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Recurrent autoimmunity is pivotal in the accelerated destruction of cultured major histo-incompatible islet grafts in nod mice
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS II), human bronchial epithelial cells, keratinocytes, peripheral blood lymphocytes, monocytes, hepatoma cells and glioma cells. Five micrograms of total RNA was subsequently used for a reverse transcriptase reaction. Semi-quantitative PCR was then used to determine presence of IL-31R␣ mRNA. Additional samples of bronchial epithelial cells and monocytes were stimulated with OSM or IFN␥ to evaluate IL-31R␣ mRNA regulation. Results: IL-31R␣ mRNA. See Table. Conclusions: Contrary to our hypothesis and previous reports, we identified constitutive expression of IL-31R␣ mRNA only in alveolar epithelial cells, bronchial epithelial cells, monocytes, and glioma cells. In addition, we examined IL-31R␣ mRNA regulation in bronchial epithelial cells and monocytes and found that its expression was upregulated by stimulation with OSM or IFN␥, respectively. Further studies will identify whether IL-31R␣ mRNA expression is inducible in the remaining cells and in additional cell types and will also further characterize its regulation.
Results: IL-31R␣ mRNA
Cells
Expression
Type II Alveolar Trace epithelial cells Bronchial epithelial cells Present Keratinocytes Peripheral blood lymphocytes Monocytes
Absent Absent
Hepatoma cells Glioma cells
Absent Present
Trace
Regulation — Stimulation with Oncostatin M increased expression — — Stimulation with IFN␥ increased expression — —
429. THE MECHANISM OF IMMUNE MEDIATED ISCHEMIAREPERFUSION INJURY IS CONSERVED BETWEEN DIFFERENT ANIMAL SPECIES. J. Afnan 1, S. Oakes 1, B. H. Williams 1, J. Rabaglia 1, M. C. Carroll 2, F. D. Moore, Jr. 1; 1 Brigham and Women’s Hospital, Boston, MA, 2CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA. Introduction: Reperfusion injury is initiated by binding of certain IgM natural antibodies to neo-antigens exposed in ischemic tissue with subsequent activation of complement and other cytolytic pathways. Peptides that mimic the site of IgM binding to these antigens prevent IgM binding and prevent reperfusion injury when given intravenously to ischemic mice before reperfusion occurs. We now wish to determine whether this pathogenic natural IgM antibody-ischemia neo-antigen biology is restricted to mice or whether it is more general. We have therefore tested the ability of the peptide mimics to prevent reperfusion injury in a dissimilar species, the rat. Methods: Sprague-Dawley rats were subjected to 40 minutes of mesenteric ischemia followed by 180 minutes of reperfusion. The 12-mer peptide mimic was administered i.v. shortly prior to reperfusion at 300 ug/mL, estimated from the effective dose in the mouse. Gut injury was quantified using a scoring system based on the H⫹E section. 125-I labeled albumin was used to assess local injury as gut permeability and remote injury as lung permeability. Results: All injured rats showed gross bowel hemorrhage and edema, but bowel from peptide treated animals appeared much less edematous and hemorrhagic. Microscopic analysis of microvilli showed a significantly reduced injury score in peptide treated animals, although injury had not been completely prevented. Permeability data indicated a significant reduction in local and remote injury in peptide treated animals. Conclusion: The data demonstrates attenuation of rat gut microvillus injury, of gut edema, and of remote injury following mesenteric ischemia-reperfusion due to administration of an i.v. peptide mimic of a murine ischemia neo-antigen prior to reperfusion, thus indicating that a second species utilizes the same ischemia neo-antigen and corresponding natural antibody specificity to amplify reperfusion
317
