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PubMed2005

MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice: implications for Toll-like receptor signaling.

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 cells.

A synthetic Nod2 agonist, muramyldipeptide (MDP), and two Nod1 agonists, FK565 and FK156, mimic the bacterial peptidoglycan moiety and are powerful adjuvants that induce cell-mediated immunity, especially delayed-type hypersensitivity. In this study, we used human dendritic cell (DC) cultures to examine possible T helper type 1 (Th1) responses induced by MDP and FK565/156 in combination with various synthetic Toll-like receptor (TLR) agonists, including synthetic lipid A (TLR4 agonist), the synthetic triacyl lipopeptide Pam3CSSNA (TLR2 agonist), poly(I:C) (TLR3 agonist), and CpG DNA (TLR9 agonist). Immature DCs derived from human monocytes expressed mRNAs for Nod1, Nod2, TLR2, TLR3, TLR4, and TLR9. The stimulation of DCs with MDP and FK565 in combination with lipid A, poly(I:C), and CpG DNA, but not with Pam3CSSNA, synergistically induced interleukin-12 (IL-12) p70 and gamma interferon (IFN-gamma), but not IL-18, in culture supernatants and induced IL-15 on the cell surface. In correlation with the cytokine induction, an upregulation of the mRNA expression of these cytokine genes was observed. Notably, IL-12 p35 mRNA expression increased >1,000-fold upon stimulation with lipid A plus either MDP or FK565 compared with stimulation with each stimulant alone. In contrast, for the expression of CD83 and costimulatory molecules such as CD40, CD80, and CD86, no synergistic effects were observed upon stimulation with Nod plus TLR agonists. The culture supernatants of DCs stimulated with lipid A plus either MDP or FK565 activated human T cells to produce high levels of IFN-gamma, and the activity was attributable to DC-derived IL-12. These findings suggest that Nod1 and Nod2 agonists in combination with TLR3, TLR4, and TLR9 agonists synergistically induce IL-12 and IFN-gamma production in DCs to induce Th1-lineage immune responses.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2005

Sensitization of human aortic endothelial cells to lipopolysaccharide via regulation of Toll-like receptor 4 by bacterial fimbria-dependent invasion.

Toll-like receptors (TLRs) are differentially up-regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. Epidemiological data support the idea that periodontal disease may be a risk factor for acceleration of atherosclerosis. Porphyromonas gingivalis, the etiological agent of periodontal disease, invades endothelium, has been detected in human atheromatous tissue, and accelerates atheroma formation in apolipoprotein E-/- mice with concurrent induction of TLRs in the aorta. As endothelial cells can present antigen via TLRs and play an important role in the development of atherosclerosis, we examined TLR expression in human aortic endothelial cells (HAEC) cultured with wild-type P. gingivalis, a fimbria-deficient mutant, and purified antigens. We observed increased TLR expression in HAEC infected with wild-type P. gingivalis by fluorescence-activated cell sorter, but not with noninvasive, fimbria-deficient mutant or purified P. gingivalis antigens. Following a wild-type P. gingivalis challenge, functional TLR2 and TLR4 activation was assessed by subsequent stimulation with TLR agonists Staphylococcus aureus lipoteichoic acid (SLTA; TLR2 ligand) and Escherichia coli lipopolysaccharide (LPS; TLR4 ligand). Unchallenged HAEC failed to elicit monocyte chemoattractant protein 1 (MCP-1) in response to LPS or SLTA but did so when cultured with wild-type P. gingivalis. P. gingivalis-induced TLR2 and -4 expression on HAEC functionally reacted to SLTA and E. coli LPS as measured by a further increase in MCP-1 production. Furthermore, MCP-1 expression elicited by E. coli LPS was inhibitable with TLR4-specific antibody and polymyxin B. These results indicate that invasive P. gingivalis stimulates TLR expression on the surface of endothelium and these primed cells respond to defined TLR-specific ligands.

Antigens, Bacterial↗
PubMed2005

Synthetic toll-like receptor 4 agonists stimulate innate resistance to infectious challenge.

A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.

Animals↗
PubMed2004

Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus.

