University Times

Male births declining

A study published in the online edition of Environmental Health Perspectives reports that during the past 30 years the number of male births has decreased each year in the United States and Japan. In a review of all births in both countries, the Pitt-led study found significantly fewer boys being born relative to girls in the U.S. and Japan, and that an increasing proportion of fetuses that die are male. They note that the decline in births is equivalent to 135,000 fewer white males in the U.S. and 127,000 fewer males in Japan over the past three decades and suggest that environmental factors are one explanation for these trends.

“The pattern of decline in the ratio of male to female births remains largely unexplained,” said Devra Lee Davis, lead investigator of the study, director of the University of Pittsburgh Cancer Institute’s (UPCI) Center for Environmental Oncology and professor of epidemiology in the Graduate School of Public Health.

“We know that men who work with some solvents, metals and pesticides father fewer baby boys. We also know that nutritional factors, physical health and chemical exposures of pregnant women affect their ability to have children and the health of their offspring. We suspect that some combination of these factors, along with older age of parents, may account for decreasing male births.”

Davis explained that environmental factors, such as prenatal exposure to endocrine-disrupting environmental pollutants may impact the SRY gene — a gene on the Y chromosome that determines the sex of a fertilized egg. Other environmental factors that also may affect the viability of a male fetus include the parents’ weight, nutrition and the use of alcohol and drugs.

Davis and her colleagues reported an overall decline of 17 males per 10,000 births in the U.S. and a decline of 37 males per 10,000 births in Japan since 1970. They also found that while fetal death rates generally have decreased, the proportion of male fetuses that die has continued to increase. In Japan, among the fetuses that die, two-thirds are male, up from just over half in 1970.

The study also examined the ratio of African-American male-to-female births to that of whites in the U.S. The researchers found that while the number of African-American male births has increased modestly over time, the ratio of male-to-female births for African Americans remains lower than that of whites. In addition, they noted that African-Americans have a higher fetal mortality rate overall and a higher proportion of male fetuses that die.

Davis added, “Given the importance of reproduction for the health of any species, the trends we observed in the U.S. and Japan merit concern. In light of our findings, more detailed studies should be carried out that examine sex ratio in smaller groups with defined exposures as a potential indicator of environmental contamination.”

The study was supported by a grant from the Heinz Endowments, UPCI, DSF Charitable Trust, the U.S. Centers for Disease Control and Prevention and the W. Alton Jones Foundation.

Pamela Smith Webster of UPCI was among the study’s co-investigators.

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Gene-lipid links sought

The National Heart, Lung and Blood Institute of the National Institutes of Health has awarded a $2.9 million grant to researchers at the Graduate School of Public Health (GSPH) to uncover the genetic basis of high-density lipoprotein (HDL) cholesterol, the so-called “good cholesterol,” in African and U.S. white populations.

Higher levels of HDL have been shown to provide protection against the risk of coronary heart disease (CHD). Generally, African or African-derived populations have higher HDL levels than whites, which may ameliorate the risk of CHD among Africans.

According to principal investigator M. Ilyas Kamboh, acting chair of the Department of Human Genetics at GSPH, blood HDL cholesterol levels are determined both by environmental and genetic factors, with genes contributing more than 50 percent.

“The task of identifying underlying genetic factors is not easy because there are multiple genes involved as well as interactions between genes and the environment. All of these have to be factored into studies to determine what contributes to variation in plasma HDL cholesterol levels between individuals and populations,” Kamboh said.

He added that most previous efforts to identify genes for HDL cholesterol have focused on screening for a few selected single nucleotide polymorphisms (SNPs) on each potential candidate gene, which is not optimal.

However, the recent availability of high-throughput DNA sequencing technology has provided researchers the tools they need to build “dense” maps of markers for each gene being examined.

The four-year study will sequence more than 40 candidate genes from individuals who have extremely high or low HDL levels in order to find significant genetic variations.

Other GSPH researchers involved in the project include Yesim Demirci and Candace Kammerer of human genetics and Clareann Bunker of epidemiology.

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State startup grant awarded

Barry Gold, chair of pharmaceutical sciences, received a $208,335 Pennsylvania Keystone Innovation Starter Kit grant to establish a research base in which the study of membrane-associated proteins will be developed.

