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The entorhinal cortex-dentate gyrus circuit is centrally involved in memory processing conveying to the hippocampus spatial and nonspatial context information via, respectively, medial and lateral perforant path (MPP and LPP) excitatory projections onto dentate granule cells (GCs). Here, we review work of several years from our group showing that astrocytes sense local synaptic transmission and exert in turn a presynaptic control at PP-GC synapses. Modulation of neurotransmitter release probability by astrocytes sets basal synaptic strength and dynamic range for long-term potentiation of PP-GC synapses. Intriguingly, this astrocyte control is circuit-specific, being present only at MPP-GC (not LPP-GC) synapses, which selectively express atypical presynaptic N-methyl-D-aspartate receptors (NMDAR) suitable to activation by astrocyte-released glutamate. Moreover, the astrocytic control is peculiarly dependent on the cytokine TNFα, which at constitutive levels acts as a gating factor for the astrocyte signaling. During inflammation/infection processes, increased levels of TNFα lead to uncontrolled astrocyte glutamate release, altered PP-GC circuit processing and, ultimately, impaired contextual memory performance. The TNFα-dependent pathological switch of the synaptic control from astrocytes and its deleterious consequences are observed in animal models of HIV brain infection and multiple sclerosis, conditions both known to cause cognitive disturbances in up to 50% of patients. The review also discusses open issues related to the identified astrocytic pathway: its role in contextual memory processing, potential damaging role in Alzheimer's disease, the existence of vesicular glutamate release from DG astrocytes, and the possible synaptic-like connectivity between astrocytic output sites and PP receptive sites.

Background Autism, a childhood behavioral disorder, belongs to a large suite of diseases, collectively referred to as autism spectrum disorders (ASD). Though multifactorial in etiology, approximately 10% of ASD are associated with atopic dermatitis (AD). Moreover, ASD prevalence increases further as AD severity worsens, though these disorders share no common causative mutations. We assessed here the link between these two disorders in the standard, valproic acid mouse model of ASD. In prior studies, there was no evidence of skin involvement, but we hypothesized that cutaneous involvement could be detected in experiments conducted in BALB/c mice. BALB/c is an albino, laboratory-bred strain of the house mouse and is among the most widely used inbred strains used in animal experimentation. Methods We performed our studies in valproic acid (VPA)-treated BALB/c hairless mice, a standard mouse model of ASD. Mid-trimester pregnant mice received a single intraperitoneal injection of either valproic acid sodium salt dissolved in saline or saline alone on embryonic day 12.5 and were housed individually until postnatal day 21. Only the brain and epidermis appeared to be affected, while other tissues remain unchanged. At various postnatal time points, brain, skin and blood samples were obtained for histology and for quantitation of tissue sphingolipid content and cytokine levels. Results AD-like changes in ceramide content occurred by day one postpartum in both VPA-treated mouse skin and brain. The temporal co-emergence of AD and ASD, and the AD phenotype-dependent increase in ASD prevalence correlated with early appearance of cytokine markers (i.e., interleukin [IL]-4, 5, and 13), as well as mast cells in skin and brain. The high levels of interferon (IFN)γ not only in skin, but also in brain likely account for a significant decline in esterified very-long-chain N-acyl fatty acids in brain ceramides, again mimicking known IFNγ-induced changes in AD. Conclusion Baseline involvement of both AD and ASD could reflect concurrent neuro- and epidermal toxicity, possibly because both epidermis and neural tissues originate from the embryonic neuroectoderm. These studies illuminate the shared susceptibility of the brain and epidermis to a known neurotoxin, suggesting that the atopic diathesis could be extended to include ASD.

Innate immunity is critical for host defence against pathogen and environmental challenge and this involves the production and secretion of immune mediators, such as antimicrobial peptides and pro-inflammatory cytokines. However, when dysregulated, innate immunity can contribute to multifactorial diseases, including inflammatory rheumatic disorders, type 2 diabetes, cancer, neurodegenerative and cardiovascular diseases and even septic shock. During an innate immune response, antimicrobial peptides and cytokines are trafficked via Rab11 multivesicular endosomes, and then sorted into Rab11 vesicles for traffic to the plasma membrane and secretion. In this study, a cyclin-dependent kinase inhibitor CDKI-73 was used to determine its effect on the innate immune response, based on previously identified targets for this compound. Our results showed that CDKI-73 inhibited the delivery of Rab11 vesicles to the plasma membrane, resulting in the accumulation of large multivesicular Rab11 endosomes near the cell periphery. In addition to the effect on endosome delivery, CDKI-73 down-regulated the amount of innate immune cargo, including the antimicrobial peptide Drosomycin and pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF&alpha, ). We concluded that CDKI-73 has the potential to regulate the delivery and secretion of certain innate immune cargo, which could be used to control inflammation.

