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J F Elliott

Publications and source records attributed to J F Elliott.

At least 73 records · Page 4Linked to original sources
PubMed1995

Retroviral vector with a CMV-IE/HIV-TAR hybrid LTR gives high basal expression levels and is up-regulated by HIV-1 Tat.

We have constructed a new retroviral vector by making modifications to the commonly used Moloney murine leukemia virus (MoMLV) based vector in the long terminal repeat (LTR). The changes include replacement of a portion of the U3 region of the MoMLV LTR with a hybrid regulatory element consisting of the human cytomegalovirus immediate-early enhancer/promoter (CMV-IE) together with the human immunodeficiency virus transactivation response element (HIV-TAR). Transfection of chloramphenicol acetyl transferase (CAT) reporter constructs into a variety of human cell lines showed that the hybrid LTR with the CMV-IE/HIV-TAR enhancer/promoter exhibited basal expression levels which were 10- to 50-fold higher than those obtained from the wild-type MoMLV-LTR enhancer/promoter. Expression from the recombinant LTR was further increased in the presence of the HIV-Tat protein, and surprisingly, Tat up-regulated transcription from both the HIV and the MoMLV TATA boxes. In contrast, a MoMLV enhancer/promoter containing only the HIV-TAR element in the LTR did not respond to Tat. When stably transfected into an amphotropic packaging cell line, the modified retroviral vector containing the hybrid LTR plus an extended packaging signal consistently gave higher titres of retrovirus than did the parental MoMLV based vector. Higher basal expression levels which can be further upregulated by Tat, together with more efficient virion production, suggests that the modified vector should be superior for anti-HIV gene therapy applications as well as for other more general applications in human gene therapy.

Animals↗
PubMed1994

IL-10 inhibits cytokine production, vascular leakage, and swelling during T helper 1 cell-induced delayed-type hypersensitivity.

The Th1 group of cytokines (IL-2, IFN-gamma, and lymphotoxin (LT)) is usually associated with delayed-type hypersensitivity (DTH), whereas Th2 cytokines (IL-4, IL-5, IL-6, and IL-10) often accompany Ab production. Strong DTH and Ab responses are often mutually exclusive, possibly because of negative regulation between the cytokine patterns. In vitro, IL-10 inhibits the production of cytokines by Th1 cells, and inflammatory mediators by monocytes/macrophages, suggesting that IL-10 may be a negative regulator for DTH. We have investigated the effect of systemic IL-10 on the DTH reaction (nonindurated, with edema and granulocytic infiltration) induced by injection of Th1, clones into mouse footpads. Mammalian mouse rIL-10 was purified to > 90% homogeneity. The apparent in vivo half-life of i.p. injected IL-10 was approximately 2 h. Systemically administered IL-10 inhibited the 24-h footpad swelling induced by allo- and Ag-specific Th1 clones to a variable degree, with the strongest effect coinciding with peak DTH swelling. IL-10 also inhibited footpad swelling induced by a secondary challenge in SRBC-primed mice by 25 to 40%. Inhibition of Th1-induced swelling was accompanied by a similar inhibition of vascular permeability, as measured by leakage of Evans blue. Levels of IL-2, IL-6, IL-10, IFN-gamma, and TNF-alpha/LT were elevated in footpads undergoing a DTH reaction. IL-10 treatment reduced the levels of Th1 cytokines (IL-2, IFN-gamma, and TNF-alpha/LT) as well as IL-6, that was probably synthesized by other cells as a result of Th1 activation. The correlation between the inhibition of footpad swelling and cytokine production suggested that the effect of IL-10 on DTH may be mediated through suppression of cytokine synthesis.

Animals↗
PubMed1994

Alternatively spliced, germline J alpha 11-2-C alpha mRNAs are the predominant T cell receptor alpha transcripts in mouse kidney.

