On the Trail of a Stealthy Parasite Biologist Shows Why Some Strains of Toxoplasma Are More Dangerous Than Others

ScienceDaily (Jan. 5, 2011) — About one-third of the human population is infected with a parasite called Toxoplasma gondii, but most of them don't know it. Though Toxoplasma causes no symptoms in most people, it can be harmful to individuals with suppressed immune systems, and to fetuses whose mothers become infected during pregnancy.Toxoplasma spores are found in dirt and easily infect farm animals such as cows, sheep, pigs and chickens. Humans can be infected by eating undercooked meat or unwashed vegetables.

This image shows two human skin cells. The cell at left is infected with the Toxoplasma gondii parasite expressing type II GRA15, which can be seen in green. The cell on the right (with a blue nucleus) is not infected. The nucleus of the infected cell contains many copies of the NF-kB transcription factor (shown in pink). (Credit: Image courtesy of the Saeij Laboratory) 

Jeroen Saeij, an assistant professor of biology at MIT is investigating a key question: why certain strains of the Toxoplasma parasite (there are at least a dozen) are more dangerous to humans than others. He and his colleagues have focused their attention on the type II strain, which is the most common in the United States and Europe, and is also the most likely to produce symptoms. In a paper appearing in the Jan. 3 online edition of theJournal of Experimental Medicine, the researchers report the discovery of a new Toxoplasma protein that may help explain why type II is more virulent than others.

Toxoplasma infection rates vary around the world. In the United States, it's about 10 to 15 percent, while rates in Europe and Brazil are much higher, around 50 to 80 percent. However, these are only estimates -- it is difficult to calculate precise rates because most infected people don't have any symptoms.

After an infection is established, the parasite forms cysts, which contain many slowly reproducing parasites, in muscle tissue and the brain. If the cysts rupture, immune cells called T cells will usually kill the parasites before they spread further. However, people with suppressed immune systems, such as AIDS patients or people undergoing chemotherapy, can't mount an effective defense.

"In AIDS patients, T cells are essentially gone, so once a cyst ruptures, it can infect more brain cells, which eventually causes real damage to the brain," says Saeij.

The infection can also cause birth defects, if the mother is infected for the first time while pregnant. (If she is already infected before becoming pregnant, there is usually no danger to the fetus.)

There are drugs that can kill the parasite when it first infects someone, but once cysts are formed, it is very difficult to eradicate them.

A few years ago, Saeij and colleagues showed that theToxoplasma parasite secretes two proteins called rhoptry18 and rhoptry16 into the host cell. Those proteins allow the parasite to take over many host-cell functions.

In the new study, the MIT team showed that the parasite also secretes a protein called GRA15, which triggers inflammation in the host. All Toxoplasma strains have this protein, but only the version found in type II causes inflammation, an immune reaction that is meant to destroy invaders but can also damage the host's own tissues if unchecked. In the brain, inflammation can lead to encephalitis. This ability to cause inflammation likely explains why the type II strain is so much more hazardous for humans, says Saeij.

Saeij and his team, which included MIT Department of Biology graduate students Emily Rosowski and Diana Lu, showed that type II GRA15 leads to the activation of the transcription factor known as NF-kB, which eventually stimulates production of proteins that cause inflammation. The team is now trying to figure out how that interaction between GRA15 and NF-kB occurs, and why it is advantageous to the parasite.

Ultimately, Saeij hopes to figure out how the parasite is able to evade the immune system and establish a chronic infection. Such work could eventually lead to new drugs that block the parasite from establishing such an infection, or a vaccine that consists of a de-activated form of the parasite.

MIT Department of Biology graduate students Emily Rosowski and Diana Lu are the paper's two first authors and contributed equally to the research. Other authors include MIT postdoc Kirk Jensen, lab technician Lindsay Julien, MIT undergraduate student Lauren Rodda, and Rogier Gaiser, a graduate student at Wageningen University in the Netherlands.

Source: "Strain-specific activation of the NF-kB pathway by GRA15, a novel Toxoplasma gondii dense granule protein," by Emily E. Rosowski, Diana Lu, Lindsay Julien, Lauren Rodda, Rogier A. Gaiser, Kirk D.C. Jensen, Jeroen P.J. Saeij. Journal of Experimental Medicine, 3 January 2011.

