Induction of specific tolerance by intrathymic injection of recipient muscle cells transfected with donor class I major histocompatibility complex.

Induction of tolerance to allogeneic MHC antigens has been a goal in the field of transplantation because it would reduce or eliminate the need for generalized immunosuppression. Although encouraging results have been obtained in experimental models by exposing recipient thymus to donor cells before transplantation, donor cells are not typically available at that time, and the donor antigens responsible for the effect are poorly defined. In the present study, thymic tolerance was demonstrated without using donor cells. Recipient thymus was injected before transplantation with autologous myoblasts and myotubes that were genetically modified to express allogeneic donor-type MHC class I antigen. Donor-specific unresponsiveness was induced to a completely MHC-disparate liver transplant and to a subsequent donor-type cardiac allograft, but not a third-party allograft. In vitro, recipient CTL demonstrated a 10-fold reduction in killing of donor cells, but not of third-party cells. Our results demonstrate: (1) that recipient muscle cells can be genetically engineered to induce donor-specific unresponsiveness when given intrathymically, and (2) transfected recipient cells expressing only donor MHC class I antigen can induce tolerance to a fully allogeneic donor

Immunity to MHC class I antigen after direct DNA transfer into skeletal muscle.

Plasmid cDNA encoding the alpha-chain of either membrane-bound (pcRT.45) or secreted (pcRQ.B3) RT1Aa MHC class I Ag were transferred to Lewis (RT1(1)) rat skeletal muscle by direct injection. Rats were challenged 7 days later with an ACI (RT1a) heterotropic heart transplant, and cardiac allograft survival, RT1Aa-specific antibody levels, and frequency of ACI-specific CTL were monitored. Graft rejection was accelerated by > or = 2 days in an Ag-specific and dose-dependent manner in pcRT.45-injected rats. The pcRQ.B3-injected rats also rejected grafts more rapidly; however, graft rejection was accelerated by only 1 day, and graft infiltrates were less pronounced than in pcRT.45-injected rats. Injection of pcRT.45 resulted in an increase in ACI-specific CTL precursor frequency 3 days post-transplant, whereas there was no significant change in rats pretreated with pcRQ.B3 injection. Compared with rats injected with a control plasmid encoding firefly luciferase, transfer of pcRT.45 resulted in an increase in RT1Aa-specific IgG and IgM antibody 3 days after heart transplantation. Transfer of pcRQ.B3 resulted in a similar mean increase in RT1Aa-specific IgG and IgM antibody after transplantation, but the variability from rat to rat was greater, with some animals exhibiting strong priming, and others showing little or no priming by gene injection. Our results suggest that skeletal muscle can express either membrane-bound or secreted MHC class I Ag after gene transfer, but that the membrane-bound form is more immunogenic than the secreted form in the high responder Lewis rat. Direct DNA transfer to skeletal muscle provides a rapid and specific approach to studying immunity to allogeneic MHC Ag

Use of donor serum to prevent passive transfer of hyperacute rejection

Organ transplantation in presensitized recipients continues to be contraindicated for heart and kidney recipients due to the risk of hyperacute rejection, which has no known treatment at this time. We tested whether donor serum, which contains soluble MHC class I antigen, is able to neutralize the effect of anti-donor antibody in the recipient and prevent hyperacute or accelerated rejection. A rat model of passive immunization was used to test the role of anti-donor antibody in hyperacute rejection. Seven of 10 recipients of hyperimmune serum (HyS), derived from Lewis rats (RT1l) following 3 ACI (RT1a) skin grafts, developed hyperacute or accelerated rejection. Intravenous injection of ACI serum prior to the HyS administration prevented hyperacute rejection in all recipients tested. When third-party (Wistar-Furth, RT1u) serum was given to Lewis rats injected with HyS, hyperacute rejection was not abrogated. When examining the mechanism of this effect, a simple antibody blocking phenomenon was found to be unlikely since flow cytometry analysis showed that ACI serum needed to be present at > or = 256-fold excess compared to HyS to block anti-ACI antibody binding to RT1.Aa+cells by 50%. We tested whether the RT1.Aa class I antigen in ACI serum had other biologic properties that resulted in the prolonged graft survival. However, removal of RT1.Aa antigen from ACI serum prior to use in the passive transfer model did not abrogate the graft prolongation observed previously. These data suggest that components of donor serum other than MHC class I antigen may be useful for preventing the antibody-mediated component of hyperacute rejection

Interleukin-17A upregulates receptor activator of NF-kappaB on osteoclast precursors.

