published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

Heparan sulfate (HS) is a cell-surface sulfated polysaccharide that binds to multiple proteins and has been implicated in cancer, viral infection and Alzheimer's disease. Due to the heterogeneity of HS, the structural requirements for protein binding are ill- defined. Chemical synthesis of structurally-defined HS oligosaccharides, which are tunable in terms of length, order of monosaccharides and sulfation pattern, is required for the investigation of HS-protein binding. Single molecule methods have been utilised in biophysics to study dynamic processes and can allow observation of rare events which would be 'averaged out' in ensemble measurements. Access to fluorescently labelled HS oligosaccharides should allow investigation of interactions with proteins at the single molecule level using methods such as single molecule FRET, providing a method complementary to NMR studies (ensemble) and X-ray crystallography (non-dynamic).This thesis presents the development of a method for the fluorescent labelling of a chemically synthesised HS disaccharide utilising a reducing-end amine tag. Analysis of the fluorescence properties of the labelled disaccharide at ensemble and single molecule level indicated no perturbation of the fluorophore when attached to the sugar. Fluorescence correlation spectroscopy measurements of the fluorescent HS disaccharide with the protein FGF-1 showed no binding, which is attributed to the low concentration (1 nM) of disaccharide required in the experiment. Additional work is presented in this thesis on the development of a method for atom-specific 13C labelling of HS oligosaccharides, which has been initiated by synthesis of a 13C labelled L-iduronate monosaccharide and a 13C labelled disaccharide. New strategies for the synthesis of HS oligosaccharides based on orthogonal thioglycoside-based glycosylations employing S-benzoxazolyl and S-thiazolyl donors have been investigated. Development of a chemoselective glycosylation strategy for HS oligosaccharide synthesis utilising a 'super-disarmed' [2.2.2] L-iduronic lactone is presented.

No description available.

Abstract Heparan Sulfate, A New Target for Platinum in Metastatic TNBC Author: Samantha J. Katner, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Virginia Commonwealth University, 2018 Advisor: Dr. Nicholas Farrell, Professor, Chemistry Department Heparan sulfate proteoglycans (HSPGs), composed of the linear polysaccharide heparan sulfate (HS) conjugated to a protein core, are located on the cell surface and extracellular matrix. The HS chains display varying degrees of sulfation, which constitutes the molecular recognition motif for many HS-protein interactions. HSPGs, associated growth factors, and heparanase promote tumor progression by facilitating invasion, angiogenesis, and metastasis.1 Sulfate clusters on the glycan backbone also mediate the interaction of polynuclear platinum complexes (PPCs) with HSPG through a “sulfate clamp.” Such PPC-HS interactions can be conceptualized as “polyarginine” mimics. Strong HS-PPC binding protects the oligosaccharide against sulfate loss through metalloshielding.2 The biological consequences of metalloshielding will in principle affect HS interactions with relevant enzymes and proteins such as heparanase and growth factors, similar in concept to the inhibition of DNA-protein binding through modification of DNA structure and conformation. The end-point of functional modulation of HS interactions is inhibition of angiogenesis and metastasis. PPCs are dual-function agents through their interactions with both nucleic acids and HS. The novel Pt-HS interactions open up new areas of metalloglycomics and potential anti-angiogenic activity. Here, we report PPC interactions with HS-like models: Fondaparinux (FPX)3 and heparin4. We demonstrate TriplatinNC high affinity to heparin in biophysical studies and