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Biomedical subjects

Barbara A Osborne

Publications and source records attributed to Barbara A Osborne.

16 recordsLinked to original sources
PubMed2007

Defects of immune regulation in the presenilin-1 mutant knockin mouse.

Mutations in the presenilin-1 (PS1) gene are causally linked to early-onset Alzheimer's disease (AD). Studies of neurons suggest that PS1 mutations result in a gain-of-function, which perturbs calcium regulation and increases cell vulnerability to apoptosis. Alterations in immune cell function have also been demonstrated in AD, and a role for PS1 in immune regulation has been suggested recently. We now report that splenocytes from PS1-mutant (M146V) knockin mice exhibit increased caspase activity, abnormal calcium regulation and aberrant mitochondrial function. Isolated splenic T cells from PS1-mutant mice respond poorly to proliferative signals and have downregulated cluster designation 3 and interleukin (IL)- 2-receptor expression necessary for a normal T-cell immune response. Thus, adverse effects of a mutation that causes AD on immune function that involves perturbed calcium regulation and cytokine signaling in lymphocytes, and associated sensitivity of lymphocytes to apoptosis are demonstrated. These findings suggest that abnormalities in immune function might play major roles in the pathogenesis of AD.

Alzheimer Disease↗
PubMed2006

Notch signalling during peripheral T-cell activation and differentiation.

For many years, researchers have focused on the contribution of Notch signalling to lymphoid development. Only recently have investigators begun to ask what role, if any, Notch has during the activation and differentiation of naive CD4(+) T cells in the periphery. As interest in this issue grows, it is becoming increasingly clear that the main role of Notch signalling, to regulate cell-fate decisions, might also be influential in peripheral T cells.

Animals↗
PubMed2005

Notch1 augments NF-kappaB activity by facilitating its nuclear retention.

Notch1 specifically upregulates expression of the cytokine interferon-gamma in peripheral T cells through activation of NF-kappaB. However, how Notch mediates NF-kappaB activation remains unclear. Here, we examined the temporal relationship between Notch signaling and NF-kappaB induction during T-cell activation. NF-kappaB activation occurs within minutes of T-cell receptor (TCR) engagement and this activation is sustained for at least 48 h following TCR signaling. We used gamma-secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members. We demonstrate that GSI blocked the later, sustained NF-kappaB activation, but did not affect the initial activation of NF-kappaB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF-kappaB and competes with IkappaBalpha, leading to retention of NF-kappaB in the nucleus. Additionally, we show that N1IC can directly regulate IFN-gamma expression through complexes formed on the IFN-gamma promoter. Taken together, these data suggest that there are two 'waves' of NF-kappaB activation: an initial, Notch-independent phase, and a later, sustained activation of NF-kappaB, which is Notch dependent.

Animals↗
PubMed2005

Inhibitors of gamma-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21.

Notch receptors are processed by gamma-secretase acting in synergy with T cell receptor signaling to sustain peripheral T cell activation. Activated CD4+ T cells differentiate into T helper type 1 (TH1) or TH2 subsets. Molecular cues directing TH1 differentiation include expression of the TH1-specific transcription factor T-bet, encoded by Tbx21. However, the regulation of Tbx21 remains incompletely defined. Here we report that Notch1 can directly regulate Tbx21 through complexes formed on the Tbx21 promoter. In vitro, gamma-secretase inhibitors extinguished expression of Notch, interferon-gamma and Tbx21 in TH1-polarized CD4+ cells, whereas ectopic expression of activated Notch1 restored Tbx21 transcription. In vivo, administration of gamma-secretase inhibitors substantially impeded TH1-mediated disease progression in the mouse experimental autoimmune encephalomyelitis model of multiple sclerosis. Thus, using gamma-secretase inhibitors to modulate Notch signaling may prove beneficial in treating TH1-mediated autoimmunity.

Amyloid Precursor Protein Secretases↗
PubMed2004

p53 regulates thymic Notch1 activation.

Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP)/CD8+ single positive (SP) transition, but regulation of Notchactivation is not well understood. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. We hypothesize that p53 regulates Notch activity during T cell development. We found that Notch1 expression and activation were negatively regulated by p53in several thymoma lines. Additionally, NIC was elevated in Trp53(-/-) thymocytes as compared to Trp53(+/+) thymocytes. To determine if elevated Notch1 activation in Trp53(-/-) thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP/CD8+ SP T cell ratio was decreased in Trp53(-/-) splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8+SP T cells in Trp53(-/-) mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, we suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation.

Animals↗
PubMed2003

Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents.

Notch signaling controls cell fate decisions including during development and stem cell renewal and differentiation in many postnatal tissues. Increasing evidence suggests that the Notch signaling network is frequently deregulated in human malignancies and that genetic or pharmacological manipulation of Notch signaling is a novel potential strategy for the treatment of human neoplasms. This review article summarizes the most recent preclinical and clinical evidence linking Notch signaling to cancer, delineates questions that remain unanswered and explores potential biopharmacological strategies to manipulate Notch signaling in vivo.

Animals↗
PubMed2003

TCR-mediated Notch signaling regulates proliferation and IFN-gamma production in peripheral T cells.

Notch genes encode membrane receptors that regulate cell fate decisions in metazoa. Notch receptors and ligands are expressed in developing lymphoid tissue and mature lymphocytes and the role of Notch signaling in early T and B cell development has been studied extensively. However, its contribution to mature T cell function is unknown. TCR-mediated T cell activation is a fundamental process of the adaptive immune system that has been studied for decades; however, the details of this process are incompletely understood. In this study, we present evidence that Notch is required for TCR-mediated activation of peripheral T cells. Inhibition of Notch activation dramatically decreases T cell proliferation in both CD4 and CD8 cells and blocks both NF-kappaB activity and IFN-gamma production in peripheral T cells. Our data reveal a new, nondevelopmental function of Notch as a previously unknown key link in peripheral T cell activation and cytokine secretion.

Amyloid Precursor Protein Secretases↗
PubMed2003

Notch signaling is necessary but not sufficient for differentiation of dendritic cells.

The Notch family of receptors plays an important role in regulation of cell differentiation via direct contact between hematopoietic progenitor cells (HPCs) and bone marrow stroma (BMS). However the precise contribution of Notch in dendritic cell (DC) differentiation is controversial. In 2 different experimental systems using Notch-1-null embryonic stem cells and Notch-1-deficient HPCs we have found that Notch-1 is necessary for DC differentiation. However, activation of Notch-1 and Notch-2 with cell-bound Notch ligand did not result in differentiation of mature DCs or macrophages. Instead, it caused accumulation of immature myeloid cells. Removal of feeder cells resulted in rapid differentiation of DCs and macrophages. Addition of interleukin 4 (IL-4) into the culture dramatically increased accumulation of functionally potent DCs. Lipopolysaccharide was not able to reproduce this effect. Thus, these data indicate that Notch signaling prevents differentiation of mature myeloid cells. Instead, it results in accumulation of precursors readily able to differentiate into mature DCs once the Notch signal is stopped (eg, after cell emigration from bone marrow) and in the presence of other additional differentiation signals provided by IL-4. Thus, Notch is required but not sufficient for DC differentiation.

Animals↗
PubMed2003

Glucocorticoid-induced apoptosis of thymocytes: requirement of proteasome-dependent mitochondrial activity.

