• Antibodies against C-reactive protein cross-react with 60-kilodalton heat shock proteins.

      Udvarnoki, Katalin; Cervenak, László; Uray, Katalin; Hudecz, Ferenc; Kacskovics, Imre; Spallek, Ralf; Singh, Mahavir; Füst, George; Prohászka, Zoltán; Third Department of Medicine, Semmelweis University, H-1125 Budapest, Kútvölgyi st. 4, Hungary. (2007-04)
      C-reactive protein (CRP) is an acute-phase reactant frequently used in histochemistry as a marker of ongoing inflammation. Furthermore, CRP is a powerful biomarker for the prediction of coronary artery disease risk. Heat-shock protein 60 (Hsp60) and CRP are complement-activating molecules, and the effect of their interactions on the regulation of complement activation was studied. However, during the first experiments, we learned that polyclonal anti-CRP antibodies cross-react with Hsp60. Therefore, the aim of our present study was to analyze the cross-reactivity of anti-CRP antibodies (Ab) with Hsp60 in solid-phase enzyme immune assays, in epitope studies using a series of overlapping synthetic peptides, and in Ouchterlony analyses. We found that three different commercial rabbit polyclonal antibodies and two monoclonal (9C9 and CRP-8) anti-CRP antibodies specifically recognize recombinant human Hsp60 and recombinant Mycobacterium tuberculosis Hsp65, respectively. Hsp60 was found to inhibit the binding of anti-CRP polyclonal Ab to Hsp60. Six epitope regions of Hsp60 were recognized by the anti-CRP antibodies, and one region (amino acids [AA] 218 to 232) was recognized by monoclonal antibodies CRP-8 and 9C9. This epitope region of Hsp60 displays 26.6% amino acid identity to CRP AA region 77 to 90. These data suggest that the B-cell epitopes shared between CRP and Hsp60 give rise to a true mimicry-based cross-reaction and the induction of cross-reactive antibodies. Our study underlines the importance of thorough study design and careful interpretation of results while using polyclonal anti-CRP antibodies for histochemistry, especially at low dilutions. Furthermore, analytical interference with Hsp60 in CRP assays should also be tested.
    • Cellular retinaldehyde-binding protein (CRALBP) is a direct downstream target of transcription factor Pax6.

      Boppana, Sridhar; Scheglov, Alexander; Geffers, Robert; Tarabykin, Victor; Max-Planck-Institute for Experimental Medicine, Hermann-Rein Strasse 3, 37075 Göttingen, Germany. boppansr@umdnj.edu (2012-02)
      Transcription factor Pax6 plays an essential role in the expression of other transcription factors, cell adhesion molecules and is crucial for neurogenesis in the developing forebrain. Analysis of gene expression profiles through microarray experiments in Pax6 mutants allowed us to focus on CRALBP, one of the many genes that were downregulated.
    • Deletion of Irf3 and Irf7 Genes in Mice Results in Altered Interferon Pathway Activation and Granulocyte-Dominated Inflammatory Responses to Influenza A Infection.

      Hatesuer, Bastian; Hoang, Hang Thi Thu; Riese, Peggy; Trittel, Stephanie; Gerhauser, Ingo; Elbahesh, Husni; Geffers, Robert; Wilk, Esther; Schughart, Klaus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
      The interferon (IFN) pathway plays an essential role in the innate immune response following viral infections and subsequent shaping of adaptive immunity. Infections with influenza A viruses (IAV) activate the IFN pathway after the recognition of pathogen-specific molecular patterns by respective pattern recognition receptors. The IFN regulatory factors IRF3 and IRF7 are key players in the regulation of type I and III IFN genes. In this study, we analyzed the role of IRF3 and IRF7 for the host response to IAV infections in Irf3-/-, Irf7-/-, and Irf3-/-Irf7-/- knockout mice. While the absence of IRF3 had only a moderate impact on IFN expression, deletion of IRF7 completely abolished IFNα production after infection. In contrast, lack of both IRF3 and IRF7 resulted in the absence of both IFNα and IFNβ after IAV infection. In addition, IAV infection of double knockout mice resulted in a strong increase of mortality associated with a massive influx of granulocytes in the lung and reduced activation of the adaptive immune response.
    • Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.

      Günther, Katharina; Rust, Mareike; Leers, Joerg; Boettger, Thomas; Scharfe, Maren; Jarek, Michael; Bartkuhn, Marek; Renkawitz, Rainer; Institute for Genetics, Justus-Liebig-University, D35392 Giessen, Germany. (2013-03-01)
      The heterogeneous collection of nucleosome remodelling and deacetylation (NuRD) complexes can be grouped into the MBD2- or MBD3-containing complexes MBD2-NuRD and MBD3-NuRD. MBD2 is known to bind to methylated CpG sequences in vitro in contrast to MBD3. Although functional differences have been described, a direct comparison of MBD2 and MBD3 in respect to genome-wide binding and function has been lacking. Here, we show that MBD2-NuRD, in contrast to MBD3-NuRD, converts open chromatin with euchromatic histone modifications into tightly compacted chromatin with repressive histone marks. Genome-wide, a strong enrichment for MBD2 at methylated CpG sequences is found, whereas CpGs bound by MBD3 are devoid of methylation. MBD2-bound genes are generally lower expressed as compared with MBD3-bound genes. When depleting cells for MBD2, the MBD2-bound genes increase their activity, whereas MBD2 plus MBD3-bound genes reduce their activity. Most strikingly, MBD3 is enriched at active promoters, whereas MBD2 is bound at methylated promoters and enriched at exon sequences of active genes.
    • Enantiomer-specific and paracrine leukemogenicity of mutant IDH metabolite 2-hydroxyglutarate.

      Chaturvedi, A; Araujo Cruz, M M; Jyotsana, N; Sharma, A; Goparaju, R; Schwarzer, A; Görlich, K; Schottmann, R; Struys, E A; Jansen, E E; Rohde, C; Müller-Tidow, C; Geffers, R.; Göhring, G; Ganser, A; Thol, F; Heuser, M; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-08)
      Canonical mutations in IDH1 and IDH2 produce high levels of the R-enantiomer of 2-hydroxyglutarate (R-2HG), which is a competitive inhibitor of α-ketoglutarate (αKG)-dependent enzymes and a putative oncometabolite. Mutant IDH1 collaborates with HoxA9 to induce monocytic leukemia in vivo. We used two mouse models and a patient-derived acute myeloid leukemia xenotransplantation (PDX) model to evaluate the in vivo transforming potential of R-2HG, S-2HG and αKG independent of the mutant IDH1 protein. We show that R-2HG, but not S-2HG or αKG, is an oncometabolite in vivo that does not require the mutant IDH1 protein to induce hyperleukocytosis and to accelerate the onset of murine and human leukemia. Thus, circulating R-2HG acts in a paracrine manner and can drive the expansion of many different leukemic and preleukemic clones that may express wild-type IDH1, and therefore can be a driver of clonal evolution and diversity. In addition, we show that the mutant IDH1 protein is a stronger oncogene than R-2HG alone when comparable intracellular R-2HG levels are achieved. We therefore propose R-2HG-independent oncogenic functions of mutant IDH1 that may need to be targeted in addition to R-2HG production to exploit the full therapeutic potential of IDH1 inhibition.
    • Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes.

      Terrados, Gloria; Finkernagel, Florian; Stielow, Bastian; Sadic, Dennis; Neubert, Juliane; Herdt, Olga; Krause, Michael; Scharfe, Maren; Jarek, Michael; Suske, Guntram; Institute of Molecular Biology and Tumor Research, Philipps-University, Emil-Mannkopff-Str. 2, D-35032 Marburg, Germany. (2012-09)
      The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes.
    • Gut bacterial communities across tadpole ecomorphs in two diverse tropical anuran faunas.

