Aggressive brain tumors can originate from a range of nervous system cells

ScienceDaily (Oct. 22, 2012) — Scientists have long believed that glioblastoma multiforme (GBM), the most aggressive type of primary brain tumor, begins in glial cells that make up supportive tissue in the brain or in neural stem cells. In a paper published October 17 in Science, however, researchers at the Salk Institute for Biological Studies have found that the tumors can originate from other types of differentiated cells in the nervous system, including cortical neurons.

GBM is one of the most devastating brain tumors that can affect humans. Despite progress in genetic analysis and classification, the prognosis of these tumors remains poor, with most patients dying within one to two years of diagnosis. The Salk researcher's findings offer an explanation for the recurrence of GBM following treatment and suggest potential new targets to treat these deadly brain tumors.

"One of the reasons for the lack of clinical advances in GBMs has been the insufficient understanding of the underlying mechanisms by which these tumors originate and progress," says Inder Verma, a professor in Salk's Laboratory of Genetics and the Irwin and Joan Jacobs Chair in Exemplary Life Science.

To better understand this process, Verma's team harnessed the power of modified viruses, called lentiviruses, to disable powerful tumor suppressor genes that regulate the growth of cells and inhibit the development of tumors. With these tumor suppressors deactivated, cancerous cells are given free rein to grow out of control.

To do that, Verma and his colleagues attached small RNA molecules, known as short hairpin RNAs, to the modified viruses and injected them directly into very few cells in the brains of genetically engineered mice that express an enzyme known as CRE specifically in neurons, astrocytes or neural stem cells. The modified viruses target two genes -- -neurofibromatosis 1 (NF1) and p53 -- -that, when mutated, are implicated in severe gliomas like GBM. Using sophisticated analytical techniques, they discovered that neurons genetically converted by the lentiviruses that also produce green fluorescent protein (GFP) as a marker to track the progression of tumors are capable of forming malignant gliomas.

Because the origin of glioblastomas from neurons has not been previously reported, the Salk scientists provided further evidence that mature neurons can be transformed by these oncogenes by isolating cortical neurons from genetically engineered mice and transducing them with one of the lentiviruses. The neurons that were transplanted back into the mice developed the same tumors as the ones in the laboratory.

"Our findings," says lead author Dinorah Friedmann-Morvinski, a postdoctoral researcher in the Laboratory of Genetics, "suggest that, when two critical genes -- -NF-1 and p53 -- -are disabled, mature, differentiated cells acquire the capacity to reprogram [dedifferentiate] to a neuroprogenitor cell-like state, which can not only maintain their plasticity, but also give rise to the variety of cells observed in malignant gliomas."

If scientists can block the process of dedifferentiation or proliferation of dedifferentiated neuroprogenitor cells, they may be able to stop tumor progression. That's important in an aggressive disease like GBM because of its high rate of recurrence.

"Our results offer an explanation of recurrence of gliomas following treatment," says Verma, "because any tumor cell that is not eradicated can continue to proliferate and induce tumor formation, thereby perpetuating the cycle of continuous cell replication to form malignant gliomas."

The scientists say the tumors in their mouse model are similar to GBMs that affect humans. Because they have the same pathology and characteristic genetic signature, scientists can study potential therapies in mice that should, theoretically, work in humans. While they may not eradicate GBM, these therapies may slow the progression of the disease and improve patients' quality of life.

Other researchers on the study were Eugene Ke, Yasushi Soda, Tomotoshi Marumoto and Oded Singer of the Salk Institute; and Eric Bushong and Mark Ellisman of the University of California, San Diego.

The work was supported by the National Institutes of Health, Ipsen/Biomeasure, the Leona M. and Harry B. Helmsley Charitable Trust, the H.N. and Frances C. Berger Foundation, and the National Center for Research Resources.

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The above story is reprinted from materials provided by Salk Institute for Biological Studies, via Newswise.

