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.     

Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells: implications for gene therapy

Astrocytic tumors frequently express Fas/APO-1 (Fas), in sharp contrast to surrounding normal brain cells, providing a potential window through which selective killing of tumor cells could be pursued. To assess this possibility, we transduced Fas into U251, a glioma cell line resistant to anti-Fas antibody-mediated apoptosis, and obtained transfectants with high levels of Fas expression. Anti-Fas antibody showed significantly enhanced cytotoxicity for the transfectants, suggesting that U251 cells maintained an intercellular cascade of Fas-mediated apoptosis. When U251 transfectants with high-level Fas expression were transduced with Fas ligand-encoding gene via retrovirus, they were unaffected by exposure to anti-Fas antibody or Fas ligand adenovirus (Adeno-FL). Thus, retroviral induction of Fas ligand into the glioma cells with high levels of Fas led to the selection of cells that were resistant to Fas-dependent apoptosis. These resistant U251 transfectants were susceptible to FADD adenovirus (Adeno-FADD)-induced apoptosis, indicating that a cascade of death signals was blocked at the steps between Fas ligand and FADD. As for adenoviral transduction of Fas ligand into gliomas, gliomas with a relatively high level of expression of Fas were remarkably sensitive to Adeno-FL-induced apoptosis. Besides, Adeno-FADD induced pronounced apoptosis in all glioma cells. Our data suggest the possibility of using adenovirus-mediated transduction of Fas ligand and FADD genes as a therapeutic approach to target gliomas.     

Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response

Proliferation of vascular smooth muscle cells (VSMCs) in response to injury plays a key role in the pathogenesis of vascular disorders. Fas ligand (FasL) induces apoptosis in Fas-bearing cells, and its expression on activated T cells contributes to the regulation of the immune response and physiological cell turnover. Here, we show that a replication-defective adenovirus encoding FasL (Ad-FasL) induced apoptosis in Fas-bearing VSMCs. When introduced locally to balloon-injured rat carotid arteries, a well characterized model of a VSMC-derived lesion, Ad-FasL functioned as a potent inhibitor of neointima formation. In rats immunized with an empty adenoviral vector, robust T cell infiltration of the vessel wall was detected after local delivery of a beta-galactosidase-expressing virus (Ad-betagal), whereas T cell infiltrates were not detected after local delivery of Ad-FasL. Prior immunization prevented beta-galactosidase expression from Ad-betagal, whereas the expression of the FasL transgene was unaffected. When Ad-betagal and Ad-FasL were delivered together to preimmunized animals, T cell infiltration was reduced and beta-galactosidase expression was restored. These data demonstrate that Fas ligand gene transfer can effectively inhibit injury-induced vessel lesion formation and can allow adenovirus-harboring cells to evade immune destruction.     

Protection from endotoxemia by adenoviral-mediated gene transfer of human bactericidal/permeability-increasing protein

Sepsis represents a growing concern in high-risk patients and there has been a lack of effective preventives and therapies. Bacterial/permeability increasing protein (BPI) is a human neutrophil granule-associated defense molecule specific for Gram-negative bacteria and their products. To develop a BPI-transgene-based prophylactic or therapeutic modality, we have developed a recombinant, replication-deficient adenoviral vector expressing full-length human BPI protein (AdhBPI). The expression of BPI is under control of a murine cytomegalovirus (CMV) promoter. Using in vitro and in vivo systems, AdhBPI-mediated gene transfer led to extracellular secretion of BPI protein, which effectively neutralized endotoxin (lipopolysaccharide [LPS]) and markedly reduced the production of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) by freshly isolated murine alveolar macrophages. By using a mouse model of nonlethal sepsis elicited with LPS, we demonstrated that in vivo gene transfer of BPI was able to markedly inhibit the effect of a large dose of LPS on cytokine responses when injected intraperitoneally. Furthermore, such in vivo BPI gene transfer also improved the survival of mice suffering from lethal septic shock elicited by intraperitoneal injection of d-galactosamine and LPS. Thus, our results suggest that human BPI gene transfer vector has the potential to be used as a therapeutic agent for septic conditions.     

Synergistic effect of microencapsulation and immunoalteration on islet allograft survival in bioartificial pancreas

Recently, we reported successful transplantation (Tx) of microencapsulated (mc) islets. However, graft failure observed in several cases was associated with an increased foreign body reaction compared to long-term functioning grafts. This study was performed to investigate the impact of an immunoalterating islet pretreatment (12-14 days culture at 22 degrees C) on graft function. After microencapsulation in barium alginate beads the islets were cultured for another day. Diabetic LEWIS rats (blood glucose >19 mM) were transplanted with 3500 immunoaltered mc-Wistar islets intraperitoneally. Controls were transplanted with 3500 non-cultured syngeneic or allogeneic mc-islets. Additional syngeneic and allogeneic controls were transplanted with 6000 non-cultured, non-encapsulated islets intraperitoneally. Seventy percent of the recipients of microencapsulated, long-term low temperature cultured islets maintained normoglycemia at least for 15 weeks, while this was true in only 17% of those animals receiving microencapsulated non-pretreated allogeneic islets. Islets in non-encapsulated controls were rejected within several days. Graft function correlated with histologically proven viable islets within the capsules. Microencapsulation of islets markedly prolonged allograft survival compared to non-encapsulated islets; application of an immunoaltering low-temperature culture further improved graft function significantly. These data may support the hypothesis of induction of a reaction against microcapsules by the antigen release from the graft which may be avoided by immunoaltering islet pretreatment.     

