• Künstliche Intelligenz zur diagnostischen Unterstützung ausgewählter seltener lysosomaler Speichererkrankungen: Ergebnisse einer Pilotstudie.

      Sieg, Anna-Lena; Anibh, Martin; Muschol, Nicole Maria; Köhn, Anja; Lampe, Christina; Kortum, Xiauwei; Mehmecke, Sandra; Blöß, Susanne; Lechner, Werner; Klawonn, Frank; et al. (Thieme, 2019-02-10)
      Hintergrund: Die Diagnosestellung einer seltenen Stoffwechselerkrankung stellt eine Herausforderung für Familien und betreuende Ärzte dar. Um den Weg zur Diagnose zu unterstützen, wurde ein diagnostisches Werkzeug entwickelt, welches die Erfahrungen Betroffener nutzt.
    • LC/MS Based Monitoring of Endogenous Decay Markers for Quality Assessment of Serum Specimens

      Thumfart, Jörg Oliver; Abidi, Nada; Mindt, Sonani; Costani, Victor; Hofheinz, Ralf; Klawonn, Frank; Findeisen, Peter; 1Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany (2015-05-04)
      Preanalytical variations have major impact on most biological assays. Specifically MS-based multiparametric proteomics analyses of blood specimens are seriously affected by limited protein stability due to high intrinsic proteolytic activity of serum and plasma. However, the direct analysis of sample quality (DASQ) for serum specimens is not readily available. Here we propose the mass spectrometry based monitoring of peptide patterns that are ex vivo changing in a time dependent manner to alleviate these constrains.
    • Letter to the editor by Winter et al.: Reply

      Hoffmann, Georg E.; Klawonn, Frank; Orth, Matthias; Lichtinghagen, Ralf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (De Gruyter, 2018-04-03)
    • Lipopolysaccharide binding protein, interleukin-10, interleukin-6 and C-reactive protein blood levels in acute ischemic stroke patients with post-stroke infection.

      Worthmann, Hans; Tryc, Anita B; Dirks, Meike; Schuppner, Ramona; Brand, Korbinian; Klawonn, Frank; Lichtinghagen, Ralf; Weissenborn, Karin; Department of Neurology, Hannover Medical School, Carl-Neuberg-Str. 1, 30623, Hannover, Germany. (2015)
      Ischemic stroke patients are prone to infection by stroke-induced immunodepression. We hypothesized that levels of lipopolysaccharide binding protein (LBP), interleukin-10 (IL-10), IL-6 and C-reactive protein (CRP) are early predictors for the development of stroke-associated infection.
    • Mass-spectrometric profiling of cerebrospinal fluid reveals metabolite biomarkers for CNS involvement in varicella zoster virus reactivation.

      Kuhn, Maike; Sühs, Kurt-Wolfram; Akmatov, Manas K; Klawonn, Frank; Wang, Junxi; Skripuletz, Thomas; Kaever, Volkhard; Stangel, Martin; Pessler, Frank; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (2018-01-17)
      Varicella zoster virus (VZV) reactivation spans the spectrum from uncomplicated segmental herpes zoster to life-threatening disseminated CNS infection. Moreover, in the absence of a small animal model for this human pathogen, studies of pathogenesis at the organismal level depend on analysis of human biosamples. Changes in cerebrospinal fluid (CSF) metabolites may reflect critical aspects of host responses and end-organ damage in neuroinfection and neuroinflammation. We therefore applied a targeted metabolomics screen of CSF to three clinically distinct forms of VZV reactivation and infectious and non-infectious disease controls in order to identify biomarkers for CNS involvement in VZV reactivation.
    • Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF.

      Nicke, Tristan; Schnitzer, Tobias; Münch, Karin; Adamczack, Julia; Haufschildt, Kristin; Buchmeier, Sabine; Kucklick, Martin; Felgenträger, Undine; Jänsch, Lothar; Riedel, Katharina; et al. (2013)
      The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS-NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV-visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS.
    • Methicillin-resistant Staphylococcus pseudintermedius synthesizes deoxyadenosine to cause persistent infection.

