|T H E N I H C A T A L Y S T||M A R C H A P R I L 2006|
|P E O P L E|
Kim Hasenkrug received his Ph.D. in cell biology from the Albert Einstein College of Medicine in New York in 1991. He did postdoctoral training in the Laboratory of Persistent Viral Diseases, NIAID, and became a tenure-track investigator in 1998. He is currently a senior investigator in the Laboratory of Persistent Viral Diseases at Rocky Mountain Labs.
Despite the remarkable capacity of the immune system to recognize and clear most infectious agents, we are all chronically infected with viruses that have escaped immunological eradication.
Although most chronic viral infections are relatively innocuous, immunological escape of viruses such as hepatitis C virus and the human immunodeficiency virus causes a great deal of morbidity and mortality worldwide.
During my postdoctoral studies in Bruce
Chesebro's laboratory, I investigated genetic resistance to Friend virus
infection in mice and found that even the most resistant strains of mice were
unable to completely clear infection. http://www.niaid.nih.gov/dir/labs/lpvd.htm
I became fascinated with the problem of chronic infections and realized that a better understanding of the basic mechanisms by which this retrovirus established and maintained persistence could aid in the development of vaccines and therapies against some of our most dangerous viruses.
Much of my early work focused on using the Friend model to determine mechanisms of protection by live-attenuated vaccines. It was known that live-attenuated simian immunodeficiency virus (SIV) provided the best vaccine protection from SIV in the nonhuman primate model for HIV, but that model was not well suited for mechanistic studies.
Using live attenuated Friend virus we showed that complete protection, defined as protection from both acute disease and the establishment of chronic infection, required immune CD4+ T cells, CD8+ T cells, and also virus-neutralizing antibodies.
Each of these components provided essential, nonoverlapping functions. Although live-attenuated vaccines are considered too dangerous to use as vaccines in humans, the description of how live-attenuated Friend virus worked provides an experimental framework indicating the types of responses required for a successful retroviral vaccine.
In addition to prevention of chronic infections, I have also been very interested in immune control of established chronic infections.
We found CD4+ T cells and IFN-g to be crucial for control of chronic Friend virus and the prevention of relapses. Interestingly, although CD8+ T cells were critical for recovery from acute infection, they played no role during chronic infection.
As we began to probe more deeply the functions of CD4+ and CD8+ T cells during chronic infection, we made the intriguing discovery that chronic infection induced regulatory CD4+ T cells that suppressed CD8+ T cell functions.
Subsequently, other labs reported similar findings for HIV and other chronic viral infections in humans, suggesting that the induction of regulatory T cells may be a common mechanism of escape.
Now that we have determined why the CD8+ T cells are impotent, our studies are focused on determining the molecular mechanisms of suppression, both at the level of the CD4+ regulatory T cells and the CD8+ effector cells.
We recently developed an in vitro suppression assay to facilitate our mechanistic studies, and results recapitulate much of what we observe in vivo.
We are also using our in vivo model to determine ways to specifically inhibit the regulatory T cells, render the CD8+ T cells resistant to suppression, and reactivate immune responses during chronic infection.
Our goal is to modulate the immune response to enable the complete clearance of chronic infections.
We recently achieved thousand-fold reductions in chronic Friend virus levels using immunocytotherapy combined with CD137 co-stimulation.
We will use the data from our mechanistic studies to further develop and refine our immunotherapeutic approaches, and we hope to translate our findings to therapies for chronic infections in humans.
Lenore Launer received her Ph.D. in epidemiology and nutrition from Cornell University in Ithaca, N.Y., in 1987. After a postdoctoral fellowship at NICHD, she held academic appointments in the Netherlands at Erasmus University Medical School in Rotterdam, Free University in Amsterdam, and the National Institute for Public Health and the Environment, Bilthoven, before joining NIA in 1999 as chief of the Neuroepidemiology Section. She is currently a senior investigator and chief of that section in the Laboratory of Epidemiology, Demography, and Biometry, NIA.
