Human immunodeficiency virus type 1 stimulates the expression and production of secretory leukocyte protease inhibitor (SLPI) in oral epithelial cells: a role for SLPI in innate mucosal immunity.

The innate immune response is a key barrier against pathogenic microorganisms such as human immunodeficiency virus type 1 (HIV-1). Because HIV-1 is rarely transmitted orally, we hypothesized that oral epithelial cells participate in the innate immune defense against this virus. We further hypothesized that secretory leukocyte protease inhibitor (SLPI), a 12-kDa mucosal antiviral protein, is a component of the host immune response to this virus. Here we demonstrated constitutive expression and production of SLPI in immortalized human oral keratinocytes. Brief exposure of cells to HIV-1 BaL and HXB2 significantly increased SLPI mRNA and protein production compared to that in mock-exposed cells (P < 0.01), as evaluated by real-time quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. HIV-1-mediated stimulation of SLPI occurred at the transcriptional level, was dose and time dependent, was elicited by heat-inactivated and infectious viruses, and did not depend on cellular infection. Experiments with purified retroviral proteins showed that the stimulatory effect was induced specifically by external envelope glycoproteins from HIV-1 and simian immunodeficiency virus. SLPI responsiveness to HIV-1 was also observed in an unrelated oral epithelial cell line and in normal (nonimmortalized) human oral epithelial cells isolated from healthy uninfected gingival tissues. In this first report of SLPI regulation by HIV-1, we show that the expression and production of the antimicrobial and anti-inflammatory protein can be stimulated in oral epithelial cells by the virus through interactions with gp120 in the absence of direct infection. These findings indicate that SLPI is a component of the oral mucosal response to HIV-1.

Saliva and inhibition of HIV-1 infection: molecular mechanisms.

Oral fluids are rarely a vehicle for HIV-1 infection in vivo, unlike other mucosal secretions. This unique property raises questions regarding (1) the molecular mechanisms responsible for the lack of salivary transmission, (2) the extent to which oral immunological responses mirror responses at other mucosal sites, (3) the use of promising salivary markers of HIV-1 disease progression, (4) the relationship between oral and blood viral loads, (5) cofactors that influence oro-genital transmission, and (6) the feasibility of oral-based antibody testing for HIV-1 diagnosis in the home. This paper discusses these questions and provides background summaries, findings from new studies, consensus opinions, practical relevance to developing countries, and suggestions for future research agenda on each of the key topics.

Salivary concentration of secretory leukocyte protease inhibitor, an antimicrobial protein, is decreased with advanced age.

BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) exhibits antimicrobial activities that, in addition to other well-characterized proteins such as lysozyme and lactoferrin, is thought to play a critical role in mucosal defenses. Although elderly individuals are particularly susceptible to mucosal infections, salivary production of SLPI has not been assessed in an aged cohort. OBJECTIVES: Hypothesizing that oral SLPI concentrations are reduced with advanced age, this cross-sectional study compared SLPI concentrations to concentrations of lysozyme, lactoferrin and total protein in unstimulated salivary secretions of healthy, community-dwelling 79+-year-old and younger adults. METHODS: Study participants were 45 non-hospitalized dentate adults aged 79-89 (23 elderly) or 21-51 years (22 non-elderly). Home-based interviews and clinical examinations determined dentate status and confirmed the absence of dentures, oral mucosal disease, anti-infective medication use, irradiation therapy for head and neck cancer and self-perceived xerostomia. Whole unstimulated saliva was collected from all subjects and analyzed for antimicrobial protein concentration by enzyme-linked immunosorbent assay and for total protein content by the bicinchoninic acid method. Bivariate and multivariate (generalized linear modeling) analyses evaluated the relationships between age, gender and salivary protein concentrations. RESULTS: Mean salivary levels of SLPI and lysozyme were lower in elderly compared with non-elderly subjects (p < 0.001), unlike lactoferrin and total protein levels. Similar results were obtained when concentrations of the individual proteins were normalized to the total protein concentration, suggesting that glandular production of SLPI and lysozyme preferentially decreases with aging. Gender differences were detected only for SLPI concentrations; males had lower SLPI levels than females regardless of age (p < 0.01). Generalized linear models confirmed that age (p < 0.001) and gender (p < 0.05) were each associated with the SLPI concentration and together accounted for 50% of the variation in SLPI concentration in this population. CONCLUSION: These findings indicate that SLPI production is diminished among healthy community-dwelling older adults, particularly elderly males. Further investigation should determine the impact of decreased local SLPI production on the increased risk of oral mucosal disease with advanced age.