injury to the point of necrosis. This implies that this mechanism of inflammation is potentially applicable to higher species. 430. RECURRENT AUTOIMMUNITY IS PIVOTAL IN THE ACCELERATED DESTRUCTION OF CULTURED MAJOR HISTO-INCOMPATIBLE ISLET GRAFTS IN NOD MICE. Wang D, Shi Q, Hadley GA, Farber DL, Bartlett ST; University of Maryland School of Medicine Introduction: Recent data from Edmonton shows a disappointing 10% five-year clinical islet graft survival. Of many factors, autoimmunity may play a pivotal role. Evidence suggests a strong role for autoimmunity in MHC-matched murine islet transplants, but the data for MHC-mismatched grafts is less clear. To address this issue, we utilized pretransplant in vitro culture to delete intraislet APCs in order to diminish or eliminate alloimmunity. Then, we compared the survival time of cultured B10.BR islets in streptozocin-induced and spontaneously diabetic NOD mice using a clinically relevant intraportal islet transplant site. Methods: B10.BR mouse islets were prepared and cultured at 24°C 95% air and 5% CO2 for 7 days prior to transplantation. Cultured B10.BR mouse islets were transplanted intraportally into recipient animals. Four experimental groups were performed: Group 1, islet transplantation in streptozocin-induced diabetic BALB/c mice without further therapy; Group 2, islet transplantation in streptozocin-induced diabetic NOD mice; Group 3, islet transplantation in streptozocin-induced diabetic NOD mice with anti-lymphocyte serum (ALS) treatment on pre-transplant days ⫺6, ⫺4, and ⫺2; Group 4, islet transplantation in spontaneously-developed diabetic NOD mice with ALS treatment as above. Graft function was assessed by blood glucose monitoring. Results: Cultured B10.BR islet grafts survived indefinitely in BALB/c mice without further treatment. While the mean survival time (MST) of cultured B10.BR islets in chemically-induced diabetic NOD mice was 13.5 days, addition of ALS treatment significantly prolonged the survival time of cultured B10.BR islets in chemicallyinduced diabetic NOD mice (MST⫽51 days). However, addition of ALS treatment had little effect in prolongation of survival of cultured B10.BR islets in spontaneously developed diabetic NOD mice (MST 17 days). Conclusions: Culturing B10.BR islets can abrogate alloimmunity in BALB/c mice, but not in NOD mice. ALS is required to prolong islet graft survival in chemically-induced diabetic NOD mice, a unique finding for this strain. In addition, autoimmune diabetic NOD hosts do not allow prolonged islet graft survival despite conditions that are favorable in non-autoimmune diabetic MHC-mismatched hosts or chemically diabetic NOD mice. These data suggest that recurrent autoimmunity occurs regardless of the degree of MHC-mismatch. Autoimmunity must be overcome to achieve long term success of islet grafts.
431. INDUCTION OF BREAST CANCER REGRESSION WITH INTRA-TUMORAL RV-NEU AND RV-GMCSF NEOADJU-
Results: IL-31R␣ mRNA
Cells
Expression
Type II Alveolar Trace epithelial cells Bronchial epithelial cells Present Keratinocytes Peripheral blood lymphocytes Monocytes
Absent Absent
Hepatoma cells Glioma cells
Absent Present
Trace
Regulation — Stimulation with Oncostatin M increased expression — — Stimulation with IFN␥ increased expression — —
429. THE MECHANISM OF IMMUNE MEDIATED ISCHEMIAREPERFUSION INJURY IS CONSERVED BETWEEN DIFFERENT ANIMAL SPECIES. J. Afnan 1, S. Oakes 1, B. H. Williams 1, J. Rabaglia 1, M. C. Carroll 2, F. D. Moore, Jr. 1; 1 Brigham and Women’s Hospital, Boston, MA, 2CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA. Introduction: Reperfusion injury is initiated by binding of certain IgM natural antibodies to neo-antigens exposed in ischemic tissue with subsequent activation of complement and other cytolytic pathways. Peptides that mimic the site of IgM binding to these antigens prevent IgM binding and prevent reperfusion injury when given intravenously to ischemic mice before reperfusion occurs. We now wish to determine whether this pathogenic natural IgM antibody-ischemia neo-antigen biology is restricted to mice or whether it is more general. We have therefore tested the ability of the peptide mimics to prevent reperfusion injury in a dissimilar species, the rat. Methods: Sprague-Dawley rats were subjected to 40 minutes of mesenteric ischemia followed by 180 minutes of reperfusion. The 12-mer peptide mimic was administered i.v. shortly prior to reperfusion at 300 ug/mL, estimated from the effective dose in the mouse. Gut injury was quantified using a scoring system based on the H⫹E section. 