Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii.

ABSTRACT: BACKGROUND: Sphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000). These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses. RESULTS: Under conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the opposite effect and decreased after perturbation with PEG8000. CONCLUSIONS: A combination of growth assays, transcriptome profiling, and membrane fatty acid analyses revealed that permeating and non-permeating solutes trigger different adaptive responses in strain RW1, suggesting these solutes affect cells in fundamentally different ways. Future work is now needed that connects these responses with the responses observed in more realistic scenarios of soil desiccation.

Expression and presentation of endogenous mouse mammary tumor virus superantigens by thymic and splenic dendritic cells and B cells.

Tolerance against superantigens (SAgs) encoded by endogenous mouse mammary tumor virus (Mtv) loci involves the intrathymic deletion of SAg-reactive T cells expressing a particular TCR V beta-chain, presumably upon presentation of the SAg by specialized APC. However, although the role of dendritic cells (DC) in the induction of tolerance against conventional Ags has been demonstrated, little is known about the role played by DC in tolerance induction against Mtv SAgs. Moreover, there is conflicting evidence concerning the capacity of DC to express and present Mtv SAgs. In this report we have analyzed the expression of Mtv SAgs in highly purified thymic and splenic DC and B cells by reverse transcriptase-PCR, using primers amplifying Mtv SAg-specific spliced mRNAs. DC express Mtv SAgs at levels comparable to B cells, but display a differential expression pattern of the various Mtv loci compared with B cells. Furthermore, our results show that DC are able to induce the deletion of SAg-reactive thymocytes in an in vitro assay, indicating that Mtv SAgs are functionally expressed on the DC surface. Collectively, our data are consistent with the hypothesis that DC play a role in the induction of intrathymic tolerance to Mtv SAgs.

Phenotypic switching in Pseudomonas brassicacearum involves GacS- and GacA-dependent Rsm small RNAs.

The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes rsmX, rsmY, and rsmZ. Naturally occurring mutations in the gacS-gacA system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different N-acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits, gacS and gacA mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a gacA mutant, the expression of rsmX was completely abolished, unlike that of rsmY and rsmZ. Overexpression of any of the three sRNAs in the gacA mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in P. brassicacearum results from mutations in the gacS-gacA system.

Novel role of a family of major facilitator transporters in biofilm development and virulence of Candida albicans.

The QDR (quinidine drug resistance) family of genes encodes transporters belonging to the MFS (major facilitator superfamily) of proteins. We show that QDR transporters, which are localized to the plasma membrane, do not play a role in drug transport. Hence, null mutants of QDR1, QDR2 and QDR3 display no alterations in susceptibility to azoles, polyenes, echinocandins, polyamines or quinolines, or to cell wall inhibitors and many other stresses. However, the deletion of QDR genes, individually or collectively, led to defects in biofilm architecture and thickness. Interestingly, QDR-lacking strains also displayed attenuated virulence, but the strongest effect was observed with qdr2∆, qdr3∆ and in qdr1/2/3∆ strains. Notably, the attenuated virulence and biofilm defects could be reversed upon reintegration of QDR genes. Transcripts profiling confirmed differential expression of many biofilm and virulence-related genes in the deletion strains as compared with wild-type Candida albicans cells. Furthermore, lipidomic analysis of QDR-deletion mutants suggests massive remodelling of lipids, which may affect cell signalling, leading to the defect in biofilm development and attenuation of virulence. In summary, the results of the present study show that QDR paralogues encoding MFS antiporters do not display conserved functional linkage as drug transporters and perform functions that significantly affect the virulence of C. albicans.

The murine interleukin-2 receptor gamma chain gene: organization, chromosomal localization and expression in the adult thymus.

