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Biomedical subjects

J P Dougherty

Publications and source records attributed to J P Dougherty.

At least 37 records · Page 2Linked to original sources
PubMed1995

Both anti-CD11a (LFA-1) and anti-CD11b (MAC-1) therapy delay the onset and diminish the severity of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) induced in rodents by activated CD4+ T cells specific for various myelin proteins such as myelin basic protein and proteolipid protein. The disease is characterized by breach of the blood-brain barrier, perivascular infiltration of leukocytes into the CNS, local inflammation and demyelination in the form of plaques. In this study, we evaluated the effect of administration of antibodies to two members of the beta 2 integrin sub-family of adhesion molecules, CD11a and CD11b, on the onset and progression of EAE. CD11a and CD11b are involved in cell-cell interactions leading to T cell and macrophage extravasation to inflammatory sites and T cell activation. Our results show that anti-CD11a antibodies could completely block the induction of EAE and anti-CD11b antibodies significantly delayed the onset and diminished the severity of clinical signs of EAE even when injections were initiated at the first appearance of clinical signs.

Amino Acid Sequence↗
PubMed1995

Genetic rearrangements occurring during a single cycle of murine leukemia virus vector replication: characterization and implications.

Retroviruses evolve at rapid rates, which is presumably advantageous for responding to selective pressures. Understanding the basic mutational processes involved during retroviral replication is important for comprehending the ability of retroviruses to escape immunosurveillance and antiviral drug treatment. Moreover, since retroviral vectors are important vehicles for somatic cell gene therapy, knowledge of the mechanism of retroviral variation is critical for anticipating untoward mutational events occurring during retrovirus-medicated gene transfer. The focus of this report is to examine the spectrum of genomic rearrangements arising during a single cycle of Moloney murine leukemia virus (MoMLV) vector virus replication. An MoMLV vector containing the herpes simplex virus thymidine kinase (tk) gene was constructed. MoMLV vector virus was produced in packaging lines, and target cells were infected. From a total of 224 mutant proviruses analyzed, 114 had gross rearrangements readily detectable by Southern blotting. The remaining proviruses were of parental size. PCR and DNA sequence analysis of 73 of the grossly rearranged mutant proviruses indicated they resulted from deletions, combined with insertions, duplications, and complex mutations that were a result of multiple genomic alterations in the same provirus. Complex hypermutations distinct from those previously described for spleen necrosis virus and human immunodeficiency virus were detected. There was a correlation between the mutation breakpoints and single-stranded regions in the predicted viral RNA secondary structure. The results also confirmed that the tk gene is inactivated at an average rate of about 8.8% per cycle of retroviral replication, which corresponds to a rate of mutation of 3%/kbp.

3T3 Cells↗
PubMed1994

Pseudotyped REV/SRV retroviruses reveal restrictions to infection and host range within members of the same receptor interference group.

The reticuloendotheliosis virus (REV) group of retroviruses and type D simian retroviruses (SRV) belong to the same receptor interference group. The cellular receptor for these viruses has not yet been identified. In order to study the distribution of the receptor and to identify a receptor negative cell line, vector viral pseudotypes between REV and SRV were made. Using these viral pseudotypes, susceptibility to infection was examined in some rodent and marsupial cell lines. Infectivity assays with these envelope pseudotypes demonstrated that all cell types tested were resistant to infection. However, treatment of the rodent cells with the N-linked glycosylation inhibitor tunicamycin rendered most of the cells susceptible to infection. These results indicate that all the rodent cells tested express a nonfunctional receptor for viruses of the REV and SRV groups, which can be made functional by tunicamycin treatment. A difference in receptor host range among members of the same receptor interference group was observed which suggests that the REV/SRV receptor for this interference group might be differentially modified in different cell types. The studies also identified at least one cell line not expressing the REV/SRV receptor which should be useful for isolating the gene encoding the receptor.

Animals↗
PubMed1994

A murine model for B-lymphocyte somatic cell gene therapy.

