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Dissolving microneedle arrays (dMNAs) can be used to deliver vaccines via the intradermal route. Fabrication of dMNAs using centrifugation is the most common preparation method of dMNAs, but it results in a substantial loss of antigens. In order to solve the issue of antigen waste, we engineered an automatic dispensing system for dMNA preparation. Here, we report on the fabrication of influenza whole inactivated virus (WIV) vaccine-loaded dMNAs (WIV dMNAs) by using the automatic dispensing system. Prior to the dispensing process, polydimethylsiloxane (PDMS) moulds were treated with oxygen plasma to increase surface hydrophilicity. WIV dMNAs were prepared with 1% (w/v) trehalose and pullulan (50 : 50 weight ratio). During the dispensing process, reduced pressure was applied to the PDMS mould via a vacuum chamber to make microneedle cavities airless. After producing dMNAs, WIV was quantified and 1.9 μg of WIV was loaded per dMNA, of which 1.3 μg was in the microneedle tips. Compared to the centrifugation method, this automatic dispensing system resulted in a 95% reduction of antigen waste. A hemagglutination assay confirmed that WIV dMNA maintained the stability of the antigen for at least four weeks of storage, even at room temperature or at 37 °C. The WIV dMNAs displayed 100% penetration efficiency in human skin, and 83% of the microneedle volume was dissolved in the skin within 10 minutes. In a vaccination study, mice immunised with WIV dMNAs showed similar IgG levels to those that received WIV intramuscularly. In conclusion, using the automatic dispensing system for dMNA production strongly reduced antigen waste and yielded dMNAs with excellent physical, mechanical, and immunological properties.

Introduction: Influenza vaccines play a vital role in protecting individuals from influenza virus infection and severe illness. However, current influenza vaccines have suboptimal efficacy, which is further reduced in cases where the vaccine strains do not match the circulating strains. One strategy to enhance the efficacy of influenza vaccines is by extended antigen delivery, thereby mimicking the antigen kinetics of a natural infection. Prolonging antigen availability was shown to quantitatively enhance influenza virus-specific immune responses but how it affects the quality of the induced immune response is unknown. Therefore, the current study aimed to investigate whether prolongation of the delivery of influenza vaccine improves the quality of the induced immune responses over that induced by prime-boost immunization., Methods: Mice were given daily doses of whole inactivated influenza virus vaccine for periods of 14, 21, or 28 days; the control group received prime-boost immunization with a 28 days interval., Results: Our data show that the highest levels of cellular and humoral immune responses were induced by 28 days of extended antigen delivery, followed by 21, and 14 days of delivery, and prime-boost immunization. Moreover, prolonging vaccine delivery also improved the quality of the induced antibody response, as indicated by higher level of high avidity antibodies, a balanced IgG subclass profile, and a higher level of cross-reactive antibodies., Conclusions: Our findings contribute to a better understanding of the immune response to influenza vaccination and have important implications for the design and development of future slow-release influenza vaccines., Competing Interests: The Icahn School of Medicine at Mount Sinai has filed patent applications relating to influenza virus vaccines and therapeutics that list FK as inventor. FK has consulted for Merck, Seqirus, CureVac and Pfizer in the past, and is currently consulting for Pfizer, 3rd Rock Ventures, GSK and Avimex and he is a co-founder and scientific advisory board member of CastleVax. The Krammer laboratory is also collaborating with Pfizer on animal models for SARS-CoV-2. AH is advisory board member of Intravacc. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Beukema, Gong, Al-Jaawni, de Vries-Idema, Krammer, Zhou, Cox and Huckriede.)

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.

