A 24-week multicentre, randomized, open-label, parallel-group study comparing the efficacy and safety of ixekizumab vs. fumaric acid esters and methotrexate in patients with moderate-to-severe plaque psoriasis naive to systemic treatment.

BACKGROUND: Interleukin-17 antagonists have received a first-line label for moderate-to-severe plaque psoriasis. OBJECTIVES: We conducted the first head-to-head trial between the two most commonly used first-line therapies in Germany, fumaric acid esters (FAEs) and methotrexate, and the interleukin-17A antagonist, ixekizumab. METHODS: Systemic-naive patients were randomized in this parallel-group, active-comparator, open-label, rater-blinded trial (each group n = 54). The primary outcome was the proportion of patients achieving ≥ 75% improvement in Psoriasis Area and Severity Index (PASI 75) at 24 weeks. Key secondary outcomes included 24-week PASI 90 and 100, static Physician's Global Assessment (sPGA) score of 0 or 1, and Dermatology Life Quality Index (DLQI) score of 0 or 1. Safety events at week 24 were analysed using Fisher's exact test. Missing data were imputed using nonresponder imputation. The trial was registered at ClinicalTrials.gov (NCT02634801) and EudraCT (2015-002649-69). RESULTS: At week 24, more ixekizumab-treated patients achieved PASI 75 [91% vs. 22% FAEs (P < 0·001) and 70% methotrexate (P = 0·014)], PASI 90 [80% vs. 9% FAEs (P < 0·001) and 39% methotrexate (P < 0·001)] and PASI 100 [41% vs. 4% FAEs (P < 0·001) and 13% methotrexate (P = 0·0041)], as well as sPGA (0,1) and DLQI (0,1). CONCLUSIONS: Ixekizumab was superior in inducing PASI 75/90/100, sPGA (0,1) and DLQI (0,1) responses at week 24 compared with methotrexate and FAEs. Safety profiles for all treatments were consistent with prior studies.

Constitutive apical secretion of an 80-kD sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line.

The biosynthesis, processing, and apical secretion of a group of polypeptides (Kondor-Koch, C., R. Bravo, S. D. Fuller, D. Cutler, and H. Garoff. 1985. Cell. 43:297-306) are studied in MDCK cells using a specific polyclonal antiserum. These polypeptides are synthesized as a precursor protein which has an apparent Mr of 65,000 in its high mannose form. This precursor is converted into a protein with an apparent Mr of 80,000 containing complex carbohydrates and sulfate. After intracellular cleavage of the 80-kD protein, the 35-45-kD subunits are secreted as an 80-kD glycoprotein complex (gp 80) linked together by disulfide bonds. Secretion of the protein complex occurs by a constitutive pathway at the apical surface of the epithelial monolayer. Since the immediate post-translational precursor, the 65-kD protein, is hydrophilic in nature as shown by its partitioning behavior in a phase-separated Triton X-114 solution, gp 80 is segregated into the apical exocytotic pathway as a soluble molecule. The proteolytic maturation of gp 80 is blocked in the presence of chloroquine and its secretion is retarded. The 80-kD precursor is released at the apical cell surface, demonstrating that proteolytic processing is not necessary for the apical secretion of this protein. If N-glycosylation is inhibited by tunicamycin treatment the protein is secreted in equal amounts at both cell surfaces, indicating a role of the carbohydrate moieties in the vectorial transport of this protein.

Chromatin remodeling in development and differentiation.

During development and differentiation, early inductive processes that influence cell fate at a later stage leave marks at distinct gene loci that are maintained through several rounds of mitosis. The structure of chromatin is part of this epigenetic memory that restricts or permits differential expression of genes in descendant cells. Establishing a cell-type-specific chromatin pattern thus predestines future cell differentiation and deters cell-lineage infidelity, as it often occurs during neoplastic transformation. As such, understanding the dynamics and mechanisms underlying chromatin remodeling has been a major focus of recent molecular genetic research that holds great promise for biomedical discoveries.

C/EBP beta regulation of the tumor necrosis factor alpha gene.

Activated macrophages contribute to chronic inflammation by the secretion of cytokines and proteinases. Tumor necrosis factor alpha (TNF alpha) is particularly important in this process because of its ability to regulate other inflammatory mediators in an autocrine and paracrine fashion. The mechanism(s) responsible for the cell type-specific regulation of TNF alpha is not known. We present data to show that the expression of TNF alpha is regulated by the transcription factor C/EBP beta (NF-IL6). C/EBP beta activated the TNF alpha gene promoter in cotransfection assays and bound to it at a site which failed to bind the closely related protein C/EBP alpha. Finally, a dominant-negative version of C/EBP beta blocked TNF alpha promoter activation in myeloid cells. Our results implicate C/EBP beta as an important regulator of TNF alpha by myelomonocytic cells.

Structure of the chicken myelomonocytic growth factor gene and specific activation of its promoter in avian myelomonocytic cells by protein kinases.

In chicken myeloid cells but not in erythroid cells, kinase-type oncogenes activate expression of the chicken myelomonocytic growth factor (cMGF). The autocrine loop established this way plays a key role in lineage-specific cooperation of nuclear and kinase-type oncogenes in retrovirally induced myeloid leukemia. In this report, we describe the cloning of the cMGF gene, including its promoter. The structure of the cMGF gene is homologous to those of the granulocyte colony-stimulating factor and interleukin-6 genes. Expression from reporter constructs containing the cMGF promoter is specific to myelomonocytic cells. Kinases activate cMGF at the transcriptional level in macrophages and strongly induce reporter expression in myelomonocytic cells.

Comprehensive translational control of tyrosine kinase expression by upstream open reading frames.

Post-transcriptional control has emerged as a major regulatory event in gene expression and often occurs at the level of translation initiation. Although overexpression or constitutive activation of tyrosine kinases (TKs) through gene amplification, translocation or mutation are well-characterized oncogenic events, current knowledge about translational mechanisms of TK activation is scarce. Here, we report the presence of translational cis-regulatory upstream open reading frames (uORFs) in the majority of transcript leader sequences of human TK mRNAs. Genetic ablation of uORF initiation codons in TK transcripts resulted in enhanced translation of the associated downstream main protein-coding sequences (CDSs) in all cases studied. Similarly, experimental removal of uORF start codons in additional non-TK proto-oncogenes, and naturally occurring loss-of-uORF alleles of the c-met proto-oncogene (MET) and the kinase insert domain receptor (KDR), was associated with increased CDS translation. Based on genome-wide sequence analyses we identified polymorphisms in 15.9% of all human genes affecting uORF initiation codons, associated Kozak consensus sequences or uORF-related termination codons. Together, these data suggest a comprehensive role of uORF-mediated translational control and delineate how aberrant induction of proto-oncogenes through loss-of-function mutations at uORF initiation codons may be involved in the etiology of cancer. We provide a detailed map of uORFs across the human genome to stimulate future research on the pathogenic role of uORFs.

Tumorigenic N-terminal deletions of c-Myb modulate DNA binding, transactivation, and cooperativity with C/EBP.

Oncogenic activation of c-myb by retroviral insertion has been implicated in tumor formation in chicken and mice. These genetic alterations result in deregulated expression of the c-myb gene and frequently in N-terminal truncation of the c-Myb protein. We demonstrate that truncation of the c-Myb N-terminus affects DNA binding and reporter activation. However, all three mutants, Myb Delta N20, Myb Delta N47 and Myb Delta N71 cooperated with C/EBP beta in reporter assays. In contrast to Myb Delta N20 and Myb Delta N47, however, the Myb Delta N71 mutant failed to activate the chromatin embedded endogenous mim-1 gene together with C/EBP beta. This suggests that an N-terminal region (amino acids 47-71) within repeat 1 (R1) of the murine c-Myb DNA binding domain affects activation of chromosomal target genes in collaboration with C/EBP beta.

The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators.

The proto-oncoprotein Bcl-3 is a member of the IkappaB family and is present predominantly in the nucleus. To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain. Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3. The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation. Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase.

The BTB/POZ domain targets the LAZ3/BCL6 oncoprotein to nuclear dots and mediates homomerisation in vivo.

The LAZ3/BCL6 gene was identified by its disruption in 3q27 translocations associated with diffuse large cell lymphomas. It is predicted to be a transcription factor as it contains six Krüppel-like Zinc finger motifs and a N-terminal BTB/POZ domain, a protein/protein interaction interface that is widely conserved in Metazoans. Using two antisera raised against non overlapping regions of the predicted ORF, we demonstrate that the LAZ3/BCL6 protein appears as a close ca. 79 kDa doublet in B lymphoid cell lines with either a rearranged or a non rearranged LAZ3/BCL6 locus. By immunofluorescence experiments on transiently transfected COS-1 or NIH3T3 cells, we show that the LAZ3/BCL6 protein displays a punctuated nuclear localisation. This appears to rely on LAZ3/BCL6 proper folding and/or activities as it is impaired in a hormone reversible-fashion through fusion of LAZ3/BCL6 to the ligand-binding domain of the oestrogen receptor. Moreover, deletion of its BTB/POZ domain leads to the disappearance of the nuclear dots although the protein remains nuclear. In addition, by using the yeast two-hybrid system, we show that the LAZ3/BCL6 BTB/POZ domain homomerises in vivo. Thus, the LAZ3/BCL6 BTB/POZ domain has the capability to self-interact and target the protein to discrete nuclear substructures.

Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kappaB and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis.

BACKGROUND: Recent reports link C. pneumoniae infection of arteriosclerotic lesions to the precipitation of acute coronary syndromes, which also feature tissue factor and plasminogen activator inhibitor 1 (PAI-1) overexpression. We investigated whether or not C. pneumoniae can induce thrombogenicity by upregulation of procoagulant proteins. METHODS AND RESULTS: Human vascular endothelial and smooth muscle cells were infected with a strain of C. pneumoniae isolated from an arteriosclerotic coronary artery. Tissue factor, PAI-1, and interleukin-6 expression was increased in infected cells. Concomitantly, NF-kappaB was activated and IkappaBalpha degraded. p50/p65 heterodimers were identified as the components responsible for the NF-kappaB activity. CONCLUSIONS: These data provide evidence that C. pneumoniae infection can induce procoagulant protein and proinflammatory cytokine expression. This cellular response is accompanied by activation of NF-kappaB. Our results demonstrate how C. pneumoniae infection may initiate acute coronary syndromes.

Expression of the homeobox gene GBX2 during chicken development.

We have examined the expression pattern of the GBX2 gene during chicken embryogenesis. First transcripts are found in the epiblast of a HH st. 3+ embryo. With the onset of neurogenesis, transcripts mark the posterior neuroectoderm. Later on, expression is detectable in the isthmic region, the hindbrain and the neural tube. We show that GBX2 transcripts, as well as the protein, mark the presumptive hindbrain region. After establishment of the brain vesicles GBX2 transcripts were also detected in distinct domains of the diencephalon. In addition to neural sites of expression, GBX2 was found in several domains including the otic vesicle, the somitic mesoderm, the lateral foregut endoderm, the ventral limb bud ectoderm and in the feather buds.

Regulation of C/EBP beta/NF-M activity by kinase oncogenes.

CAAT Enhancer Binding proteins (C/EBP) belong to a family of transcription factors which are implicated in a number of developmental and growth regulatory processes. One member of this family known as C/EBP beta (called NF-M in the chicken system) is particularly important in myelomonocytic cells because it is targeted by kinases and collaborates with the Myb oncoprotein to induce the expression of myeloid specific genes. Experiments dissecting the structure of NF-M suggest that it is a repressed transcription factor. Using the yeast two-hybrid system we showed that a negative regulatory domain masks the transactivation domain. Examination of NF-M mutants suggests that kinase (proto-) oncogenes uncover the concealed transcriptional activity by phosphorylation of the negative regulatory domain.

Essential role of c-myc in ara-C-induced differentiation of human erythroleukemia cells.

The mode of action of the differentiation inducer 1-beta-D-arabinofuranosylcytosine (ara-C) is as yet unknown. We have analyzed the role of c-myc expression in ara-C-induced differentiation of K562 human erythroleukemia cells. Six hours after differentiation induction, c-myc expression is down-regulated transiently. This was followed by the onset of cell differentiation together with a final c-myc 'down-regulation' at 24 h after treatment. This regulation of expression may be caused by hypermethylation of c-myc DNA sequences, which we find to follow a similar pattern. We propose that the loss of c-myc expression might be a necessary prerequisite for ara-C-induced differentiation. This hypothesis was tested by transfecting a constitutive c-myc expression construct into K562 cells prior to ara-C treatment. By following the differentiation parameters cell morphology, benzidine staining (hemoglobin monitored), and alkaline phosphatase activity (presence of mature red blood cell antigen monitored) in cells expressing cotransfected LacZ marker gene, it was demonstrated that the introduction of the c-myc expression plasmid blocked ara-C-induced differentiation. We conclude that loss of c-myc expression mediated the differentiation found in ara-C-treated K562 cells.

B-Myb, a repressed trans-activating protein.

