• Lactate dehydrogenase B regulates macrophage metabolism in the tumor microenvironment.

      Frank, Ann-Christin; Raue, Rebecca; Fuhrmann, Dominik C; Sirait-Fischer, Evelyn; Reuse, Carsten; Weigert, Andreas; Lütjohann, Dieter; Hiller, Karsten; Syed, Shahzad Nawaz; Brüne, Bernhard; et al. (Ivyspring International publisher, 2021-06-04)
      Background: Glucose metabolism in the tumor-microenvironment is a fundamental hallmark for tumor growth and intervention therein remains an attractive option for anti-tumor therapy. Whether tumor-derived factors such as microRNAs (miRs) regulate glucose metabolism in stromal cells, especially in tumor-associated macrophages (TAMs), to hijack them for trophic support, remains elusive. Methods: Ago-RIP-Seq identified macrophage lactate dehydrogenase B (LDHB) as a target of tumor-derived miR-375 in both 2D/3D cocultures and in murine TAMs from a xenograft mouse model. The prognostic value was analyzed by ISH and multiplex IHC of breast cancer patient tissues. Functional consequences of the miR-375-LDHB axis in TAMs were investigated upon mimic/antagomir treatment by live metabolic flux assays, GC/MS, qPCR, Western blot, lentiviral knockdown and FACS. The therapeutic potential of a combinatorial miR-375-decoy/simvastatin treatment was validated by live cell imaging. Results: Macrophage LDHB decreased in murine and human breast carcinoma. LDHB downregulation increase aerobic glycolysis and lactagenesis in TAMs in response to tumor-derived miR-375. Lactagenesis reduced fatty acid synthesis but activated SREBP2, which enhanced cholesterol biosynthesis in macrophages. LDHB downregulation skewed TAMs to function as a lactate and sterol/oxysterol source for the proliferation of tumor cells. Restoring of LDHB expression potentiated inhibitory effects of simvastatin on tumor cell proliferation. Conclusion: Our findings identified a crucial role of LDHB in macrophages and established tumor-derived miR-375 as a novel regulator of macrophage metabolism in breast cancer, which might pave the way for strategies of combinatorial cancer cell/stroma cell interventions.
    • Linoleic and palmitoleic acid block streptokinase-mediated plasminogen activation and reduce severity of invasive group A streptococcal infection

      Rox, Katharina; Jansen, Rolf; Loof, Torsten G.; Gillen, Christine M.; Bernecker, Steffen; Walker, Mark J.; Chhatwal, Gursharan Singh; Müller, Rolf; Helmholtz-Institut für pharmazeutische Forschung Saarland,Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-09-18)
      In contrast to mild infections of Group A Streptococcus (GAS) invasive infections of GAS still pose a serious health hazard: GAS disseminates from sterile sites into the blood stream or deep tissues and causes sepsis or necrotizing fasciitis. In this case antibiotics do not provide an effective cure as the bacteria are capable to hide from them very quickly. Therefore, new remedies are urgently needed. Starting from a myxobacterial natural products screening campaign, we identified two fatty acids isolated from myxobacteria, linoleic and palmitoleic acid, specifically blocking streptokinase-mediated activation of plasminogen and thereby preventing streptococci from hijacking the host’s plasminogen/plasmin system. This activity is not inherited by other fatty acids such as oleic acid and is not attributable to the killing of streptococci. Moreover, both fatty acids are superior in their inhibitory properties compared to two clinically used drugs (tranexamic or ε-amino caproic acid) as they show 500–1000 fold lower IC50 values. Using a humanized plasminogen mouse model mimicking the clinical situation of a local GAS infection that becomes systemic, we demonstrate that these fatty acids ameliorate invasive GAS infection significantly. Consequently, linoleic and palmitoleic acid are possible new options to combat GAS invasive diseases.
    • A Listeria monocytogenes ST2 clone lacking chitinase ChiB from an outbreak of non-invasive gastroenteritis.

      Halbedel, Sven; Prager, Rita; Banerji, Sangeeta; Kleta, Sylvia; Trost, Eva; Nishanth, Gopala; Alles, Georg; Hölzel, Christina; Schlesiger, Friederike; Pietzka, Ariane; et al. (Springer Nature, 2019-01-01)
      An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.
    • Macrophage entrapped silica coated superparamagnetic iron oxide particles for controlled drug release in a 3D cancer model.

