Adhesion of Candida albicans, but not Candida krusei, to salivary statherin and mimicking host molecules.

The aim of the present study was to identify salivary molecules affecting adhesion of Candida albicans and Candida krusei to salivary pellicles and epithelial cells. Strains of C. albicans (GDH18, GDH3339, CA1957, ATCC 28366 and ATCC 10321), but not C. krusei (strains ATCC 14243 and Ck9), bound to saliva-coated hydroxyapatite and buccal epithelial cells. Parotid saliva fractions containing statherin, glycosylated proline-rich proteins (PRP) and as yet unidentified components mediated adhesion of strain GDH18; Fuc alpha 1-2Gal beta 1-4Glc partly inhibited the adhesion to those fractions not containing statherin. Pure statherin, but not PRP-1, mediated dose-dependent adhesion of C. albicans strain GDH18 to hydroxyapatite beads. Candida isolates (GDH18, GDH3339 and CA1957) bound somewhat more avidly to statherin/saliva relative to ATCC strains 28366 and 10321, while the opposite was true for adhesion to buccal epithelial cells. Adhesion of C. albicans strain GDH18 to saliva-coated hydroxyapatite and buccal epithelial cells was completely (93%) and partly (43%) blocked by statherin-specific immunoglobulin G (IgG) antibodies, respectively. Control IgG antibodies did not block Candida adhesion. Blockage of Candida adhesion to epithelial cells also occurred with Fuc alpha 1-2Gal beta 1-4Glc (49%) and N-acetylglucosamine (38%), while statherin specific IgG antibodies in combination with Fuc alpha 1-2Gal beta 1-4Glc almost completely eliminated Candida adhesion (79%). In addition, statherin in solution blocked the adhesion of strain GDH18 to epithelial cells by inducing aggregation of Candida cells.

Apoptosis in restenosis versus stable-angina atherosclerosis: implications for the pathogenesis of restenosis.

Decreases in programmed cell death (apoptosis) may contribute to restenotic hyperplasia by prolonging the life span of intimal cells. Apoptotic events were compared in restenotic versus primary lesions, by using atherectomy samples from 16 restenotic and 30 primary human peripheral and coronary lesions from patients presenting with stable angina. We used transmission electron microscopy to identify apoptosis, quantify its frequency, distinguish apoptosis from necrosis, and relate these events to cellular composition. Smooth muscle cell (SMC) density was higher in restenotic versus primary lesions (P<0.0001), whereas the number of macrophages was significantly reduced (P<0.01) and the number of lymphocytes was lower, but not significantly (P=0.06). As the main finding, restenotic lesions contained fewer apoptotic cells compared with primary lesions (3% versus 13%, P=0.002), whereas no differences were found for cellular necrosis. With regard to cell type, the lower frequency of apoptotic cells observed in restenotic tissue was attributable to both SMCs and macrophages. The key finding of less apoptosis in restenotic versus primary lesions was in agreement with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis (2% versus 9%, P<0.001). For all lesions analyzed, significant inverse correlations were observed between the density of SMCs and the frequency of apoptotic cell death (r=-0.60, P<0.001) as well as the density of SMCs and that of macrophages (r=-0.74, P<0.001). No relationship was seen between the frequency of apoptosis and the density of macrophages. In conclusion, the data of the present study indicate that a low level of apoptosis may be an important mechanism leading to restenotic intimal lesion development after interventional procedures.

[Decreased apoptosis as a pathogenic factor in intimal hyperplasia of human arteriosclerosis lesions]. / Verminderte Apoptose als Pathogenesefaktor der intimalen Hyperplasie humaner Arterioskleroseläsionen.

Restenosis remains a persistent problem following intravascular reconstruction. Smooth muscle cell proliferation, extracellular matrix production and remodeling are accepted mechanisms of restenotic lesion formation. Decreased programmed cell death (apoptosis) may also contribute to restenosis by prolonging the life span of intimal cells, with their subsequent accumulation and development of hyperplastic lesions. The objectives of the present study were as follows: i) to identify cell death, ii) to distinguish and quantify apoptosis from necrosis, and iii) to compare restenotic with primary lesions. To this end, human atherectomy specimens from 25 primary and 14 restenotic coronary and peripheral lesions were studied by TUNEL test (TdT-mediated dUTP Nick End Labeling; detection of cell death by the presence of fragmented DNA), transmission electron microscopy and morphometric analysis. Intimal hyperplasia was more consistent with restenosis than with primary lesion origin, and was mainly attributed to increased smooth muscle cell density (649 vs. 219 cells/mm2; p < 0.001). The main finding of the present study is that hypercellular restenotic tissue contains fewer TUNEL+ cells than hypocellular plaques (14% vs. 27%; p < 0.05). Most importantly, ultrastructural evaluation revealed a markedly reduced portion of intimal plaque cells, especially smooth muscle cells exhibiting distinct morphologic signs of apoptosis (3% vs. 13%; p < 0.001). In contrast, incidence of necroses did not differ between both lesion types (0.13 vs. 0.12 necroses/ cell; p = 0.49). Thus, our data indicate apoptosis and not necrosis to be the crucial cell death form to account for the apparent discrepancy found in both lesion types with reduced apoptosis in cell-rich restenoses. The findings of the present study suggest that decreased apoptosis is an important regulatory mechanism ultimately leading to intimal hyperplasia as commonly found in human restenosis post angioplasty.

