- Email: [email protected]
Multiple stress response genes are up-regulated in distant organs in response to minor burn injury
Vol. 191, No. 4S, October 2000
Surgical Forum Abstracts
S31
were compared by calculating the area under the secretion vs. time curve utilizing the trapezoidal rule. Insulin was measured in duplicate using ELISA presented as mean ⫾ SEM in pg/ml. Statistical analysis was by ANOVA. KO and wt mice 3 and 12 months old (n ⫽16) underwent histological examination of islets by an independent pathologist.
Conclusions: The data support the use of pancreatic acinar cells to express insulin. Future work is planned to develop vectors with regulated insulin secretion, and less immunogenicity for prolonged expression.
Results:
Critical Care II: Cell Signaling I
Basal 1st phase 2nd phase
KO 3mo
WT 3mo
KO 12mo
WT 12mo
254 ⫾ 9 248 ⫾ 10*⽧ 435 ⫾ 9⽧
279 ⫾ 5 318 ⫾ 7⽧ 457 ⫾ 9⽧
301 ⫾ 11 590 ⫾ 17⽧ 767 ⫾ 18⫹⽧
266 ⫾ 10 417 ⫾ 10⽧ 417 ⫾ 9⽧
st
* blunted 1 phase compared to all other groups, p ⬍ 0.05
increased 1st phase compared to all other groups, p ⬍ 0.05
⫹
increased 2nd phase compared to all other groups, p ⬍ 0.05
⽧
increased glucose stimulation compared to basal across all groups
Basal insulin levels across all groups were similar (p ⴝ NS). Histological sections of islets suggest there is no difference in islet cell morphology between 3mo. and 12mo. KO and wt mice. Conclusions: The data suggest that the phenotype of the SSTR-5 KO mice is a blunted 1st phase in 3mo. mice and an augmentation of glucose stimulated insulin secretion in 12mo. mice. We conclude that SSTR-5 gene regulates glucose stimulated insulin secretion and alterations of insulin release occur in the perfused pancreata of SSTR-5 genetically ablated mice.
Correction of hyperglycemia by adenoviral vectormediated insulin gene expression in the murine pancreas Alexander L Shifrin, M.D., Alberto Auricchio, M.D., James Wilson, M.D., Ph.D., Director, Steven E Raper, M.D. Institute for Human Gene Therapy, Steven E. Raper, M.D., Associate Professor of Surgery. University of Pennsylvania School of Medicine, BRB II/III, 421 Curie Blvd., Philadelphia, PA, 19104-6160, USA. Phone: 215.898.1060. Introduction: Type I diabetes causes autoimmune destruction of -cells in the islets of Langerhans. Current therapies consist of multiple insulin injections, pump, whole pancreas, or islet transplantation all of which have significant limitations. We hypothesized that the pancreatic acinar cell might be a good target for insulin gene transfer. Pancreatic acinar cells drain into the portal circulation, are not subject to immune attack, and are capable of exporting a variety of peptides. Methods: A recombinant E1-deleted human adenovirus was engineered to create a vector expressing the human insulin gene (hIns M), modified to contain furin cleavage sites, allowing proteolysis and processing to mature insulin by non--cells. Immunodeficient RAG-1 mice were made diabetic with streptozotocin (200 mg/kg i.v.) and injected with 1 ⫻ 1011 particles of the hIns M vector, using a well characterized model of pancreatic gene transfer. Serial blood glucose and insulin levels (RIA assay) were measured. Pancreatic immunohistochemistry was done with anti-human insulin monoclonal antibodies. Results: Vector administration lowered blood sugar and increased insulin levels (Figures 1&2). Immunohistochemical staining showed the presence of insulin in acinar cells throughout the pancreas.
Multiple stress response genes are up-regulated in distant organs in response to minor burn injury Kristina G Hobson, MD, Kiho Cho, PhD, David G Greenhalgh, MD. Shriner’s Hospital for Children Northern California and the University of California, Davis 2626 Stockton Blvd., Room 660 Sacramento, CA, 95817, USA (916) 453-2286 Introduction: The systemic inflammatory response to local injury often causes more morbidity than the injury itself. The mediators that arise in an injured area and subsequently induce this systemic response are not well elucidated. We hypothesized that an 18% TBSA burn in mice leads to early up-regulation of genes both in the local wound and in distant organs. Methods: Mice were subjected to 18% TBSA flame burn. Skin and lungs from both control (non-burned) and experimental mice were harvested at multiple time-points after injury. Differential display was performed using multiple arbitrary primers, and the products were analyzed by agarose gel electrophoresis. Up-regulated bands were excised and cloned for sequence analysis. The sequences were subsequently compared with the GenBank database. When similarities were found, primers for these specific genes were designed and used to analyze multiple tissues from burned and control mice by RT-PCR. Results: Several genes thought to play protective roles in the response to injury were found to exhibit increased expression within hours of injury. These differentially expressed genes included granzyme C, a cytotoxic T-cell serine protease, TIS-11d, a protein that inhibits TNF␣ by destabilizing its mRNA, nedd-4, a ubiquitin protein ligase, HAX-1s, which has been shown to induce resistance to apoptosis in T-cells, acetyl Co-A carboxylase, and mitochondrial malate dehydrogenase. Repeat RT-PCR with specific primers confirmed up-regulation of these genes in the skin, lung, thymus, and lymph nodes. Conclusions: Even a minor injury causes systemic up-regulation of several stress response genes. Understanding how these specific genes become activated will dramatically improve the treatment of the systemic inflammatory response and multiple organ failure.
