The complexity of oral physiology and its impact on salivary diagnostics.

OBJECTIVES: Saliva contains biomarkers for systemic as well as oral diseases. This study was undertaken to assess the variability in the sources of such biomarkers (plasma, cells) and attempted to identify saliva deterioration markers in order to improve saliva diagnostic outcomes. MATERIALS AND METHODS: Inter- and intrasubject variations in salivary gingival crevicular fluid levels were determined by measuring salivary albumin and transferrin levels. The purity of collected glandular secretions was determined by bacterial culture, and the variability in epithelial cell numbers by cell counting and optical density measurement. Saliva sample deterioration markers were identified by RP-HPLC and LC-ESI-MS/MS. RESULTS: Tenfold variations were observed in plasma-derived albumin and transferrin levels, emphasizing the need for biomarker normalization with respect to plasma contributions to saliva. Epithelial cell levels varied 50-fold in samples collected before and after a meal. Salivary fungal levels varied within subjects and among subjects from 0 to >1,000 colony-forming units per milliliter. In saliva samples incubated for various time intervals at 37°C, five peptides were identified that steadily increased in intensity over time and which could be explored as "deterioration markers." CONCLUSION: Taking saliva characteristics appropriately into account will help realize the promise that this body fluid is suitable to be exploited for reliable healthcare monitoring and surveillance.

Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction.

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na+] and [Cl-] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

Microbial Diversity in the Early In Vivo-Formed Dental Biofilm.

Although the mature dental biofilm composition is well studied, there is very little information on the earliest phase of in vivo tooth colonization. Progress in dental biofilm collection methodologies and techniques of large-scale microbial identification have made new studies in this field of oral biology feasible. The aim of this study was to characterize the temporal changes and diversity of the cultivable and noncultivable microbes in the early dental biofilm. Samples of early dental biofilm were collected from 11 healthy subjects at 0, 2, 4, and 6 h after removal of plaque and pellicle from tooth surfaces. With the semiquantitative Human Oral Microbiome Identification Microarray (HOMIM) technique, which is based on 16S rRNA sequence hybridizations, plaque samples were analyzed with the currently available 407 HOMIM microbial probes. This led to the identification of at least 92 species, with streptococci being the most abundant bacteria across all time points in all subjects. High-frequency detection was also made with Haemophilus parainfluenzae, Gemella haemolysans, Slackia exigua, and Rothia species. Abundance changes over time were noted for Streptococcus anginosus and Streptococcus intermedius (P = 0.02), Streptococcus mitis bv. 2 (P = 0.0002), Streptococcus oralis (P = 0.0002), Streptococcus cluster I (P = 0.003), G. haemolysans (P = 0.0005), and Stenotrophomonas maltophilia (P = 0.02). Among the currently uncultivable microbiota, eight phylotypes were detected in the early stages of biofilm formation, one belonging to the candidate bacterial division TM7, which has attracted attention due to its potential association with periodontal disease.

Proteome data set of human gingival crevicular fluid from healthy periodontium sites by multidimensional protein separation and mass spectrometry.

BACKGROUND AND OBJECTIVE: Gingival crevicular fluid has been of major interest for many decades as a valuable body fluid that may serve as a source of biomarkers for both periodontal and systemic diseases. Owing to its very small sample size, submicroliter volumes, identification of its protein composition by classical biochemical methods has been limited. The advent of highly sensitive mass spectrometric technology has permitted large-scale identification of protein components of many biological samples. This technology has been employed to identify the protein composition of gingival crevicular fluid from inflamed and periodontal sites. In this report, we present a proteome data set of gingival crevicular fluid from healthy periodontium sites. MATERIAL AND METHODS: A combination of a periopaper collection method with application of multidimensional protein separation and mass spectrometric technology led to a large-scale documentation of the proteome of gingival crevicular fluid from healthy periodontium sites. RESULTS: The approaches used have culminated in identification of 199 proteins in gingival crevicular fluid of periodontally healthy sites. The present gingival crevicular fluid proteome from healthy sites was compared and contrasted with those proteomes of gingival crevicular fluid from inflamed and periodontal sites, as well as serum. The cross-correlation of the gingival crevicular fluid and plasma proteomes permitted dissociation of the 199 identified gingival crevicular fluid proteins into 105 proteins (57%) that can be identified in plasma and 94 proteins (43%) that are distinct and unique to the gingival crevicular fluid microenvironment. Such analysis also revealed distinctions in protein functional categories between serum proteins and those specific to the gingival crevicular fluid microenvironment. CONCLUSION: Firstly, the data presented herein provide the proteome of gingival crevicular fluid from periodontally healthy sites through establishment of innovative analytical approaches for effective analysis of gingival crevicular fluid from periopapers both at the level of complete elusion and with removal of abundant albumin, which restricts identification of low-abundant proteins. Secondly, it adds significantly to the knowledge of gingival crevicular fluid composition and highlights new groups of proteins specific to the gingival crevicular fluid microenvironment.

