• DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4).

      Quentmeier, Hilmar; Eberth, Sonja; Romani, Julia; Weich, Herbert A; Zaborski, Margarete; Drexler, Hans G; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2012-01-17)
      Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation.
    • The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach.

      Poblete-Castro, Ignacio; Escapa, Isabel F; Jäger, Christian; Puchalka, Jacek; Lam, Carolyn Ming Chi; Schomburg, Dietmar; Prieto, María Auxiliadora; Martins dos Santos, Vítor A P; Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany. ignacio.pobletecastro@helmholtz-hzi.de (2012)
      Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.
    • Characterisation of bovine leukocyte Ig-like receptors.

      Hogan, Louise; Bhuju, Sabin; Jones, Des C; Laing, Ken; Trowsdale, John; Butcher, Philip; Singh, Mahavir; Vordermeier, Martin; Allen, Rachel L; Centre for Infection, Division of Clinical Sciences, St George's, University of London, Cranmer Terrace, London, United Kingdom. p0904768@sgul.ac.uk (2012)
      Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.
    • YY1-Binding Sites Provide Central Switch Functions in the PARP-1 Gene Expression Network.

      Doetsch, Martina; Gluch, Angela; Poznanović, Goran; Bode, Juergen; Vidaković, Melita; Helmholtz Centre for Infection Research/Epigenetic Regulation, Braunschweig, Germany. (2012)
      Evidence is presented for the involvement of the interplay between transcription factor Yin Yang 1 (YY1) and poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of mouse PARP-1 gene (muPARP-1) promoter activity. We identified potential YY1 binding motifs (BM) at seven positions in the muPARP-1 core-promoter (-574/+200). Binding of YY1 was observed by the electrophoretic supershift assay using anti-YY1 antibody and linearized or supercoiled forms of plasmids bearing the core promoter, as well as with 30 bp oligonucleotide probes containing the individual YY1 binding motifs and four muPARP-1 promoter fragments. We detected YY1 binding to BM1 (-587/-558), BM4 (-348/-319) and a very prominent association with BM7 (+86/+115). Inspection of BM7 reveals overlap of the muPARP-1 translation start site with the Kozak sequence and YY1 and PARP-1 recognition sites. Site-directed mutagenesis of the YY1 and PARP-1 core motifs eliminated protein binding and showed that YY1 mediates PARP-1 binding next to the Kozak sequence. Transfection experiments with a reporter gene under the control of the muPARP-1 promoter revealed that YY1 binding to BM1 and BM4 independently repressed the promoter. Mutations at these sites prevented YY1 binding, allowing for increased reporter gene activity. In PARP-1 knockout cells subjected to PARP-1 overexpression, effects similar to YY1 became apparent; over expression of YY1 and PARP-1 revealed their synergistic action. Together with our previous findings these results expand the PARP-1 autoregulatory loop principle by YY1 actions, implying rigid limitation of muPARP-1 expression. The joint actions of PARP-1 and YY1 emerge as important contributions to cell homeostasis.
    • The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis.

      Cudmore, Melissa J; Hewett, Peter W; Ahmad, Shakil; Wang, Ke-Qing; Cai, Meng; Al-Ani, Bahjat; Fujisawa, Takeshi; Ma, Bin; Sissaoui, Samir; Ramma, Wenda; et al. (2012)
      VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.
    • Multi-layered stochasticity and paracrine signal propagation shape the type-I interferon response.

      Rand, Ulfert; Rinas, Melanie; Schwerk, Johannes; Nöhren, Gesa; Linnes, Melanie; Kröger, Andrea; Flossdorf, Michael; Kály-Kullai, Kristóf; Hauser, Hansjörg; Höfer, Thomas; et al. (2012)
      The cellular recognition of viruses evokes the secretion of type-I interferons (IFNs) that induce an antiviral protective state. By live-cell imaging, we show that key steps of virus-induced signal transduction, IFN-β expression, and induction of IFN-stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular-and not viral-origin, and temporal unpredictability of IFN-β expression is largely due to cell-intrinsic noise generated both upstream and downstream of the activation of nuclear factor-κB and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all-or-nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi-layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.
    • Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci.

      Nehlsen, Kristina; da Gama-Norton, Leonor; Schucht, Roland; Hauser, Hansjörg; Wirth, Dagmar; Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany. dagmar.wirth@helmholtz-hzi.de. (2011-11-22)
    • Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci

      Nehlsen, Kristina; da Gama-Norton, Leonor; Schucht, Roland; Hauser, Hansjörg; Wirth, Dagmar (2011-11-22)
    • 22nd European Society for Animal Cell Technology (ESACT) Meeting on Cell Based Technologies Vienna, Austria. 15-18 May 2011. Abstracts.

      Hauser, Hansjörg; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2011-11-22)
    • An extended bioreaction database that significantly improves reconstruction and analysis of genome-scale metabolic networks.