TNF␣, IFN␥ and T-cell costimulation mediate the lethal bacterial superantigen response that occurs after thermal injury MR Kell FRCS, JA Lederer PhD. Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston. Rm. 425 Thorn, Brigham and Women’s Hospital, Boston, MA, USA, 617 732 6828. Introduction: A previous report from our laboratory indicated that burn injured mice undergo a lethal shock-like response when exposed to bacterial superantigen early after injury. This response was accompanied by enhanced in vivo and in vitro production of tumor necrosis factor-␣ (TNF␣) and interferon-␥ (IFN␥). This study addresses the role of these inflammatory cytokines and the CD80/CD86 T-cell costimulatory pathways in this lethal immune response. Methods: FVB mice were treated with a neutralizing monoclonal antibody specific for mouse IFN␥, a TNF antagonist [TNF receptor (p55) Fc fusion protein], murine CTLA4 Ig fusion protein or control Ig two hours before injury. 2hrs later animals were anaesthetized and given sham/burn injury and a lethal dose of Staphylococcal Enterotoxin A (SEA), 48hr mortality was observed. Next, the above protocol was repeated but serum was obtained at 4-hrs post injury to assess serum cytokine levels using ELISA. Results: We exhibited 94% (n ⫽ 16) mortality in SEA treated burninjured mice given control Ig. In contrast IFN␥, TNF antagonist or CTLA4 Ig pre-treatment significantly reduced mortality in SEA treated burn-injured mice (12.5%, p ⬍ 0.01). CTLA4 Ig decreased serum TNF␥
Surgical Forum Abstracts
S31
were compared by calculating the area under the secretion vs. time curve utilizing the trapezoidal rule. Insulin was measured in duplicate using ELISA presented as mean ⫾ SEM in pg/ml. Statistical analysis was by ANOVA. KO and wt mice 3 and 12 months old (n ⫽16) underwent histological examination of islets by an independent pathologist.
Conclusions: The data support the use of pancreatic acinar cells to express insulin. Future work is planned to develop vectors with regulated insulin secretion, and less immunogenicity for prolonged expression.
Results:
Critical Care II: Cell Signaling I
Basal 1st phase 2nd phase
KO 3mo
WT 3mo
KO 12mo
WT 12mo
254 ⫾ 9 248 ⫾ 10*⽧ 435 ⫾ 9⽧
279 ⫾ 5 318 ⫾ 7⽧ 457 ⫾ 9⽧
301 ⫾ 11 590 ⫾ 17⽧ 767 ⫾ 18⫹⽧
266 ⫾ 10 417 ⫾ 10⽧ 417 ⫾ 9⽧
st
* blunted 1 phase compared to all other groups, p ⬍ 0.05
increased 1st phase compared to all other groups, p ⬍ 0.05
⫹
increased 2nd phase compared to all other groups, p ⬍ 0.05
⽧
increased glucose stimulation compared to basal across all groups
Basal insulin levels across all groups were similar (p ⴝ NS). Histological sections of islets suggest there is no difference in islet cell morphology between 3mo. and 12mo. KO and wt mice. Conclusions: The data suggest that the phenotype of the SSTR-5 KO mice is a blunted 1st phase in 3mo. mice and an augmentation of glucose stimulated insulin secretion in 12mo. mice. We conclude that SSTR-5 gene regulates glucose stimulated insulin secretion and alterations of insulin release occur in the perfused pancreata of SSTR-5 genetically ablated mice.
