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Biomedical subjects

D A Peterson

Publications and source records attributed to D A Peterson.

At least 19 recordsLinked to original sources
PubMed1998

In vivo expression of a TCR antagonist: T cells escape central tolerance but are antagonized in the periphery.

Transgenic 3.L2 T cells are stimulated by Hb(64-76)/I-Ek and are positively selected on I-Ek plus self-peptides. To this pool of self-peptides we have added a single, well-defined 3.L2 TCR antagonist (A72) in vivo. We find that mice expressing both the 3.L2 TCR and A72 have a minimal loss of T cells expressing the clonotypic TCR in the thymus and spleen. Importantly, the proliferative response of 3.L2 x A72 splenocytes is significantly reduced compared with splenocytes from 3.L2 mice. This reduced response can be attributed to peripheral antagonism. Thus we have identified a new class of self-ligands whose predominant effect is constitutive peripheral antagonism rather than negative selection. The net effect of these ligands is to avoid potential self-reactivity while maintaining as large a repertoire as possible.

Animals↗
PubMed1998

The Fas/Fas ligand pathway and Bcl-2 regulate T cell responses to model self and foreign antigens.

We have examined the role of Fas and Bcl-2 in T cell survival and responses to antigen in vivo using T cells that express a transgenic antigen receptor specific for hen egg lysozyme (HEL) and that either lack functional Fas or Fas ligand (FasL) or overexpress Bcl-2 as a transgene. HEL-specific, Bcl-2-transgenic T cells showed prolonged responses to immunization with cognate peptide but were eliminated rapidly when exposed to HEL expressed systemically as a self antigen. In contrast, Fas- and FasL-defective T cells did not display exaggerated responses to immunization with HEL peptide, but did show increased expansion and survival in response to systemic self antigen and were able to activate anti-HEL (self) antibody-forming cells. Thus, Bcl-2 and Fas play different roles in the regulation of T cell responses to antigen in vivo and in self tolerance.

Adoptive Transfer↗
PubMed1997

Antigen receptor engagement delivers a stop signal to migrating T lymphocytes.

We investigated the role of the T cell antigen receptor (TcR) in control of T cell migration in an in vitro system. We used T cells from transgenic mice bearing a TcR for the lysozyme peptide 48-62 bound to I-A(k) (3A9). T cells from the 3A9 TcR transgenic mice crawled on purified intercellular adhesion molecule-1 substrates, but strikingly, stopped upon interaction with the physiological ligand, i.e., the mouse I-A(k) with covalently attached hen egg white lysozyme peptide residues 48-62 complex. TcR-triggered stopping was reversible by treatment with adhesion-strengthening phorbol esters. The microtubule organizing center of stopped cells was positioned adjacent to the site of stable cell anchorage. Direct conversion of lymphocyte function associated-1 to the high-affinity conformation with antibodies also stopped T cells in a similar manner to antigen. Thus, physiological TcR engagement triggers a stop signal through lymphocyte function associated-1. We propose that the stop signal is an early and essential event in T cell activation that also will play an important role in control of T cell migration.

Animals↗
PubMed1997

Endogenous FGF-2 is important for cholinergic sprouting in the denervated hippocampus.

To investigate the molecular mechanisms of cholinergic sprouting in the hippocampus after removal of entorhinal cortical inputs, we evaluated trophic factor gene expression in the denervated hippocampus. Despite the proposed role for nerve growth factor (NGF) in this sprouting, we observed no change in NGF mRNA or protein at several postlesion time points. In contrast, FGF-2 mRNA was increased within 16 hr. FGF-2 immunoreactivity was localized within GFAP-positive hypertrophic astrocytes distributed specifically within the denervated outer molecular layer after the lesion. To address the functional significance of this increase in FGF-2, we assessed the magnitude of cholinergic sprouting in animals receiving chronic intracerebroventricular infusions of neutralizing antibodies specific for FGF-2 and compared it with that observed in lesioned animals receiving infusate controls. Animals given FGF-2 antibodies displayed a marked reduction in cholinergic sprouting as compared with controls. In fact, many of these animals exhibited virtually no sprouting at all despite histological verification of complete lesions. These results suggest that endogenous FGF-2 promotes cholinergic axonal sprouting in the injured adult brain. Furthermore, immunocytochemical localization of receptors for FGF-2 (i.e., FGFR1) on projecting basal forebrain cholinergic neurons suggests that FGF-2 acts directly on these neurons to induce the lesion-induced sprouting response.

Animals↗
PubMed1997

Reversible Schwann cell hyperplasia and sprouting of sensory and sympathetic neurites after intraventricular administration of nerve growth factor.