[Incidence and localization of apoptosis bodies in human arteriosclerosis lesions]. / Inzidenz und Lokalisation von Apoptosekörpern in humanen Arterioskleroseläsionen.

Increased density of smooth muscle cells is an accepted feature of human restenosis after angioplasty. In addition to migration and proliferation, deregulated forms of programmed cell death may represent pathogenic mechanisms which lead to increased intimal cellularity. The goal of the present study was (i) to demonstrate programmed cell death in human plaque tissue by the detection of apoptotic bodies and to distinguish it from cellular necrosis, (ii) to evaluate the frequency and the localization of apoptotic bodies, and (iii) to compare restenotic and primary lesions for different expression patterns. To this end, coronary and peripheral atherectomy specimens from 14 restenotic and 25 primary lesions were examined by electron microscopy and morphometric analysis. Apoptotic bodies were distinguished from cellular necroses due to distinct morphological features, and were observed extracellularly, isolated or cell membrane-bound, as well as intracellularly in smooth muscle cells and macrophages. The main finding of this study is that hypercellular restenotic tissue from both coronary and peripheral lesions contains fewer apoptotic bodies than hypocellular plaques from primary lesions (p < 0.01 and p < 0.05, respectively). Most importantly, a highly significant, inverse correlation was seen between the density of apoptotic bodies and intimal cellularity (r = -0.67; p < 0.0001). Especially in the extracellular matrix regions, restenotic lesions showed fewer apoptotic bodies (p < 0.001). Again, these plaques exhibited a smaller number of apoptotic bodies with intracellular or membrane-bound localization; however, this observation was without statistical significance compared to primary lesions. For both plaque types, apoptotic bodies were found more frequently (by the factor 4-10) in the presence of smooth muscle cells than with macrophages. With respect to the cellular composition of the plaques, apoptotic bodies were evenly detected in 15-28% of all smooth muscle cells and macrophages. Our results document a considerable intimal density of apoptotic bodies in high-grade human arteriosclerotic lesions and, in addition, reveal nearby smooth muscle cells and macrophages exhibiting intensive phagocytotic capacity. Differences in the density of apoptotic bodies and in cellularity, coincident with an inverse correlation between these determinants, were observed for restenotic and primary tissue. These findings strongly point to deregulated forms of programmed cell death as important pathogenic mechanisms involved in human restenosis.

Ultrastructural characteristics of human atherectomy tissue from coronary and lower extremity arterial stenoses.

In animal studies, smooth muscle cell phenotype conversion has been suggested to be an essential prerequisite for subsequent migratory and proliferative events leading to (neo)intima formation. To determine ultrastructural characteristics of individual smooth muscle cells and to relate them to specific lesion types and intimal cell density, we compared atherectomy samples from 17 restenotic and 32 primary coronary and peripheral lesions using transmission electron microscopy and histology. Ultrastructural analysis of cell-rich tissue, predominantly of restenotic origin, revealed smooth muscle cells full of synthetic organelles. Moreover, these cells were frequently found to be surrounded by loose extracellular matrix and partially fragmented basement membrane components. In contrast, plaques exhibiting low cell density, as exclusively seen with primary lesions, displayed an extensive buildup of extracellular matrix containing sparse numbers of microfilament-rich smooth muscle cells. The central finding of our study is a morphometrically quantitated, twofold greater (p <0.001) volume fraction of synthetic organelles (VS) within smooth muscle cells in restenotic versus primary plaques, indicating a more dedifferentiated cellular phenotype as a typical feature of restenotic lesions. Equally enhanced VS values were seen for restenotic coronary and peripheral plaques. No VS decrease was observed during time after angioplasty (2.2 to 30 months) regardless of previous revascularization procedures (balloon angioplasty or atherectomy). Despite intra- and interlesional variability, VS and intimal cell density were strongly correlated (r = 0.74; p <0.001). This correlation was observed more often with clinical restenoses and, importantly, in a portion (10% to 15%) of primary lesions. Data from restenotic lesions indicate that a dedifferentiated smooth muscle cell phenotype, pericellular matrix disintegration, and intimal hypercellularity are long-lasting biologic responses to previous smooth muscle cell injury. Similar tissue characteristics expressed in several primary lesions suggest that comparable pathogenic mechanisms are related to the progression and/or acuity of chronic lesions.

