Microencapsulated islet allografts in diabetic NOD mice and nonhuman primates.

OBJECTIVE: Our goal was to assess the efficacy of encapsulated allogeneic islets transplanted in diabetic NOD mice and streptozotocin (STZ)-diabetic nonhuman primates (NHPs). MATERIALS AND METHODS: Murine or NHP islets were microencapsulated and transplanted in non-immunosuppressed mice or NHPs given clinically-acceptable immunosuppressive regimens, respectively. Two NHPs were treated with autologous mesenchymal stem cells (MSCs) and peri-transplant oxygen therapy. Different transplant sites (intraperitoneal [i.p.], omental pouch, omental surface, and bursa omentalis) were tested in separate NHPs. Graft function was monitored by exogenous insulin requirements, fasting blood glucose levels, glucose tolerance tests, percent hemoglobin A1c (% HbA1c), and C-peptide levels. In vitro assessment of grafts included histology, immunohistochemistry, and viability staining; host immune responses were characterized by flow cytometry and cytokine/chemokine multiplex ELISAS. RESULTS: Microencapsulated islet allografts functioned long-term i.p. in diabetic NOD mice without immunosuppression, but for a relatively short time in immunosuppressed NHPs. In the NHPs, encapsulated allo-islets initially reduced hyperglycemia, decreased exogenous insulin requirements, elevated C-peptide levels, and lowered % HbA1c in plasma, but graft function diminished with time, regardless of transplant site. At necropsy, microcapsules were intact and non-fibrotic, but many islets exhibited volume loss, central necrosis and endogenous markers of hypoxia. Animals receiving supplemental oxygen and autologous MSCs showed improved graft function for a longer post-transplant period. In diabetic NHPs and mice, cell-free microcapsules did not elicit a fibrotic response. CONCLUSIONS: The evidence suggested that hypoxia was a major factor for damage to encapsulated islets in vivo. To achieve long-term function, new approaches must be developed to increase the oxygen supply to microencapsulated islets and/or identify donor insulin-secreting cells which can tolerate hypoxia.

Engraftment of adult porcine islet xenografts in diabetic nonhuman primates through targeting of costimulation pathways.

Recent advances in human allogeneic islet transplantation have established beta-cell replacement therapy as a potentially viable treatment option for individuals afflicted with Type 1 diabetes. Two recent successes, one involving neonatal porcine islet xenografts transplanted into diabetic rhesus macaques treated with a costimulation blockade-based regimen and the other involving diabetic cynomolgus monkeys transplanted with adult porcine islet xenografts treated with an alternative multidrug immunosuppressive regimen have demonstrated the feasibility of porcine islet xenotransplantation in nonhuman primate models. In the current study, we assessed whether transplantation of adult porcine islet xenografts into pancreatectomized macaques, under the cover of a costimulation blockade-based immunosuppressive regimen (CD28 and CD154 blockade), could correct hyperglycemia. Our findings suggest that the adult porcine islets transplanted into rhesus macaques receiving a costimulation blockade-based regimen are not uniformly subject to hyperacute rejection, can engraft (2/5 recipients), and have the potential to provide sustained normoglycemia. These results provide further evidence to suggest that porcine islet xenotransplantation may be an attainable strategy to alleviate the islet supply crisis that is one of the principal obstacles to large-scale application of islet replacement therapy in the treatment of Type 1 diabetes.

A membrane-mimetic barrier for islet encapsulation.

