Catching Cancer With Carbon Nanotubes: New Device to Test Blood Can Spot Cancer Cells, HIV on the Fly

ScienceDaily (Mar. 30, 2011) — A Harvard bioengineer and an MIT aeronautical engineer have created a new device that can detect single cancer cells in a blood sample, potentially allowing doctors to quickly determine whether cancer has spread from its original site.

These posts, made of carbon nanotubes, can trap cancer cells and other tiny objects as they flow through a microfluidic device. Each post is 30 microns in diameter.

The microfluidic device, described in the March 17 online edition of the journal Small, is about the size of a dime, and could also detect viruses such as HIV. It could eventually be developed into low-cost tests for doctors to use in developing countries where expensive diagnostic equipment is hard to come by, says Mehmet Toner, professor of biomedical engineering at Harvard Medical School and a member of the Harvard-MIT Division of Health Sciences and Technology.

Toner built an earlier version of the device four years ago. In that original version, blood taken from a patient flows past tens of thousands of tiny silicon posts coated with antibodies that stick to tumor cells. Any cancer cells that touch the posts become trapped. However, some cells might never encounter the posts at all.

Toner thought if the posts were porous instead of solid, cells could flow right through them, making it more likely they would stick. To achieve that, he enlisted the help of Brian Wardle, an MIT associate professor of aeronautics and astronautics, and an expert in designing nano-engineered advanced composite materials to make stronger aircraft parts.

Out of that collaboration came the new microfluidic device, studded with carbon nanotubes, that collects cancer cells eight times better than the original version.

Captured by nanotubes

Circulating tumor cells (cancer cells that have broken free from the original tumor) are normally very hard to detect, because there are so few of them -- usually only several cells per 1-milliliter sample of blood, which can contain tens of billions of normal blood cells. However, detecting these breakaway cells is an important way to determine whether a cancer has metastasized.

"Of all deaths from cancer, 90 percent are not the result of cancer at the primary site. They're from tumors that spread from the original site," Wardle says.

When designing advanced materials, Wardle often uses carbon nanotubes -- tiny, hollow cylinders whose walls are lattices of carbon atoms. Assemblies of the tubes are highly porous: A forest of carbon nanotubes, which contains 10 billion to 100 billion carbon nanotubes per square centimeter, is less than 1 percent carbon and 99 percent air. This leaves plenty of space for fluid to flow through.

The MIT/Harvard team placed various geometries of carbon nanotube forest into the microfluidic device. As in the original device, the surface of each tube can be decorated with antibodies specific to cancer cells. However, because the fluid can go through the forest geometries as well as around them, there is much greater opportunity for the target cells or particles to get caught.

The researchers can customize the device by attaching different antibodies to the nanotubes' surfaces. Changing the spacing between the nanotube geometric features also allows them to capture different sized objects -- from tumor cells, about a micron in diameter, down to viruses, which are only 40 nm.

The researchers are now beginning to work on tailoring the device for HIV diagnosis. Toner's original cancer-cell-detecting device is now being tested in several hospitals and may be commercially available within the next few years.

Rashid Bashir, director of the Micro and Nanotechnology Laboratory at the University of Illinois at Urbana-Champaign, says that the ability to filter specific particles, cells or viruses from a blood sample so they can be analyzed is a critical step towards creating handheld diagnostic devices.

"Anything you can do to improve capture efficiency, or anything novel you can do to get the particles to interact with a surface more effectively, will help with sample preparation," says Bashir, who was not part of the research team.

Thyroid Hormone Controls the Eye‘s Visual Pigments Throughout Life

ScienceDaily (Mar. 30, 2011) — What part does the thyroid gland have in vision? Thyroid hormone is crucially involved in controlling which visual pigment is produced in the cones. Previously, it was assumed that the colour sensitivity of the cones is fixed in the adult retina. Researchers at the Max Planck Institute for Brain Research in Frankfurt/M., together with colleagues at the University of Frankfurt and universities in Vienna, have now been able to show that in mature cones of mice and rats the production of visual pigment is regulated by thyroid hormone. It is assumed that this mechanism exists in all mammals, including humans. If so, the adult-onset of thyroid hormone deficiency would affect colour vision.