Dislocation of the rotating cutter during directional coronary atherectomy: a note of caution.

Directional coronary atherectomy (DCA) has received increased attention, especially as a bail-out procedure after failed balloon angioplasty. However, this technique may also be burdened by severe pitfalls. We report a patient with a balloon-resistant left coronary artery lesion subsequently treated with DCA. Despite its over-the-wire guidance, as the rotating cutter was advanced, it deviated from its intra-housing course and intruded into the vascular wall. Dislocation of the rotating blade was due to pressure from hard plaque tissue. After having carefully pulled back the complete catheter system, a severe spasm of the left main stem occurred, which was reversed by intracoronary nitroglycerine. The final angiography showed a left coronary artery without significant, residual stenosis. The case report underscores that DCA passes must be performed under continuous fluoroscopic control, especially for balloon-resistant lesions because of the unpredictability of DCA-imminent complication.

Human salivary acidic proline-rich protein polymorphisms and biosynthesis studied by high-performance liquid chromatography.

Human salivary acidic proline-rich proteins (PRPs) constitute a significant fraction of the total salivary protein and possess important biological activities. Different genetic and post-translationally processed forms of the PRPs exhibit significant quantitative variations in several of these activities, especially the modulation of salivary calcium phosphate chemistry and oral bacterial adhesion. To quantify and understand these differences, we have developed a high-performance liquid chromatography (HPLC) method to identify and measure individual PRPs in saliva. The data obtained permit the identification of PRP polymorphisms and phenotypes, the determination of the relative amounts of PRPs derived from the two loci, PRH1 and PRH2, and the measurement of the extent of post-translational cleavage of the primary polypeptide products. Substantial inter-gland and inter-individual variations were found in relative amounts of PRPs derived from the two loci (at least two-fold), and in post-translational cleavage (greater than two-fold), both of which are likely to be biologically significant. Also in this study, the presence of what appear to be minor amounts of numerous variant PRPs in glandular secretions was observed, and two uncommon PRP polymorphisms were identified in the 127 subjects studied.

Primary structure of a novel human salivary acidic proline-rich protein.

Human salivary acidic proline-rich proteins (PRPs) form a significant fraction of the total salivary protein and fulfill several biologically important roles in the oral cavity. Five commonly occurring PRP polymorphisms, Db, Pa, PIF, Pr2 and Pr1, have been identified, their structures determined, and several uncommon polymorphisms (frequencies < 1:100) have been reported. Most PRPs occur as protein pairs, because of an unusual, limited but well-controlled post-translational cleavage. We now describe an additional uncommon polymorphism, found in the saliva of one of 127 individuals examined in a recent study, identified by high performance anion-exchange liquid chromatography. By analogy with previous terminology, we designate this protein pair as PRP-5, for the primary 150-residue polypeptide gene product, and PRP-6, for the secondary 106-residue cleavage product. Amino acid analysis of intact PRP-6 and sequence determination of PRP-6 chymotryptic peptides, residues 15-24 and 26-35, show a single difference in PRP-6, compared to the most similar, characterized PRP, PRP-4, in that residue 30 is histidine in PRP-6, rather than arginine as in PRP-4 and in all the other sequenced PRPs. This substitution may have implications for the resistance of this polymorphic variant to degradation by trypsin-like enzymes originating from the oral microflora.

Inhibition of calcium phosphate precipitation by human salivary statherin: structure-activity relationships.

