The lymphokine interleukin (IL) 4 plays a crucial role in the regulation of IgE synthesis. In the present study, the cellular and cytokine requirements for the IL4-dependent induction of IgE synthesis in humans were analyzed. Recombinant IL4 could induce IgE synthesis by peripheral blood mononuclear cells and autologous T/B cell mixtures, but not by highly purified B cells. IgE induction by IL4 was strongly decreased in monocyte-depleted peripheral blood mononuclear cells. These results show that the induction of IgE synthesis by recombinant IL4 is T cell dependent and optimal in the presence of monocytes. IL5 and IL6, but not IL2, IL1 and tumor necrosis factor-alpha, strongly up-regulated the IL4-dependent synthesis of IgE, with modest effects on cell proliferation. An anti-IL6 polyclonal antibody strongly inhibited IL4-driven IgE production. Endogenous IL6 plays, therefore, an obligatory role in the IL4-dependent induction of IgE. However, a combination of IL4, IL5 and IL6 (with or without IL1) at optimal concentrations could not induce IgE synthesis by purified normal B cells, indicating that cytokine-mediated signals, although essential, are not sufficient for the IL4-dependent induction of IgE synthesis.

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.

Mesenchymal stem cells (MSCs) have potent immunosuppressive effects in vitro and are considered as a therapeutic option for autoimmune disease and organ transplantation. While MSCs show beneficial effects on immune disease progression and transplant survival in animal models, the immunomodulatory mechanisms involved are largely unknown. In the present study, we show that intravenously infused C57BL/6- green fluorescent protein (GFP) MSCs home to the lungs in C57BL/6 recipient mice and induce an inflammatory response. This response was characterized by increased mRNA expression of monocyte chemoattractant protein-1 (MCP1), <em>IL1</em>-<em>β</em>, and TNF-α and an increase in macrophages in lung tissue 2 h after MSC infusion. Simultaneously, serum levels of proinflammatory IL6, CXCL1, and MCP1 protein increased, demonstrating systemic immune activation after MSC infusion. In liver tissue, no C57BL/6-GFP MSCs were detected, but MCP1 and TNF-α mRNA levels peaked 4 h after MSC infusion. The expression of the anti-inflammatory cytokines TGF-<em>β</em>, IL4, and <em>IL1</em>0 was only marginally affected. Nevertheless, 3 days after MSC infusion, animals developed a milder inflammatory response to lipopolysaccharides. Our results suggest that the in vivo immunomodulatory effects of MSCs originate from an inflammatory response that is induced by the infusion of MSCs, which is followed by a phase of reduced immune reactivity.

METHODS

A study of tuberculosis cases and healthy blood donor controls from the Western Region of The Gambia, West Africa.

OBJECTIVE

To investigate the potential role of candidate gene polymorphisms in host susceptibility to tuberculosis.

METHODS

Single base change polymorphisms in interleukin 1 beta (IL1 beta), interleukin 10 (IL1IL1 alpha) and IL1IL1RA) were typed in over 400 tuberculosis cases and 400 healthy blood donor controls.

RESULTS

<em>IL1</em> gene cluster polymorphisms (<em>IL1</em>RA and possibly <em>IL1</em> alpha) showed marginally significant association with tuberculosis. In particular <em>IL1</em>RA allele 2 heterozygotes were less frequent among tuberculosis cases than controls (P = 0.03). <em>IL1</em> <em>beta</em>, <em>IL1</em>0 and FUT-2 polymorphisms were not associated with tuberculosis.

CONCLUSIONS

Genetic susceptibility to tuberculosis among Gambians may be partly determined by genes in the IL1 gene cluster on chromosome 2. Further association studies will be required on other population groups to confirm whether these results are of biological significance.

We have recently identified conventional <em>B</em>2 cells as atherogenic and <em>B</em>1a cells as atheroprotective in hypercholesterolemic ApoE(-/-) mice. Here, we examined the development of atherosclerosis in <em>B</em>AFF-R deficient ApoE(-/-) mice because <em>B</em>2 cells but not <em>B</em>1a cells are selectively depleted in <em>B</em>AFF-R deficient mice. We fed <em>B</em>AFF-R(-/-) ApoE(-/-) (<em>B</em>affR.ApoE DKO) and <em>B</em>AFF-R(+/+)ApoE(-/-) (ApoE KO) mice a high fat diet (HFD) for 8-weeks. <em>B</em>2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while <em>B</em>1a cells and non-<em>B</em> lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. <em>B</em> cells were absent in atherosclerotic lesions of <em>B</em>affR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by <em>B</em>2 cells, despite low but measurable numbers of <em>B</em>2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. <em>B</em>AFF-R deficiency in ApoE(-/-) mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, <em>IL1</em>-<em>β</em> and proinflammatory chemokine MCP-1 was also reduced. <em>B</em>ody weight and plasma cholesterols were unaffected in <em>B</em>affR.ApoE DKO mice. Our data indicate that <em>B</em>2 cells are important contributors to the development of atherosclerosis and that targeting the <em>B</em>AFF-R to specifically reduce atherogenic <em>B</em>2 cell numbers while preserving atheroprotective <em>B</em>1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation.

