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Biomedical subjects

E C Veerman

Publications and source records attributed to E C Veerman.

At least 37 records · Page 2Linked to original sources

Amphotericin B- and fluconazole-resistant Candida spp., Aspergillus fumigatus, and other newly emerging pathogenic fungi are susceptible to basic antifungal peptides.

The present study shows that a number of basic antifungal peptides, including human salivary histatin 5, a designed histatin analog designated dhvar4, and a peptide from frog skin, PGLa, are active against amphotericin B-resistant Candida albicans, Candida krusei, and Aspergillus fumigatus strains and against a fluconazole-resistant Candida glabrata isolate.

Amino Acid Sequence↗

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 microg/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.

Actinomyces↗

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 microg mucin per mL were found in submandibular, sublingual, and palatine saliva, respectively.

Antibodies↗

MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage.

Mucins from human whole saliva, as well as from respiratory- and cervical-tract secretions, were subjected to density-gradient centrifugation in CsCl/0.5 M guanidinium chloride. A polydisperse population of MUC5B mucins was demonstrated in all samples using anti-peptide antisera (LUM5B-2, LUM5B-3 and LUM5B-4) raised against sequences within the MUC5B mucin. The sequences recognized by the LUM5B-2 and LUM5B-3 antisera are located within the domains flanking the highly glycosylated regions of MUC5B, and reduction increased the reactivity with these antibodies, suggesting that the epitopes are partially shielded and that these regions are folded and stabilized by disulphide bonds. Rate-zonal centrifugation before and after reduction showed MUC5B to be a large oligomeric mucin composed of disulphide-linked subunits. In saliva and respiratory-tract secretions, populations of MUC5B mucins with different charge densities were identified by ion-exchange HPLC, suggesting the presence of MUC5B 'glycoforms'. In trachea, the F2 monoclonal antibody against the sulpho-Lewis C structure reacted preferentially with the later-to-be-eluted populations. An antibody (LUM5B-4) recognizing a sequence in the C-terminal domain of MUC5B identified, after reduction, the mucin subunits as well as smaller fragments, suggesting that some of the MUC5B mucins are cleaved within the C-terminal domain. Immunohistochemistry revealed that MUC5B is produced by cells dispersed throughout the human submandibular and sublingual glands, in the airway submucosal glands as well as the goblet cells, and in the epithelium and glands of the endocervix. The F2 antibody stained a subpopulation of the MUC5B-producing cells in the airway submucosal glands, suggesting that different cells may produce different glycoforms of MUC5B in this tissue.

Amino Acid Sequence↗

On the chemical characterization of colloid cyst contents.

Colloid cysts of the third ventricle have been investigated by chemical characterization of the cyst contents using ELISA with monoclonal antibodies for certain carbohydrate epitopes as well as a polyclonal antiserum against peptide domains, and immunohistochemistry on the cyst wall using the same antibodies. Furthermore, the carbohydrate composition of one sample has been determined after gel filtration. The cyst contents reacted strongly with the monoclonal antibody for the sulfo-Lewis epitope, and with the antimucin polyclonal antiserum. In one case the cyst fluid exhibited a blood group A antigen. A sample of cyst wall obtained by biopsy showed strong immunoreactivity against sulfo-Lewis antigen, and the sialo-Lewis antigen. The presence of the S atom with its high atomic number relative to that of C, H, and O atoms, may contribute to the high density appearance of colloid cysts on CT-scans. The sulfo-Lewis and sialo-Lewis carbohydrate epitopes are known as ligands for selectins, involved in inflammatory processes, and may well account for the aseptic meningeal reaction that may follow spilling of cyst contents during operative evacuation. The carbohydrate epitopes exhibited by colloid cysts and their contents, have also been reported for the mucins of salivary glands, uterine cervix, gall bladder and colon, and therefore, are not inconsistent with the assumption of an endodermal origin of colloid cysts.

Biopsy↗

The interaction between saliva and Actinobacillus actinomycetemcomitans influenced by the zeta potential.

