Cutting edge: influence of the TCR Vbeta domain on the selection of semi-invariant NKT cells by endogenous ligands.

Invariant Valpha14 (Valpha14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Valpha14-Jalpha18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the Valpha14i TCR for CD1d-alpha-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on Valpha14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7(+), but not Vbeta8.2(+), Valpha14i NKT cells is favored in situations where endogenous ligand concentration or TCRalpha-chain avidity are suboptimal. Furthermore, thymic Vbeta7(+) Valpha14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of Valpha14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.

Anionic Sites, Fucose Residues and Class I Human Leukocyte Antigen Fate During Interaction of Toxoplasma gondii with Endothelial Cells

Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocytte antigen (HLA) before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containig vacuoles.

Different epitopes of the LACK protein are recognized by V beta 4 V alpha 8 CD4+ T cells in H-2b and H-2d mice susceptible to Leishmania major.

After inoculation of Leishmania major, a rapid production of IL-4 by LACK-specific CD4+ T cells has been shown to drive Th2 cell development in susceptible mice i.e. BALB/c and C57BL/6 mice rendered susceptible by neutralization of IFN-gamma at the onset of infection. Here, we showed that peptide AA 156-173 induced an early IL-4 mRNA expression not only in BALB/c mice but also in resistant B10.D2 mice when IFN-gamma is neutralized. Epitope mapping of LACK protein demonstrated that peptide containing AA 293-305 induced early IL-4 mRNA transcripts in susceptible H-2b mice i.e. BALB/b and resistant C57BL/6 mice when IFN-gamma is neutralized. Stringently, the early IL-4 response to the H-2d (AA 156-173) or the H-2b (AA 293-305) epitopes occurred in V beta 4 V alpha 8 CD4+ T cells from either H-2d or H-2b susceptible mice, respectively.

Development of a coculture model of encapsulated cells.

In the whole animal, metabolic regulations are set by reciprocal interactions between various organs, via the blood circulation. At present, analyses of such interactions require numerous and uneasily controlled in vivo experiments. In a search for an alternative to in vivo experiments, our work aims at developing a coculture system in which different cell types are isolated in polymer capsules and grown in a common environment. The signals exchanged between cells from various origins are, thus, reproducing the in vivo intertissular communications. With this perspective, we evaluated a new encapsulation system as an artificial housing for liver cells on the one hand and adipocytes on the other hand. Murine hepatocytes were encapsulated with specially designed multicomponent capsules formed by polyelectrolyte complexation between sodium alginate, cellulose sulphate and poly(methylene-coguanidine) hydrochloride, of which the permeability has been characterized. We demonstrated the absence of cytotoxicity and the excellent biocompatibility of these capsules towards primary culture of murine hepatocytes. Encapsulated hepatocytes retain their specific functions--transaminase activity, urea synthesis, and protein secretion--during the first four days of culture in minimum medium. Mature adipocytes, isolated from mouse epidydimal fat, were embedded in alginate beads. Measurement of protein secretion shows an identical profile between free and embedded adipocytes. We finally assessed the properties of encapsulated hepatocytes, cryopreserved over a periods of up to four months. The perspective of using encapsulated cells in coculture are discussed, since this system may represent a promising tool for fundamental research, such as analyses of drug metabolism, intercellular regulations, and metabolic pathways, as well as for the establishment of a tissue bank for storage and supply of murine hepatocytes.

Cutting edge: influence of the TCR V beta domain on the avidity of CD1d:alpha-galactosylceramide binding by invariant V alpha 14 NKT cells.

CD1d tetramers loaded with alpha-galactosylceramide (alpha-GalCer) bind selectively to mouse invariant Valpha14 (Valpha14i) NKT cells and their human counterparts. Whereas tetramer binding strictly depends on the expression of a Valpha14-Jalpha18 chain in murine NKT cells, the associated beta-chain (typically expressing Vbeta8.2 or Vbeta7) appears not to influence tetramer binding. In this study, we describe novel alpha-GalCer-loaded mouse and human CD1d-IgG1 dimers, which revealed an unexpected influence of the TCR-beta chain on the avidity of CD1d:alpha-GalCer binding. A subset of Valpha14i NKT cells clearly discriminated alpha-GalCer bound to mouse or human CD1d on the basis of avidity differences conferred by the Vbeta domain of the TCR-beta chain, with Vbeta8.2 conferring higher avidity binding than Vbeta7.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.

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.

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-g, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ.

Bone-marrow haematopoietic-stem-cell niches.

Adult stem cells hold many promises for future clinical applications and regenerative medicine. The haematopoietic stem cell (HSC) is the best-characterized somatic stem cell so far, but in vitro expansion has been unsuccessful, limiting the future therapeutic potential of these cells. Here we review recent progress in characterizing the composition of the HSC bone-marrow microenvironment, known as the HSC niche. During homeostasis, HSCs, and therefore putative bone-marrow HSC niches, are located near bone surfaces or are associated with the sinusoidal endothelium. The molecular crosstalk between HSCs and the cellular constituents of these niches is thought to control the balance between HSC self-renewal and differentiation, indicating that future successful expansion of HSCs for therapeutic use will require three-dimensional reconstruction of a stem-cell-niche unit.

Screening and fractionation of plant extracts with antiproliferative activity on human peripheral blood mononuclear cells

Three hundred and thirteen extracts from 136 Brazilian plant species belonging to 36 families were tested for their suppressive activity on phytohemaglutinin (PHA) stimulated proliferation of human peripheral blood mononuclear cells (PBMC). The proliferation was evaluated by the amount of [³H]-thymidine incorporated by the cells. Twenty extracts inhibited or strongly reduced the proliferation in a dose-dependent manner at doses between 10 and 100 µg/ml. Three of these extracts appeared to be non-toxic to lymphocytes, according to the trypan blue permeability assay and visual inspection using optical microscopy. Bioassay-guided fractionation of Alomia myriadenia extract showed that myriadenolide, a labdane diterpene known to occur in this species, could account for the observed activity of the crude extract. Using a similar protocol, an active fraction of the extract from Gaylussacia brasiliensis was obtained. Analysis of the ¹H and13C NMR spectra of this fraction indicates the presence of an acetylated triterpene whose characterization is underway. The extract of Himatanthus obovatus is currently under investigation.