Clinicopathologic features of retinoblastoma after primary chemoreduction

BACKGROUND: Cytotoxic T cells can induce target cell lysis and apoptosis by different pathways. The interactions of CD95 antigen (Fas) with its ligand (CD95L) and of tumor necrosis factor (TNF)-alpha with its receptor (TNF-R1) lead to apoptotic cell death. Recently, conflicting studies have been published concerning the expression and the role of CD95L in allograft rejection and tolerance. METHODS: In this study, the intragraft expression of CD95/CD95L and TNF-alpha and the frequency and distribution of apoptotic cells were compared in a model of heterotopic cardiac allograft in the rat in which recipients were either not treated (acute rejection) or pretreated with donor-specific blood transfusion (tolerant). RESULTS: In the acutely rejected allografts, a peak in the expression of CD95L and TNF-alpha and in the number of apoptotic cells was observed during the first week after transplantation; apoptotic cells were confined to graft-infiltrating cells. In the tolerated allografts, however, levels of graft-infiltrating cell apoptosis and CD95L and TNF-alpha expression during the same period of time were dramatically lower. The expression of Fas was constitutive and was not modulated during acute rejection or tolerance. CONCLUSION: This down-regulation of CD95L and TNF-alpha in allografts rendered tolerant by donor-specific transfusion suggests a role for apoptosis-inducing pathways in acute allograft rejection.     

Soluble and cell surface ICAM-1 as markers for disease activity in multiple sclerosis

Infiltration of a transplanted organ by host lymphoid cells is the hallmark of acute rejection. However, after intestinal transplantation, physiological lymphocyte migration may lead to host cell infiltration of the graft even in the absence of rejection. It is unclear whether this lymphocyte migration also involves the intraepithelial compartment of the graft or whether infiltration there is indicative of acute rejection. We demonstrate here that host cell infiltration of the intestinal mucosa occurs both during acute rejection of a small bowel allograft and, to a lesser extent, when rejection is prevented by immunosuppression with FK506. The infiltrating host cells consisted of CD3+ T cells with a predominant CD4-CD8+ phenotype resembling intraepithelial lymphocytes (IELs). Functional studies showed that the nonspecific cytolytic activity of IELs was not affected by acute rejection or by immunosuppression with FK506. These findings indicate that host cell infiltration of the intestinal mucosa does not connote an ongoing acute rejection. Furthermore, the decreased mucosal barrier function during acute rejection of intestinal allgrafts is probably not due to impaired cytolytic activity of IELs.     

Limb allograft in rats: studies on optimal maintenance dose of FK506 and development of graft-versus-host-disease (GVHD)

The optimal dose of FK506 on rat limb allograft was investigated across the BN-to-F344 histocompatibility barrier. BN limb allografts were rejected in untreated F344 hosts within 11 +/- 1 days (mean +/- SD) after operation. A single injection of 0.5 mg/kg, 1.0 mg/kg, or 2.0 mg/kg of FK506 on the day of transplantation significantly prolonged graft survival, mean survival times (MST) based on gross sign of skin rejection were 16 +/- 1 days, 19 +/- 1 days, 21 +/- 1 days, respectively. Maintenance doses of 0, 0.25, 0.5, 1.0, or 2.0 mg/kg/week of FK506 after a single administration of 10 mg/kg of FK506 on the day of limb allograft prolonged the graft survival, 63 +/- 10, 68 +/- 20, 87 +/- 23, 98 +/- 30, 136 +/- 20 days, respectively, and showed no evidence of infection or toxic side effects. The regimen of lower maintenance dose of FK506, however, developed chronic GVHD. In the second set of experiments, development of peripheral blood chimeras was studied in PVG-to-ACl limb allograft model. A single injection of 50 mg/kg of the day of transplantation prolonged graft survival and MST was 154 days. The average rates of peripheral blood chimeras were 2 to 6% and there was no relationship between graft survival and peripheral blood chimeras. However, GVHD developed in one of the six recipients at 201 days after operation. In the similar experiments, grafts were irradiated before operation. Peripheral blood chimeras was not observed in there experiments and GVHD was not developed in 300 days after operation. These data suggest that FK506 is quite effective for rat limb allograft survival in dose-dependent manner and GVHD could be prevented by graft irradiation before operation.     