The adhesion of Actinobacillus actinomycetemcomitans is a virulence factor in the aetiology of periodontitis and is determined by physico-chemical properties, e.g. surface charge and hydrophobicity, of the bacterial cell surface. Although oral surfaces are constantly coated with saliva, few studies have dealt with the binding of A. actinomycetemcomitans with saliva. In this report, the charge properties of A. actinomycetemcomitans have been studied through measurement of the zeta potential and the saliva-bacteria interaction investigated at different pH-values. At physiological conditions the zeta potential was negative, varying from -11 to -26 mV, for two laboratory and two fresh isolates of A. actinomycetemcomitans. Under these conditions, binding of the low-molecular-weight salivary mucin, lactoferrin, and S-IgA was confirmed using salivary samples and purified salivary fractions in liquid-phase and in ELISA. The iso-electric points of the laboratory and fresh clinical isolates of A. actinomycetemcomitans were determined at pH 4.6 and 3.8, respectively. At pH below the iso-electric point, giving positive values of the zeta potential, additional salivary protein species bound to A. actinomycetemcomitans, including the high-molecular-weight salivary mucin (MG1) and agglutinin. Binding of the low-molecular-weight salivary mucin (MG2), lactoferrin, and S-IgA, was hardly affected by this change in zeta potential. A salivary coating formed on the bacterium at pH 7 reduced the zeta potential of the laboratory strain Y4 greatly and an iso-electric point for the bacterium could not be determined. Overall, the study suggests that upon changes in environmental pH additional salivary attachment sites on the micro-organism are exposed.

Actinobacillus↗

Neutrophil-activating protein mediates adhesion of Helicobacter pylori to sulfated carbohydrates on high-molecular-weight salivary mucin.

The in vitro binding of surface-exposed material and outer membrane proteins of Helicobacter pylori to high-molecular-weight salivary mucin was studied. We identified a 16-kDa surface protein which adhered to high-molecular-weight salivary mucin. This protein binds specifically to sulfated oligosaccharide structures such as sulfo-Lewis a, sulfogalactose and sulfo-N-acetyl-glucosamine on mucin. Sequence analysis of the protein proved that it was identical to the N-terminal amino acid sequence of neutrophil-activating protein. Moreover, this adhesin was able to bind to Lewis x blood group antigen.

Amino Acid Sequence↗

Sulfomucins in the human body.

Mucins are widely distributed in mucous secretion fluids or are associated with plasma membranes. Up to now 9 genes of epithelial mucins have been identified, distributed over five chromosomes. Superposed on the genetic diversity, each type of mucin displays heterogeneity in oligosaccharide composition, including the terminal sugar residues. On top of that there is variation between individuals brought about by blood group antigens. Heterogeneity is further incited by the degree of sulfation. This tremendous structural heterogeneity endows mucin molecules with properties suggestive for a multifunctional role. The major biological function assigned to mucins is still the protection of tissues covered by the mucous gel. Current knowledge on the specific biological functions of the sulfate residues is fragmentary and periphrastic. Glycosylation including sulfation appears to be subject to modification under pathological conditions. There is evidence that sulfation rate-limits bacterial degradation of mucins. Moreover, accumulating data focus towards their involvement in recognition phenomena. Sulfate residues on blood group related structures provoke specific epitopes for selective interaction with microorganisms e.g. Helicobacter pylori. A distinct class of mucins acts as ligands for selectins, crucial in cellular recognition processes like cellular homing of lymphocytes. Whereas in earlier days mucins were only seen as water-binding molecules, protecting the underlying mucosa against harmful agents, the current picture of these molecules is characterized by the selective interaction with their environment, including epithelial-, and endothelial cells and microorganisms, thereby regulating a great number of biological processes. However, the specific role of sulfate remains to be further elucidated.

Gene Expression↗

Cystatin and cystatin-derived peptides have antibacterial activity against the pathogen Porphyromonas gingivalis.

