TB Vaccine Gets Its Groove Back

 

ScienceDaily (May 24, 2009) — A team of Vanderbilt University Medical Center investigators has cracked one of clinical medicine's enduring mysteries – what happened to the tuberculosis vaccine. The once-effective vaccine no longer prevents the bacterial lung infection that kills more than 1.7 million people worldwide each year.

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Their solution, reported in the journalPLoS One, could lead to an improved TB vaccine and also may offer a novel platform for vaccines against other pathogens.

"Our findings represent nearly a 180-degree reversal from the dogma of the last 60 years – that the TB vaccine stopped working because it became over-attenuated and was too 'wimpy' to be effective," said Douglas Kernodle, M.D., associate professor of Medicine.

Instead, Kernodle and colleagues found that the TB vaccine has acquired some traits that make it less effective in evoking a sustained immune response. When they take away these traits, the TB vaccine induces stronger immune responses in mice.

The current TB vaccine, known as BCG (bacille Calmette-Guérin), has been around since the 1920s. It was made by weakening (attenuating) a strain of bacteria that causes tuberculosis in cows and that genetically is 98 percent identical to the human TB germ.

During the early years of its use, BCG was 80 percent effective against pulmonary TB. But because there were no long-term storage options for bacterial strains until the 1960s, BCG was grown continuously in culture, with "sub-cultures" of the original BCG maintained in laboratories around the world. Over time, BCG changed – the original vaccine ceased to exist and the daughter sub-cultures lost effectiveness against pulmonary TB.

Today, although BCG no longer protects against lung disease, it is still 80 percent effective against "disseminated TB" (TB infection in many parts of the body) in early childhood. Because of this protection, BCG is given annually to 100 million newborns worldwide – not in the United States and a few other countries – and is estimated to prevent about 40,000 cases each year of TB meningitis and other disseminated TB, Kernodle said.

But the question of why BCG lost its effectiveness against pulmonary TB has not been fully investigated. Researchers accepted the notion that as BCG was grown in culture, it changed genetically and became too weak to evoke the kind of immune response needed for protection.

Kernodle and colleagues came to the problem of BCG's poor activity against pulmonary TB from a different angle. They had reported in 2001 that one way TB itself evades the immune system is by producing antioxidants. Since BCG also produces antioxidants, they suggested that removing BCG's antioxidant-producing capacity might improve the vaccine.

"Our idea to take something away from BCG – and therefore theoretically attenuate it even further – was met with a lot of skepticism," Kernodle said. "But we believed our data that we could make BCG more immunogenic and safer."

Two years ago, after the Kernodle group had modified BCG and was beginning to test it for immune responses, researchers at the Institut Pasteur in Paris published a paper describing the genomic evolution of BCG. They found that in addition to containing gene deletions consistent with attenuation of the vaccine, the BCG genome also had regions of gene duplication and increased gene expression. Some of the duplicated and over-expressed genes were for antioxidants already being targeted by the Kernodle group.

It was suddenly obvious what had happened to BCG, Kernodle said.

"It had not become too weak – instead, by making more antioxidants it had become better at suppressing immune responses."

In the current studies, first author Lakshmi Sadagopal, Ph.D., research instructor of Medicine, vaccinated mice with a modified BCG (genetically changed in three ways to reduce or eliminate the production of several antioxidants) and examined the immune response in the days following vaccination and later with a "challenge" dose of BCG.

She found that, compared to BCG, the modified BCG induced greater cytokine (immune regulatory factor) production during the early phase of the immune response, more CD8 cell-killing T cells at the peak of the primary response, and more CD4 helper T cells during the memory phase. Modified BCG also produced greater recall immune responses and was eliminated better by the vaccinated host animal than the parent BCG vaccine, which might correlate with improved safety in humans.

"At each time point of the immune response, the modified BCG vaccine worked better than the parent BCG vaccine," Kernodle said. "By targeting antioxidants that had increased in expression during decades of cultivation, we ended up making BCG more like it was back in the 1920s when it was 80 percent effective against pulmonary TB. We fixed it."

Using modern molecular techniques to reduce the activity of antioxidants below levels in naturally occurring strains, "it should be possible to make it even better than the original BCG," he added.

The Aeras Global TB Vaccine Foundation, supported by the Bill & Melinda Gates Foundation, has already licensed the modification technology developed by Kernodle and colleagues. Aeras is working to make the best possible modified BCG vaccine, and it has built the infrastructure to conduct clinical trials in South Africa, Kenya and India – countries with a high incidence of TB.

