LPS Removal Research Articles

Human leptospirosis: In search for a better vaccine.

Leptospirosis is a neglected disease caused by bacteria of the genus Leptospira and is more prevalent in tropical and subtropical countries. This pathogen infects humans and other animals, responsible for the most widespread zoonosis in the world, estimated to be responsible for 60 000 deaths and 1 million cases per year. To date, commercial vaccines against human leptospirosis are available only in some countries such as Japan, China, Cuba and France. These vaccines prepared with inactivated Leptospira (bacterins) induce a short-term and serovar-specific immune response, with strong adverse side effects. To circumvent these limitations, several research groups are investigating new experimental vaccines in order to ensure that they are safe, efficient, and protect against several pathogenic Leptospira serovars, inducing sterilizing immunity. Most of these protocols use attenuated cultures, preparations after LPS removal, recombinant proteins or DNA from pathogenic Leptospira spp. The aim of this review was to highlight several promising vaccine candidates, considering their immunogenicity, presence in different pathogenic Leptospira serovars, their role in virulence or immune evasion and other factors.

New strategies for Leptospira vaccine development based on LPS removal.

Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFNγ, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity.

Inner sensors of endotoxin - implications for sepsis research and therapy.

Despite great efforts and numerous clinical trials, there is still a major need for effective therapies for sepsis. Neutralization or elimination of bacterial toxins remains a promising approach. The understanding of the interaction of the endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria with its cellular receptor, namely the CD14/TLR4/MD2 complex, was a major breakthrough. Unfortunately, clinical trials for sepsis on the neutralization of LPS or on the inhibition of TLR4 signaling failed whereas those on LPS removal remain controversial. Recent discoveries of another class of LPS receptors localized within the cytoplasm, namely caspase-11 in mice and caspases-4/5 in humans, have renewed interest in the field. These provide new potential targets for intervention in sepsis pathogenesis. Since cytoplasmic recognition of LPS induces non-canonical inflammasome pathway, a potentially harmful host response, it is conceivable to therapeutically target this mechanism. However, a great deal of care should be used in the translation of research on the non-canonical inflammasome inhibition due to multiple inter-species differences. In this review, we summarize the knowledge on endotoxin sensing in sepsis with special focus on the intracellular sensing. We also highlight the murine versus human differences and discuss potential therapeutic approaches addressing the newly discovered pathways.

Effects of deoxynivalenol-feed contamination on circulating LPS in pigs.

Low concentration of LPS can be detected in healthy mammals without triggering systemic inflammation. Here we analysed the influence of the mycotoxin deoxynivalenol (DON) on very low LPS concentrations and the role of DON in the physiology of pigs challenged with high artificial LPS dosage mimicking septic shock. Pigs were fed for 29 d with DON-contaminated (4.59 mg/kg feed) or control feed. Samples of control animals showed 6.6 ± 13.5 pg/ml LPS in portal and 3.1 ± 7.6 pg/ml LPS in jugular serum samples. In the DON fed group, 3.4 ± 7.2 pg/ml and 0.6 ± 0.8 pg/ml were detected. The differences were statistically not significant, indicating that DON is not a trigger for enhanced LPS transfer into the blood circulation. Next, pigs were challenged with 7.5 µg LPS/kg body mass via portal or jugular route. The application route did not significantly influence the LPS concentration. We expected higher circulating LPS concentrations in the presence of DON due to the additional stress of liver metabolism and reduced liver capacity to remove LPS from circulation. This scenario is supported by tendency. In summary, we found that DON is unlikely to influence LPS transfer in the gut; DON likely reduces the capacity for LPS removal in septic shock conditions.

LPS removal reduces CD80-mediated albuminuria in critically ill patients with Gram-negative sepsis.

LPS-induced sepsis is a leading cause of acute kidney injury (AKI) in critically ill patients. LPS may induce CD80 expression in podocytes with subsequent onset of proteinuria, a risk factor for progressive chronic kidney disease (CKD) frequently observed after AKI. This study aimed to investigate the therapeutic efficacy of LPS removal in decreasing albuminuria through the reduction of podocyte CD80 expression. Between January 2015 and December 2017, 70 consecutive patients with Gram-negative sepsis-induced AKI were randomized to either have coupled plasma filtration and adsorption (CPFA) added to the standard care ( n = 35) or not ( n = 35). To elucidate the possible relationship between LPS-induced renal damage, proteinuria, and CD80 expression in Gram sepsis, a swine model of LPS-induced AKI was set up. Three hours after LPS infusion, animals were treated or not with CPFA for 6 h. Treatment with CPFA significantly reduced serum cytokines, C-reactive protein, procalcitonin, and endotoxin levels in patients with Gram-negative sepsis-induced AKI. CPFA significantly lowered also proteinuria and CD80 urinary excretion. In the swine model of LPS-induced AKI, CD80 glomerular expression, which was undetectable in control pigs, was markedly increased at the podocyte level in LPS-exposed animals. CPFA significantly reduced LPS-induced proteinuria and podocyte CD80 expression in septic pigs. Our data indicate that LPS induces albuminuria via podocyte expression of CD80 and suggest a possible role of timely LPS removal in preventing the maladaptive repair of the podocytes and the consequent increased risk of CKD in sepsis-induced AKI.

Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 1. Polymer-polymer systems

Abstract The production of recombinant biopharmaceuticals is highly dependent of a proper choice of the downstream processing stages. Particularly, the purification that must ensure that all the endotoxins (lipopolysaccharides - LPS) are efficiently removed from the final product. The efficient removal of LPS has a direct impact on the manufacturing of therapeutic biopharmaceuticals, since LPS is naturally presented in Gram-negative bacterial expression systems. In order to provide a more simple and faster technique for the purification of green fluorescent protein (GFP) and LPS removal, aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and poly(acrylic acid) (NaPA) and electrolytes were studied. Firstly, the binodal curves of PEG/NaPA + salt systems were established using NaCl, Li2SO4, KI, and KNO3 as additives. It was demonstrated that the formation of ATPS is enhanced following the anion tendency: SO42− > Cl− ≫ NO3− > I−. The stability of GFP in the presence of all different phase forming agents (polymers and salts) was evaluated, and the high biocompatibility of these ATPS demonstrated by the maintenance of GFP fluorescence in all conditions under study. Then, the GFP extraction and LPS removal aptitude of each ATPS was investigated. GFP and LPS were preferentially partitioned into the top (PEG-rich) phase (KGFP > 20), but with a removal of 35% of LPS was attained. Hydrophobic and electrostatic interactions were found to be the major driving forces for GFP partitioning and LPS removal. Moreover, a new effect was found, the presence of high loads of LPS can affect (decrease) the KGFP values (KGFP without LPS > KGFP with 104 EU/mL > KGFP with 106 EU/mL). The ATPS with best GFP extraction performance was selected for the recovery directly from the cell lysates of Escherichia coli. In this experiment, the system composed of 12 wt% PEG 1000 g/mol, 12 wt% NaPA 8000 g/mol, and 0.25 M Li2SO4 lead to a LPS removal (REMLPS) of 13%, KGFP of 20.2, and high selectivity relatively to the total proteins (S = 20), since the majority of contaminants proteins were preferentially partitioned into the bottom (NaPA-rich) phase (purification factor of 4-fold). It is here demonstrated that the ATPS composed of PEG/NaPA + salts as additives can be used as a first step for the recovery of GFP and removal of contaminants (LPS in part, and most contaminant proteins) from cell lysates by applying a system with low polymer content and using mild conditions.

Induction of human tolerogenic dendritic cells by 3'-sialyllactose via TLR4 is explained by LPS contamination.

The human milk oligosaccharide 3′-sialyllactose (3′SL) has previously been shown to activate murine dendritic cells (DC) in a Toll-like receptor (TLR) 4-mediated manner ex vivo. In this study we aimed to investigate whether 3′SL has similar immunomodulatory properties on human DC. 3′SL was shown to induce NF-κB activation via human TLR4. However, LPS was detected in the commercially obtained 3′SL from different suppliers. After the removal of LPS from 3′SL, we studied its ability to modify DC differentiation in vitro. In contrast to LPS and 3′SL, LPS-free 3′SL did not induce functional and phenotypical changes on immature DC (iDC). iDC that were differentiated in the presence of LPS or 3′SL showed a semi-mature phenotype (i.e., fewer CD83+CD86+ DC), produced IL-10 and abrogated IL-12p70 and tumor necrosis factor-alpha levels upon stimulation with several TLR ligands. Differentiation into these tolerogenic DC was completely abrogated by LPS removal from 3′SL. In contrast to previous reports in mice, we found that LPS-free 3′SL does not activate NF-κB via human TLR4. In conclusion, removing LPS from (oligo)saccharide preparations is necessary to study their potential immunomodulatory function.

Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity

ABSTRACTProkaryotes release membrane vesicles (MVs) with direct roles in disease pathogenesis. MVs are heterogeneous when isolated from bacterial cultures so Density Gradient Centrifugation (DGC) is valuable for separation of MV subgroups from contaminating material. Here we report the technical variability and natural biological heterogeneity seen between DGC preparations of MVs for Mycobacterium smegmatis and Escherichia coli and compare these DGC data with size exclusion chromatography (SEC) columns. Crude preparations of MVs, isolated from cultures by ultrafiltration and ultracentrifugation were separated by DGC with fractions manually collected as guided by visible bands. Yields of protein, RNA and endotoxin, protein banding and particle counts were analysed in these. DGC and SEC methods enabled separation of molecularly distinct MV populations from crude MVs. DGC banding profiles were unique for each of the two species of bacteria tested and further altered by changing culture conditions, for example with iron supplementation. SEC is time efficient, reproducible and cost effective method that may also allow partial LPS removal from Gram-negative bacterial MVs. In summary, both DGC and SEC are suitable for the separation of mixed populations of MVs and we advise trials of both, coupled with complete molecular and single vesicle characterisation prior to downstream experimentation.

Preparation of Pseudomonas aeruginosa alginate-flagellin immunoconjugate

Mucoid strains of Pseudomonas aeruginosa are closely associated with chronic pulmonary infections. In this report we describe a straightforward approach to conjugate high molecular weight alginate to type b-flagellin (FLB) and investigation of its bioactivity. The conjugation process was performed by using ADH and EDAC. The endotoxin was eliminated from the candidate vaccine by LPS removal resin followed by LAL test. The bioconjugate molecules were verified by simultaneously determination of polysaccharide/protein content followed by gel filtration chromatography and FTIR spectroscopy. Groups of eight BALB/c mice were injected intranasally with 5 μg (per each nostril) of purified alginate, FLB and conjugated alginate-FLB with two week intervals. The functional activity of the vaccine was evaluated by ELISA and opsonophagocytosis tests. Vaccination with the alginate-FLB conjugate induced a significant (P = 0.0033) rise in alginate specific IgG in mice. At all dilution ranges, the opsonic activity of the conjugate vaccine antisera was significantly higher than alginate alone (61.9% vs. 17.3% at 1:4 dilution; P = 0.0067). The alginate-FLB conjugate could elicit high specific antibodies titer against alginate by improving its immunogenicity. In addition, the antisera raised against conjugate vaccine act as a suitable opsonin for phagocytosis of the mucoid strains of P. aeruginosa.

Hypoxia potentiates cytotoxicity of LPS-activated microglial BV2 cells in vitro by synergistic effects on cytokine and nitric oxide secretion

Results Activation of BV2 microglial cells by LPS exposure stimulated significant and persistent production of NO, IL-1b, IL-6, and TNF-a. Even after LPS removal, ongoing NO and cytokine secretion could be observed. While hypoxia alone mediated exclusively a significant, shortterm increase of IL-1b, oxygen deprivation enhanced LPS-induced secretion of NO, IL-1b, IL-6, and TNF-a significantly. Surprisingly, pre-stimulation of BV2 cells by hypoxia prior LPS exposure abolished microglial activation suppressing LPS-induced NO production. Hereby, cell-free supernatants derived from LPS-activated microglial cells exhibited a stronger cytotoxic effect in glial and neuronal cells than LPS exposition per se (P < 0.001). Again, hypoxia potentiated LPS-induced cytotoxicity. Conclusion Present data prove that i) the outcome of hypoxia is determined by the microglial activation status and that ii) LPS-induced soluble factors rather than LPS are mediators of microglial neurotoxicity under conditions of hypoxia in vitro. Activation of pro-inflammatory pathways may sensitize microglial cells to promote hypoxia-induced injury of the developing brain. Consequently, our findings may promote neuroprotective therapeutic strategies in the field of perinatal brain injury.

The Impact of Timing of Microglial Activation by Inflammation and Hypoxia Regarding Additive Neurotoxic Effects

Introduction: Perinatal cerebral hypoxia and endotoxins are main risk factors for acquired brain injury and neurological disabilities. Here, we investigated the impact of lipopolysaccharide stimulated microglia on inflammation and cytotoxicity in vitro in the context of hypoxia. Methods: BV2 cells were exposed to LPS and hypoxia (1% O2). Expression of the transcription factor hypoxia-inducible factor (HIF)-1α as well as of iNOS, IL-1b, IL-6, and tumor necrosis factor (TNF)-α were analyzed by real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. NO production was quantified by the Griess assay. Results: LPS exposition prior hypoxia increased mRNA and protein expression of HIF-1α, iNOS, and inflammatory cytokines suggesting synergistic regulation of hypoxia and inflammatory signaling pathways in microglial cells. Whereas HIF-1α accumulation was transient depending on the presence of LPS, ongoing cytokine and NO secretion was observed for at least 3 days after LPS removal. Notably, hypoxia potentiated the LPS-induced secretion of IL-1b (22-fold), IL-6 (threefold), TNF-α (threefold), and increased NO production by approximately 60%. Cell-free supernatants derived from LPS exposed BV2 cells were highly cytotoxic. Again, hypoxia enhanced the cytotoxic effect significantly. In contrast, stimulation of BV2 cells by hypoxia prior LPS abolished additive inflammatory neurotoxic effects compared with controls (p < 0.01). Conclusion: Present in vitro data indicate that LPS induces a dose-dependent and long-lasting sensitization of BV2 cells to hypoxia. Enhanced cytokine and NO secretion may contribute to delayed glial damage following cerebral hypoxic insults during the perinatal period. As hypoxia induced accumulation of the neurotrophic transcription factor HIF-1α abolishes cytotoxic inflammatory effects, present observations may have implications for neuroprotective pharmacological strategies.

