New Cellular Targets For HIV Drug Development

Focusing HIV drug development on immune cells called macrophages instead of traditionally targeted T cells could bring us closer to eradicating the disease, according to new research from University of Florida and five other institutions.

In the largest study of its kind, researchers found that in diseased cells — such as cancer cells — that are also infected with HIV, almost all the virus was packed into macrophages, whose job is to "eat" invading disease agents.

What's more, up to half of those macrophages were hybrids, formed when pieces of genetic material from several parent HIV viruses combined to form new strains.

Such "recombination" is responsible for formation of mutants that easily elude immune system surveillance and escape from anti-HIV drugs.

"Macrophages are these little factories producing new hybrid particles of the virus, making the virus probably even more aggressive over time," said study co-author Marco Salemi, Ph.D., an assistant professor in the department of pathology, immunology and laboratory medicine at the UF College of Medicine. "If we want to eradicate HIV we need to find a way to actually target the virus specifically infecting the macrophages."

At least 1.1 million people in the United States and 33 million in the world are living with HIV/AIDS, according to the Kaiser Family Foundation.

The researchers set out to see if HIV populations that infect abnormal tissues are different from those that infect normal ones, and whether particular strains are associated with certain types of illness.

They tackled the question using frozen post-autopsy tissue samples, pathology results and advanced computational techniques. They analyzed 780 HIV sequences from 53 normal and abnormal tissues from seven patients who had died between 1995 and 2003 from various AIDS-related conditions, including HIV-associated dementia, non-Hodgkin's lymphoma and generalized infections throughout the body. Four patients had been treated with highly active antiretroviral therapy, called HAART, at or near the time of death.

The researchers compared brain and lymphoma tissues, which had heavy concentrations of macrophages, with lymphoid tissues — such as from the spleen and lymph nodes— that had a mix of HIV-infected macrophages and T cells.

The analyses revealed great diversity in the HIV strains present, with different tissues having hybrid viruses made up of slightly different sets of genes. A high frequency of such recombinant viruses was also found in tissues generally associated with disease processes, such as the meninges, spleen and lymph nodes.

The researchers concluded that HIV-infected macrophages might be implicated in tumor-producing mechanisms.

The higher frequency of recombinant virus in diseased tissues likely is because macrophages multiply as a result of an inflammatory response, the researchers said.

"The study points to macrophages as a site of recombination in active disease," said neurobiologist Kenneth C. Williams, Ph.D., a Boston College associate professor and AIDS expert who was not involved in the study. "So people can say this is one spot where these viruses come from."

T cells — the so-called conductors of the immune system orchestra, whose decline is the hallmark of HIV disease — are an obvious target for HIV drug development because they die soon after infection, and are readily sampled from the blood and cultured. But although current drugs are effective at blocking infection of new cells and lowering viral loads to barely detectable levels, they never reduce the viral level in an infected person to zero.

"Where is it coming from?" said Michael S. McGrath, the University of California, San Francisco, professor who led the research team. "We believe it's coming from these macrophages."

Macrophages, like T cells, can be infected multiple times by HIV. But unlike T cells, when they get infected, they don't die within days, but live for several months, all the while being re-infected with multiple viruses of different genetic makeup. That situation is ripe for the emergence of hybrids.

"Most people who look at viral sequences assume that evolution of the virus is linear. In the real world that doesn't happen — large parts of the virus are swapped in and out. This group has shown that in this model," Williams said. "It sort of overturns the old way of trying to match virus sequence with pathology."

McGrath's group is now developing macrophage-targeting drugs that, through a grant from the National Institute of Mental Health, should be in human clinical trials in a few years.

"This is one of the last frontiers — killing off what we believe is a so far untouched reservoir," he said.

The work was published recently in the journal PLoS One.

Gene-laden Bubbles Grow New Blood Vessels

Progress in human gene therapy -- the insertion of therapeutic DNA into tissues and cells in the human body -- has been slower than expected since the first clinical trials in 1990. One of the biggest challenges for this technology is finding ways to safely and effectively deliver genes only to the specific parts of the body that they are meant to treat.

Cardiologist Jonathan Lindner of Oregon Health and Science University will discuss his latest experiments in gene therapy, which use microscopic bubbles chemically modified to stick to the cells that line blood vessels.

This technique, ultrasound-mediated gene delivery (UMGD), exploits the properties of contrast agents, microparticles that are normally injected into the body to improve the quality of ultrasound images. In UMGD, the tiny particles are microbubbles composed of pockets of gas encapsulated by thin membranes that are coated with DNA before injection. A targeted pulse of ultrasound energy "rings" the bubbles like a bell, popping them in a specific location and releasing the DNA into the surrounding tissue.

To improve the specificity of this targeting, Lindner grafts long arm-like molecules to the outside of the bubbles. These arms, which do not interfere with the DNA attached to surface, are designed to recognize and bind to molecules on the outside of specific cells in the body, allowing the bubbles to attach to a tissue before being popped. In theory, this should improve both the specificity and efficiency of the gene therapy.

