de Paul Vos

Factors Influencing Insulin Secretion From Encapsulated Islets

Adequate regulation of glucose levels by a microencapsulated pancreatic islet graft requires a minute-to-minute regulation of blood glucose. To design such a transplant, it is mandatory to have sufficient insight in factors influencing the kinetics of insulin secretion by encapsulated islets. The present study investigates factors influencing the glucose-induced insulin response of encapsulated islets in vitro. We applied static incubations and did the following observations. (i) Small islets (90–120 μm) showed a similar instead of a lower glucose-induced insulin response, suggesting that inclusion of only small islets, which are associated with lower protrusion and failing rates, has no consequences for the functional performance of the graft. (ii) A capsule diameter of 800 μm showed identical rather than lower glucose-induced insulin responses as smaller, 500-μm capsules. (iii) Capsule membranes constructed with a conventional permeability interfered with diffusion of insulin, as illustrated by a lower response of islets in capsules with a 10-min poly-L-lysine (PLL) membrane than islets in capsules with a 5-min PLL membrane. (iv) Irrespective of the tested porosity, the capsules provided sufficient immunoprotection because the 10-min PLL membranes did block diffusion of the cytokines IL-1β (17 kDa) and TNF-α (70 kDa) while the 5-min PLL membranes interfered with the diffusion of the vast majority of the cytokines. We conclude that capsules containing small islets (90–120 μm) and a membrane with a lower permeability than routinely applied is preferred in order to obtain a graft with adequate glucose-induced insulin responses.

Pancreatic Beta-Cell Purification by Altering FAD and NAD(P)H Metabolism

Isolation of primary beta cells from other cells within in the pancreatic islets is of importance for many fields of islet research. However, up to now, no satisfactory method has been developed that gained high numbers of viable beta cells, without considerable alpha-cell contamination. In this study, we investigated whether rat beta cells can be isolated from nonbeta endocrine cells by manipulating the flavin adenine dinucleotide (FAD) and nicotinamide-adenine dinucleotide phosphate (NAD(P)H) autofluorescence. Beta cells were isolated from dispersed islets by flow cytometry, based on their high FAD and NAD(P)H fluorescence. To improve beta cell yield and purity, the cellular FAD and NAD(P)H contents were altered by preincubation in culture media containing varying amounts of D-glucose and amino acids. Manipulation of the cellular FAD and NAD(P)H fluorescence improves beta cell yield and purity after sorting. This method is also a fast and reliable method to measure beta cell functional viability. A conceivable application is assessing beta cell viability before transplantation.

Plasma of pregnant and preeclamptic women activates monocytes in vitro

OBJECTIVE The objective of the study was to test the hypothesis that factors circulating in the plasma of pregnant women and women with preeclampsia activate monocytes. STUDY DESIGN Blood samples were taken from patients with early-onset severe preeclampsia (n = 9), healthy pregnant women (n = 9), and healthy nonpregnant women (n = 9). A monocytic cell line was incubated with the plasma for 4, 16, and 24 hours. After the incubation, reactive oxygen species (ROS) production and intercellular adhesion molecule (ICAM)-1 expression (protein and messenger ribonucleic acid) were measured. RESULTS Plasma of both pregnant women and women with preeclampsia, as compared with plasma from nonpregnant women, increased the mean channel brightness (MCB) of ROS after 4 hours of incubation, whereas only plasma of pregnant women increased the percentage of cells producing ROS (after 4 and 24 hours of incubation). Plasma of pregnant women and women with preeclampsia up-regulated the percentage of ICAM-1-expressing cells after 4 hours and down-regulated the percentage of ICAM-1-expressing cells and MCB after 24 hours. CONCLUSION Plasma of both pregnant women and women with preeclampsia activated monocytes in vitro.

Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats

INTRODUCTION Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle. METHODS Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR. RESULTS ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20). DISCUSSION Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved. CONCLUSION Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy.

Impaired glucose tolerance in recipients of an intraperitoneally implanted microencapsulated islet allograft is caused by the slow diffusion of insulin through the peritoneal membrane.