Contribution of Fas ligand to cardiac allograft rejection

Effector mechanisms for allograft injury remain unclear. In the present study, we verified the contribution of Fas and Fas ligand (FasL) to cardiac allograft rejection by utilizing the Fas-deficient lpr or FasL-deficient gld mice as the donor or recipient. Cardiac myocytes prepared from normal mice, but not those from lpr mice, constitutively expressed Fas and were susceptible to FasL-mediated lysis. Survival of cardiac allografts was substantially prolonged when gld or lpr mice were used as the recipient. In contrast, cardiac allografts from lpr mice were normally rejected without a delay. Histological examination of the grafts in the gld or lpr recipients demonstrated a lesser cellular infiltration and much milder myocyte damage. Proliferative response and cytotoxic T lymphocyte induction against the donor-type alloantigens were not impaired in the gld or lpr recipients. These results indicate a substantial contribution of FasL to cardiac allograft rejection, independent of Fas in the grafts. This ralses a possibility that FasL may be more generally involved in tissue damage associated with various diseases than expected from the expression of Fas in the target organs.     

A new rat infection-heart transplant model: effect of infection on graft survival studies

Considerable progress has been made in survival rates of heart transplant recipients; however, infections continue to be a major cause of death after transplantation. Although infection itself appears to cause immunologic suppression in some nontransplantation studies, the lack of an infection-transplant animal model has limited further investigation of this observation. We evaluated the utility of a heterotopic rat infection heart-transplant model by studying the effect of infection and limited administration of two immunosuppressive agents, cyclosporine and FK506, on allograft rejection and survival. Lewis rats received ACI heart allografts, and intraperitoneal infection was induced by cecal ligation. Infection was confirmed by blood and ascitic fluid cultures. Results showed that graft survival was slightly, but significantly, higher (p < 0.05) in group II (transplantation with infection) when compared to the control group I (transplantation only). Histologic rejection scores were less (p < 0.05) in group II 6 days after transplantation. The second phase of the study compared the effect of infection after transplantation in rats given a 1-week course of cyclosporine or FK506, which were discontinued after the induction of infection. Although the cyclosporine group had prolonged survival when compared to the FK506 group (p < 0.05), the respective infection groups receiving immunosuppression revealed no significant difference in allograft survival or histologic rejection scores when compared to the control groups. In this preliminary study, infection without immunosuppression resulted in a slight, but statistically significant, increase in allograft survival and reduced acute cellular rejection. In those groups receiving immunosuppressive agents, no additive immunosuppressive effect was attributable to bacterial infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tolerance induction ameliorates allograft vasculopathy in rat aortic transplants. Influence of Fas-mediated apoptosis

Based on successful induction of donor-specific unresponsiveness by alloantigenic stimulation in several animal models of acute rejection, we hypothesized that similar immune manipulations would also inhibit the evolution of chronic rejection and transplant vasculopathy. To induce immune tolerance, DA rats received a PVG heart allograft and were immunosuppressed with cyclosporine for 30 d. At day 100 the animals were challenged with a PVG aortic allograft after either 1 or 18 h of cold ischemia. 8 wk after the aortic transplantation, the grafts were investigated for morphological changes, infiltrating cells, apoptosis, and Fas-Fas ligand expression. Control allografts showed advanced transplant arteriosclerosis, whereas tolerance-induced aortic allografts displayed reduced neointimal formation, less medial atrophy, fewer apoptotic cells, and fewer Fas- and FasL-expressing cells. Prolonged ischemic storage time did not profoundly alter the morphological changes of the allografts. Fas expression was found in T cells, macrophages, vascular smooth muscle cells, and endothelial cells, whereas FasL was expressed mainly by T cells and macrophages. FasL mRNA expression was evident throughout the entire allograft wall. In conclusion, induction of allospecific tolerance can effectively prevent transplant arteriosclerosis. Cold ischemia damage does not abrogate the beneficial effect of tolerance, but creates a separate identity of mainly endothelial lesions. Furthermore, Fas-mediated apoptosis appears to be involved in the pathological lesions seen in chronic rejection.     

