Human Milk Oligosaccharides Mediate the Crosstalk Between Intestinal Epithelial Caco-2 Cells and Lactobacillus PlantarumWCFS1in an In Vitro Model with Intestinal Peristaltic Shear Force.

BACKGROUND: The intestinal epithelial cells, food molecules, and gut microbiota are continuously exposed to intestinal peristaltic shear force. Shear force may impact the crosstalk of human milk oligosaccharides (hMOs) with commensal bacteria and intestinal epithelial cells. OBJECTIVES: We investigated how hMOs combined with intestinal peristaltic shear force impact intestinal epithelial cells and crosstalk with a commensal bacterium. METHODS: We applied the Ibidi system to mimic intestinal peristaltic shear force. Caco-2 cells were exposed to a shear force (5 dynes/cm2) for 3 d, and then stimulated with the hMOs, 2'-fucosyllactose (2'-FL), 3-FL, and lacto-N-triose II (LNT2). In separate experiments, Lactobacillus plantarumWCFS1 adhesion to Caco-2 cells was studied with the same hMOs and shear force. Effects were tested on gene expression of glycocalyx-related molecules (glypican 1 [GPC1], hyaluronan synthase 1 [HAS1], HAS2, HAS3, exostosin glycosyltransferase 1 [EXT1], EXT2), defensin ß-1 (DEFB1), and tight junction (tight junction protein 1 [TJP1], claudin 3 [CLDN3]) in Caco-2 cells. Protein expression of tight junctions was also quantified. RESULTS: Shear force dramatically decreased gene expression of the main enzymes for making glycosaminoglycan side chains (HAS3 by 43.3% and EXT1 by 68.7%) (P <0.01), but did not affect GPC1 which is the gene responsible for the synthesis of glypican 1 which is a major protein backbone of glycocalyx. Expression of DEFB1, TJP1, and CLDN3 genes was decreased 60.0-94.9% by shear force (P <0.001). The presence of L. plantarumWCFS1 increased GPC1, HAS2, HAS3, and ZO-1 expression by 1.78- to 3.34-fold (P <0.05). Under shear force, all hMOs significantly stimulated DEFB1 and ZO-1, whereas only 3-FL and LNT2 enhanced L. plantarumWCFS1 adhesion by 1.85- to 1.90-fold (P <0.01). CONCLUSIONS: 3-FL and LNT2 support the crosstalk between the commensal bacterium L. plantarumWCFS1 and Caco-2 intestinal epithelial cells, and shear force can increase the modulating effects of hMOs.

Acetate and Butyrate Improve ß-cell Metabolism and Mitochondrial Respiration under Oxidative Stress.

Islet dysfunction mediated by oxidative and mitochondrial stress contributes to the development of type 1 and 2 diabetes. Acetate and butyrate, produced by gut microbiota via fermentation, have been shown to protect against oxidative and mitochondrial stress in many cell types, but their effect on pancreatic ß-cell metabolism has not been studied. Here, human islets and the mouse insulinoma cell line MIN6 were pre-incubated with 1, 2, and 4 mM of acetate or butyrate with and without exposure to the apoptosis inducer and metabolic stressor streptozotocin (STZ). Both short-chain fatty acids (SCFAs) enhanced the viability of islets and ß-cells, but the beneficial effects were more pronounced in the presence of STZ. Both SCFAs prevented STZ-induced cell apoptosis, viability reduction, mitochondrial dysfunction, and the overproduction of reactive oxygen species (ROS) and nitric oxide (NO) at a concentration of 1 mM but not at higher concentrations. These rescue effects of SCFAs were accompanied by preventing reduction of the mitochondrial fusion genes MFN, MFN2, and OPA1. In addition, elevation of the fission genes DRP1 and FIS1 during STZ exposure was prevented. Acetate showed more efficiency in enhancing metabolism and inhibiting ROS, while butyrate had less effect but was stronger in inhibiting the SCFA receptor GPR41 and NO generation. Our data suggest that SCFAs play an essential role in supporting ß-cell metabolism and promoting survival under stressful conditions. It therewith provides a novel mechanism by which enhanced dietary fiber intake contributes to the reduction of Western diseases such as diabetes.

