Anita Vreugdenhil

Cutting Edge: Cationic Antimicrobial Peptides Block the Binding of Lipopolysaccharide (LPS) to LPS Binding Protein

We investigated the mechanism by which cationic antimicrobial peptides block the activation of macrophages by LPS. The initial step in LPS signaling is the transfer of LPS to CD14 by LPS binding protein (LBP). Because many cationic antimicrobial peptides bind LPS, we asked whether these peptides block the binding of LPS to LBP. Using an assay that measures the binding of LPS to immobilized LBP, we show for the first time that a variety of structurally diverse cationic antimicrobial peptides block the interaction of LPS with LBP. The relative ability of different cationic peptides to block the binding of LPS to LBP correlated with their ability to block LPS-induced TNF-α production by the RAW 264.7 macrophage cell line.

Lipopolysaccharide (LPS)-Binding Protein Mediates LPS Detoxification by Chylomicrons

Chylomicrons have been shown to protect against endotoxin-induced lethality. LPS-binding protein (LBP) is involved in the inactivation of bacterial toxin by lipoproteins. The current study examined the interaction among LBP, chylomicrons, and bacterial toxin. LBP was demonstrated to associate with chylomicrons and enhance the amount of LPS binding to chylomicrons in a dose-dependent fashion. In addition, LBP accelerated LPS binding to chylomicrons. This LBP-induced interaction of LPS with chylomicrons prevented endotoxin toxicity, as demonstrated by reduced cytokine secretion by PBMC. When postprandial circulating concentrations of chylomicrons were compared with circulating levels of low density lipoprotein, very low density lipoprotein, and high density lipoprotein, chylomicrons exceeded the other lipoproteins in LPS-inactivating capacity. Furthermore, highly purified lipoteichoic acid, an immunostimulatory component of Gram-positive bacteria, was detoxified by incubation with LBP and chylomicrons. In conclusion, our results indicate that LBP associates with chylomicrons and enables chylomicrons to rapidly bind bacterial toxin, thereby preventing cell activation. Besides a role in the detoxification of bacterial toxin present in the circulation, we believe that LBP-chylomicron complexes may be part of a local defense mechanism of the intestine against translocated bacterial toxin.

LPS-binding protein circulates in association with apoB-containing lipoproteins and enhances endotoxin-LDL/VLDL interaction

LPS-binding protein (LBP) and serum lipoproteins cooperate in reducing the toxic properties of LPS. In the present study, we demonstrate that LBP circulates in association with LDL and VLDL in healthy persons. ApoB was found to account at least in part for the interaction of LBP with LDL and VLDL. Although LBP interacted with purified apoA-I in vitro, no association of LBP with apoA-I or HDL was found in serum. Consistent with the observed association of LBP with LDL and VLDL, these lipoproteins also were demonstrated to be the predominant LPS-binding lipoproteins. Most interestingly, the association of LBP with LDL and VLDL strongly enhanced the capacity of these lipoproteins to bind LPS. Because this function of LBP is of utmost importance during infection, the association of LBP and LPS with lipoproteins was also studied in serum from septic patients. In septic serum containing high LBP levels and a markedly altered lipoprotein spectrum, most of the LBP is associated with LDL and VLDL, although some LBP appeared to circulate free from lipoproteins. Also in this serum, LPS was found to bind predominantly to LDL and VLDL. The observed binding of LBP and LPS to LDL and VLDL, as well as the LBP-dependent incorporation of LPS into these lipoproteins, emphasizes a crucial role for circulating LBP-LDL/VLDL complexes in the scavenging of LPS.

Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study.