Apoptosis within spontaneously accepted mouse liver allografts: evidence for deletion of cytotoxic T cells and implications for tolerance induction

MHC-mismatched liver grafts are accepted spontaneously between many mouse strains. The underlying mechanism(s) is unclear. In the B10 (H2(b)) to C3H (H2(k)) strain combination used in this study, donor T cells within the liver were rapidly replaced within 2 to 4 days of transplantation with those of the recipient. Freshly isolated liver graft-infiltrating cells harvested on days 4 and 7 exhibited strong CTL responses against donor alloantigens. CTL activity was reduced substantially, however, by day 14, although levels of CTL precursors in the spleen and liver remained high. Examination of the liver allografts by in situ terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick end labeling on days 4, 7, and 14 after transplantation revealed prominent apoptotic cells dispersed throughout the nonparenchymal cell population. When acute liver allograft rejection was induced by administration of IL-2 from days 0 to 4 post-transplant (median survival time, 5 days), apoptotic activity (day 4) was reduced substantially, whereas CTL activity was enhanced. Nonparenchymal cells isolated from allografts of unmodified recipients 4, 7, and 14 days after transplantation exhibited significantly higher DNA fragmentation after 18-h culture than cells from liver isografts. Moreover, the level was 4 to 5 times higher than that of cells from IL-2-treated mice (on day 4). These observations suggest that T cell deletion, not regulation, may be responsible for spontaneous liver allograft acceptance. The molecular recognition events that cause apoptosis of infiltrating T cells and why this occurs within liver grafts, but not heart or skin grafts, remain to be elucidated.     

Clinical and histopathologic examination of renal allografts treated with tacrolimus (FK506) for at least one year

BACKGROUND: We clinically and pathologically analyzed renal allografts from 1 9 renal transplant patients treated with tacrolimus (FK506) for more than 1 year. METHODS: Twenty-six renal allograft biopsy specimens from 1 9 renal transplant patients who underwent transplantations between 1991 and 1993 were evaluated. Thirteen biopsies were performed from stable functioning renal allografts with informed consent (nonepisode biopsy) and the other 13 were from dysfunctional renal allografts with a clinical indication for biopsy (episode biopsy). RESULTS: The main pathologic diagnoses (some overlap) were acute rejection (AR; n = 4), chronic rejection (CR; n=5), AR+CR (n =4), recurrent IgA nephropathy (n =5), normal findings (n =2), minimal-type chronic FK506 nephropathy (n = 9), and mild-type FK506 nephropathy (n = 11). Of the nonepisode biopsies, 7 and 4 biopsies showed minimal-type and mild-type chronic FK506 nephropathy, respectively. Chronic FK506 nephropathy consisted of rough and foamy tubular vacuolization (5 biopsies), arteriolopathy (angiodegeneration of the arteriolar wall; 20 biopsies), focal segmental glomerulosclerosis (4 biopsies) and the striped form of interstitial fibrosis (11 biopsies). The serum creatinine levels of patients in the mild-type chronic FK506 nephropathy group, which included 7 episode biopsies, were statistically higher than those in the minimum-type chronic FK506-nephropathy group (P< 0.001). CONCLUSIONS: This study demonstrates that chronic FK506 nephropathy consists primarily of arteriolopathy manifesting as insudative hyalinosis of the arteriolar wall, and suggests that mild-type chronic FK506 nephropathy is a condition which may lead to deterioration of renal allograft function.     