My main contributions to the study of the epidemiology of dementia have been to identify cardiovascular risk factors of late-life dementia and the value of studying these risk factors in midlife. Another research focus has been to elucidate the relationship of migraine to structural brain changes in midlife.
I started my research on the epidemiology of brain aging in 1990, when I accepted an appointment at Erasmus University in the Netherlands. There I worked as a research scientist on a community-based study of dementia. I was also the scientific coordinator of multicenter prospective studies on the epidemiology of dementia involving a consortium of major European centers.
While in the Netherlands, I established a population-based cohort of individuals with and without migraine for the study of migraine-related risk factors and structural brain changes.
My research program since joining NIA in 1999 has focused on the interaction between the vascular and neuronal systems as reflected in biomarkers of subclinical and clinical disease. Related cognitive function studies and structural measures of brain neuropathology in large population-based studies have been important in these investigations.
Thus far, my research has shown that vascular dementia and Alzheimer's diseasecurrently diagnosed as two different types of dementiashare common risk factors, such as smoking, hypertension, diabetes, and elevated C-reactive protein.
This clinical picture is further corroborated by findings from an autopsy database, which I maintain in collaboration with investigators in the Honolulu Asia Aging Study (HAAS).
The clinical and pathologic characteristics of brains in older persons suggest that by old age, a human brain has accumulated different types of vascular and neurodegenerative lesions, all of which may contribute to the clinical picture of dementia.
Further, based on the HAAS, I have shown that risk factor profiles in midlife are associated with the risk for late-life brain aging, suggesting that neurodegenerative processes begin earlier than previously thought.
My research aims in the coming years include disentangling the heterogeneous pathology, on the one hand, and the shared risk factors, on the other, of subtypes of dementia.
Using bioimaging, molecular markers, and clinical measures, I intend to identify brain-aging traits that cluster within and across biologic systems.
I will also investigate the interaction between genetic and environmental markers of vascular health, inflammation, cellular nutrition, and oxidation as they relate to brain-aging traits, as well as explore the role of early-life experiences in shaping the trajectory of brain aging.
These questions will be tested not only in the HAAS, but also in two studies I have been involved in since coming to NIA. The first, which is jointly led with Tamara Harris, NIA, is AGES-RS (Age Gene-Environment SusceptibilityReykjavik Study). This is a large population-based study established in 1967 by the Icelandic Heart Association and conducted in Reykjavik.
Beside the advantage of a relatively genetically homogeneous population and excellent collaborators, we have data on early-life experiences of the cohort, through both the Reykjavik Study exams in midlife and the archival material available in Iceland.
AGES-RS focuses on four systems that are vulnerable to aging and diseasethe neurocognitive, cardiovascular, musculoskeletal, and metabolic/body composition systems. Base-line data collection on about 5,800 men and women has just been completed, and a follow-up is in planning. This study is also supported in part by other collaborators in the NIA, NEI, NIDCD, NHLBI, and NINDS programs.
The second study I'm involved in is ACCORD, the largest randomized treatment trial in older people with diabetes; it's funded by NHLBI to test the efficacy of intensive vs standard treatment in reducing cardiovascular disease and mortality in diabetics.
I have added a cognitive and an MRI component to investigate whether interventions that aim to maximize improvement in hyperglycemia, blood pressure, and lipid levels result in a change in brain function and structure.
Thus, through observational studies and randomized trials, I hope to better understand how vascular factors play a role in common late-life dementia and whether there are avenues to pursue for intervention.
Zheng-Gang Liu received his Ph.D. from the University of Massachusetts, Amherst, in 1995 and carried out his postdoctoral training in the laboratory of Michael Karin at the University of San Diego. He joined the Department of Cell and Cancer Biology, NCI, in 1998 as a tenure-track investigator and is currently a senior principal investigator at the Cell and Cancer Biology Branch, CCR, NCI.
My training has been in the field of apoptosis and signal transduction, focusing first on the regulation of activation-induced apoptosis of T cells and then on cellular stress-induced JNK activation and TNF signaling.
