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Coeliac disease, epilepsy and cerebral calcifications (CEC) syndrome is a rare clinical condition. One hundred and seventy-one patients have been reported in the literature. Patients are mostly from Italy, Spain, and Argentina, suggesting a geographically restricted condition. Epilepsy is more frequently characterized by occipital seizures. It may be benign or drug-resistant, sometime evolving into severe epileptic encephalopathy. Gluten free diet (GFD) efficacy seems to be inversely related to the duration of epilepsy and the young age of the patient. Patients with cerebral calcifications (CC) and coeliac disease (CD) without epilepsy are considered as having an incomplete form of CEC syndrome. Some patients with epilepsy and CC without CD are supposed to have a CEC syndrome with silent or latent CD.Whether CEC syndrome is a genetic condition, or whether epilepsy and/or CC are a consequence of an untreated CD is unknown yet. Since histopathological findings seem to be the expression of vascular calcifyied malformation, CEC syndrome may be considered a genetically determined entity, such as a type of Sturge-Weber-like phacomatosis. Moreover, CEC, as well as CD, is associated with HLA-DQ2 and HLA-DQ8 phenotype and genotype. The progressive growth and late occurrence of CC before beginning a GFD, the demonstration of anti-gliadin antibodies in the cerebro-spinal fluid and the association with HLA class II genes, suggest that an immune reaction originating from the jejunal mucosa, triggered by gliadin in gluten intolerance predisposed subjects (HLA phenotype) may be responsible for seizures and CC. Moreover, a long-lasting untreated CD folic acid deficiency may cause calcifications.Probably, CEC is considered a genetic, non-inherited, ethnically and geographically restricted syndrome associated with environmental factors. [Copyright &y& Elsevier]

Abstract: We studied the effect of TNF-α stimulation on a cholangiocarcinoma cell line, CCKS1. CCKS1 expressed only one type TNF receptor, TNFR2. Treatment of CCKS1 with TNF-α substantially activated NFκB, MAPK and Akt signalings which in turn activated matrix metalloproteinase-9 (MMP-9) secretion and in vitro invasiveness of CCKS1. Pretreatment of cells with anti-TNFR2 neutralizing antibody inhibited the TNF-α-dependent signaling and MMP-9 secretion and subsequently blocked invasion in vitro. Moreover, an inhibitor for matrix metalloproteinase, Galardin, suppressed the invasion in a dose-dependent manner. Similarly, pharmacological inhibition of signaling clearly suppressed the TNF-α dependent MMP-9 secretion. These results strongly suggest that TNF-α-TNFR2 signaling plays an important role to convert the cholangiocarcinoma cells to be more aggressive one. [Copyright &y& Elsevier]

The earliest defense against microbial infection is represented by the responses of the innate (or natural) immune system, that also profoundly regulates the adaptive (or acquired) T- and B-cell immune responses.Activation of the innate immune system is primed by microbial invasion in response to conserved structures present in large groups of microorganisms (LPS, peptidoglycan, double-stranded RNA), and is finely tuned by different cell types (including dendritic cells, macrophages, natural killer cells, natural killer T cells, and γδ T cells). In addition, several soluble factors (complement components, defensins, mannose-binding lectins, interferons, cytokines and chemokines) can play a major role in the regulation of both the innate and adaptive immunity.In this review, we will briefly overview the regulation of some cellular subsets of the innate immune system particularly involved in human immunodeficiency virus (HIV) infection and then focus our attention on those cytokines and chemokines whose levels of expression are more profoundly affected by HIV infection and that, conversely, can modulate virus infection and replication. [Copyright &y& Elsevier]

Exposure of the gastric mucosa to back-diffusing concentrations of HCl (0.25 M, pH 0.51) stimulates vagal afferent input to the brainstem. Here we have examined whether pretreatment of rats with the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α causes sensitization of vagal afferent pathways to HCl. Rats were pretreated i.p. with interleukin-1β, tumor necrosis factor-α (10 μg/kg) or their vehicle (sterile saline) 24, 48 and 96 h before intragastric administration of HCl (0.25 M, 1 ml/100 g). Activation of neurons in the nucleus tractus solitarii was visualized by c-Fos immunohistochemistry 2 h after the HCl challenge. I.p. administration of interleukin-1β and tumor necrosis factor-α alone induced c-Fos in the brainstem, an effect that was gone after 24 h. At this time, however, the effect of HCl to cause expression of c-Fos in the nucleus tractus solitarii was significantly enhanced by pretreatment with interleukin-1β and tumor necrosis factor-α. The sensitizing effect of i.p.-administered interleukin-1β was sustained for more than 48 h and prevented by the interleukin-1 receptor antagonist anakinra. Intracisternal administration of interleukin-1β and tumor necrosis factor-α (100 ng) failed to amplify the HCl-evoked expression of c-Fos in the brainstem.These results show that peripheral administration of the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α induces prolonged sensitization of vagal afferent pathways to gastric HCl challenge. This effect seems to arise from a peripheral action on vagal afferents and may be of relevance to gastric chemonociception. [Copyright &y& Elsevier]

The M-CSF and its receptor (M-CSFR, CSF-1R or c-fms proto-oncogene) system were initially implicated as essential in mammals for normal monocyte development as well as for pregnancy. To allow a comparison with the M-CSF and M-CSFR system of an oviparous animal, we cloned a M-CSFR-like gene from rainbow trout (Oncorhynchus mykiss). The gene was cloned from a cDNA library of head kidney. It contained an open reading frame encoding 967 amino acids with a predicted size of 109kDa. The putative amino acid sequence of rainbow trout M-CSFR showed 54% amino acid identity to fugu (Takifugu rubripes) M-CSFR, 52% to zebrafish (Danio rerio) M-CSFR and 40% to mouse (Mus musculus) and human (Homo sapiens) M-CSFR. The M-CSFR-like gene was constitutively expressed in head kidney, kidney, intestine, spleen and blood. The gene was detected especially in the ovary of immature female rainbow trout. These results suggest that a M-CSFR-like receptor may be involved in female reproductive tracts even in an oviparous animal like fish. [Copyright &y& Elsevier]

Abstract: Interleukin (IL)-8 plays a central role in the initiation and maintenance of inflammatory responses in the inflammatory bowel disease. The proinflammatory cytokine-mediated production of IL-8 requires activation of various kinases, which leads to the IκB degradation and NF-κB activation. In this study, we investigated the role of luteolin, a major flavonoid of Lonicera japonica, on TNF-α-induced IL-8 production in human colonic epithelial cells. HT29 cells were stimulated with TNF-α in the presence or absence of luteolin. IL-8 production was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase–polymerase chain reaction (RT–PCR) analysis, and the mitogen-activated protein kinases (MAPKs) activation and IκB degradation were determined by Western blot analysis. NF-κB activation was assessed by the electrophoretic motility shift assay (EMSA). Luteolin suppressed TNF-α-induced IL-8 production in dose-dependent manner. In addition, luteolin inhibited TNF-α-induced phosphorylation of p38 MAPK and extracellular-regulated kinases (ERK), IκB degradation, and NF-κB activation. These results suggest that luteolin has the inhibitory effects on TNF-α-induced IL-8 production in the intestinal epithelial cells through blockade in the phosphorylation of MAPKs, following IκB degradation and NF-κB activation. [Copyright &y& Elsevier]