Thymocytes undergo negative and positive selection during development in the thymus. During this selection process, the majority of thymocytes are eliminated by apoptosis through signaling via TCR or die by neglect, possibly mediated through glucocorticoids. In this study, we report that thymocytes require molecular oxygen to undergo apoptosis induced by dexamethasone (DEX), a synthetic glucocorticoid, and treatment with N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibits thymocyte apoptosis in vivo as well as ex vivo. We detected elevated intracellular levels of hydrogen peroxide (H(2)O(2)) during DEX-induced apoptosis, which is reduced by NAC treatment, indicating that the elevated levels of intracellular H(2)O(2) are proapoptotic. We also show that loss of mitochondrial membrane potential, cytochrome c release, as well as caspase-3 activation induced by DEX are attenuated by NAC treatment. We identified the production site for H(2)O(2) as the ubiquinone cycle at complex III of mitochondria by using various inhibitors of the mitochondrial electron transport chain, and we show that the cell death events mediated by mitochondria are also significantly reduced when the inhibitors were used. Through inhibition of the proteasome, we also show that the production of H(2)O(2) and the cell death events mediated by mitochondria are regulated by proteosomal activities in DEX-induced thymocyte apoptosis. We conclude that in DEX-treated thymocytes, the increased production of H(2)O(2) originates from mitochondria and is proapoptotic for cell death mediated by mitochondria. We also conclude that all the apoptotic events mediated by mitochondria are regulated by proteasomes.

Acetylcysteine↗
PubMed2003

Cell death in the thymus--it' s all a matter of contacts.

Apoptosis, or programmed cell death, plays a critical role in shaping the T cell repertoire, deleting unproductive as well as potentially autoreactive T cells. Our understanding of how thymocyte apoptosis is regulated is continually evolving, as new essential modulators of this process are discovered. A conundrum that remains, however, is how signaling through essentially the same receptors and cascades evokes distinct biological responses: death by neglect, positive or negative selection. We hypothesize that the immunological synapse (IS) may be critical to transducing survival signals during thymocyte development, and suggest that factors affecting IS assembly may also influence T cell selection.

Animals↗
PubMed2002

Epstein-Barr virus EBNA2 blocks Nur77- mediated apoptosis.

Epstein-Barr virus infection in vitro immortalizes primary B cells. EBNA2 is an Epstein-Barr virus-encoded transcriptional transactivator that mimics the effects of activated Notch signaling and is essential for this proliferative response. An assay using Sindbis virus (SV) as a cell death inducer revealed that, like Notch, EBNA2 also has antiapoptotic activity. We show that Nur77 is a mediator of SV-induced cell death and that EBNA2 antiapoptotic activity results from interaction with Nur77. EBNA2 colocalized with Nur77 in transfected cells and coprecipitated with Nur77 in IB4 B cells. EBNA2 binds to Nur77 through sequences in the EBNA2 amino acid 123-147 conserved domain and an EBNA2 mutant unable to bind Nur77 also lost the ability to protect cells from SV-induced apoptosis. EBNA2 exerted its antideath function by retaining Nur77 in the nucleus and preventing Nur77 from targeting mitochondria in response to apoptotic stimuli. Thus, targeting of Nur77 can be added to the list of strategies used by viruses to counter apoptosis.

Animals↗
PubMed2002

Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells.

Truncated Notch receptors have transforming activity in vitro and in vivo. However, the role of wild-type Notch signaling in neoplastic transformation remains unclear. Ras signaling is deregulated in a large fraction of human malignancies and is a major target for the development of novel cancer treatments. We show that oncogenic Ras activates Notch signaling and that wild-type Notch-1 is necessary to maintain the neoplastic phenotype in Ras-transformed human cells in vitro and in vivo. Oncogenic Ras increases levels and activity of the intracellular form of wild-type Notch-1, and upregulates Notch ligand Delta-1 and also presenilin-1, a protein involved in Notch processing, through a p38-mediated pathway. These observations place Notch signaling among key downstream effectors of oncogenic Ras and suggest that it might be a novel therapeutic target.

Animals↗
PubMed2002

Cloned transchromosomic calves producing human immunoglobulin.

