In vivo localization of [(111)In]-DTPA-D-Phe1-octreotide to human ovarian tumor xenografts induced to express the somatostatin receptor subtype 2 using an adenoviral vector.

Adenoviral vectors, encoding genes for cell surface antigens or receptors, have been used to induce their high level expression on tumor cells in vitro and in vivo. These induced antigens and receptors can then be targeted with radiolabeled antibodies or peptides for potential radiotherapeutic applications. The purpose of this study was to determine a dosing schema of an adenoviral vector encoding the human somatostatin receptor subtype 2 (AdCMVhSSTr2) for achieving the highest tumor localization of [(111)In]-DTPA-D-Phe1-octreotide, which binds to this receptor, in a human ovarian cancer model as a prelude to future therapy studies. AdCMVhSSTr2 was produced and used to induce hSSTr2 on A427 human nonsmall cell lung cancer cells and on SKOV3.ipl human ovarian cancer cells in vitro, as demonstrated by competitive binding assays using [125I]-Tyr1-somatostatin and [(111)In]-DTPA-D-Phe1-octreotide. Mice bearing i.p. SKOV3.ip1 tumors administered 1 x 10(9) plaque-forming units of AdCMVhSSTr2 i.p. 5 days after tumor cell inoculation, followed by an i.p. injection of [(111)In]-DTPA-D-Phe1-octreotide 2 days later, showed a range of 15.3-60.4% median injected dose/gram (ID/g) in tumor at 4 h after injection compared with 3.5% ID/g when [125I]-Tyr1-somatostatin was administered and 0.3% ID/g when the negative control peptide [125I]-mIP-bombesin was administered. Mice administered a control adenoviral vector encoding the gastrin-releasing peptide receptor did not have tumor localization of [(111)In]-DTPA-D-Phe1-octreotide (<1.6% ID/g), demonstrating specificity of [(111)In]-DTPA-D-Phe1-octreotide for the AdCMVhSSTr2 induced tumor cells. In another set of experiments, the tumor localization of [(111)In]-DTPA-D-Phe1-octreotide was not different 1, 2, or 4 days after AdCMVhSSTr2 injection (31.8, 37.7, and 40.7% ID/g, respectively; P = 0.88), indicating that multiple injections of radiolabeled peptide can be administered with equivalent uptake over a 4-day period. [(111)In]-DTPA-D-Phe1-octreotide tumor localization in animals administered AdCMVhSSTr2 on consecutive days or 2 days apart was 22.4% ID/g and 53.2% ID/g, respectively (P = 0.009) when [(111)In]-DTPA-D-Phe1-octreotide was given 1 day after the second AdCMVhSSTr2 injection. There was no difference in [(111)In]-DTPA-D-Phe1-octreotide localization after a single AdCMVhSSTr2 injection (40.7% ID/g) or two injections of AdCMVhSSTr2 given 1 (45.9% ID/g) or 2 (53.2% ID/g) days apart, where [(111)In]-DTPA-D-Phe1-octreotide was given in each case 4 days after the first AdCMVhSSTr2 injection (P = 0.65). Therefore, two AdCMVhSSTr2 injections did not increase [(111)In]-DTPA-D-Phe1-octreotide tumor localization compared with one injection, which eliminates concerns about an immune response to a second dose of AdCMVhSSTr2. This will be the basis for a therapeutic protocol with multiple administrations of an octreotide analogue labeled with a therapeutic radioisotope.

Evidence that rapamycin inhibits interleukin-12-induced proliferation of activated T lymphocytes.

Interleukin 12 is a heterodimeric cytokine involved in the regulation of natural killer cell and T lymphocyte responses. In previous studies, we found that IL-12 induces proliferation of T cells only after co-stimulation with lectin, alloantigen, or anti-CD3 antibody. The IL-2-mediated proliferation of long-term T cell lines generated in this fashion is typically insensitive to the immunosuppressive agent, cyclosporine but sensitive to rapamycin. In this study, we examined the effect of cyclosporine and rapamycin on T cells responsive to IL-12. For long-term cultured T cell lines stimulated with phytohemagglutinin, alloantigen, or solid-phase anti-CD3 antibody, rapamycin blocked IL-12-induced proliferation to background levels. Culture in cyclosporine produced minimal inhibition of IL-12-induced T cell proliferation. Freshly isolated CD3+ cells did not proliferate in response to IL-12, nor did culture of these cells in IL-12 lead to upregulation of IL-2 receptor. These data suggest that the effect of IL-12, an important growth regulator for activated T lymphocytes, may involve late cellular activation events.

