Peter In't Veld

Poly-L-Lysine induces fibrosis on alginate microcapsules via the induction of cytokines.

Alginate – poly-l-lysine (PLL) microcapsules can be used for transplantation of insulin-producing cells for treatment of type I diabetes. In this work we wanted to study the inflammatory reactions against implanted microcapsules due to PLL. We have seen that by reducing the PLL layer, less overgrowth of the capsule is obtained. By incubating different cell types with PLL and afterwards measuring cell viability with MTT, we found massive cell death at concentrations of PLL higher than 10 μg/ml. Staining with annexin V and propidium iodide showed that PLL induced necrosis but not apoptosis. The proinflammatory cytokine, tumor necrosis factor (TNF), was detected in supernatants from monocytes stimulated with PLL. The TNF response was partly inhibited with antibodies against CD14, which is a well-known receptor for lipopolysaccharide (LPS). Bactericidal permeability increasing protein (BPI) and a lipid A analogue (B-975), which both inhibit LPS, did not inhibit PLL from stimulating monocytes to TNF production. This indicates that PLL and LPS bind to different sites on monocytes, but because they both are inhibited by a p38 MAP kinase inhibitor, they seem to have a common element in the signal transducing pathway. These results suggest that PLL may provoke inflammatory responses either directly or indirectly through its necrosis-inducing abilities. By combining soluble PLL and alginate both the toxic and TNF-inducing effects of PLL were reduced. The implications of these data are to use alginate microcapsules with low amounts of PLL for transplantation purposes.

Screening for Insulitis in Adult Autoantibody-Positive Organ Donors

Antibodies against islet cell antigens are used as predictive markers of type 1 diabetes, but it is unknown whether they reflect an ongoing autoimmune process in islet tissue. We investigated whether organs from adult donors that are positive for autoantibodies (aAbs) against islet cell antigens exhibit insulitis and/or a reduced β-cell mass. Serum from 1,507 organ donors (age 25–60 years) was analyzed for islet cell antibodies (ICAs), glutamate decarboxylase aAbs (GADAs), insulinoma-associated protein 2 aAbs (IA-2As), and insulin aAbs. Tissue from the 62 aAb+ donors (4.1%) and from matched controls was examined for the presence of insulitis and for the relative area of insulin+ cells. Insulitis was detected in two cases; it was found in 3 and 9% of the islets and consisted of CD3+/CD8+ T-cells and CD68+ macrophages; in one case, it was associated with insulin+ cells that expressed the proliferation marker Ki67. Both subjects belonged to the subgroup of three donors with positivity for ICA, GADA, and IA-2-Ab and for the susceptible HLA-DQ genotype. Comparison of relative β-cell area in aAb+ and aAb− donors did not show a significant difference. Insulitis was found in two of the three cases that presented at least three aAbs but in none of the other 59 antibody+ subjects or 62 matched controls. It was only detected in <10% of the islets, some of which presented signs of β-cell proliferation. No decrease in β-cell mass was detected in cases with insulitis or in the group of antibody+ subjects.

Insulitis in human type 1 diabetes: The quest for an elusive lesion.

The histopathology of type 1 diabetes is defined by a decreased β-cell mass in association with insulitis, a characteristic lymphocytic infiltration limited to the islets of Langerhans and prominent in early stage disease in children. A cytotoxic T-cell mediated destruction of insulin-producing β-cells is thought to be initiated by an unknown (auto)antigen, leading to the destruction >75% of β-cell mass at clinical diagnosis. Although considered to be pathognomonic for recent onset disease, insulitis has only been described in approximately 150 cases over the past century. This review describes the quest for this elusive lesion and gives its incidence in various patient subpopulations stratified for age of onset and duration of the disease. It discusses recent new insights into the regenerative capacity of the β-cell mass in the pre-clinical stages of the disease and relates these findings to the inflammatory processes within the islet tissue.

β-Cell Replication Is Increased in Donor Organs From Young Patients After Prolonged Life Support

OBJECTIVE This study assesses β-cell replication in human donor organs and examines possible influences of the preterminal clinical conditions. RESEARCH DESIGN AND METHODS β-Cell replication was quantified in a consecutive series of n = 363 human organ donors using double immunohistochemistry for Ki67 and insulin. Uni- and multivariate analysis was used to correlate replication levels to clinical donor characteristics and histopathologic findings. RESULTS β-Cell replication was virtually absent in most donors, with ≤0.1% Ki67-positive β-cells in 72% of donors. A subpopulation of donors, however, showed markedly elevated levels of replication of up to 7.0% Ki67-positive β-cells. β-Cell replication was accompanied by the increased replication of glucagon-, somatostatin-, and CA19.9-positive cells. Prolonged life support, kidney dysfunction, relatively young donor age, inflammatory infiltration, and prolonged brain death before organ retrieval were all found to be significantly associated with an increased level (≥90th percentile) of β-cell replication, with the first three risk factors being independent predictors. Increased β-cell replication was most often noted in relatively young donors (≤25 years) who received prolonged (≥3 days) life support (68%); in contrast, it was rare in donors with a short duration of life support regardless of age (1%). Prolonged life support was accompanied by increased levels of CD68+ and LCA/CD45+ infiltration in the pancreatic parenchyma. CONCLUSION These results indicate that preterminal clinical conditions in (young) organ donors can lead to increased inflammatory infiltration of the pancreas and to increased β-cell replication.

