Y Namii

Clonal analysis of nodular parathyroid hyperplasia in renal hyperparathyroidism

Abstract. Although it is well known that chronic renal failure induces parathyroid hyperplasia, the pathogenesis and development of this parathyroid lesion in this disease are poorly understood. Histopathologically, there is progression from diffuse to nodular hyperplasia, and each nodule consists of a single cell type with aggressive proliferative potential. Pathophysiologic and clinical investigations have suggested that neoplastic tumors may emerge from nodular hyperplasia. In this study the clonality of parathyroid tissue in nodular and diffuse hyperplasia in renal hyperparathyroidism was analyzed by a method based on restriction fragment length polymorphism of the X chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. DNA of peripheral lymphocytes was screened in 43 women undergoing parathyroidectomy for advanced renal hyperparathyroidism, and 10 of these patients appeared to be heterozygous. Fourteen specimens from these patients were available for clonal analysis. The analysis showed that all four specimens of diffuse hyperplasia were polyclonal, whereas all seven specimens from nodules in nodular hyperplasia and all three samples representing parathyroid tissue removed from forearm because of graft-dependent recurrence were revealed to be monoclonal. It is likely that the clonal origin of each nodule is independent. These results suggest that in renal hyperparathyroidism parathyroid glands initially grow diffusely and polyclonally, and then the cells in the nodules are later transformed monoclonally and proliferate aggressively. From the present study it can be concluded that nodular hyperplasia represents monoclonal parathyroid neoplasia, which might explain why patients with nodular hyperplasia in renal hyperparathyroidism are refractory to medical treatment, requiring parathyroidectomy. To prevent recurrences, nodular hyperplastic tissue should not be left at surgery.

Evidence that the adenoviral vector containing the CTLA4-Ig gene improves transgene expression and graft survival

Abstract Rejection of organ allografts is dependent on T cell activation, which requires T-cell recepter engagement by antigen and costimulatory signals delivered by T-cell surface molecules such as CD28. CTLA4-Ig is a recombinant fusion protein that contains the extracellular domain of human CTLA4 (a homologue of CD28) fused to a human IgG 1 heavy chain. It has previously been shown to block the CD28-mediated costimulatory signal and to inhibit immune responses in vitro and in vivo. 1,2 Recently, the replication-defective adenovirus vector has been shown to be efficient in transferring exogenous genes into a variety of cells both in vitro and in vivo. 3 In this study, we examined the ability of adenovirus vector containing CTLA4-Ig (Adex/CTLA4-1g) to modify the rejection process observed in ACI to LEW rats liver transplantation.

Inhibitory effect on the establishment of hepatic metastasis by transduction of the tissue plasminogen activator gene to murine colon cancer.

Hepatic metastasis is a major factor in limiting the prognosis of patients with colon carcinoma. Recent investigations indicate a correlation between plasminogen activator profiles and hepatic metastasis. We examined the effectiveness of tissue plasminogen activator (tPA) gene therapy using a hepatic metastasis model of murine colon carcinoma. Murine colon carcinoma Colon 26 cells transduced with an MFGtPA retroviral vector (Colon 26/tPA) or an MFGLacZ retroviral vector (Colon 26/LacZ) were injected into the liver via the superior mesenteric vein of BALB/c mice, whose survival rates were checked daily. The mean survival rate of mice with hepatic metastasis induced by Colon 26/LacZ was 23.1 days, whereas that of mice with Colon 26/tPA was >100 days. The in vitro proliferation of Colon 26/tPA was comparable with that of Colon 26/LacZ, and antitumor immunity to wild-type Colon 26 cells was not induced after an intrahepatic injection of Colon 26/tPA. We suggest that transduction of the tPA gene to murine colon cancer is useful against the establishment of hepatic metastasis.

Adenovirus‐mediated gene transfer of CTLA4lg gene results in prolonged survival of heart allograft

Abstract It has been demonstrated that the administration of CTLA4Ig protein can induce the suppression of allograft and xenograft rejection. The purpose of this study is to determine the effect of adenovirus‐mediated gene transfer with CTLA4Ig gene on the transgene expression and suppression of alloimmune response in allogeneic cardiac transplantation of rats. Adenoviral vectors with β galactosidase or human CTLA4Ig cDNA (Adex/LacZ, Adex/hCTLA4Ig) were constructed. These vectors were transduced to the liver of Lewis (LEW) rats by intravenous injection. Then LEW rats received heterotopic cardiac transplantation from Dark Agouti rats. Experiments were performed using both CTLA4Ig gene transduction and/or immunosuppressant FK506. The transgene expression after adenovirus‐mediated transfer with CTLA4Ig cDNA lasted for several months, compared with several weeks after that in controls, which was proportional to the blood concentration of CTLA4Ig protein. By the administration of 1 × 109 PFU of Adex/hCTLA4Ig, the survival of cardiac grafts was significantly prolonged, compared with controls or the use of 1 × 108 PFU of Adex/hCTLA4Ig. In the rats with beating grafts over 100 days, the blood concentrations of CTLA4Ig were undetectable. The combination therapy using a low titer Adex/hCTLA4Ig and low‐dose of FK506 was synergistically effective on this cardiac transplantation model. In conclusion, adenovirus‐mediated gene transfer with CTLA4Ig gene was efficient for the prolongation of both transgene expression and allograft survival.

