Screening a panel of drugs with diverse mechanisms of action yields potential therapeutic agents against neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite current aggressive therapy, the survival rate for high risk NB remains less than 40%. To identify novel effective chemo-agents against NB, we screened a panel of 96 drugs against two NB cell lines, SK-N-AS and SH-SY5Y. We found 30 compounds that were active against NB cell lines at < or =10 microM concentration. More interestingly, 17 compounds are active at < or =1 microM concentration, and they act through a wide spectrum of diverse mechanisms such as mitotic inhibition, topoisomerase inhibition, targeting various biological pathways, and unknown mechanisms. The majority of these active compounds also induced caspase 3/7 by more than 2-fold. Of these 17 active compounds against NB cell lines at sub-micromolar concentration, eleven compounds are not currently used to treat NB. Among them, nine are FDA approved compounds, and three agents are undergoing clinical trials for various malignancies. Furthermore, we identified four agents active against these NB cell lines that have not yet been tested in the clinical setting. Finally we demonstrated that Cucurbitacin I inhibits neuroblastoma cell growth through inhibition of STAT3 pathway. These drugs thus represent potential novel therapeutic agents for patients with NB, and further validation studies are needed to translate them to the clinic.

Blockade of the CD154-CD40 costimulatory pathway prevents the development of experimental autoimmune glomerulonephritis.

BACKGROUND: Experimental autoimmune glomerulonephritis (EAG) was induced in Wistar-Kyoto (WKY) rats by immunization with rat glomerular basement membrane (GBM) in adjuvant. This model is characterized by anti-GBM antibody production, accompanied by focal necrotizing glomerulonephritis with crescent formation. There is also glomerular infiltration by T cells and macrophages. Our hypothesis was that blocking the interaction between CD154 (CD40L) on Th cells and CD40 on antigen-presenting cells should inhibit T-cell activation, and thus the development of EAG. METHODS: The in vivo effects of a hamster anti-rat monoclonal antibody to CD154 (AH.F5) were examined in EAG starting at day -1 prior to immunization, day +7 after immunization, or day +14 after immunization. RESULTS: When administered from day -1 at a dose of 10 mg/kg intraperitoneally three times per week for the duration of the study (4 weeks), AH.F5 resulted in a marked reduction in circulating anti-alpha3(IV)NC1 antibodies, deposits of IgG on the GBM, albuminuria, deposits of fibrin in the glomeruli, severity of glomerular abnormalities, and numbers of glomerular T cells and macrophages. When administered from day +7 at the same dose, AH.F5 resulted in a moderate reduction in the severity of disease, while administration from day +14 had no significant effect. CONCLUSION: These studies demonstrate for the first time that early blockade of the CD154-CD40 T-cell costimulatory pathway can prevent the development of crescentic nephritis, and that delayed treatment can reduce the severity of disease. This confirms the importance of T cell mediated immunity in the pathogenesis of EAG, and suggests that strategies targeting T-cell costimulation may provide a novel approach in the treatment of human glomerulonephritis.

The contribution of Fc effector mechanisms in the efficacy of anti-CD154 immunotherapy depends on the nature of the immune challenge.

Blockade of the CD154-CD40 co-stimulatory pathway with anti-CD154 mAbs has shown impressive efficacy in models of autoimmunity and allotransplantation. Clinical benefit was also demonstrated in systemic lupus erythematosus (SLE) and idiopathic thrombocytopenia patients with the humanized anti-CD154 mAb, 5C8 (hu5C8). However, thromboembolic complications that occurred during the course of the hu5C8 clinical trials have proven to be a major setback to the field and safe alternative therapeutics targeting the CD154-CD40 pathway are of great interest. Recently, effector mechanisms have been shown to play a part in anti-CD154 mAb-induced transplant acceptance in murine models, while this issue remains unresolved for humoral-mediated models. Herein, aglycosyl anti-CD154 mAbs with reduced binding to FcgammaR and complement were used as a novel means to test the role of effector mechanisms in non-human primate and murine models not amenable to gene knockout technology. While aglycosyl hu5C8 mAb was relatively ineffective in rhesus renal and islet allotransplantation, it inhibited primary and secondary humoral responses to a protein immunogen in cynomolgus monkeys. Moreover, an aglycosyl, chimeric MR1 mAb (muMR1) prolonged survival and inhibited pathogenic auto-antibody production in a murine model of SLE. Thus, the mechanisms required for efficacy of anti-CD154 mAbs depend on the nature of the immune challenge.