Funding: American Heart Association, Massachusetts Life Sciences Center, Singapore-MIT Alliance for Research and Technology (SMART), National Institutes of Health, Cleo and Paul Schimmel Fund, Cancer Research Institute, MIT UROP office and the John Reed Fund.

Malfunctioning Gene Associated With Lou Gehrig's Disease Leads to Nerve-Cell Death in Mice

ScienceDaily (Jan. 5, 2011) — Lou Gehrig's disease, or amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD) are characterized by protein clumps in brain and spinal-cord cells that include an RNA-binding protein called TDP-43. This protein is the major building block of the lesions formed by these clumps.

Neurons expressing the ALS-associated human protein TDP-43 (green color) show an absence of normal TDP-43 protein (red). Virginia Lee and colleagues have shown that pertubation of normal TDP-43 expression is linked to neuron death. Edward Lee, MD, PhD; University of Pennsylvania School of Medicine. (Credit: Edward Lee)

In a study published in the Journal of Clinical Investigation, a team led by Virginia M.-Y. Lee, PhD, director of Penn's Center for Neurodegenerative Disease Research, describes the first direct evidence of how mutated TDP-43 can cause neurons to die. Although normally found in the nucleus where it regulates gene expression, TDP-43 was first discovered in 2006 to be the major disease protein in ALS and FTLD by the Penn team led by Lee and John Q. Trojanowski, MD, PhD, director of the Institute on Aging at Penn. This discovery has transformed research on ALS and FTLD by linking them to the same disease protein.

"The discovery of TDP-43 as the pathological link between mechanisms of nervous system degeneration in both ALS and FTLD opened up new opportunities for drug discovery as well as biomarker development for these disorders," says Lee. "An animal model of TDP-43-mediated disease similar to ALS and FTLD will accelerate these efforts."

In the case of TDP-43, neurons could die for two reasons: One, the clumps themselves are toxic to neurons or, two, when TDP-43 is bound up in clumps outside the nucleus, it depletes the cell of normally functioning TDP-43. Normally a cell regulates the exact amount of TDP-43 in itself -- too much is bad and too little is also bad. The loss of function of TDP-43 is important in regulating disease because it regulates gene expression.

To determine the effects of misplaced TDP-43 on the viability of neurons, the researchers made transgenic mice expressing human mutated TDP-43 in the cytoplasm and compared them to mice expressing normal human TDP-43 in the nucleus of nerve cells. Expression of either human TDP-43 led to neuron loss in vulnerable forebrain regions; degeneration of part of the spinal cord tract; and muscle spasms in the mice. These effects recapitulate key aspects of FTLD and a subtype of ALS known as primary lateral sclerosis.

The JCI study showed that a dramatic loss of function causes nerve-cell death because normal mouse TDP-43 is eliminated when human mutated TDP-43 genes are put into the mice. Since cells regulate the exact amount of TDP-43, over-expression of the human TDP-43 protein prevents the mouse TDP-43 from functioning normally. Lee and colleagues think this effect leads to neuron death rather than clumps of TDP-43 because these clumps were rare in the mouse cells observed in this study. Lee says that it is not yet clear why clumps were rare in this mouse model when they are so prevalent in human post-mortem brain tissue of ALS and FTLD patients.

Neurodegeneration in the mouse neurons expressing TDP-43 -- both the normal and mutated human versions -- was accompanied by a dramatic downregulation of the TDP-43 protein mice are born with. What's more, mice expressing the mutated human TDP-43 exhibited profound changes in gene expression in neurons of the brain's cortex.

The findings suggest that disturbing the normal TDP-43 in the cell nucleus results in loss of normal TDP-43 function and gene regulatory pathways, culminating in degeneration of affected neurons.

Next steps, say the researchers, will be to look for the specific genes that are regulated by TDP-43 and how mRNA splicing is involved so that the abnormal regulation of these genes can be corrected.