Minimal hepatic encephalopathy is associated with changes in the peripheral immune system which are transferred to the brain, leading to neuroinflammation and thus to cognitive and motor impairment. Mechanisms by which changes in the immune system induce cerebral alterations remain unclear. Extracellular vesicles (EVs) seem to play a role in this process in certain pathologies. The aim of this work was to assess whether EVs play a role in the induction of neuroinflammation in cerebellum and motor incoordination by chronic hyperammonemia. We characterized the differences in protein cargo of EVs from plasma of hyperammonemic and control rats by proteomics and Western blot. We assessed whether injection of EVs from hyperammonemic to normal rats induces changes in neuroinflammation in cerebellum and motor incoordination similar to those exhibited by hyperammonemic rats. We found that hyperammonemia increases EVs amount and alters their protein cargo. Differentially expressed proteins are mainly associated with immune system processes. Injected EVs enter Purkinje neurons and microglia. Injection of EVs from hyperammonemic, but not from control rats, induces motor incoordination, which is mediated by neuroinflammation, microglia and astrocytes activation and increased IL-1b, TNF&alpha, its receptor TNFR1, NF-kB in microglia, glutaminase I, and GAT3 in cerebellum. Plasma EVs from hyperammonemic rats carry molecules necessary and sufficient to trigger neuroinflammation in cerebellum and the mechanisms leading to motor incoordination.

Background: Annexin A1 (ANXA1) and Translocator Protein-18KDa (TSPO) down-regulate neuroinflammation. We investigated the role of recombinant ANXA1 (rANXA) on TSPO functions on Toll Like Receptor (TLR) activated microglia. Methods: BV-2 cells (murine microglia), were stimulated by E. coli Lipopolysaccharide (LPS) and treated with rANXA1 in order to measure TSPO expression and inflammatory parameters. Anti-sense ANXA1 and TLR4 and TSPO shRNA, as well as pharmacological treatments, were employed to assess the mechanisms involved. Results: LPS-stimulated BV-2 cells caused overexpression of TSPO, which was inhibited by: pharmacological blockade of TLR4 or TLR4 mRNA silencing; inhibition of myeloid differentiation primary response gene 88 (MyD88) dimerization; or blocking of nuclear factor kappa B (NF-kappa B) activation. rANXA1 treatment impaired LPS-induced TSPO upregulation by down-modulating MyD88 and NF-kappa B signaling; the effect was abolished by WRW4, an antagonist of formyl peptide receptor 2 (FPR2). rANXA1 treatment also downregulated interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) secretion in LPS-stimulated BV-2 cells. TSPO knockdown in BV-2 cells augmented LPS-induced TNFa secretion and abolished the inhibitory effect of rANXA1 on TNF alpha secretion evoked by LPS. Conclusions: exogenous ANXA1 down-modulates LPS-induced TSPO via MyD-88/NF-kappa B pathways, and constitutive TSPO is pivotal for the control of ANXA1 on TNF alpha secretion. TSPO actions may be involved with the mechanisms of ANXA1 on inflammatory brain diseases.

// Dino Kocijancic 1 , Sara Leschner 1 , Sebastian Felgner 1 , Ronja-Melinda Komoll 1 , Michael Frahm 1 , Vinay Pawar 1 , Siegfried Weiss 1, 2 1 Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany 2 Institute of Immunology, Medical School Hannover, Hannover, Germany Correspondence to: Dino Kocijancic, email: dino.kocijancic@helmholtz-hzi.de Keywords: TNF alpha, LPS, cancer immune therapy, salmonella, bacteria mediated tumor therapy Received: June 15, 2016 Accepted: March 29, 2017 Published: April 06, 2017 ABSTRACT The potential of bacteria-mediated tumor therapy (BMTT) is highlighted by more than a century of investigation. Attenuated Salmonella has prevailed as promising therapeutic agents. For BMTT - categorized as an immune therapy - the exact contribution of particular immune reactions to the therapeutic effect remains ambiguous. In addition, one could argue for or against the requirement of bacterial viability and tumor targeting. Herein we evaluate the isolated therapeutic efficacy of purified LPS and TNF-α, which together account for a dominant immunogenic pathway of gram negative bacteria like Salmonella . We show that therapeutic efficacy against CT26 tumors does not require bacterial viability. Analogous to viable Salmonella SL7207, tumor regression by a specific CD8 + T cell response can be induced by purified LPS or recombinant TNF-α (rTNF-α). Conversely, therapeutic effects against RenCa tumors were abrogated upon bacterial avitalization and limited using isolated adjuvants. This argues for an alternative mechanistic explanation for SL7207 against RenCa that depends on viability and persistence. Unable to boost bacterial therapies by co-injection of rTNF-α suggested therapeutic effects along this axis are exhausted by the intrinsic adjuvanticity of bacteria alone. However, the importance of TNF-α for BMTT was highlighted by its support of tumor invasion and colonization in concert with lower infective doses of Salmonella . In consideration, bacterial therapeutic effectiveness along the axis of LPS and TNF-α appears limited, and does not offer the necessary plasticity for different tumors. This emphasizes a need for recombinant strengthening and vehicular exploitation to accommodate potency, plasticity and distinctiveness in BMTT.

Mechanisms thought of as homeostatic must exist to maintain neuronal activity in the brain within the dynamic range in which neurons can signal. Several distinct mechanisms have been demonstrated experimentally. Three mechanisms that act to restore levels of activity in the primary visual cortex of mice after occlusion and restoration of vision in one eye, which give rise to the phenomenon of ocular dominance plasticity, are discussed. The existence of different mechanisms raises the issue of how these mechanisms operate together to converge on the same set points of activity. This article is part of the themed issue ‘Integrating Hebbian and homeostatic plasticity’.