Citrus pectins have demonstrated health benefits through direct interaction with Toll-like receptor 2. Methyl-ester distribution patterns over the homogalacturonan were found to contribute to such immunomodulatory activity, therefore molecular interactions with TLR2 were studied. Molecular-docking analysis was performed using four GalA-heptamers, GalA 7 Me 0 , GalA 7 Me 1,6 , GalA 7 Me 1,7 and GalA 7 Me 2,5 . The molecular relations were measured in various possible conformations. Furthermore, commercial citrus pectins were characterized by enzymatic fingerprinting using polygalacturonase and pectin-lyase to determine their methyl-ester distribution patterns. The response of 12 structurally different pectic polymers on TLR2 binding and the molecular docking with four pectic oligomers clearly demonstrated interactions with human-TLR2 in a structure-dependent way, where blocks of (non)methyl-esterified GalA were shown to inhibit TLR2/1 dimerization. Our results may be used to understand the immunomodulatory effects of certain pectins via TLR2. Knowledge of how pectins with certain methyl-ester distribution patterns bind to TLRs may lead to tailored pectins to prevent inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Although vaccination is still considered to be the cornerstone of public health care, the increase in vaccination coverage has stagnated for many diseases. Most of these vaccines require two or three doses to be administered across several months or years. Single-injection vaccine formulations are an effective method to overcome the logistical barrier to immunization that is posed by these multiple-injection schedules. Here, we developed subcutaneously (s.c.) injectable microspheres with a sustained release of the model antigen bovine serum albumin (BSA). The microspheres were composed of blends of two novel biodegradable multi-block copolymers consisting of amorphous, hydrophilic poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL) blocks and semi-crystalline poly(dioxanone) (PDO) blocks of different block sizes. In vitro studies demonstrated that the release of BSA could be tailored over a period of approximately four to nine weeks by changing the blend ratio of both polymers. Moreover, it was found that BSA remained structurally intact during release. Microspheres exhibiting sustained release of BSA for six weeks were selected for the in vivo study in mice. The induced BSA-specific IgG antibody titers increased up to four weeks after administration and were of the same magnitude as found in mice that received a priming and a booster dose of BSA in phosphate-buffered saline (PBS). Determination of the BSA concentration in plasma showed that in vivo release probably took place up to at least four weeks, although plasma concentrations peaked already one week after administration. The sustained-release microspheres might be a viable alternative to the conventional prime-boost immunization schedule, but a clinically relevant antigen should be incorporated to assess the full potential of these microspheres in practice.

The emergence of SARS-CoV-2 variants raised questions regarding the durability of immune responses after homologous or heterologous boosters after Ad26.COV2.S-priming. We found that SARS-CoV-2-specific binding antibodies, neutralizing antibodies, and T cells are detectable 5 months after boosting, although waning of antibodies and limited cross-reactivity with Omicron BA.1 was observed., Competing Interests: Potential conflicts of interest. L. G. V. reports research grants from U Needle, MyLife Technologies, and Bavarian Nordic, all outside of the submitted work; consulting fees from Emergent for expert meeting travel vaccines; participation on a scientific advisory board for Geosentinel; and serving as committee member for the National Board Guideline Development Travel Medicine. A. G. reports participation on a data and safety monitoring board (DSMB) IDSCOVA (Establishing the tolerability, safety and immunogenicity of intradermal delivery of mRNA SARS-CoV-2 vaccine in healthy adults) study on intradermal COVID vaccinations and serving as member of national advisory board on coronavirus disease vaccinations for immunocompromised patients. A. L. W. H. reports support for the present research from the Dutch Research Council (ZonMw) to their organization. D. F. P. reports participation on the DSMB of the COBRA-KAI trial (COVID-19 vaccination in patients with reduced B-cell and T-cell immunity: response after vaccination in a kaleidoscopic group of hematologic patients: what's the impact?). V. A. S. H. D. reports research funding outside of the submitted work from ZonMw and EU Horizon 2020, consulting fees for an advisory board meeting from GSK, and honoraria for lectures from Pharming, Takeda. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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.