Fluorescence micrographs of the cone cells in the retina of an adult healthy rat (top) and of an adult rat with thyroid hormone deficiency (bottom). The cones were labeled with antibodies against their opsins; green opsin is shown in green and UV/blue opsin in magenta. The healthy rat has many green cones and few UV/blue cones. The rat with thyroid hormone deficiency expresses UV/blue opsin in all cones and reduces expression of green opsin. Appearing in lighter magenta in the bottom image are cones that contain some green opsin in addition to the dominant UV/blue opsin

Thyroid hormone has a crucial role during development of the body and also of the nervous system. Children born with a thyroid hormone deficiency have serious defects of physiological and mental development, hence newborns are routinely checked for thyroid hormone deficiency, and hormone substitution therapy is given when indicated.

Studies in mice have shown that thyroid hormone also plays an important role in the development of the eye and particularly the cone visual cells. In the retina of the eye, the cones are the visual cells responsible for colour vision. Most mammals have two spectral cone types containing either of two visual pigments (opsins), one sensitive to shortwave light (UV/blue opsin), the other to middle-to-longwave light (green opsin). Cones express a thyroid hormone receptor. Its activation by the hormone suppresses the synthesis of UV/blue opsin and activates the production of green opsin.

Until now, the control of opsin production by thyroid hormone was considered a developmental phenomenon. Experts assumed that in mature cones the developmentally established 'opsin program' is fixed and needs no further regulation. This perception is now challenged by a study carried out by lead authors Martin Glösmann and Anika Glaschke in Leo Peichl's team at the Max Planck Institute for Brain Research, Frankfurt, and their colleagues at the universities of Frankfurt and Vienna. The study shows that opsin production in mature cones continues to depend on the thyroid hormone level.

The researchers had started with an analysis of thyroid hormone involvement in the early postnatal development of mouse cones. "Then we wanted to know how long the time window for the hormone effect was, at what point the hormone's influence on opsin production stopped," says Anika Glaschke. "To our surprise we did not find such an endpoint, even several weeks after birth there was a hormone effect." So the team analysed the cones in adult mice and rats that had been rendered hypothyroid for several weeks. In these mice all cones switched to the production of UV/blue opsin and reduced green opsin production. After termination of the treatment, hormone levels returned to normal and the cones reverted to the production of their 'regular' opsin -- one cone type to green opsin, the other to UV/blue opsin. The researchers conclude that the spectral cone types, which are defined by the opsin they express, are dynamically and reversibly controlled by thyroid hormone throughout life.

"In addition to their importance for basic retinal research, our findings may also have clinical relevance," says Martin Glösmann, who currently examines the genetic foundations of the process at the University of Veterinary Medicine, Vienna. "If this mechanism also acts in human cones, the adult-onset of thyroid hormone deficiency -- e.g. as a consequence of dietary iodine deficiency or removal of the thyroid -- would also affect the cone opsins and colour vision." There are no such reports in the clinical literature, presumably because the general symptoms of thyroid hormone deficiency are so severe that therapy is initiated before the cone opsin shifts would show up.

Cholesterol Regulator Plays Key Role in Development of Liver Scarring, Cirrhosis

ScienceDaily (Mar. 30, 2011) — UCLA researchers have demonstrated that a key regulator of cholesterol and fat metabolism in the liver also plays an important role in the development of liver fibrosis -- the build-up of collagen scar tissue that can develop into cirrhosis. Cirrhosis, in turn, is a major cause of premature death and is incurable without a liver transplant.

Treated with a control substance, livers from normal and LXR-deficient mice appear identical and undamaged (top left and right). The bottom images show the greater degree of fibrosis (blue bands) in the livers of mice lacking LXRs (right) compared to normal mice (left) after liver injury.

Published in the March issue of the journal Gastroenterology, the study shows that liver X receptors (LXRs), master regulators of cholesterol, fat and inflammatory gene expression, also control the fibrosis-making cells of the liver, known as hepatic stellate cells.

In the face of chronic liver injury -- due to excess fat, chronic viral hepatitis or alcohol abuse, for example -- stellate cells become activated and launch an inflammatory and fibrotic cascade that eventually results in the build-up of collagen scar tissue in the liver.

LXRs, when stimulated, "turn on" several hundred genes that hold instructions to create proteins for carrying out bodily processes in cells, from transporting and excreting cholesterol to synthesizing fat in the liver. They have also been shown to suppress inflammatory processes in several contexts.

"Our work sets the stage for looking at new ways to modulate cholesterol and/or fat metabolism in order to have therapeutic potential for the treatment of fibrosing liver diseases," said lead author Dr. Simon Beaven, an assistant professor of digestive diseases at the David Geffen School of Medicine at UCLA.

The research was done in the laboratory of senior author Dr. Peter Tontonoz, a professor of pathology and laboratory medicine at the Geffen School of Medicine and a Howard Hughes Medical Institute investigator.

