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BACKGROUND: Short-chain fatty acids (SCFAs), fermentation products of undigested fibers, are considered beneficial for colonic health. High plasma concentrations are potentially harmful; therefore, information about systemic SCFA clearance is needed before therapeutic use of prebiotics or colonic SCFA administration. OBJECTIVE: The aim of this study was to investigate the effect of rectal butyrate administration on SCFA interorgan exchange. METHODS: Twelve patients (7 men; age: 66.4 +/- 2.0 y; BMI 24.5 +/- 1.4 kg/m(2)) undergoing upper abdominal surgery participated in this randomized placebo-controlled trial. During surgery, 1 group received a butyrate enema (100 mmol sodium butyrate/L; 60 mL; n = 7), and the other group a placebo (140 mmol 0.9% NaCl/L; 60 mL; n = 5). Before and 5, 15, and 30 min after administration, blood samples were taken from the radial artery, hepatic vein, and portal vein. Plasma SCFA concentrations were analyzed, and fluxes from portal-drained viscera, liver, and splanchnic area were calculated and used for the calculation of the incremental area under the curve (iAUC) over a 30-min period. RESULTS: Rectal butyrate administration led to higher portal butyrate concentrations at 5 min compared with placebo (92.2 +/- 27.0 mumol/L vs. 14.3 +/- 3.4 mumol/L, respectively; P < 0.01). In the butyrate-treated group, iAUCs of gut release (282.8 +/- 133.8 mumol/kg BW . 0.5 h) and liver uptake (-293.7 +/- 136.0 mumol/kg BW . 0.5 h) of butyrate were greater than in the placebo group [-16.6 +/- 13.4 mumol/kg BW . 0.5 h (gut release) and 16.0 +/- 13.8 mumol/kg BW . 0.5 h (liver uptake); P = 0.01 and P < 0.05, respectively]. As a result, splanchnic butyrate release did not differ between groups. CONCLUSION: After colonic butyrate administration, splanchnic butyrate release was prevented in patients undergoing upper abdominal surgery. These observations imply that therapeutic colonic SCFA administration at this dose is safe. The trial was registered at clinicaltrials.gov as NCT02271802.

BACKGROUND: COPD is accepted to be a multicomponent disease with various comorbidities. The contribution of the gastrointestinal tract to the systemic manifestation of COPD has never been investigated. This metabolically active organ may experience recurring local oxygen deficits during daily life, leading to disturbed intestinal integrity in COPD patients. METHOD: 18 patients with moderate COPD (mean FEV1: 55+/-3%predicted) and 14 matched healthy controls were tested on two occasions, a baseline measurement at rest and, at another day, during the performance of activities of daily living (ADLs). To assess enterocyte damage, plasma intestinal fatty acid binding protein (IFABP) levels were determined, whereas urinary excretion of orally ingested sugar probes was measured using liquid chromatography and mass spectrometry to assess gastrointestinal permeability. RESULTS: Plasma IFABP concentrations were not different between COPD patients and healthy controls at rest. In contrast, 0-3h urinary lactulose/rhamnose and sucralose/erythritol ratios and 5-24h urinary sucralose/erythritol ratios were significantly higher in COPD patients compared to controls, indicating increased permeability of the small intestine and colon. Furthermore, the performance of ADLs led to significantly increased plasma IFABP concentrations in COPD patients but not in control subjects. In line, the intestinal permeability difference between COPD patients and controls was intensified. CONCLUSION: Besides an altered intestinal permeability in COPD patients at rest, performing ADLs led to enterocyte damage in addition to intestinal hyperpermeability in COPD patients but not in controls, indicating functional alteration in the gastrointestinal tract. Hence, intestinal compromise should be considered as a new component of the multisystem disorder COPD. CLINICAL TRIAL REGISTRATION: ISRCTN33686980.

