Frequent occurrence of transgene deletion in transgenic plants

The status of the transgene in tobacco plants transformed by Agrobacterium was analyzed with PCR. Twelve percent of the transgenic plants with the nptII gene showed different levels of transgene deletion, which was also found in transgenic watermelon (10-30%) and carrot (40-60%). It appeared that the percentage of transgenic plants carrying deleted transgenes depended on both the transgene and the plant. It is suggested that the transgene should be inserted between a right border and a selection marker to reduce the number of transgenic plants containing deleted transgenes after selection.     

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.     

Localization of inducible nitric oxide synthase in acute renal allograft rejection in the rat

There is increasing evidence for a role for nitric oxide (NO) in the alloimmune response and induction of NO synthesis occurs during allograft rejection. The aim of this study was to investigate the source of NO synthesis in rejecting allografts. Localization of inducible nitric oxide synthase (iNOS) was studied by immunohistochemistry, in a rat model of acute renal allograft rejection, in unmodified Lewis recipients in which rejection is complete 7 days after transplantation of F1 hybrid Lewis-Brown Norway kidneys. High levels of iNOS expression were found in infiltrating mononuclear cells in glomeruli and interstitium of rejecting kidneys; there was no expression in parenchymal renal cells, or in control isografts of either rat strain. Expression of iNOS in the cortex was present from 4 to 6 days posttransplantation, and had declined by the 7th day, where expression was principally in the medulla. The pattern of iNOS staining was similar to ED1 staining, a marker for rat macrophages. These findings suggest that infiltrating macrophages in the graft reaction are a prominent source of NO; this iNOS expression supports a role for NO in the modulation of local allogeneic responses, and possibly as a mediator of cytotoxic graft damage.     

Is acute rejection deleterious to long-term liver allograft function?

AIMS: The aim of the study was to evaluate the effects on systemic and coronary haemodynamics and myocardial substrate utilization of a new calcium sensitizer, levosimendan, after coronary artery bypass grafting. METHODS AND RESULTS: Twenty-three low-risk patients were included in this randomized and double-blind study. They received placebo (n = 8), 8 (n = 8) or 24 (n = 7) micrograms.kg-1 of levosimendan after coronary artery bypass operation. Systemic and coronary sinus haemodynamics with thermodilution and myocardial substrate utilization were measured. The heart rate increased 11 beats.min-1 after the higher dose (P < 0.05). Cardiac output increased by 0.7 and 1.61.min-1 (P < 0.05 for both) after 8 and 24 micrograms.kg-1 of levosimendan, respectively. Systemic and pulmonary vascular resistance decreased significantly after both doses. Coronary sinus blood flow increased by 28 and 42 ml/(P = 0.054 for the combined effect) after the lower and higher dose, respectively. Myocardial oxygen consumption or substrate extractions did not change statistically significantly. CONCLUSION: Despite improved cardiac performance, levosimendan did not increase myocardial oxygen consumption or change myocardial substrate utilization. Thus levosimendan has the potential to treat low cardiac output states after cardiopulmonary bypass surgery.     

The contribution of terminal complement components to acute and hyperacute allograft rejection in the rat

Acute rejection and antibody-mediated hyperacute allograft rejection are affected by activation of the complement cascade. Split products of early complement components influence the localization, activation, and effector functions of platelets, granulocytes, monocytes, and lymphocytes, while the formation of membrane attack complex (C5b-C9) can lead to rapid cell destruction. Therefore, we compared acute and Ab-mediated hyperacute allograft rejection in a recently described model of C6 deficient PVG (C-) (RT1c) rats and their normal counterpart PVG (C+) (RT1c) rats. Cardiac allografts from fully MHC disparate ACI donors were heterotopically grafted into naive and skin graft sensitized PVG (C-) and PVG (C+) rats. ACI cardiac allografts were rejected acutely (8.3 +/- 2 days; n = 7) by naive PVG (C+) recipients, but survived significantly longer in PVG (C-) recipients (22 +/- 10 days; n = 10). Presensitized PVG (C+) rats rejected ACI cardiac allografts hyperacutely in 6.1 +/- 2.4 hr (n = 5). In contrast, ACI cardiac allografts transplanted into presensitized (PVG (C-) rats had markedly longer survival of 91 +/- 14 hr (n = 5). The alloantibody responses of naive PVG (C+) and PVG (C-) recipients 7 days after cardiac allografting, and of presensitized PVG (C+) and PVG (C-) recipients at time of cardiac allografting were not significantly different as measured by flow cytometry against ACI lymphocytes. Immunofluorescence demonstrated deposition of IgM, IgG and C3 in ACI allografts in PVG (C-) as well as in PVG (C+) recipients. Deposition of C6 was only found in grafts rejected by PVG (C+). The significantly longer survival of ACI cardiac allografts in C6-deficient PVG (C-) rats indicates that the membrane attack complex contributes to acute as well as antibody-mediated hyperacute allograft rejection.     

