Structure-function effects of different pectin chemistries and its impact on the gastrointestinal immune barrier system.

The gastrointestinal immune system is crucial for overall health, safeguarding the human body against harmful substances and pathogens. One key player in this defense is dietary fiber pectin, which supports the gut's immune barrier and fosters beneficial gut bacteria. Pectin's composition, including degree of methylation (DM), RG-I, and neutral sugar content, influences its health benefits. This review assesses how pectin composition impacts the gastrointestinal immune barrier and what advantages specific chemistries of pectin has for metabolic, cardiovascular, and immune health. We delve into recent findings regarding pectin's interactions with the immune system, including receptors like TLRs and galectin 3. Pectin is shown to fortify mucosal and epithelial layers, but the specific effects are structure dependent. Additionally, we explore potential strategies for enhancing the gut immune barrier function. Understanding how distinct pectin chemistries affect the gastrointestinal immune system is vital for developing preventive and therapeutic solutions for conditions related to microbiota imbalances and immune issues. Ultimately, this review offers insights into strategies to boost the gut immune barrier's effectiveness, fostering better overall health by using specific pectins in the diet.

Unbiased random mutagenesis contributes to a better understanding of the virulent behaviour of Paenibacillus larvae.

AIMS: American foulbrood, caused by the Gram-positive bacteria Paenibacillus larvae, is one of the most severe bacterial diseases of the European honey bee. The bacterium has been known for long, but only the last decade the mechanisms used by the pathogen to cause disease in its host are starting to unravel. In this study, the knowledge of this virulent behaviour is expanded and several possible virulence factors are suggested. METHODS AND RESULTS: Identification of possible virulence factors has been done by random mutagenesis to ensure an unbiased approach. A library of mutants was tested for a significant difference in virulence using in vitro exposure assays. Affected loci were characterized and their potential to contribute in virulence of the pathogen was assessed. CONCLUSIONS: The identified mutated loci dacB, dnaK, metN, ywqD, lysC, serC and gbpA are known to encode for virulence factors in other bacteria and are suggested to play a similar role in P. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The study identified new possible virulence factors for P. larvae genotype ERIC I in an unbiased way. This contributes to the knowledge and understanding of the possible mechanisms used by this pathogen to colonize and kill its host.

Impact of plant growth-promoting rhizobacteria on root colonization potential and life cycle of Rhizophagus irregularis following co-entrapment into alginate beads.

AIMS: This study aimed at evaluating the impact of seven plant growth-promoting rhizobacteria (PGPR) on root colonization and life cycle of Rhizophagus irregularis MUCL 41833 when co-entrapped in alginate beads. METHODS AND RESULTS: Two in vitro experiments were conducted. The first consisted of the immobilization of R. irregularis and seven PGPR isolates into alginate beads to assess the effect of the bacteria on the pre-symbiotic growth of the fungus. In the second experiment, the best performing PGPR from experiment 1 was tested for its ability to promote the symbiotic development of the AMF in potato plantlets from three cultivars. Results showed that only one isolate identified as Pseudomonas plecoglossicida (R-67094) promoted germ tube elongation and hyphal branching of germinated spores during the pre-symbiotic phase of the fungus. This PGPR further promoted the symbiotic development of the AMF in potato plants. CONCLUSIONS: The co-entrapment of Ps. plecoglossicida R-67094 and R. irregularis MUCL 41833 in alginate beads improved root colonization by the AMF and its further life cycle under the experimental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Co-entrapment of suitable AMF-PGPR combinations within alginate beads may represent an innovative technology that can be fine-tuned for the development of efficient consortia-based bioformulations.

Immunomodulating properties of protein hydrolysates for application in cow's milk allergy.

