Claudio Counoupas

Bio: Claudio Counoupas is an academic researcher from University of Sydney. The author has contributed to research in topics: Vaccination & Immune system. The author has an hindex of 10, co-authored 27 publications receiving 466 citations. Previous affiliations of Claudio Counoupas include Centenary Institute & University of Pisa.

Direct Binding of Human NK Cell Natural Cytotoxicity Receptor NKp44 to the Surfaces of Mycobacteria and Other Bacteria

Abstract: Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guerin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56bright. In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium, to the mycobacterium-related species Nocardia farcinica, and to Pseudomonas aeruginosa. Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

Interaction of Mycobacterium tuberculosis Cell Wall Components with the Human Natural Killer Cell Receptors NKp44 and Toll-Like Receptor 2

Abstract: We have previously demonstrated that a soluble form of the human NK cell natural cytotoxicity receptor NKp44, binds to the surface of Mycobacterium tuberculosis (MTB). Herein, we investigated the interaction of MTB cell wall components (CWC) with NKp44 or with Toll-like receptor 2 (TLR2) and the role of NKp44 and TLR2 in the direct activation of NK cells upon stimulation with MTB CWC. By using several purified bacterial CWC in an ELISA, we demonstrated that NKp44 was able to bind to the MTB cell wall core mycolyl-arabinogalactan-peptidoglycan (mAGP) as well as to mycolic acids (MA) and arabinogalactan (AG), while soluble TLR2 bound to MTB peptidoglycan (PG), but not to MA or AG. The mAGP complex induced NK cell expression of CD25, CD69, NKp44 and IFN-γ production at levels comparable to M. bovis Bacillus Calmette-Guerin-stimulated (BCG) cells. While AG and MA used alone failed to induce NK cell activation, mycobacterial PG-exhibited NK cell stimulatory capacity. Activation of resting NK cells by mAGP and IFN-γ production were inhibited by anti-TLR2 MAb, but not by anti-NKp44 MAb. Differently, anti-NKp44 MAb partially inhibited CD69 expression on NK cells pre-activated with IL-2 and then stimulated with mAGP or whole BCG. Overall, these results provide evidence that components abundant in mycobacterial cell wall are able to interact with NKp44 (AG, MA) and TLR-2 (PG), respectively. While interaction of TLR2 with mycobacterial cell wall promotes activation of resting NK cells and IFN-γ production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.

Delta inulin-based adjuvants promote the generation of polyfunctional CD4+ T cell responses and protection against Mycobacterium tuberculosis infection.

Abstract: There is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis. Advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. In this report we determined if Advax displays broad applicability against important human pathogens by assessing protective immunity against infection with M. tuberculosis. The fusion protein CysVac2, comprising the M. tuberculosis antigens Ag85B (Rv1886c) and CysD (Rv1285) formulated with Advax provided significant protection in the lungs of M. tuberculosis-infected mice. Protection was associated with the generation of CysVac2-specific multifunctional CD4+ T cells (IFN-γ+TNF+IL-2+). Addition to Advax of the TLR9 agonist, CpG oligonucleotide (AdvaxCpG), improved both the immunogenicity and protective efficacy of CysVac2. Immunisation with CysVac2/AdvaxCpG resulted in heightened release of the chemoattractants, CXCL1, CCL3, and TNF, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of CysVac2-specific CD4+ T cells. As delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, CysVac2/AdvaxCpG is a strong candidate for further preclinical evaluation for progression to human trials.

The ESX-5 Associated eccB5-eccC5 Locus Is Essential for Mycobacterium tuberculosis Viability

Abstract: The recently described ESX-5 secretion system of Mycobacterium tuberculosis is one of the most important modulators of host-pathogen interactions due to its crucial impact on PPE protein secretion, cell wall stability and virulence. Although various components of the ESX-5 secretion machinery have been defined, other ESX-5 core components still remain to be characterized. In this study, we focused on EccB5 and EccC5, a transmembrane protein (EccB5) and a membrane-bound ATPase (EccC5), both predicted to be building blocks of the M. tuberculosis ESX-5 membrane-associated complex. In vitro expression studies demonstrated that EccB5 and EccC5 encoding genes constitute an operon. The expression of this operon is essential for M. tuberculosis, since the deletion of the eccB5-eccC5 genomic segment at the ESX-5 locus is possible only after the integration of a second functional copy of eccB5-eccC5 genes into the M. tuberculosis chromosome. The characterization of two M. tuberculosis conditional mutant strains (MtbPptreccB5 and MtbPptreccC5), in which the eccB5-eccC5 operon or the eccC5 gene, respectively, were expressed under the control of an anhydrotetracycline-repressible promoter, confirmed that the repression of eccB5-eccC5 genes is detrimental for growth of M. tuberculosis both in vitro and in THP-1 human macrophage cell line. Moreover, analysis of the secretome of MtbPptreccB5-eccC5 and MtbPptreccC5 strains revealed that both EccB5 and EccC5 are required for secretion of ESX-5 specific substrates, thus confirming that they are indeed components of the ESX-5 secretion machinery. Taken together these findings demonstrate the importance of an intact and functional ESX-5 system for viability of M. tuberculosis, thus opening new interesting options for alternative antimycobacterial control strategies.