compare HS interactions with DNA and HS using competition assays.3,4 these approaches may be extended to a range of metal-ammine compounds.4 The biological consequences of PPC-HS interactions include modulation of heparanase cleavage of FPX,3 growth factor binding to HS, and growth factor-induced migration and signaling in breast cancer and endothelial cells, as potential anti-metastatic and anti-angiogenic effects in vivo. We report proof-of-principle of strong in vivo anti-metastatic activity of PPCs in triple negative breast cancer (TNBC) models.5–7 Already, PPC-HS interactions have major biological consequences in the aggressive metastatic TNBC mouse models. Impressively, PPCs reduce overall tumor metastases with emphasis in lung, bone, and liver locations in both immunocompetent and immunosuppressive mouse models. PPCs demonstrated permeability through the blood brain barrier (BBB) implying further applications for PPCs. PPCs represent a novel class of intrinsically dual-function agents combining platinum cytotoxicity through DNA targeting with anti-angiogenic effects through glycan targeting. Together, these results suggest that strong PPC-HS interactions have a significant role in the inhibition of breast cancer metastases, particularly in metastatic TNBC patients. 1. Peterson, E. J. et al. Antiangiogenic platinum through glycan targeting. Chem. Sci. 8, 241–252 (2017). 2. Mangrum, J. B. et al. A new approach to glycan targeting: enzyme inhibition by oligosaccharide metalloshielding. Chem. Commun. (Camb). 50, 4056–8 (2014). 3. Gorle, A. K. et al. Substitution-Inert Polynuclear Platinum Complexes as Metalloshielding Agents for Heparan Sulfate. Chem. Eur. J (2018). doi:10.1002/chem.201706030 4. Katner, S. J., Johnson, W. E., Peterson, E. J., Page, P. & Farrell, N. P. Comparison of Metal–Ammine Compounds Binding to DNA and Heparin. Glycans as Ligands in Bioinorganic Chemistry. Inorg. Chem. acs.inorgchem.7b03043 (2018). doi:10.1021/acs.inorgchem.7b03043 5. Katsuta, E., Peterson, E. J., Katner, S. J., Farrell, N. P. & Takabe, K. Triplatin preferably suppress lung metastasis of breast cancer, and peritoneal carcinomatosis of colon and pancreatic cancer. Proc. AACR Washingt. D.C. Abstract #5117 (2017). 6. Katner, S. J. et al. Heparan sulfate , a new target for platinum in metastatic TNBC. Proc. AACR Chicago, Abstract #3941 (2018). 7. Katner, S. J. et al. Anti-metastatic platinum through glycan targeting in breast cancer. Proc. AACR Washingt. D.C. Abstract #17 (2017). 8. Silva, H. et al. Heparan sulfate proteoglycan-mediated entry pathway for charged tri-platinum compounds. Differential cellular accumulation mechanisms for platinum. Mol. Pharmacol. 9, 1795–1802 (2012). 9. Peterson, E. J. et al. Nucleolar targeting by platinum: P53-independent apoptosis follows rRNA inhibition, cell-cycle arrest, and DNA compaction. Mol. Pharm. 12, 287–297 (2015).

Proteoglycans containing Heparan Sulfate (HS), a sulfated glycosaminoglycan (GAG), play a major role in the cell signaling process, interacting with many different proteins. HS is over expressed on the surface of many cancer cells. Enzymatic cleavage of HS-GAGs by heparanase causes release of angiogenic growth factors leading to tumor cell migration. Heparanase is also over-expressed in tumors with significant correlation between metastatic potential and heparanase activity. Proteoglycans and their associated enzymes are thus significant drug targets of high biological relevance. A functional consequence of strong PPC-HS binding has been shown in proof-of-concept studies confirming inhibition of the model pentasaccharide, Fondaparinux, by bacterial Heparinase. Such metalloshielding by PPCs may also protect HS from enzymatic cleavage by the mammalian heparanase; preventing growth factors from binding to HS and/or preventing release of bound growth factors and thus inhibiting the metastatic response in the cancer cells. HS-GAGs are also receptors