Defects in the interleukin-2 receptor gamma (IL-2R gamma) chain in the man result in an X-linked severe combined immunodeficiency, SCIDX1, characterized by an absence of T-cell differentiation. This phenotype may result from pertubations in IL-2, IL-4-, IL-7- or IL-15-mediated signaling, as the IL-2R gamma chain forms an integral component of these receptor systems. We have isolated and characterized cDNA and genomic clones for the murine IL-2R gamma. The gene (Il2rg) is well conserved between mouse and man with respect to overall structure and size, and contains regions of high conservation in the promoter region as well. Il2rg maps to mouse X chromosome region 40, in a region of synteny with human Xq12-13.1. We have also explored the expression of the IL-2R gamma during thymocyte development. IL-2R gamma transcripts are detected in the earliest thymocyte precursor cells and persist throughout intrathymic development into the mature peripheral compartment. Genomic clones for the murine IL-2R gamma will allow for further studies on the regulation and function of this gene in vivo.

Mir211 Is Dysregulated In Conjunctival Melanocytic proliferations

Purpose Downregulation of TRPM1 mRNA, a transient receptor potential cation channel, has been identified in highly metastatic cutaneous melanoma cell lines. TRPM1 mRNA expression is inversely correlated with skin melanoma metastases. Recent evidence has demonstrated that the tumor suppressive activity of TRPM1 is due to miR211 situated in intron 6 of TRPM1. As we have previously identified a downregulation of TRPM1 mRNA expression in conjunctival melanoma, we decided to assess miR211 expression and its potential target gene IGF2R and KCNMA1 in conjunctival melanocytic proliferations. As MITF has been shown to regulate both TRPM1 and mir211 expression, we also assessed MITF expression in our series. Methods Expression of miR211 was assessed by in situ hybridization in 14 conjunctival naevi and 14 conjunctival melanoma. Integrity of miRNA in tissues was evaluated in each sample with the preservation of miR126 expression in endothelial cells. Protein expression of MITF, IGF2R and KCNMA1 was assessed by immunohistochemistry. Statistical analysis was performed with JUMP 8,0 software. In situ hybridization and immunohistochemistry were assessed independently by two observers. Results There were 7 subepithelial nevi and 7 compound nevi. There were 5 female and 9 male. The population mean age was 48.7 ± 6.4 years (SEM). miR211 was found in 11 nevi (79%). MITF was expressed in all the nevi. IGF2R was found in 13 nevi. KCNMA1 was found in 57% of the nevi.The melanoma group was composed of 9 females and 5 males with a mean age of 67 ± 4.8 years (SEM). Using the recent TNM classification, 5 tumors were belonging to the T1, 3 to theT2 and 6 to the T3 categories. miR211 was found in 5 melanoma (36%). There was a significant downregulation of miR211 in the melanoma compared to the nevi (p=0,0219). MITF was found in 13 melanoma (93%). IGF2R and KCNMA1 were respectively found in 71% and 77% of the melanoma. There was no significant differential expression of MITF, KCNMA1 and IGF2R between the nevi and the melanoma as well as no association between miR211 expression and protein expression of two potential target genes Conclusions In vivo miR211 is significantly reduced in conjunctival melanoma compared to conjunctival nevi. No correlation between mir211 expression and two potential target genes KCNMA1 and IGF2R was observed.

Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes.

Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. & Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases.

Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision.

The elucidation of mechanisms underlying telencephalic neural development has been limited by the lack of knowledge regarding the molecular and cellular aspects of the ganglionic eminence (GE), an embryonic structure that supplies the brain with diverse sets of GABAergic neurons. Here, we report a comprehensive transcriptomic analysis of this structure including its medial (MGE), lateral (LGE) and caudal (CGE) subdivisions and its temporal dynamics in 12.5 to 16 day-old rat embryos. Surprisingly, comparison across subdivisions showed that CGE gene expression was the most unique providing unbiased genetic evidence for its differentiation from MGE and LGE. The molecular signature of the CGE comprised a large set of genes, including Rwdd3, Cyp26b1, Nr2f2, Egr3, Cpta1, Slit3, and Hod, of which several encode cell signaling and migration molecules such as WNT5A, DOCK9, VSNL1 and PRG1. Temporal analysis of the MGE revealed differential expression of unique sets of cell specification and migration genes, with early expression of Hes1, Lhx2, Ctgf and Mdk, and late enrichment of Olfm3, SerpinE2 and Wdr44. These GE profiles reveal new candidate regulators of spatiotemporally governed GABAergic neuronogenesis.