Since coming to NCI, my research has focused on two themes:
1) Molecular mechanisms of TNF signaling. TNF is a proinflammatory cytokine that plays a critical role in diverse cellular events. Under the influence of TNF signaling, cells may variously undergo proliferation, differentiation, and apoptosis.
In the past few years, my group has made several critical discoveries about TNF signaling. For instance, we found that the key effector molecule of TNF signaling, RIP, is cleaved by Casps-8 during apoptosis and that this cleavage plays a major role in modulating the outcome of life and death in TNF-treated cells.
Moreover, RIP cleavage is a key factor in switching the path of cell death from necrosis to apoptosis. In addition, we also found that TRAF2, another key effector of TNF signaling, recruits IKK complex to TNFR1 complex to activate the NF-kB pathway.
Currently, my group is studying mechanisms of TNF-induced necrosis. I am especially interested in what controls the switch between apoptosis and necrosis in cells after TNF treatment.
2) Regulation of apoptosis. Apoptosis, or programmed cell death, is a common phenomenon during development and occurs to rid the organism of harmful or unwanted cells. Apoptosis is crucial in enabling organisms to maintain cellular homeostasis. Deregulation of apoptosis is involved in many diseases; for instance, inefficient apoptosis has been found in many different cancers.
Since all cells have the genetic machinery required to commit suicide, the ability to selectively regulate this process has profound implications for treating disease.
Because more and more evidence indicates that irregular cell growth often leads to apoptosis, we believe that in addition to promoting growth signals, inactivation of apoptosis is essential for normal cells to become tumor cells. This process can be achieved by either increasing a signal that actively blocks apoptosis or generating a defective mutation in the cell death machinery.
Identification of these apoptosis-inactivating targets in different cancers will greatly enrich our knowledge of tumorigenesis and help inform the development of new cancer therapies.
To that end, a major research interest of mine is to identify the genes that protect cancer cells from apoptosis and decipher the mechanisms of their actions.
Tom Misteli received his Ph.D. from the University of London, U.K., in 1995 and was a postdoctoral fellow at the Cold Spring Harbor Laboratory, N.Y. He was recruited to the Laboratory of Receptor Biology and Gene Expression, NCI, in 1999. He now leads the Cell Biology of Genomes Group.
Much progress has been made during recent decades in deciphering genome sequences and elucidating the basic molecular mechanisms involved in gene regulation. Although these efforts have been very successful, they have also made it clear that these pieces of information are insufficient to understand how genomes work in vivo.
To do so, we must understand genome function at a global scale, and we need to uncover how genomes function in the context of the cell nucleus in living cells.
My laboratory seeks to elucidate the fundamental principles of how genomes are organized in vivo and how this organization contributes to gene regulation.
To begin to analyze the cell biological properties of genomes, we developed in vivo imaging methods to study the dynamics of gene expression for the first time in living cells.
Using these tools, we discovered that almost all aspects of nuclear organization and function are highly dynamic. For example, we were able to show that transcription factors find their specific binding sites within the genome by simple 3-D diffusion during which they scan the genome for their preferred binding sites.
Our measurements of interaction dynamics of proteins revealed that most transcription factors interact very transiently and rapidly with chromatin inside of living cells. These findings have led to a paradigm shift in how we think about gene expression in that they indicate that most regulatory gene expression events are stochastic.
The methods we developed have now become standard tools in the field and are powerful approaches to interrogate how genomes function in vivo. Our current efforts are aimed at visualizing and measuring the dynamic interplay of a complete transcription complex in a living cell and understanding how polymerases are dynamically regulated in vivo.
To this end, we are combining in vivo imaging methods with computational stimulation and modeling techniques to gain a quantitative view of gene expression in a living cell.
A second aspect of our work addresses the fundamental question of how genomes are spatially organized inside the cell nucleus. This is of great relevance because it is now clear that the position of chromosomes and of single genes within the nuclear space is nonrandom.