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D. Friedmann-Morvinski, E. A. Bushong, E. Ke, Y. Soda, T. Marumoto, O. Singer, M. H. Ellisman, I. M. Verma. Dedifferentiation of Neurons and Astrocytes by Oncogenes Can Induce Gliomas in Mice. Science, 2012; DOI: 10.1126/science.1226929

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Immune cells make flexible choices

ScienceDaily (Oct. 22, 2012) — Our immune system must be tremendously complex to respond to the unending assault of viruses, bacteria and cancerous cells. One of the mechanisms used by the immune system to cope with the huge variety of possible threats is to randomly combine DNA segments for the production of receptors on lymphocytes -- a type of white blood cell. The number of possible receptors that can be produced in this way is about 1000 times the number of stars in our galaxy -- one followed by 15 zeroes. And yet, the actual array of receptors produced does not conform to this picture of random chance: Some receptors are produced at a higher rate than others.

New research at the Weizmann Institute can help explain how the immune system maintains its complexity while giving preference to certain receptors.

The research team headed by Dr. Nir Friedman, including postdoctoral fellows Drs. Wilfred Ndifon and Hilah Gal, together with Prof. Ruth Arnon and Dr. Rina Aharoni, all of the Immunology Department, looked at the DNA sequences for receptors in immune cells called T lymphocytes. These receptors identify disease agents so they can be destroyed by the immune system. The genetic sequences encoding these receptors are each composed of three random DNA segments -- something like the random lineups in a slot machine. Each of those segments is taken from a different area of the lymphocyte cell genome; each area has a full "menu" of segments to choose from. The assembly of the sequence takes place when the DNA strand folds, bringing a segment from the first area close to those in the second and third areas. The sequence is then cut and pasted together, and the excess bits of DNA in between discarded, thus creating a new and unique genetic sequence for the receptors in each lymphocyte cell.

In a study that appeared recently in the Proceedings of the National Academy of Sciences (PNAS), USA, the team used a system they developed based on advanced high-throughput sequencing techniques to investigate the genetic sequences of an entire array of lymphocyte receptors in mice. With this "panoramic view," the researchers were able to assess how widespread each receptor was and even to suggest a reason for the uneven distribution. It appears that the secret is in the pieces of DNA that eventually get discarded: Both the length of these segments and their flexibility -- a function of the protein "packaging" that gives them shape -- determine how likely it is that two distant segments will meet.

With this insight, the researchers created a model that can predict the production distribution of receptors based on the distance between segments and the flexibility of the DNA. They then looked at small groups of individuals -- up to five -- to see if they could find common lymphocyte receptor sequences among them. Surprisingly, the team discovered that a group of five was more likely to all share a common sequence than smaller sub-groups. That may seem like saying there is a higher chance of winning at the slot machine five times in a row than twice, but the scientists can explain this finding based on the preferences revealed. The common sequences may simply be situated in the genome in such a way that they are more likely to be integrated into the receptor sequences. Such sequences may have been selected by evolution for their ability to fight common disease agents or prevent autoimmune disease.

Friedman: "While our immune system often seems to rely on 'luck' to produce random receptors against a long list of threats, the shared receptors suggest that this mechanism is finely tuned to ensure a response to common diseases."

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W. Ndifon, H. Gal, E. Shifrut, R. Aharoni, N. Yissachar, N. Waysbort, S. Reich-Zeliger, R. Arnon, N. Friedman. Chromatin conformation governs T-cell receptor J  gene segment usage. Proceedings of the National Academy of Sciences, 2012; 109 (39): 15865 DOI: 10.1073/pnas.1203916109

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New glow for electron microscopy: Protein-labeling technique allows high-resolution visualization of molecules inside cells

ScienceDaily (Oct. 22, 2012) — The glowing green molecule known as green fluorescent protein (GFP) has revolutionized molecular biology. When GFP is attached to a particular protein inside a cell, scientists can easily identify and locate it using fluorescence microscopy. However, GFP can't be used with electron microscopy, which offers much higher resolution than fluorescence microscopy.