We recently reported the expression of a truncated T cell receptor (TCR) alpha mRNA in kidney and brain of normal mice. In the kidney, the truncated TCR alpha transcript was expressed by bone marrow-dependent, non-T large interstitial cells located predominantly in the medulla. Here, we report the molecular characterization of the truncated TCR alpha transcript from kidney. Using a modified anchored-PCR (A-PCR) technique and directional cloning, 37 cDNA clones extending 5' of the C alpha region were generated. cDNA sequencing showed that 29 of the clones (78%) originated in the J alpha 11-2 region. Of these clones, 17 started upstream or in the J alpha 11-2 exon and contained the entire J alpha 11-2 sequence correctly spliced to the first C alpha exon. Analysis of the sequence revealed the presence of multiple stop codons in all three reading frames. The other 12 clones originated further upstream of the J alpha 11-2 exon and did not include the J alpha 11-2 exon, but rather arose from the joining of a cryptic splice donor signal to the usual TCR alpha C splice acceptor. This alternatively spliced transcript contained an open reading frame extending from the upstream J alpha 11-2 region to 82 nucleotides downstream of the beginning of the TCR C alpha region, and potentially encoded a 36 amino acid polypeptide. The remaining eight clones all contained the J alpha TA61 region correctly spliced to C alpha with two of these extending upstream of the J alpha TA61 exon. The predominance of J alpha 11-2-C alpha containing clones was confirmed by RNase protection assay using total RNA from kidney and spleen of scid mice. The 3' region of the transcript contained a fully conserved, correctly spliced TCR alpha C region which was polyadenylated at the 3' end. The truncated TCR alpha mRNA could be detected in preparations of cytoplasmic RNA, indicating that this transcript follows a normal RNA processing pathway. Our results demonstrate that the truncated TCR alpha mRNA expressed in normal mouse kidney is a germline J-C transcript resulting from transcription initiated predominantly upstream of the J alpha 11-2 region. This germline transcript in the kidney is undergoing alternative splicing leading to the appearance of an open reading frame coding for a short polypeptide. These results suggest that the product of this transcript may be functionally relevant.

Alternative Splicing↗
PubMed1994

Polyclonal expansion of peripheral gamma delta T cells in human Plasmodium falciparum malaria.

Plasmodium falciparum malaria in humans is associated with an increase in the percentage and absolute number of gamma delta T cells in the peripheral blood. This increase begins during the acute infection phase and persists for at least 4 weeks during convalescence. In the present study, 25 to 30% of the gamma delta T cells expressed HLA-DR antigens in vivo and in some patients they proliferated in response to further stimulation by purified human interleukin 2 in vitro. However, there was no in vitro proliferative response to various malarial antigens, including a 75-kDa heat shock protein and a 72-kDa glucose-regulated protein of P. falciparum during the acute infection phase. Cytofluorographic studies showed that although an increase of V delta 1- gamma delta T cells was largely responsible for the expansion of the total number of gamma delta T cells, there was also a proportional increase in V delta 1+ cells. These results were confirmed with anchored PCR and by DNA sequencing to characterize at the molecular level the set of T-cell receptor (TCR) delta mRNAs expressed in the peripheral blood of two patients with high levels of gamma delta T cells. In each case, most of the TCR delta mRNA transcripts corresponded to nonproductively rearranged delta genes (unrearranged J delta or near J delta spliced to C delta). In those sequences which did represent productively rearranged genes, most of the transcripts originated from a V delta 2/J delta 1 joining, as in normal individuals. A minority of transcripts originated from a V delta 1/J delta 1 rearrangement, and one originated from a V alpha 4/J delta 1 rearrangement. Polyclonal activation of gamma delta T cells was inferred from the extensive junctional diversity seen in the delta mRNAs analyzed. Expansion of a heterogeneous set of both V delta 1(-)- and V delta 1(+)-bearing T cells suggests that the elevated levels of gamma delta T cells seen during acute P. falciparum malaria arose from immune responses to multiple distinct parasite antigens or unidentified host factors.

Adult↗
PubMed1994

Immunization with the larger isoform of mouse glutamic acid decarboxylase (GAD67) prevents autoimmune diabetes in NOD mice.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) has been implicated in autoimmune diabetes in NOD mice, but the role of the 67-kDa GAD isoform (GAD67) is less clear. We found that immunization of 4-week-old NOD mice with purified recombinant mouse GAD67 prevented or significantly delayed the onset of diabetes. To further explore this phenomenon, we characterized anti-GAD67 immune responses in naive and GAD-immunized NOD mice. Anti-GAD67 antibodies titers were relatively low in naive mice at all ages, but a single immunization with GAD67 at 4 weeks induced high titers of anti-GAD antibodies by 6 weeks of age. In both 4-week-old and diabetic NOD mice, there were significant endogenous T-cell proliferative responses against purified recombinant mouse GAD67. These T-cell proliferative responses were blocked by anti-I-ANOD and anti-CD4 antibodies. To characterize the anti-GAD T-cell responses in the NOD mice, we established T-cell lines and T-cell clones which recognized GAD67, and we used recombinant subfragments of GAD to localize the predominant T-cell epitopes in GAD67. T-cells from naive NOD mice proliferated in response to all GAD subfragments, whereas T-cells from diabetic mice responded primarily to the COOH-terminal 83 amino acids of GAD67. These results suggest that GAD67 is an autoantigen in IDDM and immunization of prediabetic NOD mice with GAD67 can prevent the onset of diabetes.