The state program is designed to recruit top faculty researchers by providing discretionary funds for researchers in selected growth sector fields to build up labs and staff, acquire equipment and leverage their research.

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Comp sci prof fights botnets

José Brustoloni has received grants totaling $258,000 to research defenses against botnets. Botnets comprise bots (also known as zombies or infected computers) under the control of an attacker.

It is estimated that 7 percent of the computers currently connected to the Internet are infected by at least one botnet, and 250,000 new bots are detected each day. Attackers use botnets in a variety of attacks, including identity or intellectual property theft, sending denial-of-service (DoS) attacks or spam or committing click fraud.

Brustoloni’s lab will build prototypes and evaluate new devices for detecting and mitigating botnets. A first device will detect bots, disable their communication with bot controllers, notify the owners of the infected computers and optionally quarantine the latter until they are disinfected. A second device will detect and block botnet DoS attacks. Such attacks maliciously increase traffic in Internet servers, crowding out legitimate clients.

The grants are funded by The Technology Collaborative, a state economic development agency; ECI Telecom, a manufacturer of service-provider edge routers, and Netronome, a producer of high-performance network processor cards.

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Asian Studies Center gets Toshiba grant

The Toshiba International Foundation has announced a $15,000 one-year grant to Pitt’s Asian Studies Center for a research project titled “Shifting Cultural Landscape: Globalization of Japanese Popular Culture.” Richard J. Cohen, the center’s associate director, will be the project director.

The research encompasses three faculty members’ projects: Akiko Hashimoto, “Global Heroes and Villains in Japanese Popular Culture”; Keiko McDonald, “Japanese Pop Culture Comes to the Screen: Cross-cultural Approach,” and Gabriella Lukacs, “Eating Japan: ‘Iron Chef’ and the Globalization of Japanese Television.”

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Children with ADHD at risk for alcohol problems

Children with attention deficit hyperactivity disorder (ADHD) are at risk for alcohol abuse or dependence — as well as other substance-related problems — as they grow older, new research confirms. Two Pitt-led studies show that ADHD is indeed a risk factor for alcohol problems, with parental alcoholism and stressful experiences in the family playing an important role.

Results were published in the April issue of Alcoholism: Clinical & Experimental Research.

“Children with ADHD are believed to be at risk for alcoholism because of their impulsivity and distractibility, as well as other problems that often accompany ADHD, such as school failure and behavior problems,” explained Brooke Molina, an associate professor of psychiatry and psychology at Pitt, and corresponding author for both studies. “Our studies show that adolescents with ADHD were much more likely to use alcohol than their peers without ADHD.”

In the first of the two studies on age specificity, researchers interviewed 364 children diagnosed with ADHD as adolescents (11-17 years of age) or as young adults (18-28 years of age). Demographically and age-matched individuals without ADHD also were recruited to serve as a comparison.

“We found that the children with ADHD were more likely than the comparison group to drink heavily and to have enough problems related to their drinking that they were diagnosed with alcohol abuse or dependence,” said Molina.

“The 15-to-17-year-olds with childhood ADHD reported being drunk an average of 14 times in the previous year, versus only 1.8 times for peers in the study who did not have childhood ADHD. Whereas 14 percent of that age group with childhood ADHD were diagnosed with alcohol abuse or dependence, none of the 15-to-17-year-olds without childhood ADHD had alcohol problems.”

Researchers note that it appears that one of the reasons for the past inconsistencies in research is that the ADHD-alcohol relationship does not become solid until at least mid-adolescence. It may be that only a subset of kids with ADHD — namely, those with more aggressive or antisocial behavior patterns — are at risk by young adulthood.

Molina said her findings support this theory. “For example, 42 percent of those children with ADHD who also had serious, persistent behavior problems later had alcohol abuse or dependence by the age of 18 to 25.”

In the second study, on “life stress,” researchers interviewed 142 adolescents who had been diagnosed with childhood ADHD, as well as 100 demographically matched adolescents without childhood ADHD. Participants were asked about their drinking behavior and negative life events; in addition, parents reported their own drinking histories.

Molina said, “We found that parental alcoholism predicted heavy problem drinking among the teenagers, that the association was partly explained by higher rates of stress in these families, and these connections were stronger when the adolescent had ADHD in childhood.”

“However,” noted Molina, “we need to put these findings in perspective. It is important to recognize that not all children with ADHD will have problems with alcohol.”