Chemists from MIT have now designed a GFP equivalent for electron microscopy -- a tag that allows scientists to label and visualize proteins with unprecedented clarity.

"With things that may appear only a few pixels across by fluorescence microscopy -- for example, a mitochondrion -- you can't make out any of the internal features. But with electron microscopy it's very easy to discern the intricate internal structures," says Jeff Martell, a graduate student in chemistry at MIT and lead author of a paper describing the new tag in the Oct. 21 online edition of Nature Biotechnology.

The new tag could help scientists pinpoint the locations of many cell proteins, providing new insight into those proteins' functions, according to the researchers.

Improving on nature

Dubbed APEX, the new tag is similar to naturally occurring proteins that have been tried as imaging labels for electron microscopy. Horseradish peroxidase (HRP) is one commonly used tag, but it works only in a few compartments of a cell. Other recently developed tags work throughout a cell but are technically challenging to use because they require light to be shined on the sample and oxygen to be bubbled through it.

To improve on these methods, the researchers started with a protein similar to HRP, called ascorbate peroxidase (APX). APX is more versatile than HRP because it can function within a cell's cytosol, in the main cavity of a cell.

Both HRP and APX belong to a class of enzymes called peroxidases, which remove an electron and a proton from other molecules in a process known as oxidation. Every peroxidase has different targets, and one of HRP's main targets is a molecule called DAB, which when oxidized can be visualized with electron microscopy. The researchers genetically engineered APX so that it would also target DAB.

To use this new APEX tag (for "engineered APX"), the researchers deliver, into a living cell, a small ring of DNA containing the APEX gene joined to the gene for the protein they plan to image. The cell then produces the target protein, bound to the APEX protein.

Next, the researchers need to deliver DAB, which is not normally found in cells. This delivery takes place during the process of "fixing," or stabilizing cells, which must be done before they can be imaged with electron microscopy.

When the APEX protein oxidizes DAB, it generates radicals that rapidly clump together into a tarlike polymer. That polymer can be detected through electron microscopy, allowing the researchers to pinpoint the location of the target protein.

Biological question resolved

To demonstrate the usefulness of their new tag, the researchers set out to resolve an open question regarding the location of a calcium channel protein discovered last year. Two research groups identified the protein and reported that it is located within mitochondria, but they had conflicting theories as to its precise location and orientation. Using the new imaging technique, the MIT-led team labeled the protein and determined that it is embedded in the inner mitochondrial membrane and faces into the innermost part of mitochondria, the mitochondrial matrix.

The team also showed that the new tag can label proteins throughout the cell -- not only within mitochondria but also in the nucleus, the endoplasmic reticulum and the cytosol.

Martell and Alice Ting, the Ellen Swallow Richards Associate Professor of Chemistry at MIT and senior author of the Nature Biotechnology paper, invented the new technology. Other authors who helped to test the tag and explore biological applications are Mark Ellisman, Thomas Deerinck and Gina Sosinsky of the University of California at San Diego, Yasemin Sancak and Vamsi Mootha of Harvard Medical School, and Thomas Poulos of the University of California at Irvine.

In current studies, the researchers are working on filling entire cells, such as neurons, with their imaging agent. This allows certain neurons in an electron microscope image to stand out, making it easier to trace the connections they make with other neurons. For that project, the MIT researchers are collaborating with Joshua Sanes, a professor of molecular and cellular biology at Harvard University, who says he believes the new labeling technology will be very useful.

"We want to find the exact connections that these cells are making, and APEX is a good way to label cells for electron microscopy. We can label specific types of cells and figure out how they fit into the neural circuitry," Sanes says.

Ting and Martell have filed for a patent on their imaging technology and are now working on making the APEX molecule more stable and better able to bind heme (an iron atom embedded in an organic compound), which is necessary for it to function properly.

The research was funded by the National Institutes of Health.

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The above story is reprinted from materials provided by Massachusetts Institute of Technology. The original article was written by Anne Trafton.

