Recent findings by medical researchers indicate that naturally occurring nanotubes may serve as tunnels that protect retroviruses and bacteria in transit from diseased to healthy cells — a fact that may explain why vaccines fare poorly against some invaders.
To better study the missions of these intercellular nanotubes, scientists have sought the means to form them quickly and easily in test tubes.
Sandia National Laboratories researchers have now learned serendipitously to form nanotubes with surprising ease.
"Our work is the first to show that the formation of nanotubes is not complicated, but can be a general effect of protein-membrane interactions alone," says Darryl Sasaki of Sandia's Bioscience and Energy Center.
Sandia is a National Nuclear Security Administration laboratory.
The tunnel-like structures have been recognized only recently as tiny but important bodily channels for the good, the bad, and the informational.
In addition to providing protected transport to certain diseases, the nanotubes also seem to help trundle bacteria to their doom in the tentacles of microphages. Lastly, the nanotubes may provide avenues to send and receive information (in the form of chemical molecules) from cell to cell far faster than their random dispersal into the bloodstream would permit.
Given the discovery of this radically different transportation system operating within human tissues, it was natural for researchers to attempt to duplicate the formation of the nanotubes. In their labs, they experimented with giant lipid vesicles that appeared to mimic key aspects of the cellular membrane.
Giant lipid vesicles resemble micron-sized spherical soap bubbles that exist in water. They are composed of a lipid bilayer membrane only five nanometers thick.
The object for experimenters was to create conditions in which the spheres would morph into cylinders of nanometer radii.
But researchers had difficulties, says Sasaki, perhaps because they used a composite lipid called egg PC that requires unnecessarily high energies to bend into a tubular shape.
Egg PC is inexpensive, readily available, and offers good, stable membrane properties. It is the usual lipid of choice in forming nanocylinders via mechanical stretching techniques.
But Sandia postdoctoral researcher Haiqing Lui instead used POPC — a single pure lipid requiring half the bending energy of egg PC.
She was trying to generate nanotubes by a completely different approach that involved the use of motor proteins to stretch naturally occurring membranes into tubes.
Working with Sandia researcher George Bachand, she serendipitously found that interaction of the POPC membrane with a high affinity protein called streptavidin alone was enough to form the nanotubes.
"Perhaps this information — linking membrane bending energy with nanotube formation — may provide some clue about the membrane structure and the cell's ability to form such intercellular connections," Sasaki says.
The formation was confirmed by Sandia researcher Carl Hayden, who characterized the nanotube formation through a confocal imaging microscope. The custom instrument allows pixel-by-pixel examination of the protein interaction with the membranes comprising the nanotubes by detecting the spectrum and lifetimes of fluorescent labels on the proteins.
Nanotube formation had been noticed previously by cell biologists, but they had dismissed the tiny outgrowths as "junk — an aberration of cells growing in culture," says Sasaki. "The reason they were only noticed recently as trafficking routes is because of labeling studies that marked organelles and proteins. This allowed a focused look at what these nanostructures might be used for."
It became clear, says Sasaki, that the organelles were being transported with "specific directionality" on the backs of motor proteins within the tubes, rather than randomly.
Three-dimensional networks of nanotubes also are found to be created by macrophages — part of the police force of the body — grown in culture, says George. The tubes in appearance and function resemble a kind of spider web, capturing bacterium and transporting them to the macrophages, which eat them.
Source : DOE/Sandia National Laboratories
Showing posts with label vaccines. Show all posts
Showing posts with label vaccines. Show all posts
Sunday, August 24, 2008
Tuesday, May 8, 2007
Defenceless against a tiny bug
Andrew Pollack
The E.coli still has the last laugh.Despite many approaches, prevention still seems the best way out of a deadly infection.
Shousun C. Szu, a scientist at the National Institutes of Health, says the best way to prevent people from being poisoned by deadly E. coli would be to vaccinate all infants against the bacteria. Graeme McRae, a Canadian biotechnology executive, says it would be more practical to inoculate cows instead. Vaccines for people and for cattle are just two approaches under development to prevent or treat food poisoning by the strain E. coli O157:H7.
