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. The dietary fiber pectin plays several immunity-related roles in the gastrointestinal tract and could help manage inflammatory conditions according to a review of recent research. Pectin, found in many fruits and vegetables, takes various structural forms that have different impacts on gut health. Martin Beukema and co-workers at University Medical Centre Groningen in The Netherlands reviewed how these pectin structures interact with the gastrointestinal immune barrier. Studies have shown that pectins can strengthen mucous membranes and epithelial tissue, and activate or inhibit immune cells such as macrophages. Pectins also have a positive influence on the diversity and abundance of microbiota that inhabit the gut, for example helping beneficial bacteria to adhere to epithelial cells while blocking the adhesion of pathogens. Beukema and co-workers point to an urgent need for more studies in humans. Several meta-analyses have illustrated that higher dietary fiber intake is correlated with a reduced incidence of disease and mortality3. These findings are corroborated by other studies showing that reduced dietary fiber intake is correlated with a higher incidence of Western diseases with anomalies in immunity5, indicating that dietary fiber plays an important role in immune homeostasis. One mechanism by which fibers may protect against the development of disease is by preserving the gastrointestinal immune barrier. The gastrointestinal immune barrier is the gatekeeper of the human body. It is composed of a mucus layer and a layer of epithelial cells and prevents luminal molecules from entering the underlying lamina propria containing immune cells7. Intestinal barrier dysfunction may result in immune disorders such as autoimmune and inflammatory diseases, including Crohn’s disease and ulcerative colitis9. Improving intestinal barrier function by increasing dietary fiber consumption may therefore be an effective strategy to prevent or delay Western immune-related diseases10.Dietary fibers play an important role in the maintenance of health11. The small intestine contains a thin and loose mucus layer12 that not only facilitates nutrient uptake but also allows dietary molecules such as fibers to interact with the intestinal epithelial and immune cells directly11. The consequences of these direct interactions with cells of the intestinal immune barrier include strengthening of the mucus layer13, enhancing barrier function of epithelial cells14 or modulating intestinal immune responses15. This direct interaction between dietary fibers and the intestinal immune system may be one of the mechanisms by which dietary fibers improve health and prevent disease16.Before dietary fibers are degraded by microbial enzymes in the colon, they may interact directly with the cells of the immune barrier in the small intestine17. Dietary fibers cannot be digested by human enzymes and are subject to fermentation by the intestinal microbiota mainly in the colon18. Some dietary fibers stimulate the growth of specific microbial communities and serve as substrates for the production of fermentation products, such as short-chain fatty acids (SCFAs)17. Dietary fibers can exert health-promoting effects through stimulation of the production of SCFAs. Numerous recent publications have shown that these SCFAs play an important role in metabolism20 and immunity22. In addition to the fermentation products of dietary fibers, microbial communities can secrete other metabolites, such as tryptophan metabolites or secondary bile acids, which have the potential to regulate intestinal immunity  segments and branched rhamnogalacturonan segments25. Pectins can have many different structural characteristics contributing to their functional properties, including the degree of methyl-esterification, molecular weight or neutral side chain structures25. Several studies involving animal models and human subjects have shown that pectins protect the intestinal barrier from damage28. These effects may be exerted by direct interactions with the gastrointestinal immune barrier, as some types of pectins are known to interact with immune receptors30. Furthermore, pectins may protect the intestinal barrier indirectly by stimulating the growth and diversity of microbiota communities as pectins are known to stimulate microbial communities31. These effects are strongly dependent on the chemical structure of pectins31. In recent years, many studies have demonstrated the impact of specific structural features of pectins on their health benefits to consumers32. The main aim of this review article was to overview the current knowledge of the modulatory properties of specific chemical structures of pectins and their beneficial effects on the gastrointestinal immune barrier; for example, for a dietary fiber that might be instrumental in preventing many Western diseases5. We will focus on pectin structures that influence the immune system by direct effects on immune cells or by indirectly interacting with the immune system by modulating intestinal microbial communities and the metabolites they produce. This article may provide insight into the structural features responsible for intestinal health benefits. Ultimately, this might lead to tailoring of pectin formulation or products that might contribute to prevention or delay of human disease development.The soluble dietary fiber pectin is an important dietary fiber with known modulatory effects on the gastrointestinal immune barrier. Pectins have been isolated from the primary and secondary cell walls of many fruits and vegetables, including citrus fruits, apples, sugar beets, and potatoes25. Pectins are heteropolysaccharides consisting mainly of α-1,4-linked galacturonic acid  residues . Pectins can be classified as low DM pectins (DM < 50%) or high DM pectins (DM > 50%)34. These methyl-esterified GalA residues can be differently distributed along the pectin molecule. The degree of blockiness (DB) determines the distribution of non-esterified galacturonic acid residues. Pectins with a high DB contain a more blockwise distribution of non-esterified GalA residues, whereas low DB pectins have a more random distribution of non-esterified GalA residues35. Homogalacturonan pectins have been isolated from several fruits and vegetables origins, including sunflower, citrus fruits, sisal, rice endosperm cell walls or apples33.Pectins have been isolated from the primary and secondary cell walls of many fruits and vegetables. The structure of pectin is dependent on several parameters, including the origin, the endogenous enzyme activity or the method of extraction36. Pectins containing regions with such a high number of neutral side chains have been isolated from many fruits and vegetables, including apples, sugar beets, apricots, carrots, cabbage, onions or pears33.In addition to homogalacturonan regions, pectins can contain other structural regions, such as xylogalacturonan, apiogalacturonan, rhamnogalacturonan I (RG-I) or rhamnogalacturonan II (RG-II) Fig. . Xylogal37, although some studies have reported effects independent of those exerted on the gastrointestinal tract, including the direct uptake of pectic oligosaccharides in blood and the direct interaction of pectins with systemic organs38. Currently, however, the pronounced health benefits of pectins are considered to result from modulating the intestinal immune barrier37. As pectins might impact different aspects of the gastrointestinal immune barrier37, we first briefly discuss the current understanding of the composition of the gastrointestinal immune barrier. The gastrointestinal immune barrier consists of multiple layers, including a mucus layer, a cellular layer of epithelial cells and the lamina propria containing immune cells  or stimulating SCFA production43. Mucin secretion from goblet cells can also be enhanced by immune cell-derived cytokines44. In addition to mucins, the mucus layer contains immunoglobin A (IgA) and Paneth cell-derived antimicrobial components, which provide a defense against invading, undesired microbial species45. Luminal bacteria or dietary components can stimulate the secretion of these antimicrobial components after PRR activation in Paneth cells46.The mucus layer forms a physical barrier that separates the epithelium from the microbiota and luminal content47. The epithelial cells consist of enterocytes and secretory epithelial cells, such as the aforementioned Paneth cells or goblet cells48. Furthermore, the cellular layer contains an additional epithelial cell type called microfold (M) cells, which are present on subepithelial lymphoid structures called Peyer’s patches (PP). M cells are specialized antigen sampling cells that present antigens to underlying immune cells in the PP49. Together, these epithelial cells form a physical barrier that separates the luminal content from the lamina propria. The integrity of the gastrointestinal barrier relies on the permeability of the epithelial cell layer50. A network of desmosomes, tight junctions and adherens junctions regulates epithelial permeability and thereby the paracellular passage of the luminal content to the underlying lamina propria50. Epithelial integrity can be enhanced by bacteria or luminal dietary components through the activation of PRRs, such as TLR2, or through SCFA-induced GPR activation52.Underneath the mucus layer resides the cellular layer, which is composed of a single layer of epithelial cells and intraepithelial lymphocytes. The intraepithelial lymphocytes secrete factors that support and maintain the epithelial layer39. These immune cells must recognize incoming antigens and provide protection against harmful substances, but also induce tolerance for commensal bacteria53. The lamina propria is the homing site for a large proportion of innate immune cells, such as DCs, macrophages, innate lymphoid cells and granulocytes, and adaptive immune cells, such as B cells and T cells39. DCs and macrophages are innate immune cells that can recognize luminal antigens with PRRs54. DCs are capable of sampling antigens and food components through their epithelial-penetrating dendrites55. After recognition, DCs and macrophages can phagocytose microbes or other molecules and present them as antigens to adaptive immune cells in the lamina propria, PP or mLN. Via this route, DCs and macrophages regulate the induction of IgA-producing plasma cells or induce the differentiation of naïve T cells into regulatory T cells (Treg) or effector T cells 56. In addition, the differentiation of naïve T cells can be induced by microbial-derived SCFAs or aryl hydrogen receptor ligands57. These effector T cells induce pro-inflammatory responses against specific antigens, whereas their counterparts, Tregs, suppress these pro-inflammatory responses57.A large proportion, 70–80%, of all immune cells in the human body are located in gut-associated lymphoid tissues (GALT), which include the lamina propria, PP and mesenteric lymph nodes (mLN)49. DCs present luminal antigens to underlying adaptive immune cells, leading to specific T cell differentiation or the formation of IgA-producing plasma cells58.PPs are