[Prevalence and prognosis of aspirin resistance in critical limb ischemia patients]. / Prévalence et valeur pronostique de la résistance à l'aspirine chez les patients en ischémie critique chronique des membres inférieurs.

OBJECTIVES: To assess the prevalence and the association between aspirin resistance in critical limb ischemia patients using the VerifyNow® bed-side platelet test, and occurrence of cardiovascular morbidity and/or death at one year. MATERIALS AND METHODS: National multicenter prospective observational study related to COPART II centers. From 2010 through 2014, 64 subjects hospitalized for critical limb ischemia and already treated by aspirin before the VerifyNow® test were included. A VerifyNow® test>550 ARU was defined as aspirin resistance. Critical limb ischemia was defined according to the TASC I criteria. The primary outcome was a composite including death, acute coronary syndrome, stroke and major amputation during the one-year follow-up period. RESULTS: In all, 9/64 patients were aspirin resistant, the status was confirmed in one case. The prevalence of aspirin resistance was 14.06%. There was no significant difference between aspirin resistant and aspirin non-resistant groups in terms of cardiovascular history and glycemia status. Neither was there significant difference between the two groups in terms of survival. CONCLUSION: Aspirin resistance was not predictive of poorer survival in critical limb ischemia patients. However, our population was limited. Considering that a clear definition of aspirin resistance and standardized diagnostic tests are lacking, complementary studies might be useful.

Inhibition of histone deacetylation induces constitutive derepression of the beta interferon promoter and confers antiviral activity.

The induction of alpha/beta interferon (IFN-alpha/beta) genes constitutes one of the first responses of the cell to virus infection. The IFN-beta gene is constitutively repressed in uninfected cells and is transiently activated after virus infection. In this work we demonstrate that histone deacetylation regulates the silent state of the murine IFN-beta gene. Using chromatin immunoprecipitation (ChIP) assays, we show a direct in vivo correlation between the transcriptionally silent state and a state of hypoacetylation of histone H4 on the IFN-beta promoter region. Trichostatin A (TSA), a specific inhibitor of histone deacetylases, induced strong, constitutive derepression of the murine IFN-beta promoter stably integrated into a chromatin context, as well as the hyperacetylation of histone H4, without requiring de novo protein synthesis. We also show in this work that TSA treatment strongly enhances the endogenous IFN level and confers an antiviral state to murine fibroblastic L929 cells. Inhibition of histone deacetylation with TSA protected the cells against the lost of viability induced by vesicular stomatitis virus (VSV) and inhibited VSV multiplication. Using antibodies neutralizing IFN-alpha/beta, we show that the antiviral state induced by TSA is due to TSA-induced IFN production. The demonstration of the predominant role of histone deacetylation during the regulation of the constitutive repressed state of the IFN-beta promoter constitutes an interesting advance on the understanding of the negative regulation of this gene and opens up the possibility of new therapeutic perspectives.

Repression of virus-induced interferon A promoters by homeodomain transcription factor Ptx1.

Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by substitutions in the proximal positive virus responsive element A (VRE-A) of their promoters and by the presence or absence of a distal negative regulatory element (DNRE). The functional feature of the DNRE is to specifically act by repression of VRE-A activity. With the use of the yeast one-hybrid system, we describe here the identification of a specific DNRE-binding protein, the pituitary homeobox 1 (Ptx1 or Pitx1). Ptx1 is detectable in different cell types that differentially express IFN-A genes, and the endogenous Ptx1 protein binds specifically to the DNRE. Upon virus induction, Ptx1 negatively regulates the transcription of DNRE-containing IFN-A promoters, and the C-terminal region, as well as the homeodomain of the Ptx1 protein, is required for this repression. After virus induction, the expression of the Ptx1 antisense RNA leads to a significant increase of endogenous IFN-A gene transcription and is able to modify the pattern of differential expression of individual IFN-A genes. These studies suggest that Ptx1 contributes to the differential transcriptional strength of the promoters of different IFN-A genes and that these genes may provide new targets for transcriptional regulation by a homeodomain transcription factor.

Specific binding of high-mobility-group I (HMGI) protein and histone H1 to the upstream AT-rich region of the murine beta interferon promoter: HMGI protein acts as a potential antirepressor of the promoter.

