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.

Histatins are the major wound-closure stimulating factors in human saliva as identified in a cell culture assay.

Wounds in the oral cavity heal much faster than skin lesions. Among other factors, saliva is generally assumed to be of relevance to this feature. Rodent saliva contains large amounts of growth factors such as epidermal growth factor (EGF) and nerve growth factor (NGF). In humans, however, the identity of the involved compounds has remained elusive, especially since EGF and NGF concentrations are approximately 100,000 times lower than those in rodent saliva. Using an in vitro model for wound closure, we examined the properties of human saliva and the fractions that were obtained from saliva by high-performance liquid chromotography (HPLC) separation. We identified histatin 1 (Hst1) and histatin 2 (Hst2) as major wound-closing factors in human saliva. In contrast, the d-enantiomer of Hst2 did not induce wound closure, indicating stereospecific activation. Furthermore, histatins were actively internalized by epithelial cells and specifically used the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway, thereby enhancing epithelial migration. This study demonstrates that members of the histatin family, which up to now were implicated in the antifungal weaponry of saliva, exert a novel function that likely is relevant for oral wound healing.

In vivo release of the antimicrobial peptide hLF1-11 from calcium phosphate cement.

We studied the release of human lactoferrin 1-11 (hLF1-11), a potent antimicrobial peptide, in an animal model. Calcium phosphate cement with 50 mg/g hLF1-11 was injected into the femoral canal of 12 rabbits. One, 3, and 7 days later, four animals were terminated, and the femora excised. Sections of bone and cement were removed for histological analysis. We used liquid chromatography-mass spectrometry/mass spectrometry for semiquantitative determination of the hLF1-11 concentration. Blood samples were drawn for leukocyte count and differentiation to identify a potential immunomodulating effect of hLF1-11. After an initial burst release, the hLF1-11 concentration in cement and bone decreased steadily. This in vivo release profile is consistent with earlier in vitro studies. Tissue ingrowth into the cement, without signs of inflammation or necrosis, was observed. Leukocytosis or a shift in leukocyte differentiation did not occur. The carrier released over 99% of the hLF1-11, resulting in peak concentrations at the cement-bone interface. This indicates that hLF1-11 could become a valuable prophylactic agent in osteomyelitis treatment.

DMBT1 confers mucosal protection in vivo and a deletion variant is associated with Crohn's disease.

BACKGROUND & AIMS: Impaired mucosal defense plays an important role in the pathogenesis of Crohn's disease (CD), one of the main subtypes of inflammatory bowel disease (IBD). Deleted in malignant brain tumors 1 (DMBT1) is a secreted scavenger receptor cysteine-rich protein with predominant expression in the intestine and has been proposed to exert possible functions in regenerative processes and pathogen defense. Here, we aimed at analyzing the role of DMBT1 in IBD. METHODS: We studied DMBT1 expression in IBD and normal tissues by quantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and mRNA in situ hybridization. Genetic polymorphisms within DMBT1 were analyzed in an Italian IBD case-control sample. Dmbt1(-/-) mice were generated, characterized, and analyzed for their susceptibility to dextran sulfate sodium-induced colitis. RESULTS: DMBT1 levels correlate with disease activity in inflamed IBD tissues. A highly significant fraction of the patients with IBD displayed up-regulation of DMBT1 specifically in the intestinal epithelial surface cells and Paneth cells. A deletion allele of DMBT1 with a reduced number of scavenger receptor cysteine-rich domain coding exons is associated with an increased risk of CD (P = .00056; odds ratio, 1.75) but not for ulcerative colitis. Dmbt1(-/-) mice display enhanced susceptibility to dextran sulfate sodium-induced colitis and elevated Tnf, Il6, and Nod2 expression levels during inflammation. CONCLUSIONS: DMBT1 may play a role in intestinal mucosal protection and prevention of inflammation. Impaired DMBT1 function may contribute to the pathogenesis of CD.

Histatin-derived monomeric and dimeric synthetic peptides show strong bactericidal activity towards multidrug-resistant Staphylococcus aureus in vivo.

Homodimerization of histatin-derived peptides generally led to improved bactericidal activity against Staphylococcus aureus in vitro. In vivo, monomers and dimers were equally active in killing bacteria in mice with a soft tissue infection. Altogether, these peptides are promising compounds for the development of novel therapeutics against infections with drug-resistant bacteria.

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.

Oral diseases: from detection to diagnostics.

In addition to saliva, other oral components such as gingival crevicular fluid, epithelial cells, bacteria, breath, and dental plaque have diagnostic potential. For oral diseases such as caries and periodontal disease, visual diagnosis is usually adequate, but objective diagnostic tests with predictive value are desired. Therefore, prediction models like the Cariogram have been developed that also include oral aspects such as saliva secretion, buffering capacity, and Streptococcus mutans counts for the prediction of caries. Correlation studies on salivary components and caries have not been conclusive, but correlation studies on functional aspects, such as saliva-induced bacterial aggregation and caries, look promising. Modern proteomic techniques make it possible to study simultaneously the many salivary components involved in these functions.

The effect of saliva composition on texture perception of semi-solids.

Saliva is expected to be of significance for the perception of food stimuli in the mouth. Mixing the food with saliva, including breakdown and dilution, is considered to be of large importance for semi-solids as these products are masticated without chewing. It is known that there are large variations in composition of saliva originating from different glands and different subjects. In this study we investigated how variations in salivary characteristics affect sensory perception. Eighteen trained subjects participated in the study. Saliva was collected at rest and during three types of stimulation (odour, parafilm chewing and citric acid), and flow rates were determined. The collected saliva was analyzed for protein concentration, buffer capacity, mucin level and alpha-amylase activity. The salivary components measured in this study varied considerably among subjects, but also within subjects as a result of different means of stimulation. Variations in salivary components were correlated with sensory perception of a number of flavour, mouth feel and after feel attributes in the semi-solids mayonnaise and custard dessert. Total protein concentration and alpha-amylase activity were observed to correlate most strongly with texture perception.