Citrus pectins were studied by enzymatic fingerprinting using a simultaneous enzyme treatment with endo-polygalacturonase (endo-PG) from Kluyveromyces fragilis and pectin lyase (PL) from Aspergillus niger to reveal the methyl-ester distribution patterns over the pectin backbone. Using HILIC-MS combined with HPAEC enabled the separation and identification of the diagnostic oligomers released. Structural information on the pectins was provided by using novel descriptive parameters such as degree of blockiness of methyl-esterified oligomers by PG (DB PGme ) and degree of blockiness of methyl-esterified oligomers by PL (DB PLme ). This approach enabled us to clearly differentiate citrus pectins with various methyl-esterification patterns. The simultaneous use of PG and PL showed additional information, which is not revealed in digests using PG or PL alone. This approach can be valuable to differentiate pectins having the same DM and to get specific structural information on pectins and therefore to be able to better predict their physical and biochemical functionalities., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Human milk oligosaccharides (hMOs) are unique bioactive components in human milk. 3-Fucosyllactose (3-FL) is an abundantly present hMO that can be produced in sufficient amounts to allow application in infant formula. Lacto- N -triaose II (LNT2) can be obtained by acid hydrolysis of lacto- N -neotetraose (LNnT). Both 3-FL and LNT2 have been shown to have health benefits, but their impact on infant microbiota composition and microbial metabolic products such as short-chain fatty acids (SCFAs) is unknown. To gain more insight in fermentability, we performed in vitro fermentation studies of 3-FL and LNT2 using pooled fecal microbiota from 12-week-old infants. The commonly investigated galacto-oligosaccharides (GOS)/inulin (9 : 1) served as control. Compared to GOS/inulin, we observed a delayed utilization of 3-FL, which was utilized at 60.3% after 36 h of fermentation, and induced the gradual production of acetic acid and lactic acid. 3-FL specifically enriched bacteria of Bacteroides and Enterococcus genus. LNT2 was fermented much faster. After 14 h of fermentation, 90.1% was already utilized, and production of acetic acid, succinic acid, lactic acid and butyric acid was observed. LNT2 specifically increased the abundance of Collinsella , as well as Bifidobacterium. The GOS present in the GOS/inulin mixture was completely fermented after 14 h, while for inulin, only low DP was rapidly utilized after 14 h. To determine whether the fermentation might lead to enhanced colonization of commensal bacteria to gut epithelial cells, we investigated adhesion of the commensal Lactobacillus plantarum WCFS1 to Caco-2 cells. The fermentation digesta of LNT2 collected after 14 h, 24 h, and 36 h, and GOS/inulin after 24 h of fermentation significantly increased the adhesion of L. plantarum WCFS1 to Caco-2 cells, while 3-FL had no such effect. Our findings illustrate that fermentation of hMOs is very structure-dependent and different from the commonly applied GOS/inulin, which might lead to differential potencies to stimulate adhesion of commensal cells to gut epithelium and consequent microbial colonization. This knowledge might contribute to the design of tailored infant formulas containing specific hMO molecules to meet the need of infants during the transition from breastfeeding to formula.

Objectives: As coronavirus disease 2019 is a novel disease, treatment strategies continue to be debated. This provides the intensive care community with a unique opportunity as the population of coronavirus disease 2019 patients requiring invasive mechanical ventilation is relatively homogeneous compared with other ICU populations. We hypothesize that the novelty of coronavirus disease 2019 and the uncertainty over its similarity with noncoronavirus disease 2019 acute respiratory distress syndrome resulted in substantial practice variation between hospitals during the first and second waves of coronavirus disease 2019 patients., Design: Multicenter retrospective cohort study., Setting: Twenty-five hospitals in the Netherlands from February 2020 to July 2020, and 14 hospitals from August 2020 to December 2020., Patients: One thousand two hundred ninety-four critically ill intubated adult ICU patients with coronavirus disease 2019 were selected from the Dutch Data Warehouse. Patients intubated for less than 24 hours, transferred patients, and patients still admitted at the time of data extraction were excluded., Measurements and Main Results: We aimed to estimate between-ICU practice variation in selected ventilation parameters (positive end-expiratory pressure, Fio 2 , set respiratory rate, tidal volume, minute volume, and percentage of time spent in a prone position) on days 1, 2, 3, and 7 of intubation, adjusted for patient characteristics as well as severity of illness based on Pao 2 /Fio 2 ratio, pH, ventilatory ratio, and dynamic respiratory system compliance during controlled ventilation. Using multilevel linear mixed-effects modeling, we found significant ( p ≤ 0.001) variation between ICUs in all ventilation parameters on days 1, 2, 3, and 7 of intubation for both waves., Conclusions: This is the first study to clearly demonstrate significant practice variation between ICUs related to mechanical ventilation parameters that are under direct control by intensivists. Their effect on clinical outcomes for both coronavirus disease 2019 and other critically ill mechanically ventilated patients could have widespread implications for the practice of intensive care medicine and should be investigated further by causal inference models and clinical trials., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

