The review showed, for example, that for every 50-year-old woman whose life is prolonged by mammography, dozens are treated unnecessarily — some with harmful consequences — or treated without benefit. Hundreds are told they have breast cancer when they do not.

Cindy M. Grimm, PhD, associate professor of computer science and engineering in the School of Engineering & Applied Science at Washington University in St. Louis, was not surprised by the review, a  Cochrane review of the scientific evidence for a medical treatment.

“It’s not just the mammogram that’s the problem,” she says, “it’s accurately interpreting the mammogram.

“People aren’t good at it. Even expert radiologists aren’t good at it. Results vary widely from person to person, even when people have gone through the same training.”

But Grimm thought a perceptual trick she and colleagues had invented, called subtle gaze direction, might be used to improve training.

An experiment showed that a novice could be subtly guided to follow an expert’s scanpath across a mammogram and that this subtle nudging improved the novice’s accuracy.

The experimental results will be presented at the Eye Tracking Research & Application Symposium this March.

Grimm and her colleagues say the technique, should it prove durable, is widely applicable to visual search tasks. Not only might it improve the reading of mammograms and other types of medical images, such as MRIs and PET scans, but it might also be used to improve the accuracy of airport screening and learning in virtual environments.

Directing the gaze

Grimm invented subtle gaze direction together with colleagues Reynold Bailey, PhD, then her graduate student, and Ann McNamara, PhD, then of Saint Louis University, a conference acquaintance.

“I had double-majored in art and computer science as an undergraduate at the University of California, Berkeley,” Grimm says. “So I was aware that artists have all sorts of tricks for guiding viewers to look at particular areas in a painting, sometimes, in the case of narrative art, in a particular sequence.

“They might make an area brighter than the background, increase the contrast or have strong edges (borders) that attract the eye.

“Movie producers do the same thing in post processing,” Grimm says. “For example, when one actor is talking and others are listening, the audience tends to watch the talker. But the producer can direct attention to a listener’s reaction instead by changing the color or brightness of that part of the image.”

Subtle gaze direction is a high-tech version of this time-honored craft. It works, says Grimm, by exploiting the difference between peripheral and central (foveal) vision.

“We use a small area in the central part of our retina called the fovea to see detail,” she says. “But foveal vision doesn’t actually cover much of our field of view.

“If you hold out your thumb, your foveal vision — the part of your surroundings you’re actually seeing in detail — covers about the same area as your thumbnail.

“We use our foveal vision to read or drive or for other detail-oriented tasks. At the same time, we are monitoring the rest of our environment with our peripheral vision, which has lower resolution but responds faster than our foveal vision.

“When our peripheral vision picks up a stimulus, our eyes move to focus our foveal vision on it so that we can see it clearly.

“During those quick eye movements, called saccades, vision is suppressed, or masked, so that the motion of the eye, the motion blur of the image and the gap in visual perception are not noticeable to the viewer. We lose an astonishing 40 minutes of vision a day to saccadic masking.”

“Perhaps in that case, as well, gaze direction could be used to train novice pollen identifiers.”

To direct the gaze, Grimm and her colleagues changed the brightness or “warmth” of an area in the peripheral field of view to draw the novice’s focus to this area.

The stimulus remained subtle, however, because the viewer’s gaze is monitored in real-time by an eye-tracking device and the modulations to the peripheral vision are terminated before the eye fixates on them.

“The idea,” says Grimm “is to get someone to look in a particular direction while altering their experience of viewing the image as little as possible.”

“In the case of mammograms,” for example, “you want to get a learner to look at the tumor region but you don’t want to do anything that makes the tumor region look different than it does on the mammogram itself.”

The mammography study

Reading mammograms is a good target for computer assistance because training is time-consuming and expensive, typically requiring a four-year residency and a two-year fellowship.

Despite advances in technology, novices are still trained by working as an apprentice to an expert.

The mammography study, led by Bailey, now an assistant professor of computer science at the Rochester Institute of Technology, brought together the same group of scientists as the subtle gaze direction experiment. McNamara is now assistant professor of visualization at Texas A&M University.

For the study, Grimm and her colleagues used a database of images provided by the Mammographic Image Analysis Society that includes both images and text files that contains coordinates of abnormalities and their size.

“Expert diagnostic radiologists have a particular search pattern that is not the same as that of a novice,” Grimm says. “We don’t know exactly what they’re doing, but they tend to do a fairly broad scan and then fixate on parts of the image that have a tumor-like texture. A novice might instead attend to brighter spots in the image or fail to scan all of it.”