Familial combined hyperlipidemia (FCHL) is the most commonly inherited hyperlipidemia in man, with a frequency of +/-1% in the general population and approximately 10% in myocardial infarction survivors. A genomic scan in 18 Dutch FCHL families resulted in the identification of several loci with evidence for linkage. One of these regions, 1p36.2, contains TNFRSF1B which encodes one of the tumor necrosis factor receptors. An intron 4 polymorphic CA-repeat was used to confirm linkage to FCHL. Linear regression analysis using 79 independent sib pairs showed linkage with a quantitative FCHL discriminant function (P = 0.032), and, borderline, with apolipoprotein B levels (P = 0.064). Furthermore, in a case-control study, association was demonstrated since the overall CA-repeat genotype distribution was significantly different among 40 unrelated FCHL patients and 48 unrelated healthy spouse controls (P = 0.029). This difference was due to a significant increase in allele CA271 homozygotes in the FCHL patients (P = 0.019). Mutation analysis of exon 6 in 73 FCHL family members demonstrated the presence of a single nucleotide polymorphism with two alleles, coding for methionine (196M) and arginine (196R). Complete linkage disequilibrium between CA267, CA271 and CA273 and this polymorphism was detected. In 85 hyperlipidemic FCHL subjects, an association was demonstrated between soluble TNFRSF1B plasma concentrations and the CA271-196M haplotype. In conclusion, TNFRSF1B was found to be associated with susceptibility to FCHL. Our data suggest that an as yet unknown disease-associated mutation, linked to alleles 196M and CA271, plays a role in the pathophysiology of FCHL.

OBJECTIVE: Intestinal microbiota have been suggested to contribute to the development of obesity, but the mechanism remains elusive. The relationship between microbiota composition, intestinal permeability, and inflammation in nonobese and obese subjects was investigated. DESIGN AND METHODS: Fecal microbiota composition of 28 subjects (BMI 18.6-60.3 kg m-2 ) was analyzed by a phylogenetic profiling microarray. Fecal calprotectin and plasma C-reactive protein levels were determined to evaluate intestinal and systemic inflammation. Furthermore, HbA1c , and plasma levels of transaminases and lipids were analyzed. Gastroduodenal, small intestinal, and colonic permeability were assessed by a multisaccharide test. RESULTS: Based on microbiota composition, the study population segregated into two clusters with predominantly obese (15/19) or exclusively nonobese (9/9) subjects. Whereas intestinal permeability did not differ between clusters, the obese cluster showed reduced bacterial diversity, a decreased Bacteroidetes/Firmicutes ratio, and an increased abundance of potential proinflammatory Proteobacteria. Interestingly, fecal calprotectin was only detectable in subjects within the obese microbiota cluster (n = 8/19, P = 0.02). Plasma C-reactive protein was also increased in these subjects (P = 0.0005), and correlated with the Bacteroidetes/Firmicutes ratio (rs = -0.41, P = 0.03). CONCLUSIONS: Intestinal microbiota alterations in obese subjects are associated with local and systemic inflammation, suggesting that the obesity-related microbiota composition has a proinflammatory effect

TNF mediates multiple biologic activities through two distinct cell surface receptors, TNFR-p55 and TNFR-p75. TNF plays an important role in nonspecific resistance against the fungus Candida albicans. We used transgenic mice deficient for TNFR-p55 or TNFR-p75 to investigate the role of the TNFR in antifungal defense. Mice deficient for TNFR-p55 have highly impaired ability to clear infection with C. albicans and readily succumb to the infection. Also mice deficient for TNFR-p75 had a significant reduction in their ability to clear the fungus although lethality was not increased. These data demonstrate that TNFR-p55 in particular, but also TNFR-p75, plays a definite role in defense against infection with C. albicans. In NMRI mice, infection with C. albicans resulted in a significant systemic release of soluble (s)TNFR-p75. Cyclophosphamide-induced granulocytopenia led to a reduction of sTNFR-p75 release, whereas levels of bioactive TNF in response to fungal infection were increased. Release of sTNFR-p55 was not affected by induction of granulocytopenia. These observations suggest that granulocytes are a source of sTNFR-p75, possibly contributing to regulation of TNF activity during infection with C. albicans.