Kernodle and colleagues say the results are also encouraging for other vaccine development. Because the modified BCG produces a better immune response profile than existing vaccine technologies, it could be a useful vector for vaccines directed against other pathogens, including HIV and the parasites that cause malaria.

The National Institutes of Health, a VUMC Discovery Award and the Aeras Global TB Vaccine Foundation supported the research. Kernodle is the David E. Rogers Associate Professor of Medicine.

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Adapted from materials provided by Vanderbilt University Medical Center.

High Blood Pressure COuld Be Caused BY A Common Virus, Study Suggests

ScienceDaily (May 16, 2009) — A new study suggests for the first time that cytomegalovirus (CMV), a common viral infection affecting between 60 and 99 percent of adults worldwide, is a cause of high blood pressure, a leading risk factor for heart disease, stroke and kidney disease.

Led by researchers at Beth Israel Deaconess Medical Center (BIDMC) and published in the May 15, 2009 issue of PLoS Pathogens, the findings further demonstrate that, when coupled with other risk factors for heart disease, the virus can lead to the development of atherosclerosis, or hardening of the arteries.
"CMV infects humans all over the world," explains co-senior author Clyde Crumpacker, MD, an investigator in the Division of Infectious Diseases at BIDMC and Professor of Medicine at Harvard Medical School. "This new discovery may eventually provide doctors with a whole new approach to treating hypertension, with anti-viral therapies or vaccines becoming part of the prescription."
A member of the herpes virus family, CMV affects all age groups and is the source of congenital infection, mononucleosis, and severe infection in transplant patients. By the age of 40, most adults will have contracted the virus, though many will never exhibit symptoms. Once it has entered the body, CMV is usually there to stay, remaining latent until the immune system is compromised, when it then reemerges.
Previous epidemiological studies had determined that the CMV virus was linked to restenosis in cardiac transplant patients, a situation in which the heart's arteries "reblock." The virus had also been linked to the development of atherosclerosis, the hardening of the heart's arteries. But, in both cases, the mechanism behind these developments remained a mystery. This new study brought together a team of researchers from a variety of disciplines – infectious diseases, cardiology, allergy and pathology – to look more closely at the issue.
"By combining the insights of investigators from different medical disciplines, we were able to measure effects of a viral infection that may have been previously overlooked," explains Crumpacker.
In the first portion of the study, the scientists examined four groups of laboratory mice. Two groups of animals were fed a standard diet and two groups were fed a high cholesterol diet. After a period of four weeks, one standard diet mouse group and one high-cholesterol diet mouse group were infected with the CMV virus.
Six weeks later, the animals' blood pressures were measured by the cardiology team using a small catheter inserted in the mouse carotid artery. Among the mice fed a standard diet, the CMV-infected mice had increased blood pressure compared with the uninfected group. But even more dramatically, 30 percent of the CMV-infected mice that were fed a high-cholesterol diet not only exhibited increased blood pressure, but also showed signs of having developed atherosclerosis.
"This strongly suggests that the CMV infection and the high-cholesterol diet might be working together to cause atherosclerosis," says Crumpacker. In order to find out how and why this was occurring, the investigators went on to conduct a series of cell culture experiments.
Their first analysis demonstrated that CMV stimulated production of three different inflammatory cytokines – IL6, TNF, and MCP1 – in the infected mice, an indication that the virus was causing inflammation to vascular cells and other tissues.
A second analysis found that infection of a mouse kidney cell line with murine CMV led to an increase in expression of the renin enzyme, which has been known to activate the renin-angiotensin system and lead to high blood pressure. Clinical isolates of human CMV in cultured blood vessel cells also produced increased renin expression.
"Viruses have the ability to turn on human genes and, in this case, the CMV virus is enhancing expression of renin, an enzyme directly involved in causing high blood pressure," says Crumpacker. When the scientists inactivated the virus through the use of ultraviolet light, renin expression did not increase, suggesting that actively replicating virus was causing the increase in renin.
In their final experiments, the researchers demonstrated that the protein angiotensin 11 was also increased in response to infection with CMV. "Increased expression of both renin and angiotensin 11 are important factors in hypertension in humans," says Crumpacker. "What our study seems to indicate is that a persistent viral infection in the vessels' endothelial cells is leading to increased expression of inflammatory cytokines, renin and angiotensin 11, which are leading to increased blood pressure."
According to recent figures from the American Heart Association, one in three U.S. adults has high blood pressure, and because there are no known symptoms, nearly one-third of these individuals are unaware of their condition. Often dubbed "the silent killer," uncontrolled high blood pressure can lead to stroke, heart attack, heart failure or kidney failure, notes Crumpacker.
"We found that CMV infection alone led to an increase in high blood pressure, and when combined with a high-cholesterol diet, the infection actually induced atherosclerosis in a mouse aorta," says Crumpacker. "This suggests that further research needs to be directed at viral causes of vascular injury. Some cases of hypertension might be treated or prevented by antiviral therapy or a vaccine against CMV."
This study was funded by grants from the National Heart, Lung and Blood Institute of the National Institutes of Health.
Study co-authors include Jielin Zhang of BIDMC's Division of Infectious Diseases (co-senior author); Jilin Cheng formerly of BIDMC's Division of Infectious Diseases and now at Fudan University, Shanghai, China (first author); Qingen Ke of BIDMC's Division of Cardiology; Zhuang Jin and Haiban Wang of BIDMC's Division of Allergy; Olivier Kocher of BIDMC's Division of Pathology; and James Morgan of Caritas St. Elizabeth's Medical Center, Boston.
Adapted from materials provided by Beth Israel Deaconess Medical Center.