BACKGROUND

Sargramostim, granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, stimulates cells of the intestinal innate immune system. Clinical trials show that sargramostim induces clinical response and remission in patients with active Crohn's disease. To study the mechanism, we examined the effects of GM-CSF in the dextran sulfate sodium (DSS)-induced acute colitis model. We hypothesized that GM-CSF may work through effects on dendritic cells (DCs).

METHODS

Acute colitis was induced in Balb/c mice by administration of DSS in drinking water. Mice were treated with daily GM-CSF or phosphate-buffered saline (PBS). To probe the role of plasmacytoid DCs (pDCs) in the response to GM-CSF, we further examined the effects of monoclonal antibody 440c, which is specific for a sialic acid-binding immunoglobulin (Ig)-like lectin expressed on pDCs.

RESULTS

GM-CSF ameliorates acute DSS-induced colitis, resulting in significantly improved clinical parameters and histology. Microarray analysis showed reduced expression of proinflammatory genes including TNF-alpha and IL1-beta; the results were further confirmed by real-time reverse-transcriptase polymerase chain reaction and serum Bio-plex analysis. GM-CSF treatment significantly expands pDCs and type 1 IFN production. Administration of mAb 440c completely blocked the therapeutic effect of GM-CSF. GM-CSF is also effective in RAG1(-/-) mice, demonstrating activity-independent effects on T and B cells. IFN-beta administration mimics the therapeutic effect of GM-CSF in DSS-treated mice. GM-CSF increases systemic and mucosal type 1 IFN expression and exhibits synergy with pDC activators, such as microbial cytosine-phosphate-guanosine (CpG) DNA.

CONCLUSIONS

GM-CSF is effective in the treatment of DSS colitis in a mechanism involving the 440c(+) pDC population.

Interleukin 10 (IL1IL1, TNFA, TGFB, IL6, IL8 and IL1BV cohort revealed that one of IL1IL1IL1BV progression from chronic hepatitis to liver cirrhosis and HCC among hepatitis B patients. In addition, survival analysis clearly showed that the onset age of HCC was also accelerated among chronic hepatitis B patients who were carrying IL1IL1IL1IL1BV infection, especially to HCC development.

OBJECTIVE

To determine whether IL1IL1 beta and TNF alpha.

METHODS

Resting T cells were isolated from peripheral blood of healthy donors, and stimulated with 2 microg/ml phytohaemagglutinin (PHA) and 0.5 ng/ml IL2 for 24 hours. Synovial T cells were isolated from RA synovial tissue. The levels of soluble receptor activator of the NF-kappa B ligand (RANKL), osteoprotegerin (OPG), IFN gamma, M-CSF, and GM-CSF were determined by ELISA. Membrane bound RANKL expression was analysed by flow cytometry. Commercially available human osteoclast precursors were cocultured with T cells to induce osteoclast formation, which was determined with tartrate resistant acid phosphatase staining and pit formation assay.

RESULTS

In PHA prestimulated T cells or RA synovial T cells, IL1IL1 beta, or TNFalpha increased soluble RANKL production and membrane bound RANKL expression in a dose dependent manner. IL1IL1 beta, and TNF alpha did not induce M-CSF, GM-CSF, IFN gamma, or OPG production in PHA prestimulated T cells or RA synovial T cells. IL1IL1 beta, but less potent than that of TNF alpha. In the coculture system, OPG completely blocked osteoclast induction by IL1IL1 beta, and greatly inhibited induction by TNF alpha.

CONCLUSIONS

IL1IL1 beta, or TNF alpha can indirectly stimulate osteoclast formation through up regulation of RANKL production from T cells in RA synovitis; IL1IL1 beta, but less potent than TNF alpha.

