Haemorrhagic shock and encephalopathy syndrome: report of two cases with special reference to hypoglycaemia.

Haemorrhagic shock and encephalopathy syndrome (HSES) is a devastating disorder affecting infants. So far no cases have been reported in Switzerland. It is characterised by the abrupt onset of hyperpyrexia, shock, encephalopathy, diarrhoea, disseminated intravascular coagulation (DIC) and renal and hepatic failure in previously healthy infants. Severe hypoglycaemia has been repeatedly reported in association with HSES. However, the pathophysiology of the hypoglycaemia is not clear. We report on two infants (2 and 7 months old) with typical HSES, both of whom were presented with nonketotic hypoglycaemia. In the first case, plasma insulin was 23 pmol/l at the time of hypoglycaemia (0.1 mmol/l). In the second case, increased values for interleukin-6 (IL-6) (319 pg/ml) and IL-8 (1382 pg/ml) were found 24 hours after admission, whereas IL-1 and tumour necrosis factor-alpha (TNF-alpha) were not measurable. Alpha-1-antitrypsin was decreased (0.6 g/l). In hyperpyrexic, unconscious and shocked infants, HSES should be considered and hypoglycaemia should be specifically looked for. Hypoglycaemia is not caused by hyperinsulinism but may be secondary to the release of cytokines.

Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.

Activation of the mouse TATA-less and human TATA-containing UDP-glucuronosyltransferase 1A1 promoters by hepatocyte nuclear factor 1.

UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1alpha and HNF1beta in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human.

Role of hepatocyte wounding on " Plasmodium " liver-stage development and survival

RESUME La première étape primordiale au cycle de vie du Plasmodium dans un hôte mammifère est l'invasion des hepatocytes par des sporozoites. L'infection finale des hepatocytes est précédée de la traversée de plusieurs cellules hôtes, rompant les membranes plasmiques et ayant comme résultat la sécrétion des facteurs cytotoliques dans le micro-environnement. Ce matériel endogène libéré est fortement stimulant/immunogène et peut servir de signal de danger initiant des réponses distinctes dans diverses cellules. De nos jours, le caractère essentiel et salutaire de la migration des sporozoites comme étape d'infection du Plasmodium est vivement controversée. Ainsi, notre étude a visé à caractériser l'effet de l'interaction du parasite avec ses cellules hôtes d'un point de vue immunologique. En particulier, nous avons voulu évaluer l'effet de la perte de matériel cellulaire pendant l'infection de Plasmodium sur les hepatocytes primaires de souris et sur des cultures cellulaires HepG2. Nous avons observé que les facteurs cytotoxiques dérivés des cellules endommagés activent NF-κB - un important régulateur de réponse inflammatoires -dans des cellules voisines des cellules endommagés, qui sont des cellules hôtes potentielles pour l'infection finale du parasite. Cette activation de NF-κB s'est produite peu de temps après l'infection et a mené in vitro et in vivo à une réduction d'infection de façon dépendante du temps, un effet qui a pu être compensé par l'addition de BAY11-7082, un inhibiteur spécifique de NF-κB. De plus, aucune activation de NF-κB avec des parasites SPECT-/-, incapables de traverser les hepatocytes, n'a été observée. Nous avons montré parla suite que l'activation de NF-κB induit l'expression de l'enzyme iNOS dans les hepatocytes, qui est responsable d'une diminution des hepatocytes infectés. En outre, les hepatocytes primaires des souris MyD88-/- n'ont montré ni activation de NF-κB, ni expression d'iNOS lors de l'infection, ce qui suggère la participation des membres de famille du Toll/IL-1 récepteur dans la reconnaissance des facteurs cytosoxiques. En effet, le manque de MyD88 a augmenté significativement l'infection in vitro et in vivo. D'autre part, un rôle bénéfique pour l'activation de NF-κB a été évalué. Les cellules infectées étaient plus résistantes contre l'apoptose induite par Fas (CD95/Apo-1) que les cellules non infectées ou les cellules infectées dans lesquelles NF-κB a été bloqué par BAY11-7082 in vitro. Paradoxalement, l'expression d'iNOS contribue à la protection des cellules infectées contre l'apoptose pax Fas, puisque le traitement avec l'inhibiteur spécifique SMT (S-methylisothiourea) a rendu les cellules infectées plus susceptibles à l'apoptose. Un effet bénéfique additionnel pour le parasite est que la plupart des cellules hôtes traversées présentent des peptides du parasite aux cellules T cytotoxiques spécifiques et peuvent donc réorienter la réaction immune spécifique sur les cellules non infectées. Nous montrons que les cellules hôtes endommagés par la migration du parasite induit l'inflammation, qui limite l'ampleur de l'infection. D'autre part, nos données soutiennent que la survie du parasite Plasmodium dans le foie est assurée par une augmentation de la résistance des hepatocytes contre l'apoptose. SUMMARY The first obligatory step of the Plasmodium life cycle in the mammalian host is the invasion of hepatocytes by sporozoites. Final hepatocyte infection involves the penetration of several host cells, whose plasma membranes are ruptured in the process, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory / immunogenic and can serve as a danger signal initiating distinct responses in various cells. To date, it is highly controversial whether sporozoite migration through hepatocytes is an essential and beneficial step for Plasmodium infection. Thus, our study aimed at characterizing the effect of the interaction of the parasite with its host cells from an immunological point of view In particular, we wanted to evaluate the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-κB - a main regulator of host inflammatory responses - in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-κB occurred shortly after infection and led to a reduction of infection load in a time dependent manner in vitro and in viva, an effect that could be reverted by addition of the specific NF-κB inhibitor BAY11-7082. In addition, no NF-κB activation was observed when SPECT-/- parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-κB activation causes the induction of inducible nitric oxide synthase (iNOS) expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88-/- mice showed no NF-κB activation and iNOS expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. In a further complementary series of experiments, we assessed a possible beneficial role for the activation of NF-κB. Infected cells were more resistant to Fas (CD95/Apo-1)-mediated apoptosis than uninfected cells or infected cells in which NF-κB was blocked by BAYl1-7082 in vitro. Paradoxically, iNOS expression contributes to the protection of infected cells from Fas-induced apoptosis, since treatment with the specific iNOS inhibitor SMT (S-Methylisothiourea Sulfate) rendered the infected cells more susceptible to apoptosis. An additional beneficial effect of host cell traversal for the parasite is the fact that mainly traversed cells present parasite-derived peptides to specific cytotoxic T cells and therefore may redirect the specific immune response to uninfected cells. In summary, we have shown that host cells wounded by parasite migration induce inflammation, which limits the extent of parasite infection. In addition, our data support the notion that survival of Plasmodium parasites in the liver is mediated by increasing the resistance of hepatocytes to Fas-induced apoptosis.