HIV in body fluids during primary HIV infection: implications for pathogenesis, treatment and public health.

OBJECTIVE: To describe initial viral dissemination to peripheral tissues and infectious body fluids during human primary HIV infection. DESIGN: Observational cohort study. METHODS: Blood plasma, cerebrospinal fluid (CSF), seminal plasma, cervicovaginal lavage fluid and/or saliva were sampled from 17 individuals with primary HIV infection (range of time from symptoms onset to sampling, 8--70 days) and one individual with early infection (168 days). Subjects' HIV-1 RNA levels in each fluid were compared with levels from antiretroviral-naive controls with established HIV infection. For study subjects, correlations were assessed between HIV-1 RNA levels and time from symptoms onset. Responses to antiretroviral therapy with didanosine + stavudine + nevirapine +/- hydroxyurea were assessed in each compartment. RESULTS: HIV-1 RNA levels were highest closest to symptoms onset in blood plasma (18 patients) and saliva (11 patients). CSF HIV-1 RNA levels (five patients) appeared lower closer to symptoms onset, although they were higher overall in primary versus established infection. Shedding into seminal plasma (eight patients) and cervicovaginal fluid (two patients) was established at levels observed in chronic infection within 3--5 weeks of symptoms onset. High-level seminal plasma shedding was associated with coinfection with other sexually transmitted pathogens. Virus replication was suppressed in all compartments by antiretroviral therapy. CONCLUSIONS: Peak level HIV replication is established in blood, oropharyngeal tissues and genital tract, but potentially not in CSF, by the time patients are commonly diagnosed with primary HIV infection. Antiretroviral therapy is unlikely to limit initial virus spread to most tissue compartments, but may control genital tract shedding and central nervous system expansion in primary infection.

Hyper-excretion of human immunodeficiency virus type 1 RNA in saliva.

Anatomical compartments (e.g., the reproductive tract) are reservoirs of human immunodeficiency virus type-1 (HIV-1) and potential sites of residual infection in patients receiving anti-retroviral therapy (ART). Viral hyper-excretion relative to blood is a hallmark of reservoirs. To determine whether hyper-excretion can occur in the oral cavity, we compared viral loads in blood plasma and saliva of 67 adults. Salivary viral hyperexcretion was defined as a four-fold or higher viral load in saliva than in plasma. HIV-1 RNA was detected in 79% of plasma samples, in 44% of unfiltered saliva samples, in 16% of filtered saliva samples, and in 59% of saliva-derived cell pellets. Compared with non-hyper-excretors (n = 62), hyper-excretors (n = 5) had elevated levels of viral RNA in unfiltered saliva and saliva-derived cells, HIV-associated periodontal disease, gingival inflammation, and no combination ART. Morphological characterization of cell pellets identified lymphocytes as a likely HIV-1 source. These collective findings are consistent with an oral HIV-1 reservoir in selected individuals.

Characterization of human immunodeficiency virus type 1 in saliva and blood plasma by V3-specific heteroduplex tracking assay and genotype analyses.

The gp120 V3-encoding region of human immunodeficiency virus type 1 (HIV-1) RNA derived from the saliva and blood plasma of 11 individuals was characterized by heteroduplex tracking assay and sequence analyses. R5-like viral variants were identified in both fluids of all subjects. X4-like variants were detected in the plasma and/or saliva of three subjects, indicating that X4-like variants are not excluded from the saliva compartment. Viral subpopulations were similar in both fluids of most subjects, suggesting that HIV-1 in oral fluids and blood may stem from a common source. These findings raise the possibility of using saliva as a noninvasive fluid for evaluating and monitoring viral evolution in infected persons.

