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Biomedical subjects

M D Eisenbraun

Publications and source records attributed to M D Eisenbraun.

11 recordsLinked to original sources
PubMed2001

Unexpected effects of a heterozygous dnmt1 null mutation on age-dependent DNA hypomethylation and autoimmunity.

DNA methylation modifies gene expression. Methylation patterns are established during ontogeny, but they change with aging, usually with a net decrease in methylation. The significance of this change in T cells is unknown, but it could contribute to autoimmunity, senescence, or both. We examined the effects of a null mutation in DNA methyltransferase 1 (Dnmt1), a gene maintaining DNA methylation patterns, on immune aging. Whereas aged control mice developed hypomethylated DNA, autoimmunity, and signs of immune senescence as predicted, the knockout mice surprisingly increased DNA methylation and developed signs of autoimmunity and senescence more slowly. To identify potential mechanisms, we compared transcripts of DNA methyltransferase and methylcytosine binding protein family members in control and knockout mice. MeCP2, a methylcytosine binding protein involved in gene suppression and chromatin inactivation, was the only transcript differentially expressed between old knockout mice and controls, and thus it is a candidate for a gene product mediating these effects.

Aging↗
PubMed2000

Age-dependent alterations in the assembly of signal transduction complexes at the site of T cell/APC interaction.

TCR interaction with peptide-MHC complexes triggers migration of protein kinases, actin-binding proteins, and other accessory molecules to the T cell/APC synapse. We used confocal immunofluorescence methods to show that the adapter protein LAT (linker for activation of T cells) and the guanine nucleotide exchange factor Vav also move to the APC interface in mouse CD4 T cells conjugated to anti-CD3 hybridoma cells, and in TCR-transgenic CD4 cells conjugated to APC bearing agonist (but not closely related nonagonist) peptides. The proportion of CD4+ T cells able to relocalize LAT or Vav, or to relocate cytoplasmic NT-AT (NF-ATc) from cytoplasm to nucleus, declines about 2-fold in aged mice. The decline in LAT relocalization is accompanied by a similar decline in tyrosine phosphorylation of LAT in CD4 cells stimulated by CD3/CD4 cross-linking. Two-color experiments show that LAT redistribution is strongly associated with relocalization of both NF-ATc and protein kinase C-theta among individual cells. LAT migration to the immunological synapse depends on actin polymerization as well as on activity of Src family kinases, but aging leads to only a small change in the percentage of CD4 cells that redistribute F-actin to the site of APC contact. These results suggest that defects in the ability of T cells from aged donors to move kinase substrates and coupling factors, including LAT and Vav, into the T cell/APC contact region may contribute to the decline with age in NF-ATc-dependent gene expression, and thus to defects in T cell clonal expansion.

Actins↗
PubMed2000

Altered composition of the immunological synapse in an anergic, age-dependent memory T cell subset.

In young mice, memory CD4 T lymphocytes with high P-glycoprotein activity (P-gp(high)) are unresponsive to TCR stimulation in vitro but can be activated by PMA plus ionomycin. The proportion of these hyporesponsive cells increases considerably with age. The earliest events in T cell activation were studied in P-gp(high) and P-gp(low) CD4 memory cells at the single-cell level using confocal immunofluorescence methods. Recruitment of both linker for activation of T cells (LAT) and protein kinase C-theta to the immunological synapse, i.e., the site of T cell interaction with stimulator cells, was greatly impaired in P-gp(high) cells from both young and old mice. Translocation of NF-AT to the nucleus, CD69 expression, and proliferative capacity were also diminished to a similar extent in P-gp(high) cells under the same activation conditions. In contrast, movement of c-Cbl to the synapse region occurred in a high proportion of CD4 memory T cells regardless of P-gp subset or age. Moreover, although P-gp(low) cells frequently recruited both c-Cbl and LAT to the APC synapse, cells in the less responsive P-gp(high) subset frequently relocated c-Cbl, but not LAT, to the interface region. In some systems, c-Cbl can act as a negative regulator of receptor-dependent tyrosine kinases, and alterations of c-Cbl to LAT ratios in the P-gp(high) subset may thus contribute to the hyporesponsiveness of this age-dependent, anergic memory cell population.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Altered development of intestinal intraepithelial lymphocytes in P-glycoprotein-deficient mice.

