Hyaluronan Regulates Neuronal and Immune Function in the Rat Small Intestine and Colonic Microbiota after Ischemic/Reperfusion Injury.

BACKGROUND: Intestinal ischemia and reperfusion (IRI) injury induces acute and long-lasting damage to the neuromuscular compartment and dysmotility. This study aims to evaluate the pathogenetic role of hyaluronan (HA), a glycosaminoglycan component of the extracellular matrix, as a modulator of the enteric neuronal and immune function and of the colonic microbiota during in vivo IRI in the rat small intestine. METHODS: mesenteric ischemia was induced in anesthetized adult male rats for 60 min, followed by 24 h reperfusion. Injured, sham-operated and non-injured animals were treated with the HA synthesis inhibitor, 4-methylumbelliferone (4-MU 25 mg/kg). Fecal microbiota composition was evaluated by Next Generation Sequencing. Neutrophil infiltration, HA homeostasis and toll like receptor (TLR2 and TLR4) expression in the small intestine were evaluated by immunohistochemical and biomolecular approaches (qRT-PCR and Western blotting). Neuromuscular responses were studied in vitro, in the absence and presence of the selective TLR2/4 inhibitor, Sparstolonin B (SsnB 10, 30 µM). RESULTS: 4-MU significantly reduced IRI-induced enhancement of potentially harmful Escherichia and Enterococcus bacteria. After IRI, HA levels, neutrophil infiltration, and TLR2 and TLR4 expression were significantly enhanced in the muscularis propria, and were significantly reduced to baseline levels by 4-MU. In the injured, but not in the non-injured and sham-operated groups, SsnB reduced both electrical field-stimulated (EFS, 0.1-40 Hz) contractions and EFS-induced (10 Hz) non-cholinergic non-adrenergic relaxations. CONCLUSIONS: enhanced HA levels after intestinal IRI favors harmful bacteria overgrowth, increases neutrophil infiltration and promotes the upregulation of bacterial target receptors, TLR2 and TLR4, in the muscularis propria, inducing a pro-inflammatory state. TLR2 and TLR4 activation may, however, underlay a provisional benefit on excitatory and inhibitory neuronal pathways underlying peristalsis.

Mucinous breast cancer: A narrative review of the literature and a retrospective tertiary single-centre analysis.

Mucinous carcinoma (MC) is a rare breast cancer characterized by the presence of large extracellular mucin amount. Two main subtypes can be distinguished: pure (PMC) and mixed (MMC). We conducted a retrospective MC analysis in our prospective maintained database, calculating disease-free survival (DFS) and 5-year overall survival (OS). We found a global 92.1% OS (higher in MMC group and statistically significative) and a DFS of 95.3% (higher in MMC group but not statistically significative).

The medicinal leech as a valuable model for better understanding the role of a TLR4-like receptor in the inflammatory process.

Despite extensive investigation focused on both the molecular characteristics and the expression level of Toll-like receptors (TLRs) during the inflammatory response in vertebrates, few data are available in the literature on the role of these proteins in invertebrate's immune response. Here, we propose the medicinal leech as a valuable model to better elucidate the role of TLR4 and its related products, such as tumor necrosis factor (TNF-α), after activation of the leech peripheral immune system with the endogenous medicinal leech recombinant allograft inflammatory factor-1 (rHmAIF-1) or with an exogenous stimulus, such as lipopolysaccharide (LPS). Our results indicate that activated macrophages (HmAIF-1+) and granulocytes (CD11b+) express both TLR4 and its coreceptor CD14. Moreover, functional studies performed by injecting a cyanobacterium selective TLR4 antagonist CyP demonstrated that only the TLR4 pathway was blocked, while the immune response caused by lipoteichoic acid (LTA) treatment is not affected. These results are consistent with literature on vertebrates, indicating that TLR4 functions as a LPS receptor while the recognition of LTA may involve other pathways.

The Effect of Cyanobacterial LPS Antagonist (CyP) on Cytokines and Micro-RNA Expression Induced by Porphyromonas gingivalis LPS.

Lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg-LPS) is a key bacterial structure involved in the maintenance of a chronic pro-inflammatory environment during periodontitis. Similar to other gram-negative LPS, Pg-LPS induces the release of pro-inflammatory cytokines through interaction with Toll-Like Receptor 4 (TLR4) and is able to stimulate negative TLR4 regulatory pathways, such as those involving microRNA (miRNA). In this work, we employed CyP, an LPS with TLR4-MD2 antagonist activity obtained from the cyanobacterium Oscillatoria planktothrix FP1, to study the effects on pro-inflammatory cytokine production and miRNA expression in human monocytic THP-1 cells stimulated with Pg-LPS or E. coli LPS (Ec-LPS). Results showed that CyP inhibited TNF-α, IL-1ß and IL-8 expression more efficiently when co-incubated with Pg-LPS rather than with Ec-LPS. The inhibition of pro-inflammatory cytokine production was maintained even when CyP was added 2 h after LPS. The analysis of the effects of CyP on miRNA expression showed that, although being an antagonist, CyP did not inhibit miR-146a induced by Pg-LPS or Ec-LPS, whereas it significantly inhibited miR-155 only in the cultures stimulated with Ec-LPS. These results suggest that CyP may modulate the pro-inflammatory response induced by Pg-LPS, not only by blocking TLR4-MD2 complex, but also by preserving miR-146a expression.

MiR-146a induction by cyanobacterial lipopolysaccharide antagonist (CyP) mediates endotoxin cross-tolerance.

Endotoxin tolerance is a phenomenon characterized by a reduced capacity of monocytes and macrophages to respond to repeated stimulation with lipopolysaccharide (LPS) which has been suggested to represent a way of controlling the intensity and duration of innate immune response. During endotoxin tolerance, monocytes undergo functional re-programming primarily by epigenetic regulation. Recently, micro-RNA (miR)-146a has been demonstrated to be the major player of the negative regulation of the pro-inflammatory response, affecting TNF-α production. In this study, we have employed CyP, a cyanobacterial LPS antagonist acting on TLR4-MD2 complex, for priming human monocytes and evaluating their response to a subsequent challenge with E. coli LPS. Results show that CyP is able to induce cross-tolerance to E. coli LPS by inhibiting TNF-α production. The mechanism of action is mediated by a specific induction of miR-146a and reduction of IRAK1 and TRAF6 expressions in human monocytes by CyP priming. Up-regulation of miR-146a by CyP alone, affects subsequent cell response in term of TNF-α production even when monocytes are incubated with other TLR ligands, as lipoteichoic acid (LTA), thus confirming miR-146a as a critical player mediating TNF-α regulation during cross-tolerance with CyP.

Natural Products with Toll-Like Receptor 4 Antagonist Activity.

Toll-Like Receptors (TLRs) are the innate immunity receptors that play an activating role when interacting with molecules released by bacteria and viruses (PAMPs, pathogen-associated molecular patterns) or with molecules released by injured cells and tissues (DAMPs, danger-associated molecular patterns). TLR triggering leads to the induction of proinflammatory cytokines and chemokines, driving the activation of both innate and adaptive immunity. In particular, Toll-Like Receptor 4 (TLR4) has been described to be involved in the inflammatory processes observed in several pathologies (such as ischemia/reperfusion injury, neuropathic pain, neurodegenerative diseases, and cancer). Molecules obtained by natural sources have been discovered to exert an anti-inflammatory action by targeting TLR4 activation pathways. This review focuses on TLR4 antagonists obtained from bacteria, cyanobacteria, and plants.

Effects of the gut-liver axis on ischaemia-mediated hepatocellular carcinoma recurrence in the mouse liver.