Bailey hired an expert radiologist at the Rochester Institute of Technology to view and mark 65 images from the database. The expert’s scanpath was recorded during this process by an eye-tracking system.

During the experiment, subtle gaze direction was used to guide a group of novices along the expert scanpath. A control group viewed the mammograms without gaze manipulation.

Novices who were guided were significantly more accurate than the control group or a third group guided along a random path. Moreover, even though the training session was brief, the effect lingered even after gaze manipulation was disabled.

Grimm says more work must be done to show that more extensive training will stick long-term. In the meantime, she can think of many ways gaze manipulation could be used to improve performance on visual search tasks.

“One simple use of the technology would be to make sure readers look at every part of the image. If you’re using eye tracking,” she says, “you know where people are looking, so you can make sure they don’t skip part of the image.”

Gaze manipulation might also be used to assist tumor-recognition software. “Suppose you had a software program that was reasonably good at spotting possible tumor areas but, erring on the side of caution, flagged too many areas as suspicious.

“Such software might be paired with gaze direction to ensure the radiologist looked at all of the flagged areas,” she says. “That wouldn’t necessarily be a training application; it could be a routine element of reading mammograms.”

The mammogram study is widely applicable, Grimm says, because there are so many visual search tasks. She mentions airport scanners, but they are just at the top of a long list.

“I work with someone who identifies pollen species,” she says. “Apparently, it takes a novice a year to learn, and they spend hours and hours looking through a microscope at these pollen grains. Again, some people are good at it and others struggle for competence.
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“What is most exciting to me about this stunning discovery is that it may finally give us a handle by which to discover the physical laws of cellular motion as they apply to biology,” says Mina Bissell, a leading authority on breast cancer and Distinguished Scientist with Berkeley Lab’s Life Sciences Division.

Bissell is a corresponding author of a paper describing this work in theProceedings of the National Academy of Sciences (PNAS), along with Kandice Tanner, a post-doctoral physicist in Bissell’s research group. The PNAS paper is titled “Coherent angular motion in the establishment of multicellular architecture of glandular tissues.” Other authors were Hidetoshi Mori, Rana Mroue and Alexandre Bruni-Cardoso, also members of Bissell’s research group.

Healthy human epithelial cells in breast and other glandular tissue form either sphere-shaped acini or tube-shaped ducts. The cell and tissue polarity (function-enabling spatial orientations of cellular and tissue structures) that comes with the formation of acini is essential for the health and well-being of the breast. Loss of this polarity as a result of cells not forming spheres is one of the earliest signs of malignancy. However, despite all that is known about cell morphogenesis, the fundamental question as to how epithelial cells are able to assemble into spheres that are similar in size and shape to organs in vivo has until now been a mystery.

“We’ve discovered a novel type of cell motility where single cells undergo multiple rotations and cohesively maintain that rotational motion as they divide and assemble into acini,” says Tanner. “We’ve also demonstrated that this CAMo is a critical function for the establishment of spherical architecture and not simply a consequence of multicellular aggregates. If CAMo is disrupted, the final geometry is not a sphere.”

Working with both immortalized and primary human epithelial cells, cultured in a unique 3D gel that serves as a surrogate for the basement membrane (an assay developed by Bissell and colleagues two decades ago), and using 4D live-imaging (3D plus time) confocal microscopy, Tanner, Bissell and their colleagues found that CAMo arises from a centripetal force generated by the flexing of crescent-shaped muscle-like molecules called actomyosin in the cell’s cytoskeleton. This centripetal force sets the cell to rotating about an axis. The rotation is slow, barely once an hour, it may run clockwise or counterclockwise, and its axis might shift, but this rotational motion is cohesive. It continues as the cell divides and the subsequent progeny form into acini, bestowing on cells and acini the polarity and the cavity needed for proper form and function.

“Without CAMo, the cells lose their way and do not form structures that allow mammary cells to make and secrete milk,” says Tanner. “In order to form a polarized sphere, the cells have to be properly oriented so that certain components are up and certain components are down. The CAMo rotation provides the cells with this orientation.”

Bissell is renowned for her pioneering work that elucidated the critical role in breast cancer development played by the extracellular matrix (ECM), a network of fibrous and globular proteins in the microenvironment that surrounds a breast cell. Her experiments have shown that when the nucleus of a breast cell fails to receive the proper biochemical cues and signals from the ECM and other components of the microenvironment, cells and tissue lose structure, which opens the door to malignancy. The discovery of CAMo now provides an important missing mechanism that facilitates the reception and response of a breast cell to the cues and signals from the ECM.

