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Retroviral immunology has been a 25-year interest of Chesebro's. The Friend virus, which is in the same family of viruses as HIV, causes fatal leukemia in a high percentage of susceptible strains of mice. Remarkably, other strains become infected but "cure" their own leukemia. "We know more about a protective response to this virus than to any other retrovirus," Chesebro says. He is closing in from two directions on understanding effective immune response to Friend by attempting to develop a vaccine and by attempting to clone the Rfv-3 gene on mouse chromosome 15 that appears to confer the effective immune response. Ultimately, Chesebro expects, there will be three essential components: correct responses in CD8+ cells and CD4+ cells and in humoral antibody production.
For his work on HIV-dementia, Chesebro collaborates with colleagues at the Department of Neurology at Johns Hopkins University School of Medicine in Baltimore, who have used careful cognitive testing to differentiate HIV patients with true AIDS dementia from those with other neurological deficits caused by opportunistic infections or other factors in the disease. Through long-term studies, they have collected blood and virus samples from living patients and brain samples from deceased participants. These materials have revealed that although almost 100 percent of patients die with active virus present in their brains, just 20 percent have AIDS dementia. Cloning of the virus from these patients has revealed that there are clear sequence differences in the virus from demented and nondemented patients. Because the indirect viral effects on neuronal cultures correlate with effects in living patients, Chesebro is exploring, in vitro, what specific changes in HIV envelope sequenceor the host's response to the virusresult in damage to neurons. "It could be a combination of cytokines, lipids, and envelope proteins produced by infected microglia that leads to damage to neurons," Chesebro speculates.
Two other scientists in the LPVD interviewed by The CatalystByron Caughey and tenure-track investigator Sue Priolajoin Chesebro in his third interest in TSE diseases. While the three appear to be very collegial, they don't even start out with the same opinion of the prion hypothesis, namely that abnormal prion protein, PrP-res, is sufficient to transmit TSEs, which include bovine spongiform encephalopathy, scrapie in sheep and goats, and kuru and Creutzfeldt-Jakob disease in humans. Caughey comes closest to being a believer: "I don't believe it's proven, but it might be the case," he says. Priola, a virologist, acknowledges that there is a lot of supporting evidence for the hypothesis, "But it is not entirely convincing. . . . It is hard to find viruses sometimes, but in this case, no one is even looking." Chesebro comes down gently on the side of the nonbelievers: "I'm not convinced that the transmissible agent is a protein only. In the case of the genetic TSE diseases, where people express mutant PrP genes, these genes may be a susceptibility factor, but by no means do I believe we know the agent. I believe the causative agent of TSE diseases is a virus; the fact that normal PrP interacts with PrP-res to form amyloid is like processes that occur in many amyloid diseases, including Alzheimer's. But in those diseases the protein alone won't transmit the disease," as seems to be the case in TSE diseasesat least under artificial conditions in which injection of the appropriate PrP-res directly into the brains of animals will lead to disease.
One of the most important breakthroughs in TSE research was made in 1994 by Caughey, who first induced self-propagating conversion of normally folded PrP to abnormally folded PrP-res (so called because it is resistant to proteases) in the test tube. This achievement was simultaneously an important proof-of-principle for the prion hypothesis and the basis for fundamental techniques for working with PrP. The lab is now exploiting these techniques in basic and applied research. Caughey used this approach to demonstrate that the in vitro reaction is "incredibly specific." In some instances in the test tube, PrP-res derived from one species of animal will convert PrPderived from a different speciesbut only in instances in which the same is true in vivo, and cross-species transmission of disease is possible. The lab is finding that single- amino-acid differences between different species' strains of PrP are sufficient to block cross-species reactions. Now using infrared spectroscopy, Caughey has identified different b-sheet conformations in PrP-res that might account for different strains of TSE agents. He is now trying to increase the efficiency of the test-tube reaction so that a measurement can be made of whether new infectivity is generated by PrP-res formation. The TSE research group is also studying inhibitors of the reaction, including Congo red, sulfated glycans, peptide fragments of PrP, and phthalocyanines. These might yield clues to therapeutic targets in TSE diseases and possibly other amyloidoses.
Priola is using scrapie-infected cell cultures and the in-vitro reaction to study the control of the passage of TSE from one animal to another and to look at the effects of human familial TSE-associated PrP mutations. Working with mouse and hamster versions of TSE as models, Priola has pinpointed parts of the PrP amino-acid sequence that are critical in determining the compatibility of PrP isoforms from different species. In addition, Priola has described aberrations in the metabolism of mutant PrP molecules that may underlie pathogenesis in familial TSE diseases. Currently one of Priola's goals is to develop a diagnostic test based on Caughey's test-tube reaction that could be used to identify animals that have BSE very early, before they show behavioral signs of mad cow disease.
Marshall Bloom, also of the LPVD, has studied diverse aspects of the immunologically peculiar Aleutian mink disease parvovirus (ADV). ADV produces different types of disease in newborn kits vs. adult mink. Infected kits typically succumb to fulminant respiratory infection, similar to hyaline membrane disease of premature human infants. Adults develop a persistent infection, forming massive numbers of lymphocytes and extremely high concen trations of gamma globulin and antibody to the virus. Virus-antibody complexes deposit in the kidneys, leading to kidney failure and death.
