Growth of Candida albicans in human saliva is supported by low-molecular-mass compounds.

Saliva plays a key role in the maintenance of a stable oral microflora. It contains antimicrobial compounds but also functions as a substrate for growth of bacteria under conditions of low external nutrient supply. Besides bacteria, yeasts, in particular Candida albicans, commonly inhabit the oral cavity. Under immunocompromised conditions, instantaneous outgrowth of this yeast occurs in oral carriers of C. albicans, suggesting that this yeast is able to survive in the oral cavity with saliva as sole source of growth substrate. The aim of the present study was to identify the salivary constituents that are used by C. albicans for growth and survival in saliva. In addition, we have explored the effect of growth in saliva on the susceptibility of C. albicans to histatin 5, a salivary antifungal peptide. It was found that C. albicans was able to grow in human saliva without addition of glucose, and in the stationary phase could survive for more than 400 h. Candida albicans grown in saliva was more than 10 times less susceptible for salivary histatin 5 than C. albicans cultured in Sabouraud medium.

Sphingoid bases inhibit acid-induced demineralization of hydroxyapatite.

Calcium hydroxyapatite (HAp), the main constituent of dental enamel, is inherently susceptible to the etching and dissolving action of acids, resulting in tooth decay such as dental caries and dental erosion. Since the prevalence of erosive wear is gradually increasing, there is urgent need for agents that protect the enamel against erosive attacks. In the present study we studied in vitro the anti-erosive effects of a number of sphingolipids and sphingoid bases, which form the backbone of sphingolipids. Pretreatment of HAp discs with sphingosine, phytosphingosine (PHS), PHS phosphate and sphinganine significantly protected these against acid-induced demineralization by 80 ± 17%, 78 ± 17%, 78 ± 7% and 81 ± 8%, respectively (p < 0.001). On the other hand, sphingomyelin, acetyl PHS, octanoyl PHS and stearoyl PHS had no anti-erosive effects. Atomic force measurement revealed that HAp discs treated with PHS were almost completely and homogeneously covered by patches of PHS. This suggests that PHS and other sphingoid bases form layers on the surface of HAp, which act as diffusion barriers against H(+) ions. In principle, these anti-erosive properties make PHS and related sphingosines promising and attractive candidates as ingredients in oral care products.

Phytosphingosine kills Candida albicans by disrupting its cell membrane.

The mechanism of action of phytosphingosine (PHS), a member of the sphingosine family which has candidacidal activity when added externally, was investigated. Previously, it has been reported that the fungicidal activity of PHS is based on the induction of caspase-independent apoptosis. In contrast, we found that addition of PHS causes a direct permeabilization of the plasma membrane of yeast, highlighted by the influx of the membrane probe propidium iodide, and the efflux of small molecules (i.e., adenine nucleotides) as well as large cellular constituents such as proteins. Freeze-fracture electron microscopy revealed that PHS treatment causes severe damage of the plasma membrane of the cell, which seems to have lost its integrity completely. We also found that PHS reverts the azide-induced insensitivity to histatin 5 (Hst5) of Candida albicans. In a previous study, we had found that the decreased sensitivity to Hst5 of energy-depleted cells is due to rigidification of the plasma membrane, which could be reverted by the membrane fluidizer benzyl alcohol. In line with the increased membrane permeabilization and ultrastructural damage, this reversal of the azide-induced insensitivity by PHS also points to a direct interaction between PHS and the cytoplasmic membrane of C. albicans.

Energy depletion protects Candida albicans against antimicrobial peptides by rigidifying its cell membrane.

Inhibitors of the energy metabolism, such as sodium azide and valinomycin, render yeast cells completely resistant against the killing action of a number of cationic antimicrobial peptides, including the salivary antimicrobial peptide Histatin 5. In this study the Histatin 5-mediated killing of the opportunistic yeast Candida albicans was used as a model system to comprehensively investigate the molecular basis underlying this phenomenon. Using confocal and electron microscopy it was demonstrated that the energy poison azide reversibly blocked the entry of Histatin 5 at the level of the yeast cell wall. Azide treatment hardly induced depolarization of the yeast cell membrane potential, excluding it as a cause of the lowered sensitivity. In contrast, the diminished sensitivity to Histatin 5 of energy-depleted C. albicans was restored by increasing the fluidity of the membrane using the membrane fluidizer benzyl alcohol. Furthermore, rigidification of the membrane by incubation at low temperature or in the presence of the membrane rigidifier Me(2)SO increased the resistance against Histatin 5, while not affecting the energy charge of the cell. In line, azide induced alterations in the physical state of the interior of the lipid bilayer. These data demonstrate that changes in the physical state of the membrane underlie the increased resistance to antimicrobial peptides.

The human cathelicidin peptide LL-37 and truncated variants induce segregation of lipids and proteins in the plasma membrane of Candida albicans.

