Researchers Identify Anti-HIV Proteins

San Diego—Scientists have long sought to learn why in some HIV-infected individuals, known as long-term nonprogressors, the immune system is able to successfully keep the virus in check and substantially delay or prevent progression to AIDS. As far back as 1986, AIDS researcher Jay Levy, MD, of the University of California, San Francisco, had evidence that white blood cells of long-term nonprogressors (but not those of progressors) produce an unknown chemical or chemicals that inhibit viral replication. But the identity of such a substance—called CD8 antiviral factor, or CAF—remained elusive.

Now, researchers at Rockefeller University's Aaron Diamond AIDS Research Center in New York City say they have identified components of that elusive factor: a family of HIV-suppressing proteins produced by the CD8 T cells of long-term nonprogressors. The new findings, reported here at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy by David D. Ho, MD, were also published online by Science, in the September 26 edition of Science Express (http://www.sciencexpress.org).

If the new findings are verified, the work could open up new possibilities for research involving treatments for patients infected with HIV.

In the study, Ho, Linqi Zhang, PhD, and colleagues at Aaron Diamond and Ciphergen Biosystems Inc, a Fremont, Calif, biotechnology company, used protein chips (arrays of proteins) and mass spectrometry to analyze substances produced by CD8 T cells from three long-term nonprogressors, as well as from four progressors and 15 healthy controls. They found that only cells from nonprogressors and uninfected people, but not progressors, made three similar proteins. Further studies revealed that the proteins were identical to three previously identified proteins called α-defensin-1, -2, and -3—proteins that are primarily produced by neutrophils and that help defend against many types of infections.

The investigators then discovered that when they removed the defensins from the fluids harvested from cultures of CD8 T cells of long-term nonprogressors, anti-HIV activity was sharply reduced. And when β-chemokines—immune messengers produced by CD8 T cells that can prevent HIV entry into host cells—were also removed, much of the remaining anti-HIV activity disappeared. They also found that defensins purified from the neutrophils of a healthy person and synthetic forms of α-defensin-1 and α-defensin-2 inhibited the replication of HIV in cell cultures.

The new findings are not without controversy. Some suggest that the defensins isolated by the Aaron Diamond investigators could have been produced by other cells that the researchers added to the CD8 T cells to help them grow. But Ho said that additional experiments performed without these other cell types yielded defensins, confirming that the proteins are indeed produced by CD8 T cells.

Others say that other studies with defensins indicate that their anti-HIV activity is modest, and that defensins are likely to be just one of many factors that help nonprogressors stay healthy. Ho, Zhang, and colleagues discovered that a combination of α-defensin-1 and -2 had more potent anti-HIV activity than either one tested alone, and noted, "It is possible that the anti–HIV-1 activity is mediated by different defensins interacting together."

Factors other than defensins also may play a role. "We already know from other studies that there doesn't have to be one uniform mechanism existing in all long-term nonprogressors," noted Carl Dieffenbach, PhD, associate director of the basic sciences program in the AIDS division at the National Institute of Allergy and Infectious Diseases (NIAID).

For example, some long-term nonprogressors have genetic factors that help them fight HIV. Some lack a receptor on immune cells that HIV uses to bind to and infect the cells, while others have combinations of human lymphocyte antigens that are associated with nonprogression. NIAID scientists reported on October 7 in the advanced online issue of Nature Immunology (DOI: 10.1038/ni845) that the CD8 T cells of nonprogressors are able to divide more rapidly and to a greater extent and produce higher levels of perforin, a molecule known to kill HIV-infected cells (http://www.nature.com/ni/).

The next step in the defensin story may be to see whether the findings are generalizable beyond the small group of patients investigated by Ho and colleagues, by studying other long-term nonprogressor populations to determine whether this finding holds up. If so, said Dieffenbach, "there would be something here, in terms of basic biology and drug targets, that companies could get into," such as finding ways to boost expression of defensins or blocking their degradation.

Dorothy Y. H. Tuan, PhD, a molecular biologist at the Medical College of Georgia in Augusta, is pondering some basic questions regarding sickle cell disease. If she gets the right answers, she hopes to uncover findings that could lead to a cure or successful treatment of a disease that affects about 72 000 people in the United States—primarily African Americans.

Clinical manifestations of sickle cell disease (or more properly sickle cell anemia) are caused by anemia and vaso-occlusive events causing "crises"—episodes of severe pain, bone infarction, leg ulcers, and, in the long-term, atrophy of the spleen. The disease is caused by a mutation in the β-globin gene that changes the structure of the β-globin protein inducing sickle hemoglobin (HbS). The HbS then polymerizes into long strings when deoxygenated. Cells containing these deformed, sickled HbS can then occlude microvascular circulation.

"We want to study whether there is some way of preventing this hemoglobin from polymerizing into this long, rigid rod within your cell," said Tuan, one of a cadre of researchers who are seeking genetic solutions to cure or effectively treat sickle cell disease.

Tuan, whose work is supported by a 4-year grant from the National Institutes of Health, wants to understand how different globin genes get "turned off and on" and whether there is a way to alter the results of mutation in the β-globin gene.

Tuan, along with other investigators, is also trying to find ways to reactivate the production of fetal hemoglobin (HbF), which can prevent HbS from polymerizing into sickle-shaped cells. The HbF is produced in the fetus. Shortly after birth the body switches to production of adult hemoglobin (HbA), which allows for the creation of HbS.