At the same time, notes Lee, "We soon will launch studies of novel strategies to prevent TDP-43-mediated nervous system degeneration using this mouse model of ALS and FTLD."

The study was funded in part by funds from the National Institutes of Health.

Vaccine Blocks Cocaine High in Mice: Approach Could Also Stop Addiction to Other Drugs, Including Heroin and Nicotine

ScienceDaily (Jan. 5, 2011) — Researchers have produced a lasting anti-cocaine immunity in mice by giving them a safe vaccine that combines bits of the common cold virus with a particle that mimics cocaine.

Laboratory mouse.

In their study, published Jan. 4 in the online edition of Molecular Therapyand funded by the National Institute on Drug Abuse, the researchers say this novel strategy might be the first to offer cocaine addicts a fairly simple way to break and reverse their habit, and it might also be useful in treating other addictions, such as to nicotine, heroin and other opiates.

"Our very dramatic data shows that we can protect mice against the effects of cocaine, and we think this approach could be very promising in fighting addiction in humans," says the study's lead investigator, Dr. Ronald G. Crystal, chairman and professor of genetic medicine at Weill Cornell Medical College.

He says the antibody immune response produced in lab mice by the vaccine binds to, and sequesters, cocaine molecules before the drug reached the brains of these animals -- and prevents any cocaine-related hyperactivity. The vaccine effect lasted for at least 13 weeks, the longest time point evaluated.

"While other attempts at producing immunity against cocaine have been tried, this is the first that will likely not require multiple, expensive infusions, and that can move quickly into human trials," Dr. Crystal says. "There is currently no FDA-approved vaccine for any drug addiction."

"An approach that works is desperately needed for cocaine addiction, which is an intransigent problem worldwide," he adds. "There are no therapies now."

The novelty of this possible treatment is that it hooks a chemical that is very similar in structure to cocaine, onto components of the adenovirus, a common cold virus. In this way, the human immune system is alerted to an infectious agent (the virus) but also learns to "see" the cocaine as an intruder as well, Dr. Crystal says. Once the structure of the new intruder is recognized, natural immunity builds to cocaine particles, so any time cocaine is snorted or used in any way, antibodies to the substance are quickly produced and the cocaine molecules are engulfed by the antibodies and prevented from reaching the brain.

"The human immune system doesn't naturally tag cocaine as something to be destroyed -- just like all small-molecule drugs are not eliminated by antibodies," he says. "We have engineered this response so that it is against the cocaine mimic."

In this study, a team of researchers -- scientists from Weill Cornell Medical College, Cornell University in Ithaca, and the Scripps Research Institute in La Jolla, Calif. -- ripped apart an adenovirus, retrieving only the components that elicit an immune response and discarding those that produce sickness. They then hooked the cocaine analog on to these proteins to make the vaccine. "We used the cocaine analog because it is a little more stable than cocaine, and it also elicits better immunity," Dr. Crystal says.

The researchers then injected billions of these viral concoctions into "garden variety" laboratory mice (mice that are not genetically engineered). They found a strong immune response was generated against the vaccine, and that these antibodies, when put in test tubes, gobbled up cocaine.

They then tested the vaccine's effect on behavior, and found that mice that received the vaccine before cocaine were much less hyperactive while on the drug than mice that were not vaccinated. The effect was even seen in mice that received large, repetitive doses of cocaine. Proportionally, the cocaine doses reflected amounts that humans might use.

The vaccine needs to be tested in humans, of course, says Dr. Crystal, but he predicts that if it works, it will function best in people who are already addicted to cocaine and who are trying to stop using the drug. "The vaccine may help them kick the habit because if they use cocaine, an immune response will destroy the drug before it reaches the brain's pleasure center."

In addition to Dr. Crystal, the study's authors included Martin J. Hicks, Bishnu P. De, Jonathan B. Rosenberg, Jesse T. Davidson, Neil R. Hackett, Stephen M. Kaminsky and Miklos Toth of Weill Cornell Medical College; Jason G. Mezey of Weill Cornell Medical College and Cornell University in Ithaca, N.Y.; Amira Y. Moreno, Kim D. Janda, Sunmee Wee and George F. Koob of the Scripps Research Institute in La Jolla, Calif.

