Faculty Bibliography

Studies have shown that the transmission of HIV is most likely to occur via rectal or vaginal routes, and rarely through oral exposure. However, the mechanisms of virus entry at mucosal surfaces remain incompletely understood. Prophylactic strategies against HIV infection may be attainable once gaps in current knowledge are filled. To address these gaps, we evaluated essentially normal epithelial surfaces and mapped the periluminal distribution of CD4+ HIV target cells, including T cells and antigen-presenting cells, and an HIV-binding molecule gp340 that can be expressed by epithelial cells in secreted and cell-associated forms. Immunohistochemistry for CD4, CD16, CD3, CD1a and gp340 in human oral, rectal/sigmoid and cervical mucosal samples from HIV-negative subjects demonstrated that periluminal HIV target cells were more prevalent at rectal/sigmoid and endocervical surfaces lined by simple columnar epithelium, than at oral and ectocervical surfaces covered by multilayered stratified squamous epithelium (p<0.001). gp340 expression patterns at these sites were also distinct and strong in oral minor salivary gland acini and ducts, including ductal saliva, in individual rectum/sigmoid and endocervix periluminar columnar cells, and in ectocervix squamous cells. Only weak expression was noted in the oral non-ductal squamous epithelium. We conclude that periluminal HIV target cells, together with periluminal epithelial cell-associated gp340 appear to be most accessible for HIV transmission at rectal/sigmoid and endocervical surfaces. Our data help define vulnerable structural features of mucosal sites exposed to HIV.

The field of "salivary diagnostics" includes studies utilizing samples obtained from a variety of sources within the oral cavity. These samples include; whole unstimulated saliva, stimulated whole saliva, duct saliva collected directly from the parotid, submandibular/sublingual glands or minor salivary glands, swabs of the buccal mucosa, tongue or tonsils, and gingival crevicular fluid. Many publications state "we collected saliva from subjects" without fully describing the process or source of the oral fluid. Factors that need to be documented in any study include the time of day of the collection, the method used to stimulate and collect the fluid, and how much fluid is being collected and for how long. The handling of the oral fluid during and post-collection is also critical and may include addition of protease or nuclease inhibitors, centrifugation, and cold or frozen storage prior to assay. In an effort to create a standard protocol for determining a biomarker's origin we carried out a pilot study collecting oral fluid from 5 different sites in the mouth and monitoring the concentrations of pro-and anti-inflammatory cytokines detected using MesoScaleDiscovery (MSD) electrochemiluminesence assays. Our data suggested that 3 of the cytokines are primarily derived from the submandibular gland, while 7 of the cytokines come from a source other than the major salivary glands such as the minor salivary glands or cells in the oral mucosae. Here we review the literature on monitoring biomarkers in oral samples and stress the need for determining the blood/saliva ratio when a quantitative determination is needed and suggest that the term oral diagnostic be used if the source of an analyte in the oral cavity is unknown.

Limited information is available about the effect of human immunodeficiency virus (HIV) and subsequent antiretroviral treatment on host-microbe interaction. This study aimed to determine the salivary microbial composition in 10 HIV-seropositive subjects, before and 6 months after highly active antiretroviral therapy (HAART), compared with that of 10 HIV-seronegative subjects. Both a conventional culture and two culture-independent analyses were used and consistently demonstrated differences in microbial composition among the three sets of samples. HIV+ subjects had higher levels of total cultivable microbes, including oral streptococci, lactobacilli, S. mutans, and Candida, in saliva as compared to HIV- subjects. The total cultivable microbial level was significantly correlated with CD4+ T cell counts. Denaturing gradient gel electrophoresis (DGGE), which compared the overall microbial profiles, showed distinct fingerprinting profiles for each group. Human oral microbe identification microarray (HOMIM), which compared the 16S rRNA genes, showed a clear separation among the three sample groups. Veillonella, Synergistetes, and Streptococcus, were present in all 30 saliva samples. Only minor changes or no changes were observed in the prevalence of Neisseria, Haemophilus, Gemella, Leptotrichia, Solobacterium, Parvimonas and RothiaI. Severn genera were detected only in HIV- samples, including Capnocytophaga, Slackia, Porphyromonas, Kingella, Peptostreptococcaceae, Lactobacillus, and Atopobium. The prevalence of Fusobacterium, Campylobacter, Prevotella, Capnocytophaga, Selenomonas, Actinomyces, and Atopobium was increased after therapy with HAART. In contrast, the prevalence of Aggregatibacter was significantly decreased after HAART. Findings of this study suggest that HIV infection and therapy with HAART could have a significant effect on salivary microbial colonization and composition.

