Faculty Bibliography

The interaction and mixing between a bilayer sample of mucus and vaginal formulation was evaluated through viscosity measurements with respect to time and shear. Physical mixtures of mucus and vaginal formulation were used as controls. Three test protocols were designed: (1) constant shear, (2) intermittent shear, and (3) delayed shear. Several marketed vaginal products (Gynol II, KY Plus, KY, and Advantage-S) and experimental formulations (C31G with hydroxyethylcellulose [HEC]) were evaluated and compared by these tests. The results of the constant shear test showed that the shear stress profile of the bilayer approached that of the corresponding physical mixture, consistent with complete mixing of the bilayer under shear. The time taken for the bilayer to mix completely was in the following order: KY Plus > Gynol II and C31G > KY > Advantage-S. Under the intermittent shear protocol, the following order for complete mixing was observed: KY Plus > C31G > Gynol II > KY > Advantage-S. The 2 products evaluated by the delayed shear test, C31G and Gynol II, were both completely mixed at 180 minutes. The development of an in vitro test, when coupled with in vivo data, should serve in the screening and evaluation of future vaginal formulations.

Three-dimensional imaging of the human vagina demonstrates that the cross section can be a "W," rather than an "H," and that intravaginal gel can ascend into the endocervix and presumably into the endometrium.

A microbicide is designed to coat the vaginal epithelium and prevent transmission of HIV. Complete coverage is desired for optimal protection. In vivo factors affecting coverage have not yet been studied. This randomized crossover trial evaluates the effect of gel volume and patient activity upon vaginal epithelial coating. Gynol II gel was mixed with a magnetic resonance imaging (MRI) contrast agent. Ten women self-inserted, on separate visits, 3 or 5 mL of gel and underwent serial MRI scanning both before and after simulated intercourse. Gel spread was dependent upon time and volume. There was modest spread during the first hour and greater spread 6 h after insertion. Five milliliters of gel resulted in statistically significantly greater coverage immediately following insertion, within the first 30 min and at 6 h after insertion. Simulated intercourse greatly enhances gel spread. After simulated intercourse, the distribution of the gel at each volume was similar. Less leakage of gel was reported with the smaller volume.

A self-actuated, flow-cycling polymerase chain reaction (PCR) reactor that takes advantage of buoyancy forces to continuously circulate reagents in a closed loop through various thermal zones has been constructed, tested, and modeled. The heating required for the PCR is advantageously used to induce fluid motion without the need for a pump. Flow velocities on the order of millimeters per second are readily attainable. In our preliminary prototype, we measured a cross-sectionally averaged velocity of 2.5 mm/s and a cycle time of 104 s. The flow velocity is nearly independent of the loop's length, making the device readily scalable. Successful amplifications of 700- and 305-bp fragments of Bacillus cereus genomic DNA have been demonstrated. Since the device does not require any moving parts, it is particularly suitable for miniature systems

Orally based diagnostic testing is emerging as an alternative, noninvasive method for analyzing a variety of analytes. These analytes include pathogens, antibodies, drugs, and nucleic acids. In the present study we developed a protocol for evaluation of collectors that could be used in orally based, point-of-care diagnostics. A performance comparison was carried out with a number of commercially available collectors, and their ability to deliver fluid, proteins, bacteria, and nucleic acid from pathogens compatible with PCR was assessed. The collectors were all capable of picking up and delivering test materials, albeit at various levels

Human saliva contains multiple components that inhibit HIV-1 infection in vitro, which may contribute to low oral HIV-1 transmission. Salivary agglutinin (SAG) is a high-molecular-weight glycoprotein encoded by DMBT-1 and identical to gp340, a member of the lung scavange receptor, cysteine-rich receptor family. gp340 binds to surfactants A and D, which is believed to function in the clearance of microorganisms from the lung, as part of the innate immune response. Previously we reported that SAG (gp340) specifically inhibits HIV-1 infection with broad activity against diverse HIV-1 isolates. This gp340 inhibitory activity is mediated by binding to viral gp120 and involves a region different from the CD4-binding site on gp120. Here, we report that the gp340-binding region is localized to a linear, highly conserved sequence near the stem of the V3 loop that is critical for chemokine receptor interaction during viral binding and infection. The interaction of gp340 with gp120 is enhanced by prebinding of sCD4 to gp120, suggesting that gp340 inhibitory activity is mediated by blocking access of the gp120 to the chemokine receptor

Salivary agglutinin (SAG) is a high molecular mass glycoprotein (340 kDa) that plays important roles in innate immunity. SAG has been found to specifically inhibit HIV-1 infectivity and to bind to virus through the envelope protein gp120. Although SAG binds to gp120 of the virus, the exact nature of this binding has not been characterized. Using surface plasmon resonance technology, we have found that SAG interacts with recombinant envelopes derived from diverse HIV-1 isolates with K(D) values ranging from 10(-7) to 10(-10) M, comparable to gp120-sCD4 binding. Furthermore, SAG binding to gp120 is Ca(2+) dependent. sCD4 prebound to gp120 failed to abrogate SAG binding, suggesting a distinct mechanism for SAG inhibition of HIV-1 infectivity. Inhibition by monoclonal antibodies specific for carbohydrates also implicates the involvement of carbohydrates in the interaction between SAG and gp120. These results argue that the anti-HIV-1 activity of SAG is due to carbohydrate-mediated binding to gp120. A demonstration that SAG is related to lung scavenger receptor, gp-340, further suggests the roles of SAG in preventing pathogen invasion at the entry portal and raises its potential as an anti-HIV-1 drug candidate

In experiments to assess the in vitro impact of the candidate microbicides nonoxynol 9 (N-9), C31G, and sodium dodecyl sulfate (SDS) on human immune and epithelial cell viability, cell lines and primary cell populations of lymphocytic and monocytic origin were generally shown to be equally sensitive to exposures ranging from 10 min to 48 h. However, U-937 cells were more sensitive to N-9 and C31G after 48 h than were primary monocyte-derived macrophages. Cytokine activation of monocytes and lymphocytes had no effect on cell viability following exposure to these microbicidal compounds. Primary and passaged vaginal epithelial cultures and cell lines differed in sensitivity to N-9 and C31G but not SDS. These studies provide a foundation for in vitro experiments in which cell lines of human immune and epithelial origin can be used as suitable surrogates for primary cells to further investigate the effects of microbicides on cell metabolism, membrane composition, and integrity and the effects of cell type, proliferation, and differentiation on microbicide sensitivity.