Polymer scaffolds for pancreatic islet transplantation - Progress and challenges.

Pancreatic-islet transplantation is a safe and noninvasive therapy for type 1 diabetes. However, the currently applied site for transplantation, ie, the liver, is not the optimal site for islet survival. Because the human body has shortcomings in providing an optimal site, artificial transplantation sites have been proposed. Such an artificial site could consist of a polymeric scaffold that mimics the pancreatic microenvironment and supports islet function. Recently, remarkable progress has been made in the technology of engineering scaffolds. The polymer-islet interactions, the site of implantation, and scaffold prevascularization are critical factors for success or failure of the scaffolds. This article critically reviews these factors while also discussing translation of experimental studies to human application as well as the steps required to create a clinically applicable prevascularized, retrievable scaffold for implantation of insulin-producing cells for treatment of type 1 diabetes mellitus.

A Retrievable, Efficacious Polymeric Scaffold for Subcutaneous Transplantation of Rat Pancreatic Islets.

OBJECTIVE: We aim on developing a polymeric ectopic scaffold in a readily accessible site under the skin. SUMMARY BACKGROUND DATA: The liver as transplantation site for pancreatic islets is associated with significant loss of islets. Several extrahepatic sites were tested in experimental animals, but many have practical limitations in the clinical setting and do not have the benefit of easy accessibility. METHODS AND RESULTS: Functional survival of rat islets was tested during 7 days of culture in the presence of poly(D,L-lactide-co-ε-caprolactone) (PDLLCL), poly(ethylene oxide terephthalate)/polybutylene terephthalate (PEOT/PBT) block copolymer, and polysulfone. Tissue responses were studied in vivo after subcutaneous implantation in rats. Culture on PEOT/PBT and polysulfone profoundly disturbed function of islets, and induced severe tissue responses in vivo. Modification of their hydrophilicity did not change the suitability of the polymers. PDLLCL was the only polymer that promoted functional survival of rat islets in vitro and was associated with minor tissue reactions after 28 days. Rat islets were transplanted in the PDLLCL scaffold in a diabetic rat model. Before islet seeding, the scaffold was allowed to engraft for 28 days to allow the tissue response to dampen and to allow blood vessel growth into the device. Islet transplantation into the scaffold resulted in normoglycemia within 3 days and for the duration of the study period of 16 weeks. CONCLUSIONS: In conclusion, we found that some polymers such as PEOT/PBT and polysulfone interfere with islet function. PDLLCL is a suitable polymer to create an artificial islet transplantation site under the skin and supports islet survival.

Necrostatin-1 releasing nanoparticles: In vitro and in vivo efficacy for supporting immunoisolated islet transplantation outcomes.

Immunoisolation of pancreatic islets in alginate microcapsules allows for transplantation in the absence of immunosuppression but graft survival time is still limited. This limited graft survival is caused by a combination of tissue responses to the encapsulating biomaterial and islets. A significant loss of islet cells occurs in the immediate period after transplantation and is caused by a high susceptibility of islet cells to inflammatory stress during this period. Here we investigated whether necrostatin-1 (Nec-1), a necroptosis inhibitor, can reduce the loss of islet cells under stress in vitro and in vivo. To this end, we developed a Nec-1 controlled-release system using poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) as the application of Nec-1 in vivo is limited by low stability and possible side effects. The PLGA NPs stably released Nec-1 for 6 days in vitro and protected beta cells against hypoxia-induced cell death in vitro. Treatment with these Nec-1 NPs at days 0, 6, and 12 post-islet transplantation in streptozotocin-diabetic mice confirmed the absence of side effects as graft survival was similar in encapsulated islet grafts in the absence and presence of Nec-1. However, we found no further prolongation of graft survival of encapsulated grafts which might be explained by the high biocompatibility of the alginate encapsulation system that provoked a very mild tissue response. We expect that the Nec-1-releasing NPs could find application to immunoisolation systems that elicit stronger inflammatory responses, such as macrodevices and vasculogenic biomaterials.

