Carol Tucker-Burden

Prolonged acceptance of concordant and discordant xenografts with combined CD40 and CD28 pathway blockade

BACKGROUND The prompt and vigorous immune response to xenogenic tissue remains a significant barrier to clinical xenotransplantation. Simultaneous blockade of the CD28 and CD40 costimulatory pathways has been shown to dramatically inhibit the immune response to alloantigen. METHODS . In this study, we investigated the ability of simultaneous blockade of the CD28 and CD40 pathways to inhibit the immune response to xenoantigen in the rat-to-mouse and pig-to-mouse models. RESULTS Simultaneous blockade of the CD28 and CD40 pathways produced marked inhibition of the cellular response to xenoantigen in vivo and produced long-term acceptance of xenogeneic cardiac and skin grafts (rat-to-mouse), and markedly suppressed an evoked antibody response to xenoantigen. In addition, this strategy significantly prolonged the survival of pig skin on recipient mice. CONCLUSIONS Long-term hyporesponsiveness to xenoantigen across both a concordant and discordant species barrier, measured by the stringent criterion of skin grafting, can be achieved using a noncytoablative treatment regimen.

Vigorous Allograft Rejection in the Absence of Danger

Tolerance to self is a necessary attribute of the immune system. It is thought that most autoreactive T cells are deleted in the thymus during the process of negative selection. However, peripheral tolerance mechanisms also exist to prevent development of autoimmune diseases against peripheral self-Ags. It has been proposed that T cells develop tolerance to peripheral self-Ags encountered in the absence of inflammation or “danger” signals. We have used immunodeficient Rag 1−/− mice to study the response of T cells to neo-self peripheral Ags in the form of well-healed skin and vascularized cardiac allografts. In this paper we report that skin and cardiac allografts without evidence of inflammation are vigorously rejected by transferred T cells or when recipients are reconstituted with T cells at a physiologic rate by nude bone graft transplantation. These results provide new insights into the role of inflammation or “danger” in the initiation of T cell-dependent immune responses. These findings also have profound implications in organ transplantation and suggest that in the absence of central deletional tolerance, peripheral tolerance mechanisms are not sufficient to inhibit alloimmune responses even in the absence of inflammation or danger.

Epidermal Growth Factor Receptor and PTEN Modulate Tissue Factor Expression in Glioblastoma through JunD/Activator Protein-1 Transcriptional Activity

Hypoxia and necrosis are fundamental features of glioblastoma (GBM) and their emergence is critical for the rapid biological progression of this fatal tumor; yet, underlying mechanisms are poorly understood. We have suggested that vaso-occlusion following intravascular thrombosis could initiate or propagate hypoxia and necrosis in GBM. Tissue factor (TF), the main cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Epidermal growth factor receptor (EGFR) amplification and PTEN loss are two common genetic alterations seen in GBM but not in lower-grade astrocytomas that could be responsible for TF up-regulation. The most frequent EGFR mutation in GBM involves deletion of exons 2 to 7, resulting in the expression of a constitutively active receptor, EGFRvIII. Here, we show that overexpression of EGFR or EGFRvIII in human glioma cells causes increased basal TF expression and that stimulation of EGFR by its ligand, EGF, leads to a marked dose-dependent up-regulation of TF. In all cases, increased TF expression led to accelerated plasma coagulation in vitro. EGFR-mediated TF expression depended most strongly on activator protein-1 (AP-1) transcriptional activity and was associated with c-Jun NH(2)-terminal kinase (JNK) and JunD activation. Restoration of PTEN expression in PTEN-deficient GBM cells diminished EGFR-induced TF expression by inhibiting JunD/AP-1 transcriptional activity. PTEN mediated this effect by antagonizing phosphatidylinositol 3-kinase activity, which in turn attenuated both Akt and JNK activities. These mechanisms are likely at work in vivo, as EGFR expression was highly correlated with TF expression in human high-grade astrocytoma specimens.