      Stelzer, Michael; Sun, Jibin; Kamphans, Tom; Fekete, Sándor P; Zeng, An-Ping (2011-11)
      The bioreaction database established by Ma and Zeng (Bioinformatics, 2003, 19, 270-277) for in silico reconstruction of genome-scale metabolic networks has been widely used. Based on more recent information in the reference databases KEGG LIGAND and Brenda, we upgrade the bioreaction database in this work by almost doubling the number of reactions from 3565 to 6851. Over 70% of the reactions have been manually updated/revised in terms of reversibility, reactant pairs, currency metabolites and error correction. For the first time, 41 spontaneous sugar mutarotation reactions are introduced into the biochemical database. The upgrade significantly improves the reconstruction of genome scale metabolic networks. Many gaps or missing biochemical links can be recovered, as exemplified with three model organisms Homo sapiens, Aspergillus niger, and Escherichia coli. The topological parameters of the constructed networks were also largely affected, however, the overall network structure remains scale-free. Furthermore, we consider the problem of computing biologically feasible shortest paths in reconstructed metabolic networks. We show that these paths are hard to compute and present solutions to find such paths in networks of small and medium size.
    • Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach.

      Koutinas, Michalis; Kiparissides, Alexandros; Silva-Rocha, Rafael; Lam, Ming-Chi; Martins Dos Santos, Vitor A P; de Lorenzo, Victor; Pistikopoulos, Efstratios N; Mantalaris, Athanasios; Centre for Process Systems Engineering, Department of Chemical Engineering, South Kensington Campus, Imperial College London, UK. (2011-07)
      The majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses.
    • A limited LCA of bio-adipic acid: manufacturing the nylon-6,6 precursor adipic acid using the benzoic acid degradation pathway from different feedstocks.

      van Duuren, J B J H; Brehmer, B; Mars, A E; Eggink, G; Dos Santos, V A P Martins; Sanders, J P M; Wageningen UR Food & Biobased Research, Wageningen, The Netherlands. joost.vanduuren@helmholtz-hzi.de (2011-06)
      A limited life cycle assessment (LCA) was performed on a combined biological and chemical process for the production of adipic acid, which was compared to the traditional petrochemical process. The LCA comprises the biological conversion of the aromatic feedstocks benzoic acid, impure aromatics, toluene, or phenol from lignin to cis, cis-muconic acid, which is subsequently converted to adipic acid through hydrogenation. Apart from the impact of usage of petrochemical and biomass-based feedstocks, the environmental impact of the final concentration of cis, cis-muconic acid in the fermentation broth was studied using 1.85% and 4.26% cis, cis-muconic acid. The LCA focused on the cumulative energy demand (CED), cumulative exergy demand (CExD), and the CO(2) equivalent (CO(2) eq) emission, with CO(2) and N(2) O measured separately. The highest calculated reduction potential of CED and CExD were achieved using phenol, which reduced the CED by 29% and 57% with 1.85% and 4.26% cis, cis-muconic acid, respectively. A decrease in the CO(2) eq emission was especially achieved when the N(2) O emission in the combined biological and chemical process was restricted. At 4.26% cis, cis-muconic acid, the different carbon backbone feedstocks contributed to an optimized reduction of CO(2) eq emissions ranging from 14.0 to 17.4 ton CO(2) eq/ton adipic acid. The bulk of the bioprocessing energy intensity is attributed to the hydrogenation reactor, which has a high environmental impact and a direct relationship with the product concentration in the broth.
    • High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

      Hemmen, Katherina; Reinl, Tobias; Buttler, Kerstin; Behler, Friederike; Dieken, Hauke; Jänsch, Lothar; Wilting, Jörg; Weich, Herbert A; Department of Gene Regulation, HZI, Build. D, Inhoffenstr. 7, 38124, Braunschweig, Germany. katharina.hemmen@ewetel.net (2011-05)
      Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.
    • Novel nonviral bioassays for mouse type I and type III interferon.

      Kugel, Daniela; Pulverer, Julia Elisabeth; Köster, Mario; Hauser, Hansjörg; Staeheli, Peter; Department of Virology, University of Freiburg, Freiburg, Germany. (2011-04)
      We used embryo fibroblasts from Mx2-Luc transgenic mice that express Firefly luciferase under control of the interferon (IFN)-regulated mouse Mx2 promoter to develop simple nonviral bioassays for type I and type III IFN. Since type III IFN is acid-labile, Mx2-Luc fibroblasts detected the presence of type I IFN in acid-treated biological samples with high sensitivity and selectivity. For selective detection of type III IFN, we employed embryo fibroblasts from Mx2-Luc mutant mice that lack functional receptors for type I IFN. The sensitivity of this latter assay remained comparatively low, presumably because type III IFN receptors are not abundantly present on fibroblasts. The main advantages of our novel IFN assays are that they are easy to perform, yield fast results, and can be used in laboratories that are not licensed for work with infectious agents. Further, the type I IFN assay has superior sensitivity than commercially available enzyme-linked immunosorbent assay systems.
    • Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration.