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-1β 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.

Introduction: Pectins have anti-inflammatory properties on intestinal immunity through direct interactions on Toll-like receptors (TLRs) in the small intestine or via stimulating microbiota-dependent effects in the large intestine. Both the degree of methyl-esterification (DM) and the distribution of methyl-esters (degree of blockiness; DB) of pectins contribute to this influence on immunity, but whether and how the DB impacts immunity through microbiota-dependent effects in the large intestine is unknown. Therefore, this study tests pectins that structurally differ in DB in a mouse model with Citrobacter rodentium induced colitis and studies the impact on the intestinal microbiota composition and associated attenuation of inflammation., Methods and Results: Both low and high DB pectins induce a more rich and diverse microbiota composition. These pectins also lower the bacterial load of C. rodentium in cecal digesta. Through these effects, both low and high DB pectins attenuate C. rodentium induced colitis resulting in reduced intestinal damage, reduced numbers of Th1-cells, which are increased in case of C. rodentium induced colitis, and reduced levels of GATA3 + Tregs, which are related to tissue inflammation., Conclusion: Pectins prevent C. rodentium induced colonic inflammation by lowering the C. rodentium load in the caecum independently of the DB., (© 2021 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

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.

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., (© 2021 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

Background: The identification of risk factors for adverse outcomes and prolonged intensive care unit (ICU) stay in COVID-19 patients is essential for prognostication, determining treatment intensity, and resource allocation. Previous studies have determined risk factors on admission only, and included a limited number of predictors. Therefore, using data from the highly granular and multicenter Dutch Data Warehouse, we developed machine learning models to identify risk factors for ICU mortality, ventilator-free days and ICU-free days during the course of invasive mechanical ventilation (IMV) in COVID-19 patients., Methods: The DDW is a growing electronic health record database of critically ill COVID-19 patients in the Netherlands. All adult ICU patients on IMV were eligible for inclusion. Transfers, patients admitted for less than 24 h, and patients still admitted at time of data extraction were excluded. Predictors were selected based on the literature, and included medication dosage and fluid balance. Multiple algorithms were trained and validated on up to three sets of observations per patient on day 1, 7, and 14 using fivefold nested cross-validation, keeping observations from an individual patient in the same split., Results: A total of 1152 patients were included in the model. XGBoost models performed best for all outcomes and were used to calculate predictor importance. Using Shapley additive explanations (SHAP), age was the most important demographic risk factor for the outcomes upon start of IMV and throughout its course. The relative probability of death across age values is visualized in Partial Dependence Plots (PDPs), with an increase starting at 54 years. Besides age, acidaemia, low P/F-ratios and high driving pressures demonstrated a higher probability of death. The PDP for driving pressure showed a relative probability increase starting at 12 cmH 2 O., Conclusion: Age is the most important demographic risk factor of ICU mortality, ICU-free days and ventilator-free days throughout the course of invasive mechanical ventilation in critically ill COVID-19 patients. pH, P/F ratio, and driving pressure should be monitored closely over the course of mechanical ventilation as risk factors predictive of these outcomes.

