This approach ensures high efficacy and cell specificity fordifferent anti-cancer agents, as they are delivered into the most vulnerablesubcellular compartment within the cells of interest and makes it possible forantibody mimetics to penetrate into a compartment of interest within the targetcells (“diving antibodies”). Furthermore, polyplexes, complexes ofpolycationic block copolymers of DNA, have been developed and characterized.These complexes are efficient both  The cell nucleus, where the main program of cell function is stored, is anatural target for many biologically active substances. These substances can bedivided into two large groups . ThefirPhotosensitizers (PSs) and radionuclides emitting particles with a short pathlength  or Auger electrons (AEs))exemplify agents from the first group. Both of them are cytotoxic agents thatare widely used in medical practice to treat cancer, but they are not limitedto this type of diseases. The cell nucleus is extremely sensitive to thecytotoxic agent of PSs, reactive oxygen species  . As for is little doubt that DNA needs to be delivered into the cell nucleus if itsexpression is to be achieved. The second group of biologically activesubstances also includes regulatory polypeptides whose effect mani fests uponinteraction with the macromolecules of the cell nucleus.Therefore, for the purposes of translational medicine, biologically activeagents must be delivered to the nuclei of target cells so that their propertiescan be deployed, since most of these agents cannot reach the nuclei bythemselves.key for delivering macromolecules or other biologically active substances  is to usenatural intracellular transport processes, such as receptor-mediatedendocytosis or nucleocytoplasmic transport . AccFig. 1)consists of a ligandmodule, an endosomolytic module, a nuclear localization module, and a carriermodule. The ligand module ensures interaction with the internalizable surfacereceptor and, therefore, recognition of the target cell and transport of anagent inside this cell via receptor-mediated endocytosis. The endosomolyticmodule has the function of pH-dependent pore formation in the endosomes, andthus it ensures release of the MNT, with the active component being deliveredfrom these compartments with weakly acidic contents to the target cell cytosol.The nuclear localization module contains an amino acid sequence that acts as anuclear localization signal (NLS) specifically interacting with the importincomplex in the cytosol and ensuring transport of the agent through the nuclearpore. The carrier module is used to join other modules into an integral wholeand attach the “cargo.” Along with the aforementioned four modules,the MNT can also contain other modules if interaction with some additionalintra- and extracellular components is required. Thus far, the properties ofthe following MNTs are the ones that have been studied most thoroughly bothin vitro and in vivo:The modular nanotransporters (MNTs) being developed in our laboratory meetthese criteria and can be regarded as a technological platform for the deliveryof therapeutic agents to a given compartment of target cells of the desiredtype , 7-16. TEGF), which exhibit specificity with respect to cells  as a ligand module ovelcancer , 19, 20,MSH), which exhibit specificity with respect to cells (Anexample: melanoma cells . The measured constants of theaffinity of MNTs to importin dimers were close to that of a free naturalpolypeptide carrying the same NLS. Hence, it was demonstrated that all themodules within a chimeric artificial MNT molecule had retained their functions.This conclusion was convincingly confirmed by tests performed to evaluate theperformance of the MNT modules of all developed types. To save space, let usjust provide few examples. Thus, the dissociation constant  by measuFig. 2), as was actuallyplanned by the authors in order to solve the problem related to“cargo” delivery into the nucleus.Therefore, all the full-length MNTs penetrated the target cells viareceptor-mediated endocytosis (as confirmed by the fact that the specificligands of the respective receptors inhibited their penetration) and localizedwithin their cell nuclei , 27, 28e6 and bacteriochlorinp attached to MNTEGF or MNTMSH arehundreds and thousands of time more cytotoxic than the free ones. Thus, in theexperiments on A431 cells overexpressing EGFR, the half-maximal effectiveconcentration (EC50) of MNTEGF-chlorine6 was 0.53 nM, while EC50 of free chlorine6 was 1780 nM . In othMSH [Qualitatively similar results were obtained in the experiments withMNTMSH , where iin vivo experimentson tumor-carrying mice that PSs are efficiently delivered to the nuclei ofcancer cells using MNTs [EGF and MNTMSH injected intravenouslyto tumor-carrying mice revealed that MNTs preferentially accumulate in cancercells . The experimentinvolving intravenous injection of MNTMSH for the treatment ofexperimentally induced melanomas showed that photosensitizer bacteriochlorinp delivered by MNTMSH inhibits B16-F1 tumor growth85–89% more efficiently compared to free bacteriochlorinp; the inhibition of the growth of Cloudman S90 melanoma was93% more efficient. The ratio between the PS concentrations in the tumor and inthe skin was as high as 9.8, some 4.5 times higher than that observed for freebacteriochlorin p [EGF with photosensitizer chlorin e6in a model of A431 human epidermoid carcinoma grafted into immunodeficientmice: 75% of the mice survived by day 92, while only 20% of the mice treatedwith free chlorin e6 (positive control) and none of theuntreated animals survived by day 23.It has been convincingly demonstrated in ing MNTs , 31. An hlorin p .A signiAPs and AEs cause dense ionization and thus efficiently damage the moleculesalong their tracks; the path length of these particles in tissues is rathershort: 50–100 µm  for APs and severaldozens or hundreds nanometers for AEs . These features of emitters of APs and AEs arerather attractive owing to the fact that in the case when they are selectivelydelivered to the target cells, one can expect the damage to the surroundingnormal cells to be minimal. Meanwhile, both types of radiation cause multipledouble-strand DNA breaks that are hardly repairable. In fact, nuclear DNA isthe main target of the cytotoxic activity of these radiation types , 34. TheEGF was 8- to 18-fold higher thanthat of 211At not delivered to the nuclei of these cells [The α-particle emitter 211At, which has been used as a source of APs inexperiments with MNTs, is considered one of the most promising radionuclidesfor therapeutic purposes . The APse cells . It alsoThe following emitters of AEs, which are widely used in medicine as sources ofgamma radiation, were employed in the experiments with MNTs: 125I, 67Ga, and111In. On average, they emit 24.9, 4.7, and 14.7 AEs per decay, respectively. The yie125I or 67Ga delivered by MNTEGF accumulatedrather intensively in the nuclei of A431 human epidermoid carcinoma cells[EGF was 3,500 times more cytotoxic to A431 cells than the125I-iodinated control polypeptide, which had not penetrated the cells [ma cells, 28: by he cells . Similarhe cells : the cythe cells .Safety testing of MNTs during preclinical studies conducted at the NationalMedical Research Radiological Center of the Ministry of Health of the RussianFederation showed that the studied MNTs injected intratumorally exhibited avery low toxicity (both acute and chronic) in mice and rats, lowimmunogenicity/ allergenicity in mice and guinea pigs, and were not pyrogenicin rabbits , 44, 45.111In-MNTEGF administered as a single dose into humanbladder carcinoma (EJ) grafted subcutaneously to immunodeficient Balb/cnu/nu mice was retained inside the tumor for a rather longtime  [EGFexhibited a pronounced dose-dependent therapeutic effect on EJ tumors (up to90% compared to the untreated control (both non-labeled MNTEGF andfree 111In) at the same dose) [Fig. 3)..5 days) ; no moreme dose) Another variant of MNTs, 111In-MNTF, exhibited a similar therapeutic effect, 24. 111The results obtained using different cytotoxic agents  have motivated researchers to view MNTs asprospective agents for the delivery of a much wider range of biologicallyactive molecules. In this sense, bioactive polypeptides are particularlyattractive because MNTs are actually chimeric polypeptides and inclusion ofadditional polypeptide fragments into their composition is a problem that canbe solved using genetic engineering methods.EGF, is oneof the variants of such MNTs. The p21 protein exhibits a broad range ofactivities: it affects DNA repair and controls the DNA replication fork byforming a complex with the PCNA protein, and it regulates the cell cycle byinteracting with cyclins and cyclin-dependent kinases [EGF that contained theC-terminal fragment of protein p21 (amino acid residues 87–164), with thesite through which p21 binds to PCNA, was synthesized based on these startingpoints [EGF statistically significantly inhibits DNA repair comparedto the control, MNTEGF  [The MNT carrying a fragment of the p21 protein, p21-MNT kinases . This magpoints . DNA wasgpoints . The comragment) .tert-butylhydroperoxide. It was shown for the mouse model of oxidative stress induced byhepatotoxin acetaminophen that the preliminary administration of MNTs withanti-Keap1 monobody activating the Nrf2/ARE signaling pathway inhibits thehepatotoxic action of the acetaminophen that is detected according to elevatedserum aspartate aminotransferase and alanine aminotransferase activity [in vitro and invivo.The encouraging results of this study have contributed to further progresstowards the targeted intracellular delivery of biologically activepolypeptides. MNTs carrying an antibody mimetic anti-Keap1 monobody  as the effector part weredesigned under the Russian Science Foundation Grant No. 17-14-01304 . The traactivity . These rFor decades, the potential opportunity to change the function of cells bymodifying their genetic program has been stimulating researchers who focus ongene therapy  orbioengineering for the production of the target macromolecules, etc. As oftenhappens when trying to solve such important problems consisting of severallarge tasks, finding the optimal solution to one of them is far from obvious.Targeted delivery of genetic material is one such task. A natural solution tothis problem could involve viruses, as they are the supramolecular structuresbest suited for overcoming the barriers at the organism and cellular levelsduring targeted delivery of virus’s own genetic material. The largestnumber of gene therapy preclinical and clinical studies has centered on theseviruses. However, viral vectors are associated with a risk of unexpected, andoften severe, adverse events, which will actually remain a problem for quite along time , 55. TheOne of the variants of non-viral delivery is to use polycations, which formcomplexes with the nucleic acids known as polyplexes. In polyplexes, DNA (orRNA) is packaged and protected against hydrolytic enzymes, so that thesecomplexes remain sufficiently stable in biological environments. Polyplexes arenon-pathogenic. Many of them are also non-immunogenic and low-toxic. Bymodifying the original polymers, one can obtain particles with differentproperties, as well as attach different functional components to impart suchproperties as cellular specificity or other tailored properties to thecomplexes .It is obvious that in order to achieve these favorable properties, thepolymeric vehicles within the polyplexes need to be supplemented with theaforementioned functional