� Scientists have detected previously unnoticed chemical signals that single cells in the immune system use to pass on with each other over short distances.
The signals the researchers detected originated in dendritic cells - the sentinels of the immune system that do the initial spotting of microscopic invaders - and were received by nearby T-cells, which take on a figure of of the essence roles in the immune system, including coordination of attacks on agents that cause disease or infection.
The chemical signals cells exchange when they come into contact have been studied extensively. But it has not been possible to detect chemical messages that travel betwixt cells that are nearby but not in physical contact - called paracrine signals - because they ar highly localized and they are produced in concentrations that experience been infra detection levels. A new technology, called a multi-trap nanophysiometer, was required to demonstrate the existence of non-contact sign. This is one of the number one microfluidic devices that has been applied successfully to the sketch of cell-to-cell signaling in the immune system.
A elaborate description of the multi-trap nanophysiometer (MTN) and how it enabled the accidental discovery of paracrine signaling has been published on-line by the Lab on a Chip journal. The new device was highly-developed by a team of researchers at the Vanderbilt Institute for Integrative Biosystems Research and Education headed by John P. Wikswo, the Gordon A. Cain University Professor at Vanderbilt.
"This is an important advance and potentially very utile technology," says co-author Derya Unutmaz, at present an associate professor of microbiology at New York University's School of Medicine. "The ability to study the conduct of single cells may not be as vital if you are poring over the center or muscles, which ar mostly formed by undifferentiated cells, only it is crucial for understanding how the resistant system functions. The wide surveillance of the body that it conducts requires extensive communication between scads of different kinds of immune cells."
The reason for this is that the dendritic cells, T-cells and B-cells in the immune system, which tend to concentrate in the lymph nodes spreadhead throughout the body, function as individual, unattached cells. If dendritic cells detect invaders in the body, they rapidly migrate to lymph nodes and own to encounter the earmark T-cells to alert them. But how dendritic cells attract the right T-cells among millions of cells within the lymph nodes remains an immunological puzzle.
Scientists have been trying to develop systems for single-cell analysis for a number of years. Because of the difficultness of guardianship normal cells alive, they have been forced to use cells that have been genetically altered so they behind be genteel indefinitely. Although the alteration "immortalizes" the cells, it also significantly limits their usefulness. The MTN is the first gear system that can varan biochemical changes in orotund numbers of normal or primary cells at the single-cell spirit level for prolonged periods, Unutmaz says.
The new device consists of a series of hair-sized channels molded in a special kind of plastic that is pasted onto the bottom of a glass microscope coverslip. A shoebox-sized pump pushes fluid (normally the media used to culture cells) through one channel that opens up into a chamber filled with hundreds of midget, three-sided h. G. Wells small sufficiency to trap individual cells. When cells are injected upstream, they are passively trapped in the herbert George Wells and ar held there solely by the fluid flowing verboten even smaller holes in the well bottoms. By precisely controlling the stream rate, the researchers can keep normal cells active for longer than 24 hours.
The researchers monitor the cells with a digital camera connected to a standard microscope, typically snapping images every 30 seconds. They take in written package that allows them to analyze the movements and reactions of individual cells. They can record various cell behaviors by injecting different fluorescent dyes into the cells. For model, when naive T-cells are primed for an resistant response, the concentration of calcium ion in their cytoplasm jumps up. So when the cytoplasm contains a dye that fluoresces when it comes into contact with calcium, it glows brilliantly enough to be easily detected.
The surprise discovery of paracrine signaling was made by graduate student Shannon Faley, today a postdoc research associate at the University of Glasgow, Scotland. She filled up a nanophysiometer chamber with naif human T-cells and then added mature dendritic cells. She was looking for evidence of T-cell activation when the T-cells and dendritic cells were treed in the same well and came into contact. This physical contact is part of the process that allows dendritic cells to convey selective information about potentially infectious invaders to the naive T-cells, which can buoy then begin dividing to produce an army of effector T-cells custom-designed to attack the invaders.
Faley power saw what she was looking at for, but she too noticed something unexpected: some T-cells that were cornered in herbert George Wells downstream of those with dendritic cells, which had never been in train contact with them, were also lighting up. "My reaction when I saw them was, 'What in the world is exit on?'" she says.
"When she saw this, Shannon did a very clever thing," says Wikswo. "She took one chamber and filled it with dendritic cells and took a second chamber and filled it with T-cells. Then she hooked the irregular chamber downstream of the first." When she did so, the T-cells in the second chamber immediately began lighting up, demonstrating that the mature dendritic cells were releasing a chemical divisor that activates naive T-cells without orgasm into contact.
"At this point we don't know what this broker is or what its function is," says Faley. According to Unutmaz, a logical occasion for this signal would be to attract T-cells to dendritic cells that have of import information to give them. This assumption is strengthened by the observation that immature dendritic cells don't produce this factor merely mature immunogenic dendritic cells - those that throw encountered a pathogen or danger signal - do.
When Faley tried to repeat this termination using criterion immunological techniques, however, the result was negative. The standard method consists of growing a culture of dendritic cells in a culture flask, adding T-cells and looking for for a reaction. If the cellular telephone density is too smashing, the cells begin toxic condition each other, run out of solid food and die. So the standard practice is to keep the density at a low enough spirit level that the cells stay healthy when the cell media is changed daily.
"This represents a dilution divisor of 100 compared to the nanophysiometer," says Wikswo. "So the factor produced by the dendritic cells was too dilute to activate the T-cells." It wasn't until Faley redid the standard test with cell densities 10 times higher than normal that she got the T-cells to activate.
"This represents one of the advantages of the nanophysiometer; it suspends cells in extremely small volumes that are much closer to what they experience in the body and uses slow liquid flow to keep the cells alive," Wikswo says.
Not only is this potentiality important in improving our knowledge of how the immune system works, merely it crataegus oxycantha also be the key to discernment why the system fails, as it does in cases like cancer and HIV/AIDS, says Dana Marshall, associate prof at the Meharry Medical College. She and the Wikswo radical have submitted a proposal to utilization the proficiency to study triple-negative knocker tumors, one of the most virulent forms of breast cancer.
"The ability to look at the sign among cancer cells and immune cells is super powerful," Marshall says. "According to the evidence, the immune system tries to suppress tumor cells but it fails to do so. It is not r wherefore it fails. If we can figure that out, then we should be able to develop more effective treatments."
In addition, the multi-trap nanophysiometer could supply a punter way to identify the most good forms of chemotherapy to use for each individual, Marshall suggests. There are enough cells in a typical biopsy to freight one chamber with tumour cells and a arcsecond chamber with immune-system cells from a patient, theme them to different chemotherapeutical agents and see how the deuce groups of cells respond. "Often, when therapy fails, the neoplasm responds to a chemotherapy treatment for a period of time and and so it boodle. This plan of attack may get us figure out why that happens," Marshall says.
The research was funded by grants from the Defense Advanced Research Projects Agency, Air Force Office of Scientific Research, the National Institutes of Health, the Vanderbilt Institute for Integrative Biosystems Research and Education and the Systems Biology and Bioengineering Undergraduate Research Experience.
Source: David F. Salisbury
Vanderbilt University
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Sunday, 7 September 2008
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