I NTRAPERITONEAL transplantation of microencapsulated islets has been reported to restore normoglycemia both in chemically induced and in autoimmune diabetic animal models,‘-5 and recently also in man.6 But a previous report from our laboratory showed that glucose tolerance remains disturbed in spite of normoglycemia, and that there is no increase of plasma insulin levels in response to a physiologic stimulus such as the intake of a meal5 This may be the consequence of several factors such as a gradual decrease of the number of viable and functioning islets after transplantation as a consequence of fibrotic overgrowth of the capsules, or a slow release of insulin in response to a glycemic stimulus as a consequence of overly large capsule diameters.’ The transplantation site as such may be another factor. Previous studies on the kinetics of intraperitoneal insulin absorption report a short delay between the intraperitoneal administration of insulin and rise of plasma insulin levels. *-‘i However, these studies were often performed with insulin bolus injections in unphysiologically high doses which may well be associated with a more rapid absorption of insulin than should be expected with a gradual release of insulin from encapsulated pancreatic islets. Therefore, the present study investigates whether the intraperitoneal implantation site as such interferes with optimal transport kinetics between the islets and the bloodstream.

Impaired trophoblast invasion and increased numbers of immune cells at day 18 of pregnancy in the mesometrial triangle of type 1 diabetic rats

INTRODUCTION Type 1 diabetes (T1D) is associated with adverse pregnancy outcome, usually attributed to hyperglycemia. Recently, we showed that pregnancy outcome in normoglycemic T1D rats was characterized by decreased fetal and placental weight, suggesting impaired placental development. In the present study, we tested the hypothesis that trophoblast invasion and spiral artery (SA) remodeling is impaired in T1D rats ant that this is associated with aberrant local presence of NK cells and macrophages in the mesometrial triangle (MT). METHODS Placentae with MT from pregnant biobreeding diabetes-prone (BBDP; T1D model) rats, control biobreeding diabetes-resistant (BBDR) and Wistar-rats were dissected at day 18 of gestation and stained for trophoblast invasion, SA remodeling, uNK cells and macrophages. RESULTS Interstitial trophoblast invasion and SA remodeling was impaired in BBDP-rats vs. control rats, coinciding with increased presence of NK cells and an increased iNOS+/CD206+ ratio of macrophages. DISCUSSION Decreased fetal and placental weight in BBDP-rats was associated with diminished interstitial trophoblast invasion and less optimal SA remodeling, increased numbers of NK cells and increased iNOS+/CD206+ macrophage ratio in the MT of BBDP-rats. CONCLUSIONS The impaired trophoblast invasion and SA remodeling may be due to an aberrant local immune-response and may result in damage to the fetal placenta and insufficient supply of nutrients towards the fetus with eventually decreased fetal weight as a consequence.

An artificial transplantation site for pancreatic islets

MICROENCAPSULATED islets still have their functional limitations. Transplantation near blood vessels may improve the function. An intraperitoneally located and prevascularized expanded polytetrafluouroethylene (ePTFE) solid support is potentially a suitable transplantation site for encapsulated pancreatic islets because it allows both for the implantation of a large volume islet graft in the immediate vicinity of blood vessels and for its complete removal. ePTFE seems a suitable material for the construction of solid supports as it is biocompatible and can be coated to induce vascularization. The present study investigates the efficacy of an ePTFE solid support for the implantation of nonencapsulated islet isografts in streptozotocin-induced diabetic rat recipients.

Impaired glucose tolerance in rat islet isograft recipients after cytomegalovirus infection

M.J. Smelt, B.J. de Haan, M.M. Faas, A. de Haan, P. de Vos. Impaired glucose tolerance in rat islet isograft recipients after cytomegalovirus infection. Transpl Infect Dis 2013: 15: E44–E47. All rights reserved M.J. Smelt, B.J. de Haan, M.M. Faas, A. de Haan, P. de Vos Department of Pathology and Medical Biology, Section of Medical Biology, University Medical Center Groningen, Groningen, The Netherlands, Department of Medical Microbiology, Section of Molecular Virology, University Medical Center Groningen, Groningen, The Netherlands