Limits on oral transmission of HIV-1.

PIP: This article discusses the potential of acquiring an HIV-1 infection through an oral route, with a view of offering clues for its prevention. In a study of adult animals given low concentration cell-free simian immunodeficiency virus (SIV) orally, histological examination suggested that SIV infected lymphoid tissue through the antigen-transporting crypt epithelium rather than through dendritic cells. The investigators found no evidence of acquiring SIV via the gastrointestinal tract. For humans, HIV transmission from saliva or intimate family contact seems to be extremely rare. This could be because of the low concentration of HIV-1 in saliva. A study of 40 people found that significantly less HIV was found in salivary secretions than in plasma. Another possible explanation for inefficient oral transmission might be that HIV-1 in the oropharynx is inhibited by components found in salivary secretions. Conversely, studies have noted that risk of oral transmission of HIV from contaminated breast milk and semen is higher than from saliva. Breast-feeding by an HIV-infected woman puts the baby at substantial risk of infection and receptive fellatio cannot be considered a safe sex act.^ieng

Oral and systemic factors associated with increased levels of human immunodeficiency virus type 1 RNA in saliva.

OBJECTIVE: The purpose of this investigation was to quantify human immunodeficiency virus type-1 (HIV-1) RNA in saliva and plasma and identify factors associated with increased salivary viral load. STUDY DESIGN: Forty HIV-1-seropositive adults underwent oral examinations to assess mucosal and periodontal health. Whole saliva was evaluated for HIV-1 RNA titer and occult blood. Plasma viral load, CD4 cell count, HIV-1 staging, and antiretroviral therapy data were obtained from medical records. Associations between salivary titers and oral/systemic parameters were analyzed by means of t tests, Wilcoxon signed rank tests, Pearson's correlation coefficient, and analysis of covariance. RESULTS: Forty-two percent of the subjects had detectable salivary HIV-1 RNA. Oral titers were highly correlated with plasma viral levels (r = 0.51, P <.01). HIV-associated periodontal disease (in particular, linear gingival erythema), severe gingival inflammation, and absence of antiretroviral therapy were associated with high salivary titers (P <.01). CONCLUSIONS: Substantial quantities of HIV-1 can be shed in the oral cavity, particularly when inflammatory conditions are present. Salivary titer may be a useful indicator of systemic viral burden.

Quantitation of human immunodeficiency virus type 1 RNA in different biological compartments.

Little information is available describing viral loads in body fluids other than blood. In addition, the suitability of commercially available assays for human immunodeficiency virus type 1 (HIV-1) RNA quantitation has not been evaluated in most nonblood fluids. We compared Organon Teknika's nucleic acid sequence-based amplification method (NASBA) and Roche's Amplicor HIV-1 Monitor (reverse transcriptase PCR [RT-PCR]) for quantitating HIV-1 RNA in cerebrospinal fluid (CSF), saliva, breast milk, seminal plasma, and cervical-vaginal lavage fluid (CVL). Saliva and breast milk frequently demonstrated some inhibition in the RT-PCR assay, similar to the inhibition previously described in seminal plasma. Inhibition of the RT-PCR assay was not observed with CSF or CVL, nor in any of the NASBA assays. When fluids from HIV-infected individuals were tested by RT-PCR and NASBA, 73 and 27% of CSF samples and 60 and 40% of breast milk specimens had detectable RNA, respectively. These differences were not statistically significant. In cross-sectional studies using RT-PCR to measure viral RNA in paired blood plasma and CSF samples, 71% of blood plasma samples and 42% of CSF samples were positive. A similar analysis using NASBA with paired blood plasma and CVL, saliva, or seminal plasma samples revealed 91% were blood plasma positive and 55% were CVL positive, 76% were blood plasma positive and 46% were saliva positive, and 83% were blood plasma positive and 63% were seminal plasma positive. NASBA worked fairly well to quantitate HIV-1 RNA from all fluids without apparent inhibition. RT-PCR performed well on CVL and CSF, frequently with greater sensitivity, although its use in other fluids appears limited due to the presence of inhibitors. These studies demonstrate that viral loads in nonblood fluids were generally lower than in blood.