Bioactive Glass and Glass-Ceramic Scaffolds for Bone Tissue Engineering

Abstract: Traditionally, bioactive glasses have been used to fill and restore bone defects. More recently, this category of biomaterials has become an emerging research field for bone tissue engineering applications. Here, we review and discuss current knowledge on porous bone tissue engineering scaffolds on the basis of melt-derived bioactive silicate glass compositions and relevant composite structures. Starting with an excerpt on the history of bioactive glasses, as well as on fundamental requirements for bone tissue engineering scaffolds, a detailed overview on recent developments of bioactive glass and glass-ceramic scaffolds will be given, including a summary of common fabrication methods and a discussion on the microstructural-mechanical properties of scaffolds in relation to human bone (structure-property and structure-function relationship). In addition, ion release effects of bioactive glasses concerning osteogenic and angiogenic responses are addressed. Finally, areas of future research are highlighted in this review.

Current development of biodegradable polymeric materials for biomedical applications

Abstract: In the last half-century, the development of biodegradable polymeric materials for biomedical applications has advanced significantly. Biodegradable polymeric materials are favored in the development of therapeutic devices, including temporary implants and three-dimensional scaffolds for tissue engineering. Further advancements have occurred in the utilization of biodegradable polymeric materials for pharmacological applications such as delivery vehicles for controlled/sustained drug release. These applications require particular physicochemical, biological, and degradation properties of the materials to deliver effective therapy. As a result, a wide range of natural or synthetic polymers able to undergo hydrolytic or enzymatic degradation is being studied for biomedical applications. This review outlines the current development of biodegradable natural and synthetic polymeric materials for various biomedical applications, including tissue engineering, temporary implants, wound healing, and drug delivery.

Immunology of Tuberculosis

Abstract: MTB ranks as the first worldwide pathogen latently infecting one third of the population and the second leading cause of death from a single infectious agent, after the human immunodeficiency virus (HIV). The development of vigorous and apparently appropriate immune response upon infection with M.tuberculosis in humans and experimental animals conflict with failure to eradicate the pathogen itself and with its ability to undergo clinical latency from which it may exit. From a clinical standpoint, our views on MTB infection may take advantage from updating the overall perspective, that has quite changed over the last decade, following remarkable advances in our understanding of the manipulation of the immune system by M.tuberculosis and of the role of innate components of the immune response, including macrophages, neutrophils, dendritic cells and NK cells in the initial spread of MTB and in its exit from latency. Scope of this review is to highlight the the major mechanisms of MTB escape from immune control and to provide a supplementary translational perspective for the interpretation of innate immune mechanisms with particular impact on clinical aspects.

Overview of natural hydrogels for regenerative medicine applications

Abstract: Hydrogels from different materials can be used in biomedical field as an innovative approach in regenerative medicine. Depending on the origin source, hydrogels can be synthetized through chemical and physical methods. Hydrogel can be characterized through several physical parameters, such as size, elastic modulus, swelling and degradation rate. Lately, research is focused on hydrogels derived from biologic materials. These hydrogels can be derived from protein polymers, such as collage, elastin, and polysaccharide polymers like glycosaminoglycans or alginate among others. Introduction of decellularized tissues into hydrogels synthesis displays several advantages compared to natural or synthetic based hydrogels. Preservation of natural molecules such as growth factors, glycans, bioactive cryptic peptides and natural proteins can promote cell growth, function, differentiation, angiogenesis, anti-angiogenesis, antimicrobial effects, and chemotactic effects. Versatility of hydrogels make possible multiple applications and combinations with several molecules on order to obtain the adequate characteristic for each scope. In this context, a lot of molecules such as cross link agents, drugs, grow factors or cells can be used. This review focuses on the recent progress of hydrogels synthesis and applications in order to classify the most recent and relevant matters in biomedical field.