T H E   N I H   C A T A L Y S T     N O V E M B E R  –  D E C E M B E R  2005



Glinda Cooper

Glinda Cooper received her Ph.D. in epidemiology from the University of North Carolina at Chapel Hill in 1993. She then joined NIEHS as a staff fellow, became a tenure-track investigator in 1998, and is currently a senior investigator in the Epidemiology Branch.

Autoimmune diseases affect 5 to 8 percent of the United States population and are a leading cause of death among women younger than 65 years old.

Despite the impact of these diseases, they have been relatively neglected with respect to epidemiologic research — the study of the distribution of disease within a population — an approach that can provide insights into disease mechanisms, prevention, and prognosis.

My research has focused on the prototypical disease, systemic lupus erythematosus, or lupus. 

Lupus is a severe, disabling disease that can lead to significant morbidity and mortality, particularly from renal and cardiovascular disease. The vast majority (more than 85 percent) of lupus patients are female. Although it has long been known that genetic susceptibility plays an important role in lupus, there is growing interest in environmental influences on the development and progression of this disease.

A central piece of my research is the Carolina Lupus Study, the first population-based case-control study of hormonal and occupational risk factors for lupus conducted in the United States.

Quite surprising, given the female predominance in lupus in humans and the role of estrogen in disease progression seen in lupus mouse models, none of the markers of estrogen exposure we examined were associated with risk of lupus.

These markers included earlier age at menarche and later age at menopause, use of hormone replacement therapy or hormonal contraceptives, and number of pregnancies and live births. Many of these exposures are associated with the risk of breast cancer, suggesting that lupus does not fit that model of an "estrogen-related"  disease.  

Several occupational cohort studies have examined the association between silica exposure and the risk of various systemic autoimmune diseases, but ours was the first group to include an assessment of silica exposure in a population-based study of lupus. Our findings were striking. We observed a silica dust dose-response across levels of exposure, with an approximate twofold increased risk in the medium-exposure category and a fourfold increased risk in the high-exposure category. As expected, the prevalence of high exposure was lower among women than men, but the associations were seen in both groups.

Our success in carrying out a population-based study of lupus that contains a strong environmental component has encouraged others to consider the contribution that environment can make to lupus and to other autoimmune diseases.  Several new studies are being developed, intramurally and extramurally, by other investigators incorporating aspects of our data-collection instruments and hypotheses.

I also continue to pursue an interest in ovarian function, an area that includes menstrual cycle patterns and the timing of ovarian failure or menopause. The effect of potential environmental endocrine disruptors on ovarian function is an issue that is particularly relevant to the mission of NIEHS.

We were the first to report an association between serum levels of the persistent organochlorine DDE (a breakdown product of the pesticide DDT), and earlier age at menopause, with effects of similar magnitude to that seen with smoking.

In the Agricultural Health Study, a joint NCI-NIEHS prospective cohort study of more than 50,000 licensed pesticide applicators and their spouses, we found an association between pesticide use and an increased risk of long cycles, missed periods, and intermenstrual bleeding among premenopausal women.

Menstrual cycle characteristics and timing of natural menopause may be sensitive markers of biological effects of environmental exposures, and I am continuing to examine these outcomes in studies of environmental pollutants.

Patricia Gearhart

Patricia Gearhart received her Ph.D. in immunology in 1974 from the University of Pennsylvania in Philadelphia. She did postdoctoral work at Johns Hopkins University, Baltimore, on a Helen Hay Whitney fellowship. She was a staff associate at the Carnegie Institution of Washington in Baltimore and an associate professor in the Bloomberg School of Public Health at Johns Hopkins University before joining the Laboratory of Molecular Gerontology, NIA, in 1995. She became a tenure-track investigator in 2001 and is currently a senior investigator in the Unit on Antibody Diversity.

I like to start out my seminars with a slide from the "War of the Worlds"  movie that came out in the summer of 2005. In the movie, humans are being pulverized by invading Martians, and extinction is imminent. Then, suddenly, the Martians drop dead! They died because they didn't have the activation-induced deaminase (AID) molecule, which would have allowed them to become immune to earth's microorganisms. 

The idea of having an unlimited repertoire of proteins able to tackle any foreign antigen, even ones the human race has never seen, is mind-boggling. Immunity is partly based on a random slew of mutations that occur in antibody genes, followed by selection of those B lymphocytes that can express the mutated proteins with the highest affinity. 