“In addition to wanting to know how a single cell and its progeny assemble into polar tissue, we also wanted to know whether cellular dynamics are corrupted by malignant transformation,” Bissell says. “In this study, we found that malignant cells do not display CAMo but instead become randomly motile and do not form spheres.”

In recent research, Bissell and her group demonstrated that through manipulation of the ECM, malignant cells cultured in an ECM enriched with laminin — a protein that they had shown induces cell quiescence — can undergo a reversion in which their normal phenotype is restored despite their malignant genome. In this new study, Tanner, Bissell and their colleagues found that when malignant cells cultured in the 3D ECM surrogate gel underwent phenotypic reversion in response to signaling inhibitors, CAMo was restored. When CAMo was restored, the reverted cancer cells formed polarized spheres.

“These results complement our early hypothesis that signaling and support by the ECM when cells are in proper context informs both form and function in cells,” Bissell says. “The results also suggest that in response to microenvironmental cues from the ECM, cells execute a program of cytoskeletal movements that dictate different kinds of motilities. We hypothesize that these motilities direct the formation of a given type of tissue and preclude other multicellular geometries. We believe this is a crucial evolutionary phenomena for multicellular organisms.”

In this new study, Tanner and Bissell and their colleagues were surprised to observe a significant delay between the second and third round of breast cell divisions in the 3D ECM surrogate gel. This mitotic delay is similar to the mitotic delay that’s been observed during human blastocyst formation and is critical for normal embryogenesis. Tanner says the delay is probably necessary for the progeny to acquire sufficient adhesion so that the CAMo can be maintained for the adhere cells. This finding may provide a possible explanation for how the mammary gland reorganizes after each pregnancy and involution.

“Once the cells are sufficiently adhered to one another, they can continue CAMo as a cohesive unit,” Tanner says. “We postulate that this cohesive CAMo motility is the mechanism by which the original structure of the breast tissue is restored following lactation and breast feeding.”

The next step for the research team will be to study the effects of CAMo from the perspective of the ECM.

“We would like to look at the interaction of the ECM with a single cell as it undergoes CAMo and show the in vivorelevance,” Tanner says.

This research was supported by the U.S. Department of Defense Breast Cancer Program, the National Cancer Institute and the DOE Office of Science.
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The research, led by graduate student Benjamin J. Frisch in the James P. Wilmot Cancer Center laboratory of corresponding author Laura M. Calvi, M.D., is featured in the journal Blood. It is accompanied by an editorial — “Bad to the Bone” — written by another leading investigator in the field, Steven W. Lane, M.D., of Queensland Institute of Medical Research. Lane says that the URMC’s unexpected laboratory finding provokes new clinical questions, such as whether screening for osteoporosis could provide any useful information for how to manage acute leukemia in newly diagnosed patients.

Leukemia is a devastating disease that results in the disruption of normal blood production. Blood stem cells (hematopoietic stem cells or HSCs) give rise to all mature blood cells and maintain a balance of self-renewal and expansion. However, in this study, even when leukemia is barely traceable in the blood, leukemic cells implant in the bone marrow and attack the body’s natural process of making healthy blood stem cells.

In this hematopoietic microenvironment, or niche, investigators have been searching for clues. In 2003 Calvi introduced the concept that osteoblasts, which actively work to form bone in this same microenvironment, might have a key role in expanding and supporting the production of normal blood cells. Published in the journal Nature, that study served as the basis for the current investigation.

Frisch began focusing on the impact of the leukemia cells, which reside on the inside surface of bones adjacent to bone marrow activity. Until now, according to the Blood paper, no one had defined the important interactions that take place between leukemia cells and osteoblasts (bone forming cells) and osteoclasts, which continually break down bone. Frisch and colleagues used a mouse model and human leukemia tissue samples to show that:

• The way in which leukemia alters the balance and cycles of osteoblast and osteoclast activity is complex and counterintuitive, and results in several measurable changes to the skeleton.

For example, since bone formation and bone resorption are usually tightly knit functions, researchers expected to see that dramatic bone loss due to leukemia would also be consistent with a breakdown of bone and minerals, or resorption. Instead, they saw a mild increase in osteoclastic cells responsible for bone resorption, suggesting that leukemia uncouples these two bone cell functions. Ultimately, researchers would like to understand more about osteoclasts during the disease process, so that they can perhaps target those cells for treatment.

