Biomedical subjects

H L Robinson

Publications and source records attributed to H L Robinson.

At least 73 records · Page 4Linked to original sources

PubMed1994

Establishment of interference in osteoblasts by an osteopetrosis-inducing avian leukosis virus.

Avian leukosis virus (ALV)-induced osteopetrosis is characterized by the presence of unintegrated viral DNA is diseased bone. To learn more about this phenomenon, ALVs with high (Br21) and low (RAV-1) osteopetrotic potential have been compared for the ability to establish superinfection resistance in cultures of calvarial-derived osteoblasts. Both viruses could establish interference in osteoblast cultures that were maintained as growing cells. This interference was maintained when cultures were induced to differentiate. These results suggest that the unintegrated viral DNA in osteopetrotic bone is not due to the inability of osteopetrosis-inducing viruses to establish superinfection resistance in osteoblasts.

Animals↗

PubMed1993

DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations.

Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.

Animals↗

PubMed1993

Tissue tropism of avian leukosis viruses: analyses for viral DNA and proteins.

Analyses of six tissues from Rous-associated virus type 1-infected chickens have revealed a number of tissue-specific differences in virus synthesis and distribution. Chicks were infected at 1 day of age. Tissue (bursa, thymus, liver, kidney, muscle, and brain) were harvested 1 month later. Each of the tissues contained an average of 1.8 to 4 copies of viral DNA per cell. Most of this DNA was integrated. In brain, about one-third of the total viral DNA was in an unintegrated, linear form. Bursa, thymus, liver, and kidney expressed both Gag and Env proteins. In contrast, muscle expressed more Gag than Env, and brain expressed neither Gag nor Env. Tissues that were producing both Gag and Env contained higher levels of mature virus particles than tissues that were not producing both of these proteins.

Animals↗

PubMed1993

Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA.

Direct DNA inoculations have been used to demonstrate that in vivo transfections can be used to elicit protective immune responses. The direct inoculation of an H7 haemagglutinin-expressing DNA protected chickens against lethal challenge with an H7N7 influenza virus. Three-week-old chickens were vaccinated by inoculating 100 micrograms of plasma DNA by each of three routes (intravenous, intraperitoneal and subcutaneous). One month later, chickens were boosted with 100 micrograms of DNA by each of the three routes. At 1-2 weeks postboost, chickens were challenged via the nares with 100 lethal doses of an H7N7 virus. Low to undetectable levels of H7-specific antibodies were present postvaccination and boost. High titres of H7-specific antibodies appeared within 1 week of challenge. In a series of four experiments, 50% (28/56) of the DNA-vaccinated and < 2% (1/67) of the control chickens survived the challenge. This exceptionally simple method of immunization holds high promise for the development of subunit vaccines.

Amantadine↗

PubMed1993

Use of DNA encoding influenza hemagglutinin as an avian influenza vaccine.

Recently, we demonstrated that direct inoculation of a hemagglutinin 7 (H7)-expressing DNA could vaccinate chickens against a lethal H7 influenza virus challenge. These experiments used a defective-retroviral-based vector to express H7 (p188) (Robinson et al., 1993). Here, we report protective immunizations using a non-retroviral-based vector for H7 expression (pCMV/H7). Unlike the previously used retroviral-based vector, this vector cannot be transmitted as an infectious agent (as a consequence of phenotypic mixing with exogenous or endogenous virus proteins). Vaccination was accomplished by inoculating young, immunocompetent chickens by each of three routes (intravenous, intraperitoneal, and intramuscular) with 100 micrograms of cesium chloride-purified pCMV/H7 DNA in saline. After two immunizations, birds were challenged via the nares with a lethal dose of a highly virulent chicken influenza virus of the H7 subtype. The results of five independent vaccine trials demonstrated protective immunizations in approximately 60% of the pCMV/H7 DNA-inoculated chickens. By contrast, only 3% of the chickens inoculated with control DNA survived the lethal challenge.

Animals↗

PubMed1993

Context-dependent role of human immunodeficiency virus type 1 auxiliary genes in the establishment of chronic virus producers.