Chemical characterization and complement fixation of pectins from Cola cordifolia leaves

Defatted leaves from the medicinal tree Cola cordifolia were extracted with 50% EtOH, 50°C and 100°C water. The polysaccharide rich extracts were fractionated and the structure of the polysaccharides elucidated. Linkage analysis of the polysaccharides indicates a rhamnogalacturonan type I backbone where both Rha and parts of GalA are substituted in position 3, indicating a highly branched polymer with short side chains. The purified fractions were tested for complement fixation, macrophage stimulating activity and anti-adhesion activity towards Helicobacter pylori. Here we report on complex and polydisperse types of pectins (Mw: 3–1300kDa) as well as the presence of low Mw (<3kDa) acidic oligosaccharides. The fractions showed a moderate complement fixing activity and no macrophage activating effects after LPS removal. Anti-adhesion activity towards H. pylori was not found.

Green fluorescent protein extraction and LPS removal from Escherichiacoli fermentation medium using aqueous two-phase micellar system

The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, since endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. This work aimed to study the use of aqueous two-phase micellar systems (ATPMS) from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv), which works as a biological indicator. The GFPuv extraction and LPS removal were evaluated in ATPMS, partition assays were carried out using pure GFPuv and cell lysate from Escherichia coli. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase ( K GFPuv > 1), and LPS removal into the micelle-rich phase (% REM LPS > 98%). GFPuv was partitioned preferentially into the micelle-poor phase due to excluded-volume interactions in the micelle-rich phase. Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.

Synthesis and endotoxin removal properties of a novel affinity sorbent with poly(1-vinylimidazole) as the ligand

In this paper, a novel silica gel affinity sorbent with poly(1-vinylimidazole) (PVI) ligand was prepared for the selective removal of endotoxin (LPS) from a water or a BSA solution. The vinyl silane-modified porous silica gel particle SiO 2-g-KH570 was used as macromonomer, and PVI was grafted on by heterogeneous graft copolymerization. First, the effect of reaction conditions on the grafted degree (GD) of PVI was investigated in detail by thermogravimetry. Then, the influences of initial LPS concentration, pH and ionic strength of the tested aqueous solutions on LPS removal ability of PVI-g-SiO 2 affinity sorbent were studied and compared with those of His-g-SiO 2 affinity sorbent. The results showed that maximum LPS removal capacity was obtained with a GD about 2.5 wt% and the PVI-g-SiO 2 affinity sorbent had better selective removal of LPS compared to His-g-SiO 2 affinity sorbent in both water and BSA solution. The affinity mechanism between PVI and LPS is also discussed in this paper. All of our results indicate that PVI is a good ligand for LPS removal.

LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two-phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (K(GFPuv) < 1.00), and LPS removal into the micelle-rich phase (%REM(LPS) > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.

Endotoxin activity dosage in septic patients

Endotoxin (LPS), present in the outer membrane of Gram negative bacteria, is responsible for a series of biological effects, primarily linked to the lipid fraction (Lipid A).The aim of our work was to correlate the endotoxin activity with the risk of severe sepsis in patients showing severe bacterial infection and hospitalized in the three Intensive Care Units of the AUSL BAT. Fifty-three endotoxin activity (EA) blood tests were performed on 28 septic patients. 17/28 (60.7%) patients showed EA &gt; 0.60 (associated with high risk of severe sepsis). Endotoxin activity (EA) blood tests were repeated in 11/17 cases after LPS removal by extracorporeal blood treatment with Polymyxin B (Toraymyxin). 6/11 patients had a reduction of LPS in the bloodstream after 1 treatment with Toraymyxin; in 5 of these 6 patients there was a further reduction after 2 treatments with Toraymyxin. EA dosage has been proven either a method for the diagnosis of severe sepsis due to Gram negative bacteria or helpful means in selecting patients for treatment with Toraymyxin.