      Ullah, Sami; Seidel, Katja; Türkkan, Sibel; Warwas, Dawid Peter; Dubich, Tatyana; Rohde, Manfred; Hauser, Hansjörg; Behrens, Peter; Kirschning, Andreas; Köster, Mario; et al. (2018-12-23)
      Targeted delivery of drugs is a major challenge in treatment of diverse diseases. Systemically administered drugs demand high doses and are accompanied by poor selectivity and side effects on non-target cells. Here, we introduce a new principle for targeted drug delivery. It is based on macrophages as transporters for nanoparticle-coupled drugs as well as controlled release of drugs by hyperthermia mediated disruption of the cargo cells and simultaneous deliberation of nanoparticle-linked drugs. Hyperthermia is induced by an alternating electromagnetic field (AMF) that induces heat from silica-coated superparamagnetic iron oxide nanoparticles (SPIONs). We show proof-of-principle of controlled release by the simultaneous disruption of the cargo cells and the controlled, AMF induced release of a toxin, which was covalently linked to silica-coated SPIONs via a thermo-sensitive linker. Cells that had not been loaded with SPIONs remain unaffected. Moreover, in a 3D co-culture model we demonstrate specific killing of associated tumour cells when employing a ratio as low as 1:40 (SPION-loaded macrophage: tumour cells). Overall, our results demonstrate that AMF induced drug release from macrophage-entrapped nanoparticles is tightly controlled and may be an attractive novel strategy for targeted drug release.
    • MAIT cells are enriched and highly functional in ascites of patients with decompensated liver cirrhosis.

      Niehaus, Christian E; Strunz, Benedikt; Cornillet, Martin; Falk, Christine S; Schnieders, Ansgar; Maasoumy, Benjamin; Hardtke, Svenja; Manns, Michael P; Rm Kraft, Anke; Björkström, Niklas K; et al. (Wiley Online Open, 2020-02-03)
      Patients with advanced liver cirrhosis have an increased susceptibility to infections. As part of the cirrhosis-associated immune dysfunction, mucosal associated invariant T (MAIT) cells, that have the capacity to respond towards bacteria, are severely diminished in circulation and liver tissue. However, MAIT cell presence and function in the peritoneal cavity, a common anatomical site for infections in cirrhosis, remain elusive. To study this, matched peripheral blood and ascites fluid were collected from 35 patients with decompensated cirrhosis, with or without spontaneous bacterial peritonitis (SBP). MAIT cell phenotype and function were analyzed using high-dimensional flow cytometry and obtained data was compared to blood samples of healthy controls (n=24) and patients with compensated cirrhosis (n=11). We found circulating MAIT cells to be severely decreased in cirrhotic patients as compared to controls. In contrast, in ascites fluid, MAIT cells were significantly increased together with CD14+ CD16+ monocytes, ILCs, and NK cells. This was paralleled by elevated levels of several pro-inflammatory cytokines and chemokines in ascites fluid as compared to plasma. Peritoneal MAIT cells displayed an activated tissue-resident phenotype and this was corroborated by increased functional responses following stimulation with E. coli or lL-12 + IL-18 as compared to circulating MAIT cells. During SBP, peritoneal MAIT cell frequencies increased most among all major immune cell subsets, suggestive of active homing of MAIT cells to the site of infection. CONCLUSIONS: Despite severely diminished MAIT cell numbers and impaired phenotype in circulation, peritoneal MAIT cells remain abundant, activated, and highly functional in decompensated cirrhosis and are further enriched in SBP. This suggests that peritoneal MAIT cells could be of interest for immune intervention strategies in patients with decompensated liver cirrhosis and SBP.
    • miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity.

      Łyszkiewicz, Marcin; Winter, Samantha J; Witzlau, Katrin; Föhse, Lisa; Brownlie, Rebecca; Puchałka, Jacek; Verheyden, Nikita A; Kunze-Schumacher, Heike; Imelmann, Esther; Blume, Jonas; et al. (PLOS, 2019-03-01)
      The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.
    • Misinterpretation of the odds ratios.

      Fernández, Nathalie; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2020-05-11)
      No abstract available
    • More than just a metabolic regulator--elucidation and validation of new targets of PdhR in Escherichia coli.

      Göhler, Anna-Katharina; Kökpinar, Öznur; Schmidt-Heck, Wolfgang; Geffers, Robert; Guthke, Reinhard; Rinas, Ursula; Schuster, Stefan; Jahreis, Knut; Kaleta, Christoph; Department of Genetics, University of Osnabrück, Osnabrück, Germany. (2011)
      The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulatory network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach.
    • Multi-omics examination of Q fever fatigue syndrome identifies similarities with chronic fatigue syndrome.