• In this study, leukemia caused low-level and widespread bone thinning and bone loss, similar to osteoporosis, particularly in the long bones. Preliminary lab experiments showed that treatment with bisphosphonates, a commonly used class of drugs for people who suffer from bone loss, partially restored bone loss in mice with leukemia.
• Leukemia results in the expression of a protein, known as CCL3, which slows bone formation. Thus, elevated CCL3 levels in leukemia make it a tempting treatment target. Theoretically, newer drugs that block the CCL3 pathway might be able to restore the low-level, net loss of bone observed in many leukemia patients. A few drug compounds that act on the CCL3 pathway are under study in early-stage clinical trials, Frisch said.

Another interesting question, the study noted, is the way in which dysfunction in the bone marrow microenvironment might delay a patient’s recovery after chemotherapy, or be the catalyst for relapse.

“Our findings are quite provocative and we hope they will lead to new approaches to promote normal blood production in patients with blood cancers,” said Calvi, associate professor of Medicine. “Because the loss of normal hematopoietic function is the chief cause of serious illness and death among leukemia patients, it is critical that we understand all aspects of how this occurs and find new strategies to accelerate the recovery of these defects.”

Funding was provided by the Wilmot Scholar Cancer Research Award and the Pew Scholar in Biomedical Sciences Award. Co-authors include John M. Ashton, Ph.D., URMC Department of Genetics; Lianping Xing, Ph.D., URMC Department of Pathology and Laboratory Medicine; Michael W. Becker, M.D., URMC Department of Medicine, and Craig T. Jordan, Ph.D., the Philip and Marilyn Wehrheim Professor of Medicine at Wilmot.
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The specially engineered probiotic bacteria, like those found in many yogurts, were intravenously injected into mice with tumors, after which the researchers took full body bioluminescent images. The 3-D images revealed information about the number and location of the bacteria, to the level of precisely revealing where within the tumor the bacteria were living, providing much more information on the interaction of bacteria and tumors than was previously available using similar two-dimensional imaging methods.

According to the authors, led by Mark Tangney of University College Cork in Ireland, “before now, researchers used luminescence to provide an approximation of where a test organism was within the body, and would then follow up with multiple further experiments using different techniques to try to find a precise location.”

This new research suggests that such bacteria can be engineered to contain diagnostic or therapeutic agents that would be produced specifically within the tumor for targeted treatment.
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This new magnetic resonance spectroscopy (MRS) technique provides a definitive diagnosis of cancer based on imaging of a protein associated with a mutated gene found in 80 percent of low- and intermediate-grade gliomas. Presence of the mutation also means a better prognosis.

“To our knowledge, this is the only direct metabolic consequence of a genetic mutation in a cancer cell that can be identified through noninvasive imaging,” said Dr. Elizabeth Maher, associate professor of internal medicine and neurology at UT Southwestern and senior author of the study, available online in Nature Medicine. ”This is a major breakthrough for brain tumor patients.”

UT Southwestern researchers developed the test by modifying the settings of a magnetic resonance imaging (MRI) scanner to track the protein’s levels. The data acquisition and analysis procedure was developed by study lead author Dr. Changho Choi, associate professor of the Advanced Imaging Research Center (AIRC) and radiology. Previous research linked high levels of this protein to the mutation, and UT Southwestern researchers already had been working on MRS of gliomas to find tumor biomarkers.

“Our next step is to make this testing procedure widely available as part of routine MRIs for brain tumors. It doesn’t require any injections or special equipment,” said Dr. Maher, medical director of UT Southwestern’s neuro-oncology program.

To substantiate the test as a diagnostic tool, biopsy samples from 30 glioma patients enrolled in the UT Southwestern clinical trial were analyzed; half had the mutation and expected high levels of the protein. MRS imaging of these patients had been done before surgery and predicted, with 100 percent accuracy, which patients had the mutation.

For Thomas Smith of Grand Prairie, the test helped determine the best time to begin chemotherapy. When an MRS scan showed a sharp rise in the 25-year-old’s protein levels, this indicated to his health care team that his tumor was moving from dormancy to rapid growth.

“We treated him with chemotherapy and his protein levels came down,” Dr. Maher said.

Before participating in the study, Mr. Smith had tumor removal surgery in 2007. Because part of the tumor could not be safely removed, however, he continued to suffer seizures and had other neurological problems. Since chemotherapy, his symptoms have diminished.

“I did six rounds of chemo, every six weeks,” Mr. Smith said. “My seizures stopped and all my symptoms improved. I am only on anti-seizure medication now.”