Natural Science Lecture

Title: Behavioural Study Of Porcupines In Sarawak Planted Forest Project

Speaker: Alex Jukie

Date: 16th May 2009 (Saturday)

Time: 2.00-4.00pm

Registration 2.00-2.30pm

Venue: New World Suite Bintulu

Free Admission

Climate Adds Fuel To Asian Wildfire Emissions

ScienceDaily (May 10, 2009) — In the last decade, Asian farmers have cleared tens of thousands of square miles of forests to accommodate the world's growing demand for palm oil, an increasingly popular food ingredient. Ancient peatlands have been drained and lush tropical forests have been cut down.

As a result, the landscape of equatorial Asia now lies vulnerable to fires, which are growing more frequent and having a serious impact on the air as well as the land.

A team of NASA-sponsored researchers have used satellites to make the first series of estimates of carbon dioxide (CO₂) emitted from these fires -- both wildfires and fires started by people -- in Malaysia, Indonesia, Borneo, and Papua New Guinea. They are now working to understand how climate influences the spread and intensity of the fires.

Using data from a carbon-detecting NASA satellite and computer models, the researchers found that seasonal fires from 2000 to 2006 doubled the amount of carbon dioxide (CO₂) released from the Earth to the atmosphere above the region. The scientists also observed through satellite remote sensing that fires in regional peatlands and forests burned longer and emitted ten times more carbon when rainfall declined by one third the normal amount. The results were presented in December 2008 in Proceedings of the National Academy of Sciences.

Tropical Asian fires first grabbed the attention of government officials, media, and conservationists in 1997, when fires set to clear land for palm oil and rice plantations burned out of control. The fires turned wild and spread to dry, flammable peatlands during one of the region's driest seasons on record. By the time the flames subsided in early 1998, emissions from the fires had reached 40 percent of the global carbon emissions for the period.

"In this region, decision makers are facing a dichotomy of demands, as expanding commercial crop production is competing with efforts to ease the environmental impact of fires," said Jim Collatz, an Earth scientist at NASA's Goddard Space Flight Center in Greenbelt, Md., and a co-author of the study. "The science is telling us that we need strategies to reduce the occurrence of deforestation fires and peatlands wildfires. Without some new strategies, emissions from the region could rise substantially in a drier, warmer future."

Since the 1997 event, the region has been hit by two major dry spells and a steady upswing in fires, threatening biodiversity and air quality and contributing to the buildup of CO₂ in the atmosphere. As more CO2 is emitted, the global atmosphere traps more heat near Earth's surface, leading to more drying and more fires.

Until recently, scientists knew little about what drives changes in how fires spread and how long they burn. Collatz, along with lead author Guido van der Werf of Vrije University, Amsterdam, and other colleagues sought to estimate the emissions since the devastating 1997-98 fires and to analyze the interplay between the fires and drought.

They used the carbon monoxide detecting Measurements of Pollution in the Troposphere (MOPITT) instrument on NASA's Terra satellite -- as well as 1997-2006 fire data and research computer models -- to screen for and differentiate between carbon emissions from deforestation versus general emissions. Carbon monoxide is a good indicator of the occurrence of fire, and the amounts of carbon monoxide in fire emissions are related to the amount of carbon dioxide. They also compared the emissions from different types of plant life (peat land vs. typical forest) by examining changes in land cover and land use as viewed by Terra's Moderate Resolution Imaging Spectradiometer (MODIS) and by Landsat 7.