Beaven noted that the recent rise in obesity has resulted in a surge in the prevalence of a condition known as fatty liver, which can be a precursor to fibrosis and chronic liver disease. Simple fatty liver, also known as non-alcoholic fatty liver disease, or NAFLD, is one of the most common reasons patients consult a liver doctor in the United States. Cirrhosis due to fatty liver is skyrocketing and within a decade may become the most common indication for liver transplantation.

Beaven said the need to find better treatments for liver disease is crucial.

"A 'holy grail' for liver researchers is to develop anti-fibrotic treatments that target activated stellate cells in order to slow or prevent the development of cirrhosis," Beaven said. "Our study offers the first detailed look at how LXRs specifically impact the activation of hepatic stellate cells and the subsequent development of liver fibrosis in animal models."

UCLA researchers have found that LXRs normally play a role in helping to reduce the collagen-producing actions of stellate cells when the cells are "activated" by liver damage. For the study, UCLA scientists first tested how activated stellate cells taken from mice would react when a chemical that induces LXR activity was added to the cell culture.

In stellate cells from normal mice, LXRs suppressed the inflammatory and fibrosis-promoting program. But in those taken from mice genetically lacking LXRs, that same program of genes significantly increased because the inhibitory effect of LXRs was no longer present.

"We showed that LXRs dampen stellate cell activation by repressing inflammatory and collagen-producing genes," Beaven said.

To further gauge the strength of the response, scientists took the medium from the cultures of LXR-deficient cells and added it to stellate cells from normal mice. These cells then showed a markedly exaggerated inflammatory and collagen-producing response, suggesting that LXR-deficient stellate cells are secreting signals to promote fibrosis.

The researchers noted that these experiments demonstrate that LXRs control a fibrotic response in stellate cells that can have a wide influence on neighboring cells.

The scientists also found that after replicating chronic liver injury, mice without LXRs had dramatically more liver fibrosis than normal mice.

"The genetic loss of LXRs rendered the mice susceptible to developing fibrotic liver disease," Beaven said.

But LXRs are also known to have important functions in the immune system. The researchers then wanted to know whether the effects they were seeing in animals were due to changes in stellate cell activity specifically or whether immune cells -- derived from bone marrow -- accounted for most of the effect. After extensive testing, the researchers found no differences

in the level of liver fibrosis among normal mice and animals lacking LXRs, suggesting that the contribution from the immune system was negligible.

"This finding, along with the cell culture studies, suggests that LXRs' influence on fibrosis most likely resides in altering stellate cell function in the liver," Beaven said. "This is a critical finding and opens an entire new field of study for stellate cell biologists."

Additional studies will further identify which genes in stellate cells are activated by LXRs and help researchers better understand the role of cholesterol metabolism in the fibrotic response.

This study was funded primarily by grants from the National Institutes of Health and the Howard Hughes Medical Institute. Collaborators from the University of Southern California were funded by core grants from the NIH and the Southern California Research Center for ALPD and Cirrhosis.

Other study authors included senior investigator Dr. Peter Tontonoz of the Howard Hughes Medical Institute; Kevin Wroblewski and Cynthia Hong from Tontonoz's lab; Jiaohong Wang and Hide Tsukamoto of the Southern California Research Center for ALPD and Cirrhosis, USC's Keck School of Medicine and the Department of Veterans Affairs Greater Los Angeles Healthcare System; and Steven Bensinger of the department of pathology at the David Geffen School of Medicine at UCLA.

Greater Versatility of Adult Stem Cells Thanks to 3-D Lab Experiments

ScienceDaily (Mar. 30, 2011) — A type of adult stem cell is now proving itself more versatile for research and therapies thanks to revolutionary 3D experiments. These cells have already shown great promise for repairing damaged bone and cartilage but until now have been fairly limited in the types of cells they can form in the laboratory.

MSCs are common in children and adults and quite easy to find in blood, bone marrow, and many other tissues. They are already being used to repair bone in a small number of patients with severe fractures or bone disease.Dr Paul Genever from the University of York spoke March 31 at the annual UK National Stem Cell Network science meeting. He told the gathered audience of scientists about his work to grow mesenchymal stem cells (MSCs) -- currently one of the leading candidates to be used in stem cell therapies -- as tiny spheres. Under these conditions MSCs show potential to become a variety of different cell types including, possibly, the early precursors to heart muscle cells.

Dr Genever's experiments hope to recreate the microscopic 3D environment that stem cells would normally occupy inside our bodies and so give an accurate approximation of the factors that might influence the ability of MSCs to eventually produce different types of cell for regenerative medicine.