Cow's milk proteins cause allergic symptoms in 2-3% of all infants. In these individuals, the tolerogenic state of the intestinal immune system is broken, which can lead to sensitization against antigens and eventually to allergic responses. Although a true treatment for food allergy is not available, symptoms can be avoided by providing the infants with hydrolyzed proteins. Hydrolyzed proteins are proteins that are enzymatically degraded. They lack typical allergenic IgE-binding epitopes but are also thought to play a pertinent role in other mechanisms inducing hypoallergenic effects. This review discusses the mechanisms and evidence for immunomodulating properties of cow's milk hydrolysates. Hydrolysates are found to strengthen the epithelial barrier, modulate T-cell differentiation, and decrease inflammation. Some studies suggest a role for hydrolysates in manipulating pathogen recognition receptors signaling as underlying mechanism. Peptides from hydrolysates have been shown to bind to TLR2 and TLR4 and influence cytokine production in epithelial cells and macrophages. Current insight suggests that hydrolysates may actively participate in modulating the immune responses in subjects with cow's milk allergy and those at risk to develop cow's milk allergy. However, more research is required to design effective and reproducible means to develop targeting strategies to modulate the immune response.

Recent developments in basophil research: do basophils initiate and perpetuate type 2 T-helper cell responses?.

Basophils account for only 0.1-1% of all peripheral blood leukocytes. They were considered to be a redundant cell type for a long time. However, several findings show a non-redundant role for basophils in type 2 T-helper cell (Th2) immune responses in helminth infections, allergy and autoimmunity. Both immunoglobulin-E-dependent and -independent pathways have been described to contribute to basophil activation. In addition, several recent studies reported that basophils can function as antigen-presenting cells and are important in the initiation of Th2 immune responses. However, there are also conflicting studies that do not corroborate the importance of basophils in Th2 immune responses. This review discusses the role of basophils in Th2 immune responses in view of these recent findings.

Vegephy: impact of vegetable oils used as extemporaneous additives on pesticide residues and their crop protection potential.

Within the framework of the VEGEPHY project set up to assess the effect of refined oils used as extemporaneous additives, both alone and in combination with oligosaccharides (guar, xanthan and carboxymethyl cellulose), on the quality of plant protection treatments for wheat, a study was conducted on the effect of combinations of additives on the level of pesticide residues in wheat plants and grain and their impact on treatment efficiency. The use of some of these additives gave efficiency results that were comparable with those obtained with additives that are currently used but are not bio-based. The use of refined oils as extemporaneous additives, both alone and combined with oligosaccharides, led to better penetration by the pesticide as well as longer persistence of its activity. The experiments were conducted with fungicides, herbicides, plant growth regulators and various types of formulation (EC, SC and WG). The results obtained were conclusive, but they did depend on the physico-chemical properties of the active substance. The risk of exceeding the maximum residue levels (MRLs) of pesticides was also evaluated. Even where the MRLs had not been not exceeded, the experiments showed that in some cases the treatment with these additives could lead to pesticide residues up to two times higher than those measured for the treatment without additives. It would be necessary, therefore, to reduce the treatment dose associated with the additive and/or have a long enough pre-harvest interval to avoid exceeding the MRLs. The use of green additives could be a useful and easy way to attain the European Union (EU) goal of reducing pesticide use by improving the retention of active substances on the plant and reducing the number of treatments.

Type of intrinsic resistant starch type 3 determines in vitro fermentation by pooled adult faecal inoculum.

Resistant starch (RS) results in relatively high health-beneficial butyrate levels upon fermentation by gut microbiota. We studied how physico-chemical characteristics of RS-3 influenced butyrate production during fermentation. Six highly resistant RS-3 substrates (intrinsic RS-3, 80-95 % RS) differing in chain length (DPn 16-76), Mw distribution (PI) and crystal type (A/B) were fermented in vitro by pooled adult faecal inoculum. All intrinsic RS-3 substrates were fermented to relatively high butyrate levels (acetate/butyrate ≤ 2.5), and especially fermentation of A-type RS-3 prepared from polydisperse α-1,4 glucans resulted in the highest relative butyrate amount produced (acetate/butyrate: 1). Analysis of the microbiota composition after fermentation revealed that intrinsic RS-3 stimulated primarily Lachnospiraceae, Bifidobacterium and Ruminococcus, but the relative abundances of these taxa differed slightly depending on the RS-3 physico-chemical characteristics. Especially intrinsic RS-3 of narrow disperse Mw distribution stimulated relatively more Ruminococcus. Selected RS fractions (polydisperse Mw distribution) obtained after pre-digestion were fermented to acetate and butyrate (ratio ≤ 1.8) and stimulated Lachnospiraceae and Bifidobacterium. This study indicates that especially the α-1,4 glucan Mw distribution dependent microstructure of RS-3 influences butyrate production and microbiota composition during RS-3 fermentation.