IntroductionThe interaction between the immune and skeletal systems is evidenced by the bone loss observed in autoimmune diseases such as rheumatoid arthritis. In this paper we describe a new mechanism by which the immune cytokine IL-17A directly affects osteoclastogenesis.MethodsHuman CD14+ cells were isolated from healthy donors, cultured on dentine slices and coverslips and stimulated with IL-17A and/or receptor activator of NF-kappaB ligand (RANKL). Osteoclast differentiation was evaluated by gene expression, flow cytometry, tartrate-resistant acid phosphatase staining, fluorescence and electron microscopy. Physiologic bone remodelling was studied in wild-type (Wt) and IL-17A-/- mice using micro-computer tomography and serum RANKL/osteoprotegerin concentration. Functional osteoclastogenesis assays were performed using bone marrow macrophages isolated from IL-17A-/- and Wt mice.ResultsIL-17A upregulates the receptor activator for NF-kappaB receptor on human osteoclast precursors in vitro, leading to increased sensitivity to RANKL signalling, osteoclast differentiation and bone loss. IL-17A-/- mice have physiological bone homeostasis indistinguishable from Wt mice, and bone marrow macrophages isolated from these mice develop fully functional normal osteoclasts.ConclusionsCollectively our data demonstrate anti-IL-17A treatment as a selective therapeutic target for bone loss associated with autoimmune diseases

DX5+NKT cells display phenotypical and functional differences between spleen and liver as well as NK1.1-Balb/c and NK1.1+ C57Bl/6 mice

These results show that DX5+NKT cells are a heterogeneous population, depending on the dedicated organ and mouse strain, that has diverse functional capacity

Augmenter of Liver Regeneration Reduces Ischemia Reperfusion Injury by Less Chemokine Expression, Gr-1 Infiltration and Oxidative Stress

Hepatic ischemia reperfusion injury (IRI) is a major complication in liver resection and transplantation. Here, we analyzed the impact of recombinant human augmenter of liver regeneration (rALR), an anti-oxidative and anti-apoptotic protein, on the deleterious process induced by ischemia reperfusion (IR). Application of rALR reduced tissue damage (necrosis), levels of lipid peroxidation (oxidative stress) and expression of anti-oxidative genes in a mouse IRI model. Damage associated molecule pattern (DAMP) and inflammatory cytokines such as HMGB1 and TNF alpha, were not affected by rALR. Furthermore, we evaluated infiltration of inflammatory cells into liver tissue after IRI and found no change in CD3 or gamma delta TCR positive cells, or expression of IL17/IFN gamma by gamma delta TCR cells. The quantity of Gr-1 positive cells (neutrophils), and therefore, myeloperoxidase activity, was lower in rALR-treated mice. Moreover, we found under hypoxic conditions attenuated ROS levels after ALR treatment in RAW264.7 cells and in primary mouse hepatocytes. Application of rALR also led to reduced expression of chemo-attractants like CXCL1, CXCL2 and CCl2 in hepatocytes. In addition, ALR expression was increased in IR mouse livers after 3 h and in biopsies from human liver transplants with minimal signs of tissue damage. Therefore, ALR attenuates IRI through reduced neutrophil tissue infiltration mediated by lower expression of key hepatic chemokines and reduction of ROS generation

Interleukin-17A upregulates receptor activator of NF-κB on osteoclast precursors

IntroductionThe interaction between the immune and skeletal systems is evidenced by the bone loss observed in autoimmune diseases such as rheumatoid arthritis. In this paper we describe a new mechanism by which the immune cytokine IL-17A directly affects osteoclastogenesis.MethodsHuman CD14+ cells were isolated from healthy donors, cultured on dentine slices and coverslips and stimulated with IL-17A and/or receptor activator of NF-kappaB ligand (RANKL). Osteoclast differentiation was evaluated by gene expression, flow cytometry, tartrate-resistant acid phosphatase staining, fluorescence and electron microscopy. Physiologic bone remodelling was studied in wild-type (Wt) and IL-17A-/- mice using micro-computer tomography and serum RANKL/osteoprotegerin concentration. Functional osteoclastogenesis assays were performed using bone marrow macrophages isolated from IL-17A-/- and Wt mice.ResultsIL-17A upregulates the receptor activator for NF-kappaB receptor on human osteoclast precursors in vitro, leading to increased sensitivity to RANKL signalling, osteoclast differentiation and bone loss. IL-17A-/- mice have physiological bone homeostasis indistinguishable from Wt mice, and bone marrow macrophages isolated from these mice develop fully functional normal osteoclasts.ConclusionsCollectively our data demonstrate anti-IL-17A treatment as a selective therapeutic target for bone loss associated with autoimmune diseases