Our studies have contributed to the idea that how genomes are organized in the nucleus is related to their functional status.We showed that chromosomes are arranged differently in different tissues and that their position within the nucleus changes during differentiation.
One of our most import findings was the discovery that the position of chromosomes near each other contributes to the formation of cancer translocations in which chromosomes break and undergo illegitimate joining events, giving rise to fused chromosomes.
We are now expanding these studies by developing experimental systems in which we can induce and follow in single cells the fate of damaged chromosomes. These systems will allow us to query the cell biological mechanisms that lead to formation of cancer translocations.
These studies have clearly demonstrated that the cellular organization of genomes is critically important for proper genome function. One of the most important questions now is to understand how the fundamental principles of nuclear organization contribute to physiological genome function.
To address this problem, we are investigating how genome organization is established, maintained, and altered in physiological processes, including various diseases and during differentiation. These efforts will ultimately lead to an understanding of how genomes actually work inside of living cells.
David Waugh received his Ph.D. in biochemistry from Indiana University, Bloomington, in 1989 and was a post-doctoral fellow at the Massachusetts Institute of Technology before becoming director of the Macromolecular Engineering Laboratory at Hoffmann-La Roche in 1991. In 1996, he established the Protein Engineering Section at the NCI-FCRDC. He is currently head of that section and a senior investigator in the Macromolecular Crystallography Laboratory, NCI-FCRDC.
My research is divided roughly equally between two main projects: 1) "maximum likelihood" methods for protein expression and purification and 2) structural proteomics of type III secretion (the transport of virulence factors from the pathogen directly into the host cell) in Yersinia pestis, the causative agent of plague.
It is widely recognized that poor solubility of recombinant proteins in heterologous expression systems is a major bottleneck in structural and functional proteomics projects.
Although I was not the first to recognize that the solubility of recombinant proteins can sometimes be improved by fusing them to highly soluble partners, my section conducted the first systematic study of this phenomenon under rigorously controlled experimental conditions.
This work led to the discovery that Escherichia coli maltose binding protein (MBP) has an amazing ability to improve the solubility and promote the proper folding of its fusion partners.
These early experiments also shattered the dogma that any highly soluble protein can function as a solubility enhancer; the two other soluble fusion partners that were tested in this study, glutathione S-transferase (GST) and thioredoxin, were far less effective than MBP.
The current objective of my research in this area is to learn why some highly soluble proteins are much more effective solubility enhancers than others. I believe that understanding the underlying mechanism of the solubilizing effect is the key to realizing its full potential as a means of circumventing the inclusion body problem.
Although MBP is a powerful solubility enhancer, it is not a particularly good affinity tag for protein purification. For this reason, my group experimented with the incorporation of supplemental affinity tags within the framework of an MBP fusion protein.
The main challenge, from an engineering standpoint, was to identify locations in which accessory tags could be placed without interfering with the ability of MBP to enhance the solubility of its fusion partners.
Fortunately, it was possible to identify several permissive sites for the addition of accessory tags. This enabled us to explore various strategies for generic protein purification, and our efforts have recently culminated in the development of a simple process using a dual His6-MBP tag that appears to be suitable for automation.
The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His-tag facilitates its purification.
Recognizing that most affinity tags have the potential to interfere with structural and functional studies, we also assumed a leading role in the development of tobacco etch virus (TEV) protease as a reagent for removing affinity tags.
We have shown that, contrary to popular belief, many different amino acid side chains can be accommodated in the P1¢ site of a TEV protease recognition site with little or no impact on the efficiency of processing.
The wild-type protease readily cleaves itself at a specific site to yield a truncated enzyme with greatly diminished activity, but we managed to overcome this problem by making amino acid substitutions in the vicinity of the internal cleavage site.
We also determined the crystal structures of TEV protease complexed with a peptide substrate and an inhibitor, which revealed the structural basis of its stringent sequence specificity.
Our success in producing large quantities of crystallization-grade proteins led to a small-scale structural genomics project aiming to solve the three-dimensional structures of proteins involved in type III secretion in Y. pestis.