Characterization of Tetragenococcus strains from sugar thick juice reveals a novel species, Tetragenococcus osmophilus sp. nov., and divides Tetragenococcus halophilus into two subspecies, T. halophilus subsp. halophilus subsp. nov. and T. halophilus subsp. flandriensis subsp. nov.

Most bacteria recovered so far from sugar thick juice during storage represent strains of the species Tetragenococcus halophilus. Recently, several Gram-positive, non-motile, non-spore-forming cocci with other physiological and genetic traits were isolated from sugar thick juice samples from different origins. In this study, representative isolates were investigated using a polyphasic taxonomic approach. The 16S rRNA gene sequence similarity between these isolates and their closest relative, Tetragenococcus muriaticus, was 97.4%. The level of DNA-DNA relatedness between isolate T1(T), representing the newly found Tetragenococcus isolates, and T. muriaticus was 57%. Isolate T1(T) had a DNA G+C content of 36.7 mol%. Phylogenetic data and genomic and phenotypic features demonstrated that the isolates represent a novel species, for which the name Tetragenococcus osmophilus sp. nov. is proposed with T1(T) as the type strain (=LMG 26041(T) =DSM 23765(T)). Additionally, T. halophilus isolates from high-salt and high-sugar environments showed clear differences in several physiological and genetic characteristics like RAPD fingerprints and 16S rRNA gene sequences. DNA-DNA hybridizations, however, showed 79 to 80% relatedness between osmophilic and halophilic T. halophilus isolates, demonstrating that the different strains belong to the same species. Based on the phenotypic and genotypic differences observed, as well as the different origins of the strains and the industrial relevance of thick juice degradation, two subspecies of T. halophilus are described in this manuscript: T. halophilus subsp. halophilus subsp. nov. for the strains isolated from salt media and T. halophilus subsp. flandriensis subsp. nov. for the strains isolated from sugar-rich environments, which were first isolated in Flanders, Belgium. The type strains for the subspecies are IAM 1676(T) (=LMG 11490(T) =DSM 20339(T)) and T5(T) (=LMG 26042(T) =DSM 23766(T)), respectively.

The level and distribution of methyl-esters influence the impact of pectin on intestinal T cells, microbiota, and Ahr activation.

Pectins are dietary fibres that modulate T cell immunity, microbiota composition, and fermentation profiles, but how this is influenced by the degree of methyl-esterification (DM) and degree-of-blockiness (DB) of pectin is unknown. Here, we demonstrate that supplementation of DM19(high-DB), DM49(low-DB) and DM43(high-DB) pectins at a low dose increased the frequencies of intestinal T-helper (Th)1 and Th2 cells after 1 week of pectin supplementation in mice, whereas DM18(low-DB) did not. After 4 weeks of supplementation with those pectins, Th1 and Th2 frequencies returned to control levels, whereas Rorγt+ regulatory T-cell frequencies increased. These structure-dependent effects could derive from induced shifts in microbiota composition that differed between DM18(low-DB) pectin and the other pectins. T-cell-modulating effects were not short-chain-fatty acid-dependent, but rather through an increase in Aryl-hydrocarbon-receptor-activating components. Thus, pectins with a specific combination of DM and DB have an impact on intestinal T cell-immunity in mice, when supplemented at a low dose.

ß(2→6)-Type fructans attenuate proinflammatory responses in a structure dependent fashion via Toll-like receptors.