Evidence of hepatocyte apoptosis in rat liver after the administration of carbon tetrachloride

In acute liver injury induced by the injection of CCl4, cell death has been attributed to the necrosis of hepatocytes in the centrilobular area. In the present study, we re-examined the hepatic injury evoked by CCl4 in rats and explored the possibility that apoptosis may also contribute to its pathogenesis. Apoptotic hepatocytes were identified and quantified by light and electron microscopy, the in situ immunohistochemical labeling of nuclear DNA fragmentation, flow cytometry, and DNA gel electrophoresis. We found that a substantial number of hepatocytes underwent apoptosis. Apoptotic changes were also observed in ballooned hepatocytes. Apoptotic hepatocytes increased in number at 3 hours and peaked at 6 hours after the CCl4 injection. Apoptotic bodies were sequestrated in the adjacent hepatocytes and sinusoidal cells. Double staining of the cells with immunostaining for phagocytes and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining for labeling of DNA fragmentation showed that the majority of apoptotic hepatocytes were phagocytosed by Kupffer cells and macrophages. The results indicated that apoptosis occurs in the ballooned and injured hepatocytes of the centrilobular area. What occurs after CCl4 administration may be important in reducing inflammation, shortening the course of acute hepatic injury, and preventing the development of fibrosis.     

Induction of Fas-mediated apoptosis in a human renal epithelial cell line by interferon-gamma: involvement of Fas-mediated apoptosis in acute renal rejection

To examine the role of the Fas ligand/Fas (FasL/Fas) system and apoptosis in renal allograft rejection, we analyzed FasL/Fas expression and apoptosis in 63 renal allograft biopsy specimens obtained from 56 renal transplant recipients in Tokyo, Japan. Sixteen biopsy specimens were diagnosed (Banff criteria) as acute rejection (AR), 17 as AR plus chronic rejection, 10 as borderline stages, and 20 as no rejection (NR). Immunohistochemically, Fas antigen was highly expressed in the renal tubular epithelium in tissues from patients with rejection. The mean number of Fas-positive tubular epithelium was significantly higher in biopsy specimens with AR than in those with NR, but FasL expression was highly expressed in infiltrating lymphocytes in the interstitium of allografts with cellular rejection. The mean number of FasL-positive infiltrating lymphocytes was significantly higher in specimens with AR than in those with NR or borderline stages. For detection of apoptotic cells, the specimens were subjected to terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling, which showed that the mean number of tubular epithelial cells positive for this labeling was highest for the specimens with AR, the number being significantly higher than in those with AR plus chronic rejection or with NR. Thus, Fas expression on epithelial cells might actively trigger their apoptosis by the interaction between Fas and FasL. Studies of human normal renal-derived cell lines (RPTEC 2601 [epithelial] and NHMC 5155 [mesangial]) showed that both constitutively expressed Fas (but not FasL) mRNA. After pretreatment with interferon-gamma, Fas-induced apoptosis was observed in the RPTEC 2601 line, but without interferon-gamma pretreatment, anti-Fas-mediated apoptosis was not seen. Under identical conditions, the NHMC 5155 line was resistant to anti-Fas-mediated apoptosis regardless of interferon-gamma pretreatment. This suggested that AR might be associated with increased apoptosis in the renal tubular epithelial cells mediated by the Fas/FasL system.     

HLA class-I-soluble antigen serum levels in liver transplantation. A predictor marker of acute rejection

The serum levels of sHLA-I have been determined in 16 patients following liver transplantation. sHLA-I levels did not show remarkable variations in six patients without evidence of transplant-related complications. sHLA-I levels strongly increased in 10 patients undergoing acute rejection episodes. In these patients, an average 20% daily increase of sHLA-I levels was detected on the 6 days preceding and on the 2 days following the rejection episode. A fast decrease of sHLA-I levels was observed in seven patients following treatment of acute rejection with anti-CD3 mAb. The serum level of sHLA-I antigens positively correlated with ALT serum level and inversely correlated with PT. The determination of sHLA-I in serum may therefore be proposed as a useful marker in the monitoring of patients following liver transplantation. The increase of sHLA-I antigens may predict the onset of acute rejection whereas their decrease may be related to a good response of acute rejection to immunosuppressive treatment.