Right now, scientists can do little medically to fight the pathogen, which was responsible for two severe outbreaks in the US. The main approach has been to try to prevent contamination through careful handling of food, rigorous inspections and government regulation. Slaughterhouses have already sharply reduced contamination through practices like washing carcasses with hot water, steam or acids. Now the focus is on new procedures and regulations for the fresh-produce industry. On the animal side, a vaccine for cattle developed by McRae’s company, Bioniche Life Sciences, was approved in December for distribution to veterinarians in Canada. Studies have shown that the vaccine can reduce but not eliminate the E. coli shed into manure. Not only does that make the cows cleaner as they go into the slaughterhouse, but it could also conceivably reduce the risk that the germ will spread from a feedlot to a nearby produce field though water or wild animals. Cows and their manure are considered the major sources of the pathogen. “If we can reduce the likelihood that animals are going to carry the bacteria, then we might reduce over time what they put out into the environment,” said Guy Loneragan, a veterinary epidemiologist at West Texas A&M University. Other methods being tested include cattle antibiotics, an industrial chemical, bacterial-killing viruses and friendly bacteria to displace the evil ones. Efforts to develop drugs and vaccines for people also face barriers. Because outbreaks are rare and sporadic, for instance, it would be difficult to test such treatments in clinical trials. E. coli O157:H7 causes 75,000 cases of infection and 61 deaths in the United States each year, according to a 1999 estimate by the Centers for Disease Control and Prevention posted on its Web site. Dr. Phillip I. Tarr, an expert at Washington University in St. Louis, says treatment is difficult because the bloody diarrhea that signals infection may not occur until three to four days after ingestion of the bacteria. By then, a patient could be well on the way to kidney failure. Antibiotics, the usual treatment for bacterial infection, only make things worse by killing the bacteria and releasing more of their toxin, Tarr said. He added that the sole treatment shown to reduce the severity of kidney problems was intravenous fluids. Other scientists are trying. Thallion Pharmaceuticals of Montreal and Teijin Pharma of Japan have separately developed monoclonal antibodies that can latch on to the toxin molecules and neutralize them. Monoclonal antibodies, a synthetic version of the body's own infection fighters, are commonly used to treat cancer and other diseases. Thallion and Teijin have shown that the antibodies can protect laboratory animals from lethal doses and have conducted preliminary safety testing in people. But at the recent FDA advisory committee meeting, both said it would be prohibitively expensive to test whether their drugs could prevent hemolytic uremic syndrome. Some outside scientists question whether a treatment that starts after the toxin is already in the bloodstream would be effective. One approach already in use is probiotics, the idea that friendly bacteria fed to cattle will displace O157. The Nutrition Physiology Corp. of Guymon, Okla., sells a feed additive with lactobacillus, the same type of bacterium used in yogurt. The additive is sold to aid cattle digestion, but some studies suggest that it also reduces O157 in manure. An experimental approach is to feed cows sodium chlorate, a chemical used in the pulp and paper industry. This idea takes advantage of the fact that O157 has an enzyme that allows it to survive without oxygen, which is not true for most desirable bacteria. That enzyme will convert sodium chlorate to sodium chlorite, which poisons the pathogen. “It’s like a suicide pill to the E. coli,” said Robin C. Anderson, a microbiologist for the Agriculture Department in College Station, Texas. Anderson said the treatment did not harm the cow. The antibiotic neomycin has also been shown to reduce O157 levels in manure. Using antibiotics in animals raises concerns of spurring development of human pathogens resistant to the medicines. Another approach being studied involves phages, viruses that infect and kill bacteria. Experts say multiple approaches might be used in parallel, because no single approach works perfectly. Michael T. Osterholm, director of the Center for Infectious Disease Research and Policy at the University of Minnesota, said: “What really is a concern to me about this issue is we always have a tendency to want high-tech responses to what in many cases are common-sense low-tech solutions,” Osterholm said. In any case, even if a high-tech solution was desired, there does not seem to be a vaccine for spinach as there is for cattle. Greens are now often rinsed in chlorine solution, but that is not always effective because surface nooks and crannies can shelter the bacteria, said James Gorney, senior vice president for food safety and technology at the United Fresh Produce Association, a trade group. A possible alternative is to use a gas like chlorine dioxide instead of a liquid wash, Gorney said. Irradiation can also kill the bacteria. But he said the amount of radiation needed could damage fruits or vegetables. And some consumers object to the technique. “Any