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May 1, 2014

Research Notes

Malfunction in molecular “proofreader” prevents repair of UV-induced DNA damage

Malfunctions in the molecular “proofreading” machinery, which repairs structural errors in DNA caused by ultraviolet (UV) light damage, help explain why people who have the disease xeroderma pigmentosum (XP) are at an extremely high risk for developing skin cancer, according to researchers at the School of Medicine and the University of Pittsburgh Cancer Institute (UPCI). Their findings will be published in the early online version of the Proceedings of the National Academy of Sciences.

Previous research has shown that a DNA-repair protein called human UV-damaged DNA-binding protein, or UV-DDB, signals for a repair when two UV-DDB molecules bind to the site of the problem, said senior investigator Bennett Van Houten, a faculty member in the School of Medicine.

“Our new study shows UV-DDB makes stops along the DNA strand and transiently attaches to it, causing a proofreading change in the protein’s conformation, or shape. If the DNA is damaged the protein stays; if the DNA is not damaged the protein leaves,” he said. “When it comes to a spot that has been damaged by UV radiation, two molecules of UV-DDB converge and stay tightly bound to the site, essentially flagging it for the attention of repair machinery.”

The researchers followed the trail of single molecules of UV-DDB by tagging them with light-emitting quantum dots, enabling them to watch the molecules jump from place to place in real time on both normal and UV-exposed DNA strands.

They also tracked a mutant UV-DDB protein associated with XP, an inherited and incurable disease of light sensitivity that affects about one in 250,000 people. They found that the mutant UV-DDB molecules still are capable of binding to DNA, but continued to slide along the DNA rather than staying put to signal where the fix was needed.

“Without this important damage control, UV-induced errors could accumulate to cause cell alterations that foster cancer development,” Van Houten said. “Like a bus with no brakes, the XP-associated UV-DDB complex stays on the road and sees possible passengers, but keeps going past the stop.”

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Climate change study in Pacific NW lakes may presage future drought

Sorting through oxygen isotopes in muddy sediment at the bottom of 10 Pacific Northwest lakes has led a research team, including Pitt scientists, to conclude that droughts in that region match natural regional warming for various periods over the past 2,000 years.

The team — which includes Mark Abbott, a faculty member in the Department of Geology and Planetary Sciences, and Pitt alumnus Byron Steinman, now a postdoctoral fellow at Penn State — says in a paper published in Geophysical Research Letters online that tying long-ago droughts to protracted natural climate change may show what can be expected as man-made climate change warms the Earth.

Abbott and Steinman, along with their collaborators, took cores from the lake bottoms that penetrated into the lake mud as much as 30 feet.

They measured the sediments that contain limestone for two oxygen isotopes — oxygen 16 and oxygen 18. Oxygen 18, the heavier of the two, is known to be present in greater abundance during periods of drought.

“This work contributes to our understanding of how the climate system has worked in the past with the goal of improving our ability to predict future droughts,” Abbott said. “And this knowledge should give us a better idea of how often droughts might occur in the future as the climate system changes.”

He also noted that the United Nations Intergovernmental Panel on Climate Change recently released its 2014 report, which predicts dire consequences, including drought, as a consequence of rapidly advancing man-made climate change.

The paper, “Ocean-atmosphere Forcing of Centennial Hydroclimate Variability in the Pacific Northwest,” is available online.

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Sanofi Pasteur,  Pitt collaborate to assess effectiveness of dengue vaccine

Pitt’s Center for Vaccine Research (CVR) and Sanofi Pasteur, the vaccines division of Sanofi, have entered a scientific collaboration to help assess the effectiveness of a dengue vaccine once introduced for immunization programs.

CVR is creating the new test to help assess the effectiveness of Sanofi Pasteur’s dengue vaccine candidate, which aims to reduce cases of dengue and the circulation of the virus in the population. The new test will tell if a person’s immunity to the mosquito-borne virus is due to a previous natural infection or from vaccination.

“Distinguishing whether a person’s immune response is from the vaccine or from infection by a mosquito can play an important role in the assessment of a candidate vaccine,” said Ernesto Marques, a faculty member in the Department of Infectious Diseases and Microbiology.

“The goal of this test is to provide additional support in assessing the effectiveness of the vaccine after introduction.”

Dengue disease is caused by four types of dengue virus. It occurs mostly in tropical and subtropical countries, putting about half the world’s population at risk. It is endemic in Puerto Rico and locally acquired cases re-emerged recently in the Florida Keys and Texas. There is no treatment for dengue and no vaccine to prevent it.