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Journal Reference:

Jeffrey D Martell, Thomas J Deerinck, Yasemin Sancak, Thomas L Poulos, Vamsi K Mootha, Gina E Sosinsky, Mark H Ellisman, Alice Y Ting. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nature Biotechnology, 2012; DOI: 10.1038/nbt.2375

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No survival advantage with peripheral blood stem cells versus bone marrow, study suggests

ScienceDaily (Oct. 19, 2012) — Claudio Anasetti, M.D., chair of the Department of Blood & Marrow Transplant at Moffitt Cancer Center, and colleagues from 47 research sites in the Blood and Marrow Transplant Clinical Trials Network conducted a two-year clinical trial comparing two-year survival probabilities for patients transplanted with peripheral blood stem cells or bone marrow stem cells from unrelated donors. The goal was to determine whether graft source, peripheral blood stem cells or bone marrow, affects outcomes in unrelated donor transplants for patients with leukemia or other hematologic malignancies.

Fifty transplant centers in the United States and Canada participated in this phase III study, which randomized 278 patients to receive bone marrow and 273 patients to receive peripheral blood stem cells as the graft source for transplant. The results of the study are in the Oct. 18 issue of The New England Journal of Medicine.

According to the trial analyses, there were no observed differences in overall survival, relapse, non-relapse mortality, or acute graft-versus-host disease (GHVD) between the patients receiving peripheral blood stem cells or bone marrow stem cells from unrelated donors. GVHD is a serious and often deadly post-transplant complication that occurs when the newly transplanted donor cells attack the transplant recipient's body. While engraftment was faster in patients receiving peripheral blood stem cells, there was a higher incidence of overall chronic GVHD in these patients (53 percent) than in those transplanted with bone marrow stem cells (40 percent). Patients receiving transplants of peripheral blood stem cells from unrelated donors also had a higher incidence of chronic GVHD affecting multiple organs (46 percent) than patients who received bone marrow stem cells (31 percent).

"Although peripheral blood stem cells from related donors have demonstrated clinical benefits, our trial demonstrates that when these stem cells originate from unrelated donors, they are not superior to bone marrow stem cells in terms of patient survival, and they increase the risk for chronic GVHD," said Anasetti, lead study author. "More effective strategies to prevent GVHD are needed to improve outcomes for all patients receiving unrelated donor transplants."

Peripheral blood stem cells are stem cells originally found in the bone marrow that have been moved into the blood stream by a special regimen of drugs. Unlike bone marrow stem cells, which must be extracted from the bones in an operating room, peripheral blood stem cells are more easily obtained through apheresis, a process similar to regular blood donation, which collects the peripheral blood stem cells through a tube inserted in a vein. A critical step before the transplant involves finding a donor that is tissue matched to the recipient.

About one-third of patients who need a peripheral blood stem cell or bone marrow transplant for treatment of leukemia or another blood disease are able to secure a related donor. According to the National Marrow Donor Program, for the 70 percent who cannot find a donor within their family, most will be able to find an unrelated donor. Because the majority of transplant patients need cells from unrelated donors, it's necessary to better understand the risks associated with transplants of unrelated donor cells.

Clinical trials on related donor transplants have demonstrated that peripheral blood stem cell transplants in patients with leukemia and other blood diseases result in better engraftment, lower relapse rates, and increased survival compared with transplants with bone marrow stem cells. However, those trials also found that peripheral blood stem cell transplants carry an increased risk of GVHD. Patients who survive early post-transplant may develop chronic GVHD, a disabling condition managed with long-term immunosuppressant therapy.

Many transplant centers are increasingly using peripheral blood stem cells as a source for adult stem cells because of their superiority in clinical trials that have directly compared outcomes between peripheral blood stem cells and bone marrow stem cells from related donors. However, there has not been a comparative study of the two transplant sources that has prospectively analyzed patient outcomes in unrelated donor transplants.

The study was funded by the National Heart, Lung and Blood Institute (U10HL069294), the National Cancer Institute and the National Marrow Donor Program.

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The above story is reprinted from materials provided by H. Lee Moffitt Cancer Center & Research Institute.