The expression vector pTNF-GFP containing the 2351 bp 5′ flanking region of Japanese flounder tumor necrosis factor (TNF) gene was linked with the green fluorescence protein (GFP) gene and was introduced into YO-K cells derived from Japanese flounder kidney and HINAE cells derived Japanese flounder embryos. YO-K cells and HINAE cells were incubated with three concentrations (250, 500, 1000 μg/ml) of lipopolysaccharide (LPS) at 20 °C for 24 h. The number of cells expressing GFP, as well as the amount of GFP protein was increased by LPS stimulation in both cell lines. GFP mRNA transcription was also induced by LPS stimulation in both YO-K cells and HINAE cells after 1 h stimulation. In YO-K cells, expression level of GFP decreased gradually from 3 to 6 h post-stimulation, while a reverse trend was observed in HINAE cells. A deletion assay of TNF gene promoter showed that the 5′ flanking region, -1783 to -1300 bp, containing cis-acting regulatory elements mediated LPS induction. An electrophoretic mobility shift assay using 2 fragments (-1783 to -1541 bp and -1540 to -1300 bp) revealed that only LPS-stimulated nuclear extracts bound to the -1540 to -1300 bp fragment. These results suggest that transcription of the TNF gene promoter in homologous cultured cells exhibited an inducible pattern and was regulated under the control of the immune system. [Copyright &y& Elsevier]

Immune regulation, induction of various inflammatory and growth regulatory genes such as IL-1β, IL-6, TNF-α and GM-CSF require activation of transcription factors such as nuclear factor-κB (NF-κB), activated transcription factor (ATF-2), c-Fos and cAMP response element-binding protein (CREB). Untreated B16F-10 cells produce very high amount of proinflammatory cytokines such as IL-1β, IL-6, TNF-α and GM-CSF. Piperine treatment significantly reduced the above proinflammatory cytokines. We also found that piperine could reduce the expression of IL-1β, IL-6, TNF-α, GM-CSF and IL-12p40 genes. Piperine at a concentration of 2.5, 5 and 10 μg/ml inhibited the collagen matrix invasion of B16F-10 melanoma cells in a dose-dependent manner. Piperine could inhibit the matrix metalloproteinase production which was demonstrated by zymographic analysis. We found that the nuclear translocation of p65, p50, c-Rel subunits of NF-κB and other transcription factors such as ATF-2, c-Fos and CREB were inhibited by the treatment of piperine. [Copyright &y& Elsevier]

Abstract: The anti-inflammatory effects of caffeic acid (CA), caffeic acid methyl ester (CM) and di-O-acetylcaffeic acid (DAC) were investigated in rats using the carrageenin-induced edema model and the antinociceptive effects of these compounds were also assessed in mice by means of the acetic acid-induced abdominal constriction test and hot plate test. CM (10mg/kg, p.o.) showed the most potent anti-inflammatory and antinociceptive effects in these animal models. To investigate the mechanism of the anti-inflammatory action, we examined the effects of these compounds on the lipopolysaccaride (LPS)-induced NO and PGE2 responses in the murine macrophage cell line, RAW 264.7. Our data indicate that CM is the most potent inhibitor of NO and PGE2 production and it also significantly decreased tumor necrosis factor-alpha (TNF-α) release. Consistent with these observations, the protein and mRNA expression levels of iNOS and COX-2 were found to be inhibited by CM in a dose-dependent manner. Furthermore, CM inhibited the nuclear factor-κB (NF-κB) activation induced by LPS, which was associated with the prevention of the degradation of the inhibitor κB, and subsequently with decreased p65 protein levels in the nucleus. Taken together, our data indicate that the anti-inflammatory properties of CM might result from the inhibition of iNOS, COX-2 and TNF-α expression through the down-regulation of NF-κB binding activity. [Copyright &y& Elsevier]

Oncogenic transformation leads to cell cycle aberration and apoptosis dysregulation. Targeting cell cycle and apoptosis pathways has emerged as an attractive approach for the treatment of cancer. The activity of cdks can be modulated by targeting these kinases with small molecules that bind to the ATP binding pocket of cdks, or by altering the composition of the cdk/endogenous cdk inhibitor complexes by different mechanisms. Apoptosis can be modulated by targeting pro-apoptotic or pro-survival pathways. Several proteins relevant to oncogenic and proliferative processes, such as p53, bcl-2, AKT, ras and epidermal growth factor receptor, are also important in blocking apoptosis. Several small molecules that modulate cell cycle control and apoptosis have been approved recently and many will be approved in the near future. Several challenges remain, including finding ways of targeting these agents specifically to tumors (sparing normal cells), and the development of rationales for combining these new agents with standard therapies and for prioritizing the development of an overwhelming number of novel small molecules targeting cell cycle and apoptosis. Novel technologies such as genomics and proteomics will be instrumental in designing combinatorial regimens tailored to patients on the basis of the genetic makeup of tumors. Irrespective of all shortcomings, the future of modulation of apoptosis and cell cycle machinery for oncology therapy is quite exciting. [Copyright &y& Elsevier]

Apoptosis is the principal mechanism by which cells are physiologically eliminated in metazoan organisms. During apoptotic death, cells are neatly carved up by caspases and packaged into apoptotic bodies as a mechanism to avoid immune activation. Recently, necrosis, once thought of as simply a passive, unorganized way to die, has emerged as an alternate form of programmed cell death whose activation might have important biological consequences, including the induction of an inflammatory response. Autophagy has also been suggested as a possible mechanism for non-apoptotic death despite evidence from many species that autophagy represents a survival strategy in times of stress. Recent advances have helped to define the function of and mechanism for programmed necrosis and the role of autophagy in cell survival and suicide. [Copyright &y& Elsevier]

Rheumatoid arthritis (RA) is a common, systemic autoimmune disease of which the exact etiology is not known. In the past 10 years, substantial progress has been made in the identification of the antigens specifically recognized by the autoantibodies of RA patients. A central factor in this respect is citrullination, a form of post-translational modification that is strongly associated with autoimmunity in RA. Here, we summarize and discuss our current knowledge on (i) autoantibody systems in RA, (ii) the occurrence of peptidylarginine deiminases and (iii) citrullinated proteins in natural and diseased environments, and (iv) genetic factors involved in RA that may influence the generation and presentation of citrullinated proteins and the resulting antibody production against these modified proteins. Citrullination of proteins may play a key role in the initiation and/or the progression of RA. The onset of citrulline-specific autoimmunity in RA is probably mediated by both environmental and genetic factors, and future studies will learn whether therapeutic intervention at the level of citrullination may provide new possibilities to treat RA. [Copyright &y& Elsevier]

Systemic lupus erythematosus (SLE) is a systemic autoimmune inflammatory disease with partially understood etiology, which can affect virtually any organ. Despite suggestions to the contrary, SLE is proving to be a reliable phenotype for genetic studies. Similar to many other autoimmune diseases, SLE demonstrates a complex pattern of inheritance that is consistent with the involvement of multiple susceptibility genes as well as environmental risk factors. During the past several years, some new candidate genes have been implicated in induction of SLE through association studies, and multiple susceptibility regions have been detected through genome-wide linkage studies. Many of the susceptibility effects have been confirmed by independent studies. [Copyright &y& Elsevier]