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The use of total parenteral nutrition (TPN) in the treatment of critically ill patients has been the subject of debate because it has been associated with disturbances in intestinal homeostasis. Important factors in maintaining the intestinal homeostasis are the intestinal microbiota and Paneth cells, which exist in a mutually amendable relationship. We hypothesized that the disturbed intestinal homeostasis in TPN-fed individuals results from an interplay between a shift in microbiota composition and alterations in Paneth cells. We studied the microbiota composition and expression of Paneth cell antimicrobial proteins in rats receiving TPN or a control diet for 3, 7, or 14 d. qPCR analysis of DNA extracts from small intestinal luminal contents of TPN-fed rats showed a shift in Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes after 14 d (P < 0.05) compared with the control group. This finding coincided with greater staining intensity for lysozyme and significantly greater mRNA expression of Paneth cell antimicrobial proteins lysozyme (P < 0.05), rat alpha-defensin 5 (P < 0.01), and rat alpha-defensin 8 (P < 0.01). Finally, 14 d of TPN resulted in greater circulating ileal lipid-binding protein concentrations (P < 0.05) and greater leakage of horseradish peroxidase (P < 0.01), which is indicative of enterocyte damage and a breached intestinal barrier. Our findings show a shift in intestinal microbiota in TPN-fed rats that correlated with changes in Paneth cell lysozyme expression (r(s) = -0.75, P < 0.01). Further studies that include interventions with microbiota or nutrients that modulate them may yield information on the involvement of microbiota and Paneth cells in TPN-associated intestinal compromise.

Adenosine is an endogenous nucleoside that can modulate the function of cells involved in the inflammatory response, such as polymorphonuclear leukocytes (PMN) and monocytes. Production and release of cytokines by activated mononuclear phagocytes is an important event in the pathogenesis of ischemia-reperfusion injury, a pathologic phenomenon that is associated with excessive ATP catabolism and subsequent local release of adenosine. The "retaliatory" metabolite adenosine has been shown to interfere with PMN function, thereby attenuating the deleterious consequences of ischemia and reperfusion. In this study, we demonstrate that adenosine inhibits the production of TNF-alpha, IL-6, and IL-8 by LPS-activated human monocytes with a differential potency. The A2 receptor-specific adenosine analogues 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine (NECA) were most effective in attenuating LPS-induced cytokine production, whereas the A1-selective adenosine analogue N6-cyclopentyladenosine (CPA) was less effective, indicating that inhibition of cytokine production by adenosine is primarily an A2 receptor-mediated event. The observed inhibitory effects were not restricted to endotoxin-induced cytokine production, because adenosine also inhibited TNF-alpha production by monocytes stimulated with the proinflammatory cytokine IL-1 beta. Again, 2-chloroadenosine and NECA reduced IL-beta-induced TNF-alpha production more potently than CPA. In contrast, adenosine enhanced production of IL-6 and IL-8 by monocytes stimulated with IL-1 beta. Furthermore, only 2-chloroadenosine, but not NECA, strongly inhibited cytokine-induced IL-6 and IL-8 production. These results suggest an additional A2 receptor-mediated mechanism of retaliatory action of adenosine under pathologic conditions where cytokine production by activated mononuclear phagocytes is involved, such as ischemia-reperfusion injury and septic shock.