Tumor necrosis factor alpha (TNFα) is an adipokine involved in the regulation of cell differentiation and lipid metabolism, but its specific role has not been clearly understood. We validated a hypothesis that loss of TNFα function would inhibit Wnt/β-catenin signaling and accelerate adipogenesis in adolescent genetic obese mice. Epididymal white adipose tissues (eWAT) from TNFα deficient (TNFα−/−), leptin receptor deficient (db/db) and double gene mutant (db/db/TNFα−/−, DT) male mice were used for comparative analysis of key molecules in Wnt/β-catenin signaling and adipogenic markers by qRT-PCR and western blot techniques. Compared with TNFα−/− and WT mice of 28 days old, an obese trait was observed in both db/db and DT mice, while the latter showed more significant body weight gain and eWAT hypertrophy. The mRNA level of key molecules in Wnt/β-catenin pathway was reduced in both obese groups, while the DT group was the lowest. Expression of adipocyte-specific genes was up-regulated during obese development in the two obese groups, while the DT group revealed more correlation than that of db/db group. At the protein level, a down regulation of Wnt10b and β-catenin in obese eWAT showed similar tendency with that of mRNA level. Compared with the lean groups, the levels of adiponectin and PPARγ2 for the obese groups were down-regulated at 21-day-old age, while they were elevated at older age. Our results suggested that deficiency in TNFα inhibited Wnt/β-catenin signaling of the obese eWAT and up-regulated expression of adipokines, and accelerated adipogenesis in genetic obese mice on a chow diet. Electronic supplementary material The online version of this article (doi:10.1007/s11010-014-1987-5) contains supplementary material, which is available to authorized users.

alper, murat/0000-0001-7069-0623; Uzun, Feyzahan/0000-0002-3050-0714 WOS: 000338073500001 PubMed: 25165412 Purpose. We investigated the effect of eritoran, a Toll-like receptor 4 antagonist, on retinochoroidal inflammatory damage in an endotoxin-induced inflammatory rat model. Methods. Endotoxin-induced inflammatory model was obtained by intraperitoneal injection of 1.5mg/kg lipopolysaccharide (LPS). Group 1 had control rats; in groups 2-3 LPS and 0.5mg/kg sterile saline were injected; and in groups 4-5 LPS and 0.5mg/kg eritoran were injected. Blood samples were taken and eyes were enucleated after 12 hours (h) (groups 2 and 4) or 24 hours (Groups 3 and 5). Tumor necrosis factor-alpha (TNF-alpha) and malondialdehyde (MDA) levels in the serum and retinochoroidal tissue and nuclear factor kappa-B (NF kappa B) levels in retinochoroidal tissue were determined. Histopathological examination was performed and retinochoroidal changes were scored. Results. Eritoran treatment resulted in lower levels of TNF-alpha, MDA, and NF kappa B after 12 h which became significant after 24 h. Serum TNF-alpha and retinochoroidal tissue NF kappa B levels were similar to control animals at the 24th h of the study. Eritoran significantly reversed histopathological damage after 24 h. Conclusions. Eritoran treatment resulted in less inflammatory damage in terms of serum and retinochoroidal tissue parameters. Gazi University Scientific Projects and Research CenterGazi University [01/2007-75] Gazi University Scientific Projects and Research Center supported this work with a Project no. 01/2007-75.

Background Early-life stress and exposure to stressful stimuli play a major role in the development of chronic widespread pain in adults. However, how they interact in chronic pain syndromes remains unclear. Methods Dams and neonatal litters were submitted to a restriction of nesting material (neonatal limited bedding [NLB]) for 1 week. As adults, these rats were exposed to a painless sound stress protocol. The involvement of sympathoadrenal catecholamines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) in nociception was evaluated through behavioral and enzyme-linked immunosorbent assays, surgical interventions, and intrathecal antisense treatments. Results Adult NLB rats exhibited mild muscle hyperalgesia, which was markedly aggravated by sound stress (peaking 15 days after exposure). Adrenal medullectomy did not modify hyperalgesia in NLB rats but prevented its aggravation by sound stress. Sustained administration of epinephrine to NLB rats mimicked sound stress effect. Intrathecal treatment with antisense directed to IL-6 receptor subunit gp130 (gp130), but not to tumor necrosis factor receptor type 1 (TNFR1), inhibited hyperalgesia in NLB rats. However, antisense against either gp130 or TNFR1 inhibited sound stress-induced enhancement of hyperalgesia. Compared with control rats, NLB rats exhibit increased plasma levels of IL-6 but decreased levels of TNFα, whereas sound stress increases IL-6 plasma levels in control rats but not in NLB rats. Conclusions Early-life stress induces a persistent elevation of IL-6, hyperalgesia, and susceptibility to chronic muscle pain, which is unveiled by exposure to stress in adults. This probably depends on an interaction between adrenal catecholamines and proinflammatory cytokines acting at muscle nociceptor level.