Objectives: We sought to determine the relationship between osteoprotegerin (OPG) and clinical outcomes in patients with heart failure (HF) after acute myocardial infarction (AMI). Background: Arterial calcification is a prominent feature of arterial atherosclerosis and is associated with the occurrence of AMI. Osteoprotegerin is a recently discovered member of the tumor necrosis superfamily that may link the skeletal with the vascular system. Methods: We assayed plasma OPG levels in 234 patients with AMI complicated with HF and their relation to adverse outcomes during follow-up in patients randomly assigned to angiotensin-converting enzyme inhibition or angiotensin II antagonism. Blood was sampled at baseline (median three days after AMI), one month, and at one and two years. Results: Elevated plasma levels of OPG at baseline were associated with adverse outcomes during a median of 27 months follow-up; OPG remained an independent prognostic indicator also after adjustment for other known predictors of mortality and cardiovascular events after AMI (e.g., creatinine clearance, N-terminal B-type natriuretic peptide, high-sensitivity C-reactive protein). In non-survivors, plasma OPG levels were persistently elevated during longitudinal testing, suggesting that OPG may be of value for monitoring patients at risk. Conclusions: Osteoprotegerin is a novel marker for cardiovascular mortality and clinical events in patients with AMI complicated with HF. These findings are compatible with the hypothesis suggesting a possible association between mediators of bone homeostasis and cardiovascular disease. [Copyright &y& Elsevier]

Antioxidants can inhibit the proliferation of T lymphocytes induced by mitogens. This has been postulated to be due to their scavenging of reactive oxygen species which may act as second messengers in the antigen-induced signaling cascade leading to cell proliferation. When added concurrently with various mitogens, the thiol pyrrolidine dithiocarbamate (PDTC) inhibited the subsequent proliferation of lymphocytes. The extracellular copper chelator bathocuproine disulfonic acid (BCPS) increased the amount of PDTC needed for inhibition. We sought to determine the mechanism by which the two different treatments, PDTC (0.4 μM, copper-dependent) and PDTC (20 μM with BCPS, redox-sensitive) affected proliferation. We found that both inhibited the increase in expression of many of the genes, including IL-2 and MKP-2, that were induced early after stimulation of lymphocytes with phorbol myristate acetate and ionomycin. The inhibition of MKP-2 may have contributed to the enhancement observed by the thiol of mitogen-induced ERK phosphorylation. Of the two redox-sensitive, IL-2 regulating transcription factors, NF-κB and AP-1, the mitogen-induced activity of the former was inhibited by PDTC. Treatment of unstimulated cells with PDTC induced the expression of many genes, most notably several metallothioneins and heat shock proteins, and this may provide an alternative explanation for the inhibition of cellular proliferation. [Copyright &y& Elsevier]

Bacillus Calmette Guérin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which render mice highly sensitive to endotoxin-mediated hepatotoxicity. This work investigates the role of inducible nitric oxide synthase (iNOS) in liver damage induced by BCG and endotoxins in BCG-infected mice.Liver injury and cytokine activation induced by BCG and by LPS upon BCG infection (BCG/LPS) were compared in wild-type and iNOS-/- mice.iNOS-/- mice infected with living BCG are protected from hepatic injury when compared to wild-type mice which express iNOS protein in macrophages forming hepatic granulomas. In addition, iNOS-/- mice show a decrease in BCG-induced IFN-γ serum levels. LPS challenge in BCG-infected mice strongly activates iNOS in the liver and spleen of wild-type mice which show important liver damage associated with a dramatic increase in TNF and IL-6 and also Th1 type cytokines. In contrast, iNOS-/- mice are protected from liver injury after BCG/LPS challenge and their TNF, IL-6 and Th1 type cytokine serum levels raise moderately.These results demonstrate that nitric oxide (NO) from iNOS is involved in hepatotoxicity induced by both mycobacterial infection and endotoxin effects upon BCG infection and that inhibition of NO from iNOS protects from liver injuries. [Copyright &y& Elsevier]

Dietary fish oil (rich in ω-3 fatty acids: eicosapentaenoic acid and docosahexaenoic acid) suppresses synthesis and activity of proinflammatory cytokines that induce anorexia. We hypothesized that dietary fish oil reverses the feeding pattern of tumor anorexia, increasing food intake and retarding tumor growth.Thirty-two Fischer rats were placed in Automated Eater Meter cages and randomly divided into four groups: tumor bearing (TB) rats eating normal chow diet (TB-Chow); TB rats eating chow diet supplemented with ω-3 fatty acids (TB-ω-3FA); Controls, non-tumor bearing (NTB) rats eating normal chow (NTB-Chow); and NTB rats with ω-3 fatty acid supplementation (NTB-ω-3FA). Doses of 106 methylcholanthrene (MCA) sarcoma cells were subcutaneously injected in TB rats. Daily food intake, meal size (MZ), meal number (MN), body weight, and tumor volume were measured, and rats were euthanized at onset of anorexia. Data were statistically analyzed using analyses of variance (ANOVA) and t-tests. Data are reported as mean ± SE.Tumor appeared significantly earlier in TB-Chow than in TB-ω-3FA rats (7.5 ± 0.3 days versus 11.6 ± 0.8 days, p < 0.05). Daily food intake declined significantly in TB-Chow versus TB-ω-3FA rats 18 days after tumor inoculation and, at onset of anorexia, was 9.41 ± 1.77 g/day versus 13.32 ± 0.81 g/day, p < 0.05. Food intake decreased initially by decrease in meal number (at day 15) followed by a decrease in meal size (at day 18). At onset of anorexia, meal size and meal number were significantly decreased in TB-Chow versus TB-ω-3FA rats (0.75 ± 0.067 g/meal versus 1.05 ± 0.08 g/meal, p < 0.05) and (9.5 ± 1.32 versus 12.79 ± 0.93 meals/day, p < 0.05), respectively. Tumor volume was significantly smaller in TB-ω-3FA versus TB-Chow rats (7.6 ± 0.6 cm3 versus 16.5 ± 1.0 cm3, p < 0.05), as was tumor weight (7.5 ± 2.2 g versus 18.1 ± 1.6 g, p < 0.05).In TB rats, ω-3FA improved food intake; restored normal eating pattern, delayed onset of anorexia, tumor appearance, and growth; and prevented body weight loss. Supplementation of ω-3 fatty acids has therapeutic potential in cancer anorexia. [Copyright &y& Elsevier]

Antiphospholipid-mediated endothelium perturbation plays a role in antiphospholipid syndrome (APS)-associated vasculopathy. Antiphospholipid antibodies activate endothelium both in vitro and in vivo experimental models by inducing a pro-inflammatory/-coagulant phenotype; the antibodies recognize β2 glycoprotein I (β2GPI) on human endothelial cells (EC) from different parts of the vasculature.In spite of such large in vitro evidence, few studies have addressed the issue whether or not a comparable endothelial perturbation might be detectable in vivo.We investigated several indirect ex vivo parameters of endothelial dysfunction: plasma levels of soluble adhesion molecules (sADM), soluble thrombomodulin (sTM), von Willebrand factor (vWF) and tissue plasminogen activator (t-PA) by solid-phase assays. The study included: patients with primary antiphospholipid syndrome (n=32), with the syndrome secondary to non-active systemic lupus erythematosus (SLE, n=10), six patients with persistent antiphospholipid positivity at medium/high titre without any clinical manifestation of the syndrome. Fifty-two age and sex matched healthy subjects have been enrolled as controls. In addition, circulating endothelial cells identified by flow cytometry and the brachial artery flow-mediated vasodilation (FMV) were evaluated in 26 patients (20 primary and 6 lupus syndromes) and 30 healthy controls.Plasma levels of soluble adhesion molecules did not differ from controls, while a significant increase in von Willebrand factor titres (P<0.05) was found. No significant difference was found regarding the number of circulating endothelial cells and flow-mediated vasodilation.As a whole, these findings do suggest that antiphospholipid antibodies per se are not able to support a full-blown endothelial perturbation in vivo. As shown in antiphospholipid syndrome experimental animal models, a two-hit hypothesis is suggested. [Copyright &y& Elsevier]