Genetic polymorphism of proline-rich human salivary proteins.

In randomly collected saliva samples from 120 Caucasians, 79 Blacks, and 40 Chinese, three phenotypes were observed by electrophoresis in alkaline slab polyacrylamide gels. The proteins showing polymorphism were identical with four previously characterized proline-rich proteins. Inheritance is controlled by two autosomal codominant alleles. The gene frequencies were for Caucasians, Pr(l)=0.73, Pr(2)=0.27; for Blacks, Pr(1)=0.80, Pr(2)=0.20; for Chinese, Pr(l)=0.84, Pr(2)=0.16.

Multiple components contribute to ability of saliva to inhibit influenza viruses.

INTRODUCTION: Saliva is a potentially important barrier against respiratory viral infection but its mechanism of action is not well studied. METHODS: We tested the antiviral activities of whole saliva, specific salivary gland secretions, and purified salivary proteins against strains of influenza A virus (IAV) in vitro. RESULTS: Whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibited IAV based on hemagglutination inhibition and neutralization assays. This differs from human immunodeficiency virus (HIV), for which only submandibular/sublingual secretions are reported to be inhibitory. Among purified salivary proteins, MUC5B, scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), histatins, and human neutrophil defensins (HNPs) inhibited IAV at the concentrations present in whole saliva. In contrast, some abundant salivary proteins (acidic proline-rich proteins and amylase) had no activity, nor did several other less abundant salivary proteins with known activity against HIV (e.g. thrombospondin or serum leukocyte protease inhibitor). Whole saliva and MUC5B did not inhibit neuraminidase activity of IAV and viral neutralizing and aggregating activity of MUC5B was potentiated by the neuraminidase inhibitor oseltamivir. Hence, MUC5B inhibits IAV by presenting a sialic acid ligand for the viral hemagglutinin. The mechanism of action of histatins requires further study. CONCLUSIONS: These findings indicate that saliva represents an important initial barrier to IAV infection and underline the complexity of host defense activity of oral secretions. Of interest, antiviral activity of saliva against IAV and HIV differs in terms of specific glandular secretions and proteins that are inhibitory.

Proteome of human minor salivary gland secretion.

Recent research efforts in oral biology have resulted in elucidation of the proteomes of major human salivary secretions and whole saliva. One might hypothesize that the proteome of minor gland secretions may show significantly different characteristics when compared with the proteomes of parotid or submandibular/sublingual secretions. To test this hypothesis, we conducted the first exploration into the proteome of minor salivary gland secretion. Minor gland secretion was obtained from healthy volunteers, and its components were subjected to liquid-chromatography-electrospray-ionization-tandem-mass-spectrometry. This led to the identification of 56 proteins, 12 of which had never been identified in any salivary secretion. The unique characteristics of the minor salivary gland secretion proteome are related to the types as well as the numbers of components present. The differences between salivary proteomes may be important with respect to specific oral functions.

Acquired enamel pellicle and its potential role in oral diagnostics.

The acquired enamel pellicle (AEP) is a protein film with unique composition and properties, which is formed by the selective adsorption of a variety of oral fluid-derived proteins onto tooth enamel surfaces. Since events leading to caries and periodontal disease occur in close proximity to the tooth surface, pellicle constituents are likely to contain biomarkers valuable for diagnostic applications. Despite the importance of this oral structure, progress in understanding its formation and composition has been slow because of difficulties in efficient pellicle collection methods and limitations of biochemical techniques for the characterization of microgram amounts of proteins/peptides. Recent developments in both pellicle collection methods and nanoscale sensing technologies have brought the exploitation of pellicle analysis into the realm of point-of-care oral diagnostics.

Saliva: a dynamic proteome.