one of these technologies doesn't offer us a pasteurization step," Gorney said. "So we are left with prevention, prevention and prevention, preventing the contamination from ever occurring.” Szu of the health institutes and colleagues have developed a vaccine made of the complex sugar that is on the surface of the bacteria, the very O-type polysaccharide that gives O157 its name. The sugar is linked to a protein taken from another bacterium to make it more potent in stimulating the immune system. Szu and collaborators have tested the vaccine on adult volunteers and on children 2 to 5 years old. The volunteers were not exposed to O157 -- that would be unethical -- but they developed antibodies to it. Moreover, when the bacteria were exposed in the laboratory to blood samples from vaccinated people, the microbes were killed. Szu said the next test would be in infants. The vaccine is years from the market. As with drugs, testing effectiveness would be difficult, and some experts say it may not make sense to vaccinate every child to protect a small number. The cattle vaccine developed by Bioniche is based on the work of B. Brett Finlay of the University of British Columbia, who helped discover how O157 bacteria attach themselves to the cattle intestines, where they can then multiply. The bacteria use a type of microscopic syringe to shoot proteins into the cells lining the intestine, and the cells erect a protein pedestal, to which the bacteria can bind. The Bioniche vaccine consists of proteins involved in the attachment. The idea is that the cow's immune system would make antibodies to attack the proteins, thereby blocking the attachment. The bacteria could still pass through the cow and into manure. But if they could not colonize, their levels should remain low. Tests at the University of Nebraska found that the vaccine reduced by 70 percent the number of cows shedding O157 into their manure, said Rodney A. Moxley, a professor of veterinary science there. As few as 10 bacteria can make someone ill. The bacteria release one or two potent toxins that cause bloody diarrhea. In 15 percent of children younger than 10, and more rarely for adults, the infection causes hemolytic uremic syndrome, in which red blood cells are destroyed and the kidneys fail. In a small percentage of such cases, the syndrome proves fatal. NYT News Service
The E.coli still has the last laugh.Despite many approaches, prevention still seems the best way out of a deadly infection.
Shousun C. Szu, a scientist at the National Institutes of Health, says the best way to prevent people from being poisoned by deadly E. coli would be to vaccinate all infants against the bacteria. Graeme McRae, a Canadian biotechnology executive, says it would be more practical to inoculate cows instead. Vaccines for people and for cattle are just two approaches under development to prevent or treat food poisoning by the strain E. coli O157:H7.
Right now, scientists can do little medically to fight the pathogen, which was responsible for two severe outbreaks in the US. The main approach has been to try to prevent contamination through careful handling of food, rigorous inspections and government regulation. Slaughterhouses have already sharply reduced contamination through practices like washing carcasses with hot water, steam or acids. Now the focus is on new procedures and regulations for the fresh-produce industry. On the animal side, a vaccine for cattle developed by McRae’s company, Bioniche Life Sciences, was approved in December for distribution to veterinarians in Canada. Studies have shown that the vaccine can reduce but not eliminate the E. coli shed into manure. Not only does that make the cows cleaner as they go into the slaughterhouse, but it could also conceivably reduce the risk that the germ will spread from a feedlot to a nearby produce field though water or wild animals. Cows and their manure are considered the major sources of the pathogen. “If we can reduce the likelihood that animals are going to carry the bacteria, then we might reduce over time what they put out into the environment,” said Guy Loneragan, a veterinary epidemiologist at West Texas A&M University. Other methods being tested include cattle antibiotics, an industrial chemical, bacterial-killing viruses and friendly bacteria to displace the evil ones. Efforts to develop drugs and vaccines for people also face barriers. Because outbreaks are rare and sporadic, for instance, it would be difficult to test such treatments in clinical trials. E. coli O157:H7 causes 75,000 cases of infection and 61 deaths in the United States each year, according to a 1999 estimate by the Centers for Disease Control and Prevention posted on its Web site. Dr. Phillip I. Tarr, an expert at Washington University in St. Louis, says treatment is difficult because the bloody diarrhea that signals infection may not occur until three to four days after ingestion of the bacteria. By then, a patient could be well on the way to kidney failure. Antibiotics, the usual treatment for bacterial infection, only make things worse by killing the bacteria and releasing more of their toxin, Tarr said. He added that the sole treatment shown to reduce the severity of kidney problems was intravenous fluids. Other scientists are trying. Thallion Pharmaceuticals of Montreal and Teijin Pharma