BACKGROUND & AIMS: There is growing evidence that liver graft ischemia-reperfusion (I/R) is a risk factor for hepatocellular carcinoma (HCC) recurrence, but the mechanisms involved are unclear. Herein, we tested the hypothesis that mesenteric congestion resulting from portal blood flow interruption induces endotoxin-mediated Toll-like receptor 4 (Tlr4) engagement, resulting in elevated liver cancer burden. We also assessed the role of remote ischemic preconditioning (RIPC) in this context. METHODS: C57Bl/6j mice were exposed to standardized models of liver I/R injury and RIPC, induced by occluding the hepatic and femoral blood vessels. HCC was induced by injecting RIL-175 cells into the portal vein. We further evaluated the impact of the gut-liver axis (lipopolysaccharide (LPS)-Tlr4 pathway) in this context by studying mice with enhanced (lipopolysaccharide infusion) or defective (Tlr4-/- mice, gut sterilization, and Tlr4 antagonist) Tlr4 responses. RESULTS: Portal triad clamping provoked upstream mesenteric venous engorgement and increased bacterial translocation, resulting in aggravated tumor burden. RIPC prevented this mechanism by preserving intestinal integrity and reducing bacterial translocation, thereby mitigating HCC recurrence. These observations were linked to the LPS-Tlr4 pathway, as supported by the high and low tumor burden displayed by mice with enhanced or defective Tlr4 responses, respectively. CONCLUSIONS: Modulation of the gut-liver axis and the LPS-Tlr4 response by RIPC, gut sterilization, and Tlr4 antagonism represents a potential therapeutic target to prevent I/R lesions, and to alleviate HCC recurrence after liver transplantation and resection. LAY SUMMARY: Cancer recurrence can occur after liver resection or liver transplantation for hepatocellular carcinoma (HCC). This study suggests that intestinal venous congestion, which often occurs during liver surgery, favors the translocation of gut-derived bacterial products in the portal vein, thereby facilitating cancer recurrence by enhancing the signaling of Toll-like receptor 4 in the liver. Using a mouse model of HCC recurrence, we show that strategies that (i) reduce bacterial translocation (by gut decontamination, or by protecting the intestine from venous ischemia damage) or (ii) inhibit Tlr4 signaling in the liver, could reduce cancer recurrence.

Neurodegenerative diseases: The immunological perspective.

Increasing evidence supports the notion that the neurodegenerative process occurring in Alzheimer's disease (AD), Parkinson's disease (PD) and Amyotrophic Lateral Sclerosis (ALS) does not only imply the neuronal compartment but also involves a strong interaction with the immunological cells of the Central Nervous System (CNS), primarily microglia. Starting from the observation that the neurodegenerative disorders are frequent in elderly individuals, who have an immunological background that possibly favors this process, it is evident that a dysregulation of innate immune response triggered by misfolded and aggregated proteins, or by endogenous molecules released by injured neurons, directly contributes to disease pathogenesis and progression. There are important differences in the immunological processes occurring in AD, PD, ALS involving microglial function. Furthermore, although the contribution of adaptive immune cells in AD seems to be modest, in PD and especially in ALS models, T cells can influence microglial phenotype, inducing neuroprotection. A better understanding of the immunological mechanisms involved in the different phases of the neurodegenerative processes observed in AD, PD, ALS could effectively contribute to the development of new preventive and therapeutic strategies for such diseases.

Toll-like receptor 4-dependent glial cell activation mediates the impairment in memory establishment induced by ß-amyloid oligomers in an acute mouse model of Alzheimer's disease.

BACKGROUND: Amyloid-ß oligomers (AßO) are species mainly involved in the synaptic and cognitive dysfunction in Alzheimer's disease. Although their action has been described mainly at neuronal level, it is now clear that glial cells govern synaptic activity in their resting state, contributing to new learning and memory establishment. In contrast, when activated, they may lead to synaptic and cognitive dysfunction. Using a reliable acute AßO-mediated mouse model of AD, we explored whether the memory alteration AßOs induce relies on the activation of glial cells, and if Toll-like receptor 4 (TLR4), pivotal in the initiation of an immune response, is involved. METHODS: C57 naïve mice were given a single intracerebroventricular injection of synthetic AßO-containing solution (1µM), which induces substantial impairment in the establishment of recognition memory. Then, first we assessed glial cell activation at different times post-injection by western blot, immunohistochemistry and ELISA in the hippocampus. After that we explored the efficacy of pre-treatment with anti-inflammatory drugs (indomethacin and an IL-1ß receptor antagonist) to prevent impairment in the novel object recognition task, and compared AßO's effects in TLR4 knockout mice. RESULTS: A single AßO injection rapidly activated glial cells and increased pro-inflammatory cytokine expression. Both anti-inflammatory drugs prevented the AßO-mediated impairment in memory establishment. A selective TLR4 receptor antagonist abolished AßO's action on memory, and in TLR4 knockout mice it had no effect on either memory or glial activation. CONCLUSIONS: These data provide new information on AßO's mechanism of action, indicating that besides direct action at the synapses, they also act through the immune system, with TLR4 playing a major role. This suggests that in a potential therapeutic setting inflammation must be considered as well.