Immunologic and viral markers of HIV-1 disease progression: implications for dentistry.

BACKGROUND: Current medical care for patients infected with the human immunodeficiency virus type 1, or HIV-1, involves monitoring laboratory assays for CD4+ lymphocyte cell count and plasma viral load. TYPES OF STUDIES REVIEWED: The authors reviewed recent medical and dental studies that contribute to our current understanding of these immunologic and viral markers and their relevance to systemic and oral health. RESULTS: Dramatic reduction in plasma viral load resulting from more potent antiretroviral drug combinations is the goal of medical management for HIV. These protease inhibitor-containing regimens, although complex, expensive and associated with substantial side effects, have decreased the morbidity and mortality associated with HIV in the United States. Although reduction in viral load can result in increases in CD4+ counts, which restores some level of immune competence, a cure for AIDS has not yet been found. CLINICAL IMPLICATIONS: Patients with low CD4+ cell counts (and often high viral loads) are more likely to develop destructive periodontal infections and other oral manifestations of HIV. Partial recovery of the immune system after viral load reduction may affect both the prognosis and oral disease experience of these patients.

Endogenous salivary inhibitors of human immunodeficiency virus.

The human immunodeficiency virus type 1 (HIV-1) is rarely transmitted through salivary secretions, due in part to the presence of endogenous inhibitors. Here, the protective characteristics of the intraoral environment are summarized and inhibitory factors that reduce HIV-1 infectivity in vitro described, focusing on secretory leucocyte protease inhibitor (SLPI), a 12-kDa mucosal protein that blocks HIV infection in several cell-culture systems. SLPI appears to interact with a cellular surface molecule to limit viral entry into target cells. To determine whether the inhibitor has a similar role in vivo, the contribution of salivary SLPI to anti-HIV-1 activity was assessed. Whole unstimulated filtered salivas from infected and uninfected donors contained similar concentrations of the inhibitor. Depletion from SLPI filtered saliva produced a corresponding loss of inhibitory activity. In general, filtered whole salivas obtained from 10 donors had antiviral activities that correlated positively with SLPI concentrations. However, some samples having SLPI well below the concentration required for inhibitory activity in vitro exhibited modest inhibition, suggesting the presence of other anti-HIV-1 components in oral fluids. Thus, SLPI is a major but not sole inhibitor of this virus in saliva.

Endogenous mucosal antiviral factors of the oral cavity.

The oral cavity represents a unique site for mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Unlike other mucosal sites, the oral cavity is rarely a site of HIV transmission despite detectable virus in saliva and oropharyngeal tissues of infected persons. One reason for this apparent paradox is the presence of endogenous mucosal antiviral factors. Innate inhibitory molecules, such as virus-specific antibodies, mucins, thrombospondin, and soluble proteins, have been identified and partially characterized from saliva. A recent addition to the growing list is secretory leukocyte protease inhibitor (SLPI), an approximately 12-kDa non-glycosylated protein found in serous secretions. Physiologic concentrations of SLPI potently protect adherent monocytes and activated peripheral blood mononuclear cells against HIV-1 infection. SLPI levels in saliva and semen but not breast milk approximate levels required for inhibition in vitro. Characterization of SLPI and other endogenous antiviral molecules may enhance our understanding of factors influencing mucosal HIV-1 transmission.

The role of the oral environment in HIV-1 transmission.

Human immunodeficiency virus type 1, or HIV-1, is infrequently transmitted through the mouth, unlike other mucosal sites. Factors such as low salivary viral titers, low numbers of CD4-positive target cells, anti-HIV antibodies and endogenous salivary antiviral factors work in concert to protect oral tissues from infection and reduce the risk of viral transmission through salivary secretions. This review summarizes the various factors thought to influence oral transmission HIV-1, focusing on the mucosal protein secretory leukocyte protease inhibitor, or SLPI.

