http://repub.eur.nl/res/aut/7048/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/37825/ 2012-08-01T00:00:00Z IL-10 is an anti-inflammatory cytokine that regulates the extent of host immunity to infection by exerting suppressive effects on different cell types. Herpes viruses induce IL-10 to modulate the virus-host balance towards their own benefit, resulting in prolonged virus persistence. To define the cellular and molecular players involved in IL-10 modulation of herpes virus-specific immunity, we studied mouse cytomegalovirus (MCMV) infection. Here we demonstrate that IL-10 specifically curtails the MCMV-specific CD4 T cell response by suppressing the bidirectional crosstalk between NK cells and myeloid dendritic cells (DCs). In absence of IL-10, NK cells licensed DCs to effectively prime MCMV-specific CD4 T cells and we defined the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 to regulate this bidirectional NK/DC interplay. Consequently, markedly enhanced priming of MCMV-specific CD4 T cells in Il10-/-mice led to faster control of lytic viral replication, but this came at the expense of TNF-α mediated immunopathology. Taken together, our data show that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive immune cell crosstalk, thereby impacting beneficially on the ensuing virus-host balance for both the virus and the host. http://repub.eur.nl/res/pub/2352/ 1994-01-01T00:00:00Z We have isolated a human collagen alpha 1(I)-like gene from a cosmid library. The clone which contains 37kb of human DNA has been shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations. The collagen gene sequence extends through most of the cloned DNA and must, therefore, be at least 35kb in length. http://repub.eur.nl/res/pub/2374/ 1984-01-01T00:00:00Z The transplantation antigens H-2K, H-2D and H-2L are developmentally regulated, highly polymorphic cell surface proteins encoded by the major histocompatibility gene complex (MHC). First detectable on the early embryo, they are subsequently expressed on most somatic cells of the adult mouse in association with the protein beta2-microglobulin (beta 2 M; ref. 5). Cultured F embryonal carcinoma (EC) cells can be induced to differentiate along alternative pathways to form either parietal or visceral9 extra-embryonic endoderm, each concomitant with a change in morphology and pattern of gene expression. Previous reports have demonstrated an increased level of transplantation antigens in differentiated F9 EC cells, but the cell types expressing them were not defined. Here we show that the level of MHC H-2Kb and beta 2 M transcripts is increased during both pathways of this differentiation. Expression of a foreign MHC H-2Kbm1 gene was found to be regulated in a similar manner when the gene was introduced into EC cells. In contrast, an introduced rabbit beta-globin gene was not regulated but expressed constitutively. http://repub.eur.nl/res/pub/2375/ 1984-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/2376/ 1984-01-01T00:00:00Z We have introduced into murine erythroleukaemia (MEL) cells a series of human globin gene cosmids and two sets of hybrid genes constructed from the human beta-globin gene and the human gamma-globin or murine H-2Kbm1 genes. S1-nuclease analysis of the mRNA products from these genes before and after MEL cell differentiation showed that the human beta-globin gene, but not the human epsilon- or gamma-globin or H-2Kbm1 genes, is induced specifically. Hybrid genes containing human beta-globin DNA sequences from either 5' or 3' side of the translation initiation site were both inducible. Measurement of the relative rate of transcription showed this induction to be the result of transcriptional activation. We therefore suggest that DNA sequences which regulate beta-globin gene expression during MEL differentiation are located both 5' and 3' to the translation initiation site. http://repub.eur.nl/res/pub/2356/ 1983-01-01T00:00:00Z http://repub.eur.nl/res/pub/2364/ 1983-01-01T00:00:00Z The beta-globin gene present on the deletion locus in a Dutch gamma beta-thalassaemic patient was found to be identical to the normal beta-globin gene with respect to DNA sequence and its transcription in HeLa cells. DNase I sensitivity and methylation experiments show that the affected beta-globin gene is present in an inactive configuration in vivo. This is the result of a translocation of a normally inactive locus next to the beta-globin gene on the affected chromosome, or the deletion of sequences which are normally required for the maintenance of the active