We investigated whether cystatins and cystatin-derived peptides, encompassing sequences of secondary structures of cystatin S and papain binding domains of cystatin C, display antimicrobial properties. Of the different microorganisms tested, only the growth of P. gingivalis was inhibited by chicken cystatin and cystatin C. Cystatin S, cystatin S:1-14, cystatin S:61-73 and cystatin S:108-121 also inhibited its growth, whereas cystatin S:21-38, cystatin S:39-55, cystatin S:81-95, cystatin S:94-109, and cystatin C: 9-12/55-60/106-107 did not. No inhibition of the cysteine proteinase activity of P. gingivalis was observed for all cystatin-derived peptides. On the other hand, leupeptin and antipain inhibited P. gingivalis proteinase activity, but had no effect on the growth. These data suggest that cystatins contain antibacterial sequences active against P. gingivalis and that the growth inhibition does not depend on the inhibition of P. gingivalis cysteine proteinases.

Amino Acid Sequence↗

Synthetic histatin analogues with broad-spectrum antimicrobial activity.

Histatins are salivary histidine-rich cationic peptides, ranging from 7 to 38 amino acid residues in length, that exert a potent killing effect in vitro on Candida albicans. Starting from the C-terminal fungicidal domain of histatin 5 (residues 11-24, called dh-5) a number of substitution analogues were chemically synthesized to study the effect of amphipathicity of the peptide in helix conformation on candidacidal activity. Single substitutions in dh-5 at several positions did not have any effect on fungicidal activity. However, multi-site substituted analogues (dhvar1 and dhvar2) exhibited a 6-fold increased activity over dh-5. In addition, dhvar1 and dhvar2 inhibited the growth of the second most common yeast found in clinical isolates, Torulopsis glabrata, of oral- and non-oral pathogens such as Prevotella intermedia and Streptococcus mutans, and of a methicillin-resistant Staphylococcus aureus. In their broad-spectrum activity, dhvar1 and dhvar2 were comparable to magainins (PGLa and magainin 2), antimicrobial peptides of amphibian origin. Both the fungicidal and the haemolytic activities of dhvar1, dhvar2 and magainins increased at decreasing ionic strength.

Amino Acid Sequence↗

The salivary lipocalin von Ebner's gland protein is a cysteine proteinase inhibitor.

The lipocalins make up a heterogeneous superfamily of proteins. Although showing almost no sequence homology, they share very similar secondary and tertiary structures. Their ability to bind hydrophobic ligands is well established, but the physiological function of most lipocalins remains unclear. The lipocalin from the human Von Ebner's Gland of the tongue (VEGh) contains three sequence motifs corresponding with the papain-binding domains of cystatins, a family of naturally occurring cysteine proteinase inhibitors. We found that VEGh inhibited papain activity to a similar extent as salivary cystatin S. Furthermore, synthetic peptides derived from VEGh and cystatin C, comprising these three motifs, inhibited papain, too. We conclude that VEGh is a physiological inhibitor of cysteine proteinases and therefore can play a role in the control of inflammatory processes in oral and ocular tissues.

Amino Acid Sequence↗

A monoclonal antibody directed against high M(r) salivary mucins recognizes the SO3-3Gal beta 1-3GlcNAc moiety of sulfo-Lewis(a): a histochemical survey of human and rat tissue.

Using a panel of synthetic oligosaccharides attached to a polyacrylamide carrier, the epitope of monoclonal antibody F2, evoked to high M(r) salivary mucins, was mapped to the SO3-3Gal beta 1-3GlcNAc- moeity of the sulfo-Le(a) antigen. Using immunochemical techniques, the expression of the F2-epitope was investigated in a number of different isolated human mucin species, as well as in human and rat tissue specimens. The mAb F2 bound to high M(r) salivary mucins, cervical mucins, colon mucins and gallblader mucins, but not to low M(r) salivary mucins nor to gastric mucins. Immunohistochemical screening of human tissues with mAb F2 revealed a positive reaction with a number of epithelia, including the (sero)mucous salivary glands, the goblet cells of the colon, submucosal glands of the lung, the lining epithelium of cervical and esophageal glands, the suprabasal skin keratinocytes, and Hassall's corpuscles of the thymus. No staining was found in normal breast, pancreas, small intestine, spleen, and lymph nodes. Normal gastric glands were negative, but gastric intestinal metaplastic glands strongly stained with the antibody. In rat tissues, mAb F2 labeled epithelial cells of salivary glands, colon and stomach. In addition to epithelial cells, extracellular matrix components in rat thymus and skin were labeled by mAb F2. No labeling of erythrocytes, granulocytes, lymphocytes or bone marrow cells was found by FACScan analysis. The present data shows a tissue specific distribution of the F2-epitope in cells from the epithelial lineage in human and rat.