Tumour necrosis factor alpha (TNFα) is a cytokine which is pivotal in the inflammatory response. Blockade of TNFα has been shown to be effective in a number of human autoimmune diseases, including rheumatoid arthritis, raising the question whether this approach may be effective in inflammatory kidney disease, such as ANCA-associated vasculitis (AAV). In AAV, there is considerable evidence for the role of TNFα in the pathophysiology of disease, including increased expression of TNFα mRNA in leucocytes and in renal tissue. Importantly, TNFα can induce leucocyte cell membrane expression of the autoantigens involved in vasculitis (proteinase 3 [PR3] and myeloperoxidase [MPO]), thus priming cells for the effects of ANCA. In rodent models of anti-GBM disease (nephrotoxic nephritis), TNF enhances glomerular injury and TNF blockade using soluble TNFαreceptor or anti-TNFα antibody ameliorates disease. Mice deficient in TNFα are protected from nephrotoxic nephritis and this effect is dependent mainly on intrinsic renal cells. A mouse model of anti-MPO antibody induced glomerulonephritis is enhanced by LPS, and this effect is blocked by anti-TNFα antibody. In a rat model of AAV induced by MPO (experimental autoimmune vasculitis) anti-TNFα antibody improves renal pathology and also reduces leucocyte transmigration, as shown by intravital microscopy. In clinical studies, the Wegener’s Granulomatosis Etanercept Trial (WGET) showed no benefit of additional etanercept versus standard therapy. However, there are several reasons why the results of the WGET study do not rule out the use of anti-TNFα antibody in acute renal AAV, including the study design and the considerable biological differences between the effects of etanercept and anti-TNFα antibody. There are several clinical studies demonstrating a response to anti-TNFα antibody in patients with AAV refractory to conventional treatment, and in some of these, the addition of anti-TNFα antibody was the only change in treatment. We suggest that further investigation of TNFα blockade in AAV is warranted.

The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4 and CD8 T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cells Fil: Zirger, Jeffrey M.. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Puntel, Mariana. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bergeron, Josee. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Wibowo, Mia. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos Fil: Moridzadeh, Rameen. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos Fil: Bondale, Niyati. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Barcia, Carlos. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Kroeger, Kurt M.. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos Fil: Liu, Chunyan. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos Fil: Castro, Maria Graciela. University of California at Los Angeles. School of Medicine; Estados Unidos. Cedars Sinai Medical Center; Estados Unidos. University of Michigan; Estados Unidos Fil: Lowenstein, Pedro R.. Cedars Sinai Medical Center; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados Unidos. University of Michigan; Estados Unidos

This second section of the first ECCO pathogenesis workshop on anti-TNF therapy failures in inflammatory bowel diseases addresses the biological roles of TNFα and the effects and mechanisms of action of TNFα antagonists. Mechanisms underlying their failure, including induction of TNF-independent inflammatory pathways and phenomena of paradoxical inflammation are discussed.

The liver plays a central role in the transformation and degradation of endogenous and exogenous chemicals, and in the removal of unwanted cells such as damaged, genetically mutated and virus-infected cells. Because of this function, the liver is susceptible to toxicity caused by the products generated during these natural occurrences. Hepatocyte death is the major feature of liver injury. In response to liver injury, specific intracellular processes are initiated to maintain liver integrity. Inflammatory cytokines including tumor necrosis factor (TNF)α and interleukin-6 (IL-6) are key mediators of these processes and activate different cellular response such as proliferation, survival and death. TNFα induces specific signaling pathways in hepatocytes that lead to activation of either pro-survival mediators or effectors of cell death. Whereas activation of transcription factor NF-κB promotes survival, c-Jun N-terminal kinases (JNKs) and caspases are strategic effectors of cell death in the TNFα-mediated signaling pathway. This review summarizes recent advances in the mechanisms of TNFα-induced hepatotoxicity and suggests that NF-κB plays a protective role against JNK-induced hepatocyte death. Identification of the mechanisms regulating interplay between the NF-κB and JNK pathways is required in the search for novel targets for the treatment of liver disease, including hepatitis and hepatocellular carcinoma.

Synovial inflammation, angiogenesis and joint degradation are the hallmarks of inflammatory arthritis progression. Angiostatic targeting is an extensively studied potential therapeutic option for inflammatory arthritis,. Studies have confirmed that surface-active phospholipids (SAPLs), predominantly phosphatidylcholines (PCs), are responsible for the lubricating properties of lubricin in joints. Paclitaxel, a potent antineoplastic agent in cancer chemotherapy, has been shown to inhibit several processes associated with arthritis development such as angiogenesis, neutrophil activation and collagenase expression but is limited by systemic toxicity. This study was aimed at designing a surface-active phospholipid mimetic nanocarrier system and assessing its efficacy for intra-articular delivery of paclitaxel in rat joints. Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes were prepared using a thin-film hydration method and characterized for size, morphology, drug encapsulation and in vitro release. DPPC liposomes of a size of 311 +/- 57 nm and 92 +/- 0.6% paclitaxel encapsulation were developed. In vitro release studies showed a short initial burst phase and a sustained release profile with a cumulative release of 18 +/- 0.36% of the drug by 60 h in phosphate-buffered saline (PBS). The efficacy of the intra-articular formulation was evaluated in antigen-induced arthritic rat models and compared with direct injections of paclitaxel. After a 28-day period, intra-articular paclitaxel delivered in liposomes led to a significant improvement in gait scores and synovial inflammation in rats compared to the control, as seen in histopathology studies. Reduction in inflammation in the experimental group was confirmed by evaluating TNF alpha levels in serum samples. This study suggests feasibility of using surface-active phospholipid based carriers for local, intra-articular therapy of paclitaxel in arthritis.