Culturing Conditions Dictate the Composition and Pathways Enrichment of Human and Rat Perirenal Adipose-Derived Stromal Cells' Secretomes.

Understanding the impact of various culturing strategies on the secretome composition of adipose-derived stromal cells (ASC) enhances their therapeutic potential. This study investigated changes in the secretome of perirenal ASC (prASC) under different conditions: normoxia, cytokine exposure, high glucose, hypoxia, and hypoxia with high glucose. Using mass spectrometry and enrichment clustering analysis, we found that normoxia enriched pathways related to extracellular matrix (ECM) organization, platelet degranulation, and insulin-like growth factor (IGF) transport and uptake. Cytokine exposure influenced metabolism, vascular development, and protein processing pathways. High glucose affected the immune system, metabolic processes, and IGF transport and uptake. Hypoxia impacted immune and metabolic processes and protein processing. Combined hypoxia and high glucose influenced the immune system, IGF transport and uptake, and ECM organization. Our findings highlight the potential of manipulating culturing conditions to produce secretomes with distinct protein and functional profiles, tailoring therapeutic strategies accordingly.

Exopolysaccharide ß-(2,6)-levan-type fructans have a molecular-weight-dependent modulatory effect on Toll-like receptor signalling.

SCOPE: Fructans are a group of dietary fibers which are known to have many beneficial effects including immune-modulating effects. A family of fructans are ß-(2,6)-linked levan-type fructans that are known to serve as exopolysaccharides in the cell wall of many species of bacteria including commensal bacteria and probiotics. It is still largely unknown whether and how they can serve as immunomodulating molecules. RESULTS: Microbial ß-(2,6)-fructans were found to induce TLR-dependent activation of THP-1 cells, in a dose-dependent fashion. Low molecular weight (Mw), medium Mw and high Mw ß-(2,6)-fructans activated both TLR2 and 4 in a dose- and molecular weight-dependent fashion. In addition, it was found that ß-(2,6)-fructans were able to inhibit signalling of various TLRs with the strongest effect on TLR5 and 8, which were inhibited by all the ß-(2,6)-fructans in a dose- and molecular weight-dependent fashion. The final effect of this activation and inhibition of TLRs on cytokine responses in human dendritic cells (DCs) was minor which may be explained by the counter-activating effects of the different ß-(2,6)-linked levan-type fructans on inhibition of TLR signalling in the DCs. CONCLUSION: A mechanism by which exopolysaccharide levan ß-(2,6)-fructans can be immune-modulating is by impacting TLR signalling. This knowledge could lead to food in which exopolysaccharide levan ß-(2,6)-fructans are added for preventing disorders where TLR-signalling is modulated.

Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

Efficacy of pectins with different degrees of methyl-esterification and of blockiness in preventing gut epithelial cell barrier disruption and the impact on sodium-glucose co-transporter expression under low and high glucose conditions.

Pectins support intestinal barrier function and have anti-diabetic effects, and can differ in the degree of methyl-esterification (DM) and the distribution of non-esterified galacturonic acid residues (DB). The mechanisms and effects of pectin type at different glucose levels are unknown. Pectins with different DM/DB on T84 cells were tested in the presence and absence of the barrier disruptor A23187 at 5 mM and 20 mM glucose. DM19 and DM43 pectins with high DB do rescue the intestinal barrier from disruption. Their effects were as strong as those of the barrier-rescuing anti-diabetic drug metformin, but effects with metformin were restricted to high glucose levels while pectins had effects at both low and high glucose levels. At high glucose levels, DM43HB pectin, which enhanced trans-epithelial electrical resistance, also increased the expressions of claudin1, occludin, and ZO-1. Low and high DM pectins decrease the apical expression of the sodium-glucose co-transporter (SGLT-1) and thereby influence glucose transport, explaining the anti-diabetogenic effect of pectin. Higher DB pectins had the strongest effect. Their impact on SGLT-1 was stronger than that of metformin. Pectin's rescuing effect on barrier disruption and its impact on glucose transportation and anti-diabetogenic effects depend on both the DB and the DM of pectins.

Microfluidic Generation of Thin-Shelled Polyethylene Glycol-Tyramine Microgels for Non-Invasive Delivery of Immunoprotected ß-Cells.