Traumatic brain injury (TBI) initiates a cascade of cellular and molecular responses including both pro- and anti-inflammatory. Although post-traumatic hypothermia has been shown to improve outcome in various models of brain injury, the underlying mechanisms responsible for these effects have not been clarified. In this study, inflammation cDNA arrays and semi-quantitative RT-PCR were used to detect genes that are differentially regulated after TBI. In addition, the effect of post-traumatic hypothermia on the expression of selective genes was also studied. Rats (n = 6-8 per group) underwent moderate fluid-percussion (F-P) brain injury with and without hypothermic treatment (33 degrees C/3 h). RNA from 3-h or 24-h survival was analyzed for the expression of <em>IL1</em>-<em>beta</em>, IL2, IL6, TGF-<em>beta</em>2, growth-regulated oncogene (GRO), migration inhibitory factor (MIF), and MCP (a transcription factor). The interleukins IL-1<em>beta</em>, IL-2, and IL-6 and TGF-<em>beta</em> and GRO were strongly upregulated early and transiently from 2- to 30-fold over sham at 3 h, with normalization by 24 h. In contrast, the expressions of MIF and MCP were both reduced by TBI compared to sham. Post-traumatic hypothermia had no significant effect on the acute expression of the majority of genes investigated. However, the expression of TGF-<em>beta</em>2 at 24 h was significantly reduced by temperature manipulation. The mechanism by which post-traumatic hypothermia is protective may not involve a general genetic response of the inflammatory genes. However, specific genes, including TGF-<em>beta</em>2, may be altered and effect cell death mechanisms after TBI. Hypothermia differentially regulates certain genes and may target more delayed responses underlying the secondary damage following TBI.

Diabetes mellitus (DM) may alter bone remodeling, as osteopenia and osteoporosis are among the complications. Moreover, DM increases the risk and severity of chronic inflammatory periodontal disease, in which bone resorption occurs. Broad evidence suggests that chronic inflammation can contribute to the development of DM and its complications. Hyperglycemia is a hallmark of DM that may contribute to sustained inflammation by increasing proinflammatory cytokines, which are known to cause insulin resistance, via toll-like receptor (TLR)-4-mediated mechanisms. However, the mechanisms by which bone-related complications develop in DM are still unknown. Studies done on the effect of high glucose concentrations on osteoblast functions are contradictory because some suggest increases (although others suggest reductions) in the biomineralization process. Therefore, we evaluated the effect of high glucose levels on biomineralization and inflammation markers in a human osteoblastic cell line. Cells were treated with either physiological 5.5 mM or increasing concentrations of glucose up to 24 mM, and we determined the following: i) the quantity and quality of calcium-deposit crystals in culture and ii) the expression of the following: a) proteins associated with the process of biomineralization, b) the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG), c) cytokines IL1, IL6, IL8, IL1biomineralization process in osteoblastic cells and provoke the following: i) a rise in mineralization, ii) an increase in the mRNA expression of RANKL and a decrease of OPG, iii) an increase in the mRNA expression of osteocalcin, bone sialoprotein and the transcription factor Runx2, iv) a diminished quality of the mineral, and v) an increase in the expression of IL1beta, IL6, IL8, MCP-1 and IL1both high glucose levels and hyperosmotic conditions provoked TLR-2, -3, -4 and -9 overexpression in osteoblastic cells, suggesting that they are susceptible to osmotic stress.

As immune defects in amyloid-β (Aβ) phagocytosis and degradation underlie Aβ deposition and inflammation in Alzheimer's disease (AD) brain, better understanding of the relation between Aβ phagocytosis and inflammation could lead to promising preventive strategies. We tested two immune modulators in peripheral blood mononuclear cells (PBMCs) of AD patients and controls: 1α,25(OH)2-vitamin D3 (1,25D3) and resolvin D1 (RvD1). Both 1,25D3 and RvD1 improved phagocytosis of FAM-Aβ by AD macrophages and inhibited fibrillar Aβ-induced apoptosis. The action of 1,25D3 depended on the nuclear vitamin D and the protein disulfide isomerase A3 receptors, whereas RvD1 required the chemokine receptor, GPR32. The activities of 1,25D3 and RvD1 commonly required intracellular calcium, MEK1/2, PKA, and PI3K signaling; however, the effect of RvD1 was more sensitive to pertussis toxin. In this case study, the AD patients: a) showed significant transcriptional up regulation of IL1RN, ITGB2, and NFκB; and b) revealed two distinct groups when compared to controls: group 1 decreased and group 2 increased transcription of TLRs, IL-1, IL1R1 and chemokines. In the PBMCs/macrophages of both groups, soluble Aβ (sAβ) increased the transcription/secretion of cytokines (e.g., IL1 and IL6) and chemokines (e.g., CCLs and CXCLs) and 1,25D3/RvD1 reversed most of the sAβ effects. However, they both further increased the expression of IL1 in the group 1, sβ-treated cells. We conclude that in vitro, 1,25D3 and RvD1 rebalance inflammation to promote Aβ phagocytosis, and suggest that low vitamin D3 and docosahexaenoic acid intake and/or poor anabolic production of 1,25D3/RvD1 in PBMCs could contribute to AD onset/pathology.