state. http://repub.eur.nl/res/pub/2366/ 1983-01-01T00:00:00Z MspI essentially fails to cut the sequence GGCmCGG at enzyme concentrations which give total digestion of CCGG, CmCGG and GGCCGG sites. This result explains why certain sites in mammalian DNA are resistant to both MspI and HpaII and shows that this results from an idiosynchracy of MspI rather than a novel form of DNA methylation at this site in mammalian cells. http://repub.eur.nl/res/pub/2367/ 1983-01-01T00:00:00Z Chemically induced differentiation of cultured murine erythroleukaemia (MEL) cells results in a several hundred-fold increase in transcription of the adult mouse globin genes and thus serves as a model for gene activation during erythropoiesis. One approach to study gene regulation in this system has been to analyse the expression of foreign globin genes introduced into MEL cells. By introducing cosmid DNA containing the human adult(beta), fetal(gamma) and embryonic(epsilon)-globin genes, we have shown here that expression of the beta, but not the gamma or epsilon genes, is regulated during MEL differentiation. Regulated expression of the human beta-globin gene was observed when it was introduced either as part of the intact globin gene cluster or as an individual gene with 1.5 kilobases (kb) of 5' flanking DNA. Transcription from a herpes simplex virus (HSV) promoter adjacent to the thymidine kinase (tk) gene is also inducible in MEL cells. http://repub.eur.nl/res/pub/2349/ 1982-01-01T00:00:00Z A procedure has been developed to allow the recovery of an integrated plasmid genome from a transformed cell, together with large areas of the flanking DNA sequences. DNA from Saccharomyces cerevisiae BAS2, in which the pBR322--ura 3 plasmid (Y1p5) is integrated at the yeast histone H2A and H2B locus, was used to generate a cosmid library, using a new cosmid vector (pTL5) that is ampicillin sensitive and tetracycline resistant. Colonies were selected for ampicillin resistance, which was conferred by the incorporation of the integrated pBR322 beta-lactamase gene into the recombinant cosmid. Restriction enzyme and blot hybridization analyses show that the rescued clones contain the yeast histone genes in addition to the Y1p5 sequences; a total of approximately 50 kilobase pairs of DNA sequences flanking the plasmid was recovered as a series of overlapping cosmids. This approach should allow the recovery of most genes that can be linked to a marker pBR322 sequence and for which a specific phenotype can be selected in a recipient eukaryotic cell. http://repub.eur.nl/res/pub/2350/ 1982-01-01T00:00:00Z http://repub.eur.nl/res/pub/2353/ 1982-01-01T00:00:00Z http://repub.eur.nl/res/pub/2362/ 1982-01-01T00:00:00Z Cosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E. coli guanine phosphoribosyltransferase gene). The design of the cosmids allows the exchange of the eukaryotic markers in recombinant cosmids. Human and mouse cosmid libraries containing DNA inserts of about 40kb have been generated by an improved method. Several clones from the human beta-globin locus were isolated. These cosmids transform mouse L cells at high efficiency in both circular and linear form. The newly introduced genes are expressed accurately in L cells. http://repub.eur.nl/res/pub/2342/ 1981-04-01T00:00:00Z 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. http://repub.eur.nl/res/pub/2347/ 1981-01-01T00:00:00Z We have cloned the single beta-globin gene from an Italian patient who is a double heterozygote for beta o/delta beta o thalassaemia. RNA isolated from nucleated red cells from this patient can be translated in vitro to give detectable levels of A gamma- G gamma and alpha-globin, but no beta-globin. S1-mapping transcription studies show that beta-globin mRNA is present at about 1-3% of the level of alpha- and gamma-globin mRNA. In addition, the expression of this gene has been studied by reversed genetics. SV40-plasmid-beta o-globin gene recombinants have been transfected into Hela cells and analysed for beta-globin mRNA. In contrast to the results obtained with mRNA isolated directly from the blood of this patient, in the transfected Hela cells the same level of beta-globin mRNA is seen for the beta o thalassaemic globin gene and for a normal beta-globin gene. To elucidate the nature of the lesion, the entire DNA sequence of the beta-globin gene of this patient has been determined. The sequence shows that this gene contains a termination codon at position 39 (CAG - greater than UAG). Otherwise, there is a remarkable conservation of the entire DNA sequence.