Two molecularly cloned viruses, human immunodeficiency virus type 1 (HIV-1)-NL4-3 (NL4-3) and HIV-1-HXB-2 (HXB-2), have been used to study the role of HIV-1 auxiliary genes in the establishment of chronic virus producers. NL4-3 encodes all known HIV-1 proteins, whereas HXB-2 is defective for three auxiliary genes: vpr, vpu, and nef. Studies were done in H9 cells, a T-cell line unusually permissive for the establishment of chronic virus producers. NL4-3 and HXB-2 undergo lytic phases of infection in H9 cultures with HXB-2, but not NL4-3, supporting the efficient establishment of chronic virus producers. Tests of mutant NL4-3 genomes containing various combinations of defective auxiliary genes revealed that both vpr and nef limited the ability of NL4-3 to establish chronic virus producers. Tests of a series of recombinants between NL4-3 and HXB-2 revealed that 5' internal sequences as well as fragments containing defective auxiliary genes affected the establishment of chronic virus producers. Viral envelope sequences and levels of virus production did not correlate with the ability to establish chronic virus producers. These results suggest that complex interactions of viral auxiliary and nonauxiliary gene functions with the host cell determine the ability to establish chronic virus producers.

Cloning, Molecular↗

PubMed1993

Induction of renal adenocarcinoma by a nonmutated erbB oncogene.

Oncogenicity tests have revealed that a nonmutated erbB oncogene induces renal adenocarcinoma in addition to erythroblastosis. The erbB oncogene is a truncated form of the chicken epidermal growth factor receptor that lacks the extracellular ligand-binding domain. Previously, the nonmutated erbB oncogene has been reported to cause only erythroblastosis. The expansion of the disease potential of erbB to additional neoplasms has been associated with mutations (truncations, deletions, and point mutations) within the erbB gene. Our results indicate that a nonmutated virally expressed erbB oncogene (REB-c) causes a 100% incidence of renal neoplasia.

Adenocarcinoma↗

PubMed1992

Increased oncogenic potential of ErbB is associated with the loss of a COOH-terminal domain serine phosphorylation site.

The erbB oncogene encodes an altered form of the epidermal growth factor (EGF) receptor that lacks the extracellular ligand binding domain. This oncogene is exclusively leukemogenic. However, an increase in oncogenic potential and a broadening of the tissue specificity of tumor formation occurs after retroviral transduction of erbB. The increased oncogenic potential correlates with structural alterations within the erbB gene. One common event is the deletion of a serine phosphorylation site located within the COOH-terminal domain. This site of phosphorylation has been demonstrated to be required for EGF-induced desensitization of signaling by the EGF receptor (Countaway, J. L., Nairn, A. C., and Davis, R.J. (1992) J. Biol. Chem. 267, 1129-1140). Here we show that the mutation of erbB at this negative regulatory serine phosphorylation site causes fibroblast transformation in vitro and is associated with an increased oncogenic potential in vivo.

3T3 Cells↗

PubMed1992

Replication of patient isolates of human immunodeficiency virus type 1 in T cells: a spectrum of rates and efficiencies of entry.

Isolates of human immunodeficiency virus type 1 (HIV-1) undergo many different rates of replication, with the time course of replication being determined by the host cell and the virus. Recently, we demonstrated that the permissiveness of four CD4+ T-cell lines for the laboratory strain NL4-3 correlated with the rate and efficiency of virus entry. In this study, we have analyzed the replication of a "slow/low" isolate from the pre-AIDS period of infection and two "rapid/high" isolates from the AIDS period of infection to determine which steps in the virus life cycle determine differences in the growth characteristics of patient isolates. Differences in the growth of the patient isolates correlated with differences in entry but not postentry steps of the virus life cycle. The two rapid/high patient isolates (SF33 and SF216) underwent 50% entry in less than or equal to 0.5 hr in C8166 cells, in less than or equal to 1 hr in mitogen-stimulated peripheral blood mononuclear cells, and in greater than or equal to 2.5 hr in H9 cells. In contrast, a class 3 slow/low patient isolate required 1 hr for 50% entry into C8166 cells, 3 hr for 50% entry into peripheral blood mononuclear cells, and 5-6 hr for 50% entry into H9 cells. Entry efficiency correlated with entry rate, with the rapid/high viruses having a 2-fold higher titer and the slow/low virus having a 5-fold higher titer on C8166 than H9 cells. The laboratory strain NL4-3 displayed intermediate rates and efficiencies of entry. These data demonstrate that entry characteristics are major determinants of the pathogenic potential of patient isolates.