Among the pleiotropic effects of statins, their antioxidant action may be involved in their protective effects. Thus, we investigated the antioxidant effect of simvastatin, associated or not with α-tocopherol, on levels of electronegative low-density lipoprotein (LDL−), nitrotyrosine, thiols (homocysteine, glutathione, cysteine, methionine), and lipid-soluble antioxidants in blood plasma of hypercholesterolemic subjects. In this study, 25 hypercholesterolemic subjects were treated for 2 months with simvastatin (20 mg/day) and with simvastatin (20 mg/day) + α-tocopherol (400 IU/day). Concentrations of thiols were determined by high-performance capillary electrophoresis–laser-induced fluorescene. Lipid-soluble antioxidants were determined by HPLC, and LDL−, and nitrotyrosine by ELISA. Simvastatin, independent of its association with α-tocopherol, reduced plasma concentrations of LDL−, nitrotyrosine, total cholesterol, and LDL cholesterol and the LDL cholesterol/HDL cholesterol ratio. Neither simvastatin nor simvastatin plus α-tocopherol altered plasma levels of the thiols analyzed. α-Tocopherol did not change the antioxidant effect of simvastatin on the levels of LDL− and nitrotyrosine in hypercholesterolemic subjects. The reduction of LDL− and nitrotyrosine by simvastatin seems to be related to the pleiotropic effects of this statin, and it may have an important protective effect against endothelial dysfunction and atherosclerosis. [Copyright &y& Elsevier]

Lipopolysaccharide (LPS) induces a release of tumor necrosis factor (TNF)-α, endothelin (ET)-1, interleukin (IL)-lα, IL-6 and IL-10 in rat liver macrophages (Kupffer cells). Prostaglandin (PG)E2 inhibits the release of the fibrogenic mediators TNF-α, ET-1 and IL-lα, and enhances the release of the anti-fibrogenic mediators IL-6 and IL-10. This effect of PGE2 is mimicked by specific agonists for the PGE2 receptors EP2 and EP4; whereas, agonists for the PGE2 receptors EP1 and EP3 are inactive. Rat liver macrophages express mRNA encoding the PGE2 receptors EP2 and EP4 but not the PGE2 receptors EP1 and EP3. These data suggest that PGE2 exerts its anti-fibrogenic effect through the EP2 and EP4 receptor by inhibiting the release of the fibrogenic mediators TNF-α, ET-1 and IL-lα, and by enhancing the release of the anti-fibrogenic mediators IL-6 and IL-10 in liver macrophages. [Copyright &y& Elsevier]

We have previously demonstrated that mouse cytomegalovirus (MCMV) aggravates atherosclerosis in apolipoprotein E knockout (apoE–/–) mice, most likely by enhancing both systemic and local (e.g. in the vascular wall) cytokine production. However, until now it was unclear which cell type is responsible for this enhanced pro-inflammatory cytokine production. In this study we focused on the macrophage (mΦ), which besides being an important source of such cytokines, is known to be an important player in both atherosclerosis and viral clearance. We investigated whether MCMV could induce a pro-inflammatory immune mΦ phenotype, which ultimately may contribute to the development of atherosclerosis. To this end, peritoneal exudate cells (PEC) were elicited in apoE–/– mice by either MCMV or thioglycolate injection, and mΦ were phenotyped at 1 week post-intraperitoneal injection. MCMV-induced peritoneal mΦ contained MCMV DNA but had limited MCMV mRNA expression, indicating latent infection. These mΦ showed increased production of interferon-γ (IFNγ), exclusive production of interleukin-18 (IL-18) and increased expression of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86, when compared with thioglycolate-induced mΦ. From these results, we conclude that intraperitoneal injection of MCMV induces an immune-responsive exudate in which at 7 days post-infection, MCMV-infected mΦ express a pro-inflammatory immune phenotype. As such, the MCMV-induced mΦ may be an important player in aggravating atherosclerosis through systemic and/or local immune activation. [Copyright &y& Elsevier]

Mammalian tooth development has served as an excellent model system to investigate the intricate, interactive mechanisms of patterning, morphogenesis and cytodifferentiation during organogenesis. Teeth develop from interactions between epithelium and neural crest-derived (ecto)mesenchyme that are largely mediated by ligand–receptor signalling. It is well-established that signalling molecules of the Bmp, Fgf, Wnt and Hedgehog families, are involved at multiple stages of tooth development. Recently, however, a specific role for molecules belonging to the TNF-family of ligands in tooth morphogenesis has been identified, suggesting that this pathway, acting to activate NF-κB, has played an important role in the development and evolution of tooth number and shape. [Copyright &y& Elsevier]

The potential of amyloid-β (Aβ) immunization as a disease-modifying therapy for Alzheimer’s disease is limited by the occurrence of encephalitic side effects in a subset of treated patients. The encephalitis was not predicted from immunization studies in transgenic, Aβ-depositing mice. More recently, studies in these same mice indicate that passive immunization with certain anti-Aβ antibodies can induce microhemorrhage. Cerebral amyloid angiopathy (CAA) may play a key role in determining the risk for these complications. Because aged nonhuman primates (NHPs) have a more human-like immune system than rodents, and because NHPs naturally develop senile plaques and CAA with age, NHPs appear to be important, adjunctive models for assessing the efficacy and safety of immunotherapeutics for Alzheimer’s disease. Conversely, the ability to model the complications of Aβ immunotherapy will be important for elucidating the bases of these complications, and for developing protocols that minimize or eliminate the risks of these serious adverse effects. [Copyright &y& Elsevier]

We assessed the significance of serum cytokine levels in patients with fulminant myocarditis.Although many investigations have demonstrated the crucial role of cytokines in the development of myocarditis, it remains uncertain whether serum levels of cytokines enable one to predict the prognosis of human myocarditis, especially concerning cardiogenic shock (CS) requiring a mechanical cardiopulmonary support system (MCSS).We studied 22 consecutive patients with fulminant myocarditis and compared them with 15 patients with acute myocardial infarction (AMI) requiring MCSS. The patients with myocarditis were classified into three groups: eight patients with CS requiring MCSS on admission (group 1); six patients who unexpectedly lapsed into CS requiring MCSS more than two days after catecholamine had been initiated (group 2); and eight patients without MCSS (group 3). Furthermore, 14 patients with myocarditis requiring MCSS were divided into a fatal group (n = 5) and a survival group (n = 9). Biochemical markers, including serum cytokine levels and hemodynamic variables on admission, were analyzed.Serum levels of interleukin (IL)-10 and tumor necrosis factor-alpha, but not other cytokines, were significantly higher in myocarditis than in AMI. Only serum levels of IL-10 were significantly higher in group 1 and 2 than in group 3 (49.1 ± 37.5/20.7 ± 17.6 pg/ml vs. 2.4 ± 1.1 pg/ml; p = 0.0008/0.0012). Serum IL-10 levels were also significantly higher in the fatal group than in the survival group with myocarditis (74.0 ± 27.0 pg/ml vs. 16.4 ± 8.8 pg/ml; p = 0.003).Serum IL-10 levels on admission enabled one to predict subsequent CS requiring MCSS and mortality of fulminant myocarditis patients. [Copyright &y& Elsevier]