BACKGROUND

A large number of activated T cells are found in the joints of patients with rheumatoid arthritis (RA). Interleukin 7 (IL7), a T cell growth factor and a regulator of Th1 and Th2 cytokine production, is produced by synoviocytes from patients with RA.

OBJECTIVE

To investigate the effect on proinflammatory cytokine production of synovial fluid mononuclear cells (SFMC) and the mechanism by which IL7 influences CD4+ T cell activity in patients with RA.

METHODS

In a cross sectional group of patients with RA, IL7 levels were compared with those of healthy controls and related to disease activity. The effect of IL7 on cytokine production was tested by RA SFMC and on SF CD4+ T cells in the presence of mononuclear cells (MC). Production of tumour necrosis factor alpha (TNF alpha), <em>IL1</em> <em>beta</em>, interferon gamma (IFN gamma), and IL4 was measured by enzyme linked immunosorbent assay (ELISA) and by single cell FACS analysis. Expression of the IL7 receptor alpha chain on CD4+ T cells (essential for IL7 signalling) was assessed. Direct effects of IL7 on isolated synovial fluid (SF) CD4+ T cells were studied by cytokine analysis. By neutralisation of <em>IL1</em>2 in MC cultures, indirect effects of IL7 on T cells through accessory cells were studied.

RESULTS

IL7 serum levels were higher in patients with RA than in healthy controls and correlated positively with C reactive protein levels. IL7 stimulated TNFalpha production by SFMC and very potently stimulated IFN gamma and TNF alpha production by SF CD4+ T cells. These effects were probably mediated through the IL7 receptor alpha chain, which was abundantly expressed on SF CD4+ T cells. Besides the direct stimulation of T cell cytokine production by IL7, its action was partly dependent on <em>IL1</em>2, indicating that IL7 also stimulates accessory cell function, leading to T cell activation.

CONCLUSIONS

IL7 stimulates proinflammatory cytokine production of intra-articular CD4+ T cells and accessory cells from patients with RA. The correlation with measures of disease activity indicates that IL7 might substantially contribute to the perpetuation of Th1 and TNF alpha mediated proinflammatory responses in patients with RA.

BACKGROUND

The clinical management of leprosy Type 1 (T1R) and Type 2 (T2R) reactions pose challenges mainly because they can cause severe nerve injury and disability. No laboratory test or marker is available for the diagnosis or prognosis of leprosy reactions. This study simultaneously screened plasma factors to identify circulating biomarkers associated with leprosy T1R and T2R among patients recruited in Goiania, Central Brazil.

METHODS

A nested case-control study evaluated T1R (n = 10) and TR2 (n = 10) compared to leprosy patients without reactions (n = 29), matched by sex and age-group (+/- 5 years) and histopathological classification. Multiplex bead based technique provided profiles of 27 plasma factors including 16 pro inflammatory cytokines: tumor necrosis factor-alpha (TNF-alpha), Interferon-gamma (IFN-gamma), interleukin (IL)- IL1IL1IL1 beta, IL6, IL1beta (MIP1beta), regulated upon activation normal T-cell expressed and secreted (RANTES), monocyte chemoattractrant protein 1 (MCP1), CC-chemokine 11 (CCL11/Eotaxin), CXC-chemokine 10 (CXCL10/IP10); 4 anti inflammatory interleukins: IL4, IL1IL1IL1Ralpha and 7 growth factors: IL7, IL9, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), platelet-derived growth factor BB (PDGF BB), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF).

RESULTS

Elevations of plasma CXCL10 (P = 0.004) and IL6 (p = 0.013) were observed in T1R patients compared to controls without reaction. IL6 (p = 0.05), IL7 (p = 0.039), and PDGF-BB (p = 0.041) were elevated in T2R. RANTES and GMCSF were excluded due to values above and below detection limit respectively in all samples.