      Vences, Miguel; Lyra, Mariana L; Kueneman, Jordan G; Bletz, Molly C; Archer, Holly M; Canitz, Julia; Handreck, Svenja; Randrianiaina, Roger-Daniel; Struck, Ulrich; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; McKenzie, Valerie J; Tebbe, Christoph C; Haddad, Célio F B; Glos, Julian; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-04)
      Animal-associated microbial communities can play major roles in the physiology, development, ecology, and evolution of their hosts, but the study of their diversity has yet focused on a limited number of host species. In this study, we used high-throughput sequencing of partial sequences of the bacterial 16S rRNA gene to assess the diversity of the gut-inhabiting bacterial communities of 212 specimens of tropical anuran amphibians from Brazil and Madagascar. The core gut-associated bacterial communities among tadpoles from two different continents strongly overlapped, with eight highly represented operational taxonomic units (OTUs) in common. In contrast, the core communities of adults and tadpoles from Brazil were less similar with only one shared OTU. This suggests a community turnover at metamorphosis. Bacterial diversity was higher in tadpoles compared to adults. Distinct differences in composition and diversity occurred among gut bacterial communities of conspecific tadpoles from different water bodies and after experimental fasting for 8 days, demonstrating the influence of both environmental factors and food on the community structure. Communities from syntopic tadpoles clustered by host species both in Madagascar and Brazil, and the Malagasy tadpoles also had species-specific isotope signatures. We recommend future studies to analyze the turnover of anuran gut bacterial communities at metamorphosis, compare the tadpole core communities with those of other aquatic organisms, and assess the possible function of the gut microbiota as a reservoir for protective bacteria on the amphibian skin.
    • Haplotypes of the porcine peroxisome proliferator-activated receptor delta gene are associated with backfat thickness.

      Meidtner, Karina; Schwarzenbacher, Hermann; Scharfe, Maren; Severitt, Simone; Blöcker, Helmut; Fries, Ruedi; Chair of Animal Breeding, Technical University of Munich, Hochfeldweg 1, 85354 Freising - Weihenstephan, Germany. karina.meidtner@tierzucht.tum.de (2009)
      BACKGROUND: Peroxisome proliferator-activated receptor delta belongs to the nuclear receptor superfamily of ligand-inducible transcription factors. It is a key regulator of lipid metabolism. The peroxisome proliferator-activated receptor delta gene (PPARD) has been assigned to a region on porcine chromosome 7, which harbours a quantitative trait locus for backfat. Thus, PPARD is considered a functional and positional candidate gene for backfat thickness. The purpose of this study was to test this candidate gene hypothesis in a cross of breeds that were highly divergent in lipid deposition characteristics. RESULTS: Screening for genetic variation in porcine PPARD revealed only silent mutations. Nevertheless, significant associations between PPARD haplotypes and backfat thickness were observed in the F2 generation of the Mangalitsa x Piétrain cross as well as a commercial German Landrace population. Haplotype 5 is associated with increased backfat in F2 Mangalitsa x Piétrain pigs, whereas haplotype 4 is associated with lower backfat thickness in the German Landrace population. Haplotype 4 and 5 carry the same alleles at all but one SNP. Interestingly, the opposite effects of PPARD haplotypes 4 and 5 on backfat thickness are reflected by opposite effects of these two haplotypes on PPAR-delta mRNA levels. Haplotype 4 significantly increases PPAR-delta mRNA levels, whereas haplotype 5 decreases mRNA levels of PPAR-delta. CONCLUSION: This study provides evidence for an association between PPARD and backfat thickness. The association is substantiated by mRNA quantification. Further studies are required to clarify, whether the observed associations are caused by PPARD or are the result of linkage disequilibrium with a causal variant in a neighbouring gene.
    • A human-horse comparative map based on equine BAC end sequences.