Transplantation of microencapsulated pancreatic cells is emerging as a promising therapy to replenish ß-cell mass lost from auto-immune nature of type I diabetes mellitus (T1DM). This strategy intends to use micrometer-sized microgels to provide immunoprotection to transplanted cells to avoid chronic application of immunosuppression. Clinical application of encapsulation has remained elusive due to often limited production throughputs and body's immunological reactions to implanted materials. This article presents a high-throughput fabrication of monodisperse, non-immunogenic, non-degradable, immunoprotective, semi-permeable, enzymatically-crosslinkable polyethylene glycol-tyramine (PEG-TA) microgels for ß-cell microencapsulation. Monodisperse ß-cell laden microgels of ≈120 µm, with a shell thickness of 20 µm are produced using an outside-in crosslinking strategy. Microencapsulated ß-cells rapidly self-assemble into islet-sized spheroids. Immunoprotection of the microencapsulated is demonstrated by inability of FITC-IgG antibodies to diffuse into cell-laden microgels and NK-cell inability to kill microencapsulated ß-cells. Multiplexed ELISA analysis on live blood immune reactivity confirms limited immunogenicity. Microencapsulated MIN6ß1 spheroids remain glucose responsive for 28 days in vitro, and able to restore normoglycemia 5 days post-implantation in diabetic mice without notable amounts of cell death. In short, PEG-TA microgels effectively protect implanted cells from the host's immune system while being viable and functional, validating this strategy for the treatment of T1DM.

Successful Islet Transplantation Into a Subcutaneous Polycaprolactone Scaffold in Mice and Pigs.

Islet transplantation is a promising treatment for type 1 diabetes. It has the potential to improve glycemic control, particularly in patients suffering from hypoglycemic unawareness and glycemic instability. As most islet grafts do not function permanently, efforts are needed to create an accessible and replaceable site, for islet grafts or for insulin-producing cells obtained from replenishable sources. To this end, we designed and tested an artificial, polymeric subcutaneous transplantation site that allows repeated transplantation of islets. Methods: In this study, we developed and compared scaffolds made of poly(D,L,-lactide-co-ε-caprolactone) (PDLLCL) and polycaprolactone (PCL). Efficacy was first tested in mice' and then, as a proof of principle for application in a large animal model, the scaffolds were tested in pigs, as their skin structure is similar to that of humans. Results: In mice, islet transplantation in a PCL scaffold expedited return to normoglycemia in comparison to PDLLCL (7.7 ± 3.7 versus 16.8 ± 6.5 d), but it took longer than the kidney capsule control group. PCL also supported porcine functional islet survival in vitro. Subcutaneous implantation of PDLLCL and PCL scaffolds in pigs revealed that PCL scaffolds were more stable and was associated with less infiltration by immune cells than PDLLCL scaffolds. Prevascularized PCL scaffolds were therefore used to demonstrate the functional survival of allogenic islets under the skin of pigs. Conclusions: To conclude, a novel PCL scaffold shows efficacy as a readily accessible and replaceable, subcutaneous transplantation site for islets in mice and demonstrated islet survival after a month in pigs.

Human milk oligosaccharides and non-digestible carbohydrates reduce pathogen adhesion to intestinal epithelial cells by decoy effects or by attenuating bacterial virulence.

This work investigated the effects of different chemical structures of human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) on pathogen adhesion by serving as decoy receptors. Pre-exposure of pathogens to inulins and low degree of methylation (DM) pectin prevented binding to gut epithelial Caco2-cells, but effects were dependent on the molecules' chemistry, pathogen strain and growth phase. Pre-exposure to 3-fucosyllactose increased E. coli WA321 adhesion (28%, p < 0.05), and DM69 pectin increased E. coli ET8 (15 fold, p < 0.05) and E. coli WA321 (50%, p < 0.05) adhesion. Transcriptomics analysis revealed that DM69 pectin upregulated flagella and cell membrane associated genes. However, the top 10 downregulated genes were associated with lowering of bacteria virulence. DM69 pectin increased pathogen adhesion but bacterial virulence was attenuated illustrating different mechanisms may lower pathogen adhesion. Our study illustrates that both hMOs and NDCs can reduce adhesion or attenuate virulence of pathogens but that these effects are chemistry dependent.

