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


by Aarthi Ashok

Annie Nguyen

Karen Ross


Brittany Copp


Effects of Early Rearing Experience on Stress and Dominance in Rhesus Macaques
Brittany Copp, Tulane University, New Orleans, Louisiana
Preceptor: James Higley, Laboratory of Clinical and Translational Studies, NIAAA

The stress response to a perceived threat and the coping mechanism used to return to normal behavior is thought to be heavily influenced by early experiences such as the quality of maternal care.

Copp looked at stress levels (measured by cortisol levels) in rhesus macaques that were either reared normally by protective mothers (MR) or raised by surrogates with minimal social interactions (SPR). She found that as stereotypies, or repetitive motor movements, increased, the SPR animals showed increased cortisol, while MR animals that showed low levels of these stereotypic movements exhibited high cortisol. This was indicative of an abnormal response to stress in the SPR group.

Interestingly, while MR animals used social contact as a coping mechanism during stressful conditions, the SPR animals avoided social contact. Copp believes that this may be due to SPR monkeys' finding it difficult to initiate social behavior and also because they may not find such contact comforting.

The physiological response to stress has also been shown to differ based on the dominance rank of the monkeys. All female rhesus monkeys inherit their rank from their mothers, and Copp was therefore interested in understanding ranking in the SPR group, whose members have no clear inheritance. In a short study during a previous summer, she was able to demonstrate that the SPR animals consistently ranked lowest in the group of MR and SPR animals. She used this information in her current study to correlate stress levels with rank or perception of rank in the case of SPR animals.

By measuring stress using MHPG (monoamine metabolite of norepinephrine) levels in these monkeys, Copp found that in the MR group, increasing MHPG levels correlated with increasing rank, while in the SPR group, increasing MHPG levels correlated with decreasing rank. This suggested that the lower-ranking animals of the SPR group perceived normal activities as more stressful than the MR animals. This response may provide the reason for their lower ranking within the group. However, larger studies are needed to confirm these findings, as the small sample size of Copp's study meant that these correlations did not reach significance.

"I will miss the monkeys,"  Copp said, talking of her plans for graduate school while pointing to a picture of Stella, a mother macaque, "but I have had two excellent summers here." 

Aarthi Ashok


Megan McCain

Immunofluorescent Analysis of Chemokine Trafficking
Megan McCain, Washington University, St. Louis, Missouri
Preceptor: Fred Indig, Confocal Imaging Facility, Research Resources Branch, NIA

Chemokines are a class of cytokines that are secreted by cells that play an important role in human immune response against foreign antigens. Chemokines that are endocytosed by cells such as macrophages are degraded and presented as antigens on their cell surface. These chemokine antigens can be recognized by either CD8+ or CD4+ T cells, which then become activated to participate in the immune reaction. Recognition by the CD8+ or CD4+ T cells requires that the antigen be presented on the cell surface by MHC class I and class II molecules, respectively.

Antigens presented by MHC class I molecules are degraded by cytoplasmic proteasomes, whereas MHC class II antigens are processed in the lysosomes. Which pathway is used in the presentation of chemokine antigens on macrophages?

With the assistance of Purevdorj Olkhanud  and Arya Biragyn of the Laboratory of Immunology, NIA, McCain addressed this question in her study of the intracellular trafficking of a fluorescently tagged chemokine, MIP3a, in mouse macrophage cells. She followed the fluorescent chemokine in these cells by first allowing them to bind MIP3a at 4° C and then inducing endocytosis of the chemokine by shifting the cells to 37° C. McCain fixed the cells at various time points after the shift to 37° C and stained them with antibodies against clathrin, proteasomes, and lysosomes.

Her confocal microscopy images show that MIP3a co-localized with clathrin within two minutes of the 37° C shift, demonstrating that the chemokine is internalized by clathrin-dependent endocytosis. Ten minutes after internalization, the chemokine co-localized with both lysosomes and proteasomes but no longer with clathrin.

This result led McCain to the unique finding that by gaining access to proteasomes and lysosomes, the chemokine could be presented by both the MHC class I and the class II pathways.  By 60 minutes, almost all chemokine staining was lost, suggesting that the cells are able to completely degrade this chemokine within an hour.

"We could specifically target cancer cells by tagging a toxic molecule onto a specific chemokine whose receptors are highly expressed on tumor cells," McCain suggested, noting the implications of the research for cancer immunotherapy.

Aarthi Ashok

Amy Zolko

The Functional Performance of Children with Smith-Magenis Syndrome
Amy Zolko, University of Maryland, College Park
Preceptor: Michaele Smith, Rehabilitation Medicine Department, CC

Smith-Magenis Syndrome (SMS) arises from a partial or full deletion of band p11.2 on chromosome 17 and is characterized by a distinct pattern of physical, behavioral, cognitive, and functional abnormalities.

