Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

HIV-associated disruption of mucosal epithelium facilitates paracellular penetration by human papillomavirus.

The incidence of human papillomavirus (HPV)-associated epithelial lesions is substantially higher in human immunodeficiency virus (HIV)-infected individuals than in HIV-uninfected individuals. The molecular mechanisms underlying the increased risk of HPV infection in HIV-infected individuals are poorly understood. We found that HIV proteins tat and gp120 were expressed within the oral and anal mucosal epithelial microenvironment of HIV-infected individuals. Expression of HIV proteins in the mucosal epithelium was correlated with the disruption of epithelial tight junctions (TJ). Treatment of polarized oral, cervical and anal epithelial cells, and oral tissue explants with tat and gp120 led to disruption of epithelial TJ and increased HPV pseudovirion (PsV) paracellular penetration in to the epithelium. PsV entry was observed in the basal/parabasal cells, the cells in which the HPV life cycle is initiated. Our data suggest that HIV-associated TJ disruption of mucosal epithelia may potentiate HPV infection and subsequent development of HPV-associated neoplasia.

Periodontal disease in HIV/AIDS.

Since the early 1990's, the death rate from AIDS among adults has declined in most developed countries, largely because of newer antiretroviral therapies and improved access to these therapies. In addition, from 2006 to 2011, the total number of new cases of HIV infection worldwide has declined somewhat and has remained relatively constant. Nevertheless, because of the large numbers of existing and new cases of HIV infection, the dental practitioner and other healthcare practitioners will still be required to treat oral and periodontal conditions unique to HIV/AIDS as well as conventional periodontal diseases in HIV-infected adults and children. The oral and periodontal conditions most closely associated with HIV infection include oral candidiasis, oral hairy leukoplakia, Kaposi's sarcoma, salivary gland diseases, oral warts, other oral viral infections, linear gingival erythema and necrotizing gingival and periodontal diseases. While the incidence and prevalence of these oral lesions and conditions appear to be declining, in part because of antiretroviral therapy, dental and healthcare practitioners will need to continue to diagnose and treat the more conventional periodontal diseases in these HIV-infected populations. Finding low-cost and easily accessible and acceptable diagnostic and treatment approaches for both the microbiological and the inflammatory aspects of periodontal diseases in these populations are of particular importance, as the systemic spread of the local microbiota and inflammatory products of periodontal diseases may have adverse effects on both the progression of HIV infection and the effectiveness of antiretroviral therapy approaches. Developing and assessing low-cost and accessible diagnostic and treatment approaches to periodontal diseases, particularly in developing countries, will require an internationally coordinated effort to design and conduct standardized clinical trials.

Differential transmission of HIV traversing fetal oral/intestinal epithelia and adult oral epithelia.

While human immunodeficiency virus (HIV) transmission through the adult oral route is rare, mother-to-child transmission (MTCT) through the neonatal/infant oral and/or gastrointestinal route is common. To study the mechanisms of cell-free and cell-associated HIV transmission across adult oral and neonatal/infant oral/intestinal epithelia, we established ex vivo organ tissue model systems of adult and fetal origin. Given the similarity of neonatal and fetal oral epithelia with respect to epithelial stratification and density of HIV-susceptible immune cells, we used fetal oral the epithelium as a model for neonatal/infant oral epithelium. We found that cell-free HIV traversed fetal oral and intestinal epithelia and infected HIV-susceptible CD4(+) T lymphocytes, Langerhans/dendritic cells, and macrophages. To study the penetration of cell-associated virus into fetal oral and intestinal epithelia, HIV-infected macrophages and lymphocytes were added to the surfaces of fetal oral and intestinal epithelia. HIV-infected macrophages, but not lymphocytes, transmigrated across fetal oral epithelia. HIV-infected macrophages and, to a lesser extent, lymphocytes transmigrated across fetal intestinal epithelia. In contrast to the fetal oral/intestinal epithelia, cell-free HIV transmigration through adult oral epithelia was inefficient and virions did not infect intraepithelial and subepithelial HIV-susceptible cells. In addition, HIV-infected macrophages and lymphocytes did not transmigrate through intact adult oral epithelia. Transmigration of cell-free and cell-associated HIV across the fetal oral/intestinal mucosal epithelium may serve as an initial mechanism for HIV MTCT.

HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells.

Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells - and not those that passed through the adult cells - remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission.

Epstein-Barr virus (EBV)-infected monocytes facilitate dissemination of EBV within the oral mucosal epithelium.

Epstein-Barr virus (EBV) causes hairy leukoplakia (HL), a benign lesion of oral epithelium that occurs primarily in the setting of human immunodeficiency virus (HIV)-associated immunodeficiency. However, the mechanisms of EBV infection of oral epithelium are poorly understood. Analysis of HL tissues shows a small number of EBV-positive intraepithelial macrophages and dendritic/Langerhans cells. To investigate a role for these cells in spreading EBV to epithelial cells, we used tongue and buccal explants infected ex vivo with EBV. We showed that EBV first infects submucosal CD14(+) monocytes, which then migrate into the epithelium and spread virus to oral epithelial cells, initiating productive viral infection within the terminally differentiated spinosum and granulosum layers. Incubation of EBV-infected monocytes and oral explants with antibodies to CCR2 receptor and monocyte chemotactic protein 1 prevented entry of monocytes into the epithelium and inhibited EBV infection of keratinocytes. B lymphocytes played little part in the spread of EBV to keratinocytes in our explant model. However, cocultivation of EBV-infected B lymphocytes with uninfected monocytes in vitro showed that EBV may spread from B lymphocytes to monocytes. Circulating EBV-positive monocytes were detected in most HIV-infected individuals, consistent with a model in which EBV may be spread from B lymphocytes to monocytes, which then enter the epithelium and initiate productive viral infection of keratinocytes.

Longitudinal evaluation of loss of attachment in HIV-infected women compared to HIV-uninfected women.

BACKGROUND: The Women's Interagency HIV Study (WIHS) is the largest, most detailed, controlled longitudinal collection of data to evaluate the influence of human immunodeficiency virus (HIV) disease and its therapies on the periodontium. METHODS: This report evaluates periodontal probing depth (PD), attachment loss (AL), and tooth loss from 584 HIV-seropositive and 151 HIV-seronegative women, recorded at 6-month intervals from 1995 to 2002. Using the random split-mouth method, PD and AL were recorded from four sites per tooth: mesial-buccal, buccal, distal-buccal, and lingual. Influence of viral load, CD4 count, race, smoking, drug use, low income, and level of education were evaluated. RESULTS: At baseline, AL was 1.6 versus 1.1 mm (P = 0.003) and PD was marginally deeper (2.1 versus 2.0 mm; P = 0.02) in HIV-seropositive versus HIV-seronegative women. Adjusted longitudinal analysis showed that HIV infection did not increase the mean PD (rate ratio [RR], 1.00; 95% confidence interval [CI], 0.96 to 1.04), worst PD (RR, 1.03; 95% CI, 0.98 to 1.09), mean AL (RR, 0.97; 95% CI, 0.96 to 1.02), worst AL (RR, 1.01; 95% CI, 0.94 to 1.07), or tooth loss (RR, 1.02; 95% CI, 1.0 to 1.05). CONCLUSIONS: CD4 count and viral load had no consistent effects on PD or AL. Among HIV-infected women, a 10-fold increase in viral load was associated with a marginal increase in tooth loss. The progression of periodontal disease measured by PD and AL did not significantly differ between HIV-infected and HIV-uninfected women. The HIV-seropositive women lost more teeth. Race, smoking, drug use, income, and education level did not influence the results for either group.

The effect of HAART on salivary microbiota in the Women's Interagency HIV Study (WIHS).