CONCLUSIONS

Potential biomarkers of T1R identified were CXCL10 and IL6 whereas IL7, PDGF-BB and IL6, may be laboratory markers of TR2. Additional studies on these biomarkers may help understand the immunopathologic mechanisms of leprosy reactions and indicate their usefulness for the diagnosis and for the clinical management of these events.

Trypanosoma cruzi, the aetiological agent of Chagas disease, invades nucleated mammalian cells including macrophages. In this study, we investigated the crosstalk between T. cruzi-induced immune activation of reactive oxygen species (ROS) and pro-inflammatory responses, and their role in myocardial pathology. Splenocytes of infected mice (C3H/HeN) responded to Tc-antigenic stimulus by more than a two-fold increase in NADPH oxidase (NOX) activity, ROS generation, cytokine production (IFN-γ>> IL-4>> TNFα>> IL1-β≈ IL6), and predominant expansion of CD4(+) and CD8(+) T cells. Inhibition of NOX, but not of myeloperoxidase and xanthine oxidase, controlled the ROS (>98%) and cytokine (70-89%) release by Tc-stimulated splenocytes of infected mice. Treatment of infected mice with apocynin (NOX inhibitor) in drinking water resulted in a 50-90% decline in endogenous NOX/ROS and cytokine levels, and splenic phagocytes' proliferation. The splenic percentage of T cells was maintained, though more than a 40% decline in splenic index (spleen weight/body weight) indicated decreased T-cell proliferation in apocynin-treated/infected mice. The blood and tissue parasite burden were significantly increased in apocynin-treated/infected mice, yet acute myocarditis, ie inflammatory infiltrate consisting of macrophages, neutrophils, and CD8(+) T cells, and tissue oxidative adducts (eg 8-isoprostanes, 3-nitrotyrosine, and 4-hydroxynonenal) were diminished in apocynin-treated/infected mice. Consequently, hypertrophy (increased cardiomyocytes' size and β-MHC, BNP, and ANP mRNA levels) and fibrosis (increased collagen, glycosaminoglycans, and lipid contents) of the heart during the chronic phase were controlled in apocynin-treated mice. We conclude that NOX/ROS is a critical regulator of the splenic response (phagocytes, T cells, and cytokines) to T. cruzi infection, and bystander effects of heart-infiltrating phagocytes and CD8(+) T cells resulting in cardiac remodelling in chagasic mice.

BACKGROUND

Psoriasis is one of the most frequent skin diseases world-wide. The disease impacts enormously on affected patients and poses a huge financial burden on health care providers. Several lines of evidence suggest that the nuclear hormone receptor peroxisome proliferator activator (PPAR) beta/delta, known to regulate epithelial differentiation and wound healing, contributes to psoriasis pathogenesis. It is unclear, however, whether activation of PPARbeta/delta is sufficient to trigger psoriasis-like changes in vivo.

RESULTS

Using immunohistochemistry, we define the distribution of PPARbeta/delta in the skin lesions of psoriasis. By expression profiling, we confirm that PPARbeta/delta is overexpressed in the vast majority of psoriasis patients. We further establish a transgenic model allowing inducible activation of PPARbeta/delta in murine epidermis mimicking its distribution in psoriasis lesions. Upon activation of PPARbeta/delta, transgenic mice sustain an inflammatory skin disease strikingly similar to psoriasis, featuring hyperproliferation of keratinocytes, dendritic cell accumulation, and endothelial activation. Development of this phenotype requires the activation of the Th17 subset of T cells, shown previously to be central to psoriasis. Moreover, gene dysregulation in the transgenic mice is highly similar to that in psoriasis. Key transcriptional programs activated in psoriasis, including IL1-related signalling and cholesterol biosynthesis, are replicated in the mouse model, suggesting that PPARbeta/delta regulates these transcriptional changes in psoriasis. Finally, we identify phosphorylation of STAT3 as a novel pathway activated by PPARbeta/delta and show that inhibition of STAT3 phosphorylation blocks disease development.

CONCLUSIONS

Activation of PPARbeta/delta in the epidermis is sufficient to trigger inflammatory changes, immune activation, and signalling, and gene dysregulation characteristic of psoriasis.