The proteome of whole saliva, in contrast to that of serum, is highly susceptible to a variety of physiological and biochemical processes. First, salivary protein secretion is under neurologic control, with protein output being dependent on the stimulus. Second, extensive salivary protein modifications occur in the oral environment, where a plethora of host- and bacteria-derived enzymes act on proteins emanating from the glandular ducts. Salivary protein biosynthesis starts with the transcription and translation of salivary protein genes in the glands, followed by post-translational processing involving protein glycosylation, phosphorylation, and proteolysis. This gives rise to salivary proteins occurring in families, consisting of structurally closely related family members. Once glandular secretions enter the non-sterile oral environment, proteins are subjected to additional and continuous protein modifications, leading to extensive proteolytic cleavage, partial deglycosylation, and protein-protein complex formation. All these protein modifications occur in a dynamic environment dictated by the continuous supply of newly synthesized proteins and removal by swallowing. Understanding the proteome of whole saliva in an environment of continuous turnover will be a prerequisite to gain insight into the physiological and pathological processes relevant to oral health, and be crucial for the identification of meaningful biomarkers for oral disease.

Saliva and Serum Protein Exchange at the Tooth Enamel Surface.

The acquired enamel pellicle is an oral, fluid-derived protein layer that forms on the tooth surface. It is a biologically and clinically important integument that protects teeth against enamel demineralization, and abrasion. Tooth surfaces are exposed to different proteinaceous microenvironments depending on the enamel location. For instance, tooth surfaces close to the gingival sulcus contact serum proteins that emanate via this sulcus, which may impact pellicle composition locally. The aims of this study were to define the major salivary and serum components that adsorb to hydroxyapatite, to study competition among them, and to obtain preliminary evidence in an in vivo saliva/serum pellicle model. Hydroxyapatite powder was incubated with saliva and serum, and the proteins that adsorbed were identified by mass spectrometry. To study competition, saliva and serum proteins were labeled with CyDyes, mixed in various proportions, and incubated with hydroxyapatite. In vivo competition was assessed using a split-mouth design, with half the buccal tooth surfaces coated with serum and the other half with saliva. After exposure to the oral environment for 0 min, 30 min and 2 h, the pellicles were analyzed by SDS-PAGE. In pure saliva- or serum-derived pellicles, 82 and 84 proteins were identified, respectively. When present concomitantly, salivary protein adsorbers effectively competed with serum protein adsorbers for the hydroxyapatite surface. Specifically, acidic proline-rich protein, cystatin, statherin and protein S100-A9 proteins competed off apolipoproteins, complement C4-A, haptoglobin, transthyretin and serotransferrin. In vivo evidence further supported the replacement of serum proteins by salivary proteins. In conclusion, although significant numbers of serum proteins emanate from the gingival sulcus, their ability to participate in dental pellicle formation is likely reduced in the presence of strong salivary protein adsorbers. The functional properties of the acquired enamel pellicle will therefore be mostly dictated by the salivary component.

Oral fluid proteolytic effects on histatin 5 structure and function.

Histatins are human salivary antifungal proteins that are prone to extensive enzymatic degradation upon their release into the oral cavity. Histatin proteolysis, leading to the disappearance of the intact protein can be expected to have functional consequences. Histatin 5, comprising 24 residues, is the smallest of the major salivary histatins and the most active in terms of its antifungal properties. The rate and mode of histatin 5 degradation were determined by incubating the protein in whole saliva supernatant for various time intervals. Fragmentation products were collected by reversed-phase high performance liquid chromatography (RP-HPLC), characterised structurally by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and functionally in a fungal growth inhibition assay. Of the 19 fragments identified, 16 were derived from single proteolytic cleavage events in histatin 5. A remarkable finding was the inter-subject consistency in the histatin 5 degradation pattern. Added histatin 5 disappeared from whole saliva supernatant at an average rate of 105+/-22 microg/ml/h, which in part could explain the virtual absence of histatin 5 in whole saliva. Despite the rapid proteolysis of histatin 5, the early degradation mixture was as active in antifungal assays as intact histatin 5. These data demonstrate that the oral-fluid mediated proteolysis of histatin 5 represents an intrinsic biological property of whole saliva. The data also reveal that the early proteolysis phase of histatin 5 does not abolish the antifungal properties associated with this protein.

Multi-component adsorption model for pellicle formation: the influence of salivary proteins and non-salivary phospho proteins on the binding of histatin 5 onto hydroxyapatite.