Previously we demonstrated that two soluble(s) tumor necrosis factor receptors, TNF-R55 as well as sTNF-R75, are constitutively released in vitro by monocytes, and that this release was markedly enhanced after activation. Because LPS is an important activator of monocytes, we investigated the effect of LPS on sTNF-R release by monocytes. It was found that release of sTNF-R75, but not (or minimally) release of sTNF-R55, was enhanced after activation with LPS, reaching plateau levels after approximately 2 days. CD14, one of the membrane receptors for LPS, is an intermediate in this process, as shown in experiments using mAb directed against CD14. Under serum-free conditions, LPS-induced sTNF-R75 release was less as compared with release in the presence of serum, suggesting involvement of serum proteins. Addition of LPS binding protein (LBP) enhanced the LPS-induced sTNF-R75 release under serum-free conditions, but had no effect in the presence of serum. On the other hand, bactericidal/permeability-increasing protein (BPI), known to possess LPS neutralizing activity, inhibited LPS-induced sTNF-R75 release. Furthermore, cell surface expression of both types of TNF-R was shown to be controlled by LPS, LBP, and BPI. LPS caused, within 1 h, a complete reduction of TNF-R55 as well as TNF-R75 expression, followed by enhanced re-expression of both receptors after 24 h. The down-modulation of expression was increased by LBP, whereas BPI counteracted the LPS-induced down-regulation. The LPS-enhanced release of sTNF-R75, capable of inactivation of TNF, as well as LPS-induced initial down-modulation of TNF-R expression leading to postulated temporary unresponsiveness to TNF may share in a physiological mechanism to carefully control the effects of TNF.

The intestinal microbiota is increasingly acknowledged to play a crucial role in the development of obesity. A shift in intestinal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been observed in obese subjects. A similar shift has been reported in mice with deficiency of active Paneth cell alpha-defensins. We aimed at investigating changes in Paneth cell antimicrobial levels in the gut of obese subjects. Next, we studied activation of the unfolded protein response (UPR) as a possible mechanism involved in altered Paneth cell function. Paneth cell numbers were counted in jejunal sections of 15 severely obese (BMI > 35) and 15 normal weight subjects. Expression of Paneth cell antimicrobials human alpha-defensin 5 (HD5) and lysozyme were investigated using immunohistochemistry, qPCR, and western blot. Activation of the UPR was assessed with western blot. Severely obese subjects showed decreased protein levels of both HD5 and lysozyme, while Paneth cell numbers were unchanged. Lysozyme protein levels correlated inversely with BMI. Increased expression of HD5 (DEFA5) and lysozyme (LYZ) transcripts in the intestine of obese subjects prompted us to investigate a possible translational block caused by UPR activation. Binding protein (BiP) and activating transcription factor 4 (ATF4) levels were increased, confirming activation of the UPR in the gut of obese subjects. Furthermore, levels of both proteins correlated with BMI. Involvement of the UPR in the lowered antimicrobial protein levels in obese subjects was strongly suggested by a negative correlation between BiP levels and lysozyme levels. Additionally, indications of ER stress were apparent in Paneth cells of obese subjects. Our findings provide the first evidence for altered Paneth cell function in obesity, which may have important implications for the obesity-associated shift in microbiota composition. In addition, we show activation of the UPR in the intestine of obese subjects, which may underlie the observed Paneth cell compromise. Copyright (c) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

To ensure a sufficient barrier between a host and noxious luminal content, the intestinal epithelium must be equipped with efficient mechanisms to limit damage to the epithelial lining. Using a human model, we were able to investigate these mechanisms in the human gut exposed to ischaemia-reperfusion (IR) over the time course of 150 min. In 10 patients a part of jejunum, to be removed for surgical reasons, was selectively exposed to IR. Control tissue was collected, as well as tissue exposed to 30 min of ischaemia with 0, 30 or 120 min of reperfusion. Haematoxylin/eosin staining demonstrated the appearance of subepithelial spaces following 30 min of ischaemia, while the epithelial lining remained intact at this stage. Western blot for myosin light chain kinase (MLCK) revealed a significant increase in protein levels after ischaemia (p < 0.01), and selective staining of MLCK and phosphorylated MLC (pMLC) in lamina propria muscle fibres indicated that appearance of subepithelial spaces was a consequence of active villus contraction. Early during reperfusion, accumulation of pMLC was observed exclusively at the basal side of enterocytes that had lost contact with the collagen-IV-positive basement membrane. These epithelial sheets were pulled together like a zipper, even before these cells were shed. This constriction, verified by increased F-actin and pMLC double staining, accounted for a 45% reduction in virtual wound surface (p < 0.001) at 30 min of reperfusion. In addition, these mechanisms were involved in resealing remaining small epithelial defects, resulting in a fully restored epithelial lining within 120 min of reperfusion. In conclusion, we show in a human in vivo model that the human jejunum has the ability to preserve the epithelial lining during intestinal IR by rapid lamina propria contraction and zipper-like constriction of epithelial cells that are to be shed into the lumen. These newly described phenomena limit exposure to noxious luminal content. Copyright (c) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

