Some of the early events underlying Th2 cell maturation and susceptibility to Leishmania major infection in BALB/c mice.

The first experimental evidence for the development of polarized CD4+ Th1 and Th2 responses in vivo has been obtained using the murine model of infection with Leishmania major, an intracellular parasite of macrophages in their vertebrate host. Genetically determined resistance and susceptibility to infection with this parasite have been clearly demonstrated to result from the development of polarized Th1 and Th2 responses, respectively. Using this model system, the dominant role of cytokines in the induction of polarized CD4+ responses has been validated in vivo. The requisite role of IL-4 in mediating both Th2 differentiation and susceptibility to infection in BALB/c mice has directed interest towards the search for evidence of IL-4 production early after infection and identification of its cellular source. We have been able to demonstrate a burst of IL-4 production in susceptible BALB/c mice within the first day of infection with L. major and could establish that this rapidly produced IL-4 instructed Th2 lineage commitment of subsequently activated CD4+ T cells and stabilized this commitment by downregulating IL-12 Rbeta2 chain expression, resulting in susceptibility to infection. Strikingly, this early IL-4 response to infection resulted from the cognate recognition of a single epitope in a distinctive antigen, LACK, from this complex microorganism by a restricted population of CD4+ T cells that express Vbeta4-Valpha8 T cell receptors.

One naive T cell, multiple fates in CD8+ T cell differentiation.

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR-pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.

Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation.

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. We show that keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation.

Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation.

Jagged1-mediated Notch signaling has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. Unexpectedly, we report here that inducible Cre-loxP-mediated inactivation of the Jagged1 gene in bone marrow progenitors and/or bone marrow (BM) stromal cells does not impair HSC self-renewal or differentiation in all blood lineages. Mice with simultaneous inactivation of Jagged1 and Notch1 in the BM compartment survived normally following a 5FU-based in vivo challenge. In addition, Notch1-deficient HSCs were able to reconstitute mice with inactivated Jagged1 in the BM stroma even under competitive conditions. In contrast to earlier reports, these data exclude an essential role for Jagged1-mediated Notch signaling during hematopoiesis.

High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism.

Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

Peroxisome proliferator-activated receptor mediates cross-talk with thyroid hormone receptor by competition for retinoid X receptor. Possible role of a leucine zipper-like heptad repeat.

The peroxisome proliferator-activated receptors (PPAR) and thyroid hormone receptors (TR) are members of the nuclear receptor superfamily, which regulate lipid metabolism and tissue differentiation. In order to bind to DNA and activate transcription, PPAR requires the formation of heterodimers with the retinoid X receptor (RXR). In addition to activating transcription through its own response elements, PPAR is able to selectively down-regulate the transcriptional activity of TR, but not vitamin D receptor. The molecular basis of this functional interaction has not been fully elucidated. By means of site-directed mutagenesis of hPPAR alpha we mapped its inhibitory action on TR to a leucine zipper-like motif in the ligand binding domain of PPAR, which is highly conserved among all subtypes of this receptor and mediates heterodimerization with RXR. Replacement of a single leucine by arginine at position 433 of hPPAR alpha (L433R) abolished heterodimerization of PPAR with RXR and consequently its trans-activating capacity. However, a similar mutation of a leucine residue to arginine at position 422 showed no alteration of heterodimerization, DNA binding, or transcriptional activation. The dimerization deficient mutant L433R was no longer able to inhibit TR action, demonstrating that the selective inhibitory effect of PPAR results from the competition for RXR as well as possibly for other TR-auxiliary proteins. In contrast, abolition of DNA binding by a mutation in the P-box of PPAR (C122S) did not eliminate the inhibition of TR trans-activation, indicating that competition for DNA binding is not involved. Additionally, no evidence for the formation of PPAR:TR heterodimers was found in co-immunoprecipitation experiments. In summary, we have demonstrated that PPAR selectively inhibits the transcriptional activity of TRs by competition for RXR and possibly non-RXR TR-auxiliary proteins. In contrast, this functional interaction is independent of the formation of PPAR:TR heterodimers or competition for DNA binding.

The human epidermal differentiation complex: cornified envelope precursors, S100 proteins and the 'fused genes' family.