components. Furthermore, the polymer composition alsoneeds to be optimized to bring the properties of the polyplexes closer to thoseof virions capable of delivering genetic material.Let us consider the example of the well-known polyethyleneimine (PEI)-basedpolyplexes. Particles of different sizes and charges form when PEI is mixedwith DNA in different proportions . Toincrease the time during which the polyplexes circulate in the blood and toreduce the toxicity of PEI, PEG is attached to PEI, yielding PEG-PEI blockcopolymers. Since both the N/P and PEG/PEI ratios can be variegated, theresulting problem to be solved involves finding the optimal ratio between thecomponents in the polyplex. To solve this problem, polyplex variants withdifferent ratios between the components were tested on 11 cell lines;transfection efficiency was assessed according to the activity of the expressedreporter gene . It was in vivo transfection ofthese cancer cells [Modifying block copolymers with ligands specific to internalizable receptors onthe target cells impart cellular specificity to the polyplexes. Thus, thepolyplexes containing αMSH acquired specificity with respect to melanomacells overexpressing melanocortin 1 receptors (αMSH is their ligand) andshowed a much greater efficiency in er cells .Fig. 4A).Therefore, if polyplexes need to be delivered into a tumor to agreater depth, there should be some additional impact on the tumor. One of thevariants allowing one to increase both the penetration depth of polyplexes andtheir concentration in the tumor is to modify the tumor interstitium  [Fig. 4B).The size of PEI-based polyplexes ensuring the most efficient transfection casts doubt on whether nanoparticles ofthis size can penetrate through nuclear pores into the nucleus of anon-dividing cell, because the known size limit is ap proximately 40 nm evenfor NLS-carrying particles . Theexp type I) , 64 Regarding the delivery of cytotoxic agents using modular nanotransportersfor cancer therapy: Modular nanotransporters  havebeen developed. This technology makes it possible to impart cellularspecificity and high efficiency to a large number of antitumor agents bydelivering them to the cell nucleus using the natural processes ofintracellular transport.in vitro and in vivo has been designed.We believe that the approach being currently developed can lead to abreakthrough in the design of tools for the study of the function of livingcells and, possibly, in the development of therapeutic agents.(B) Regarding the delivery of biologically active polypeptides: Modularnanotransporters have been used to design antibody mimetics  capable of penetrating living cells andaffecting the function of target molecules; furthermore, a new type of modularnanotransporters that affect the functions of transcription factors in cellsboth in vitro and in vivo.(C) Regarding the delivery of genetic material using polyplexes: It has beendemonstrated that polyplexes preferentially transfect dividing cells, whichshould be taken into account during the potential practical use of polyplexes.The efficiency of transfection using polyplexes has been demonstrated bothThe hope is that the range of biologically active agents delivered into thecell  will be subsequently broadened: novel“diving antibodies” could be designed, and humanized MNTs forpotential systemic use could be obtained. These studies have already started, 69.


Celiac disease is an autoimmune disorder characterized by a heightened immune response to gluten proteins in the diet, leading to gastrointestinal symptoms and mucosal damage localized to the small intestine. Despite its prevalence, the only treatment currently available for celiac disease is complete avoidance of gluten proteins in the diet. Ongoing clinical trials have focused on targeting the immune response or gluten proteins through methods such as immunosuppression, enhanced protein degradation and protein sequestration. Recent studies suggest that polyphenols may elicit protective effects within the celiac disease milieu by disrupting the enzymatic hydrolysis of gluten proteins, sequestering gluten proteins from recognition by critical receptors in pathogenesis and exerting anti-inflammatory effects on the system as a whole. This review highlights mechanisms by which polyphenols can protect against celiac disease, takes a critical look at recent works and outlines future applications for this potential treatment method. Gluten, a protein found in wheat, barley and rye, is the antigenic trigger for celiac disease, an autoimmune enteropathy localized in the small intestine. Initial symptoms of celiac disease are gastrointestinal discomfort as well as increased permeability and inflammation of the small bowel. Long-term exposure to gluten can cause extensive damage of the small intestine, leading to impaired nutrient absorption and chronic conditions related to malnutrition . Celiac Despite heightened public awareness and understanding of celiac disease, the only reliable treatment strategy is eliminating gluten from the diet altogether, which can be both inconvenient and a financial burden . A variePolyphenols are a structurally diverse class of secondary plant metabolites that have been shown to have both beneficial and detrimental effects on human health. As potent antioxidant and anti-inflammatory agents, polyphenols have been investigated as natural therapeutics for chronic inflammatory diseases such as allergies and inflammatory bowel disease (IBD); however, they also can elicit anti-nutritional effects upon consumption by humans and animals alike due to their ability to inhibit digestive enzymes and interact with other dietary compounds, including proteins ,15. InteRecent studies suggest that the anti-nutritional characteristics of polyphenols including flavonoids and procyanidins can be used to physically sequester gliadin proteins, disrupting key steps in disease pathogenesis in vitro. This potential treatment strategy features a variety of advantages including ease of incorporation into the diet, developed understanding of toxicity risks and a large body of work supporting supplementation of these compounds to treat other ailments. The objective of this review is to discuss gliadin sequestration via polyphenol interaction as a therapeutic strategy for managing celiac disease as well as potential mechanisms and future applications of polyphenols as bioactive therapeutic agents.Triticeae tribe of Poaceae cereal grasses including food grains such as wheat, barley and rye [Gluten is a protein found primarily in grains of the  and rye . It is h and rye .Prolamins are storage proteins which are soluble in organic solvents and are found in a variety of cereal seeds. They make up approximately half of the total protein found in mature grains, with the exception of oats and rice ,24. WhilThe sulfurous component of prolamins is cysteine. Cysteine frequency in the S-rich class of prolamins is 2–3% compared to 30–40% glutamine and 15–20% proline , but it Gliadins, rather than glutenins, have been implicated as being responsible for the onset of the inflammatory and immune responses in celiac disease pathogenesis, though repeat motifs conserved in both subunits of gluten are recognized as immunostimulatory epitopes . In partCeliac disease manifests in the small intestine, which is the site of gluten protein digestion and absorption in the gastrointestinal tract. As luminal and brush border enzymes are secreted, digestive enzymes hydrolyze gluten proteins into free amino acids and di- and tripeptides by targeting specific cleavage sites dependent on the enzyme . DigestiOne mechanism of gliadin transport across the brush border is protected transcytosis due to abnormal expression of CD71 in the enterocytes of individuals with celiac disease. CD71 has been shown to protect gliadin from lysosomal degradation during transcytosis upon complexation with IgA, blocking the further degradation of immunogenic peptide fragments and allowing them to enter the lamina propria intact. This has been shown to occur in active cases of celiac disease where patients demonstrate elevated luminal anti-gliadin IgA as a response to exposure, as opposed to individuals adhering to a gluten-free diet . ParacelThough dysregulated expression of IL-15 has been implicated in many inflammatory autoimmune diseases , it is cZonulin proteins, which are MyD88 dependent and produced by IECs , can triDisassembly of TJs and AJs occurs via vesicular transport of proteins from the cell membrane to alternative locations within the cell, or by rearrangement of the actin cytoskeleton. Gliadin-stimulated zonulin release has been shown to modify junctional properties of IEC lines IEC-6 and Caco-2, altering the localization of TJ proteins claudin-3 and -4 and causing the polymerization of actin filament ,46. ExpoThe adaptive immune response associated with celiac disease is largely based on the interaction between gliadin peptides and an endogenous enzyme called transglutaminase 2 (TG2). Early studies implicating TG2 in celiac disease pathogenesis showed that jejunal tissue samples taken from individuals with treated and untreated celiac disease demonstrated increased TG2 activity compared to healthy controls . While t+ cells follow the Th1/Th0 pathways and release anti-inflammatory cytokine IL-10 and pro-inflammatory cytokine interferon (IFN)-γ, which activates signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1) [+ cytotoxic T cells, which are stimulated by the release of cytokines from the CD4+ T cells [+ T cells further propagate the immune response by activating effector B cells, or plasma cells, which produce anti-gliadin and anti-TG2 IgA and IgG antibodies. In contrast to most other instances of T cell-dependent activation of B cells, B cell activation in celiac disease does not result in the production of memory B cells. As a result, gliadin and TG2 antibodies disappear from circulation after approximately one month of following a gluten-free diet [The adaptive immune response associated with celiac disease is initiated by MHC class II molecules on the surface of APCs in the lamina propria. These MHC class II molecules are able to bind to deamidated gliadin as well as gliadin–TG2 complexes, presenting each as antigens to naïve T cells. MHC class II molecules have been found to preferentially bind to the main chain of peptide ligands through hydrogen bonding ,54. Pres1 (IRF1) . IL-10 i1 (IRF1) , and IFN1 (IRF1) ,58. Muco T cells . CD4+ T ree diet .Presentation of symptoms of celiac disease function on a gradient of exposure. For some individuals with celiac disease, as little as 50 mg of gluten per day is enough to elicit a physiological response  in the bThe most telling diagnostic feature of celiac disease is the degradation of small intestinal mucosal architecture including surface damage to enterocytes, infiltration of IELs and the blunting of villous structure, which results in the loss of small intestinal surface area . As celiAs of 2021, the only reliable method for avoiding the symptoms and intestinal damage associated with celiac disease is adherence to a gluten-free diet. Despite the inconvenience and financial cost of a gluten-free diet , it has The downsides of the gluten-free diet include increased food costs and nutritional implications . One stuMany strategies have been explored to help individuals with celiac disease maintain normal diets without the restrictive nature of the gluten-free diet. These strategies have come in the form of both synthetic and naturally-derived options, and