It is estimated that around 100 million clinical cases of dengue occur annually, but a larger number of additional cases are so mild that the people who are infected don’t even realize they have it. Each year 500,000 people, including children, develop severe dengue, characterized by high fever, uncontrolled bleeding, respiratory distress and organ failure.

For every symptomatic case of dengue, there could be as many as three asymptomatic, or “silent” cases, according to recent international research.

Nicholas Jackson, head of dengue research and development for Sanofi Pasteur, said: “The new dengue test will be important to fully understand the impact of vaccination by providing additional support in assessing symptomatic versus silent infections, ultimately helping officials gauge how much a vaccine reduces disease transmission.”

“This test also could be used by the government and health agencies to manage an immunization program,” added Marques. “It will give evidence that the vaccine works and could allow doctors to determine which populations still need vaccination so they can most effectively target their immunization outreach efforts.”

The Sanofi Pasteur dengue vaccine candidate was found to be safe and demonstrated protection against three of the four dengue virus types in the first efficacy clinical study, with results reported in 2012 in The Lancet. The study, which included 4,002 children, was conducted in a region of Thailand where dengue is highly endemic, and it was the first time a dengue vaccine candidate showed protection against the virus.

Data from Sanofi Pasteur’s ongoing phase III clinical studies with over 31,000 volunteers are expected to be available later this year and will document efficacy of their vaccine in a broader population and different epidemiological environments.

Pitt has a strong history in dengue research, most notably the first isolation and characterization of two of the four types of dengue virus in 1958 by William M. Hammon, then a faculty member in microbiology and epidemiology at the Graduate School of Public Health.

In 1980, Donald S. Burke, currently the CVR co-director and dean of the public health school, isolated dengue type 2 viruses in Bangkok.

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UPCI awarded nearly $10M in NCI grants

The University of Pittsburgh Cancer Institute (UPCI) has been awarded two grants from the National Cancer Institute (NCI), totaling nearly $10 million, that will aid in bringing the latest research developments from bench to bedside and accelerate research into such things as rare tumors. UPCI is one of only 12 centers in the country to receive the NCI Experimental Therapeutics-Clinical Trials Network with Phase I Emphasis grant and the only center in Pennsylvania to receive a Lead Academic Participating Site (LAPS) grant under the NCI’s new clinical trials network.

UPCI is the only NCI-designated comprehensive cancer center in western Pennsylvania and through the network of its clinical partner, UPMC CancerCenter, enables several thousand patients to participate in clinical trials each year.

“Participating in a clinical trial is the optimal form of therapy for patients who are willing and able and allows us to learn something for the future along the way,” said Nancy E. Davidson, a faculty member in the Department of Medicine and director of UPCI and the UPMC CancerCenter.

She added: “We are grateful for the support of our patients and providers who have been an integral part of our success and helped us attain these two very important awards.”

The NCI Experimental Therapeutics-Clinical Trials Network with Phase I Emphasis grant is led by Edward Chu, a faculty member in the Department of Medicine. The  $4.25  million,  five-year  grant  funds complex research into new drug therapies.

“Our focus is on developing completely novel agents and combination regimens,” Chu said. “We also are trying to understand how some of these new targeted therapies work and how we can apply science to individually tailor these new treatments to specific cancers.”

UPCI is uniquely qualified to lead efforts in drug development because of the team approach that goes into the research, he noted, with expertise in pharmacokinetics, pharmacodynamics and basic science.

“We have a large patient base that allows us to do these novel first-in-man studies. The large majority of the patients who are referred to us have failed standard-of-care therapies, and they are looking for new treatments. There is only a small handful of cancer centers across the country that can offer the types of phase I clinical studies available to our patients here in Pittsburgh and the western Pennsylvania region,” he said.

The LAPS grant is part of the new National Clinical Trials Network (NCTN), designed to speed up the time it takes research to get from the lab to patients through technological advances and enhanced cooperation. The nearly $5 million award is led by Adam Brufsky, a faculty member in the Department of Medicine and UPCI’s associate director for clinical investigation. The grant will fund the costs of maintaining a clinical trials infrastructure that permits patients to enroll in national trials led by the NCTN at more than a dozen sites across the UPMC CancerCenter network.

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Pharmacy study awarded grant

Sean H. J. Kim, a faculty member in the Department of Therapy and Therapeutics, has been awarded a grant from the American Association of Colleges of Pharmacy for his study “Quantitative Mechanistic Modeling of Drug Disposition With Variable Interactions.”