Animals↗
PubMed1993

Cloning of a novel surface antigen from the insect stages of Trypanosoma brucei by expression in COS cells.

Trypanosoma brucei cDNA libraries constructed in the vector pCDM8 were screened selectively for insect (procyclic) stage surface antigen cDNAs by transient expression in mammalian COS-7 fibroblasts and "panning" with a rabbit polyclonal antiserum. This strategy yielded two surface antigen cDNAs termed PSSA-1 and PSSA-2. The PSSA-1 cDNA encoded an isotype of procyclin, the major phosphatidylinositol-linked stage-specific glycoprotein antigen of the tsetse fly infective forms of T. brucei. The PSSA-2 cDNA encoded a new and previously unidentified stage-specific surface antigen with the features of a typical transmembrane glycoprotein but with an unusual cytoplasmic tail composed of a proline-rich tandem repeat. Fluorescent antibody staining of PSSA-1 transfected COS cells with a panel of procyclin-specific monoclonal antibodies confirmed that the protein was located on the outer surface of the plasma membrane. Furthermore, the antigen on COS cells was insensitive to treatment with phosphatidylinositol-specific phospholipase C suggesting that the inositol of the glycosylinositol phospholipid-lipid anchor contained the same fatty acyl modification reported recently for the procyclin molecule in cultured procyclic trypanosomes. In contrast the PSSA-2 antigen on COS cells was stained very weakly by whole parasite antisera. Northern blot hybridization revealed that the PSSA-2 antigen was encoded by a single 1.7-kilobase transcript which was present in parasites from the insect procyclic stage of the life cycle but not from the animal bloodstream stage. Southern blot hybridization analysis of DNA from procyclic stage trypanosomes indicated that the gene for PSSA-2 may be present in more than one copy in procyclic trypanosomes.

Amino Acid Sequence↗
PubMed1993

Induction of a proteoglycan core protein mRNA in mouse T lymphocytes.

The mouse T lymphocyte cell line EL4.E1 synthesizes a proteoglycan core protein (PGCP) mRNA which is identical to serglycin mRNA found in mouse bone marrow-derived mast cells and a mouse mastocytoma cell line. PGCP mRNA was strongly induced in EL4.E1 cells by phorbol myristate acetate, which also induces mRNAs for several cytokines in these cells. In contrast to the induction of cytokine mRNAs, however, the induction of PGCP mRNA was not inhibited by Cyclosporine. PGCP mRNA was also inducible by allogeneic stimulation of normal mouse spleen cells, and by Con A stimulation of an Interleukin 2-producing T hybridoma cell line. A number of other cell lines expressed an identical or similar, mRNA, including two cytotoxic T cell lines, and three tumor cell lines related to bone marrow-derived cells. The levels of several proteoglycans have previously been reported to increase in cells of bone marrow origin under activating conditions, but this appears to be the first report of an induction of the corresponding PGCP mRNA by immune stimulation of T lymphocytes.

Animals↗
PubMed1992

Duck hepatitis B virus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcriptase activities.

Activities of the hepadnavirus polymerases are known to include those of DNA polymerase, reverse transcriptase and RNase H. To date, it has been difficult or impossible to clone and express the product as an active enzyme. In this study, full length capped RNA encoding Duck Hepatitis B Virus (DHBV) polymerase was produced by in vitro transcription from a T7 promoter. The RNA was translated in a rabbit reticulocyte lysate system and produced an 35S-Methionine labelled 79 Kd band on SDS-polyacrylamide gel electrophoresis. The translation product showed DNA polymerase and reverse transcriptase activities on exogenous templates (respectively) of DNA or RNA with random DNA hexamer primers. The same RNA transcripts were also microinjected into Xenopus oocytes, but appeared to be toxic and gave no detectable translation product. Production of hepadnavirus polymerase by in vitro transcription/translation may provide a useful tool for structure/function and pharmacological studies on this important group of polymerases.

Animals↗
PubMed1992

Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci.