AIM To assesses the safety and efficacy of Aspergillus niger prolyl endoprotease (AN-PEP) to mitigate the immunogenic effects of gluten in celiac patients. METHODS Patients with initial diagnosis of celiac disease as confirmed by positive serology with subtotal or total villous atrophy on duodenal biopsies who adhere to a strict gluten-free diet (GFD) resulting in normalised antibodies and mucosal healing classified as Marsh 0 or I were included. In a randomised double-blind placebo-controlled pilot study, patients consumed toast (approximately 7 g/d gluten) with AN-PEP for 2 wk (safety phase). After a 2-wk washout period with adherence of the usual GFD, 14 patients were randomised to gluten intake with either AN-PEP or placebo for 2 wk (efficacy phase). Measurements at baseline included complaints, quality-of-life, serum antibodies, immunophenotyping of T-cells and duodenal mucosa immunohistology. Furthermore, serum and quality of life questionnaires were collected during and after the safety, washout and efficacy phase. Duodenal biopsies were collected after the safety phase and after the efficacy phase. A change in histological evaluation according to the modified Marsh classification was the primary endpoint. RESULTS In total, 16 adults were enrolled in the study. No serious adverse events occurred during the trial and no patients withdrew during the trial. The mean score for the gastrointestinal subcategory of the celiac disease quality (CDQ) was relatively high throughout the study, indicating that AN-PEP was well tolerated. In the efficacy phase, the CDQ scores of patients consuming gluten with placebo or gluten with AN-PEP did not significantly deteriorate and moreover no differences between the groups were observed. During the efficacy phase, neither the placebo nor the AN-PEP group developed significant antibody titers. The IgA-EM concentrations remained negative in both groups. Two patients were excluded from entering the efficacy phase as their mucosa showed an increase of two Marsh steps after the safety phase, yet with undetectable serum antibodies, while 14 patients were considered histologically stable on gluten with AN-PEP. Also after the efficacy phase, no significant deterioration was observed regarding immunohistological and flow cytometric evaluation in the group consuming placebo compared to the group receiving AN-PEP. Furthermore, IgA-tTG deposit staining increased after 2 wk of gluten compared to baseline in four out of seven patients on placebo. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits. CONCLUSION AN-PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN-PEP.

Serum I-FABP as marker for enterocyte damage in coeliac disease and its relation to villous atrophy and circulating autoantibodies

Enterocyte damage is the hallmark of coeliac disease (CD) resulting in malabsorption. Little is known about the recovery of enterocyte damage and its clinical consequences. Serum intestinal fatty acid binding protein (I‐FABP) is a sensitive marker to study enterocyte damage.

Lipopolysaccharide-binding protein is vectorially secreted and transported by cultured intestinal epithelial cells and is present in the intestinal mucus of mice.

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host’s response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.

The duration of effect of infliximab maintenance treatment in paediatric Crohn's disease is limited

Aliment Pharmacol Ther 2011; 33: 243–250

Enteral administration of high-fat nutrition before and directly after hemorrhagic shock reduces endotoxemia and bacterial translocation

Objective:To determine whether potential enhancement of endotoxin neutralization via high-fat enteral nutrition affects endotoxemia and bacterial translocation after hemorrhage. Summary Background Data:Endotoxin and bacterial translocation due to gut barrier failure are important initiating events in the pathogenesis of sepsis after hemorrhage. Systemic inhibition of endotoxin activity attenuates bacterial translocation and distant organ damage. Triacylglycerol-rich lipoproteins constitute a physiological means of binding and neutralizing endotoxin effectively. We hypothesized that enhancement of triacylglycerol-rich lipoproteins via high-fat enteral nutrition would reduce endotoxemia and prevent bacterial translocation. Methods:A rat model of nonlethal hemorrhagic shock was used. Hemorrhagic shock (HS) rats were divided into 3 groups: rats starved overnight (HS-S); rats fed with a low-fat enteral diet (HS-LF), and rats receiving a high-fat enteral diet (HS-HF). Results:Circulating triacylglycerol and apolipoprotein B, reflecting the amount of triacylglycerol-rich lipoproteins, were elevated in HS-HF rats compared with both HS-S rats (P ≤ 0.005 and P ≤ 0.05, respectively) and HS-LF rats (P ≤ 0.005 and P ≤ 0.05). Circulating endotoxin was lower in HS-HF rats (7.2 ± 10.2 pg/ml) compared with both HS-S rats (29.1 ± 13.4 pg/ml, P ≤ 0.005) and HS-LF rats (29.9 ± 5.2 pg/ml, P ≤ 0.005). In line, bacterial translocation was lower in HS-HF rats (incidence 4/8 rats; median 3 [range 0–144] cfu/g) compared with both HS-S rats (8/8; 212 [60–483] cfu/g; P = 0.006), and HS-LF rats (8/8; 86 [30–209] cfu/g; P = 0.002). Conclusion:This study is the first to show that high-fat enteral nutrition, leading to increased plasma triacylglycerol and apolipoprotein B levels, significantly decreases endotoxemia and bacterial translocation after hemorrhage.