We sought to determine whether a novel, non-pharmacological form of immune modulation therapy (IMT), shown experimentally to reduce inflammatory and increase anti-inflammatory cytokines, improved outcomes in patients with advanced heart failure (HF).Immune activation contributes to the progression of HF, but treatments directed against inflammation have been largely unsuccessful.Seventy-five HF patients (New York Heart Association [NYHA] functional class III to IV) were randomized to receive either IMT (n = 38) or placebo (n = 37) in a double-blind trial for six months, with continuation of standard HF therapy. Patients were evaluated using the 6-min walk test, changes in NYHA functional class, cardiac function, and quality of life assessments, as well as occurrence of death and hospitalization.There was no between-group difference in 6-min walk test, but 15 IMT patients (compared with 9 placebo) improved NYHA functional classification by at least one class (p = 0.140). The Kaplan-Meier survival analysis showed that IMT significantly reduced the risk of death (p = 0.022) and hospitalization (p = 0.008). Analysis of a clinical composite score demonstrated a significant between-group difference (p = 0.006). There was no difference in left ventricular ejection fraction, but there was a trend toward improved quality of life (p = 0.110).These preliminary findings are consistent with the hypothesis that immune activation is important in the pathogenesis of HF and establish the basis for a phase III trial to define the benefit of IMT in chronic HF. [Copyright &y& Elsevier]

Adrenomedullin (AM) is a peptide that possesses potentially beneficial properties. Since the initial discovery of the peptide by Kitamura et al. in 1993, the literature has been awash with reports describing its novel mechanisms of action and huge potential as a therapeutic target. Strong evidence now exists that AM is able to act as an autocrine, paracrine, or endocrine mediator in a number of biologically significant functions, including the endothelial regulation of blood pressure, protection against organ damage in sepsis or hypoxia, and the control of blood volume through the regulation of thirst. Its early promise as a potential mediator/modulator of disease was not, however, entirely as a result of the discovery of physiological functions but due more to the observation of increasing levels measured in plasma in direct correlation with disease progression. In health, AM circulates at low picomolar concentrations in plasma in 2 forms, a mature 52-amino acid peptide and an immature 53-amino acid peptide. Plasma levels of AM have now been shown to be increased in a number of pathological states, including congestive heart failure, sepsis, essential hypertension, acute myocardial infarction, and renal impairment. These earliest associations have been further supplemented with evidence of a role for AM in other pathologies including, most intriguingly, cancer. In this review, we offer a timely review of our current knowledge on AM and give a detailed account of the putative role of AM in those clinical areas in which the best therapeutic opportunities might exist. [Copyright &y& Elsevier]

In this study, we investigated the roles of CD4 T cell cytokines IL-17 and IL-17F in GM-CSF production from lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not induce GM-CSF release from LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1β- and TNF-α-induced GM-CSF mRNA expression and production, whereas IL-17F had an enhancing effect on IL-1β-induced GM-CSF production, but a marked inhibitory effect on TNF-α-induced secretion. GM-CSF production was further enhanced with the combination of three cytokines IL-1β, TNF-α and IL-17 or IL-17F. Additionally, when Th1 or Th2 cytokine was combined with IL-1β or TNF-α, both Th1 and Th2 cytokines had a modest stimulatory effect on TNF-α-induced GM-CSF production, whereas IL-4 and IFN-γ profoundly attenuated IL-1β-induced secretion. Moreover, the regulation by IL-17 plus Th1 or Th2 cytokine of GM-CSF production from LMVECs treated with IL-1β or TNF-α was dependent on the concentration of IL-17. Our findings indicate that IL-17 and IL-17F play a differential regulatory role in GM-CSF production by LMVECs stimulated with IL-1β and/or TNF-α, which is sensitive to Th1 and Th2 cytokine modulation. [Copyright &y& Elsevier]

C1q is the first subcomponent of the classical pathway of the complement system and a major connecting link between innate and acquired immunity. As a versatile charge pattern recognition molecule, C1q is capable of engaging a broad range of ligands via its heterotrimeric globular domain (gC1q) which is composed of the C-terminal regions of its A (ghA), B (ghB) and C (ghC) chains. Recent studies using recombinant forms of ghA, ghB and ghC have suggested that the gC1q domain has a modular organization and each chain can have differential ligand specificity. The crystal structure of the gC1q, molecular modeling and protein engineering studies have combined to illustrate how modular organization, charge distribution and the spatial orientation of the heterotrimeric assembly offer versatility of ligand recognition to C1q. Although the biochemical and structural studies have provided novel insights into the structure-function relationships within the gC1q domain, they have also raised many unexpected issues for debate. [Copyright &y& Elsevier]

We developed an assay allowing the detection and quantification of cell death after transient expression of apoptotic genes in B- and T-lymphocytes. For efficient gene transfer, B- and T-cells were electroporated under optimized conditions. To blind out the high background of non-transfected cells and cell death caused by the electroporation procedure itself, the green fluorescent protein (GFP) was co-transfected with the gene of interest. However, if the gene of interest was a potent apoptosis inducer, most successfully transfected cells were killed before GFP was expressed to levels sufficient for standard flow cytometry analysis or apoptosis assays. After staining of the transfected cells with propidium iodide (PI), very few GFP+/PI+ cells were detectable. To overcome this problem, the cell death rate induced by the transiently expressed gene was determined as the reduction of living green cells in the apoptotic versus a reference sample. This was achieved by an advanced flow cytometrical analysis quantifying the number of surviving green cells in normalised sample volumes directly relating to the number of initially transfected cells. Functioning of the assay was demonstrated by transient transfection of the potent apoptosis inducers TNF-receptor-associated death domain protein (TRADD) and a fusion protein of the transmembrane domain of the latent membrane protein 1 (LMP1) of Epstein–Barr virus and the signaling domain of TNF-receptor 1. We successfully applied the assay to the Burkitt lymphoma cell line BJAB and the T-leukemia cell line Jurkat. [Copyright &y& Elsevier]

We investigated the effect of Type D personality on the occurrence of adverse events at nine months in patients with ischemic heart disease (IHD) after percutaneous coronary intervention (PCI) with sirolimus-eluting stents (SESs) or bare stents. Type D patients experience increased negative emotions and tend not to express these emotions in social interactions.The SES is a new advent in interventional cardiology that reduces the restenosis rate and the risk of a major adverse cardiac event, but the SES has not been shown to confer any benefits on death or myocardial infarction (MI).Consecutive patients with IHD (n = 875) enrolled in the Rapamycin-Eluting Stent Evaluated At Rotterdam Cardiology Hospital (RESEARCH) registry completed the Type D Personality Scale (DS14) six months after PCI. The end point was a composite of death and MI. Events occurring before administration of the DS14 were excluded from analyses.At nine months'' follow-up, there were 20 events. Type D patients were at a cumulative increased risk of adverse outcome compared with non-Type D patients: 5.6% versus 1.3% (p < 0.002). Type D personality (odds ratio [OR] 5.31; 95% confidence interval [CI] 2.06 to 13.66) remained an independent predictor of adverse outcome adjusting for all other variables, including SES versus bare-stent implantation.Type D personality was an independent predictor of adverse events in patients optimally treated with the latest advent in interventional cardiology. The DS14 could be used as a screening instrument in routine clinical practice to optimize risk stratification in IHD patients. [Copyright &y& Elsevier]

