Collin J. Weber

Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways

We evaluated the ability of neonatal porcine islets to engraft and restore glucose control in pancreatectomized rhesus macaques. Although porcine islets transplanted into nonimmunosuppressed macaques were rapidly rejected by a process consistent with cellular rejection, recipients treated with a CD28-CD154 costimulation blockade regimen achieved sustained insulin independence (median survival, >140 days) without evidence of porcine endogenous retrovirus dissemination. Thus, neonatal porcine islets represent a promising solution to the crucial supply problem in clinical islet transplantation.

Serum 25-Hydroxyvitamin D Levels and the Prevalence of Peripheral Arterial Disease Results from NHANES 2001 to 2004

Objective—The purpose of this study was to determine the association between 25-hydroxyvitamin D (25(OH)D) levels and the prevalence of peripheral arterial disease (PAD) in the general United States population. Methods and Results—We analyzed data from 4839 participants of the National Health and Nutrition Examination Survey 2001 to 2004 to evaluate the relationship between 25(OH)D and PAD (defined as an ankle-brachial index <0.9). Across quartiles of 25(OH)D, from lowest to highest, the prevalence of PAD was 8.1%, 5.4%, 4.9%, and 3.7% (P trend <0.001). After multivariable adjustment for demographics, comorbidities, physical activity level, and laboratory measures, the prevalence ratio of PAD for the lowest, compared to the highest, 25(OH)D quartile (<17.8 and ≥29.2 ng/mL, respectively) was 1.80 (95% confidence interval: 1.19, 2.74). For each 10 ng/mL lower 25(OH)D level, the multivariable-adjusted prevalence ratio of PAD was 1.35 (95% confidence interval: 1.15, 1.59). Conclusions—Low serum 25(OH)D levels are associated with a higher prevalence of PAD. Several mechanisms have been invoked in the literature to support a potential antiatherosclerotic activity of vitamin D. Prospective cohort and mechanistic studies should be designed to confirm this association.

The role of CD4+ helper T cells in the destruction of microencapsulated islet xenografts in nod mice.

Islet transplants for large numbers of patients with diabetes will require xenografts. Microencapsulation is an appealing method for islet xenografting. However, graft function has been limited by a cellular reaction, particularly intense in spontaneously diabetic, NOD mice. The purpose of this study was to elucidate the mechanism of this reaction. Poly-1-lysine-alginate microcapsules containing 4000-12,000 dog or 1800-2000 rat islets were xenografted intraperitoneally into streptozotocin (SZN)-diabetic C57BL/6J and NOD mice, with or without recipient treatment with GK 1.5 (anti-CD4 monoclonal antibody) (20-30 microliters i.p. every 5 days, begun on day -7. Grafts were considered technically successful if random blood glucose (BG) was normalized (less than 150 mg/dl) within 36 hr. Graft failure was defined as BG greater than 250 mg/dl. Dog and rat islets in microcapsules normalized BG in both SZN and NOD mice within 24 hr routinely. Empty microcapsules and GK 1.5 treatments alone did not affect BG. NODs destroyed both microencapsulated dog and rat islets more rapidly than did SZN-diabetic mice (P less than .01). Graft biopsies showed an intense cellular reaction, composed of lymphocytes, macrophages and giant cells, and no viable islets. GK 1.5 treatment significantly prolonged both dog-to-NOD and rat-to-NOD grafts (P less than 0.01). Biopsies of long-term functioning grafts (on days 65-85) demonstrated viable islets and no cellular reaction around microcapsules; 1/4 rat and 1/8 dog islet xenografts continued to function indefinitely in NOD recipients, even after cessation of GK 1.5 therapy. Prediabetic NODs receiving encapsulated dog or rat islets mounted a moderate cellular reaction to grafts. Empty microcapsules excited no cellular reaction in diabetic or prediabetic NODs. We conclude that the NOD reaction to microencapsulated xenogeneic islets is helper T cell-dependent, and that the target of this reaction is not the microcapsule itself, but the donor cells within.

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.

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

Recent advances in human allogeneic islet transplantation have established β‐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.

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.