Additional value of serum I-FABP levels for evaluating celiac disease activity in children

Abstract Objectives. Addition of a non-invasive marker for intestinal damage to the currently used parameters for celiac disease activity (symptoms, serologic tests and biopsy) might further improve clinical management of celiac disease (CD). Intestinal fatty acid binding protein (I-FABP) is a cytosolic enterocyte protein and sensitive marker for enterocyte damage in the small intestine. We investigated whether serum I-FABP levels can reliably identify villous atrophy in children with a positive CD antibody screening. Moreover, the recovery of I-FABP levels after gluten free diet (GFD) was studied. Methods. I-FABP levels were analyzed retrospectively in 49 children with biopsy proven CD and in 19 patients with a positive screening but without histological confirmation of CD. Blood was collected before biopsy and repeatedly after the onset of GFD. Results. Initial I-FABP concentrations in CD (median 458 pg/ml) were significantly (p < 0.001) elevated compared to controls (median 20 pg/ml). In the control group, only two of 19 children were found to have elevated I-FABP levels, of which one was subsequently diagnosed with CD after gluten challenge. I-FABP concentrations correlated with severity of villous atrophy. In all CD patients, I-FABP levels decreased quickly after GFD and normalized in 80% of patients within 12 weeks. Conclusions. Elevated I-FABP levels accurately predict villous atrophy in children with a positive serologic test for CD (positive predictive value 98%). In addition, measurement of I-FABP enables monitoring the response to GFD.

Arginine appearance and nitric oxide synthesis in critically ill infants can be increased with a protein-energy–enriched enteral formula

BACKGROUND Arginine is considered an essential amino acid during critical illness in children, and supplementation of arginine has been proposed to improve arginine availability to facilitate nitric oxide (NO) synthesis. Protein-energy-enriched enteral formulas (PE-formulas) can improve nutrient intake and promote anabolism in critically ill infants. However, the effect of increased protein and energy intake on arginine metabolism is not known. OBJECTIVE We investigated the effect of a PE-formula compared with that of a standard infant formula (S-formula) on arginine kinetics in critically ill infants. DESIGN A 2-h stable-isotope tracer protocol was conducted in 2 groups of critically ill infants with respiratory failure because of viral bronchiolitis, who received either a PE-formula (n = 8) or S-formula (n = 10) in a randomized, blinded, controlled setting. Data were reported as means ± SDs. RESULTS The intake of a PE-formula in critically ill infants (aged 0.23 ± 0.14 y) resulted in an increased arginine appearance (PE-formula: 248 ± 114 μmol · kg(-1) · h(-1); S-formula: 130 ± 53 μmol · kg(-1) · h(-1); P = 0.012) and NO synthesis (PE-formula: 1.92 ± 0.99 μmol · kg(-1) · h(-1); S-formula: 0.84 ± 0.36 μmol · kg(-1) · h(-1); P = 0.003), whereas citrulline production and plasma arginine concentrations were unaffected. CONCLUSION In critically ill infants with respiratory failure because of viral bronchiolitis, the intake of a PE-formula increases arginine availability by increasing arginine appearance, which leads to increased NO synthesis, independent of plasma arginine concentrations. This trial was registered at www.trialregister.nl as NTR515.