Genetic factors appear to be important in the restenotic process after percutaneous coronary intervention (PCI), as well as in inflammation, a pivotal factor in restenosis. TNFα, a key regulator of inflammatory responses, may exert critical influence on the development of restenosis after PCI. The GENetic DEterminants of Restenosis (GENDER) project included 3104 patients who underwent a successful PCI. Systematic genotyping for six polymorphisms in the TNFα gene was performed. The role of TNFα in restenosis was also assessed in ApoE*3-Leiden mice, TNFα knockout mice, and by local delivery of a TNFα biosynthesis inhibitor, thalidomide. The -238G-1031T haplotype of the TNFα gene increased clinical and angiographic risk of restenosis (P=0.02 and P=0.002, respectively). In a mouse model of reactive stenosis, arterial TNFα mRNA was significantly time-dependently up-regulated. Mice lacking TNFα or treated locally with thalidomide showed a reduction in reactive stenosis (P=0.01 and P=0.005, respectively). Clinical and preclinical data indicate that TNFα plays an important role in restenosis. Therefore, TNFα genotype may be used as a risk marker for restenosis and may contribute to individual patient screening prior to PCI in clinical practice. Inhibition of TNFα may be an anti-restenotic target strategy. ©FASEB. Chemicals / CAS: thalidomide, 50-35-1; RNA, Messenger; Thalidomide, 50-35-1; Tumor Necrosis Factor-alpha

Pro-inflammatory cytokines play an important role in both recruitment and activation of leukocytes migrating into tissues in response to invading pathogens. In this study the production of pro-inflammatory cytokines, determined by ELISA assays, and the recruitment of leukocytes into the lungs of BALBIc mice infected with Paracoccidioides brasiliensis conidia were evaluated during the early stages of infection. The results showed that infected mice had a significant increase in leukocytes in the lung during the first 4 days with a peak at day 2 post-challenge; infiltrates were composed mainly of polymorphonuclear neutrophils (PMN). Pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-a), interleukin (IL) 6, IL-1P and macrophage inflammatory protein (MIP) 2 were produced at elevated levels during the first 4 days post-challenge, but only in pulmonary samples and not in sera. Additionally, during the early stages of infection, overall weight loss was recorded in infected mice. These results suggest that pro-inflammatory cytokines could be responsible for the recruitment of leukocytes into the lung during the early stages of 19 brasiliensis infection. In addition, both pro-inflammatory cytokine production and leukocyte recruitment may participate in the control of infection by influencing the organization of the immune response in the host exposed to P brasiliensis conidia. COL0013709

Lipopolysaccharide (LPS) may cause sepsis when it enters the blood circulation. The toxic moiety of LPS is the well-preserved lipid A part. Lipid A contains two phosphate groups attached to diglucosamine, which are crucial for the many biological activities of LIPS. In previous studies we found that alkaline phosphatase (AP) was able to dephosphorylate LPS. To test whether LPS-dephosphorylation can be used for intervention during sepsis, we investigated the effects of Salmonella minnesota Re 595 LIPS and its dephosphorylated counterpart monophosphoryl lipid A (MPLA) on macrophage activation in vivo and in vitro. Exposure of RAW264.7 cells to LPS induced high levels of tumor necrosis factor (TNFalpha) and nitric oxide (NO), whereas MPLA elicited no response. LPS in vivo induced a significant rise in TNFa levels in mice and an enhanced inflammatory cell influx in the lung, whereas MPLA did not. Having shown the relevance of this particular phosphate group of LIPS, we subsequently explored the ILPS-dephosphorylating ability of AP in different tissues, and the effect of AP administration in mice challenged with LIPS. Histochemical data show that AP dephosphorylated native LPS in all tissues examined, whereas MPLA was not dephosphorylated. When mice received AP immediately after the LIDS challenge, the survival rate was 100%, over 57% in the control group. We conclude that the enzymatic removal of phosphate groups from LPS by AP represents a crucial detoxification reaction, which may provide a new strategy to treat LPS-induced diseases like sepsis.

In aged mice, new B-cell development is diminished and the antibody repertoire becomes more autoreactive. Our studies suggest that (i) apoptosis contributes to reduced B lymphopoiesis in old age and preferentially eliminates those B-cell precursors with higher levels of the surrogate light chain (SLC) proteins (λ5/VpreB) and (ii) λ5(low) B-cell precursors generate new B cells which show increased reactivity to the self-antigen/bacterial antigen phosphorylcholine (PC). Pro-B cells in old bone marrow as well as pro-B cells from young adult λ5-deficient mice are resistant to cytokine-induced apoptosis (TNFα; TGFβ), indicating that low λ5 expression in pro-B cells is sufficient to cause increased survival. Transfer of TNFα-producing 'age-associated B cells' (ABC; CD21/35(-) CD23(-)) or follicular (FO) B cells from aged mice into RAG-2 KO recipients led to preferential loss of λ5(high) pro-B cells, but retention of λ5(low), apoptosis-resistant pro-B cells. In old mice, there is increased reactivity to PC in both immature bone marrow B cells and mature splenic FO B cells. In young mice, absence of λ5 expression led to a similar increase in PC reactivity among bone marrow and splenic B cells. We propose that in old age, increased apoptosis, mediated in part by TNFα-producing B cells, results in preferential loss of SLC(high) pro-B cells within the bone marrow. Further B-cell development then occurs via an 'SLC(low)' pathway that not only impairs B-cell generation, but promotes autoreactivity within the naïve antibody repertoires in the bone marrow and periphery.