FK-506 delays corneal graft rejection in a model of corneal xenotransplantation

FK-506 is a relatively new immunosuppressant similar in action to cyclosporine A, but is much more potent. Its primary action is against T lymphocytes, the major cellular component in corneal allograft rejection. The purpose of this study was the evaluation of the ability of topical and systemic FK-506 in preventing corneal xenograft rejection in an experimental animal model. Cross-species xenotransplants were used as the most vigorous stimulus to induce corneal rejection. Corneas derived from Hartley guinea pigs were transplanted into the left eyes of 32 male Lewis rats. Topical treatment was administered by using FK-506 0.3 mg/ml in a cyclodextrin suspension or vehicle (cyclodextrin suspension) four times per day. For systemic treatment, 0.5 mg/kg/day of FK-506 or vehicle (saline) was administered intraperitoneally. Treatments were started 60 minutes after surgery and continued for 21 days. The grafts underwent a double-masked examination, and a score was given for clarity, edema, and vascularization. The animals were sacrificed 21 days after transplantation. The control groups had allograft rejection after 6.75 +/- 0.31 (topical vehicle) and after 7.37 +/- 0.32 (systemic vehicle) days. The FK-506-treated groups showed allograft rejection after 14 +/- 0.88 (topical FK-506) or after 16.25 +/- 1.23 (systemic FK-506) days. In addition, FK-506-treated rats manifested less corneal neovascularization than control animals. We conclude that systemic or topical FK-506 is effective in prolonging xenograft survival in the rat keratoplasty model.     

Strength and pain measures associated with lateral epicondylitis bracing

BACKGROUND: In the experiment described here, we investigated the effects of the immunosuppressants FK506 and leflunomide (Lef) on the survival of hamster hearts and liver xenografts in Lewis rats. METHODS: Lewis rats were used as recipients of hamster heart or liver grafts using different regimens of FK506 and Lef. Donor-matched heart grafts were transplanted into long-term surviving Lewis rat recipients of hamster xenografts to test donor-specific prolongation of xenograft survival. Hyperimmune, late xenograft rejection, and naive sera were transferred into long-term surviving Lewis rat recipients of hamster heart xenografts to determine whether these sera could inhibit the efficacy of donor-specific long-term survival. Anti-donor-specific antibodies were analyzed by flow cytometry. RESULTS: After a short induction with FK506 plus Lef, maintenance treatment with FK506 alone was sufficient to prolong survival of hamster xenografts. All hamster heart and four of six hamster liver xenografts survived for more than 3 months. Second hamster hearts were permanently accepted by Lewis rats bearing long-term surviving hamster heart xenografts when rats were treated with FK506 monotherapy (mean survival time >60 days, n=4). Long-term surviving hamster heart grafts were rejected after transfer of hyperimmune serum but not late xenograft rejection serum or naive serum. Lef and FK506 significantly reduced the production of anti-donor-specific antibodies in Lewis rats transplanted with hamster liver and heart xenografts. CONCLUSION: Long-term survival of hamster liver and heart xenografts in Lewis rats could be induced by a regimen of short-term FK506 in combination with Lef followed by FK506 monotherapy. The acquired sensitivity of late xenoreactivity to FK506 reflects primarily a modification in the host immune response to the hamster graft.     