located in the small intestine and are located under the epithelium. PPs consist of DCs, macrophages, B cells, and T cells. Luminal antigens from the microbiota or dietary components can be taken up through M cells and are presented to subepithelial dendritic cells+-expressing DCs mainly migrate to the mLN via lymph vessels, after which they stimulate naïve T cells and B cells. CD103+-expressing DCs together with mLN stromal cells generate gut-homing T cells60.The immune cells in the lamina propria and in the PP can communicate with immune cells in the mLN. CD10361. However, tolerance to dietary antigens appears to develop in a different compartment of the gastrointestinal tract than tolerance to intestinal microbiota62. This is because intestinal immunity in the small intestine is more directed towards exposure to dietary antigens, whereas immune responses in the large intestine are more adapted to the greater bacterial load62. Pectins may therefore stimulate the immune system in the small intestine through direct interactions with immune cells, whereas they may stimulate large intestinal immunity in a microbiota-dependent manner.A balanced regulation between pro-inflammatory and anti-inflammatory responses in the gastrointestinal tract leads to protection against pathogens and tolerance to food antigens or commensal microbiotaDietary fibers such as pectins can impact the aforementioned gastrointestinal immune barrier at different levels, as discussed in the following sections.63. A strengthened mucus layer may limit the passage of harmful substances into the underlying tissues and thereby prevent activation of inflammatory responses. Several studies have shown that pectins have beneficial effects on the integrity of the gastrointestinal mucus layer. Under both healthy and diseased conditions, it has been shown that pectins can strengthen the intestinal mucus layer by influencing goblet cells or by the mucoadhesive effects of pectins66.Dietary fibers such as pectins can strengthen the mucus layer through several mechanisms65. In vitro, both DM30 and DM59 pectins stimulated mucin secretion in HT-29MTX cells41. However, in vivo in rats, only DM30 pectins, and not DM59 pectins, were able to stimulate jejunal mucus secretion by goblet cells without affecting the number of mucus-secreting goblet cells41. The different effects of low and high DM pectins on mucus secretion could be explained by the different mucoadhesive properties of low and high DM pectins. Due to the negative charge of their non-esterified galacturonic acid residues, DM38 pectins cannot interact with the negatively charged mucins65. Low DM pectins are thereby able to penetrate the mucus layer and reach the epithelium, where they may stimulate the production and secretion of mucus by goblet cells67. DM70 pectins, however, can form hydrogen bonds with mucins and form gel networks that strengthen the mucus layer65. High DM pectins are not thought to reach goblet cells to induce mucus secretion, but they may protect the epithelium from harmful mucus-penetrating agents by strengthening the mucus layer. In addition, the pectin structure of RG-I showed protective effects on the mucus layer in mice with DSS-induced colitis66. RG-I protected the mucus layer by preserving the number of goblet cells and the expression of Muc-166. Together, these findings suggest that low DM pectins, high DM pectins or RG-I pectin structures can strengthen gut barrier function by stimulating mucin secretion by goblet cells or through mucus adhesive properties.The effects of pectins on the mucus layer are dependent on the structure of pectin68. Several studies have highlighted the importance of pectins in the preservation of epithelial integrity under healthy and diseased conditions69. In healthy young adults and elderly individuals, four weeks of pectin supplementation did not increase epithelial barrier integrity, as tight junction-related genes were unchanged after pectin administration28. Under diseased conditions, pectins lowered epithelial permeability in Bangladeshi children with persistent diarrhea69. In addition, pectins were able to maintain epithelial integrity in rats after disruption by a high-fat diet27. These studies suggest that pectins cannot enhance epithelial integrity under healthy conditions, but they can restore or maintain epithelial integrity under diseased conditions.Dietary fibers are known to have positive effects on epithelial cell layer integrity in the gastrointestinal tract. They can enhance epithelial integrity by stimulating gut bacteria to produce SCFAs that are derived from microbial fermentation of dietary fibers or through direct interaction with pattern recognition receptors (PRRs), such as TLR228. However, Vogt et al. demonstrated in vitro that DM30 or DM74 pectins were more protective against the epithelial barrier disruptor PMA than DM56 pectins29. Moreover, DM7 pectins maintained epithelial integrity in mice with caerulein-induced pancreatitis by preserving tight junction structures26. The findings of Vogt et al. and Sun et al. suggest that compared to DM63 pectins, low DM pectins (DM < 30) or very high DM pectins (DM > 74) might enhance epithelial integrity in young adults and elderly individuals28. In addition to the DM-dependent effects, the in vitro effect of RG-I pectins was investigated. RG-I pectins reduced intestinal permeability in Caco-2 cells by maintaining claudin-1 structures66. These studies demonstrate that pectins can protect epithelial integrity against barrier-disrupting agents by maintaining tight junction structures. Low DM pectins and very high DM pectins or pectins with RG-I structures may be responsible for these effects66.Recent research indicates that not all pectins but specific structural characteristics of pectins induce the protective effects of pectins on epithelial integrity, which might cause sometimes contradictory findings in humans. DM63 pectins were not able to enhance epithelial integrity in intestinal biopsies from healthy young adults and elderly individuals using Ussing chambers73. It has been suggested that dietary fibers are transported into the PP through M cells and activate immune cells, and this effect has been demonstrated for pectins76. In addition, in vitro studies have demonstrated that pectins activate or inhibit the responses of DCs and macrophages through direct interaction with immune receptors77. The activation and inhibition of macrophage and DC responses may be beneficial to the host under different circumstances. Under healthy conditions, mild activation of innate immune responses by pectins can be beneficial because it promotes intestinal immunity. Furthermore, under conditions with aberrant immune activation, inhibition of immune activation can be beneficial78. Attenuating immune activation may prevent the initiation of excessive immune responses and thereby limit inappropriate activation of immune cells and inflammation78.Many dietary fibers, including pectins, can directly interact with innate immune cells, such as macrophages or DCs, and influence their responses77. Suh et al. demonstrated that pectins must have an intact backbone to exhibit their activating effects, as pectin hydrolysates with low molecular weights have no activating properties76. Furthermore, other studies demonstrated that RG-I and RG-II pectins activate macrophages and dendritic cells, but RG-I pectins activate these cells more than RG-II pectins77. Galactan and arabinan structures seem to regulate the strong activation of immune responses by RG-I pectins77. It has also been demonstrated that the DM of pectin plays an important role in immune activation. Partial removal of methyl esters from DM57 pectins to DM21 pectins enhanced the macrophage-activating properties of pectins72. This was, however, not corroborated by another study that showed that partial removal of methyl esters from DM85 pectins to DM17 pectins strongly decreased its macrophage-activating effects79. This could, however, be explained by activating properties of RG-I side chains in this DM85 pectin, which were removed together with the methyl esters by a specific procedure79. Current insight is that the molecular weight, DM and RG-I side chain structures of pectin influence the ability of pectins to activate macrophages and DCs. RG-II side chains may be involved but activate immune cells to a lower extent79.Several in vitro studies have demonstrated that specific pectin structures can activate DC and macrophage responses72. Ishisono et al. suggested that the highly branched side chains of pectins are required for the anti-inflammatory effects of pectins on LPS-induced IL-6 secretion in macrophages70. This study, however, did not specify which specific side chains of RG-I or RG-II pectins were required. Another study showed that removing the RG-I side chains of pectins enhanced the inhibitory effect of pectins on LPS-induced cytokine secretion in macrophages71. The anti-inflammatory effects as measured by Ishisono et al. may therefore have been derived from RG-II side chain structures. Furthermore, another study confirmed that DM21 pectins, with a certain degree of acetylation of 9%, have anti-inflammatory properties and inhibit macrophage responses71. Together, these studies suggest that the inhibitory effect of pectins is orchestrated through structural characteristics such as DM, acetylation, RG-I, and RG-II structures.Several in vitro studies have demonstrated that specific pectin structures can inhibit DC and macrophage responses80. Molecules derived from damaged cells are known as damage-associated molecular patterns (DAMPs). PRRs play an important role in initiating innate immune responses80. In recent years, many studies have reported that PRRs interact with dietary fibers82. Pectins are known to affect immune responses through interactions with the PRRs galectins and Toll-like receptors83.Innate immune cells can recognize dietary fibers through interaction with extracellular PRRs. PRRs function as sensors that recognize molecular patterns from microbes, known as pathogen-associated molecular patterns (PAMPs). PRRs are expressed by innate immune cells, such as macrophages, dendritic cells, neutrophils and epithelial cells83. Galectin-3 is expressed extracellularly or intracellularly by different cell types and plays a role in health and disease84. Extracellular Galectin-3 is known to interact with carbohydrates, but intracellular Galectin-3 is considered to bind to intracellular proteins84. The effects of pectins on Galectin-3 are suggested to initially occur via extracellular Galectin-3. However, as pectin and Galectin-3 can be endocytosed by macrophages, pectins may also affect intracellular pathways by binding to Galectin-385. The binding of pectins to Galectin-3 is established through the interaction between the lectin domain of Galectin-3 and galactose residues or arabinan side chains of RG-I and RG-II pectins83. By binding to Galectin-3, pectins can stimulate immunity against cancer cells. Cancer cells express Galectin-3, limiting T-cell mediated immunity against cancer cells. Galectin-3 binds to T-cells, leading to inhibition of T cell responses or apoptosis. By binding to Galectin-3, pectins block the inhibition of T-cell mediated immunity against cancer cells and can therefore be used in anticancer therapy87. In addition to cancer cells, Galectin-3 is expressed on immune cells, such as monocytes, macrophages, DCs, neutrophils and epithelial cells. These immune cells use Galectin-3 as a PRR to induce innate immune responses against pathogens88. Pectins might enhance these responses by binding to Galectin-3. Thus, RG-I and RG-II pectins with galactose or arabinan structures can bind to Galectin-3, and pectins might exert anti-metastatic or anti-pathogenic effects through this interaction88.Pectin has been reported to bind to Galectin-389. Specific pectin structures can activate signaling of TLR2 and TLR4, which are extracellularly expressed, and thereby stimulate innate immune responses. Pectins containing RG-II and homogalacturonan structures induce strong activation of TLR4 in DCs91. The TLR4-activating effect of homogalacturonan pectins is independent of the DM, as DM30, DM56 and DM74 pectins activate TLR4 at a similar level29. In contrast to TLR4, TLR2 activation by pectins is exerted in a DM-dependent manner. Only very high-DM pectins (DM74) activate TLR2, whereas lower DM pectins (DM30 and DM56) do not activate TLR229. This indicates that the activation of TLR2 is induced after recognition of very high-DM pectins. TLR4 may be induced after recognition of the specific patterns of the non-esterified galacturonic acid backbone or RG-II structures.Toll-like receptors are another type of PRR that can interact with pectins. Toll-like receptors are intracellularly or extracellularly expressed on most immune and epithelial cells92. These TLR4-inhibiting effects were suggested to be exerted through neutral side chains of RG-I or RG-II structures70. In addition, the DM can explain the TLR4-inhibiting effects of pectins because very high DM (DM90) pectin inhibited LPS-induced TLR4 activation in macrophages more effectively than lower DM pectins (DM60 and DM30)93. Furthermore, an opposite trend was observed for TLR2-1 inhibition. TLR2-1 was more effectively inhibited by low DM pectins, and this TLR2-1-inhibiting effect decreased with higher DM pectins30. Together, these results suggest that TLR4 inhibition is exerted through recognition of high DM structures or neutral side chain structures of RG-I or RG-II pectins. TLR2-1 is more effectively inhibited by low DM pectins30.In addition to their TLR-activating properties, pectins are recognized for their TLR-inhibiting properties on TLR2 and TLR4. Several studies have shown that pectins inhibit LPS-induced TLR4 activation in monocytes or dendritic cells18. These intestinal species interact with the intestinal immune barrier, contributing to the health status of the host18. Given the importance of health, maintaining a balanced intestinal microbiota composition is crucial. Dysbiosis of intestinal microbiota composition may disturb host-microbe interactions and contribute to the development of diseases5. A balanced composition of the intestinal microbiota is therefore vital to maintain health and prevent disease development.The gastrointestinal tract harbors trillions of microorganisms, including bacteria, fungi, viruses, protozoa and archaea, which together form the intestinal microbiota94. For example, dietary fibers can increase the abundance of beneficial intestinal microbiota17. As a consequence, dietary fibers reduce the abundance of pathogens or mucin-degrading bacteria, which in turn protect the epithelium against invading pathogens, as has been shown for Citrobacter rodentium in a mouse study24. Another mechanism by which dietary fibers have beneficial effects on the intestinal immune barrier is through stimulating gut bacteria to produce SCFAs18. Dietary fibers can be used by anaerobic bacteria as fermentation substrates to produce SCFAs, including acetate, propionate and butyrate18. SCFAs help maintain a low pH in the intestine, thereby inhibiting pathogen growth and favoring the growth of healthy intestinal microbiota95. In addition, SCFAs can dampen immune responses by acting on DCs, macrophages, neutrophils, T regulatory cells and epithelial cells21. They influence immune responses through interactions with GPR41, GPR43 or GPR109a, by inhibition of histone deacetylases or by direct interaction with transcription factors97. The influence of SCFAs on epithelial cells is mainly through stimulation of butyrate production in bacteria, as epithelial cells use butyrate as an energy substrate that stimulates epithelial cell growth98. Moreover, dietary fibers can have beneficial effects on the intestinal immune barrier, favoring adhesion of commensal or beneficial bacteria to the epithelium100. Thus, dietary fibers can have beneficial effects on the intestinal immune barrier through stimulation of microbial communities and SCFA production and modulation of bacterial adhesion to epithelial cells100. Proof that pectin can influence gut microbiota via one or a combination of the above described mechanisms is discussed in the next sections.Dietary fibers, including pectin, are components that can contribute to a balanced microbiota composition. Dietary fibers can have beneficial effects on the intestinal immune barrier via the intestinal microbiota through several mechanisms107. The genera Bacteroides and Prevotella are the primary pectin degraders, as they possess carbohydrate-active enzymes (CAZymes) within their polysaccharide utilization loci (PUL)108. These bacteria use lyases, methylesterases and acetylases to breakdown pectin molecules109. The breakdown substrates can be used by surrounding bacteria as growth substrates and thereby influence the composition of the microbial community110. Several in vitro and in vivo studies have reported that pectins induce distinct microbial compositions107. In vitro, it was shown that pectins can increase the abundance of Lactobacilli, Bacteroides and Prevotella103. Furthermore, some in vivo studies showed that pectins increase the abundance of Bacteroidetes, Clostridiales, Bacteroides, Lactobacilli and Prevotella, whereas other in vivo studies report that pectins decrease the abundance of Lactobacillus and Bacteroides spp.107. The inconsistencies in the composition of the microbial communities between the different studies may be related to the variations in initial microbiota composition, fermentation substrates or the influence of the host; however, a recent correlation study found that the impact by which pectins affect the composition of the microbial community strongly depends on the structural characteristics of pectins31.Many studies have demonstrated that dietary pectin can influence the composition and diversity of the gut microbiota31. This study suggested that DM is the most important regulator of microbiota composition31. The effect of DM has been demonstrated in other in vitro and in vivo studies112. DM8 and DM35 pectins were preferably metabolized by gut microbiota and induced a higher abundance of Bifidobacteria and Bacteroides than DM66, DM71 and DM93 pectins in fermentation studies112. In addition, both DM29 and DM53 pectins induced a higher abundance of Prevotella spp. and a lower abundance of Lactobacillus in the pig colon, but these effects were stronger with low DM pectins than with high DM pectins107. The differences in microbiota composition between low and high DM pectins may be related to their differences in digestibility. Low DM pectins are digested faster than high DM pectins, which is suggested to be due to the production of pectate lyases and methyl esterases that digest low DM pectins faster than high DM pectins112. Enzymatic degradation higher in the gastrointestinal tract of low DM pectins may lead to earlier availability of growth substrates for the microbiota than high DM pectins. Consequently, low DM pectins start inducing the growth of microbiota communities earlier in the gastrointestinal tract than high DM pectins113. In addition to DM-dependent effects of pectins, other in vitro fermentation studies confirmed that the RG-I structures and RG-I side chain structures, i.e., arabinan, galactan, oligoarabinosides, and oligogalactosides, of pectins were very efficient in stimulating the growth of Bifidobacteria113. Together, these results show that pectins can induce shifts in microbiota composition and that these shifts are mainly dependent on the DM or RG-I structures of pectins.In a recent in vitro fermentation study, five structural characteristics of pectins were strongly linked to shifts in microbiota composition. The five structural characteristics included the DM of homogalacturonan regions, the composition of neutral sugars, the distribution of homogalacturonan and rhamnogalacturonan fractions, the degree of branching and the presence of amide groups95. Particularly, Bacteroidetes and Firmicutes can utilize dietary fibers and are the main producers of the SCFAs acetate, butyrate and propionate in the gastrointestinal tract115. The generation of SCFAs strongly depends on substrate availability, microbiota composition and intestinal transit time95.SCFAs are metabolic products derived from the fermentation of dietary fibers under anaerobic conditions117. Each individual structural pattern of pectin induces distinct profiles of acetate, propionate and butyrate in the gastrointestinal tract. Larsen et al. showed that after in vitro fermentation of RG-I structures, high levels of propionate and butyrate were induced31. This effect of RG-I structures on SCFA production was confirmed in rats, showing an increased the production of SCFAs production in rats fed with RG-I-enriched diets113. Furthermore, a correlation study by Larsen et al. confirmed an association between high DM pectins and high levels of propionate31. The higher production of propionate induced by DM53 pectins compared to DM29 pectins was also reported in another fecal batch fermentation study116. In contrast, an opposite pattern was observed in rats, showing that total SCFA concentrations were higher in the cecum of rats fed with low DM pectins  than in rats fed with high DM pectins 117. The contradicting findings on the impact of pectin structures on SCFA production may result from a different initial microbiota composition, a different pectin dosage, a difference in pectin digestibility or the influence of the host on microbiota composition31. Overall, the current understanding is that pectins can be used as fermentation substrates for gut microbiota and that the production of SCFAs is strongly influenced by the RG-I structures or the DM of pectins.Pectins can be used as fermentation substrates to stimulate the production of SCFAs118. Several studies have demonstrated that pectins and pectic-oligosaccharides (POS) have the ability to block the adhesion of pathogens to epithelial cells121. POS are known to reduce the adhesion of pathogenic bacteria, such as Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes, and Campylobacter jejuni, to epithelial cells124. This anti-adhesive effect of pectins and POS on pathogens was higher with pectins with a lower molecular weight or DM3 or DM5 pectins121.Dietary fibers are known to inhibit