High frequencies of functionally impaired cytokeratin 18-specific CD8+ T cells in healthy HLA-A2+ donors.

Combining cell surface phenotyping with functional analysis, human CD8+ T cells have been divided into several subsets which are being studied extensively in diverse physiological situations, such as viral infection, cancer and ageing. In particular, so-called terminally differentiated effector cells possess a CD45RA+ CCR7- CD27- CD28- phenotype, contain perforin and, in different models, have been shown to exert direct ex vivo killing and to release interleukins upon both antigen-nonspecific and -specific stimulation. Using HLA class I multimers, we have identified a high frequency of peripheral CD8+ T cells that recognize a peptide derived from the self protein cytokeratin 18 presented by the HLA-A*0201 molecule. These cells can be detected in approximately 15% of the HLA-A2-positive healthy donors tested. A detailed analysis revealed that they must have divided extensively in vivo, have an effector cell phenotype and express various natural killer cell-associated receptors. Interestingly, however, they remained unresponsive to antigen-specific stimulation in vitro in terms of cytotoxicity and cytokine secretion. Thus, cytokeratin 18-specific cells constitute a frequently encountered, new CD8+ T lymphocyte subpopulation without classical effector status and with so far unknown function.

Messenger RNA expression of transporter and ion channel genes in undifferentiated and differentiated Caco-2 cells compared to human intestines.

PURPOSE: The purpose of this work was to study the influence of cell differentiation on the mRNA expression of transporters and channels in Caco-2 cells and to assess Caco-2 cells as a model for carrier-mediated drug transport in the intestines. METHOD: Gene mRNA expression was measured using a custom-designed microarray chip with 750 deoxyoligonucleotide probes (70mers). Each oligomer was printed four times on poly-lysine-coated glass slides. Expression profiles were expressed as ratio values between fluorescence intensities of Cy3 and Cy5 dye-labeled cDNA derived from poly(A) + RNA samples of Caco-2 cells and total RNA of human intestines. RESULTS: Significant differences in the mRNA expression profile of transporters and channels were observed upon differentiation of Caco-2 cells from 5 days to 2 weeks in culture, including changes for MAT8, S-protein, and Nramp2. Comparing Caco-2 cells of different passage number revealed few changes in mRNAs except for GLUT3, which was down-regulated 2.4-fold within 13 passage numbers. Caco-2 cells had a similar expression profile when either cultured in flasks or on filters but differed more strongly from human small and large intestine, regardless of the differentiation state of Caco-2 cells. Expression of several genes highly transcribed in small or large intestines differed fourfold or more in Caco-2 cells. CONCLUSIONS: Although Caco-2 cells have proven a suitable model for studying carrier-mediated transport in human intestines, the expression of specific transporter and ion channel genes may differ substantially.

Involvement of nuclear factor-kappaB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin- 1 beta in ectopic endometrial cells.

OBJECTIVE: To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Nine women with endometriotic lesions. INTERVENTION(S): Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S): This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S): This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.

Eliminació autotròfica de nitrogen en una pila biològica (microbial fuel cells )

L’aigua i l’energia formen un binomi indissociable. En relació al cicle de l’aigua, des de fa varies dècades s’han desenvolupat diferents formes per recuperar part de l’energia relacionada amb l’aigua, per exemple a partir de centrals hidroelèctriques. No obstant, l’ús d’aquesta aigua també porta associat un gran consum energètic, relacionat sobretot amb el transport, la distribució, la depuració, etc... La depuració d’aigües residuals porta associada una elevada demanda energètica (Obis et al.,2009). En termes energètics, tot i que la despesa elèctrica d’una EDAR varia en funció de diferents paràmetres com la configuració i la capacitat de la planta, la càrrega a tractar, etc... es podria considerar que el rati mig seria d’ aproximadament 0.5 KWh•m-3.Els principals costos d’explotació estan relacionats tant amb la gestió de fangs (28%) com amb el consum elèctric (25%) (50% tractament biològic). Tot i que moltes investigacions relacionades amb el tractament d’aigua residual estan encaminades en disminuir els costos d’operació, des de fa poques dècades s’està investigant la viabilitat de que l’aigua residual fins i tot sigui una font d’energia, canviant la perspectiva, i començant a veure l’aigua residual no com a una problemàtica sinó com a un recurs. Concretament s’estima que l’aigua domèstica conté 9.3 vegades més energia que la necessària per el seu tractament mitjançant processos aerobis (Shizas et al., 2004). Un dels processos més desenvolupats relacionats amb el tractament d’aigües residuals i la producció energètica és la digestió anaeròbia. No obstant, aquesta tecnologia permet el tractament d’altes càrregues de matèria orgànica generant un efluent ric en nitrogen que s’haurà de tractar amb altres tecnologies. Per altre banda, recentment s’està investigant una nova tecnologia relacionada amb el tractament d’aigües residuals i la producció energètica: les piles biològiques (microbial fuel cells, MFC). Aquesta tecnologia permet obtenir directament energia elèctrica a partir de la degradació de substrats biodegradables (Rabaey et al., 2005). Les piles biològiques, més conegudes com a Microbial Fuel Cells (acrònim en anglès, MFC), són una emergent tecnologia que està centrant moltes mirades en el camp de l’ investigació, i que es basa en la producció d’energia elèctrica a partir de substrats biodegradables presents en l’aigua residual (Logan., 2008). Els fonaments de les piles biològiques és molt semblant al funcionament d’una pila Daniell, en la qual es separa en dos compartiments la reacció d’oxidació (compartiment anòdic) i la de reducció (compartiment catòdic) amb l’objectiu de generar un determinat corrent elèctric. En aquest estudi, bàsicament es mostra la posada en marxa d'una pila biològica per a l'eliminació de matèria orgànica i nitrogen de les aigües residuals.

Role of the sub-cellular localization of RasGAP fragment N2 for its ability to sensitize cancer cells to genotoxin-induced apoptosis.

The specific sensitization of tumor cells to the apoptotic response induced by genotoxins is a promising way of increasing the efficacy of chemotherapies. The RasGAP-derived fragment N2, while not regulating apoptosis in normal cells, potently sensitizes tumor cells to cisplatin- and other genotoxin-induced cell death. Here we show that fragment N2 in living cells is mainly located in the cytoplasm and only minimally associated with specific organelles. The cytoplasmic localization of fragment N2 was required for its cisplatin-sensitization property because targeting it to the mitochondria or the ER abrogated its ability to increase the death of tumor cells in response to cisplatin. These results indicate that fragment N2 requires a spatially constrained cellular location to exert its anti-cancer activity.

Neutrophil-derived CCL3 is essential for the rapid recruitment of dendritic cells to the site of Leishmania major inoculation in resistant mice.

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3(-/-) mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.