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Claudio Anasetti, Brent R. Logan, Stephanie J. Lee, Edmund K. Waller, Daniel J. Weisdorf, John R. Wingard, Corey S. Cutler, Peter Westervelt, Ann Woolfrey, Stephen Couban, Gerhard Ehninger, Laura Johnston, Richard T. Maziarz, Michael A. Pulsipher, David L. Porter, Shin Mineishi, John M. McCarty, Shakila P. Khan, Paolo Anderlini, William I. Bensinger, Susan F. Leitman, Scott D. Rowley, Christopher Bredeson, Shelly L. Carter, Mary M. Horowitz, Dennis L. Confer. Peripheral-Blood Stem Cells versus Bone Marrow from Unrelated Donors. New England Journal of Medicine, 2012; 367 (16): 1487 DOI: 10.1056/NEJMoa1203517

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A New Way to Make Reprogrammed Stem Cells

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New technique for sorting live cells may expedite biomedical research

ScienceDaily (Oct. 17, 2012) — Researchers from North Carolina State University and University of North Carolina at Chapel Hill have developed a new technique that uses sound waves to rapidly separate selected collections of cells for use in biomedical research.

"We think this is important because it will make it faster and easier for researchers to sort out the live cells they need for research ranging from disease study to drug development," says Dr. Xiaoning Jiang, an associate professor of mechanical and aerospace engineering and adjunct professor of biomedical engineering at NC State and co-author of a paper on the work.

Biomedical research often focuses on how specific cell types respond to various chemicals or environmental factors. These cells are often grown in a liquid medium and on top of a collection of "micropallets," which are essentially small plastic platforms that sit on the substrate at the bottom of the container. Researchers then select the cells they want and detach the relevant micropallets, which can be removed for additional experimentation or analysis.

Current techniques for removing these micropallets rely on lasers or physical manipulation to separate the pallets from the substrate. But each approach has its drawbacks. Physical manipulation is a slow process, while the energy produced by lasers to release larger micropallets (e.g., a micropallet 500 micrometers in diameter) can inadvertently kill a significant number of the cells. Neither technique is efficient at detaching a significant number of large micropallets quickly.

The new technique from NC State uses ultrasound technology to release the micropallets. Specifically, it uses focused, relatively high-frequency sound waves that are translated into a wave of pressure within the substrate itself. When that wave of force hits a targeted micropallet, the pallet is lifted off the substrate and can be removed, together with its attached cells, for further study.

Using this technique, micropallets can be selectively released in less than a millisecond. This is not as fast as laser-based techniques, but is much faster than physical manipulation. However, the ultrasound technique has a viability rate of better than 90 percent, meaning that more than 90 percent of live cells survive the process. This is significantly better than existing techniques for the release of large-sized pallets, which can have viability rates of less than 50 percent.

Video: https://www.youtube.com/watch?v=pVIeabhU1OY

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The above story is reprinted from materials provided by North Carolina State University.

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Sijia Guo, Yuli Wang, Nancy Allbritton and Xiaoning Jiang. Ultrasound-induced release of micropallets with cells. Applied Physics Letters, Volume 101 / Issue 16 (2012) DOI: 10.1063/1.4757648

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Could Stem Cells Treat Autism? Newly Approved Study May Tell

By Mary Brophy Marcus
HealthDay Reporter

FRIDAY, Oct. 12 (HealthDay News) -- Autism researchers have been given the go-ahead by the U.S. Food and Drug Administration to launch a small study in children with autism that evaluates whether a child's own umbilical cord blood may be an effective treatment.

Thirty children with the disorder, aged 2 to 7, will receive injections of their own stem cells from umbilical cord blood banked by their parents after their births. All of the cord blood comes from the Cord Blood Registry, the world's largest stem cell bank.

Scientists at Sutter Neuroscience Institute, in Sacramento, Calif., said the placebo-controlled study will evaluate whether the stem cell therapy helps improve language and behavior in the youngsters.

