Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein beta

Background. Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPß in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPß-null glial cultures. Methods. Due to fertility and mortality problems associated with the C/EBPß-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPß-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPß DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. Results. C/EBPß mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon ¿ (IFN¿). Quantitative chromatin immunoprecipitation showed binding of C/EBPß to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFN¿ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1ß and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPß. In addition, neurotoxicity elicited by LPS+IFN¿-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPß in microglia.

Polimorfismos genéticos (en los genes OPRM1, BETA-ARRESTINA2,STAT6 y COMT) y su influencia en la tolerancia al tratamiento con opiáceos mayores en pacientes oncológicos

La morfina es l’opioid majoritàriament utilitzat en dolor oncològic, però existeix elevada variabilitat de resposta. Vam intentar correlacionar aquesta variabilitat amb polimorfismes genètics (Opmr-1, Beta-arrestina2, Stat6 i COMT, relacionats amb mecanismes d’acció opioids). Hem estudiat 29 pacients amb dolor (EVA superior o igual a 6) que van iniciar tractament amb morfina i vam avaluar eficacia i tolerancia a la morfina correlacionant-ho amb els polimorfismos que presentaven. Vam observar que els genotips CC/TC per β-arrestina2 i AA/GA per COMT i Oprm1 es podrien associar a millor resposta i menor toxicitat a la morfina, i els genotips AA/GA per STAT6 s’associaven significativament a menor toxicitat

Dexamethasone inhibition of leucocyte adhesion to rat mesenteric postcapillary venules: role of intercellular adhesion molecule 1 and KC

BACKGROUND A previous study showed that the glucocorticoid dexamethasone, at doses of 100 ¿g/kg and above, inhibited leucocyte adhesion to rat mesenteric postcapillary venules activated with interleukin 1ß (IL-1ß), as assessed by videomicroscopy. AIMS To identify whether the adhesion molecule, intercellular adhesion molecule 1 (ICAM-1), or the chemokine KC could be targeted by the steroid to mediate its antiadhesive effect. METHODS Rat mesenteries were treated with IL-1ß (20 ng intraperitoneally) and the extent of leucocyte adhesion measured at two and four hours using intravital microscopy. Rats were treated with dexamethasone, and passively immunised against ICAM-1 or KC. Endogenous expression of these two mediators was validated by immunohistochemistry, ELISA, and the injection of specific radiolabelled antibodies. RESULTS Dexamethasone greatly reduced IL-1ß induced leucocyte adhesion, endothelial expression of ICAM-1 in the postcapillary venule, and release of the mast cell derived chemokine KC. Injection of specific antibodies to the latter mediators was also extremely effective in downregulating (>80%) IL-1ß induced leucocyte adhesion. CONCLUSIONS Induction by IL-1ß of endogenous ICAM-1 and KC contributes to leucocyte adhesion to inflamed mesenteric vessels. Without excluding other possible mediators, these data clearly show that dexamethasone interferes with ICAM-1 expression and KC release from mast cells, resulting in suppression of leucocyte accumulation in the bowel wall, which is a prominent feature of several gastrointestinal pathologies.

Microdiàlisis cerebral aplicada a l'estudi del metabolisme energètic i a la resposta neuroinflamatòria dels pacients amb traumatisme cranioencefàlic

