Modified AdCTLA-4 vector blocks alloimmune response in vitro.

BACKGROUND: Gene transfer of the costimulatory blockade molecule CTLA-4Ig into cold-preserved rat liver allografts results in indefinite allograft survival. Despite local delivery, this mode of immunomodulation is associated with systemic immunosuppression. In an effort to restrict immunosuppression to the graft, we have constructed a novel adenoviral vector, AdCTLA-4ex-TAG, in which the Ig sequence of CTLA-4Ig DNA has been deleted to destabilize the gene product to promote rapid extrahepatic degradation while maintaining its immunosuppressive activity within the graft milieu. METHODS: (1) Vector construction. CTLA-4 extracellular binding domain (CTLA-4ex) was prepared by PCR-based cloning methods and fused in frame to a genetic element encoding an epitope TAG allowing identification of the transgene product CTLA-4exTAG. CTLA-4exTAG was subcloned into a shuttle vector enabling isolation of AdCTLA-4exTAG. (2) In vitro transfection: AdCTLA-4exTAG was transfected into MH(1)C(1) cells and then supernatant was recovered for Western blot analysis. (3) In vitro alloimmune response was characterized by CFSE proliferation assay. (4) Extrahepatic effect of AdCTLA-4exTAG was characterized by the ability to control tumor growth after implantation of a regressive, immune sensitive cancer cell line (REGb) in the skin of BDIX rats after liver transduction with AdCTLA-4exTAG. RESULTS: Expression and secretion of the recombinant protein were documented by Western blot after infection of the MH(1)C(1) cell line() with AdCTLA-4exTAG. Addition of infected MH(1)C(1) cell supernatant resulted in abrogation of alloimmune response as shown by markedly diminished division of CD4(+) T cells in a CFSE proliferation assay. Extrahepatic tumor regressed normally after liver transduction with AdCTLA-4exTAG. CONCLUSIONS: These results show efficient in vitro expression of CTLA-4exTAG after transfection with AdCTLA-4exTAG. The modified protein retains its ability to abrogate in vitro alloimmune response. Efficient control of extrahepatic tumor growth after liver-directed delivery of AdCTLA-4exTAG suggests that the immunosuppressive effect of this vector is restricted to the liver. These results set the ground for the utilization of this novel adenoviral vector in the transplant setting.

Flow cytometric measurement of HLA-DR expression on circulating monocytes in healthy and sick neonates using monocyte negative selection.

UNLABELLED: The aim of this study was to investigate the effect of prematurity, neonatal sepsis, respiratory distress syndrome (RDS) and perinatal asphyxia on monocyte HLA-DR expression of neonates using a flow cytometric method based on monocyte negative selection. The subjects were one hundred and thirty-one neonates (59 healthy, 44 septicaemic, 20 with RDS and eight with perinatal asphyxia) and 20 healthy adults. Monocyte HLA-DR expression was measured using one-colour HLA-DR labelling in a gate for monocytes obtained using the combination of CD3-CD19--PE/CD15--FITC MoAbs. In addition, the common dual staining method using MoAbs against two CD14 epitopes (TUK4, MO2) was evaluated. With the one-colour HLA-DR labelling higher purity and recovery values of monocytes were achieved than with the dual labelling METHOD: Healthy neonates had significantly lower percentages of HLA-DR(+) monocytes than adults (69 +/- 13% versus 91.5 +/- 2.5%) and comparable mean fluorescence intensity (MFI) (119 +/- 25 versus 131 +/- 26). Values did not differ significantly between healthy term and preterm neonates. Preterm neonates with RDS had a significantly lower percentage of HLA-DR(+) monocytes than the healthy preterm neonates. In neonates with asphyxia both parameters were comparable to those of the healthy ones. Septicaemic neonates presented significantly lower values of both parameters than the healthy, RDS and asphyxiated neonates. Monocyte negative selection provides a reliable estimation of HLA-DR expression on monocytes. Expression of monocyte HLA-DR is lower in healthy neonates in comparison with adults and is further decreased in neonates with sepsis and RDS, but it is not influenced by prematurity and perinatal asphyxia.

Activation of interleukin-6/STAT3 and liver regeneration following transplantation.

BACKGROUND: Every liver that is procured, stored, and transplanted experiences injury from cold ischemia and reperfusion. Most recover quickly, but some grafts sustain enough injury to result in prolonged organ dysfunction or require retransplantation. The molecular mechanisms involved in early graft function and recovery following cold ischemia and reperfusion (I/R) after liver transplantation have not been well defined. Interleukin (IL)-6 is a critical factor in the mitogenic response within the liver, and is important for cell cycle progression and protection from injury. Activation of the latent transcription factor, STAT3, is dependent on IL-6 release. The role of the IL-6/STAT3 pathway and hepatocellular regeneration in graft recovery and cell cycle progression following cold ischemia and reperfusion was studied in a rat liver transplant orthotopic (OLT) model. Methods. Rat OLT was performed in a syngeneic model. The presence, time course, and magnitude of expression of IL-6, STAT3 activation, and upregulation of target immediate early genes were determined in liver grafts with minimal (<1 h) and prolonged (12 h) cold preservation times followed by transplantation. Progression of the cell cycle and replication was confirmed by BrdU uptake. RESULTS: Prolonged cold ischemia resulted in increased IL-6 expression and STAT3 activation. This correlated with upregulation of junB, c-fos, c-myc, and c-jun, immediate early genes associated with hepatic regeneration. Extensive DNA replication was present in livers with 12-h ischemia, demonstrating successful completion of the cell cycle. CONCLUSIONS: The participation of the IL-6/STAT3 pathway leading to cell cycle progression and regeneration is an important component in the recovery of organs immediately following cold preservation and transplantation.

Pattern of alloimmune response in second same donor allografts after induction of tolerance using CTLA4Ig.

BACKGROUND: The pattern of allograft acceptance in the presence of costimulatory blockade is manifested by the sequential appearance of Th1 cells, followed by Th2 cells. The aim of this study was to examine whether this phenomenon repeats itself after second same donor allotransplantation, hoping to determine whether acceptance in this setting provokes a predominance of the Th2 response. METHODS: Tolerance was achieved by transplantation of CTLA4Ig-transduced ACI liver allografts in Lewis recipients. Recipient long-term survivors received a second transplant, consisting of a cervical heterotopic heart from the same ACI donor strain. Animals were sacrificed at predetermined intervals following the second transplant and the heart and liver were processed for histology and cytokine mRNA expression. RESULTS: Recipients of CTLA4Ig-transduced livers survived indefinitely. Rechallenge with same donor strain second allograft was manifested by an anergic immune response in the second cardiac allograft, and a very mild transient infiltrate within the first accepted liver graft. Cardiac function was maintained with resolution of all infiltrates. The cytokine cascade was activated within the allografts; however, the pattern of acceptance was not associated with predominance of a specific Th subtype. CONCLUSIONS: The pattern of acceptance of an allograft following CTLA4Ig-mediated costimulatory blockade is not related to long-term predominance of Th2 cells, a phenomenon that may be unique to the setting of a tolerant liver. It may be likely that the infiltrating lymphocytes that are dominant in the second graft are suppressed by other memory mechanisms.