OBJECTIVES: Fatty acid-binding proteins (FABPs) have relatively high tissue concentrations and low plasma concentrations and are released into the circulation following organ injury. We explored the utility of intestinal-(I)-FABP and liver-(L)-FABP for the diagnosis of abdominal injury in patients with multiple trauma. METHODS: This prospective study included 102 trauma patients and 30 healthy volunteers. Plasma I-FABP and L-FABP levels were measured in the emergency department (ED) by enzyme-linked immunosorbent assay (ELISA). Forty-one patients suffered from serious or severe abdominal trauma (Abbreviated Injury Score [AIS] code "ai" for abdominal injury, AISai > or = 3) and nine were moderately abdominally injured (AISai < 3). Fifty-two had no abdominal injury. RESULTS: Median I-FABP and L-FABP levels in the AISai > or = 3 group (516 pg/mL and 135 ng/mL, respectively) were significantly higher compared to the AISai < 3 group (154 pg/mL and 13 ng/mL, respectively) or those without abdominal injury (207 pg/mL and 21 ng/mL, respectively) or normal controls (108 pg/mL and 13 ng/mL, respectively). The cutoff to distinguish the ai > or = 3 is 359 pg/mL for I-FABP and 54 ng/mL for L-FABP, with 93% specificity and 75% sensitivity for I-FABP and 93% and 82% for L-FABP, respectively. CONCLUSIONS: High I-FABP and L-FABP levels correlate with relevant severity of abdominal tissue damage in patients with multiple trauma. I-FABP and L-FABP could be useful as markers for the early detection of significant abdominal injury in acute multiple trauma and identify patients who require rapid intervention.

Human intestinal ischemia-reperfusion (IR) is a frequent phenomenon carrying high morbidity and mortality. Although intestinal IR-induced inflammation has been studied extensively in animal models, human intestinal IR induced inflammatory responses remain to be characterized. Using a newly developed human intestinal IR model, we show that human small intestinal ischemia results in massive leakage of intracellular components from ischemically damaged cells, as indicated by increased arteriovenous concentration differences of intestinal fatty acid binding protein and soluble cytokeratin 18. IR-induced intestinal barrier integrity loss resulted in free exposure of the gut basal membrane (collagen IV staining) to intraluminal contents, which was accompanied by increased arteriovenous concentration differences of endotoxin. Western blot for complement activation product C3c and immunohistochemistry for activated C3 revealed complement activation after IR. In addition, intestinal IR resulted in enhanced tissue mRNA expression of IL-6, IL-8, and TNF-alpha, which was accompanied by IL-6 and IL-8 release into the circulation. Expression of intercellular adhesion molecule-1 was markedly increased during reperfusion, facilitating influx of neutrophils into IR-damaged villus tips. In conclusion, this study for the first time shows the sequelae of human intestinal IR-induced inflammation, which is characterized by complement activation, production and release of cytokines into the circulation, endothelial activation, and neutrophil influx into IR-damaged tissue.