Anemia has recently been recognized as an important comorbid condition and potentially novel therapeutic target in patients with heart failure (HF). Anemia is common in HF patients, with a prevalence ranging from 4% to 55% depending on the population studied. Multiple potential mechanisms of interaction exist between anemia and the clinical syndrome of HF, including hemodilution, inflammatory activation, renal insufficiency, and malnutrition. A growing body of literature from observational databases and clinical trials suggests that anemia is an independent risk factor for adverse outcomes in patients with HF. Although preliminary data suggest that treatment of anemia may result in significant symptomatic improvement in HF, aggressive treatment of anemia may also be associated with increased risk of hypertension or thrombosis. Multiple ongoing studies will provide definitive data on the balance of risks and benefits of anemia treatment in chronic HF. [Copyright &y& Elsevier]

We examined the effects of early treatment with a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor pravastatin on the progression of glucose intolerance and cardiovascular remodeling in a model of spontaneously developing type II diabetes mellitus (DM), the Otsuka Long-Evans Tokushima Fatty (OLETF) rats.Clinical trials showed that pravastatin prevented new-onset DM in hypercholesterolemic patients, and that it was effective in prevention of cardiovascular events in diabetics.The OLETF rats were treated with pravastatin (100 mg/kg/day) from 5 weeks of age and compared with age-matched untreated OLETF rats and normal Long-Evans Tokushima Otsuka (LETO) rats on serial oral glucose tolerance tests (OGTT) and Doppler echocardiography and on histopathological/biochemical analyses of the heart at 30 weeks.The OGTT revealed that 40% and 89% of untreated OLETF rats were diabetic at 20 and 30 weeks, respectively, but 0% and only 30%, respectively, were diabetic in the treated OLETF. Left ventricular diastolic function was found impaired from 20 weeks in untreated OLETF but remained normal in the treated-OLETF. The wall-to-lumen ratio and perivascular fibrosis of coronary arteries were increased in untreated-OLETF but were limited in the treated-OLETF at 30 weeks. Moreover, cardiac expressions of a fibrogenic growth factor, transforming growth factor-β1 (TGF-β1), and a proinflammatory chemokine, monocyte chemoattractant protein-1 (MCP-1), were increased in untreated-OLETF. However, in the treated-OLETF, overexpressions of TGF-β1 and MCP-1 were attenuated, which was associated with overexpression of endothelial nitric oxide synthase (eNOS) (2.5-fold of control LETO).Early pravastatin treatment prevented cardiovascular remodeling in the spontaneous DM model by retarding the progression of glucose intolerance, overexpressing cardiac eNOS, and inhibiting overexpressions of fibrogenic/proinflammatory cytokines. [Copyright &y& Elsevier]

The role of the interleukin (IL)-17 family members in the regulation of G-CSF production by lung microvasculature has not been elucidated yet. We therefore investigated the effects of IL-17 and IL-17F on the regulation of G-CSF production by lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not up-regulate G-CSF production from primary human LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1β- and TNF-α-induced G-CSF production, whereas IL-17F had an enhancing effect on IL-1β-induced production, but an inhibitory effect on TNF-α-induced secretion. G-CSF production was further enhanced with the combination of three cytokines IL-1β, TNF-α and IL-17. In contrast, three cytokines IL-1β, TNF-α and IL-17F were combined together, G-CSF production was less than that induced by IL-1β or IL-1β plus TNF-α or IL-17F. Moreover, IL-17 plus Th1 or Th2 cytokine had a modest stimulatory effect on TNF-α-induced G-CSF production, whereas IL-17 plus IFN-γ had an inhibitory effect on IL-1β-induced release. Similarly, IL-17F plus IL-10, IL-13 or IFN-γ had an inhibitory effect on IL-1β-induced production. Our findings indicate that CD4 T cell cytokines IL-17 and IL-17F play a differential regulatory role in G-CSF production by LMVECs stimulated with IL-1β and/or TNF-α, which is also sensitive to Th1 and Th2 cytokine modulation. [Copyright &y& Elsevier]

We sought to investigate the role of endogenous monocyte chemoattractant protein (MCP)-1 in ischemia-induced neovascularization.Roles of inflammatory changes including macrophage infiltration are suggested in ischemic neovascularization.Unilateral hindlimb ischemia was induced by excising surgically the entire femoral artery and vein in mice. Immediately after operation, plasmid deoxyribonucleic acid encoding a dominant negative mutant of MCP-1 (7ND) or the empty plasmid (mock) was injected into the ipsilateral thigh adductor muscle.In mock-treated mice, MCP-1 was upregulated transiently in ischemic hindlimb peaking at day 3. Serial laser Doppler blood flow (LDBF) analysis showed an abrupt decrease in blood flow, followed by a recovery to the near-normal levels in mock-treated mice; 7ND treatment had no effects on the initial decrease in LDBF but deteriorated the recovery. At day 3, macrophage infiltration and inductions of tumor necrosis factor (TNF)-alpha and vascular endothelial growth factor (VEGF) were prominent in the ischemic adductor muscle in mock-treated mice; 7ND treatment significantly reduced macrophage infiltration and suppressed TNF-alpha and VEGF inductions in response to ischemia. At day 21, postmortem angiography and anti-CD31 immunohistostaining revealed well-developed collateral vessels and capillary formation, respectively, in the ischemic muscle of mock-treated mice; 7ND overexpression remarkably suppressed the collateral vessel formation and capillary formation.Endogenous MCP-1 may play a role in ischemia-induced neovascularization by recruiting macrophages that activate TNF-alpha and VEGF inductions. [Copyright &y& Elsevier]

Alzheimer’s disease (AD) is a neurogenetic condition that affects the processes via which the brain functions. Major observable hallmarks of AD are accumulated clusters of proteins in the brain. These clusters, termed neurofibrillary tangles (NFT), resemble pairs of threads wound around each other in a helix fashion accumulating within neurons. These tangles consist of a protein called Tau, which binds to tubulin, thus forming microtubules. Unlike NFTs, deposits of amyloid precursor protein (β-APP) gather in the spaces between nerve cells. The nearby neurons often look swollen and deformed, and the clusters of protein are usually accompanied by reactive inflammatory cells, microglia, which are part of the brain’s immune system responsible for degrading and removing damaged neurons or plaques. Since phosphorylation/dephosphorylation mechanisms are crucial in the regulation of Tau and β-APP, a superfamily of mitogen-activated protein kinases (MAPKs) has recently emerged as key regulators of the formation of plagues, eventually leading to dementia and AD. The complex molecular interactions between MAPKs and proteins (plagues) associated with the evolution of AD form a cornerstone in the knowledge of a still burgeoning field of neurodegenerative diseases and ageing. This review overviews current understanding of the molecular pathways related to MAPKs and their role in the development of AD and, possibly, dementia. MAPKs, therefore, may constitute a neurogenetic, therapeutic target for the diagnosis and evolution of a preventative medical strategy for early detection, and likely treatment, of Alzheimer’s. [Copyright &y& Elsevier]