  The skin is essential for survival and protects our body against biological attacks, physical stress, chemical injury, water loss, ultraviolet radiation and immunological impairment. The epidermal barrier constitutes the primordial frontline of this defense established during terminal differentiation. During this complex process proliferating basal keratinocytes become suprabasally mitotically inactive and move through four epidermal layers (basal, spinous, granular and layer, stratum corneum) constantly adapting to the needs of the respective cell layer. As a result, squamous keratinocytes contain polymerized keratin intermediate filament bundles and a water-retaining matrix surrounded by the cross-linked cornified cell envelope (CE) with ceramide lipids attached on the outer surface. These cells are concomitantly insulated by intercellular lipid lamellae and hold together by corneodesmosmes. Many proteins essential for epidermal differentiation are encoded by genes clustered on chromosomal human region 1q21. These genes constitute the 'epidermal differentiation complex' (EDC), which is divided on the basis of common gene and protein structures, in three gene families: (i) CE precursors, (ii) S100A and (iii) S100 fused genes. EDC protein expression is regulated in a gene and tissue-specific manner by a pool of transcription factors. Among them, Klf4, Grhl3 and Arnt are essential, and their deletion in mice is lethal. The importance of the EDC is further reflected by human diseases: FLG mutations are the strongest risk factor for atopic dermatitis (AD) and for AD-associated asthma, and faulty CE formation caused by TG1 deficiency causes life-threatening lamellar ichthyosis. Here, we review the EDC genes and the progress in this field.

The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-mum thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.

Topical and intravitreous administration of cationic nanoemulsions to deliver antisense oligonucleotides directed towards VEGF KDR receptors to the eye.

Antisense oligonucleotides (ODNs) specific for VEGFR-2-(17 MER) and inhibiting HUVEC proliferation in-vitro were screened. One efficient sequence was selected and incorporated in different types of nanoemulsions the potential toxicity of which was evaluated on HUVEC and ARPE19 cells. Our results showed that below 10 microl/ml, a 2.5% mid-chain triglycerides cationic DOTAP nanoemulsion was non-toxic on HUVEC and retinal cells. This formulation was therefore chosen for further experiments. In-vitro transfection of FITC ODNs in ARPE cells using DOTAP nanoemulsions showed that nanodroplets do penetrate into the cells. Furthermore, ODNs are released from the nanoemulsion after 48 h and accumulate into the cell nuclei. In both ex-vivo and in-vivo ODN stability experiments in rabbit vitreous, it was noted that the nanoemulsion protected at least partially the ODN from degradation over 72 h. The kinetic results of fluorescent ODN (Hex) distribution in DOTAP nanoemulsion following intravitreal injection in the rat showed that the nanoemulsion penetrates all retinal cells. Pharmacokinetic and ocular tissue distribution of radioactive ODN following intravitreal injection in rabbits showed that the DOTAP nanoemulsion apparently enhanced the intraretinal penetration of the ODNs up to the inner nuclear layer (INL) and might yield potential therapeutic levels of ODN in the retina over 72 h post injection.

Development and selection of Valpha l4i NKT cells.

Valpha14 invariant natural killer T (Valpha14i NKT) cells are a unique lineage of mouse T cells that share properties with both NK cells and memory T cells. Valpha14i NKT cells recognize CDld-associated glycolipids via a semi-invariant T cell receptor (TCR) composed of an invariant Valpha14-Jalpha 18 chain paired preferentially with a restricted set of TCRbeta chains. During development in the thymus, rare CD4+ CD8+ (DP) cortical thymocytes that successfully rearrange the semi-invariant TCR are directed to the Valpha14i NKT cell lineage via interactions with CD d-associated endogenous glycolipids expressed by other DP thymocytes. As they mature, Valphal4i NKT lineage cells upregulate activation markers such as CD44 and subsequently express NK-related molecules such as NKI.1 and members of the Ly-49 inhibitory receptor family. The developmental program of Valpha l4i NKT cells is critically regulated by a number of signaling cues that have little or no effect on conventional T cell development, such as the Fyn/SAP/SLAM pathway, NFkappaB and T-bet transcription factors, and the cytokine IL-15. The unique developmental requirements of Valphal4i NKT cells may represent a paradigm for other unconventional T cell subsets that are positively selected by agonist ligands expressed on hematopoietic cells.

BCL9 is an essential component of canonical Wnt signaling that mediates the differentiation of myogenic progenitors during muscle regeneration.