The study was funded primarily by the National Institute on Alcohol Abuse and Alcoholism.

Other Pitt researchers in involved included Amanda L. Thompson of psychology and Michael P. Marshal of the School of Medicine.

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New Crohn’s disease genes found

On the heels of an article in Science magazine reporting the discovery of strong links between Crohn’s disease and variations in a gene involved in controlling inflammation, a group of researchers in the United States and Canada reports finding more genetic variations linked to an increased risk for the disease.

The IBD Genetics Consortium, funded by the National Institutes of Health, includes members from Pitt, Cedars-Sinai Medical Center in Los Angeles, the University of Chicago, Johns Hopkins University, Montreal Heart Institute, Université de Montréal, the University of Toronto and Yale.

The discovery of these genetic variants has identified several key biological pathways that will be the focus of further research to understand how the debilitating inflammatory process starts and progresses in many cases of the disease.

“As we collect more and more data from these genome-wide association studies, we continue to discover susceptibility genes for Crohn’s and other inflammatory bowel diseases (IBDs). More importantly, those genes are leading us to the biological pathways that relate to IBD pathogenesis. By understanding these pathways, we may be better able to develop more effective therapies for IBD,” said consortium member Richard H. Duerr, associate professor of medicine and human genetics at Pitt, co-director of the Inflammatory Bowel Disease Center and director of the IBD Genetics Program.

These latest results are helping to fill in the gaps in knowledge about Crohn’s and other IBDs, which affect more than 1 million Americans. Because IBDs tend to run in families and are more frequent in certain ethnic populations, scientists long have suspected that they have a significant genetic component.

Previous genetic studies have found strong links between Crohn’s disease and mutations in two genes, one known as CARD15 and a gene known as IL23R, which codes for the immune cell receptor for interleukin-23, an important biochemical mediator of inflammation in the body.

However, mutations in these two genes alone cannot account for the entire genetic component of the disease. To identify additional genes that are associated with Crohn’s, the researchers scanned more than 300,000 single nucleotide polymorphisms, or SNPs, in the genomes of people with Crohn’s disease and in healthy controls. (SNPs are subtle variations in the genetic code found in all genes.)

The comparison of these SNPs between patients with Crohn’s disease and people without the disease identified multiple variations in several genes that were strongly associated with Crohn’s disease. The consortium identified variations in three genes as contributing significantly to the risk for Crohn’s disease.

Corresponding author John D. Rioux of the Montreal Heart Institute and the Université de Montréal said the identification of the PHOX2B gene in this study suggests that neuroendocrine cells of the intestinal epithelium have a key role to play in Crohn’s. The identification of the NCF4 gene indicates that the production of altered reactive oxygen species, which are important in the generation of a protective response against microbes, may lead to an increased risk of developing the condition.

Particularly interesting to the authors was the association between Crohn’s and variations in the ATG16L1 gene, which they found is essential for the cell’s normal mechanism for degrading worn-out cellular components and helping to eliminate some pathogenic bacteria — a pathway known as autophagy.

Finding ATG16L1’s involvement in Crohn’s disease provides confirmatory evidence for what doctors have long suspected: that an individual’s ability to respond effectively to harmful microbes influences his or her susceptibility to the disease.

Co-author Ramnik Xavier of Massachusetts General Hospital’s Center for the Study of IBD said, “We propose that genetic variation in the ATG16L1 gene leads to alterations in how the body uses autophagy and therefore may result in increased persistence of [toxic] cellular and bacterial components. This may lead to an inappropriate immune activation and increased risk of Crohn’s disease.”

In addition to finding the additional Crohn’s disease susceptibility genes, the researchers found strong signals for genetic risk factors located in areas of the genome where there are no known genes. “Further work will be necessary to identify the causal genes in these regions,” the authors said.

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UPCI presents cancer research

Researchers from the University of Pittsburgh Cancer Institute recently presented findings from several studies at the annual meeting of the American Association for Cancer Research, held April 14-18.

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Female hormones in river water?

A study from Pitt’s School of Medicine and the UPCI Center for Environmental Oncology suggests that fish caught in Pittsburgh rivers contain substances that mimic the actions of estrogen, the female hormone. Because fish concentrate chemicals from their habitat in their bodies, these results suggest that feminizing chemicals may be making their way into the region’s waterways.