Contains fulltext : 80930.pdf (Publisher’s version ) (Closed access) An unassigned and prominent resonance in the region from delta 2.0-2.1 ppm has frequently been found in the in vivo MR spectra of cancer patients. We demonstrated the presence of this resonance with in vivo MRS in the cyst fluid of a patient with an ovarian tumor. (1)H-NMRS on the aspirated cyst fluid of this patient confirmed the observation. A complex of resonances was observed between 2.0 and 2.1 ppm. It was also present in 11 additional ovarian cyst fluid samples randomly chosen from our biobank. The resonance complex was significantly more prominent in samples from mucinous tumors than in samples from other histological subtypes. A macromolecule (>10 kDa) was found responsible for this complex of resonances. A correlation spectroscopy (COSY) experiment revealed cross peaks of two different types of bound sialic acid suggesting that N-glycans from glycoproteins and/or glycolipids cause this resonance complex. In the literature, plasma alpha-1 acid glycoprotein (AGP), known for its high content of N-linked glycans, has been suggested to contribute to the delta 2.0-2.1 spectral region. The AGP cyst fluid concentration did not correlate significantly with the peak height of the delta 2.0-2.1 resonance complex in our study. AGP may be partly responsible for the resonance complex but other N-acetylated glycoproteins and/or glycolipids also contribute. After deproteinization of the cyst fluid, N-acetyl-L-aspartic acid (NAA) was found to contribute significantly to the signal in this spectral region in three of the 12 samples. GC-MS independently confirmed the presence of NAA in high concentration in the three samples, which all derived from benign serous tumors. We conclude that both NAA and N-acetyl groups from glycoproteins and/or glycolipids may contribute to the delta 2.0-2.1 ppm resonance complex in ovarian cyst fluid. This spectral region seems to contain resonances from biomarkers that provide relevant clinical information on the type of ovarian tumor.

BACKGROUND: Warm ischemia-reperfusion (I/R) injury plays an important role in posttransplant organ failure. In particular, organs from marginal donors suffer I/R injury. Although iron has been implicated in the pathophysiology of renal I/R injury, the mechanism of iron-mediated injury remains to be established. The authors therefore investigated the role of circulating redox-active iron in an experimental model for renal I/R injury. METHODS: Male Swiss mice were subjected to unilateral renal ischemia for 45 min, followed by contralateral nephrectomy and reperfusion. To investigate the role of circulating iron, mice were treated with apotransferrin, an endogenous iron-binding protein, or iron-saturated apotransferrin (holotransferrin). RESULTS: Renal ischemia induced a significant increase in circulating redox-active iron levels during reperfusion. Apotransferrin, in contrast to holotransferrin, reduced the amount of circulating redox-active iron and abrogated renal superoxide formation. Apotransferrin treatment did not affect I/R-induced renal apoptosis, whereas holotransferrin aggravated apoptotic cell death. Apotransferrin, in contrast to holotransferrin, inhibited the influx of neutrophils. Both apo- and holotransferrin reduced I/R-induced complement deposition, indicating that the effects of transferrin are differentially mediated by its iron and protein moiety. Finally, apotransferrin, in contrast to holotransferrin, dose-dependently inhibited the loss of renal function induced by ischemia. CONCLUSIONS: Redox-active iron is released into the circulation in the course of renal I/R. Reducing the amount of circulating redox-active iron by treatment with apotransferrin protects against renal I/R injury, inhibiting oxidative stress, inflammation, and loss of function. Apotransferrin could be used in the treatment of acute renal failure, as seen after transplantation of ischemically damaged organs.

