Evidence presented in this paper indicates that nitric oxide (NO), generated by a nonspecific "wound"-type of inflammation, is an important mediator of the early dysfunction of transplanted islets in rodents. Although allogeneic islets stimulate NO production to a greater degree than syngeneic islets, the amounts of NO produced after either are significantly elevated above baseline. Inhibition of NO production by N(G)-monomethyl-L-arginine (NMA), markedly decreases the time needed to restore euglycemia after intraportal transplantation of syngeneic islets in diabetic rats. The dose of NMA used was not observably toxic, with no significant changes in blood pressure, hepatic artery blood flow, serum hepatic enzyme levels, or in weight compared with control animals. In rat recipients of intraportal syngeneic transplants, evidence that NO is produced at the site of implantation includes (1) an early and transient increase in posttransplant hepatic vein nitrate levels (pretransplant, 90 microM; 24 hr, 230 microM; 48 hr, 250 microM; 72 hr, 170 microM; and 96 hr, 140 microM), (2) concurrent appearance of inducible NO synthase mRNA in liver extracts, and (3) immunohistochemical localization of inducible NO synthase within the transplanted islets. Suppression of NO production or inhibition of NO activity is a potential strategy to increase the early function and engraftment transplanted islets in the clinical setting.

Experimental animal models of cancer have provided invaluable information about the mechanisms of tumor development, progression and metastasis. Despite the high prevalence of prostate cancer and the burden of disease on society, few laboratory and preclinical models of prostate cancer have been developed that approximate the spectrum of tumor progression and metastatic disease. Notwithstanding improvements in the diagnosis and treatment of localized prostate cancer in recent years, metastatic disease presents a formidable challenge and is lethal for men with advanced prostate cancer. 1 This has spurred efforts to understand the mechanisms responsible for the switch from the indolent to the metastatic tumor phenotype. 2 Elucidation of the molecular pathways that regulate metastasis depends on the availability of clinically relevant in vivo animal models of prostate cancer that serve as laboratory surrogates to assess the efficacy of therapeutic or preventive modalities in vivo.Xenograft models of prostate cancer that mimic features of the human disease have been developed in immunodeficient mice. 3 These models have been critical in the identification of genes involved in tumor growth and progression and are employed extensively for testing novel drug, irradiation and emerging gene therapies. Moreover, orthotopic implantation of prostate tumor cells or tissue in nude mice resulted in the derivation of sublines with the capacity for enhanced tumor take and increased lymph node and distant metastasis. 4 -7 Transgenic and reconstitution models of prostate cancer have been used to study prostate carcinogenesis, metastasis, and to test novel therapeutic and chemoprevention regimens. 8,9 The mouse prostate reconstitution (MPR) model utilizes fetal mouse urogenital sinus tissue that is infected ex vivo with recombinant retroviruses then implanted into mice. 9 Transgenic mouse models of prostate cancer have been developed in which the rat probasin promoter targets prostate epithelial cell-specific expression of SV40 T antigen (Tag) leading to the development of spontaneous autochthonous prostate cancer that is regulated temporally and spatially. 10,11 In the transgenic adenocarcinoma mouse prostate (TRAMP) model, all males develop primary prostate tumors and metastases to distant sites between 18 -24 weeks of age. 12 We demonstrated previously that treatment of mice bearing ectopic transplantable TRAMP-C1 tumors with flt3 ligand (L), a hematopoietic growth factor, resulted in transient tumor regression without long-term antitumor immunity. 13 We describe the establishment of an orthotopic model of primary prostate cancer and lymph node metastasis in immunocompetent mice. To establish the use of this model for immunotherapy, the therapeutic efficacy of flt3L was assessed in mice with orthotopic, metastatic prostate cancer. MATERIAL AND METHODS Cell lines and cultureThe parental transplantable epithelial prostate cancer cell line, TRAMP-C1, derived from a prostate adenocarcinoma in transAbbreviations: TRAMP, transgenic a...

A novel orthotopic metastatic model of mouse prostate cancer was developed using MHC-negative TRAMP-C1P3 (transgenic adenocarcinoma of mouse prostate) cells derived by serial passage of the parental TRAMP-C1 line in mouse prostate glands. TRAMP-C1P3 cells grew efficiently in mouse prostate glands and reproducibly metastasized to draining lymph nodes. Using this model, we show that Fms-like tyrosine kinase-3 ligand (flt3-L) dramatically inhibited growth of preexisting orthotopic TRAMP-C1P3 tumors and the development of metastatic disease. Mice remained in remission for several months following termination of flt3-L treatment but eventually relapsed and died of progressive disease. flt3-ligand treatment induced a pronounced mixed inflammatory cell infiltrate that consisted of CD8alpha-CD4- dendritic cells (CD11c+), macrophages, granulocytes (Gr-1+) and to a lesser extent T cells (CD4+ and CD8+). Dendritic cells isolated from TRAMP-C1P3 tumors were phenotypically immature (CD11c+ B7.2-I-A-CD40-), and this phenotype was also predominant in peripheral organs of mice treated with flt3-L alone or in combination with the DC maturation factor, CD40-L. Diminished expression of TCR-beta, CD3-epsilon, and CD3-zeta was also observed on intratumoral T cells, although these signaling proteins were reexpressed following in vitro culture with IL-2. The TCR/CD3 complex remained intact on peripheral T cells except in mice treated with flt3-L where CD3-zeta loss was observed. In contrast to alphabeta-T cells, tumor-infiltrating gammadelta-T cells maintained expression of their antigen receptors but not CD3epsilon. Thus, TRAMP-C1P3 tumors quickly establish a microenvironment that profoundly diminishes expression of molecules critical for normal dendritic cell and T cell function, thus limiting the efficacy of flt3-L and CD40-L immunotherapy. Overall, these data suggest that long-term cures of established MHC-negative tumors may not be achieved until therapeutic interventions are engineered to overcome this immunosuppressive microenvironment.

Postsurgery, pancreas transplantation results in alterations of carbohydrate metabolism. Additionally, immunosuppressive therapy impacts on glucose regulation. We evaluated the hormonal and metabolic responses of pancreas allografts, utilizing the hyperglycemic clamp technique coupled with the tritiated glucose methodology, in 11 volunteers who had received simultaneous pancreas-kidney transplantation (P-K) with systemic drainage. Their responses were compared with seven volunteers who had received only a kidney (K) graft and with seven normal control (C) volunteers. Although basal glucose and hepatic glucose output were similar in all three groups, basal insulin, C-peptide, glucagon, and pancreatic polypeptide were highest in the P-K group and lowest in normal subjects. During hyperglycemia, all groups showed a similar characteristic, initial complete suppression of hepatic glucose production, with recovery followed by a later suppression. Peripheral glucose uptake was similar in P-K and C subjects but decreased in K patients. Systemic insulin levels were fourfold higher in the pancreas transplant patients than in healthy subjects. Thus, under basal and hyperglycemic stimulation, 1) hepatic glucose homeostasis is regulated normally, even with pancreatic drainage into the systemic circulation; 2) overall glucose disposal is normal in P-K patients because of marked hyperinsulinemia; and 3) there is loss of tonic inhibition of endocrine pancreatic function secondary to pancreatic denervation.