Graminan-type fructans (GTFs) have demonstrated immune benefits. However, mechanisms underlying these benefits are unknown. We studied GTFs interaction with Toll-like receptors (TLRs), performed molecular docking and determined their impact on dendritic cells (DCs). Effects of GTFs were compared with those of inulin-type fructans (ITFs). Whereas ITFs only contained ß(2→1)-linked fructans, GTFs showed higher complexity as it contains additional ß(2→6)-linkages. GTFs activated NF-κB/AP-1 through MyD88 and TRIF pathways. GTFs stimulated TLR3, 7 and 9 while ITFs activated TLR2 and TLR4. GTFs strongly inhibited TLR2 and TLR4, while ITFs did not inhibit any TLR. Molecular docking demonstrated interactions of fructans with TLR2, 3, and 4 in a structure dependent fashion. Moreover, ITFs and GTFs attenuated pro-inflammatory cytokine production of stimulated DCs. These findings demonstrate immunomodulatory effects of GTFs via TLRs and attenuation of cytokine production in dendritic cells by GTFs and long-chain ITF.

Pectin limits epithelial barrier disruption by Citrobacter rodentium through anti-microbial effects.

SCOPE: C. rodentium is the murine equivalent of Enteropathogenic Escherichia. coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) which induce damage to the intestinal epithelial barrier that results in diarrhea and intestinal inflammation. Dietary fibre intake can be an effective approach to limit epithelial damage by these enteric pathogens. Therefore, the protective effect of dietary fibre pectin against dysfunction of epithelial barrier integrity upon C. rodentium infection was investigated. METHODS AND RESULTS: Pectins that structurally differed in the degree and distribution of methylesters were tested on barrier protective effects on epithelial cells against C. rodentium by measuring transepithelial electrical resistance and lucifer yellow fluxes. All three pectins protected the epithelial barrier from C. rodentium induced damage in a structure-independent manner. These barrier protective effects were also independent of pectin-induced TLR2 activation. Furthermore, the pectins induced anti-adhesive effects on C. rodentium by interacting with C. rodentium and not with epithelial cells. This may be explained by antimicrobial effects of pectins on C. rodentium and not on other enteric bacteria including Lactobacillus plantarum and E. coli. A competition ELISA for binding of C. rodentium to pectin supported this finding as it showed that pectin interacts strongly with C. rodentium, whereas it interacts weakly or not with L. plantarum or E. coli. CONCLUSION: These findings demonstrate that pectin protects the epithelial barrier from C. rodentium induced damage by inducing anti-microbial effects.

Digestibility of resistant starch type 3 is affected by crystal type, molecular weight and molecular weight distribution.

Resistant starch type 3 (RS-3) holds great potential as a prebiotic by supporting gut microbiota following intestinal digestion. However the factors influencing the digestibility of RS-3 are largely unknown. This research aims to reveal how crystal type and molecular weight (distribution) of RS-3 influence its resistance. Narrow and polydisperse α-glucans of degree of polymerization (DP) 14-76, either obtained by enzymatic synthesis or debranching amylopectins from different sources, were crystallized in 12 different A- or B-type crystals and in vitro digested. Crystal type had the largest influence on resistance to digestion (A >>> B), followed by molecular weight (Mw) (high DP >> low DP) and Mw distribution (narrow disperse > polydisperse). B-type crystals escaping digestion changed in Mw and Mw distribution compared to that in the original B-type crystals, whereas A-type crystals were unchanged. This indicates that pancreatic α-amylase binds and acts differently to A- or B-type RS-3 crystals.

The impact of the level and distribution of methyl-esters of pectins on TLR2-1 dependent anti-inflammatory responses.

Pectins have anti-inflammatory effects via Toll-like receptor (TLR) inhibition in a degree of methyl-esterification-(DM)-dependent manner. However, pectins also vary in distribution of methyl-esters over the galacturonic-acid (GalA) backbone (Degree of Blockiness - DB) and impact of this on anti-inflammatory capacity is unknown. Pectins mainly inhibit TLR2-1 but magnitude depends on both DM and DB. Low DM pectins (DM18/19) with both low (DB86) and high DB (DB94) strongly inhibit TLR2-1. However, pectins with intermediate DM (DM43/DM49) and high DB (DB60), but not with low DB (DB33), inhibit TLR2-1 as strongly as low DM. High DM pectins (DM84/88) with DB71 and DB91 do not inhibit TLR2-1 strongly. Pectin-binding to TLR2 was confirmed by capture-ELISA. In human macrophages, low DM and intermediate DM pectins with high DB inhibited TLR2-1 induced IL-6 secretion. Both high number and blockwise distribution of non-esterified GalA in pectins are responsible for the anti-inflammatory effects via inhibition of TLR2-1.