The study was funded by the National Institute on Drug Abuse (NIDA) of the National Institutes of Health.

Birch Bark Ingredient Comes With Many Metabolic Benefits

ScienceDaily (Jan. 5, 2011) — An ingredient found in abundance in birch bark appears to have an array of metabolic benefits, according to new studies in animals that are reported in the January issue of Cell Metabolism, a Cell Press publication. In mice, the compound known as betulin lowered cholesterol, helped prevent diet-induced obesity, and improved insulin sensitivity. Betulin-treated mice were also more resistant to developing atherosclerotic plaques in their arteries.

Betulin works by targeting so-called sterol regulatory element-binding proteins (SREBPs), transcription factors that are known to be important for activating the expression of genes involved in the biosynthesis of cholesterol, fatty acids, and triglycerides.

"Our study shows that the SREBP pathway is a good target for several metabolic diseases," said Bao-Liang Song of the Shanghai Institutes for Biological Sciences. "We also identify a leading compound."

In the new study, Song and his colleagues went in search of a compound that might act directly on SREBP. That chemical screen turned up betulin as a top contender. They then confirmed in cells that betulin lowered the activity of genes that are normally switched "on" by SREBP. It also lowered lipid levels within cells.

Song's team then treated mice on a high-fat, Western diet with betulin, the cholesterol-lowering statin known as lovastatin, or a placebo (saline) for 6 weeks. Compared to placebo, both drugs led the mice to gain less weight on the high-fat diet, though by different means. Betulin caused the animals to burn more calories while lovastatin appeared to reduce the amount of lipid taken up from the diet.

Further investigations showed that betulin also lowered lipid levels in blood, liver, and fat tissue. Betulin also made the animals more sensitive to insulin. Mice with a mutation that makes them prone to develop atherosclerosis showed fewer plaques when treated with either lovastatin or betulin.

"Betulin has several major metabolic effects," Song said.

The researchers say that their findings suggest that betulin may have similar or even better effects than lovastatin, a member of the most widely prescribed drug class for treating high cholesterol. For instance, in their studies betulin decreased lipids in liver and fat to a greater extent than lovastatin did. Betulin also improved insulin resistance through its effects on fatty acid and triglyceride synthesis.

Song notes that betulin is a readily available compound and is already in use as a precursor in the manufacture of other drugs.

Although betulin appears to have very low toxicity, he says future studies will need to further investigate the safety of betulin and its metabolic effects. Researchers will also explore the possibility that a derivative of betulin might have even greater potency. "That may be the path forward to move this clinically," Song said.

Infant Hydrocephalus, Seasonal and Linked to Farm Animals in Uganda

ScienceDaily (Jan. 5, 2011) — Hydrocephalus in Ugandan children and other developing countries is seasonal, linked to farm animals and in part, caused by previous bacterial infection, according to an international team of researchers from Uganda and the United States, who believe that the best approach to this problem is prevention.

"Hydrocephalus in infants in developing countries is a grand medical mystery," said Steven Schiff, the Brush Chair professor of engineering and director, Penn State Center for Neural Engineering.

Hydrocephalus is a build up of the fluid that normally surrounds the brain. The increased pressure causes the head to swell and damages brain tissue. Treatment includes placing a shunt to drain the fluid, but inevitably these shunts become plugged and require emergency care, not always available in rural Africa and other resource-limited regions of the developing world. Surgeons vigorously explore the use of new brain endoscopes to divert fluid buildup internally in such children, but this approach addresses the fluid and does not fix previous infection damage to the brain.

"Brains of children with hydrocephalus can be completely or mostly destroyed either by the scarring from the disease or by the pressure of the cerebrospinal fluid that cannot escape," said Schiff. "Many of these children with the worst aftereffects of infection will be mentally deficient and survive only as long as their mothers can adequately care for them. Understanding the causes could eliminate or prevent the enormous costs to lives and families that hydrocephalus brings."

