a double-edged sword in human physiology provides a positive role during protection of fetus and grafts after transplantations but becomes a negative factor during autoimmune disorders, cancer, sepsis and mood disorders.

Prevention of allogeneic fetal rejection is possible by tryptophan metabolism (26) rejecting with lack of IDO but allocating if IDO present (29; 28; 114). These studies lead to find “the natural regulation mechanism” for protecting the transplants from graft versus host disease GVHD (128) and getting rid of tumors.

The plasticity of  mammary and uterus during reproduction may hold some more answers to prevent GVHD and tumors of cancer with good understanding of IDO and tryptophan mechanism (129; 130). After allogeneic bone marrow transplants the risk of solid tumor development increased about 80% among 19,229 patients even with a greater risk among patients under 18 years old (117).  The adaptation of tolerance against host mechanism is connected to the IDO expression (131). During implantation and early pregnancy IDO has a role by making CD4+CD25+Foxp3+ regulatory T cells (Tregs) and expressing in DCs and  MQs  (114; 132; 133).

Clonal deletion mechanism prevents mother to react with paternal products since female mice accepted the paternal MHC antigen-expressing tumor graft during pregnancy and rejected three weeks after delivery (134). CTLA-4Ig gene therapy alleviates abortion through regulation of apoptosis and inhibition of spleen lymphocytes (135).  

 Immune System and IDO DCs are the orchestrator of the immune response (56; 57; 58) with list of functions in uptake, processing, and presentation of antigens; activation of effector cells, such as T-cells and NK-cells; and secretion of cytokines and other immune-modulating molecules to direct the immune response. The differential regulation of IDO in distinct DC subsets is widely studied to delineate and correct immune homeostasis during autoimmunity, infection and cancer and the associated immunological outcomes. Genesis of antigen presenting cells (APCs), eventually the immune system, require migration of monocytes (MOs), which is originated in bone marrow. Then, these MOs move from bloodstream to other tissues to become macrophages and DCs (59; 60).

Initiation of immune response requires APCs to link resting helper T-cell with the matching antigen to protect body. DCs are superior to MQs and MOs in their immune action model. When DCs are first described (61) and classified, their role is determined as a highly potent antigen-presenting cell (APC) subset with 100 to 1000-times more effective than macrophages and B-cells in priming T-cells. Both MQs and monocytes phagocytize the pathogen, and their cell structure contains very large nucleus and many internal vesicles. However, there is a nuance between MQ and DCs, since DCs has a wider capacity of stimulation, because MQs activates only memory T cells, yet DCs can activate both naïve and memory T cells.

DCs are potent activators of T cells and they also have well controlled regulatory roles. DC properties determine the regulation regardless of their origin or the subset of the DCs. DCs reacts after identification of the signals or influencers for their inhibitory, stimulatory or regulatory roles, before they express a complex repertoire of positive and negative cytokines, transmembrane proteins and other molecules. Thus, “two signal theory” gains support with a defined rule.  The combination of two signals, their interaction with types of cells and time are critical.

In short, specificity and time are matter for a proper response. When IDO mRNA expression is activated with CTL40 ligand and IFNgamma, IDO results inhibition of T cell production (4).  However, if DCs are inhibited by 1MT, an inhibitor of IDO, the response stop but IgG has no affect (10).  In addition, if the stimulation is started by a tryptophan metabolite, which is downstream of IDO, such as 3-hydroxyantranilic or quinolinic acids, it only inhibits Th1 but not Th2 subset of T cells (62).

Furthermore, inclusion of signal molecules, such as Fas Ligand, cytochrome c, and pathways also differ in the T cell differentiation mechanisms due to combination, time and specificity of two-signals.  The co-culture experiments are great tool to identify specific stimuli in disease specific microenvironment (63; 12; 64) for discovering the mechanism and interactions between molecules in gene regulation, biochemical mechanism and physiological function during cell differentiation.