Mouse mammary tumor virus (MMTV) is a milk-borne retrovirus that exploits the adaptive immune system. It has recently been shown that MMTV activates B cells via Toll-like receptor 4 (TLR4), a molecule involved in innate immune responses. Here, we show that direct virus binding to TLR4 induced maturation of bone marrow-derived dendritic cells and up-regulated expression of the MMTV entry receptor (CD71) on these cells. In vivo, MMTV increased the number of dendritic cells in neonatal Peyer's patches and their expression of CD71; both these effects were dependent on TLR4. Thus, retroviral signaling through TLRs plays a critical role in dendritic-cell participation during infection.

Animals↗
PubMed2005

Epithelioid granulomas, pattern recognition receptors, and phenotypes of Crohn's disease.

INTRODUCTION: Crohn's disease is a chronic inflammatory disorder of the gut. It is assumed that a defective interaction between the bacterial flora of the gut and the innate immune system plays a key role in the pathogenesis of the disease. This may lead to specific histological lesions. The epithelioid granuloma is particularly interesting in this regard as it is also observed in several bacterial infections of the gut. AIMS AND METHODS: We hypothesised that genetic or environmental factors with a known influence on inflammation or immunity would lead to an increased prevalence of granulomas. Therefore, surgical specimens from 161 patients were evaluated for the presence of granulomas. Patients were genotyped for the three single nucleotide polymorphisms in caspase recruitment domain 15 (CARD15)/NOD2 associated with CD and for Asp299Gly in Toll-like receptor 4 (TLR4). RESULTS: The overall prevalence of granulomas was 68.9%. We did not find a significant correlation between granulomas and TLR4 or CARD15 variants. The frequency of granulomas increased with more distal disease (63% small bowel, 72% right colon, 88% left colon, 90% rectum; p=0.01). Granulomas were more frequent in younger patients (odds ratio 0.95 (95% confidence interval 0.92-0.98) p=0.007). CONCLUSION: In this study of 161 well documented CD patients, we found no significant association between CARD15 and TLR4 variants and granulomas. This finding seems to refute our initial hypothesis. However, it may be that additional factors are needed for granuloma development. Granulomas may develop only when specific bacterial components are present. Therefore, future research on granuloma pathogenesis should be orientated towards detection and identification of bacterial components in these lesions.

Adult↗
PubMed2001

Lipopolysaccharide endotoxins.

Bacterial lipopolysaccharides (LPS) typically consist of a hydrophobic domain known as lipid A (or endotoxin), a nonrepeating "core" oligosaccharide, and a distal polysaccharide (or O-antigen). Recent genomic data have facilitated study of LPS assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and have established the importance of lateral gene transfer in generating structural diversity of O-antigens. Many enzymes of lipid A biosynthesis like LpxC have been validated as targets for development of new antibiotics. Key genes for lipid A biosynthesis have unexpectedly also been found in higher plants, indicating that eukaryotic lipid A-like molecules may exist. Most significant has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells. TLR4 belongs to a family of innate immunity receptors that possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment, and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88. The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking inflammation associated with infection.

ATP-Binding Cassette Transporters↗
PubMed2005

Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Galpha(i) proteins.

Heterotrimeric G(i) proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of G(i) proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of Galpha(i2) or Galpha(i1/3) protein in Galpha(i2)-knockout (Galpha(i2)-/-) or Galpha(i1/3)-knockout (Galpha(i1/3)-/-) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (MPhi) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF-alpha and IFN-gamma in splenocytes from Galpha(i2)-/- mice compared with wild-type (WT) mice. Also, LPS-induced TNF-alpha was increased in splenocytes from Galpha(i1/3)-/- mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-alpha, IL-10, and thromboxane B(2) (TxB(2)) production was decreased in MPhi harvested from Galpha(i2)-/- mice. Also, LPS-induced production of IL-10 and TxB(2) was decreased in MPhi from Galpha(i1/3)-/- mice. In subsequent in vivo studies, TNF-alpha levels after LPS challenge were significantly greater in Galpha(i2)-/- mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated Galpha(i2)-/- mice compared with WT mice. These data suggest that G(i) proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli.

Animals↗
PubMed2005

Toll-like receptor-4 regulation of hepatic Cyp3a11 metabolism in a mouse model of LPS-induced CNS inflammation.