      Bünsow, Dorothea; Tantawy, Eshraq; Ostermeier, Tjorven; Bähre, Heike; Garbe, Annette; Larsen, Jesper; Winstel, Volker; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor & Francis, 2021-03-29)
      Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an emerging zoonotic pathogen of canine origin that causes an array of fatal diseases, including bacteremia and endocarditis. Despite large-scale genome sequencing projects have gained substantial insights into the genomic landscape of MRSP, current knowledge on virulence determinants that contribute to S. pseudintermedius pathogenesis during human or canine infection is very limited. Using a panel of genetically engineered MRSP variants and a mouse abscess model, we here identified the major secreted nuclease of S. pseudintermedius designated NucB and adenosine synthase A (AdsA) as two synergistically acting enzymes required for MRSP pathogenesis. Similar to Staphylococcus aureus, S. pseudintermedius requires nuclease secretion along with the activity of AdsA to degrade mammalian DNA for subsequent biosynthesis of cytotoxic deoxyadenosine. In this manner, S. pseudintermedius selectively kills macrophages during abscess formation thereby antagonizing crucial host immune cell responses. Ultimately, bioinformatics analyses revealed that NucB and AdsA are widespread in the global S. pseudintermedius population. Together, these data suggest that S. pseudintermedius deploys the canonical Nuc/AdsA pathway to persist during invasive disease and may aid in the development of new therapeutic strategies to combat infections caused by MRSP.
    • Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.

      Wiesselmann, Milan; Hebecker, Stefanie; Borrero-de Acuña, José M; NIMTZ, MANFRED; Bollivar, David; Jänsch, Lothar; Moser, Jürgen; Jahn, Dieter; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Portland Press, 2020-11-19)
      During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.
    • Modulation of TAP-dependent antigen compartmentalization during human monocyte-to-DC differentiation.

      Döring, Marius; Blees, Hanna; Koller, Nicole; Tischer-Zimmermann, Sabine; Müsken, Mathias; Henrich, Frederik; Becker, Jennifer; Grabski, Elena; Wang, Junxi; Janssen, Hans; et al. (American Society of Hematology, 2019-03-26)
      Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)–dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.
    • The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

      Hartmann, Tobias; Schrapers, Peer; Utesch, Tillmann; Nimtz, Manfred; Rippers, Yvonne; Dau, Holger; Mroginski, Maria Andrea; Haumann, Michael; Leimkühler, Silke; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-04-26)
      Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.
    • The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes.

      Thedieck, Kathrin; Hain, Torsten; Mohamed, Walid; Tindall, Brian J; Nimtz, Manfred; Chakraborty, Trinad; Wehland, Jürgen; Jänsch, Lothar; Helmholtz Centre for Infection Research, Division of Cell and Immune Biology, Cellular Proteomics Group, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2006-12)
      Pathogenic bacteria have to cope with defence mechanisms mediated by adaptive and innate immunity of the host cells. Cationic antimicrobial peptides (CAMPs) represent one of the most effective components of the host innate immune response. Here we establish the function of Lmo1695, a member of the VirR-dependent virulence regulon, recently identified in Listeria monocytogenes. Lmo1695 encodes a membrane protein of 98 kDa with strong homology to the multiple peptide resistance factor (MprF) of Staphylococcus aureus. Like staphylococcal MprF, we found that Lmo1695 is involved in the synthesis of the membrane phospholipid lysylphosphatidylglycerol (L-PG). In addition, Lmo1695 is also essential for lysinylation of diphosphatidylglycerol (DPG), another phospholipid widely distributed in bacterial membranes. A Deltalmo1695 mutant lacking the lysinylated phospholipids was particularly susceptible to CAMPs of human and bacterial origin. The mutant strain infected both epithelial cells and macrophages only poorly and was attenuated for virulence when tested in a mouse model of infection. Lmo1695 is a member of a growing list of survival factors which enable growth of L. monocytogenes in different environments.
    • Neurobeachin and the Kinesin KIF21B Are Critical for Endocytic Recycling of NMDA Receptors and Regulate Social Behavior.

      Gromova, Kira V; Muhia, Mary; Rothammer, Nicola; Gee, Christine E; Thies, Edda; Schaefer, Irina; Kress, Sabrina; Kilimann, Manfred W; Shevchuk, Olga; Oertner, Thomas G; et al. (Elsevier, 2018-05-29)
      Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology.
    • New Insights into the Respiratory Chain of Plant Mitochondria. Supercomplexes and a Unique Composition of Complex II1

      Eubel, Holger; Jänsch, Lothar; Braun, Hans-Peter (The American Society for Plant Biologists, 2003-09)
    • The nuclear export inhibitor aminoratjadone is a potent effector in extracellular-targeted drug conjugates.

