Cholera toxin inhibits IL-12 production and CD8α+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

Prior studies have demonstrated that cholera toxin (CT) and other cAMP-inducing factors inhibit interleukin (IL)-12 production from monocytes and dendritic cells (DCs). We show that CT inhibits Th1 responses in vivo in mice infected with Toxoplasma gondii. This correlated with low serum IL-12 levels and a selective reduction in the numbers of CD8α+ conventional DCs (cDCs) in lymphoid organs. CT inhibited the function of interferon (IFN) regulatory factor (IRF) 8, a transcription factor known to positively regulate IL-12p35 and p40 gene expression, and the differentiation of CD8α+ and plasmacytoid DCs (pDCs). Fluorescence recovery after photobleaching analysis showed that exposure to CT, forskolin, or dibutyryl (db) cAMP blocked LPS and IFN-γ–induced IRF8 binding to chromatin. Moreover, CT and dbcAMP inhibited the binding of IRF8 to the IFN-stimulated response element (ISRE)–like element in the mouse IL-12p40 promoter, likely by blocking the formation of ISRE-binding IRF1–IRF8 heterocomplexes. Furthermore, CT inhibited the differentiation of pDCs from fms-like tyrosine kinase 3 ligand–treated bone marrow cells in vitro. Therefore, because IRF8 is essential for IL-12 production and the differentiation of CD8α+ cDCs and pDCs, these data suggest that CT and other Gs-protein agonists can affect IL-12 production and DC differentiation via a common mechanism involving IRF8

The Oncoprotein EVI1 and the DNA Methyltransferase Dnmt3 Co-Operate in Binding and De Novo Methylation of Target DNA

EVI1 has pleiotropic functions during murine embryogenesis and its targeted disruption leads to prenatal death by severely affecting the development of virtually all embryonic organs. However, its functions in adult tissues are still unclear. When inappropriately expressed, EVI1 becomes one of the most aggressive oncogenes associated with human hematopoietic and solid cancers. The mechanisms by which EVI1 transforms normal cells are unknown, but we showed recently that EVI1 indirectly upregulates self-renewal and cell-cycling genes by inappropriate methylation of CpG dinucleotides in the regulatory regions of microRNA-124-3 (miR-124-3), leading to the repression of this small gene that controls normal differentiation and cell cycling of somatic cells. We used the regulatory regions of miR-124-3 as a read-out system to investigate how EVI1 induces de novo methylation of DNA. Here we show that EVI1 physically interacts with DNA methyltransferases 3a and 3b (Dnmt3a/b), which are the only de novo DNA methyltransferases identified to date in mouse and man, and that it forms an enzymatically active protein complex that induces de novo DNA methylation in vitro. This protein complex targets and binds to a precise region of miR-124-3 that is necessary for repression of a reporter gene by EVI1. Based on our findings, we propose that in cooperation with Dnmt3a/b EVI1 regulates the methylation of DNA as a sequence-specific mediator of de novo DNA methylation and that inappropriate EVI1 expression contributes to carcinogenesis through improper DNA methylation

A glycosylated recombinant human granulocyte colony stimulating factor produced in a novel protein production system (AVI-014) in healthy subjects: a first-in human, single dose, controlled study

BACKGROUND: AVI-014 is an egg white-derived, recombinant, human granulocyte colony-stimulating factor (G-CSF). This healthy volunteer study is the first human investigation of AVI-014. METHODS: 24 male and female subjects received a single subcutaneous injection of AVI-014 at 4 or 8 mcg/kg. 16 control subjects received 4 or 8 mcg/kg of filgrastim (Neupogen, Amgen) in a partially blinded, parallel fashion. RESULTS: The Geometric Mean Ratio (GMR) (90% CI) of 4 mcg/kg AVI-014/filgrastim AUC(0-72 hr) was 1.00 (0.76, 1.31) and Cmax was 0.86 (0.66, 1.13). At the 8 mcg/kg dose, the AUC(0-72) GMR was 0.89 (0.69, 1.14) and Cmax was 0.76 (0.58, 0.98). A priori pharmacokinetic bioequivalence was defined as the 90% CI of the GMR bounded by 0.8-1.25. Both the white blood cell and absolute neutrophil count area under the % increase curve AUC(0-9 days) and Cmax (maximal % increase from baseline)GMR at 4 and 8 mcg/kg fell within the 0.5-2.0 a priori bound set for pharmacodynamic bioequivalence. The CD 34+ % increase curve AUC(0-9 days) and Cmax GMR for both doses was approximately 1, but 90% confidence intervals were large due to inherent variance, and this measure did not meet pharmacodynamic bioequivalence. AVI-014 demonstrated a side effect profile similar to that of filgrastim. CONCLUSION: AVI-014 has safety, pharmacokinetic, and pharmacodynamic properties comparable to filgrastim at an equal dose in healthy volunteers. These findings support further investigation in AVI-014

Zinc finger transcription factor ecotropic viral integration site 1 is induced by all-trans retinoic acid (ATRA) and acts as a dual modulator of the ATRA response