OBJECTIVE: Study the prevalence of potentially pathogenic microorganisms in saliva of HIV-positive women in the Women's Interagency HIV Study. STUDY DESIGN: 157 HIV-positive and 31 HIV-negative women were studied. At baseline and every 6 months over 4 years, information was collected on socioeconomic and educational status, oral and systemic health, including HIV markers and antiretroviral therapy, and frequency of professional oral care utilization. Bacterial and yeast pathogenic isolates from stimulated whole saliva were tentatively identified using standard methodologies. RESULTS: The prevalence of microorganisms in stimulated saliva of HIV-positive women was not significantly different from that of HIV-negative women. In HIV-positive women, highly active antiretroviral therapy (HAART) was independently and significantly associated with the presence of a variety of salivary bacterial species. HAART increased the risk for recovering Fusobacterium species (P < .001), enteric gram-negative rods (P < .05), Peptostreptococcus micros (P < .05), Campylobacter species (P < .0001), Eubacterium species (P < .001), and Tannerella forsythia (P < .01). In contrast, HAART led to decreased recovery rate of yeasts (Candida albicans and Candida dubliniensis) (P < .0001). CONCLUSION: The present findings suggest that the institution of HAART promotes an increasingly pathogenic salivary microbiota, at least temporarily. Similar findings have been reported for various nonoral microbial ecosystems.

Highly tissue substructure-specific effects of human papilloma virus in mucosa of HIV-infected patients revealed by laser-dissection microscopy-assisted gene expression profiling.

Human papilloma virus (HPV) causes focal infections of epithelial layers in skin and mucosa. HIV-infected patients on highly active antiretroviral therapy (HAART) appear to be at increased risk of developing HPV-induced oral warts. To identify the mechanisms that allow long-term infection of oral epithelial cells in these patients, we used a combination of laser-dissection microscopy (LDM) and highly sensitive and quantitative, non-biased, two-step multiplex real-time RT-PCR to study pathogen-induced alterations of specific tissue subcompartments. Expression of 166 genes was compared in three distinct epithelial and subepithelial compartments isolated from biopsies of normal mucosa from HIV-infected and non-infected patients and of HPV32-induced oral warts from HIV-infected patients. In contrast to the underlying HIV infection and/or HAART, which did not significantly elaborate tissue substructure-specific effects, changes in oral warts were strongly tissue substructure-specific. HPV 32 seems to establish infection by selectively enhancing epithelial cell growth and differentiation in the stratum spinosum and to evade the immune system by actively suppressing inflammatory responses in adjacent underlying tissues. With this highly sensitive and quantitative method tissue-specific expression of hundreds of genes can be studied simultaneously in a few cells. Because of its large dynamic measurement range it could also become a method of choice to confirm and better quantify results obtained by microarray analysis.

Self-care intervention to reduce oral candidiasis recurrences in HIV-seropositive persons: a pilot study.

OBJECTIVES: This single-blind randomized controlled pilot study evaluated the efficacy of a behavioral intervention program, PRO-SELF: Candidiasis, to reduce time to recurrence of oral candidiasis over 6 months in susceptible HIV-seropositive persons. The intervention involved instruction by dentists on improving oral hygiene, minimizing sugar intake, and self-diagnosing candidiasis. METHODS: Participants were adults with oral candidiasis responsive to antifungals who presented to the UCSF Stomatology Clinic between 1997 and 2000. At 2-3 weeks of follow-up visits, a dentist "examiner", masked to group assignment, quizzed participants as to the presence of candidiasis, and assessed candidiasis status. A second, unmasked dentist "instructor" then delivered the program to intervention participants. Participants recorded dietary and oral hygiene practices in 24-h recall diaries: intervention participants at each visit and controls at initial and final visits. RESULTS: At randomization, CD4+ cell counts (cells/mm(3)) were 298 +/- 188 among 18 intervention participants and 396 +/- 228 among 17 controls. The candidiasis recurrence rates at 6 months were 78% among intervention compared with 88% among control participants (hazard ratio 0.72; 95% CI 0.35-1.50). Performing oral hygiene after meals/snacks showed the largest relative improvement: intervention-control difference in proportion of meals/snacks affected was 24% (95% CI -1 to 48%). Self-diagnoses of candidiasis were inaccurate, possibly because of mild episodes. CONCLUSIONS: The results weakly indicate that regular instruction from healthcare professionals helps patients delay candidiasis recurrence by improving oral hygiene. Among HIV-seropositive persons, those with poor oral hygiene, and high-sugar diets are most likely to benefit.

Baseline characteristics of participants in the oral health component of the Women's Interagency HIV Study.

