Bart J. de Haan

Effect of the alginate composition on the biocompatibility of alginate-polylysine microcapsules

Alginate-polylysine (PLL) capsules are commonly applied for immunoprotection of endocrine tissues. Alginate is composed of mannuronic acid (M) and guluronic acid (G). Different types of alginate have different ratios of G to M, but little is known of the influence of these differences on biocompatibility. Therefore, we have investigated in vivo the effect of the G-content of the alginate on the biocompatibility of the capsules. Capsules prepared of commercially available alginates with either a high or an intermediate G-content were implanted in the peritoneal cavity of rats and retrieved one month later for histological evaluation. The fibrotic reaction was more severe against high-G alginate capsules than to intermediate-G alginate capsules. The majority of the high-G capsules proved to be overgrown and adherent to the abdominal organs whereas with intermediate-G alginate most capsules were found freely floating in the peritoneal cavity and free of any adhesion of cells. This was not caused by the alginate as such but rather by inadequate binding of high-G alginate to PLL since in the absence of PLL, i.e. with beads instead of capsules, no fibrotic reaction was observed. As high-G alginates have beneficial effects for islet encapsulation, efforts should be made to apply polycations which more effectively interact with high-G alginate than PLL.

Specific inulin-type fructan fibers protect against autoimmune diabetes by modulating gut immunity, barrier function and microbiota homeostasis

SCOPE Dietary fibers capable of modifying gut barrier and microbiota homeostasis affect the progression of type 1 diabetes (T1D). Here, we aim to compare modulatory effects of inulin-type fructans (ITFs), natural soluble dietary fibers with different degrees of fermentability from chicory root, on T1D development in nonobese diabetic mice. METHODS AND RESULTS Female nonobese diabetic mice were weaned to long- and short-chain ITFs [ITF(l) and ITF(s), 5%] supplemented diet up to 24 weeks. T1D incidence, pancreatic-gut immune responses, gut barrier function, and microbiota composition were analyzed. ITF(l) but not ITF(s) supplementation dampened the incidence of T1D. ITF(l) promoted modulatory T-cell responses, as evidenced by increased CD25+ Foxp3+ CD4+ regulatory T cells, decreased IL17A+ CD4+ Th17 cells, and modulated cytokine production profile in the pancreas, spleen, and colon. Furthermore, ITF(l) suppressed NOD like receptor protein 3 caspase-1-p20-IL-1β inflammasome in the colon. Expression of barrier reinforcing tight junction proteins occludin and claudin-2, antimicrobial peptides β-defensin-1, and cathelicidin-related antimicrobial peptide as well as short-chain fatty acid production were enhanced by ITF(l). Next-generation sequencing analysis revealed that ITF(l) enhanced Firmicutes/Bacteroidetes ratio to an antidiabetogenic balance and enriched modulatory Ruminococcaceae and Lactobacilli. CONCLUSION Our data demonstrate that ITF(l) but not ITF(s) delays the development of T1D via modulation of gut-pancreatic immunity, barrier function, and microbiota homeostasis.

L. plantarum, L. salivarius, and L. lactis Attenuate Th2 Responses and Increase Treg Frequencies in Healthy Mice in a Strain Dependent Manner

Many studies on probiotics are aimed at restoring immune homeostasis in patients to prevent disease recurrence or reduce immune-mediated pathology. Of equal interest is the use of probiotics in sub-clinical situations, which are characterized by reduced immune function or low-grade inflammation, with an increased risk of infection or disease as a consequence. Most mechanistic studies focus on the use of probiotics in experimental disease models, which may not be informative for these sub-clinical conditions. To gain better understanding of the effects in the healthy situation, we investigated the immunomodulatory effects of two Lactobacillus probiotic strains, i.e. L. plantarum WCFS1 and L. salivarius UCC118, and a non-probiotic lactococcus strain, i.e. L. lactis MG1363, in healthy mice. We studied the effect of these bacteria on the systemic adaptive immune system after 5 days of administration. Only L. plantarum induced an increase in regulatory CD103+ DC and regulatory T cell frequencies in the spleen. However, all three bacterial strains, including L. lactis, reduced specific splenic T helper cell cytokine responses after ex vivo restimulation. The effect on IFN-γ, IL5, IL10, and IL17 production by CD4+ and CD8+ T cells was dependent on the strain administered. A shared observation was that all three bacterial strains reduced T helper 2 cell frequencies. We demonstrate that systemic immunomodulation is not only observed after treatment with probiotic organisms, but also after treatment with non-probiotic bacteria. Our data demonstrate that in healthy mice, lactobacilli can balance T cell immunity in favor of a more regulatory status, via both regulatory T cell dependent and independent mechanisms in a strain dependent manner.

Upscaling the production of microencapsulated pancreatic islets.

