Three-dimensional (3D) culture systems are critical to investigate cell physiology and to engineer tissue grafts. In this study, we describe a simple yet innovative bioreactor-based approach to seed, expand, and differentiate bone marrow stromal cells (BMSCs) directly in a 3D environment, bypassing the conventional process of monolayer (two-dimensional [2D]) expansion. The system, based on the perfusion of bone marrow-nucleated cells through porous 3D scaffolds, supported the formation of stromal-like tissues, where BMSCs could be cocultured with hematopoietic progenitor cells in proportions dependent on the specific medium supplements. The resulting engineered constructs, when implanted ectopically in nude mice, generated bone tissue more reproducibly, uniformly, and extensively than scaffolds loaded with 2D-expanded BMSCs. The developed system may thus be used as a 3D in vitro model of bone marrow to study interactions between BMSCs and hematopoietic cells as well as to streamline manufacture of osteoinductive grafts in the context of regenerative medicine.

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Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70–86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.

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Allergen-specific immunotherapy is a clinically proven effective treatment for many allergic diseases, including asthma; however, it is not currently available for latex allergy because of the high risk of anaphylaxis. There is, therefore, a crucial need for an animal model of latex allergy in which to develop effective immunotherapy. Previous mouse models of latex allergy either did not characterize the allergic pulmonary immune response or used crude latex extracts, making it difficult to quantify the contribution of individual proteins and limiting their usefulness for developing specific immunotherapy. We immunized mice with recombinant Hev b 5, a defined major latex allergen, or latex glove protein extract, representing the range of occupationally encountered processed latex allergens. The immune response was compared with that seen in ovalbumin-immunized mice. Immunization with Hev b 5 or glove extract elicits hallmarks of allergic pulmonary Th2-type immune responses, comparable to those for ovalbumin, including (1) serum antigen-specific IgE, (2) an eosinophilic inflammatory infiltrate in the lung, (3) increased interleukin-5 in lung bronchoalveolar lavage fluid, and (4) mucus hypersecretion by epithelial cells in the lung airways. This mouse model will aid the development of potentially curative treatments for latex-sensitized individuals, including those with occupational asthma.

Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8 ؉ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8 ؉ cells, but the analogous cells in humans have not been identified. Here IntroductionClinical success of bone marrow (BM) transplantation is critically dependent on rapid hematopoietic and immune reconstitution. 1,2 Murine studies identified rare BM cells that enhance stem-cell engraftment and induce donor-specific tolerance. These facilitating cells (FCs) were defined as CD8 ϩ TCR Ϫ plasmacytoid dendritic cell precursors, 3,4 or as a subset of CD8 ϩ CD3⑀ ϩ cells, which lack conventional ␣␤-and ␥␦-T-cell receptor (TCR) heterodimers. 5,6 With mouse CD8 ϩ TCR Ϫ CD3 ϩ FCs, CD3 is essential for enhanced reconstitution across the allogeneic barriers. [6][7][8] Patients receiving grafts depleted of CD3 ϩ lymphocytes to avoid graft-versus-host disease (GVHD) are at high risk of graft failure. 9 The engraftment-facilitating function associated with CD8 ϩ but not CD4 ϩ T-cell subsets in both clinical transplantation 10,11 and the NOD/SCID mouse xenotransplantation model. 12 This indicates that the human CD8 ϩ population, harboring alloreactive T cells responsible for GvHD, 1 also contains cells with graft-facilitating capacity; however these immunologic properties have not yet been dissected in either clinical or experimental studies. We identified a rare population of human CD8 ϩ TCR Ϫ CD3 ϩ cells and used the NOD/SCID assay to show their graft-facilitating potential. MethodsSample collection, cell purification, and culture Granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (mPB), cord blood (CB), and BM collections from healthy donors were approved by the Ethical Committee of University Hospitals Basel. Informed consent was obtained in accordance with the Declaration of Helsinki. Mononuclear cells (MNCs) were isolated by Ficoll-Histopaque (Sigma-Aldrich, St Louis, MO). CB CD34 ϩ cells were isolated by magnetic-assisted cell separation (MACS; Miltenyi Biotec, Bergish Gladbach, Germany) to a purity of more than 98%. CD8 ϩ , CD8 ϩ TCR Ϫ , and CD8 ϩ TCR Ϫ CD3 ϩ cells were purified from mPB by sorting with MoFlo (Dako, Fort Collins, CO). Colony forming unit (CFU) assays of CD34 ϩ cells were performed in 1% methylcellulose containing stem-cell factor (SCF), interleukin (IL)-3, G-CSF, granulocyte-macrophage-CSF (GM-CSF), and erythropoietin (EPO) 13 after coculture with CD8 ϩ cells or subsets thereof for 2 hours at 37°C, in the presence of anti-flt3 ligand (Flt3L) monoclonal antibody (mAb; M5, Immunex, Seattle, WA) where indicated. CD8 ϩ TCR Ϫ CD3 ϩ cells were maintained in RPMI/10% fetal calf serum (FCS) and IL-7 (100 ng/mL; Novartis Basel, Basel, Switzerland) for 5 days to measure Flt3L, 14 and in IL-7 on anti-CD3 mAb-coated dishes for 3 days to isolate mRNA, or 14 days to examine cell phenotype. Flo...

T-cell production throughout life depends on efficient colonization and intrathymic expansion of BM-derived hematopoietic precursors. After irradiation-induced thymic damage, thymic recovery is facilitated by Flt3 ligand (FL), expressed by perivascular fibroblasts surrounding the thymic entry site of

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