Blockade of the IL-1R1/TLR4 pathway mediates disease-modification therapeutic effects in a model of acquired epilepsy.

We recently discovered that forebrain activation of the IL-1 receptor/Toll-like receptor (IL-1R1/TLR4) innate immunity signal plays a pivotal role in neuronal hyperexcitability underlying seizures in rodents. Since this pathway is activated in neurons and glia in human epileptogenic foci, it represents a potential target for developing drugs interfering with the mechanisms of epileptogenesis that lead to spontaneous seizures. The lack of such drugs represents a major unmet clinical need. We tested therefore novel therapies inhibiting the IL-1R1/TLR4 signaling in an established murine model of acquired epilepsy. We used an epigenetic approach by injecting a synthetic mimic of micro(mi)RNA-146a that impairs IL1R1/TLR4 signal transduction, or we blocked receptor activation with antiinflammatory drugs. Both interventions when transiently applied to mice after epilepsy onset, prevented disease progression and dramatically reduced chronic seizure recurrence, while the anticonvulsant drug carbamazepine was ineffective. We conclude that IL-1R1/TLR4 is a novel potential therapeutic target for attaining disease-modifications in patients with diagnosed epilepsy.

The Role of Toll-Like Receptor 4 in Infectious and Noninfectious Inflammation.

Toll-like receptor 4 (TLR4) belongs to the family of pattern recognition receptors (PRRs). They are highly conserved receptors that recognize conserved pathogen-associated molecular patterns (PAMPs), thus representing the first line of defense against infections. TLR4 has been long recognized as the sensing receptor for gram-negative lipopolysaccharide (LPS). In addition, it also binds endogenous molecules produced as a result of tissue injury. Hence, TLR4 represents a key receptor on which both infectious and noninfectious stimuli converge to induce a proinflammatory response. TLR4-mediated inflammation, triggered by exogenous or endogenous ligands, is also involved in several acute and chronic diseases, having a pivotal role as amplifier of the inflammatory response. This review focuses on the research progress about the role of TLR4 activation in infectious and noninfectious (e.g., sterile) inflammation and the effects of TLR4 signaling in some pathological conditions.

Phase I-II study of hypofractionated simultaneous integrated boost using volumetric modulated arc therapy for adjuvant radiation therapy in breast cancer patients: a report of feasibility and early toxicity results in the first 50 treatments.

