A.V. Nieuw Amerongen

Effects of histatin 5 and derived peptides on Candida albicans.

Three anti-microbial peptides were compared with respect to their killing activity against Candida albicans and their ability to disturb its cellular and internal membranes. Histatin 5 is an anti-fungal peptide occurring naturally in human saliva, while dhvar4 and dhvar5 are variants of its active domain, with increased anti-microbial activity. dhvar4 has increased amphipathicity compared with histatin 5, whereas dhvar5 has amphipathicity comparable with that of histatin 5. All three peptides caused depolarization of the cytoplasmic and/or mitochondrial membrane, indicating membranolytic activity. For the variant peptides both depolarization and killing occurred at a faster rate. With FITC-labelled peptides, no association with the cytoplasmic membrane was observed, contradicting the formation of permanent transmembrane multimeric peptide pores. Instead, the peptides were internalized and act on internal membranes, as demonstrated with mitochondrion- and vacuole-specific markers. In comparison with histatin 5, the variant peptides showed a more destructive effect on mitochondria. Entry of the peptides and subsequent killing were dependent on the metabolic state of the cells. Blocking of the mitochondrial activity led to complete protection against histatin 5 activity, whereas that of dhvar4 was hardly affected and that of dhvar5 was affected only intermediately.

Cystatins S and C in Human Whole Saliva and in Glandular Salivas in Periodontal Health and Disease

Cystatins are inhibitors of cysteine proteinases and could play a protective and regulatory role under inflammatory conditions. Since total cystatin activity of whole saliva was increased in periodontal patients (Henskens et al., 1993), we wanted to investigate the types or origins of cystatins involved in this increase. Distinct types of cystatins were identified by isoelectric focusing and immunoblotting with specific antibodies against one of the salivary acidic isoforms, cystatin S, and the widely distributed basic cystatin C. Clarified human whole saliva (CHWS) of healthy subjects contained cystatin S, whereas cystatin C was barely detectable. In contrast, in CHWS of gingivitis and periodontitis patients, both cystatin C and S levels were higher. The origin of cystatin activity was investigated by collecting submandibular (SM), sublingual (SL), and parotid (PAR) saliva from seven subjects with mild gingivitis. Total cystatin activity was about five times higher in SM saliva than in PAR saliva. In SM and SL saliva, both cystatins S and C were demonstrated. In contrast, in PAR samples, solely cystatin C was detectable. The introduction of experimental gingivitis in one periodontally healthy subject resulted in the appearance of a cystatin C band in PAR saliva and in an increase of cystatins S and C in SM saliva. We conclude that the previously observed increase of cystatin activity in whole saliva in inflammatory periodontal disease is, at least in part, due to an increased glandular output of both the isof orm cystatin S (pI 4.7) and the basic cystatin C (pI 9.0).

Detection and Quantification of MUC7 in Submandibular, Sublingual, Palatine, and Labial Saliva by Anti-peptide Antiserum

The large carbohydrate moiety of low-Mr salivary mucin MUC7 (originally referred to as MG2) is subject to variations. Biochemical analysis and quantification of MUC7 in saliva samples require recognition tools that are independent of the carbohydrate moiety. Therefore, we have evoked three antisera to synthetic peptides of MUC7. One of these (CpMG2), raised against the C-terminal peptide, recognized native MUC7 in saliva and was characterized further. Recognition of MUC7 by CpMG2 turned out to be specific, resistant to dissociating and reductive treatments, and independent of glycosylation differences, as indicated by Western analysis and ELISA. The antiserum could be used to monitor MUC7 during purification procedures. MUC7 was demonstrated in small volumes of saliva from all (sero)mucous glands, including the palate and lip. Analysis with antibodies and lectins indicated large variations in amount as well as in glycosylation of MUC7. An ELISA was developed to determine the relative quantity of MUC7 in the glandular salivas: mean values of approximately 220, 980, and 100 μg mucin per mL were found in submandibular, sublingual, and palatine saliva, respectively.

Oral and salivary changes in patients with end stage renal disease (ESRD): a two year follow-up study

Objectives To compare oral health, salivary flow rate, xerostomia and thirst in end stage renal disease (ESRD) patients remaining on dialysis treatment and after renal transplantation. Design Longitudinal observation. Setting ESRD patients recruited from dialysis centres in Amsterdam, The Hague and Utrecht, The Netherlands. Method At baseline and after two years, salivary flow rates, xerostomia and thirst were determined in 43 ESRD patients. The number of decayed missing filled teeth/surfaces (DMFT/DMFS) was recorded, and periodontal status assessed. Results After renal transplantation (n = 20), the salivary flow rate increased significantly from UWS = 0.30 ± 0.21 ml/min to 0.44 ± 0.29 ml/min (p <0.001) and the level of xerostomia and thirst decreased. After two years, the percentage of bleeding on probing in dialysis patients (n = 23) decreased from 29.5 ± 25.4% to 10.3 ± 12.3%, (p <0.05). No differences in DMFT and DMFS were observed between dialysis and renal transplant patients. Conclusions DMFT, dental plaque, gingival bleeding and periodontal indices did not change remarkably after two years, comparing dialysis and renal transplant patients. Renal transplantation enhances salivary flow and decreases symptoms of xerostomia and thirst, and hence enhances the potential to improve the quality of life of affected individuals.

