The type 1 diabetes susceptibility locus Idd5 favours robust neonatal development of highly autoreactive regulatory T cells in the NOD mouse.

Regulatory T lymphocytes expressing the transcription factor Foxp3 (Tregs) play an important role in the prevention of autoimmune diseases and other immunopathologies. Aberrations in Treg-mediated immunosuppression are therefore thought to be involved in the development of autoimmune pathologies, but few have been documented. Recent reports indicated a central role for Tregs developing during the neonatal period in the prevention of autoimmune pathology. We therefore investigated the development of Tregs in neonatal NOD mice, an important animal model for autoimmune type 1 diabetes. Surprisingly, we found that, as compared with seven other commonly studied inbred mouse strains, in neonatal NOD mice, exceptionally large proportions of developing Tregs express high levels of GITR and PD-1. The latter phenotype was previously associated with high Treg autoreactivity in C57BL/6 mice, which we here confirm for NOD animals. The proportions of newly developing GITRhighPD-1+ Tregs rapidly drop during the first week of age. A genome-wide genetic screen indicated the involvement of several diabetes susceptibility loci in this trait. Analysis of a congenic mouse strain confirmed that Idd5 contributes to the genetic control of GITRhighPD-1+ Treg development in neonates. Our data thus demonstrate an intriguing and paradoxical correlation between an idiosyncrasy in Treg development in NOD mice and their susceptibility to type 1 diabetes.

IL-2 and IL-15 drive intrathymic development of distinct periphery-seeding CD4+Foxp3+ regulatory T lymphocytes.

Development of Foxp3-expressing regulatory T-lymphocytes (Treg) in the thymus is controlled by signals delivered in T-cell precursors via the TCR, co-stimulatory receptors, and cytokine receptors. In absence of IL-2, IL-15 or their receptors, fewer Treg apparently develop in the thymus. However, it was recently shown that a substantial part of thymic Treg are cells that had recirculated from the periphery back to the thymus, troubling interpretation of these results. We therefore reassessed the involvement of IL-2 and IL-15 in the development of Treg, taking into account Treg-recirculation. At the age of three weeks, when in wt and IL-15-deficient (but not in IL-2-deficient) mice substantial amounts of recirculating Treg are present in the thymus, we found similarly reduced proportions of newly developed Treg in absence of IL-2 or IL-15, and in absence of both cytokines even less Treg developed. In neonates, when practically no recirculating Treg were found in the thymus, the absence of IL-2 led to substantially more reduced Treg-development than deficiency in IL-15. IL-2 but not IL-15 modulated the CD25, GITR, OX40, and CD73-phenotypes of the thymus-egress-competent and periphery-seeding Treg-population. Interestingly, IL-2 and IL-15 also modulated the TCR-repertoire expressed by developing Treg. Upon transfer into Treg-less Foxp3sf mice, newly developed Treg from IL-2- (and to a much lesser extent IL-15-) deficient mice suppressed immunopathology less efficiently than wt Treg. Taken together, our results firmly establish important non-redundant quantitative and qualitative roles for IL-2 and, to a lesser extent, IL-15 in intrathymic Treg-development.

The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small-cell lung cancer via an AKT-dependent mechanism.

Although lung cancer patients harboring EGFR mutations benefit from treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKI), most of them rapidly relapse. RHOB GTPase is a critical player in both lung carcinogenesis and the EGFR signaling pathway; therefore, we hypothesized that it could play a role in the response to EGFR-TKI In a series of samples from EGFR-mutated patients, we found that low RHOB expression correlated with a good response to EGFR-TKI treatment while a poor response correlated with high RHOB expression (15.3 versus 5.6 months of progression-free survival). Moreover, a better response to EGFR-TKI was associated with low RHOB levels in a panel of lung tumor cell lines and in a lung-specific tetracycline-inducible EGFRL858R transgenic mouse model. High RHOB expression was also found to prevent erlotinib-induced AKT inhibition in vitro and in vivo Furthermore, a combination of the new-generation AKT inhibitor G594 with erlotinib induced tumor cell death in vitro and tumor regression in vivo in RHOB-positive cells. Our results support a role for RHOB/AKT signaling in the resistance to EGFR-TKI and propose RHOB as a potential predictor of patient response to EGFR-TKI treatment.

Single-strand DNA translation initiation step analyzed by Isothermal Titration Calorimetry.

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K(d)=3.62+/-2.1 x 10(-8)M) or the RNA corresponding sequence (K(d)=2.7+/-0.82 x 10(-8) M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.

Epigenetic regulation of RhoB loss of expression in lung cancer.