Central nervous system (CNS) infection and inflammation severely reduce the capacity of cytochrome P-450 metabolism in the liver. We developed a mouse model to examine the effects of CNS inflammation on hepatic cytochrome P-450 metabolism. FVB, C57BL/6, and C3H/HeouJ mice were given Escherichia coli LPS (2.5 microg) by intracerebroventricular (ICV) injection. The CNS inflammatory response was confirmed by the elevation of TNF-alpha and/or IL-1beta proteins in the brain. In all mouse strains, LPS produced a 60-70% loss in hepatic Cyp3a11 expression and activity compared with saline-injected controls. Adrenalectomy did not prevent the loss in Cyp3a11 expression or activity, thereby precluding the involvement of the hypothalamic-adrenal-pituitary axis. Endotoxin was detectable (1-10 ng/ml) in serum between 15 and 120 min after ICV dosing of 2.5 microg LPS. Peripheral administration of 2.5 microg LPS by intraperitoneal injection produced similar serum endotoxin levels and a similar loss (60%) in Cyp3a11 expression and activity in the liver. The loss of Cyp3a11 in response to centrally or peripherally administered LPS could not be evoked in Toll-like receptor-4 (TLR4)-mutant (C3H/HeJ) mice, indicating that TLR4 signaling pathways are directly involved in the enzyme loss. In summary, we conclude that LPS is transferred from the brain to the circulation in significant quantities in a model of CNS infection or inflammation. Subsequently, LPS that has reached the circulation stimulates a TLR4-dependent mechanism in the periphery, evoking a reduction in Cyp3a11 expression and metabolism in the liver.

Animals↗
PubMed2006

Regulation of Toll-like receptor 4-induced proasthmatic changes in airway smooth muscle function by opposing actions of ERK1/2 and p38 MAPK signaling.

Activation of Toll-like receptors (TLRs) on immune surveillance cells in the lung has been implicated in the pathobiology of allergic asthma, a condition associated with altered airway smooth muscle (ASM) contractility. Because ASM is known to directly respond to various proasthmatic stimuli, the potential role of TLR signaling in ASM in regulating airway expression of the proasthmatic phenotype was investigated. Cultured human ASM cells were found to express TLR4 and TLR9 mRNA transcripts and, whereas TLR9 stimulation had little effect, TLR4 activation with LPS elicited significant increases in IL-6 release and evoked proasthmatic-like changes in the constrictor and relaxation responsiveness of isolated rabbit ASM tissues. Complementary studies further demonstrated that the ASM responses to LPS were associated with activation of the ERK1/2 and p38 MAPK signaling pathways, IKK-mediated activation of NF-kappaB, and coupling of phosphorylated ERK1/2 with the p65 subunit of NF-kappaB. Moreover, the induced NF-kappaB activity and changes in ASM responsiveness were prevented in LPS-exposed ASM that were pretreated with inhibitors of ERK1/2 signaling, whereas inhibition of p38 MAPK augmented the proasthmatic responses to LPS. Finally, activation of p38 MAPK with anisomycin prevented both the LPS-induced stimulation of ERK1/2-mediated NF-kappaB activity and associated changes in ASM responsiveness. Collectively, these data support the novel concept that TLR4 activation in ASM elicits changes in ASM function that are regulated by opposing effects of MAPK signaling, wherein LPS-induced ERK1/2 activation mediates NF-kappaB-dependent proasthmatic-like changes in ASM function, whereas coactivation of p38 MAPK serves to homeostatically downregulate the proasthmatic effects of ERK1/2 activation.

Animals↗
PubMed2005

TLR-4 pathway mediates the inflammatory response but not bacterial elimination in E. coli pneumonia.