The acquired enamel pellicle formed by selective adsorption of proteins in whole saliva is a protective integument on the tooth surface. The purpose of the present study was to investigate the formation of human acquired enamel pellicle using an in vitro hydroxyapatite (HA) model and 3H-histatin 5 to allow accurate measurement of histatin 5 binding in a multi-component experimental system. A binary system was employed by mixing 3H-histatin 5 with one unlabeled protein prior to incubation with HA or by first incubating 3H-histatin 5 with the HA which had been pre-coated with one of a panel of unlabeled proteins (human albumin, salivary amylase, lysozyme, acidic PIFs, statherin, the N-terminal fragment of statherin, and egg yolk phosvitin). A ternary system was employed by mixing 3H-histatin 5 with HA sequentially pre-coated with two different unlabeled proteins, including recombinant histatin 1. The results showed that only salivary statherin and egg yolk phosvitin promote histatin 5 adsorption significantly. The amount of histatin 5 adsorbed was also found to increase as a function of the amount of phosvitin and statherin used to pre-coat HA up to a maximum level that was two- to four-fold greater than that observed on untreated HA. These data suggest that specific protein-protein interactions may play important roles in pellicle formation in vivo.

Immunological comparison of proline-rich proteins from human and primate parotid secretion.

Antisera raised in response to proline-rich proteins purified from parotid secretions of man and the primate Macaca fascicularis were employed to investigate the interrelationships of these proteins by immunodiffusion, immunoelectrophoresis and the combined use of disc gel acrylamide electrophoresis with radial immunodiffusion. The major human proline-rich proteins, PRP I, PRP II, PRP III and PRP IV as well as several minor proline-rich proteins cross-react with antiserum to PRP I or PRP III. Similarly primate parotid saliva contains several components cross-reacting with antiserum directed against a purified primate proline-rich protein, MPRP. Antiserum to PRP I or PRP III cross-reacted with MPRP and primate parotid saliva protein, whereas antiserum to MPRP cross-reacted only with human parotid saliva protein and not with the isolated human proline-rich proteins. The immunological relationships of these salivary proline-rich proteins within and between species suggest their origin from a common precursor molecule.

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.

Transfer of a gene encoding the anticandidal protein histatin 3 to salivary glands.

Mucosal candidiasis, the most common opportunistic fungal infection in human immunodeficiency virus (HIV)-infected patients, is an early sign of clinically overt acquired immunodeficiency syndrome (AIDS) and an important cause of morbidity, particularly in HIV-infected children. The appearance of azole-resistant strains of Candida albicans had made clinical management of candidiasis increasingly difficult. We propose a novel approach to the management of candidal infections that involves the use of naturally occurring antifungal proteins, such as the histatins. Histatins are a family of small proteins that are secreted in human saliva. We have constructed recombinant adenovirus vectors that contain the histatin 3 cDNA. These vectors are capable of directing the expression of histatin 3 in the saliva of rats at up to 1,045 micrograms/ml, well above the levels found in normal human saliva. The adenovirus-directed histatin demonstrated a 90% candidacidal effect in the timed-kill assay against both fluconazole-susceptible and fluconazole-resistant strains of C. albicans and inhibited germination by 45% in the same strains. These studies suggest that a gene transfer approach to overexpress naturally occurring antifungal proteins may be useful in the management of mucosal candidiasis.

Recombinant histatins: functional domain duplication enhances candidacidal activity.

Histatin 3 (Hst3) is a 32-amino-acid (aa) His-rich protein with antimicrobial activity found in human salivary secretions. To explore further the structure/function relationship of Hst, we utilized a bacterial system for the efficient production of recombinant Hst3 (re-Hst3) and Hst variants. Previously, we demonstrated that the middle portion of Hst3 (aa 13-24) contains the functional domain responsible for killing Candida albicans. Using PCR and splice overlap extension, a Hst variant (re-Hst3rep) was made in which the functional domain was repeated in tandem. Using the pRSET bacterial expression system, re-Hst3 and the variant re-Hst3rep were produced as chimeric fusions and were isolated from bacterial sonicates by affinity chromatography. Affinity purified fusion proteins were digested with CNBr and re-Hst were separated from their fusion partners by reverse-phase high-performance liquid chromatography. The activity of re-Hst3 and re-Hst3rep was compared to that of native Hst3 from human salivary secretions in the C. albicans killing assay. The LD50 values for candidacidal activity of native Hst3, re-Hst3 and re-Hst3rep were 7.2, 6.8 and 4.1 nmol/ml, respectively. At lower concentrations re-Hst3rep was five times more active than native Hst3 or re-Hst3 and at even lower concentrations re-Hst3rep exhibited significant candidacidal activity while native Hst3 and re-Hst3 were inactive. These results demonstrate an expression system for production of biologically active functional Hst and Hst variants and shows that repetition of the functional domain of Hst3 enhances candidacidal activity.