As a result, the simplest differential cell development from the early development of DCs impact the outcome of the data. For example, collection of MOs from peripheral blood mononuclear cells (PBMCs) with IL4 and GM-CSF leads to immature DCs (iDCs). On next step, treatment of iDCs with tumor necrosis factor (TNF) or other plausible cytokines (TGFb1, IFNgamma, IFNalpha,  IFNbeta, IL6 etc.) based on the desired outcome differentiate iDCs  into mature DCs (mDCs). DCs live only up to a week but MOs and generated MQs can live up to a month in the given tissue. B cells inhibit T cell dependent immune responses in tumors (65).

AutoImmune Disorders:

The Circadian Clock Circuitry and the AHR

The balance of IDO expression becomes necessary to prevent overactive immune response self-destruction, so modulation in tryptophan and NDA metabolisms maybe essential.  When splenic IDO-expressing CD11b (+) DCs from tolerized animals applied, they suppressed the development of arthritis, increased the Treg/Th17 cell ratio, and decreased the production of inflammatory cytokines in the spleen (136).

The role of Nicotinamide prevention on type 1 diabetes and ameliorates multiple sclerosis in animal model presented with activities of  NDAs stimulating GPCR109a to produce prostaglandins to induce IDO expression, then these PGEs and PGDs converted to the anti-inflammatory prostaglandin, 15d-PGJ(2) (137; 138; 139).  Thus, these events promotes endogenous signaling mechanisms involving the GPCRs EP2, EP4, and DP1 along with PPARgamma. (137).

Modulating the immune response at non-canonical at canonocal pathway while keeping the non-canonical Nf-KB intact may help to mend immune disorders. As a result, the targeted blocking in canonical at associated kinase IKKβ and leaving non-canonocal Nf-kB pathway intact, DCs tips the balance towards immune supression. Hence, noncanonical NF-κB pathway for regulatory functions in DCs required effective IDO induction, directly or indirectly by endogenous ligand Kyn and negative regulation of proinflammatory cytokine production. As a result, this may help to treat autoimmune diseases such as rheumatoid arthritis, type 1 diabetes, inflammatory bowel disease, and multiple sclerosis, or allergy or transplant rejection.

While the opposite action needs to be taken during prevention of tumors, that is inhibition of non-canonical pathway.  Inflammation induces not only relaxation of veins and lowering blood pressure but also stimulate coagulopathies that worsen the microenvironment and decrease survival rate of patients after radio or chemotherapies.Cancer Generating tumor vaccines and using adjuvants underway (140).

Clinical correlation and genetic responses also compared in several studies to diagnose and target the system for cancer therapies (127; 141; 131).  The recent surveys on IDO expression and human cancers showed that IDO targeting is a candidate for cancer therapy since IDO expression recruiting Tregs, downregulates MHC class I and creating negative immune microenvironment for protection of development of tumors (125; 27; 142).  Inhibition of IDO expression can make advances in immunotherapy and chemotherapy fields (143; 125; 131; 144).

IDO has a great importance on prevention of cancer development (126). There are many approaches to create the homeostasis of immune response by Immunotherapy.  However, given the complexity of immune regulations, immunomodulation is a better approach to correct and relieve the system from the disease.  Some of the current IDO targeted immunotherapy or immmunomodulations with RNA technology for cancer prevention (145; 146; 147; 148; 149; 150) or applied on human or animals  (75; 151; 12; 115; 152; 9; 125) or chemical, (153; 154) or  radiological (155).  The targeted cell type in immune system generally DCs, monocytes (94)T cells (110; 156)and neutrophils (146; 157). On this paper, we will concentrate on DCvax on cancer treatments.

 T-reg, regulatory T cells; Th, T helper; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; TCR, T cell receptor; IDO, indoleamine 2,3-dioxygenase. (refernece: http://www.pnas.org/content/101/28/10398/suppl/DC)

T-reg, regulatory T cells; Th, T helper; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; TCR, T cell receptor; IDO, indoleamine 2,3-dioxygenase. (refernece: http://www.pnas.org/content/101/28/10398/suppl/DC)

IDO and the downstream enzymes in tryptophan pathway produce a series of immunosuppressive tryptophan metabolites that may lead into Tregs proliferation or increase in T cell apoptosis (62; 16; 27; 158), and some can affect NK cell function (159).