Cobalamin inhibition of HIV-1 integrase and integration of HIV-1 DNA into cellular DNA.

Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibited in vitro by hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50 values of approximately 17, 17, 17, and 4 microM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50 values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 microM, respectively) and of cultured lymphocytes (IC50 values of 60, 50, 60, and 11 microM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase.

Determinants of human immunodeficiency virus type 1 resistance to gp41-derived inhibitory peptides.

A synthetic peptide, DP178, containing amino acids 127 to 162 of the human immunodeficiency virus type 1 (HIV-1) gp41 Env glycoprotein, is a potent inhibitor of virus infection and virus mediated cell-to-cell fusion (C. Wild, T. Greenwell, and T. Matthews, AIDS Res. Hum. Retroviruses 9:1051-1053, 1993). In an effort to understand the mechanism of action of this peptide, we derived resistant variants of HIV-1(IIIB) and NL4-3 by serial virus passage in the presence of increasing doses of the peptide. Sequence analysis of the resistant isolates suggested that a contiguous 3-amino-acid sequence within the amino-terminal heptad repeat motif of gp41 was associated with resistance. Site-directed mutagenesis studies confirmed this observation and indicated that changes in two of these three residues were necessary for development of the resistant phenotype. Direct binding of DP178 to recombinant protein and synthetic peptide analogs containing the wild-type and mutant heptad repeat sequences revealed a strong correlation between DP178 binding and the biological sensitivity of the corresponding virus isolates to DP178. The results are discussed from the standpoints of the mechanism of action of DP178 and recent crystallographic information for a core structure of the gp41 ectodomain.

Inhibition of human immunodeficiency virus type 1 infectivity by secretory leukocyte protease inhibitor occurs prior to viral reverse transcription.

Infection of monocytes with human immunodeficiency virus type 1(Ba-L) (HIV-1(Ba-L)) is significantly inhibited by treatment with the serine protease inhibitor, secretory leukocyte protease inhibitor (SLPI). SLPI does not appear to act on virus directly, but rather the inhibitory activity is most likely due to interaction with the host cell. The current study was initiated to investigate how SLPI interacts with monocytes to inhibit infection. SLPI was found to bind to monocytes with high affinity to a single class of receptor sites (approximately 7,000 receptors per monocyte, K(D) = 3.6 nmol/L). The putative SLPI receptor was identified as a surface protein with a molecular weight of 55 +/- 5 kD. A well-characterized function of SLPI is inhibition of neutrophil elastase and cathepsin G. However, two SLPI mutants (or muteins) that contain single amino acid substitutions and exhibit greatly reduced protease inhibitory activity still bound to monocytes and retained anti-HIV-1 activity. SLPI consists of two domains, of which the C-terminal domain contains the protease inhibiting region. However, when tested independently, neither domain had potent anti-HIV-1 activity. SLPI binding neither prevented virus binding to monocytes nor attenuated the infectivity of any virus progeny that escaped inhibition by SLPI. A polymerase chain reaction (PCR)-based assay for newly generated viral DNA demonstrated that SLPI blocks at or before viral DNA synthesis. Therefore, it most likely inhibits a step of viral infection that occurs after virus binding but before reverse transcription. Taken together, the unique antiviral activity of SLPI, which may be independent of its previously characterized antiprotease activity, appears to reside in disruption of the viral infection process soon after virus binding.

Detecting, diagnosing, and preventing oral cancer.