target multiple stages of celiac disease pathogenesis by interacting directly with either the body or with gluten proteins. Celiac disease pathophysiology is based on an overreactive immune response upon exposure to gluten proteins. The sensitization of the digestive system to gluten, initiation of intestinal permeability and upregulation of immune signaling observed in celiac disease are all therapeutic targets which have been explored in clinical trials over the last two decades. + T cells in individuals with the HLA-DQ2 haplotype that is administered subcutaneously on a weekly basis [Gluten sensitization and tolerance development has been explored with Nexvax2, a vaccine comprising peptides recognized by CD4ly basis . While Ply basis  and decrly basis . Howeverly basis , and itsly basis .The disruption of the gut barrier is a primary step in celiac disease pathogenesis. Enhancement of barrier function and prevention of gut permeability have both been explored as potential therapeutic options for celiac disease.Bifidobacterium longum CECT 7347 and Lactobacillus casei ATCC 9595 have been shown to improve gut barrier function, reduce inflammation and repair gliadin-mediated intestinal damage in gluten-sensitized mice [Probiotics have also been shown to improve barrier function in both in vitro and in vivo models of celiac disease. zed mice ,79, and zed mice .As perhaps the most promising pharmaceutical option for treating celiac disease, larazotide acetate also targets barrier function as a protective mechanism. Larazotide acetate is a synthetic 8-amino acid peptide that preserves gut barrier function by acting as a zonulin antagonist and preventing the disruption of tight junction proteins stimulated by gliadin. In vitro, larazotide acetate has been shown to prevent gliadin-mediated permeability in Caco-2 cells by preserving tight junction proteins, an effect which also prevented the paracellular transport of FITC-labeled gliadin . Based oBifidobacterium bifidum IATA-ES2 and Bifidobacterium longum ATCC 5707, which have been demonstrated to suppress IFN-γ secretion by peripheral blood mononuclear cells (PBMCs) in the presence of gliadin in vitro [Bifidobacterium infantis showed reduced serologic markers after three weeks of supplementation alongside a gluten-containing diet compared to the controls, and patients noted an improvement of gastrointestinal symptoms [Necator americanus, or hookworms, have been shown to reduce gluten-mediated IFN-γ prevent mucosal damage in celiac disease patients. In a small clinical trial of 12 adults with celiac disease, infection with N. americanus preserved intestinal histopathology upon gluten challenge as measured by Marsh scores and IEL counts per 100 enterocytes [Suppression of the immune system has been explored via the administration of both natural and synthetic agents. Natural immunosuppressants including probiotics such as in vitro ,86. Datasymptoms . Naturalerocytes .+ T cells in transgenic mice as well as improve villous architecture and reduce mucosal lesions [+ and CD8+ T cells that is involved with the recruitment and homing of those cells to the small intestine, which ultimately results in the destruction of the small intestinal mucosa during celiac disease [Immunosuppression has also been achieved in pre-clinical and clinical trials exploring direct antagonists to a mounted immune response. Injection with anti-IL-15 antibody TM-β1 has been shown to reverse abnormally high CD8 lesions . Similar lesions ,91. Thou disease . The eff disease .Once gliadins have passed the gut barrier and an inflammatory response is mounted, celiac disease pathogenesis proceeds by an adaptive immune response where gliadins, alone or bound to TG2, are presented as antigens. Inhibiting or otherwise impairing antigen presentation has been shown to be an effective strategy through several approaches. One approach focuses on impairing the processing of gliadin peptides required for antigen presentation. As previously discussed, deamidation of glutamine residues by TG2 greatly enhances the affinity of the peptide for the MHC class II binding pocket. One study found that incubation of gliadins with synthetic “blocking peptides” reduced TG2 activity in vitro by up to 36% . SynthesDirect inhibition of TG2 has been explored ex vivo with active-site TG2 inhibitor R283. Pre-treatment of tissue with R283 prevented gliadin-mediated T cell activation in duodenal biopsies, but the effect was diminished when gliadin had been deamidated prior to addition to the culture, suggesting that the prevention of deamidation was the critical factor at play . A similWhile many of the pathogenesis-focused therapies target events after passage of gliadin to the lamina propria, gliadin-focused therapies instead modify the immunological potential of gluten. Methods for detoxification of gliadin proteins include enzymatic supplementation to further hydrolyze immunostimulatory proteins or prevention of breakdown and recognition altogether through protein sequestration. Lactobacillus during sourdough bread fermentation to degrade gluten peptides further than typical processing procedures [Aspergillus niger prolyl endopeptidase (AN-PEP) and two proteases from Sphingomonas capsulate  have demonstrated prevention of histopathological changes upon gluten challenge in clinical trials [The incomplete digestion of gliadin in the lumen observed in individuals with celiac disease has been explored as a therapeutic target with varying levels of success. Despite the proteolytic efficacy of ocedures , no therocedures . Howeverl trials ,105. OthIn contrast to the method of assisting complete breakdown of gliadin, inhibition of luminal processing has also been shown to be an effective method for reducing gliadin immunogenicity. This has been demonstrated by both natural and synthetic agents.Neutralization of gliadin proteins has been achieved in vivo using an anti-gliadin IgY antibody (“AGY”) derived from egg yolk powder, which has the legal status of being “Generally Recognized as Safe”. Oral administration of this powder concurrently with gluten challenge decreased patient-reported symptoms, anti-TG2 antibodies and intestinal permeability as measured by lactulose-mannitol excretion ratios in a small-scale clinical trial. Complexation of gliadins by IgY is thought to prevent the celiac disease immune response by preventing absorption of the proteins into the bloodstream ,107.This sequestration effect has also been achieved by oral administration of a synthetic polymer of hydroxyethyl methacrylate-styrene sulfonate, known as poly(HEMA)-co-SS, or commercially as BL-7010 . BL-7010A research area that remains neglected in the treatment of celiac disease is the potential impact of dietary supplements and naturally-derived compounds on gluten digestion and processing. Of interest are polyphenols, which have been shown to have anti-inflammatory properties within the context of chronic diseases of the GI tract and anti-nutritional properties with respect to proteins.Polyphenols are a structurally diverse class of secondary metabolites produced by plants to aid non-growth processes such as defense against external stressors, hormone release and signaling within the plant during ripening . UbiquitPolyphenols are organic compounds composed of multiple phenol groups a. Some cPolyphenolic interaction with proteins has been well-characterized, particularly with respect to the role that these interactions play in food quality. The ability of polyphenols to contribute to important qualities such as color, flavor and mouthfeel require an understanding of their physicochemical interactions with other compounds in the food system being studied and with the consumer. Tea provides a simple examples for how this phenomenon can affect a product. When milk is added to tea, the tea polyphenols in solution can become bound by milk proteins, which alters polyphenol bioavailability and, thus, the nutritional value of both proteins and polyphenols to consumption .Protein–polyphenol interactions can occur via covalent or non-covalent mechanisms. Covalent interactions typically occur between functional groups on the proteins and phenolic oxidation products , which can occur naturally or during processing . Non-covProtein structure can be used to predict the characteristics of interaction between proteins and polyphenols. Structurally-defined proteins will often display specific binding pockets whereas non-specific interactions are observed with proteins that have only secondary structural motifs . In eithEpidemiological evidence suggests that polyphenols can beneficially impact human health, demonstrating anti-inflammatory, anti-carcinogenic and anti-obesity properties in vitro and in vivo. Often contributing to each of these is the antioxidative capacity of flavonoids. As a class of compounds, polyphenols have the ability to both scavenge free radicals and prevent radical formation, although antioxidative potential can vary between compounds and applications . EpigallThe bioactivity of flavonoids and other polyphenols is not limited to the neutralization or prevention of free radicals, or protection against oxidative damage. The role that flavonoids can play in cell signaling and anti-inflammatory compounds has been investigated in vitro and in vivo through their ability to induce or inhibit enzymes that play important roles in cell maintenance . FlavonoκB signaling [κB signaling by EGCG also influences the immune system in obese individuals, reducing the impact of the obesity-induced inflammatory state by promoting the proliferation of regulatory T cells and secretion of anti-inflammatory cytokine IL-10 [The anti-inflammatory capabilities of flavonoids have been investigated with respect to a variety of disease states. Obesity is characterized by chronic, low-grade inflammation, which plays a role in the development of other diseases such as type-2 diabetes and cardiovascular disease. EGCG has the ability to decrease tumor necrosis factor (TNF)-α signaling in vitro and in vivo. In the development of atherosclerosis, TNF-α induces endothelial cell secretion of monocyte chemoattractant protein (MCP)-1, which is associated with the development of atherosclerotic plaques. Pre-treatment of porcine endothelial cells with EGCG prevented TNF-α-induced MCP-1 upregulation . In liveignaling . The supne IL-10 .Despite their numerous health benefits, polyphenols are sometimes referred to as “anti-nutrients”, as polyphenol-rich diets have been associated with reduced absorption and digestibility of micro- and macronutrients. Underlying mechanisms driving the anti-nutritional effects of polyphenols in the diet include digestive enzyme inhibition and protein sequestration.The inhibition of digestive enzymes by extracts of plant products including grape seed, berries and tea has been demonstrated in vitro. This effect does not only apply to endogenous enzymes; bacterial enzymes can also be affected . Enzyme Inhibition of digestive proteases has also been studied, with polyphenols showing inhibitory effects on trypsin activity, while conflicting data exist with regard to pepsin . In vitrPolyphenols have been shown to influence the immune system within the context of food-related and digestive diseases both directly, as immunomodulators, and indirectly as sequestrants of allergenic proteins. The immunomodulatory activity of polyphenols is demonstrated by their ability to disrupt cell signaling pathways, modify cytokine production and concomitantly affect T cell proliferation and