Acquired Immunodeficiency Syndrome↗

PubMed1992

5' avian leukosis virus sequences and osteopetrotic potential.

Recombinants of Rous-associated virus-0 and Br21 have been used to localize 5' viral sequences that affect the osteopetrotic potential of avian leukosis viruses. Rous-associated virus-0 is a benign subgroup E virus of endogenous origin that does not cause osteopetrosis. Br21 is a constructed subgroup E virus with high osteopetrotic potential. 5' sequences that affected osteopetrotic potential resided in an 834-bp region near the 5' LTR. Sequence analysis of this region revealed differences between Br21 and RAV-0 in the mRNA leader and codons for MA.

Animals↗

PubMed1992

Human immunodeficiency virus type 1 entry into T cells: more-rapid escape from an anti-V3 loop than from an antireceptor antibody.

The entry of human immunodeficiency virus type 1 into two T-cell lines has been analyzed to determine the relative time courses with which virus entry can be blocked (i) by washing, (ii) by adding a monoclonal antibody to the V3 loop of gp120 that neutralizes without blocking CD4 binding (0.5 beta), or (iii) by adding an antireceptor monoclonal antibody that competes for virus binding (leu3a). During entry into C8166 cells, 50% escape from the wash as well as the anti-V3 loop antibody required 20 min, whereas 50% escape from the leu3a block required 45 minutes. In contrast, during entry into H9 cells, 50% escape from the wash block required 50 min, 50% escape from the anti-V3 loop antibody required 110 min, and 50% escape from the antireceptor antibody required 190 min. These results demonstrate that the times required for entering virus to escape each of the blocks were cell type specific. They also demonstrate that V3 loop-dependent steps occur relatively early in entry and suggest that binding of gp120 to CD4 is important for late as well as early steps in human immunodeficiency virus type 1 entry.

Antibodies, Monoclonal↗

PubMed1992

Protein tyrosine kinase activities of the epidermal growth factor receptor and ErbB proteins: correlation of oncogenic activation with altered kinetics.

We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2+, ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and approximately 2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higher Km(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphorylation suggested that the ErbB proteins had lower catalytic efficiencies for the exogenous substrate than did the chEGFR. The three tested ErbB proteins had cytoplasmic domain mutations that conferred distinctive disease potentials. These mutations did not affect the kinetics for the phosphorylation of the exogenous substrate TK6. Two of the ErbB proteins contained all of the sites used for autophosphorylation. In these, a mutation that broadened oncogenic potential to endothelial cells caused an additional increase in Vmax(app) for autophosphorylation. Thus, mutations that change the EGFR into an ErbB oncogene cause multiple changes in the kinetics of protein tyrosine kinase activity.

3T3 Cells↗

PubMed1992

Oncogene transduction and activation.

This article explains what a transducing virus is, summarizes what is currently known about the origins of transducing viruses, considers the frequency of the generation of transducing viruses, and discusses how transduced oncogenes undergo activation. Most of the data come from work with avian leukosis viruses, as research with this group of viruses has set many of the precedents for work on retroviral transduction.

Animals↗

PubMed1992

Characterization of an angiosarcoma-inducing mutation in the erbB oncogene.

The erbB oncogenes of two transducing viruses that arose in chicken 5005 have been molecularly characterized. One of these viruses, AEV-5005, caused erythroblastosis. The other, AAV-5005, caused angiosarcoma. Both viruses had identical 5' junctions of viral and host sequences, indicating that both arose from the same proviral insertion. The erbB oncogenes of both viruses encoded transmembrane, kinase and C-terminal domains of the chicken epidermal growth factor receptor. The C-terminal domain of the AEV-5005 erbB was complete, whereas that of AAV-5005 contained a 59 amino acid internal deletion (amino acids 993-1051 of the chicken epidermal growth factor receptor). Oncogenicity tests using retroviral constructs containing the erbB sequences from AEV-5005 and AAV-5005 demonstrated that both erbB genes caused erythroblastosis and that the 59 amino acid deletion conferred the ability to induce angiosarcoma. The 59 amino acid deletion also caused increased levels of erbB autophosphorylation in cells grown in the presence of sodium orthovanadate.