Intragraft gene and protein expression in rat liver allografts treated with costimulatory blockade alone or in combination with CyA.

BACKGROUND: Costimulatory blockade has been shown to prevent acute rejection (AR) and promote long-term graft survival in a number of animal models including nonhuman primates. The effect of concomitant administration of conventional immunosuppressives on long-term liver allograft survival and intragraft expression of immune mediators has not previously been examined. MATERIALS AND METHODS: A high-responding Dark Agouti to Lewis orthotopic liver transplant (LEW OLT) model was used to compare anti-CD154 alone, or in combination with cyclosporin (CyA) on allograft survival. Donor-specific reactivity was assessed by mixed lymphocyte reaction (MLR) and allogeneic skin grafts. Surviving rats were euthanized on day 150 and intragraft gene (CD80, 86, 152, 154, IFN-gamma, IL-2, IL-6, IL-10, IL-13, TNF-alpha, TGF-beta, IL-7, Fas-ligand, Granzyme B, bax, and bcl(2)) and protein (CD4, CD8, ED1, CD154, CD80, CD86) expression was measured. RESULTS: Untreated control recipients had a median survival time of 5 days. Recipients treated with anti-CD154 survived to beyond 150 days with no evidence of AR. Concomitant administration of CyA did not alter the long-term survival. There was no difference in the serum aspartate aminotransferase between treatment groups or a change over time. All treated recipients showed a reduction in donor-specific MLR at day 40 and 60 but had persistence of donor reactivity to skin grafts at day 100. Histologically, liver architecture was well preserved despite the presence of a nondestructive mononuclear cell infiltrate. Analysis of intragraft gene expression revealed an inverse relationship between the duration of anti-CD154 therapy and the gene expression of costimulatory molecules and Th1 cytokine transcripts. The pro-apoptotic gene, bax, was increased in recipients treated with anti-CD154, but not CyA, compared with normal liver. CONCLUSIONS: These data demonstrate that anti-CD154 therapy either alone or in combination with CyA allows for the long-term survival of liver allografts in the rat despite there being a difference in the intragraft gene and protein profile.

Tolerance induction in rats, using a combination of anti-CD154 and donor splenocytes, given once on the day of transplantation.

BACKGROUND: Donor-specific tolerance induction remains an attractive objective that generates much research in the field of transplantation. Unfortunately, most of the protocols available involve pregraft conditioning, making these treatments incompatible with clinical applications. METHODS: LEW.1A rats were grafted with histoincompatible LEW.1W hearts. On the day of transplantation, recipients were treated with anti-CD40L combined with donor splenocytes. The hearts were evaluated for graft survival; cellular infiltrate and intragraft cytokines were determined using real-time reverse transcriptase-polymerase chain reaction. Tolerance induction was assessed by skin grafting and adoptive transfers. RESULTS: The combination of a single injection of anti-CD40L and donor splenocytes, given on the day of surgery, allowed 40% of cardiac allografts to survive long-term (mean survival time=66.3 day). The cellular composition or the extent of graft infiltrate was not modified but was associated with a massive decrease of proinflammatory cytokines expression within the graft. Long-term survivors accepted donor-matched skin grafts, and leukocytes harvested from these animals transferred tolerance into irradiated freshly grafted recipients. CONCLUSION: A combination of costimulation blockade and donor cells, given once at the time of transplantation, is sufficient to induce allograft tolerance in rats.