Distinct bactericidal activities of bovine lactoferrin peptides LFampin 268-284 and LFampin 265-284: Asp-Leu-Ile makes a difference.

Two lactoferrampin (LFampin) peptides derived from bovine lactoferrin were compared with respect to their bactericidal activities. LFampin 265-284 killed a set of Gram-positive bacteria that were resistant to LFampin 268-284. The presence of 265Asp-Leu-267Ile did not simply lead to an overall increased potency, since higher concentrations of LFampin 265-284 than LFampin 268-284 were needed to kill the Gram-negative bacteria that were tested. The Asp-Leu-Ile sequence enhances the propensity of LFampin to adopt an alpha-helix, as shown by circular dichroism spectroscopy. These results suggest that the helical conformation of the peptide is an important determinant of the susceptibility of Gram-positive bacteria.

A generalized physiological hyperreactivity to acute stressors in hypertensives.

Hypertensives have consistently been found to have a more reactive cardiovascular system than normotensives. In the present study, it was examined whether this enhanced cardiovascular stress reactivity generalizes to the hypothalamus-pituitary-adrenal (HPA) axis and the immune system. Forty-two unmedicated hypertensives and 21 normotensive controls performed five passive coping and active coping stressful tasks in the laboratory. In addition to the expected greater mean diastolic blood pressure reactivity to the tasks, hypertensives exhibited enhanced (baseline corrected) task salivary cortisol and secretory immunoglobulin A (S-IgA) levels. Moreover, correlations were found between blood pressure responses and task related cortisol activity and between baseline blood pressure levels and task-induced S-IgA levels. These results indicate that hypertensives not only have a hyperreactive cardiovascular system, but also an enhanced HPA axis and immune system reactivity to stress. A central stress mechanism may be responsible for the heightened generalized stress response in hypertensives.

Histatin and lactoferrin derived peptides: antimicrobial properties and effects on mammalian cells.

In order to analyze the clinical potential of two antimicrobial peptides, human lactoferrin 1-11 (hLF1-11) and synthetic histatin analogue Dhvar-5, we measured the killing effect on bacteria, and the potential toxicity on erythrocytes and bone cells. The antimicrobial activity was determined in a killing assay on six strains, including methicillin resistant Staphylococcus Aureus. The effect on human erythrocytes and MC3T3 mouse bone cells was measured with a hemolysis assay and a viability assay, respectively. Both hLF1-11 and Dhvar-5 dose-dependently killed all bacterial strains, starting at concentrations of 6 microg/mL. hLF1-11 had no effect on mammalian cells at concentrations up to 400 microg/mL, but Dhvar-5 induced significant hemolysis (37% at 200 microg/mL) and bone cell death (70% at 400 microg/mL). This indicates that both peptides are able to kill various resistant and non-resistant bacteria, but Dhvar-5 may exert a cytotoxic effect on host cells at higher concentrations.

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).

Neuroendocrine regulation of salivary IgA synthesis and secretion: implications for oral health.

Secretory immunoglobulin A (S-IgA) represents the main adaptive immune mechanism in the oral cavity. The regulation of secretion and synthesis of S-IgA is not only dependent on prior antigenic stimulation, but is also under strong neuroendocrine control. Thus, alterations in neuroendocrine functioning (such as induced by stress, exercise, pregnancy, menstrual cycle, and pharmacological interventions) may affect salivary IgA levels. This review deals with the neuroendocrine regulation of synthesis and secretion of salivary IgA and its potential role in the maintenance of oral health.

Continuous-release or burst-release of the antimicrobial peptide human lactoferrin 1-11 (hLF1-11) from calcium phosphate bone substitutes.

OBJECTIVES: In order to identify possible drug delivery systems against resistant bone infection, we determined the release of the antimicrobial peptide (AMP) human lactoferrin 1-11 (hLF1-11) from commercially available bone substitutes. METHODS: We combined six calcium phosphate cements and six granule-types with 5 mg/g hLF1-11 and measured its availability and release in vitro from cements (7 days) and granules (3 days). The integrity and antimicrobial activity of the hLF1-11 that was released during the first 24 h were measured, using mass spectrometry, and a killing assay on methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: Most of the cements showed burst release followed by low-level continuous release, whereas the coated granules showed high burst release for 24 h. After release the peptide was active (in nine of 12 materials) and intact. CONCLUSIONS: Different release profiles may be obtained by choosing the appropriate carrier, which supports the feasibility of biodegradable carriers releasing AMPs against resistant infections.

Toothbrushing affects the protein composition of whole saliva.

Saliva is increasingly used for diagnostic purposes, but little is known about the potential effect of toothbrushing on the composition of saliva. Therefore, saliva collected before and 0, 15, 30 and 45 min after toothbrushing according to the Bass method was analysed for relevant protein composition. The flow rate initially increased (+15%) after toothbrushing, followed by a decrease after 15 min (-15%). The concentration of salivary immunoglobulin A (S-IgA) decreased (-40%) whereas the albumin concentration increased (+400%) within 20 min. Flow rate and S-IgA concentration returned to baseline values within 30 min. The albumin concentration, however, was still increased after 45 min. No significant increases were observed in the concentrations of total protein and amylase. Our results show that toothbrushing has a significant effect on the protein composition of saliva, by contamination with serum constituents. This should be taken into account when saliva is used for diagnostic purposes.