Human polyclonal antibodies (hPABs) are useful therapeutics, but because they are available only from human donors, their supply and application is limited. To address this need, we prepared a human artificial chromosome (HAC) vector containing the entire unrearranged sequences of the human immunoglobulin (hIg) heavy-chain (H) and lambda (lambda) light-chain loci. The HAC vector was introduced into bovine primary fetal fibroblasts using a microcell-mediated chromosome transfer (MMCT) approach. Primary selection was carried out, and the cells were used to produce cloned bovine fetuses. Secondary selection was done on the regenerated fetal cell lines, which were then used to produce four healthy transchromosomic (Tc) calves. The HAC was retained at a high rate (78-100% of cells) in calves and the hIg loci underwent rearrangement and expressed diversified transcripts. Human immunoglobulin proteins were detected in the blood of newborn calves. The production of Tc calves is an important step in the development of a system for producing therapeutic hPABs.

Animals↗
PubMed2002

DNA-containing extracellular 50-nm particles in the ileal Peyer's patch of sheep.

Follicles of the ileal Peyer's patch are sites of B cell proliferation and of diversification of the primary immunoglobulin repertoire in ruminants. We demonstrate here that 50-nm carbonic anhydrase-reactive particles released in the intercellular space in the follicle-associated epithelium of the ileal Peyer's patch of lambs contain DNA protected with a detergent-resistant membrane. We named these particles DiCAPs (DNA in carbonic anhydrase particles). DiCAPs can be purified from a suspension collected from ileal Peyer's patch follicles by sedimentation in a sucrose gradient. The DiCAP membrane is resistant to several ionic and non-ionic detergents alone, but can be disrupted by a combination of Triton X-100 and proteinase K. Differential nuclease treatment of purified DiCAPs indicates that they contain DNA. Digestion of DiCAP DNA with six-base pair restriction enzymes produces smears, suggesting that individual DiCAPs contain unique sequences. Nonetheless, the size of DiCAP DNA is smaller (approximately 16 kb) than that of lamb genomic DNA. Polymerase chain reaction and sequence analysis of DiCAP DNA reveals the presence of light and heavy chain variable genes as well as housekeeping genes. The data demonstrate the presence of DNA in these extracellular particles, and suggest a role of DiCAPs in transfer of DNA between cells within the ileal Peyer's patch. This raises the possibility of a novel form of communication between cells mediated by nucleic acids.

Animals↗
PubMedDate not supplied

Hyperbaric oxygen inhibits benign and malignant human mammary epithelial cell proliferation.

BACKGROUND: Hyperbaric oxygenation (HBO) therapy is the administration of 100%-inhaled oxygen to patients at increased atmospheric pressure. MATERIALS AND METHODS: We used an in vitro model to examine the effects of HBO on mammary cell proliferation. Normal mammary epithelia, primary tumor and metastatic tumor cells derived from the same patient and immortalized by transfection with the human papilloma virus E6 oncogene, as well as the MCF7 human mammary adenocarcinoma cell line, were studied. RESULTS: HBO (97.9% O2, 2.1% CO2, 2.4 atmospheres absolute) inhibited the proliferation of all 4 cell types as measured by light microscopy, [3H]thymidine uptake, a tetrazolium-based colorimetric assay and a clonogenicity assay. The anti-proliferative effect of HBO was time-dependent (p < 0.01 for all 4 cell types). Hyperoxia alone (95% O2, 5% CO2, 1 atmosphere absolute) and increased atmospheric pressure alone (8.75% O2, 2.1% CO2, 2.4 atmospheres absolute) also inhibited proliferation, but their effects were not as profound as HBO (p < 0.01 when either hyperoxia or increased pressure was compared to HBO for all 4 cell types). HBO enhanced the anti-proliferative effects of melphalan (p < 0.05), gemcitabine (p < 0.001) and paclitaxel (p < 0.001). The clonogenicity assay demonstrated that the effects of HBO were still evident 2 weeks after the exposure (p < 0.01 for all 4 cell types). Experiments using Hoechst-propidium iodide or annexin V-propidium iodide staining showed no HBO-induced increases in necrosis or apoptosis. CONCLUSION: HBO inhibits benign and malignant mammary epithelial cell proliferation, but does not enhance cell death.

Adenocarcinoma↗