Expression of MYC, IgM, As Well As Non-Germinal Centre B-Cell Like Immunophenotype and Positive Immunofish Index Predict a Worse Progression Free Survival and Overall Survival in a Series of 670 De Novo Diffuse Large B-Cell Lymphomas Included in Clinical Trials: A GELA Study of the 2003 Program

Introduction: Diffuse large B-cell lymphomas (DLBCL) represent a heterogeneous disease with variable clinical outcome. Identifying phenotypic biomarkers of tumor cells on paraffin sections that predict different clinical outcome remain an important goal that may also help to better understand the biology of this lymphoma. Differentiating non-germinal centre B-cell-like (non-GCB) from Germinal Centre B-cell-like (GCB) DLBCL according to Hans algorithm has been considered as an important immunohistochemical biomarker with prognostic value among patients treated with R-CHOP although not reproducibly found by all groups. Gene expression studies have also shown that IgM expression might be used as a surrogate for the GCB and ABC subtypes with a strong preferential expression of IgM in ABC DLBCL subtype. ImmunoFISH index based on the differential expression of MUM-1, FOXP1 by immunohistochemistry and on the BCL6 rearrangement by FISH has been previously reported (C Copie-Bergman, J Clin Oncol. 2009;27:5573-9) as prognostic in an homogeneous series of DLBCL treated with R-CHOP. In addition, oncogenic MYC protein overexpression by immunohistochemistry may represent an easy tool to identify the consequences of MYC deregulation in DLBCL. Our aim was to analyse by immunohistochemistry the prognostic relevance of MYC, IgM, GCB/nonGCB subtype and ImmunoFISH index in a large series of de novo DLBCL treated with Rituximab (R)-chemotherapy (anthracyclin based) included in the 2003 program of the Groupe d'Etude des Lymphomes de l'Adulte (GELA) trials. Methods: The 2003 program included patients with de novo CD20+ DLBCL enrolled in 6 different LNH-03 GELA trials (LNH-03-1B, -B, -3B, 39B, -6B, 7B) stratifying patients according to age and age-adjusted IPI. Tumor samples were analyzed by immunohistochemistry using CD10, BCL6, MUM1, FOXP1 (according to Barrans threshold), MYC, IgM antibodies on tissue microarrays and by FISH using BCL6 split signal DNA probes. Considering evaluable Hans score, 670 patients were included in the study with 237 (35.4%) receiving intensive R-ACVBP regimen and 433 (64.6%) R-CHOP/R-mini-CHOP. Results: 304 (45.4%) DLBCL were classified as GCB and 366 (54.6%) as non-GCB according to Hans algorithm. 337/567 cases (59.4%) were positive for the ImmunoFISH index (i.e. two out of the three markers positive: MUM1 protein positive, FOXP1 protein Variable or Strong, BCL6 rearrangement). Immunofish index was preferentially positive in the non-GCB subtype (81.3%) compared to the GCB subtype (31.2%), (p<0.001). IgM was recorded as positive in tumor cells in 351/637 (52.4%) DLBCL cases with a preferential expression in non-GCB 195 (53.3%) vs GCB subtype 100(32.9%), p<0.001). MYC was positive in 170/577 (29.5%) cases with a 40% cut-off and in 44/577 (14.2%) cases with a cut-off of 70%. There was no preferential expression of MYC among GCB or non-GCB subtype (p>0.4) for both cut-offs. Progression-free Survival (PFS) was significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p<0.0001), MYC positive tumor with a 40% threshold (p<0.001), ImmunoFISH positive index (p<0.002), non-GCB DLBCL subtype (p<0.0001). Overall Survival (OS) was also significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p=0.02), MYC positive tumor with a 40% threshold (p<0.01), ImmunoFISH positive index (p=0.02), non-GCB DLBCL subtype (p<0.0001). All significant parameters were included in a multivariate analysis using Cox Model and in addition to IPI, only the GCB/non-GCB subtype according to Hans algorithm predicted significantly a worse PFS among non-GCB subgroup (HR 1.9 [1.3-2.8] p=0.002) as well as a worse OS (HR 2.0 [1.3-3.2], p=0.003). This strong prognostic value of non-GCB subtyping was confirmed considering only patients treated with R- CHOP for PFS (HR 2.1 [1.4-3.3], p=0.001) and for OS (HR 2.3 [1.3-3.8], p=0.002). Conclusion: Our study on a large series of patients included in trials confirmed the relevance of immunohistochemistry as a useful tool to identify significant prognostic biomarkers for clinical use. We show here that IgM and MYC might be useful prognostic biomarkers. In addition, we confirmed in this series the prognostic value of the ImmunoFISH index. Above all, we fully validated the strong and independent prognostic value of the Hans algorithm, daily used by the pathologists to subtype DLBCL.