Other UT Southwestern researchers involved in the study included Sandeep Ganji, a doctorate student in radiological sciences; Dr. Ralph DeBerardinis, assistant professor of pediatrics and with the Eugene McDermott Center for Human Growth and Development; Dr. Kimmo Hatanpaa, associate professor of pathology; Dr. Dinesh Rakheja, assistant professor of pathology; Dr. Zoltan Kovacs, assistant professor in the AIRC; Drs. Xiao-Li Yang and Tomoyuki Mashimo, both senior research scientists in internal medicine; Dr. Jack Raisanen, professor of pathology; Dr. Isaac Marin-Valencia, resident in pediatrics; Dr. Juan Pascual, assistant professor of neurology and neurotherapeutics, pediatrics, and physiology; Dr. Christopher Madden, associate professor of neurological surgery; Dr. Bruce Mickey, professor of neurological surgery and otolaryngology-head and neck surgery, and radiation oncology; Dr. Craig Malloy, professor in the AIRC and of internal medicine and radiology; and Dr. Robert Bachoo, assistant professor in neurology and neurotherapeutics, and internal medicine.

The research was supported by grants from the National Institutes of Health, the Cancer Prevention and Research Institute of Texas and financial support from the Annette G. Strauss Center for Neuro-oncology at UT Southwestern.
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The research is presented in the first issue of the new Advanced Healthcare Materials, a Wiley journal.

“Hollow gold nanoparticles can generate heat if they are hit with a near-infrared laser,” said Research Institute Assistant Member Haifa Shen, M.D., Ph.D., the report’s lead author. “Multiple investigators have tried to use gold nanoparticles for cancer treatment, but the efficiency has not been very good — they’d need a lot of gold nanoparticles to treat a tumor.”

Instead, Shen and his colleagues turned to a technology developed by the study’s principal investigator, Mauro Ferrari, Ph.D., The Methodist Hospital Research Institute (TMHRI) president and CEO, to amplify the gold particles’ response to infrared light.

“We developed a system based on Dr. Ferrari’s multi-stage vector technology platform to treat cancers with heat,” Shen said. “We found that heat generation was much more efficient when we loaded gold nanoparticles into porous silicon, the carrier of the multistage vectors.”

Shen and his team found that in the presence of 808 nanometer light, the gold-filled silicon particles heated up a surrounding solution by about 20 deg C (35 deg F) in seven minutes. Water particles immediately around the particles were presumed to have been hotter. And experiments showed that tumor cell growth was lowest in the presence of gold-loaded silicon nanoparticles in three types of breast cancer cells — MDA-MB-231 and SK-BR-3 (human), and 4T1 (mouse).

The silicon wafers the scientists are using are the result of painstaking work by Ferrari’s group to design nanoparticles that preferentially bind to breast cancer cells, rather than, say, healthy liver or immune system cells. The shape and size of the silicon particles, as well as their surface chemistry, are all crucial, Ferrari’s group found. Too big or the wrong shape, and the silicon nanoparticles bind to multiple cell types — or none at all. Polyamine structures are attached to the wafers to improve their attraction to cancer cell surfaces and their solubility. The wafers are about one micrometer in diameter (one-thousandth of a millimeter). By contrast, the typical breast cancer cell is about 10 to 12 times that size.

Shen says the gold particles, too, must be designed with a specific use in mind, albeit for indirect reasons.

“The hollow gold particles we load into the porous silicon must be the right size and have the correct-sized space inside them to interact with the infrared light we are using,” he said. “But the wavelength of infrared we use will have to change depending on where the tumor is. If it’s close to the skin, we can use shorter wavelengths. Deeper inside the body, we have to use longer wavelengths of infrared to penetrate the tissue. The hollow space of the gold particles must be modified in response to that.”

Both silicon and gold have low toxicity profiles in the human body, and are popular materials in current investigations using medical nanotechnology. Silicon is steadily broken down by physiological processes into an acid that is removed through the kidneys. And gold is chemically inert.

And infrared — the type of light used by TV remote controls and garage door openers — is also far less dangerous than light with shorter wavelengths, such as ultraviolet, which can cause DNA damage, and x-rays.

Understanding why hollow gold particles heat up in the presence of certain wavelengths of infrared is complex enough to require some background in physical chemistry. But the upshot is that the energy of certain wavelengths of light is largely absorbed by the particles, and that energy is released as vibrational (heat) energy. Absorption is influenced both by the diameter of the space within the hollow gold particles, and by the properties of gold itself.

Shen says he’d like to know whether the silicon-gold nanotechnology can be used to wipe out whole tumors, rather than just cancerous cells.

“We are planning pre-clinical studies to study the technology’s impact on whole tissues, breast cancer cells and possibly pancreatic cancer cells,” Shen said. “We would also like to see whether this approach makes chemotherapy more effective, meaning you could use less drugs to achieve the same degree of success in treating tumors. These investigations are next.”

