Comparative Study on Biologic and Immunologic Characteristics of the Pancreas Islet Cell between 24degrees C and 37degrees C Culture in the Rat / 대한이식학회지

PURPOSE: Islet cell transplantation, as an alternative approach to endocrine cell replacement to treat the diabetes mellitus, has received significant attention because it holds several advantages over whole gland transplantation. However cell damage from islet isolation and immunologic rejection after transplantation prevent from successful clinical application for diabetic patients. Culture of cells at low temperature has known to stabilize the cell viability, and to decrease the immunologic antigenicity. Aim of this study is to investigate the effect of culture at 24oC on cell viability, cellular function, immunogenicity and cytokine profiles in rat pancreas islet. METHODS: Pancreas islets were isolated from Lewis rat and cultured at 24oC or 37oC during 14 days. Islet recovery after culture period was counted as islet equivalent number, and islet viability was examined with fluorescent vital staining (FDA/PI). Islet function was measured with glucose stimulation test. Annexin V expression and MHC class I and II expression were measured with flow cytometric assay for apoptosis and immunogenicity respectively. Lymphocyte cell proliferation through mixed lymphocyte islet culture was examined with WST-1 proliferation assay. Cytokine profiles were analyzed with quantitative real time RT-PCR. All these parameters were measured on 1, 3, 5, 7, 14 culture days after islet isolation. RESULTS: Islet recovery was higher in islet cultured at 24oC than in islet cultured at 37oC without change of viability. Insulin secretion after glucose stimulation was more effective in 24oC culture condition. Decrease of apoptotic cell death was demonstrated in 24oC cultured islet. MHC class I and II expression on islets and lymphocyte proliferation when cocultured with islets were less prominent in 24oC cultured islet. TNF-alpha and IL-4 cytokine expression was higher in islet cultured at 24oC than in islet cultured at 37oC. IL-1beta and IL-10 cytokine expression were similar in both culture condition. CONCLUSION: This study demonstrated that cell recovery and function are increased in islet cultured at 24oC than in islet cultured at 37oC while antigenicity and proinflammatory cytokine expression are decreased. Low temperature culture can be a good approach to prevent the loss of islet mass, and to reduce the immunologic rejection of transplanted islet for successful clinical islet transplantation.

The Effect of Intravenous Immunoglobulin on Hyperacute and Acclerated Rejection in Heart Transplantation of the Rat / 대한이식학회지

Hyperacute or acute accelerated rejection caused by preformed antibody in sensitized patients resulted in increased waiting period and complicated posttransplant hospital course. Intravenous immunoglobulin (IVIG) has known to have anti cytotoxic effect by blocking the anti HLA antibody. PURPOSE: We investigated the effect of IVIG on hyperacute and acclerated rejection of the heart graft in the presensitized rat. METHODS: Recipients (Wistar) were sensitized from repeated allo (Lewis) skin graft and followed by heterotopic allo cardiac transplantation. A guinea pig was used for the xenotransplantation model. IVIG (Green Cross kappa, 400 mg/kg in allotransplantation, 800 mg/kg in xenotransplantation) was given just before heart transplantation. Graft survival and donor specific IgG, IgM and complement were measured. RESULTS: Graft survival was 7.2 days in non sensitized allogenic heart transplantation (n=9), 1.3 days in sensitized allogenic recipients (n=7). Graft survival was prolonged from 1.3 days to 4.4 days with IVIG treatment (n=5). As for xenogenic transplantation, graft survival was prolonged from 30 min to 7.4 hr with IVIG treatment (n=5). Donor specific IgG and IgM and complement increment were blocked by IVIG during the IVIG treatment. Donor specific IgG and Ig M and complement were increased after the cessation of IVIG treatment. CONCLUSION: IVIG was able to prolong the graft survival of the sensitized allograft and xenograft. Suppression of the donor specific IgG, IgM and complement might be one of the underlying mechanisms. A further studies have to follow to clarify the more detailed mechanism.