We propose that the tryptophan catabolites produced through the kynurenine pathway (KP), and more particularly quinolinic acid (QUIN), may play an important role in the pathogenesis of Alzheimer's disease (AD). In this study, we demonstrated that after 72 hours amyloid peptide (Abeta) 1-42 induced indoleamine 2,3-dioxygenase (IDO) expression and in a significant increase in production of QUIN by human macrophages and microglia. In contrast, Abetabetabetabeta alone). Abeta has been shown to induce IL1-beta mRNA expression by human foetal astrocytes and macrophages. We demonstrate that QUIN has the same effect. Interestingly, IL-1beta has been found in association with plaques in AD. All together these data imply that QUIN may be, locally, one of the factors involved in the pathogenesis of neuronal damage in AD.

OBJECTIVE

Interleukin-1 (IL-1) and interleukin-6 (IL-6) are determining factors in the immune and inflammatory responses to tumors cells. Experimental data suggest that interleukin-1 and interleukin-6 play important roles in the development and progression of breast cancer. We designed a broad study to investigate the susceptibility and prognostic implications of the genetic variation in IL-1alpha, IL-1beta and IL-6 in breast carcinoma.

METHODS

We used the polymerase chain reaction and restriction enzyme digestion to characterize the genetic variation of IL-1alpha, IL-1beta and IL-6 in 305, unrelated Tunisian patients with breast carcinoma and 200 healthy control subjects. Associations between the genetic markers and the clinicopathological parameters, the specific overall survival rate (OVS) of breast carcinoma and the disease free-survival rate (DFS) were assessed using univariate and multivariate analyses.

RESULTS

Both IL-6 (-597) GA and IL-6 (-174) GC heterozygous genotypes were found to be significantly associated with breast carcinoma (OR = 1.59, p = 0.024 and OR = 1.61, p = 0.022 respectively). A highly significant association was found between the (+3954) T allele of IL1-B gene and the aggressive phenotype of breast carcinoma as defined by the high histological grade, axillary lymph node metastasis and large tumor size. The IL-1alpha (-889) TT homozygous genotype showed a significant association with reduced disease-free survival and/or overall survival rate. The IL-1beta (+3954) TT, IL-6 (-597) GG and IL-6 (-174) GG homozygous genotypes were found to be associated with reduced DFS but not with overall survival.

CONCLUSIONS

The polymorphisms in the promoter region of the IL-6 gene may represent a marker for the increased risk of breast carcinoma. Genetic variations in IL-1alpha, IL-1beta and IL-6 may predict the clinical outcome of breast carcinoma.

To better understand the role of inflammatory responses in temporal lobe epilepsy, we characterized Interleukin1-beta (IL1-beta), Nuclear Factor-kappaB (NF-kappaB), and Cyclooxygenase-2 (COX-2) expression together with neurodegeneration in the rat lithium-pilocarpine model. The immunohistochemical expression of IL1-beta, NF-kappaB, and COX-2 started by 12 h post-injection, persisted for 24 h (status epilepticus period), and returned to basal levels by 3 and 6 days (latent period). The regional distribution of IL1-beta, NF-kappaB, and COX-2 occurred mainly in structures prone to develop neuronal damage. Using double-staining protocols, we detected IL1-beta expression in glial cells, COX-2 expression in neurons, and NF-kappaB in both cell types. The presence of Fluoro-Jade-B-positive degenerating neurons was associated with IL1-beta, NF-kappaB, and COX-2 proteins expression during status epilepticus but not during the latent period while neurons were still degenerating. These data suggest that seizure-related IL1-beta, NF-kappaB, and COX-2 expression may contribute to the pathophysiology of epilepsy by inducing neuronal death and astrocytic activation.

BACKGROUND

Osteoclasts, specialised bone resorbing cells regulated by RANKL and M-CSF, are implicated in rheumatoid joint erosion. Lymphocyte-monocyte interactions activate bone resorption, this being attributed to tumour necrosis factor alpha (TNFalpha) and interleukin 1 beta (IL1beta) enhanced osteoblast expression of RANKL. In animal studies, TNF potently increases osteoclast formation in the presence of RANKL. RANKL-independent osteoclastogenesis also occurs, though IL1 is required for resorptive function in most studies. These inflammatory cytokines have a pivotal role in rheumatoid arthritis,

OBJECTIVE

To study the interactions of TNFalpha and IL1beta with RANKL, particularly the time course of the interactions and the role of lymphocytes.

METHODS

Cultures of lymphocytes and monocytes (osteoclast precursors) or of purified CD14(+) cells alone (osteoclast precursors) were exposed to various combinations of TNFalpha, RANKL, and IL1beta or the inhibitors osteoprotegerin, IL1 receptor antagonist, or neutralising antibodies to RANKL or to IL1. Osteoclastogenesis and resorptive activity were assessed on microscopy of dentine slices.