of Japan have separately developed monoclonal antibodies that can latch on to the toxin molecules and neutralize them. Monoclonal antibodies, a synthetic version of the body's own infection fighters, are commonly used to treat cancer and other diseases. Thallion and Teijin have shown that the antibodies can protect laboratory animals from lethal doses and have conducted preliminary safety testing in people. But at the recent FDA advisory committee meeting, both said it would be prohibitively expensive to test whether their drugs could prevent hemolytic uremic syndrome. Some outside scientists question whether a treatment that starts after the toxin is already in the bloodstream would be effective. One approach already in use is probiotics, the idea that friendly bacteria fed to cattle will displace O157. The Nutrition Physiology Corp. of Guymon, Okla., sells a feed additive with lactobacillus, the same type of bacterium used in yogurt. The additive is sold to aid cattle digestion, but some studies suggest that it also reduces O157 in manure. An experimental approach is to feed cows sodium chlorate, a chemical used in the pulp and paper industry. This idea takes advantage of the fact that O157 has an enzyme that allows it to survive without oxygen, which is not true for most desirable bacteria. That enzyme will convert sodium chlorate to sodium chlorite, which poisons the pathogen. “It’s like a suicide pill to the E. coli,” said Robin C. Anderson, a microbiologist for the Agriculture Department in College Station, Texas. Anderson said the treatment did not harm the cow. The antibiotic neomycin has also been shown to reduce O157 levels in manure. Using antibiotics in animals raises concerns of spurring development of human pathogens resistant to the medicines. Another approach being studied involves phages, viruses that infect and kill bacteria. Experts say multiple approaches might be used in parallel, because no single approach works perfectly. Michael T. Osterholm, director of the Center for Infectious Disease Research and Policy at the University of Minnesota, said: “What really is a concern to me about this issue is we always have a tendency to want high-tech responses to what in many cases are common-sense low-tech solutions,” Osterholm said. In any case, even if a high-tech solution was desired, there does not seem to be a vaccine for spinach as there is for cattle. Greens are now often rinsed in chlorine solution, but that is not always effective because surface nooks and crannies can shelter the bacteria, said James Gorney, senior vice president for food safety and technology at the United Fresh Produce Association, a trade group. A possible alternative is to use a gas like chlorine dioxide instead of a liquid wash, Gorney said. Irradiation can also kill the bacteria. But he said the amount of radiation needed could damage fruits or vegetables. And some consumers object to the technique. “Any one of these technologies doesn't offer us a pasteurization step," Gorney said. "So we are left with prevention, prevention and prevention, preventing the contamination from ever occurring.” Szu of the health institutes and colleagues have developed a vaccine made of the complex sugar that is on the surface of the bacteria, the very O-type polysaccharide that gives O157 its name. The sugar is linked to a protein taken from another bacterium to make it more potent in stimulating the immune system. Szu and collaborators have tested the vaccine on adult volunteers and on children 2 to 5 years old. The volunteers were not exposed to O157 -- that would be unethical -- but they developed antibodies to it. Moreover, when the bacteria were exposed in the laboratory to blood samples from vaccinated people, the microbes were killed. Szu said the next test would be in infants. The vaccine is years from the market. As with drugs, testing effectiveness would be difficult, and some experts say it may not make sense to vaccinate every child to protect a small number. The cattle vaccine developed by Bioniche is based on the work of B. Brett Finlay of the University of British Columbia, who helped discover how O157 bacteria attach themselves to the cattle intestines, where they can then multiply. The bacteria use a type of microscopic syringe to shoot proteins into the cells lining the intestine, and the cells erect a protein pedestal, to which the bacteria can bind. The Bioniche vaccine consists of proteins involved in the attachment. The idea is that the cow's immune system would make antibodies to attack the proteins, thereby blocking the attachment. The bacteria could still pass through the cow and into manure. But if they could not colonize, their levels should remain low. Tests at the University of Nebraska found that the vaccine reduced by 70 percent the number of cows shedding O157 into their manure, said Rodney A. Moxley, a professor of veterinary science there. As few as 10 bacteria can make someone ill. The bacteria release one or two potent toxins that cause bloody diarrhea. In 15 percent of children younger than 10, and more rarely for adults, the infection causes hemolytic uremic syndrome, in which red blood cells are destroyed and the kidneys fail. In a small percentage of such cases, the syndrome proves fatal. NYT News Service
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