The Implication of Monocyte/Macrophage into the Graft Following Heart Transplantation in Rat

BACKGROUND: In organ transplantation, the cellular immune reaction, namely T-cell immunity, plays a major role in rejecting the graft. While T & B cell activities in organ transplantation have been studied extensively, monocytes/macrophages have not because of their a minor role in innate immunity. Monocytes act as immunologically active cells in several aspects in organ transplantation, such as antigen-presenting cells, cells releasing many substance, such as IL-1, IL-2, TNF-alpha, and many growth factors, and cells phagocytosing foreign antigens and tissues in the effector phase of immune reaction. METHODS: We attempted to study the role of monocytes/ macrophages in graft rejection following allogenic organ transplantation in rodents. RESULTS: While graft survivals following a cardiac allograft were more then 100 days in all the singenic Wistar to Wistar transplants, the graft survival for Lewis to Wistar allografts were 7 to 12 days with a mean of 9.2 days. In the histology of the transplanted hearts, cellular infiltration developed from posttransplantation day 1, and all the histologic findings, such as myocardial ischemia, interstitial bleeding, and endocardial changes, were more progressive around the days of graft rejection. Macrophage infiltration analyzed by immunohistochemstry using the spectific antibody ED1, was noticed from postoperative day 1, and the macrophages were distributed all through the layer of the heart. In the study on the intragraft monokine gene by using RT-PCR, mRNA of IL-1 expressed on day 1 and reappearedon day 7. mRNA of TNF-alphaexpressed on day 3 and MCP-1 on day 1. All the monokine gene expressions progressed up to the days of rejection. CONCLUSION: From these results showing the concurrent pattern of cell infiltration and intragraft cytokine gene expression of monocytes/macrophages with the lymphocyte, we suggest that intervention of monocytes in organ transplantation may prolong graft survival with or without the anti T cell strategy.

Study for the Improvement of Early Implantation and Long Term Graft Survival in Pancreatic Islet Cell Transplantation by Induction of Angiogenesis with Gene Transfection of Vascuar Endothelial Growth Factor / 대한이식학회지

PURPOSE: Transplantation of pancreas islet has been worldwidely studied as a one of therapeutic modalities to achieve the insulin independence. We studied whether the expression of vascular endothelial growth factor (VEGF) on pancreas islets with liposomal VEGF gene transfer could improve the efficacy of early implantation and long term graft survival in pancreatic islet cell transplantation. METHODS: Syngenic pancreas islets were transplanted beneath the renal capsule. Islets were transfected with plasmid VEGF c-DNA using cationic liposome DMRIE-C. Glucose metabolism and histologic findings were compared between the groups transplanted with VEGF DNA containing islets (n=5) and the control group with (n=5) or without (n=4) local recombinant VEGF adminstration during islet transplant. RESULTS: Glucose was controlled at 5.5 days after transplantation in control group without r-VEGF adminstration, at 4 days in group with recombinant VEGF adminstration, and at 6.6 days in group with VEGF DNA transfected islets. Euglycemia was maintained over 150 days in control group. However, graft failure was developed in 22 days after transplantation in group with VEGF DNA transfected islet. Histologically there were severe infiltrations of neutrophil and lymphocyte in VEGF DNA transfected grafts from 5 days after transplantation. CONCLUSION: Although VEGF could be a favorable angiogenic factor in pancreas islet transplantation, VEGF expression following VEGF DNA transfection into islets could not increase the graft survival due to inflammatory process. More investigations are needed to clarify the mechanism on destructive process of islets after gene transfection into islets, and another approaches to get the effect of gene transfection should be followed.

In Vitro Analysis of Co-cultured Pancreatic Islet with Lymphocyte / 대한이식학회지

BACKGROUND: Transplantation of Pancreatic islet represents one of the most exciting treatment modalities for Type I diabetes mellitus. To achieve better graft survival, it is important to protect the graft from alloantigen-specific immune response. It was emphasized that islets, as with other forms of cellular transplants, have the potential advantage of being immunologically altered before transplantation, resulting in tolerance to the host without using long-term, nonspecific immunosuppression. The mixed islet-lymphocyte culture is an excellent tool to evaluate the immunogenicity of a pancreatic islets. Therefore, we co-cultured pancreatic islet and lymphocyte to investigate cytokine gene expression from the lymphocyte, and to investigate pancreatic islet viability and functional changes after co-culture. MATERIALS AND METHODS: Lewis rat islets were purified from pancreas by collagenase type XI and dextran gradient method. Afterwards, Lewis rat islets were co-cultured as a stimulator and Wistar rat lymphocyte as a reponder. As a control group lymphocyte alone and islet alone were cultured in a same condition. For estimating islets viability, we counted islet viability as an IEQ (Islet equivalent). For evaluation of islets function, insulin release assay was perfomed by RIA under the glucose challenge. used by RIA. We studied cytokines gene expression of cultured cells by reverse transcriptase polymerase chain reaction (RT-PCR). RESULT AND CONCLUSION: 1) Islet yield was 1229.8 420.1 per rat, and counted as 2615.4 548.2 IEQ per rat. 2) Islet viability was decreased gradually with the lapse of time, more rapid in allogenic co-culture group than islet alone or isogenic co-culture group. 3) Release of insulin increased until day 3, and then decreased gradually. Insulin release was positively correlated with glucose gradient. The amount of released insulin was greater in co-cultured group than islet alone group. 4) Interleukine-2 and interferron-gamma gene expression increased in allo co-culture group, however transforming growth factor-beta gene expression was not affected by co-culture.