The interesting part of the mechanism is even without presence of IDO itself, downstream enzymes of IDO in the kynurenine tryptophan degradation still show immunosuppressive outcome (160; 73) due to not only Kyn but also TGFbeta stimulated long term responses. DC vaccination with IDO plausible (161) due to its power in immune response changes and longevity in the bloodstream for reversing the system for Th17 production (162).

Clinical Interventions are taking advantage of the DC’s central role and combining with enhancing molecules for induction of immunity may overcome tolerogenic DCs in tumors of cancers (163; 164).

The first successful application of DC vaccine used against advanced melanoma after loading DCs with tumor peptides or autologous cell lysate in presence of adjuvants keyhole limpet hematocyanin (KLH) (165).  Previous animal and clinical studies show use of DCs against tumors created success (165; 166; 167) as well as some problems due to heterogeneity of DC populations in one study supporting tumor growth rather than diminishing (168).

DC vaccination applied onto over four thousand clinical trial but none of them used siRNA-IDO DC vaccination method. Clinical trials evaluating DCs loaded ex vivo with purified TAAs as an anticancer immunotherapeutic interventions also did not include IDO (Table from (169). This table presented the data from 30 clinical trials, 3 of which discontinued, evaluating DCs loaded ex vivo with TAAs as an anticancer immunotherapy for 12 types of cancer [(AML(1), Breast cancer (4), glioblastoma (1), glioma (2), hepatocellular carcinoma (1), hematological malignancies (1), melanoma (6), neuroblastoma sarcoma (2), NSCLC (1), ovarian cancer (3), pancreatic cancer (3), prostate cancer (10)] at phase I, II or I/II.

Tipping the balance between Treg and Th17 ratio has a therapeutic advantage for restoring the health that is also shown in ovarian cancer by DC vaccination with adjuvants (161).  This rebalancing of the immune system towards immunogenicity may restore Treg/Th17 ratio (162; 170) but it is complicated. The stimulation of IL10 and IL12 induce Treg produce less Th17 and inhibiting CTL activation and its function (76; 171; 172) while animals treated with anti-TGFb before vaccination increase the plasma levels of IL-15 for tumor specific T cell survival in vivo (173; 174) ovarian cancer studies after human papilloma virus infection present an increase of IL12 (175).

Opposing signal mechanism downregulates the TGFb to activate CTL and Th1 population with IL12 and IL15 expression (162; 173).  The effects of IL17 on antitumor properties observed by unique subset of CD4+ T cells (176) called also CD8+ T cells secrete even more IL17 (177).

Using cytokines as adjuvants during vaccination may improve the efficacy of vaccination since cancer vaccines unlike infections vaccines applied after the infection or disease started against the established adoptive immune response.  Adjuvants are used to improve the responses of the given therapies commonly in immunotherapy applications as a combination therapy (178).

Enhancing cancer vaccine efficacy via modulation of the microenvironment is a plausible solution if only know who are the players.  Several molecules can be used to initiate and lengthen the activity of intervention to stimulate IDO expression without compromising the mechanism (179).  The system is complicated so generally induction is completed ex-vivo stimulation of DCs in cell lysates, whole tumor lysates, to create the microenvironment and natural stimulatory agents. Introduction of molecules as an adjuvants on genetic regulation on modulation of DCs are critical, because order and time of the signals, specific location/ tissue, and heterogeneity of personal needs (174; 138; 180). These studies demonstrated that IL15 with low TGFb stimulates CTL and Th1, whereas elevated TGFb with IL10 increases Th17 and Tregs in cancer microenvironments.

IDO and signaling gene regulation

For example Ret-peptide antitumor vaccine contains an extracellular fragment of Ret protein and Th1 polarized immunoregulator CpG oligonucleotide (1826), with 1MT, a potent inhibitor of IDO, brought a powerful as well as specific cellular and humoral immune responses in mice (152).

The main idea of choosing Ret to produce vaccine in ret related carcinomas fall in two criterion, first choosing patients self-antigens for cancer therapy with a non-mutated gene, second, there is no evidence of genetic mutations in Ret amino acids 64-269. Demonstration of proliferating hemangiomas, benign endothelial tumors and often referred as hemangiomas of infancy appearing at head or neck, express IDO and slowly regressed as a result of immune mediated process.