Thus, when you're injected with a vaccine, some of your B cells can bind to the antigen and produce low-affinity antibodies of the IgM type. Soon after, the B cells start a process called somatic  hypermutation — somatic because these mutations are not present in your germ-line genes and hypermutation because the genes undergo mutation at a frequency that is a million times as high as any other gene in your body. 

I became interested in the generation of antibody diversity as a graduate student at the University of Pennsylvania.  This was in the pre–molecular biology era, so we studied different proteins with anti-antibodies. The hybridoma era came next, and it was possible to sequence antibodies at the protein level to identify different molecules.

In 1980, I showed that B cells initially make IgM antibodies using a few germ-line genes and then switch to making IgG antibodies with many mutations in their genes. 

As a staff associate at the Carnegie Institution of Washington, I had the opportunity to learn gene cloning and DNA sequencing. I was able to show in 1983 that the range of mutations extended over a two-kilobase region around the antibody variable gene. In other words, hypermutation was targeted to a small region of the chromosome.

While on the faculty at Johns Hopkins University, I realized the mechanism producing hypermutation would only be solved by studying DNA repair. I had the good fortune to come to NIA in Baltimore in 1995 and work in a repair lab headed by Vilhelm Bohr.

In 1998, we published the first paper to show that a mismatch repair protein, MSH2, played a powerful role in the mutation of certain nucleotides. Then in 2001, we were the first to identify the involvement of the error-prone DNA polymerase h in the hypermutation process. But we still didn't know the mechanism.

The whole story came together with the separate contributions of researchers from Japan, England, and elsewhere.

First, in 2000, the Japanese team discovered that the AID protein (which the Martians didn't have) initiates hypermutation and heavy chain class recombination. AID is expressed only in B cells; people who don't have AID have recurring infections because they can't mutate their variable genes or make IgG.

Then, in 2002, the mechanism was solved when researchers in England reported that AID works by deaminating cytosine in DNA to form uracil. Uracil is a foreign nucleotide and will cause mutations. MSH2 and its partner, MSH6, enter the pathway by binding to the uracil mismatches and then recruiting DNA polymerase to synthesize errors.

The most satisfying moment in my career came this year when we published a paper that tied these proteins together using biochemistry, genetics, and biology. (T.M Wilson, A. Vaisman, S.A. Martomo, et al. "MSH2-MSH6 stimulates DNA polymerase h, suggesting a role for A:T mutations in antibody genes,"  J. Exp. Med. 201:637, 2005).

The final frontier is to determine how AID is targeted to immunoglobulin genes. We are trying a variety of approaches to solve this enigmatic puzzle. Inappropriate targeting by overexpression of AID can produce mutations in other genes and DNA strand breaks. B cells therefore walk a fine line between generating antibody diversity and tumors.

Kim Green


Kim Y. Green received her Ph.D. in 1986 from the University of Tennessee at the Center for Health Sciences in Memphis. She joined the Laboratory of Infectious Diseases (LID), NIAID, that year as a postdoctoral fellow in the Epidemiology Section. In 1993, she became a tenure-track investigator in the LID and is currently a senior investigator. She is also an adjunct associate professor at the University of Maryland, College Park.

When I joined the Epidemiology Section, the rotavirus vaccine candidate developed in the laboratory under the guidance of my mentor, Albert Kapikian, was already in clinical trials. It was an exciting time, because data from the Phase III trials were emerging to show that the vaccine was effective in preventing severe diarrhea in infants and young children.

My initial research in the laboratory as a postdoctoral fellow addressed the molecular mechanisms for serotypic diversity among the predominant rotaviruses associated with diarrheal illness. I developed assays that enabled the dissection of the serotype specificity of the antibody response to the vaccine in young vaccinees. These studies showed the importance of serotype-specific antibodies and supported the rationale for the use of a multivalent rotavirus vaccine. 

In 1993, I was asked to initiate a research program in the Epidemiology Section to address the role of the Norwalk virus and related viruses (now called noroviruses) in human diarrheal illness. Norwalk virus had been discovered by Kapikian in 1972 but had taken a back seat to the vigorous rotavirus vaccine development effort in the section. My research program began with nothing but frozen stool samples and sera collected from volunteers who had participated in challenge studies at NIH in the early 1970s, along with the newly available sequence of the Norwalk virus genome published by scientists at Baylor University in Waco, Texas.