La tècnica de la microdiàlisis cerebral (MDC) és un instrument que proporciona informació rellevant en la monitorització del metabolisme cerebral en els pacients neurocrítics. El lactat i l’índex lactat-piruvat (ILP) són dos marcadors utilitzats per a la detecció de la hipòxia cerebral en pacients que han patit un traumatisme cranioencefàlic (TCE). Aquests dos marcadors poden estar anormalment elevats en circumstàncies que no cursen amb hipòxia tissular. Per una altra banda la recent aparició dels catèters de MDC amb porus de major mida denominats d’”alta resolució”, permet ampliar el rang de molècules que es poden detectar en el dialitzat. Objectius: 1) descriure les característiques del metabolisme energètic cerebral que s’observa en la fase aguda dels pacients que han patit un TCE en base als dos indicadors del metabolisme anaeròbic: lactat i ILP, i 2) determinar la recuperació relativa (RR) de les molècules implicades en la resposta neuroinflamatòria: de IL-1β, IL- 6, IL-8 i IL-10. Material i mètodes: Es van seleccionar 46 pacients d’una cohort de pacients amb TCE moderat o greu ingressats a la Unitat de Cures Intensives de l’Hospital Universitari de la Vall d’Hebron i monitoritzats amb MDC. Es van analitzar els nivells de lactat i ILP i es va correlacionar amb els nivells de PtiO2. Es van realitzar experiments in vitro per estudiar la recuperació de les membranes de 100 KDa per tal de poder interpretar posteriorment els nivells reals de les molècules estudiades en l’espai extracel•lular del teixit cerebral. Resultats: La concordança entre el lactat i l’índex LP per a determinar episodis de disfunció metabòlica va ser dèbil (índex de kappa = 0,36, IC 95%: 0,34-0,39). Més del 80% dels casos en què el lactat i l’índex LP es trobaven incrementats, els valors de la PtiO2 es van trobar dins els rangs de normalitat (PtiO2&15mmHg). La recuperació de les citoquines a través de la membrana de microdiàlisis va ser menor de l’esperat tenint en compte la mida dels porus de la membrana. Conclusions: el lactat i l’índex LP elevats va ser una troballa freqüent després d’un TCE i no es va relacionar, en la majoria de casos, amb episodis d’hipòxia tissular. Per un altra part la mida del porus de la membrana no és l’únic paràmetre indicador de la RR de macromolècules.

Common Variants of TLR1 Associate with Organ Dysfunction and Sustained Pro- Inflammatory Responses during Sepsis

Background: Toll-like receptors (TLRs) are critical components for host pathogen recognition and variants in genes participating in this response influence susceptibility to infections. Recently, TLR1 gene polymorphisms have been found correlated with whole blood hyper-inflammatory responses to pathogen-associated molecules and associated with sepsis-associated multiorgan dysfunction and acute lung injury (ALI). We examined the association of common variants of TLR1 gene with sepsis-derived complications in an independent study and with serum levels for four inflammatory biomarker among septic patients. Methodology/Principal Findings: Seven tagging single nucleotide polymorphisms of the TLR1 gene were genotyped in samples from a prospective multicenter case-only study of patients with severe sepsis admitted into a network of intensive care units followed for disease severity. Interleukin (IL)-1 b, IL-6, IL-10, and C-reactive protein (CRP) serum levels were measured at study entry, at 48 h and at 7th day. Alleles -7202G and 248Ser, and the 248Ser-602Ile haplotype were associated with circulatory dysfunction among severe septic patients (0.001<=p <= 0.022), and with reduced IL-10 (0.012<= p <=0.047) and elevated CRP (0.011<= p <=0.036) serum levels during the first week of sepsis development. Additionally, the -7202GG genotype was found to be associated with hospital mortality (p =0.017) and ALI (p =0.050) in a combined analysis with European Americans, suggesting common risk effects among studies Conclusions/Significance: These results partially replicate and extend previous findings, supporting that variants of TLR1 gene are determinants of severe complications during sepsis.

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

Loss of intestinal epithelial barrier function in Salmonella Enteritidis infection

Intestinal infection with Salmonella enterica serotype Enteritidis, a food-borne infection spread to humans especially through contaminated eggs and egg-products as well as undercooked contaminated fresh meat, is the most common cause of intestinal inflammation in the European Union. Enteritis caused by Salmonella Enteritidis is characterized by fever, diarrhoea and abdominal pain. The disruption of the intestinal epithelial barrier function contributes to diarrhoea and is responsible for the perpetuation of the inflammatory process. In this sense, oxidative stress and the proinflammatory cytokines TNF-α, IFN-γ and IL-1β are described to induce the disorganization of the tight junctions (TJ), the most apical epithelial intercellular junctions and responsible for the paracellular permeability. The interest of this chapter relies not only in the investigation dealing with the mechanisms of TJ regulation but also in the contribution to the development of new tools for the prevention of epithelial barrier disruption in enteritis caused by Salmonella Enteritidis.