Ecotropic viral integration site 1 (EVI1) plays important roles in leukaemia and development, and its expression is temporally and spatially highly restricted during the latter process. Nevertheless, the only physiological agent that to date has been shown to regulate transcription of this gene in mammalian cells is all-trans retinoic acid. Here we describe the identification of a retinoic acid response element that was located in the most distal of several alternative first exons of the human EVI1 gene and was constitutively bound by canonical retinoid receptors in NTERA-2 teratocarcinoma cells. Furthermore, it was the target of negative feedback by EVI1 on the induction of its own promoter by retinoic acid. This process required a previously described transcription repression domain of EVI1. Extending its role as a modulator of the retinoic acid response, EVI1 had the opposite effect on the RARβ retinoic acid response element, whose induction by all-trans retinoic acid it enhanced through a mechanism that involved almost all of its known functional domains. Augmentation of the retinoic acid response by EVI1 was also observed for the endogenous RARβ gene. Thus, we have established EVI1 as a novel type of modulator of the retinoic acid response, which can both enhance and repress induction by this agent in a promoter-specific manner

EVI1 Impairs myelopoiesis by deregulation of PU.1 function

EVI1 is an oncogene inappropriately expressed in the bone marrow (BM) of approximately 10% of myelodysplastic syndrome (MDS) patients. This disease is characterized by severe anemia and multilineage myeloid dysplasia that are thought to be a major cause of mortality in MDS patients. We earlier reported on a mouse model that constitutive expression of EVI1 in the BM led to fatal anemia and myeloid dysplasia, as observed in MDS patients, and we subsequently showed that EVI1 interaction with GATA1 blocks proper erythropoiesis. Whereas this interaction could provide the basis for the erythroid defects in EVI1-positive MDS, it does not explain the alteration of myeloid differentiation. Here, we have examined the expression of several genes activated during terminal myelopoiesis in BM cells and identified a group of them that are altered by EVI1. A common feature of these genes is their regulation by the transcription factor PU.1. We report here that EVI1 interacts with PU.1 and represses the PU.1-dependent activation of a myeloid promoter. EVI1 does not seem to inhibit PU.1 binding to DNA, but rather to block its association with the coactivator c-Jun. After mapping the PU.1-EVI1 interaction sites, we show that an EVI1 point mutant, unable to bind PU.1, restores the activation of PU.1-regulated genes and allows a normal differentiation of BM progenitors in vitro

Structure-activity analysis of C-terminal endothelin analogues

Several synthetic endothelin (ET) analogues of the C-terminal ET hexapeptide (ET16-21) were analyzed by radio-receptor competition binding assays and biologic activity using both ETA and ETB receptor subtypes. In addition, we produced a hybridoma monoclonal antibody, anti-ET15-21, that appeared to crossreact with the entire ET molecule and was able to neutralize its biologic activity. Antibody binding was measured with competition enzyme-linked immunosorbent assays and a surface plasmon resonance-based biosensor (BIA technology). The ET16-21 moiety was modified with systematic replacement of each residue by alanine (Ala-scan). Whereas the C-terminal residues (Asp(18), Ile(20), and particularly Trp(21)) were very important for both receptor binding and immunologic activity, Ala substitution in positions 16, 17, and 19 hardly affected such activities. Analysis of another series of synthetic ET16-21 analogues with the His(16) residue replaced by a non-amino-acidic block confirmed that the last two C-terminal residues are essential for receptor and antibody binding, whereas the central region of this hexapeptide is much more tolerant to modification. However, a critical steric conformation of the active hexapeptide is necessary

NATURALLY OCCURRING AND THERAPY-INDUCED ANTIBODIES TO HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF) IN HUMAN SERUM

Sera were obtained from two groups of patients. Group A included 7 patients with low-grade non-Hodgkin's lymphoma treated with three or more cycles of standard-dose chemotherapy and recombinant human granulocyte-colony stimu- lating factor (rhG-CSF). The cytokine was administered to half the patients after the ®rst chemotherapy cycle and to the other half after the second according to a randomized design and then to all patients from the third chemotherapy cycle on, until documented hemopoietic reconstitution. Group B included 3 patients with high-grade non-Hodgkin's lymphoma, 1 patient with resistant Hodgkin's disease, and 1 patient with multiple myeloma who received high-dose chemotherapy and rhG-CSF. Anti-G-CSF antibodies were detected in the sera of 4 patients. Both immunoglobulin IgM and IgG antibodies were detected at low levels in pretreatment sera from one group A patient. IgG antibody titers increased markedly during the ®rst and second periods of G-CSF administration. IgG class antibodies developed in 3 group B patients during the ®rst course of rhG-CSF administration. Circulating anti-G-CSF antibodies did not seem to affect hematological recovery. Low levels of anti-G-CSF antibodies were also detected in sera (15/135) from different healthy adults and in sera (5/40) from umbilical cord blood. Saturable antibody binding and competition enzyme-linked immunosorbent assay (ELISA) and immunoblotting con®rmed antibody speci®city