Array comparative genomic hybridization analysis does not show genetic alterations in spermatozoa and offspring generated after spermatogonial stem cell transplantation in the mouse.

BACKGROUND: The most promising procedure to restore fertility in male childhood cancer patients is spermatogonial stem cell transplantation (SSCT). Although the efficiency of SSCT has been proven in the mouse model, its safety needs to be investigated too before considering any implementation in the clinic. To examine the incidence of genetic abnormalities after SSCT, the karyotypes of donor-derived spermatozoa and offspring were analyzed. METHODS: Donor cells were obtained from prepubertal mice and introduced in the seminiferous tubules of genetically sterile W/W(v) mice. Five to 10 months after SSCT, DNA was extracted from epididymal sperm to perform array comparative genomic hybridization (aCGH) analysis. In addition, spermatozoa, liver and kidney from the offspring were subjected to aCGH analysis. RESULTS: Numerical chromosomal aberrations could not be detected in spermatozoa from transplanted males, nor in their offspring. The few genetic deviations (deletions, amplifications) observed were all polymorphisms. CONCLUSIONS: No major genetic alterations could be detected after SSCT. These data are supportive for further development of SSCT as a strategy for fertility restoration.

Mitochondrial function in immune cells in health and disease.

One of the main functions of mitochondria is production of ATP for cellular energy needs, however, it becomes more recognized that mitochondria are involved in differentiation and activation processes of immune cells. Upon activation, immune cells have a high need for energy. Immune cells have different strategies to generate this energy. In pro-inflammatory cells, such as activated monocytes and activated T and B cells, the energy is generated by increasing glycolysis, while in regulatory cells, such as regulatory T cells or M2 macrophages, energy is generated by increasing mitochondrial function and beta-oxidation. Except for being important for energy supply during activation, mitochondria also induce immune responses. During an infection, they release mitochondrial danger associated molecules (DAMPs) that resemble structures of bacterial derived pathogen associated molecular patterns (PAMPs). Such mitochondrial DAMPS are for instance mitochondrial DNA with hypomethylated CpG motifs or a specific lipid that is only present in prokaryotic bacteria and mitochondria, i.e. cardiolipin. Via release of such DAMPs, mitochondria guide the immune response towards an inflammatory response against pathogens. This is an important mechanism in early detection of an infection and in stimulating and sustaining immune responses to fight infections. However, mitochondrial DAMPs may also have a negative impact. If mitochondrial DAMPs are released by damaged cells, without the presence of an infection, such as after a trauma, mitochondrial DAMPs may induce an undesired inflammatory response, resulting in tissue damage and organ dysfunction. Thus, immune cells have developed mechanisms to prevent such undesired immune activation by mitochondrial components. In the present narrative review, we will describe the current view of mitochondria in regulation of immune responses. We will also discuss the current knowledge on disturbed mitochondrial function in immune cells in various immunological diseases.

Microencapsulated islet allografts in diabetic NOD mice and nonhuman primates.