OBJECTIVES: This study described baseline sociodemographic and oral health characteristics of a subset of HIV sero-positive and sero-negative women who participated in the oral health component of the Women's Interagency HIV Study (WIHS). METHODS: In 1995-96, 584 HIV sero-positive and 151 sero-negative women from five WIHS core sites were enrolled in the oral study. Data on oral mucosa, salivary glands, dentition and periodontium, along with demographics, socioeconomics, and behavioral characteristics, were used to characterize this population. RESULTS: Mean (SD) age was 37 (8) years for HIV sero-positive and 36 (8) years for sero-negative women; 27% of sero-positive women had CD4 counts < or =200 and 34% had viral loads >50,000 copies/ml. Sero-positive and sero-negative women were similar demographically, as well as on plaque index, gingival bleeding, linear gingival banding, and numbers of DMF teeth and surfaces, but sero-positive women had more abnormal gingival papilla (P = 0.004) and fewer teeth (P = 0.01). Among sero-positive women, those with <200 CD4 counts had more DMF teeth (P = 0.007), and the number of DMF surfaces increased with decreasing CD4 counts (P = 0.04). Sero-positive women who fit the Center for Disease Control (CDC) AIDS criteria were also more likely to have more DMF teeth (P = 0.004), DMF surfaces (P = 0.003), and decayed and/or filled (DF) root surfaces (P = 0.0002) compared to sero-positive women without AIDS. CONCLUSIONS: Dental and periodontal variables showed little difference between HIV sero-positive and sero-negative women. Among sero-positive women, there were significant differences in coronal and root caries by AIDS diagnostic criteria, but no periodontal indicators by either AIDS diagnostic criteria or CD4 status, were observed.

Kaposi sarcoma-associated herpesvirus (KSHV) induces heme oxygenase-1 expression and activity in KSHV-infected endothelial cells.

Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study.

Differential expression of Candida albicans secreted aspartyl proteinase and phospholipase B genes in humans correlates with active oral and vaginal infections.

The in vivo expression of Candida albicans secreted aspartyl proteinase (SAP1-SAP8) and phospholipase B (PLB1 and PLB2) genes was analyzed in 137 human subjects with oral and vaginal candidiasis or carriage. Total RNA was isolated from whole unstimulated saliva or vaginal swabs, and the expression of SAP1-8 and PLB1-2 was evaluated by reverse-transcriptase polymerase chain reaction using specific primer sets. A spectrum of SAP gene expression profiles was obtained from different C. albicans strains during symptomatic disease and asymptomatic carriage. SAP2 and SAP5 were the most common genes expressed during both infection and carriage. SAP1, SAP3, SAP4, SAP7, SAP8, and PLB1 expression was correlated with oral disease, whereas SAP1, SAP3, and SAP6-SAP8 expression was correlated with vaginal disease. Furthermore, SAP1, SAP3, and SAP8 were preferentially expressed in vaginal, rather than oral, infections. This study demonstrates the differential expression of the hydrolytic enzyme genes in humans and correlates the expression of specific Candida species virulence genes with active disease and anatomical location.

A 4-year longitudinal evaluation of xerostomia and salivary gland hypofunction in the Women's Interagency HIV Study participants.