Animals↗

Sulfated glycans on oral mucin as receptors for Helicobacter pylori.

Helicobacter pylori is able to colonize gastric epithelia, causing chronic active gastritis, gastric and duodenal ulcers and presumably gastric malignancies. Attempts to identify the natural reservoir for this microorganism other than the stomach have been unsuccessful. It is suspected that H. pylori can be transmitted orally, since the microorganism has been detected at various sites of the oral cavity. The aim of the present study was to determine whether H. pylori can bind to salivary mucins, which in vivo coat the oral epithelia, and characterize further the interaction. Binding of salivary mucins and of synthetic oligosaccharides was studied in ELISA and immunoblotting, using specific mono- and polyclonal antibodies, and synthetic neoglycoconjugates. H. pylori bound most avidly to a highly sulfated subpopulation of high molecular weight salivary mucins, secreted from the palatine salivary glands, and with less avidity to mucin species secreted by the sublingual and submandibular salivary glands, which are less sulfated. Binding was strongly enhanced upon decreasing pH from 6.0 to 5.0. Using synthetic polyacrylamide coupled oligosaccharides it was found that SO3-3-Gal and the SO3-3-Lewis(a) blood group antigen bound to H. pylori. In contrast, binding of sialylated Lewis(a) and Lewis(b) antigens was much weaker. This study indicates that sulfated oligosaccharides on salivary mucins may provide receptor structures for adhesion of H. pylori to oral surfaces.

Bacterial Adhesion↗

Cystatin A in gingival crevicular fluid of periodontal patients.

Cystatins are physiological inhibitors of cysteine proteinases which are widely distributed in human tissues and fluids. In the present study we analysed both the cystatin activity and the different cystatin isoforms in gingival crevicular fluid and saliva samples of nine periodontitis patients. All crevicular fluid samples, which were collected with filter paper points, showed cystatin activity ranging from 7-67 units/mg protein. The mean cystatin activity (24 units/mg protein) was significantly lower (p < 0.05) than that of the saliva samples (mean 93 units/mg protein). The cystatin isoforms in the crevicular fluid were further characterized by immunoblotting with specific antibodies against cystatin C, S, SN and A. While they were clearly present in saliva, cystatin C, cystatin S and cystatin SN could not be detected in any of the crevicular fluid samples. Remarkably, cystatin A was found in all the crevicular fluids as well as in the saliva samples. It is concluded that the cystatin activity found in crevicular fluid is caused, at least partially, by cystatin A. Furthermore, the gingival crevicular fluid is not a major contributor of cystatin C, S and SN activity in saliva.

Adult↗

Influence of polymers for use in saliva substitutes on de- and remineralization of enamel in vitro.

