F Fu

Allografts of the liver, which has a comparatively heavy leukocyte content compared with other vascularized organs, are accepted permanently across major histocompatibility complex barriers in many murine strain combinations without immunosuppressive therapy. It has been postulated that this inherent tolerogenicity of the liver may be a consequence of the migration and perpetuation within host lymphoid tissues of potentially tolerogenic donor-derived ("chimeric") leukocytes, in particular, the precursors of chimeric dendritic cells (DC). In this study, we have used granulocyte/macrophage colony-stimulating factor to induce the propagation of progenitors that give rise to DC (CD45+, CD11c+, 33D1+, nonlymphoid dendritic cell 145+, major histocompatibility complex class II+, B7-1+) in liquid cultures of murine bone marrow cells. Using this technique, together with immunocytochemical and molecular methods, we show that, in addition to cells expressing female host (C3H) phenotype (H-2Kk+; I-E+; Y chromosome-), a minor population of male donor (B10)-derived cells (H-2Kb+; I-A+; Y chromosome+) can also be grown in 10-d DC cultures from the bone marrow of liver allograft recipients 14 d after transplant. Highly purified nonlymphoid dendritic cell 145+ DC sorted from these bone marrow-derived cell cultures were shown to comprise approximately 1-10% cells of donor origin (Y chromosome+) by polymerase chain reaction analysis. In addition, sorted DC stimulated naive, recipient strain T lymphocytes in primary mixed leukocyte cultures. Evidence was also obtained for the growth of donor-derived cells from the spleen but not the thymus. In contrast, donor cells could not be propagated from the bone marrow or other lymphoid tissues of nonimmunosuppressed C3H mice rejecting cardiac allografts from the same donor strain (B10). These findings provide a basis for the establishment and perpetuation of cell chimerism after organ transplantation.

ABSTRACT In vivo anterior cruciate ligament (ACL) bundle (anteromedial bundle [AMB] and posterolateral bundle [PLB]) relative elongation during walking and running remain unknown. In this study, we aimed to investigate in vivo ACL relative elongation over the full gait cycle during walking and running. Ten healthy volunteers walked and ran at a self‐selected pace on an instrumented treadmill while biplane radiographs of the knee were acquired at 100 Hz (walking) and 150 Hz (running). Tibiofemoral kinematics were determined using a validated model‐based tracking process. The boundaries of ACL insertions were identified using high‐resolution magnetic resonance imaging (MRI). The AMB and PLB centroid‐to‐centroid distances were calculated from the tracked bone motions, and these bundle lengths were normalized to their respective lengths on MRI to calculate relative elongation. Maximum AMB relative elongation during running (6.7 ± 2.1%) was significantly greater than walking (5.0 ± 1.7%, p = 0.043), whereas the maximum PLB relative elongation during running (1.1 ± 2.1%) was significantly smaller than walking (3.4 ± 2.3%, p = 0.014). During running, the maximum AMB relative elongation was significantly greater than the maximum PLB relative elongation ( p < 0.001). ACL relative elongations were correlated with tibiofemoral six degree‐of‐freedom kinematics. The AMB and PLB demonstrate similar elongation patterns but different amounts of relative elongation during walking and running. The complex relationship observed between ACL relative elongation and knee kinematics indicates that ACL relative elongation is impacted by tibiofemoral kinematic parameters in addition to flexion/extension. These findings suggest that ACL strain is region‐specific during walking and running. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1920–1928, 2019

77 Transforming growth factor β (TGFβ) inhibits the functional maturation of dendritic cells (DC). It has been shown that administration of mouse bone marrow (BM)-derived DC propagated in GM-CSF and TGFβ1(`TGFβ DC'), that are MHC class II+, but deficient in B7 family and CD40 molecule expression, prolongs donor-specific mouse cardiac allograft survival, associated with inhibition of anti-donor cytotoxic T lymphocyte(CTL) activity. The purpose of this study was to determine whether genetic engineering of these DC to overexpress TGFβ might enhance their tolerogenicity. Methods: genes encoding mouse TGFβ1 were transduced into B10 (H-2b) BM-derived TGFβ DC using replication-deficient adenoviral (Ad) vectors, and LacZ as reporter genes. Expression of co-stimulatory molecules on DC was monitored by flow cytometry.Results: the immune stimulatory effects of DC were determinedin vitro by MLR and CTL generation assays, and in vivo by their influence on B10 cardiac graft survival in C3H (H-2k) recipients. The transduction rate of Ad-LacZ into TGFβ DC was 86%, 83%, 78% and 76% at 100, 50, 20, and 10 MOI (multiplicity of infection), respectively. Ad-LacZ gene transfer did not affect the in vivo DC migration pattern, as detected by both donor MHC class II and X-gal staining of tissues following footpad injection into C3H mice. Transduction with Ad-LacZ at 50 MOI did not significantly affect TGFβ DC B7 or CD40 molecule expression or their allostimulatory activity in MLR, but enhanced the generation of CTL activity (51Cr release: 57.8 ± 7.5% vs 29.1±% in controls at E:T ratio of 100:1). TGFβ DC transduced with Ad-TGFβ1 producing 5 ng/106/d TGFβ1 did not show alterations, compared with Ad-LacZ controls, in MHC class II (40.5% vs 46.6%), CD80 (B7-1; 36.4% vs 39.6%), CD86 (B7-2: 28.1% vs 27.2%), or CD40 (14.5% vs 13.6%) expression, but showed reduced allostimulatory activity in MLR (5132 ± 139 vs 7154 ± 1074 cpm in Ad-LacZ controls) and generation of donor-specific CTL activity (51Cr release 20.4 ± 0.5% vs 57.8± 7.5% in Ad-LacZ controls). The life span of Ad-TGFβ1-transduced DC appeared prolonged in allogeneic recipients, as shown by histoimmunochemical staining of lymphoid tissue following footpad injection. Administration of B10 BM-derived Ad-TGFβ1 transduced TGFβ DC (2× 106) into C3H recipients of B10 cardiac grafts further prolonged graft survival (median graft survival time 21 days vs 16 days in Ad-LacZ-transduced DC injection controls, and 11 days in normal controls p<0.05). In conclusion, mouse BM-derived TGFβ DC can be efficiently transduced to express functional murine TGFβ1 genes using Ad vectors. This transduction does not appear to alter DC expression of MHC class II and co-stimulatory molecules, but increases their tolerogenicity in vivo as shown by further prolongation of cardiac graft survival in allogeneic recipients.