Previous studies of human statherin showed the active region for inhibition of secondary calcium phosphate precipitation (crystal growth) to reside in the highly charged amino-terminal one-third of this molecule, and the neutral tyrosine-, glutamine- and proline-rich carboxy-terminal two-thirds of the molecule is required for maximal inhibition of primary (spontaneous) precipitation. The purpose of the present study was to define more clearly the activities of these different molecular segments of statherin with respect to the two kinds of inhibitory activities. Peptides from statherin were prepared by specific proteolysis using trypsin, endoproteinase Arg-C, and activated factor X to produce the amino-terminal hexa-, nona- and decapeptides, respectively, and carboxypeptidase-A was used to obtain a peptide extending from residue 1 to about residues 32-37. The peptides were purified by anion exchange and gel filtration chromatography, and characterized and quantified by amino-acid analysis. Serially diluted samples of statherin and derived peptides were assayed to determine the concentrations, giving a standard 50% inhibition of precipitation (C50%) in assay systems designed for this purpose using polyaspartate as a standard. Results are expressed as (C50% statherin)/(C50% peptide). For inhibition of primary precipitation, these values were peptide(1-6), 0.20; peptide(1-9), 0.15; peptide(1-31/35), 0.24. For inhibition of secondary precipitation, the values were peptide(1-6), 3.8; peptide(1-9), 2.8; peptide(1-10), 1.9; peptide(1-32/37), 1.5. These quantitative findings show that maximum inhibition of primary precipitation by statherin requires the entire molecule. Thus, removal of a relatively small segment of its carboxy-terminal region results in a substantial reduction in inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The primary structures of six human salivary acidic proline-rich proteins (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f).

Human glandular salivary secretions contain several acidic proline-rich phosphoproteins (PRPs). These proteins have important biological functions related to providing a protective environment for the teeth, and appear to possess other activities associated with modulation of adhesion of bacteria to oral surfaces. These functions and activities depend on the primary structures of the PRPs. Previously determined amino acid sequences of two 150-residue molecules, PRP-1 and PRP-2, and two related 106-residue proteins, PRP-3 and PRP-4, indicated that residue 4 was Asn in PRP-1 and PRP-3, and Asp in PRP-2 and PRP-4, and position 50 was Asn in all four proteins. Recent data from cDNA sequence studies and further structural studies, however, showed that the previously proposed sequences cannot be completely correct. The present work has shown that the protein previously designated as PRP-1 actually consisted of two positional isomers, PIF-s, which has Asn and Asp at positions 4 and 50 respectively, and authentic PRP-1, which has the reverse arrangement. The same isomerism is present in the smaller proteins, PIF-f and PRP-3. Since the isomeric pairs have identical compositions and charges, their presence was not previously detected. Also, by using a more highly purified preparation, it has been found that position 50 in PRP-2 and PRP-4 is Asp, rather than Asn previously reported. These new findings for the six PRPs define their complete primary structures, which are now consistent with those proposed for PRP-1 and PIF-s from cDNA data, and are also consistent with the chromatographic and electrophoretic behaviours of the six PRPs and their derived peptides. These corrected structures are important for understanding the biological functions and activities of these unusual proteins.

Inhibition of calcium phosphate precipitation by human salivary acidic proline-rich proteins: structure-activity relationships.

Absence of precipitation of calcium phosphate salts onto tooth surfaces from human saliva, which is supersaturated with respect to calcium phosphate salts, has been attributed in part to the presence in the salivary secretions of a group of acidic proline-rich phosphoproteins (PRP). These macromolecules are considered to act by adsorbing onto dental enamel where they inhibit surface-induced precipitation of calcium phosphate salts. The inhibitory activity is known to be associated primarily with the amino-terminal region of the PRP. The aim of this study was to determine the features of the primary structure of this molecular segment responsible for inhibitory activity. The 30-residue, amino-terminal segment of PRP-3, which contains the two phosphoserines and 11 of the 13 carboxyl groups present in PRP-3, was obtained by tryptic digestion. This peptide, designated PRP-3(TI), was treated with thermolysin to give the monophosphopeptides, Val-PSer-Gln-Glu-Asp-Val-Pro and Leu-Val-Ile-Ser-Asp-Gly-Gly-Asp-PSer-Glu-Gln, and with alkaline phosphatase to give the dephosphorylated analog, PRP-3(TI)DP. The inhibitory activities of PRP-3(TI) and the derived peptides, a synthetic peptide, phosphoseryl-phosphoserine (PSer-PSer), and O-phosphoserine (PSer), were determined using an assay based on inhibition of seeded precipitation of calcium phosphate. Inhibitory activities, expressed as concentrations of inhibitors required to give standard inhibitory activities, were PRP-3(TI), 0.59 microM; PSer-PSer, 3.5 microM; the two monophosphopeptides, 29 and 32.5 microM; PRP-3(TI)DP, 56 microM; PSer, 329 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Saturation of human salivary secretions with respect to calcite and inhibition of calcium carbonate precipitation by salivary constituents.