the growth of pathogens by preventing their adhesion to epithelial cells. Blocking pathogenic adhesion to epithelial cells may be beneficial because it prevents pathogen-induced epithelial damage118. Several studies have shown that pectins or pectic oligosaccharides enhance the adherence of commensal or probiotic Lactobacilli strains to epithelial cells124. The effects were species dependent, as pectins enhanced the adhesive effects of some Lactobacillus species, such as Lactobacillus plantarum 0981, 0995 or Lactobacillus brevis 0983, whereas pectins decreased the adhesion of other Lactobacillus species, such as Lactobacillus paracasei 0985, Lactobacillus plantarum 0989, 0990, and 0996122. The structural characteristics were, however, not specified in those studies. The exact mechanisms by which pectins or dietary fibers affect bacterial attachment are not fully understood and are thought to be multifaceted125. Fibers may resemble the saccharides from glycoproteins on epithelial cells that pathogens normally adhere to126. Furthermore, pectins may target other mechanisms in epithelial cells, including the regulation of transcription factors, chaperone proteins, glycosidic hydrolysates or other adhesion-related proteins125.In contrast to the anti-adhesive effects of pectins on pathogens, stimulating adhesion of commensal bacteria to epithelial cells may be favorable, as commensal bacteria may compete with pathogens for attachment or secrete other beneficial products77. However, most of these studies were performed in vitro. Some in vivo studies have confirmed the microbiota-independent effects of pectins on the gastrointestinal immune barrier. A recent study showed that pectins ameliorate TNSB-induced colitis in a microbiota-independent manner, as pectins had colitis-attenuating effects after deletion of microbiota by antibiotics127. Moreover, to show the direct microbiota-independent effects of pectins on the gastrointestinal immune barrier, it is recommended to perform in vivo studies with germ-free mice, such as those previously performed by Fransen et al., for inulin-type fructans128. Furthermore, additional experiments with human intestinal organoid models are needed to further extrapolate these direct effects of pectins on the gastrointestinal immune barrier of humans129.This review discusses the current knowledge on the impact of specific pectin structures on the intestinal immune barrier Fig. . Several100. However, due to the individual differences of the human microbiota, achieving similar effects of the pectin structures in humans may be challenging130. To better understand the microbiota-dependent impact of pectins on the gastrointestinal immune barrier, human studies are needed in which diet, age, gender, use of medication and host milieu are controlled131.In addition, the ability of pectins to reinforce the gastrointestinal immune barrier by impacting the gut microbiota and its fermentation products was reviewed. Specific pectin structures may contribute to intestinal homeostasis by stimulating the growth of microbial communities and enhancing their metabolic activity or by affecting the attachment of bacteria to epithelial cells25. Most studies that investigated the health effects of pectins used pectins with a combination of several structural characteristics. It is therefore hard if not impossible to determine which specific structural patterns of pectins induced health-promoting effects in these studies. To better understand how specific pectin structures can stimulate the intestinal immune barrier, future research should address the following: (1) The impact of the degree of single structural components of pectins  on the gastrointestinal immune barrier; (2) The impact of the distribution of a single structural component  of pectins on the gastrointestinal immune barrier; (3) The combined impact of the degree and distribution of single structural characteristics of pectin on the gastrointestinal immune barrier. This may lead to the development of specific pectin formulations with beneficial effects on the gastrointestinal immune barrier in specific target groups. Because of its wide variety of structural features and specific health effects, pectin is a molecule that can be tailored for personalized medicine. Pectins may, for instance, be useful in preventing or restoring dysbiotic microbiota compositions associated with obesity, inflammatory bowel disease and metabolic syndrome94. Several studies have shown that supplementation of dietary fibers can prolong remission periods in patients with IBDs133, but no human studies have demonstrated that dietary fibers can be used to treat active IBDs134, suggesting that dietary fibers can prevent but not cure IBD symptoms. In Crohn’s disease, ulcerative colitis and IBS food processing are dysregulated and might lead to disturbed fermentation of dietary fibers with possible negative effects on disease progression as a consequence134. This should always be considered when administering dietary fibers in these disorders. Furthermore, the microbiota-independent effects of pectins, including reducing mucositis by blocking TLR2-1, may be useful in limiting chemically induced intestinal inflammation after chemotherapeutic or radiotherapeutic treatments135. Pectin-based nutraceutical or functional foods may therefore be used to limit the development of diseases and maintain health in a microbiota-dependent and microbiota-independent manner.The dietary fiber pectin is a complex molecule with many structural characteristics


Protecting food crops from viral pathogens is a significant challenge for agriculture. An integral approach to genome-editing, known as CRISPR/Cas9 , is used to produce virus-resistant cultivars. The CRISPR/Cas9 tool is an essential part of modern plant breeding due to its attractive features. Advances in plant breeding programs due to the incorporation of Cas9 have enabled the development of cultivars with heritable resistance to plant viruses. The resistance to viral DNA and RNA is generally provided using the Cas9 endonuclease and sgRNAs (single-guide RNAs) complex, targeting particular virus and host plant genomes by interrupting the viral cleavage or altering the plant host genome, thus reducing the replication ability of the virus. In this review, the CRISPR/Cas9 system and its application to staple food crops resistance against several destructive plant viruses are briefly described. We outline the key findings of recent Cas9 applications, including enhanced virus resistance, genetic mechanisms, research strategies, and challenges in economically important and globally cultivated food crop species. The research outcome of this emerging molecular technology can extend the development of agriculture and food security. We also describe the information gaps and address the unanswered concerns relating to plant viral resistance mediated by CRISPR/Cas9. Food crops are vulnerable to various diseases including bacteria, fungi and viruses, resulting in major economic losses. Thus, crop resistance can be improved against various pathogens ,2. DiseaGenetic diversity with enhanced virus protection is an indispensable part of virus resistance . New breR) genes incorporated into cultivars with superior agronomic characteristics  and and24] af a gene . Moreove S genes . The fun species . This tocultivar . This apMultiple sgRNA sequences can be utilized to drive the numerous double-strand breaks (DSBs) through CRISPR/Cas9 at the appropriate site of the target gene. Two sgRNA bindings will generate two DSBs at the respective sites before the start codon and after the stop codon of the gene of interest. These DSBs then remove the DNA fragments carrying the gene of interest before repairing the cellular non-homologous end-joining (NHEJ) machinery  28]. Th. Th28]. S) genes. These S genes disrupt the plant proteins, which are essential and primarily multifunctional. Thus, plant health and productivity are also retarded. An alternate approach, such as cis-regulatory component and promoter editing, can alter gene expression instead of gene disruption. It is sometimes crucial to utilize resistance (R) genes against virulent pathogens. In contrast, the host–pathogen interaction has not been fitted well, and the S genes have not been explored extensively [R genes. Insertion of the CRISPR-mediated gene operates through an alternative pathway that works when Cas9 generates the sgRNA-directed DSBs; this pathway uses cellular homology-directed repair (HDR) instead of non-homologous end-joining (NHEJ) machinery  ge regions .The CRISPR/Cas9 technology evaluates the high specificity in targeting the genomic regions, such as irradiation-induced mutagenesis. This tool raises questions about how a sgRNA can target the entirely complementary genomic DNA sequences and other genomic sites (off-target regions) and the extent of targeting and potentially provoke unexpected damage. There are two types of expected off-target effects in genomic sites: higher sequence similarities to the target and the unexpected off-target in irrelevant genomic sites. Gene sequence information is essential to predict the expected off-target outcomes . Random To enhance the specificity and effectiveness of the CRISPR/Cas9 system, it is vital to analyze and address its possible off-target mutations. Off-target effects generated by the CRISPR/Cas9 system can be identified in plants via qRT-PCR resequencing . The offOryzae sativa). Several Cas9-sgRNAs were designed to effectively delete the small fragments of the dense and erect panicle1 (DEP1) gene in the Indica rice line. These improvements in traits that contribute to yield, such as reduced plant height and dense and upright panicles, have been executed in the production of mutant plants [GmFT2a gene, an essential integrator in photoperiodism of the flowering pathway of soybean plants. The newly developed soybean cultivars exhibited late flowering, which results in the increased size of the vegetative growth. This mutation was also firmly inherited among the following generations [SELF-PRUNING5G (SP5G) in tomato plants to manipulate the photoperiod response. These mutations were achieved using Cas9 and caused rapid flowering and augmented the dense growth habit of tomato plants, resulting in an immediate increase in early yield [Xanthomonas citri subsp. citri, which causes bacterial citrus canker, was undertaken using Cas9 technology. A promoter gene CsLOB1 was targeted, which promotes the development of canker in citrus plants. The developed transgenic lines exhibited improved resistance against canker in citrus compared to the wild types [Blumeria graminis f. sp. tritici. This finding also showed the development of powdery mildew resistance in wheat [Due to its potentiality, low cost, and simplicity, Cas9 has become a widely adopted genome-editing tool in numerous living organisms, including plant species. Editing genes allow the contemporaneous alteration of multiple genetic loci, thus accelerating various commercial crop improvements and enhancing food safety globally . A numbet plants . This meerations . CRISPR/ly yield . The devld types . Bread win wheat .R) genes, in which genetic