      Raijmakers, Ruud P H; Roerink, Megan E; Jansen, Anne F M; Keijmel, Stephan P; Gacesa, Ranko; Li, Yang; Joosten, Leo A B; van der Meer, Jos W M; Netea, Mihai G; Bleeker-Rovers, Chantal P; et al. (BMC, 2020-11-26)
      Inflammatory markers, including 4E-BP1 (P = 9.60-16 and 1.41-7) and MMP-1 (P = 7.09-9 and 3.51-9), are significantly more expressed in both QFS and CFS patients compared to HC. Blood metabolite profiles show significant differences when comparing QFS (319 metabolites) and CFS (441 metabolites) patients to HC, and are significantly enriched in pathways like sphingolipid (P = 0.0256 and 0.0033) metabolism. When comparing QFS to CFS patients, almost no significant differences in metabolome were found. Comparison of microbiome taxonomy of QFS and CFS patients with that of HC, shows both in- and decreases in abundancies in Bacteroidetes (with emphasis on Bacteroides and Alistiples spp.), and Firmicutes and Actinobacteria (with emphasis on Ruminococcus and Bifidobacterium spp.). When we compare QFS patients to CFS patients, there is a striking resemblance and hardly any significant differences in microbiome taxonomy are found.
    • Nasal DNA methylation profiling of asthma and rhinitis.

      Qi, Cancan; Jiang, Yale; Yang, Ivana V; Forno, Erick; Wang, Ting; Vonk, Judith M; Gehring, Ulrike; Smit, Henriëtte A; Milanzi, Edith B; Carpaij, Orestes A; et al. (2020-01-14)
    • Non-Targeted Mass Isotopolome Analysis Using Stable Isotope Patterns to Identify Metabolic Changes.

      Dudek, Christian-Alexander; Schlicker, Lisa; Hiller, Karsten; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
      Gas chromatography coupled with mass spectrometry can provide an extensive overview of the metabolic state of a biological system. Analysis of raw mass spectrometry data requires powerful data processing software to generate interpretable results. Here we describe a data processing workflow to generate metabolite levels, mass isotopomer distribution, similarity and variability analysis of metabolites in a nontargeted manner, using stable isotope labeling. Using our data analysis software, no bioinformatic or programming background is needed to generate results from raw mass spectrometry data.
    • Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila.

      Rasch, Janine; Theuerkorn, Martin; Ünal, Can; Heinsohn, Natascha; Tran, Stefan; Fischer, Gunter; Weiwad, Matthias; Steinert, Michael; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015)
      Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung-epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30-40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L. pneumophila.
    • OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes.

      Wang, Xu; Mulas, Floriana; Yi, Wenjing; Brunn, Anna; Nishanth, Gopala; Just, Sissy; Waisman, Ari; Brück, Wolfgang; Deckert, Martina; Schlüter, Dirk; et al. (EMBO Press, 2019-04-03)
      Astrocytes are critical regulators of neuroinflammation in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Growing evidence indicates that ubiquitination of signaling molecules is an important cell-intrinsic mechanism governing astrocyte function during MS and EAE Here, we identified an upregulation of the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) in astrocytes during MS and EAE Mice with astrocyte-specific OTUB1 ablation developed more severe EAE due to increased leukocyte accumulation, proinflammatory gene transcription, and demyelination in the spinal cord as compared to control mice. OTUB1-deficient astrocytes were hyperactivated in response to IFN-γ, a fingerprint cytokine of encephalitogenic T cells, and produced more proinflammatory cytokines and chemokines than control astrocytes. Mechanistically, OTUB1 inhibited IFN-γ-induced Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling by K48 deubiquitination and stabilization of the JAK2 inhibitor suppressor of cytokine signaling 1 (SOCS1). Thus, astrocyte-specific OTUB1 is a critical inhibitor of neuroinflammation in CNS autoimmunity.
    • Pathogen-induced ubiquitin-editing enzyme A20 bifunctionally shuts off NF-κB and caspase-8-dependent apoptotic cell death.