BACKGROUND: To report results in terms of feasibility and early toxicity of hypofractionated simultaneous integrated boost (SIB) approach with Volumetric Modulated Arc Therapy (VMAT) as adjuvant treatment after breast-conserving surgery. METHODS: Between September 2010 and May 2011, 50 consecutive patients presenting early-stage breast cancer were submitted to adjuvant radiotherapy with SIB-VMAT approach using RapidArc in our Institution (Istituto Clinico Humanitas ICH). Three out of 50 patients were irradiated bilaterally (53 tumours in 50 patients). All patients were enrolled in a phase I-II trial approved by the ICH ethical committee. All 50 patients enrolled in the study underwent VMAT-SIB technique to irradiate the whole breast with concomitant boost irradiation of the tumor bed. Doses to whole breast and surgical bed were 40.5 Gy and 48 Gy respectively, delivered in 15 fractions over 3 weeks. Skin toxicities were recorded during and after treatment according to RTOG acute radiation morbidity scoring criteria with a median follow-up of 12 months (range 8-16). Cosmetic outcomes were assessed as excellent/good or fair/poor. RESULTS: The median age of the population was 68 years (range 36-88). According to AJCC staging system, 38 breast lesions were classified as pT1, and 15 as pT2; 49 cases were assessed as N0 and 4 as N1. The maximum acute skin toxicity by the end of treatment was Grade 0 in 20/50 patients, Grade 1 in 32/50, Grade 2 in 0 and Grade 3 in 1/50 (one of the 3 cases of bilateral breast irradiation). No Grade 4 toxicities were observed. All Grade 1 toxicities had resolved within 3 weeks. No significant differences in cosmetic scores on baseline assessment vs. 3 months and 6 months after the treatment were observed: all patients were scored as excellent/good (50/50) compared with baseline; no fair/poor judgment was recorded. No other toxicities or local failures were recorded during follow-up. CONCLUSIONS: The 3-week course of postoperative radiation using VMAT with SIB showed to be feasible and was associated with acceptable acute skin toxicity profile. Long-term follow-up data are needed to assess late toxicity and clinical outcomes.

Neuroprotective effects of toll-like receptor 4 antagonism in spinal cord cultures and in a mouse model of motor neuron degeneration.

Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.

Coadministration of the cyanobacterial lipopolysaccharide antagonist CyP with antibiotic inhibits cytokine production by an in vitro meningitis model infected with Neisseria meningitidis.

OBJECTIVES: In this study, the objective was to determine the anti-inflammatory properties of CyP, a cyanobacterial lipopolysaccharide (LPS) antagonist, used in combination with antibiotic chemotherapy during infection of an in vitro meningitis model infected with Neisseria meningitidis (meningococcus). METHODS: Monocultures of human meningioma cells and meningioma-primary human macrophage co-cultures were infected with meningococci (10(2)-10(8) cfu/monolayer) or treated with isolated outer membranes or purified LPS (0.1-100 ng/monolayer) from N. meningitidis. CyP (1-20 µg/monolayer) was added at intervals from t = 0 to 4 h, with and without benzylpenicillin (1-20 µg/monolayer). The antagonistic effect of CyP and its adjunctive properties to benzylpenicillin administration was determined by measuring cytokine levels in culture supernatants after 24 h. RESULTS: CyP significantly inhibited (P < 0.05) the secretion of interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 and RANTES ('regulated upon activation, normal T cell expressed and secreted') (overall reduction levels from 50% to >95%) by meningioma cell lines and meningioma-macrophage co-cultures challenged with either live meningococci or bacterial components. Inhibition was effective when CyP was added within 2 h of challenge (P < 0.05) and was still pronounced by 4 h. In the co-culture model, CyP alone partially inhibited IL-1ß secretion, but did not prevent tumour necrosis factor (TNF)-α secretion, whereas penicillin alone inhibited IL-1ß and TNF-α but conversely did not reduce MCP-1 and RANTES secretion. However, coadministration of CyP and penicillin in both models had an additive effect and restored the overall inhibitory profile. CONCLUSIONS: CyP inhibits cytokine production in an in vitro meningitis model and augments the anti-inflammatory response when combined with benzylpenicillin. Administration of an LPS antagonist with antibiotic merits consideration in the emergency treatment of patients presenting with meningococcal infection.

Therapeutic potential of new antiinflammatory drugs.

Experimental and clinical findings have shown in the past decade that specific proinflammatory mediators and their cognate receptors are upregulated in epileptic brain tissue. In particular, the IL-1 receptor (R)/Toll-like receptor (TLR) signaling pathways are activated in experimental models of seizures and in human epileptic tissue from drug-resistant patients. Pharmacological targeting of these proinflammatory pathways using selective receptor antagonists, or the use of transgenic mice with perturbed cell signaling, demostrated that the activation of IL-1R type 1 and TLR4 by their respective endogenous ligands, i.e., interleukin (IL)-1b and High Mobility Group Box 1, is implicated in the precipitation and recurrence of experimentally induced seizures in rodents. This evidence highlights a new target system for pharmacological intervention to inhibit seizures by interfering with mechanisms involved in their genesis and recurrence.