Candidacidal activity of human salivary histatin recombinant variants produced by site-directed mutagenesis.

Histatin 5 (Hst5) is a 24-amino acid (aa) member of the Hst family that is found in human salivary secretions and exhibits candidacidal activity. Hst5 contains a 13-aa region that alone is capable of killing fungal pathogens and is referred to as the functional domain. To investigate the role of specific aa located within the functional domain, the pRSET bacterial expression system was used to produce recombinant Hst5 (re-Hst5) and several re-variants that were generated by site-directed mutagenesis. The vector pRSETC expresses genes of interest as fusion proteins attached to the carboxy end of an N-terminal His6 tag that binds to nickel (Ni2+). The re-variants were generated using the polymerase chain reaction (PCR) and had Gly substituted for either the His, Glu or Lys/Arg within the functional domain. PCR products that encoded either the wild-type or variant forms of re-Hst5 were inserted into pRSETC and produced as fusion proteins which were affinity purified from cell lysates by Ni(2+)-Sepharose chromatography. Fusion proteins were digested with CNBr and re-Hsts were purified by reversed-phase high performance liquid chromatography (RP-HPLC). Re-Hsts were tested in bioassays to measure the ability to kill both Candida albicans (C. albicans) blastoconidia and spheroplasts which were generated by removal of the cell wall. In both assays, re-Hst5 displayed dose-dependent candidacidal activity that was nearly identical to that of native Hst5 purified from human salivary secretions. Re-Hst5 variants with either Glu or Lys/Arg substitutions demonstrated significantly lower candidacidal activity in both assays, while the variant with His mutated showed essentially no activity at physiological concentrations. These results indicate that acidic and basic aa within the functional domain contribute to candidacidal activity and that the His are essential for candidacidal activity. Additionally, since C. albicans spheroplasts were also susceptible to Hsts, the cell wall is not an essential component in the Hst mechanism of candidacidal action.

Zinc and copper bind to unique sites of histatin 5.

Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5, a human salivary protein. Proton nuclear magnetic resonance (NMR) spectra were obtained to investigate the specificity of metal binding to the seven histidyl, one aspartyl and one glutamyl amino acid side-chains of histatin 5 in aqueous solutions. Three C(epsilon1)-H histidyl and the C(gamma)-H glutamyl resonances of histatin 5 were selectively altered in spectra of solutions containing three equivalents of zinc. Copper binding to histatin 5 resulted in a reduced intensity of C(beta)-H aspartyl resonances, while no evidence for calcium binding was found. These results indicate that zinc binding to histatin 5 involves His-15 present within the -H-E-X-X-H- zinc binding motif, and copper binding occurs within the N-terminal D-S-H-, ATCUN motif.

Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain.

Histatin 5 is a low molecular weight salivary protein which is known to exhibit inhibitory activity against several proteinases, including the cysteine proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine proteinase clostripain derived from the pathogen Clostridium histolyticum. Using a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to that of leupeptin, a known competitive inhibitor of clostripain. It was found that the concentration of histatin 5 required to inhibit 50% of clostripain activity was 23.6+/-1.6 nM. Kinetic analysis revealed that histatin 5 is a competitive inhibitor of clostripain with an inhibition constant (K(i)) of 10 nM. The K(i) for the inhibition of clostripain activity against nitroanilide substrate by leupeptin was found to be 60 nM, significantly higher than that of histatin 5. Thus, histatin 5 inhibits clostripain more effectively than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incubated at physiologic concentrations with clostripain. The potent inhibition of clostripain by histatin 5 points towards the possibility that this protein may prevent establishment of clostridial infections and therefore may have significant potential for the treatment of diseases associated with this enzyme.

Elevation of salivary antimicrobial proteins following HIV-1 infection.

Thirty-seven HIV-1-positive patients contributed salivary samples from individual major salivary glands. Nineteen patients were unmedicated and asymptomatic, and 18 patients had developed signs of AIDS. Salivas from 15 healthy males served as controls. Levels of four salivary antimicrobial proteins (lactoferrin, lysozyme, secretory IgA, and histatins) were determined, as well as total fluid output of the major salivary glands. Concentrations of all four salivary antimicrobial proteins were found to be increased in the stimulated submandibular/sublingual saliva of all HIV-1-positive patients as well as the subset of unmediated HIV-1-positive patients. Those patients with evidence of oral candidiasis had the highest concentrations of lysozyme and histatins, potent antifungal proteins, in their saliva. Although the etiology of these protein increases is still unknown, these results further document salivary changes following HIV-1 infection.