Mauricio C. De Marzi

Superantigen natural affinity maturation revealed by the crystal structure of staphylococcal enterotoxin G and its binding to T‐cell receptor Vβ8.2

The illnesses associated with bacterial superantigens (SAgs) such as food poisoning and toxic shock syndrome, as well as the emerging threat of purpura fulminans and community‐associated methicillin‐resistant S. aureus producer of SAgs, emphasize the importance of a better characterization of SAg binding to their natural ligands, which would allow the development of drugs or biological reagents able to neutralize their action. SAgs are toxins that bind major histocompatibility complex class II molecules (MHC‐II) and T‐cell receptors (TCR), in a nonconventional manner, inducing T‐cell activation that leads to production of cytokines such as tumor necrosis factor and interleukin‐2, which may result in acute toxic shock. Previously, we cloned and expressed a new natural variant of staphylococcal enterotoxin G (SEG) and evaluated its ability to stimulate in vivo murine T‐cell subpopulations. We found an early, strong, and widespread stimulation of mouse Vβ8.2 T‐cells when compared with other SAgs member of the SEB subfamily. In search for the reason of the strong mitogenic potency, we determined the SEG crystal structure by X‐ray crystallography to 2.2 Å resolution and analyzed SEG binding to mVβ8.2 and MHC‐II. Calorimetry and SPR analysis showed that SEG has an affinity for mVβ8.2 40 to 100‐fold higher than that reported for other members of SEB subfamily, and the highest reported for a wild type SAg‐TCR couple. We also found that mutations introduced in mVβ8.2 to produce a high affinity mutant for other members of the SEB subfamily do not greatly affect binding to SEG. Crystallographic analysis and docking into mVβ8.2 in complex with SEB, SEC3, and SPEA showed that the deletions and substitution of key amino acids remodeled the putative surface of the mVβ8.2 binding site without affecting the binding to MHC‐II. This results in a SAg with improved binding to its natural ligands, which may confer a possible evolutionary advantage for bacterial strains expressing SEG. Proteins 2007.

Production of monoclonal antibodies from hybridoma cells immobilized in 3D sol–gel silica matrices

The immobilization of mammalian cells in suitable matrices that can retain their viability and capability to produce certain metabolites has gained attention in recent years. In this work, hybridoma cells were immobilized in sol–gel silica matrices for in vitro production of monoclonal antibodies. For that purpose, different matrices were evaluated in terms of cell viability, antibody diffusion to surrounding media and physicochemical properties of the polymeric material. Tetrakis (2-ethoxyethyl) orthosilicate (THEOS) matrices were found to be the best option for hybridoma immobilization. The concentrations of the silica precursor as well as the number of immobilized cells were also optimized. Three hundred mM of THEOS precursor and 5 × 105 hybridoma cells appear to be the most suitable alternative. Hybridoma cells immobilized in THEOS matrices were able to produce monoclonal antibodies to the same extent as free cells, thus introducing the possibility of using them in the design of bioreactors for large-scale production.

Peptidoglycan recognition protein-peptidoglycan complexes increase monocyte/macrophage activation and enhance the inflammatory response.

Peptidoglycan recognition proteins (PGRP) are pattern recognition receptors that can bind or hydrolyse peptidoglycan (PGN). Four human PGRP have been described: PGRP‐S, PGRP‐L, PGRP‐Iα and PGRP‐Iβ. Mammalian PGRP‐S has been implicated in intracellular destruction of bacteria by polymorphonuclear cells, PGRP‐Iα and PGRP‐Iβ have been found in keratinocytes and epithelial cells, and PGRP‐L is a serum protein that hydrolyses PGN. We have expressed recombinant human PGRP and observed that PGRP‐S and PGRP‐Iα exist as monomer and disulphide dimer proteins. The PGRP dimers maintain their biological functions. We detected the PGRP‐S dimer in human serum and polymorphonuclear cells, from where it is secreted after degranulation; these cells being a possible source of serum PGRP‐S. Recombinant PGRP do not act as bactericidal or bacteriostatic agents in the assayed conditions; however, PGRP‐S and PGRP‐Iα cause slight damage in the bacterial membrane. Monocytes/macrophages increase Staphylococcus aureus phagocytosis in the presence of PGRP‐S, PGRP‐Iα and PGRP‐Iβ. All PGRP bind to monocyte/macrophage membranes and are endocytosed by them. In addition, all PGRP protect cells from PGN‐induced apoptosis. PGRP increase THP‐1 cell proliferation and enhance activation by PGN. PGRP‐S–PGN complexes increase the membrane expression of CD14, CD80 and CD86, and enhance secretion of interleukin‐8, interleukin‐12 and tumour necrosis factor‐α, but reduce interleukin‐10, clearly inducing an inflammatory profile.

Controlled adhesion and proliferation of a human osteoblastic cell line by tuning the nanoporosity of titania and silica coatings

The engineering of surfaces to control cell adhesion represents an active area of biomaterials research. Herein, we demonstrate that it is possible to tune the adhesion and proliferation of a human osteoblastic cell line (Saos-2) by tailoring the nanopore size of an oxide film coating.