The Effects of Histatin-derived Basic Antimicrobial Peptides on Oral Biofilms

Susceptibility of bacteria to antimicrobial agents is strongly reduced by the formation of complex biofilms. We investigated whether synthetic histatin analogs with broad-spectrum antibacterial activity in vitro were also active against these complex mixtures of bacteria, as present in saliva and plaque. In a simplified model system for dental plaque, hydroxyapatite discs were placed in a continuous culture system comprised of Streptococcus mutans, S. sanguis, S. salivarius, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, and Prevotella intermedia. Ex situ treatment of the biofilms formed on these discs with 100 μg/mL of peptide dhvar4 significantly reduced facultative anaerobic, total anaerobic, and obligate anaerobic Gram-negative counts with 0.8, 0.5, and 0.5 log units, respectively. Ex vivo treatment of salivary bacteria gave reductions of 0.4, 0.7, and 1.5 log units, respectively. For ex vivo treatment of plaque bacteria, reductions of 0.4, 0.4, and 1.4 log units, respectively, were found. In both saliva and plaque samples, obligate anaerobic Gram-negative bacteria were significantly more susceptible to dhvar4 than facultatively anaerobic or anaerobic bacteria as a whole (p = 0.013 and p = 0.018, for salivary bacteria, and p = 0.021 and p = 0.020 for plaque bacteria, respectively). Although the oral bacteria are protected by biofilm formation, the synthetic histatin analog caused a significant reduction of viable counts in a model for oral biofilm as well as in isolated oral biofilms.

Immunohistochemical Detection of Salivary Agglutinin/gp-340 in Human Parotid, Submandibular, and Labial Salivary Glands

Salivary agglutinin is a Streptococcus mutans binding protein and a member of the scavenger receptor cysteine-rich superfamily. It is identical to lung gp-340 and brain DMBT1, which possibly play a role in innate immunity and tumor suppression, respectively. The goal of this study was to localize salivary agglutinin in human salivary glands. Two monoclonal antibodies, directed against gp-340, were characterized. mAb 213-1 reacted with sialic acid epitopes and cross-reacted with MUC7. The reaction with mAb 213-6 disappeared after reduction, suggesting that a protein epitope was recognized. In the parotid gland, immunohistochemical labeling with mAb 213-6 was found in the duct cells. In the submandibular gland and labial gland, both serous acini and demilune cells were labeled. In the labial gland, labeling was found at the luminal side of the duct cells. Salivary agglutinin was distinctly localized in salivary glands, but in distinct glandular secretions, no differences in electrophoretic behavior were observed.

Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.

A Role for Lewis a antigens on salivary agglutinin in binding to Streptococcus mutans

Streptococcus mutans is a major etiological agent in dental caries. Salivary agglutinin is one of the main salivary components binding to S.mutans. To learn more about the interaction of salivary agglutinin with S.mutans, parotid, submandibular, sublingual and palatal saliva samples were incubated with S. mutans suspension. Both depleted saliva samples and bacterial extracts were analyzed by SDS-PAGE and immunoblotting. Salivary agglutinin was present in all types of glandular saliva and in all cases bound to S.mutans, also to PC337C, a P1− mutant of S.mutans. Agglutinin was separated by SDS-PAGE under reducing and non-reducing conditions and then transferred to nitrocellulose. Non-reduced agglutinin bound S.mutans, but reduced agglutinin did not. Adhesion of S.mutans to agglutinin-coated microplates was inhibited by amine-containing components, 1 M NaCl or KCl and EDTA. Adhesion decreased with decreasing pH with no adhesion below pH 5.0. These data suggest that calcium-dependent electrostatic interactions play a role in binding. By immunoblotting was demonstrated that blood group antigens and Lewis antigens were present on agglutinin. Synthetic blood group antigens and Lewis antigens covalently coupled to polyacrylamide were tested for binding to S.mutans. Only Lea(Galβ1,3(Fucα1,4)GlcNAc) bound to S.mutans, whereas the blood group antigens Leb, Lex, Ley, H1, H2, A, B and sialylated Lea did not. Lea without galactose (Fucα1,4GlcNAc) still bound to S. mutans, but Lea without fucose (Galβ1,3GlcNAc) did not. Binding of agglutinin to S. mutans was not inhibited by Lea. In conclusion, S. mutans can bind to Lea carbohydrate epitopes in which the fucose is an essential residue. Lea carbohydrate epitopes are present on salivary agglutinin but play no major role in binding.

SDS-PAGE analysis of the protein layers adsorbing in vivo and in vitro to bone substituting materials

The composition of the protein layer adsorbed to the bone substituting materials, hydroxyapatite, beta-whitlockite, titanium and aluminium, in vivo (intramuscularly in guinea pig) and in vitro, was investigated using SDS-gel electrophoresis (SDS-PAGE). After in vivo implantation for 1 d mainly proteins with molecular weights between 10,000 and 20,000 were adsorbed. After 3 months the biolayer of the implanted biomaterials also contained proteins with molecular weights 35,000, 45,000, 60,000 and 200,000. No large qualitative differences in protein composition of the biolayers on the various implanted materials were found. In vitro incubation with human serum resulted in binding of proteins with estimated molecular weights of 30,000, 60,000 (albumin), 200,000 and greater than 200,000. It is suggested that the differences between in vivo and in vitro protein adsorption are due to proteolysis occurring in vivo in the vicinity of the implanted material.

Histatin 5 and derivatives: Their localization and effects on the ultra-structural level

Histatins, a family of cationic peptides present in saliva, are active against the opportunistic yeast Candida albicans. The mechanism of action is still unclear. Histatin 5 and more potent synthetic variants, dhvar4 and dhvar5, were used to study localization and effects on morphology on the ultra-structural level. Although all peptides induced leakage, no association with the plasma membrane, indicative for permanent pores, was observed with immuno-gold-labeling. Freeze-fracturing showed severe changes of the plasma membrane. Together with, for the dhvars, the loss of intracellular integrity, this suggests that leakage may be a secondary effect rather than an effect of formation of permanent pores.