RESULTS

TNFalpha potently increased osteoclast proliferation/differentiation in the presence of RANKL. This effect was greatest when RANKL was present before but not after exposure of osteoclast precursor cells to TNFalpha. The resorptive activity of osteoclasts generated by TNFalpha in the absence of RANKL was critically dependent upon IL1, which was expressed by lymphocyte-monocyte interaction.

CONCLUSIONS

TNFalpha potently enhances RANKL mediated osteoclast activity. Interactions between TNFalpha and IL1 also result in osteoclastic activity independently of RANKL. These findings will inform therapeutic approaches to the prevention of joint erosion in rheumatoid arthritis.

A model for NF kappa B activation involving reactive oxygen intermediates has recently been proposed. We have explored this model in three cell lines, Jurkat T cells, EL4.NOB-1 T cells and KB epidermal cells using hydrogen peroxide and two physiological activators of NF kappa B, interleukin-1 (IL1) and tumor necrosis factor (TNF) as stimuli. In agreement with earlier studies hydrogen peroxide activated NF kappa B in Jurkat, although only at much higher concentrations (10 mM) than those previously reported. However, hydrogen peroxide failed to activate in the two other cell lines under a range of conditions. Similarly, N-acetylcysteine only proved inhibitory in hydrogen peroxide and TNF treated Jurkat and failed to inhibit IL1 and TNF-activated NF kappa B in EL4.NOB-1 and KB cells respectively. N-Acetylcysteine inhibited IL1-induced interleukin-2 in EL4, however, demonstrating that N-acetylcysteine was biologically active. These results suggest that the reactive oxygen model of NF kappa B activation may be cell-type restricted. In contrast to the results with N-acetylcysteine, the antioxidant and metal chelator, pyrolidine dithiocarbamate (PDTC) inhibited NF kappa B activation, although these effects may be unrelated to any antioxidant properties. PDTC also inhibited IL1-induced interleukin-2. Finally, studies with the pro-oxidant diamide showed that this could not activate NF kappa B in any of the cells and in contrast proved inhibitory. The results from this study therefore suggest that the reactive oxygen model of NF kappa B activation may be restricted to certain cell types and that the presence of such a system is not required for the activation of NF kappa B by IL1 and TNF.

Symptoms commonly associated with sleep loss and chronic inflammation include sleepiness, fatigue, poor cognition, enhanced sensitivity to pain and kindling stimuli, excess sleep and increases in circulating levels of tumor necrosis factor α (TNF) in humans and brain levels of interleukin-1 β (IL1) and TNF in animals. Cytokines including IL1 and TNF partake in non-rapid eye movement sleep (NREMS) regulation under physiological and inflammatory conditions. Administration of exogenous IL1 or TNF mimics the accumulation of these cytokines occurring during sleep loss to the extent that it induces the aforementioned symptoms. Extracellular ATP associated with neuro- and glio-transmission, acting via purine type 2 receptors, e.g., the P2X7 receptor, has a role in glia release of IL1 and TNF. These substances in turn act on neurons to change their intrinsic membrane properties and sensitivities to neurotransmitters and neuromodulators such as adenosine, glutamate and GABA. These actions change the network input-output properties, i.e., a state shift for the network. State oscillations occur locally within cortical columns and are defined using evoked response potentials. One such state, so defined, shares properties with whole animal sleep in that it is dependent on prior cellular activity--it shows homeostasis. The cortical column sleep-like state is induced by TNF and is associated with experimental performance detriments. ATP released extracellularly as a consequence of cellular activity is posited to initiate a mechanism by which the brain tracks its prior sleep-state history to induce/prohibit sleep. Thus, sleep is an emergent property of populations of local neural networks undergoing state transitions. Specific neuronal groups participating in sleep depend upon prior network use driving local network state changes via the ATP-cytokine-adenosine mechanism. Such considerations add complexity to finding biochemical markers for sleepiness.

BACKGROUND

Patients with clinically active Crohn's disease (CD), defined by a Crohn's Disease Activity Index (CDAI)>150, may have normal C-reactive protein (CRP) serum levels. In such cases, it is difficult to know whether these patients have really active disease or rather functional symptoms. This distinction is important to decide the most appropriate treatment. The aim of our work was to assess intestinal and colonic lesions in such patients and to look for biological markers potentially associated with endoscopic activity of the disease.