Mature primary B lymphocytes represent a potentially important cellular target for somatic cell gene therapy, which could prove advantageous for the treatment of certain metabolic and immunologic disorders. Their capacity to serve as antigen-presenting cells could be utilized for triggering and/or potentiating immune responses to tumors and viruses. Alternatively, B cells expressing an autoantigen could be manipulated to induce antigen-specific unresponsiveness for treatment of autoimmune diseases. Efficient expression of an exogenous gene product in long-lived B lymphocytes could be particularly useful for providing a corrected gene product in the bloodstream. Despite these advantages, efficient gene transfer into mature primary B cells has not been reported. One reason for this is that current protocols for retroviral vector-mediated gene transfer into lymphocytes rely on in vitro expansion and/or drug selection. This precludes the use of mature primary B cells as targets, since they cannot be readily cultured for long periods of time. In this report, we describe an efficient and rapid protocol for the introduction of exogenous genes into primary B cells without the need for drug selection. We have used retroviral vectors containing the human adenosine deaminase gene as a marker gene, since the biological activity of this enzyme is easy to measure and is readily distinguishable from that of the endogenous mouse adenosine deaminase. Upon adoptive transfer into SCID mice, infected B cells continuously expressing one to three copies of the human adenosine deaminase gene could be found in the spleens of recipient animals for at least 3 months.

Adenosine Deaminase↗
PubMed1993

In vivo antigen presentation by both brain parenchymal cells and hematopoietically derived cells during the induction of experimental autoimmune encephalomyelitis.

A fundamental issue in the etiology of autoimmune diseases of the central nervous system such as multiple sclerosis and its animal counterpart experimental autoimmune encephalomyelitis (EAE) concerns the identity of cells capable of presenting autoantigen to the T cells that mediate these diseases. The prevailing dogma is that only bone marrow-derived cells function as APC during EAE induction. We have addressed this issue by studying EAE induction in mouse bone marrow chimeras, and have found that although bone marrow-derived APC such as macrophages and brain microglial cells are more efficient at presenting autoantigen, brain parenchymal cells such as astrocytes and endothelial cells are also capable of inducing disease. EAE was induced in these chimeras by the adoptive transfer of encephalitogenic T cell lines designed to be MHC-histocompatible with APC contained either within the hematopoietic system of the chimera or with APC resident to the brain of the chimera. The subsequent development of EAE in these chimeras then indicated which population of cells served as in vivo APC during EAE pathogenesis. Possible effects of alloreactivity between the host chimera and the adoptively transferred T cells were eliminated by using encephalitogenic T cell lines made tolerant to the haplotype(s) of the recipient chimera.

Amino Acid Sequence↗
PubMed1993

Efficient gene transfer into primary murine lymphocytes obviating the need for drug selection.

The efficient introduction of exogenous genes into primary lymphocytes is potentially important both for somatic cell gene therapy and for studying lymphocyte biology. We describe the use of retroviral vectors to efficiently introduce exogenous genes into primary, mature murine lymph node T and B cells, and primary, immature murine CD4- CD8- double-negative (DN) thymocytes. Efficient infection of primary cells was achieved by cocultivation of target cells with lethally irradiated helper cells that produce high titers of retroviral vectors containing either the neomycin phosphotransferase II (neo) gene, or both the neo and the human adenosine deaminase (ADA) genes, in the presence of lymphokines and/or mitogens. Two days postinfection, without neomycin selection, one to five copies of the exogenous genes per cell were detected by Southern blot analysis. Expression of the exogenous human ADA protein was detected at levels comparable to the endogenous murine ADA protein in the mature T and B lymphocytes, and was somewhat lower for the immature DN thymocytes.

Adenosine Deaminase↗
PubMed1993

High rate of genetic rearrangement during replication of a Moloney murine leukemia virus-based vector.

A protocol was designed to measure the forward mutation rate over an entire gene replicated as part of a Moloney murine leukemia virus-based vector. For these studies, the herpes simplex virus thymidine kinase (tk) gene under the control of the spleen necrosis virus U3 promoter was used as target sequence since it allows selection for either the functional or the inactivated gene. Our results indicate that after one round of retroviral replication, the tk gene is inactivated at an average rate of 0.08 per cycle of replication. Southern blotting revealed that the majority of the mutant proviruses resulted from gross rearrangements and that deletions of spleen necrosis virus and tk sequences were the most frequent cause of the gene inactivation. Sequence analysis of the mutant proviruses suggested that homologous as well as nonhomologous recombination was involved in the observed rearrangements. Some mutations consisted of simple deletions, and others consisted of deletions combined with insertions. The frequency at which these mutations occurred during one cycle of retroviral replication provides evidence indicating that Moloney murine leukemia virus-based vectors may undergo genetic rearrangement at high rates. The high rate of rearrangement and its relevance for retrovirus-mediated gene transfer are discussed.

3T3 Cells↗
PubMed1992

Comparison of Moloney murine leukemia virus mutation rate with the fidelity of its reverse transcriptase in vitro.