The high-mobility-group I (HMGI) protein is a nonhistone component of active chromatin. In this work, we demonstrate that HMGI protein specifically binds to the AT-rich region of the murine beta interferon (IFN-beta) promoter localized upstream of the murine virus-responsive element (VRE). Contrary to what has been described for the human promoter, HMGI protein did not specifically bind to the VRE of the murine IFN-beta promoter. Stably transfected promoters carrying mutations on this HMGI binding site displayed delayed virus-induced kinetics of transcription. When integrated into chromatin, the mutated promoter remained repressed and never reached normal transcriptional activity. Such a phenomenon was not observed with transiently transfected promoters upon which chromatin was only partially reconstituted. Using UV footprinting, we show that the upstream AT-rich sequences of the murine IFN-beta promoter constitute a preferential binding region for histone H1. Transfection with a plasmid carrying scaffold attachment regions as well as incubation with distamycin led to the derepression of the IFN-beta promoter stably integrated into chromatin. In vitro, HMGI protein was able to displace histone H1 from the upstream AT-rich region of the wild-type promoter but not from the promoter carrying mutations on the upstream high-affinity HMGI binding site. Our results suggest that the binding of histone H1 to the upstream AT-rich region of the promoter might be partly responsible for the constitutive repression of the promoter. The displacement by HMGI protein of histone H1 could help to convert the IFN-beta promoter from a repressed to an active state.

Type I interferons: expression and signalization.

Type I interferon (IFN-A and IFN-B) genes encode a large family of multifunctional secreted proteins involved in antiviral defence, cell growth regulation and immune activation. These cytokines, as a consequence of their biological activities, have been established as effective therapeutic molecules for malignant and viral diseases. Virus infection is the main inducer leading to transient expression of type I IFN (A and B) and the antiviral response appears to proceed through a two-step pathway requiring, first, induction of type I IFN gene expression and, second, transcriptional activation by the synthesized IFN proteins, binding to their specific cell surface receptors, of a large number of genes. The proteins they encode are responsible, in part, for the pleiotropic multiple biological activities of the IFN. In this two-step pathway, the virus-induced IFN genes and the IFN-stimulated gene (ISG) expression seem to share common factors. Even if IFN-A genes are structurally related and very often coordinately induced in virus-infected cells, differences in the expression of the individual IFN-A messenger RNAs of the multigenic IFN-A gene family are observed in human as well as in murine cells, reflecting, in a particular cell type, the transcriptional activity of the corresponding promoter regions. Important studies on interferon regulatory factors and ISG factors have been made in the last decade. However, some factors involved in IFN-A gene regulation remain to be identified. Our goal has been to review the factors involved in the control of the type I IFN gene expression to understand the mechanisms of induction and repression of their transcription and to explain the properties of these cytokines through their signal transduction pathway.

Silencer activity in the interferon-A gene promoters.

Interferon-A (IFN-A) differential gene expression is modulated by a complex interplay between cis-acting DNA elements and the corresponding specific trans-regulating factors. Substitutions in the proximal virus-responsive element of the interferon-A (IFN-A) promoters contribute to their differential gene expression. The 5' distal silencing region in the weakly virus-inducible murine IFN-A11 gene has been previously delimited. DNase I footprinting experiments and transient gene expression assays demonstrate identical silencing activity in equivalent regions of the genes for IFN-A11 and IFN-A4 promoters. A minimal 20-mer distal negative regulatory element (DNRE) in both promoters is necessary and sufficient for the silencing and a region in the highly inducible IFN-A4 promoter located between the silencer and the virus-responsive element overrides the silencer activity. Mutations in the central region of the DNRE, causing derepression, also altered the formation of one of the two major DNA-protein complexes. One of these contains a protein related to or identical to the high mobility group I(Y) proteins, while the other complex contains a major protein present in uninduced and virus-induced cells with a molecular mass of 38 kDa, which may be related to the silencer activity. Similar DNREs are present in other virus-uninducible IFN-A promoters, and these data suggest that a common silencer may mediate the transcriptional repression in different genes of this family.

Synergism between multiple virus-induced factor-binding elements involved in the differential expression of interferon A genes.

Comparative transfection analysis of murine interferon A4 and interferon A11 promoter constructs transiently transfected in mouse L929 and human HeLa S3 cells infected with Newcastle disease virus showed that the second positive regulatory domain I-like domain (D motif), located between nucleotides -57 and -46 upstream of the transcription start site, contributes to the activation of virus-induced transcription of the interferon (IFN)-A4 gene promoter by cooperating with the positive regulatory domain I-like and TG-like domains previously described. Electrophoretic mobility shift assay performed with the virus-inducible fragments containing these motifs indicated that the binding activity that we have denoted as virus-induced factor (Génin, P., Bragança, J., Darracq, N., Doly, J., and Civas, A. (1995) Nucleic Acids Res. 23, 5055-5063) is different from interferon-stimulated gene factor 3. It binds to the D motif but not to the virus-unresponsive form of the D motif disrupted by a G-57 --> C substitution. We show that the low levels of IFN-A11 gene expression are caused essentially by the lack of two inducible enhancer domains disrupted by the A-78 --> G and the G-57 --> C substitutions. These data suggest a model taking account of the differential regulation of IFN-A gene family members. They also suggest that virus-induced factor may correspond to the primary transcription factor directly activated by virus that is involved in the initiation of IFN-A gene transcription.

Identification of distal silencing elements in the murine interferon-A11 gene promoter.

The murine interferon-A11 (Mu IFN-A11) gene is a member of the IFN-A multigenic family. In mouse L929 cells, the weak response of the gene's promoter to viral induction is due to a combination of both a point mutation in the virus responsive element (VRE) and the presence of negatively regulating sequences surrounding the VRE. In the distal part of the promoter, the negatively acting E1E2 sequence was delimited. This sequence displays an inhibitory effect in either orientation or position on the inducibility of a virus-responsive heterologous promoter. It selectively represses VRE-dependent transcription but is not able to reduce the transcriptional activity of a VRE-lacking promoter. In a transient transfection assay, an E1E2-containing DNA competitor was able to derepress the native Mu IFN-A11 promoter. Specific nuclear factors bind to this sequence; thus the binding of trans-regulators participates in the repression of the Mu IFN-A11 gene. The E1E2 sequence contains an IFN regulatory factor (IRF)-binding site. Recombinant IRF2 binds this sequence and anti-IRF2 antibodies supershift a major complex formed with nuclear extracts. The protein composing the complex is 50 kDa in size, indicating the presence of IRF2 or antigenically related proteins in the complex. The Mu IFN-A11 gene is the first example within the murine IFN-A family, in which a distal promoter element has been identified that can negatively modulate the transcriptional response to viral induction.

A novel PRD I and TG binding activity involved in virus-induced transcription of IFN-A genes.

Comparative analysis of the inducible elements of the mouse interferon A4 and A11 gene promoters (IE-A4 and IE-A11) by transient transfection experiments, DNase 1 footprinting and electrophoretic mobility shift assays resulted in identification of a virus-induced binding activity suggested to be involved in NDV-induced activation of transcription of these genes. The virus-induced factor, termed VIF, is activated early by contact of virions with cells. It specifically recognizes the PRD I-like domain shared by both inducible elements, as well as the TG-like domain of IE-A4. This factor, distinct from the IRF-1, IRF-2 and the alpha F1 binding proteins and presenting a different affinity pattern from that of the TG protein, is proposed as a candidate for IFN-type I gene regulation.

Induction of interferon-beta gene expression by dexamethasone in murine L929 cells.

Glucocorticoids bind to their receptors and trigger the transcriptional activation or repression of target genes by binding to DNA sequences, the glucocorticoid responsive element (GRE). The murine interferon-beta (Mu-IFN beta) gene in L929 cells can be induced by dexamethasone to give both transcription and translation products specific to murine IFN beta. The 3'-noncoding region of the Mu-IFN beta gene was found to contain a GRE very similar to the consensus GRE sequence involved in glucocorticoid-regulated genes. Gel retardation assays showed that the oligonucleotide corresponding to that GRE competed with the MMTV GRE oligonucleotide for glucocorticoid receptor binding and was supershifted by human antiglucocorticoid receptor antibodies. Transiently transfected murine cells (L929) with the GRE-IFN beta 3' sequence inserted upstream of the thymidine kinase promoter and the chloramphenicol acetyl transferase gene treated with dexamethasone with or without the antiglucocorticoid RU486 and their chloramphenicol acetyl transferase activity assayed, show that this GRE is efficient. We conclude that the Mu-IFN beta gene in L929 murine cells can be induced by dexamethasone, and that the hormone effect may be mediated by the 3'-GRE sequence.

Developmental control of IFN-alpha expression in murine embryos.

The expression of IFN-alpha transcripts was investigated in murine embryos, fetuses, and fetal annexes in mid and late pregnancy. We have shown by Northern blot analysis, reverse transcription-polymerase chain reaction, and in situ hybridization the presence of IFN-alpha transcripts in mouse placenta, fetus, and newborn. From the 14th day of gestation until birth, a typical IFN-alpha transcript (1.2 kb) is found in the fetus. A transcript of larger size (2.2 kb) appears near birth and is present in the newborn mouse. Fetal annexes between the 10th and 21st days of gestation also express IFN-alpha. From the 10th day until birth, the 1.2-kb IFN-alpha mRNA species is present, as well as unusually large transcripts: 4 and 7 kb. To localize IFN-alpha transcripts, in situ hybridization was performed, using 35S-IFN-alpha antisense RNA probe in comparison with the sense RNA probe. The tissue pattern of IFN-alpha transcription in fetuses shows a clear labeling of many epithelia, such as skin, ependyme, and intestine glandular epithelium. A possible relation with cellular differentiation is discussed.

IL-6 induced by IL-1 inhibits malaria pre-erythrocytic stages but its secretion is down-regulated by the parasite.

The capacity of IL-6 to mediate the antiparasitic activity of IL-1 on intrahepatic development of malaria parasite was demonstrated. The comparisons of IL-6 levels in infected and noninfected hepatocyte cultures, either purified or enriched with nonparenchymal cells and stimulated by IL-1 or IL-6, indicate that subtle interactions exist between intrahepatocytic development of Plasmodium yoelii and liver synthesis of IL-6. During its intrahepatic multiplication, the parasite causes a decline in IL-6 production. IL-6 mRNA was not detected in the livers of infected mice during development of either hepatic or blood stage parasites although IL-6 activity was found in the sera during both stages.

The genes for the inter-alpha-inhibitor family share a homologous organization in human and mouse.

Inter-alpha-inhibitor (I alpha I) and related molecules in human are comprised of three evolutionarily related, heavy (H) chains and one light (L) chain, also termed bikunin. The latter originates from a precursor molecule that is cleaved to yield the bikunin and another protein designated alpha-1-microglobulin (A1m). The four H and L chains are encoded by four distinct genes designated H1, H2, H3, and L. The L and H2 genes are localized onto human chromosomes (chr) 9 and 10, respectively, whereas the H1 and H3 genes are tandemly arranged on chr 3. Mouse poly(A)+ RNAs or endonuclease-restricted mouse DNA were analyzed by standard and pulsed-field gel electrophoresis (PFGE) techniques in agarose gels and blot-hybridized with human H1, H2, H3 or L cDNA probes. The variable sized transcripts and unique restriction fragment patterns detected with each probe indicate that four genes, including one common L gene for A1m and bikunin also exist in mouse. The co-migration of H1- and H3-hybridizing fragments on PFGE suggests that the mouse H1 and H3 genes are also tandemly arranged. An Msp I restriction fragment length polymorphism (RFLP) in the mouse L gene (proposed symbol, Intin-4) links this gene to other genes already mapped at mouse Chr 4 near the brown (b) locus, a homologous region to the human chr 9q32-34 band where the human I alpha I L gene is located. Therefore, a similar number and arrangement of I alpha I genes is found in mouse and human, including the triplication of an H gene ancestor. These results point to an ancient origin of this complex set of genes.

Isolation and characterization of a novel interferon-alpha-encoding gene, IFN-alpha 11, within a murine IFN cluster.

Two recombinant cosmids containing three complete murine interferon-alpha-encoding genes (Mu IFN-alpha) have been isolated from a mouse cosmid library. The cluster organization of these genes has been determined. A new Mu IFN-alpha gene (Mu IFN-alpha 11) has been isolated and studied with respect to its structure and inducible transcription pattern. The nucleotide and deduced amino acid sequences of the Mu IFN-alpha 11 gene, as compared to the two other IFN-alpha 7 and IFN-alpha 8 genes, show that, albeit highly homologous, these genes are all different. The transient expression of the three genes gave rise to proteins showing antiviral properties which were neutralized with murine anti-IFN-alpha antibodies. The transcription of the Mu IFN-alpha genes was studied in two uninduced or Newcastle-disease-virus-induced murine cell types. Mu IFN-alpha 11, as well as alpha 7 and alpha 8, are not expressed in L929 nor in C243 cells upon viral induction and therefore constitute an interesting model to study Mu IFN-alpha gene repression.

Repression of the murine interferon alpha 11 gene: identification of negatively acting sequences.

The uninducible murine interferon alpha 11 gene (Mu IFN-alpha 11) shows strong homology with the highly inducible Mu IFN-alpha 4 gene in the promoter region. Negative regulatory sequences located between positions -470 and -145 were characterized in the Mu IFN-alpha 11 promoter. The removal of these sequences leads to virus-inducibility of Mu IFN-alpha 11 while their insertion in Mu IFN-alpha 4 corresponding region significantly reduced the inducibility of Mu IFN-alpha 4 promoter. On the other hand, the virus-responsive element (VRE) of the Mu IFN-alpha 11 differs by a single nucleotide substitution at position -78 from the VRE alpha 4. Constructions carrying either VRE alpha 11 or VRE alpha 4 upstream a heterologous promoter displayed different virus inducibilities. The -78 A/G substitution affects the inducibility by decreasing the affinity of VRE-binding trans-regulators. Our results suggest that the combined effect of the negative regulatory sequences and of the mutation in the VRE alpha 11, completely silences the Mu IFN-alpha 11 gene.

Interleukin-6 and its receptor are expressed by human megakaryocytes: in vitro effects on proliferation and endoreplication.

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an important role in the megakaryocytic differentiation. Recently, we have observed that IL-6 is synthesized by several human cell lines with megakaryocytic features. In this study, we have investigated whether a similar phenomenon occurs during normal megakaryocytic differentiation. Human megakaryocytes (MK) were obtained by culturing normal marrow in liquid culture with aplastic plasma (AP). First, an IL-6 secretion in bone marrow culture enriched in MK as well as in purified MK populations was demonstrated by a biologic assay. Second, IL-6 mRNA was detected in a purified population of MK by the polymerase chain reaction and dot blot analysis. IL-6 mRNA and protein were undetectable in platelets. Third, in situ hybridization procedure demonstrated the presence of IL-6 mRNA in individual immature MK. Fourth, IL-6 protein was detected in MK at the unicellular level by an immunoalkaline phosphatase technique using a monoclonal antibody against IL-6. Furthermore, the presence of IL-6 receptor (IL-6-R) on MK was demonstrated by in situ hybridization using an IL-6-R probe and in situ autoradiography after binding with [125I]-labeled recombinant IL-6. The IL-6 endogenously produced in liquid cultures containing normal human plasma or AP was subsequently neutralized. This resulted in a 50% decrease of the MK growth with a minor shift in the ploidy distribution toward lower values. In semisolid cultures the addition of anti-IL-6 antibodies led to a 42% decrease in colony number in cultures stimulated by IL-3 but not in other conditions of culture. These results suggest that normal human megakaryocytopoiesis might be regulated in part by an IL-6 autocrine loop.

Expression of interleukin 6 and its specific receptor by untreated and PMA-stimulated human erythroid and megakaryocytic cell lines.