BACKGROUND: Enhanced control of both transport properties and surface physiochemical characteristics will be important steps in the development of an effective immunoisolation barrier critical to the success of pancreatic islet cell transplantation. We hypothesize that the cell membrane establishes an important paradigm for the design of a biomimetic immunoisolation barrier with improved performance characteristics because of its capacity to control interfacial mass transport, as well as its ability to act as a template for more complex structures with other immunoregulatory macromolecules. METHODS: Islets were isolated from Wistar rats using collagenase digestion and a discontinuous Ficoll-Histopaque gradient and subsequently encapsulated in 2% alginate. After coating with a polyelectrolyte multilayer of polylysine and alginate, a polymeric membrane-mimetic coating was applied to the capsule surface. Individual islet viability was evaluated at each stage of the encapsulation procedure by use of a two-color live/dead cell assay. Preservation of islet function was determined by transplanting 1000 encapsulated islets into the peritoneal cavity of streptozotocin-induced diabetic nonobese diabetic NOD/Scid mice. RESULTS: At the end of the coating procedure, the proportion of viable cells within each islet was >50% in 88% of encapsulated rat islets and >75% in over half of the encapsulated cohort. Nonfasting blood glucose levels normalized within 24 hours after transplantation (n = 8). Normoglycemia has been maintained in all mice with the longest time course being 73 days thus far. CONCLUSIONS: We have demonstrated that microencapsulated islets coated with a membrane-mimetic thin film can be generated with high viability in vitro and persistent function in vivo.

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.

The presentation of class I and class II epitopes of listeriolysin O is regulated by intracellular localization and by intercellular spread of Listeria monocytogenes.

The hemolysin, listeriolysin 0 (LLO), produced by Listeria monocytogenes is both a virulence factor and an immunodominant Ag. In this study, we investigated how the lytic activity of LLO effects the context of presentation of two known LLO epitopes by either class I or class II MHC molecules. T cell hybridomas were used to monitor each peptide/MHC ligand. APCs infected with strains of Listeria expressing hemolytic LLO strongly presented the class I MHC epitope; however, this ligand was not well presented by cells infected with nonhemolytic Listeria. In contrast, there was almost no presentation of the class II-binding LLO epitope in cells infected with fully hemolytic Listeria. Only hemolysin-deficient Listeria were presented by class II MHC. Listeria expressing wild-type LLO but deficient in other virulence factors showed a presentation pattern equivalent to that of hemolytic Listeria. To address further the divergence of presentation, we used an intercellular spread assay to detect Ag presentation by cells neighboring the primarily infected one. We found that hemolytic Listeria were presented by both class I and II MHC on cells adjacent to the initially infected one(s). Finally, our kinetic analysis of presentation revealed that the class II ligand is presented over 4 h before the class I ligand. We have demonstrated that LLO's lytic activity potentiates presentation of listerial Ags by class I MHC and inhibits presentation via class II MHC. LLO-mediated intracellular localization (cytoplasmic vs endosomal) of bacteria corresponds to the operative presentation pathway.

Mechanisms of T cell epitope immunodominance analyzed in murine listeriosis.

We demonstrate conclusively that the bacterial exotoxin listeriolysin O (LLO) is a target Ag for eliciting CD4+ T cell responses following infection with Listeria monocytogenes. The minimal I-Ek-restricted immunodominant CD4+ T cell epitope was identified as peptide 215-226 (p215-226). Most LLO-specific T cell hybridomas recognized p203-226, p208-226, p215-226, and p215-234, although each exhibited a characteristic pattern of preferential reactivity. One hybridoma (IIIC5) reacted to p203-226 but not to p208-226 or any other LLO peptide tested. With APCs from B10 congenic mice and cells transfected with either I-Ak or I-Ek, IIIC5 recognized p203-216 with I-Ak, while a different hybridoma (IB5) recognized p215-226 with I-Ek. Competitive binding studies demonstrated that of 15 LLO peptides tested, only p203-226, p215-226, and p215-234 had high affinity for I-Ek, while p203-226 could also bind to isolated I-Ak. Of nine LLO peptides tested, only p215-234 bound multiple class II MHC alleles. These findings suggest that the immunodominance of p203-226 may be due in part to the presence of multiple T cell epitopes with I-Ek- and I-Ak-binding capability. Many of the rules of immunodominance observed with model Ags are also operative in our murine model of bacterial infectious disease. Furthermore, a novel mechanism of immunodominance based on newly defined structural features of MHC molecules is implicated. This information is crucial for rational vaccine development.

Human T-cell recognition of Listeria monocytogenes: recognition of listeriolysin O by TcR alpha beta + and TcR gamma delta + T cells.