Myeloid differentiation factor-88/interleukin-1 signaling controls cardiac fibrosis and heart failure progression in inflammatory dilated cardiomyopathy.

RATIONALE: The myeloid differentiation factor (MyD)88/interleukin (IL)-1 axis activates self-antigen-presenting cells and promotes autoreactive CD4(+) T-cell expansion in experimental autoimmune myocarditis, a mouse model of inflammatory heart disease. OBJECTIVE: The aim of this study was to determine the role of MyD88 and IL-1 in the progression of acute myocarditis to an end-stage heart failure. METHODS AND RESULTS: Using alpha-myosin heavy chain peptide (MyHC-alpha)-loaded, activated dendritic cells, we induced myocarditis in wild-type and MyD88(-/-) mice with similar distributions of heart-infiltrating cell subsets and comparable CD4(+) T-cell responses. Injection of complete Freund's adjuvant (CFA) or MyHC-alpha/CFA into diseased mice promoted cardiac fibrosis, induced ventricular dilation, and impaired heart function in wild-type but not in MyD88(-/-) mice. Experiments with chimeric mice confirmed the bone marrow origin of the fibroblasts replacing inflammatory infiltrates and showed that MyD88 and IL-1 receptor type I signaling on bone marrow-derived cells was critical for development of cardiac fibrosis during progression to heart failure. CONCLUSIONS: Our findings indicate a critical role of MyD88/IL-1 signaling in the bone marrow compartment in postinflammatory cardiac fibrosis and heart failure and point to novel therapeutic strategies against inflammatory cardiomyopathy.

Interleukin-1β inhibition prevents choroidal neovascularization and does not exacerbate photoreceptor degeneration.