Combining galacto-oligosaccharides and 2'-fucosyllactose alters their fermentation kinetics by infant fecal microbiota and influences AhR-receptor dependent cytokine responses in immature dendritic cells.

Galacto-oligosaccharides (GOS) and 2'-fucosyllactose (2'-FL) are non-digestible carbohydrates (NDCs) that are often added to infant formula to replace the functionalities of human milk oligosaccharides (HMOs). It is not known if combining GOS and 2'-FL will affect their fermentation kinetics and subsequent immune-modulatory effects such as AhR-receptor stimulation. Here, we used an in vitro set-up for the fermentation of 2'-FL and GOS, either individually or combined, by fecal microbiota of 8-week-old infants. We found that GOS was fermented two times faster by the infant fecal microbiota when combined with 2'-FL, while the combination of GOS and 2'-FL did not result in a complete degradation of 2'-FL. Fermentation of both GOS and 2'-FL increased the relative abundance of Bifidobacterium, which coincided with the production of acetate and lactate. Digesta of the fermentations influenced dendritic cell cytokine secretion differently under normal conditions and in the presence of the AhR-receptor blocker CH223191. We show that, combining GOS and 2'-FL accelerates GOS fermentation by the infant fecal microbiota of 8-week-old infants. In addition, we show that the fermentation digesta of GOS and 2'-FL, either fermented individually or combined, can attenuate DC cytokine responses in a similar and in an AhR-receptor dependent way.

Inclusion of extracellular matrix molecules and necrostatin-1 in the intracapsular environment of alginate-based microcapsules synergistically protects pancreatic ß cells against cytokine-induced inflammatory stress.

Immunoisolation of pancreatic islets in alginate-based microcapsules is a promising approach for grafting of islets in absence of immunosuppression. However, loss and damage to the extracellular matrix (ECM) during islet isolation enhance susceptibility of islets for inflammatory stress. In this study, a combined strategy was applied to reduce this stress by incorporating ECM components (collagen type IV/RGD) and necroptosis inhibitor, necrostatin-1 (Nec-1) in alginate-based microcapsules in vitro. To demonstrate efficacy, viability and function of MIN6 ß-cells and human islets in capsules with collagen type IV/RGD and/or Nec-1 was investigated in presence and absence of IL-1ß, IFN-γ and TNF-α. The combination of collagen type IV/RGD and Nec-1 had higher protective effects than the molecules alone. Presence of collagen type IV/RGD and Nec-1 in the intracapsular environment reduced cytokine-induced overproduction of free radical species and unfavorable shifts in mitochondrial dynamics. In addition, the ECM components collagen type IV/RGD prevented a cytokine induced suppression of the FAK/Akt pathway. Our data indicate that the inclusion of collagen type IV/RGD and Nec-1 in the intracapsular environment prevents islet-cell loss when exposed to inflammatory stress, which might contribute to higher survival of ß-cells in the immediate period after transplantation. This approach of inclusion of stress reducing agents in the intracapsular environment of immunoisolating devices may be an effective way to enhance the longevity of encapsulated islet grafts. STATEMENT OF SIGNIFICANCE: Islet-cells in immunoisolated alginate-based microcapsules are very susceptible to inflammatory stress which impacts long-term survival of islet grafts. Here we show that incorporation of ECM components (collagen type IV/RGD) and necrostatin-1 (Nec-1) in the intracapsular environment of alginate-based capsules attenuates this susceptibility and promotes islet-cell survival. This effect induced by collagen type IV/RGD and Nec-1 was probably due to lowering free radical production, preventing mitochondrial dysfunction and by maintaining ECM/integrin/FAK/Akt signaling and Nec-1/RIP1/RIP3 signaling. Our study provides an effective strategy to extend longevity of islet grafts which might be of great potential for future clinical application of immunoisolated cells.

Species-dependent impact of immunosuppressive squalene-gusperimus nanoparticles and adipose-derived stem cells on isolated human and rat pancreatic islets.