We examined the role of Toll-like receptor (TLR)-4 in modifying the lung inflammatory response and its effects on the bacterial recovery from the lungs following inhaled Escherichia coli in two different strains of TLR-4 mutant mice that are hyporesponsive to LPS. The C57BL/10ScN(tlr4(lps-del)) mice containing a deletion mutation in the TLR-4 gene showed lower proinflammatory cytokine levels, lower lung MPO activity, and less parenchymal and peribronchial inflammation compared with the C57BL/10ScSn mice, a related TLR-4 wild-type substrain. However, the C57BL/10ScN(tlr4(lps-del)) mutant showed lower bacterial recovery in the lungs following inhaled E. coli associated with a rapid but transient increase in air space neutrophil counts at 6 h. In comparison, the C3H/HeJ(tlr4(Lps-d)) mutant mice containing a Pro712His substitution in TLR-4 demonstrated lower proinflammatory cytokine levels, lower lung MPO activity, and lower neutrophil accumulation in the air spaces but showed no differences in the bacterial burden of inhaled E. coli at 6 h, when compared with the TLR-4 wild-type C3H/HeSnJ mice. Thus two different TLR-4 mutants showed attenuated inflammatory responses in the lungs, but the reduced inflammatory responses were not consistently associated with either improved or impaired bacterial elimination from the lungs. Our findings indicate that the inflammatory response to inhaled E. coli is TLR-4 dependent, but bacterial elimination depends on other factors in addition to TLR-4.

Administration, Inhalation↗
PubMed2003

Toll-like receptor 4 and CD14 mRNA expression are lower in resistive exercise-trained elderly women.

The purpose of this study was to examine the influence of resistive exercise training and hormone status on mRNA expression of toll-like receptor 4 (TLR4), CD14, IL-1beta, IL-6, and TNF-alpha. Resistive exercise-trained women on "traditional" hormone replacements [hormone replacement therapy (HRT), n = 9], not taking hormones (NHR, n = 6), or taking medications known to influence bone (MIB, n = 7) were compared with untrained subjects not taking supplemental hormones (Con, n = 6). Blood was taken from trained subjects before, immediately after, and 2 h after resistive exercise (same time points for resting Con). TLR4 mRNA expression (RT-PCR) was not different among groups or across time but was significantly (P = 0.044) lower (1.9-fold) when trained groups were collapsed and compared with Con. There was also a significant group effect (P < 0.0001) for TLR4 mRNA when expressed per monocyte. CD14 expression was significantly (P = 0.006) lower (2.3-fold) for training groups collapsed and compared with Con. CD14 mRNA, expressed per monocyte, was significantly lower immediately after resistive exercise for NHR, HRT, and MIB compared with Con. There were few significant effects detected for IL-6, IL-1beta, and TNF-alpha mRNA, but there was a significant group effect (P < 0.0001) for TNF-alpha mRNA expressed per monocyte (Con > HRT, NHR, MIB). These findings suggest that there may be a resistive exercise training-induced reduction in TLR4/CD14 expression in older women. Further research is needed to determine whether lower TLR4/CD14 could explain the lower LPS-stimulated inflammatory cytokines observed in these women.

Aged↗
PubMed2004

Adjuvant-mediated tumor regression and tumor-specific cytotoxic response are impaired in MyD88-deficient mice.

The Mycobacterium bovis bacillus Calmette-Guérin cell-wall skeleton (BCG-CWS) activates Toll-like receptor (TLR) 2 and TLR4, but unlike the typical TLR4 agonist bacterial lipopolysaccharide barely induces type 1 IFN. BCG-CWS has been used for adjuvant immunotherapy for patients with cancer. We investigated the adjuvant potential of BCG-CWS for induction of CTLs subsequent to TLR-mediated dendritic cell (DC) maturation, using a syngeneic mouse tumor model (B16 melanoma in C57BL/6). We evaluated the retardation of tumor growth and cytotoxic response in wild-type and MyD88-/- mice immunized with tumor debris and/or BCG-CWS. Delays in tumor growth and cytotoxic response were induced by immunization with a mixture of BCG-CWS emulsion and the tumor. BCG-CWS was capable of activating DCs ex vivo by the criteria of CD80/CD86 up-regulation and cytokine (interleukin-12, tumor necrosis factor-alpha) induction. Efficient tumor suppression and ex vivo cytokine induction did not occur in MyD88-deficient mice and cells, suggesting that the MyD88 adapter is crucial for induction of tumor cytotoxicity. Because TLR4 is involved in both MyD88-dependent and -independent pathways and the latter affects DC maturation, our findings indicate that both pathways cooperate to induce CTL-based tumor immunity.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Sequence variants of toll-like receptor 4 are associated with prostate cancer risk: results from the CAncer Prostate in Sweden Study.