INTRODUCTION: The impaired host defense system in HIV infection impacts the oral and gastrointestinal microbiota and associated opportunistic infections. Antiretroviral treatment is predicted to partially restore host defenses and decrease the oral manifestation of HIV/AIDS. Well-designed longitudinal studies are needed to better understand the interactions of soluble host defense proteins with bacteria and virus in HIV/AIDS. "Crosstalk" was designed as a longitudinal study of host responses along the gastrointestinal (GI) tract and interactions between defense molecules and bacteria in HIV infection and subsequent therapy. PURPOSE: The clinical core formed the infrastructure for the study of the interactions between the proteome, microbiome and innate immune system. The core recruited and retained study subjects, scheduled visits, obtained demographic and medical data, assessed oral health status, collected samples, and guided analysis of the hypotheses. This manuscript presents a well-designed clinical core that may serve as a model for studies that combine clinical and laboratory data. METHODS: Crosstalk was a case-control longitudinal clinical study an initial planned enrollment of 170 subjects. HIV+ antiretroviral naive subjects were followed for 9 visits over 96 weeks and HIV uninfected subjects for 3 visits over 24 weeks. Clinical prevalence of oral mucosal lesions, dental caries and periodontal disease were assessed. RESULTS: During the study, 116 subjects (47 HIV+, 69 HIV-) were enrolled. Cohorts of HIV+ and HIV- were demographically similar except for a larger proportion of women in the HIV- group. The most prevalent oral mucosal lesions were oral candidiasis and hairy leukoplakia in the HIV+ group. DISCUSSION: The clinical core was essential to enable the links between clinical and laboratory data. The study aims to determine specific differences between oral and GI tissues that account for unique patterns of opportunistic infections and to delineate the differences in their susceptibility to infection by HIV and their responses post-HAART.

A rapid test for an infectious disease that can be used at point-of-care at a physician's office, a pharmacy, or in the field is critical for the prompt and appropriate therapeutic intervention. Ultimately by treating infections early on will decrease transmission of the pathogen. In contrast to metabolic diseases or cancer where multiple biomarkers are required, infectious disease targets (e. g. antigen, antibody, nucleic acid) are simple and specific for the pathogen causing the disease. Our laboratory has focused on three major infectious disease; HIV, Tuberculosis, and Malaria. These diseases are pandemic in much of the world thus putting natives, tourists and military personnel at risk for becoming infected, and upon returning to the U.S., transmitting these diseases to their contacts. Our devices are designed to detect antigens, antibodies or nucleic acids in blood or saliva samples in less than 30 minutes. An overview describing the current status of each of the three diagnostic platforms is presented. These microfluidic point-of-care devices will be relatively inexpensive, disposable, and user friendly.

The amphoteric C31G solution contains equimolar alkyl dimethlyglycine and alkyl dimethyl amine oxide buffered with citric acid. C31G acts as a broad spectrum antiviral and an antibacterial. No previous in vivo studies have been done to test C31G in an animal model of HSV-1 ocular keratitis. We assessed the anti-herpetic activity of C31G in the rabbit eye model using three treatment groups: (1) 1% trifluorothymidine (TFT); (2) 0.25% C31G plus 0.5% hydroxypropyl methylcellulose (HPMC); and (3) vehicle, 0.5% HPMC. Scarified rabbit corneas were inoculated with the HSV-1 strain McKrae. On post inoculation (PI) day 3, rabbits were placed in three balanced groups based on slit-lamp examination (SLE) scores. Treatment began on PI day 3, five times a day for five consecutive days. In addition to the daily, masked SLE scoring, the eyes were assessed daily for stromal opacity, scleral inflammation, neovascularization, eyelid inflammation, inflammatory discharge, and epiphora. C31G and TFT were very effective in reducing the lesions and pathogenesis associated with HSV-1 ocular keratitis. The vehicle control scores were significantly higher and did not effectively treat HSV-1 keratitis. C31G has the potential to be used to treat herpetic keratitis as well as other herpetic topical lesions in humans.

gp340 is a member of the scavenger receptor cysteine-rich family of innate immune molecules and also functions as a tumor suppressor. This study describes a picogram level assay using electrochemiluminescent technology on the MesoScale Discovery (MSD) platform. Antibodies were evaluated and the best pair used to assay whole mouth stimulated saliva (WMSS) and cervical vaginal lavage (CVL). The assay was tested using specimens obtained from healthy volunteers in order to determine if gp340 concentration in saliva correlates with levels in vaginal lavage fluid. Interestingly, no correlation was determined between gp340 content in these two fluids.

In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.

BACKGROUND: A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. METHODS: A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. RESULTS: This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. CONCLUSIONS: The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples.