Soluble CD83 ameliorates experimental colitis in mice.

The physiological balance between pro- and anti-inflammatory processes is dysregulated in inflammatory bowel diseases (IBD) as in Crohn's disease and ulcerative colitis. Conventional therapy uses anti-inflammatory and immunosuppressive corticosteroids to treat acute-phase symptoms. However, low remission rate and strong side effects of these therapies are not satisfying. Thus, there is a high medical need for new therapeutic strategies. Soluble CD83, the extracellular domain of the transmembrane CD83 molecule, has been reported to have interesting therapeutic and immunosuppressive properties by suppressing dendritic cell (DC)-mediated T-cell activation and inducing tolerogenic DCs. However, the expression and function of CD83 in IBD is still unknown. Here, we show that CD83 expression is upregulated by different leukocyte populations in a chemical-induced murine colitis model. Furthermore, in this study the potential of sCD83 to modulate colitis using an experimental murine colitis model was investigated. Strikingly, sCD83 ameliorated the clinical disease symptoms, drastically reduced mortality, and strongly decreased inflammatory cytokine expression in mesenteric lymph nodes and colon. The infiltration of macrophages and granulocytes into colonic tissues was vigorously inhibited. Mechanistically, we could show that sCD83-induced expression of indolamine 2,3-dioxygenase is essential for its protective effects.

Dendritic cells for active immunotherapy: optimizing design and manufacture in order to develop commercially and clinically viable products.

Dendritic cell (DC) active immunotherapy is potentially efficacious in a broad array of malignant disease settings. However, challenges remain in optimizing DC-based therapy for maximum clinical efficacy within manufacturing processes that permit quality control and scale-up of consistent products. In this review we discuss the critical issues that must be addressed in order to optimize DC-based product design and manufacture, and highlight the DC based platforms currently addressing these issues. Variables in DC-based product design include the type of antigenic payload used, DC maturation steps and activation processes, and functional assays. Issues to consider in development include: (a) minimizing the invasiveness of patient biological material collection; (b) minimizing handling and manipulations of tissue at the clinical site; (c) centralized product manufacturing and standardized processing and capacity for commercial-scale production; (d) rapid product release turnaround time; (e) the ability to manufacture sufficient product from limited starting material; and (f) standardized release criteria for DC phenotype and function. Improvements in the design and manufacture of DC products have resulted in a handful of promising leads currently in clinical development.

Nuclear import/export of hRPF1/Nedd4 regulates the ubiquitin-dependent degradation of its nuclear substrates.

The ubiquitin-protein ligase (E3), hRPF1/Nedd4, is a component of the ubiquitin-proteasome pathway responsible for substrate recognition and specificity. Although previously characterized as a regulator of the stability of cytoplasmic proteins, hRPF1/Nedd4 has also been suggested to have a role in the nucleus. However, in light of the cytoplasmic localization of hRPF1/Nedd4, it is unclear whether bona fide nuclear substrates of hRPF1/Nedd4 exist, and if so, what mechanism may allow a cytoplasmic ubiquitin ligase to manifest nuclear activity. Our search for nuclear substrates led to the identification of the human proline-rich transcript, brain-expressed (hPRTB) protein, the ubiquitination and degradation of which is regulated by hRPF1/Nedd4. Interestingly, hPRTB colocalizes with the splicing factor SC35 in nuclear speckles. Finally, we demonstrate that hRPF1/Nedd4 is indeed capable of entering the nucleus; however, the presence of a functional Rev-like nuclear export sequence in hRPF1/Nedd4 ensures a predominant cytoplasmic localization. Cumulatively, these findings highlight a nuclear role for the ubiquitin ligase hRPF1/Nedd4 and underscore cytoplasmic/nuclear localization as an important regulatory component of hRPF1/Nedd4-substrate recognition.

Aspartic proteases from the nematode Caenorhabditis elegans. Structural organization and developmental and cell-specific expression of asp-1.

A Caenorhabditis elegans gene (asp-1) and cDNA that encode a homologue of cathepsin D aspartic protease were cloned and characterized. The asp-1 mRNA is transcribed from a single exon, and it begins with the SL1 trans-splice leader sequence. The protein (ASP-1) is expressed as a 396-amino acid, 42.7-kDa pre-pro-peptide that is post-translationally processed into a approximately 40-kDa lysosomal protein. ASP-1 shares approximately 60% sequence identity with the aspartic protease precursor from the nematode Strongyloides stercoralis. The amino acid sequences adjacent to the two active site aspartic acid residues in ASP-1 are 100% identical to those in other eukaryotic aspartic proteases. In addition, ASP-1 contains conserved, potential disulfide bond-forming cysteine residues and N-glycosylation sites. The asp-1 gene is exclusively transcribed in the intestinal cells, with the highest levels of expression observed at late embryonic and early larval stages of development. asp-1 transcription is not observed in adult nematodes or mature larvae. Furthermore, transcription predominantly occurs in eight anterior cells of the intestine (int6-int8). Analyses of ASP-1 nucleotide and amino acid sequences revealed the presence of five additional C. elegans aspartic proteases.