      Leeb, Tosso; Vogl, Claus; Zhu, Baoli; de Jong, Pieter J; Binns, Matthew M; Chowdhary, Bhanu P; Scharfe, Maren; Jarek, Michael; Nordsiek, Gabriele; Schrader, Frank; Blöcker, Helmut; Institute of Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559 Hannover, Germany. Tosso.Leeb@itz.unibe.ch (2006-06)
      In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E
    • Hypoxia Enhances Immunosuppression by Inhibiting CD4+ Effector T Cell Function and Promoting Treg Activity.

      Westendorf, Astrid M; Skibbe, Kathrin; Adamczyk, Alexandra; Buer, Jan; Geffers, Robert; Hansen, Wiebke; Pastille, Eva; Jendrossek, Verena; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Hypoxia occurs in many pathological conditions, including inflammation and cancer. Within this context, hypoxia was shown to inhibit but also to promote T cell responses. Due to this controversial function, we aimed to explore whether an insufficient anti-tumour response during colitis-associated colon cancer could be ascribed to a hypoxic microenvironment.
    • Interleukin-2 improves amyloid pathology, synaptic failure and memory in Alzheimer's disease mice.

      Alves, Sandro; Churlaud, Guillaume; Audrain, Mickael; Michaelsen-Preusse, Kristin; Fol, Romain; Souchet, Benoit; Braudeau, Jérôme; Korte, Martin; Klatzmann, David; Cartier, Nathalie; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-03-01)
      Interleukin-2 (IL-2)-deficient mice have cytoarchitectural hippocampal modifications and impaired learning and memory ability reminiscent of Alzheimer's disease. IL-2 stimulates regulatory T cells whose role is to control inflammation. As neuroinflammation contributes to neurodegeneration, we investigated IL-2 in Alzheimer's disease. Therefore, we investigated IL-2 levels in hippocampal biopsies of patients with Alzheimer's disease relative to age-matched control individuals. We then treated APP/PS1ΔE9 mice having established Alzheimer's disease with IL-2 for 5 months using single administration of an AAV-IL-2 vector. We first found decreased IL-2 levels in hippocampal biopsies of patients with Alzheimer's disease. In mice, IL-2-induced systemic and brain regulatory T cells expansion and activation. In the hippocampus, IL-2 induced astrocytic activation and recruitment of astrocytes around amyloid plaques, decreased amyloid-β42/40 ratio and amyloid plaque load, improved synaptic plasticity and significantly rescued spine density. Of note, this tissue remodelling was associated with recovery of memory deficits, as assessed in the Morris water maze task. Altogether, our data strongly suggest that IL-2 can alleviate Alzheimer's disease hallmarks in APP/PS1ΔE9 mice with established pathology. Therefore, this should prompt the investigation of low-dose IL-2 in Alzheimer's disease and other neuroinflammatory/neurodegenerative disorders.
    • Recruitment of the ATP-dependent chromatin remodeler dMi-2 to the transcribed region of active heat shock genes.

      Mathieu, Eve-Lyne; Finkernagel, Florian; Murawska, Magdalena; Scharfe, Maren; Jarek, Michael; Brehm, Alexander; Institute for Molecular Biology and Tumor Research, Philipps-University, Emil-Mannkopff-Strasse 2, 35037 Marburg, Germany. (2012-06)
      The ATP-dependent chromatin remodeler dMi-2 can play both positive and negative roles in gene transcription. Recently, we have shown that dMi-2 is recruited to the hsp70 gene in a heat shock-dependent manner, and is required to achieve high transcript levels. Here, we use chromatin immunoprecipitation sequencing (ChIP-Seq) to identify other chromatin regions displaying increased dMi-2 binding upon heat shock and to characterize the distribution of dMi-2 over heat shock genes. We show that dMi-2 is recruited to the body of at least seven heat shock genes. Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs. We find that dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A. Rather, dMi-2 binding is restricted to transcribed regions. Our results suggest that dMi-2 distribution over active heat shock genes are determined by transcriptional activity.