Vasculostatin inhibits intracranial glioma growth and negatively regulates in vivo angiogenesis through a CD36-dependent mechanism

Angiogenesis is a critical physiologic process that is appropriated during tumorigenesis. Little is known about how this process is specifically regulated in the brain. Brain angiogenesis inhibitor-1 (BAI1) is a brain-predominant seven-transmembrane protein that contains five antiangiogenic thrombospondin type-1 repeats (TSR). We recently showed that BAI1 is cleaved at a conserved proteolytic cleavage site releasing a soluble, 120 kDa antiangiogenic factor called vasculostatin (Vstat120). Vstat120 has been shown to inhibit in vitro angiogenesis and suppress subcutaneous tumor growth. Here, we examine its effect on the intracranial growth of malignant gliomas and further study its antitumor mechanism. First, we show that expression of Vstat120 strongly suppresses the intracranial growth of malignant gliomas, even in the presence of the strong proangiogenic stimulus mediated by the oncoprotein epidermal growth factor receptor variant III (EGFRvIII). This tumor-suppressive effect is accompanied by a decrease in tumor vascular density, suggesting a potent antiangiogenic effect in the brain. Second, and consistent with this interpretation, we find that treatment with Vstat120 reduces the migration of cultured microvascular endothelial cells in vitro and inhibits corneal angiogenesis in vivo. Third, we show that these antivascular effects critically depend on the presence of the cell surface receptor CD36 on endothelial cells in vitro and in vivo, supporting the role of Vstat120 TSRs in mediating these effects. These results advance the understanding of brain-specific angiogenic regulation, and suggest that Vstat120 has therapeutic potential in the treatment of brain tumors and other intracerebral vasculopathies.

Inhibition of cellular immune responses to encapsulated porcine islet xenografts by simultaneous blockade of two different costimulatory pathways.

Background. Transplantation of human islets has been successful clinically. Since human islets are scarce, we are studying microencapsulated porcine islet xenografts in nonobese diabetic (NOD) mice. We have evaluated the cellular immune response in NOD mice with and without dual costimulatory blockade. Methods. Alginate-poly-L-lysine-encapsulated adult porcine islets were transplanted i.p. in untreated diabetic NODs and NODs treated with CTLA4-Ig to block CD28/B7 and with anti-CD154 mAb to inhibit CD40/CD40-ligand interactions. Groups of mice were sacrificed on subsequent days; microcapsules were evaluated by histology; peritoneal cells were analyzed by FACS; and peritoneal cytokines were quantified by ELISA. Controls included immunoincompetent NOD-Scids and diabetic NODs given sham surgery or empty microcapsules. Results. Within 20 days, encapsulated porcine islets induced accumulation of large numbers of macrophages, eosinophils, and significant numbers of CD4+ and CD8+ T cells at the graft site, and all grafts were rejected. During rejection, IFN&ggr;, IL-12 and IL-5 were significantly elevated over sham-operated controls, whereas IL-2, TNF&agr;, IL-4, IL-6, IL-10, IL-1&bgr; and TGF&bgr; were unchanged. Treatment with CTLA4-Ig and anti-CD154 prevented graft destruction in all animals during the 26 days of the experiment, dramatically inhibited recruitment of host inflammatory cells, and inhibited peritoneal IFN&ggr; and IL-5 concentrations while delaying IL-12 production. Conclusions. When two different pathways of T cell costimulation were blocked, T cell-dependent inflammatory responses were inhibited, and survival of encapsulated islet xenografts was significantly prolonged. These findings suggest synergy between encapsulation of donor islets and simultaneous blockade of two host costimulatory pathways in prolonging xenoislet transplant survival.

Long-term Metabolic Control of Autoimmune Diabetes in Spontaneously Diabetic Nonobese Diabetic Mice by Nonvascularized Microencapsulated Adult Porcine Islets