Substantial dysfunction and loss of cholinergic neurons occur in Alzheimer's disease (AD). Nerve growth factor (NGF) is a potent neurotrophic factor for cholinergic basal forebrain neurons, and the use of NGF to stimulate residual dysfunctional cells in AD is being considered. To define the effects of NGF on other cell populations in the brain, NGF was continuously infused into the lateral ventricle of rats for 7 weeks. At the end of treatment, Schwann cell hyperplasia and abundant sensory and sympathetic neurite sprouting were observed in the subpial region of the medulla oblongata and the spinal cord. Following withdrawal of NGF, the Schwann cell hyperplasia and sprouting of sensory and sympathetic neurites disappeared completely. These findings suggest that better temporal and spatial delivery systems for NGF must be explored to limit potential undesirable side effects while maintaining the survival and function of diseased basal forebrain cholinergic neurons.

Animals↗
PubMed1997

Diversity of response in vascular smooth muscle cells to changes in oxygen tension.

Hypoxia causes pulmonary vasoconstriction (HPV), but also dilation of systemic vessels and the ductus arteriosus. In the adult animal. HPV is initiated by inhibition of potassium current (IK) in the smooth muscle cells of small resistance arteries, which results in membrane depolarization and calcium entry through voltage-gated calcium channels. The oxygen-sensitive channels that initiate HPV are 4-aminopyridine (4-AP)-sensitive delayed rectifier channels (KDR), the most prominent of which has a conductance of 37 pS. In the fetus, hypoxia causes pulmonary vasoconstriction through inhibition of a calcium-sensitive potassium channel (KCa). In smooth muscle cells from the rabbit ductus arteriosus, which dilates in response to hypoxia, whole-cell potassium current is reversibly enhanced, rather than inhibited, by hypoxia. The principal oxygen-sensitive channel is inhibited by 4-AP and has a conductance of about 58 pS. There are morphological and electrophysiological differences between individual pulmonary artery smooth muscle cells, for example, in some cells IK is predominantly carried by KDR channels and in others by KCa channels. KDR cells are more common in the resistance pulmonary arteries and KCa in the conduit arteries. Responses of specific vessels (conduit, resistance; pulmonary, systemic, ductus) at different stages of development (fetal, neonatal and adult) to changes in oxygen tension may be determined by the distribution of a variety of ion channels in the smooth muscle cells.

Animals↗
PubMed1997

Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.

Successful gene therapy approaches will require efficient gene delivery and sustained expression of the transgene in recipients. A variety of methods, ranging from direct DNA delivery to infection with recombinant viruses containing foreign genes, have been developed, but they all have some major limitations that restrict their utility. We have described a human lentiviral (HIV)-based vector that can transduce non-dividing cells in vitro and deliver genes in vivo. With this vector, expression of transgenes in the brain has been detected for more than six months--the longest period tested so far. Because lentiviral vectors are pseudotyped with vesicular stomatitis virus G glycoprotein (VSVG; ref. 8), they can transduce a broad range of tissues and cell types. We now describe the ability of lentiviral vectors to introduce genes directly into liver and muscle. Sustained expression of green fluorescent protein (GFP), used as a surrogate for therapeutic protein, can be observed for more than 22 weeks in the liver. Similar long-term expression (more than eight weeks) was observed in transduced muscle. In contrast, little or no GFP could be detected in liver or muscle transduced with the Moloney murine leukaemia virus (M-MLV), a prototypic retroviral based vector. At a minimum, 3-4% of the total liver tissue was transduced by a single injection of 1-3 x 10(7) infectious units (I.U.) of recombinant HIV vector. Furthermore, no inflammation of recruitment of lymphocytes could be detected at the site of injection. Animals previously transduced with a lentiviral vector can be efficiently re-infected with lentiviral vectors. Additionally, we show that the requirement for lentiviral accessory proteins to establish efficient transduction in vivo is tissue dependent.

Animals↗
PubMed1997

Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is one of the most frequently used methods for measuring cell proliferation and neural cytotoxicity. It is widely assumed that MTT is reduced by active mitochondria in living cells. By using isolated mitochondria from rat brain and B12 cells, we indeed found that malate, glutamate, and succinate support MTT reduction by isolated mitochondria. However, the data presented in this study do not support the exclusive role of mitochondria in MTT reduction by intact cells. Using a variety of approaches, we found that MTT reduction by B12 cells is confined to intracellular vesicles that later give rise to the needle-like MTT formazan at the cell surface. Some of these vesicles were identified as endosomes or lysosomes. In addition, MTT was found to be membrane impermeable. These and other results suggest that MTT is taken up by cells through endocytosis and that reduced MTT formazan accumulates in the endosomal/lysosomal compartment and is then transported to the cell surface through exocytosis.

Animals↗
PubMed1997

Differential effects of nuclear receptor corepressor (N-CoR) expression levels on retinoic acid receptor-mediated repression support the existence of dynamically regulated corepressor complexes.