Hydrocephalus in infants in sub-Saharan Africa is thought to be caused most often by meningitis-type infections during the first month of life. The U.S. and Ugandan researchers looked at the fluid from the brains of three sets of 25 consecutive infant hydrocephalus patients during January, July and October to try to determine the cause of the disease. By the time parents bring infants with rapidly growing heads to the CURE Children's Hospital in Mbale, Uganda, the underlying infection is gone. The researchers were unable to culture any bacteria from the samples.

To identify traces of previous bacterial infection, the researchers used DNA sequencing to look for 16S ribosomal DNA that exists in all bacteria. They reported their findings in the current issue of the Journal of Neurosurgery: Pediatrics, showing that 94 percent of the samples contained bacterial remnants. The researchers found a seasonal difference between samples representing infection during the dry season that were predominantly Betaproteobacteria and Gammaproteobacteria, that resulted from rainy season infection. Acinetobacter appeared in the majority of patients following rainy season infection.

Some sequences that appeared in the DNA analysis were from unknown bacteria and in many cases the bacterial fragments were not identifiable as to the type of Acinetobacter they represented.

In the United States and other industrialized countries, infant hydrocephalus is usually due to either a congenital anomaly or, in low birthweight premature infants, due to brain hemorrhages from immature blood vessels. At one time, Group B Streptococcus was a common cause of postinfectious hydrocephalus in infants in industrialized countries, but now physicians test mothers for the infection and treat with antibiotics before they give birth and the infections are rare. Surprisingly, according to Schiff, in Uganda, none of the remnant DNA in the infants was from Group B Streptococcus.

Looking for the source of the neonatal infections, the researchers targeted the living environment from infants with evidence of prior acinetobacter infection and located patients' homes. What they found were villages of huts where cow dung was pounded into the hut floors to keep water and ants out and used in patios around the huts where vegetation is cleared to protect against snakes. Newborns enter an environment where they not only live near animals, but also are surrounded by their material.

The researchers sampled both the cow dung floors and excrement from cattle, goats and chickens. They found similar genetic sequences from the bacteria retrieved from the infants as in the hut floors and nearby dung.

"It is really hard to keep infants to an adequate standard of cleanliness in this environment," said Schiff. "The bacteria we found reflects, I think, a significant environmental influence."

While the researchers have not yet proven that these bacterial infections are the cause of the devastating hydrocephalus occurrences, they believe that in part, bacterial infections from animals are the cause.

Historically, certain East African peoples have applied cow dung to stem bleeding in umbilical cord stumps, which caused newborn infections. Although such infections are now rare, the scope of newborn bacterial infections related to living in close proximity to domestic animals remains poorly categorized.

"As far as we can tell, these types of environmental newborn infections are the dominant cause of hydrocephalus on the planet," said Schiff. "We may be dealing with bacteria that we can't culture, viruses or parasites, and we may be dealing with different organisms in different locations"

The researchers are continuing their work and forming an African Hydrocephalus Consortium with Rwanda, Kenya, Tanzania and Zambia. They are conducting follow-up clinical trials at the Mbarara University of Science and Technology in southwest Uganda on mother-infant pairs with new neonatal infections, and at the CURE Children's Hospital of Uganda on older infants with postinfectious hydrocephalus. These trials use next generation technologies and high quality microbiology to sort out the causative agents affecting these infants. They are also continuing to explore the environmental connection so that public health strategies toward preventing the initial infections might be found.

Other Penn State researchers on the project include Linguine Li, postdoctoral fellow, veterinary and biomedical sciences; Abinash Padhi, postdoctoral fellow, biology; and Sylvia L. Ranjeva, undergraduate, engineering science and mechanics, who were first authors on the paper; Bhushan Jayaro, director, Animal Diagnostic Lab and professor of veterinary and biomedical sciences; Vivek Kapur, head and professor, veterinary and biomedical sciences; Mary Poss, professor, biology and veterinary and biomedical sciences, all at Penn State .

Also part of the project were Benjamin C. Warf, associate professor of surgery, Harvard Medical School, Children's Hospital Boston and Derek Johnson, executive director; John Mugamba, medical director and Zephania Opio, laboratory director, CURE Children's Hospital of Uganda.

The Penn State Clinical and Translational Sciences Institute and Harvey F. Brush endowment funds supported this work.