We previously showed that peripheral noxious input after spinal cord injury (SCI) inhibits beneficial spinal plasticity and impairs recovery of locomotor and bladder functions. These observations suggest that noxious input may similarly affect the development and maintenance of chronic neuropathic pain, an important consequence of SCI. In adult rats with a moderate contusion SCI, we investigated the effect of noxious tail stimulation, administered one day after SCI, on mechanical withdrawal responses to von Frey stimuli from 1 to 28 days, post-treatment. In addition, because the pro-inflammatory cytokine tumor necrosis factor α (TNFα) is implicated in numerous injury-induced processes including pain hypersensitivity, we assessed the temporal and spatial expression of TNFα, TNF receptors, and several downstream signaling targets after stimulation. Our results showed that unlike sham surgery or SCI only, nociceptive stimulation following SCI induced mechanical sensitivity by 24 hours. These behavioral changes were accompanied by increased expression of TNFα. Cellular assessments of downstream targets of TNFα revealed that nociceptive stimulation increased the expression of caspase 8 and the active subunit (12 kDa) of caspase 3 at a time point consistent with the onset of mechanical allodynia, indicative of active apoptosis. In addition, immunohistochemical analysis revealed distinct morphological signs of apoptosis in neurons and microglia at 24 hours post-stimulation. Interestingly, expression of the inflammatory mediator NFκB was unaltered by nociceptive stimulation. These results suggest that noxious input caudal to the level of SCI can increase the onset and expression of behavioral responses indicative of pain, potentially involving TNFα signaling.

Estrogen has a plethora of effects in the cardiovascular system. Studies of estrogen and the heart span human clinical trials and basic cell and molecular investigations. Greater understanding of cell and molecular responses to estrogens can provide further insights into the findings of clinical studies. Differences in expression and cellular/intracellular distribution of the two main receptors, estrogen receptor (ER) α and β, are thought to account for the specificity and differences in responses to estrogen. Much remains to be learned in this area, but cellular distribution within the cardiovascular system is becoming clearer. Identification of GPER as a third ER has introduced further complexity to the system. 17β-estradiol (E2), the most potent human estrogen, clearly has protective properties activating a signaling cascade leading to cellular protection and also influencing expression of the protective heat shock proteins (HSP). E2 protects the heart from ischemic injury in basic studies, but the picture is more involved in the whole organism and clinical studies. Here the complexity of E2's widespread effects comes into play and makes interpretation of findings more challenging. Estrogen loss occurs primarily with aging, but few studies have used aged models despite clear evidence of differences between the response to estrogen deficiency in adult and aged animals. Thus more work is needed focusing on the effects of aging vs. estrogen loss on the cardiovascular system.

While TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in neoplastic cells, many tumors, including pancreatic ductal adenocarcinoma (PDA), display intrinsic resistance, highlighting the need for TRAIL-sensitizing agents. Here we report that TRAIL-induced apoptosis in PDA cell lines is enhanced by pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) or by shRNA-mediated depletion of either GSK-3 alpha or GSK-3 beta. In contrast, depletion of GSK-3 beta, but not GSK-3 alpha, sensitized PDA cell lines to TNF alpha-induced cell death. Further experiments demonstrated that TNF alpha-stimulated I kappa B alpha phosphorylation and degradation as well as p65 nuclear translocation were normal in GSK-3 beta-deficient MEFs. Nonetheless, inhibition of GSK-3 beta function in MEFs or PDA cell lines impaired the expression of the NF-kappa B target genes Bcl-xL and cIAP2, but not I kappa B alpha. Significantly, the expression of Bcl-xL and cIAP2 could be reestablished by expression of GSK-3 beta targeted to the nucleus but not GSK-3 beta targeted to the cytoplasm, suggesting that GSK-3 beta regulates NF-kappa B function within the nucleus. Consistent with this notion, chromatin immunoprecipitation demonstrated that GSK-3 inhibition resulted in either decreased p65 binding to the promoter of BIR3, which encodes cIAP2, or increased p50 binding as well as recruitment of SIRT1 and HDAC3 to the promoter of BCL2L1, which encodes Bcl-xL. Importantly, depletion of Bcl-xL but not cIAP2, mimicked the sensitizing effect of GSK-3 inhibition on TRAIL-induced apoptosis, whereas Bcl-xL overexpression ameliorated the sensitization by GSK-3 inhibition. These results not only suggest that GSK-3 beta overexpression and nuclear localization contribute to TNF alpha and TRAIL resistance via anti-apoptotic NF-kappa B genes such as Bcl-xL, but also provide a rationale for further exploration of GSK-3 inhibitors combined with TRAIL for the treatment of PDA. This work was supported, in part by the Mayo Clinic Pancreatic Cancer SPORE P50CA102701 (DDB), Mayo Clinic Prostate Cancer SPORE Developmental Project (J-SZ), American Cancer Society Institutional New Investigator Award (J-SZ), R01 CA69008 (SHK), funds from Universidad del Pais Vasco and CIBERehd (MHV) a Mildred-Scheel fellowship of German Cancer Society (VE and AK), and Reserve Talent of Universities Overseas Research Program of Heilongjiang (ZD).

A knockin (KI) mouse model with the R192Q missense mutation in the Cacna1a gene commonly detected in familial hemiplegic migraine was used to study whether trigeminal ganglia showed a basal inflammatory profile that could be further enhanced by the lipopolysaccharide (LPS) toxin. Adenosine-5′-triphosphate (ATP)-gated purinergic ionotropic receptor 3 (P2X3) currents expressed by the large majority of trigeminal sensory neurons were taken as functional readout. Cultured R192Q KI trigeminal ganglia showed higher number of active macrophages, basal release of tumor necrosis factor alpha (TNFα), and larger P2X3 receptor currents with respect to wild type (WT) cells. After 5 h application of LPS in vitro, both WT and R192Q KI cultures demonstrated significant increase in macrophage activation, very large rise in TNFα mRNA content, and ambient protein levels together with fall in TNFα precursor, suggesting potent release of this inflammatory mediator. Notwithstanding the unchanged expression of P2X3 receptor protein in WT or R192Q KI cultures, LPS evoked a large rise in WT neuronal currents that recovered faster from desensitization. Basal R192Q KI currents were larger than WT ones and could not be further augmented by LPS. These data suggest that KI cultures had a basal neuroinflammatory profile that might facilitate the release of endogenous mediators (including ATP) to activate constitutively hyperfunctional P2X3 receptors and amplify nociceptive signaling by trigeminal sensory neurons.