Intraepithelial lymphocytes (IEL) that reside in the intestinal epithelium are known to exhibit phenotypic and functional characteristics that are distinct from other T cells. We have recently shown that peripheral T cells exclusively express an isoform of P-glycoprotein (P-gp) encoded by the mdr1a gene, but do not require mdr1a expression for normal proliferative, cytokine, or cytotoxic responses. In the present study, we have used mdr1-type knockout (KO) mice to demonstrate that IEL also utilize mdr1a, but only preferentially, in that the mdr1b isoform can be expressed in the absence of mdr1a expression. We also report that a high level of P-gp activity appears to be necessary for the normal development of certain IEL subpopulations. In specific, while the total number of IEL was relatively unaffected by the absence of mdr1a expression, the proportions of CD8 alpha beta and TCR alpha beta+ IEL increased significantly in mdr1a and mdr1a/b KO mice at the expense of CD8 alpha alpha and TCR gamma delta+ IEL, respectively. Moreover, these subset alterations also appeared to have functional consequences, in that proliferative, IL-2, and IFN-gamma responses of IEL from KO mice were distinct from those of normal IEL. In summary, our data suggest that mdr1a expression is required for the development of certain IEL subpopulations, most notably TCR gamma delta+ cells, and thereby indirectly influences the balance of T cell subsets in the intestinal epithelium.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Antigen-specific immunity. Th cell-dependent B cell responses.

Helper T cell-regulated B cell responses constitute a major component of the primary immune response to many pathogens. The subsequent development of antigen-specific immune memory is one critical outcome of this primary adaptive immune response. Antigen-specific immunity develops through a series of intercellular information exchanges organized around cognate T cell receptor-peptide/MHC interactions. Here, we discuss these complex molecularevents andtheircellularconsequences in a serial synapsis model of adaptive immunity. Our laboratory has developed strategies to isolate antigen-specific Th cells and B cells to analyze gene expression and cellular function in single responding lymphocytes directly ex vivo. These studies provide insight into the regulation and cellular organization of antigen-specific immune responses in vivo.

Animals↗
PubMed1999

mdr1a-encoded P-glycoprotein is not required for peripheral T cell proliferation, cytokine release, or cytotoxic effector function in mice.

The plasma membrane transport protein P-glycoprotein (P-gp) is expressed by subsets of both CD4+ and CD8+ T cells in mice. The proportion of T cells that express P-gp goes up with age, and the P-gp-expressing subset of the CD4 memory population is hyporesponsive in many in vitro assays. The significance of P-gp expression for T cell function has not been well established, although several reports have suggested that it may promote cytokine export and/or cytotoxic T cell function. To elucidate which T cell functions may require P-gp, we have compared a variety of responses using T cells from wt and P-gp knockout mice. Protein expression and rhodamine-123 efflux studies revealed that peripheral T cells exclusively utilize the mdr1a-encoded isoform rather than the homologous mdr1b or mdr2 isoforms. Comparisons of T cells from mdr1a+/+ and mdr1a-/- mice showed no differences in proliferation or in secretion of IL-2, IL-4, IL-5, IL-10, or IFN-gamma in response to polyclonal stimulation. Moreover, mdr1a-/- T cells produced strong allospecific cytotoxic responses comparable to those of wt T cells. Our results show that P-gp is not a necessary component of peripheral T cell functional responses. Further investigation will be needed to determine the significance of P-gp expression in T lymphocytes.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1995

Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA.