The colony-stimulating factors (CSFs) are a group of cytokines central to the hematopoiesis of blood cells, the modulation of their functional responses, as well as the maintenance of homeostasis and overall immune competence. This group consists of the macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF), and multi-CSF (IL-3). M-CSF and G-CSF are relatively lineage-specific, having a role in the proliferation, differentiation, and survival of macrophages, neutrophils, and their precursors. In contrast, GM-CSF and multi-CSF function at earlier stages of lineage commitment regulating the expansion and maturation of primitive hematopoietic progenitors. Colony stimulating factor production and degradation are strictly controlled, thus allowing for effective modulation of their biological functions in steady-state conditions as well as under periods of stress. Moreover, the mechanisms behind their expression and that of their cognate receptors ensures that their actions are tightly coordinated, within the context of a network of complex but finely tuned regulatory pathways derived from a variety of local and endocrine hematopoietic regulators.In this review we present some of the most salient information on CSF biology collected over the last three decades. We examine the gene and protein structure of each of the four CSFs and their corresponding receptors, and consider the main determinants behind their biological activities. The components responsible for their functional redundancy as well as the mechanisms that mediate their specificity are also discussed. Although most of available knowledge about CSFs is on human and mouse CSFs, an attempt was made to integrate recent findings in other systems in order to highlight a more widespread role for CSFs throughout evolution. [Copyright &y& Elsevier]

The cytokine network is an important homeostatic system with potent activities in immune surveillance, growth, developmental and repair processes. Although interleukin-1β (IL-1β) is considered a pivotal pro-inflammatory cytokine, merely focussing on its inflammatory role would be too narrow an approach. Elucidation of the human, the mouse and the Fugu rubripes (pufferfish) genome now enables a more comprehensive overview of this cytokine family and its receptors in several vertebrate classes. Phylogenetic analyses of the IL-1 family members, comprising over 80 sequences of various fish, amphibian, avian and mammalian species, reveal that for only a few mammalian IL-1 family members unambiguous orthologues have been found in fish, indicating a recent origin of some of the mammalian IL-1 family members. Interestingly, the Fugu genome did reveal teleost orthologues for IL-18 and its putative receptor complex. All teleost IL-1β sequences cluster separately from IL-1β sequences of other species. In contrast, a number of IL-1 receptor family members have well conserved fish orthologues. This supports the concept of an ancestral role of this family, possibly in the brain. [Copyright &y& Elsevier]

Hemophagocytic syndrome (HPS) is a clinicopathologic entity characterized by increased proliferation and activation of benign macrophages with hemophagocytosis throughout the reticuloendothelial system. Uncontrolled T-lymphocyte activation is responsible for increased TH1 cytokines secretion such as IFN-γ, IL-12 and IL-18 that promotes macrophage activation. Genetic defects specific for cytotoxic T lymphocytes (CTL) and natural killer (NK) cells have been identified in patients with primary HPS that are responsible for altered cell death and apoptosis induction or target killing. HPS may be secondary to malignancy, infection or autoimmune disease, and mechanisms involved are poorly understood. However, in adult-onset Still''s disease, juvenile chronic arthritis and probably systemic lupus erythematosus, IL-18 might play a role in initiating macrophage activation. [Copyright &y& Elsevier]

The toxic effects of air pollution are widely documented. In recent years, however, there has been an increasing interest in the study of the health effects of particulate matter (PM), a previously unexplored constituent of urban air pollution. Exposure to increased levels of PM of respirable size is strongly and consistently associated with increased cardiopulmonary morbidity and mortality. Conversely, improved air quality appears to correlate with decreased mortality. Particulate matter is a mixture of inorganic and organic components that vary in size, origin, and composition. The mechanisms of PM health effects are still poorly understood. However, studies in cellular and animal models suggest a variety of possible mechanisms including direct effects of particle components on the intracellular sources of reactive oxygen species (ROS), indirect effects due to pro-inflammatory mediators released from PM-stimulated macrophages, and neural stimulation after particle deposition in the lungs. The involvement of ROS in each one of these possible pathways is discussed. [Copyright &y& Elsevier]

Activation of the innate immune response involves recognition of the infectious non-self and subsequent activation of cellular and humoral reactions. Insect humoral reactions depend on primary and secondary responses. The primary response is mediated by the activation of cascades of constitutive proteins present in the hemolymph, such as the prophenoloxidase (proPO) cascade. The secondary response requires transcriptional activation of defense proteins, such as the induction of antimicrobial peptides. Drosophila possess specific mechanisms to discriminate between microbes and respond to infection by inducing the appropriate reactions. In innate immunity, pathogen-associated molecular patterns are recognized. The mechanisms for microbial recognition in Drosophila, however, are largely unknown. Recent data suggest that, in insect immunity, diverse peptidoglycan recognition protein members are involved in distinguishing between invading bacteria and activation of appropriate immune reactions. [Copyright &y& Elsevier]

Tumor necrosis factor (TNF) is a key mediator in regulating the inflammatory response. Previously two TNF genes have been cloned and sequenced from rainbow trout, Oncorhynchus mykiss. In this study, the mature peptides of the two TNF molecules were produced in bacteria, purified under native conditions and their bioactivities evaluated in vitro. Both trout rTNF1 and rTNF2 induced gene expression of a number of proinflammatory factors including IL1β, TNF1, TNF2, IL8 and COX2 in freshly isolated head kidney leucocytes and the macrophage cell line RTS11. The stimulatory doses of both rTNFs were ≥10 ng/ml. Moreover, leucocyte migration and phagocytic activity were enhanced in vitro by the rTNFs in a dose dependent manner. Western blot analysis revealed the presence of multiple forms of rTNF structures including monomeric, dimeric and trimeric forms, suggesting that formation of a homotrimeric structure may be essential for the TNF bioactivities. [Copyright &y& Elsevier]

Recently it has been found that osteoclast differentiation is induced by tumor necrosis factor (TNF)-α. Interleukin (IL)-4 was reported to suppress osteoclast differentiation and bone resorption. However, no study has investigated the effect of IL-4 on TNF-α-induced osteoclast formation. In this study, we investigated whether IL-4 inhibits TNF-α-mediated osteoclast formation in mouse bone marrow derived macrophages (BMM). First, IL-4 suppresses RANKL-induced osteoclast formation and bone resorption. Next, when BMM were cultured with TNF-α, osteoclast-like cells were formed. When they were cultured with both TNF-α and IL-4, osteoclast formation and bone resorption was suppressed by IL-4 in a dose-dependent manner. It has been recently found that TNF-α and RANKL synergistically promote osteoclastogenesis. Finally, we investigated whether IL-4 had the ability to inhibit synergistic TNF-α and RANKL-induced osteoclastogenesis, with the result that it effectively inhibited the synergistic osteoclast formation in a dose-dependent manner. We conclude that IL-4 can strongly inhibit osteoclast formation that is related to both physiological bone resorption induced by RANKL and pathological bone resorption induced by TNF-α. [Copyright &y& Elsevier]

Background: Sarcoidosis is a multi-system disease of unknown etiology. Interferons (IFN) have been implicated in its pathogenesis. The objective of this study was to examine the causal relationship between sarcoidosis and IFN therapy. Methods: Patients admitted for sarcoidosis (n=60) were reviewed for a history of IFN therapy. In addition, all cases of sarcoidosis in a cohort of hepatitis C-infected patients treated with IFN-α (n=1159) were analyzed. Results: Five patients with prior IFN-α therapy and sarcoidosis were identified; an additional 23 have been reported in the literature. The median age of the 28 reported patients was 50 years, 16 (57%) were female, and 16 (57%) had isolated cutaneous disease. The median time to diagnosis was 4 months (range 1–16 months) following the introduction of IFN. A remission was observed in all patients with adequate follow-up (n=27): 15 (53%) upon dosage reduction or IFN discontinuation, seven (25%) with systemic corticosteroids, three (11%) with topical treatment, and three (11%) despite ongoing IFN therapy. Relapse was observed in both of the patients rechallenged with IFN. Conclusions: There is a potential causal relationship between IFN therapy and sarcoidosis. In patients with sarcoidosis in the setting of IFN therapy, the majority respond to IFN withdrawal or dosage reduction; however, some require corticosteroid therapy. [Copyright &y& Elsevier]