Induction of tumor necrosis factor alpha was studied in the brain of rats after focal cerebral ischaemia by occlusion of the left middle cerebral artery. Using a specific antisense riboprobe for in situ hybridization histochemistry, cells positive for tumor necrosis factor alpha messenger RNA were detected within 30 min in the brain regions known to be necrotic within one to two days after onset of ischaemia. Their number increased over a time period of 1-8 h and then declined. Only a few tumor necrosis factor alpha messenger RNA positive cells could be detected four days after the onset of ischaemia. Reverse-transcription polymerase chain reaction experiments showed that maximal increase of tumor necrosis factor alpha messenger RNA level in the ischaemic brain hemisphere occurred 3 h after occlusion of the middle cerebral artery. Immunocytochemical experiments using an anti-tumor necrosis factor alpha antibody showed the presence of tumor necrosis factor alpha immunopositive cells as early as 30 min after occlusion of the middle cerebral artery in the same brain regions where tumor necrosis factor alpha messenger RNA positive cells were detected. Tumor necrosis factor alpha positive cells were highly abundant in the infarcted brain 8-24 h, but only few of them were detectable four days after the onset of ischaemia. Specificity of the anti-tumor necrosis factor alpha antibody and of the induction of tumor necrosis factor alpha protein was confirmed by western blot analysis. Tumor necrosis factor alpha messenger RNA- and protein-positive cells were also detected in the watershed zone and in some structures of the contralateral brain hemisphere. According to their morphology, tumor necrosis factor alpha-positive cells could be identified as microglial cells and macrophages at different states of activation. This assumption was further confirmed by double-labeling studies using the isolectin B-4 from Griffonia simplicifolia, a specific microglial/macrophage cell marker. These results demonstrate that expression of tumor necrosis factor alpha is part of an intrinsic inflammatory reaction of the brain after ischaemia.

Objectives: In the present study, we evaluated functional activity of monocytes in experimental preeclampsia induced by low-dose endotoxin infusion.Study design: Pregnant (n = 12) and cyclic rats (n = 12) were equipped with a permanent jugular vein cannula and infused with either low-dose endotoxin or saline. One day before the infusion, and 4, 24, 72, and 168 hours after the infusion, blood samples (400 muL) were taken and white blood cell (WBC) and differential cell counts were measured. Samples were (re)stimulated with endotoxin, and the percentage of tumor necrosis factor-alpha (TNFalpha) producing monocytes was measured.Results: During experimental preeclampsia, monocyte TNFa production is persistently decreased, while total WBC and granulocyte counts are persistently increased compared with normal pregnant rats. No persistent effect of endotoxin was found in cyclic rats.Conclusion: Because decreased endotoxin-induced TNFa production is a feature of activated monocytes, the present results indicate that monocytes are persistently activated in experimental preeclampsia. Increased WBC counts and granulocyte numbers in these rats also point to an activated inflammatory response. (C) 2004 Elsevier Inc. All rights reserved.

Pregnancy in the rat may be associated with an activated innate immune system. Therefore, we investigated monocyte function as well as total white blood cell (WBC) counts during the follicular phase of the ovarian cycle, pregnancy and pseudopregnancy in the rat. Rats were equipped with a permanent jugular vein cannula, and 0.43 ml blood samples were taken from this cannula during the 4 days of the regular oestrus cycle of the rat (n = 12). Thereafter, six rats were rendered pregnant, and the other six rats were rendered pseudopregnant according to standard methods. Blood samples were withdrawn from the cannula on days 4, 7 and I I of pseudopregnancy and on days 4, 7, 11 and 20 of pregnancy. From each blood sample, 0.4 ml was stimulated with lipopolysaccharide (LPS) and monocyte intracellular cytokine production measured using flow cytometry. 30 mul of the blood was used to measure WBC counts and differential WBC counts. The results showed that the number of WBC was significantly increased only on day 11 of pregnancy compared with the follicular phase, and that this was due to the increased numbers of polymorphonuclear (PMN) cells. The percentage of TNFalpha-producing monocytes was increased on all days of pseudopregnancy and on day 11 of pregnancy. The fact that the percentage of monocytes producing TNFalpha upon an LPS stimulus was increased during the post-implantation phase of pregnancy and during pseudopregnancy as compared to the follicular phase may indicate that these conditions are proinflammatory conditions. For the post-implantation phase of pregnancy, this is once more stressed by the increased numbers of WBC and PMN. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

International audience; Tumor necrosis factor α(TNF-α) plays a crucial role in the pathogenesis of the central nervous system infections. In an in vitro reconstructed blood–brain barrier model, a significant dysregulation of receptor mediated endocytosis of low density lipoproteins (LDL) and transferrin (Tf) is demonstrated at delayed phase of direct TNF-α activation. Concomitant with the increase in LDL uptake, we demonstrate a decrease of Tf-receptor mediated endocytosis. The potential role of TNF action in the differential or opposite routing of macromolecules is also characterized by a stimulation of their transcytosis. These findings may provide a new insight into theinflammatory effect of TNF-α on brain capillary endothelial cells.

