Faculty Bibliography

Oral samples, including saliva, offer an attractive alternative to serum or urine for diagnostic testing. This is particularly true for point-of-use detection systems. The various types of oral samples that have been reported in the literature are presented here along with the wide variety of analytes that have been measured in saliva and other oral samples. The paper focuses on utilizing point-detection of infectious disease agents, and presents work from our group on a rapid test for multiple bacterial and viral pathogens by monitoring a series of targets. It is thus possible in a single oral sample to identify multiple pathogens based on specific antigens, nucleic acids, and host antibodies to those pathogens. The value of such a technology for detecting agents of bioterrorism at remote sites is discussed.

During sexual transmission of HIV in women, the first cells likely to be infected are submucosal CD4(+) T cells and dendritic cells of the lower genital tract. HIV is segregated from these target cells by an epithelial cell layer that can be bypassed even when healthy and intact. To understand how HIV penetrates this barrier, we identified a host protein, gp340, that is expressed on genital epithelium and binds the HIV envelope via a specific protein-protein interaction. This binding allows otherwise subinfectious amounts of HIV to efficiently infect target cells and allows this infection to occur over a longer period of time after binding. Our findings suggest a mechanism of viral entry during heterosexual transmission where HIV is bound to intact genital epithelia, which then promotes the initial events of infection. Understanding this step in the initiation of infection will allow for the development of tools and methods for blocking HIV transmission

Low-penetrance breast cancer susceptibility alleles seem to play a significant role in breast cancer risk but are difficult to identify in human cohorts. A genetic screen of 176 N2 backcross progeny of two Trp53(+/-) strains, BALB/c and C57BL/6, which differ in their susceptibility to mammary tumors, identified a modifier of mammary tumor susceptibility in an approximately 25-Mb interval on mouse chromosome 7 (designated SuprMam1). Relative to heterozygotes, homozygosity for BALB/c alleles of SuprMam1 significantly decreased mammary tumor latency from 70.7 to 61.1 weeks and increased risk twofold (P = 0.002). Dmbt1 (deleted in malignant brain tumors 1) was identified as a candidate modifier gene within the SuprMam1 interval because it was differentially expressed in mammary tissues from BALB/c-Trp53(+/-) and C57BL/6-Trp53(+/-) mice. Dmbt1 mRNA and protein was reduced in mammary glands of the susceptible BALB/c mice. Immunohistochemical staining demonstrated that DMBT1 protein expression was also significantly reduced in normal breast tissue from women with breast cancer (staining score, 1.8; n = 46) compared with cancer-free controls (staining score, 3.9; n = 53; P < 0.0001). These experiments demonstrate the use of Trp53(+/-) mice as a sensitized background to screen for low-penetrance modifiers of cancer. The results identify a novel mammary tumor susceptibility locus in mice and support a role for DMBT1 in suppression of mammary tumors in both mice and women.

A novel assay is described for multiplex detection of antibodies against different pathogens from a single sample. The assay employs a modified lateral flow format (consecutive flow, CF) together with a sensitive reporter particle technology (up-converting phosphor technology, UPT) that allows for fully instrumented assay analysis. Lateral flow (LF) strips developed for the detection of human antibodies against human immunodeficiency virus type-1 and -2 (HIV-1 and -2) with additional capture zones to detect antibodies against Myobacterium tuberculosis (TB) and hepatitis C Virus (HCV) provided the strips to test multiplexing. Data are presented that show the performance of the TB and HCV test, as well as two multiplex assays, TB with HIV and HCV with HIV. The TB/HCV assays demonstrate excellent detection capability, and HIV multiplexing does not affect the qualitative test result. The bench-top CF format was converted to a microfluidic platform and a first prototype semiautomated chip capable of performing CF is presented here

A 'lab-on-a-chip' system for detecting bacterial pathogens in oral fluid samples is described. The system comprises: (1) an oral fluid sample collector; (2) a disposable, plastic microfluidic cassette ('chip') for sample processing including immunochromatographic assay with a nitrocellulose lateral flow strip; (3) a platform that controls the cassette operation by providing metered quantities of reagents, temperature regulation, valve actuation; and (4) a laser scanner to interrogate the lateral flow strip. The microfluidic chip hosts a fluidic network for cell lysis, nucleic acid extraction and isolation, PCR, and labeling of the PCR product with bioconjugated, upconverting phosphor particles for detection on the lateral flow strip

Confirmatory detection of diseases, such as HIV and HIV-associated pathogens in a rapid point-of-care (POC) diagnostic remains a goal for disease control, prevention, and therapy. If a sample could be analyzed onsite with a verified result, the individual could be counseled immediately and appropriate therapy initiated. Our group is focused on developing a microfluidic 'lab-on-a-chip' that will simultaneously identify antigens, antibodies, RNA, and DNA using a single oral sample. The approach has been to design individual modules for each assay that uses similar components (e.g., valves, heaters, metering chambers, mixers) installed on a polycarbonate base with a common reporter system. Assay miniaturization reduces the overall analysis time, increases accuracy by simultaneously identifying multiple targets, and enhances detector sensitivity by upconverting phosphor technology (UPT). Our microfluidic approach employs four interrelated components: (1) sample acquisition-OraSure UPlink collectors that pick-up and release bacteria, soluble analytes, and viruses from an oral sample; (2) microfluidic processing-movement of microliter volumes of analyte, target analyte extraction and amplification; (3) detection of analytes using UPT particles in a lateral flow system; and (4) software for processing the results. Ultimately, the oral-based microscale diagnostic system will detect viruses and bacteria, associated pathogen antigens and nucleic acids, and antibodies to these pathogens

Recent studies indicate that mucosal innate immune factors modulate HIV-1 infection in vitro. Our interest was to examine the levels of innate mucosal factors for their potential association with HIV-1 shedding in the female genital tract. Vaginal lavages were collected from HIV-1-infected women who had vaginal viral loads (VVL) that were below, within, or above the 90% confidence interval (CI) predicted by their matched plasma viral loads. Innate immune factors [cathepsin D, lactoferrin (Lf), myeloid related protein (MRP)-8, MRP-8/14, secretory leukocyte protease inhibitor, and gp340], cytokines (IL-1beta and TNF-alpha), and chemokines (MIP-1alpha, MIP-1beta, RANTES, and SDF-1alpha) were quantified by ELISA. Leukocyte levels were determined using a leukocyte reagent strip for urinalysis. Lf, MRP-8/14, gp340, and IL-1beta levels were significantly higher in vaginal lavages above the 90% CI and generally correlated with each other and with VVL. Leukocyte levels were significantly higher in the lavages that had virus shedding above the 90% CI and correlated strongly with Lf levels and VVL. In this group of women, these results suggest that the levels of certain innate immune factors are more closely associated with HIV-1 shedding in the genital mucosa than plasma virus concentrations.