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Biomedical subjects

David Baltimore

Publications and source records attributed to David Baltimore.

At least 19 recordsLinked to original sources
PubMed2005

Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells.

A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both alpha and beta chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

Animals↗
PubMed2005

Physiological functions for brain NF-kappaB.

Members of the nuclear factor kappaB (NF-kappaB) family of transcription factors are activated within the CNS in pathological settings of apoptosis and neurological disease. Recent work using several model systems provides accumulating evidence that these transcription factors also participate in the regulation of neuronal activity-dependent transcription and behavior under physiological conditions. This review highlights advances in our understanding of the mechanisms of Ca(2+)-responsive activation and synaptic signaling to the nucleus by NF-kappaB transcription factors within the CNS, and the relevance of this transcription factor family for learning and memory.

Animals↗
PubMed2004

Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth.

The Ryk receptor belongs to the atypical receptor tyrosine kinase family. It is a new member of the family of Wnt receptor proteins. However, the molecular mechanisms by which the Ryk receptor functions remain unknown. Here, we report that mammalian Ryk, unlike the Drosophila Ryk homolog Derailed, functions as a coreceptor along with Frizzled for Wnt ligands. Ryk also binds to Dishevelled, through which it activates the canonical Wnt pathway, providing a link between Wnt and Dishevelled. Transgenic mice expressing Ryk siRNA exhibit defects in axon guidance, and Ryk is required for neurite outgrowth induced by Wnt-3a and in the activation of T cell factor (TCF) induced by Wnt-1. Thus, Ryk appears to play a crucial role in Wnt-mediated signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2004

One nucleotide in a kappaB site can determine cofactor specificity for NF-kappaB dimers.

The transcription factor NF-kappaB regulates a wide variety of genes involved in multiple processes. Although the apparent consensus sequence of DNA binding sites for NF-kappaB (kappaB sites) is very broad, the sites active in any one gene show remarkable evolutionary stability. Using a lentivirus-based methodology for implantation of gene regulatory sequences we show that for genes with two kappaB sites, both are required for activity. Swapping sites between kappaB-dependent genes altered NF-kappaB dimer specificity of the promoters and revealed that two kappaB sites can function together as a module to regulate gene activation. Further, although the sequence of the kappaB site is important for determining kappaB family member specificity, rather than determining the ability of a particular dimer to bind effectively, the sequence affects which coactivators will form productive interactions with the bound NF-kappaB dimer. This suggests that binding sites may impart a specific configuration to bound transcription factors.

Animals↗
PubMed2004

Unique CD40-mediated biological program in B cell activation requires both type 1 and type 2 NF-kappaB activation pathways.

B lymphocytes can be activated by many different stimuli. However, the mechanisms responsible for the signaling and functional specificities of individual stimuli remain to be elucidated. Here, we have compared the contribution of the type 1 (p50-dependent) and type 2 (p52-dependent) NF-kappaB activation pathways to cell survival, proliferation, homotypic aggregation, and specific gene regulation of murine primary B lymphocytes. Whereas lipopolysaccharide (LPS) and B cell activation factor (BAFF) mainly activate the type 1 or type 2 pathways, respectively, CD40 ligand (CD40L) strongly activates both. Rescue of spontaneous apoptosis is diminished in p52(-/-) B cells after BAFF stimulation and in p50(-/-)c-Rel(-/-) B cells after LPS stimulation. Interestingly, significant CD40-induced B cell survival is still observed even in p50(-/-)c-Rel(-/-)p65(-/+) B cells, which is correlated with the ability of CD40L to up-regulate Bcl-x(L) expression in these cells. CD40L- and LPS-induced B cell proliferation, as well as up-regulation of proliferation-related genes, however, are greatly reduced in c-Rel(-/-) and p50(-/-)c-Rel(-/-) B cells but are normal in p52(-/-) B cells. We have further demonstrated that both c-Rel and p52 are required for CD40-mediated B cell homotypic aggregation, which explains well why neither LPS nor BAFF has this function. Overall, our studies suggest that both type 1 and type 2 NF-kappaB pathways contribute to the gene expression and biological program unique for CD40 in B cell activation.

Animals↗
PubMed2003

Genetic analysis of NF-kappaB/Rel transcription factors defines functional specificities.