Lindner created an arm designed to attach to endothelial cells lining blood vessels. He will present data evaluating the behavior of these "targeted" bubbles in living tissue. The ability to stick these gene-laden microbubbles to the lining of blood vessels increased the amount of gene transfection. This strategy may be particularly important for delivering therapeutic DNA to the walls of blood vessels. For example, Dr. Lindner and collaborators have successfully stimulated the growth of new blood vessels using UMGD with microbubbles carrying a gene for vascular endothelial growth factor. This therapeutic use could be important for treating ischemia in patients who have had a heart attack, peripheral artery disease, or stroke.

The team is also investigating using the bubbles to transport small doses of drugs. "If you're trying to deliver a nasty drug to part of the body, this may be a way to improve safety," says Lindner.

The talk "Targeted microbubble technology and ultrasound-mediated gene delivery" by Jonathan Lindner will be presented at the 157th  Acoustical Society of America Meeting to be held May 18-22 in Portland, Ore.

Fish Oil Protects Against Diseases Like Parkinson's

Dr. Nicolas Bazan, Director of the Neuroscience Center of Excellence, Boyd Professor, and Ernest C. and Yvette C. Villere Chair of Retinal Degenerative Diseases Research at LSU Health Sciences Center New Orleans, will present new research findings showing that an omega three fatty acid in the diet protects brain cells by preventing the misfolding of a protein resulting from a gene mutation in neurodegenerative diseases like Parkinson's and Huntington's.

He will present these findings for the first time on April 19, 2009 at the Ernest N. Morial Convention Center, Nouvelle C Room, at the American Society for Nutrition, Experimental Biology 2009 Annual Meeting.

With funding from the National Eye Institute of the National Institutes of Health, Dr. Bazan and his colleagues developed a cell model with a mutation of the Ataxin-1 gene. The defective Ataxin-1 gene induces the misfolding of the protein produced by the gene. These misshapened proteins cannot be properly processed by the cell machinery, resulting in tangled clumps of toxic protein that eventually kill the cell. Spinocerebellar Ataxia, a disabling disorder that affects speech, eye movement, and hand coordination at early ages of life, is one disorder resulting from the Ataxin-1 misfolding defect. The research team led by Dr. Bazan found that the omega three fatty acid, docosahexaenoic acid (DHA), protects cells from this defect.

Dr. Bazan's laboratory discovered earlier that neuroprotectin D1 (NPD1), a naturally-occurring molecule in the human brain that is derived from DHA also promotes brain cell survival. In this system NPD1 is capable of rescue the dying cells with the pathological type of Ataxin-1, keeping their integrity intact.

"These experiments provide proof of principle that neuroprotectin D1 can be applied therapeutically to combat various neurodegenerative diseases," says Dr. Bazan. "Furthermore, this study provides the basis of new therapeutic approaches to manipulate retinal pigment epithelial cells to be used as a source of NPD1 to treat patients with disorders characterized by this mutation like Parkinson's, Retinitis Pigmentosa and some forms of Alzheimer's Disease."

Earliest Evidence Of Domesticated Maize Discovered: Dates Back 8,700 Years

This is so fascinating. According to Ranere, recent studies have confirmed that maize derived from teosinte, a large wild grass that has five species growing in Mexico, Guatemala and Nicaragua. The teosinte species that is closest to maize is Balsas teosinte, which is native to Mexico's Central Balsas River Valley.

"We went to the area where the closest relative to maize grows, looked for the earliest maize and found it," said Ranere. "That wasn't surprising since molecular biologists had determined that Balsas teosinte was the ancestral species to maize. So it made sense that this was where we would find the earliest domestication of maize."

The study began with Piperno, a Temple University anthropology alumna, finding evidence in the form of pollen and charcoal in lake sediments that forests were being cut down and burned in the Central Balsas River Valley to create agricultural plots by 7000 years ago. She also found maize and squash phytoliths -- rigid microscopic bodies found in many plants -- in lakeside sediments.

Ranere, an archaeologist, joined in the study to find rock shelters or caves where people lived in that region thousands of years ago. His team carried out excavations in four of the 15 caves and rock shelters visited in the region, but only one of them yielded evidence for the early domestication of maize and squash.

Ranere excavated the site and recovered numerous grinding tools. Radiocarbon dating showed that the tools dated back at least 8700 years. Although grinding tools were found beneath the 8700 year level, the researchers were not able to obtain a radiocarbon date for the earliest deposits. Previously, the earliest evidence for the cultivation of maize came from Ranere and Piperno's earlier research in Panama where maize starch and phytoliths dated back 7600 years.

Ranere said that maize starch, which is different from teosinte starch, was found in crevices of many of the tools that were unearthed.

"We found maize starch in almost every tool that we analyzed, all the way down to the bottom of our site excavations," Ranere said. "We also found phytoliths that comes from maize or corn cobs, and since teosinte doesn't have cobs, we knew we had something that had changed from its wild form."

Ranere said that their findings also supported the premise that maize was domesticated in a lowland seasonal forest context, as opposed to being domesticated in the arid highlands as many researchers had once believed.