Inhibition of complement factor C5 protects against renal ischemia-reperfusion injury: inhibition of late apoptosis and inflammation. De Vries B, Matthijsen RA, Wolfs TG, Van Bijnen AA, Heeringa P, Buurman WA. Department of General Surgery, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, Maastricht, The Netherlands. BACKGROUND: Complement has been implicated in the pathophysiology of renal ischemia-reperfusion (I/R) injury. However, the mechanism underlying complement-mediated renal I/R injury is thus far unknown. To investigate the involvement of complement in I/R injury, we studied the activation and deposition of complement in a murine model of renal I/R injury. Furthermore, we examined the effect of inhibition of complement-factor C5 on renal I/R injury. METHODS: Mice were subjected to 45 min of unilateral ischemia and subsequent contralateral nephrectomy and reperfusion for 2, 12, or 24 hr. Mice were control treated or treated with BB5.1, a monoclonal antibody that prevents cleavage of complement factor C5, thereby preventing C5a generation and formation of the membrane attack complex (MAC). RESULTS: Renal I/R induced extensive deposition of C3 early after reperfusion, whereas C6 and C9 deposition (MAC formation) occurred relatively late. I/R-induced complement deposition was mainly localized to tubular epithelium. Treatment with BB5.1 totally prevented MAC formation but also reduced C3 deposition. Inhibition of C5 strongly inhibited late inflammation, as measured by neutrophil influx and induction of the murine CXC chemokines macrophage inflammatory protein-2, KC, and lipopolysaccharide-induced CXC chemokine. Anti-C5 treatment furthermore abrogated late I/R-induced apoptosis, whereas early apoptosis was not affected. Moreover, BB5.1 treatment significantly protected against I/R-induced renal dysfunction. CONCLUSIONS: Renal I/R is followed by activation of the complement system and intrarenal deposition of C3 and MAC. Complement activation plays a crucial role in the regulation of inflammation and late apoptosis. Complement inhibition, by preventing C5 activation, abrogates late apoptosis and inflammation, being strongly protective against renal function loss

Prestorage leucocyte reduction of red cell components prevents release of bactericidal permeability increasing protein and defensins. Fransen EJ, Rombout-Sestrienkova E, van Pampus EC, Buurman WA, Reutelingsperger CP, Maessen JG. Department of Cardiothoracic Surgery, University Hospital Maastricht, P. Debyelaan 25, PO Box 5800, 6202 AZ Maastricht, the Netherlands. efr@scpc.azm.nl BACKGROUND AND OBJECTIVES: In this study we examined whether prestorage leucocyte reduction prevents the accumulation of bioreactive substances in red cell units. MATERIALS AND METHODS: Measurements were performed in the supernatants of buffy-coat-depleted (standard red cells) and leucocyte-reduced (filtered red cells) red cell units. The effect of storage was evaluated by taking repetitive samples up to 35 days after donation. We determined the concentrations of polymorphonuclear neutrophil (PMN)-derived bactericidal permeability increasing protein (BPI), defensins and annexin A5. In addition, leucocyte counts (using nageotte chamber) were performed on days 0 and 35. RESULTS: During storage, the concentrations of BPI, defensins and annexin A5 in standard red cells gradually increased. However, in the filtered red cells BPI and defensins were found in only a few samples, whereas the annexin A5 concentration in these units did not change during storage. Haemolysis data in both types of red cell components were similar at all time-points, except prestorage. Significant correlations were found between the release of BPI, defensins and annexin A5 into red cell units and the loss of leucocytes during storage. CONCLUSIONS: PMNs lose their membrane integrity during cold storage and release their contents into red cell components. Prestorage leucocyte reduction of red cell components prevents the accumulation of BPI, defensins and annexin A5.

Identification of single amino acid residues essential for the binding of lipopolysaccharide (LPS) to LPS binding protein (LBP) residues 86-99 by using an Ala-scanning library. Reyes O, Vallespi MG, Garay HE, Cruz LJ, Gonzalez LJ, Chinea G, Buurman W, Arana MJ. Division of Physical Chemistry, Center for Genetic Engineering and Biotechnology, Havana, Cuba. oreyes@cigb.edu.cu Lipopolysaccharide binding protein (LBP) is a 60 kDa acute phase glycoprotein capable of binding to LPS of Gram-negative bacteria and facilitating its interaction with cellular receptors. This process is thought to be of great importance in systemic inflammatory reactions such as septic shock. A peptide corresponding to residues 86-99 of human LBP (LBP86-99) has been reported to bind specifically with high affinity the lipid A moiety of LPS and to inhibit the interaction of LPS with LBP. We identified essential amino acids in LBP86-99 for binding to LPS by using a peptide library corresponding to the Ala-scanning of human LBP residues 86-99. Amino acids Trp91 and Lys92 were indispensable for peptide-LPS interaction and inhibition of LBP-LPS binding. In addition, several alanine-substituted synthetic LBP-derived peptides inhibited LPS-LBP interaction. Substitution of amino acids Arg94, Lys95 and Phe98 by Ala increased the inhibitory effect. The mutant Lys95 was the most active in blocking LPS binding to LBP. These findings emphasize the importance of single amino acids in the LPS binding capacity of small peptides and may contribute to the development of new drugs for use in the treatment of Gram-negative bacterial sepsis

