SUMMER POSTERS DAY:

AN AUGUST SHOWING ALONG THE "ROAD TO DISCOVERY"

by Aarthi Ashok and Fran Pollner

Double Take: Rebecca Burke (left) and Christina Nelson Perez

Inhibiting HIV with Alloimmunized Lymphocyte

Preceptors: Chad Womack, Viral Pathogenesis Laboratory, NIAID-VRC, and Lauren Wood, HIV & AIDS Malignancy Branch, NCI

Previous work by NCI’s Gene Shearer demonstrated that stimulation by foreign lymphocytes generates an alloresponse in ordinary lymphocytes that results in their ability to inhibit HIV replication in vitro.

The current study—comprising three experiments—seeks to characterize the alloresponse and identify the source of anti-HIV activity.

First, the team isolated CD8+ T-cells and stimulated them with foreign B-cells to establish whether the activated CD8 cells would inhibit HIV replication in the presence of HIV+ CD4 cells. This experiment, Nelson Perez noted, proved inconclusive because both the experimental and the control cultures came back negative for HIV.

The second experiment examined the surface markers of the CD8 cells before and after alloactivation. FACS analysis showed that CD27 and, to a lesser extent, CD45RA increased post-alloactivation, while CCR7 and HLA-DR decreased.

The third experiment, soon to be undertaken, will expose HIV-infected cells to each of these CD8 subpopulations separately to see to what extent each factor contributes to HIV inhibition

The ability eventually to identify correlates of alloimmunity with CD8 T-cell effector function may lead to the design of immunotherapies for transplant, cancer, and HIV patients.

Nelson Perez, who just graduated from medical school, says she’s been "bitten by the HIV research bug." She’s interested in pediatrics and public health. Burke, now applying to medical school, is drawn to pediatrics.

Vishal Sidhar

When a Pathway Meets a Pathway

Vishal Sidhar, New York Medical College. Characterization of a Clathrin-Independent Pathway in a Variety of Cell Types

Preceptors: Julie Donaldson and Roberto Weigert, Laboratory of Cell Biology, NHLBI

The transport of proteins and lipids to and from the plasma membrane is achieved through different cellular trafficking systems that are critical for interactions of all cells with their environment and hence for cell survival.

These transport pathways can be broadly classified as either clathrin-dependent or clathrin-independent, based on whether or not the transport vesicles are coated with the protein clathrin.

The clathrin-dependent endocytic pathway has been extensively studied in many cell types, whereas studies describing the clathrin-independent pathway are just emerging.

In HeLa cells, previous studies showed that these two different pathways can function independently of each other to transport distinct cargo but converge at the early endosomes compartment and that the GTPase Rab22a is involved in the recycling of the clathrin-independent cargo back to the membrane.

Sidhar sought to understand whether the convergence of these two pathways and the GTPases involved in the regulation of the clathrin-independent pathway are conserved in cell types other than the HeLa cell.

Using transferrin as a marker for the clathrin-dependent pathway and MHC class I molecules as a marker for the clathrin-independent pathway, Sidhar examined these pathways in human, canine, and monkey cell types by confocal microscopy.

There was very little overlap observed between transferrin and MHC I molecules in all cell types examined when fluorescently tagged forms of these markers were added to cells for short time periods, indicating that each marker was internalized by a distinct pathway.

However, not all the cell types showed significant overlap of these markers at later times, suggesting that these pathways may not converge in all cell types as was seen in the HeLa cell.

MHC I molecules were present in unique membrane tubules that extended out to the plasma membrane in all these cell types. Sidhar observed that the Rab22a GTPase was present in membrane tubules reminiscent of the MHC I localization pattern.

He further noted that when a constitutively active form of Rab22a (Rab22a-Q64L) was overexpressed in these cells, there was an increase in the number of long tubules, while overexpression of an inactive form of Rab22a (S19N) led to an absence of tubules.

This result suggested that Rab22a did indeed control the recycling pathway in these cell types similar to HeLa cells; it also implicated Rab22a function in the recycling of material internalized through the clathrin-independent pathway.