In Vitro Analysis of Co-cultured Pancreatic Islet with Lymphocyte / 대한이식학회지

BACKGROUND: Transplantation of Pancreatic islet represents one of the most exciting treatment modalities for Type I diabetes mellitus. To achieve better graft survival, it is important to protect the graft from alloantigen-specific immune response. It was emphasized that islets, as with other forms of cellular transplants, have the potential advantage of being immunologically altered before transplantation, resulting in tolerance to the host without using long-term, nonspecific immunosuppression. The mixed islet-lymphocyte culture is an excellent tool to evaluate the immunogenicity of a pancreatic islets. Therefore, we co-cultured pancreatic islet and lymphocyte to investigate cytokine gene expression from the lymphocyte, and to investigate pancreatic islet viability and functional changes after co-culture. MATERIALS AND METHODS: Lewis rat islets were purified from pancreas by collagenase type XI and dextran gradient method. Afterwards, Lewis rat islets were co-cultured as a stimulator and Wistar rat lymphocyte as a reponder. As a control group lymphocyte alone and islet alone were cultured in a same condition. For estimating islets viability, we counted islet viability as an IEQ (Islet equivalent). For evaluation of islets function, insulin release assay was perfomed by RIA under the glucose challenge. used by RIA. We studied cytokines gene expression of cultured cells by reverse transcriptase polymerase chain reaction (RT-PCR). RESULT AND CONCLUSION: 1) Islet yield was 1229.8 420.1 per rat, and counted as 2615.4 548.2 IEQ per rat. 2) Islet viability was decreased gradually with the lapse of time, more rapid in allogenic co-culture group than islet alone or isogenic co-culture group. 3) Release of insulin increased until day 3, and then decreased gradually. Insulin release was positively correlated with glucose gradient. The amount of released insulin was greater in co-cultured group than islet alone group. 4) Interleukine-2 and interferron-gamma gene expression increased in allo co-culture group, however transforming growth factor-beta gene expression was not affected by co-culture.

Impact of Islet Purification in Canine Pancreas Islet Cell Transplantation

Purification of islets in pancreas islet cell transplantation has some potential advantages ,such as more safety, improved islet cell implantation, and reduced immunogenicity, compared with an unpurified pancreas islet cell transplantation. We evaluated the effect of islet cell purification on islet yields, hemodynamics, and graft outcome following intraportal canine pancreas islet cell transplantation. The baseline characteristics, including body weight, pancreas weight and collagenase recirculation time for the unpurified group(n=12) and the purified group(n=17) did not show any significant difference(P>0.05). The mean cell pellet volume before intraportal injection was 25.4 ml in the unpurified group and 7.2ml in the purified group(P0.05). Mean recovery rate of islet after purification was 66.8%. The portal pressure change after intraportal islet injection was significantly less in the purified group(16.2+/-11.3 cmH2O vs 6.2+/-2.0 cmH2O, P OR =70%, n=4 vs low purity <70%, n=13) in the purified group, showed significant hemodynamic stability in the high purity group, but no significant difference in the islet recovery rate between the low and high purity group(58.6% vs 61.7%). Glucose was controlled in 3 cases(25.0%) in the unpurified group and 7 cases(41.2%) in the purified group. Death due to portal hypertension occurred in 2 cases(16.7%) in unpurified group and 2 cases(11.7%) in the purified group. Interestingly, in the highly purified group, all the animals were alive with normoglycemia during the follow up period. We conclude that purified pancreas islet cell transplantation, especially in a highly purified group, has distinct hemodynamic advantages compared with unpurified islet transplantation following intraportal islet injection. However further research to develop the methods that will minimize the loss of islet yields during purification and enhance the purity is neccessary to achieve a successful islet transplantation with a long-term good result.

Study on the Immunologic Mechanism in the Xenogenic Transplantation / 대한면역학회지

Organ transplantation has become a' widely accepted treatment modality for end-stage organ disease. The shortage of allogenic donors for organ transplantation has brought about the necessity of xenotransplantation as an unlimited source of organ donation. However, organ transplantation between different species have never been successful because of hyperacute rejection. Although the mechanism of this phenomenon is not fully understood, many researchers believe that the natural antibodies present in the recipient's serum may bind to the graft and induce the activation of complement cascade triggering the process of hyperacute rejection. ...continue...