Transplantation of pancreatic islets is a promising approach to controlling glucose levels in type 1 diabetes mellitus (T1DM), but islet survival is still limited. To overcome this, islet co-culture with mesenchymal stromal cells (MSCs) together with safe immunosuppressive agents like squalene-gusperimus nanoparticles (Sq-GusNPs) may be applied. This could support islet survival and engraftment. Here, we studied how Sq-GusNPs and adipose-derived stem cells (ASCs) influence islets response under pro-inflammatory conditions. Through qRT-PCR, we studied the expression of specific genes at 24 hours in human and rat islets and ASCs in co-culture under indirect contact with or without treatment with Sq-GusNPs. We characterized how the response of islets and ASCs starts at molecular level before impaired viability or function is observed and how this response differs between species. Human islets and ASCs responses showed to be principally influenced by NF-κB activation, whereas rat islet and ASCs responses showed to be principally mediated by nitrosative stress. Rat islets showed tolerance to inflammatory conditions due to IL-1Ra secretion which was also observed in rat ASCs. Human islets induced the expression of cytokines and chemokines with pro-angiogenic, tissue repair, and anti-apoptotic properties in human ASCs under basal conditions. This expression was not inhibited by Sq-GusNPs. Our results showed a clear difference in the response elicited by human and rat islets and ASCs in front of an inflammatory stimulus and Sq-GusNPs. Our data support the use of ASCs and Sq-GusNP to facilitate engraftment of islets for T1DM treatment.

Human milk oligosaccharides and non-digestible carbohydrates prevent adhesion of specific pathogens via modulating glycosylation or inflammatory genes in intestinal epithelial cells.

Human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) are known to inhibit the adhesion of pathogens to the gut epithelium, but the mechanisms involved are not well understood. Here, the effects of 2'-FL, 3-FL, DP3-DP10, DP10-DP60 and DP30-DP60 inulins and DM7, DM55 and DM69 pectins were studied on pathogen adhesion to Caco-2 cells. As the growth phase influences virulence, E. coli ET8, E. coli LMG5862, E. coli O119, E. coli WA321, and S. enterica subsp. enterica LMG07233 from both log and stationary phases were tested. Specificity for enteric pathogens was tested by including the lung pathogen K. pneumoniae LMG20218. Expression of the cell membrane glycosylation genes of galectin and glycocalyx and inflammatory genes was studied in the presence and absence of 2'-FL or NDCs. Inhibition of pathogen adhesion was observed for 2'-FL, inulins, and pectins. Pre-incubation with 2'-FL downregulated ICAM1, and pectins modified the glycosylation genes. In contrast, K. pneumoniae LMG20218 downregulated the inflammatory genes, but these were restored by pre-incubation with pectins, which reduced the adhesion of K. pneumoniae LMG20218. In addition, DM69 pectin significantly upregulated the inflammatory genes. 2'-FL and pectins but not inulins inhibited pathogen adhesion to the gut epithelial Caco-2 cells through changing the cell membrane glycosylation and inflammatory genes, but the effects were molecule-, pathogen-, and growth phase-dependent.

In vivovascularization and islet function in a microwell device for pancreatic islet transplantation.

Islet encapsulation in membrane-based devices could allow for transplantation of donor islet tissue in the absence of immunosuppression. To achieve long-term survival of islets, the device should allow rapid exchange of essential nutrients and be vascularized to guarantee continued support of islet function. Recently, we have proposed a membrane-based macroencapsulation device consisting of a microwell membrane for islet separation covered by a micropatterned membrane lid. The device can prevent islet aggregation and support functional islet survivalin vitro. Here, based on previous modeling studies, we develop an improved device with smaller microwell dimensions, decreased spacing between the microwells and reduced membrane thickness and investigate its performancein vitroandin vivo. This improved device allows for encapsulating higher islet numbers without islet aggregation and by applying anin vivoimaging system we demonstrate very good perfusion of the device when implanted intraperitoneally in mice. Besides, when it is implanted subcutaneously in mice, islet viability is maintained and a vascular network in close proximity to the device is developed. All these important findings demonstrate the potential of this device for islet transplantation.