Cytokine secretion from monocytes persists differentially after activator removal—One mechanism of long‐term biological response to implants

Biomedical implants significantly improve the quality of life in an ever-increasing number of patients. However, inflammation of tissues around implants remains a long-term, post-placement sequelae that may contribute to implant failure. Infection-mediated failure is partly a consequence of inappropriate host response and chronic inflammation, and is mediated primarily by the secretory products of monocytes and macrophages. Although the secretion of inflammatory mediators from activated monocytes is well characterized, the resolution of mediator levels post-activation is relatively unstudied. The current study defines the time course of cytokine secretion by activated human monocytes after the activator has been removed. THP1 human monocytes were activated by LPS, and cytokine secretion was monitored over time after LPS removal using enzyme-linked immunosorbent assays (TNFalpha or IL8) or a cytokine array. The release of cytokines was compared with conditions without LPS removal. As expected, secretion of nearly all cytokines was reduced when LPS was removed, but the amount of the reduction was highly cytokine-dependent. Furthermore, levels of cytokines were stable in medium alone but not in cell-culture, suggesting an active process to either degrade or internalize secreted cytokines. Our results are consistent with clinical experience that inflammation resolves rapidly after treatment to remove bacteria or inflamed tissue. However, the differential cytokine regulation indicates a sophisticated coordination of cytokine levels probably associated with management of the wound healing response after removal of the bacterial insult. This wound healing response is one critical component of the long-term success of biomedical implants.

Removal of lipopolysaccharides from protein–lipopolysaccharide complexes by nonflammable solvents

During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein–LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein–LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein–LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein–LPS complexes, while the separation of LPS from protein–LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes.

Hyaluronan Fragments Stimulate Endothelial Recognition of Injury through TLR4

Tissues must quickly recognize injury to respond to the rapid pace of microbial growth. In skin, dermal microvascular endothelial cells must also react to danger signals from the surrounding tissue and immediately participate by initiating the wound repair process. Components of the extracellular matrix such as hyaluronan are rapidly broken down into smaller molecular weight oligosaccharides in a wound, and these can activate a variety of biological processes. This study set out to determine if hyaluronan fragments released following injury can stimulate endothelial cells and what mechanism is responsible for this response. Using genechip microarray analysis, a response to hyaluronan fragments was detected in endothelial cells with the most significant increase observed for the chemokine IL-8. This observation was verified with qualitative reverse transcriptase-PCR and ELISA in human endothelial cell culture, and in a mouse model by observing serum levels of MIP-2 and KC following hyaluronan fragment administration in vivo. Activation was TLR4-dependent, as shown by use of TLR4 blocking antibody and TLR4-deficient mice, but not due to the presence of undetected contaminants as shown by inactivation following digestion with the hyaluronan-degrading enzyme chondroitinase ABC or incubation with the hyaluronan-specific blocking peptide Pep-1. Inactivation of LPS activity failed to diminish the action of hyaluronan fragments. These observations suggest that endogenous components of the extracellular matrix can stimulate endothelia to trigger recognition of injury in the initial stages of the wound defense and repair response.

A new endotoxin adsorption device in Gram-negative sepsis: use of immobilized albumin with the MATISSE ® adsorber

MATISSE ® is a new endotoxin (ET) adsorption system. It comprises the FRESENIUS hemoadsorption machine 4008 ADS to maintain the extracorporeal circuits and disposables including the MATISSE ®-adsorber based on macroporous beads immobilized with human serum albumin. In vivo and in vitro experiments with this new compound had shown favourable ET elimination properties. In a first study 19 healthy volunteers were included for the safety and tolerability of the endotoxin adsorber system. One single veno-venous hemoperfusion with this system was performed. Apheresis treatment with the human albumin immobilized adsorber seems to be safe and well tolerable in healthy volunteers. A slight decrease in platelet count as well as insulin level was observed. No serious adverse events were mentioned. Similar findings were made in an open uncontrolled trial in six patients with suspected Gram-negative sepsis. Here endotoxin adsorption seems to be safe and well tolerated without adsorber related major adverse events. In the last clinical trial including 145 patients mainly peritonitis patients (104), again no safety concerns were found during the apheresis. Trends in improvement of morbidity and organ dysfunction were found as well as efficient LPS removal in peritonitis patients. LPS whole blood immunoadsorption is a promising new method. No relevant side effects were observed so far. A large controlled study to prove clinical efficacy in patients with severe sepsis and confirmed endotoxemia (e.g. peritonitis) should be necessary.