migration. These characteristics have been explored extensively within the context of IBD as well as food allergies, both of which share overlapping characteristics with celiac disease.In general, polyphenols have been implicated as potentially beneficial in the mediation of IBD, which includes ulcerative colitis (UC) and Crohn’s disease. Both of these diseases are characterized by chronic inflammation within the gastrointestinal tract. While UC primarily affects the colon, Crohn’s disease can manifest along the entirety of the gastrointestinal tract, most commonly appearing in the terminal ileum . A key dκB suppression—are also observed in the intestinal mucosa, resulting in overall amelioration of the inflammatory state of IBD [κB, tea polyphenol EGCG has been shown to reduce TNF-α expression by peritoneal macrophages [Studies on the potential for polyphenols as a complementary therapy for IBD have shown that the anti-inflammatory and immunomodulatory effects of tea flavonoids observed in liver tissue and endothelial cells—inhibition of COX-2, decreased NO production, NF-e of IBD ,143,145.e of IBD . Decreasrophages , and IECrophages ,147. EGCrophages , which prophages ,150.Mangifera indica L.) polyphenols on IBD have shown decreases in Simple Clinical Colitis Activity Index over the course of 8 weeks, along with decreases in plasma IL-8, growth-related oncogene and granulocyte macrophage colony-stimulating factor [Clinical trials focusing on the impact of dietary polyphenols on IBD demonstrate the therapeutic efficacy of these compounds in practice. The recent European Prospective Cohort (EPIC) study delineated an inverse association between the intake flavones and incidence of Crohn’s disease over the course of the study . Interveg factor . Similarg factor . These cWhile not a classic food allergy, celiac disease is similar to a food allergy in that it is stimulated by a food-based protein antigen. The efficacy of polyphenols in the mitigation of allergic responses has been investigated as both part of food matrices and as natural treatments for protein-stimulated inflammatory responses.Apple polyphenols have shown anti-allergenic effects by dose dependently suppressing the expression of MHC class II molecules when tested in ovalbumin-stimulated dendritic cells via upregulation of the membrane associated ring-CH type finger 1 (MARCH1) gene, which downregulates surface molecules. However, TNF-α was upregulated and IL-10 was downregulated , demonstThe use of polyphenols as protective agents against food allergens has been explored most notably with the creation of hypoallergenic peanut butter ,155,156.The mechanistic explanation for decreased binding and recognition of allergenic proteins has been explored using an array of analytical techniques and purified forms of the allergenic proteins. Studies characterizing the interactions between EGCG and peanut allergens Ara h 2 and Ara h 6 demonstrate binding specificity upon formation of protein–polyphenol complexes. Additionally, binding elicits a structural change on the allergens, modifying the frequencies of α-helices and β-sheets within each. In silico analyses revealed potential binding sites on each Ara protein, which is useful in predicting the impact of binding on epitope recognition by immune cells . The mecPolyphenols have demonstrated efficacy in protection against diseases with similar etiologies as celiac disease in vitro and in vivo due to their abilities to both interact with immunostimulatory proteins and to mediate inflammatory responses. Recently, a limited number of studies have investigated how these effects may translate to a celiac disease model, building a strong foundation for the therapeutic potential of polyphenolic supplementation as a complementary or alternative treatment for celiac disease. While interactions between gliadin and polyphenols have been demonstrated in a variety of matrix-specific contexts over the last decade, the first evidence of biological efficacy for polyphenols in the treatment of celiac disease was delineated in 2017. In this seminal study, gliadin–polyphenol complexes were characterized and shown to be stable throughout digestion and prevent gliadin-mediated inflammation and permeability in vitro . This woThe potential benefits of polyphenol supplementation for individuals with celiac disease have been shown to stem from both the anti-nutritional and anti-inflammatory properties of polyphenols. Polyphenols are not only potent anti-inflammatory compounds, as discussed with respect to IBD, but they also possess the ability to interact with proteins, allowing for direct sequestration of gliadin and disruption of pathogenesis through inhibition of digestive enzymes. The protective effects of polyphenols within the context of celiac disease that have been demonstrated to date are summarized in Gliadin–polyphenol interactions are dependent on a variety of factors including the specific nature of gliadins, the structure of polyphenols and the reaction matrix. Gliadin–polyphenol complexes that exist naturally within food matrices ,158 or tThe trend of non-specificity when it comes to interactions between gliadins and polyphenols has been shown to be consistent across pH levels and polyphenolic structures, likely due to the absence of a “true”  gliadin structure, though binding conformation and affinities are affected by polyphenol structure. Structural characteristics of polyphenols that influence binding include molecular weight, degree of galloylation and degree of polymerization. Saturation transfer difference NMR has shown that while increasing each of these characteristics in a given polyphenols tends to increase binding affinity to gliadins, increasing degree of galloylation and/or polymerization tends to be more influential than increasing molecular weight ,163,164.Direct interactions of polyphenols with gliadins have the potential to influence celiac disease pathogenesis by sequestering gliadins in order to inhibit recognition and absorption by the body as well as modifying the structure of immunostimulatory gliadin peptides, which may prevent their ability to bind to key receptors and enzymes.The formation of gliadin–polyphenol complexes can be beneficial in protecting against celiac disease if these complexes allow sequestration of the protein and prevent it from interacting in the celiac disease milieu. Physical sequestration of gliadins via complexation has been shown to be effective in clinical trials with the synthetic, non-bioavailable polymer BL-7010 . Early mGreen tea polyphenols (including EGCG) are able to precipitate both native and pepsin-trypsin-digested gliadins via the formation of insoluble complexes throughout in vitro digestion processes, resulting in an overall reduction in total protein levels of a sample digest . Similar2-gliadin (57–89), and EGCG have also been characterized at pH 2.0, 6.8 and 7.2, demonstrating similar formation of insoluble complexes and interactions between EGCG and specific proline and glutamine residues, which play an important role in antigen presentation in celiac disease pathogenesis [2-gliadin (57–89) has been extensively studied for its dominant role in stimulating the celiac disease immune response. With the sequence LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, this peptide features six overlapping epitopes for enzymatic binding and immune recognition [2-gliadin (57–89) has not yet been investigated, these findings suggest that EGCG may physically block immunostimulatory epitopes on this peptide in a phenomenon known as “epitope masking”. This phenomenon has been demonstrated within the context of food allergies. Perot and others showed that pre-treatment of native gliadins with polyphenol extracts from cranberry and apple reduced gliadin recognition by IgE and IgG antibodies, which are relevant in wheat allergy [Interactions between a uniquely immunostimulatory gliadin, αogenesis . The 33-ognition . While t allergy . This prA key feature in epitope masking and disruption of protein-mediated immune responses as a result of physical sequestration is thought to be structural change. In many cases of food allergy mitigation with polyphenols, structural changes in allergenic proteins have been predicted or observed ,166,167.2-gliadin (57–89) have been shown to cause the protein to transition from disordered to ordered by increasing relative helicity of the protein at a range of digestive pH levels [Interactions between EGCG and aH levels , a findiH levels . This stH levels . These sH levels . FurtherThe digestion of gliadins into smaller, immunostimulatory fragments in the lumen of the small intestine is a critical first step in celiac disease pathogenesis. Prevention of this step has been shown to reduce the immune response to gliadin in vivo . InhibitDespite a large number of studies focusing on the interactions between gliadin proteins and peptides with polyphenols, only a limited number have investigated the biological implications of these interactions and all of these studies have used an in vitro model of the disease. The Caco-2 monolayer model is a simplified model of the intestinal barrier wherein human colonocytes are seeded onto a semi-permeable membrane and grown to confluence. This cell model expresses characteristics of the human small intestine including the brush border enzymes, tight junction proteins and microvilli , and hasAs previously discussed, the initiation of intestinal permeability is an early step in celiac disease pathogenesis leading to the adaptive immune response mucosal damage. The ability of polyphenols to prevent intestinal permeability has been demonstrated in the Caco-2 monolayer model by measuring transepithelial electrical resistance (TEER). The decrease in TEER, or increase in intestinal permeability, observed upon treatment of cells with pepsin-trypsin-digested gliadin can be prevented by concurrent treatment with green tea extract . The preTransport of gliadins from the lumen to the lamina propria follows the initiation of intestinal permeability in the celiac disease milieu. Procyanidins B3 and C2 have both been shown to effectively prevent the transport of a gliadin peptide from the apical to basolateral compartment of the Caco-2 monolayer model over the course of 3 h , and treThe celiac disease inflammatory response which drives mucosal damage and the overactive immune response associated with the condition is the most popular target for celiac disease interventions. The innate immune response is associated with the production of pro-inflammatory cytokines associated with mucosal damage and celiac disease “hallmark” IL-15 ,40. FollMediation of the celiac-related inflammatory response by polyphenols has been demonstrated in Caco-2 cells with both green tea extract , which iThe procyanidin B2-rich cocoa extract applied to Caco-2 cells in the presence of bioactive gliadin peptide p31-43 intervened in the celiac-specific inflammatory cascade, decreasing secretion of IL-15, IL-1β, IL-6 and IL-8 in comparison to p31-43 alone. The extract was also effective in decreasing the inflammatory response when stimulated by IFN-γ , though Most striking from this study was that the inhibition of the gliadin-mediated inflammatory response resulted in a significant decrease in TG2 production by Caco-2 cells . TG2 is Polyphenols are widely known for their varied effects on human health, including anti-inflammatory and anti-nutritional activities. These characteristics make polyphenols a uniquely interesting therapeutic option for