Inflammation has been implicated as an etiological factor in several human cancers. Growing evidence suggests that chronic inflammation may also play a role in the etiology of prostate cancer. Considering that genetic susceptibility is a major risk factor for this disease, we hypothesize that sequence variants in genes that regulate inflammation may modify individual susceptibility to prostate cancer. The lipopolysaccharide receptor Toll-like receptor 4 (TLR4) is a central player in the signaling pathways of the innate immune response to infection by Gram-negative bacteria and is an important candidate inflammatory gene. We performed a systematic genetic analysis of TLR4 sequence variants by evaluating eight single-nucleotide polymorphisms that span the entire gene among 1383 newly diagnosed prostate cancer patients and 780 age- and residence-matched controls in Sweden. We found an association between a sequence variant (11381G/C) in the 3'-untranslated region of the TLR4 gene and prostate cancer risk. The frequency of the variant genotypes (CG or CC) was significantly higher in the patients (24.1%) than in the controls (19.7%; P = 0.02). The frequency of risk genotypes among patients diagnosed before the age of 65 years was even higher (26.3%). Compared with men who had the wild-type genotype of this single-nucleotide polymorphism (GG), those with GC or CC genotypes had a 26% increased risk for prostate cancer (odds ratio, 1.26; 95% confidence interval, 1.01-1.57) and 39% increased risk increased risk for early onset prostate cancer (before age 65 years; odds ratio, 1.39; 95% confidence interval, 1.02-1.91). The risk attributable to this variant for prostate cancer in Sweden was estimated to be 4.9%. Although the biological mechanism of the observed association remains to be elucidated, our finding supports a role for a bacteria-associated response pathway, possibly acting via inflammation, in the development of prostate cancer.

3' Untranslated Regions↗
PubMed2004

Anterior pituitary cells express pattern recognition receptors for fungal glucans: implications for neuroendocrine immune involvement in response to fungal infections.

OBJECTIVES: Hormones and cytokines are known to act as regulatory messengers between the neuroendocrine and immune systems. The innate immune system identifies infectious agents by means of pattern-recognition receptors. These receptors recognize pathogen-specific macromolecules called pathogen-associated molecular patterns. Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity via interaction with membrane receptors on immune-competent cells. Glucans are also released into the systemic circulation of patients with fungal infections. Recent evidence confirms the existence of glucan-specific receptors on cells outside the immune system. We hypothesized that glucans may directly interact with pituitary cells as an early signaling event in fungal infections. METHODS: We characterized the receptor-mediated interaction of glucan derived from Candida albicans with pituitary cells using surface plasmon resonance. Prolactin levels were assayed by commercial ELISA. TLR2, TLR4 and CD14 mRNA levels were assessed by RT-PCR. RESULTS: A single glucan-specific binding site was identified on rodent somatomammotroph (K(D) = 3.9 microM) and human folliculostellate cell (K(D) = 3.6 microM) membranes. Coincubation of glucan with somatomammotroph cells for 72 h significantly (p < 0.01) increased prolactin accumulation by 56-62% over that observed in cells treated with media alone. Glucan also increased TLR4 and CD14 gene expression in human folliculostellate cells. CONCLUSIONS: Pituitary cells directly recognize and respond to fungal cell wall glucans resulting in stimulation of pituitary cell TLR4 and CD14 gene expression. In addition, glucan stimulates secretion of prolactin, a hormone that plays an important role in the response to infection.

Animals↗
PubMed2005

Genetic and environmental factors in monozygotic twins with Crohn's disease and their first-degree relatives: a case report.

BACKGROUND/AIMS: Familial Crohn's disease has shown concordance concerning location and clinical type of the disease especially among monozygotic twins. Susceptibility to Crohn's disease is both based on genetic and environmental factors. We investigated polymorphisms of CARD15, TLR4, and OCTN, and environmental factors in a monozygotic twin pair with Crohn's disease and their first-degree relatives. METHODS: 22-year-old monozygotic female twins with ileocolonic Crohn's disease and their healthy brother and parents were examined. DNA samples from patients and relatives were genotyped for CARD15, TLR4,and OCTN polymorphisms. ASCA and p-ANCA analyses were performed. Additionally, patients and relatives filled out a questionnaire concerning multiple environmental factors. RESULTS: Both twins presented in the same year with identical Vienna Classification phenotypes: stenotic behavior (B2) and localization in terminal ileum and colon (L3). Both carried a CARD15 R702W variant, but had normal alleles in TLR4 and OCTN. They were smokers since the age of 15, used oral contraceptives and had undergone appendectomy. The healthy father and brother were CARD15 R702W positive, were non-smokers and had not undergone appendectomy. CONCLUSION: This case report is the first to describe complete concordance in CARD15 status, phenotypic appearance, and smoking, appendectomy and oral contraceptive use in a pair of monozygotic twins with CD.