Coauthors of the Advanced Healthcare Materials paper were Jian You, whose contributions were equal to Shen’s, Guodong Zhang, Arturas Ziemys, Qingpo Li, Litao Bai, Xiaoyong Deng, Donald R. Erm, Xuewu Liu, Chun Li, and Mauro Ferrari. The research was supported with grants to Ferrari from the Department of Defense and the National Institutes of Health.
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Ever since Nobel laureate Alexander Fleming found that human tears contain antiseptic proteins called lysozymes about a century ago, scientists have tried to solve the mystery of how they could relentlessly wipe out far larger bacteria. It turns out that lysozymes have jaws that latch on and chomp through rows of cell walls like someone hungrily devouring an ear of corn, according to findings that will be published Jan. 20 in the journalScience.

“Those jaws chew apart the walls of the bacteria that are trying to get into your eyes and infect them,” said molecular biologist and chemistry professor Gregory Weiss, who co-led the project with associate professor of physics & astronomy Philip Collins.

The researchers decoded the protein’s behavior by building one of the world’s smallest transistors — 25 times smaller than similar circuitry in laptop computers or smartphones. Individual lysozymes were glued to the live wire, and their eating activities were monitored.

“Our circuits are molecule-sized microphones,” Collins said. “It’s just like a stethoscope listening to your heart, except we’re listening to a single molecule of protein.”

It took years for the UCI scientists to assemble the transistor and attach single-molecule teardrop proteins. The scientists hope the same novel technology can be used to detect cancerous molecules. It could take a decade to figure out but would be well worth it, said Weiss, who lost his father to lung cancer.

“If we can detect single molecules associated with cancer, then that means we’d be able to detect it very, very early,” Weiss said. “That would be very exciting, because we know that if we treat cancer early, it will be much more successful, patients will be cured much faster, and costs will be much less.”

The project was sponsored by the National Cancer Institute and the National Science Foundation. Co-authors of the Science paper are Yongki Choi, Issa Moody, Patrick Sims, Steven Hunt, Brad Corso and Israel Perez.
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The cholesterol receptor offers a promising new target for anti-viral therapy, for which an approved drug may already exist, say the researchers, whose findings were reported online in advance of publication in Nature Medicine.

An estimated 4.1 million Americans are infected with hepatitis C virus, or HCV, which attacks the liver and leads to inflammation, according to the National Institutes of Health. Most people have no symptoms initially and may not know they have the infection until liver damage shows up decades later during routine medical tests.

Previous studies showed that cholesterol was somehow involved in HCV infection. The UIC researchers suspected that a receptor called NPC1L1, known to help maintain cholesterol balance might also be transporting the virus into the cell.

The receptor is common in the gut of many species — but is found on liver cells only in humans and chimpanzees, says Susan Uprichard, assistant professor in medicine and microbiology and immunology and principal investigator in the study. These primates, she said, are the only animals that can be infected by HCV.

Uprichard and her coworkers showed that knocking down or blocking access to the NPC1L1 receptor prevented the virus from entering and infecting cells.

Bruno Sainz, Jr., UIC postdoctoral research associate in medicine and first author of the paper, said because the receptor is involved in cholesterol metabolism it was already well-studied. A drug that “specifically and uniquely targets NPC1L1″ already exists and is approved for use to lower cholesterol levels, he said.

The FDA-approved drug ezetimibe (sold under the trade-name Zetia) is readily available and perfectly targeted to the receptor, Sainz said, so the researchers had an ideal method for testing NPC1L1′s involvement in HCV infection.

They used the drug to block the receptor before, during and after inoculation with the virus, in cell culture and in a small-animal model, to evaluate the receptor’s role in infection and the drug’s potential as an anti-hepatitis agent.

The researchers showed that ezetimibe inhibited HCV infection in cell culture and in mice transplanted with human liver cells. And, unlike any currently available drugs, ezetimibe was able to inhibit infection by all six types of HCV.

The study, Uprichard said, opens up a number of possibilities for therapeutics.

Hepatitis C is the leading cause for liver transplantation in the U.S., but infected patients have problems after transplant because the virus attacks the new liver, Uprichard said.

While current drugs are highly toxic and often cannot be tolerated by transplant patients taking immunosuppressant drugs, ezetimibe is quite safe and has been used long-term without harm by people to control their cholesterol, Uprichard said. Because it prevents entry of the virus into cells, ezetimibe may help protect the new liver from infection.

For patients with chronic hepatitis C, ezetimibe may be able to be used in combination with current drugs.