B-Myb belongs to a family of related transcription factors which share a unique DNA binding domain. B-Myb plays an important role in regulation of the cell cycle. Its expression is upregulated by the human papilloma virus HPV16 E7 oncoprotein. Overexpression of B-Myb can bypass p53-mediated cell cycle arrest. The founding member of the myb gene family, c-Myb, and A-Myb are involved in hematopoiesis and neurogenesis, respectively, and are both activators of gene transcription. Whether B-Myb is a transactivator or a repressor, however, has remained a matter of discussion. We reviewed the transactivation potential of B-Myb in yeast, taking advantage of the fact that inducible gene activation is an evolutionarily conserved process. By mutational analysis we localized a conserved activation domain in B-Myb. In vertebrate cells the transactivation potential of B-Myb is concealed by the C-terminal part of the protein. We show that the cell cycle regulators cyclin A and cyclin E activate B-Myb by eradicating the inhibition mediated by its carboxy-terminus. Our data suggest that in vertebrates the trans-activating function of B-Myb is regulated during the cell cycle and link Myb functions to cell cycle progression.

Cloning of two novel human importin-alpha subunits and analysis of the expression pattern of the importin-alpha protein family.

The import of many proteins into the nucleus is mediated by the importin-alpha/beta heterodimer. While only one importin-beta gene has been found, several forms of importin-alpha have been described. In addition to the three human importin-alphas already identified, we report here the primary structure of two new human importin-alpha proteins. The five known human importin-alpha subunits can be classified into three subfamilies that appear conserved in higher eukaryotic organisms. We show by immunoblotting that the different importin-alpha subfamilies are expressed in a variety of human tissues and mammalian cell lines.

Cell type-specificity of epidermal growth factor (EGF) binding in primary cultures of early postnatal mouse cerebellum.

Binding of 125I-labeled epidermal growth factor (EGF) to cells of cultured early postnatal mouse cerebellar cells was investigated by autoradiography in conjunction with cell type-specific immunolabeling of neurons, astrocytes and oligodendrocytes. By use of tetanus toxin for recognition of neurons and glial fibrillary acidic (GFA) protein and 04 antigen as markers for astrocytes and oligodendrocytes, respectively, it could be shown that oligodendrocytes do not express receptors for EGF within the limits of sensitivity of the autoradiographic method, and that less than 1% of all small neurons (mostly granule cells) and 50-90% of all GFA protein-sensitive astrocytes show detectable levels of EGF binding.

Cultivation of immature astrocytes of mouse cerebellum in a serum-free, hormonally defined medium. Appearance of the mature astrocyte phenotype after addition of serum.

A hormonally defined culture medium is described which supports survival and proliferation of astroglia from primary cultures of early postnatal mouse cerebellum. This medium consists of bovine serum albumin, insulin, transferrin, selenium, hyaluronic acid, protease inhibitor aprotinin, and epidermal growth factor. Trypsin-dissociated single cerebellar cell suspensions are plated in this medium on poly-l-coated glass coverslips and maintained for two weeks before subcultivation. After subcultivation into defined medium more than 99% of all cells are vimentin-positive and fibronectin- and almost completely glial fibrillary acid (GFA) protein-negative, indicating that these cells are less mature astrocytes. After replacement of defined medium by culture medium containing 10% horse serum, expression of GFA protein is detectable in addition to vimentin by indirect immunofluorescence.

Notch pathway inhibition controls myeloma bone disease in the murine MOPC315.BM model.

Despite evidence that deregulated Notch signalling is a master regulator of multiple myeloma (MM) pathogenesis, its contribution to myeloma bone disease remains to be resolved. Notch promotes survival of human MM cells and triggers human osteoclast activity in vitro. Here, we show that inhibition of Notch through the γ-secretase inhibitor XII (GSI XII) induces apoptosis of murine MOPC315.BM myeloma cells with high Notch activity. GSI XII impairs murine osteoclast differentiation of receptor activator of NF-κB ligand (RANKL)-stimulated RAW264.7 cells in vitro. In the murine MOPC315.BM myeloma model GSI XII has potent anti-MM activity and reduces osteolytic lesions as evidenced by diminished myeloma-specific monoclonal immunoglobulin (Ig)-A serum levels and quantitative assessment of bone structure changes via high-resolution microcomputed tomography scans. Thus, we suggest that Notch inhibition through GSI XII controls myeloma bone disease mainly by targeting Notch in MM cells and possibly in osteoclasts in their microenvironment. We conclude that Notch inhibition is a valid therapeutic strategy in MM.