METHODS

We included 28 consecutive CD patients with CDAI>150 and a normal CRP level. These patients underwent a full colonoscopy with Crohn's Disease Endoscopy Index of Severity (CDEIS) calculation, fecal calprotectin, blood fibrinogen, acid alpha-1 glycoprotein, and erythrocyte sedimentation rate measurement. The Harvey-Bradshaw score was also calculated. Serum IL1 beta, IL6, IL8, sIL2R, and sTNFR2 were measured.

RESULTS

The median CDAI was 181 (151-485). Almost all (92.9%) these patients had endoscopic lesions, but the majority had only mild lesions (CDEIS<or=6). No correlation was found between CDEIS and any of the clinical or biological markers. However, all the patients with significant endoscopic lesions (defined by a CDEIS>6) had previous surgical intestinal resection and lesions involving the anastomosis.

CONCLUSIONS

Patients with elevated CDAI and normal CRP have only mild mucosal lesions of CD. Most significant lesions may be observed at the anastomosis and proximal to it in previously operated patients. None of the biological markers tested was associated with these endoscopic lesions.

OBJECTIVE

Recent studies suggest that crystals of monosodium urate (MSU), deposited in joints of patients with acute gouty arthritis, activate the NACHT domain, leucine-rich repeat and pyrin domain-containing protein (NALP)3 inflammasome. In the present study we have investigated whether production of proinflammatory cytokines by crystals was exacerbated during costimulation with Toll-like receptor (TLR) ligands.

METHODS

Mononuclear cells of 22 healthy donors were stimulated by various concentrations of MSU crystals in the absence or presence of lipopolysaccharide (LPS), Pam3Cys and flagellin. Production of tumour necrosis factor alpha (TNFalpha), interleukin (IL)1 beta and IL6, as well as the intracellular concentrations of proIL1 beta were measured by ELISA. mRNA transcripts of TNFalpha and IL1 beta were assessed by real-time PCR. Stimulation experiments were also performed with peripheral blood mononuclear cells (PBMCs) of one patient carrying a NALP3 mutation.

RESULTS

MSU induced a moderate release of IL1 beta and IL6, but not of TNFalpha. Urate crystals amplified IL1 beta production stimulated by the TLR4 ligand LPS, while no synergy was apparent for IL6 production. In addition, no synergy between urate crystals and Pam3Cys (TLR2 ligand) or flagellin (TLR5 ligand) was apparent. The synergy between urate crystals and LPS was directed at the level of the NALP3 inflammasome, as it was present only when active IL1 beta was measured, but not at the level of IL1 mRNA or proIL1 beta. The synergy between LPS and MSU crystals ceased to exist in the presence of a caspase 1 inhibitor.

CONCLUSIONS

MSU crystals act in synergy with LPS for the induction of enhanced release of IL1 beta. Increased cleavage of proIL1 beta by urate-activated caspase 1 is proposed as the underlying mechanism.

Intestinal dendritic cells (DCs) send processes between epithelial cells into the gut lumen to sample pathogens. Noninvasive enteropathogenic Escherichia coli (EPEC) colonize the gut using a type three secretion system (T3SS) to inject effector proteins into epithelial cells. We hypothesized that EPEC might also inject proteins into DC processes to dampen immune recognition. Using a T3SS-linked fluorescence resonance energy transfer-based system we show that EPEC injects effectors into in vitro grown human myeloid DCs. Injected cells emit a blue signal due to cleavage of the green fluorescence resonance energy transfer-based substrate CCF2/AM by β-lactamase. When cultured with a mutant EPEC unable to translocate effector proteins, myeloid DCs show rapid activation of NF-κB, secrete large amounts of proinflammatory cytokines and increase expression of CD80, CD83, and CD86, whereas wild-type EPEC barely elicits cytokine production and shuts off nuclear translocation of NF-κB p65. By deleting effector protein genes, we identified NleE as being critical for this effect. Expression of NleE in HeLa cells completely prevented nuclear p65 accumulation in response to IL1-β, and luciferase production in an NF-κB reporter cell line. DCs cocultured with wild-type EPEC or NleE-complemented strains were less potent at inducing MLR. EPEC was also able to inject effectors into DCs sending processes through model gut epithelium in a transwell system and into Peyer's patch myeloid DCs. Thus, EPEC translocate effectors into human DCs to dampen the inflammatory response elicited by its own pathogen-associated molecular patterns.