Comprehensive analysis of proteins secreted by dermatophytes in a protein medium which to some extent mimic "in vivo" growth parameters

RESUME : Les dermatophytes sont les agents infectieux les plus fréquents responsables de la plupart des mycoses superficielles chez les humains et chez les animaux. Ces infections, dermatophytoses, également appelées tineas ou teignes, sont fréquentes et causent des problèmes de santé publique au niveau mondial. La capacité d'envahir et de progresser au sein des structures kératinisées est probablement liée à la sécrétion de différentes enzymes kératinolytiques, qui sont considérées comme la principale caractéristique liée à la pathogénicité de ces champignons. L'objectif de ma thèse a été premièrement de progresser dans l'identification et la caractérisation des nouvelles protéines sécrétées, afin de mieux comprendre a) la capacité globale des dermatophytes à envahir les structures kératinisées, et b) les différences dans la virulence et la spécificité d'hôte que présentent les espèces étudiées .Pour progresser dans l'identification et la caractérisation de ces nouvelles protéines, les secretomes de six espèces de dermatophytes (Trichophyton rubrum, Trichophyton violaceum, Trichophyton soudanense, Trichophyton equinum, Arthroderma vanbreuseghemii et Trichophyton tonsurans) ont été étudiés. Bien qu'il y ait un niveau globalement élevé de similitude entre les protéases sécrétées, les différentes espèces de dermatophytes sécrètent des profiles protéiques distincts lorsqu'elles sont cultivées dans les mêmes conditions de culture, et donc une signature spécifique a pu être associé à chaque espèce. Ces profiles ont été un outil avantageux pour identifier et cartographier les protéines orthologues aux six espèces et ont aussi permit la discrimination d'espèces très proches comme T. tonsurans et T. equinum qui ne peuvent pas être différenciées par l'ADN ribosomal. Ce travail également présente ce que l'on croit être la première identification global des protéines sécrétées par les dermatophytes dans des conditions de culture que incitent l'activité protéolytique extracellulaire. Ce catalogue de protéines, comprenant des endo- and exo- proteases, autres hydrolases, oxydoreductases et des protéines avec fonction inconnue, représente probablement le spectre d'enzymes qui permettent la dégradation des tissus kératinisés en composés qui peuvent être assimilés par le champignon. Les résultats suggèrent qu'un changement écologique pourrait être associé à une expression différentielle des gènes codant les protéines sécrétées, en particulier, les protéases, plutôt qu'à des divergences génétiques au niveau des gènes codant les protéines orthologues. Une sécrétion différentielle des protéines par les dermatophytes pourrait également être responsable de la variabilité inflammatoire qui causent ces agents infectieux chez les différents hôtes. Par conséquent, les protéines identifiées ici sont également importantes pour faire la lumière sur la réponse immunitaire de l'hôte au cours du processus infectieux. SUMMARY : Dermatophytes are the most common infectious agents responsible for superficial mycosis in humans and animals. Dermatophytoses, also called tineas or ringworm, are frequent and cause public health problems worldwide. The secretion of different keratinolytic enzymes is believed to be a key pathogenicity-related characteristic of these fungi. The aim of this work was first to progress in the identification and characterization of novel secreted proteins, in order to better understand a) the overall capability of dermatophytes to invade keratinised structures, and b) differences in virulence and host-specificity of the investigated species. To progress in the identification and characterization of novel proteins, the secretomes from Trichophyton rubrum, Trichophyton violaceum, Trichophyton soudanense, Trichophyton equinum, Arthroderma vanbreuseghemii and Trichophyton tonsurans were studied. Although there is a high global level of similarity among the secreted proteases, different dermatophyte species produce distinct patterns of proteins when grown in the same culture medium, and so a specific signature could be associated to each species. These patterns were useful to identify and map orthologous proteins among the six species, as well as to discriminate the closely related species T. tonsurans and T. equinum, which cannot be differentiated by ribosomal DNA. This work also presents the first in-depth identification of the major proteins secreted by dermatophytes growing under conditions promoting extracellular proteolytic activity. This catalogue of proteins, which include several endo- and exo- proteases, other hydrolases, oxydoreductases, and proteins of unknown function, probably represents the spectrum of enzymes that allow the degradation of keratinized tissues into compounds which can be assimilated by the fungus. The results suggest that ecological switching could be related to a differential expression of genes encoding secreted proteins, particularly, proteases, rather than genetic divergences of the genes encoding orthologous proteins. Differential secretion of proteins by Dermatophyte species could also be responsible for the variable inflammation caused by the infectious agent within the host. Therefore, the proteins here identified are also important to shed light into the immune response of the host during the infection process.