Degenerate oligonucleotides based on the published Escherichia coli glutamate decarboxylase (GAD) protein sequence were used in a polymerase chain reaction to generate a DNA probe for the E. coli GAD structural gene. Southern blots showed that there were two cross-hybridizing GAD genes, and both of these were cloned and sequenced. The two GAD structural genes, designated gadA and gadB, were found to be 98% similar at the nucleotide level. Each gene encoded a 466-residue polypeptide, named, respectively, GAD alpha and GAD beta, and these differed by only five amino acids. Both GAD alpha and GAD beta contain amino acid residues which are highly conserved among pyridoxal-dependent decarboxylases, but otherwise the protein sequences were not homologous to any other known proteins. By restriction mapping and hybridization to the Kohara miniset library, the two GAD genes were located on the E. coli chromosome. gadA maps at 4046 kb and gadB at 1588 kb. Neither of these positions is in agreement with the current map position for gadS as determined by genetic means. Analysis of Southern blots indicated that two GAD genes were present in all E. coli strains examined, including representatives from the ECOR collection. However, no significant cross-hybridizing gene was found in Salmonella species. Information about the DNA sequences and map positions of gadA and gadB should facilitate a genetic approach to elucidate the role of GAD in E. coli metabolism.

Amino Acid Sequence↗
PubMed1991

Generation of monoclonal antibodies against soluble human T cell receptor polypeptides.

One approach to the diagnosis and therapy of T cell-mediated diseases is to develop reagents specific for T cell receptor (TcR) variable (V) regions. To date, however, TcR expressed on the surface of antigen-specific T lymphocytes have proven to be poorly immunogenic. As a result, few monoclonal antibodies (mAb) recognizing human variable regions are available. In this report, we have used the "phosphatidylinositol linkage" strategy to generate soluble forms of two human allogeneic TcR derived from human cytotoxic T lymphocytes (CTL) known to be specific for HLA-A2 and HLA-Aw68/HLA-Aw69, respectively. Monomeric TcR alpha and beta chains from the HLA-A2-specific CTL were purified in large quantities from CHO cells and each was used to immunize mice to generate mAb. In particular, the anti-beta chain mAb, denoted anti-V beta 13, stain a significant (approximately 5%) fraction of human peripheral blood alpha/beta T lymphocytes, immunoprecipitate native anti-A2 TcR molecules, and activate T cells transfected with the relevant alpha and beta chain cDNA. Anti-alpha chain mAb were also obtained against a constant region determinant which can immunoprecipitate detergent-solubilized polypeptides. In general, we find that immunizations with soluble protein are far superior to those with cells bearing TcR chimeras or in combination with the purified protein.

Amino Acid Sequence↗
PubMed1991

Assay of mutation induced in human lymphoblastoid cells by combustion-generated soot particles.

A human lymphoblastoid cell line has been used to test for mutations caused by combustion-generated soot particles and their constituent components, which are substrate carbon-black and adsorbed condensate, principally in the form of polycyclic aromatic hydrocarbons (PAH). It was found that the mutagenicity of the PAH fraction is higher when it is contacted with cells as a liquid extract than when it is supplied as a coating on soot particles. The substrate particles were found to be nonmutagenic. The rate of transfer of mutagens from the surface of particles, combined with the retention time of respirable aerosol particles, are deemed to define their mutagenic potential.

Adsorption↗
PubMed1990

Expression of T cell antigen receptor heterodimers in a lipid-linked form.

The interaction of the T cell receptor for antigen (TCR) with its antigen-major histocompatibility complex ligand is difficult to study because both are cell surface multimers. The TCR consists of two chains (alpha and beta) that are complexed to the five or more nonpolymorphic CD3 polypeptides. A soluble form of the TCR was engineered by replacing the carboxyl termini of alpha and beta with signal sequences from lipid-linked proteins, making them susceptible to enzymatic cleavage. In this manner, TCR heterodimers can be expressed independently of the CD3 polypeptides and in significant quantities (0.5 milligram per week). This technique seems generalizable to biochemical and structural studies of many other cell surface molecules as well.

Alkaline Phosphatase↗
PubMed1990

Genes for Plasmodium falciparum surface antigens cloned by expression in COS cells.

Two genes encoding membrane antigens of Plasmodium falciparum were isolated by transient expression in mammalian cells and selection with human immune sera from African adults exposed to P. falciparum malaria. COS-7 cells were transfected with a plasmid expression library constructed from P. falciparum genomic DNA, and cells expressing reactive malaria antigens on their surface were enriched by adherence to antibody-coated dishes. One of the genes isolated is distinctive in that it does not contain repeat sequences typical of many malarial genes cloned by immunoscreening of bacterial expression libraries. The second gene apparently encodes a polymorphic version of the P. falciparum merozoite surface antigen Ag513, since the two sequences are identical in the 5' and 3' coding regions but diverge completely in the center. The COS-7 expression system provides an alternate means for cloning genes encoding malarial membrane antigens by using those antibodies in complex immune sera that bind membrane-associated, nondenatured molecules.

Amino Acid Sequence↗