Phosphorylation of the enantiomers of the carbocyclic analog of 2'-deoxyguanosine in cells infected with herpes simplex virus type 1 and in uninfected cells. Lack of enantiomeric selectivity with the viral thymidine kinase.

CdG, the carbocyclic analog of 2'-deoxyguanosine, is active against herpes, hepatitis B, and human cytomegaloviruses. We have studied the interaction of the tritiated enantiomers of CdG with the herpes simplex virus type 1-specific thymidine kinase (HSV-1 TK) and have examined their metabolism in uninfected and HSV-1-infected cells. D- and L-CdG were equally effective competitive inhibitors of the phosphorylation of thymidine (dThd) by the partially purified HSV-1 TK (Ki values were 2.1 and 3.4 microM, respectively) and were also equal as substrates (Km values were 17 and 26 microM, respectively, and Vmax values of the enantiomers were equal and about 50% greater than the Vmax for dThd). The partially purified enzyme preparation, which contained cellular nucleotide kinase activities (pyruvate kinase also was present in the assay medium), converted D-CdG almost exclusively to the triphosphate and L-CdG almost exclusively to the monophosphate. Similarly, in virus-infected cells the D-enantiomer was converted predominantly to the triphosphate and the L-enantiomer predominantly to the monophosphate. In uninfected cells the results were qualitatively similar. In CEM cells deoxycytidine (dCyd) kinase (EC 2.7.1.74) seemed to be the enzyme principally responsible for the phosphorylation of both enantiomers, as shown by competition studies. Thus, both the HSV-1 TK and cellular dCyd kinase (of CEM cells) showed no selectivity for the enantiomers of CdG. This lack of enantiomeric specificity has obvious implications for the design of inhibitors of both viral proliferation and cellular metabolism.

The New England Organ Bank--lessons from running a regional organ bank.

1. The NEOB is a very large organ procurement organization (OPO) recovering multiple organs and tissues on behalf of 15 transplant centers serving 11.5 million people scattered over 6 states. 2. The database maintained by the New England Organ Bank (NEOB) demonstrates a changing pattern of donors within our region. Trauma is decreasing as a cause of death and the age of donors is steadily increasing. 3. A flexible system for the allocation and distribution of kidneys is described. This system emphasizes waiting time as the primary criterion for allocation. This emphasis has not disadvantaged the highly sensitized patient and is equitable for minority recipients. The use of the longest waiting unsensitized patient to assign donors to transplant centers maintains the patient-based nature of the system, while allowing transplant centers to recover kidneys for their own patients and reducing ischemic time. The system is highly adaptable to a variety of local situations. 4. Extensive public education and research projects not feasible for small organizations are made possible through the resources available to a large OPO.

Prevalence of hepatitis C virus RNA in organ donors positive for hepatitis C antibody and in the recipients of their organs.

BACKGROUND: There is a high prevalence of liver disease among the recipients of organs from donors with antibodies to hepatitis C virus (HCV). We undertook a study to determine the frequency of persistent HCV infection, as indicated by the presence of HCV RNA, among both cadaveric organ donors positive for antibodies to HCV (anti-HCV) and the recipients or organs from these donors. METHODS: Serum samples from donors and recipients were tested for HCV RNA with the reverse transcriptase polymerase chain reaction, with use of primers from the 5' untranslated region of the HCV genome, and for anti-HCV with the first-generation enzyme-linked immunosorbent assay (ELISA) and two second-generation tests. RESULTS: HCV RNA was detected in 9 of the 11 organ donors (82 percent) with a positive first-generation ELISA for anti-HCV. Among the organ recipients, the prevalence of HCV RNA increased after transplantation: 7 of 26 patients (27 percent) had positive samples before transplantation, as compared with 23 of 24 patients (96 percent) after transplantation (P less than 0.001). Among 13 recipients who were HCV RNA-negative before receiving organs from the nine HCV RNA-positive donors, HCV infection was detected in all 13 after transplantation, and anti-HCV developed in 8 (62 percent). On the basis of a positive test for HCV RNA, the maximal sensitivity of the three anti-HCV tests was 57 percent (positive in 4 of 7 patients with end-stage organ failure) before transplantation and 70 percent (positive in 16 of 23 patients) after transplantation. CONCLUSIONS: Nearly all the recipients of organs from anti-HCV-positive donors become infected with HCV. The current tests for anti-HCV antibodies underestimate the incidence of transmission and the prevalence of HCV infection among immunosuppressed organ recipients.