OBJECTIVE: Our goal was to assess the efficacy of encapsulated allogeneic islets transplanted in diabetic NOD mice and streptozotocin (STZ)-diabetic nonhuman primates (NHPs). MATERIALS AND METHODS: Murine or NHP islets were microencapsulated and transplanted in non-immunosuppressed mice or NHPs given clinically-acceptable immunosuppressive regimens, respectively. Two NHPs were treated with autologous mesenchymal stem cells (MSCs) and peri-transplant oxygen therapy. Different transplant sites (intraperitoneal [i.p.], omental pouch, omental surface, and bursa omentalis) were tested in separate NHPs. Graft function was monitored by exogenous insulin requirements, fasting blood glucose levels, glucose tolerance tests, percent hemoglobin A1c (% HbA1c), and C-peptide levels. In vitro assessment of grafts included histology, immunohistochemistry, and viability staining; host immune responses were characterized by flow cytometry and cytokine/chemokine multiplex ELISAS. RESULTS: Microencapsulated islet allografts functioned long-term i.p. in diabetic NOD mice without immunosuppression, but for a relatively short time in immunosuppressed NHPs. In the NHPs, encapsulated allo-islets initially reduced hyperglycemia, decreased exogenous insulin requirements, elevated C-peptide levels, and lowered % HbA1c in plasma, but graft function diminished with time, regardless of transplant site. At necropsy, microcapsules were intact and non-fibrotic, but many islets exhibited volume loss, central necrosis and endogenous markers of hypoxia. Animals receiving supplemental oxygen and autologous MSCs showed improved graft function for a longer post-transplant period. In diabetic NHPs and mice, cell-free microcapsules did not elicit a fibrotic response. CONCLUSIONS: The evidence suggested that hypoxia was a major factor for damage to encapsulated islets in vivo. To achieve long-term function, new approaches must be developed to increase the oxygen supply to microencapsulated islets and/or identify donor insulin-secreting cells which can tolerate hypoxia.

Attenuated atherosclerosis upon IL-17R signaling disruption in LDLr deficient mice.

Atherosclerosis is an inflammatory disease characterized by the influx of macrophages and T cells and IL-17 may connect innate and adaptive immune responses involved in atherogenesis. We investigated the role of IL-17 receptor signaling in atherosclerosis and transplanted LDLr deficient recipient mice with IL-17R deficient bone marrow. Induction of atherosclerosis by Western-type diet induced a 46% reduction in lesion size in the aortic root and the plaque composition revealed no significant changes in collagen content and neutrophil counts, but a reduction in mast cell number and an increase in macrophage number. In addition, we observed a decrease in anti-oxLDL antibodies of the IgG class upon IL-17R BMT, while introduction of IL-17R deficient bone marrow resulted in a reduced IL-6 production and an increased IL-10 production. In conclusion, signaling via the IL-17 receptor in bone marrow derived cells enhances the process of atherosclerosis.

TaxonGap: a visualization tool for intra- and inter-species variation among individual biomarkers.

UNLABELLED: Selection of optimal biomarkers for the identification of different operational taxonomic units (OTUs) may be a hard and tedious task, especially when phylogenetic trees for multiple genes need to be compared. With TaxonGap we present a novel and easy-to-handle software tool that allows visual comparison of the discriminative power of multiple biomarkers for a set of OTUs. The compact graphical output allows for easy comparison and selection of individual biomarkers. AVAILABILITY: Graphical User Interface; Executable JAVA archive file, source code, supplementary information and sample files can be downloaded from the website: http://www.kermit.ugent.be/taxongap

Recovery from hemophilia B Leyden: an androgen-responsive element in the factor IX promoter.

One form of the inherited, X-linked, bleeding disorder, hemophilia B, resolves after puberty. Mutations at -20 and -26 in the clotting factor IX promoter impair transcription by disrupting the binding site for the liver-enriched transcription factor LF-A1/HNF4. The -26 but not the -20 mutation also disrupts an androgen-responsive element, which overlaps the LF-A1/HNF4 site. This explains the improvement seen in patients with the -20 mutation and the failure of the -26 patient to recover.

An alternative real-time PCR method to detect the Bacillus cereus group in naturally contaminated food gelatine: a comparison study.

AIMS: Comparison of an internally controlled real-time PCR assay with the standard plate-based assay (ISO 21871) for the detection of Bacillus cereus group cells in gelatine. METHODS AND RESULTS: A comprehensive TaqMan probe was designed allowing the real-time PCR assay to be fully inclusive and exclusive. An internal amplification control was designed and implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (kappa = 0.99) was observed between the performance of the real-time PCR and the standard plate-based method on naturally contaminated gelatines (n = 197). Relative accuracy, relative sensitivity and relative specificity were > or =99%. CONCLUSIONS: The real-time PCR assay is a valid alternative of the standard plate-based assay. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h, while analysis cost did not increase. The gelatine-producing industry can ensure gelatine safety and quality in a much faster way.