"For a long time, I though it strange that researchers argued about the location and age of maize domestication yet never looked in the Central Balsas River Valley, the homeland for the wild ancestor," said Ranere. "Dolores was the first one to do it.'

In addition to Ranere and Piperno, other researchers in the study included Irene Holst of the Smithsonian Tropical Research Institute, Ruth Dickau of Temple, and Jose Iriarte of the University of Exeter. The study was funded by the National Science Foundation, National Geographic Society, Wenner-Gren Foundation, Smithsonian National Museum of Natural History, Smithsonian Tropical Research Institute and the Temple University College of Liberal Arts.

Glucose-To-Glycerol Conversion In Long-lived Yeast Provides Anti-aging Effects

Cell biologists have found a more filling substitute for caloric restriction in extending the life span of simple organisms. In a study published May 8 in the open-access journal PLoS Genetics, researchers from the University of Southern California Andrus Gerontology Center show that yeast cells maintained on a glycerol diet live twice as long as normal -- as long as yeast cells on a severe caloric-restriction diet. They are also more resistant to cell damage.

Many studies have shown that caloric restriction can extend the life span of a variety of laboratory animals. Caloric restriction is also known to cause major improvements in a number of markers for cardiovascular diseases in humans. This study is the first to propose that "dietary substitution" can replace "dietary restriction" in a living species.

"If you add glycerol, or restrict caloric intake, you obtain the same effect," said senior author Valter Longo. "It's as good as calorie restriction, yet cells can take it up and utilize it to generate energy or for the synthesis of cellular components."

Longo and colleagues Min Wei and Paola Fabrizio introduced a glycerol diet after discovering that genetically engineered long-lived yeast cells that survive up to 5-fold longer than normal have increased levels of the genes that produce glycerol. In fact, they convert virtually all the glucose and ethanol into glycerol. Notably, these cells have a reduced activity in the TOR1/SCH9 pathway, which is also believed to extend life span in organisms ranging from worms to mice.

When the researchers blocked the genes that produce glycerol, the cells lost most of their life span advantage. However, Longo and colleagues believe that the "glucose to glycerol" switch represents only a component of the protective systems required for the extended survival. The current study indicates that glycerol biosynthesis is an important process in the metabolic switch that allows this simple organism to activate its protective systems and live longer.

"This is a fundamental observation in a very simple system," Longo said, "that at least introduces the possibility that you don't have to be calorie-restricted to achieve some of the remarkable protective effects of the hypocaloric diet observed in many organisms, including humans. It may be sufficient to substitute the carbon source and possibly other macronutrients with nutrients that do not promote the "pro-aging" changes induced by sugars."

Funding for the study came from the American Federation for Aging Research and the National Institute on Aging (NIH).

Groundbreaking Study Reveals Intermediary Steps Of Genetic Encoding For The First Time

The scientists report that they were able to crystallize a very large complex of a macromolecular "machine" in the human cell and determine its structure or what it actually looks like, thereby zeroing in on the process of genetic encoding. Importantly, 15 to 20 percent of all human genetic disorders, including muscular dystrophy, are caused by defects in this genetic encoding process known as RNA splicing.

Using x-ray crystallography, the scientists for the first time were able to create a three-dimensional structure of an integral complex of the human spliceosome, which consists of specialized RNA and protein subunits. The spliceosome's job is to modify the message relayed from our genetic material—DNA—by clipping, or splicing, genetic bits in such a manner that they are acceptable for translation into protein. Importantly, the spliceosome also rearranges the genetic bits of the message in such a way that it can generate multiple and varied proteins which can and do have dramatic effects on human development, said lead author and Brandeis biochemist Daniel Pomeranz Krummel.

"The process of RNA splicing is vital to human cell development and survival," said Pomeranz Krummel. "In this process, the regions of our DNA encoding for protein are removed from non-encoding regions and brought together—quite often in alternative arrangements. Defects in this process can have disasterous repercussions in the form of genetic disorders," said Pomeranz Krummel, adding that neuronal development can be particularly affected when things go awry. Indeed, defects in this process have recently been implicated in various human neurological disorders, including epilepsy.

Specifically, this macromolecular machine clips, or splices, gene sequences transcribed as part of a precursor to the mRNA, removing them before the final mRNA product is translated into protein. The spliceosome must clip these sequences, known as introns, at the right place in the precursor mRNA.

"In human cells one gene can be made into a variety of proteins, so if the process just goes slightly wrong, the genetic alteration can lead to incredible disaster; yet on the other hand, this incredible complexity has led to our amazing evolutionary progress," said Pomeranz Krummel. "The human genome is not terribly different from the earthworm's with regards to its size, but the process of RNA splicing that occurs in our cells is different. The fundamental difference between us and the earthworm is that our cells have evolved to utilize this process of RNA splicing to generate a whole other dimension to the transmission of genetic information."

Pomeranz Krummel's lab will next focus on understanding how this complex interacts with other macromolecular machines in the human cell. The study was funded by the Medical Research Council (U.K.) and the Human Frontier Science Program.