N-methyl-2-pyrrolidone (NMP) is a versatile water-miscible polar aprotic solvent. It is used as a drug solubilizer and penetration enhancer in human and animal, yet its bioactivity properties remain elusive. Here, we report that NMP is a bioactive anti-inflammatory compound well tolerated in vivo, that shows efficacy in reducing disease in a mouse model of atherosclerosis.
View Article and Find Full Text PDFThe type I interferons, interferon-alpha (IFN-alpha) and interferon-beta (IFN-beta), are situated on the short arm of chromosome 9, specifically 9p21-22. This locus lies very close to an area that is deleted or rearranged in nearly half of all melanomas tested. The identification of 9p rearrangements in both melanoma precursor lesions (dysplastic naevi) and primary lesions has implicated the 9p locus in the early stages of melanoma development.
View Article and Find Full Text PDFWe have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood, thymus, and spleen of IFN-beta-/- mice, activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production, relative to IFN-beta+/+ mice. Notably, constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages, respectively, of IFN-beta-/- mice.
View Article and Find Full Text PDFCD8+ T cell responses can be generated against antigens that are not expressed directly within antigen-presenting cells (APCs), through a process known as cross-priming. To initiate cross-priming, APCs must both capture extracellular antigen and receive specific activation signals. We have investigated the nature of APC activation signals associated with virus infection that stimulate cross-priming.
View Article and Find Full Text PDFObjective: Bone mineral density (BMD) in later life is a major determinant of osteoporotic fracture risk and has been shown to be under strong genetic influence. Transforming growth factor beta 1 (TGF-beta 1) is an important regulatory cytokine, is found in high concentrations in the bone matrix, and is a plausible candidate for the genetic regulation of BMD.
Methods: This study investigated whether a novel polymorphism within the TGF-beta 1 gene is associated with BMD in a large normal female population of 1706 dizygotic (DZ) twins (age range 18-76 yr).
We have generated mice lacking the gene for beta interferon and report that they are highly susceptible to vaccinia virus infection. Furthermore, in cultured embryo fibroblasts, viral induction of alpha interferon and of 2-5A synthetase genes is impaired. We also show that beta interferon does not prime its own expression.
View Article and Find Full Text PDFType I interferons constitute an important part of the innate immune response against viral infection. Unlike the expression of interferon (IFN) B gene, the expression of IFNA genes is restricted to the lymphoid cells. Both IFN regulatory factor 3 and 7 (IRF-3 and IRF-7) were suggested to play positive roles in these genes expression.
View Article and Find Full Text PDFThe 2'-5' oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein-Barr virus-encoded RNA EBER-1 on the regulation of 2-5(A) synthetase activity.
View Article and Find Full Text PDFWe have expressed recombinant human interferon-alpha 2b in mammalian cells and isolated cell lines constitutively secreting very high levels of biologically active protein. The expression system takes advantage of the strong human cytomegalovirus immediate early promoter in mouse myeloma NSo cells and glutamine synthetase as a selectable marker; spontaneous mutants with amplified gene copy numbers were selected by growth of primary transfectants in the presence of methionine sulfoximine. Using this procedure, we have isolated a recombinant NSo cell line which secretes human interferon at the rate of 20 micrograms/10(6) cells/24 h and accumulates up to 120 micrograms/ml (approximately 2.
View Article and Find Full Text PDFType-I interferons are encoded by a multigene family, the major members of which are at least 13 IFN A subtypes and a single IFN B gene. IFNs A and B are induced in response to similar stimuli, such as virus infection and double-stranded RNA, but in different cell types: the induction of IFN A is almost exclusively restricted to cells of lymphoid origin, while IFN B has been found to be induced in a variety of cell types including fibroblasts. The virus-responsive enhancer element in the promoter region of IFN A family members is largely responsible for the differential expression of individual subtypes in responsive cells.
View Article and Find Full Text PDFJ Interferon Cytokine Res
February 1996
This paper addresses the role of transcriptional regulation in the determination of the levels of expression of different interferon-alpha subtypes secreted from Namalwa cells following infection with Sendai virus. Using RT-PCR to determine the relative abundance of mRNA species coding for the various subtypes, we found a general correlation with corresponding protein levels, indicative of a role for transcriptional control in the determination of levels of individual subtypes. We have used reporter gene constructs to compare the inducibility of the virus-response elements from the IFNA1, A2, A4, and A14 subtype genes cloned upstream of a secreted alkaline phosphatase gene.
View Article and Find Full Text PDFIt has been previously shown that the genomic DNA of both the lymphoblastoid cell line (Namalwa) and certain human donors (of African origin) contain sequences corresponding to two allelic variants, b and c, of the interferon-alpha 2 gene (IFNA2). Little is known however about the relative expression of these two alleles in heterozygous cells. We have therefore examined the transcription of allelic variants of the human IFNA2 locus by both normal human leucocytes (from a heterozygous donor) and Namalwa cells.
View Article and Find Full Text PDFWe have examined the ability of melanoma cell lines and normal human melanocytes, which have demonstrable intact IFN genes, to secrete both IFN-alpha and IFN-beta in response to induction with virus. Normal melanocytes were found to secrete both IFN-alpha and IFN-beta after virus induction. In contrast, although all but one of the melanoma lines tested were capable of secreting IFN-beta, none were capable of IFN-alpha secretion.
View Article and Find Full Text PDFThe interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a tumour suppressor gene product. Catalytically inactive mutants of PKR give rise to a tumorigenic phenotype when overexpressed in NIH-3T3 fibroblasts and this has been attributed to a dominant negative effect on the activity of the wild-type enzyme. Here we show that the mutant with Lys296 replaced by Arg, [K296R]PKR, not only inhibits the protein kinase activity of wild-type PKR but is also inhibitory towards another double-stranded RNA-dependent enzyme, the 40-kDa form of (2'-5')oligo(adenylate) synthetase.