OBJECTIVES: Our purpose was to conduct a longitudinal investigation of xerostomia and salivary gland hypofunction in a national cohort of HIV-positive and at-risk HIV-negative participants in the Women's Interagency HIV Study. Study design. Data included responses to a dry mouth questionnaire, clinical evaluations of major salivary glands, and unstimulated and chewing-stimulated whole salivary flow rates. Repeated measures regression models were used to determine factors associated with xerostomia and salivary gland hypofunction. RESULTS: Significant univariate associations were found between HIV status and reports of "too little saliva" (P <.0001), < or = 0.1 mL/min, unstimulated saliva (P =.01), and lack of saliva upon palpation of parotid (P =.02) and submandibular/sublingual salivary glands (P =.03). Adjusted odds of reports of "too little saliva" were significantly higher for HIV-positive participants (odds ratio [OR] = 2.44; 95% CI, 1.49 - 3.97; P =.0004) than for HIV-negative participants. Among HIV-positive women, adjusted odds of reports of "too little saliva" and of < or = 0.7 mL/min chewing-stimulated saliva were significantly higher for those with CD4 < 200 (OR = 1.58; 95% CI, 1.07-2.34; P =.022; and OR = 1.53; 95% CI, 1.05-2.23; P =.027, respectively) and for those with CD4 200-500 (OR = 1.47; 95%CI, 1.07-2.02; P = 0.016; and OR = 1.37; 95% CI, 1.01-2.31; P =.001, respectively) than for those with CD4 > 500. Also, adjusted odds of < or = 0.1mL/min unstimulated saliva and < or = 0.7 mL/min chewing-stimulated saliva were significantly higher in women on highly active antiretroviral therapy (HAART) (OR = 1.25; 95% CI, 1.05 - 1.50; P =.014) than in women not on HAART (OR = 1.34; 95% CI, 1.01 - 1.79; P =.044). CONCLUSIONS: HIV-positive women are at a significantly higher risk for xerostomia and salivary gland hypofunction than HIV-negative women, and low CD4 cell counts and HAART use are significant risk factors for these conditions.

Radiation-induced mucositis: a randomized clinical trial of micronized sucralfate versus salt & soda mouthwashes.

Oral mucositis is one of the major toxicities caused by radiation therapy (RT) treatments to the head and neck. The clinical efficacy of sucralfate (Carafate R) mouthwash for head and neck cancer patients (HNC) is not consistent across studies. In this study, it was hypothesized that if the particles in the original sucralfate suspension were micronized (i.e., < or = 25 microns) then the coating action of the mouthwash in the oral cavity would be enhanced. The purpose of this pilot study was to compare the efficacy of micronized sucralfate (Carafate R) mouthwash and salt & soda mouthwash in terms of the severity of the mucositis, the severity of mucositis-related pain, and the time required to heal RT-induced mucositis in patients with HNC. Severe mucositis and related pain can interfere with the ingestion of food and fluids, so patients' body weights were measured as well. All patients in this randomized clinical trial carried out a systematic oral hygiene protocol called the PRO-SELF: Mouth Aware (PSMA) Program. Patients who developed RT-induced mucositis anytime during their course of RT were randomized to one of the two mouthwashes and followed to the completion of RT and at one month following RT. Two referral sites were used for the study. Repeated measures occurred with the following instruments/variables: MacDibbs Mouth Assessment and weight. Demographic, disease, and cancer treatment information was also obtained. Thirty patients successfully completed the study. The typical participant was male (70%), married/partnered (70%), White (63%), not working or retired (73%), and had an average of 14.5 years of education (SD = 3.7). T-tests and Chi-square analyses with an alpha set at 0.05 were used to compare differences between the two mouthwashes. No significant differences were found in the number of days to onset of mucositis (i.e., 16 +/- 8.4 days). When patients had their worst MacDibbs score, (i.e., the most severe mucositis), there were no significant differences between the mouthwashes as to MacDibbs score, the RT dose received, or ratings of pain (upon swallowing). Similarly, at the end of RT, no significant differences were found between mouthwashes as to MacDibbs scores or ratings of pain (upon swallowing). At the one-month follow-up assessment no significant differences were found between the mouthwashes in MacDibbs scores or pain ratings (upon swallowing). The analysis of the efficacy of the two mouthwashes revealed no significant differences in the time to heal (in days) from the RT-induced mucositis. The findings from this trial provide important clinical information regarding cost analysis of RT mucositis management. Given that there is no significant difference in efficacy between micronized sucralfate and salt & soda, use of the less costly salt & soda is prudent and cost-effective.

Risk of HIV infection attributable to oral sex among men who have sex with men and in the population of men who have sex with men.

We examined HIV infection and estimated the population-attributable risk percentage (PAR%) for HIV associated fellatio among men who have sex with other men (MSM). Among 239 MSM who practised exclusively fellatio in the past 6 months, 50% had three partners, 98% unprotected; and 28% had an HIV-positive partner; no HIV was detected. PAR%, based on the number of fellatio partners, ranges from 0.10% for one partner to 0.31% for three partners. The risk of HIV attributable to fellatio is extremely low.