Different human hematopoietic cell lines were analyzed for the presence of interleukin 6 (IL-6) and IL-6 receptor (IL-6-R). Both IL-6 mRNA and secreted active IL-6 protein were detectable in untreated cell lines with erythroid or megakaryoblastic features (K562, HEL, KU 812, MEG-01, and Dami), but they were not expressed constitutively in other leukemic cell lines (KG1, HL60, and U937). IL-6-R production, studied by the presence of its mRNA and specific binding sites for iodinated recombinant IL-6, was detected in most cell lines except K562, HEL, and Dami. Therefore, only KU 812 and MEG-01 coexpress both IL-6 and IL-6-R. After phorbol ester myristate acetate (PMA) treatment, all the cell lines studied expressed or overexpressed IL-6. In the erythroid K562 cell line, IL-6-R was not detectable before induction, but was promptly expressed after stimulation with PMA, suggesting that some of the new features of K562 cells induced by PMA may be mediated by IL-6. However, neutralizing antibodies against IL-6 did not block either the growth arrest or the loss of the erythroid phenotype induced by PMA. The presence of IL-6 and IL-6-R in erythroid and megakaryocytic leukemic cell lines suggests that their synthesis may occur during normal hematopoietic differentiation.

Autocrine regulation of terminal differentiation by interleukin-6 in the pluripotent KU812 cell line.

The human KU812 leukemic cell line is a model for studying cell commitment towards different hematopoietic lineages. Indeed, this cell line is characterized by both a capacity for self-renewal and the ability to differentiate spontaneously along erythroid and basophilic cell lineages. In this study we show that interleukin-6 (IL-6) and its specific receptor (IL-6-R) are spontaneously expressed in the human KU812 cell line. Addition of antibody against IL-6 weakly inhibited its cell proliferation (20 to 30%) suggesting that the endogenous production of IL-6 was partially responsible for the growth of the cell line. In contrast, the spontaneous terminal differentiation of this cell line towards the erythroid and basophilic lineages was inhibited by an antibody against IL-6 and this effect was reversed by addition of recombinant human IL-6 (rIL-6). These results suggest that IL-6 is involved more in differentiation than in the proliferation of KU812 cells. After several passages, KU812 cells lose their capacity to differentiate spontaneously. In these cells, the IL-6-R was no more detectable. We therefore suggest that this loss of spontaneous differentiation is associated with an interruption of the IL-6 autocrine loop.

Unusually large interferon-alpha-like mRNAs and high expression of interleukin-6 in human fetal annexes.

Endogenous interferons (IFNs) with antiviral activity have been detected in human fetal annexes, without any apparent induction. Some are of unusually high molecular weight and antigenic properties (Duc-Goiran P., Robert-Galliot, B., Lopez, J., and Chany, C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5010-5014). We study here the expression of IFN genes from the three antigenically distinct groups, in placental tissues taken during third-trimester pregnancies. We show by Northern blot analysis with RNA probes that IFN-alpha-like transcripts correlate with the presence of functionally active protein, purified by immunoaffinity chromatography. We fractionate on a formamide sucrose gradient, identify, and characterize a large 4.3-kb IFN-alpha-like transcript, which may be specifically expressed during fetal development. Despite the absence of IFN-beta transcript, a protein with an antiviral activity which was neutralized with a polyclonal anti-IFN-beta serum was detected in the placenta and could be related to a beta-like IFN. Besides IFNs, various growth factors and cytokines can be found in the placenta. We report here the presence of interleukin-6 expressed at high levels in fetal annexes. The coexpression of Interleukin-6 and endogenous IFNs suggests that they play an important role during development.

Isolation and characterization of a functional murine interferon alpha gene which is not expressed in fibroblasts upon virus induction.

A mouse genomic segment containing three new members of the murine interferon alpha (MuIFN-alpha) gene family was isolated from a fibroblastic cosmid library. A 4 kb EcoRI fragment contained a new MuIFN-alpha gene named MuIFN-alpha 8. The nucleotide sequence of the coding and flanking regions of this gene showed a high level of homology to those of known members of the MuIFN-alpha family. Transient expression of the MuIFN-alpha 8 gene in COS cells and oocyte translation of in vitro transcripts both led to a biologically active protein. The antiviral activity was neutralized by monoclonal and polyclonal MuIFN-alpha antibodies. Although the 5' flanking sequence shows features characteristic of an IFN regulatory region, the MuIFN-alpha 8 gene is not expressed in murine fibroblasts treated with Newcastle disease virus or poly(I).poly(C).