There is anecdotal evidence that stem cell infusions may have a benefit in other conditions such as cerebral palsy, said lead study investigator Dr. Michael Chez, director of pediatric neurology at the institute.

"We're hoping we'll see in the autism population a group of patients that also responds," Chez said. Other autism and stem cell research is going on abroad, but this study is the first to use a child's own cord blood stem cells.

Chez said the study will involve only patients whose autism is not linked to a genetic syndrome or brain injury, and all of the children will eventually receive the stem cells.

Two infusions will take place during the 13-month study. At the start of the research, the children will be split into two groups, half receiving an infusion of cord blood stem cells and half receiving a placebo. At six months, the groups will swap therapies. The infusions will be conducted on an outpatient basis with close monitoring, Chez said.

"We're working with Sutter Children's Hospital, who does our oncology infusions with the same-age children," he said. "They are very experienced nurses who work with preschool and school-age kids to help them get through medical experiences."

Each child and his or her parents will be given a private room with a television and videos, beverages, and perhaps a visit from the hospital's canine therapy dog, and then a topical anesthetic will be applied to the arm to numb the skin before intravenous needle placement. A hematology expert will be giving the infusions and monitoring for safety, said Chez, who noted that each child will be watched closely for an hour and a half before heading home. They will be seen the next day as well for a safety check.

At six, 12 and 24 weeks, the researchers will measure behavioral and language changes in the children, and other changes noted by parents and the children's doctors will be logged as well.

"Parental observations like socialization and irritability scale as secondary measures are important," Chez said. Brain electrical activity and immune markers such as levels of pro-inflammatory proteins in the blood also will be tracked.

The FDA-approved study is being funded by a grant from the Cord Blood Registry Corporation, but they are not involved in the hands-on research, Chez said. The Sutter Institute for Medical Research is supporting the research with supplies and staff as well, he added.

One in 88 children -- about five times as many boys as girls -- are diagnosed with autism, according to the U.S. Centers for Disease Control and Prevention. But its cause is unknown and there is no cure for the disorder, which begins in early childhood and affects social, behavioral and language development.

Genetics and environmental and immunological factors are thought to play a role, and possibly the interaction of these and other variables, Chez said. He pointed out that autism is not actually one disorder, but stretches across a spectrum in which some children have more severe symptoms than others. One theory suggests autism occurs because cells in the brain, called neurons, are not connecting normally. Chez said stem cells may address this issue.

Chez is particularly interested in the relationship between the nervous and immune systems, and said stem cells from cord blood have been used to treat some cancers and immune disorders. In some children with autism, spinal fluid tests and brain tests have indicated that immune problems exist, and, he said, "We hope this therapy may correct some of those deficiencies."

Dr. Andrew Adesman, chief of developmental and behavioral pediatrics at Steven and Alexandra Cohen Children's Medical Center of New York, said this is a well-designed preliminary study looking at whether or not cord blood can be helpful. Adesman was not involved in the study.

"There are mixed opinions about this approach," Adesman said. "A lot of research suggests that there may be an immunological component involved in autism, but some are skeptical this specific approach will be effective. If nothing else, this study is designed such that we should have preliminary results in about a year's time."

Even if the results are positive, he said, it could take a while before the general population has access to treatments.

"The media often trumpets new findings that are hopeful, but obviously there can be significant time before research translates to the bedside. Not every finding is applicable to all patients," he said. "There are elements of premature hope in many stories."

Chez also cautioned that this is very early research.

"You can't rush good science," he said. "We don't want to give people false hope. Part of doing this is to decide whether we should be doing this."

Copyright © 2012 HealthDay. All rights reserved. SOURCES: Michael Chez, M.D., director, pediatric neurology, Sutter Neuroscience Institute, Sacramento, Calif.; Andrew Adesman, M.D., chief, developmental and behavioral pediatrics, Steven and Alexandra Cohen Children's Medical Center of New York, New Hyde Park; Sutter Neuroscience Institute/Cord Blood Registry, news release, Aug. 21, 2012

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