View Article and Find Full Text PDFJ Interferon Cytokine Res
May 1995
We have analyzed human donor DNA for the presence of sequences corresponding to allelic variants of the IFN-alpha 2 locus. Using both restriction enzyme digestion of PCR-amplified fragments and sequence analysis of these fragments, we have identified the three reported allelic variants, IFN-alpha 2a, IFN-alpha 2b, and IFN-alpha 2c, in genomic DNA derived from donors of African or Afro-Caribbean origin. This is the first report of the IFN-alpha 2a and IFN-alpha 2c alleles occurring in human donor DNA and supports the view that these are variants of the predominant IFN-alpha 2b allele rather than arising from mutations occurring in cultured cells.
View Article and Find Full Text PDFJ Biol Chem
February 1995
Distinct NF-kappa B subunit combinations contribute to the specificity of NF-kappa B-mediated transcriptional activation and to the induction of multiple cytokine genes including interferon-beta (IFN-beta). To evaluate the regulatory influence of different homo- and heterodimers, NF-kappa B subunits were analyzed for transcriptional activity in vitro using test templates containing two types of NF-kappa B recognition elements (the human immunodeficiency virus type 1 enhancer and the IFN-beta-positive regulatory domain-II (PRDII) as well as IFN-beta PRDIII-PRDI-PRDII linked to the -56 minimal promoter of rabbit beta-globin. Recombinant NF-kappa B subunits (p50, p65, c-Rel, p52, and I kappa B alpha) and interferon regulatory factor 1 were produced from either Escherichia coli or baculovirus expression systems.
View Article and Find Full Text PDFInterferon (IFN) regulatory factor 1 (IRF-1) and IRF-2 are structurally similar but functionally distinct transcription factors that bind to the positive regulatory domains I and III (PRDI/III) within the human IFN-beta promoter. To begin structure-function analysis of IRF-1 and IRF-2, the regulatory potential of carboxyl-terminal deletion mutants was analyzed by co-transfection studies in human cells and was correlated with DNA binding capacity. Transcriptional repression by IRF-2 was contained within the first 125 amino-terminal amino acids and correlated directly with IRF-2 DNA binding; deletion to a protein of 100 amino acids resulted in loss of repression and IRF-2 DNA binding.
View Article and Find Full Text PDFSixteen Chinese chronic hepatitis B virus (HBV)-infected patients were treated with recombinant interferon-alpha 2a (rIFN-alpha 2a). Of these, 8 made a response to IFN, with titers of neutralizing antibody of 141-4525 as determined by an antiviral neutralization bioassay. To determine whether the immunogenicity of the IFN was directly linked to the patients' genotype, their genomic DNA was analyzed for the presence of the human IFN-alpha 2a gene.
View Article and Find Full Text PDFHum Antibodies Hybridomas
March 1995
We report a detailed comparison of two commonly used stable, amplifiable mammalian expression systems (Chinese Hamster Ovary cells/dihydrofolate reductase and Mouse NSO myeloma/glutamine synthetase) used to express a humanized IgG1 monoclonal antibody. We compare copy number and steady state mRNA levels of both the selectable marker and heavy chain of the antibody throughout the selection and amplification process. In both cell lines, copy number and steady state levels of heavy chain and selectable marker increased during selection and were further increased during amplification.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
December 1993
HLA-A and -B transplantation antigens can be expressed differentially at the basal level and in response to interferons (IFNs). To determine which DNA control elements and nuclear factors are responsible for these differences, HLA-A and -B upstream regulatory regions were used in expression and mobility-shift analyses. The HLA-A enhancer was found to contain two Rel (KBF/NF-kappa B) binding motifs, while the HLA-B enhancer has only one and is transactivated less well by overexpression of the NF-kappa B p65 subunit.
View Article and Find Full Text PDFDuring the molecular cloning of the human dsRNA activated-p68 kinase (PKR), polyclonal antibodies against PKR selected, in addition to cDNAs corresponding to PKR, another cDNA presenting only slight homology with PKR cDNA. This cDNA recognized an mRNA species of 2 kilobases induced by both alpha- and gamma-interferons. Its transcription did not require protein synthesis.
View Article and Find Full Text PDFThe interferon-inducible double-stranded-RNA(dsRNA)-dependent protein kinase PKR has been implicated in both the antiviral and cell growth-regulatory effects of the interferons. Over-expression of the wild-type form of this protein inhibits cell proliferation, whereas over-expression of inactive mutant forms transforms cells to a tumourigenic phenotype. It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild-type protein kinase.
View Article and Find Full Text PDFWe have previously reported three types of DNA-protein complexes, formed specifically with the interferon-stimulable response elements (ISRE) in the 5' flanking DNA of the interferon-inducible 6-16 and 9-27 genes, a type-I interferon-inducible early complex involving factor E (ISGF3), M and G complexes induced more slowly in response to type-I and type-II interferons, respectively and C1/C2, a constitutive complex(s). Similar complexes have been reported by others. The operationally defined band-shift complexes M, G and C1/C2 are shown here to be heterogeneous and to differ in their factor content, depending on the ISRE probe.
View Article and Find Full Text PDFWe have analyzed the genomic DNA sequence corresponding to the human interferon-alpha 2 (IFN-alpha 2) gene locus. In human lymphoblastoid Namalwa cells, we have detected sequences corresponding to IFN-alpha 2b and -2c, while in human KG-1 cells both IFN-alpha 2a and -2b were present. However, in 100 independent IFN-alpha 2 clones derived from 20 unrelated Caucasian volunteers, we found only sequences corresponding to IFN-alpha 2b.
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