“We forsee future HCV therapy as a drug-cocktail approach, like that used against AIDS,” Uprichard said. “Based on cell culture and mouse model data, we expect ezetimibe, an entry inhibitor, may have tremendous synergy with current anti-HCV drugs resulting in an improvement in the effectiveness of treatment.”

The study was supported by NIH Public Health Service grants, the American Cancer Society Research Scholar grant, the UIC Center for Clinical and Translational Science NIH grant, the UIC Council to Support Gastrointestinal and Liver Disease, and a grant from the Ministry of Health, Labor and Welfare of Japan.

Naina Barretto, Danyelle Martin, Snawar Hussain, Katherine Marsh and Xuemei Yu, of UIC; Nobuhiko Hiraga, Michio Imamura and Kazuaki Chayama, of Hiroshima University in Japan; and Waddah Alrefai of UIC and the Jesse Brown VA Medical Center in Chicago also contributed to the study.

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Regulatory T cells (Tregs), which are part of the body’s immune system, downregulate the activity of other immune cells, thus preventing the development of autoimmune diseases or allergies. Scientists at the German Cancer Research Center (DKFZ) have now found the activation steps that are blocked by Tregs in immune cells. Since Tregs can also suppress the body’s immune defense against cancer, the findings obtained by the DKFZ researchers are important for developing more efficient cancer treatments.

It is vital that the body’s own immune system does not overreact. If its key players, the helper T cells, get out of control, this can lead to autoimmune diseases or allergies. An immune system overreaction against infectious agents may even directly damage organs and tissues.

Immune cells called regulatory T cells (“Tregs”) ensure that immune responses take place in a coordinated manner: They downregulate the dividing activity of helper T cells and reduce their production of immune mediators. “This happens through direct contact between regulatory cell and helper cell,” says Prof. Peter Krammer of DKFZ. “But we didn’t know yet what this contact actually causes in helper cells.” The researchers’ hypothesis was that the contact with the Tregs affects certain steps in the complex signaling cascade that leads to the activation of the helper T cells.

If the T cell receptor, a sensor molecule on the surface of helper cells, senses foreign or damaged protein molecules, this will trigger a cascade of biochemical activation reactions. At the end of this signaling cascade, genes that are required for an immune attack will be read in the nucleus of helper cells.

Jointly with colleagues from several German research institutes, Peter Krammer, Angelika Schmidt and co-workers have now compared the signaling cascades in helper cells with and without contact to Tregs. The immunologists found out that a short contact of the two types of cells in the culture dish is sufficient to suppress the helper cells. Following Treg contact, the typical release of calcium ions into the plasma of helper cells does not occur. As a result, two important transcription factors, NFkappaB and NFAT, do no longer function. They normally activate genes for immune mediators, thus alerting the immune system.

“The mode of action of Tregs is of great importance for cancer medicine. Many of our colleagues have shown in various types of cancer that Tregs can downregulate the immune response against tumors so that transformed cells escape the immune defense. This can contribute to the development and spread of cancer. We are therefore searching for ways to reactivate such suppressed helper cells,” said Krammer, explaining the goals of his work. For developing immune therapies against cancer it is also crucial to understand how Tregs work. The researchers are trying to prevent that immune cells which have been painstakingly activated against cancer in the culture dish are immediately suppressed again by Tregs.

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“Our data show that at lower doses of ionizing radiation, DNA repair mechanisms work much better than at higher doses,” says Mina Bissell, a breast cancer researcher with Berkeley Lab’s Life Sciences Division. “This non-linear DNA damage response casts doubt on the general assumption that any amount of ionizing radiation is harmful and additive.”

Bissell was part of a study led by Sylvain Costes, a biophysicist also with Berkeley Lab’s Life Sciences Division, in which DNA damage response to low dose radiation was characterized simultaneously across both time and dose levels. This was done by measuring the number of RIF, for “radiation induced foci,” which are aggregations of proteins that repair double strand breaks, meaning the DNA double helix is completely severed.

Berkeley Lab biophysicist Sylvain Costes is generating 3D time lapse of DNA repair centers in human cells to understand better how cancer may arise from DNA damage. (Photo by Roy Kaltschmidt, Berkeley Lab)

“We hypothesize that contrary to what has long been thought, double strand breaks are not static entities but will rapidly cluster into preferred regions of the nucleus we call DNA repair centers as radiation exposure increases,” says Costes. “As a result of this clustering, a single RIF may reflect a center where multiple double strand breaks are rejoined. Such multiple repair activity increases the risks of broken DNA strands being incorrectly rejoined and that can lead to cancer.”