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

A genome-wide screen for interactions reveals a new locus on 4p15 modifying the effect of waist-to-hip ratio on total cholesterol.

Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain ∼25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened for variants that modify the relationship between known epidemiological risk factors and circulating lipid levels in a meta-analysis of genome-wide association (GWA) data from 18 population-based cohorts with European ancestry (maximum N = 32,225). We collected 8 further cohorts (N = 17,102) for replication, and rs6448771 on 4p15 demonstrated genome-wide significant interaction with waist-to-hip-ratio (WHR) on total cholesterol (TC) with a combined P-value of 4.79×10(-9). There were two potential candidate genes in the region, PCDH7 and CCKAR, with differential expression levels for rs6448771 genotypes in adipose tissue. The effect of WHR on TC was strongest for individuals carrying two copies of G allele, for whom a one standard deviation (sd) difference in WHR corresponds to 0.19 sd difference in TC concentration, while for A allele homozygous the difference was 0.12 sd. Our findings may open up possibilities for targeted intervention strategies for people characterized by specific genomic profiles. However, more refined measures of both body-fat distribution and metabolic measures are needed to understand how their joint dynamics are modified by the newly found locus.

siRNA-Mediated Knock-Down of P-Glycoprotein Expression Reveals Distinct Cellular Disposition of Anticancer Tyrosine Kinases Inhibitors.