After large scale of genomic analysis show insulin like growth factor 2 as the key regulator of hematoma growth (Ritter et al. 2003). We set out to develop new technology with our previous expertise in immunotherapy and immunomodulation (181; 182; 183; 184), correcting Th17/Th1 ratio (185), and siRNA technology (186; 187).  We developed siRNA-IDO-DCvax. Patented two technologies “Immunomodulation using Altered DCs (Patent No: US2006/0165665 A1) and Method of Cancer Treatments using siRNA Silencing (Patent No: US2009/0220582 A1).

In melanoma cancer DCs were preconditioned with whole tumor lysate but in breast cancer model pretreatment completed with tumor cell lysate before siRNA-IDO-DCvax applied. Both of these studies was a success without modifying the autanticity of DCs but decreasing the IDO expression to restore immunegenity by delaying tumor growth in breast cancer (147) and in melanoma (188).  Thus, our DCvax specifically interfere with Ido without disturbing natural structure and content of the DCs in vivo showed that it is possible to carry on this technology to clinical applications.

Furthermore, our method of intervention is more sophisticated since it has a direct interaction mechanism with ex-vivo DC modulation without creating long term metabolism imbalance in Trp/Kyn metabolite mechanisms since the action is corrective and non-invasive.

There were several reasons.

First, prevention of tumor development studies targeting non-enzymatic pathway initiated by pDCs conditioned with TGFbeta is specific to IDO1 (189).

Second, IDO upregulation in antigen presenting cells allowing metastasis show that most human tumors express IDO at high levels (123; 124).

Third, tolerogenic DCs secretes several molecules some of them are transforming growth factor beta (TGFb), interleukin IL10), human leukocyte antigen G (HLA-G), and leukemia inhibitory factor (LIF), and non-secreted program cell death ligand 1 (PD-1 L) and IDO, indolamine 2.3-dioxygenase, which promote tumor tolerance. Thus, we took advantage of DCs properties and Ido specificity to prevent the tolerogenicity with siRNA-IDO DC vaccine in both melanoma and breast cancer.

Fourth, IDO expression in DCs make them even more potent against tumor antigens and create more T cells against tumors. IDOs are expressed at different levels by both in broad range of tumor cells and many subtypes of DCs including monocyte-derived DCs (10), plasmacytoid DCs (142), CD8a+ DCs (190), IDO compotent DCs (17), IFNgamma-activated DCs used in DC vaccination.  These DCs suppress immune responses through several mechanisms for induction of apoptosis towards activated T cells (156) to mediate antigen-specific T cell anergy in vivo (142) and for enhancement of Treg cells production at sites of vaccination with IDO-positive DCs+ in human patients (142; 191; 192; 168; 193; 194). If DCs are preconditioned with tumor lysate with 1MT vaccination they increase DCvax effectiveness unlike DCs originated from “normal”, healthy lysate with 1MT in pancreatic cancer (195).  As a result, we concluded that the immunesupressive effect of IDO can be reversed by siRNA because Treg cells enhances DC vaccine-mediated anti-tumor-immunity in cancer patients.

Gene silencing is a promising technology regardless of advantages simplicity for finding gene interaction mechanisms in vitro and disadvantages of the technology is utilizing the system with specificity in vivo (186; 196).  siRNA technology is one of the newest solution for the treatment of diseases as human genomics is only producing about 25,000 genes by representing 1% of its genome. Thus, utilizing the RNA open the doors for more comprehensive and less invasive effects on interventions. Thus this technology is still improving and using adjuvants. Silencing of K-Ras inhibit the growth of tumors in human pancreatic cancers (197), silencing of beta-catenin in colon cancers causes tumor regression in mouse models (198), silencing of vascular endothelial growth factor (VGEF) decreased angiogenesis and inhibit tumor growth (199).

