Faculty Bibliography

Proteins encoded by the SRCR superfamily including gp340 recognize repeated patterns on pathogenic microorganisms and play important roles in innate immune defense as well as epithelial cell differentiation. Based upon the presence of SRCR domains in proteins with broad binding specificities and high amino acid sequence homology, it was speculated that SRCR domains may be involved in ligand binding. In this study, a truncated gp340 molecule representing the N-terminal sequence including the first SRCR and one-half of the first SID was expressed in mammalian 293 cells as a 35-kDa recombinant protein. The expressed protein was recognized by a panel of antibodies specific for human salivary agglutinin (SAG) and the full-length parental gp340 and exhibited biological properties similar to the entire 340-kDa glycoprotein. The truncated gp340 protein bound to the same HIV-1 V3 sequences previously identified to interact with full-length SAG in a Ca2+ -dependent manner. The recombinant N-terminal SRCR protein also demonstrated potent anti-HIV- 1 activity against both CCR5- and CXCR4-using isolates, similar to the full-length glycoprotein. We have, thus, demonstrated that the N-terminal SRCR of gp340 directly interacts with viral gp120 and likely mediates anti-HIV-1 activity via this interaction.

Saliva and other types of oral samples can readily be used for noninvasive diagnosis of diseases of the oral cavity and systemic diseases. Following an introduction outlining the types of oral samples and the analytes that can be measured in these samples, a detailed description of a novel oral-based diagnostic system to detect multiple bacterial and/or viral pathogens is presented. A reasonably priced, portable, point-of-care diagnostic system should be available within five years

C31G, which has potent activity against the human immunodeficiency virus type 1 (HIV-1) and an established record of safety in animal studies and human trials, is a microbicidal agent comprised of a buffered equimolar mixture of two amphoteric, surface-active agents: an alkyl amine oxide (C14AO) and an alkyl betaine (C16B). Studies of long-term in vitro exposure to C31G and its constituents have suggested that the components of C31G may contribute differentially to its toxicity and efficacy. In the present studies, in vitro assays of cytotoxicity and anti-HIV-1 activity demonstrated that C16B was slightly less cytotoxic compared to either C31G or C14AO, whereas the anti-HIV-1 activities of C31G and its individual constituents were similar. In the murine model of cervicovaginal microbicide toxicity, in vivo exposure to C14AO resulted in severe cervical inflammation followed by a delayed disruption of the columnar epithelium. In contrast, exposure to C16B caused severe cervical epithelial disruption and a secondary, less intense inflammatory response. These results demonstrate that (i) there are both mechanistic and temporal differences in toxicity associated with the components of C31G not necessarily predicted by in vitro assessments of cytotoxicity and (ii) contributions of each component to the anti-HIV-1 activity of C31G appear to be equal. In addition, these findings indicate that direct and indirect mechanisms of in vivo toxicity can be observed as separate but interrelated events. These results provide further insight into the activity of C31G, as well as mechanisms potentially associated with microbicide toxicity.

Comparative assays of in vitro cytotoxicity using nonoxynol-9 (N-9) and the candidate microbicides C31G and sodium dodecyl sulfate (SDS) demonstrated that these agents, which are, respectively, characterized as nonionic, amphoteric, and anionic surfactants, differed in their concentration-dependent effects on cell viability, especially after prolonged exposure. We hypothesized that differences in cellular sensitivity may have been due, in part, to cellular changes induced by long-term exposure to each agent. To examine this possibility, HeLa cells were exposed to N-9, C31G, or SDS for extended periods of time and subsequently reassessed for sensitivity to each of these agents. Following 10 continuous days of C31G exposure, HeLa cells were less sensitive to a subsequent C31G exposure compared to cells that had not undergone long-term C31G treatment. Interestingly, long-term C31G exposure also changed subsequent sensitivity to N-9 but not SDS. In contrast, prolonged exposure to either N-9 or SDS did not reduce sensitivity to re-exposure. The effect of long-term C31G exposure was both concentration-dependent and transient, as treated cells reverted to pre-exposure sensitivity in a time-dependent manner following the cessation of C31G exposure. Lipid analyses of cells exposed to C31G for extended durations revealed altered phospholipid profiles relative to C31G-naïve cells. Experiments examining the individual components of C31G demonstrated the involvement of the amine oxide moiety in reductions in cellular sensitivity. These studies, which provide new information concerning the cytotoxicity of surfactant microbicides, suggest that cervicovaginal epithelial cells may have greater in vivo tolerance for products containing C31G through unique interactions between C31G and components of the cellular membranes.

C31G (Savvy) has been developed as a topical vaginal microbicide with broad-spectrum antibacterial and antiviral properties. The objective of this study was to evaluate the distribution of a 1.0% concentration of (3.5 mL) C31G vaginal gel in the human pelvis using magnetic resonance imaging (MRI). Gel delivery with a standard applicator was primarily to the upper vagina and was well tolerated. Vaginal mucosal coverage at 18 min was excellent with 92% linear coverage and 75% surface contact coverage of the vagina. The upper vagina was almost completely covered and gel was also noted in the lower vagina. Coverage 6 h after application was substantially decreased, with 60% of maximal linear coverage and 41% surface contact. There was a very minimal coverage of the vaginal mucosa noted 24 h following insertion. Simulated intercourse resulted in relatively little change in overall distribution at all three time points. Repeat application of the gel may be necessary if intercourse has not occurred within the first few hours after initial insertion.

C31G is currently the focus of clinical trials designed to evaluate this agent as a microbicidal and spermicidal agent. In the following studies, the in vivo safety of C31G was assessed with a Swiss Webster mouse model of cervicovaginal toxicity and correlated with results from in vitro cytotoxicity experiments and published clinical observations. A single exposure of unformulated 1% C31G resulted in mild-to-moderate epithelial disruption and inflammation at 2 and 4 h postapplication. The columnar epithelium of the cervix was the primary site of damage, while no perturbation of the vaginal mucosa was observed. In contrast, application of unformulated 1.7% C31G resulted in greater levels of inflammation in the cervical epithelium at 2 h postapplication and severe epithelial disruption that persisted to 8 h postapplication. Application of a nonionic aqueous gel formulation containing 1% C31G resulted in no apparent cervicovaginal toxicity at any time point evaluated. However, formulation of 1.7% C31G did not substantially reduce the toxicity associated with unformulated C31G at that concentration. These observations correlate with findings gathered during a recent clinical trial, in which once-daily applications resulted in no adverse events in women receiving the formulation containing 1% C31G, compared to moderate-to-severe adverse events in 30% of women receiving the 1.7% C31G formulation. The Swiss Webster mouse model was able to effectively discriminate between concentrations and formulations of C31G that produced distinct clinical effects in human trials. The Swiss Webster animal model may be a highly valuable tool for preclinical evaluation of candidate vaginal microbicides.

Because it is a noninvasive technique, there is growing interest in replacing blood with oral-based methods of diagnostics. Oral diagnostics may be used for diagnosis and therapeutic drug monitoring of both oral diseases (eg, caries, periodontal disease,oral lesions, oral cancer) and systemic diseases (eg, infectious diseases, including HIV and AIDS, autoimmune diseases, cancer,and endocrine disorders). The authors address both existing techniques and oral-based diagnostics that will be applicable to the aging population in the future. They also highlight those techniques that are uniquely suited to point-of-care applications.