Zolko's project aimed to provide physical therapists and other involved health practitioners with detailed documentation of the level of functional motor skills attained by children with SMS compared with age-matched peers. Reasonable expectations regarding both the rate of skill development and the achievable functional level could be translated into additional interventional strategies.

The research team used two assessment methods — the Pediatric Evaluation of Disabilities Inventory (PEDI) and the Peabody Developmental Motor Scales (PDMS-2) — to assess functional motor skills in SMS patients. The PEDI is a parental questionnaire designed to monitor individual progress in daily functional activities such as brushing teeth, dressing, walking, and peer interaction. Used by clinicians, the PDMS-2 estimates a child s motor competency on subtests that measure reflexes, locomotion, ability to sustain body control, and object manipulation. Twenty-eight children, ages five months to seven years, with confirmed SMS diagnosis were enrolled in the study and monitored annually for several years.

Both the PEDI and the PDMS-2 showed that children with SMS scored below the mean for their age group for all subtests. Nonetheless, they eventually did achieve the desired milestones, such as running, skipping, and stair climbing, only at a slower rate than normal children. However, it is not certain that children with SMS will accomplish all the motor skills gained by unaffected children or at what age they can be expected to achieve them. Typically, children with SMS are not as coordinated as their unaffected peers.

The study suggested that children with SMS should be monitored annually to detect any motor delay as they grow older and that prolonging physical therapy may be effective in helping them obtain higher-level functional skills. The investigators plan more studies to determine the effects of prolonged therapy.

Zolko is in her last year at the University of Maryland at College Park and plans to pursue graduate study in physical therapy.

Annie Nguyen


Simone Berkower


Understanding the Evolution of Vaccine-Derived Polioviruses
Simone Berkower, Yale University, New Haven, Connecticut
Preceptor: Elena Cherkasova, Laboratory of Method Development, CBER, FDA

The live attenuated virus upon which the oral polio vaccine (OPV) developed by Albert Sabin is based has been observed to revert to wild-type virus after inoculation into the human host.

These strains, known as vaccine-derived polioviruses (VDPVs), have regained virulence and occasionally result in paralysis and polio outbreaks. This increased risk is especially significant in developing countries, where the oral construct is used almost exclusively due to its low cost and easy administration.

Berkower and her colleagues set out to elucidate the underpinnings of these changes. The team analyzed vaccine-derived strains isolated from healthy children after OPV administration. The children were given three doses of vaccine, and stool samples were collected approximately once a week after each dose. Viral RNA isolated from stool samples was synthesized into its complementary DNA via reverse transcription.

Subsequent DNA sequencing revealed amino acid mutations in capsid proteins in or near antigenic regions matching those of its wild-type predecessor or homotypic wild polioviruses.

The researchers point to errors made by the RNA polymerase during replication of the viral genome as one cause of the live attenuated Sabin 1 virus mutation.

They speculate that common antigenic alterations in evolving OPV strains largely reflect attempts to eliminate fitness-decreasing mutations acquired either during the original selection of the vaccine or already present in the parental strains.

Studying the patterns of mutation of VDPVs may provide new insights into viral evolution, the researchers note, as well as better approaches to crafting future vaccine constructs and vaccination policies.

Berkower, now a student at Yale University, spent the two NIH Summer Research program summers after her junior and senior years at the Sidwell Friends High School in Washington, D.C. She plans to continue her research endeavors at Yale.

Annie Nguyen

David Rogawski


The Prion Protein Protects Against Harmful Aggregation of Huntingtin
David Rogawski, Williams College, Williamstown, Massachusetts
Preceptors: Lois Greene, Kyung Jin Lee, Evan Eisenberg, Laboratory of Cell Biology, NHLBI

Rogawski worked on an intriguing connection between Huntington's disease and the prion protein PrPC, which, in its misfolded, "infectious"  form is responsible for a family of fatal neurological illnesses, including mad cow disease.

Despite its potential to cause devastating disease, PrPC is found in the brains of most mammals and may play an important role in neuronal functioning.

Rogawski investigated the normal function of PrPC and discovered that it helps protect neurons from the symptoms of Huntington's disease. Huntington's is a neurodegenerative disease caused by a mutation in the huntingtin gene.

The defective huntingtin protein aggregates inside brain cells, eventually killing them.

Working with cultured cells that carried the mutant huntingtin protein, Rogawski found that depleting PrPC accelerated the formation of huntingtin aggregates.

He observed that PrPC affected the qualitative appearance of the aggregates. Cells with a normal complement of PrPC tended to have one large hunting-tin aggregate, whereas those that lacked PrPC had numerous, smaller aggregates.

He then showed that introduction of a mouse version of PrPC into PrPC-depleted cells reduced the rate of aggregate formation.

The group's findings raise the possibility that PrPC may have a general neuroprotective function, preventing unwanted protein aggregates from forming in otherwise-healthy neurons.

Karen Ross


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