Combining siRNA IDO and DCvax from adult stem cell is a novel technology for regression of tumors in melanoma and breast cancers in vivo. Our data showed that IDO-siRNA reduced tumor derived T cell apoptosis and tumor derived inhibition of T cell proliferation.  In addition, silencing IDO made DCs more potent against tumors since treated or pretreated animals showed a delay or decreased the tumor growth (188; 147)

 

Clinical Trials:

First FDA approved DC-based cancer therapies for treatment of hormone-refractory prostate cancer as autologous cellular immunotherapy (163; 164).  However, there are many probabilities to iron out for a predictive outcome in patients.

Table 2 demonstrates the current summary of clinical trials report.  This table shows 38 total studies specifically Ido related function on cancer (16), eye (3), surgery (2), women health (4), obesity (1), Cardiovascular (2), brain (1), kidney (1), bladder (1), sepsis shock (1), transplant (1),  nervous system and behavioral studies (4), HIV (1) (Table 4).  Among these only 22 of which active, recruiting or not yet started to recruit, and 17 completed and one terminated.

Most of these studies concentrated on cancer by the industry, Teva GTC ( Phase I traumatic brain injury) Astra Zeneca (Phase IV on efficacy of CRESTOR 5mg for cardiovascular health concern), Incyte corporation (Phase II ovarian cancer) NewLink Genetics Corporation Phase I breast/lung/melanoma/pancreatic solid tumors that is terminated; Phase II malignant melanoma recruiting, Phase II active, not recruiting metastatic breast cancer, Phase I/II metastatic melanoma, Phase I advanced malignancies) , HIV (Phase IV enrolling by invitation supported by Salix Corp-UC, San Francisco and HIV/AIDS Research Programs).

Many studies based on chemotherapy but there are few that use biological methods completed study with  IDO vaccine peptide vaccination for Stage III-IV non-small-cell lung cancer patients (NCT01219348), observational study on effect of biological therapy on biomarkers in patients with untreated hepatitis C, metastasis melanoma, or Crohn disease by IFNalpha and chemical (ribavirin, ticilimumab (NCT00897312), polymorphisms of patients after 1MT drug application in treating patients with metastatic or unmovable refractory solid tumors by surgery (NCT00758537), IDO expression analysis on MSCs (NCT01668576), and not yet recruiting intervention with adenovirus-p53 transduced dendric cell vaccine , 1MT , radiation, Carbon C 11 aplha-methyltryptophan- (NCT01302821).

Among the registered clinical trials some of them are not interventional but  observational and evaluation studies on Trp/Kyn ratio (NCT01042847), Kyn/Trp ratio (NCT01219348), Kyn levels (NCT00897312, NCT00573300),  RT-PCR analysis for Kyn metabolism (NCT00573300, NCT00684736, NCT00758537), and intrinsic IDO expression of mesenchymal stem cells in lung transplant with percent inhibition of CD4+ and CD8+ T cell proliferation toward donor cells (NCT01668576), determining polymorphisms (NCT00426894). These clinical trials/studies are immensely valuable to understand the mechanism and route of intervention development with the data collected from human populations   

Future Actions for Molecular Dx and Targeted Therapies:

Viable tumor environment. Tumor survival is dependent upon an exquisite interplay between the critical functions of stromal development and angiogenesis, local immune suppression and tumor tolerance, and paradoxical inflammation. TEMs: TIE-2 expressing monocytes; “M2” TAMs: tolerogenic tumor-associated macrophages; MDSCs: myeloid-derived suppressor cells; pDCs: plasmacytoid dendritic cells; co-stim.: co-stimulation; IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; MMP: matrix metaloprotease; IL: interleukin; TGF-β: transforming growth factor-beta; TLRs: toll-like receptors.  (reference: http://www.hindawi.com/journals/cdi/2012/937253/fig1/)

Viable tumor environment. Tumor survival is dependent upon an exquisite interplay between the critical functions of stromal development and angiogenesis, local immune suppression and tumor tolerance, and paradoxical inflammation. TEMs: TIE-2 expressing monocytes; “M2” TAMs: tolerogenic tumor-associated macrophages; MDSCs: myeloid-derived suppressor cells; pDCs: plasmacytoid dendritic cells; co-stim.: co-stimulation; IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; MMP: matrix metaloprotease; IL: interleukin; TGF-β: transforming growth factor-beta; TLRs: toll-like receptors. (reference: http://www.hindawi.com/journals/cdi/2012/937253/fig1/)

Current survival or response rate is around 40 to 50 % range.  By using specific cell type, selected inhibition/activation sequence based on patient’s genomic profile may improve the efficacy of clinical interventions on cancer treatments. Targeted therapies for specific gene regulation through signal transduction is necessary but there are few studies with genomics based approach.