protein-stimulated inflammatory diseases such as celiac disease. Significant progress has been made in the understanding of the potential for polyphenols to be used as a therapeutic agent for celiac disease over the last decade, though the studies completed to date are not without limitations. Satisfactory in vivo studies testing the efficacy of these treatments have yet to be completed, and many of the in vitro studies available do not fully delineate intervention mechanisms, as proof of principle is still an important objective in each system with different polyphenolic profiles and gliadin sources. Furthermore, the versatile bioactivity of polyphenols lends them to a variety of potential beneficial effects within celiac disease pathogenesis, many of which will not be discovered until more complex disease models are used.Based on what is known about celiac disease and protein sequestration via polyphenols as a treatment option, more research is needed to determine the impact of structural modification on celiac-specific immune signaling versus classical anti-inflammatory effects of polyphenols. While the idea of polyphenolic sequestration is more novel, the bioactive properties of free polyphenols within the context of celiac disease cannot be overlooked, especially considering their efficacy as anti-inflammatory and antioxidative compounds in chronic diseases with similar etiologies, such as IBD. Potential mechanisms of interest relating to gliadin–polyphenol interactions include decreasing TG2 activity via enzyme inhibition or epitope masking, disruption of antigen presentation and anti-gliadin IgA epitope masking . Each ofA nutraceutical therapy for celiac disease treatment is an attractive option for clinicians and patients alike. Polyphenols, ubiquitous to plant-based foods, are already found in a typical diet, and are safely taken as supplements by many. To date, polyphenols are the only common dietary component being explored as a treatment option for celiac disease, in comparison to a myriad of synthetic pharmaceuticals compounds which require extensive testing for safety and tolerance before efficacy can even be approached. While there is a long way to go between the studies described here and true clinical use of this treatment option, the groundwork has been laid for further investigation towards a treatment for celiac disease that is safe, effective and well understood.


To define the growing significance of cellular targets and/or effectors of cancer drugs, we examined the fitness dependency of cellular targets and effectors of cancer drug targets across human cancer cells from 19 cancer types. We observed that the deletion of 35 out of 47 cellular effectors and/or targets of oncology drugs did not result in the expected loss of cell fitness in appropriate cancer types for which drugs targeting or utilizing these molecules for their actions were approved. Additionally, our analysis recognized 43 cellular molecules as fitness genes in several cancer types in which these drugs were not approved, and thus, providing clues for repurposing certain approved oncology drugs in such cancer types. For example, we found a widespread upregulation and fitness dependency of several components of the mevalonate and purine biosynthesis pathways  and an association between the overexpression of these molecules and reduction in the overall survival duration of patients with breast and other hard-to-treat cancers, for which such drugs are not approved. In brief, the present analysis raised cautions about off-target and undesirable effects of certain oncology drugs in a subset of cancers where the intended cellular effectors of drug might not be good fitness genes and that this study offers a potential rationale for repurposing certain approved oncology drugs for targeted therapeutics in additional cancer types. Over the past few decades, cancer treatment has witnessed tremendous progress in disease-free survival and in the delay and/or prevention of cancer recurrence in cancer patients. The first-generation of cancer chemotherapeutics and cytotoxic drugs generally target cellular processes that are fundamental to the growth of both cancer and normal cells, such as nucleic acids, protein synthesis and cell metabolism. Such drugs exhibit both anticancer and toxic side effects due to their non-specificity with respect to targets in cancer and normal cells in addition to other variables of drug metabolism etc. ,2,3,4. BCurrently available FDA-approved oncology drugs have been developed through molecule-driven empirical approaches . This haTargeted cancer therapy is still unable to inhibit the growth of all tumor cells of a given cancer-type in patients. It is possible that a new approach might be helpful for additional benefits for cancer patients. In this context, Behan et al. developed a comprehensive portrait of the gene dependency of human cancer  wherein These findings postulated that the effectiveness of approved oncology drugs in a given cancer type might be influenced by its ability to target or impair the functionality of specific cellular targets as fitness genes. However, whether these cellular targets (of approved oncology drugs) are also fitness genes in other types of cancer remains unknown. Such an analysis will result in a broader utility of targeting specific cellular targets in additional cancer types and is being investigated here using the above CRISPR-Cas9-based cellular fitness dependency datasets ,23. In thttps://www.accessdata.fda.gov/scripts/cder/daf/index.cfm, accessed on 13 September 2019). FDA approved oncology drugs searched on Bank databases  [https://score.depmap.sanger.ac.uk/gene, accessed on 13 September 2019) [U.S. Food and Drug Administration approved oncology drugs during the period of 1952–September 2019 were collected from the FDA website (er 2019) ,26,27,28er 2019) .The Drug bank database mined for the targets of 235 oncology drugs included 185 small molecules, 5 enzymes and 45 biotechnology drugs. Among these targets, 230 are approved/re-approved after January 2000. Drug accession number, type of molecule, and weight of the molecule are collected and documented for each drug .Drug associated with 232 targets were extracted from the Drug bank database with one to one and one to many relationships. Drug targets included DNA, enzymes, protein complexes and genes. Among the target 109 genes of oncology drugs, 100 genes were found to be also present in the quality-control passed list of 7460 genes in the Cancer Dependency Map dataset [Complete set of 7470 fitness genes from CRISPR-Cas9-mediated knock-out study in 324 cancer cell lines representing 19 cancer-types were compared with the oncology drug target data and obtained an 100 genes targets present in common. We analyzed the fitness dependency of these genes using Cancer Dependency Map database. A set of 47 targets of FDA approved drugs were showing significant loss of fitness in corresponding cell lines were collected and categorized for cancer types based on the cell line model.Fitness scores for each target for cancer types were plotted using in-house R implementation. Multiple analysis for fitness scores to compare the targets for same cancer type, multiple cancer type for same cancer types etc. were performed and plotted to interpret results. https://www.bioconductor.org, version 3.12, accessed on 10 August 2020) and Genepattern heatmap algorithms . Alteration graph was directly exported from the cBioPortal web browser. Genome alterations and gene expression analysis for selected genes in the corresponding cancer datasets were performed using the cBioPortal.org ,30 and XSurvival plots for selected genes for corresponding datasets were performed using the SurvExpress tool  using dehttp://sankeymatic.com, accessed on 11 December 2020).Drug Target relationship for FDA approved oncology drugs for approved cancer types as well as significant fitness cancer types was identified and represented as Sankey chart diagram using Sankey MATIC tool  in the subsequent studies. We observed that 15 of the 47 cellular targets of oncology drugs overlapped with recently identified 628 priority therapeutic targets  Figure . Both thTo determine the requirement of the 47 known cellular targets and/or effectors (of cancer drugs) for the cellular fitness of cancer types, we examined the fitness dependency of these 47 genes in the CRISPR-Cas9-derived Cancer Dependency Map . We obseIn the next set of studies, we analyzed the effect of knocking out 53 cellular genes (previously known to be targeted by oncology drugs) across 19 cancer types and found that the deletion of these genes did not result in a significant loss of cellular fitness in multiple cell types A. SimilaA number of recent reports demonstrated growth-promoting activities of oncology drugs in physiologically relevant whole animal models ,36,37,38To reveal a broader significance of the 47 cellular molecules which exhibited a significant fitness dependency in multiple cancer-types, we determined whether these molecules are required for the fitness or growth of cancer types for which drugs targeting these molecules were not approved. We found that the depletion of 43 of the 47 cellular molecules in multiple cancer types was associated with a substantial loss of cell fitness A . FurthIn general, cancer drug targets and/or effectors exhibited a widespread fitness dependency in cancer types for which drugs targeting these cellular targets are not approved (light green) compared with that in cancer types for which drugs targeting such molecules are approved (dark green) . For exaA multivariant analysis of tumors for overexpression versus underexpression of these fitness genes was associated with a highly significant reduction in the overall survival of respective cancer patients . SimilarTo study women’s cancer, we evaluated the expression of 13 targets and/or effectors of cancer drugs, of which 10 are common among breast, ovarian, and endometrial cancers, with a significant fitness dependency in breast cancer . Among tGGPS1 and FDPS are targets of nitrogen-containing bisphosphonates such as zoledronic acid derivatives that are widely used to prevent bone-related events related to breast cancer relapse. These drugs reduce mortality in postmenopausal women through the inhibition of bone metastasis by suppressing osteoclast-mediated bone resorption ,40,41,42A recently completed clinical trial, named AZURE trial, postulated certain beneficial antitumor activity of bisphosphonates against breast cancer in the adjuvant setting in a subset of postmenopausal women who were negative for MAF transcription factor ,54. PrevZoledronic acid acts by inhibiting these enzymes due to its analogous nature with naturally occurring pyrophosphates and by suppressing the geranylgeranylation and farnesylation of small GTPases . AntifolGGPS1, FDPS, and GART are upregulated in multiple cancers in addition to breast cancer  , includiAdditionally, the overexpression of GGPS1, FDPS, GART, and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) was also associated with a highly significant overall reduction in the survival duration of patients with esophageal and pancreatic cancers. Similarly, the overexpression of GGPS1, GART, and HMGCS1 reduced the overall survival duration in patients with glioblastoma, ovarian cancer, and endometrial cancer but not in those with prostate cancer . We founFDPS and GGPS1 are downstream components of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) , a rate-The present study encouraged the utilization of post-genomic data for the benefit of patients with cancer by repurposing approved cancer drugs by integrating fitness dependency of the intended target and/effector of a given oncology drug. Summary of the work presented here in To exemplify the usefulness of the findings presented here, we focused on breast cancer and demonstrate that targets of bisphosphonates such as FDPS and GGPS1 as well as statin such as HMGCS1 are excellent fitness genes in breast cancer and many hard-to-treat cancers. Because a large body of prior data suggests that use of statins may be associated with a reduced incidence of breast as well as esophageal cancer etc. ,64,65,66The findings presented here can impact the field of cancer drug repurposing and provide new hypotheses to be tested in future studies using appropriate preclinical model systems and subsequently, a novel cancer-specific clinical trial. In addition to repurposing bisphosphonates and statins, with or without pemetrexed, for breast cancer , the present analysis provided a rationale for repurposing a range of approved oncology drugs in cancer-types for which these drugs are not approved, but their cellular targets are excellent fitness genes in these cancer-types. The potential relationship between the levels of cancer therapeutic targets in serum, plasma, and tumors might also be of interest because certain targets and/or effectors of oncology drugs were also detected in extracellular fluids as secretory proteins  , in addi