Abstract Presently used single-needle air-driven droplet generators are incapable of producing sufficient numbers of islet-containing droplets in a sufficiently short time-period to allow for successfully grafting alginate-poly- l -lysine encapsulated islets in large animals or humans. We have designed an air-driven multineedle droplet generator, which increases the production rate by simultaneously producing multiple droplets. Although we have tested a four-needle device, the construction is such that the number of needles, and thereby the production rate, can be readily extended. The production rate can be further extended by increasing the number of islets per millilitre alginate in the reservoir. When tested with 500 and 800 μm capsules, an increase in the number of islets per millilitre alginate was found to be associated with an increase in the number of inadequately encapsulated islets in a diameter-dependent fashion. When small instead of large capsules are produced from a given volume of alginate, larger numbers of capsules are obtained, but also a larger portion of inadequate capsules. With 10 000 islets per millimetre alginate, these combined effects can be calculated to result in a two-fold increase in the production rate of adequate capsules when 500 μm instead of 800 μm capsules are produced. Hence, substantial upscaling of the production can be achieved by combining an increase in the number of needles with a decrease in the capsule diameter.

Factors influencing functional survival of microencapsulated islet grafts

Graft function of encapsulated islets is restricted in spite of the fact that inflammatory responses against capsules are limited to a portion less than 10%. It has been shown that dysfunction is accompanied by a gradual decrease in the glucose-induced insulin response (GIIR), a hyperproliferation of islet cells, and gradual necrosis. Also, limited survival is associated with the presence of macrophages in the overgrowth. In the present study, we investigate whether macrophages are the inducers of dysfunction of encapsulated grafts. Four weeks after successful transplantation of microencapsulated rat allografts we determined the GIIR, the rate of islet cell replication, and islet cell death. Also, we quantified the number of macrophages on the overgrown capsules. This assessment was applied to set up an in vitro coculture system of macrophages and encapsulated islets. We retrieved 93 ± 6.2% of the capsules of which 9.2 ± 0.3% was overgrown. The GIIR of the retrieved nonovergrown islets was reduced when compared with freshly encapsulated islets. The replication rate of the retrieved islet cells was eightfold higher than in the normal pancreas. Apoptosis was rarely observed but 37 ± 4% of the total islet surface was composed of necrosis. We found a mean of 1542 ± 217 macrophages per capsule. Coculture of 1500 NR8383 macrophages per encapsulated islets induced a substantial reduction in GIIR but a decrease instead of increase in replication. Necrosis was restricted to 13 ± 1.3% of the islet cells and was not increased by the presence of macrophages. Our observations indicate that we should focus on reduction of macrophage activation and on improving the nutrition of encapsulated islets to prevent islet cell death.

Adsorption of human immunoglobulin to implantable alginate-poly-L-lysine microcapsules: effect of microcapsule composition.

Alginate-poly-L-lysine-alginate (APA) microcapsules continue to be the most widely studied device for the immuno-protection of transplanted therapeutic cells. Producing APA microcapsules having a reproducible and high level of biocompatibility requires an understanding of the mechanisms of the immune response towards the implants. Here, we investigate the adsorption of immunoglobulins (IgG, IgM, and IgA) onto the surface of APA microcapsules in vitro after their exposure to human serum and peritoneal fluid. Immunoglobulins (Ig) are considered to be opsonizing proteins, thus they tend to mediate inflammation when adsorbed to foreign surfaces. Ig adsorption was monitored using direct immunofluorescence. The amount of Ig adsorbed to the microcapsule surface was not significantly influenced by the guluronic acid content nor the purity level of the alginate, although microcapsules of intermediate-G purified alginate corresponded with the lowest adsorption levels. Ig adsorption was negligible when the poly-L-lysine membrane was omitted, suggesting that positive charges at the microcapsule surface are responsible for binding Ig.

The role of pathogen-associated molecular patterns in inflammatory responses against alginate based microcapsules.

Alginate-based microcapsules are used for immunoisolation of cells to release therapeutics on a minute-to-minute basis. Unfortunately, alginate-based microcapsules are suffering from varying degrees of success, which is usually attributed to differences in tissue responses. This results in failure of the therapeutic cells. In the present study we show that commercial, crude alginates may contain pathogen-associated molecular patterns (PAMPs), which are recognized by the sensors of the innate immune system. Known sensors are Toll-like receptors (TLRs), NOD receptors, and C-type lectins. By using cell-lines with a non-functional adaptor molecule essential in Toll-like receptor signaling, i.e. MyD88, we were able to show that alginates signal mainly via MyD88. This was found for low-G, intermediate-G, and high-G alginates applied in calcium-beads, barium-beads as well as in alginate-PLL-alginate capsules. These alginates did stimulate TLRs 2, 5, 8, and 9 but not TLR4 (LPS receptor). Upon implantation in rats these alginates provoked a strong inflammatory response resulting in fibrosis of the capsules. Analysis demonstrated that commercial alginates contain the PAMPs peptidoglycan, lipoteichoic acid, and flagellin. By applying purification procedures, these PAMPs were largely removed. This was associated with deletion of the inflammatory tissue responses as confirmed by an implantation experiment in rats. Our data also show that alginate itself does not provoke TLR mediated responses. We were able to unravel the sensor mechanism by which contaminants in alginates may provoke inflammatory responses.