Muscle stem cells and their progeny play a fundamental role in the regeneration of adult skeletal muscle. We have previously shown that activation of the canonical Wnt/beta-catenin signaling pathway in adult myogenic progenitors is required for their transition from rapidly dividing transient amplifying cells to more differentiated progenitors. Whereas Wnt signaling in Drosophila is dependent on the presence of the co-regulator Legless, previous studies of the mammalian ortholog of Legless, BCL9 (and its homolog, BCL9-2), have not revealed an essential role of these proteins in Wnt signaling in specific tissues during development. Using Cre-lox technology to delete BCL9 and BCL9-2 in the myogenic lineage in vivo and RNAi technology to knockdown the protein levels in vitro, we show that BCL9 is required for activation of the Wnt/beta-catenin cascade in adult mammalian myogenic progenitors. We observed that the nuclear localization of beta-catenin and downstream TCF/LEF-mediated transcription, which are normally observed in myogenic progenitors upon addition of exogenous Wnt and during muscle regeneration, were abrogated when BCL9/9-2 levels were reduced. Furthermore, reductions of BCL9/9-2 inhibited the promotion of myogenic differentiation by Wnt and the normal regenerative response of skeletal muscle. These results suggest a critical role of BCL9/9-2 in the Wnt-mediated regulation of adult, as opposed to embryonic, myogenic progenitors.

Genetic differentiation of common shrew Sorex araneus populations among different alpine valleys revealed by microsatellites

Geographical barriers may affect the genetic structure of populations by reducing gene exchanges among them. In Switzerland, the common shrew Sorer araneus Linnaeus, 1758 is mostly confined to mountainous areas because of a competing sister species, Millet's shrew S. coronatus Millet, 1828, which occupies most of the Swiss lowlands. The structure of common shrew populations found in different alpine valleys may therefore be affected by the topography. Using microsatellites, genetic structuring of seven shrew populations is investigated among four different valleys of, the Swiss Alps. Using the exact G-test, significant genetic structuring is detected between several valleys. Isolation by distance does not fully explain our results. It appears that high mountain ridges (> 2400 m) can significantly reduce gene flow. F- and R-statistics are estimated and compared to the exact G-tests results. Mantel tests show that F-ST, unlike R-ST, is significantly correlated with differentiation. F-ST remains however low even at high differentiation levels, while R-ST has a high variance. We discuss how these results may have wider implications with regards the interpretation of microsatellite data. Finally, a new microsatellite locus, L99, appears to discriminate S. araneus of the Vaud and Cordon races from both S. araneus Valais and S. coronatus.

Cognate peptide-receptor ligand mapping by directed phage display

Background: A rapid phage display method for the elucidation of cognate peptide specific ligand for receptors is described. The approach may be readily integrated into the interface of genomic and proteomic studies to identify biologically relevant ligands.Methods: A gene fragment library from influenza coat protein haemagglutinin (HA) gene was constructed by treating HA cDNA with DNAse I to create 50 ¿ 100 bp fragments. These fragments were cloned into plasmid pORFES IV and in-frame inserts were selected. These in-frame fragment inserts were subsequently cloned into a filamentous phage display vector JC-M13-88 for surface display as fusions to a synthetic copy of gene VIII. Two well characterized antibodies, mAb 12CA5 and pAb 07431, directed against distinct known regions of HA were used to pan the library. Results: Two linear epitopes, HA peptide 112 ¿ 126 and 162¿173, recognized by mAb 12CA5 and pAb 07431, respectively, were identified as the cognate epitopes.Conclusion: This approach is a useful alternative to conventional methods such as screening of overlapping synthetic peptide libraries or gene fragment expression libraries when searching for precise peptide protein interactions, and may be applied to functional proteomics.

Membrane mu poly(A) signal and 3' flanking sequences function as a transcription terminator for immunoglobulin-encoding genes.

Developmentally regulated mechanisms involving alternative RNA splicing and/or polyadenylation, as well as transcription termination, are implicated in controlling the levels of secreted mu (mu s), membrane mu (mu m) and delta immunoglobulin (Ig) heavy chain mRNAs during B cell differentiation (mu gene encodes the mu heavy chain). Using expression vectors constructed with genomic DNA segments composed of the mu m polyadenylation signal region, we analyzed poly(A) site utilization and termination of transcription in stably transfected myeloma cells and in murine fibroblast L cells. We found that the gene segment containing the mu m poly(A) signals, along with 536 bp of downstream flanking sequence, acted as a transcription terminator in both myeloma cells and L cell fibroblasts. Neither a 141-bp DNA fragment (which directed efficient polyadenylation at the mu m site), nor the 536-bp flanking nucleotide sequence alone, were sufficient to obtain a similar regulation. This shows that the mu m poly(A) region plays a central role in controlling developmentally regulated transcription termination by blocking downstream delta gene expression. Because this gene segment exhibited the same RNA processing and termination activities in fibroblasts, it appears that these processes are not tissue-specific.