The pro-inflammatory cytokine IL-1β has been shown to promote angiogenesis. It can have a neurotoxic or neuroprotective effect. Here, we have studied the expression of IL-1β in vivo and the effect of the IL-1 receptor antagonist on choroidal neovascularization (CNV) and retinal degeneration (RD). IL-1β expression significantly increased after laser injury (real time PCR) in C57BL/6 mice, in the C57BL/6 Cx3cr1(-/-) model of age-related macular degeneration (enzyme-linked immunoabsorbent assay), and in albino Wistar rats and albino BALB Cx3cr1(+/+) and Cx3cr1(-/-) mice (enzyme-linked immunoabsorbent assay) after light injury. IL-1β was localized to Ly6G-positive, Iba1-negative infiltrating neutrophils in laser-induced CNV as determined by IHC. IL-1 receptor antagonist treatment significantly inhibited CNV but did not affect Iba1-positive macrophage recruitment to the injury site. IL-1β significantly increased endothelial cell outgrowth in aortic ring assay independently of vascular endothelial growth factor, suggesting a direct effect of IL-1β on choroidal endothelial cell proliferation. Inhibition of IL-1β in light- and laser-induced RD models did not alter photoreceptor degeneration in Wistar rats, C57BL/6 mice, or RD-prone Cx3cr1(-/-) mice. Our results suggest that IL-1β inhibition might represent a valuable and safe alternative to inhibition of vascular endothelial growth factor in the control of CNV in the context of concomitant photoreceptor degeneration as observed in age-related macular degeneration.

Myeloid-related proteins 8 and 14 contribute to monosodium urate monohydrate crystal-induced inflammation in gout.

OBJECTIVE: Monosodium urate monohydrate (MSU) crystal-induced interleukin-1β (IL-1β) secretion is a critical factor in the pathogenesis of gout. However, without costimulation by a proIL-1β-inducing factor, MSU crystals alone are insufficient to induce IL-1β secretion. The responsible costimulatory factors that act as a priming endogenous signal in vivo are not yet known. We undertook this study to analyze the costimulatory properties of myeloid-related protein 8 (MRP-8) and MRP-14 (endogenous Toll-like receptor 4 [TLR-4] agonists) in MSU crystal-induced IL-1β secretion and their relevance in gout. METHODS: MRP-8/MRP-14 was measured in paired serum and synovial fluid samples by enzyme-linked immunosorbent assay (ELISA) and localized in synovial tissue from gout patients by immunohistochemistry. Serum levels were correlated with disease activity, and MSU crystal-induced release of MRPs from human phagocytes was measured. Costimulatory effects of MRP-8 and MRP-14 on MSU crystal-induced IL-1β secretion from phagocytes were analyzed in vitro by ELISA, Western blotting, and polymerase chain reaction. The impact of MRP was tested in vivo in a murine MSU crystal-induced peritonitis model. RESULTS: MRP-8/MRP-14 levels were elevated in the synovium, tophi, and serum of patients with gout and correlated with disease activity. MRP-8/MRP-14 was released by MSU crystal-activated phagocytes and increased MSU crystal-induced IL-1β secretion in a TLR-4-dependent manner. Targeted deletion of MRP-14 in mice led to a moderately reduced response of MSU crystal-induced inflammation in vivo. CONCLUSION: MRP-8 and MRP-14, which are highly expressed in gout, are enhancers of MSU crystal-induced IL-1β secretion in vitro and in vivo. These endogenous TLR-4 ligands released by activated phagocytes contribute to the maintenance of inflammation in gout.

NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8⁺ T cells.

Memory T cells exert antigen-independent effector functions, but how these responses are regulated is unclear. We discovered an in vivo link between flagellin-induced NLRC4 inflammasome activation in splenic dendritic cells (DCs) and host protective interferon-γ (IFN-γ) secretion by noncognate memory CD8(+) T cells, which could be activated by Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that CD8α(+) DCs were particularly efficient at sensing bacterial flagellin through NLRC4 inflammasomes. Although this activation released interleukin 18 (IL-18) and IL-1β, only IL-18 was required for IFN-γ production by memory CD8(+) T cells. Conversely, only the release of IL-1β, but not IL-18, depended on priming signals mediated by Toll-like receptors. These findings provide a comprehensive mechanistic framework for the regulation of noncognate memory T cell responses during bacterial immunity.

Interleukin-1 as a therapeutic target in gout.

PURPOSE OF REVIEW: To give an overview of current evidence for interleukin (IL)-1 blockade in the management of gout. RECENT FINDINGS: Three IL-1 blockers are currently available for clinical use: anakinra, rilonacept and canakinumab. Recent studies have focused on drugs with a long half-life: rilonacept and canakinumab. For treatment of acute gouty arthritis, three randomized controlled trials (RCTs) showed efficacy of canakinumab with some safety concerns and one RCT failed to show efficacy of rilonacept. For prevention of gout flare when starting uric acid lowering therapy (ULT), four RCTs showed efficacy of rilonacept and one RCT showed efficacy of canakinumab. SUMMARY: There is sufficient evidence supporting the use of IL-1 blockers for treatment of acute gouty arthritis or for prevention of gout flares when starting ULT in selected patients, with contraindications or intolerance to conventional therapy. More data are needed to assess safety and to specify their use in routine practice.