Allorecognition in T-cell receptor (beta-chain) transgenic mice.

Recent advances in knowledge of the structure of the T-cell receptor and of major histocompatibility complex (MHC) molecules have increased our understanding of the nature of their interaction in the immune response. Nevertheless, it remains unclear how the T-cell receptor recognizes foreign MHC molecules in the process of graft rejection. One approach to this problem is to characterize the alloreactivity of a given T-cell receptor. We have chosen to take this approach in vivo by examining patterns of rejection of vascularized heart allografts in transgenic mice carrying a rearranged T-cell receptor-beta-chain gene, in which essentially all alpha beta T cells bear the rearranged gene product. Heterotopic heart allografts were performed in transgene-positive and transgene-negative recipients. The data show that transgene-positive mice will reject fully allogeneic grafts of three different haplotypes after a modest delay, but will not reject grafts from F1 mice that bear H-2 antigens from these same haplotypes and from the recipient strain. Transgene-negative animals reject all grafts promptly. These results suggest that the restricted T-cell receptor repertoire expressed by transgene-positive recipients affects their ability to respond to an alloantigen as expressed on a vascularized graft and that this response is influenced by the presence of self-MHC molecules on the graft.

Transmission of hepatitis C virus by organ transplantation.

BACKGROUND: Liver disease is a frequent and major complication after organ transplantation. We sought to determine whether hepatitis C virus (HCV) is transmitted by organ transplantation and whether it causes post-transplantation liver disease. METHODS: Serum samples from all cadaver organ donors to the New England Organ Bank between 1986 and 1990 were screened retrospectively for antibodies to HCV (anti-HCV) by enzyme-linked immunosorbent assay (ELISA). We reviewed the hospital records of all recipients of organs from anti-HCV-positive donors for evidence of liver disease. Serum samples from recipients obtained before transplantation and during follow-up were analyzed for anti-HCV. RESULTS: Of 716 organ donors, 13 (1.8 percent) were positive for anti-HCV. Their organs (19 kidneys, 6 hearts, and 4 livers) went to 29 recipients. Non-A, non-B hepatitis developed after transplantation in 14 of the 29 (48 percent), for a prevalence 7.4 times the 6.5 percent prevalence after transplantation from untested donors that was previously reported by two institutions in the organ bank (P less than 0.0001). The liver disease began a mean of 3.8 months after transplantation and became chronic in 12 patients; the other 2 had subfulminant hepatic failure. Liver disease was more frequent in the patients who had received antilymphocyte preparations (P = 0.04). HCV was the cause of the post-transplantation liver disease in 12 of the 13 recipients (92 percent) for whom serum samples were available. Anti-HCV was detected by ELISA in eight and enzyme immunoassay in one; in three others, HCV RNA was detected by polymerase chain reaction in serum samples obtained after transplantation. CONCLUSIONS: Organ transplantation can transmit hepatitis C. This raises serious questions about the continued acceptance of organs from donors positive for anti-HCV.

Ten-year experience with cyclosporine as primary immunosuppression in recipients of renal allografts.

Cyclosporine has been used as primary immunosuppression in renal allograft recipients in our unit for the past decade. The overall clinical experience and long-term effects of the agent are reviewed. There were 461 consecutive recipients of kidney grafts; 379 received grafts from cadaver donors (CAD) and 82 from living related donors (LRD). Four separate clinical protocols were used sequentially using progressively decreasing doses of CyA; azathioprine was added in group 4 recipients of LRD grafts, and in patients receiving secondary CAD grafts (group 5). The patient mortality rate was less than 5%, with sepsis being the prime contributor. The majority of kidney grafts were lost within the first 2 months after operation; those that never functioned were found almost invariably to have been irreversibly rejected. During the subsequent years of follow-up, attrition of CAD grafts was significantly greater than LRD grafts. In contrast, the attrition rate of primary and secondary CAD grafts was the same after the first 3 months, emphasizing the importance of early immunologic graft destruction. Primary nonfunction occurred in 49% of CAD kidneys and 17% of LRD grafts; however 71% of initially nonfunctioning LRD grafts never functioned at all compared to 34% of CAD grafts, the majority of such organs undergoing fulminate rejection. Individual graft loss after 1 year was almost inevitably due to chronic rejection; there were no differences in long-term allograft function among the treatment groups. Although CyA has improved overall results of kidney transplantation, chronic rejection remains a major unresolved problem.