Antibody detection employing sol-gel immobilized parasites

Immunofluorescence assay (IFA) and immunoperoxidase assay (IPA) are useful diagnostic techniques for specific antibody detection for different diseases. Both involve several alternatives for immobilization of cells, such as solvent or heat fixation. Non-covalent immobilization implies rigorous storage conditions at -20 degrees C to preserve the slides, and usually numerous cells are detached during the washing steps, which can lead to inconsistencies in the results. Sol-gel chemistry is usually used for coating different materials because of the mild conditions of the polymerization reaction and the ability to introduce functional groups to a wide variety of surfaces. We have developed a novel procedure for the attachment of Trypanosoma cruzi epimastigotes and Leishmania guyanensis promastigotes to a silicon oxide polymer covered glass surface. The film was prepared using standard microscope slides with tetraethoxysilane and 3-aminopropyl triethoxysilane as polymeric precursors. When acetone was used as the major coating solvent, the IFA showed the fluorescence of the attached parasites without matrix background interference. Similar results were observed when the IPA was evaluated. The sensitivity and specificity of the sol-gel immobilized parasite slides were comparable with the heat fixation technique. The performance of the coated slides was maintained for at least 2 months at 4 degrees C storage temperature. This immobilization method does not affect the molecular epitopes of the attached cells. Thus, homogeneous, ready to use, long lasting coated slides were obtained, which are appropriate for field conditions.

Uptake and Intracellular Trafficking of Superantigens in Dendritic Cells

Bacterial superantigens (SAgs) are exotoxins produced mainly by Staphylococcus aureus and Streptococcus pyogenes that can cause toxic shock syndrome (TSS). According to current paradigm, SAgs interact directly and simultaneously with T cell receptor (TCR) on the T cell and MHC class II (MHC-II) on the antigen-presenting cell (APC), thereby circumventing intracellular processing to trigger T cell activation. Dendritic cells (DCs) are professional APCs that coat nearly all body surfaces and are the most probable candidate to interact with SAgs. We demonstrate that SAgs are taken up by mouse DCs without triggering DC maturation. SAgs were found in intracellular acidic compartment of DCs as biologically active molecules. Moreover, SAgs co-localized with EEA1, RAB-7 and LAMP-2, at different times, and were then recycled to the cell membrane. DCs loaded with SAgs are capable of triggering in vitro lymphocyte proliferation and, injected into mice, stimulate T cells bearing the proper TCR in draining lymph nodes. Transportation and trafficking of SAgs in DCs might increase the local concentration of these exotoxins where they will produce the highest effect by promoting their encounter with both MHC-II and TCR in lymph nodes, and may explain how just a few SAg molecules can induce the severe pathology associated with TSS.

Crystal Structure of Staphylococcal Enterotoxin G (SEG) in Complex with a Mouse T-cell Receptor β Chain

Superantigens (SAgs) are bacterial or viral toxins that bind MHC class II (MHC-II) molecules and T-cell receptor (TCR) in a nonconventional manner, inducing T-cell activation that leads to inflammatory cytokine production, which may result in acute toxic shock. In addition, the emerging threat of purpura fulminans and community-associated meticillin-resistant Staphylococcus aureus emphasizes the importance of a better characterization of SAg binding to their natural ligands that may allow the development of reagents to neutralize their action. The three-dimensional structure of the complex between a mouse TCR β chain (mVβ8.2) and staphylococcal enterotoxin G (SEG) at 2.0 Å resolution revealed a binding site that does not conserve the “hot spots” present in mVβ8.2-SEC2, mVβ8.2-SEC3, mVβ8.2-SEB, and mVβ8.2-SPEA complexes. Analysis of the mVβ8.2-SEG interface allowed us to explain the higher affinity of this complex compared with the others, which may account for the early activation of T-cells bearing mVβ8.2 by SEG. This mode of interaction between SEG and mVβ8.2 could be an adaptive advantage to bestow on the pathogen a faster rate of colonization of the host.

Cellular clot formation in a sipunculan worm: entrapment of foreign particles, cell death and identification of a PGRP-related protein.

Clotting in animals having open or closed circulatory system comprises humoral and cellular mechanisms. Sipunculans are a small phylum of non-segmented marine worms that do not have a true circulatory system. These worms can form a cellular clot without transforming cell-free coelomic fluid into an insoluble mass. The clot may also contribute to immune response by entrapping foreign particles. We evaluated the formation of a cellular clot ex vivo in the sipunculan Themiste petricola after activation through glass contact and sea water, the ability to entrap magnetic beads and non-pathogen cyanobacteria particles within the clot, and the presence of a peptidoglycan recognition protein S (PGRP-S) antigen in cells forming the clot. Our results showed that the clot was formed by homotypic aggregation of large granular leukocytes (LGLs), a subtype of cells found in the coelomic fluid. Aggregated LGLs served to entrap magnetic beads and non-pathogen cyanobacteria particles, and PGRP-S antigen was detected both in non-activated LGLs and in activated homotypic aggregates through immunofluorescence, Western blot and flow cytometry. Cellular clots were found to be positive to Annexin V-FITC labelling. Complete disintegration of cytoplasm with shedding of microparticles, nuclear isolation and degradation were also observed by light microscopy and flow cytometry. In conclusion, cellular clot formation in Themiste petricola may serve both haemostatic and immune functions entailing rapid activation changes in LGLs, entrapment of foreign particles within a homotypic aggregate, and further cell death.