A number of polymers which have previously been tested for their applicability as thickening agents in saliva substitutes were studied in vitro for their caries-protective properties. These were: polyacrylic acid, carboxymethylcellulose, xanthan gum, guar gum, hydroxyethylcellulose and porcine gastric mucin. The polymers were tested for their effects on: (1) growth of hydroxyapatite crystals in a supersaturated calcium phosphate solution, (2) dissolution of hydroxyapatite crystals in 50 mM acetic acid, pH 5.2 and (3) demineralization and remineralization of bovine enamel in a pH-cycling model. Growth of hydroxyapatite crystals was strongly inhibited by polyacrylic acid and carboxymethylcellulose at very low concentrations (0.005% w/v). Other polymers displayed lower inhibition of hydroxyapatite crystal growth. Hydroxyapatite dissolution was inhibited by all polymers except by hydroxymethylcellulose and xanthan gum. This occurred both in the presence of the polymers as well as after a 30-min preincubation. In the pH-cycling experiment, bovine enamel specimens with preformed lesions were alternately exposed to a demineralization buffer and a remineralization buffer containing the polymers hydroxyethylcellulose, carboxymethylcellulose, xanthan gum, polyacrylic acid, or porcine gastric mucin. A remineralization buffer containing 1 ppm NaF was used as a positive control. Under the experimental conditions, the control experiment without additives resulted in a net mineral loss (30.6 mumol Ca/cm2 after 14 days of pH cycling). In the presence of 1 ppm NaF, a small mineral gain was observed (8.6 mumol/cm2). All polymers largely inhibited further demineralization (1.2-12.3 mumol/cm2) except polyacrylic acid which, inhibited of its high calcium-binding capacity, caused demineralization, especially in the remineralization buffer (17.1 mumol/cm2). In conclusion, polymers tested in this study, except the polyacrylic acid, reduced the demineralization of enamel in vitro. The precise mechanism of the protective effect is not clear but it is speculated that formation of an absorbed polymer layer on the hydroxyapatite or enamel surface may provide protection against acidic attacks.

Acetic Acid↗

In vivo binding of the salivary glycoprotein EP-GP (identical to GCDFP-15) to oral and non-oral bacteria detection and identification of EP-GP binding species.

Extra Parotid Glycoprotein (EP-GP) is a glycoprotein isolated from human saliva, having homologues in several other body fluids. The biological role of EP-GP and its homologues is unknown. Recently, EP-GP was shown to bind in vitro to the bacterium Streptococcus salivarius HB. In contrast, no binding to a number of other oral microorganisms could be demonstrated. In the present study we have determined whether binding of EP-GP to bacteria occurs in vivo in saliva and in other EP-GP containing body fluids. Therefore the presence of EP-GP on bacteria in vivo was determined by analyzing oral, skin and ear floras by confocal fluoresence microscopy using specific antibodies. About 12% of the in vivo oral flora had EP-GP present on their surface, while approximately 5% of the bacteria from ear canal or skin was positive for EP-GP. IgA was detected on approximately 65% of the salivary bacteria, whereas the high-molecular weight mucin (MG1) and cystatin C were not detectable on any oral bacterium. Using a replica-plate assay, a number of EP-GP binding strains in saliva were isolated and identified as Gemella haemolysans, Gemella morbillorium, Streptococcus acidominimus, Streptococcus oralis, Streptococcus salivarius and Streptococcus parasanguis. Bacteria from the ear canal and skin bacteria were identified as Staphylococcus hominis. It is concluded that EP-GP is selectively bound in vivo to several oral and non-oral bacterial species.

Body Fluids↗

Treatment of xerostomia with polymer-based saliva substitutes in patients with Sjögren's syndrome.

OBJECTIVE: To determine the efficacy of 3 types of polymer-based saliva substitutes in reducing oral dryness in patients with Sjögren's syndrome (SS). METHODS: Subjective efficacy of 3 different saliva substitutes (determined by self-administered questionnaire) was evaluated in a double-blind, placebo-controlled trial in 43 patients with primary and secondary SS. High-viscosity versus low-viscosity xanthan gum-based saliva substitutes were also compared in 33 SS patients. Salivary flow rates (SFR) were determined to examine correlations between the SFR and the subjective efficacy of the saliva substitute. RESULTS: Neither the saliva substitutes nor the placebo was truly effective. Preference for a particular saliva substitute over placebo was equally distributed among the 3 types of substitutes. The SFR of patients who preferred polyacrylic acid-based saliva substitutes was lower than that in patients who preferred the porcine mucin-based substitute (P < 0.05). Patients whose oral dryness was reduced by low-viscoelastic substitutes had a low stimulated SFR ( < 0.20 ml/minute; P < 0.05). CONCLUSION: The optimal properties of a saliva substitute are not the same for all patients with SS, but are dependent on such parameters as the individual SFR. Thus, to determine the best saliva substitute for a particular patient, it is necessary to have the patient try a number of substitutes of different viscoelastic properties.

Acrylic Resins↗