Thyroid hormone and retinoic acid receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors that stimulate the transcription of target genes in the presence of activating ligands and repress transcription in their absence. Transcriptional repression by the thyroid hormone and retinoic acid receptors has been proposed to be mediated by the nuclear receptor corepressor, N-CoR, or the related factor, SMRT (silencing mediator of retinoic acid and thyroid hormone receptors). Recent studies have suggested that transcriptional repression by N-CoR involves a corepressor complex that also contains mSin3A/B and the histone deacetylase, RPD3. In this manuscript, we demonstrate that transcriptional repression by the retinoic acid receptor can be either positively or negatively regulated by changes in the levels of N-CoR expression, suggesting a relatively strict stoichiometric relationship between N-CoR and other components of the corepressor complex. Consistent with this interpretation, overexpression of several functionally defined domains of N-CoR also relieve repression by nuclear receptors. N-CoR is distributed throughout the nucleus in a nonuniform pattern, and a subpopulation becomes concentrated into several discrete dot structures when highly expressed. RPD3 is also widely distributed throughout the nucleus in a nonuniform pattern. Simultaneous imaging of RPD3 and N-CoR suggest that a subset of each of these proteins colocalize, consistent with the existence of coactivator complexes containing both proteins. In addition, a substantial fraction of both N-CoR and mSin3 A/B appear to be independently distributed. These observations suggest that interactions between RPD3 and Sin3/N-CoR complexes may be dynamically regulated.

Animals↗
PubMed1996

Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo.

Neurogenesis continues throughout adulthood in discrete regions. Proliferative zones include the subependymal zone, from where progenitors migrate along the rostral migratory pathway to differentiate into neurons in the olfactory bulb, and the hippocampal subgranular zone, where they migrate and differentiate into granule neurons. Progenitors isolated from adult subependymal zone exhibit in vitro neurogenesis when stimulated with epidermal or fibroblast growth factor. Cultured adult rat hippocampal progenitors (AHPs) grafted to adult rat hippocampus show site-specific neuronal differentiation. Here we investigate determinants of multipotentiality in the adult central nervous system, by grafting AHPs into homotypic (hippocampus) or heterotypic (the rostral migratory pathway) neurogenic sites or a heterotypic, non-neurogenic site (the cerebellum). We found that grafts into neurogenic, but not nonneurogenic sites, showed neuronal differentiation. Furthermore, AHPs grafted in the rostral migratory pathway migrated into the olfactory bulb, differentiating into tyrosine-hydroxylase-positive neurons, a non-hippocampus phenotype. These results reveal that AHP populations can respond to persistent neuronal differentiation cues in the adult central nervous system.

Adult↗
PubMed1996

Sprouting adult CNS cholinergic axons express NILE and associate with astrocytic surfaces expressing neural cell adhesion molecule.

To assess the cellular and molecular substrates for cholinergic axon growth in the adult central nervous system (CNS), we implanted grafts of control and nerve growth factor (NGF)-producing genetically modified fibroblasts within the striatum of rats. Sprouting cholinergic axonal processes that grew into grafts of NGF-producing fibroblasts were fasciculated and followed the surface of astrocytic processes for long distances within the grafts. The close and long distance anatomical relationship between the sprouted axons and the astrocytes supported previous ultrastructural evidence that astrocytes may serve as a cellular substrate for sprouting cholinergic axons in vivo. The sprouted axon processes were associated with the expression of nerve growth factor-inducible large external (NILE) glycoprotein on their surfaces. NILE expression was not seen in control grafts where there was an absence of cholinergic ingrowth. NILE has been demonstrated to play a role in axon fasciculation in a number of other neural systems. The astrocytic processes in both control and NGF-producing fibroblast grafts expressed neural cell adhesion molecule (NCAM), suggesting that NCAM-mediated adhesion may be responsible for the close relationship between the axons and astrocytes within the grafts. NGF-induced heterotypic interactions between neuronal NILE and astroglial NCAM may also be required for adult cholinergic axonal sprouting.

Acetylcholine↗
PubMed1996

Fibroblast growth factor-2 protects entorhinal layer II glutamatergic neurons from axotomy-induced death.

