The effect of dietary phytosphingosine on cholesterol levels and insulin sensitivity in subjects with the metabolic syndrome.

BACKGROUND: Sphingolipids, like phytosphingosine (PS) are part of cellular membranes of yeasts, vegetables and fruits. Addition of PS to the diet decreases serum cholesterol and free fatty acid (FFA) levels in rodents and improves insulin sensitivity. OBJECTIVE: To study the effect of dietary supplementation with PS on cholesterol and glucose metabolism in humans. METHODS: Twelve men with the metabolic syndrome (MetS) (according to the International Diabetes Federation (IDF) criteria; age 51+/-2 years (mean+/-s.e.m.); body mass index (BMI) 32+/-1 kg/m(2)) were randomly assigned to 4 weeks of PS (500 mg twice daily) and 4 weeks of placebo (P) in a double-blind cross-over study, with a 4-week wash-out period between both interventions. At the end of each intervention anthropometric measures and serum lipids were measured and an intravenous glucose tolerance test (IVGTT) was performed. RESULTS: Phytosphingosine did not affect body weight and fat mass compared with P. PS decreased serum total cholesterol (5.1+/-0.3 (PS) vs 5.4+/-0.3 (P) mmol/l; P<0.05) and low-density lipoprotein (LDL)-cholesterol levels (3.1+/-0.3 (PS) vs 3.4+/-0.3 (P) mmol/l; P<0.05), whereas it did not alter serum triglyceride and high-density lipoprotein (HDL)-cholesterol levels. In addition, PS lowered fasting plasma glucose levels (6.2+/-0.3 (PS) vs 6.5+/-0.3 (P) mmol/l; P<0.05). PS increased the glucose disappearance rate (K-value) by 9.9% during the IVGTT (0.91+/-0.06 (PS) vs 0.82+/-0.05 (P) %/min; P<0.05) at similar insulin levels, compared with P, thus implying enhanced insulin sensitivity. PS induced only minor gastrointestinal side effects. CONCLUSION: Dietary supplementation of PS decreases plasma cholesterol levels and enhances insulin sensitivity in men with the MetS.

Development of a new test for the global fibrinolytic capacity in whole blood.

BACKGROUND: The development of global tests for the fibrinolytic capacity in blood is hampered by the low base-line fibrinolytic activity in blood, by the involvement of both plasmatic components and blood cells in the fibrinolytic system and by the loss of fibrinolytic activity as a result of the action of plasminogen activator inhibitor-1 (PAI-1). OBJECTIVE: To develop a new test for the global fibrinolytic capacity (GFC) of whole blood samples. METHODS AND RESULTS: Collection of blood in thrombin increased the subsequent generation of fibrin degradation products. This was ascribed to rapid clot formation and concomitant reduction of in vitro neutralization of tissue-type plasminogen activator (tPA) by PAI-1. On the basis of this observation, the following test was designed: blood samples were collected in thrombin with and without aprotinin and clots were incubated for 3 h at 37 degrees C. The GFC was assessed from the difference between the fibrin degradation products in the two sera. The assay was applied to blood samples from patients and healthy subjects. Other hemostasis parameters were determined in plasma samples taken simultaneously. The GFC varied considerably (normal range 0.13-13.6 microg mL(-1)); physical exercise strongly increased the GFC. Statistically significant correlations were found with tPA activity, PAI-1 activity and fibrinogen level. A mixture of antibodies against tPA and urokinase-type plasminogen activator (uPA) completely inhibited the GFC. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFI) accelerated fibrinolysis 8-fold. CONCLUSION: The new test represents a global assessment of the main fibrinolytic factors in plasma and potentially those associated with blood cells.

Kinetic characteristics of acidic and alkaline ceramidase in human epidermis.