125-I labeled albumin was used to assess local injury as gut permeability and remote injury as lung permeability. Results: All injured rats showed gross bowel hemorrhage and edema, but bowel from peptide treated animals appeared much less edematous and hemorrhagic. Microscopic analysis of microvilli showed a significantly reduced injury score in peptide treated animals, although injury had not been completely prevented. Permeability data indicated a significant reduction in local and remote injury in peptide treated animals. Conclusion: The data demonstrates attenuation of rat gut microvillus injury, of gut edema, and of remote injury following mesenteric ischemia-reperfusion due to administration of an i.v. peptide mimic of a murine ischemia neo-antigen prior to reperfusion, thus indicating that a second species utilizes the same ischemia neo-antigen and corresponding natural antibody specificity to amplify reperfusion
317
injury to the point of necrosis. This implies that this mechanism of inflammation is potentially applicable to higher species. 430. RECURRENT AUTOIMMUNITY IS PIVOTAL IN THE ACCELERATED DESTRUCTION OF CULTURED MAJOR HISTO-INCOMPATIBLE ISLET GRAFTS IN NOD MICE. Wang D, Shi Q, Hadley GA, Farber DL, Bartlett ST; University of Maryland School of Medicine Introduction: Recent data from Edmonton shows a disappointing 10% five-year clinical islet graft survival. Of many factors, autoimmunity may play a pivotal role. Evidence suggests a strong role for autoimmunity in MHC-matched murine islet transplants, but the data for MHC-mismatched grafts is less clear. To address this issue, we utilized pretransplant in vitro culture to delete intraislet APCs in order to diminish or eliminate alloimmunity. Then, we compared the survival time of cultured B10.BR islets in streptozocin-induced and spontaneously diabetic NOD mice using a clinically relevant intraportal islet transplant site. Methods: B10.BR mouse islets were prepared and cultured at 24°C 95% air and 5% CO2 for 7 days prior to transplantation. Cultured B10.BR mouse islets were transplanted intraportally into recipient animals. Four experimental groups were performed: Group 1, islet transplantation in streptozocin-induced diabetic BALB/c mice without further therapy; Group 2, islet transplantation in streptozocin-induced diabetic NOD mice; Group 3, islet transplantation in streptozocin-induced diabetic NOD mice with anti-lymphocyte serum (ALS) treatment on pre-transplant days ⫺6, ⫺4, and ⫺2; Group 4, islet transplantation in spontaneously-developed diabetic NOD mice with ALS treatment as above. Graft function was assessed by blood glucose monitoring. Results: Cultured B10.BR islet grafts survived indefinitely in BALB/c mice without further treatment. While the mean survival time (MST) of cultured B10.BR islets in chemically-induced diabetic NOD mice was 13.5 days, addition of ALS treatment significantly prolonged the survival time of cultured B10.BR islets in chemicallyinduced diabetic NOD mice (MST⫽51 days). However, addition of ALS treatment had little effect in prolongation of survival of cultured B10.BR islets in spontaneously developed diabetic NOD mice (MST 17 days). Conclusions: Culturing B10.BR islets can abrogate alloimmunity in BALB/c mice, but not in NOD mice. ALS is required to prolong islet graft survival in chemically-induced diabetic NOD mice, a unique finding for this strain. In addition, autoimmune diabetic NOD hosts do not allow prolonged islet graft survival despite conditions that are favorable in non-autoimmune diabetic MHC-mismatched hosts or chemically diabetic NOD mice. These data suggest that recurrent autoimmunity occurs regardless of the degree of MHC-mismatch. Autoimmunity must be overcome to achieve long term success of islet grafts.
431. INDUCTION OF BREAST CANCER REGRESSION WITH INTRA-TUMORAL RV-NEU AND RV-GMCSF NEOADJU-