The NF-kappaB transcription factors consist of dimeric proteins of the Rel homology family. They activate many promoters containing highly divergent kappaB-site sequences. We have generated cell lines lacking individual and multiple NF-kappaB proteins and used them to establish interactions between components of the NF-kappaB-IkappaB signaling system. Functional compensation within the family of dimers was evident in knockout cell lines. Analysis of transiently transfected genes gave an impression of promiscuity that was not borne out by analysis of endogenous genes. Using TNFalpha as an inducer, a panel of endogenous genes showed a wide range of subunit specificities as well as highly variable kinetics of induction. Comparing the function and subunit specificity of genes with the sequence of the kappaB DNA-binding site we found little correlation, indicating that NF-kappaB family member specificity for endogenous promoters is not solely encoded by the kappaB site sequence itself.

3T3 Cells↗
PubMed2003

Essential role for STAT5 signaling in CD25+CD4+ regulatory T cell homeostasis and the maintenance of self-tolerance.

A population of CD25(+)CD4(+) regulatory T cells (T regs) functions to maintain immunological self tolerance by inhibiting autoreactive T cell responses. CD25(+)CD4(+) T regs are present in low, but steady, numbers in the peripheral lymphoid tissues of healthy mice. Recent studies have shown that IL-2 is an essential growth factor for these cells. How this cytokine functions to regulate CD25(+)CD4(+) T reg homeostasis and prevent autoimmune disease remains unknown. In conventional CD4(+) T cells, IL-2 triggers signaling pathways that promote proliferation and survival by activating the STAT5 transcription factor and by increasing the expression of the antiapoptotic protein, Bcl-2. We show here that bcl-2 deficiency does not affect CD25(+)CD4(+) T reg homeostasis, and that ectopic expression of this molecule fails to rescue CD25(+)CD4(+) T reg numbers or to prevent the development of autoimmunity in IL-2-deficient mice. Furthermore, transient activation of STAT5 is sufficient to increase CD25(+)CD4(+) T reg numbers in IL-2-deficient mice. Our study uncovers an essential role for STAT5 in maintaining CD25(+)CD4(+) T reg homeostasis and self-tolerance.

Animals↗
PubMed2003

NF-kappa B functions in synaptic signaling and behavior.

Ca(2+)-regulated gene transcription is essential to diverse physiological processes, including the adaptive plasticity associated with learning. We found that basal synaptic input activates the NF-kappa B transcription factor by a pathway requiring the Ca(2+)/calmodulin-dependent kinase CaMKII and local submembranous Ca(2+) elevation. The p65:p50 NF-kappa B form is selectively localized at synapses; p65-deficient mice have no detectable synaptic NF-kappa B. Activated NF-kappa B moves to the nucleus and could directly transmute synaptic signals into altered gene expression. Mice lacking p65 show a selective learning deficit in the spatial version of the radial arm maze. These observations suggest that long-term changes to adult neuronal function caused by synaptic stimulation can be regulated by NF-kappa B nuclear translocation and gene activation.

Active Transport, Cell Nucleus↗
PubMed2003

Medicine. The need for a global HIV vaccine enterprise.

A new collaborative model of research is needed to increase resources, to prioritize the R (ii) to increase the pace, reduce the overlap, and more systematically explore the elements of and delivery systems for vaccines; (iii) to use common standards for the prompt comparative testing of vaccine candidates; (iv) to expand resources for manufacturing vaccine candidates to speed their use in human trials; and (v) to increase the capacity for international clinical trials and to focus this effort toward quickly measuring the effectiveness of vaccine protection as prototype vaccine candidates are identified.

AIDS Vaccines↗
PubMed2003

Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance.