Correction of hyperglycemia by adenoviral vectormediated insulin gene expression in the murine pancreas Alexander L Shifrin, M.D., Alberto Auricchio, M.D., James Wilson, M.D., Ph.D., Director, Steven E Raper, M.D. Institute for Human Gene Therapy, Steven E. Raper, M.D., Associate Professor of Surgery. University of Pennsylvania School of Medicine, BRB II/III, 421 Curie Blvd., Philadelphia, PA, 19104-6160, USA. Phone: 215.898.1060. Introduction: Type I diabetes causes autoimmune destruction of -cells in the islets of Langerhans. Current therapies consist of multiple insulin injections, pump, whole pancreas, or islet transplantation all of which have significant limitations. We hypothesized that the pancreatic acinar cell might be a good target for insulin gene transfer. Pancreatic acinar cells drain into the portal circulation, are not subject to immune attack, and are capable of exporting a variety of peptides. Methods: A recombinant E1-deleted human adenovirus was engineered to create a vector expressing the human insulin gene (hIns M), modified to contain furin cleavage sites, allowing proteolysis and processing to mature insulin by non--cells. Immunodeficient RAG-1 mice were made diabetic with streptozotocin (200 mg/kg i.v.) and injected with 1 ⫻ 1011 particles of the hIns M vector, using a well characterized model of pancreatic gene transfer. Serial blood glucose and insulin levels (RIA assay) were measured. Pancreatic immunohistochemistry was done with anti-human insulin monoclonal antibodies. Results: Vector administration lowered blood sugar and increased insulin levels (Figures 1&2). Immunohistochemical staining showed the presence of insulin in acinar cells throughout the pancreas.
Multiple stress response genes are up-regulated in distant organs in response to minor burn injury Kristina G Hobson, MD, Kiho Cho, PhD, David G Greenhalgh, MD. Shriner’s Hospital for Children Northern California and the University of California, Davis 2626 Stockton Blvd., Room 660 Sacramento, CA, 95817, USA (916) 453-2286 Introduction: The systemic inflammatory response to local injury often causes more morbidity than the injury itself. The mediators that arise in an injured area and subsequently induce this systemic response are not well elucidated. We hypothesized that an 18% TBSA burn in mice leads to early up-regulation of genes both in the local wound and in distant organs. Methods: Mice were subjected to 18% TBSA flame burn. Skin and lungs from both control (non-burned) and experimental mice were harvested at multiple time-points after injury. Differential display was performed using multiple arbitrary primers, and the products were analyzed by agarose gel electrophoresis. Up-regulated bands were excised and cloned for sequence analysis. The sequences were subsequently compared with the GenBank database. When similarities were found, primers for these specific genes were designed and used to analyze multiple tissues from burned and control mice by RT-PCR. Results: Several genes thought to play protective roles in the response to injury were found to exhibit increased expression within hours of injury. These differentially expressed genes included granzyme C, a cytotoxic T-cell serine protease, TIS-11d, a protein that inhibits TNF␣ by destabilizing its mRNA, nedd-4, a ubiquitin protein ligase, HAX-1s, which has been shown to induce resistance to apoptosis in T-cells, acetyl Co-A carboxylase, and mitochondrial malate dehydrogenase. Repeat RT-PCR with specific primers confirmed up-regulation of these genes in the skin, lung, thymus, and lymph nodes. Conclusions: Even a minor injury causes systemic up-regulation of several stress response genes. Understanding how these specific genes become activated will dramatically improve the treatment of the systemic inflammatory response and multiple organ failure.
TNF␣, IFN␥ and T-cell costimulation mediate the lethal bacterial superantigen response that occurs after thermal injury MR Kell FRCS, JA Lederer PhD. Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston. Rm. 425 Thorn, Brigham and Women’s Hospital, Boston, MA, USA, 617 732 6828. Introduction: A previous report from our laboratory indicated that burn injured mice undergo a lethal shock-like response when exposed to bacterial superantigen early after injury. This response was accompanied by enhanced in vivo and in vitro production of tumor necrosis factor-␣ (TNF␣) and interferon-␥ (IFN␥). This study addresses the role of these inflammatory cytokines and the CD80/CD86 T-cell costimulatory pathways in this lethal immune response. Methods: FVB mice were treated with a neutralizing monoclonal antibody specific for mouse IFN␥, a TNF antagonist [TNF receptor (p55) Fc fusion protein], murine CTLA4 Ig fusion protein or control Ig two hours before injury. 2hrs later animals were anaesthetized and given sham/burn injury and a lethal dose of Staphylococcal Enterotoxin A (SEA), 48hr mortality was observed. Next, the above protocol was repeated but serum was obtained at 4-hrs post injury to assess serum cytokine levels using ELISA. Results: We exhibited 94% (n ⫽ 16) mortality in SEA treated burninjured mice given control Ig. In contrast IFN␥, TNF antagonist or CTLA4 Ig pre-treatment significantly reduced mortality in SEA treated burn-injured mice (12.5%, p ⬍ 0.01). CTLA4 Ig decreased serum TNF␥