Dr Genever said "In the past we've grown MSCs in 2D layers in the lab and they are only really strongly inclined to become bone, fat or cartilage -- they are very useful for research and therapy, but in both cases would largely be limited to these three cell types.

"Our 3D technique aims to recreate the nutrients, oxygen levels and mechanical forces that these cells would normally experience inside our bodies. By growing the cells as 3D spheres of microscopic size instead of in a 2D layer, they specialise their roles more rapidly and more completely and also appear to be able to become a greater range of cell types. This shows that they are quite a bit more versatile than we thought and so are a very exciting prospect for the use of these cells in therapies."

The spheres used are made of aggregates of MSCs and are tiny, measuring only 200-300 micrometers across -- about half the size of a dust mite. Within these spheres it is possible to monitor the effects of interactions between several cells and between cells and other supporting structures. The MSCs can also be combined with other types of cells that they would usually be associated with such as endothelial cells, which are found on the surfaces of blood vessels.

Professor Douglas Kell, Chief Executive, BBSRC said "Stem cells are a vital part of normal development and healthy repair. Stem cell biology is subtle and complicated and this discovery will help to ensure that results from laboratory experiments offer a good approximation of what is happening with stem cells under normal circumstances inside humans and other animals."

The work is funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and Smith & Nephew.

Research Into Poison Curare May Lead to Medication Against Tobacco Addiction

ScienceDaily (Mar. 30, 2011) — For the first time, three-dimensional images of protein being paralysed by the poison curare have been made by researchers of the Laboratory for Structural Neurobiology at K.U.Leuven. Curare has a paralysing effect and the poison's active chemical component is used in lung surgery. To date, however, scientists did not know how exactly it works. 3D images have now opened new perspectives for the development of medications against sleeping disorders, tobacco addiction and muscle diseases.

The ion channel reacting to curare – comprising five different sections (pictured here in different colours) that look like the sails of a windmill. The opening in the middle is the pore through which ions flow in or out of the cell. The little globules are tubocurarine, the active chemical component of curare. (The colours indicate various chemical elements. Grey is carbon; red is oxygen and blue is nitrogen.)

The human cell membrane -- the wall of a living cell -- houses more than 7,000 proteins, but researchers have only managed to identify the structure and function of 27 of these. Ion channels are an important class of membrane proteins that are responsible for communication. The Laboratory for Structural Neurobiology at K.U.Leuven has mapped the three dimensional structure of ion channels.

Professor Ulens, director of the lab, explains what the 3D images of curare mean: "We are locksmiths who examine on an atomic scale how a key -- the poison -- fits the lock of a door -- the ion channel -- and how the key keeps the door locked. Some kinds of poison only fit one lock, but curare is a passkey that can close various ion channels. Using 3D knowledge of the structure of this lock, researchers are able to develop passkey medications for a class of disorders. Or they can develop a specific medication for one disorder, such as tobacco addiction for example, as nicotine affects one specific ion channel.

Ion channels are actually switches. The proteins are shaped like microscopic pores that can open and close. Ions -- charged particles -- flow in or out of the cells through them. Poisons are able to disrupt the communication between cells in the body by blocking ion channels. Curare is the poison the indigenous populations of the Amazon use while hunting. They apply the poison to their arrows in order to paralyse their prey. Tubocurarine -- the active chemical component of curare -- paralyses the muscles and can shut down respiration, resulting in death.

The fact that so little is known about membrane proteins is related to the fatty environment of the cell membrane. In X-ray crystallography -- the standard technique to study proteins -- crystals of proteins are grown in water and then X-rayed in order to expose and examine their structure. Forming crystals of fatty membrane proteins is difficult, however. Professor Ulens explains how his team was able to circumvent this problem: "For the past ten years, researchers were forced to get in through the backdoor: a chemical copy of a section of ion channel. Chemically similar, but not porous. As a result, the formation of crystals was much easier. For the first time, our lab has applied the back entrance to the ion channel, which is sensitive to curare. We now have an image of how this class of ion channels recognises chemical substances."

Ulens hopes to use these results to contribute to the rational development of medications: "In the past, the pharmaceutical industry developed medications by releasing hundreds of thousands of substances into ion channels. If a certain substance caused a reaction, it would be tested on patients -- a system of trial and error. Our research results in the more goal-oriented development of medications: by acquiring insight into the three-dimensional structure of an ion canal, specific medications that bind to the protein can be developed."