Objective: We report a case series of patients with prolonged but reversible unconsciousness after coronavirus disease 2019 (COVID-19)-related severe respiratory failure., Methods: A case series of patients who were admitted to the intensive care unit due to COVID-19-related acute respiratory failure is described., Results: After cessation of sedatives, the described cases all showed a prolonged comatose state. Diagnostic neurologic workup did not show signs of devastating brain injury. The clinical pattern of awakening started with early eye opening without obeying commands and persistent flaccid weakness in all cases. Time between cessation of sedatives to the first moment of being fully responsive with obeying commands ranged from 8 to 31 days., Conclusion: Prolonged unconsciousness in patients with severe respiratory failure due to COVID-19 can be fully reversible, warranting a cautious approach for prognostication based on a prolonged state of unconsciousness., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Encapsulation of pancreatic islets in alginate-microcapsules is used to reduce or avoid the application of life-long immunosuppression in preventing rejection. Long-term graft function, however, is limited due to varying degrees of host tissue responses against the capsules. Major graft-longevity limiting responses include inflammatory responses provoked by biomaterials and islet-derived danger-associated molecular patterns (DAMPs). This paper reports on a novel strategy for engineering alginate microcapsules presenting immunomodulatory polymer pectin with varying degrees of methyl-esterification (DM) to reduce these host tissue responses. DM18-pectin/alginate microcapsules show a significant decrease of DAMP-induced Toll-Like Receptor-2 mediated immune activation in vitro, and reduce peri-capsular fibrosis in vivo in mice compared to higher DM-pectin/alginate microcapsules and conventional alginate microcapsules. By testing efficacy of DM18-pectin/alginate microcapsules in vivo, we demonstrate that low-DM pectin support long-term survival of xenotransplanted rat islets in diabetic mice. This study provides a novel strategy to attenuate host responses by creating immunomodulatory capsule surfaces that attenuate activation of specific pro-inflammatory immune receptors locally at the transplantation site., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Insufficient intake of dietary fibers in Western societies is considered a major contributing factor in the high incidence rates of diabetes. The dietary fiber pectin has been suggested to be beneficial for management of both Diabetes Type 1 and Type 2, but mechanisms and effects of pectin on insulin producing pancreatic β-cells are unknown. Our study aimed to determine the effects of lemon pectins with different degree of methyl-esterification (DM) on β-cells under oxidative (streptozotocin) and inflammatory (cytokine) stress and to elucidate the underlying rescuing mechanisms, including effects on galectin-3. We found that specific pectins had rescuing effects on toxin and cytokine induced stress on β-cells but effects depended on the pectin concentration and DM-value. Protection was more pronounced with low DM5 pectin and was enhanced with higher pectin-concentrations. Our findings show that specific pectins might prevent diabetes by making insulin producing β-cells less susceptible for stress., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

High intake of dietary fibres and calcium has been correlated to a lower frequency of Western disease such as allergy, asthma and obesity. How the combined higher intake of dietary fibres and calcium reduces the incidence of these diseases is unknown. Dietary fibre pectin can interact with Toll-like receptor (TLR) 2 and calcium in a degree of methyl-esterification (DM)-dependent manner. Low DM pectins interact stronger with TLR2 than high DM pectins. Since low DM pectin are known to bind calcium strongly, we investigated how calcium influences the DM-dependent impact of pectins on TLR2 signalling. We tested TLR2 activating, inhibiting and binding properties of pectins with DM18, DM52 and DM69 under 0 mM, 1 mM and 10 mM calcium conditions. None of the pectins activated TLR2, but pectins inhibited TLR2. Under 0 mM calcium conditions, especially DM18 and DM52 strongly inhibited TLR2 and bound strongly to TLR2. Addition of 1 and 10 mM calcium to these pectins reduced TLR2 inhibition and TLR2 binding. Our study shows that calcium reduces inhibition of TLR2 by low and intermediate DM pectins, but calcium has lower impact on TLR2 inhibition by high DM pectins. Calcium may therefore beneficially influence the impact of pectin on TLR2 signalling and contribute to an improved intestinal barrier function. A combined higher intake of pectin and calcium may therefore contribute to a lower incidence of Western diseases.