B lymphocyte memory: role of stromal cell complement and FcgammaRIIB receptors.

To dissect the influence of CD21/CD35 and FcgammaRIIB in antigen retention and humoral memory, we used an adoptive transfer model in which antigen-primed B and T lymphocytes were given to sublethally irradiated wild-type mice or mice deficient in CD21/CD35 (Cr2(-/-)) or FcgammaRIIB receptors (FcgammaRIIB(-/-)). Cr2(-/-) chimeras showed impaired memory as characterized by a decrease in antibody titer, reduced frequency of antibody secreting cells, an absence of affinity maturation, and significantly reduced recall response. The impaired memory in Cr2(-/-) chimeras corresponded with the reduced frequency of antigen-specific memory B cells. Interestingly, FcgammaRIIB(-/-) chimeras showed a differential phenotype with impaired splenic but normal bone marrow responses. These data suggest that CD21/CD35 on stroma, including follicular dendritic cells, is critical to the maintenance of long-term B lymphocyte memory.

Variation in the ordered structure of complexes between CD154 and anti-CD154 monoclonal antibodies.

The cell surface co-stimulatory protein CD154 (CD40L) is a target for monoclonal antibody (mAb) inhibitors of T-cell mediated immune diseases. This protein, like most other members of the TNF ligand family, forms homotrimeric complexes on the cell surface and in solution, with a three-fold axis of symmetry. We find that several different anti-CD154 monoclonal antibodies form distinctive complexes with soluble CD154. These soluble complexes have been analyzed using size exclusion chromatography, static and dynamic light scattering, and electron microscopy and shown to consist of caged structures of various geometries. The cell surface complexes have been analyzed by confocal microscopy and, depending on the mAb, remain as small, separate complexes or form large aggregates. The formation of these complexes in solution is likely to have an impact on measures of affinity, while the cell surface complexes could affect binding potency and provoke other biological effects.

CD40 ligand (CD154) does not contribute to lymphocyte-mediated inhibition of virulent Mycobacterium tuberculosis within human monocytes.

Human monocytes displayed increased expression of CD40 following infection with virulent Mycobacterium tuberculosis. Nevertheless, soluble CD40 ligand (CD40L; also designated CD154) had no effect on the intracellular growth of the organism. Restriction of the intracellular growth of M. tuberculosis by peripheral blood lymphocytes and antigen-specific CD4+ T-cell lines likewise was not reduced by blocking anti-CD40L monoclonal antibody 5c8.

A novel CD154 monoclonal antibody in acute and chronic rat vascularized cardiac allograft rejection.

BACKGROUND: The CD40-CD154 interaction is critically important in the cell-mediated immune responses. Blockade of this costimulatory pathway has been shown to prevent acute allograft rejection in murine, as well as nonhuman primate models. However, the role of the CD40-CD154 pathway in the development of chronic rejection and the effects of CD154 targeting on progression of chronic rejection have not been evaluated. METHODS: We examined the effect of AH.F5, a new hamster anti-rat CD154 monoclonal antibody, in a fully allogeneic acute(u) into Lewis [LEW] (RT11) and chronic [WF.1L (RT1l) into LEW (RT1l)] vascularized cardiac allograft rejection model. In the chronic model, the antibody was evaluated for prevention (starting day of transplant) and interruption of progression (starting day 30 or 60 after transplant) of chronic vasculopathy. Graft survival, morphology, and immunohistology were evaluated. RESULTS: In the acute rejection model, anti-CD154 therapy alone prevented acute allograft rejection and resulted in 50% long-term allograft survival (>200 days) and donor-specific tolerance. In recipients treated with anti-CD154 monoclonal antibody in combination with a short course of cyclosporine, 100% of allografts survived long-term and all recipients achieved donor-specific tolerance. In the chronic rejection model, allografts from animals treated with the anti-CD154 antibody had a statistically significant lower score of graft arteriosclerosis and fibrosis in both the prevention and 30-day interruption groups when compared with control allografts. In addition, immunohistochemistry showed a decrease in intragraft mononuclear cell infiltration and activation. CONCLUSION: A new anti-CD154 antibody not only prevents acute allograft rejection, but also inhibits and interrupts the development of chronic rejection. In the acute rejection model cyclosporine acts synergistically with anti-CD154 therapy to prolong allograft survival and induce tolerance. In the chronic rejection model relatively early initiation of therapy is essential to prevent progression of chronic allograft vasculopathy and fibrosis.