Contributions of inflammatory mast cell mediators to alterations in macrophage function after malathion administration

FK506 a new and potent immunosuppressive agent has been shown to be effective in prolonging pancreatic islet allograft survival. The present study was to determine its efficacy in prolonging pig islet xenotransplantation in two different strains of rat recipients. A total of two dosages of FK506 at 1 or 2 mg/kg per day for 2 weeks and then at weekly intervals were tested as monotherapy for their effect on the survival of renal subcapsular xenografts of purified or impure adult pig islets in inbred ACI and outbred Wistar rats. Histological assessment indicated that FK506 at 2 mg/kg per day significantly prolonged purified pig islet xenograft survival and to 7.5 months in two of three ACI recipients. Monotherapy with a lower dosage of FK506 or transplantation with impure pig islets resulted in increased graft survival time over controls, but less than that with the 2 mg/kg per day FK506. The viable pig islet xenografts showed a normal appearance and were readily identified by immunohistochemical staining for insulin and glucagon and further confirmed by immunohistochemical staining with anti-pig islet specific monoclonal antibody clone P44, developed in our laboratory. Mononuclear cell infiltration, mainly of the CD8-positive T-cell subset, increased with the duration of the graft in the recipient. By 7.5 months the majority of the xenografted islet cells were enclosed by the cellular infiltrate. The in vitro perfusion study of pig islets that had survived for 1 or 2 months in vivo showed that they were responsive to glucose stimulation with increase in insulin secretion into the perfusate. The results demonstrated that FK506 significantly prolonged pig islet survival in two rat strains and suggested that it is an effective immunosuppressant for the xenotransplantation model.     

Importance of T cells to accelerated rejection and acceptance of renal allografts in sensitized rat recipients

BACKGROUND: Sensitized recipients often experience fulminant allograft loss by yet ill-defined cellular and/or humoral immune mechanisms. In this study, we analyzed the contribution of cellular elements, in particular T cells, to the accelerated rejection of renal allografts in sensitized rats. METHODS AND RESULTS: LEW rats sensitized with BN skin grafts died of uremia in 3.3+/-0.9 days after transplantation of a BN kidney, similarly to bilaterally nephrectomized animals. Adoptive transfer of 10(6) graft-infiltrating mononuclear cells as well as their CD25+ subset into otherwise normal LEW recipients accelerated rejection of BN test cardiac allografts (5.4+/-0.5 days to 6.6+/-0.4 days vs. 7.8+/-0.8 days in controls, P<0.0007), while the CD25- population was ineffective (8.0+/-0.6 days, NS). Furthermore, alpha/beta-T-cell receptor (TCR)-targeted therapy with R73 monoclonal antibody abrogated accelerated rejection, and produced long-term survival in sensitized animals treated before kidney engraftment (day -7 to day -1). Long-term survival was associated with an up-regulation of intragraft interleukin-4 and interleukin-10 expression in conjunction with depressed Th-1-type cytokines. In addition, alpha/beta-TCR-targeted therapy even in low subtherapeutic dose decreased IgM alloantibody levels, and prevented the switch from IgM to IgG alloantibody response. CONCLUSIONS: This is the first report that documents the striking efficacy of alpha/beta-TCR-targeted therapy in sensitized rat renal transplant recipients. The results provide evidence for a critical role of T cells for both accelerated rejection and long-term graft survival. Up-regulation of Th2-type cytokine profile may, at least in part, contribute to the acquisition of immune unresponsiveness after alpha/beta-TCR-targeted therapy in this well-defined rat renal transplant model.     

Pelvic ultrasonography in normal girls and in girls with pubertal precocity

Small bowel allograft rejection in large animals has yet to be well defined. There are no specific early signs of graft rejection. The present experiments were undertaken to compare acute small bowel allograft rejection in pigs with and without FK506 and also to examine the usefulness of mucosal biopsies. Thirty-six outbred Large-White pigs were divided into (1) group 1 (n = 9): nonimmunosuppressed recipients; (2) group 2 (n = 8): FK506-immunosuppressed recipients; (3) group 3 (n = 2): autotransplant controls; and (4) donors (n = 17). Orthotopic small bowel transplantations were performed with Thiry-Vella loops for daily biopsies. The survival rate of group 2 was significantly longer than that of group 1 (P < 0.05). One best survivor in group 2 was killed at postoperative day (POD) 365. Treatment by FK506 prevented rejection, but most of the pigs died of pneumonia. In group 1, rejection began on POD 3 and progressed to severe rejection rapidly within 7 days. In group 2, rejection began from POD 6 to POD 8, but either remained mild or spontaneously improved. The differences in the routine laboratory data and the tumor necrosis factor-alpha level were not evident between the groups. Histological studies of repeated graft biopsies are thus considered to be essential for detecting signs of graft rejection.     

Tumor necrosis factor-beta is associated with thymic apoptosis during acute rejection

BACKGROUND: Intrathymic events undergoing allograft rejection remain undefined. The present study investigated the role of tumor necrosis factor-beta on acute thymic involution in rat hepatic allograft recipients during rejection. METHODS: Apoptosis and cellular phenotypic changes in the thymus were studied after hepatic transplantation. RESULTS: Thymocytes in both the medulla and cortex were sparse during acute rejection. Phenotypically, CD4+CD8+ T cells decreased significantly, whereas there were relative increases in CD4-CD8-, CD4+CD8-, and CD4-CD8+ T cells in untreated allograft recipients. Additionally, thymic apoptosis was found by in situ DNA end labeling and electron microscopy. Apoptotic cells were predominantly distributed in the cortex. Biologic lymphotoxin (tumor necrosis factor-beta)/tumor necrosis factor-alpha cytotoxic activity in the serum was significantly increased in untreated hepatic allograft recipients. Tumor necrosis factor-beta mRNA was detected in untreated allograft livers, and intraperitoneal administration of recombinant human tumor necrosis factor-beta induced extensive apoptosis of thymocytes in vivo. In contrast, no significant thymic involution was observed in donor-specific blood transfusion-treated allograft and isograft recipients. Intraperitoneal administration of rabbit anti-human tumor necrosis factor-beta polyclonal antibody or recombinant human interleukin-10 inhibited thymic apoptosis in untreated hepatic allograft recipients. CONCLUSIONS: Allograft rejection, but not donor-specific transfusion-induced immunologic unresponsiveness, is associated with thymic involution, a process that may be mediated by tumor necrosis factor-beta.     

Flow cytometric analysis of proliferative activity of pleomorphic adenoma of salivary gland

BACKGROUND: In a previous study, it was shown that a spontaneously tolerated DA (RT1a) liver allograft in a PVG (RT1c) recipient was able to induce tolerance of a DA small bowel graft performed 17 days later in spite of infiltration of the intestinal grafts by mononuclear cells. AIMS: To compare the phenotype of graft infiltrating cells in rejecting and tolerated small bowel grafts in order to elucidate the mechanism(s) which block the graft infiltrating cells from mediating rejection. METHODS: Multiparameter immunofluorescence was used to compare the phenotype and state of activation of donor and recipient cells isolated from intestinal grafts rejected or tolerated after liver transplantation. RESULTS: Three differences were found. Firstly, there was a more rapid replacement of lamina propria (LP) cells by recipient lymphocytes in tolerated than in rejected grafts. Secondly, the proportion of LP recipient CD8alphabeta+ lymphocytes bearing the high affinity receptor for interleukin 2 was significantly less in tolerated grafts (1.1%, range 0-2%) than in rejected grafts (21.3%, range 9-26%). Finally, tolerated grafts contained significantly less NK lymphocytes (NKR-P1+) and macrophages than rejected intestinal allografts. CONCLUSIONS: These observations make it possible to delineate clear cut differences in the phenotype of cells infiltrating rejecting versus tolerated grafts. Furthermore, the data suggest that liver transplantation induces tolerance of intestinal grafts by hampering the activation of recipient TcRalphabeta+ CD8alphabeta+ T cells and subsequently the recruitment of non-specific effector cells.     