Parathyroidectomy decreases systolic and diastolic blood pressure in hypertensive patients with primary hyperparathyroidism

BACKGROUND Primary hyperparathyroidism (PHPT) is associated with an increased risk of cardiovascular disease, including hypertension. The purpose of this study was to determine the effect of parathyroidectomy on systolic and diastolic blood pressures (BP) in hypertensive patients with PHPT. METHODS A retrospective review of medical records was performed in 368 patients undergoing parathyroidectomy and 96 patients undergoing thyroidectomy by a single surgeon. We evaluated changes in serum calcium, parathyroid hormone (PTH), and BP before and 6 months after surgery. RESULTS In patients undergoing parathyroidectomy with hypertension (n = 147), a significant decrease in both systolic and diastolic BP was observed; systolic BP decreased from 152.5 +/- 10.5 to 140.3 +/- 16.2 mmHg (P < .001) and diastolic BP decreased from 94.5 +/- 6.8 to 81.7 +/- 10.3 mmHg (P < .001). In these patients, PTH decreased from 136 +/- 186 to 58.7 +/- 44.3 pg/mL (P < .001) and serum calcium decreased from 11.1 +/- 0.6 to 9.5 +/- 0.7 mg/dL (P < .001). In patients undergoing parathyroidectomy without hypertension (n = 145) and thyroidectomy alone (n = 96), no significant change in systolic or diastolic BP was observed. CONCLUSION Parathyroidectomy in hypertensive patients seems to reduce both systolic and diastolic BP . The mechanisms responsible for this effect are unknown and deserve further study.

Improved Long-Term Survival of Dialysis Patients after Near-Total Parathyroidectomy

BACKGROUND Severe secondary hyperparathyroidism, which is associated with life-threatening complications, can develop in dialysis-dependent end-stage renal disease patients. The aim of this study was to compare short- and long-term mortality in dialysis patients who underwent near-total parathyroidectomy (NTPTX) and matched nonoperated controls. STUDY DESIGN We identified 150 dialysis patients who underwent NTPTX (1993-2009) at our institution and compared them with 1,044 nonoperated control patients identified in the US Renal Data System registry, matched for age, sex, race, diabetes as cause of kidney failure, years on dialysis, and dialysis modality. Survival outcomes were estimated using multivariable Cox proportional hazards models with stratification on the matching sets, adjusted for cardiovascular comorbidities, smoking, inability to ambulate/transfer, and payor status. RESULTS During a follow-up of a mean of 3.6 years (range 0.1 month to 16.4 years), NTPTX patients had a significant reduction in the long-term risk of all-cause death (hazard ratio = 0.68; 95% CI, 0.52-0.89; p = 0.006) compared with controls. Thirty-day mortality rates for NTPTX patients and controls were 246 vs 105 per 1,000 person-years (p = 0.21). In adjusted analyses, NTPTX patients had a 37% reduced risk of all-cause death and a 33% reduced risk of cardiovascular death compared with controls. A durable reduction in mean parathyroid hormone was observed after NTPTX; from 1,776 ± 1,416.6 pg/mL to 301 ± 285.7 pg/mL (p < 0.0001). CONCLUSIONS In our center, NTPTX in dialysis patients was associated with a significant reduction in long-term risk of death compared with matched control patients, without a significantly increased short-term risk.

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.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 4: pre-clinical efficacy and complication data required to justify a clinical trial.

In 2009, the International Xenotransplantation Association (IXA) published a consensus document that provided guidelines and “recommendations” (not regulations) for those contemplating clinical trials of porcine islet transplantation. These guidelines included the IXAs opinion on what constituted “rigorous pre‐clinical studies using the most relevant animal models” and were based on “non‐human primate testing.” We now report our discussion following a careful review of the 2009 guidelines as they relate to pre‐clinical testing. In summary, we do not believe there is a need to greatly modify the conclusions and recommendations of the original consensus document. Pre‐clinical studies should be sufficiently rigorous to provide optimism that a clinical trial is likely to be safe and has a realistic chance of success, but need not be so demanding that success might only be achieved by very prolonged experimentation, as this would not be in the interests of patients whose quality of life might benefit immensely from a successful islet xenotransplant. We believe these guidelines will be of benefit to both investigators planning a clinical trial and to institutions and regulatory authorities considering a proposal for a clinical trial. In addition, we suggest consideration should be given to establishing an IXA Clinical Trial Advisory Committee that would be available to advise (but not regulate) researchers considering initiating a clinical trial of xenotransplantation.