Collatz explained that two climate phenomena drive regional drought. El Niño's warm waters in the Eastern Pacific change weather patterns around the world every few years and cause cooler water temperatures in the western Pacific near equatorial Asia that suppress the convection necessary for rainfall. Previously, scientists have used measurements from NASA's Tropical Rainfall Measurement Mission satellite to correlate rainfall with carbon losses and burned land data, finding that wildfire emissions rose during dry El Niño seasons. The Indian Ocean dipole phenomenon affects climate in the Indian Ocean region with oscillating ocean temperatures characterized by warmer waters merging with colder waters to inhibit rainfall over Indonesia, Borneo, and their neighbors.

"This link between drought and emissions should be of concern to all of us," said co-author Ruth DeFries, an ecologist at Columbia University in New York. "If drought becomes more frequent with climate change, we can expect more fires."

Collatz, DeFries, and their colleagues found that between 2000 and 2006, the average carbon dioxide emissions from equatorial Asia accounted for about 2 percent of global fossil fuel emissions and 3 percent of the global increase in atmospheric CO₂. But during moderate El Niño years in 2002 and 2006, when dry season rainfall was half of normal, fire emissions rose by a factor of 10. During the severe El Niño of 1997-1998, fire emissions from this region comprised 15 percent of global fossil fuel emissions and 31 percent of the global atmospheric increase over that period.

"This study not only updates our measurements of carbon losses from these fires, but also highlights an increasingly important factor driving change in equatorial Asia," explained DeFries. "In this part of Asia, human-ignited forest and peat fires are emitting excessive carbon into the atmosphere. In climate-sensitive areas like Borneo, human response to drought is a new dynamic affecting feedbacks between climate and the carbon cycle."

In addition to climate influences, human activities contribute to the growing fire emissions. Palm oil is increasingly grown for use as a cooking oil and biofuel, while also replacing trans fats in processed foods. It has become the most widely produced edible oil in the world, and production has swelled in recent years to surpass that of soybean oil. More than 30 million metric tons of palm oil are produced in Malaysia and Indonesia alone, and the two countries now supply more than 85 percent of global demand.

The environmental effects of such growth have been significant. Land has to be cleared to grow the crop, and the preferred method is fire. The clearing often occurs in drained peatlands that are otherwise swampy forests where the remains of past plant life have been submerged for centuries in as much as 60 feet of water. Peat material in Borneo, for example, stores the equivalent of about nine years worth of global fossil fuel emissions.

"Indonesia has become the third largest greenhouse gas emitter after the United States and China, due primarily to these fire emissions," Collatz said. "With an extended dry season, the peat surface dries out, catches fire, and the lack of rainfall can keep the fires going for months."

Besides emitting carbon, the agricultural fires and related wildfires also ravage delicate ecosystems in conservation hotspots like the western Pacific island of Borneo, home to more than 15,000 species of plants, 240 species of trees, and an abundance of endangered animals.

Smoke and other fire emissions also regularly taint regional air quality to such a degree that officials have to close schools and airports out of concern for public health and safety. Peat fires also aggravate air pollution problems in this region because they release four times more carbon monoxide than forest fires. In 1997, air pollution from the fires cost the region an estimated $4.5 billion in tourism and business.

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Adapted from materials provided by NASA/Goddard Space Flight Center.

Ultrasonic Communcation Among Frogs

Ultrasonic Communication Among Frogs

ScienceDaily (May 9, 2009) — UCLA scientists report on the only known frog species that can communicate using purely ultrasonic calls, whose frequencies are too high to be heard by humans. Known as Huia cavitympanum, the frog lives only on the Southeast Asian island of Borneo.

Ultrasounds are high-pitched sounds more than 20 kilohertz (kHz) in frequency, which exceeds the upper limit of sounds detectable by humans and is far higher than the 5 to 8 kHz frequencies most amphibians, reptiles and birds are capable of hearing or producing. Key parts of the ear must be specially adapted to detect ultrasounds.

The frogs can hear sounds up to 38 kilohertz, the highest frequency any amphibian species has been known to hear, the scientists report. Humans can hear up to about 20 kHz and typically talk at 2 or 3 kHz.

While most of the more than 5,000 frog species worldwide have eardrums that are flat on the side of the head, Huia cavitympanum has eardrums recessed in the side of the skull, similar to mammals.