for cellular accumulation of cationic Polynuclear Platinum Complexes (PPCs) through high-affinity binding to the highly anionic HS. PPCs competitively inhibit uptake of TAMRA-R9, a fluorescent nona-arginine derivative, in CHO cells. The previously reported series of Pt(II) complexes were investigated as DNA binders, initiating the apoptotic cascade. The result of PPC-DNA binding produces long range inter and intra-strand cross-links, that produce structural and conformational changes. Hydrogen bonding between phosphate oxygens and square planar Pt(II) nitrogen results in bidentate complexes by either backbone tracking or groove spanning of DNA. This complex forms a clamp like structure, called a phosphate clamp, similar to that of the arginine fork. Understanding this clamp allows us to investigate the structurally similar sulfate binding between metal complexes and target HSPG. HSPGs may allow significant research into both a novel cellular internalization of principal metals and “metalloshielding” of heparin by these compounds. Previous studies have shown that a wide range of metal ions have high affinity to heparin. The trend of metal/heparin affinity is believed to be dependent on parameters consisting of the metal’s overall size, spatial orientation of the ligands attached to each metal, the net charge and oxidation state of these metals, and number of binding sites. Studies have shown relative affinities of sulfate and carboxylate groups for the metal ions. These metal cations play an important role in the affinity, specificity, and stability of many protein/heparin interactions. The study of simple coordination compounds, like Pt, Mn, V, Ru and Co, will allow preliminary results which will extend into the PPCs mode of binding. This thesis focuses on the concept of metalloglycomics and reviews the interactions of various metal complexes with heparin. The covalent and non-covalent interactions of metal complexes with heparin resulting in strong bonding are explained through spectroscopy and calorimetry. The cleavage inhibition of heparanase by metal complexes is also described. Sulfate cluster anchoring shields the sulfates from loss as seen in mass spectrometry. The study of metalloglycomics offers potential understanding into the relevance of metal-heparin interactions and possibilities into the development of new compounds as therapeutic agents.

Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS. Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS.

Repeated cannulation of children during the course of treatment is distressing for the child, their family and to their nurses. Some paediatric units endeavour to minimise recannulation by employing strategies to reduce complications such as phlebitis and thrombosis formation. One strategy is to infuse low dose heparin and hydrocortisone (HEPHC). However, its effectiveness in prolonging cannula survival is inconclusive. There is also concern about the potential risks of administering these preparations to children. A randomised, controlled, blinded trial was conducted that examined the effectiveness of continuous infusion of low dose HEPHC in a group of children requiring long term intravenous antibiotics in a general paediatric unit. Comparisons of cannula complications and cannulae survival times were made in children receiving either continuous infusions of clear fluids or low dose HEPHC. The results demonstrated that there was no statistically significant difference (Logrank statistic=l.l, p=0.3) in cannula survival times between the two groups. It was also found that the bacterial and fungal colonisation of cannula for these children was extremely low. Based on these findings it is recommended that routine administration of low dose HEPHC to extend cannula survival time be discontinued. The findings also support current practice of removing cannula in children only when a complication occurs on completion of treatment.