Omentum Is Better Site Than Kidney Capsule for Growth, Differentiation, and Vascularization of Immature Porcine β-Cell Implants in Immunodeficient Rats

Background Rapid revascularization of islet cell implants is important for engraftment and subsequent survival and function. Development of an adequate vascular network is expected to allow adaptive growth of the &bgr;-cell mass. The present study compares omentum and kidney capsule as sites for growth and differentiation of immature &bgr;-cell grafts. Methods Perinatal porcine islet cell grafts were implanted in omentum or under kidney capsule of nondiabetic nude rats. Implants were compared over 10 weeks for their respective growth, cellular composition, number and size of &bgr; cells, their proliferative activity, and implant blood vessel density. Results In both sites, the &bgr;-cell volume increased fourfold between weeks 1 and 10 reflecting a rise in &bgr;-cell number. In the omental implants, however, the cellular insulin reserves and the percent of proliferating cells were twofold higher than in kidney implants. In parallel, the blood vessel density in omental implants increased twofold, reaching a density comparable with islets in adult pig pancreas. A positive correlation was found between the percent bromodeoxyuridine-positive &bgr; cells and the vessel density. Conclusions Growth of the &bgr;-cell volume proceeds similarly in the omentum and under the kidney capsule. However, the omentum leads to higher insulin reserves and an increased pool of proliferating cells, which might be related to a more extended vascular network. Our observations support the omentum as an alternative site for immature porcine islet cells, with beneficial effects on proliferation and implant revascularization.

Insulitis in type 1 diabetes: a sticky problem.

Lymphocytic infiltration in the islets of Langerhans is generally recognized as the defining lesion in young patients with recent-onset type 1A diabetes. In a landmark article published in Diabetes in 1965, Gepts (1) described insulitis in 70% of cases with acute diabetes and found that the lesion only affected islets with residual β-cells in pancreatic organs that had otherwise lost most of their β-cell mass. He concluded that the disease was probably caused by a protracted β-cell–specific (auto)immune process, thereby initiating a domain of study that has led to many new insights into the disease process, the identification of preclinical markers, and the definition of new intervention strategies. Unfortunately, our knowledge of the early disease processes leading to the specific destruction of β-cells is far from complete. Among other things, it is still unknown what event triggers the T-cell–mediated inflammatory process, why the infiltrate of predominantly CD8+T-cells and macrophages is limited to the islets of Langerhans and only β-cells are destroyed, against which antigen the inflammatory infiltrate is directed, and whether this antigen is of an endogenous or exogenous nature (2–5). This lack of knowledge is, to a considerable extent, caused by the scarcity of material regarding those recently diagnosed with type 1 diabetes, of which only several dozen cases have been described in the literature (6). It is especially due to the virtual absence of material regarding pre-diabetic individuals …

Heterogeneity in distribution of amyloid-positive islets in type-2 diabetic patients

Amyloid-containing (A+) islets are characteristic for type-2 diabetes (T2D), but their abundance seems variable among patients. It is unclear whether the distribution of A+ islets follows a certain pattern or occurs randomly throughout the pancreatic organ. We investigated the topography of A+ islets in eight pancreata of T2D patients and eight sex- and age-matched non-diabetic subjects. Transversal sections of head, body and tail segments were stained with synaptophysin combined with Congo red to map/quantify islet tissue and amyloid. In the eight T2D pancreata, the overall percentage of A+ islets varied from 4% to 85%. Further analysis in body and tail indicated that peripheral regions exhibited higher percentages of A+ islets than central regions (averages of, respectively, 30% and 17%, P<0.05). Non-diabetic control pancreata also exhibited A+ islets, albeit at a 25-fold lower frequency; a tendency towards higher percentage of A+ islets in peripheral versus central regions was also observed. The higher percentage A+ islets in peripheral regions was associated with a higher density and relative islet over exocrine surface area. These observations on heterogeneity in abundance and distribution of A+ islets need consideration when sampling tissue for studies on human islet amyloidosis. The present methodology allows us to further investigate the susceptibility to amyloidosis of islets in peripheral regions of the pancreas.

Preservation time dependent morphological changes in cold stored human donor pancreas

Pancreatic transplantation is being used to treat insulin-dependent diabetes. An intact structure of the graft is a prerequisite for preserved function and we therefore monitored the light microscopic and ultrastructural changes in 30 human donor pancreases stored in the cold in University of Wisconsin preservation solution. Twenty-three pancreases were stored for less than 24 h and 7 for more than 30 h. All glands stored longer than 30 h displayed cytoplasmic vacuolisation in a variable proportion of acinar cells. In addition, the glands stored over 40 h showed focal acinar necrosis. Endocrine tissue was only slightly affected, while duct cells showed no changes. It is concluded that cold preserved pancreases stored for less than 24 h are best for transplantation purposes and that acinar cells are more sensitive to ischaemic damage than endocrine and duct cells.