The study examined white bass and channel catfish caught in the Allegheny, Monongahela and Ohio rivers. These fish, all of which eat other fish and therefore concentrate contaminants from water and their prey, are among those commonly caught as a food source by local anglers.

“We know that there are hundreds, even thousands, of chemicals in the environment that can have estrogenic activity,” said Patricia K. Eagon of the Veterans Affairs Medical Center and Pitt’s School of Medicine. Eagon, co-principal investigator of the study, found that extracts from the fish acted like estrogen by binding to estrogen receptors — the proteins within cells that render the cells sensitive to estrogen.

The study also found that when the researchers treated breast cancer cells in culture with fish extracts, the cells grew at increased rates. Fish caught in areas heavily polluted by industrial and municipal wastes showed the highest degree of cell growth.

“These chemicals usually come from industrial pollution, farm animals, farm chemicals and municipal water treatment plants. What surprised us most in this study was that these estrogenic materials are present in such easily detected levels in local fish,” Eagon said.

Principal investigator Conrad D. Volz, of the Graduate School of Public Health Department of Environmental and Occupational Health, said the next step in this research is to identify the estrogenic chemicals and their sources in the local water and fish.

“These findings have significant public health implications, since we drink water from the rivers where the fish were caught,” Volz said. “Additionally, the consumption of river-caught fish, especially by semi-subsistence anglers, may increase the risk for endocrine-mediated health endpoints like some cancers and developmental problems.”

Four of six bass extracts tested for estrogenic activity displayed a strong or moderate ability to bind with the estrogen receptors, as did nine of 21 catfish extracts tested. In addition two bass extracts and five catfish extracts produced strong-to-moderate breast cancer cell growth.

Other Pitt researchers involved in the work were Maryann Donovan and Devra Davis of the UPCI Center for Environmental Oncology.

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Herbal remedy may slow pancreatic cancer cell growth

A new study from UPCI suggests that a commonly used herbal supplement, triphala, has cancer-fighting properties that prevent or slow the growth of pancreatic cancer tumors implanted in mice.

The study found that an extract of triphala, the dried and powdered fruits of three plants, caused pancreatic cancer cells to die by promoting apoptosis — a process that often is faulty in cancer cells.

Triphala is used for the treatment of intestinal-related disorders. It typically is taken with water and thought to promote appetite and digestion and to increase the number of red blood cells.

“We discovered that triphala fed orally to mice with human pancreatic tumors was an extremely effective inhibitor of the cancer process, inducing apoptosis in cancer cells,” said Sanjay K. Srivastava, lead investigator and assistant professor in the School of Medicine’s Department of Pharmacology. “Triphala triggered the cancerous cells to die off and significantly reduced the size of the tumors without causing any toxic side effects.”

Srivastava and colleagues fed mice grafted with human pancreatic tumors 1-2 milligrams of triphala five days a week and then compared tumor size and levels of apoptotic proteins in the tumors to a control group of mice that received saline only. The mice that received triphala had increased levels of proteins associated with apoptosis and significantly smaller tumor sizes when compared to the control group. Triphala-treated tumors were half the size of tumors in untreated mice. Further testing revealed that triphala activated tumor-suppressor genes, resulting in the generation of proteins that support apoptosis, but did not negatively affect normal pancreatic cells.

Co-investigator of the study is UPCI post-doctoral fellow Yan Shi.

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Vaccine helps some pancreatic cancer patients

A dendritic cell-based vaccine developed by researchers at the Pitt School of Medicine has successfully kept pancreatic cancer from progressing in a handful of patients three years post-vaccination. The results provide promising evidence that the vaccine can trigger a patient’s own immune system to rally against pancreatic cancer and offer new insights into how the vaccine could be made even more effective.

Pancreatic cancer is one of the most difficult cancers to treat because it is undetectable by a physical exam, asymptomatic and progresses quickly. Most patients die within six months of diagnosis. These factors limit the amount of data available for research, hindering significant advances in the understanding and treatment of the disease.

“Pancreatic cancer is extremely resistant to chemotherapy and radiation and, as a result, has a very high mortality rate,” said Andrew Lepisto, first author of the study and post-doctoral researcher in the medical school’s Department of Immunology. “One strategy to improve the odds of survival is to help the immune system recognize the presence of pancreatic cancer cells and attack them. Our study, although small, demonstrates that this strategy can be used with some success in pancreatic cancer patients by slowing down, or even stopping, the progression of cancer.”

