Susan A. Safley

Lack of cross-species transmission of porcine endogenous retrovirus infection to nonhuman primate recipients of porcine cells, tissues, or organs.

Background. Nonhuman primates (NHPs) have been widely used in different porcine xenograft procedures inevitably resulting in exposure to porcine endogenous retrovirus (PERV). Surveillance for PERV infection in these NHPs may provide information on the risks of cross-species transmission of PERV, particularly for recipients of vascularized organ xenografts for whom data from human clinical trials is unavailable. Methods. We tested 21 Old World and 2 New World primates exposed to a variety of porcine xenografts for evidence of PERV infection. These NHPs included six baboon recipients of pig hearts, six bonnet macaque recipients of transgenic pig skin grafts, and nine rhesus macaque and two capuchin recipients of encapsulated pig islet cells. Serologic screening for PERV antibody was done by a validated Western blot assay, and molecular detection of PERV sequences in peripheral blood mononuclear cells (PBMCs) and plasma was performed using sensitive polymerase chain reaction and reverse transcriptase-polymerase chain reaction assays, respectively. Spleen and lymph node tissues available from six bonnet macaques and three rhesus macaques were also tested for PERV sequences. Results. All plasma samples were negative for PERV RNA suggesting the absence of viremia in these xenografted animals. Similarly, PERV sequences were not detectable in any PBMC and tissue samples, arguing for the lack of latent infection of these compartments. In addition, all plasma samples were negative for PERV antibodies. Conclusion. These data suggest the absence of PERV infection in all 23 NHPs despite exposure to vascularized porcine organs or tissue xenografts and the use of immunosuppressive therapies in some animals. These findings suggest that PERV is not easily transmitted to these NHP species through these types of xenografts.

Cellular Encapsulation Enhances Cardiac Repair

Background Stem cells for cardiac repair have shown promise in preclinical trials, but lower than expected retention, viability, and efficacy. Encapsulation is one potential strategy to increase viable cell retention while facilitating paracrine effects. Methods and Results Human mesenchymal stem cells (hMSC) were encapsulated in alginate and attached to the heart with a hydrogel patch in a rat myocardial infarction (MI) model. Cells were tracked using bioluminescence (BLI) and cardiac function measured by transthoracic echocardiography (TTE) and cardiac magnetic resonance imaging (CMR). Microvasculature was quantified using von Willebrand factor staining and scar measured by Massons Trichrome. Post‐MI ejection fraction by CMR was greatly improved in encapsulated hMSC‐treated animals (MI: 34±3%, MI+Gel: 35±3%, MI+Gel+hMSC: 39±2%, MI+Gel+encapsulated hMSC: 56±1%; n=4 per group; P<0.01). Data represent mean±SEM. By TTE, encapsulated hMSC‐treated animals had improved fractional shortening. Longitudinal BLI showed greatest hMSC retention when the cells were encapsulated (P<0.05). Scar size at 28 days was significantly reduced in encapsulated hMSC‐treated animals (MI: 12±1%, n=8; MI+Gel: 14±2%, n=7; MI+Gel+hMSC: 14±1%, n=7; MI+Gel+encapsulated hMSC: 7±1%, n=6; P<0.05). There was a large increase in microvascular density in the peri‐infarct area (MI: 121±10, n=7; MI+Gel: 153±26, n=5; MI+Gel+hMSC: 198±18, n=7; MI+Gel+encapsulated hMSC: 828±56 vessels/mm2, n=6; P<0.01). Conclusions Alginate encapsulation improved retention of hMSCs and facilitated paracrine effects such as increased peri‐infarct microvasculature and decreased scar. Encapsulation of MSCs improved cardiac function post‐MI and represents a new, translatable strategy for optimization of regenerative therapies for cardiovascular diseases.

Delivery of class I and class II MHC-restricted T-cell epitopes of listeriolysin of Listeria monocytogenes by attenuated Salmonella

Using a Salmonella vaccine-Listeria infection model of intracellular infection, we studied the capacity of an attenuated strain of Salmonella carrying T-cell epitopes of listeriolysin (LLO) of L. monocytogenes to elicit epitope-specific T-cell responses. Class II (LLO 215-226) or class I (LLO 91-99) MHC-restricted T-cell epitopes of LLO were inserted within a central, hypervariable domain of the flagellin protein of an attenuated delta aroA Salmonella dublin strain. T cells from Listeria-immunized mice were activated by lysates or heat-killed preparations of Salmonella construct expressing the LLO 215-226 epitope, indicating that LLO 215-226 is processed and presented to T cells when offered to antigen-presenting cells as part of a flagellin-epitope fusion protein. The chimeric flagellin genes were integrated into the chromosome of the flagellin-negative S. dublin strain to obtain stable expression of the epitopes. Immunization with the living, chromosomally integrated Salmonella construct carrying LLO 215-226 epitope as part of the flagellin protein generated T cells reactive with the corresponding LLO peptide, indicating that this chimera can stimulate a class-specific immune response in vitro. The effect of flanking residues on the processing and presentation of MHC class I LLO 91-99 epitope was studied using Salmonella vaccine strains that express chimeric flagellins containing one of three LLO 91-99 inserts: 91-99 (normal flagellin amino acids as flanking residues); KK91-99KK (Lys-Lys flanking residues); and AAA91-99AAA (Ala-Ala-Ala flanking residues).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Evaluation of Graft‐Host Response for Various Tissue Sources and Animal Models