It has recently become evident that at least five ceramidase (CDase) isoforms are present in human epidermis, and that specifically acidic CDase (aCDase) and alkaline CDase (alkCDase) activities increase during keratinocyte differentiation, and thus might play a pivotal role(s) in permeability barrier function. Prior to investigating their possible roles in the epidermal barrier function, it is necessary to characterize basic kinetic parameters for these enzymes, as well as to determine the effects of the established CDase inhibitors and their activities. In this study, assays for both aCDase and alkCDase activities in fully differentiated human epidermis were optimized using a radiolabeled substrate. These studies revealed that aCDase activity is substantially higher than alkCDase activity, and that both isoenzymes are inhibited by a CDase inhibitor N-oleylethanolamine. These findings were also confirmed using an in situ enzyme assay.

Can we gain precision by sampling with probabilities proportional to size in surveying recent landscape changes in the Netherlands?

Seventy-two squares of 100 ha were selected by stratified random sampling with probabilities proportional to size (pps) to survey landscape changes in the period 1996-2003. The area of the plots times the urbanization pressure was used as a size measure. The central question of this study is whether the sampling with probabilities proportional to size leads to gain in precision compared to equal probability sampling. On average 1.03 isolated buildings per 100 ha have been built, while 0.90 buildings per 100 ha have been removed, leading to a net change of 0.13 building per 100 ha. The area with unspoiled natural relief has been reduced by 2.3 ha per 100 ha, and the length of linear relicts by 137 m per 100 ha. On average 74 m of linear green elements have been planted per 100 ha, while 106 m have been removed, leading to a net change of -31 m per 100 ha. For the state variables 'unspoiled natural relief', 'linear relicts', 'removed linear green elements', and 'new-removed linear green elements' there is a gain in precision due to the pps-sampling. For the remaining state variables there is no gain or even a loss of precision ('new buildings', 'removed buildings', 'new-removed buildings', 'new linear green elements'). Therefore, if many state variables must be monitored or when interest is not only in the change but also in the current totals, we recommend to keep things simple, and to select plots with equal probability.

A novel and sensitive method for the detection of T cell stimulatory epitopes of alpha/beta- and gamma-gliadin.

BACKGROUND: It is now generally accepted that coeliac disease (CD) is caused by inflammatory T cell responses to gluten peptides bound to HLA-DQ2 or -DQ8 molecules. There is overwhelming evidence that CD patients can mount T cell responses to peptides found in both alpha-gliadin and gamma-gliadin molecules. Assays that would detect the presence or absence of such peptides in food would thus be accurate indicators of safety for consumption by CD patients. AIMS: The development of a sensitive method to detect T cell stimulatory epitopes of alpha-gliadin and gamma-gliadin molecules in food products. METHODS: Monoclonal antibodies (mAb) were raised against peptides encoding the T cell stimulatory epitopes of alpha-gliadin (amino acids (aa) 59-71) and aa gamma-gliadin (aa 142-153 and aa 147-159). These mAb competition assays were developed that quantitatively detect T cell stimulatory epitopes present on both intact proteins and peptides of sizes recognisable by CD4(+) T cells. RESULTS: With the mAb based competition assays, T cell epitopes were detected in pepsin/trypsin digests of wheat proteins and ethanol extracts of various food products, with detection levels lower than those reached with gluten specific T cells. Moreover, the presence of T cell stimulatory epitopes was also detected in preparations of barley, rye, and triticale, other cereals known to be toxic for CD patients. CONCLUSIONS: A new antibody based method has been developed, detecting the presence of T cell stimulatory gluten peptides. This can be used to further ensure the safety of food consumed by CD patients.

Two monoclonal antibodies to D-dimer-specific inhibitors of fibrin polymerization.