BACKGROUND: RhoB is down-regulated in most lung cancer cell lines and tumor tissues when compared with their normal counterparts. The mechanism of this loss of expression is not yet deciphered. METHODS: Since no mutation has been reported in the RhoB sequence, we investigated the epigenetic regulation of RhoB expression by analyzing the effect of HDAC inhibitors and methyltransferase inhibitors, by direct sequencing after bisulfite treatment and by methylation specific PCR. RESULTS: We first showed that histone deacetylase (HDAC) inhibitors induce a significant RhoB re-expression in lung cancer cell lines whereas only a slight effect was observed with methyl transferase inhibitors. As promoter methylation is the most common epigenetic process in lung cancer, we performed methylation specific PCR and sequence analysis after bisulfite treatment and demonstrated that RhoB was methylated neither in lung cancer cell lines nor in tumor tissues. We also showed that a variable number of tandem repeats sequences in the 5' region of the RhoB gene was involved in HDAC response. CONCLUSION: We thus propose that RhoB regulation of expression occurs mainly by histone deacetylation rather than by promoter hypermethylation and that this process can be modulated by specific 5' sequences within the promoter.

A new lectin family with structure similarity to actinoporins revealed by the crystal structure of Xerocomus chrysenteron lectin XCL.

A newly defined family of fungal lectins displays no significant sequence similarity to any protein in the databases. These proteins, made of about 140 amino acid residues, have sequence identities ranging from 38% to 65% and share binding specificity to N-acetyl galactosamine. One member of this family, the lectin XCL from Xerocomus chrysenteron, induces drastic changes in the actin cytoskeleton after sugar binding at the cell surface and internalization, and has potent insecticidal activity. The crystal structure of XCL to 1.4 A resolution reveals the architecture of this new lectin family. The fold of the protein is not related to any of the several lectin folds documented so far. Unexpectedly, the structure similarity is significant with actinoporins, a family of pore-forming toxins. The specific structural features and sequence signatures in each protein family suggest a potential sugar binding site in XCL and a possible evolutionary relationship between these proteins. Finally, the tetrameric assembly of XCL reveals a complex network of protomer-protomer interfaces and generates a large, hydrated cavity of 1000 A3, which may become accessible to larger solutes after a small conformational change of the protein.

Inhibitory action of a new lectin from Xerocomus chrysenteron on cell-substrate adhesion.

Lectins are carbohydrate-binding proteins which potentially link to cell surface glycoconjugates and affect cell proliferation. We investigated the effect of a new lectin from the mushroom Xerocomus chrysenteron (XCL) on cell proliferation using adherent and suspension cell lines. XCL caused a dose-dependent inhibition of proliferation of the adherent cell lines NIH-3T3 and HeLa. Several experiments suggest that disruption of cell-substrate adhesion is the main factor affecting cell growth inhibition. (i) No antiproliferative effect was observed on the SF9 cell line, which does not require to be attached to grow. (ii) XCL was shown to affect the adherence of cells following their suspension by trypsin treatment. (iii) XCL was localized on the cell surface where it would act as a coating agent. (iv) XCL induced morphological changes from well spread to rounded cells and disrupted the actin cytoskeleton. By contrast, flow cytometric analysis showed that XCL does not interfere with the cell cycle, and does not induce apoptosis.

Fungal lectin, XCL, is internalized via clathrin-dependent endocytosis and facilitates uptake of other molecules.

The lectin isolated from Xerocomus chrysenteron (XCL) displays a toxic activity towards insects. In order to assess its possible mode of action and to gather useful data for its potential use in insect-resistant transgenic plants, we investigated the effects of XCL at the cellular level. Immunofluorescence microscopy studies revealed that XCL is rapidly internalized into small endocytic vesicles that further coalesce in the perinuclear region. We show that XCL is endocytosed by the clathrin-dependent pathway, and is delivered to late endosome/lysosome compartments. The internalization of XCL seems to be general since it occurs in different cell types such as insect (SF9) or mammalian (NIH-3T3 and Hela) cell lines. In the presence of XCL, the uptake of GFP and BSA is greatly enhanced, demonstrating that XCL facilitates endocytosis. Thus, XCL could serve as a delivery agent to facilitate the endocytosis of proteins that do not enter the cell alone.

Human cytomegalovirus induces drug resistance and alteration of programmed cell death by accumulation of deltaN-p73alpha.

Intrauterine transmission of human cytomegalovirus (HCMV) to the fetus following primary infection in early and late pregnancy usually results in severe neurological handicaps and sensorineural hearing loss with typical migrational anomalies, optic atrophy, disturbed myelination, cerebella hypoplasia, microcephaly, hydrocephaly, and lissencephaly. Recently, evidences raised from the phenotype of p73-deficient mice show that an association may exist between the expression of the TP53 homologous gene and HCMV tropism in the brain, suggesting an implication of p73 in viral persistence. In this study, we demonstrated that HCMV-mediated inhibition of apoptosis only occurs in p73-expressing cells. Upon infection, an accumulation of deltaN-p73alpha isoforms was observed in HCMV-infected p73-positive cells. This phenomenon was shown to be responsible for the subsequent acquired resistance to apoptosis of infected cells. Inhibition of apoptosis in p73-positive cells by HCMV may thus contribute both to virus persistency and abnormal nervous cell survival. This finding provides the first molecular basis for HCMV-associated abnormal embryonic development and neurological defects in newborns.