variability occurs that alters the plant’s suitability, raises numerous fundamental questions regarding the molecular, biochemical, cellular and physiological mechanisms involved in the plant–virus interaction [Although viruses are relatively simple genetic entities, many of their mechanisms by which diseases are generated, and by which plants can resist these effects by natural resistance, remain unknown . The moseraction . Recentleraction . Resistaeraction . HoweverPlant viruses have immense importance in agriculture because many infect different food crops such as vegetables, fruits and staple grains, causing a deterioration in productivity and quality. One virus may infect several crop species, and different kinds of viruses usually attack each species of crop. Viruses cause disease by consuming cells or killing them with toxins and utilizing cellular substances during multiplication, taking up space in cells and disrupting cellular processes. Almost all viral diseases appear to cause dwarfing or stunting of the entire crop plant and a reduction in the total yield. These effects may be severe and cause striking symptoms on the stem, fruit and roots; alternatively, they may or may not cause any symptom development. Plants may show acute severe symptoms soon after inoculation that may lead to the death of young shoots or the entire host plant. If the host survives the initial shock phase, the symptoms tend to become milder (chronic symptoms) in the subsequently developing parts of the plant, leading to partial or even total recovery.However, in some diseases, symptoms may increase progressively in severity and may result in a gradual or quick decline in the health of the plant. The most common types of plant symptoms produced by systemic virus infections are mosaics and ring spots. Mosaics are characterized by light-green, yellow or white areas with the typical green color of the leaves, flowers and fruits. Depending on the intensity or pattern of discolorations, mosaic symptoms may be described as mottling, streaks, a ring pattern, a line pattern, vein clearing, vein banding or chlorotic spotting. Ring spots are characterized by chlorotic or necrotic rings on the leaves and sometimes also on the fruits and stems. In many ring spot diseases, the symptoms tend to disappear later, but the virus remains. The extent of economic yield loss due to viral infection in many crop varieties is summarized in The CRISPR/Cas9 platform is a groundbreaking advancement that allows the engineering of interference against plant pathogens. In-depth research is required to completely develop the effectiveness of this method to globally combat hunger due to crop losses caused by viral diseases. One of the concerns of the CRISPR/Cas9 tool is the risk of newly emerging recombinant viruses that are resistant to the Cas9 endonuclease . These rCRISPR/Cas9 targeting viral open reading frames (ORFs) can generate more viral variants, resulting from the viral escape events at different levels. Cas9 may disseminate these mutant viruses from plants and with multiple sgRNAs . In addiA number of reports were found regarding resistance genes in many economically important food crops, including the 20 viruses listed in S) gene(s) available for other pathogens are usually absent for viruses. Considerable research has been conducted to identify and understand the mechanisms of virus infection, and fewer potentially relevant plant genes have also been recognized. These comprehensive reviews show that the host susceptibility factors may potentially target the S genes to develop resistance against the crop virus, which is a promising future approach for agriculture. The mutation among the plants generated using the CRISPR/Cas9 method is usually stable and heritable in nature. This mutation can be easily separated from the Cas9/sgRNA complex to avoid further alterations by Cas9/sgRNA, thus helping to develop transgene-free progeny in only one generation. The integration of Cas9/sgRNA in the CRISPR/Cas9 toolkit for virus resistance assists in the development of virus-resistant transgenic plants. The plasmid-free delivery of pre-assembled complexes is another essential technique for generating virus-resistant plants that contain no foreign DNA in the respective virus genome. Identifying potential genes or DNA sequences that can be utilized as useful targets for gene editing is also essential. For example, the fission of preserved non-coding regions with the virus genome contributes to the same taxonomic community’s resistance to specific viruses. Using the prokaryotic CRISPR/Cas9 framework means that the resistance produced would be more effective because, unlike the RNA interference method, viruses lack the machinery to prevent their inhibitory effects. The Cas9 protein and short-range in vitro transcribed RNA guide can be incorporated directly into the cells as a ribonucleo protein complex (RNP), which does not require the integration of transgenes into the genome. These approaches apply to RNA or DNA genome viruses. In addition, the rate of recombination and mutation of viral DNA also remains to be explored immediately after the cleavage virus genome enters the host cell. Among other questions, it is also unknown whether the multiplexing of sgRNAs in transgenic plants may promote resistance to diverse strains of plant viruses, because it is naturally covered with the crRNA gene within the prokaryotic CRISPR arrays.This review highlighted that CRISPR/Cas9 technology has been used to develop crop varieties resistant to numerous significant viral pathogens. Furthermore, this technique may also provide an important opportunity to research these viruses, resulting in benefits to humans. Viruses cause numerous diseases in the most economically significant crop plants, and CRISPR-mediated virus resistance has been used to target the virus DNA and RNA. This approach has some limitations; notably, viruses have the capacity to escape and generate resistance-blocking strains. Specific utilization of the host susceptibility factors involved in plant–virus interactions is a possible solution for this shortcoming. Moreover, the well-distinguished susceptible  process. At present, the impact of the CRISPR/Cas9 methodology on plant physiological traits and potential virus mutants is inadequately documented. Global collaboration using open field trial data (from multiple climates and locations) on Cas9-treated crop plant–virus–environment interactions, and the subsequent growth and yield performance of staple food crops, warrants further attention.


Intramuscular myxoma is a rare entity that may present as single or multiple lesions in patients with Mazabraud’s syndrome and is characterized by intramuscular myxomas with fibrous dysplasia. Though intramuscular myxomas occur in large muscle groups, they can very rarely occur in the chest wall. We present the case of a 41-year-old woman with an incidentally discovered intercostal mass on magnetic resonance cholangiopancreatography (MRCP). Repeat MRI demonstrated a lobulated, T2-hyperintense intercostal lesion and demonstrated adjacent fibrous dysplasia of the ribs, consistent with the patient’s history of Mazabraud's and McCune Albright syndromes. Histopathological exam following surgical resection confirmed a diagnosis of intramuscular myxoma without the presence of sarcomatous changes. Though small, slow-growing intramuscular myxomas may be observed with conservative management in the absence of significant symptoms, surgical resection is warranted to prevent complications such as osseous erosion or nerve impingement. Myxomas are rare, benign mesenchymal tumors that can occur in various anatomic locations either independently or as a constellation of pathologic syndromes. Though the most frequent site for myxomas is within the myocardium, extracardiac myxomas can occur most commonly in musculature, primarily in large muscle groups . Very raA 41-year-old woman with a history of Mazabraud’s and McCune Albright syndromes and known polyostotic fibrous dysplasia, multiple endocrinopathies , and multiple prior symptomatic myxomas, presented for the evaluation of an incidentally discovered right intercostal mass between the seventh and eighth ribs on MRCP performed for follow-up imaging of known pancreatic cysts and surveillance for intraductal papillary mucinous neoplasm. She notes that she has not had any recent pain associated with the right chest wall, and she has not noticed any enlarging, palpable mass. Relevant medical and surgical history includes multiple prior gluteal myxomas with surgical resection for worsening pain, resulting in symptomatic relief. Additionally, the patient reported ongoing evaluation for a slowly enlarging right neck myxoma causing right arm pain. Physical exam revealed a small, palpable right neck mass but did not reveal any palpable mass or tenderness along the region of interest at the chest wall.A review of the MRCP from one month prior demonstrated stability of her multiple cystic pancreatic lesions measuring up to 8 mm, without suspicious features, and revealed a significantly T2-hyperintense lobulated lesion measuring 3.1 x 2.7 x 2.5 cm in the posterior seventh to eighth intercostal space. Repeat MRI of the chest at the time of this visit confirmed this finding  antibiotics. Treatment with antibiotics  improved her clinical course with a resolution of her symptoms. Her chest tube output gradually declined, and all chest tubes were removed without complication.The patient was admitted for pain management and discharged on postoperative Day 1 following her resection with a right-sided Jackson Pratt (JP) drain terminating in her pleural cavity. Her postoperative course was complicated by right-sided empyema secondary to Intramuscular myxomas are a sub-type of soft tissue myxomas representing rare, benign tumors of mesenchymal origin with a predilection for middle-aged  and female patients, as seen in the current case . IntramuTypical imaging findings of intramuscular myxomas include homogenous, hypodense lesions with well-defined margins and soft-tissue attenuation on CT images . CorrelaHistological diagnosis of intramuscular myxoma requires a hypocellular, hypovascular lesion with an abundant myxoid component -9. CellsSmall, slow-growing intramuscular myxomas may be observed with conservative management in the absence of significant symptoms . HoweverSimilarly, surgical intervention is warranted for fibrous dysplasia in the context of worsening mass effect from growing lesions, or from rare malignant transformation to osteogenic sarcoma, as noted in case reports of patients with Mazabraud’s syndrome -13.Intramuscular myxomas are rare lesions associated with Mazabraud’s and McCune Alright syndromes. Though these lesions tend to occur in large muscle groups, they may rarely present in the chest wall. When associated with these syndromes, intramuscular myxomas tend to occur near regions of fibrous dysplasia. Small, slow-growing intramuscular myxomas may be treated with conservative management; however, surgical intervention is warranted to prevent complications such as adjacent osseous erosion or nerve impingement from tumor growth.