ABSTRACT: The efficacy of pancreatic islet transplants in correcting hyperglycemia and slowing the progression of complications in diabetics has been confirmed by many experimental and clinical studies. Unfortunately, the availability of human islets is extremely limited and, therefore, treatment of large numbers of human diabetic patients will almost certainly require either the use of islets harvested from animals (xenografts) or the use of insulin‐secreting genetically modified cells of either human or animal origin. There is currently no effective regimen which will allow long‐term survival of xenogeneic islets from widely unrelated donor‐recipient combinations, such as pig‐to‐rodent, pig‐to‐dog, or pig‐to‐primate. There is considerable interest in the development of immunoisolation techniques for protection of donor islets. However, most materials used in immunoisolation devices are relatively bio‐incompatible. Poly‐L‐lysine‐alginate microcapsules are biocompatible and provide an optimal geometry for transmembrane diffusion of insulin and nutrients. Microcapsules allow long‐term survival of xenogeneic islets in diabetic rodents or dogs with induced diabetes. However, mice and rats with spontaneous diabetes destroy encapsulated islet grafts within 2 to 3 weeks. Biopsies reveal large numbers of macrophages, immunoglobulins and limited numbers of helper and cytotoxic T‐cells in the peri‐microcapsule environment of the peritoneal cavity. Cytokines have been identified in peritoneal fluid from mice with islet grafts and may play a role in encapsulated islet destruction. Targeted immunomodulation by treatment of recipients with either anti‐helper T‐cell antibodies, or fusion proteins which block costimulatory interactions between antigen presenting cells and host T‐cells have demonstrated synergy in significant prolongation of encapsulated islet xenograft survival in NOD mice with spontaneous diabetes. Technical improvements in microcapsule design also have contributed to prolonged graft survival. “Double‐wall” microencapsulation provides a more durable microcapsule and islet pretreatment prior to encapsulation reduces the frequency of defective capsules with islets entrapped in the membrane. Long‐term durability of encapsulated islet grafts remains a concern and further improvements in microcapsule design are a prerequisite to clinical trials.

Proliferative and cytokine responses in CTLA4-Ig-treated diabetic NOD mice transplanted with microencapsulated neonatal porcine ICCs.

Our goal is to develop effective islet xenografts for treating human diabetes. We have studied microencapsulated neonatal porcine islet cell clusters (ICCs) transplanted intraperitoneally in spontaneously diabetic NOD mice, where they function to maintain normoglycemia in the autoimmune host. Nonencapsulated neonatal porcine ICCs functioned for 4.5 ± 0.5 days before being rejected; encapsulation prolonged graft function to 17 ± 2 days. CTLA4-Ig treatment did not enhance the survival of nonencapsulated ICCs. However, CTLA4-Ig treatment significantly extended the function of encapsulated ICCs to 73 ± 5 days. Histological analyses demonstrated a profuse pericapsular cellular reaction associated with rejection of encapsulated islet xenografts in untreated mice, while this reaction was significantly reduced in CTLA4-Ig-treated mice. To study mechanisms of xenograft rejection in this model, we analyzed proliferative responses to neonatal porcine ICCs and cytokines present in the peritoneal cavities of transplanted mice. Spleen cells from both CTLA4-Ig-treated and untreated rejecting NODs exhibited vigorous proliferation in the absence of antigenic stimulation, suggesting prior activation in vivo, while splenocytes from CTLA4-Ig-treated NODs with functioning grafts had low proliferative levels, equal to controls. Islet-specific proliferation was not detected in islet-rejecting mice, perhaps due to their high background levels. With the exception of elevated IL-6 levels, empty capsules did not provoke a significant peritoneal cytokine response compared with sham surgery or untransplanted control mice. However, IL-5, IL-12, TGF-β, and IL-1β were significantly elevated in NODs receiving encapsulated neonatal porcine ICCs compared with untransplanted controls. There were no significant differences between peritoneal cytokine concentrations in CTLA4-Ig-treated mice with long-term functioning grafts compared to mice that rejected grafts at earlier time points. We conclude that the combination of donor islet microencapsulation and brief treatment of the recipient with co-stimulatory blockade delays sensitization of the host, possibly by altering mechanism(s) for recruitment and/or activation of host effector cells.

Alginate microencapsulation of human mesenchymal stem cells as a strategy to enhance paracrine-mediated vascular recovery after hindlimb ischaemia

Stem cell‐based therapies hold great promise as a clinically viable approach for vascular regeneration. Preclinical studies have been very encouraging and early clinical trials have suggested favourable outcomes. However, significant challenges remain in terms of optimizing cell retention and maintenance of the paracrine effects of implanted cells. To address these issues, we have proposed the use of a cellular encapsulation approach to enhance vascular regeneration. We contained human mesenchymal stem cells (hMSCs) in biocompatible alginate microcapsules for therapeutic treatment in the setting of murine hindlimb ischaemia. This approach supported the paracrine pro‐angiogenic activity of hMSCs, prevented incorporation of hMSCs into the host tissue and markedly enhanced their therapeutic effect. While injection of non‐encapsulated hMSCs resulted in a 22 ± 10% increase in vascular density and no increase in perfusion, treatment with encapsulated hMSCs resulted in a 70 ± 8% increase in vascular density and 21 ± 7% increase in perfusion. The described cellular encapsulation strategy may help to better define the mechanisms responsible for the beneficial effects of cell‐based therapies and provide a therapeutic strategy for inducing vascular growth in the adult. As hMSCs are relatively easy to isolate from patients, and alginate is biocompatible and already used in clinical applications, therapeutic cell encapsulation for vascular repair represents a highly translatable platform for cell‐based therapy in humans. Copyright

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.