Adult↗
PubMed2003

Mechanisms of the priming effect of low doses of lipopoly-saccharides on leukocyte-dependent platelet aggregation in whole blood.

Several studies focused on the ability of bacterial lipopolysac-charides (LPS) in triggering platelet and/or leukocyte activation. The aim of this study was to investigate the molecular mechanisms involved in the aggregation of platelets and in their interaction with leukocytes in whole blood after stimulation with low doses of LPS. LPS did not directly induce platelet aggregation in whole blood, but they primed the aggregation of platelets induced by epinephrine, adenosine diphosphate and arachidonic acid. As shown by cytofluorimetry, platelets neither bind FITC-LPS, nor express the LPS-receptors CD14 and toll-like receptor 4 (TLR4). On the contrary, LPS primed monocytes and to a lesser extent polymorphonuclear neutrophils to adhere to platelets. Both platelet-leukocyte interaction and platelet aggregation in whole blood were inhibited by blockade of CD14 and TLR4. Moreover, the interaction between platelets and leukocytes was inhibited by P-selectin, and by blockade of PAF and reactive oxygen species, suggesting a role of P-selectin and of leukocyte-derived mediators. In conclusion, these results elucidate the mechanisms leading to platelet activation and interaction with leukocytes triggered by LPS. They suggest that the activation of platelets by LPS is mainly dependent on leukocytes and especially monocytes as a result of CD14 and TLR4 engagement. Moreover, we found that leukocyte-platelet interaction was triggered by the synthesis of PAF and the generation of oxygen radicals that induced upregulation of surface expression of P-selectin.

Adenosine Diphosphate↗
PubMed2005

Statins decrease Toll-like receptor 4 expression and downstream signaling in human CD14+ monocytes.

OBJECTIVE: Anti-inflammatory effects of statins contribute to their clinical benefit. Molecular mechanisms underlying these effects have not been well explored. Because statins attenuate lipopolysaccharide (LPS) responsiveness, we hypothesized that part of the pleiotropic effects are mediated through innate immunity. METHODS AND RESULTS: Toll-like receptor (TLR) 4 expression and downstream signaling in CD14+ monocytes after incubation with simvastatin and atorvastatin were quantified via flow-cytometry, quantitative RT-PCR, kinase assay, and enzyme-linked immunosorbent assay. Incubation with intermediates/ inhibitors of the mevalonate pathway was used to identify the mode of statin action. Statin incubation resulted in a dose-dependent reduction of TLR4 expression (53+/-7.6% reduction compared with untreated monocytes; P<0.005), transcript levels (68+/-6.3%; P<0.002), decreased IRAK phosphorylation (37+/-8.3%; P<0.05), and LPS-induced IL-6, IL-12, tumor necrosis factor (TNF)-alpha, and B7-1 expression (P<0.05). Four weeks of treatment with atorvastatin significantly reduced TLR4 expression on circulating CD14+ monocytes by 36.2+/-4.2% (P<0.05). Effects of statins were reversed by mevalonate (P=0.57). Incubation with specific inhibitors of geranylgeranyltransferase (54+/-4.3%), farnesyltransferase (57+/-5.1%), or with clostridium-difficile toxin B (58+/-6.1%, P<0.01) imitated the statin effects. Whereas wortmannin and LY294002 inhibited the statin effect (P=0.27), incubation with a specific RhoA kinase inhibitor had no effect (P=0.57). CONCLUSIONS: Statins influence TLR4 expression and signaling via inhibition of protein geranylgeranylation and farnesylation. These observations imply interactions with innate immunity as one pleiotropic mechanism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