Dendritic cells (DC) classically promote immune responses but can be manipulated to induce antigen-specific hyporesponsiveness in vitro. The expression of costimulatory molecules (CD40, CD86, CD80) at the DC cell surface correlates with their capacity to induce or suppress immune responses. Expression of these molecules is associated with NF-kB-dependent transcription of their genes. DC tolerogenicity has been associated with impaired NF-kB-dependent transcription of costimulatory genes as well as NF-kB translocation to the nucleus. In this report, we demonstrate that double-stranded oligodeoxyribonucleotides containing binding sites for NF-kB (NF-kB ODN) are efficiently incorporated by bone marrow-derived DC and specifically inhibit NF-kB-dependent transcription of a reporter gene. Moreover, exposure of DC to the oligonucleotide decoys inhibited lipopolysaccharide (LPS)-induced nitric oxide production, a marker of DC maturation. Treatment of bone marrow-derived DC progenitors with NF-kB ODN selectively suppressed the cell-surface expression of costimulatory molecules without interfering with MHC class I or class II expression. Furthermore, NF-kB ODN DC induced allogeneic donor-specific hyporesponsiveness in mixed leukocyte cultures, and this was associated with inhibition of Th1-type cytokine production. Finally, infusion of NF-kB ODN-modified bone marrow-derived DC into allogeneic recipients prior to heart transplantation resulted in significant prolongation of allograft survival in the absence of immunosuppression. Specific interference with NF-kB and other transcriptional pathways involved in immune stimulation in DC using ODN decoy approaches could be one means to promote tolerance induction in organ transplantation.
Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-κB-dependent mechanisms, which can be blocked by double-stranded “decoy” oligodeoxyribonucleotides (ODNs) containing binding sites for NF-κB. Herein, we describe the combined use of NF-κB ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-κB antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.
Seven distinct 14-3-3 proteins are expressed in mammals. One of the 14-3-3 genes (eta) has been previously associated with decreased expression in the prefrontal cortex (PFC) of subjects with schizophrenia. DNA microarray analysis of the PFC of 10 subjects with schizophrenia and 10 matched controls indicated that the majority of 14-3-3 genes exhibited moderate to marked decreases in expression in schizophrenia, which were significant at the group level across all 10 comparisons (p<0.021). Selected changes in gene expression were further examined using in situ hybridization (ISH) in the same subject pairs as well as in four monkeys treated chronically with haloperidol and matched control animals. All analyses were performed blind to subject identity and diagnosis, or treatment. ISH analysis and multivariate analysis of covariance confirmed the significant decreases in expression of two 14-3-3 genes: beta -31.9%, zeta -18.2%. Two other 14-3-3 genes exhibited more modest decreases in expression levels that were significant only in pairwise comparisons that did not factor in post-mortem interval or tissue storage time: gamma -11.9%, eta -15.4%. In the PFC of haloperidol-treated monkeys, there was no difference in 14-3-3 zeta expression, while 14-3-3 beta increased 28% (p<0.05) as a result of neuroleptic treatment. Our results suggest that decreased expression of selected 14-3-3 genes is a common feature of schizophrenia and that the 14-3-3 beta transcript may be unique among the 14-3-3 genes in its increase in response to haloperidol and decrease in the disease state.
The Gateway recombination system is characterized by its ability to transfer DNA sequences back and forth between an intermediate clone (the entry clone) and a variety of destination vectors. However, a number of applications do not need to exploit the advantages offered by the entry clone. Here we report reaction conditions for cloning DNA fragments into destination vectors in a single step reaction, thus reducing the cost and overall time needed to obtain an expression clone from three days to one.
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