The human cathelicidin peptide LL-37 and several truncated variants differ in their capability to transmigrate over the plasma membrane of Candida albicans. We investigated whether retention at the cell perimeter or membrane transmigration affects their membrane-disrupting activities and candidacidal properties. Using fluorescein-labeled peptides, we demonstrate that LL-37 and its C-terminally truncated peptide LL-31 remain permanently associated with the perimeter of the cell. The N-terminally truncated peptide RK-31 initially accumulated at the cell boundary, but transmigrated into the cytoplasm within 30 min. The C-terminally truncated peptide LL-25 transmigrated instantaneously into the cytoplasm. The ultrastructural effects on the plasma membrane were studied by freeze-fracture electron microscopy combined with filipin cytochemistry. All peptides, whether they transmigrated over the plasma membrane or not, induced phase separation in the plasma membrane. All peptides induced leakage of cell components, including nucleotides and proteins. Proteins were identified by SDS-PAGE in combination with mass spectrometry, which revealed that predominantly proteins smaller than 50 kDa had leaked out of C. albicans.

The management of xerostomia in patients on haemodialysis: comparison of artificial saliva and chewing gum.

Many patients on haemodialysis (HD) therapy suffer from a dry mouth and xerostomia. This can be relieved by mechanical and gustatory stimulation or palliative care. The aim of this crossover study was to investigate the effect and preferences of a sugar-free chewing gum (Freedent White) and a xanthan gum-based artificial saliva (Xialine) in the management of xerostomia in chronic HD patients. Sixty-five HD patients participated in a 6-week crossover trial. The artificial saliva was rated significantly lower than the chewing gum for effectiveness, taste and a global assessment. No preference differences were found for gender and age, although older subjects rated the artificial saliva with a higher mark. Thirty-nine subjects (60%) preferred chewing gum, 15% (n=10) preferred the artificial saliva. Therefore, both chewing gum and artificial saliva could play an important role in the palliative care of xerostomia in HD patients.

Chewing gum and a saliva substitute alleviate thirst and xerostomia in patients on haemodialysis.

BACKGROUND: Most patients on haemodialysis (HD) have to maintain a fluid-restricted diet to prevent a high interdialytic weight gain (IWG). The prevalence of xerostomia (the feeling of a dry mouth) is higher in HD patients than in controls. Recently, we demonstrated that xerostomia and thirst were positively correlated with IWG in HD patients. Thus, this may play a role as a stimulus for fluid intake between dialysis sessions. The aim of the present study was to investigate the effect of chewing gum or a saliva substitute on xerostomia, thirst and IWG. METHODS: This study was a randomized two-treatment crossover design with repeated measures. After the use of chewing gum or saliva substitute for 2 weeks, a wash-out period of 2 weeks was introduced and hereafter the other regimen was carried out. Xerostomia and thirst were assessed by validated questionnaires as xerostomia inventory (XI) and dialysis thirst inventory (DTI), at baseline and after each treatment period, as were IWG and salivary flow rates. RESULTS: Sixty-five HD patients (42 men, 54.6+/-14.1 years; 23 women, 54.7+/-16.3 years) participated in this study. Chewing gum decreased XI from 29.9+/-9.5 to 28.1+/-9.1 (P<0.05). Chewing gum as well as a saliva substitute reduced DTI significantly (P<0.05), but no differences occurred for the average IWG or salivary flow rates. CONCLUSIONS: The use of chewing gum and, to a lesser extent, a saliva substitute may alleviate thirst and xerostomia in some HD patients.

Interdialytic weight gain in patients on hemodialysis is associated with dry mouth and thirst.

BACKGROUND: Patients receiving hemodialysis (HD) have to maintain a fluid-restricted diet. Severe thirst can induce noncompliance to this diet, resulting in an increase of interdialytic weight gain (IWG = weight predialysis - postdialysis) associated with poor patient outcomes. Because oral dryness may contribute to experienced thirst, we investigated the possible relation between thirst, salivary flow rate, xerostomia, and IWG. METHODS: Unstimulated (UWS) and stimulated (CH-SWS) whole saliva were collected from 94 HD patients (64 men, 54.8 +/- 15.5 years; 30 women, 59.5 +/- 18.7 years). Secretion rates of saliva were determined gravimetrically. Xerostomia was assessed with a validated Xerostomia Inventory (XI), and thirst with a newly developed Dialysis Thirst Inventory (DTI). RESULTS: Before dialysis, 36.2% of the patients had hyposalivation (UWS < or =0.15 mL/min). The XI scores had a positive relation with IWG (r=.250, P < 0.001). Gender and age differences were observed for thirst, salivary flow rates, and xerostomia. The prevalence and severity of thirst and xerostomia were greater in younger subjects. Patients with urine output did not differ from those without urine output with respect to thirst, xerostomia, and IWG. Correlations were found between thirst (DTI) and both IWG and xerostomia (XI) (r=.329, P < 0.001, respectively; r=.740, P < 0.001). Other correlations were observed between xerostomia and both the salivary flow rate and total number of medications (r=-.252, P < 0.05, respectively; r=.235, P <.05). CONCLUSION: In HD patients, xerostomia (XI) and thirst (DTI) are associated with a higher IWG. Our data provide evidence that, in HD patients, xerostomia is related to both salivary flow rate and thirst (DTI).