The goal of this project is to establish a novel, quantitative and systems pharmacology approach to unravel common and patient-specific mechanisms of drug disposition, and develop in silico simulations that explain and predict the pharmacokinetics of complex oral formulations.

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Low cholesterol in immune cells tied to slow progression of HIV

People infected with HIV whose immune cells have low cholesterol levels experience much slower disease progression, even without medication, according to Graduate School of Public Health research that could lead to new strategies to control infection.

The researchers found that low cholesterol in certain cells, which is likely an inherited trait, affects the ability of the body to transmit the virus to other cells. The discovery, funded by the National Institutes of Health (NIH), is featured in the current issue of mBio, the journal of the American Society for Microbiology.

When HIV enters the body, it typically is picked up by immune system cells called dendritic cells, which recognize foreign agents and transport the virus to lymph nodes where it is passed to other immune system cells, including T cells. HIV then uses T cells as its main site of replication. It is through this mechanism that levels of HIV increase and overwhelm the immune system, leading to AIDS. Once a person develops AIDS, the body can no longer fight infections and cancers. Prior to effective drug therapy, the person died within one to two years after the AIDS diagnosis.

“We’ve known for two decades that some people don’t have the dramatic loss in their T cells and progression to AIDS that you’d expect without drug therapy,” said lead author Giovanna Rappocciolo, a public health faculty member.

“Instead, the disease is much slower to progress, and we believe low cholesterol in dendritic cells may be a reason.”

The discovery was made possible by using 30 years of data and biologic specimens collected through the Pitt Men’s Study, a confidential research study of the natural history of HIV/AIDS, part of the national NIH-funded Multicenter AIDS Cohort Study (MACS).

“We couldn’t have made this discovery without the MACS. Results like ours are the real pay-off of the past three decades of meticulous data and specimen collection,” said senior author Charles Rinaldo, chair of the Department of Infectious Diseases and Microbiology, and faculty member in pathology. “It is thanks to our dedicated volunteer participants that we are making such important advances in understanding HIV, and applying it to preventing and treating AIDS.”

Medications called combination antiretroviral therapy (ART) disrupt the viral replication process and can delay the onset of AIDS by decades.

However, even without taking ART, a small percentage of people infected with HIV do not have the persistent loss of T cells and increase in levels of HIV after initial infection. They sometimes can go many years, even more than a decade, without the virus seriously compromising the immune system or leading to AIDS.

Through the Pitt Men’s Study/MACS, eight such “nonprogressors” were assessed twice a year for an average of 11 years and compared to eight typically progressing HIV-positive counterparts.

Rappocciolo and her colleagues found that in nonprogressors, the dendritic cells were not transferring the virus to T cells at detectible levels.

When taking a closer look at these dendritic cells, the researchers discovered that the cells had low levels of cholesterol, even though the nonprogressors had regular levels of cholesterol in their blood. A similar finding was shown for B lymphocytes, which also pass HIV to T cells, leading to high rates of HIV replication.

Cholesterol is an essential component of the outer membranes of cells. It is required for HIV to replicate efficiently in different types of cells.

None of the study participants were taking statins, which are cholesterol-lowering medications that some people take to prevent vascular problems when cholesterol in their blood is too high.

When HIV was directly mixed with the nonprogressors’ T cells in the laboratory, those T cells became infected with the virus at the same rate as the T cells of the regularly progressing, HIV-positive participants. Indeed, T cells from the nonprogressors had normal levels of cholesterol.

“This means that the disruption is unlikely to be due to a problem with the T cells, further supporting our conclusion that the slow progression is linked to low cholesterol in the dendritic cells and B cells,” said Rappocciolo.

“What is most intriguing is that dendritic cells in the nonprogressors had this protective trait years before they became infected with HIV,” Rinaldo said. “This strongly suggests that the inability of their dendritic cells and B cells to pass HIV to their T cells is a protective trait genetically inherited by a small percentage of people. Understanding how this works could be an important clue in developing new approaches to prevent progression of HIV infection.”

Other researchers working on this study were Mariel Jais, Paolo Piazza, Todd A. Reinhart, Stella J. Berendam, Laura Garcia-Exposito and Phalguni Gupta, all of the public health school.

—Compiled by Alex Oltmanns

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The University Times Research Notes column reports on funding awarded to Pitt researchers as well as findings arising from University research.

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