Because the type III secretion system (TTSS) is essential for virulence, the resulting structural information could be used to develop effective countermeasures for this potential instrument of bioterrorism.
We have already solved 12 novel structures and are in the process of solving more, including several protein-protein complexes. In one case, we have already begun the process of structure-based drug development.
One of the cytotoxic effector proteins that Yersinia injects into mammalian cells via the TTSSYopHis a potent eukaryotic-like protein tyrosine phosphatase (PTPase). YopH dephosphorylates several proteins associated with the focal adhesion in eukaryotic cells, thereby enabling the bacterium to avoid phagocytosis and destruction by macrophages.
In collaboration with Terrence Burke Jr. (Laboratory of Medicinal Chemistry, CCR), we identified several tripeptide analogs that inhibit YopH with IC50 values in the low micromolar range. Thus far, we have managed to crystallize one of these compounds with the enzyme and solve the co-crystal structure at 2.2-Å resolution.
In addition, we determined a high-resolution structure (1.5 Å) of the YopH PTPase in complex with a nonhydrolyzable hexapeptide substrate analog, which promises to provide yet another starting point for the development of inhibitors.
Han Wen received his Ph.D. in physics from the University of Maryland, College Park, in 1994 under the mentorship of Michael Fisher and Ralph Nossal of the University of Maryland and Robert Balaban of NIH. He joined the Laboratory of Cardiac Energetics, NHLBI, in 1995 and is currently a senior investigator in that lab.
My research interest has always been the development of imaging technologies with potential applications in humans. In the early part of my NIH experience, I had the opportunity to work with a high-field MRI scanner.
The high magnetic resonance frequency, which is proportional to the field strength, yielded various interesting electromagnetic wave propagation phenomena in the body, including disruption of the uniformity of image sensitivity in the human chest.
With theoretical modeling and human scans at 1.5 tesla, 3 tesla, and 4 tesla, my colleagues and I showed that the optimal field strength for imaging the heart was around 2 tesla. I then continued to develop MRI techniques and in particular techniques for studying the biomechanics of the heart and vascular system.
One technique called DENSE (Displacement ENcoding with Stimulated Echo) is capable of mapping at high resolution the motion of the heart wall and the walls of major arteries, revealing the stress loading and compliance of the tissue in these areas. Such information is useful in diagnosing heart disease and in learning about the mechanical factors in atherosclerotic lesion formation and rupture.
A challenging problem in cardiac mechanics is how to map the muscle fiber structure of the heart noninvasively. My postdoc and I observed an interesting effect from a nanoparticle MRI contrast agent that is closely related to the capillary and fiber structure of the myocardium. We then discovered a simple and robust way to map the myofiber structure in vivo; it is based on the correlation between the image intensity and capillary orientation when the contrast agent is present.
My high magnetic field experience led to an interest in tissue electrical properties, which vary greatly among different types of tissue and therefore can provide great image contrast. I realized that in a high magnetic field an ultrasound pulse traveling in tissue should generate weak but detectable electrical signals, due to the Lorentz force on the charged molecules.
If the ultrasound is focused, as in ultrasonic imaging, then the electrical signal also forms an image that reveals electrical conduction properties of tissue. I called this Hall effect imaging and made devices that demonstrated 3-D imaging with this idea.
In the long term, I see many exciting ideas to be explored in the field of imaging. We all know that the human body is amenable to the propagation of several forms of energy waves, including ultrasound, radiofrequency electromagnetic, infrared and much higher frequency X-ray, and even gamma ray waves.
Biomedical imaging either relies on the interaction of these energy waves with tissue to provide image contrast or uses them to carry information out of the body to the detectorsand frequently both.
Contrast agents greatly widen the information content of the image and often lead to new imaging techniques. The recent progress in monochromatic X-ray sources opens new areas of X-ray imaging and contrast agent development. I hope to continue to explore new ideas and contribute to the growing capabilities of noninvasive imaging in biology and medicine.
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