Fetal calf serum (FCS) is frequently used as a growth factor and protein source in bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic product presenting multiple disadvantages including but not limited to ethical concerns. A promising alternative for FCS is human platelet lysate (hPL), which is produced out of human platelet concentrates and happens to be a stable and reliable protein source. In this study, we investigated the influence of hPL in an expansion medium (ESM) and an osteogenic differentiation medium (ODM) on the proliferation and osteogenic differentiation capacity of human BMSC. Therefore, we assessed population doublings during cell expansion, performed alizarin red staining to evaluate the calcium content in the extracellular matrix and determined the activity of alkaline phosphatase (ALP) as osteogenic differentiation correlates. The proliferation rate of BMSC cultured in ESM supplemented with hPL exceeded the proliferation rate of BMSC cultured in the presence of FCS. Furthermore, the calcium content and ALP activity was significantly higher in samples incubated in hPL-supplemented ODM, especially in the early phases of differentiation. Our results show that hPL can replace FCS as a protein supplier in cell culture media and does not negatively affect the osteogenic differentiation capacity of BMSC. Despite some limitations, fetal calf serum (FCS) is still the most common source of proteins and growth factors in cell culture media . HoweverThe most common alternative to FCS is human platelet lysate (hPL) , which iThe aim of this study was to compare the performance regarding the proliferation rate and osteogenic differentiation of BMSC cultured in ESM and ODM containing different concentrations of hPL or FCS and to evaluate the impact of the protein supplement’s concentration on the osteogenic differentiation of BMSC. As most available studies on hPL assess its effects on mesenchymal stromal cells (MSC) during the expansion period, we focused on the effects of hPL as part of ODM on the kinetics of BMSC osteogenic differentiation by evaluating alkaline phosphatase (ALP) activity and calcium deposition as osteogenic differentiation markers at multiple time points.The aims of this study were (i) to compare the impact of either FCS or hPL on the growth of BMSC under expansion conditions and (ii) to evaluate the osteogenic differentiation potential of BMSC cultivated in either FCS or hPL containing ODM. In order to compare the proliferation potential, population doublings of BMSC incubated in ESM with either 10% FCS or 10% hPL were calculated. For osteogenic differentiation experiments, the cells were incubated in ODM  and measurement of ALP activity as well as alizarin red staining as markers for osteogenic differentiation were performed after incubation periods of 1, 7, 14 and 21 days . Twelve identical samples were analyzed for each assay at each time point.BMSC were harvested from the bone marrow washouts of n = 10 patients undergoing surgery at the proximal femur at Heidelberg University Hospital. Prior to bone marrow harvesting, every patient gave written informed consent. The local ethics committee approved the study (S-443/2015) that was conducted according to the declaration of Helsinki in its present form. Cells derived from six male and four female patients with an average age of 50 years  were included in the study.BMSC were isolated from bone marrow, cultivated according to previously published protocols and pooled in order to reduce inter-individual variability as published previously ,36. DuriBMSC were split after 72 hours of expansion and cultured for a further 96 hours before they were introduced to osteogenic differentiation conditions. Cells were then stained with trypan blue and counted in a Neubauer’s cell chamber. Population doublings within a total incubation period of seven days of expansion as a parameter for the proliferation rate were calculated using Formula (1).Population doublings of the passages were added in order to obtain cumulative population doublings.β-glycerophosphate 10 mM . The growth factor sources in ODM were either FCS or hPL, added in concentrations of either 1% (according to hPL’s manufacturer’s advice for differentiation culture) or 10% according to the well-known FCS standard concentration  and incubated in ODM. The ODM was composed of 25 mM DMEM high-glucose with L-Glutamine , Penicillin/Streptomycin 100 mg/L , dexamethasone 0.1 µM , ascorbic acid-2-phosphate 2.5 mg/L  and ntration . In the rotocols ,37,38. MALP correlates with osteogenic differentiation of BMSC since it is produced during differentiation towards osteoblasts ,38. The Alizarin red stains the calcium deposits in the extracellular matrix formed by the osteoblastic (precursor) cells . The celThe experiments were performed on 12 experimental replicates per study group and two technical replicates per experimental sample. p < 0.05. Unless otherwise stated, differences mentioned in the text are non-significant. Values are shown as rounded means with standard deviation (SD) where applicable.Statistical analyses were conducted with IBM SPSS Statistics  and graphs were created using GraphPad Prism . Before further analysis, values were tested for normal distribution by a Shapiro-Wilk test. Normally distributed samples were tested with the unpaired T-Test; not normally distributed samples were tested using the Wilcoxon signed-rank test. Results were described as statistically significant for p < 0.01) more population doublings, with an average of 4.46 cumulative population doublings, than cells cultured in 10% FCS-supplemented ESM with an average of 2.22 cumulative population doublings -2,5-diphenyltetrazolium bromide (MTT) assay might provide a more detailed and standardized quantification of cell proliferation. The promoting effects of hPL on BMSC proliferation have been described before multiple times, in studies using the aforementioned more detailed assays; it has been shown that an enhanced proliferation of MSC is one of the main advantages in hPL supplementation of ESM ,23,25,26Many preliminary studies evaluated the effects of hPL-supplemented ESM on MSC in order to assess whether hPL changes MSC characteristics during expansion. Most of these studies evaluated the immunophenotype of the cells after expansion by analyzing cell surface markers with fluorescence-activated cell sorting (FACS): stem cell defining surface markers  were anaIn order to evaluate hPL as a substitute for FCS in ODM, we compared the osteogenic differentiation of BMSC cultured in ODM supplemented with 1% hPL, 10% hPL, 1% FCS and 10% FCS, respectively. Alizarin red staining and ALP activity measurement were performed at four time points that cover all relevant steps of osteogenic differentiation in the used setting . OsteogeThe ALP activity of the hPL groups as well as the F10 group reach their maxima on day seven and decrease afterwards, so the kinetics of cells incubated in hPL- and FCS-supplemented ESM are similar. Only the F1 group increases until its maximum on day 14 and remains stable until day 21. Birmingham et al.  showed tFocusing on the kinetics of extracellular calcium content, it is remarkable that both FCS groups increase to their maxima on day 21, whereas the hPL groups reach their maxima earlier, on day 14. Hoemann et al.  describeComparing FCS concentrations, it is remarkable that the commonly used concentration of 10% ,29,33,53According to our results and the data reported in the literature, hPL can be used as a supplement in ESM and ODM for BMSC, providing higher proliferation rates and higher or equal osteogenic differentiation potential. Our study was the first to evaluate the dynamics of ALP activity under the influence of two different concentrations of hPL in ODM, providing additional information about the kinetics of osteogenic differentiation under the influence of hPL. Furthermore, we reported a discrepancy of the impact of hPL in ODM on BMSC between two osteogenic differentiation parameters on a cellular/protein level, as there are hardly any studies that have evaluated two quantitative osteogenic differentiation parameters before. As we did not perform further assays evaluating osteogenic differentiation of BMSC on a genetic level, future studies should also evaluate osteogenic gene expression in order to obtain a more detailed impression of the impact of hPL in comparison to FCS on osteogenic differentiation in one and the same setting. The concentration of supplementation matters; in our case, only 10% hPL-supplemented ODM guaranteed an equal or higher osteogenic differentiation than the common approach using 10% FCS. However, we were the first study directly comparing two different concentrations of hPL in ODM, therefore further studies should follow in order to approach an optimum hPL concentration in ODM for BMSC. Considering the higher costs of hPL compared to FCS as well as the fact that the differences between the H10 and H1 groups were only detectable in the ALP activity assay, the use of hPL in smaller concentrations seems to be reasonable in order to avoid a significant increase of the budget necessary for cell culture experiments . Based oThe main aim of this study was to evaluate the influence of hPL supplementation in different concentrations in an osteogenic differentiation medium on the osteogenic differentiation of BMSC, evaluated by alizarin red staining and ALP activity measurements. As already described in the literature before, we could show a positive impact of hPL supplementation in an expansion medium on BMSC population doublings, presenting a proliferation rate almost twice as fast as under FCS supplementation. Furthermore, we showed that osteogenic differentiation is not compromised, yet, favored by a hPL-supplementation in ODM in a concentration-dependent manner; the positive effect is most visible when hPL concentration is 10% and at the beginning of the differentiation period, implying an accelerated, but absolutely comparable osteogenic differentiation potential of human BMSC.


