The Tensile Behavior of Hybrid Bonded Bolted Composite Joints: 3D-Digital Image Correlation versus Finite Element Analysis.

This study examines the behavior of hybrid bolted/bonded (HBB) joints loaded in tensile shear comprising plain weave carbon/epoxy laminates in quasi-isotropic (QI) and cross-ply (CP) layups. It proposes a combined approach of 3D digital image correlation and finite element analysis (FEA) to assess their behavior. To apply the FEA simulation accurately, a single layer of plain fabric was replaced with [0/90]s lamination. Experimental standard open-hole tension test results, as well as only bolted (OB) and HBB, along with FEA predictions, confirmed the accuracy of the substitution method. The FEA, calibrated by experimental results, provides insight into the distinctive characteristics of HBB joints in comparison with bonded and bolted joints. Critical considerations include material properties, damage modeling, adhesive characteristics, and mass scaling. The FEA results underscored the pivotal role of adhesives in HBB joints, rendering them akin solely to bonded configurations. HBB joints retain their geometry better than OB joints with considerably less out-of-plane displacement, following a sinusoidal trend. Moreover, the overall behavior of the two layups demonstrates that CP benefits from having higher strength than QI, especially at the critical hole located closer to the grip side.

Detection of regulatory cells as an assay for allograft tolerance in miniature swine.

BACKGROUND: There is currently a great need for an in vitro assay to assess the presence of tolerance following allotransplantation to determine whether immunosuppressive medications can be discontinued. Our laboratory has recently developed an assay involving coculture inhibition of cell-mediated lympholysis that correlates with tolerance to allografts in swine leukocyte antigen (SLA) Class I-mismatched miniature swine. The potential for clinical application of this assay may depend on 2 important factors: (1) whether the assay can be used in the presence of immunosuppression; and (2) whether frozen-stored naive responder cells can be utilized. METHODS: Long-term tolerant MGH miniature swine that had accepted SLA Class I-mismatched kidney transplants after a 12-day course of cyclosporine or tacrolimus were studied. Two long-term tolerant and 2 naive control animals were treated with a clinically relevant dose of cyclosporine for 2 weeks (trough level 100 to 400 ng/ml) to simulate the ongoing "chronic" immunosuppression used in human recipients of allografts. Cells from tolerant or naive, recipient-matched animals were stimulated for 6 days with donor or third-party SLA. These primed cells were then cocultured with naive unstimulated recipient major histocompatibility complex (MHC)-matched responders and irradiated stimulators. Responder cells were tested both fresh and frozen. RESULTS: Suppression of cytotoxic responses of naive responder cells was observed in all coculture assays using cells from tolerant animals primed against donor antigen in vitro, but not in assays using similarly primed cells from naive animals. Responder cells from tolerant animals receiving immunosuppression had a suppressive activity similar to that from cells of the same animals not receiving immunosuppression. Similar suppression was also observed in coculture assays using either fresh or frozen naive responder cells. CONCLUSIONS: This coculture assay appears to correlate with the presence of tolerance under conditions applicable to the clinical setting. The assay appears to identify peripheral regulatory mechanisms of tolerance in allogeneic transplant recipients, and therefore may provide an approach for determining an appropriate timepoint at which to test withdrawal of immunosuppressive medications.

Variable relationship between chimerism and tolerance after hematopoietic cell transplantation without myelosuppressive conditioning.

BACKGROUND: We have previously described a mixed chimerism protocol that avoids myelosuppressive conditioning and permits hematopoietic cell transplantation across MHC barriers without the need for whole body irradiation in miniature swine. Here, we report our current experience including animals conditioned without thymic irradiation, and we attempt to define the relationship between long-term chimerism and stable tolerance in these animals. METHODS: Recipient swine received in vivo T-cell depletion, with or without thymic irradiation on day -2. Cyclosporine was administered for 30 to 60 days beginning on day -1. A total of 1 to 2 x 10(10) /kg cytokine-mobilized donor hematopoietic cells were infused during 3 days. Chimerism was determined by flow cytometry. In vitro tolerance assays and donor-matched kidney transplantation were performed after cessation of cyclosporine. RESULTS: Most recipients maintained stable chimerism (26 of 35) and were specifically tolerant to donor-matched cells in vitro regardless of whether they received thymic irradiation. Donor-matched kidney transplantations performed in chimeric animals without in vitro antidonor immune responses were accepted without immunosuppression. Some animals developed in vitro evidence of antidonor MHC responsiveness despite the persistence of donor cells in the peripheral blood. Donor-matched kidney transplantations performed in the face of these responses were rejected. CONCLUSIONS: These data indicate that this nonmyelosuppressive protocol can induce stable chimerism and robust tolerance even in animals conditioned without thymic irradiation. However, the data also demonstrate that macrochimerism does not always correlate with tolerance. Lack of in vitro antidonor immune responses in chimeric animals is an important predictor of renal allograft acceptance in this model.

Persistent chimerism despite antidonor MHC in vitro responses in miniature swine following allogeneic hematopoietic cell transplantation.

BACKGROUND: T-cell chimerism predominates in miniature swine receiving hematopoietic-cell transplantation without myelosuppressive conditioning. Several chimeric recipients have become hyporesponsive to donor-major histocompatibility complex (MHC) in vitro and accepted donor-matched renal transplants without immunosuppression. However, some retained antidonor in vitro responses and subsequently rejected donor renal allografts despite the persistence of peripheral blood chimerism. In this study, we characterize the donor cells in both "tolerant" and "nontolerant" chimeric miniature swine. METHODS: Peripheral blood chimerism was determined by flow cytometry. In vitro antidonor responsiveness was determined by mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML). Donor cells were separated from chimeras by immunomagnetic bead separation and used as stimulators or targets in CML assays. Phenotypic analysis of donor cells in chimeras was performed using flow cytometry. RESULTS: Peripheral blood chimerism stabilized beyond 100 days and was made up almost entirely of T cells. PBMC from nontolerant chimeras could be stimulated in vitro to kill donor cells isolated from the mixed chimera itself. In contrast, PBMC from tolerant chimeras hyporesponsive to donor-type cells could not be stimulated in vitro to kill their own sorted donor cells. CONCLUSIONS: The in vivo persistence of donor T cells in mixed chimeric animals with in vitro antidonor responsiveness is not caused by an inability of these cells to be killed but rather by the poor stimulating capacity of these donor T cells. The nature of donor T cells that persist in the face of in vitro antidonor responses, has important implications for the induction of transplant tolerance by way of the generation of mixed chimerism.