Particle-mediated (gene gun) DNA transfer to the epidermis was evaluated for its ability to elicit humoral and cytotoxic T lymphocyte responses using decreasing quantities of plasmid DNA-based antigen expression vectors. Using plasmids encoding human growth hormone, human alpha-1-antitrypsin, and influenza virus nucleoprotein, strong immune responses were observed in mice following immunization with as little as 16 ng of DNA using an electric discharge gene delivery system. Significant antibody titers were observed against these antigens following a primary immunization, with responses rising dramatically following a boost. Increasing the DNA dose above 16 ng per immunization had little beneficial effect. In contrast to particle-mediated DNA delivery, intramuscular or intradermal inoculation required greater than 5000-fold more DNA to achieve comparable results. Data are also presented demonstrating that a simple, hand-held version of the Accell DNA delivery system, employing compressed helium as the particle motive force, achieves immune responses comparable to the traditional electric discharge device.

Amino Acid Sequence↗
PubMed1994

Accell particle-mediated DNA immunization elicits humoral, cytotoxic, and protective immune responses.

Accell particle-mediated gene delivery technology was employed for the intracellular delivery of antigen-encoding expression vectors in epidermal tissues in laboratory animals. Delivery of plasmid DNA-coated gold microparticles using the Accell gene delivery system resulted in de novo antigen expression in epidermal cells that stimulated the induction of antigen-specific humoral and cytotoxic cellular immune responses. Optimal DNA delivery conditions favoring maximal humoral responses required the delivery of 5 x 10(7) micron-sized gold particles containing 300 plasmid copies per particle (80 ng of vector total) into a 4-cm2 area of abdominal skin. Comparison of immune responses between animals that received intramuscular injections of relatively large quantities of vector DNA (100 micrograms) and those that received intracellular deliveries of submicrogram quantities of the same DNA to the epidermis demonstrated that the latter approach was considerably more effective at eliciting strong humoral responses. In addition, cytotoxic cellular immune responses were elicited to HIV-1 gp120 following epidermal delivery of HIV-1 gp160 or gp120 expression constructs. A qualitative shift from predominantly cytotoxic cellular to predominantly humoral immune responses with continued immunization indicated the potential for optimizing delivery conditions to favor specifically one type of response over the other.

AIDS Vaccines↗
PubMed1993

Effects of plasmid pBR322 on respiratory and ATPase activities in Escherichia coli.

The effects of antibiotic resistance plasmids on respiratory and ATPase activities were investigated in Escherichia coli. The rates of oxygen consumption coupled to the oxidation of succinate and NADH, ATP hydrolysis, and ATP-Pi exchange were measured in isolated membrane vesicles prepared from HB101 strains that contained derivatives of plasmid pBR322. The rates of oxygen consumption coupled to the oxidation of NADH were independent of the presence or absence of plasmids in the strains from which the vesicles were prepared. In contrast, the mean rates of oxygen consumption coupled to the oxidation of succinate, ATP hydrolysis, and ATP-Pi exchange were 140-292% higher in vesicles made from plasmid-containing strains than from plasmid-free HB101 and independent of the expression of the plasmid-encoded tetracycline/H+ antiporter.

Adenosine Triphosphatases↗
PubMed1993

Examination of parameters affecting the elicitation of humoral immune responses by particle bombardment-mediated genetic immunization.

A human growth hormone expression construct was delivered intracellularly into the abdominal skin of mice by particle bombardment-mediated gene transfer. Using this technology, the in vivo delivery of antigen-encoding expression vectors affixed to gold microprojectiles results in de novo antigen production in target skin and development of specific antibody responses. In this study, we examined the contribution of various delivery parameters to the resultant protein expression and related antibody responses. The highest levels of both protein expression and antibody production were correlated with particle delivery to the epidermis while deliveries extending into the dermis resulted in decreased protein and antibody production. Optimal immune responses were also shown to be dependent upon the delivery of a sufficient number of DNA-coated gold particles, indicating that a dose-response relationship exists between the number of particles delivered and the resultant protein expression and antibody production. Further, maximal protein expression and associated antibody titers were elicited with surprisingly small amounts of DNA. The practicality of targeting skin and the use of three to four orders of magnitude less DNA than is typically required in direct DNA inoculation studies demonstrates the potential utility of this emerging technology for the rapid production of antibodies in laboratory animals, and in the development of a new class of human clinical vaccines based upon direct, intracellular DNA delivery.

Administration, Topical↗