Costes and Bissell have published the results of their study in the Proceedings of the National Academy of Sciences. Also co-authoring the paper were Teresa Neumaier, Joel Swenson, Christopher Pham, Aris Polyzos, Alvin Lo, PoAn Yang, Jane Dyball, Aroumougame Asaithamby, David Chen and Stefan Thalhammer.

The authors believe their study to be the first to report the clustering of DNA double strand breaks and the formation of DNA repair centers in human cells. The movement of the double strand breaks across relatively large distances of up to two microns led to more intensely active but fewer RIF. For example, 15 RIF per gray (Gy) were observed after exposure to two Gy of radiation, compared to approximately 64 RIF/Gy after exposure to 0.1Gy. One Gy equals one joule of ionizing radiation energy absorbed per kilogram of human tissue. A typical mammogram exposes a patient to about 0.01Gy.

Corresponding author Costes says the DNA repair centers may be a logical product of evolution.

“Humans evolved in an environment with very low levels of ionizing radiation, which makes it unlikely that a cell would suffer more than one double strand break at any given time,” he says. “A DNA repair center would seem to be an optimal way to deal with such sparse damage. It is like taking a broken car to a garage where all the equipment for repairs is available rather than to a random location with limited resources.”

However, when cells are exposed to ionizing radiation doses large enough to cause multiple double strand breaks at once, DNA repair centers become overwhelmed and the number of incorrect rejoinings of double strand breaks increases.

“It is the same as when dozens of broken cars are brought to the same garage at once, the quality of repair is likely to suffer,” Costes says.

The link between exposure to ionizing radiation and DNA damage that can give rise to cancerous cells is well-established. However, the standards for cancer risks have been based on data collected from survivors of the atomic bomb blasts in Japan during World War II. The LNT model was developed to extrapolate low dose cancer risk from high dose exposure because changes in cancer incidence following low dose irradiation are too small to be measurable. Extrapolation was done on a linear scale in accordance with certain assumptions and the laws of physics.

“Assuming that the human genome is a target of constant size, physics predicts DNA damage response will be proportional to dose leading to a linear scale,” Costes explains. “Epidemiological data from the survivors of the atomic bombs was found to be in agreement with this hypothesis and showed that cancer incidence increases with an increase in ionizing radiation dose above 0.1 Gy. Below such dose, the picture is not clear.”

Previous studies failed to detect the clustering of double break strands and the formation of DNA repair centers because they were based on single-time or single-dose measurements of RIF at a discrete time after the initial exposure to ionizing radiation. This yields a net number of RIF that does not account for RIF that have not yet appeared or RIF that have already made repairs and disappeared. The time-lapse imaging used by Costes, Bissell and their co-authors showed that RIF formation continues to occur well beyond the initial radiation exposure and after earlier repair issues have been resolved. Time-lapse imaging also indicates that double strand break clustering takes place before any RIF are formed.

“We hypothesize that double strand break clustering occurs rapidly after exposure to ionizing radiation and that RIF formation reflects the repair machinery put in place around a single cluster of double strand breaks,” Costes says. “Our results provide a more accurate model of RIF dose response, and underscore fundamental concerns about static image data analysis in the dynamic environment of the living cell.”

Previous studies also mostly involved fibroblast cells whereas Costes, Bissell and their colleagues examined epithelial cells, specifically an immortalized human breast cell line known as MCF10A, which has a much higher background of RIF than fibroblasts, even without ionizing irradiation. To compensate for this higher background, Costes developed a mathematical method that enables background to be corrected for on a per- nucleus basis in unirradiated cells. Still the use of a special line of immortalized breast cells is an issue that Costes and his colleagues plan to address.

“We are now looking at primary breast epithelial cells that have been removed from healthy donors to determine if our results are repeated beyond just a single cell line and under more realistic physiological conditions,” Costes says. “We’d also like to know if our findings hold true for fibroblasts as well as epithelial cells. Also, we’d like to know if double strand break clustering is the result of a random coalescence or if there is an active transport mechanism that moves these double strand breaks towards pre-existing DNA repair centers.”

Working in collaboration with Rafael Gomez-Sjoberg of Berkeley Lab’s Engineering Division, Costes and his group are also developing a special microfluidics lab-on-a-chip device that is integrated into an X-ray microbeam. The goal is to provide a means by which cells can be kept in a controlled microenvironment while being irradiated with multiple doses. This microfluidic array will be used to characterize DNA damage response in breast and blood cells collected from human donors.

“By characterizing DNA damage response in cells from many different human donors,” Costes says, “we should be able to determine the variation across humans and gain a better understanding of how sensitivity to DNA damage from ionizing radiation might vary from individual to individual.”

This research was supported by the DOE Office of Science.

source form: sciencedaily