A Cre/lox-conditional mouse line was generated to evaluate the role of ATR in checkpoint responses to ionizing radiation (IR) and stalled DNA replication. We demonstrate that after IR treatment, ATR and ATM each contribute to early delay in M-phase entry but that ATR regulates a majority of the late phase (2-9 h post-IR). Double deletion of ATR and ATM eliminates nearly all IR-induced delay, indicating that ATR and ATM cooperate in the IR-induced G2/M-phase checkpoint. In contrast to the IR-induced checkpoint, checkpoint delay in response to stalled DNA replication is intact in ATR knockout cells and ATR/ATM and ATR/p53 double-knockout cells. The DNA replication checkpoint remains intact in ATR knockout cells even though the checkpoint-stimulated inhibitory phosphorylation of Cdc2 on T14/Y15 and activating phosphorylation of the Chk1 kinase no longer occur. Thus, incomplete DNA replication in mammalian cells can prevent M-phase entry independently of ATR and inhibitory phosphorylation of Cdc2. When DNA replication inhibitors are removed, ATR knockout cells proceed to mitosis but do so with chromosome breaks, indicating that ATR provides a key genome maintenance function in S phase.

Animals↗
PubMed2003

Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks.

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.

Cell Line↗
PubMed2002

Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5.

Double-stranded RNAs approximately 21 nucleotides long [small interfering RNA (siRNA)] are recognized as powerful reagents to reduce the expression of specific genes. To use them as reagents to protect cells against viral infection, effective methods for introducing siRNAs into primary cells are required. Here, we describe success in constructing a lentivirus-based vector to introduce siRNAs against the HIV-1 coreceptor, CCR5, into human peripheral blood T lymphocytes. With high-titer vector stocks, >40% of the peripheral blood T lymphocytes could be transduced, and the expression of a potent CCR5-siRNA resulted in up to 10-fold inhibition of CCR5 expression on the cell surface over a period of 2 weeks in the absence of selection. In contrast, the expression of another major HIV-1 coreceptor, CXCR4, was not affected. Importantly, blocking CCR5 expression by siRNAs provided a substantial protection for the lymphocyte populations from CCR5-tropic HIV-1 virus infection, dropping infected cells by 3- to 7-fold; only a minimal effect on infection by a CXCR4-tropic virus was observed. Thus, our studies demonstrate the feasibility and potential of lentiviral vector-mediated delivery of siRNAs as a general means of intracellular immunization for the treatment of HIV-1 and other viral diseases.

Acquired Immunodeficiency Syndrome↗
PubMed2002

The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation.

Nuclear localization of the transcriptional activator NF-kappaB (nuclear factor kappaB) is controlled in mammalian cells by three isoforms of NF-kappaB inhibitor protein: IkappaBalpha, -beta, and - epsilon. Based on simplifying reductions of the IkappaB-NF-kappaB signaling module in knockout cell lines, we present a computational model that describes the temporal control of NF-kappaB activation by the coordinated degradation and synthesis of IkappaB proteins. The model demonstrates that IkappaBalpha is responsible for strong negative feedback that allows for a fast turn-off of the NF-kappaB response, whereas IkappaBbeta and - epsilon function to reduce the system's oscillatory potential and stabilize NF-kappaB responses during longer stimulations. Bimodal signal-processing characteristics with respect to stimulus duration are revealed by the model and are shown to generate specificity in gene expression.

Animals↗
PubMed2002

CARD11 mediates factor-specific activation of NF-kappaB by the T cell receptor complex.

NF-kappaB is a critical target of signaling downstream of the T cell receptor (TCR) complex, but how TCR signaling activates NF-kappaB is poorly understood. We have developed an expression cloning strategy that can identify catalytic and noncatalytic molecules that participate in different pathways of NF-kappaB activation. Screening of a mouse thymus cDNA library yielded CARD11, a membrane-associated guanylate kinase (MAGUK) family member containing CARD, PDZ, SH3 and GUK domains. Using a CARD-deleted variant of CARD11 and RNA interference (RNAi), we demonstrate that CARD11 mediates NF-kappaB activation by alphaCD3/alphaCD28 cross-linking and PMA/ionomycin treatment, but not by TNFalpha or dsRNA. CARD11 is not required for TCR-mediated induction of NFAT or AP-1. CARD11 functions upstream of the IkappaB-kinase (IKK) complex and cooperates with Bcl10 in a CARD domain-dependent manner. RNAi-rescue experiments suggest that the CARD, coiled-coil, SH3 and GUK domains of CARD11 are critical for its signaling function. These results implicate CARD11 in factor- specific activation of NF-kappaB by the TCR complex and establish a role for a MAGUK family member in antigen receptor signaling.

Amino Acid Sequence↗