      Lim, Michelle C C; Maubach, Gunter; Sokolova, Olga; Feige, Michael H; Diezko, Rolf; Buchbinder, Jörn; Backert, Steffen; Schlüter, Dirk; Lavrik, Inna N; Naumann, Michael; et al. (2017-06-02)
      The human pathogen Helicobacter pylori infects more than half of the world's population and is a paradigm for persistent yet asymptomatic infection but increases the risk for chronic gastritis and gastric adenocarcinoma. For successful colonization, H. pylori needs to subvert the host cell death response, which serves to confine pathogen infection by killing infected cells and preventing malignant transformation. Infection of gastric epithelial cells by H. pylori provokes direct and fast activation of the proinflammatory and survival factor NF-κB, which regulates target genes, such as CXCL8, BIRC3 and TNFAIP3. However, it is not known how H. pylori exploits NF-κB activation and suppresses the inflammatory response and host apoptotic cell death, in order to avert the innate immune response and avoid cell loss, and thereby enhance colonization to establish long-term infection. Here we assign for the first time that H. pylori and also Campylobacter jejuni-induced ubiquitin-editing enzyme A20 bifunctionally terminates NF-κB activity and negatively regulates apoptotic cell death. Mechanistically, we show that the deubiquitinylase activity of A20 counteracts cullin3-mediated K63-linked ubiquitinylation of procaspase-8, therefore restricting the activity of caspase-8. Interestingly, another inducible NF-κB target gene, the scaffold protein p62, ameliorates the interaction of A20 with procaspase-8. In conclusion, pathogen-induced de novo synthesis of A20 regulates the shut-off of the survival factor NF-κB but, on the other hand, also impedes caspase-8-dependent apoptotic cell death so as to promote the persistence of pathogens.Cell Death and Differentiation advance online publication, 2 June 2017; doi:10.1038/cdd.2017.89.
    • The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions.

      Xiong, Qiuhong; Ünal, Can; Matthias, Jan; Steinert, Michael; Eichinger, Ludwig; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015-04)
      Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16(-) and ATG9(-)/16(-) cells and compare them to the previously reported ATG9(-) mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9(-) and ATG16(-) cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9(-)/16(-) double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9(-)/16(-) cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9(-)/16(-) cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.
    • PilY1 Promotes Legionella pneumophila Infection of Human Lung Tissue Explants and Contributes to Bacterial Adhesion, Host Cell Invasion, and Twitching Motility.

      Hoppe, Julia; Ünal, Can M; Thiem, Stefanie; Grimpe, Louisa; Goldmann, Torsten; Gaßler, Nikolaus; Richter, Matthias; Shevchuk, Olga; Steinert, Michael; Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
      Legionnaires' disease is an acute fibrinopurulent pneumonia. During infection Legionella pneumophila adheres to the alveolar lining and replicates intracellularly within recruited macrophages. Here we provide a sequence and domain composition analysis of the L. pneumophila PilY1 protein, which has a high homology to PilY1 of Pseudomonas aeruginosa. PilY1 proteins of both pathogens contain a von Willebrand factor A (vWFa) and a C-terminal PilY domain. Using cellular fractionation, we assigned the L. pneumophila PilY1 as an outer membrane protein that is only expressed during the transmissive stationary growth phase. PilY1 contributes to infection of human lung tissue explants (HLTEs). A detailed analysis using THP-1 macrophages and A549 lung epithelial cells revealed that this contribution is due to multiple effects depending on host cell type. Deletion of PilY1 resulted in a lower replication rate in THP-1 macrophages but not in A549 cells. Further on, adhesion to THP-1 macrophages and A549 epithelial cells was decreased. Additionally, the invasion into non-phagocytic A549 epithelial cells was drastically reduced when PilY1 was absent. Complementation variants of a PilY1-negative mutant revealed that the C-terminal PilY domain is essential for restoring the wild type phenotype in adhesion, while the putatively mechanosensitive vWFa domain facilitates invasion into non-phagocytic cells. Since PilY1 also promotes twitching motility of L. pneumophila, we discuss the putative contribution of this newly described virulence factor for bacterial dissemination within infected lung tissue.
    • Plasma Metabolome Signature Indicative of Germline Status Independent of Cancer Incidence.

      Penkert, Judith; Märtens, Andre; Seifert, Martin; Auber, Bernd; Derlin, Katja; Hille-Betz, Ursula; Hörmann, Philipp; Klopp, Norman; Prokein, Jana; Schlicker, Lisa; et al. (Frontiers, 2021-04-07)
      Individuals carrying a pathogenic germline variant in the breast cancer predisposition gene BRCA1 (gBRCA1+) are prone to developing breast cancer. Apart from its well-known role in DNA repair, BRCA1 has been shown to powerfully impact cellular metabolism. While, in general, metabolic reprogramming was named a hallmark of cancer, disrupted metabolism has also been suggested to drive cancer cell evolution and malignant transformation by critically altering microenvironmental tissue integrity. Systemic metabolic effects induced by germline variants in cancer predisposition genes have been demonstrated before. Whether or not systemic metabolic alterations exist in gBRCA1+ individuals independent of cancer incidence has not been investigated yet. We therefore profiled the plasma metabolome of 72 gBRCA1+ women and 72 age-matched female controls, none of whom (carriers and non-carriers) had a prior cancer diagnosis and all of whom were cancer-free during the follow-up period. We detected one single metabolite, pyruvate, and two metabolite ratios involving pyruvate, lactate, and a metabolite of yet unknown structure, significantly altered between the two cohorts. A machine learning signature of metabolite ratios was able to correctly distinguish between gBRCA1+ and controls in ~82%. The results of this study point to innate systemic metabolic differences in gBRCA1+ women independent of cancer incidence and raise the question as to whether or not constitutional alterations in energy metabolism may be involved in the etiology of BRCA1-associated breast cancer.
    • Properties of dimeric, disulfide-linked rhBMP-2 recovered from E. coli derived inclusion bodies by mild extraction or chaotropic solubilization and subsequent refolding