"Every cell in the embryo will contain the same amount of DNA, the same group of genes," Tuan said. "But why is it when cells divide in the embryo, in certain places the globin genes acquire the ability to be expressed whereas, in other places, they are going to be repressed? This is very mysterious. If we can understand that, I think we can design better vectors, better treatments for many diseases [including sickle cell]."

"We study the regulation of all this," Tuan said. "How is it turned on and how is it turned off?"

This question is important because a current treatment for sickle cell disease, hydroxyurea, is not 100% efficacious, and long-term adverse effects have not been determined. Hydroxyurea, a drug used for more than 30 years for treatment of certain kinds of cancer, was approved to treat sickle cell disease by the Food and Drug Administration in 1998 because of its ability to promote HbF in some patients. But the drug is not efficacious in approximately 25% of those treated (Blood. 1997;89:1078-1088).

However there may be some good news for the 25% of patients not responding to hydroxyurea. In a preliminary study involving eight patients who did not respond to hydroxyurea, researchers at the University of Illinois at Chicago (UIC) College of Medicine were able to maintain a therapeutic beneficial level of HbF in seven enrollees at nine months using the cancer drug decitabine (Blood. 2002;99:3905-3908).

"Decitabine is an effective therapy for patients who do not benefit from the traditional treatment. It is possibly even an improvement, although more studies will have to be done," said Joseph DeSimone, PhD, a professor of hematology and oncology at UIC. DeSimone's study was supported by SuperGen Inc.

While Tuan's and DeSimone's work offer perhaps a better outcome for patients with sickle cell disease, striking a cautionary note is Alan N. Schechter, MD, genetics section chief with the National Institute of Diabetes & Digestive & Kidney Diseases. He said uncovering the genetic secrets of sickle cell disease remains elusive.

"It's a maturing field, but the big questions have still not been elucidated despite several decades of research," Schechter said.

Washington—After an exhaustive 18-month review, an Institute of Medicine (IOM) committee has handed down broad recommendations for overhauling the nation's fractured system of human research protections. The report advocates sweeping changes at the national, institutional, and community levels, including comprehensive monitoring of all research, revamping strained institutional review boards (IRBs), and providing litigation-free compensation for participants harmed during research.

The current arrangement—federally funded institutions generally police themselves while industry answers to the Food and Drug Administration—is so disparate that estimates of the number of research volunteers in the United States vary from 2 to 20 million, said committee chair Daniel Federman, MD, distinguished professor of medicine and medical education at Harvard Medical School.

That alarming range prompted the committee's first recommendation, the establishment of a national data collection effort to track research participation. While Congress and various medical organizations ponder how to implement such a system, though, Federman said that "we must err on the side of caution" and move ahead with other reforms immediately.

Institutional review boards, now charged with keeping tabs on all of a university's or other group's human research, receive a big boost under the plan. "A theme throughout our whole report is that so much has been loaded onto [IRBs] that it is very, very difficult for them to look at ethics as well as all of the other issues they're supposed to contend with," said Timothy Jost, JD, professor of law at Washington and Lee University School of Law in Lexington, Va.

In particular, IRBs are often responsible for reviewing the scientific merit and potential conflicts of interest among investigators. The IOM plan separates these functions among three institution-based committees. One new body would review the scientific merit and risks of each proposed project while a second would analyze potential financial conflicts of interest among investigators. Both committees would then report to a "research ethics review board" (ERB), which would synthesize and scrutinize all pertinent issues. For the three-tier system to work, though, extra funds and support personnel are essential, said Jost. "One thing we heard over and over again was the inadequacy of resources [for IRBs]."

The plan also calls for the creation, when appropriate, of regional, rather than institutional, ERBs. Dedicated to keeping tabs on large multicenter or international projects, such boards should be more objective than typical IRBs, said Jost. "We felt that IRBs have sometimes become too tied up in identifying with their institution," he said, sometimes forsaking careful reviews for the sake of prestige or funding.

To help rebuild patient trust and ensure transparency of the process, patient advocates, research volunteers, and other community members should comprise at least 25% of ERB membership, said committee member Fran Visco, JD, president of the National Breast Cancer Coalition.

The report also calls for revamped informed consent practices. Instead of handing potential volunteers a written consent form and thinking the process is over after it is signed, researchers need to view informed consent as "an ongoing process rather than a discrete, isolated moment," said Federman. Repeated, clear explanations of risks are essential, he added.

The most controversial proposal may be the establishment of a no-fault compensation system for volunteers injured during research. Because no research effort "is utterly without risk," said Federman, the new system must acknowledge that participants will occasionally be harmed. The only recourse currently available to injured volunteers is litigation. A no-fault system, while not absolving researchers of their responsibility for protecting volunteers, would reimburse volunteers for medical bills, lost wages, and other costs while avoiding timely, expensive trials, said Federman.

The report suggests that initially institutions conducting research would be responsible for compensation, a situation that, it says, presumably would lead to the development of insurance specifically for this purpose. Alternatively, the report adds, the federal government might set up a compensation program that would be akin to the National Vaccine Injury Compensation Program, which since 1986 has awarded some $900 million to families of children judged to be injured by vaccinations.

In fact, it was the death of a research participant that prompted the IOM report. In 1999, gene therapy volunteer Jesse Gelsinger died in a University of Pennsylvania clinical trial. His liver failed after he received a dose of recombinant genes designed to fight a rare disorder. Federal investigators later reported that a similar reaction had occurred in monkeys and that the researchers stood to gain financially from the trial.

The IOM report, Responsible Research: A Systems Approach to Protecting Research Participants, is available at http://www.iom.edu.