The role of Moloney murine leukemia virus (MoMLV) reverse transcriptase (RT) in the generation of base substitution mutations during retroviral replication was analyzed. To that effect, the in vitro fidelity of the MoMLV RT was compared to the rate of base substitution mutations occurring during the replication of an MoMLV-based retroviral vector. Using the vector in an amber reversion assay, the base substitution mutation rate at a single locus was found to be 2 x 10(-6)/base pair in one cycle of vector virus replication. Analysis of the fidelity of the purified RT using the same template sequence revealed that, of the two mispairs (A.C and T.G) that would lead to reversion of the amber codon during replication, A.C occurs at a rate of 4.0 x 10(-6), and T.G occurs at a rate of 0.7 x 10(-4). While the rate of formation of A.C is very similar to the vector mutation rate, the rate of formation of T.G is more than 30 times higher. This discrepancy in rates suggests that there are other elements in the infected cells that contribute to the fidelity of viral replication.

3T3 Cells↗
PubMed1992

A spleen necrosis virus-based retroviral vector which expresses two genes from a dicistronic mRNA.

We have investigated a novel strategy for coexpressing two genes from a retroviral vector. The 5' nontranslated leader region of at least some picornavirus RNAs contains a sequence that can act as an internal ribosome entry site allowing initiation of translation at a downstream AUG codon in a 5' cap-independent manner. To investigate whether such a sequence can function in the context of a retroviral vector, we constructed a spleen necrosis virus-based vector carrying two selectable marker genes separated by the leader region of encephalomyocarditis virus. This vector was genetically stable and efficiently expressed both markers from a single dicistronic transcript. Since the expression of two genes by other strategies in retroviral vectors can often be problematic, these results offer a promising new approach for the design of "double gene" retroviral vectors.

Animals↗
PubMed1992

Synergy between encephalitogenic T cells and myelin basic protein-specific antibodies in the induction of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an experimentally induced demyelinating disease mediated by CD4+ T cells specific for various myelin proteins including myelin basic protein (MBP) and myelin proteolipid protein (PLP). Although myelin- and other CNS-specific antibodies are produced in EAE, B cells and antibodies are thought by most not to play a decisive role in the induction of EAE. In this report we show that B cells serve as the major antigen-presenting cells (APC) during the T cell activation stage in lymph nodes, and that MBP-specific antibodies can greatly enhance the induction of EAE. The role of B cells as APC is demonstrated in B cell-depleted mice. EAE cannot be induced by antigen/complete Freund's adjuvant immunization unless these mice are locally reconstituted with B cells prior to immunization. The enhancing effect of antibodies is demonstrated in experiments in which EAE is induced by the adoptive transfer of encephalitogenic T cells. The adoptive transfer of large numbers of encephalitogenic T cells induces EAE in 90% of normal recipient mice, but only 33% of B cell-depleted mice get EAE at the same cell dose. The efficiency of EAE induction in B cell-depleted mice can be enhanced if MBP-specific antibodies are simultaneously administered. A similar enhancement is also seen in normal mice when the number of adoptively transferred T cells is limiting. We propose that MBP-specific antibodies enhance the presentation of myelin-derived antigens by APC in the CNS to the adoptively transferred encephalitogenic T cells.

Animals↗
PubMed1992

Reticuloendotheliosis type C and primate type D oncoretroviruses are members of the same receptor interference group.

The reticuloendotheliosis viruses (REVs), originally isolated from avian species, constitute a group of retroviruses which are more closely related to mammalian retroviruses than to other avian retroviruses. The envelope glycoproteins of members of the REV group display a striking amino acid sequence identity with a group of primate oncoretroviruses which belong to a single receptor interference group and include all of the type D and some type C primate oncoretroviruses. Members of the REV group also have a broad host range which covers most avian cells and some mammalian cells, including those of simian and human origin. In view of this broad host range and the envelope sequence similarities, we investigated the cross-interference pattern between REV and primate virus groups to determine whether they utilized the same receptor. Superinfection experiments using a vector virus containing an Escherichia coli lacZ gene showed that reticuloendotheliosis and simian oncoretroviruses constitute a single receptor interference group on both human and canine cells and indicate that the viruses bind to the same receptor to initiate infection. These results suggest that this receptor binding specificity has been maintained over a wide range of retroviruses and may be responsible for the broad spread of these retroviruses between different orders of vertebrates.

