DNA sequences required for regulated expression of β-globin genes in murine erythroleukaemia cells.

We introduced into MEL cells rabbit beta-globin gene deletion mutants and two sets of hybrid genes constructed from the inducible human beta-globin gene and noninducible human gamma-globin gene or the murine H-2Kbm1 class I MHC gene. S1 nuclease analysis of gene transcripts before and after MEL differentiation showed that induction of the rabbit beta-globin gene did not require more than 58 bp of DNA 5' to the transcription initiation site. Hybrid genes were constructed with human beta-globin DNA sequences from either 5' or 3' of the translation initiation site linked to the complementary parts of the gamma or H2Kbm1 genes. Both types of constructs were inducible during MEL differentiation. The relative rates of transcription of the 5' gamma-3' beta and 5'H2-3' beta hybrid genes show that induction of the hybrid gene transcripts results at least in part from transcriptional activation of the genes. We suggest that DNA sequences that regulate beta-globin gene transcription during MEL differentiation are located both 5' and 3' to the translation initiation site

Isolation of β-globin related genes from a human cosmid library.

A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150 000 recombinant-DNA-containing colonies were screened for the presence of the human beta-globin related genes. Five recombinants were isolated containing the human beta-globin locus and encompassing approx. 70 kb of human DNA

Role of IRAK-M in Alcohol Induced Liver Injury

Increasing evidence suggests that innate immunity plays an important role in alcohol-induced liver injury and most studies have focused on positive regulation of innate immunity. The main objective of this study was to investigate the negative regulator of innate immunity, IL-1/Toll-like receptor (TLR) signaling pathways and interleukin receptor-associated kinase-M (IRAK-M) in alcoholic liver injury. We established an alcohol-induced liver injury model using wild type and IRAK-M deficient B6 mice and investigated the possible mechanisms. We found that in the absence of IRAK-M, liver damage by alcohol was worse with higher alanine transaminase (ALT), more immune cell infiltration and increased numbers of IFNγ producing cells. We also found enhanced phagocytic activity in CD68+ cells. Moreover, our results revealed altered gut bacteria after alcohol consumption and this was more striking in the absence of IRAK-M. Our study provides evidence that IRAK-M plays an important role in alcohol-induced liver injury and IRAK-M negatively regulates the innate and possibly the adaptive immune response in the liver reacting to acute insult by alcohol. In the absence of IRAK-M, the hosts developed worse liver injury, enhanced gut permeability and altered gut microbiota

IL-10 producing regulatory and helper T-cells in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a highly heterogeneous autoimmune disease characterised by the production of pathogenic autoantibodies against nuclear self-antigens. The anti-inflammatory and tolerogenic cytokine Interleukin-10 appears to play a paradoxical pathogenic role in SLE and is therefore currently therapeutically targeted in clinical trials. It is generally assumed that the pathogenic effect of IL-10 in SLE is due to its growth and differentiation factor activity on autoreactive B-cells, but effects on other cells might also play a role. To date, a unique cellular source of pathogenic IL-10 in SLE has not been identified. In this review, we focus on the contribution of different CD4+T-cell subsets to IL-10 and autoantibody production in SLE. In particular, we discuss that IL-10 produced by different subsets of adaptive regulatory T-cells, follicular helper T-cells and extra-follicular B-helper T-cells is likely to have different effects on autoreactive B-cell responses. A better understanding of the role of IL-10 in B-cell responses and lupus would allow to identify the most promising therapies for individual SLE patients in the future

Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression

CIITA activates the expression of multiple genes involved in antigen presentation and it is believed to be required for both constitutive and IFN\xce\xb3-inducible expression of these genes. To understand the role of CIITA in vivo, we have used gene targeting to generate mice that lack CIITA. CIITA-deficient (-/-) mice do not express conventional MHC class II molecules on the surface of splenic B cells and dendritic cells. In addition, macrophages resident in the peritoneal cavity do not express MHC class II molecules upon IFN\xce\xb3 stimulation nor do somatic tissues of mice injected with IFN\xce\xb3, in contrast with wild-type mice. The levels of li and H-2M gene transcripts are substantially decreased but not absent in CIITA (-/-) mice. The transcription of nonconventional MHC class II genes is, however, not affected by CIITA deficiency. A subset of thymic epithelial cells express MHC class II molecules. Nonetheless, very few mature CD4 T cells are present in the periphery of CIITA (-/-) mice despite MHC class II expression in the thymus. Consequently, CIITA (-/-) mice are impaired in T-dependent antigen responses and MHC class II-mediated allogeneic reponses

Molecular and functional heterogeneity of IL-10-producing CD4 + T cells

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4 + T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3 neg CD4 + T cells that displays regulatory activity unlike other IL-10-producing CD4 + T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4 + T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3 neg CD4 + T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation

Tomato: a crop species amenable to improvement by cellular and molecular methods

Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures. In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.

Oscillatory networks of high-level mental alignment::A perspective-taking MEG study

Mentally imagining another's perspective is a high-level social process, reliant on manipulating internal representations of the self in an embodied manner. Recently Wang et al. (2016) showed that theta-band (3–7 Hz) brain oscillations within the right temporo-parietal junction (rTPJ) and brain regions coding for motor/body schema contribute to the process of perspective-taking. Using a similar paradigm, we set out to unravel the extended functional brain network in detail. Increasing the angle between self and other perspective was accompanied by longer reaction times and increases in theta power within rTPJ, right lateral prefrontal cortex (PFC) and right anterior cingulate cortex (ACC). Using Granger-causality, we showed that lateral PFC and ACC exert top-down influence over rTPJ, indicative of executive control processes required for managing conflicts between self and other perspectives. Finally, we quantified patterns of whole-brain phase coupling in relation to the rTPJ. Results suggest that rTPJ increases its theta-band phase synchrony with brain regions involved in mentalizing and regions coding for motor/body schema; whilst decreasing synchrony to visual regions. Implications for neurocognitive models are discussed, and it is proposed that rTPJ acts as a ‘hub’ to route bottom-up visual information to internal representations of the self during perspective-taking, co-ordinated by theta-band oscillations