Peter Narins, UCLA distinguished professor of physiological science and of ecology and evolutionary biology, and Victoria Arch, a UCLA graduate student in ecology and evolutionary biology, spent several nights in the remote area where the frogs live.

"We had very little information suggesting that they would be in this location," said Arch, lead author of the study. "We found them our first night out."

Huia cavitympanum produces some audible calls and others that are entirely ultrasonic.

"You look at the frog and can see it is vocalizing — you can tell because their vocal pouches pulsate — but you don't hear any sound. It's amazing," Arch said. "Then you look down at the recording equipment and see the lights flashing, indicating sound. I have never seen that before. In a frog, it's unprecedented to have purely ultrasonic vocalizations."

Narins and Arch have an idea about why the frogs, which live along a noisy stream, use both ultrasonic communication and calls that are audible to humans.

"Our hypothesis is that these frogs have shifted to use higher frequencies in their communication to avoid the interference of sound produced by rushing water in the lower-frequency range," Arch said. "However, high-frequency sounds do not transmit as far. By producing some lower-frequency calls, they can transmit calls over a greater distance, so they can communicate with frogs that are farther away. But by also producing some calls that are high-frequency — all ultrasonic — they may be able to communicate better over that background noise.

"Producing both types of calls might be a bet-hedging strategy to be heard," Arch said. "This is how males attract females, communicate with one another and establish territorial boundaries."

The research may have implications for improving human hearing aids and addressing hearing loss. Most hearing aids, Narins said, simply amplify sound, including unnecessary background noise. Better hearing aids that are directional, for example, could result from the research.

In 1998, Kraig Adler, a professor of neurobiology and behavior who had been Narins' professor at Cornell University, told Narins he should go to China to see "a frog with an ear canal like a human," Narins recalled.

Narins took the advice. Along with colleagues, including Albert Feng from the University of Illinois, Urbana-Champaign, he discovered a frog species that lives in rushing streams and waterfalls in east-central China called Odorrana tormota(formerly known as Amolops tormotus). Odorrana tormota, Narins found, could detect and produce ultrasounds due to the highly unusual presence of an ear canal. That research was published March 16, 2006, in the journal Nature.

The frogs in China produce very loud calls that are, at the same time, both audible and ultrasonic. In Borneo, however, Huia cavitympanum produces some sounds that are purely ultrasonic.

"That was unique — and the first time that has been found in any frog species," said Narins, who has studied dozens of frog species. "We are reporting this for the first time."

"With the Chinese frog species, every time you see the vocal sac pulsate, you will hear a sound," Arch said. "With the Bornean frog, sometimes you will see it is calling and you don't hear anything; it's all outside of our hearing range."

The frogs in China and Borneo's Huia cavitympanum are the only two frog species known to have recessed ear drums, Narins said.

In China, the scientists had set up a loudspeaker in the frogs' natural habitat near a river, then played frog calls and recorded the frogs' vocal responses with a microphone and very high-frequency equipment.

Later, they learned of this second species in Borneo with similar ear morphology. Narins and Arch went to Borneo in 2007, and Arch went back last summer.

In Borneo, they lived in a camp with a roof and three walls, with one side open. They slept on a wood floor, protected by mosquito nets. Narins was bitten by leeches and woke up several mornings soaked in blood. They ate noodles and peanut butter.

"It was a huge adventure, and so exciting to study the animals in their habitat," Arch said. "You feel like you're eavesdropping on them."

"There are not many places on the planet left that are so pristine and untouched by humans," Narins said. "It's exciting to go there and wonderful to have a sense of discovering something brand-new. We were there a week and saw only two other people."

Arch and Narins studied male frogs. In the majority of frog species, the females do not vocalize, and when they do, they do so less frequently than males, Arch said.

The research was funded by the National Geographic Society's Committee for Research and Exploration, the Paul S. Veneklasen Research Foundation, the National Institute on Deafness and Other Communication Disorders (NIDCD) of the National Institutes of Health, and the National Science Foundation (through a doctoral dissertation improvement grant to Arch).

Co-authors are T. Ulmar Grafe, a faculty member in the biology department at Borneo's University Brunei Darussalam, and Marcos Gridi-Papp, a UCLA postdoctoral scholar in Narins' laboratory.

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Journal reference:

1. Arch et al. Pure Ultrasonic Communication in an Endemic Bornean Frog. PLoS ONE, 2009; 4 (4): e5413 DOI: 10.1371/journal.pone.0005413

Adapted from materials provided by University of California, Los Angeles.