Sidhar hopes these preliminary findings will lead the way for future studies that will characterize the cellular machinery required for the clathrin-independent pathway to function in all cell types.

—Aarthi Ashok

Eric Brown

Preceptor: Roland Owens, Laboratory of Molecular and Cellular Biology, NIDDK

Adeno-associated virus, a nonpathogenic human parvovirus that is being evaluated for its utility as a gene therapy vector, encodes the Rep 78 protein, which plays a role in the regulation of viral replication, integration, and gene expression. The helicase function of Rep 78 may be important for Rep 78’s abilities to inhibit cell division and regulate oncogenic transformation.

Recent studies from the labs of Robert Kotin at NHLBI and others have shown that mutation of lysine at the 340 position (K340) in the helicase domain eliminated this inhibitory effect on cell division. Brown set about to test the hypothesis that other mutations in the helicase domain would also interfere with the inhibitory effect.

To this end, he designed a plasmid construct containing the Rep 78 open reading frame (ORF) under the control of the CMV promoter and carrying the neomycin resistance gene (neo).

Other constructs carried various mutations in the Rep 78 helicase domain or the neo gene alone as a positive control. All constructs were transfected into HeLa cells.

To the surprise of Brown and his colleagues, cells transfected with the normal Rep 78 ORF grew more efficiently than the neo-transfected controls—contrary to the findings of Kotin and others.

Brown speculates that differences in construct design might explain the discrepancy. "We believe that the integration of our construct into chromosome 19 may occur in such a way that the CMV promoter is lost, leading to enhanced expression of the neo gene but loss of Rep 78 protein expression," he says.

He hopes to examine the integration of both the neo and rep genes into the chromosome 19 locus to better understand these interesting findings. He’s also contemplating a career as a physician-scientist.

—Aarthi Ashok

Jacqueline Sequoia

Jacqueline Sequoia, San Diego State University Graduate School of Public Health. A Prospective Investigation of Height and Prostate Cancer Risk in Male Smokers

Preceptors: Demetrius Albanes and Margaret Wright, Nutritional Epidemiology Branch, NCI

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in men in the United States. Increased adult height, an indication of early nutrition and genetic predisposition, has been positively associated with prostate cancer risk in several studies, although somewhat inconsistently. To address some of the discrepancies, Sequoia and her colleagues studied the association of height and prostate cancer incidence in 29,119 Finnish male smokers, aged 50 to 69 years, enrolled in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study.

During up to 17 years of follow-up, 1,346 incident prostate cancers were identified. Results indicated that the taller men had up to 30 percent higher risk of prostate cancer, particularly of advanced-stage disease, than did shorter men.

Sequoia and her colleagues suggest that exposure to higher levels of growth hormone, insulin-like growth factors, androgens, and/or nutrition during puberty may increase both adult height and the risk of prostate cancer.

—Aarthi Ashok

Matthew McConnell

Matthew McConnell, Am-herst College. Secretion of HIV-Inhibitory Peptides by Commensal Yeast.

Preceptors: Dean Hamer and Kenneth Henry, Laboratory of Biochemistry, NCI-CCR

If the yeast that lives in the mucosal compartments of the human body could passively secrete HIV inhibitors, that would be an ounce of AIDS prevention more reliable than condom usage.

Such is the driving rationale behind a study to establish a yeast expression and secretion system for C-51, a peptide that inhibits the fusion step of HIV infection. This, McConnell says, is the "very first step" on the road to clinical manipulation of yeast that live commensally in the human body, especially in the gastrointestinal tract, where much of the early infection and viral replication of HIV take place.

The team succeeded in integrating C-51 into a yeast genome and inducing its expression and secretion; the peptide demonstrated active inhibition of both CCR5- and CXCR4-tropic HIV strains. Moreover, there was evidence that the secreted peptide was glycosylated, which could enhance stability, McConnell adds.

The team’s next step is to determine whether glycosylation improves stability and affinity of C-51 in vitro and in vivo; McConnell’s next step is to begin his third year of college and his work toward a degree in chemistry. Beyond that lie M.D. and Ph.D. degrees.

—Fran Pollner