Dehydrocrotonin (DHC) is a diterpene lactone obtained from Croton cajucara (Sacaca). Dimethylamide-crotonin (DCR), a DHC derivative, has a similar inhibitory effect on leukemic HL60 cells than its parent compound evaluated by different endpoints of cytotoxicity. No cytotoxicity or morphological alterations associated with apoptosis were detected in human peripheral blood mononuclear cells (PBMC) after treatment with up to 400 μM DCR in presence of phytohemaglutinin (5 μg/ml). Based on morphological changes and the pattern of DNA fragmentation, DHC and DCR were found to induce apoptosis and terminal differentiation (assessed by nitro blue tetrazolium reduction) in HL60 cells, but these compounds did not show any toxic effect in PBMC. Thus, DCR and DHC inhibit HL60 cell growth in vitro partly by inducing apoptosis and cell differentiation, but does not cause serious damage to immune cells according to our experimental conditions. [Copyright &y& Elsevier]

LIGHT is a new member of the tumor necrosis factor superfamily, which binds to lymphotoxin β receptor, herpes virus entry mediator, or TR6. This work was carried out to elucidate the molecular mechanism of LIGHT-sensitized, interferon gamma (IFNγ)-mediated apoptosis of MDA-MB-231 cells. It was revealed that LIGHT treatment resulted in down-regulation of anti-apoptosis Bcl-2 family member: Bcl-2, Bcl-XL, Bag-1, and Mcl-1; up-regulation of pro-apoptosis Bcl-2 family member: Bak and Ser (112)-phosphor-Bad; down-regulation of pro-apoptosis Bcl-2 member Bax; the other pro-apoptosis member Bid remains unaltered. LIGHT treatment also resulted in activation of caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, DFF45, and PARP. However, caspase activation and caspase activity, especially caspase-3 activity, is not required for LIGHT-induced apoptosis of MDA-MB-231 cells, since caspase-3 inhibitor, benzyloxycarbonyl–Asp–Glu–Val–Asp-fluoromethylketone, and a broad range caspase inhibitor, benzyloxycarbonyl–val–ala–asp–fluoromethylketone failed to block the apoptosis induced by LIGHT and IFNγ in MDA-MB-231 cells. In summary, LIGHT-sensitized IFNγ-mediated apoptosis of MDA-MB-231 cells is probably through down-regulation of anti-apoptosis Bcl-2 family members; it could be caspase (especially caspase-3)-independent, even though extensive caspase activation was observed. [Copyright &y& Elsevier]

Background/Aims: Insulin- and insulin growth factor-1 stimulated signaling through the insulin receptor substrate-1 (IRS-1) promotes hepatocellular proliferation and survival. IRS-1 over-expression in transgenic (Tg) mouse livers caused constitutive activation of Erk mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) resulting in significantly increased levels of DNA synthesis and larger hepatic masses relative to non-transgenic (non-Tg) littermates. However, the livers eventually ceased to grow but remained approximately 25% larger than non-Tg livers. We hypothesized that this growth homeostasis was achieved by parallel activation of pro-apoptosis pathways.Methods: Since Fas-mediated apoptosis is a common mechanism of hepatocyte destruction, we investigated the potential role of Fas receptor as a regulator of hepatic mass in IRS-1 transgenic mice.Results: Significantly increased Fas-receptor levels were detected in the livers of IRS-1 Tg compared to non-Tg mice by Western blot analysis. Functional activation of Fas-receptor in IRS-1 Tg livers was demonstrated by increased hepatocellular apoptosis caused by intravenous injection of anti-Fas (Jo-2).Conclusions: These findings suggest that the increased growth caused by IRS-1 over-expression is balanced by constitutive activation of pro-death mechanisms. Failure of the IRS-1 Tg mice to develop liver cancer may be due to preservation of pro-growth, pro-death homeostasis mechanisms. [Copyright &y& Elsevier]

Background/Aims: When a loss of hepatic mass occurs, the expression of a large number of genes is either induced or altered, accompanying hepatocyte proliferation. In the present study, we made an in-house cDNA microarray containing 4608 elements (Liver chip), and analyzed extensively gene expression profiles of the regenerating liver after 70% partial hepatectomy (PHx) in rats.Methods: RNAs were prepared from three rat livers at each time point (taken at 0, 6, 12, 18, 24, 48, 72 h, and 1 week after PHx). Using the liver chip, we performed large-scale analysis of gene expression during liver regeneration. Elements either up- or down-regulated more than twofold at one or more time points were selected.Results: Among the 4608, 382 were identified. Using cluster analysis, we found great similarity between gene-expression profiles at 12 and 18 h after PHx as well as between 48 and 72 h after PHx. We also found that there are at least six distinct temporal patterns of gene expression in the regenerating rat liver after PHx.Conclusions: These results indicated that microarray analysis is a powerful approach for monitoring molecular events in the regenerating liver. [Copyright &y& Elsevier]

Degradation of IkappaB (IκB) is a key step for nuclear factor-kappaB (NF-κB)-induced transcription of certain proinflammatory genes, including inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. We selected seven chemopreventive agents and examined their effects on combined lipopolysaccharide- and interferon-γ-induced IκB degradation in RAW264.7 murine macrophages. IκB degradation was notably suppressed by 1′-acetoxychavicol acetate (ACA), zerumbone (ZER), and benzylisothiocyanate (BITC), however, not by auraptene (AUR), while the suppressive potencies of nobiletin (NOB), genistein (GEN), and resveratrol (RES) were low, but significant. These results suggest that ACA, ZER, and BITC suppress iNOS/COX-2 gene expression mainly by attenuating IκB degradation, while other chemopreventive agents use alternative pathway(s) to suppress the expression of proinflammatory genes. [Copyright &y& Elsevier]

Background/Aims: Idiosyncratic immune response to drugs causes two types of liver injury, cholestasis or hepatitis. However, the underlying immune mechanisms of drug-induced liver injury are presently unclear.Methods: We examined the cytokine production of peripheral blood mononuclear cells (PBMCs) from 17 patients with drug-induced liver injury and healthy controls during their incubation with and without the drug by flow cytometry. We also analyzed the cytokine production in PBMCs from eight patients after stimulation with the drug-pulsed HepG2 lysates to examine the possibility that the drug or its metabolites conjugated with a putative molecule derived from HepG2 cells might be more immunogenic.Results: Among several cytokines produced by the drug or the drug-pulsed HepG2 lysates, interferon-γ production from CD8+ cells was associated with hepatocellular injury, and tumor necrosis factor-α production from CD14+ cells was with cholestasis. Especially, the latter was apparent when the drug-pulsed HepG2 lysates were used as stimulants, suggesting that a complex consist of the drug, or its metabolite, and a putative molecule derived from HepG2 cells might be more immunogenic than the drug itself.Conclusions: The analysis of intracytoplasmic cytokine in PBMCs after stimulation with the drug or the drug-pulsed HepG2 lysates is useful to analyze the immune mechanism underlying drug-induced liver injury. [Copyright &y& Elsevier]