<p>In order to develop blood biocompatible materials a heparin surface and a phosphorylcholine (PC) functionalized polymer surface were characterized using photoelectron spectroscopy (PES). The formation of the heparin surface was studied by quartz crystal microbalance with dissipation monitoring (QCM-D). This heparin surface consists of heparin conjugates deposited on a conditioning layer, applied once or twice. The PC functionalized polymer, poly(trimethylene carbonate), was linked to a silicon substrate through 3-amino- propyltrimethoxysilane (APTMS), also studied using PES. </p><p>Synchrotron radiation based PES showed that the thicker heparin film resulted in complete coverage of the substrate, while the thinner did not. This could explain the difference in blood biocompatibility between the two films, as observed by others. It was also found that the heparin chains bend down towards the substrate (under vacuum). </p><p>For the thinner heparin film the modifications, resulting from extensive irradiation of the sample, were studied with synchrotron radiation based PES. This was done at a pressure of about 10^-7 mbar and in 0.5 mbar water vapor. It was found that the modification is slower under water vapor than at low pressures and that the damaged film incorporates water upon exposure.</p><p>The heparin coating was found to be stable and wear resistant enough to still be present on artificial heart valves after three weeks testing in circulating plasma. It then had about the same antithrombin uptake as a non-tested surface. The film was, however, partly destroyed by the durability test and plasma proteins were deposited. </p><p>The PC functionalized, APTMS linked polymer was found to be much shorter than could be expected from random reactions. One plausible explanation is an interaction between the PC group and the silane surface, favoring aminolysis close to the PC group. This is consistent with our finding that the PC group bends down towards the surface.</p>

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Globalement, un tiers de la population mondiale est infectée par Mycobacterium tuberculosis, l'agent infectieux de la tuberculose (TB). Fort heureusement, seuls 5 à 10 % des individus infectés développent un jour une TB active. Les individus non malades restent cependant infectés à vie, on parle d'infection latente. Chaque année, 8-10 millions nouveaux cas de tuberculose active sont recensés et M. tuberculosis est responsable de 1,5 à 2 millions de décès. Depuis plus d'une décennie, M. tuberculosis s'est étroitement associé à l'infection par le virus de l'immunodéficience humaine. Cette alliance néfaste représente une importante menace pour les pays en voie de développement, car ces 2 pathogènes déciment les forces vives de ces populations. Il faut malheureusement rajouter à ce triste tableau une fréquence grandissante de souches multi-résistantes, voire extensivement multi-résistantes. Face à ces souches, les avancées thérapeutiques du siècle dernier sont pratiquement réduites à néant. Considérant ces données, il est désormais crucial d'améliorer nos outils de dépistage de l'infection latente, de diagnostic de la maladie active, de prévention (vaccins) et de traitement. Pour atteindre ces objectifs, une des pistes est la caractérisation détaillée des réponses immunitaires. En comparant les réponses immunitaires des sujets infectés de manière latente à celles liées à la maladie active, nous pourrons peut-être comprendre certains mécanismes de protection. L'étude des réponses immunitaires induites par la « Heparin-Binding-Hemagglutinin » (HBHA) s'est faite dans cet objectif. La HBHA est une adhésine exprimée par le complexe M. tuberculosis. Elle est impliquée dans la dissémination extrapulmonaire du bacille et constitue donc un facteur de virulence. Par ailleurs, une vaccination de souris par seulement 3 doses de 5 µg de HBHA suffit à protéger de l'infection avec une efficacité comparable à celle du vaccin BCG. Chez l'homme, les sujets sains mais infectés développent d'importantes sécrétions d'interféron-gamma (IFN-γ) en réponse à cet antigène, alors que la majorité des patients tuberculeux ne le font pas. Cette différence est importante pour comprendre une des raisons d'échappement de M. tuberculosis au contrôle immunitaire. La HBHA est une protéine méthylée et la méthylation s’avère essentielle pour ses propriétés immunoprotectrices. Nos travaux présentés ici se sont axés sur deux éléments de la réponse immunitaire à la HBHA chez l'homme : d'une part, l'exploitation de la réponse