Expression of ribozymes in gene transfer systems to modulate target RNA levels

The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, they have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.     

Expression of CTLA4-Ig by biolistically transfected mouse islets promotes islet allograft survival

BACKGROUND: Localized delivery of immunosuppressive molecules, limited to the graft site, may allow transplantation of tissue in the absence of systemic immunosuppressive agents. We tested whether purified mouse islets that had been engineered to produce human CTLA4-Ig locally at the graft site could survive in allogeneic recipients receiving no systemic immunosuppression. METHODS: CBA (H2(k)) islets were subjected to biolistic (gene gun) transfection with a cDNA encoding human CTLA4-Ig under control of the human cytomegalovirus immediate early promoter. After 40-48 hr of culture, the transfected islets (500 per recipient) were transplanted beneath the renal capsule of alloxan-induced diabetic BALB/c (H2(d)) recipients. RESULTS: Control grafts (n=10) consisting of islets biolistically transfected with the expression plasmid alone (i.e., no gene inserted) survived for 12.8+/-3.6 (mean +/- SD) days. In contrast, islets transfected with CTLA4-Ig (n=12) survived 66.8+/-61.5 days (P=0.01), with 50% demonstrating functional survival until follow-up was concluded at 50 (n=2), 130 (n=2), or 165 (n=2) days. Immunohistochemistry on grafts that survived long term showed well-granulated, insulin-positive islets lying adjacent to, but not infiltrated by, dense aggregates of mononuclear cells. CONCLUSIONS: Transfection of allogeneic mouse islets with human CTLA4-Ig results in prolonged allograft survival. Although on histology mononuclear cells are present in the area of the transfected graft, they do not appear to infiltrate or destroy the islet graft.     

Indefinite islet allograft survival in mice after a short course of treatment with anti-CD45 monoclonal antibodies

BACKGROUND: Although islet cell transplantation is considered an ideal form of endocrine replacement for type I diabetes, clinical application in humans is still not feasible. New immunosuppressive strategies are clearly needed to control inexorable rejection. CD45 is a family of transmembrane protein tyrosine phosphatases critically involved in the regulation of lymphocyte activation signals. Anti-CD45RB monoclonal antibody can prevent rejection of murine renal allografts. METHODS: Here, we examine the consequences of targeting CD45 in murine islet cell transplantation. Diabetic mice recipients received islet allografts under the kidney capsule and were divided into seven groups. Recipients received no treatment (controls) or anti-CD45RB monoclonal antibody (mAb; MB23G2 or C363.16A) at different dosages and treatment intervals. RESULTS: All untreated control animals lost islet function, becoming hyperglycemic within 10-17 days after transplantation. Animals treated with either anti-CD45RB mAb showed a significant prolongation of islet allograft survival when compared with controls. Anti-CD45RB MB23G2 at 100 microg/day, given on days -1, 0, and 5 was particularly effective, inducing indefinite islet allograft survival in 60% of recipients. CONCLUSIONS: These results indicate that anti-CD45 mAbs are potent immunomodulatory agents, able to sustain indefinite islet allograft function after a short treatment course in the highly immunogenic model of islet transplantation.     

Levels of soluble Fas ligand in myocarditis

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Identification of amino acid residues important for ligand binding to Fas

OBJECTIVE: An autoantibody to a nucleolar RNA helicase protein (Gu) was recently discovered in a patient with gastric antral vascular ectasia or watermelon stomach, a disorder that is increasingly being described in systemic sclerosis (SSc). The present study was undertaken to determine whether anti-Gu antibodies occur in connective tissue diseases (CTD) and, if so, to determine their frequencies and any clinical or immunogenetic associations. METHODS: Anti-Gu antibodies were determined by Western blotting of glutathione-purified glutathione S transferase-Gu fusion proteins against consecutive antinucleolar antibody-positive sera (HEp-2 cell substrate) collected over a 5-year period in a rheumatology antinuclear antibody (ANA) testing laboratory. RESULTS: Anti-Gu antibodies were found in 11 (10%) of 108 antinucleolar antibody-positive sera. The subjects with anti-Gu antibodies included 3 of 46 patients with SSc (7%), 3 of 17 patients with systemic lupus erythematosus (18%), 4 of 9 patients with undifferentiated CTD (44%), and 1 healthy relative of an SSc patient. None of the anti-Gu-positive patients had any symptoms suggestive of watermelon stomach. Increased frequencies of both HLA-DQA1*0501 and DQB1*0301 were found, but only DQB1*0301 maintained statistical significance after correction. CONCLUSION: Anti-Gu (nucleolar RNA helicase) antibodies occur in low frequencies in patients with CTDs who have antinucleolar antibodies by ANA testing, but they are not specific for SSc or the watermelon stomach lesion.