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Effects of wheat germ agglutinin on human gastrointestinal epithelium: Insights from an experimental model of immune/epithelial cell 304 interaction

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Abstract

Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed, among many others, by human gastrointestinal epithelial and immune cells. WGA is a toxic compound and an anti-nutritional factor, but recent works have shown that it may have potential as an anti-tumor drug and as a carrier for oral drugs. To quantitate the toxicity threshold for WGA on normal epithelial cells we previously investigated the effects of the lectin on differentiated Caco2 cells, and showed that in the micromolar range of concentrations WGA could alter the integrity of the epithelium layer and increase its permeability to both mannitol and dextran. WGA was shown to be uptaken by Caco2 cells and only approximately 0.1% molecules were observed to cross the epithelium layer by transcytosis. Here we show that at nanomolar concentrations WGA is unexpectedly bioactive on immune cells. The supernatants of WGA-stimulated peripheral blood mononuclear cells (PBMC) can alter the integrity of the epithelium layer when administered to the basolateral side of differentiated Caco2 cells and the effects can be partially inhibited by monoclonal antibodies against IL1, IL6 and IL8. At nanomolar concentrations WGA stimulates the synthesis of pro-inflammatory cytokines and thus the biological activity of WGA should be reconsidered by taking into account the effects of WGA on the immune system at the gastrointestinal interface. These results shed new light onto the molecular mechanisms underlying the onset of gastrointestinal disorders observed in vivo upon dietary intake of wheat-based foods.