Background. The long-term metabolic function of microencapsulated xenogeneic adult porcine islets (API) was assessed in a murine model of type 1 diabetes mellitus. Methods. API were encapsulated in barium-gelled alginate and transplanted intraperitoneally in diabetic nonobese diabetic (NOD) mice given no immunosuppression or given costimulatory blockade (CoB; CTLA4-Ig+anti-CD154 mAb). Control mice received nonencapsulated API under the kidney capsule. Graft function was monitored by measurement of random blood glucose levels, serum glycosylated hemoglobin (HbA1c), serum porcine C peptide, in vivo glucose tolerance tests, and histologic analyses of host pancreas and graft biopsies. Host immune responses to the islet xenografts were characterized by phenotyping peritoneal cellular infiltrates and by measuring serum antiporcine antibody levels. Results. Without immunosuppression, nonencapsulated API functioned for less than 1 week, and microencapsulated API functioned for 35±14 days before rejection, associated with both a cellular and a humoral immune response. With continuous CoB, nonencapsulated API functioned for 27±4 days, whereas microencapsulated API functioned for >450 days with measurable levels of serum porcine C peptide, near normal in vivo glucose tolerance tests and HbA1c levels, and intact microcapsules containing viable, insulin-positive porcine islets. Conclusions. Microencapsulated API restored normoglycemia for more than 1 year in spontaneously diabetic NODs given dual CoB. To our knowledge, this is the first study to document long-term normalized HbA1c, porcine C peptide, and near normal glucose tolerance in immunosuppressed diabetic NOD mice transplanted intraperitoneally with microencapsulated API. Our study suggests that transplantation of microencapsulated porcine islet xenografts may be a future treatment for patients with type 1 diabetes mellitus.

Transplantation of the bone marrow microenvironment leads to hematopoietic chimerism without cytoreductive conditioning.

BACKGROUND It has been hypothesized that regimens to induce transplantation tolerance and long-term hematopoietic chimerism require recipient conditioning with whole body irradiation or a cytoablative regimen to create space within the marrow microenvironment to permit pluripotent stem cell engraftment. The purpose of this study was to determine if transplantation of an intact bone marrow microenvironment in the form of a bone graft would permit stable hematopoietic stem cell engraftment, shape the repertoire of developing T cells, and induce donor-specific unresponsiveness in the absence of a conditioning regimen. METHODS Fragments of femur were transplanted under the kidney capsule of recipient mice. At defined time points after bone graft transplantation recipients were assayed for chimerism, bone graft viability, and responses to donor and third party alloantigens in vitro and in vivo. RESULTS In the absence of an immunological barrier, bone graft transplantation resulted in long-term multi-lineage hematopoietic chimerism in the peripheral blood. Nude bone graft transplantation into SCID recipients resulted in development of donor- derived T cells that underwent negative selection on bone graft derived I-E+ cells within the thymus. Across a fully allogeneic barrier in immunocompetent recipients treated with combined blockade of the CD40 and CD28 pathways bone graft transplantation resulted in long-term donor-specific hyporesponsiveness in vitro and acceptance of donor specific skin grafts. CONCLUSIONS Transplantation of bone marrow in the form of a bone graft may facilitate the production of hematopoietic chimerism and lead to long-term donor-specific hyporesponsiveness in the absence of a cytoreductive conditioning regimen.

Biocompatibility and immune acceptance of adult porcine islets transplanted intraperitoneally in diabetic NOD mice in calcium alginate poly-L-lysine microcapsules versus barium alginate microcapsules without poly-L-lysine.

Background: If alginate microcapsules are to be used clinically for therapeutic cell transplants, capsule formulations must be designed to enhance optimal biocompatibility and immune acceptance. Methods: Microcapsules were generated using highly purified, endotoxin-free, ultra-low viscosity, high mannuronic acid alginate. The capsules differed with respect to gelling cation (50 mM barium or 100 mM calcium), alginate concentration (2.0% or 3.3%), alginate density (homogeneous or inhomogeneous), and the presence or absence poly-L-lysine (PLL) coating. Four types of empty capsules were implanted intraperitoneally (i.p.) in normal NOD mice, and their biocompatibility was evaluated after various time periods in vivo. Encapsulated adult porcine islets (APIs) were transplanted i.p. in diabetic NOD mice, and immune acceptance was evaluated by graft survival times, host cell adherence to capsule surfaces, and flow cytometric analysis of peritoneal host cells. Results: All empty alginate capsules were biocompatible in vivo, but barium-gelled alginate capsules without PLL were clearly the most biocompatible, since 99% of these empty capsules had no host cell adherence up to 9 months in vivo. In diabetic NOD mice, APIs functioned significantly longer in barium-alginate capsules without PLL than in calcium-alginate capsules with PLL and had strikingly less host cell adherence, although large numbers of host cells (predominantly macrophages and eosinophils) infiltrated the peritoneal cavities of recipients with APIs in both types of capsules. Addition of PLL coatings to barium-alginate capsules dramatically decreased graft survival. Conclusions: Inhomogeneous barium-gelled alginate capsules without PLL are the optimal candidates for clinical trials, based on their enhanced biocompatibility and immune acceptance in vivo.