      Klahn, Philipp; Fetz, Verena; Ritter, Antje; Collisi, Wera; Hinkelmann, Bettina; Arnold, Tatjana; Tegge, Werner; Rox, Katharina; Hüttel, Stephan; Mohr, Kathrin I; et al. (Royal Society of Chemistry, 2019-05-28)
      The concept of targeted drug conjugates has been successfully translated to clinical practice in oncology. Whereas the majority of cytotoxic effectors in drug conjugates are directed against either DNA or tubulin, our study aimed to validate nuclear export inhibition as a novel effector principle in drug conjugates. For this purpose, a semisynthetic route starting from the natural product ratjadone A, a potent nuclear export inhibitor, has been developed. The biological evaluation of ratjadones functionalized at the 16-position revealed that oxo- and amino-analogues had very high potencies against cancer cell lines (e.g. 16R-aminoratjadone 16 with IC50 = 260 pM against MCF-7 cells, or 19-oxoratjadone 14 with IC50 = 100 pM against A-549 cells). Mechanistically, the conjugates retained a nuclear export inhibitory activity through binding CRM1. To demonstrate a proof-of-principle for cellular targeting, folate- and luteinizing hormone releasing hormone (LHRH)-based carrier molecules were synthesized and coupled to aminoratjadones as well as fluorescein for cellular efficacy and imaging studies, respectively. The Trojan-Horse conjugates selectively addressed receptor-positive cell lines and were highly potent inhibitors of their proliferation. For example, the folate conjugate FA-7-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 34.3 nM, and the LHRH conjugate d-Orn-Gose-Val-Cit-pABA-16R-aminoratjadone had an IC50 of 12.8 nM. The results demonstrate that nuclear export inhibition is a promising mode-of-action for extracellular-targeted drug conjugate payloads.
    • Pain drawings as a diagnostic tool for the differentiation between two pain-associated rare diseases (Ehlers-Danlos-Syndrome, Guillain-Barré-Syndrome).

      Wester, Larissa; Mücke, Martin; Bender, Tim Theodor Albert; Sellin, Julia; Klawonn, Frank; Conrad, Rupert; Szczypien, Natasza; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2020-11-17)
      Background: The diagnosis of rare diseases poses a particular challenge to clinicians. This study analyzes whether patients' pain drawings (PDs) help in the differentiation of two pain-associated rare diseases, Ehlers-Danlos Syndrome (EDS) and Guillain-Barré Syndrome (GBS). Method: The study was designed as a prospective, observational, single-center study. The sample comprised 60 patients with EDS (3 male, 52 female, 5 without gender information; 39.2 ± 11.4 years) and 32 patients with GBS (10 male, 20 female, 2 without gender information; 50.5 ± 13.7 years). Patients marked areas afflicted by pain on a sketch of a human body with anterior, posterior, and lateral views. PDs were electronically scanned and processed. Each PD was classified based on the Ružička similarity to the EDS and the GBS averaged image (pain profile) in a leave-one-out cross validation approach. A receiver operating characteristic (ROC) curve was plotted. Results: 60-80% of EDS patients marked the vertebral column with the neck and the tailbone and the knee joints as pain areas, 40-50% the shoulder-region, the elbows and the thumb saddle joint. 60-70% of GBS patients marked the dorsal and plantar side of the feet as pain areas, 40-50% the palmar side of the fingertips, the dorsal side of the left palm and the tailbone. 86% of the EDS patients and 96% of the GBS patients were correctly identified by computing the Ružička similarity. The ROC curve yielded an excellent area under the curve value of 0.95.
    • Patient's Experience in Pediatric Primary Immunodeficiency Disorders: Computerized Classification of Questionnaires.

      Mücke, Urs; Klemann, Christian; Baumann, Ulrich; Meyer-Bahlburg, Almut; Kortum, Xiaowei; Klawonn, Frank; Lechner, Werner M; Grigull, Lorenz; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Primary immunodeficiency disorders (PIDs) are a heterogeneous group of more than 200 rare diseases. Timely diagnosis is of uttermost importance. Therefore, we aimed to develop a diagnostic questionnaire with computerized pattern-recognition in order to support physicians to identify suspicious patient histories.
    • Pattern discovery in time series using autoencoder in comparison to nonlearning approaches