We initially thought that most of the norovirus strains associated with epidemic gastroenteritis (known colloquially as "stomach flu") would be closely related to the original Norwalk virus strain. However, our work soon revealed that considerable genetic diversity existed among circulating noroviruses and that infection with these viruses was more common than expected, even among younger individuals. Noroviruses have now been established as the major cause of the acute nonbacterial gastroenteritis that often occurs in sharp outbreaks that capture headlines, such as those that have arisen on cruise ships. My laboratory played a key role in some of the early large-scale epidemiologic studies that established the importance of noroviruses in nursing homes and military settings.

We are now convinced that these viruses are worthy of concerted efforts to develop vaccine and antiviral control strategies. My future research will address some of the technical hurdles that must be overcome to pursue this line of research — especially the inability to grow the human norovirus pathogens in cell culture and the absence of an animal model for diarrheal disease. Currently, the only way to study the efficacy of a vaccine candidate, such as recombinant capsid protein, is to immunize adult volunteers and then challenge the volunteers with noroviruses present in stool material.

One approach pioneered by my laboratory was the use of related viruses within the virus family (Caliciviridae) as models to study general features of calicivirus growth, replication, and maturation in vitro. We reported the development of the first infectious RNA system ("reverse genetics") for the family Caliciviridae.

This system, modeled on the cultivatable feline calicivirus (FCV), was based on the transfection of infectious in vitro–transcribed capped RNA molecules derived from a full-length cDNA clone of the feline calicivirus genome into permissive cells and recovery of viable viruses. This system allowed us to introduce mutations into the cDNA clone and study their effect on the growth and replication of the recovered virus. 

We also found that we could engineer chimeric viruses with altered antigenic specificities. We are now working on the development of a reverse genetics system for the noroviruses.

In the meantime, we have made progress in dissecting the functions of various norovirus proteins in replication and are exploring potential antiviral inhibitors through the study of recombinant enzymes.

The noroviruses are interesting in that they have a global distribution and are ubiquitous in the environment. They seem to arise out of nowhere to cause acute gastroenteritis outbreaks. We do not yet understand how immunity is developed, and we are just beginning to identify the role of host factors in susceptibility to infection. The field of norovirus research has exciting opportunities for addressing the role of a common and sometimes severe pathogen and in gaining a better understanding of the virus and host interactions that might lead to the development of effective control strategies.

Andrew Griffith


Andrew Griffith received his M.D. degree and Ph.D. in molecular biophysics and biochemistry from Yale University in New Haven, Conn., in 1992. He completed a residency in otolaryngology–head and neck surgery and a human genetics research fellowship at the University of Michigan, Ann Arbor, before joining NIH in 1998 as a research fellow in both the Laboratory of Molecular Genetics and the Neuro-Otology Branch, NIDCD. He is currently acting chief of the Section on Gene Structure and Function and of the Hearing Section, NIDCD.

There are hundreds of genes in which mutations cause hearing loss either alone or in combination with other abnormalities as part of a syndrome. Because most hearing loss phenotypes are nonspecific, the clinical challenge is to determine which gene to test in a patient whose hearing loss may have a genetic etiology. 

Inner ear malformations seen on CT or MRI scans are an example of clinically useful markers to guide molecular genetic diagnosis. The most commonly detected malformation, enlargement of the vestibular aqueduct (EVA), is clinically important because affected children often pass newborn hearing screening but experience significant postnatal hearing loss. 

Mutations of SLC26A4 (PDS), which encodes the anion transporter pendrin, have been identified in patients with nonsyndromic EVA (NSEVA) as well as in a syndromic form of EVA called Pendred syndrome. The molecular and physiologic details of the pathogenesis of hearing loss caused by SLC26A4 mutations remain unclear.

In a study of approximately 90 EVA patients at the NIH Clinical Center, we observed that one- third of cases are associated with a thyroid iodination defect as part of Pendred syndrome and mutations of both alleles of SLC26A4.  One-third of cases are nonsyn-dromic and associated with only one detectable mutant allele of SLC26A4, while another one-third are also nonsyndromic but have no detectable mutations. Identifying the etiology of nonsyndromic EVA in these latter two genotypic groups is a focus of our current research. 

Another major goal of my laboratory is identification of the molecular function(s) of the proteins encoded by TMC genes. We used positional cloning to determine that dominant and recessive mutations of the transmembrane channel–like gene 1 (TMC1) cause hearing loss in humans and mice.

There are eight mammalian TMC genes, and their deduced amino acid sequences have no obvious similarities to other domains or proteins of known function. All of the TMC genes are predicted to encode proteins with 6 to 10 membrane-spanning domains; we are now completing a study indicating that TMC1 has 6 transmembrane domains and cytoplasmically oriented N- and C-termini. This topologic structure is shared with the large superfamily of multimeric cation channels that includes transient receptor potential (TRP) channels. 