Pectins are dietary fibers with different structural characteristics. Specific pectin structures can influence the gastrointestinal immune barrier by directly interacting with immune cells or by impacting the intestinal microbiota. The impact of pectin strongly depends on the specific structural characteristics of pectin; for example, the degree of methyl-esterification, acetylation and rhamnogalacturonan I or rhamnogalacturonan II neutral side chains. Here, we review the interactions of specific pectin structures with the gastrointestinal immune barrier. The effects of pectin include strengthening the mucus layer, enhancing epithelial integrity, and activating or inhibiting dendritic cell and macrophage responses. The direct interaction of pectins with the gastrointestinal immune barrier may be governed through pattern recognition receptors, such as Toll-like receptors 2 and 4 or Galectin-3. In addition, specific pectins can stimulate the diversity and abundance of beneficial microbial communities. Furthermore, the gastrointestinal immune barrier may be enhanced by short-chain fatty acids. Moreover, pectins can enhance the intestinal immune barrier by favoring the adhesion of commensal bacteria and inhibiting the adhesion of pathogens to epithelial cells. Current data illustrate that pectin may be a powerful dietary fiber to manage and prevent several inflammatory conditions, but additional human studies with pectin molecules with well-defined structures are urgently needed.

Dietary fibers have been shown to exert immune effects via interaction with pattern recognition receptors (PRR) such as toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors. Pectin is a dietary fiber that interacts with PRR depending on its chemical structure. Papaya pectin retains different chemical structures at different ripening stages. How this influence PRR signaling is unknown. The aim of this work was to determine how ripening influences pectin structures and their ability to interact with TLR2, 3, 4, 5 and 9, and NOD1 and 2. It was evaluated the interaction of the water-soluble fractions rich in pectin extracted from unripe to ripe papayas. The pectin extracted from ripe papayas activated all the TLR and, to a lesser extent, the NOD receptors. The pectin extracted from unripe papayas also activated TLR2, 4 and 5 but inhibited the activation of TLR3 and 9. The differences in pectin structures are the higher methyl esterification and smaller galacturonan chains of pectin from ripe papayas. Our finding might lead to selection of ripening stages for tailored modulation of PRR to support or attenuate immunity.

Dietary carbohydrate fibers are known to prevent immunological diseases common in Western countries such as allergy and asthma but the underlying mechanisms are largely unknown. Until now beneficial effects of dietary fibers are mainly attributed to fermentation products of the fibers such as anti-inflammatory short-chain fatty acids (SCFAs). Here, we found and present a new mechanism by which dietary fibers can be anti-inflammatory: a commonly consumed fiber, pectin, blocks innate immune receptors. We show that pectin binds and inhibits, toll-like receptor 2 (TLR2) and specifically inhibits the proinflammatory TLR2-TLR1 pathway while the tolerogenic TLR2-TLR6 pathway remains unaltered. This effect is most pronounced with pectins having a low degree of methyl esterification (DM). Low-DM pectin interacts with TLR2 through electrostatic forces between non-esterified galacturonic acids on the pectin and positive charges on the TLR2 ectodomain, as confirmed by testing pectin binding on mutated TLR2. The anti-inflammatory effect of low-DM pectins was first studied in human dendritic cells and mouse macrophages in vitro and was subsequently tested in vivo in TLR2-dependent ileitis in a mouse model. In these mice, ileitis was prevented by pectin administration. Protective effects were shown to be TLR2-TLR1 dependent and independent of the SCFAs produced by the gut microbiota. These data suggest that low-DM pectins as a source of dietary fiber can reduce inflammation through direct interaction with TLR2-TLR1 receptors.