Comparative study of antibody removal before pig-to-baboon and human ABO-incompatible renal transplantation

THE MECHANISM of rejection following pig-to-baboon xenotransplantation (XenoTx) has been compared with that following ABO-incompatible allotransplantation (ABO-Tx), because both involve natural antibody (Ab) against carbohydrate epitopes. Recently, ABO-Tx has shown excellent outcomes following pretreatment with double filtration plasmapheresis (DFPP). We compared changes in anti-donor Ab and histopathology of the renal graft between ABO-Tx and XenoTx. Before renal transplantation (Tx), DFPP was performed on days 26, 24, 22, and 21 in ABO-Tx (n 5 25) and on days 22 and 0 (immediately before reperfusion) in XenoTx (n 5 4). Cyclosporine (or tacrolimus), steroid, and cyclophosphamide were administered to both patients and baboons. Baboons were divided into two groups according to the extent of Ab removal: XenoTx Group I, Ab removal to the same extent as in ABO-Tx (n 5 2); XenoTx Group II, nearly total removal of Ab (n 5 2). Anti-A/B and anti-pig IgM (IgG) Abs were measured by flow cytometry. Twenty-five patients underwent ABO-Tx. All grafts remain functioning, except in two cases where the grafts were lost from recurrence of the original disease after 6 months and from chronic rejection after 5 years, although 3 had reversible humoral rejection. In pig-to-baboon renal transplantation without DFPP pretreatment, hyperacute rejection (HAR) was observed 60 and 90 minutes after transplantation. In XenoTx Group I, no HAR was observed. Grafts were rejected on days 6 and 7, and showed coagulative necrosis caused by vascular rejection; biopsies on days 1, 4, and 5 showed only minimal changes. In XenoTx Group II, the baboons could not tolerate the extra DFPP and died 4 hours and 2 days after transplantation. Immunohistochemical study of the grafts showed IgM, IgG, and C3 binding to endothelial cells within 1 hour post-Tx in XenoTx Group I, and IgM and C3 binding as early as 1 hour post-Tx in XenoTx Group II, while no Ab binding was detected at any time (1 hour to 2 years) after ABO-Tx. The same extent of Ab removal in XenoTx Group I as in ABO-Tx inhibited HAR but could not prevent delayed vascular rejection. Even when anti-pig Ab was depleted by 98% (XenoTx Group II), the small amount of remaining Ab still had the ability of binding to the pig graft within 1 hour. For successful xenografting, we suggest that the concentration of xenoantigen epitopes expressed on pig grafts may need to be reduced by genetic engineering to the level of the ABO antigens on human kidneys.

Adenovirus-mediated gene transfer of CTLA4Ig gene results in prolonged survival of heart allograft.

It has been demonstrated that the administration of CTLA4Ig protein can induce the suppression of allograft and xenograft rejection. The purpose of this study is to determine the effect of adenovirus-mediated gene transfer with CTLA4Ig gene on the transgene expression and suppression of alloimmune response in allogeneic cardiac transplantation of rats. Adenoviral vectors with beta galactosidase or human CTLA4Ig cDNA (Adex/LacZ, Adex/hCTLA4Ig) were constructed. These vectors were transduced to the liver of Lewis (LEW) rats by intravenous injection. Then LEW rats received heterotopic cardiac transplantation from Dark Agouti rats. Experiments were performed using both CTLA4Ig gene transduction and/or immunosuppressant FK506. The transgene expression after adenovirus-mediated transfer with CTLA4Ig cDNA lasted for several months, compared with several weeks after that in controls, which was proportional to the blood concentration of CTLA4Ig protein. By the administration of 1 x 10(9) PFU of Adex/hCTLA4Ig, the survival of cardiac grafts was significantly prolonged, compared with controls or the use of 1 x 10(8) PFU of Adex/hCTLA4Ig. In the rats with beating grafts over 100 days, the blood concentrations of CTLA4Ig were undetectable. The combination therapy using a low titer Adex/hCTLA4Ig and low-dose of FK506 was synergistically effective on this cardiac transplantation model. In conclusion, adenovirus-mediated gene transfer with CTLA4Ig gene was efficient for the prolongation of both transgene expression and allograft survival.

Suppression of Antigen-Antibody Reaction in Xenotransplantation

TTENTION has been focused on the potential usageof animal organs, as the shortage of organ donors isincreasing. Before the start of clinical xenotransplantation,however, certain Immunologic, physiologic, and infectiousproblems must be solved. Advancements in genetic engi-neering have made it possible to overcome severe xenograftrejection. Hyperacute rejection has reportedly been sup-pressed when organs from the transgenic pigs expressinghuman complement regulatory proteins such as DAF,CD59, or MCP were transplanted into primates.