Effect of the lipopolysaccharide antagonist Planktothrix sp. FP1 cyanobacterial extract on human polymorphonuclear leukocytes.

CyP is a lipopolysaccharide (LPS)-like molecule extracted from the freshwater cyanobacterium Oscillatoria planktothrix FP1, which has been reported to be a potent competitive inhibitor of bacterial LPS. In the present study the ability of CyP to affect human polymorphonuclear leukocyte (PMN) function was investigated. PMNs were isolated from venous blood by standard density-gradient centrifugation. Cell migration was measured by use of the Boyden chamber assay. Interleukin (IL)-8 and tumor necrosis factor (TNF)-α production was measured using a sandwich-type enzyme-linked immunosorbent assay. PMN intracellular reactive oxygen species (ROS) levels were assessed by the use of a fluorescent probe coupled to spectrophotometry. CyP 10-100 µg/ml was chemotactic for PMNs without affecting the chemotactic response to either E. coli LPS or N-formyl-Met-Leu-Phe (fMLP). CyP per se did not affect PMN production of either IL-8 or TNF-α, but concentration-dependently reduced LPS-induced production of both cytokines. On the contrary, CyP had no effect either on fMLP-induced production of IL-8 or on PMN oxidative burst (at rest and after stimulation with fMLP), a response which is known to be independent from LPS-operated pathways. In human PMNs CyP behaves as a selective and effective LPS antagonist. These findings support the therapeutic potential of CyP in endotoxin-dependent disease.

Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures.

Brain inflammation is a major factor in epilepsy, but the impact of specific inflammatory mediators on neuronal excitability is incompletely understood. Using models of acute and chronic seizures in C57BL/6 mice, we discovered a proconvulsant pathway involving high-mobility group box-1 (HMGB1) release from neurons and glia and its interaction with Toll-like receptor 4 (TLR4), a key receptor of innate immunity. Antagonists of HMGB1 and TLR4 retard seizure precipitation and decrease acute and chronic seizure recurrence. TLR4-defective C3H/HeJ mice are resistant to kainate-induced seizures. The proconvulsant effects of HMGB1, like those of interleukin-1beta (IL-1beta), are partly mediated by ifenprodil-sensitive N-methyl-d-aspartate (NMDA) receptors. Increased expression of HMGB1 and TLR4 in human epileptogenic tissue, like that observed in the mouse model of chronic seizures, suggests a role for the HMGB1-TLR4 axis in human epilepsy. Thus, HMGB1-TLR4 signaling may contribute to generating and perpetuating seizures in humans and might be targeted to attain anticonvulsant effects in epilepsies that are currently resistant to drugs.

Meconium-induced release of cytokines is mediated by the TRL4/MD-2 complex in a CD14-dependent manner.