We are currently using knockout mouse models of Tmc1 and other Tmc genes to understand the pathogenesis of hearing loss caused by TMC1 mutations, to characterize the molecular and cellular function of TMC1 protein, and, we hope, to identify other tissues and Tmc mutant model systems to study the function(s) of this gene family.

The histopathologic hallmark of Tmc1 mutant mice is early and rapid degeneration of cochlear neurosensory hair cells, where Tmc1 mRNA is expressed.  In mice that are heterozygous for the dominant Tmc1 mutation, the degeneration of cochlear hair cells depends upon the mouse strain background.

We have mapped four genetic loci with strain-specific alleles that differentially affect degeneration of hair cells. Because hair cell loss is a final common pathogenetic pathway in many mouse and human hearing loss disorders, such as age-related hearing loss and noise-induced hearing loss, identification of these modifier genes may provide critical insights into these more common but etiologically complex phenotypes. 

Indeed, previous work by others on a mouse model of polygenic hearing loss enabled us to identify a hypofunctional variant of plasma membrane calcium pump PMCA2 (encoded by ATP2B2) that modifies human age-related hearing loss caused by a mutation of the cadherin 23 (CDH23) gene.  We plan to assess the contributions of ATP2B2 — and other modifier genes that might be identified — to age-related hearing loss and noise-induced hearing loss in large case-control cohorts of human subjects.

Vittorio Sartorelli

Vittorio Sartorelli received his M.D. degree from the University of Brescia, Italy, in 1984; he completed an oncology residency at the University of Milan and postdoctoral work at Stanford University in Stanford, Calif., and the University of Southern California in Los Angeles. He was then an assistant professor of biochemistry and molecular biology at USC before joining NIAMS in 1999 to head the Muscle Gene Expression Group in the Laboratory of Muscle Biology.

The formation and development of an organism relies on the specification of discrete cell lineages. Because every cell of an organism contains — packed in its DNA — the same genetic information, it is of interest to understand the strategy a cell uses to become, for instance, a contractile muscle cell rather than a firing neuron.

My group in the Laboratory of Muscle Biology addresses the cellular and molecular mechanisms underlying specification, differentiation, and regeneration of skeletal muscle cells. Pivotal in skeletal muscle biology are the myogenic basic helix–loop–helix (bHLH) transcription factors MyoD, Myf5, myogenin, and Mrf4.

After interaction of these proteins with the ubiquitously expressed bHLH E proteins, the resulting myogenic bHLH–E heterodimers bind to and regulate expression from the E-box, a specific DNA motif present at muscle gene enhancers and/or promoters.

The interactions of the myogenic bHLH transcription factors with enzymes that modify the structure of the nucleosome — the basic unit of chromatin — enable temporally regulated formation and recruitment of specific protein complexes at the chromatin of discrete muscle gene loci.

By influencing the structure of the chromatin, such protein complexes ultimately dictate whether or not a given gene will be transcribed. Therefore, a detailed biochemical definition of the protein complexes and the mechanisms that regulate their chromatin engagement is essential to the comprehension of how muscle gene expression and cell differentiation are orchestrated.

Over the past years, we have identified and characterized proteins that are recruited on the chromatin and that either promote or inhibit muscle gene expression. A common strategy used by these proteins to regulate transcription is the enzymatically mediated addition or removal of chemical groups (acetyl, methyl, phospho groups) at defined amino acid residues of histones.

The availability of cell-permeable small molecules that interfere with the enzymatic activities of some of these proteins (histone deacetylases) has afforded us the opportunity to manipulate muscle gene expression both in cell cultures and in animal models. Using histone deacetylase inhibitors, we have been able to increase formation of mature skeletal muscle cells in cell culture and to promote the expression of markers of muscle regeneration in animals in which muscle degeneration was experimentally induced. The next challenge will be to evaluate the therapeutic efficacy of histone deacetytlase inhibitors in animal models of Duchenne and limb girdle muscular dystrophies. 

Another line of research pursued in my group relates to the mechanisms regulating the expression of muscle-specific ubiquitin ligases involved in mediating skeletal muscle atrophy. Skeletal muscle atrophy is the loss of muscle mass and function that is often associated with cancer, AIDS, diabetes, glucocorticoid treatment, chronic uremia, sepsis, and other diseases. Muscle atrophy also follows denervation, immobilization, and starvation.