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection

In a rat model of corneal transplantation, Fischer 344 (RT1(lv1)) rats received orthotopic corneal isografts or Wistar-Furth (RT1(u)) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8+ than CD4+ cells was found at days 1-3 following rejection, whereas there was a higher proportion of CD4+ cells at days 5-8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1-2, 4 and 7-10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4+ > CD8+ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1-2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes--from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4+ cells was found in both aqueous and graft at later times.     

Fas antigen expression of hepatocytes and its modification by immunosuppressants

Active cell death induced by ligation of the Fas antigen (Fas-Ag) with its antibody, Fas ligand (Fas-L), has been known to play a major role in cell killing via apoptosis by cytotoxic T lymphocytes (CTL). Thus, in liver transplantation, Fas-Ag expression of hepatocytes and its modification by immunosuppressive agents such as FK 506 or CsA can theoretically influence allograft survival. Mouse hepatocytes (BALB/c) were isolated and cultured with or without FK 506 or CsA, and Fas-Ag expression was determined by flow cytometry. Fas-Ag expression in the control was 17.2 +/- 2.5% after 24 hr of culture. When FK 506 or CsA was added, Fas-Ag expression with FK 506 at a concentration of 0.01-0.1 microg/ml was significantly lower than that with CsA (P < 0.05). When the cells were incubated with apoptosis-inducing anti-Fas-Ag monoclonal antibody, agarose gel electrophoresis of the control cells yielded a typical pattern of DNA fragmentations. The cells with FK 506 at 0.01 microg/ml yielded the least DNA fragmentation. These findings suggested that in the in vivo setting, the hepatocytes of the allograft would have a lower chance of being attacked by CTL in the host treated with FK 506.     

Localization of Six4/AREC3 in the developing mouse retina; implications in mammalian retinal development

BACKGROUND: We designed an antisense phosphorothioate oligodeoxynucleotide (oligo) to specifically inhibit the expression of rat intercellular adhesion molecule-1 (ICAM-1) mRNA (IP-9125). METHODS: IP-9125 oligo was delivered intravenously by osmotic pump alone or in combination with cyclosporine (CsA) to recipients in order to prevent the rejection of kidney or heart allografts. In additional experiments, kidney allografts were perfused with IP-9125 before grafting. RESULTS: IP-9125 inhibited ICAM-1 mRNA and ICAM-1 protein expression in rat aortic endothelial cells; scrambled controls IP-12140 and IP-13944 were ineffective. Untreated ACI (RT1a) recipients rejected Lewis (RT1l) kidney allografts at a mean survival time of 8.5+/-1.1 days. A 14-day intravenous administration of 2.5 mg/kg/day IP-9125 prolonged the survival of kidney allografts to 39.2+/-16.4 days; 5.0 mg/kg/day, to 43.0+/-17.5 days; and 10.0 mg/kg/day, to 50.4+/-21.6 days. In contrast, a scrambled control IP-12140 was not effective. A combination of 10 mg/kg/day IP-9125 and 1.0 mg/kg/day CsA delivered for 14 days synergistically extended kidney allograft survival times 88.5+/-7.5 days. In contrast, the combination of 10.0 mg/kg/day control IP-12140 with CsA was ineffective (20.7+/-3.2 days) when compared with CsA alone (20.2+/-4.0 days). Similar results were obtained for heart transplants in recipients treated with IP-9125 alone or in combination with CsA. Furthermore, in situ immunostaining showed that IP-9125 significantly reduced the expression of ICAM-1 protein in kidney allografts. Finally, perfusion of kidney grafts alone with 20.0 mg per 2 ml of IP-9125 protected kidney allografts from rejection (37.5+/-7.5 days; P < 0.001), whereas perfusion with 20 mg per 2 ml of control IP-12140 was ineffective (12.6+/-5.0 days). CONCLUSIONS: Rat ICAM-1 IP-9125 oligo inhibits ICAM-1 protein expression in vitro and in vivo as well as blocks allograft rejection when used for pretreatment of donors, graft perfusion, or postoperative treatment of recipients.