Nanotechnologies are rapidly increasing their role in immuno-oncology in line with the need for novel therapeutic strategies to treat patients unresponsive to chemotherapies and immunotherapies. The tumor immune microenvironment (TIME) has emerged as critical for tumor classification and patient stratification to design better treatments. Notably, the tumor infiltration of effector T cells plays a crucial role in antitumor responses and has been identified as the primary parameter to define hot, immunosuppressed, excluded, and cold tumors. Organic and inorganic nanoparticles (NPs) have been applied as carriers of new targeted therapies to turn cold or altered  tumors into more therapeutically responsive hot tumors. This mini-review discusses the significant advances in NP-based approaches to turn immunologically cold tumors into hot ones. Over the last decades, scientists have made a tremendous effort to design new nanotechnologies to fight cancer. Nanotechnologies represent a promising therapeutic tool to reduce cancer’s social and economic burden, which remains a dreadful cause of death worldwide. Various nanotechnologies have demonstrated an improved therapeutic outcome over existing antitumor therapies and made possible targeted therapies by significantly reducing their systemic toxicity .+ lymphocyte populations and ranging between 0 (hot immune tumors) and 4 (cold immune tumors) . Recent advances in cancer immunology revealed that specific traits of the TIME correlate with the efficacy of chemotherapy and immunotherapies . Based o tumors) .+ T cells, such as interleukin 15 (IL-15), can influence CD8+ T cell presence and persistence within the TIME , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and other immune checkpoints, and the extracellular-potassium-mediated T cell suppression . Peculiathe TIME .In this review, we aim to summarize the most recent antitumor strategies that use nanotechnologies to favorably alter the immunosuppressive nature of the TIME, changing immune-defective tumors (cold) into immunogenic and immunoreactive environments (hot) as schematically depicted in Nanotechnology-enabled drug delivery approaches have been designed and formulated for overcoming major bottlenecks of chemotherapies and immunotherapies. Organic and inorganic nanocarriers have improved tumor targeting and preserved chemical or biological payloads from degradation. Since nanoparticles (NPs) can be recognized and internalized by the mononuclear phagocyte system (MPS), some NPs leverage their uptake by immune cells to reach and accumulate within the tumor site, also referred to as the Trojan horse method . Other NTumor immune microenvironment is usually characterized by physical barriers impeding immune cells and therapies to reach the tumor site. Since the penetration of immune cells into the tumor positively correlates with relapse-free survival and immunotherapies efficacy in preclinical and clinical studies, various strategies aiming to reduce physical barriers have been investigated. The predominant physical barrier within the TIME is the ECM . Tumor ETwo main strategies have been conceived to modulate the ECM promoting a better infiltration of immune cells and therapies within the TIME.in vivo studies targeting CAFs. The first study considered photosensitizer-loaded NPs combined with phototherapy for targeted CAF depletion. In contrast, the other study performed CAF reprogramming via gold NPs delivering siRNA against heat shock protein 47  combined with all-trans retinoic acid to turn CAFs quiescent. Both studies showed a reduced ECM deposition allowing a better tumor penetration of CD8+ T cells and therapeutic NPs  therapies are an undoubtful step forward in the fight against cancer, they might not reach the desired effectiveness in cold tumors due to the presence of cytokines inhibiting both maturation and activity of antigen-presenting cells, especially dendritic cells (DCs), and inducing polarization of TAMs . ImmunosToll-like receptors (TLRs) are Type I transmembrane glycoproteins expressed by cells of the innate immune system and malignant cells. Activation of TLRs leads to the production of proinflammatory cytokines such as type I interferons that mediate an adaptive immune response through activation of DCs. TLRs can be classified into two subgroups according to the type of ligands they respond to and their cellular localization: TLRs recognizing mainly bacterial components located on the plasma membrane such as TLR1/2/4/5/6 and TLRs recognizing DNA and RNA such as TLR3/7/8/9 which are instead located intracellularly on the endosome .Toll-like receptor stimulation may play a role in tumor development via either a protumor or antitumor effect on the mutated cells. In different settings, TLR stimulation was shown to cause either an antitumor effect by directly inducing gene-related programmed cell death or, conversely, to have a protumor effect promoting cell proliferation, mutagenesis, invasion, and even resistance to apoptosis in malignant cells mainly via activation of NF-kB, MAPK cascades and PI3K-Akt pathway . Antitum2 releasing), peptide nanocomplexes, or forming immunoconjugates with other antibodies. NPs were designed to preserve the agonist, target specific cells within the TIME and release their immunostimulant cargo. These NP-based therapies mainly targeted TAMs and APCs, aiming to reduce the regulatory phenotype of TAMs and induce presentation and co-stimulatory molecules on APCs. Other studies have demonstrated that targeting cancer cells with NPs delivering agonists for TLR3 induces direct apoptosis of mutated cells  superfamily is a class of receptors involved in several T cell activities. In particular, the engagement of these receptors determines the frequency of effector and memory T cells in response to antigen stimulation . Indeed,It is known that TNFRs deliver co-stimulatory signals, as their effects largely depend on antigen recognition and TCR signaling . Thanks Cytosolic exogenous or endogenous DNA represents a danger signal for cells. The cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) rapidly detects the cytosolic DNA to mount an innate response through the activation of the stimulator of interferon genes (STING). STING activation has been investigated as a co-stimulatory strategy to boost the immune response against cancer by mediating IFN type I responses in both innate and adaptive immune systems and by activating cell death in some cancer cells . Scientitrans-presentation of IL-15 to lymphocytes resulting in their activation and related enhancement of antitumor immune response.Cytokines represent soluble mediators of cell communication and play a fundamental role in several pathophysiological conditions. The strategic modulation of cytokine secretion, circulation, and binding to their dedicated receptors is a successful approach to treating several diseases . In a mein vivo preclinical models. IFN-γ loaded backpacks binding to macrophages continuously guided the polarization of macrophages toward antitumor phenotypes (+ T cells and human chimeric antigen receptor (CAR)-T cell therapy in a mouse model  and short hairpin RNA (shRNA) are also applied in the modification of the TIME from cold to hot, as shown by the administration of siRNA/shRNA/miRNA encapsulated in various NPs that successfully reduced the expression of key proteins in cancer cells, endothelial cells, TAMs, DCs, monocytes and CAFs , have gained attention as potential therapeutic tools in many diseases, including cancer . To achiand CAFs . For exaand CAFs . In two  T cells . Similar T cells , VEGFR o T cells , indolea T cells  showed p T cells , and of  T cells  enriched T cells . Both RNin situ release of endogenous antigens and DAMPs, to boost antitumoral responses, especially if combined with ICB and to lower the systemic toxicity (in vitro by NP-delivered compounds causing cellular stress such as low concentration of zoledronate loaded into liposomes (Cellular stress and immunogenic cell death (ICD) promote intra- and extracellular danger signals that mediate the activation of inflammatory pathways involved in the onset and maintenance of antitumor responses. Several molecules for cancer treatment can induce cellular stress  that can be sensed by immunocompetent cells, such as macrophages and DCs, sustaining inflammation and antigen presentation, thus, in turn, promoting a hot TIME. NP-targeted delivery of well-known chemotherapeutic drugs  or NP-assisted photothermal and radiotherapies provide toxicity . DAMPs riposomes . Alternaiposomes . TAMs deiposomes . Other Tiposomes . Interesiposomes .Scientific evidence in favor of autophagy manipulation as cancer treatment is promising but sometimes contradictory. Studies showing iron oxide nanoparticle-mediated activation of tumoricidal autophagy in cancer cells and M1 polarization of macrophages  are in cEpigenetic modulators are another category of molecules that might benefit from the development of NP-based delivery strategies. For example, histone deacetylase inhibitors (HDACi) have been implemented within tumor-targeting particles showing significant inhibitory effects on tumor growth . StudiesThe recognition of the TIME predominant role in mediating the effectiveness of chemotherapies and immunotherapies has propelled the development of new strategies to overcome tumor resistance by turning immunologically cold tumors into hot ones. NPs have greatly helped this purpose as nanocarriers of chemical and biological factors, improving their targeted delivery, accumulation, and retention at the tumor site. However, it is a long way to demonstrate the clinical reliability of novel NP-based therapeutic approaches to regulate the immune processes within the TIME. With the support of recent nanotechnologies, the immunotherapy field might aim to identify new applications of pre-validated drugs and their combinations. For example, marketed drugs called statins primarily used against hypercholesterolemia were re-purposed to treat inflammatory and immune-mediated conditions when loaded on NPs that reduced their marked myotoxicity . SimilarGG and GA: conceptualization. GG: drafting. GA and AP: editing and revision. AP: figure. GG and GA: figure revision. All authors contributed to the article and approved the submitted version.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.