      Noering, Fabian Kai Dietrich; Schroeder, Yannik; Jonas, Konstantin; Klawonn, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (IOS Press, 2021-01-01)
      In technical systems the analysis of similar situations is a promising technique to gain information about the system's state, its health or wearing. Very often, situations cannot be defined but need to be discovered as recurrent patterns within time series data of the system under consideration. This paper addresses the assessment of different approaches to discover frequent variable-length patterns in time series. Because of the success of artificial neural networks (NN) in various research fields, a special issue of this work is the applicability of NNs to the problem of pattern discovery in time series. Therefore we applied and adapted a Convolutional Autoencoder and compared it to classical nonlearning approaches based on Dynamic Time Warping, based on time series discretization as well as based on the Matrix Profile. These nonlearning approaches have also been adapted, to fulfill our requirements like the discovery of potentially time scaled patterns from noisy time series. We showed the performance (quality, computing time, effort of parametrization) of those approaches in an extensive test with synthetic data sets. Additionally the transferability to other data sets is tested by using real life vehicle data. We demonstrated the ability of Convolutional Autoencoders to discover patterns in an unsupervised way. Furthermore the tests showed, that the Autoencoder is able to discover patterns with a similar quality like classical nonlearning approaches. © 2021 - IOS Press. All rights reserved.
    • PD-1 Blockade Aggravates Epstein-Barr Virus Post-Transplant Lymphoproliferative Disorder in Humanized Mice Resulting in Central Nervous System Involvement and CD4 T Cell Dysregulations.

      Volk, Valery; Theobald, Sebastian J; Danisch, Simon; Khailaie, Sahamoddin; Kalbarczyk, Maja; Schneider, Andreas; Bialek-Waldmann, Julia; Krönke, Nicole; Deng, Yun; Eiz-Vesper, Britta; et al. (Frontiers, 2021-01-12)
      Post-transplant lymphoproliferative disorder (PTLD) is one of the most common malignancies after solid organ or allogeneic stem cell transplantation. Most PTLD cases are B cell neoplasias carrying Epstein-Barr virus (EBV). A therapeutic approach is reduction of immunosuppression to allow T cells to develop and combat EBV. If this is not effective, approaches include immunotherapies such as monoclonal antibodies targeting CD20 and adoptive T cells. Immune checkpoint inhibition (ICI) to treat EBV+ PTLD was not established clinically due to the risks of organ rejection and graft-versus-host disease. Previously, blockade of the programmed death receptor (PD)-1 by a monoclonal antibody (mAb) during ex vivo infection of mononuclear cells with the EBV/M81+ strain showed lower xenografted lymphoma development in mice. Subsequently, fully humanized mice infected with the EBV/B95-8 strain and treated in vivo with a PD-1 blocking mAb showed aggravation of PTLD and lymphoma development. Here, we evaluated vis-a-vis in fully humanized mice after EBV/B95-8 or EBV/M81 infections the effects of a clinically used PD-1 blocker. Fifteen to 17 weeks after human CD34+ stem cell transplantation, Nod.Rag.Gamma mice were infected with two types of EBV laboratory strains expressing firefly luciferase. Dynamic optical imaging analyses showed systemic EBV infections and this triggered vigorous human CD8+ T cell expansion. Pembrolizumab administered from 2 to 5 weeks post-infections significantly aggravated EBV systemic spread and, for the M81 model, significantly increased the mortality of mice. ICI promoted Ki67+CD30+CD20+EBER+PD-L1+ PTLD with central nervous system (CNS) involvement, mirroring EBV+ CNS PTLD in humans. PD-1 blockade was associated with lower frequencies of circulating T cells in blood and with a profound collapse of CD4+ T cells in lymphatic tissues. Mice treated with pembrolizumab showed an escalation of exhausted T cells expressing TIM-3, and LAG-3 in tissues, higher levels of several human cytokines in plasma and high densities of FoxP3+ regulatory CD4+ and CD8+ T cells in the tumor microenvironment. We conclude that PD-1 blockade during acute EBV infections driving strong CD8+ T cell priming decompensates T cell development towards immunosuppression. Given the variety of preclinical models available, our models conferred a cautionary note indicating that PD-1 blockade aggravated the progression of EBV+ PTLD.
    • Peptidases released by necrotic cells control CD8+ T cell cross-priming.

      Gamrekelashvili, Jaba; Kapanadze, Tamar; Han, Miaojun; Wissing, Josef; Ma, Chi; Jaensch, Lothar; Manns, Michael P; Armstrong, Todd; Jaffee, Elizabeth; White, Ayla O; et al. (2013-10-08)
      Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.
    • Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes

      Vanz, Ana L; Lünsdorf, Heinrich; Adnan, Ahmad; Nimtz, Manfred; Gurramkonda, Chandrasekhar; Khanna, Navin; Rinas, Ursula (2012-08-08)
      Abstract Background Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. Results The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation. Conclusions In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.