Origanum vulgare possesses multiple pharmacological characteristics and its antioxidant and antibacterial properties are particularly interesting. Besides, a recent study aimed at evaluating the effects of oregano aqueous extract supplementation in poultry nutrition gave encouraging results regarding the secretion of glycoconjugates in the gut which increases tissue hydration and protects the intestinal mucosa from pathogenic bacteria, viruses, and parasites. Therefore, we investigated the effects of oregano feed supplementation on antioxidant and defense ability of pig gut. Our results showed that there was improved production of glycoconjugates in the duodenum and colon of pigs fed with supplementation of oregano aqueous extract, enhancing protection of the mucosa of these sections of the intestine. Also, we observed an enhanced antioxidant action in the two examined gut tract samples of the group supplemented with oregano. Findings can be used in further research to identify ways to improve endogenous defense ability with to reduce antibiotic use and prevent antimicrobial resistance.The European ban towards antibiotics is increasing the number of studies on the effects of feed additives, such as plant extracts, in order to enhance the health and welfare status of domestic animals intended for human consumption. This study evaluated the effects of adding oregano aqueous extract (OAE) to the diet of pig slaughtered at finisher stage. Study was performed to identify glycoconjugates and evaluate the oxidative stress levels in the duodenum and colon intestinal tracts. Glycohistochemistry was performed by staining with Periodic acid–Schiff (PAS), Alcian blue (AB) pH 2.5, AB-PAS, AB pH 1, AB pH 0.5, low iron diamine, and high iron diamine. Serial sections were pre-treated with sialidase V before staining with AB pH 2.5  preceded or not by saponification. To study oxidative stress, an immunohistochemical analysis was applied to investigate the presence of the oxidative stress target molecule Bcl-2 Associate X protein (BAX). Findings show that oregano aqueous extract supplementation improves the production of the secretion glycoconjugates involved in direct and indirect defense, thus enhancing the protection of the pig intestinal mucosa. Moreover, the reduced BAX protein immunostaining observed in both duodenum and colon of swine of the oregano-supplemented group respect to that observed in the control group suggests an enhanced antioxidant action by oregano adding. Findings could be useful for other studies aiming to reduce antibiotic use and prevent antimicrobial resistance. Origanum vulgare, native to Europe and Western Asia, possesses multiple pharmacological characteristics such as antioxidant, antibacterial, antimycotic, and anti-inflammatory properties [Origanum vulgare [p-Coumaric acids characterize the polyphenolic profile of oregano [Origanum vulgare of caffeic acid, eriodictyol, and apigenin was demonstrated [The European ban towards antibiotics has increased the number of studies on the effects of feed additives—such as plant extracts, trace minerals, organic acids, probiotics, and polyunsaturated fatty acids ,3,4,5,6—operties ,13. The operties  and its  vulgare , but the vulgare . Even if oregano , its hignstrated . The antnstrated ,18.bcl-2 family, it is considered as a stimulating factor for apoptosis [A recent study aimed at evaluating the effects of oregano aqueous extract (OAE) supplementation in poultry nutrition found encouraging results regarding the secretion of glycoconjugates in the gut which increases tissue hydration and protects the intestinal mucosa from pathogenic bacteria, viruses, and parasites . In factpoptosis  a phenomThe animal care procedures were in accordance with Legislative Decree No. 146, implementing Directive 98/58/EC of 20 July 1998 concerning the protection of animals kept for farming purposes. The research was approved with Protocol E81AC. 10/A by the Ethics Committee of the University of Camerino.Thirty-two Duroc x Large White pigs , previously randomly divided into two homogenous  groups of 16 pigs each and housed in a commercial farm in 3 × 3 m pens containing 4 pigs each, were used. The numerousness of animals for each group was calculated and considered optimal for a significance level of 0.05, a test power of 0.8, and an effect size of 1. The animals were fed according to a three-phase feeding program as summarized in The supplementation level of OAE was chosen based on our previous data in swine ,2 and poClostridium perfringens) before staining with AB pH 2.5 . Prior to enzymatic treatment, some sections had been incubated with 1% KOH in 70% ethanol for 20 min at room temperature to remove the acetyl groups  [Samples of the duodenum (4 cm removed from the pyloric sphincter) and ascending colon (in the second outer turn) were collected from thesubjects belonging to the two groups. Duodenum was chosen because shows two different secretory structures  and it is the intestinal tract receiving the stomach content, while the ascending colon represents the longest tract of large intestine acting as fermentation chamber and hosting the vitamins releasing bacteria. The samples intended for conventional glycohistochemistry were immediately fixed by immersion in Carnoy’s fluid for 24 h followed by post-fixation in 2% calcium acetate and 4% paraformaldehyde solution (1:1) for 3 h at room temperature , while tSial-AB) ; the con2O2 in order to quench endogenous peroxidase activity and then irradiated in a microwave oven at 750 Watts in 0.1M citrate buffer, pH 6.0 to retrieve the antigens. The sections were then blocked with normal goat serum  for 30 min and incubated overnight with 1:100 mouse monoclonal anti-BAX antibody . The following day, the sections were incubated with 1:200 biotin-conjugated goat anti-mouse secondary antibody . The immunohistochemical reaction was visualized using an avidin-biotin system  and diaminobenzidine (DAB) as chromogen . Some sections incubated with PBS and/or normal goat IgG instead of the primary antibody were used as controls of unspecific staining. All sections were observed under a photomicroscope . For each animal, three independent observers, unaware of the treatments carried out, evaluated five microscopic fields of each intestinal tract and the intensity of the staining was graded in numerical units as follows: negative (0), weak (0.5), moderate (1) and strong (2).In order to study oxidative stress, immunohistochemical staining for the BAX protein was performed . RehydraConcerning the histochemical data, numerical units of reactivity, sorted in a scale ranging from 0 (negative) to 3 (strong), were converted into ranks ,35,36,37CTR and EXP groups did not show differences in growth performances, as reported in The morphological features of both duodenum and colon were well preserved and no appreciable differences between the two groups were observed at light microscopy. In short, the duodenum tunica mucosae appeared raised in villi covered by a simple columnar epithelium with scattered goblet cells; at the base of the villi the epithelium forms small, deep, glandular crypts; duodenal glands were observed in the tunica submucosa . The colPositive histochemical results were obtained at the goblet cell level in both duodenum and colon, and at duodenal glands. Differences emerged between CTR and EXP groups concerning histochemical treatment response. The response to the Sial-AB and KOH-Sial-AB histochemical treatments, which induced differentiated reactivity of goblet cells in both duodenum  and colop for each histochemical treatment are reported in The responses of the samples, as reactivity intensities, to the various glycohistochemical treatments in duodenum and colon, are shown in The significance of the differences among the serial treatments AB pH 2.5, Sial-AB, and KOH-Sial-AB for CTR and EXP groups are shown in Based on the histochemical evidence, the variety of glycoconjugates produced by duodenal and colon secretory structures are illustrated in The immunohistochemistry revealed BAX protein staining in both duodenum and colon. The mucosal epithelium ranged from strong to slightly positive in the colon and the bottom of duodenal villi and it was negative in the apical part of these structures. Immunohistochemical staining was observed in the enterocytes while the goblet cells were always negative. BAX staining was also observed in some scattered cells of duodenal glands. Besides the epithelium, BAX was detected in the vascular endothelium and the neuronal cell bodies of the ganglia localized in submucous and myenteric plexuses. No staining was observed in the negative controls . As regaOregano diet supplementation did not produce differences in growth performances respect to that CTR group.4 level [On the basis of reactivity towards the histochemical treatments, it can be affirmed that both the CTR and EXP groups produced large amounts of high sulphated glicoconjugates represented by heparin/heparansulphated-like material and a smaller amount of carboxylated glicosaminoglycans from the goblet cells of both pig duodenum and colon; while duodenal glands produce neutral and sialilated glycoproteins showing sialic acid not acetylated at C4 level ,27,29,354 acetylated sialic acid in both intestinal tracts under study, especially in the duodenum, where the chondroitinsulphate A/B/C-like glycosaminoglycans production is also induced. No difference in duodenal gland glycoconjugate production was observed between the two groups.OAE diet supplementation promotes the production of sialilated glycoproteins with and without C4 acetylated sialoderivatives produced by duodenal goblet cells could be able to confer resistance to bacterial sialidase [On comparing the glycohistochemical profiles of the EXP and CTR groups, a clear increase in acid glycoconjugate production was observed, which allows for enhancement in water attraction, thus improving tissue hydration and mucosae lubrication. Moreover, the chondroitinsulphate A/B/C-like material increases the negative charge of the viscoelastic barrier produced by the duodenal goblet cells and could act as additional hapten-like sites for pathogen agents; therefore, the duodenal mucous membrane could have indirect defense advantages performed by these additional glycoderivatives, due to both steric hindrance effect and masking action against true pathogen attack sites. A direct defense response is promoted by the production of sialilated glycoproteins by both the duodenal and colon goblet cells in the EXP group; in particular, the Cialidase ,26. All Considering that the goblet cells produce a great variety of glycoconjugates which seemed to be affected by oregano supplementation and that the duodenal glands secrete neutral and sialilated glycoproteins that are not modified by dietary regime, it can be hypothesized that duodenal gland secretion could be involved in buffer and/or enzymatic functions, while secreted products from goblet cells of both duodenum and colon could be mainly involved in lubrication and defense responses.The above-mentioned considerations suggest that oregano food supplementation enhances both the quality and quantity of the secretion glycoconjugates involved in direct and indirect defense, thus better preserving mucous membranes of pig gut. Finding of the presence of BAX further support this hypothesis; in fact, lower levels of BAX immunostaining intensity were observed in both the studied intestinal tracts in the swine belonging to the EXP group compared with the CTR group.The BAX protein in the normal human intestine has already been reported ; as a meIn this study, the reduced expression of BAX protein in EXP group swine supports glycohistochemical results showing an OAE positive action on duodenum and colon mucosae. It can be supposed that the diet supplemented with OAE induced an enhanced anti-oxidative effect on tissues, including ROS generation and cellular constituent damage onset prevention.Even if the CTR diet group shows a good situation regarding both the glycoconjugate and BAX expression, the addition of oregano aqueous extract to the diet induces the production of a greater  variety of glycoderivatives and decreases the BAX presence. Findings allow us to hypothesize that the oregano supplementation strongly enhances the levels of endogenous defense and the antioxidant ability in both duodenum and colon of pig.