Triticum aestivum L.) phase  of a long-term pulse intensification field trial in the semi-arid region of the Canadian Prairies. Our results revealed pulse frequency had a minimal impact on microbial α-diversity, but caused a significant shift in the composition of the fungal (rhizosphere and bulk soil) and bacterial (bulk soil) communities. This effect was the most pronounced in the Ascomycete and Bacteroidete communities. Increasing pulse frequency also promoted a higher proportion of fungal pathotrophs in the bulk soil, particularly those putatively identified as plant pathogens. The network analysis revealed that rotations with higher pulse frequency promoted increased competition within the soil microbial networks in the rhizosphere and bulk soil. However, we also detected more negative interactions among the dominant pathotrophic taxa with increased pulse frequency, suggesting higher soil-borne disease potential. These findings highlight the potential drawbacks and reduced sustainability of increasing pulse frequency in crop rotations in semiarid environments.Including pulse crops in cereal-based cropping systems has become a widely accepted and useful agronomic practice to increase crop diversification and biologically fixed nitrogen in agroecosystems. However, there is a lack of knowledge regarding how the intensification of pulses in crop rotations influence soil microbial communities. In this study, we used an amplicon sequencing approach to examine the bulk and rhizosphere soil bacterial and fungal communities from the wheat ( Among agronomic practices used in annual cropping systems, crop rotation is one of the most common management tools used to enhance soil nutrient and water availability, control weeds and pests, and improve the ecological and economic sustainability of cropping systems . ConsidePisum sativum L.), lentil (Lens culinaris Medik.), and chickpea (Cicer arietinum L.), which are common grain legume crops that have been used to diversify cereal and oilseed cropping systems  has shown that pulse frequency in annual crop rotations is an important factor driving shifts in the composition, diversity and functional guilds of soil and root-associated microbial communities . There iIn this study, we used a long term (8 years) crop rotation study in the semiarid region of the Canadian Prairies to explore the impacts of pulse frequency on the diversity, composition and interactions of soil microbial communities. We hypothesized that increasing the pulse frequency in crop rotations will alter the diversity, composition, and network interactions of soil microbial communities.Triticum aestivum L.).A long-term field experiment was established in 2010 with a 4 years crop rotation design located at the Swift Current Research and Development Centre was utilized for this study. The experiment included low (one-pulse phase), medium (two-pulse phases) and high (three-pulse phases) pulse frequency rotations in the long-term rotation design. For this study we selected the following treatments: (i) low pulse frequency rotations, Pea-Wheat-Wheat-Wheat (PWWW) and Chickpea-Wheat-Wheat-Wheat (CWWW); (ii) medium pulse frequency rotations, Pea-Wheat-Pea-Wheat (PWPW) and Chickpea-Wheat-Chickpea-Wheat (CWCW); and (iii) high pulse frequency rotations, Pea-Pea-Pea-Wheat (PPPW), Chickpea-Chickpea-Chickpea-Wheat (CCCW) and Pea-Lentil-Chickpea-Wheat (PLCW) rotations. The first cycle of the rotations was completed in 2013 and repeated again starting in 2014. Due to the development of serious root diseases issues associated with growing chickpea in the same plots for three continuous years during the first cycle, mustard was grown during the second year of the CCCW rotation during the second cycle  to limit disease issues. Each treatment was planted into a 4 × 12 m plot and replicated four times in a randomized complete block design. This study was conducted during the 2017 growing season, which was the fourth year of the rotations during the second cycle and all rotations were seeded to wheat  annually. All plots were under no-till management practices and were planted with a no-till plot seeder.All plots received glyphosate spray at 0.45 kg/ha as a pre-seeding burn-off treatment and Trifluralin 10G at 8.5 kg/ha prior to seeding (except for wheat plots) to eliminate weed effects in each year. At the beginning of each rotation cycle (2010 and 2014) all wheat plots received a total of 80 Kg/ha actual N using 46-0-0 mineral fertilizer, and pulse crop plots received 22 kg/ha of actual P using 11-51-0 mineral fertilizer. All pulse plots also received inoculation prior to seeding with a granular rhizobium inoculant  were randomly collected in each plot to a depth of 15 cm, pooled together and homogenized in a large Ziploc bag to form one composite soil sample per plot. At the same time, four wheat plants were carefully excavated from the same sampling locations as the bulk soil cores in each plot. The loose soil was shaken from the roots and the remaining soil adhering to the roots was carefully brushed off, pooled together and homogenized as one rhizosphere soil sample per plot. There were a total of 28 bulk soil and 28 rhizosphere soil samples collected for this study. Sub-samples were immediately taken from the composite bulk and rhizosphere soil samples and flash frozen in a liquid nitrogen cryo-shipper for molecular analyses. The remaining soil was stored in a cooler with ice for transportation and then stored at 4°C for up to 48 h until further processing. Soil samples were sieved through a 2 mm sieve, and a subsample was used to determine soil moisture content (gravimetric). The remaining soil was air-dried and ground for chemical analyses.For all soil samples, soil organic carbon and total carbon were determined using the dry combustion method (after acidification with HCl) using an Elementar vario MICRO cube elemental analyzer . Soil niPRJNA694704, PRJNA694839, PRJNA694691, PRJNA694475, PRJNA717779, and PRJNA694448. Raw reads were processed  using the UPARSE pipeline of USEARCH v.9 (Genomic DNA was extracted from 0.25 g of soil in duplicate for each bulk and rhizosphere soil sample using the DNeasy PowerSoil kit (Qiagen) using the QIAcube automated nucleic acid extraction system (Qiagen). DNA was quantified using a Qubit dsDNA BR Assay Kit  and shipped on dry ice to the Genome Quebec Innovation Center  for amplicon library preparation and Illumina MiSeq sequencing. The bacterial 16S rRNA genes were sequenced using primers 515-F and 806-R  and fungARCH v.9 . SequencARCH v.9  and 16S ARCH v.9 . FUNGuilARCH v.9 . See BaiPost hoc comparisons of means were completed using Tukey’s HSD procedure at a 5% level of probability. Permutational multivariate analysis of variance (permanova) was used to test the effect of pulse frequency on the wheat rhizosphere and bulk soil microbial communities. Sequence read variability was normalized among samples by randomly resampling to the lowest number of reads for each dataset . To eliminate influences of low abundance OTUs which are in many cases artifacts generated through the sequencing process and better compare treatment effects, unclassified OTUs and rare OTUs  were removed from downstream analysis. Network analysis was constructed for soil microbial communities related to different pulse frequencies based on their OTU relative abundances, and was performed using the Molecular Ecological Network Analyses (MENA) pipeline: http://ieg4.rccc.ou.edu/mena  vegan, breakaway, ecodist and ggplot2 . To determine the effect of pulse frequency on α-diversity we estimated the total OTU richness using the package breakaway . One-wayedu/mena  and visuedu/mena . OTUs wiPulse frequency in the rotations had a minimal impact on the bulk and rhizosphere soil properties. Total C and organic C in rhizosphere soil was significantly higher in rotations with medium pulse frequency compared to rotations with low and high pulse frequency, and soil moisture was highest in rotations with low pulse frequency . In the Pulse frequency did not significantly affect the α–diversity  of the bacterial community in the rhizosphere or bulk soil . HoweverThe permanova indicated that pulse frequency had a significant effect on the fungal community in both the rhizosphere and bulk soil . This apThe network analysis revealed that increasing the pulse frequency in crop rotations modified the interactions of the soil microbial community . The topIncreasing pulse frequency also increased phylogenetic diversity of dominant nodes in the rhizosphere , especiaIn the bulk soil, high pulse frequency led to an increased total phylogenetic diversity of dominant nodes along with a shift in composition compared with low and medium pulse frequency rotations . For exaModule-EigenGene network analysis grouped soil microorganisms into different modules based on their interactions with environmental factors . In bothIn the rhizosphere, S, pH and EC had the strongest influence on the soil microbial community . This waIn the bulk soil, S, N, and K had the strongest influence on the soil microbial community in low pulse frequency rotations , while NTo evaluate the topological role of identified nodes in the network, we used within-module connectivity (Zi) and among-module connectivity (Pi) values to separate each network into four ecological modules . From thDespite the important role that crop rotations play in sustainable agriculture , there iSoil bacterial diversity did not significantly change in response to increased pulse frequency in the crop rotations. Similar results were observed during the first rotation cycle at the same field trial and phase of the rotation , but theFusarium sp. and Alternaria sp.  can be found below: NCBI BioProject; LB and BE designed and were responsible for conducting the field and lab work. TY and LB conducted the data analysis and wrote the manuscript. All authors contributed to editing the manuscript and approved the final version.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.


