Caterina Vizzardelli

Endotoxin-mediated delayed islet graft function is associated with increased intra-islet cytokine production and islet cell apoptosis

Background. Primary nonfunction resulting in immediate graft loss is responsible for the failure of a large number of islet transplants. Evidence is accumulating to single out endotoxin contamination of the various reagents needed for islet isolation as a major cause of early graft loss. Methods. Islets isolated with endotoxin-containing (400 endotoxin units/ml) collagenase type V and “endotoxin-free” (3.1 endotoxin units/ml) Liberase™ were compared. Graft function was assessed using a syngeneic murine model of marginal islet mass transplantation. Pro-inflammatory cytokine production by islets was measured by ELISA in culture supernatants, and quantitative reverse transcriptase-PCR. Islet cell apoptosis was measured using the annexin assay. Results. Graft function was significantly delayed when islets were isolated with endotoxin-containing collagenase. Addition of endotoxin to the Liberase™ solution similarly delayed graft function. After 18 hr in culture, collagenase-isolated islets released higher amounts of proinflammatory cytokines compared with Liberase™-isolated islets (interleukin-6: 2185±1174 pg/ml vs. 520±201 pg/ml; tumor necrosis factor-&agr;: 304±298 pg/ml vs. 0; IL-1&bgr;: 12.5 pg/ml±12.5 vs. 0). This observation correlated with higher cytokine mRNA expression in collagenase-isolated islets. The percentage of apoptotic islet cells immediately after isolation was 17.2%±9.4 in collagenase-isolated islets and 7.1%±2.1 in Liberase™-isolated islets. Conclusions. We propose that endotoxin contamination is the primum movens of a chain of events that involves intra-islet cytokine production and release and islet cell apoptosis, and endotoxin contamination can ultimately lead to primary nonfunction in vivo. This emphasizes the fact that using endotoxin-free reagents during isolation is a key factor for successful islet transplantation.

Early assessment of apoptosis in isolated islets of langerhans

There is substantial evidence to link early graft loss after islet transplantation to isolation-induced islet cell apoptosis. Measurement of caspase 3 activity and detection of the lost cell membrane asymmetry, revealed by annexin V binding, are newly available assays that allow the analysis of early events of apoptosis.

Neonatal porcine pancreatic cell clusters as a potential source for transplantation in humans: characterization of proliferation, apoptosis, xenoantigen expression and gene delivery with recombinant AAV.

Abstract: Neonatal porcine islets are characterized by reproducible isolation success and high yields, sizable advantages over adult islets. In this work we have analyzed selected phenotypic and functional characteristics of porcine neonatal islets relevant to their possible use for transplant in humans. We show that porcine islet cells proliferate in culture, and synthesize and store islet‐specific hormones. Proliferating beta cells can be easily identified. Implant of cultured neonatal islets in immunodeficient rodents results in the reversal of diabetes, albeit with delay. We also show that measurable apoptosis occurs in cultured neonatal porcine islets. Further, antigens recognized by human natural antibodies are expressed in a dynamic fashion over the culture period analyzed and are not limited to the alpha‐Gal epitope. Lastly, we demonstrate that a recombinant Adeno‐Associated virus can be used to efficiently deliver a reporter gene in porcine islets. This characterization might be helpful in the definition of the potential use of neonatal porcine islets for human transplantation.

Automated method for isolation of adrenal medullary chromaffin cells from neonatal porcine glands

An automated method for the isolation of neonatal porcine adrenal chromaffin cells is described. Adrenal chromaffin cells are potentially useful for therapeutic transplantation, but current isolation methodology suffers from labor intensiveness and variability in yield and viability due to imprecision of manual techniques, enzyme purity, and gland age and species. The described approach utilizes an adaptation of an automated procedure previously described for isolation of pancreatic islets. Results from neonatal porcine adrenal glands revealed consistent cell yields with high (approximately 99%) viability. Catecholamine assays showed that the resultant cultures continue to produce and secrete norepinephrine and epinephrine. Immunocytochemical analysis indicated that the majority of cells in the preparation are chromaffin cells and adrenal cortical cells. The procedure meets the following requirements: 1) minimal traumatic action on the adrenal chromaffin cells, 2) continuous digestion in which the adrenal cells that are progressively liberated can be saved from further mechanical action, 3) minimal human intervention in the digestion process, and 4) high yield and viability of the isolated adrenal chromaffin cells.