Amino Acid Sequence↗
PubMed1991

In vivo analysis of a new lacZ retrovirus vector suitable for cell lineage marking in avian and other species.

To obtain a replication-defective retrovirus vector well suited for cell lineage marking in early avian embryos, we have constructed and tested a derivative of the avian spleen necrosis virus (SNV) carrying the marker gene lacZ. Consistently high titers of this virus, designated CXL, were produced from retroviral packaging cells with no evidence of contaminating helper virus even after 12 months of continuous culture. CXL expresses lacZ strongly and stably in avian cells and has a host range that extends to other avian and some mammalian species. We show that CXL has the potential to mark a wide variety of chick embryo cell types by infection in ovo. The high titers obtainable with this virus can provide a significant advantage over alternative lacZ vectors, especially in lineage marking of early stage embryos. As an example of this, we show that CXL can be used to mark cells of the precardiac mesoderm in stage 4-5 chick embryos.

Animals↗
PubMed1991

Spleen necrosis virus, an avian retrovirus, can infect primate cells.

Spleen necrosis virus (SNV) is an avian retrovirus that can infect some mammalian cells such as dog cells as well as all avian cells tested to date. We were interested in testing whether SNV could also infect primate cells. For these experiments, we used HeLa and COS-7 cells. Initially, we determined whether the SNV long terminal repeat promoter was functional in HeLa and COS-7 cells. In transient transfection assays, the SNV promoter efficiently directed chloramphenicol acetyltransferase gene expression in both HeLa and COS-7 cells. Using SNV- and murine leukemia virus-derived retroviral vectors containing the neomycin phosphotransferase gene, we found that SNV established a provirus in HeLa and COS-7 cells as efficiently as did an amphotropic murine leukemia virus, as judged by the number of G418-resistant HeLa and COS-7 cell colonies obtained after infection and selection. Although SNV formed a provirus in both HeLa and COS-7 cells, productive infection of these cells was not obtained with use of replication-competent SNV. These results suggest that SNV can infect, form a provirus, and stably express a transduced gene in primate cells, but there is a posttranscriptional block to its replication in these cells.

Animals↗
PubMed1990

Inhibition of Ly-6A antigen expression prevents T cell activation.

Antisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein. Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells. It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, and mAb to Ly-6. In contrast, stimulation of normal spleen cells with the pharmacological agents PMA + ionomycin were unaffected by the inhibition of Ly-6 expression. Similar results were obtained with the D10 T cell clone; stimulation with Con A + interleukin 1 (IL-1), antigen-presenting cells (APC), or the clonotypic antibody + IL-1 was greatly reduced in the presence of antisense oligonucleotides to Ly-6. Stimulation with PMA + ionomycin was again unaffected. We also studied the effect of antisense oligonucleotides on stimulation of preactivated D10 cells. Preactivation of D10 cells with Con A + IL-1 renders them receptive to secondary stimulation by other lymphokines. In this case, antisense oligonucleotides to Ly-6 had no effect on secondary activation with IL-2, IL-4 + IL-1, or PMA + ionomycin. We conclude from these studies that Ly-6 expression is required for T cell receptor (TCR)-mediated T cell activation.

Animals↗
PubMed1989

New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors.

We prepared retrovirus packaging cell lines containing gag-pol genes from spleen necrosis virus (expressed from a cytomegalovirus promoter and the simian virus 40 (SV40) polyadenylation sequences) and, on a separate vector, either the env gene from spleen necrosis virus (expressed from the Rous sarcoma virus promoter and the SV40 polyadenylation sequences) or the env gene from amphotropic murine leukemia virus (expressed from a cytomegalovirus promoter and the SV40 polyadenylation sequences). The nucleotide sequences in these packaging cell lines have almost no homology to the retrovirus vectors we used. Retrovirus vectors were produced from these new helper cell lines without any genetic interactions between the vectors and sequences in the helper cells and without transfer of the packaging sequences.

Animals↗
PubMed1988

The thermal stability of oligonucleotide duplexes is sequence independent in tetraalkylammonium salt solutions: application to identifying recombinant DNA clones.

In solutions of tetraalkylammonium salts the melting temperature of oligonucleotide duplexes is independent of nucleotide sequence and thus GC content. Data quantitating the destabilizing effects of various mismatches in these solvents are also presented. The results are in accord with theories on DNA melting and establish conditions under which oligonucleotides can be used as hybridization probes with predictable and controllable specificity.

Base Sequence↗