D-dimer of human fibrin was used as antigen to obtain monoclonal antibodies (mAbs). We have obtained 16 hybridomas producing mAbs of different specificity. Only two of these mAbs inhibited fibrin polymerization. They are of the IgG-class. One mAb (II-4d) inhibited fibrin polymerization to 100% and another (II-3b) to 60% at a molar ratio mAb/fibrin=1.0. Fab-fragments of these mAbs inhibited fibrin polymerization completely at the same molar ratio. The epitopes for the mAbs studied are situated in the NH2-terminal part of the gamma-chain in fibrin D-domain. Electron microscopy showed that fibrin was in monomeric form in the presence of these mAbs or their Fab-fragments. Thus, these mAbs stop the initial step of fibrin polymerization, i.e. protofibril formation. Only one site of protofibril formation is known now in COOH-terminal half of the D-domain gamma chain named "a" site, which is complementary to the "A" site in the central E-domain of fibrin molecule. Our experiment with immobilized GPRP showed that the "a" site in fibrin D-fragment preserved its binding activity to GPRP when the D-fragment was complexed with mAbs-inhibitors of fibrin polymerization. Thus, these two mAbs inhibit fibrin polymerization not by blocking the sites "a" but either by blocking another (not "a") specific site in D-domain or by steric hindrance of highly organized fibrin polymerization process.

Associations of plasma fibrinogen assays, C-reactive protein and interleukin-6 with previous myocardial infarction.

BACKGROUND: The association of plasma fibrinogen with myocardial infarction (MI) may (like that of C-reactive protein, CRP) be a marker of subclinical inflammation, mediated by cytokines such as interleukin-6 (IL-6). There are well-recognized discrepancies between commonly performed fibrinogen assays. Increased ratio of clottable fibrinogen to intact fibrinogen (measured by a recently developed immunoassay) has been proposed as a measure of hyperfunctional fibrinogen, and is elevated in acute MI. OBJECTIVE: To compare the associations of intact fibrinogen and four routine fibrinogen assays (two von Clauss assays; one prothrombin-time derived; and one immunonephelometric) in a case-control study of previous MI. PATIENTS/METHODS: Cases (n=399) were recruited 3-9 months after their event; 413 controls were age- and sex- matched from the case-control study local population. Intact fibrinogen was measured in 50% of subjects. RESULTS: All routine fibrinogen assays showed high intercorrelations (r=0.82-0.93) and significant (P<0.0001) increased mean levels in cases vs. controls. These four routine assays correlated only moderately with intact fibrinogen (r=0.45-0.62), while intact fibrinogen showed only a small, nonsignificant increase in cases vs. controls. Consequently, the ratio of each of the four routine assays to the intact fibrinogen assay was significantly higher (P<0.0003) in cases vs. controls. Each fibrinogen assay correlated with plasma levels of CRP and IL-6 (which were also elevated in cases vs. controls). Each routine fibrinogen assay remained significantly elevated in cases vs. controls after further adjustment for C-reactive protein and interleukin-6. CONCLUSIONS: These data provide evidence for acquired, increased hyperfunctional plasma fibrinogen in MI survivors, which is not associated with markers of inflammatory reactions. The causes and significance of these results remain to be established in prospective studies.

Is Candida albicans a trigger in the onset of coeliac disease?

Coeliac disease is a T-cell-mediated autoimmune disease of the small intestine that is induced by ingestion of gluten proteins from wheat, barley, or rye. We postulate that Candida albicans is a trigger in the onset of coeliac disease. The virulence factor of C albicans-hyphal wall protein 1 (HWP1)-contains aminoacid sequences that are identical or highly homologous to known coeliac disease-related alpha-gliadin and gamma-gliadin T-cell epitopes. HWP1 is a transglutaminase substrate, and is used by C albicans to adhere to the intestinal epithelium. Furthermore, tissue transglutaminase and endomysium components could become covalently linked to the yeast. Subsequently, C albicans might function as an adjuvant that stimulates antibody formation against HWP1 and gluten, and formation of autoreactive antibodies against tissue transglutaminase and endomysium.

A neoantigenic determinant in the D-dimer fragment of fibrin.

Monoclonal antibody (mAb) III-3b binds D-dimer with K(d)=1.4 x 10(-10) M without cross-reaction with fibrin(ogen). The epitope for this mAb is in Bbeta134-190, presumably in Bbeta155-160. The latter site is buried in the coiled coil structure of fibrin(ogen) but it is exposed as a neoantigenic determinant in D-dimer upon plasmic lysis of fibrin. mAb III-3b may be used as a tool for immunodiagnostic quantification of D-dimer in blood plasma.