Amino Acid Sequence↗

PubMed1991

Human immunodeficiency virus type 1 NL4-3 replication in four T-cell lines: rate and efficiency of entry, a major determinant of permissiveness.

Single-cycle infections have been used to study the human immunodeficiency virus type 1 (HIV-1) life cycle in CD4+ T-cell lines that differ in their permissiveness for infection. In single-cycle infections of highly permissive C8166 cells, 50% of the infectious units escaped being blocked by a monoclonal antibody against the virus binding site on CD4 (leu3a) within 30 min. In contrast, 50% of the infectious units for three less permissive cell lines (H9, A3.01, and Jurkat) required 4 h to escape the leu3a block. Entry was also more efficient in the highly permissive cells, with NL4-3 stocks having three times more infectious units for C8166 cells than for H9, A3.01, or Jurkat cells. Postentry steps up through reverse transcription required approximately 3.5 h in each of the cell lines. The times lapsing between reverse transcription and the expression of reverse transcripts ranged from 17 to 25 h in the different cell lines. Virus production per cell was also similar in the different cell lines (within 1.5-fold of each other). These results indicate that a major determinant of the permissiveness of growing T cells for HIV-1 is the rate and efficiency of virus entry.

CD4-Positive T-Lymphocytes↗

PubMed1990

Correction: copy numbers of HIV-1 RNA.

In Barbara J. Culliton's article "Gore Tex organoids and organoids and genetic drugs" (News & Comment, 10 Nov., p. 747), it should have been noted that the structural analysis of the implant connections, induding the micrographs shown on page 747, were performed by Christian C. Haudenschild at the Mallory Institute of Pathology, Boston University of Medicine, Boston, MA. His name should have been listed in the photo credit. The legend for the bottom photomicrograph should have read, "Below, a view in cross section confirms the presence of abundant vessels lined with endothelial cells and surrounded by several layers of smooth musclc cells. Two profiles without lumen resemble neural structures, but their precise identity remains to be investigated."

Cell Line↗

PubMed1990

Accumulation of human immunodeficiency virus type 1 DNA in T cells: results of multiple infection events.

Human immunodeficiency virus type 1 DNA synthesis was followed in a CD4+ line of T cells (C8166) grown in the presence or absence of a monoclonal antibody to CD4 that blocks infection By 48 h after infection, cultures grown in the presence of the antibody contained approximately 4 copies of human immunodeficiency virus type 1 DNA per cell, whereas those grown in the absence of the antibody contained approximately 80 copies of viral DNA per cell. Most of the viral DNA in cultures grown in the absence of the antibody was present in a broad smear of apparently incomplete viral sequences. In cultures grown in the presence or absence of the antibody, the 9.6-kilobase linear duplex of viral DNA appeared to undergo integration within 24 h of its appearance. These results demonstrate that T cells accumulate unintegrated human immunodeficiency virus type 1 DNA as a result of multiple virions entering cells.

Blotting, Southern↗

PubMed1990

Infrequent involvement of c-fos in avian leukosis virus-induced nephroblastoma.

To determine whether c-fos is involved in avian leukosis virus-induced nephroblastoma, 28 tumors from chickens were analyzed for novel fos fragments. DNA from 1 of 16 clonal outgrowths (in chicken 6561) contained novel fos-related EcoRI and KpnI fragments which hybridized to both v-fos and viral probes. Oncogenicity tests using filtered 6561 tumor cell homogenates did not reveal a tumor-inducing transduction of c-fos. We conclude that c-fos is only an occasional target for proviral insertions or new transductions in avian leukosis virus-induced nephroblastoma. The results also identify a polymorphism in c-fos in K28 chickens and demonstrate that unintegrated viral DNA is not a general characteristic of avian leukosis virus-induced nephroblastoma.

Animals↗