The entorhinal cortex is a major relay between the hippocampus and other cortical and subcortical regions. Glutamatergic axons from layer II neurons form the entorhinal cortical projection to the hippocampus via the perforant pathway. We have demonstrated previously that lesion of the perforant pathway causes the death of approximately 30% of entorhinal layer II (ECL2) neurons. To elucidate mechanisms contributing to neuronal death and to investigate strategies preventing it, we identified the phenotype of the vulnerable neuronal population. Sections were immunolabeled with antibodies to the neuronal markers NeuN, glutamate, and calbindin-D28k, and to receptors for fibroblast growth factor-2 (FGFR1) and NMDA (NMDAR1) and were examined using confocal microscopy. Calbindin immunoreactivity was strikingly lamina-specific to ECL2, where one-third of all ECL2 neurons were calbindin-positive. Localization of glutamate revealed that half of the glutamatergic ECL2 neurons coexpressed calbindin. Quantification using unbiased stereology at 9 weeks after lesion of the perforant pathway revealed that the only ECL2 neuronal population that experienced a significant (70%) loss (20% of the total) was the population of glutamatergic ECL2 neurons that did not coexpress calbindin. All ECL2 neurons expressed FGFR1; therefore, we tested the role of FGF-2 in the survival of glutamatergic ECL2 neurons. We grafted fibroblasts genetically engineered to express nerve growth factor or FGF-2 and found that only FGF-2 grafts prevented loss of the vulnerable glutamatergic/calbindin-negative neurons. We present a hypothesis for the selective vulnerability of these glutamatergic/calbindin-negative ECL2 neurons and address the role of FGF-2 in neuronal rescue.

Afferent Pathways↗
PubMed1996

Improved resolution of fibronectin mRNA expression in the inner ear using laser scanning confocal microscopy.

We describe a modified in situ hybridization protocol for localizing and quantifying fibronectin gene expression at the cellular level in paraffin sections of rat temporal bone. When combined with a novel analytical approach using laser scanning confocal microscopy (LSCM), this protocol significantly improved the resolution, sensitivity, and specificity of existing procedures for evaluating fibronectin synthesis in developing inner ear. For simultaneous viewing of cochlear anatomy and the autoradiographic signal, transmitted light images of the cochlea were collected separately from LSCM reflected light images of the autoradiographic silver grains and then the two images were electronically merged. Within the first 2 microns below the surface of the emulsion, silver grains were clustered specifically over hybridized cells. In contrast, nonspecific silver grain development (i.e., background noise) was confined primarily to the lower 5 microns of the emulsion adjacent to the tissue section. Limiting the volume of the emulsion examined in the LSCM analysis, i.e., restricting the range of optical sectioning to the first 2 micron below the surface of the emulsion, effectively minimized nonspecific background noise and maximized the specificity of the hybridization signal. The improvements offered by the described methodological approaches are equally appropriate for non-calcified tissues.

Animals↗
PubMed1995

Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.

The dentate gyrus of the hippocampus is one of the few areas of the adult brain that undergoes neurogenesis. In the present study, cells capable of proliferation and neurogenesis were isolated and cultured from the adult rat hippocampus. In defined medium containing basic fibroblast growth factor (FGF-2), cells can survive, proliferate, and express neuronal and glial markers. Cells have been maintained in culture for 1 year through multiple passages. These cultured adult cells were labeled in vitro with bromodeoxyuridine and adenovirus expressing beta-galactosidase and were transplanted to the adult rat hippocampus. Surviving cells were evident through 3 months postimplantation with no evidence of tumor formation. Within 2 months postgrafting, labeled cells were found in the dentate gyrus, where they differentiated into neurons only in the intact region of the granule cell layer. Our results indicate that FGF-2 responsive progenitors can be isolated from the adult hippocampus and that these cells retain the capacity to generate mature neurons when grafted into the adult rat brain.

Animals↗
PubMed1995

Opposing effects of oxidants and antioxidants on K+ channel activity and tone in rat vascular tissue.

K+ channels regulate tone in both the systemic and pulmonary circulations. K+ channel inhibition leads to membrane depolarization, Ca2+ influx and vasoconstriction; K+ channel activation leads to hyperpolarization and vasodilatation. The sulfhydryl oxidant diamide opens K+ channels in pulmonary smooth muscle and acts as a potent vasodilator in perfused lungs. We examined the hypothesis that antioxidants cause constriction and oxidants cause relaxation through their effects on K+ channels in vascular smooth muscle. The oxidant diamide (380 microM and 3.8 mM) inhibited the reduction of cytochrome C by ferrous sulphate in vitro whilst the antioxidants co-enzyme Q10 (770 microM) and duroquinone (700 microM) increased the rate of reduction. Both antioxidants caused dose-dependent constriction of endothelium-intact and -denuded rat pulmonary artery and aortic rings. This constriction could be reversed by 1 microM diamide. Co-enzyme Q10 and duroquinone (both at 100 microM) partially inhibited (approximately 30%) whole-cell K+ channel currents and depolarized membranes of isolated pulmonary artery smooth muscle cell recorded using the amphotericin-perforated-patch-clamp technique. Diamide (100 microM) increased whole-cell K+ channel currents and hyperpolarized the membrane. The data suggest that oxidants and antioxidants may modulate vascular tone via an effect on K+ channels.

Animals↗