The cytokines TNFalpha and IFNgamma have previously been shown to activate caprine arthritis encephalitis virus (CAEV) transcription. Increased viral titers correlate with increased lesion severity. Therefore, TNFalpha and IFNgamma may augment the caprine arthritis lesion by increasing viral titers. CAEV transcription is under the control of the viral promoter within the U3 region of the long terminal repeat. A set of U3 deletion mutants was generated and used to establish stably integrated, U937-based cell lines. These cell lines were utilized to define the required promoter sequences for cytokine-induced transcriptional activation. Here we have identified a novel 17 nucleotide TNF-activated site within the U3 region 70 bp repeat which is both required and sufficient in a minimal construct for TNFalpha-induced CAEV transcriptional activation. In contrast to the results of previous studies with IFNgamma, we found that multiple sequences within the U3 region 70 bp repeat were required for IFNgamma-activation of the CAEV promoter. The results identify previously unrecognized complexity in the CAEV promoter that may be relevant to viral replication and disease.

Apurinic/apyrimidinic endonuclease 1 (APE1), a ubiquitous multipurpose protein, is also known as redox effector factor-1 (Ref-1). It is involved in DNA repair and redox signaling and, in turn, oxidative stress-induced neurodegeneration. Although previous studies have demonstrated that APE1/Ref-1 functions as a negative regulator of inflammatory response via several mechanisms in neuronal cells, little is known about the roles of APE1/Ref-1 in glial cells. In this study, we found that cytoplasmic APE1/Ref-1 expression was upregulated in reactive astrocytes of the kainic acid-or lipopolysaccharide (LPS)-injected hippocampus. Analysis of the inflammatory response induced by extranuclear APE1/Ref-1 (Delta NLS-Ref-1) in cultured primary astrocytes revealed that it markedly suppressed inducible nitric oxide synthase (iNOS) expression and tumor necrosis factor-alpha (TNF-alpha) secretion induced by LPS to a similar extent as did wild type APE1/Ref-1 (WT-Ref-1), supporting the concept an anti-inflammatory role of extranuclear APE1/Ref-1 in astrocytes. Additionally, overexpression of WT- and Delta NLS-Ref-1 suppressed the transcriptional activity of nuclear factor-kappa B (NF-kappa B), although it effectively enhanced activator protein 1 (AP-1) activity. The blunting effect of APE1/Ref-1 on LPS-induced NF-kappa B activation was not mediated by I kappa B kinase (IKK) activity. Instead, APE1/Ref-1 inhibited p300-mediated acetylation of p65 by suppressing intracellular reactive oxygen species (ROS) levels following LPS treatment. Taken together, our results showed that altered expression and/or subcellular distribution of APE1/Ref-1 in activated astrocytes regulated the neuroinflammatory response to excitotoxin and endotoxin insults used in model of neurodegenerative brain diseases.

Background Subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. It is suggested that the associated inflammation is mediated through activation of the mitogen-activated protein kinase (MAPK) pathway which plays a crucial role in the pathogenesis of delayed cerebral ischemia after SAH. The aim of this study was first to investigate the timecourse of altered expression of proinflammatory cytokines and matrix metalloproteinase in the cerebral arteries walls following SAH. Secondly, we investigated whether administration of a specific mitogen-activated protein kinase kinase (MEK)1/2 inhibitor, U0126, given at 6 h after SAH prevents activation of the MEK/extracellular signal-regulated kinase 1/2 pathway and the upregulation of cerebrovascular inflammatory mediators and improves neurological function. Methods SAH was induced in rats by injection of 250 μl of autologous blood into basal cisterns. U0126 was given intracisternally using two treatment regimens: (A) treatments at 6, 12, 24 and 36 h after SAH and experiments terminated at 48 h after SAH, or (B) treatments at 6, 12, and 24 h after SAH and terminated at 72 h after SAH. Cerebral arteries were harvested and interleukin (IL)-6, IL-1β, tumor necrosis factor α (TNF)α, matrix metalloproteinase (MMP)-9 and phosphorylated ERK1/2 (pERK1/2) levels investigated by immunohistochemistry. Early activation of pERK1/2 was measured by western blot. Functional neurological outcome after SAH was also analyzed. Results Expression levels of IL-1β, IL-6, MMP-9 and pERK1/2 proteins were elevated over time with an early increase at around 6 h and a late peak at 48 to 72 h post-SAH in cerebral arteries. Enhanced expression of TNFα in cerebral arteries started at 24 h and increased until 96 h. In addition, SAH induced sensorimotor and spontaneous behavior deficits in the animals. Treatment with U0126 starting at 6 h after SAH prevented activation of MEK-ERK1/2 signaling. Further, U0126 significantly decreased the upregulation of inflammation proteins at 48 and 72 h following SAH and improved neurological function. We found no differences between treatment regimens A and B. Conclusions These results show that SAH induces early activation of the MEK-ERK1/2 pathway in cerebral artery walls, which is associated with upregulation of proinflammatory cytokines and MMP-9. Inhibition of the MEK-ERK1/2 pathway by U0126 starting at 6 h post-SAH prevented upregulation of cytokines and MMP-9 in cerebral vessels, and improved neurological outcome.