      Schucht, Roland; Lydford, Simon; Andzinski, Lisa; Zauers, Jeannette; Cooper, James; Hauser, Hansjörg; Wirth, Dagmar; May, Tobias; Department of Gene Regulation and Differentiation, HZI-Helmholtz Centre for Infection Research, Braunschweig, Germany. roland.schucht@inscreenex.com (2011-03)
      The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.
    • Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience.

      Charbord, Pierre; Livne, Erella; Gross, Gerhard; Häupl, Thomas; Neves, Nuno M; Marie, Pierre; Bianco, Paolo; Jorgensen, Christian (2011-03)
      Genostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side") has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.
    • Integrated strategy for the production of therapeutic retroviral vectors.

      Carrondo, Manuel; Panet, Amos; Wirth, Dagmar; Coroadinha, Ana Sofia; Cruz, Pedro; Falk, Haya; Schucht, Roland; Dupont, Francis; Geny-Fiamma, Cécile; Merten, Otto-Wilhelm; et al. (2011-03)
      The broad application of retroviral vectors for gene delivery is still hampered by the difficulty to reproducibly establish high vector producer cell lines generating sufficient amounts of highly concentrated virus vector preparations of high quality. To enhance the process for producing clinically relevant retroviral vector preparations for therapeutic applications, we have integrated novel and state-of-the-art technologies in a process that allows rapid access to high-efficiency vector-producing cells and consistent production, purification, and storage of retroviral vectors. The process has been designed for various types of retroviral vectors for clinical application and to support a high-throughput process. New modular helper cell lines that permit rapid insertion of DNA encoding the therapeutic vector of interest at predetermined, optimal chromosomal loci were developed to facilitate stable and high vector production levels. Packaging cell lines, cultivation methods, and improved medium composition were coupled with vector purification and storage process strategies that yield maximal vector infectivity and stability. To facilitate GMP-grade vector production, standard of operation protocols were established. These processes were validated by production of retroviral vector lots that drive the expression of type VII collagen (Col7) for the treatment of a skin genetic disease, dystrophic epidermolysis bullosa. The potential efficacy of the Col7-expressing vectors was finally proven with newly developed systems, in particular in target primary keratinocyte cultures and three-dimensional skin tissues in organ culture.
    • Reconciliation of genome-scale metabolic reconstructions for comparative systems analysis.

      Oberhardt, Matthew A; Puchałka, Jacek; Martins dos Santos, Vítor A P; Papin, Jason A; Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States of America. (2011-03)
      In the past decade, over 50 genome-scale metabolic reconstructions have been built for a variety of single- and multi- cellular organisms. These reconstructions have enabled a host of computational methods to be leveraged for systems-analysis of metabolism, leading to greater understanding of observed phenotypes. These methods have been sparsely applied to comparisons between multiple organisms, however, due mainly to the existence of differences between reconstructions that are inherited from the respective reconstruction processes of the organisms to be compared. To circumvent this obstacle, we developed a novel process, termed metabolic network reconciliation, whereby non-biological differences are removed from genome-scale reconstructions while keeping the reconstructions as true as possible to the underlying biological data on which they are based. This process was applied to two organisms of great importance to disease and biotechnological applications, Pseudomonas aeruginosa and Pseudomonas putida, respectively. The result is a pair of revised genome-scale reconstructions for these organisms that can be analyzed at a systems level with confidence that differences are indicative of true biological differences (to the degree that is currently known), rather than artifacts of the reconstruction process. The reconstructions were re-validated with various experimental data after reconciliation. With the reconciled and validated reconstructions, we performed a genome-wide comparison of metabolic flexibility between P. aeruginosa and P. putida that generated significant new insight into the underlying biology of these important organisms. Through this work, we provide a novel methodology for reconciling models, present new genome-scale reconstructions of P. aeruginosa and P. putida that can be directly compared at a network level, and perform a network-wide comparison of the two species. These reconstructions provide fresh insights into the metabolic similarities and differences between these important Pseudomonads, and pave the way towards full comparative analysis of genome-scale metabolic reconstructions of multiple species.
    • Osseointegration by bone morphogenetic protein-2 and transforming growth factor beta2 coated titanium implants in femora of New Zealand white rabbits.

      Thorey, Fritz; Menzel, Henning; Lorenz, Corinna; Gross, Gerhard; Hoffmann, Andrea; Windhagen, Henning; Department of Orthopaedic Surgery, Hannover Medical School, Hannover, Germany. (2011-01)
      Intramembranous bone formation is essential in uncemented joint replacement to provide a mechanical anchorage of the implant. Since the discovery of bone morphogenic proteins (BMPs) by Urist in 1965, many studies have been conducted to show the influence of growth factors on implant ingrowth. In this study, the influence of bone morphogenetic protein-2 (rhBMP-2) and transforming growth factor β2 (TGF-β2) on implant osseointegration was investigated.
    • Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states.

      Sester, Urban; Fousse, Mathias; Dirks, Jan; Mack, Ulrich; Prasse, Antje; Singh, Mahavir; Lalvani, Ajit; Sester, Martina; Department of Internal Medicine IV, Saarland University, Homburg, Germany. (2011)
      T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p = 0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.