Mycoplasma hyopneumoniae  is a geographically widespread and economically devastating pathogen that colonizes ciliated epithelium; the infection of Mhp can damnify the mucociliary functions as well as leading to Mycoplasma pneumonia of swine (MPS). MPS is a chronic respiratory infectious disease with high infectivity, and the mortality can be increased by secondary infections as the host immunity gets down-regulated during Mhp infection. The host immune responses are regarded as the main driving force for the disease development, while MPS is prone to attack repeatedly in farms even with vaccination or other treatments. As one of the smallest microorganisms with limited genome scale and metabolic pathways, Mhp can use several mechanisms to achieve immune evasion effect and derive enough nutrients from its host, indicating that there is a strong interaction between Mhp and porcine organism. In this review, we summarized the immune evasion mechanisms from genomic variability and post-translational protein processing. Besides, Mhp can induce the immune cells apoptosis by reactive oxygen species production, excessive nitric oxide (NO) release and caspase activation, and stimulate the release of cytokines to regulate inflammation. This article seeks to provide some new points to reveal the complicated interaction between the pathogen and host immune system with Mhp as a typical example, further providing some new strategies for the vaccine development against Mhp infection. Mycoplasma hyopneumoniae  is one of the primary pathogens of Mycoplasma pneumonia of swine (MPS), also known as enzootic pneumonia (EP) (in vitro culture of Mhp is strenuous because of its fastidious requirement for culture medium (nia (EP) . This chnia (EP) . The in e medium , 4, whice medium .in vivo/in vitro) to acquire adequate nutrients like nucleic acid, lipid, and amino acid for its proliferation  production, excessive nitric oxide (NO) release and caspase activation, and stimulate the release of cytokines to regulate inflammation. This article aims to use Mhp as an example to outline the comprehensive interaction between pathogen and the host immune system, and new strategies for the next-generation of efficient vaccine development for the control of MPS are also proposed.Mollicutes genomes had increased recombination and mutation rate as the environment changed  were sequenced and uploaded into the database of the National Center for Biotechnology Information (NCBI). In general, the average genomic size of Mhp is 0.9199 Mb, encoding 744 genes and 645 proteins, the mean GC content is 28.54%, which is a significantly low value compared with other species.  changed .Genomic diversity can be associated with discrepant pathogenicity of pathogens . A comprIn brief, genomic variability of Mhp has been identified, and the effects for controlling MPS are summarized as follows. (1) Gene mutations in immunogens like P97 and P146  have a negative impact for the vaccine development , 25. (2)It is generally accepted that bacteria can use protein hydrolysis processing to accommodate the changing environment and manipulate host immune responses . Mhp celThe Post-translational protein processing events in Mhp proteomics can expand protein functions and expose more epitopes , which mMycoplasma immunoglobulin binding protein (MIB) and Mycoplasma immunoglobulin protease (MIP), respectively. The MIB-MIP system had been reported in Mycoplasma mycoides subspecies capri, and the multiple copies of MIB-MIP genes were also found in Mhp genome  G, which is mediated by p genome , while tIn short, as a genome-reduced pathogen, Post-translational protein processing and genomic variability complicate the Mhp cell surface antigen appearance and expand protein functions, which may aggravate disease development and regulate lung homeostasis to promote the proliferation of Mhp. For the immune system, abundant exposed epitopes can affect the Pathogen-Associated Molecular Patterns (PAMPs) to reduce immune efficiency, and more functional virulence factors can contribute to the negative regulation of the immune system.Different immune cells can form a complex and sophisticated network to recognize pathogens and provide direct defense against their invasion. In the respiratory tract, the mucosal immune system is constructed by epithelial barriers, mucosal dendritic cells, innate lymphoid cells, natural killer cells, natural killer T cells, mast cells, and eosinophils . Mhp canStudies had reported that PAMs could recognize and provide immune defense during Mhp infection . Before Mus musculus chemokine (C-C motif) ligand3 (Ccl3) and M. musculus serum amyloid A 3 (Saa3) were associated with the inflammatory responses  was down-regulated in lung tissues during Mhp infection; further studies found that the overexpression of CYP1A1 could activate the PPAR-γ signaling pathway and up-regulated the IL-1β, IL-6, IL-8, and TNF-α levels in Mhp-treated PAMs . Moreoveesponses . In genein vitro , 73 and  and found that older pigs possessed a higher antibody level during Mhp infection . Follow-The mucosal surface of the respiratory tract is the first defense line to prevent pathogens infection and eliminate microorganisms  or foreign bodies by producing active components like SIgA , 95. On The deviation of IgG subclass during Mhp infection indicates a Th1/Th2 response bias, in general, the IL-12 is related to the Th1 immune response, the IL-4 and IL-6 are related to the Th2 immune response, while the specific immune type that Mhp induced remains ambiguous . StudiesoriC) sequences from Mhp could be useful genetic engineering vectors for strain improvement, which had expressed EGFP in Mhp cells successfully  is a commonly used and effective method to prevent Mhp infection, while these vaccines provide partial protection and cannot cure MPS or eliminate Mhp from farms or bodies completely. Vaccines preparation need excellent strains with high immunogenicity and adequate supply of bacteria, besides, the product price, immunization efficiency and vaccination method also influence the popularization of such vaccines. In order to improve the immunogenicity and biological characteristics of Mhp strains, the self-replicating plasmids with the origin of replication  can evaluate the genomic diversity and classify the core (conserved), accessory (dispensable), and unique (strain-specific) genes of different strains, which can be used for the choice of conserved and dominant immunogens . On the Mhp is an important respiratory pathogen that can cause MPS, and brings enormous economic losses worldwide. MPS can develop into a recessive disease with the immune defense and other treatments; the isolation of Mhp from other tissues has been reported, which indicates that Mhp can turn into an internal organ and exist within its host without causing disease , 108–110Haemophilus parasuis and Mycoplasma hyorhinis, which indicated that the pathological importance of Mycoplasma hyorhinis had been underestimated  infection is immunosuppressive by damaging lymphoid tissues; the coinfection of Mhp and PCV2 contributes to a range of polymicrobial disease syndromes . What isstimated . Earlierultocida . Particu surface . In addisymptoms . In geneThe main contents of this article are briefly summarized in Mhp is an important pathogen that leads to MPS and causes major economic losses to pig industry, some commercial vaccines are already available, while the limited immune protective effect is unable to eliminate Mhp from farms or bodies. The balance between immune evasion of the pathogen and immune defense of the host determines the disease development, and based on the current research status, we proposed the genomic variability and Post-translational protein processing are important immune evasion mechanisms of Mhp, but it still needs direct concept proof and deeper research on the mechanisms of immune evasion. In order to reduce the adverse effects of immune evasion on vaccine development and increase the general applicability of vaccines, some novel research methods, such as metabolic engineering techniques and computer-aided reverse vaccinology, can improve the feasibility of novel vaccines as well as reducing the research and development cycle. Vaccine development based on reverse vaccinology has been applied to pathogens with immune evasion ability like COVID-19 , while tGL and YH conceived the idea. GL wrote the manuscript. EO drew the JS was employed by Zhejiang Hom-Sun Biosciences Co., Ltd. The remaining 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.