On the other hand, there are surveys, observational or evaluations (listed in clinical trials section) registered with www.clinicaltrials.gov that will provide a valuable short-list of molecules.  Preventing stimulation of Ido1 as well as Tgfb-1gene expression by modulating receptor mediated phosphorylation between TGFb/SMAD either at Mad-Homology 1 (MH1) or Mad-Homology 1 (MH2) domains maybe possible (79; 82; 80). Within Smads are the conserved Mad-Homology 1 (MH1) domain, which is a DNA binding module contains tightly bound Zinc atom.

Smad MH2 domain is well conserved and one the most diverse protein-signal interacting molecule during signal transduction due to two important Serine residues located extreme distal C-termini at Ser-Val-Ser in Smad 2 or at pSer-X-PSer in RSmads (80). Kyn activated orphan G protein–coupled receptor, GPR35 with unknown function with a distinct expression pattern that collides with IDO sites since its expression at high levels of the immune system and the gut (63) (200; 63).  

The first study to connect IDO with cancer shows that group (75).  The directly targeting to regulate IDO expression is another method through modulating ISREs in its promoter with RNA-peptide combination technology. Indirectly, IDO can be regulated through Bin1 gene expression control over IDO since Bin1 is a negative regulator of IDO and prevents IDO expression.  IDO is under negative genetic control of Bin1, BAR adapter–encoding gene Bin1 (also known as Amphiphysin2). Bin1 functions in cancer suppression since attenuation of Bin1 observed in many human malignancies (141; 201; 202; 203; 204; 205; 206) .  Null Bin-/- mice showed that when there is lack of Bin1, upregulation of IDO through STAT1- and NF-kB-dependent expression of IDO makes tumor cells to escape from T cell–dependent antitumor immunity.

This pathway lies in non-enzymatic signal transducer function of IDO after stimulation of DCs by TGFb1.  The detail study on Bin1 gene by alternative spicing also provided that Bin1 is a tumor suppressor.  Its activities also depends on these spliced outcome, such as  Exon 10, in muscle, in turn Exon 13 in mice has importance in role for regulating growth when Bin1 is deleted or mutated C2C12 myoblasts interrupted due to its missing Myc, cyclinD1, or growth factor inhibiting genes like p21WAF1 (207; 208).

On the other hand alternative spliced Exon12A contributing brain cell differentiation (209; 210). Myc as a target at the junction between IDO gene interaction and Trp metabolism.  Bin1 interacts with Myc either early-dependent on Myc or late-independent on Myc, when Myc is not present. This gene regulation also interfered by the long term signaling mechanism related to Kynurenine (Kyn) acting as an endogenous ligand to AHR in Trp metabolite and TGFb1 and/or IFNalpha and IFNbeta up regulation of DCs to induce IDO in noncanonical pathway for NF-kB and myc gene activations (73; 74).  Hence, Trp/Kyn, Kyn/Trp, Th1/Th17 ratios are important to be observed in patients peripheral blood. These direct and indirect gene interactions place Bin1 to function in cell differentiation (211; 212; 205).

Regulatory T-cel generation via reverse and non-canonical signaliing to pDCs

Table 3 contains the microarray analysis for Kyn affect showed that there are 25 genes affected by Kyn, two of which are upregulated and 23 of them downregulated (100). This list of genes and additional knowledge based on studies creating the diagnostics panel with these genes as a biomarker may help to analyze the outcomes of given interventions and therapies. Some of these molecules are great candidate to seek as an adjuvant or co-stimulation agents.  These are myc, NfKB at IKKA, C2CD2, CREB3L2, GPR115, IL2, IL8, IL6, and IL1B, mir-376 RNA, NFKB3, TGFb, RelA, and SH3RF1. In addition, Lip, Fox3P, CTLA-4, Bin1, and IMPACT should be monitored.