Studies on the cellular disposition of targeted anticancer tyrosine kinases inhibitors (TKIs) have mostly focused on imatinib while the functional importance of P-glycoprotein (Pgp) the gene product of MDR1 remains controversial for more recent TKIs. By using RNA interference-mediated knockdown of MDR1, we have investigated and compared the specific functional consequence of Pgp on the cellular disposition of the major clinically in use TKIs imatinib, dasatinib, nilotinib, sunitinib and sorafenib. siRNA-mediated knockdown in K562/Dox cell lines provides a unique opportunity to dissect the specific contribution of Pgp to TKIs intracellular disposition. In these conditions, abrogating specifically Pgp-mediated efflux in vitro revealed the remarkable and statistically significant cellular accumulation of imatinib (difference in cellular levels between Pgp-expressing and silenced cells, at high and low incubation concentration, respectively: 6.1 and 6.6), dasatinib (4.9 and 5.6), sunitinib (3.7 and 7.3) and sorafenib (1.2 and 1.4), confirming that these TKIs are all substrates of Pgp. By contrast, no statistically significant difference in cellular disposition of nilotinib was observed as a result of MDR1 expression silencing (differences: 1.1 and 1.5) indicating that differential expression and/or function of Pgp is unlikely to affect nilotinib cellular disposition. This study enables for the first time a direct estimation of the specific contribution of one transporter among the various efflux and influx carriers involved in the cellular trafficking of these major TKIs in vitro. Knowledge on the distinct functional consequence of Pgp expression for these various TKIs cellular distribution is necessary to better appreciate the efficacy, toxicity, and potential drug-drug interactions of TKIs with other classes of therapeutic agents, at the systemic, tissular and cellular levels.