The Pitt team created a therapeutic vaccine for pancreatic cancer made up of a synthetic version of MUC1 — a tumor-associated protein that is expressed by pancreatic tumor cells — combined with the patient’s own dendritic cells, which coordinate the immune system’s attack against foreign invaders.

The current study included 12 patients with pancreatic cancer who received the vaccine by injection once every three weeks for a total of three doses and were given a booster dose six months later. Four patients demonstrated a stable and continuous presence of antibodies against MUC1 and have no evidence of disease more than three years after the vaccination was completed and close to five years after diagnosis and surgery.

The research team also examined the specific immune response to the vaccine by sampling the blood of the patients involved in the study. They found that all the patients had an active immune response to the vaccine. They also learned the number of suppressor T cells, a special type of T cell that stifles the activation of the immune system, increased following each vaccine injection, potentially limiting the greater efficacy of the vaccine.

“We will be looking at ways to improve the vaccine by preventing the activation of suppressor T cells,” said Lepisto. “One way to do this is to use additional therapies that specifically target these cells in combination with the vaccine.”

Co-investigators of the study and senior authors are Olivera Finn, leader of the UPCI immunology program and chair of the School of Medicine’s Department of Immunology, and former UPCI researcher Ramesh Ramanathan.

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New strategy for fighting head and neck cancer

UPCI researchers, in collaboration with the Gunma University School of Medicine in Japan, have developed a vaccine strategy for head and neck cancer that targets multiple peptides (parts of proteins) to activate the immune system to attack tumors.

The researchers created the vaccine to target a tumor suppressor gene called p53, which is mutated in most cancers and associated with poor clinical outcomes.

“The key to our strategy is to select those p53 peptides that can best activate the immune system and induce it to produce immune cells able to recognize and eliminate the tumor,” said Theresa Whiteside, who, in collaboration with Albert DeLeo, directed the study. Both are professors of pathology and immunology at the School of Medicine.

“Through animal models and human cells in culture, we have found that this strategy is very effective at stimulating T cells into action,” said Whiteside, who also directs UPCI’s Immunologic Monitoring and Cellular Products Laboratory.

According to DeLeo, the vaccine based on these findings also could be relevant to other types of cancer given that the loss of p53 function is a common feature across many cancers.

Given that the proof of principle for the vaccine has been determined, a phase I clinical trial of the vaccine has begun at UPCI to assess its safety in head and neck cancer patients.

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Veggie component may fight prostate tumors

Chemicals in cruciferous vegetables such as broccoli, watercress, cabbage and cauliflower appear not only to stop human prostate cancer cells from growing in mice but also may cut off the formation of blood vessels that “feed” tumors, according to a UPCI study.

“The contribution of diet and nutrition to cancer risk, prevention and treatment has been a major focus of research in recent years because certain nutrients in vegetables and dietary agents appear to protect the body against diseases such as cancer,” said Shivendra Singh, lead investigator and professor of pharmacology and urology at the School of Medicine. “We know that increased consumption of vegetables reduces the risk for certain types of cancer, but now we are beginning to understand the mechanisms by which certain vegetables like broccoli may help our bodies fight cancer and other diseases.”

Singh’s study is based on phytochemicals, called isothiocyanates (ITCs), found in several cruciferous vegetables and generated when vegetables are either cut or chewed. His laboratory has found that phenethyl-ITC, or PEITC, is highly effective in suppressing the growth of human prostate cancer cells at concentrations achievable through dietary intake.

The current study follows previous research in which Singh’s laboratory found that mice grafted with human prostate tumors that received a small amount of PEITC daily for 31 days had significantly reduced tumor size when compared to a control group of mice.

Now the researchers have shown that treating cells in culture with PEITC inhibits angiogenesis, a process that plays an important role in the growth and spread of cancer by forming new blood vessels that pass oxygen and nutrients to tumor cells.

“Angiogenesis is a major issue in cancer metastases,” said Singh. “Our results provide promising preliminary evidence that constituents of many edible cruciferous vegetables may slow down, or even halt, this process.”

UPCI’s Dong Xiao is co-investigator of the study.