In addition, Table 4 presents the other possible mechanisms. The highlights of possible target/biomarkers are specific TLRs, conserved sequences of IDO across its homologous structures, CCR6, CCR5, RORgammat, ISREs of IDO, Jak, STAT, IRFs, MH1 and MH2 domains of Smads. Endothelial cell coagulation activation mechanism and pDC maturation or immigration from lymph nodes to bloodstream should marry to control not only IDO expression but also genesis of preferred DC subsets. Stromal mesenchymal cells are also activated by these modulation at vascular system and interferes with metastasis of cancer. First, thrombin (human factor II) is a well regulated protein in coagulation hemostasis has a role in cell differentiation and angiogenesis.

Protein kinase activated receptors (PARs), type of GPCRs, moderate the actions. Second, during hematopoietic response endothelial cells produce hematopoietic growth factors (213; 214). Third, components of bone marrow stroma cells include monocytes, adipocytes, and mesenchymal stem cells (215). As a result, addressing this issue will prevent occurrence of coagulapathologies, namely DIC, bleeding, thrombosis, so that patients may also improve response rate towards therapies. Personal genomic profiles are powerful tool to improve efficacy in immunotherapies since there is an influence of age (young vs. adult), state of immune system (innate vs. adopted or acquired immunity). Table 5 includes some of the current studies directly with IDO and indirectly effecting its mechanisms via gene therapy, DNA vaccine, gene silencing and adjuvant applications as an intervention method to prevent various cancer types.

CONCLUSION

IDO has a confined function in immune system through complex interactions to maintain hemostasis of immune responses. The genesis of IDO stem from duplication of bacterial IDO-like genes.  Inhibition of microbial infection and invasion by depleting tryptophan limits and kills the invader but during starvation of trp the host may pass the twilight zone since trp required by host’s T cells.  Thus, the host cells in these small pockets adopt to new microenvironment with depleted trp and oxygen poor conditions. Hence, the cell metabolism differentiate to generate new cellular structure like nodules and tumors under the protection of constitutively expressed IDO in tumors, DCs and inhibited T cell proliferation.

On the other hand, having a dichotomy in IDO function can be a potential limiting factor that means is that IDOs impact on biological system could be variable based on several issues such as target cells, IDO’s capacity, pathologic state of the disease and conditions of the microenvironment. Thus, close monitoring is necessary to analyze the outcome to prevent conspiracies since previous studies generated paradoxical results.

Current therapies through chemotherapies, radiotherapies are costly and effectiveness shown that the clinical interventions require immunotherapies as well as coagulation and vascular biology manipulations for a higher efficacy and survival rate in cancer patients. Our siRNA and DC technologies based on stem cell modulation will provide at least prevention of cancer development and hopefully prevention in cancer.

11.       References

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3. Importance of the Two Interferon-stimulated Response Element. Konan KV, Taylor, MW. 1996, J. Biol. Chem.-, pp. 19140-5.

4. Induction of indolamine 2,3 dioxygenase: A mechanism of the anti-tumor activity of interferon gamma. Ozaki, Y., Edelstein, M.P., Duch, D.S. 1998, PNAS USA., pp. vol:85, 1242-1246.

5. Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome . Burkin, D. J., Kimbro, K. S., Barr, B. L., Jones, C., Taylor, M. W., Gupta, S. L. 1993, Genomics , pp. 17: 262-263.

6. Localization of indoleamine 2,3-dioxygenase gene (INDO) to chromosome 8p12-p11 by fluorescent in situ hybridization. Najfeld, V., Menninger, J., Muhleman, D., Comings, D. E., Gupta, S. L. 1993, Cytogenet. Cell Genet. , pp. 64: 231-232.

7. Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA.  Dai, W., Gupta, S. L. 1990, Biochem. Biophys. Res. Commun. , pp. 168: 1-8.

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