Molecular mechanisms of follicular associated epithelium differentiation

Les muqueuses respiratoires, genitales et digestives sont continuellement exposées aux antigènes de l?alimentation, à la flore intestinale et aux pathogènes. Cela implique une activité immunologique intense et finement régulée dans ces tissus. On admet que la modulation de ces réponses immunitaires muqueuses s?effectue dans des organes sentinels spécifiques appelés o-MALT (organized mucosal associated lymphoid tissues). Ces processus de modulation et la biologie de ces sites immuno-inducteurs sont peu connus. Ceci est pourtant d?une grande relevance si l?on veut faire un design rationnel de drogues et de vaccins muqueux. Dans l?intestin grèle, ces organes sont composés de follicules multiples et sont appelés plaques de Peyer. Ils sont constitués de follicules enrichis en cellules B comprenant ou non un centre germinatif, de regions interfolliculaires comprenant des cellules T, et d?une région en d ome riche en cellules dendritiques, cellules B naives et cellules T CD4+, surmontée par un epithelium specialisé, le FAE (epithelium associé aux follicules). Le FAE contient des cellules M spécialisées dans le transport de macromolécules et micro-organismes de la lumière intestinale au tissu lymphoide sous-jacent. Ce transport des antigènes est une condition obligatoire pour induire une réponse immunitaire. Les cellules du FAE, outre les cellules M, expriment un programme de différenciation distinct de celui des cellules associées aux villosités. Ceci est characterisé par une baisse des fonctions digestives et de défenses, et l?expression constitutive des chimiokines: CCL20 et CCL25. Le but de l?étude présentée ici est de rechercher les facteurs cellulaires et/ou moléculaire responsables de cette différenciation. Certaines études ont démontré l?importance du contact entre le compartiment mésenchymateux et l?épithelium pour la morphogenèse de ce dernier. En particulier, les molécules de la matrice extracellulaire peuvent activer des gènes clefs qui, à leur tour, vont controler l?adhésion et la differenciation cellulaire. Dans l?intestin, les cellules mésenchymateuses différencient en myofibroblastes qui participent à l?élaboration de la matrice extracellulaire. Dans cette étude, nous avons décrit les différences d?expression de molécules de la matrices sous le FAE et les villosités. Nous avons également montré une absence de myofibroblastes sous le FAE. Suite à plusieurs évidences expérimentales, certains ont proposé une influence des composés présents dans la lumière sur la différenciation et/ou la maturation des plaques de Peyer. La chimiokine CCL20, capable de recruter des cellules initiatrices de la réponse immunitaire, constitue notre seul marqueur positif de FAE. Nous avons pu montrer que la flagelline, un composé du flagelle bactérien, était capable d?induire l?expression de CCL20 in vitro et in vivo. Cet effet n?est pas limité aux cellules du FAE mais est observé sur l?ensemble de l?épithelium intestinal. Molecular mechanisms of FAE differenciation. La signalement induit par la lymphotoxine ß est critique pour l?organogenèse des plaques de Peyer, car des souris déficientes pour cette molécules ou son récepteur n?ont ni plaque de Peyer, ni la plupart des ganglions lymphatiques. Nous avons obtenus plusieurs évidences que la lymphotoxine ß était impliquée dans la régulation du gène CCL20 in vitro et in vivo.<br/><br/>Mucosal surfaces of the respiratory, genital and digestive systems are exposed to food antigens, normal bacterial flora and oral pathogens. This justifies an intense and tuned immunological activity in mucosal tissues. The modulation of immune responses in the mucosa is thought to occur in specific sentinel sites, the organized mucosa associated lymphoid tissues (o-MALT). This immune modulation and the biology of these immune-inductive sites are poorly understood but highly important and relevant in the case of drugs and vaccines design. In the small intestine, these organs (gut associated lymphoid tissue : GALT) consists of single or multiple lymphoid follicles, the so-called Peyer?s patches (PP), with typical B cell-enriched follicles and germinal centers, inter-follicular T cell areas, and a dome region enriched in dendritic cells, naive B cells, and CD4+ T cells under a specialized follicle associated epithelium (FAE). To trigger protective immunity, antigens have to cross the mucosal epithelial barrier. This is achieved by the specialized epithelial M cells of the FAE that are able to take up and transport macromolecules and microorganisms from the environment into the underlying organized lymphoid tissue. The ontogeny of M cells remains controversial: some data are in favor of a distinct cell lineage, while others provide evidence for the conversion of differentiated enterocytes into M cells. In this study we mapped the proliferative, M cells and apoptotic compartments along the FAE. Enterocytes acquire transient M cell features as they leave the crypt and regain enterocyte properties as they move towards the apoptotic compartment at the apex of the FAE, favouring the hypothesis of a plastic phenotype. The follicle-associated epithelium (FAE) is found exclusively over lymphoid follicles in mucosal tissues, including Peyer?s patches. The enterocytes over Peyer?s patches express a distinct phenotype when compared to the villi enterocytes, characterized by the down regulation of digestive and defense functions and the constitutive expression of chemokines, i.e. CCL20 and CCL25. The purpose of this study was to investigate and identify the potential cells and/or molecules instructing FAE differentiation. Contact between the epithelial and the mesenchymal cell compartment is required for gut morphogenesis. Extracellular matrix molecules (ECM) can activate key regulatory genes which in turn control cell adhesion and differentiation. In the gut, mesenchymal cells differentiate into myofibroblats that participate to the elaboration of ECM. We have described a differential expression of extracellular matrix components under the FAE, correlating with the absence of subepithelial myofibroblats. Molecular mechanisms of FAE differenciation. Different studies proposed an influence of the luminal compartment in the differentiation and/or the maturation of PP. CCL20, a chemokine able to recruit cells that initiate adaptive immunity constitutes our first positive FAE molecular marker. We have shown that CCL20 gene expression is inducible in vitro and in vivo in intestinal epithelium by flagellin, a component of bacterial flagella. This effect was not restricted to the FAE. Lymphotoxin ß (LTß) signaling is critical for PPs organogenesis as LT deficient mice as well as LTß-receptor-/- mice lack PPs and most of the lymph nodes (LN). The continuous signaling via LTßR-expressing cells appears necessary for the maintenance throughout the life of PP architecture. We obtained in vitro and in vivo evidence that LTß signalling is involved in CCL20 gene expression.

Pathogenic attack and carbon reallocation in soybean leaves (Glycine max. L.): Reinitiation of the glyoxylate cycle as a defence reaction

Pathogenic attack by the fungus Botrytis cinerea (primary pathogen) on soybean leaves (Glycine max. L.; cv. Maple arrow) results in a hypersensitive response (necrotising infected leaves), in the establishment of local acquired resistance, as well as in the systemic induction of genes coding for pathogenesis-related proteins. It now appears that, concomitantly with these already well documented defence reactions, the pathogenic attack also induces the carbon reallocation mechanism based on the reinitiation of the glyoxylate cycle (pseudo-senescence of the infected leaves).