While the pathophysiology of all these conditions is often unrelated, reduced protein synthesis and increased protein degradation invariably accompany muscle atrophy, regardless of the triggering cause. Two genes have been identified that are rapidly upregulated during the course of skeletal muscle atrophy and are causally linked to its pathogenesis. These genes, Atrogin-1/MAFbx (muscle atrophy F box) and MurF1 (muscle RING finger 1), encode for two E3 ubiquitin ligases selectively expressed in skeletal muscle and heart and involved in modifying proteins fated to be degraded by the proteasome.

We are interested in understanding how transcription of these two genes is regulated during muscle atrophy. Our objective is to be able to modulate their expression to spare muscle mass in diseased conditions.

Lino Tessarollo

Lino Tessarollo received his Ph.D. in Biological Sciences from the University of Padua, Italy, in 1987. After postdoctoral training at the Institute of Oncology, Padua, and at NCI-Frederick, he was recruited in 1994 as an investigator at the ABL-Basic Research Program, NCI-Frederick, to develop targeted gene-manipulation technology in the mouse. In 1999, he joined the Mouse Cancer Genetics Program at the NCI Center for Cancer Research, where he directs the Neural Development Section and the Gene Targeting core facility.

My research focuses on the dissection of signals that control cell proliferation and survival in the mammalian system. Using mouse models and in vitro approaches, we aim to identify specific pathways that can be activated to promote survival of cell populations affected in neurodegeneration — or that can be inhibited to avoid uncontrolled cell proliferation leading to cancer.

We study the neurotrophin family of peptide growth factors (NGF, BDNF, NT-3, and NT-4/5) and their high-affinity Trk tyrosine kinase receptors (TrkA, TrkB, and TrkC) because they are critical players in the development and maintenance of the mammalian central and peripheral nervous system. These genes have attracted great interest as potential therapeutic targets for the management of neurodegenerative disorders such as Parkinson's and Alzheimer's disease.

Neurotrophin receptors are also frequently overexpressed in human cancer, including pancreatic and prostate carcinoma, Wilm's tumor, and neuroblastoma, particularly those with aggressive behavior and poor prognosis. Thus, while suppression of Trk-activated pathways may contribute to tumor management, strategies aimed at improving neurotrophin-mediated activities may also help stem neurodegenerative diseases.

Over the past decade, many laboratories have contributed to our understanding of neurotrophin receptor signaling. However, most of this information has been generated in vitro. The situation is far more complex in vivo because cellular context and environment may influence how cells respond to signals. To dissect the cell type– and environment-specific effects of the neurotrophin-activated pathways in vivo, we use a mouse model to introduce specific targeted mutations.

For the development of our in vivo systems, we focus on the following molecular properties: a) docking sites on the intracellular portion of the neurotrophin receptors to evaluate whether altering specific pathways can affect cell type– specific responsiveness to neurotrophins; b) truncated Trk isoforms that lack the kinase domain and may have positive or negative modulatory roles on the kinase active receptors; and c) tissue-specific neurotrophin or receptor deletion to dissect both neuronal and non-neuronal functions.

These studies have led us to the identification of a specific docking site in the Trk receptor juxtamembrane region, which in vivo has the ability to potentiate neurotrophin signaling without affecting cell proliferation. Similar results were obtained by the targeting of physiological truncated Trk receptors.

In vivo reduction of these truncated receptors can increase ligand activation of the kinase active receptors and avert neural cell death in a mouse model of neurodegeneration. We are now planning to assess whether these Trk mutations, which potentiate neurotrophin signaling in vivo, may affect tumor development and/or metastatic properties in established tumor mouse models.

These approaches have validated our strategies and helped us to shed more light on the role of Trk isoforms and Trk signaling in vivo. They will also be essential in assessing the risks and benefits associated with augmenting or inhibiting neurotrophin signaling in the clinic.

Moreover, our results suggest new strategies to affect Trk receptor activation in vivo by small-molecule–based approaches — which could circumvent the challenge of delivery to the CNS and specific pharmacokinetic problems associated with the classic neurotrophin peptide–based approach. Clearly, there is still insufficient knowledge on the physiological roles of these genes and their underlying molecular mechanisms of action.

We anticipate that our model systems will help amass more detailed information on how Trk-activated pathways control the maintenance and function of the mature organism.

This expanded knowledge base may help identify potential therapeutic targets within the neurotrophin signaling pathways for the treatment of neurodegenerative diseases and cancer.      n


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