Two clinical trials have shown that adeno-associated virus (AAV) delivery of bNAbs is safe and capable of sustained bNAb expression for over 18 months following a single intramuscular administration. Here, we review key concepts of effector functions mediated by bNAbs against HIV infection and the potential for vectored immunoprophylaxis as a means of producing bNAbs in patients.HIV-1 broadly neutralizing antibodies (bNAbs) targeting the viral envelope have shown significant promise in both HIV prevention and viral clearance, including pivotal results against sensitive strains in the recent Antibody Mediated Prevention (AMP) trial. Studies of bNAb passive transfer in infected patients have demonstrated transient reduction of viral load at high concentrations that rebounds as bNAb is cleared from circulation. While neutralization is a crucial component of therapeutic efficacy, numerous studies have demonstrated that bNAbs can also mediate effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent complement deposition (ADCD). These functions have been shown to contribute towards protection in several models of HIV acquisition and in viral clearance during chronic infection, however the role of target epitope in facilitating these functions, as well as the contribution of individual innate functions in protection and viral clearance remain areas of active investigation. Despite their potential, the transient nature of antibody passive transfer limits the widespread use of bNAbs. To overcome this, we and others have demonstrated vectored antibody delivery capable of yielding long-lasting expression of bNAbs  Among teservoir . Broadlyeservoir –8. Numereservoir –11.In addition to direct neutralization of virus entry, antibodies are capable of mediating non-neutralizing functions that are important in the context of HIV prevention and viral clearance, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent complement deposition (ADCD), through engagement of the Fragment crystallizable (Fc) region with various Fc receptors expressed on the surface of innate cells or complement proteins in the circulation –16. TherRecent results from the AMP study, in which patients were passively transferred with VRC01 to prevent HIV acquisition, has shown that transmission risk is increased as antibody concentrations fall  Clinica. To maxiHumanized mice are a valuable model in which to test both prophylactic and therapeutic interventions for HIV. These mice are the product of genetic engineering to express human genes or xenografting of immunocompromised mice with stem human cells and tissues. Humanized mouse models have shown long-lasting expression of various HIV-1 bNAbs delivered through VIP resulting in protection from viral challenge , 22. Giv50 and IC80, respectively) were also strong predictors of protection. Other work has shown that the protective concentration of an antibody in vivo is often 50-200 times greater than the in vitro IC50 value calculated in an in vitro neutralization assay suggesting that the more potent an antibody is, the lower the concentration required to protect  which can engage with the Fc-region of antibodies to drive Fc-mediated effector functions –41. FourThe role of Fc-mediated effector functions in HIV prevention in patients has been suggested by the analysis of the partially successful RV144 vaccine trial in Thailand, in which patients were administered a heterologous prime-boost vaccine regimen . AnalysiIn addition to HIV prevention, polyfunctional antibodies have also been found to be associated with HIV control . SimilarUnderstanding the role effector functions play in both prevention and control in chronic infection has largely focused on non-neutralizing antibodies elicited by patients. Although the vaccine regimen administered in the RV144 trial showed promising results, when an analogous regimen was given in South Africa in the HVTN02 trial it failed to achieve statistically significant protection, demonstrating the need for continued development of alternative strategies. Additionally, a study by Dugast et al.  demonstrA number of groups have independently performed assays designed to measure the protective and therapeutic efficacy of bNAbs across a wide-range of animal models . InteresIn addition to prevention, Fc-mediated effector functions of bNAbs have also been investigated in the context of driving viral clearance of established HIV infection. Recently, Asokan et al. measured the contribution of bNAb-mediated effector function in chronically infected rhesus macaques. SHIV-infected monkeys were treated with a CD4bs-directed bNAb, VRC07-LS, or variants harboring Fc mutations that either enhanced or diminished engagement with FcγRs. Using this panel of mutant antibodies, they determined that innate effector functions, such as ADCC, ADCP and complement fixation contributed approximately 21% of the observed rate of viral clearance . Interes-/-Il2gnullRag1 (NRG) mice given the CD4-binding site targeting bNAb 3BNC117 and challenged with HIV were found to have enhanced viral clearance 24 hours-post challenge compared to the control mice given a non-specific antibody  against diverse viruses that span various clades , 64. Intin vivo stability of hinge variants of IgG1 were measured and the rate of plasma decay in mice was similar to that of the wild-type, suggesting that such alterations may be viable approaches to harness Fc mediated effector function by bNAbs.Other avenues of investigation have looked to optimize bNAbs to elicit more potent Fc effector function. One potential approach has been the use of bNAbs expressed as different isotypes that have enhanced effector function, such as IgG3. Recent work by Richardson et al.  comparedIn addition to understanding how hinge length, isotype and subclass contribute to optimal Fc effector function, considerable effort has identified point mutations in the Fc region capable of enhancing FcγR engagement and downstream function . In 2001Given the potential for bNAb-mediated protection in HIV prevention and viral clearance, the question of implementation has come to the forefront. The AMP clinical trials utilized passive transfer of VRC01, a CD4bs bNAb, to measure HIV prevention in different populations, including HIV-uninfected men who have sex with men (MSM), transgender men who have sex with men, and sexually active women in sub-Saharan Africa , 84–86 Crep and cap gene flanked by two inverted terminal repeats (ITRs)  and membrane-associated accessory protein (MAAP) in alternative reading frames  . The repg frames , 92–97. g frames . Currentg frames . During g frames –102. Theg frames  As a cong frames . In placg frames . Followig frames , 106.-/- mice  vector that expressed SIV-specific immunoadhesins composed of recombinant antibody fragments . They adr region . These cr region . This syr region . NHP stur region , 112. AsIn one study, AAV was used to deliver PG9, a V1V2-directed bNAb, chosen for its potent neutralization capacity  . This clin vivo. This trial represents the first demonstration of long-lived systemic production of a broadly neutralizing antibody in humans, providing strong evidence for the potential of vectored antibody delivery. Despite this success, some participants developed anti-drug antibodies against the VRC07 antibody variable regions, leading to loss of antibody expression. As such, additional improvements to the AAV transgene may be necessary to enable widespread use of VIP for HIV prevention. Looking forward, this system could be utilized for delivery of optimized bNAbs that contain altered Fc-regions with hinge length changes or even different isotypes to improve FcγR binding. The use of such optimized transgenes has the potential to reduce the concentrations of bNAbs necessary to prevent HIV acquisition.Independently, a second on-going clinical trial by the NIH is testing the safety and efficacy of VRC07 delivery through an rAAV8 viral vector to HIV-infected adults aged 18-60 . The vector design used in this study was analogous to those described in previous papers from our laboratory and include the CASI promoter and optimized F2A-containing VRC07 transgene (in vivo studies measured the contribution of these effector functions in protection, but questions remain as to contributions of individual functions, such as ADCC, to mediating protection. Future work could help to identify which effector functions contribute most to bNAb-mediated protection, and armed with this improved understanding, specific Fc mutations or hinge length modifications could be made to the antibody to improve therapeutic outcomes. These are especially relevant in the context of vectored antibody delivery, which represents a promising approach capable of integrating advances made in our understanding of antibody-mediated innate immune function into future clinical products. However, transduction efficiency is relatively low as compared to pre-clinical models and there appears to be a significant proportion of recipients who mount an immune response against the antibody transgene. Future studies will be needed to improve the consistency of antibody delivery using this system, including optimizing the vector to improve antibody concentrations and minimizing anti-drug antibodies elicited in response to existing rAAV vectors.Broadly neutralizing antibodies have been shown to be promising candidates for HIV prevention, therapy and possibly cure. Passive administration of bNAbs have demonstrated that antibody concentration and potency are important parameters correlated with protection against HIV acquisition. Other non-neutralizing functions, including ADCC, ADCP and ADCD have also been shown to be critical in this context. As a result, there is growing interest in understanding whether there are optimal viral epitopes that antibodies can target to effectively elicit protective responses. Early findings from the RV144 vaccine study suggested that non-neutralizing antibodies targeting the V2 site were implicated in preventing disease acquisition. However, when bNAbs targeting different sites of vulnerability were tested for their ability to mediate these same non-neutralizing functions, results were variable and largely dependent upon the challenge virus. Further work is needed to understand if there is an optimal site that can facilitate these functions across a wide range of diverse strains. Additionally, several MP and AB conceived of and wrote the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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