périphérique d'IFN-γ à la HBHA comme outil de dépistage de l'infection latente et, d'autre part, l'étude des raisons de la faible sécrétion d'IFN-γ spécifique de la HBHA lors de la maladie active. L'évaluation de la sécrétion périphérique d'IFN-γ en réponse à la HBHA a permis de démontrer rétrospectivement que celle-ci permet de détecter plus de 90 % des sujets réagissant positivement à l'injection intradermique de tuberculine. De manière intéressante, l'utilisation d'un test commercial, le QuantiFERON TB Gold IT (QFT-IT) n'a permis de détecter que la moitié des sujets infectés sains. De notre point de vue, le QFT-IT ne peut être recommandé seul pour le dépistage systématique de l'infection latente par M. tuberculosis. De manière parallèle, un test de stimulation basé uniquement sur la sécrétion d’IFN-γ suite à une stimulation à l'ESAT-6, composant du QFT-IT, n'a pas permis d'augmenter la sensibilité, ni d'ajouter une plus-value au test basé sur la HBHA. A l'instar de l'intradermoréaction à la tuberculine, le dépistage de la maladie active reste décevant que ce soit par l'utilisation de la HBHA ou de l'ESAT-6. La TB active est caractérisée par une basse sécrétion périphérique d'IFN-γ en réponse à la stimulation par la HBHA. Cette faible sécrétion est cependant réversible, puisque un traitement efficace permet d'atteindre des taux d'IFN-γ significativement plus élevés. Ceci nous démontre qu'il s'agit d'une suppression associée à la phase active de l'infection. Nous avons d'abord évalué l'importance de la modulation de la sécrétion d'IFN-γ en réponse à la HBHA par 2 cytokines immunomodulatrices, l'interleukine-10 (IL-10) et le Transforming-Growth-Factor-Beta (TGF-ß). De manière intéressante, alors que ces 2 cytokines sont associées à l'infection par M. tuberculosis, la HBHA n'est inductrice ni d'IL-10, ni de TGF-ß. Les lymphocytes T régulateurs (Treg) expriment 2 marqueurs d'intérêt : le CD25, composant du récepteur à l'IL-2, et Foxp3, un gène régulateur majeur des cellules Treg. Ces cellules sont décrites comme suppressives de réponses immunitaires déclenchées par des antigènes du Soi et du non-Soi. Nous avons montré que la proportion de lymphocytes Treg périphériques est augmentée en cas de TB active. Par ailleurs, nous avons également démontré que ces cellules suppriment la sécrétion d'IFN-γ et la prolifération induite par la HBHA après stimulation des cellules mononucléées sanguines périphériques de patients tuberculeux in vitro. Cependant, la réponse anti-HBHA des patients tuberculeux, qui est démasquée par la déplétion des lymphocytes Treg, n'est pas dirigée contre des épitopes protecteurs. En effet, la méthylation n'influence pas leur sécrétion d'IFN-γ. De ce point de vue, les lymphocytes Treg sont impliqués dans la maladie tuberculeuse et influencent négativement les réponses dirigées contre un antigène protecteur. Cependant, il semble que la TB active soit également associée à une ignorance d'épitopes protecteurs. Enfin, nous avons également démontré qu'il était possible d'induire des lymphocytes Treg au départ de cellules sanguines périphériques de sujets infectés sains. En effet, la stimulation in vitro des cellules sanguines périphériques en présence de BCG et de TGF-ß est un moyen rapide pour induire l'apparition de lymphocytes Treg fonctionnels in vitro. Ceci nous interroge quant aux rôles des lymphocytes Treg dans la pathogenèse de la maladie. En effet, un excès de TGF-ß circulant est observé dans certaines conditions cliniques à haut-risque de TB post-primaire. De ce point de vue, les lymphocytes Treg pourraient être des acteurs déterminant dans la perte du contrôle à long terme de l'infection et, par là, pourraient être des cibles thérapeutiques d'intérêts lors de l'infection par M. tuberculosis. /Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). It is estimated approximately one third of the World’s population is infected with M. tuberculosis. Fortunately, only 5 to 10 % of the infected individuals will develop the disease throughout their life. However, the other healthy infected individuals remain infected for life: this is the latent TB infection (LTBI). Every year, 8 to 10 million new cases of TB are recorded globally, and about 2 to 3 million of people die from the disease. During the last several decades the co-infection of M. tuberculosis and the human immunodeficiency virus have worsened the picture. This dreadful association currently affects mostly the poorest people of the World. Unfortunately, bad news never stands alone. We now witness increasing emergence of multi-drug-resistant