OBJECTIVE: Meconium, the first intestinal discharge of the newborn, contains material accumulated during fetal life. Meconium activates complement and CD14 and may induce a systemic inflammatory response. Toll-like receptors are classical pattern-recognition receptors recognizing both exogenous and host-derived ligands. The cyanobacterial product CyP is a potent LPS antagonist binding to the TLR4/MD-2 complex. The aim of the present study was to investigate the role of the CD14/TLR4/MD-2 complex in meconium-induced inflammation. METHODS: Whole blood from six donors was preincubated with anti-CD14 or CyP. Meconium was added and the samples were incubated for 4h. Twenty-seven inflammatory mediators were measured in a Bioplex Array Reader. Human embryonic kidney cells transfected with plasmids containing NF-kappaB dependent luciferase reporter, human MD-2, TLR4, TLR2 and/or CD14, were incubated with meconium or LPS for 18 h. Luciferase activity in cytoplasmic extracts was measured using a Luciferase Assay System kit. RESULTS: Meconium induced formation of a broad panel of inflammatory mediators. CyP and anti-CD14 significantly (p<0.001) inhibited meconium-induced formation of (a) proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6, IFN-gamma) by 60-80% and 72-94%, respectively, (b) anti-inflammatory cytokines (IL-10, IL-1Ra) by 58-59% and 50-65%, respectively, (c) chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, eotaxin, IP-10) by 43-77% and 57-87%, respectively, and (d) growth factors (G-CSF, GM-CSF, basic FGF, PDGFbb) by 53-71% and 40-78%, respectively, with no statistical significant difference between Cyp and anti-CD14. The inflammatory response could only partly be explained by LPS, suggesting that endogenous components of meconium contribute to the inflammatory response. Meconium activated NF-kappaB dose-dependently in cells expressing TLR4/MD-2 together with CD14, while no effect was seen in cells expressing TLR4/MD-2 alone or in TLR2/CD14 transfected cells. CONCLUSIONS: The results indicate that the CD14-dependent meconium-induced inflammatory reaction is mediated through the TLR4/MD2 complex. These data may have implications for future therapeutic strategies for meconium aspiration syndrome.

A biosensor for all D-amino acids using evolved D-amino acid oxidase.

Determination of the D-amino acid content in foods and in biological samples is a very important task. In order to achieve this goal we developed a biosensor employing the flavoenzyme D-amino acid oxidase from the yeast Rhodotorula gracilis. To produce a device in which the D-amino acid composition does not alter the results, both the wild-type and a number of mutants obtained by rational design and directed evolution approaches were used. An analysis of D-amino acid oxidase mutants activity on D-amino acid mixtures containing various ratios of neutral, acidic, and basic substrates identified the Amberzyme-immobilized T60A/Q144R/K152E and M213G mutants as the best choice: their response shows an only limited dependence on the solution composition when at least 20% of the D-amino acid is made up of D-alanine (standard error is approximately 5-9%). This is the first report, to our knowledge, demonstrating that the entire D-amino acid content can be determined by using a screen-printed electrode amperometric biosensor, with a detection limit of 0.25 mM and a mean response time of 10-15 min. The D-amino acid assay based on R. gracilis DAAO-biosensor is inexpensive, simple to perform, and rapid: the D-amino acid concentration of a variety of biological samples can be investigated using this assay.

Cyanobacterial LPS antagonist (CyP)-a novel and efficient inhibitor of Escherichia coli LPS-induced cytokine response in the pig.

Toll-like receptors are essential pattern-recognition receptors of the innate immune system. They recognize a range of conserved molecules of invading microorganisms. The innate immune system is developed to protect the host, but can be deleterious if activated uncontrolled or inappropriate, such as in sepsis with Gram-negative bacteria. New approaches for treatment, like inhibition of innate immune responses, may be beneficial for the outcome of such conditions. Toll-like receptor 4 associated with CD14 and MD-2, is the lipopolysaccharide (LPS)-receptor and one of the candidates for such intervention. We investigated the newly described cyanobacterial LPS analogue CyP as a potential inhibitor of Escherichia coli (E. coli) LPS-induced inflammatory response in porcine whole blood. Pro-inflammatory cytokines and soluble terminal complement complex, sC5b-9, were used as read-outs. CyP, in contrast to E. coli LPS, did not induce cytokine production using doses up to 1mug/mL whole blood, indicating a lack of agonistic effect of CyP. In contrast, CyP was an efficient LPS antagonist, dose-dependently and completely inhibiting E. coli LPS-induced TNF-alpha, IL-1beta and IL-8 production. CyP was a modest activator of porcine complement compared to LPS from other Gram-negative bacteria. When CyP was pre-incubated in porcine whole blood before adding whole E. coli bacteria, a modest, variable and non-significant inhibition of cytokines were seen, reaching an average inhibition of 44% for IL-1beta. We have demonstrated for the first time that the cyanobacterial LPS analogue, CyP, is an efficient inhibitor of E. coli LPS-induced cytokines in whole blood and may be a candidate for therapeutic LPS-inhibition.