Costimulatory blockade prevents early rejection, promotes lymphocyte apoptosis, and inhibits the upregulation of intragraft interleukin-6 in an orthotopic liver transplant model in the rat.

Costimulatory pathways have a pivotal role in the T-cell response to alloantigen. The role of costimulatory blockade with anti-CD154 in orthotopic liver transplantation (OLT) has not been examined previously. This study aims to investigate effects of anti-CD154 and CTLA4-immunoglobulin (Ig) in the early post-OLT period using a major histocompatibility complex-disparate fully arterialized OLT model in the rat. Lewis rats underwent OLT with Dark Agouti liver allografts. Recipients were randomized to receive (1) isotype control, (2) anti-CD154, (3) CTLA4-Ig, or (4) cyclosporine A (CyA). Rats were killed day 8, and specimens were obtained for histological examination, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, immunohistochemistry, and quantitative reverse-transcriptase polymerase chain reaction. An additional five transplant recipients were treated with anti-CD154 for 14 days postoperatively to assess long-term allograft survival. All isotype control animals died on or before day 6 of acute rejection. Apart from four deaths caused by nonimmunologic causes, all treated recipients survived to day 8. The median survival of rats treated for 14 days with anti-CD154 was greater than 150 days. Serum aspartate aminotransferase and bilirubin levels normalized by day 3 in the CyA group and day 5 in transplant recipients treated with costimulatory blockade. Histologically, there was no difference between isotype controls and CTLA4-Ig-treated animals, whereas anti-CD154-treated transplant recipients had a lower Banff score. CD4+ and CD8+ T-cell infiltrates were prominent in transplant recipients treated with costimulatory blockade. Intragraft analysis showed an increase in lymphocyte apoptosis, Fas ligand messenger RNA expression, and reduction in interleukin-6 gene expression in transplant recipients treated with costimulatory blockade. Costimulatory blockade did not alter intragraft gene expression of other mediators of T-cell priming, differentiation, and effector function compared with isotype control animals. In conclusion, costimulatory blockade prevented acute rejection, enabled long-term survival, and increased intragraft lymphocyte apoptosis in a high-responding rat OLT model.

Dual role for TWEAK in angiogenic regulation.

Angiogenic regulators modulate endothelial cell functions, including proliferation, migration, secretion, and adhesion, through their action on endothelial cells or other cell types. TWEAK, a novel member of the tumor necrosis factor family, appears to be a pro-angiogenic agent on the basis of previous studies demonstrating its ability to induce interleukin-8 production by epithelial tumor lines, stimulate proliferation of human vascular cell types and neovascularization in rat corneas. Here, we further characterized the angiogenic potential of TWEAK, revealing a dual role for TWEAK as an angiogenic regulator. We demonstrate that TWEAK is a potent inducer of endothelial cell survival and cooperates with basic fibroblast growth factor to induce the proliferation and migration of human endothelial cells and morphogenesis of capillary lumens. In contrast, TWEAK antagonizes the morphogenic response of endothelial cells to vascular endothelial growth factor (VEGF) without inhibiting VEGF-induced survival or proliferation. Thus, our observations suggest that TWEAK may differentially regulate microvascular growth, remodeling and/or maintenance in vivo, depending upon the angiogenic context.