      Quaas, Bastian; Burmeister, Laura; Li, Zhaopeng; NIMTZ, MANFRED; Hoffmann, Andrea; Rinas, Ursula; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
    • Purification of the human fibroblast growth factor 2 using novel animal-component free materials

      Bolten, Svenja Nicolin; Knoll, Anne-Sophie; Li, Zhaopeng; Gellermann, Pia; Pepelanova, Iliyana; Rinas, Ursula; Scheper, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier BV, 2020-08)
      This paper analyzes the use of animal-component free chromatographic materials for the efficient purifi- cation of the human fibroblast growth factor 2 (hFGF-2). hFGF-2 is produced in Escherichia coli and pu- rified via three different chromatography steps, which include a strong cation exchange chromatography as a capture step, followed by heparin affinity chromatography and an anion exchange chromatography as a polishing step. The affinity chromatography step is based on the animal-derived material heparin. Chemically produced ligands provide a viable alternative to animal-derived components in production processes, since they are characterized by a defined structure which leads to reproducible results and a broad range of applications. The alternative ligands can be assigned to adsorber of the mixed-mode chromatography (MMC) and pseudo-affinity chromatography. Eight different animal-component free materials used as adsorbers in MMC or pseudo-affinity chromatog- raphy were tested as a substitute for heparin. The MMCs were cation exchangers characterized with fur- ther functional residues. The ligands of the pseudo-affinity chromatography were heparin-like ligands which are based on heparin’s molecular structure. The alternative methods were tested as a capture step and in combination with another chromatographic step in the purification procedure of hFGF-2. In each downstream step purity, recovery and yield were analysed and compared to the conventional downstream process. Two types of MMC –the column Foresight TM Nuvia TM cPrime TM from Bio-Rad Laboratories and the col- umn HiTrap TM Capto TM MMC from GE Healthcare Life Sciences - can be regarded as effective animal- component free alternatives to the heparin - based adsorber.
    • Quantitation of large, middle and small hepatitis B surface proteins in HBeAg-positive patients treated with peginterferon alfa-2a.

      Rinker, Franziska; Bremer, Corinna M; Schröder, Kathrin; Wiegand, Steffen B; Bremer, Birgit; Manns, Michael P; Kraft, Anke R; Wedemeyer, Heiner; Yang, Lei; Pavlovic, Vedran; et al. (Wiley, 2019-11-13)
      BACKGROUND & AIMS: Hepatitis B virus (HBV) contains three viral surface proteins, large, middle and small hepatitis B surface protein (LHBs, MHBs, SHBs). Proportions of LHBs and MHBs are lower in patients with inactive versus active chronic infection. Interferon alfa may convert HBeAg-positive chronic hepatitis B (CHB) to an inactive carrier state, but prediction of sustained response is unsatisfactory. The aim of this study was to test the hypothesis that quantification of MHBs and LHBs may allow for a better prognosis of therapeutic response than total hepatitis B surface antigen (HBsAg) concentration. METHODS: HBs proteins were measured before and during peginterferon alfa-2a therapy in serum from 127 Asian patients with HBeAg-positive CHB. Sustained response was defined as hepatitis B e antigen (HBeAg) seroconversion 24 weeks post-treatment. RESULTS: Mean total HBs levels were significantly lower in responders versus nonresponders at all time points (P<.05) and decreased steadily during the initial 24 weeks' treatment (by 1.16 versus 0.86 ng/mL in responders/nonresponders, respectively) with unchanged relative proportions. Genotype B had a twofold higher proportion of LHBs than genotype C (13% versus 6%). HBV DNA, HBeAg, HBsAg, and HBs protein levels predicted response equally well but not optimally (area under the ROC curve values >0.70). CONCLUSIONS: HBs proteins levels differ by HBV genotype. However, quantification of HBs proteins has no advantage over the already established HBsAg assays to predict response to peginterferon alfa-2a therapy in HBeAg-positive patients.