Activated caspase-1 is not a central mediator of inflammation in the course of ischemia-reperfusion. Daemen MA, Denecker G, van't Veer C, Wolfs TG, Vandenabeele P, Buurman WA. Department of General Surgery, University of Maastricht, The Netherlands. BACKGROUND: Upon transplantation, donor organs subjected to prolonged ischemia suffer from reperfusion injury. Recent observations suggest that caspase activation is involved in inducing the deleterious inflammatory reaction that mediates reperfusion injury. Release of cytokines like interleukin (IL)-1 and IL-18 may occur during apoptosis through activation of caspase-1/IL-1beta-converting enzyme. We hypothesized that caspase-1 activation is a key event in apoptosis/ caspase-dependent inflammation during the development of renal reperfusion injury. METHODS: Caspase-1-/-, caspase-1+/+ as well as Swiss mice were subjected to 45 min of renal ischemia and 24 hr of reperfusion. Animals were administered agents capable of neutralizing the pro-inflammatory activation products of caspase-1 (IL-1 receptor antagonist, anti-IL-1 receptor antibody, and anti-IL-18 antibody). The extent of renal functional deterioration, inflammation, and apoptosis were compared. RESULTS: No improvement in renal function as reflected by serum ureum and creatinine were found in caspase-1-/- mice as compared to wild type controls. Caspase-1-/- mice showed slightly attenuated renal inflammation as indicated by decreased renal neutrophil influx, but failed to show changes in intrarenal tumor necrosis factor-alpha production. Moreover, caspase-1-/- mice clearly exhibited reperfusion-induced apoptosis as reflected by renal terminal deoxynucleotidyltransferase histology and internucleosomal DNA cleavage. Treatment with IL-1 receptor antagonist, anti-IL-1 receptor antibody, or anti-IL-18 antibody minimally reduced renal functional deterioration, inflammation, and apoptosis. CONCLUSIONS: These findings suggest that activated caspase-1 and its inflammatory products are involved in, but not crucial to, the induction of inflammation after renal ischemia-reperfusion. Hence, apart from caspase-1, other (combinations of) activated caspases are likely to be more prominently involved in renal reperfusion injury

The mechanisms involved in the antimetastatic effect of CpG-containing DNA were investigated in a mouse model of experimental metastasis. Tumor cell colony formation in lungs or livers of mice after i.v. inoculation with syngeneic fibrosarcoma or thymoma cells was determined. The i.v. injection of plasmid DNA or synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs before tumor cell application strongly inhibited metastasis. Because synthetic CpG-ODN was not directly tumor cytotoxic, the target cells for this CpG-ODN effect were determined. The cytotoxic activity on standard natural killer (NK) targets as well as on fibrosarcoma cells of splenic NK cells and NKT cell-containing liver mononuclear cells derived from CpG-ODN-treated mice was strongly enhanced. Participation of NK/NKT cells in the CpG-induced antimetastatic effect was demonstrated by reduction of the antimetastatic effect in mice depleted of NK/NKT cells and beta2-microglobulin-deficient mice. Neutralization of interleukin 12, interleukin 18, or IFN-gamma did not interfere with the CpG-induced antimetastatic effect. However, in sera of CpG-ODN-treated mice, high levels of IFN-alpha were detected, and in IFN-alpha/beta receptor-deficient mice, the CpG-ODN-induced antimetastatic effect was strongly reduced. These data indicate that CpG-ODNs activate NK/NKT cells for antimetastatic activity indirectly via IFN-alpha/beta receptor activation. The exploitation of the stimulatory activity of CpG-ODN for the innate immune system might be a useful strategy for antimetastatic therapy