The state of saturation of human salivary secretions with respect to calcite has been investigated. This property cannot be calculated exactly because of uncertainties in the values of the solubility product constant of calcite, the dissociation constants of carbonic acid, and PCO2 values of saliva. Minimum and maximum limits for this saturation, however, can be established using appropriate values for the constants and salivary PCO2. Values that give the minimum degree of saturation show that 8 of the 70 samples of human saliva investigated would be supersaturated with respect to calcite, while 64 of the 70 samples appeared to be supersaturated when values giving the maximum degree of saturation were used. In the latter case, the ratio of ionic activity products to solubility product was above 10 for several samples and over 18 for the most supersaturated sample. Since these results show that supersaturation of saliva with respect to calcite may be a common condition, human salivary secretions were investigated for the presence of inhibitors of calcite precipitation. Inorganic phosphate and the acidic proline-rich proteins, known to be inhibitors of calcite precipitation, and human salivary statherin, now shown to have a similar activity, are present in saliva at concentrations considerably higher than those required to inhibit calcite precipitation under salivary conditions. Quantitatively, phosphate is by far the most important inhibitor of calcite precipitation present in saliva, suggesting that inhibition of calcite precipitation by the macromolecules may be of secondary significance. It seems more likely that the function of these molecules is to inhibit precipitation of calcium phosphate salts, as previously proposed. These different inhibitory activities, however, are likely to be factors in the differences in composition of oral and dental calculi in different species, and may need to be considered in the formation of calcite stones in the pancreas.

Relationship between concentration of human salivary statherin and inhibition of calcium phosphate precipitation in stimulated human parotid saliva.

Human salivary secretions are supersaturated with respect to the calcium phosphate salts which form dental enamel, a property which provides important protection for the teeth. We previously proposed that statherin, a 43-residue phosphopeptide, plays a key role in this protective system by inhibiting or delaying potentially harmful precipitation of calcium phosphate salts in the salivary glands and mouth. The purpose of the present study was to determine if the concentrations of statherin in saliva, despite their wide normal range, are high enough to fulfill this function. Concentrations of statherin in stimulated human parotid saliva samples from 36 female and 32 male subjects, aged from 17 to 30 years, were determined by a single radial immunodiffusion method. Values found ranged from 3.0 to greater than 27.3 microM, with a mean value of 12.8 (S.D. +/- 5.46) microM. At concentrations below these values, statherin inhibited spontaneous precipitation of calcium phosphate salts from an assay system which was more supersaturated with respect to dicalcium phosphate dihydrate, and comparably supersaturated with respect to hydroxyapatite, than were human saliva samples. The inhibitory activities of five of the 65 stimulated parotid saliva samples assayed were greater than would be anticipated from their statherin concentrations. This unexplained discrepancy is not associated with the presence of the acidic proline-rich proteins in saliva, although these proteins also affect calcium phosphate precipitation. The results of this study show that statherin is present in stimulated human parotid saliva at concentrations and levels of activity which are consistent with its proposed biological function, and support the proposal that statherin plays a significant role in a system which provides a protective and reparative but stable environment for the teeth.

Equilibrium dialysis and ultrafiltration studies of calcium and phosphate binding by human salivary proteins. Implications for salivary supersaturation with respect to calcium phosphate salts.

Previous ultrafiltration studies indicated that up to one-half of the calcium and two-thirds of the phosphate in human salivary secretions may be bound by salivary proteins. Since this binding is an important variable in determining the extent of salivary supersaturation with respect to calcium phosphate salts, and since the amount of binding reported is surprisingly large, calcium and phosphate ion-binding by salivary macromolecules has been reexamined. From experiments using equilibrium dialysis, it was found that (1) the fraction of salivary calcium involved in macromolecular complexes ranges from a few percent for unstimulated secretions, to no more than about 10% for stimulated glandular salivas, and (2) salivary proteins do not bind phosphate ions to any significant extent. These findings, and experiments using an improved ultrafiltration membrane, indicate that the earlier results were artifacts of the ultrafiltration technique. Fractionation of salivary proteins, followed by equilibrium dialysis measurements, showed that the anionic proline-rich proteins and a basic proline-rich glycoprotein are responsible for most of the calcium binding now observed. The finding that macromolecular complexes of salivary calcium and phosphate have been overestimated in the past, leads to the conclusion that salivary calcium and phosphate ion activities in stimulated salivary secretions may be up to 50 to 100% higher than previously thought. Revised values were therefore used to recalculate the degree of salivary supersaturation with respect to calcium phosphate salts. The results indicate that stimulated salivary secretions are supersaturated with respect to dicalcium phosphate dihydrate; this is a substantially greater degree of supersaturation than previously reported.