Unlike many other malignancies, cancers of the mouth and surrounding tissues continue to cause considerable mortality and morbidity in this country. Therefore, early detection, diagnosis, and treatment of patients with oral cancer must be a high priority for all health care providers. This review is aimed at heightening the awareness of clinicians to the early signs and symptoms of oral cancer. Recognition of early lesions is crucial for improved long-term patient survival. Factors such as advanced age, tobacco and/or alcohol use, chronic sum exposure, and a previous diagnosis of cancer can alert clinicians to patients who may be at risk for developing oral cancer. Because most oral malignancies are asymptomatic and may mimic benign conditions, any suspicious lesion should be carefully examined and, if appropriate, referred immediately for histological examination. Measures such as annual oral cancer screening examinations and patient education that stress early signs and symptoms of oral cancer can also help to reduce the risk in high-risk individuals.

Secretory leukocyte protease inhibitor blocks infectivity of primary monocytes and mononuclear cells with both monocytotropic and lymphocytotropic strains of human immunodeficiency virus type I.

Saliva contains factors that inhibit infection with the human immunodeficiency virus type 1 (HIV-1) in vitro. One of these factors was recently identified as secretory leukocyte protease inhibitor (SLPI), a salivary protein which blocked HIV-1 infectivity of monocytes and primary T cells at physiologic concentrations (J Clin Invest 1995; 96: 456). Here, we confirm and extend the original report by demonstrating that SLPI protects primary monocytes and peripheral blood mononuclear cells against infection with HIV-1 Ba-L, IIIB and NL4-3. Thus, SLPI may provide a natural barrier against oral transmission of HIV-1.

Biophysical characterization of recombinant proteins expressing the leucine zipper-like domain of the human immunodeficiency virus type 1 transmembrane protein gp41.

Envelope oligomerization is thought to serve several crucial functions during the life cycle of human immunodeficiency virus type 1 (HIV-1). We recently reported that virus entry requires coiled-coil formation of the leucine zipper-like domain of the HIV-1 transmembrane envelope glycoprotein gp41 (C. Wild, T. Oas, C. McDanal, D. Bolognesi, and T. Matthews, Proc. Natl. Acad. Sci. USA 89:10537-10541, 1992; C. Wild, J. W. Dubay, T. Greenwell, T. Baird, Jr., T. G. Oas, C. McDanal, E. Hunter, and T. Matthews, Proc. Natl. Acad. Sci. USA 91:12676-12680, 1994). To determine the oligomeric state mediated by this region of the envelope, we have expressed the zipper motif as a fusion partner with the monomeric maltose-binding protein of Escherichia coli. The biophysical properties of this protein were characterized by velocity and equilibrium sedimentation, size exclusion chromatography, light scattering, and chemical cross-linking analyses. Results indicate that the leucine zipper sequence from HIV-1 is capable of multimerizing much larger and otherwise monomeric proteins into extremely stable tetramers. Recombinant proteins containing an alanine or a serine substitution at a critical isoleucine residue within the zipper region were also generated and similarly analyzed. The alanine- and serine-substituted proteins behaved as tetrameric and monomeric species, respectively, consistent with the influence of these same substitutions on the helical coiled-coil structure of synthetic peptide models. On the basis of these findings, we propose that the fusogenic gp4l structure involves tetramerization of the leucine zipper domain which is situated approximately 30 residues from the N-terminal fusion peptide sequence.

Inhibition of productive human immunodeficiency virus-1 infection by cobalamins.

Various cobalamins act as important enzyme cofactors and modulate cellular function. We investigated cobalamins for their abilities to modify productive human immunodeficiency virus-1 (HIV-1) infection of hematopoietic cells in vitro. We show that hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), and adenosylcobalamin Ado-Cbl (Ado-Cbl) inhibit HIV-1 infection of normal human blood monocytes and lymphocytes. The inhibitory effects were noted when analyzing the monocytotropic strains HIV-1-BaL and HIV-1-ADA as well as the lymphocytotropic strain HIV-1-LAI. Cobalamins did not modify binding of gp120 to CD4 or block early steps in viral life cycle, inhibit reverse transcriptase, inhibit induction of HIV-1 expression from cells with established or latent infection, or modify monocyte interferon-alpha production. Because of the ability to achieve high blood and tissue levels of cobalamins in vivo and the general lack of toxicity, cobalamins should be considered as potentially useful agents for the treatment of HIV-1 infection.