Bloom expects various aspects of the ADV system could shed light on chronic infection states and autoimmune diseasesincluding human diseases such as lupus. In the mink system, the viral capsid plays a key role in the disease. In sharp contrast with other parvoviruses, "inoculating" mink with empty capsid of ADV not only fails to protect the animalsit actually leads to accelerated, hyperacute disease when the animals are challenged with live virus. Bloom's research group is now comparing nonpathogenic and pathogenic ADV isolates to understand precisely what structural features of the capsid convert the disease into such a bizarre killer. As with Dengue fever, a key to the disease's pathogenesis is ADV's ability to infect macrophages in the presence of antibody, and Bloom and his colleagues are studying the mink's cytokine responsesespecially an IL-6 homologuefor clues to this phenomenon. As much as he still has to learn, Bloom reckons ADV is the best-studied of all parvovirus diseases, and he values the collegial relations of a small field. "The advantage of working on Aleutian mink disease is that everyone working on it in the world came out of or through my laball five of them!"
The research of Tom Schwan, acting lab chief for the Laboratory of Microbial Structure and Function (LMSF), carries on the historic focus of RML on blood-feeding arthropods and the pathogenic bacteria they transmit to humans. But he gives the work a 21st-century twist by applying contemporary techniques to understanding the bacteria's molecular adaptations as they move from the midgut, salivary glands, and other parts of cold-blooded ticks and fleas into a dramatically different environment: the warm bloodstream of mammals.
A primary interest for Schwan is in spirochetes: developing an improved blood test for Lyme disease and relapsing fever and understanding the biology of the spirochete inside the tick. Relapsing fever, like Lyme, results when ticks transmit a spirochete, Borrelia hermsii, to humans. Though uncommon, relapsing fever is an insidious foe. Ticks that transmit the bacteria are endemic to the mountains in the West and are "fast feeders" that typically attach to a host at night and complete their feeding in 10 to 90 minutes. Victims are unlikely to know they've been bitten or to attribute their flu-like symptoms three to 10 days later to a tick bite; thus, their doctors are unlikely to take a blood sample, which would reveal the spirochete in the blood. This first stage of the disease passes as antibodies clear the infection. But that is not the last of Borrelia. Some days later the bacteria turns to its genetic closet, stuffed with at least 40 different genes for outer surface proteins, switches to an antigenically fresh exterior, and again flourishes in the bloodstream, again making the victim sick. This process has been known to repeat itself up to 12 times. Schwan believes the best hope for a vaccine lies in the surface of the spirochete as it is first transmitted from the tick.
Another research interest for Schwan is how the bacteria that cause plague sense and adapt to the molecular world in their arthropod hostthe flea. Key stimuli that turn off and on suites of genes in Yersinia pestis are temperature, oxygen concentration, pH, and the arthropod's ingestion of a blood meal. Work with RML postdoc Joe Hinnebusch and Robert Perry of the University of Kentucky in Lexington, published in Science, demonstrated that the bacteria must possess and activate a certain set of genes to successfully infect the flea. Schwan points out that the similar genes and processeswhich may present attractive vaccine and diagnostic targetsare also likely to be present other disease-causing agents transmitted by arthropods.
investigator Patti Rosa says that to begin to understand gene regulation
and adaptation in Borrelia burgdorferi, the agent that causes Lyme
disease, will first require developing
molecular tools for manipulating the spirochete's genome similar to tools
that have made Escherichia coli. and Y. pestis tractable.
An important step is finding a plasmid that replicates reliably in the
spirochete and can serve as a ferry for genes. With a small set of genetic
tools in hand, Rosa has begun to knock out genes to answer biological
questions: What genes play a critical role in the ability of the Lyme
disease spirochete to adapt to various environments? How does its gene
expression change between tick and mammalian hosts?
Steve Porcella works on bacterial
expression and host response in vivo to spirochetes that cause Lyme disease,
relapsing fever, and syphilis (Treponema pallidum). The latter
work is daunting because the spirochete can only be grown in the rabbit
testis and development of a molecular genetic system for manipulating
the bacteria has proven intractable. The labile bacteria deteriorate rapidly
outside of the rabbit testis, which Porcella uses as his model for
exploring how host and spirochete respond to one another. Porcella, a
life-long rock-climbing and mountaineering enthusiast, says, "I like
big challenges. Working with syphilis is a lot like climbing a big granite
seems impossible at first, yet if you chip away at it, you eventually
reach your goal." Another big challenge is a project perfecting what
Porcella calls "bionic mice." These animals are implanted interperitoneally
with an FM transmitter that provides an instantaneous and continuous record
of the animals' activity and body temperature. The system produces an
intimate record sensitive enough to demonstrate physiological effects
of simply changing the animal's bedding material. If some technical bugs
can be ironed out, long-term data from the bionic mice,