Modulation of estrogen receptor-alpha transcriptional activity by the coactivator PGC-1.

A transcriptional coactivator of the peroxisome proliferator-activated receptor-gamma (PPARgamma), PPARgamma-coactivator-1(PGC-1) interacts in a constitutive manner with the hinge domain of PPARgamma and enhances its transcriptional activity. In this study we demonstrate that PGC-1 is a coactivator of estrogen receptor-alpha (ERalpha)-dependent transcriptional activity. However the mechanism by which PGC-1 interacts with ERalpha is different from that of PPARgamma. Specifically, it was determined that the carboxyl terminus of PGC-1 interacts in a ligand-independent manner with the ERalpha hinge domain. In addition, an LXXLL motif within the amino terminus of PGC-1 was shown to interact in an agonist-dependent manner with the AF2 domain within the carboxyl terminus of ERalpha. The ability of PGC-1 to associate with and potentiate the transcriptional activity of an ERalpha-AF2 mutant that is unable to interact with the p160 class of coactivators suggests that this coactivator may have a unique role in estrogen signaling. It is concluded from these studies that PGC-1 is a bona fide ERalpha coactivator, which may serve as a convergence point between PPARgamma and ERalpha signaling.

Structure and expression of the Caenorhabditis elegans protein kinase C2 gene. Origins and regulated expression of a family of Ca2+-activated protein kinase C isoforms.

The molecular and cellular basis for concerted Ca2+/lipid signaling in Caenorhabditis elegans was investigated. A unique gene (pkc-2) and cognate cDNAs that encode six Ca2+/diacylglycerol-stimulated PKC2 isoenzymes were characterized. PKC2 polypeptides (680-717 amino acid residues) share identical catalytic, Ca2+-binding, diacylglycerol-activation and pseudosubstrate domains. However, sequences of the N- and C-terminal regions of the kinases diverge. PKC2 diversity is partly due to differential activation of transcription by distinct promoters. Each promoter precedes an adjacent exon that encodes 5'-untranslated RNA, an initiator AUG codon and a unique open reading frame. PKC2 mRNAs also incorporate one of two 3'-terminal exons via alternative splicing. Cells that are capable of receiving and propagating signals carried by Ca2+/diacylglycerol were identified by assessing activities of pkc-2 gene promoters in transgenic C. elegans and visualizing the distribution of PKC2 polypeptides via immunofluorescence. Highly-selective expression of certain PKC2 isoforms was observed in distinct subsets of neurons, intestinal and muscle cells. A low level of PKC2 isoforms is observed in embryos. When L1 larvae hatch and interact with the external environment PKC2 content increases 10-fold. Although 77- and 78-kDa PKC2 isoforms are evident throughout post-embryonic development, an 81-kDa isoform appears to be adapted for function in L1 and L2 larvae.

Effect of number and position of EBNA-1 binding sites in Epstein-Barr virus oriP on the sites of initiation, barrier formation, and termination of replication.

DNA replication intermediates of three plasmids containing all or part of a modified Epstein-Barr virus cis-acting plasmid maintenance region (oriP) were examined to further investigate oriP function. Replication intermediates were analyzed in vivo and in vitro by neutral-neutral two-dimensional gel electrophoresis. The major functional components of the wild-type oriP are a 140-bp dyad symmetry region (single dyad) and 20 tandem copies of a repeat with a 30-bp consensus sequence (family of repeats). A modified oriP was constructed by replacing the family of repeats with three tandem copies of the single dyad (D. A. Wysokenski and J. L. Yates, J. Virol. 63:2657-2666, 1989). Initiation was observed in vivo near the single dyad in the modified oriP, as seen in the wild-type oriP (T. A. Gahn and C. L. Schildkraut, Cell 58:527-535, 1989), but was not observed near the tandem dyads. A replication barrier and termination were observed near the tandem dyads and were similar to those observed at the family of repeats of the wild-type oriP (Gahn and Schildkraut, Cell 58:527-535, 1989). In vitro experiments indicate that the viral trans-acting factor EBNA-1 contributes to efficient barrier formation at the tandem dyads as observed in the family of repeats of the wild-type oriP (V. Dhar and C. L. Schildkraut, Mol. Cell. Biol. 11:6268-6278, 1991). The tandem dyads thus appear to function in a manner similar to the family of repeats. There are significant structural differences between the family of repeats and tandem dyads. The relationship between the number and relative positions of EBNA-1 binding sites in relation to the functions of the family of repeats and the dyad symmetry element is discussed.