Attenuation of Doxorubicin-Induced Small Intestinal Mucositis by Pectins is Dependent on Pectin's Methyl-Ester Number and Distribution.

SCOPE: Intestinal mucositis is a common side effect of the chemotherapeutic agent doxorubicin, which is characterized by severe Toll-like receptor (TLR) 2-mediated inflammation. The dietary fiber pectin is shown to prevent this intestinal inflammation through direct inhibition of TLR2 in a microbiota-independent manner. Recent in vitro studies show that inhibition of TLR2 is determined by the number and distribution of methyl-esters of pectins. Therefore, it is hypothesized that the degree of methyl-esterification (DM) and the degree of blockiness (DB) of pectins determine attenuating efficacy on doxorubicin-induced intestinal mucositis. METHODS AND RESULTS: Four structurally different pectins that differed in DM and DB are tested on inhibitory effects on murine TLR2 in vitro, and on doxorubicin-induced intestinal mucositis in mice. These data demonstrate that low DM pectins or intermediate DM pectins with high DB have the strongest inhibitory impact on murine TLR2-1 and the strongest attenuating effect on TLR2-induced apoptosis and peritonitis. Intermediate DM pectin with a low DB is, however, also effective in preventing the induction of doxorubicin-induced intestinal damage. CONCLUSION: These pectin structures with stronger TLR2-inhibiting properties may prevent the development of doxorubicin-induced intestinal damage in patients undergoing chemotherapeutic treatment with doxorubicin.

Chicory inulin enhances fermentation of 2'-fucosyllactose by infant fecal microbiota and differentially influences immature dendritic cell and T-cell cytokine responses under normal and Th2-polarizing conditions.

Scope: Non-digestible carbohydrates (NDCs) such as native chicory inulin and 2'-fucosyllactose (2'-FL) are added to infant formula to mimic some of the human milk oligosaccharide (HMO) functions. It is unknown whether combining inulin and 2'-FL influences their fermentation kinetics and whether the immune-modulatory effects of these NDCs are different under normal and inflammatory-prone Th2-polarizing conditions. Methods and results: We investigated the in vitro fermentation of 2'-FL and native chicory inulin, fermented individually and combined, using fecal inocula of 8-week-old infants. Native inulin was fermented in a size-dependent fashion and expedited the fermentation of 2'-FL. Fermentation of both native inulin and 2'FL increased the relative abundance of Bifidobacterium, which coincided with the production of acetate and lactate. The fermentation digesta of all fermentations differentially influenced both dendritic cell and T-cell cytokine responses under normal culture conditions or in presence of the Th2-polarizing cytokines IL-33 and TSLP, with the most pronounced effect for IL-1ß in the presence of TSLP. Conclusions: Our findings show that native inulin can expedite the fermentation of 2'-FL by infant fecal microbiota and that these NDC fermentation digesta have different effects under normal and Th2-polarizing conditions, indicating that infants with different immune backgrounds might benefit from tailored NDC formulations.

In vitro determination of the immunosuppressive effect, internalization, and release mechanism of squalene-gusperimus nanoparticles for managing inflammatory responses.

Gusperimus is an anti-inflammatory drug that has shown to be effective in managing autoimmunity and preventing graft rejection. This is unstable and easily broken down into cytotoxic components. We encapsulated gusperimus binding it covalently to squalene obtaining squalene-gusperimus nanoparticles (Sq-GusNPs). These nanoparticles enhanced the immunosuppressive effect of gusperimus in both mouse macrophages and T cells. The half-maximal inhibitory concentration in macrophages was 9-fold lower for Sq-GusNPs compared with the free drug. The anti-inflammatory effect of the Sq-GusNPs was maintained over time without cytotoxicity. By studying nanoparticles uptake by cells with flow cytometry, we demonstrated that Sq-GusNPs are endocytosed by macrophages after binding to low-density lipoprotein receptors (LDLR). In presence of cathepsin B or D release of gusperimus is increased demonstrating the participation of proteases in the release process. Our approach may allow the application of Sq-GusNPs for effective management of inflammatory disorders including autoimmunity and graft rejection.