and even of extensively-multi-drug-resistant M. tuberculosis strains. Against these strains current therapeutics are virtually useless. The development of new tools for prevention (vaccines), diagnostics and treatment is crucial. In order to fulfill these objectives, detailed studies on the immune responses is one of the main tracks to explore. Indeed, the comparison of immune responses in LTBI subjects with those in TB patients may provide some clues to understand immune mechanisms of protection. Studies of the immune responses that are specific to Heparin-Binding-Hemagglutinin (HBHA) may be one of these clues. HBHA is an adhesin, which is expressed by the micro-organisms of the M. tuberculosis complex. It largely contributes to the extrapulmonary dissemination of the tubercle bacilli. Hence, HBHA may be qualified as an important virulence factor. Furthermore, vaccination of mice with three doses of only 5 µg HBHA each affords the same level of protection as vaccination with BCG. In humans, peripheral blood mononuclear cells (PBMC) from LTBI subjects secrete significant levels of IFN-γ in response to HBHA, whereas PBMC from TB patients do not. This discrepancy may be a cornerstone in the understanding of some of the mechanisms underlying the immune escape mediated by M. tuberculosis. HBHA is a methylated protein, and the methylation is crucial for its immuno-protective properties. This work focused on 2 major issues of the HBHA-specific immune response in humans: the use of the peripheral IFN-γ secretion in response to HBHA as a diagnostic tool for LTBI and the analysis of the underlying mechanisms to the low IFN-γ secretion during active TB. In our study, the measurement of HBHA-specific IFN-γ secretion resulted in the detection of more than 90 % of the tuberculin-skin-test (TST) positive LTBI. Strikingly, the QuantiFERON TB Gold IT (QFT-IT), a commercial test, failed to identify those LTBI subjects in more than 50 % of the cases. Therefore, we cannot recommend the use of QFT-IT alone instead of the TST for the detection of LTBI. Similarly, a test relying on the detection of IFN-γ secretion upon ESAT-6 stimulation, one of the antigens used in the QFT-IT, was not sufficiently sensitive for the LTBI detection, nor did it improve the sensitivity or the specificity of the HBHA-based test. In contrast to the diagnosis of LTBI, the tests based on HBHA- or ESAT-6-induced IFN-γ secretions displayed poor sensitivity for the diagnosis of active TB. During active TB, the HBHA-specific IFN-γ secretion in the periphery is low. However, this weak secretion is reversible upon effective treatment, as the IFN-γ response to HBHA is increased after completion of chemotherapy. This is strongly suggestive of an immune suppression during active disease. Therefore, we have first evaluated the role of two immunomodulatory cytokines, interleukin-10 (IL-10) and Transforming-Growth-Factor-Beta (TGF-ß), in the suppression of the HBHA-specific IFN-γ secretion. We found that neutralization of neither IL-10 nor TGF-ß with specific antibodies induced HBHA-specific IFN-γ secretion by PBMC of TB patients in vitro. In contrast, depletion of regulatory T cells (Treg) that express 2 major markers, CD25, a constituent of the IL-2 receptor, and Foxp3, a master regulatory gene, resulted in increased HBHA-specific IFN-γ secretion by the PBMC of TB patients. These cells are known to be involved in the suppression of immune responses to both Self and non-Self antigens. We further show that the size of the peripheral Treg cell population increases during active disease. In addition to suppressing the HBHA-specific IFN-γ secretion these cells suppress T cell proliferation in response to HBHA in vitro. However, even after depletion of the Treg cells, the uncovered HBHA-specific immune responses are not directed to the methylated epitopes during TB disease. Finally, we show that Treg cells can be induced (or expanded) from the PBMC of LTBI subjects. Stimulation of those PBMC with BCG in the presence of TGF-ß resulted in a quick appearance of functional Treg cells in vitro. This observation strongly suggests a role of Treg cells in the pathogenesis of TB, in particular in the progression of latency to reactivation. Interestingly, excessive concentration of TGF-ß, associated with various clinical conditions, is high risk factor for post-primary TB. Thus, Treg cells may result in the loss of immune control against latent M. tuberculosis infection. Therefore, Treg cells may represent potential therapeutic targets during M. tuberculosis infection.