Basic calcium phosphate crystals induce IL-1B secretion in vitro through activation of the Nalp3 inflammasome

Objectives: We tested the effects of the three forms of basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbonate-substituted apatite (CA) and hydroxyapatite (HA)) on monocytes and macrophages on IL-1β secretion. The requirement for the NALP3 inflammasome and TLR2 and TLR4 receptors in this acute response was analyzed.

Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function.

Inflammasomes are caspase-1-activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1β and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity. Surprisingly, the absence of ASC resulted in IL-1β cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11(-/-) cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR.

Sporozoite-mediated hepatocyte wounding limits Plasmodium parasite development via MyD88-mediated NF-kappa B activation and inducible NO synthase expression

Plasmodium sporozoites traverse several host cells before infecting hepatocytes. In the process, the plasma membranes of the cells are ruptured, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory/immunogenic and can serve as a danger signal initiating distinct responses in various cells. Thus, our study aimed at characterizing the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-kappaB, a main regulator of host inflammatory responses, in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-kappaB occurred shortly after infection and led to a reduction of infection load in a time-dependent manner in vitro and in vivo, an effect that could be reverted by addition of the specific NF-kappaB inhibitor BAY11-7082. Furthermore, no NF-kappaB activation was observed when Spect(-/-) parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-kappaB activation causes the induction of inducible NO synthase expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88(-/-) mice showed no NF-kappaB activation and inducible NO synthase expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. Thus, host cell wounding due to parasite migration induces inflammation which limits the extent of parasite infection

Effects of particulate matter on inflammatory markers in the general adult population.

BACKGROUND: Particulate air pollution is associated with increased risk of cardiovascular disease and stroke. Although the precise mechanisms underlying this association are still unclear, the induction of systemic inflammation following particle inhalation represents a plausible mechanistic pathway.¦METHODS: We used baseline data from the CoLaus Study including 6183 adult participants residing in Lausanne, Switzerland. We analyzed the association of short-term exposure to PM10 (on the day of examination visit) with continuous circulating serum levels of high-sensitive C-reactive protein (hs-CRP), interleukin 1-beta (IL-1β), interleukin 6 (IL-6), and tumor-necrosis-factor alpha (TNF-α) by robust linear regressions, controlling for potential confounding factors and assessing effect modification.¦RESULTS: In adjusted analyses, for every 10 μg/m3 elevation in PM10, IL-1ß increased by 0.034 (95 % confidence interval, 0.007-0.060) pg/mL, IL-6 by 0.036 (0.015-0.057) pg/mL, and TNF-α by 0.024 (0.013-0.035) pg/mL, whereas no significant association was found with hs-CRP levels.¦CONCLUSIONS: Short-term exposure to PM10 was positively associated with higher levels of circulating IL-1ß, IL-6 and TNF-α in the adult general population. This positive association suggests a link between air pollution and cardiovascular risk, although further studies are needed to clarify the mechanistic pathway linking PM10 to cardiovascular risk.

Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation.

Interleukin-1β (IL-1β) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1β activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1β that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1β by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1β and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.

Induction of p38, tumour necrosis factor-α and RANTES by mechanical stretching of keratinocytes expressing mutant keratin 10R156H.

Background : Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma), characterized by ichthyotic, rippled hyperkeratosis, erythroderma and skin blistering, is a rare autosomal dominant disease caused by mutations in keratin 1 or keratin 10 (K10) genes. A severe phenotype is caused by a missense mutation in a highly conserved arginine residue at position 156 (R156) in K10. Objectives: To analyse molecular pathomechanisms of hyperproliferation and hyperkeratosis, we investigated the defects in mechanosensation and mechanotransduction in keratinocytes carrying the K10R156H mutation. Methods: Differentiated primary human keratinocytes infected with lentiviral vectors carrying wild-type K10 (K10wt) or mutated K10R156H were subjected to 20% isoaxial stretch. Cellular fragility and mechanosensation were studied by analysis of mitogen-activated protein kinase activation and cytokine release. Results: Cultured keratinocytes expressing K10R156H showed keratin aggregate formation at the cell periphery, whereas the filament network in K10wt cells was normal. Under stretching conditions K10R156H keratinocytes exhibited about a twofold higher level of filament collapse compared with steady state. In stretched K10R156H cells, higher p38 activation, higher release of tumour necrosis factor-alpha and RANTES but reduced interleukin-1 beta secretion compared with K10wt cells was observed. Conclusions: These results demonstrate that the R156H mutation in K10 destabilizes the keratin intermediate filament network and affects stress signalling and inflammatory responses to mechanical stretch in differentiated cultured keratinocytes.