Human Brat Ortholog TRIM3 Is a Tumor Suppressor That Regulates Asymmetric Cell Division in Glioblastoma

Cancer stem cells, capable of self-renewal and multipotent differentiation, influence tumor behavior through a complex balance of symmetric and asymmetric cell divisions. Mechanisms regulating the dynamics of stem cells and their progeny in human cancer are poorly understood. In Drosophila, mutation of brain tumor (brat) leads to loss of normal asymmetric cell division by developing neural cells and results in a massively enlarged brain composed of neuroblasts with neoplastic properties. Brat promotes asymmetric cell division and directs neural differentiation at least partially through its suppression on Myc. We identified TRIM3 (11p15.5) as a human ortholog of Drosophila brat and demonstrate its regulation of asymmetric cell division and stem cell properties of glioblastoma (GBM), a highly malignant human brain tumor. TRIM3 gene expression is markedly reduced in human GBM samples, neurosphere cultures, and cell lines and its reconstitution impairs growth properties in vitro and in vivo. TRIM3 expression attenuates stem-like qualities of primary GBM cultures, including neurosphere formation and the expression of stem cell markers CD133, Nestin, and Nanog. In GBM stem cells, TRIM3 expression leads to a greater percentage dividing asymmetrically rather than symmetrically. As with Brat in Drosophila, TRIM3 suppresses c-Myc expression and activity in human glioma cell lines. We also demonstrate a strong regulation of Musashi-Notch signaling by TRIM3 in GBM neurospheres and neural stem cells that may better explain its effect on stem cell dynamics. We conclude that TRIM3 acts as a tumor suppressor in GBM by restoring asymmetric cell division.

Interleukin-6 production and secretion by human parathyroids

Parathyroid hormone (PTH) stimulates osteoblasts to produce the proinflammatory cytokine interleukin‐6 (IL‐6), causing bone resorption. In patients with primary hyperparathyroidism, elevated serum levels of IL‐6 normalize after resection of parathyroid tumours. Because IL‐6 is also expressed in normal parathyroids and in other endocrine cells (adrenal and islet), we hypothesized that parathyroid tumours might contribute directly to the elevated serum IL‐6 levels in patients with hyperparathyroidism. Immunohistochemistry identified IL‐6, PTH, and chromogranin‐A (an endocrine and neuroendocrine tumour marker) in normal, adenomatous and hyperplastic parathyroids. Using immunofluorescence and confocal microscopy, IL‐6 co‐localized with PTH and with chromogranin‐A in parathyroid cells. All cultured parathyroid tumours secreted IL‐6 at levels markedly higher than optimally stimulated peripheral blood mononuclear cells. Supernates from cultured parathyroids stimulated proliferation of an IL‐6‐dependent cell line, and anti‐IL‐6 MoAb abolished this stimulatory effect. IL‐6 mRNA was documented in cultured parathyroid tumours, cultured normal parathyroids, fresh operative parathyroid tumours and fresh operative normal specimens. In conclusion, these data show that parathyroid tumours and normal parathyroids contain, produce and secrete IL‐6. Our findings present a novel pathway by which human parathyroids may contribute markedly to IL‐6 production and elevation of serum IL‐6 levels in patients with hyperparathyroidism. The physiological relevance of IL‐6 production by human parathyroids remains to be determined, but IL‐6 secretion by parathyroid tumours may contribute to bone loss and to other multi‐system complaints observed in these patients.