Absence of CSF-1-dependent macrophages does not improve function of transplanted islets of Langerhans

A role of macrophage-mediated inflammatory events in early islet graft loss is increasingly acknowledged. Osteopetrotic mice (op/op) have a complete absence of CSF-1, and thus of most tissue macrophages. We have investigated whether the absence of CSF-1-dependent macrophages in the graft itself or at the transplant site could decrease the delay to function of a syngeneic marginal islet mass. Islets transplanted into op/op or control recipients reversed diabetes in 59 days vs. 10 days (p = 0.28, NS). Islets isolated from op/op or control mice reversed diabetes in 11 days vs. 10 days. IL-1 and TNF-α release by cultured islets was markedly decreased for op/op islets compared with control islets (IL-1: 0 vs. 4.2 pg/ml, p = 0.07; TNF-α: 67 vs. 311 pg/ml, p = 0.002). In contrast, IL-6 release by op/op islets was significantly increased (11.1 vs. 4.3 ng/ml, p = 0.006). CSF-1-dependent tissue macrophages may not be critical in the inflammatory insult to islet transplants. Alternate patterns of intraislet release of deleterious proinflammatory cytokines may exist.

Induced Heme Oxygenase-1 Upregulation Protects Pancreatic Beta Cells from Apoptosis In Vitro

INTRODUCTION. Transplantation of islets of Langerhans represents a viable therapeutic approach for the treatment of Type I diabetes mellitus (1). Unfortunately, transplanted islets are susceptible to allogeneic recognition and rejection, recurrence of autoimmunity and destruction by local inflammation at the site of implant. The latter phenomenon is characterized by microenvironment activation with production of proinflammatory cytokines (IL-1, IFN-γ, TNF-α) and oxygen radicals that might result in functional impairment and death of islet cells, and also contribute to amplifying the subsequent specific immune response (2,3). Islet β−cells are exquisitely susceptible to oxidative stress because of their insufficient anti-oxidant pool. Induction of islet cell protection against inflammation could therefore represent a viable strategy to improve overall graft fate. Heme oxygenase 1 (HO-1) has been described as an inducible stress protein capable of cytoprotection via radical scavenging and apoptosis prevention (4). The purpose of the present study was to analyze whether upregulation of HO-1 in a beta cell line (β-TC3) and in freshly isolated murine islets could result in protection from apoptosis. METHODS AND RESULTS. HO-1 upregulation was reproducibly induced, in a dose dependent fashion, by incubating the beta cell line and isolated islets for 24 hours in the presence of iron protoporphyrin (FePP), as assessed by western blot and quantitative RT-PCR analysis. A 24-hr incubation in the presence or absence of FePP was performed in both BetaTC3 cells and islets before induction of apoptosis. Subsequently, β-TC3 cells were incubated for 24-hrs in the presence of IL-1α (10U/ml) and IFN-γ (100U/ml) to induce the expression of Fas on their surface. A pro-apoptotic stimulus was then delivered via engagement of Fas by an agonistic anti-Fas antibody, followed by addition of protein G to induce cross-linking, and cells were cultured for 48-hrs before assay. In freshly isolated murine islets, the pro-apoptotic signal was delivered by addition of TNF-α (5000U/ml) and cyclohexamide (CHX, 50 µg/ml) for additional 48 hours. Control islets were similarly treated in the presence or absence of FePP, without TNF-α and CHX. At the end of culture, β-TC3 and islet cell suspension were obtained by trypsinization, stained with propidium iodide, and analyzed by flow cytometry on a linear scale, considering apoptotic the cells within the distinct sub-G1 peak, in which DNA condensation and fragmentation results in decreased staining. Analysis of apoptosis in the β-TC3 showed that the rate of apoptotic cells was inversely correlated to the concentration of FePP in the media, strongly suggesting a dose