The cell-mediated immune response to Listeria monocytogenes has been well characterized in the mouse. Listeriolysin O (LLO) is a major antigen in murine T-cell recognition of L. monocytogenes. In this study, we show that LLO is also recognized by human TcR alpha beta T cells and TcR gamma delta T cells. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with live listeriae and then expanded with interleukin 2 were shown to respond to purified LLO. The generation of LLO-responsive T cells was dependent on the use of live bacteria during the initial in vitro challenge. LLO-induced proliferation of T cells expanded by exposure of PBMC to live listeriae was major histocompatibility complex restricted. PBMC cultured with formalin-fixed listeriae and subsequently expanded by interleukin 2 gave high proliferative responses to fixed bacteria but failed to respond to LLO. PBMC stimulated in vitro with fixed listeriae contained predominantly TcR alpha beta + T cells. In contrast, PBMC obtained from 85% of the donors studied generated high numbers of TcR gamma delta + T cells following in vitro culture with live listeriae. Using a panel of synthetic amphipathic LLO peptides, we found that LLO-specific T cells from different individuals recognized both common and unique peptides. LLO 470-508 was recognized by three of five individuals, while LLO 203-226 and LLO 107-126 were recognized by two of six individuals. A TcR gamma delta + T-cell line was established from PBMC stimulated with live listeriae and was shown to recognize LLO 470-508. Proliferative responses could be induced in this cell line by peptide-pulsed autologous PBMC but not by peptide-pulsed allogeneic PBMC. Our results establish the importance of LLO in human T-cell recognition of listeriae and show that both TcR alpha beta + T cells and TcR gamma delta + T cells recognize this antigen. Finally, since LLO 470-508 has a high degree of homology with other gram-positive bacterial toxins, the recognition of this peptide by TcR gamma delta + T cells suggests that an important role of these T cells in host defense is the recognition of bacterium-derived toxins.

Role of listeriolysin-O (LLO) in the T lymphocyte response to infection with Listeria monocytogenes. Identification of T cell epitopes of LLO.

Using a murine model, we investigated the role of the bacterial exotoxin listeriolysin O (LLO) in cellular immunity to Listeria monocytogenes. A correlation between LLO production by infecting bacteria and generation of protective immunity to virulent LLO-producing bacteria was noted. Using isogeneic hemolysin (Hly+ or Hly-) strains of L. monocytogenes, we demonstrated that LLO production by infecting bacteria is required to elicit T cells reactive both to bacteria-associated Ag and to the secreted LLO molecule as measured by IL-2 production in vitro. Distinct sets of T cells specific for largely nonoverlapping pools of antigenic determinants represented by LLO and cell-associated Ag (heat-killed L. monocytogenes) are generated after infection. We have used models for prediction of T cell epitopes based on primary structure of LLO, and synthetic amphipathic LLO peptides were evaluated as Ag in vitro or as immunogenes in vivo. Infection of several strains of mice (H-2k and H-2d) with LLO-producing L. monocytogenes resulted in the generation of T cells that could respond consistently to two peptides, LLO 215-234 and LLO 354-371. Mouse strains lacking expression of I-E molecules (e.g., B10.A(4R) and C57BL/6) responded to LLO but not to the peptides tested. With C3HeB/FeJ mice, antibodies to I-Ek blocked the presentation of LLO 215-234. The importance of the N-terminal portion of LLO 215-234 was evidenced by the drastic reduction in antigenic activity of truncated peptides (e.g., LLO 221-234 and LLO 224-234). LLO 215-234, the strongest and most consistent activator of T cells from L. monocytogenes-immune mice, fit well some models for antigenic peptides in several ways. It could be predicted to form an amphipathic alpha-helix, it contained multiple "Rothbard motifs" (charged residue or glycine, two or three hydrophobic amino acids and then a glycine or polar residue), it had a net charge of +2, and it contained the correct spacing of amino acids (five to six residues between a hydrophobic and basic amino acid) that is characteristic of I-Ek-binding peptides. Immunization with 8 of 10 synthetic LLO peptides generated T cells that recognized the immunizing peptide in vitro, but such T cells were only poorly reactive with LLO. Our results indicate that LLO is an important target Ag for stimulation of CD4+ L. monocytogenes-specific T cells, and that LLO 215-234 is antigenically dominant in C3HeB/FeJ mice.