Extracellular matrix components supporting human islet function in alginate‐based immunoprotective microcapsules for treatment of diabetes

In the pancreas, extracellular matrix (ECM) components play an import role in providing mechanical and physiological support, and also contribute to the function of islets. These ECM-connections are damaged during islet-isolation from the pancreas and are not fully recovered after encapsulation and transplantation. To promote the functional survival of human pancreatic islets, we tested different ECMs molecules in alginate-encapsulated human islets. These were laminin derived recognition sequences, IKVAV, RGD, LRE, PDSGR, collagen I sequence DGEA (0.01 - 1.0 mM), and collagen IV (50 - 200 µg/mL). Interaction with RGD and PDSGR promoted islet viability and glucose induced insulin secretion (GIIS) when it was applied at concentrations ranging from 0.01 - 1.0 mM (p < 0.05). Also the laminin sequence LRE contributed to enhanced GIIS but only at higher concentrations of 1 mM (p < 0.05). Collagen IV also had beneficial effects but only at 50 µg/ml and no further improvement was observed at higher concentrations. IKVAV and DGEA had no effects on human islets. Synergistic effects were observed by adding Collagen(IV)-RGD, Collagen(IV)-LRE, and Collagen(IV)-PDSGR to encapsulated human islets. Our results demonstrate the potential of specific ECM components in support of functional survival of human encapsulated and free islet grafts.

Factors influencing isolation of functional pancreatic rat islets.

Yields and function of isolated islets vary considerably in spite of the introduction of new or improved methods for isolation. In most studies, these variations have been attributed to inadequacies of the applied collagenase preparations. However, when we retrospectively analyzed our rat islet isolations, we found large variations in yield and function in spite of application of identical collagenase sources. Therefore, in the present study, we determined the effect of rat donor strain, the source of inhibition of proteolytic activity (by bovine serum albumin), and the culture conditions on yield and function. AO rats showed a twofold higher islet yield than Wistar and Lewis rats. However, a higher yield was not associated with a higher response on glucose load since this response was more pronounced with Lewis islets than with Wistar and AO islets. Rats with a higher weight donate more islets but have a lower insulin secretory capacity. Islet yield and function also vary with application of different sources of bovine serum albumin during digestion. Moreover, the culture conditions influence the functional survival of isolated rat islets. CMRL1066 preserves the insulin secretory capacity of rat islets better than RPMI1640. Finally, the number of islets surviving the culture is higher when 4 instead of 12 and 24 islets were applied per square centimeter. Our observations indicate that strain and weight of the rat donor, the source of bovine serum albumin, and the culture conditions of islets are pertinent factors in efficacious isolation of islets.

Susceptibility of human pancreatic β cells for cytomegalovirus infection and the effects on cellular immunogenicity.

Objectives Human cytomegalovirus (HCMV) infection has been suggested to be a causal factor in the development of type 1 diabetes, posttransplantation diabetes, and the failure of islet allografts. This effect of CMV has been interpreted as an indirect effect on the immune system rather than direct infection–induced cell death. In the present study, we investigated (i) the susceptibility of &bgr; cells to HCMV infection, (ii) regulation of immune cell–activating ligands, (iii) release of proinflammatory cytokines, and (iv) the effects on peripheral blood mononuclear cell (PBMC) activation. Methods CM insulinoma cells and primary &bgr; cells were HCMV-infected in vitro using a laboratory and a clinical HCMV strain. The susceptibility to infection was measured by the expression of viral genes and proteins. Furthermore, expression levels of Major Histocompatibility Complex I, Intracellular Adhesion Molecule-1, and Lymphocyte Function Associated Antigen-3 and the release of proinflammatory cytokines were determined. In addition, PBMC activation to HCMV-infected &bgr; cells was determined. Results &bgr; Cells were susceptible to HCMV infection. Moreover, the infection increased the cellular immunogenicity, as demonstrated by an increased MHC I and ICAM-1 expression and an increased proinflammatory cytokine release. Human cytomegalovirus–infected CM cells potently activated PBMCs. The infection-induced effects were dependent on both viral “sensing” and viral replication. Conclusions In vivo &bgr;-cell HCMV infection and infection-enhanced cellular immunogenicity may have important consequences for native or transplanted &bgr;-cell survival.