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... In addition to molecular mimicry, food components such as lectins and agglutinins may contribute to autoimmune diseases by directly binding to human tissue and components of the gut microbiome [13][14][15][16][17]. Lectins/agglutinins are ubiquitous carbohydrate-binding proteins that are found in animals, plants, and microorganisms. ...
... The surface of many prokaryotic and eukaryotic cells is covered with a dense coating of glycoconjugates or glycocalyx. The binding of WGA to Neu5Ac at the glycocalyces of human cells and pathogens expressing Neu5Ac allows for cell entry and could induce a proinflammatory immune response, especially at the gastrointestinal surfaces [16,[34][35][36]. ...
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The aim of this study was to examine the direct reaction of specific lectin/agglutinin antibodies to different tissue antigens to confirm the theory that reactivity between them may contribute to autoimmunities. Lectins are carbohydrate-binding proteins found in nearly all fruits and vegetables. Undigested lectins can penetrate the gut barriers, provoking an immune response that results in the production of antibodies against them. Using an enzyme-linked immunosorbent assay, we reacted lectin-specific antibodies with 62 different tissue antigens. Wheat germ agglutinin-specific antibody was the most reactive with the tissue antigens (37 tissues out of 62), followed by red kidney bean phytohemagglutinin-specific antibody (20), soybean agglutinin-specific antibody (20), and peanut agglutinin-specific antibody (15). This reaction between anti-lectin antibodies and many human tissue antigens may be due to possible molecular mimicry and cross-reactivity. After our results confirmed that anti-lectin antibodies bind with human tissues, we wanted to determine the prevalence of these antibodies in the blood of 500 nominally healthy donors. The percentage elevation of antibodies against different lectins ranged from 12 to 16% (Immunoglobulin G), 9.7-14.7% (Immunoglobulin A), 12-18% (Immunoglobulin M), and 7.8-14.6% (Immunoglobulin E). Serial dilutions and inhibition study confirmed that these reactions were specific. Finally, we tested the lectin-specific antibody level in sera both negative and positive for RF and ANA and found that IgM anti-lectin antibody levels were highly correlated with RF but not with ANA level. The reaction of anti-lectin antibodies with human tissue components and their detection in RF-positive samples may describe mechanisms by which the production of antibodies against undigested lectins may contribute to the pathogenesis of some autoimmune diseases.
... In human basophils, WGA induced interleukin 4 (IL-4) and interleukin 13 (IL-13) release [88]. In an experimental model of human intestinal immune/epithelial cell interaction, it exhibited toxic and inflammatory effects by disrupting epithelial integrity and inducing the synthesis of pro-inflammatory cytokines, including interleukin 1, interleukin 6 and interleukin 8 by peripheral blood mononuclear cells (PBMCs) [89]. In murine spleen cells, WGA induced a T and B cell-independent production of interleukin 12 (IL12) and, in turn, the production of interferon gamma (IFN gamma) by T/natural killer lymphocytes [90]. ...
... Involvement of an adaptive immune response by the migration of DCs to mesenteric lymph nodes and interaction with primed T cells could exacerbate the ongoing inflammation [84]. In vitro studies and in vivo animal models showed that WGA induces the release of pro-inflammatory cytokines and epithelial barrier disruption [89]. In vivo human studies are needed to better support the role of ATIs and WGA as triggering factors of NCG/WS. ...
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The gluten-free diet (GFD) has gained increasing popularity in recent years, supported by marketing campaigns, media messages and social networks. Nevertheless, real knowledge of gluten and GF-related implications for health is still poor among the general population. The GFD has also been suggested for non-celiac gluten/wheat sensitivity (NCG/WS), a clinical entity characterized by intestinal and extraintestinal symptoms induced by gluten ingestion in the absence of celiac disease (CD) or wheat allergy (WA). NCG/WS should be regarded as an “umbrella term” including a variety of different conditions where gluten is likely not the only factor responsible for triggering symptoms. Other compounds aside from gluten may be involved in the pathogenesis of NCG/WS. These include fructans, which are part of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs), amylase trypsin inhibitors (ATIs), wheat germ agglutinin (WGA) and glyphosate. The GFD might be an appropriate dietary approach for patients with self-reported gluten/wheat-dependent symptoms. A low-FODMAP diet (LFD) should be the first dietary option for patients referring symptoms more related to FODMAPs than gluten/wheat and the second-line treatment for those with self-reported gluten/wheat-related symptoms not responding to the GFD. A personalized approach, regular follow-up and the help of a skilled dietician are mandatory.
... Lectins are molecules normally produced by plants as a defense mechanism against fungi or other plants [49]. Interestingly, WGA induced an increase of epithelial permeability when administrated to Caco-2 cells, enhancing the passage of small molecules through the barrier [50]. ...
... In addition, WGA has an important effect on the immune system, as it can stimulate the immune system inducing the production of pro-inflammatory cytokines both in human peripheral blood mononuclear cells (PBMC) [50,51] and in animals [52,53]. ...
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Gluten-free diets are increasingly chosen in the Western world, even in the absence of a diagnosis of celiac disease. Around 10% of people worldwide self-report gluten-related complaints, including intestinal and extra-intestinal symptoms. In most cases, these subjects would be labeled as patients suffering from irritable bowel syndrome (IBS) who place themselves on a gluten-free diet even in the absence of celiac disease. In some instances, patients report a clear benefit by avoiding gluten from their diet and/or symptom worsening upon gluten reintroduction. This clinical entity has been termed non-celiac gluten sensitivity (NCGS). The symptoms referred by these patients are both intestinal and extra-intestinal, suggesting that similarly to functional gastrointestinal disorders, NCGS is a disorder of gut–brain interaction. It remains unclear if gluten is the only wheat component involved in NCGS. The mechanisms underlying symptom generation in NCGS remain to be fully clarified, although in the past few years, the research has significantly moved forward with new data linking NCGS to changes in gut motility, permeability and innate immunity. The diagnosis is largely based on the self-reported reaction to gluten by the patient, as there are no available biomarkers, and confirmatory double-blind challenge protocols are unfeasible in daily clinical practice. Some studies suggest that a small proportion of patients with IBS have an intolerance to gluten. However, the benefits of gluten-free or low-gluten diets in non-celiac disease-related conditions are limited, and the long-term consequences of this practice may include nutritional and gut microbiota unbalance. Here, we summarize the role of gluten in the clinical features, pathophysiology, and management of NCGS and disorders of gut–brain interaction.
... Wheat germ agglutinin (WGA) is a lectin, which binds to N-acetyl D-glucosamine sites on the endothelial membrane, and this binding triggers transport through the BBB, as demonstrated electron microscopically with a WGAhorseradish peroxidase (HRP) conjugate (80). Similar to the cationic ligands, WGA is sequestered within the lysosomal compartment of cells with minimal transcytosis (81). WGA administration in vivo stimulates an inflammatory reaction with production of multiple cytokines (81). ...
... Similar to the cationic ligands, WGA is sequestered within the lysosomal compartment of cells with minimal transcytosis (81). WGA administration in vivo stimulates an inflammatory reaction with production of multiple cytokines (81). Pegylated nanoparticles conjugated with WGA cause cytoxicity in Calu-3 cells, which was not observed with pegylated nanoparticles lacking the WGA ligand (82). ...
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Non-viral gene therapy of the brain is enabled by the development of plasmid DNA brain delivery technology, which requires the engineering and manufacturing of nanomedicines that cross the blood-brain barrier (BBB). The development of such nanomedicines is a multi-faceted problem that requires progress at multiple levels. First, the type of nanocontainer, e.g., nanoparticle or liposome, which encapsulates the plasmid DNA, must be developed. Second, the type of molecular Trojan horse, e.g., peptide or receptor-specific monoclonal antibody (MAb), must be selected for incorporation on the surface of the nanomedicine, as this Trojan horse engages specific receptors expressed on the BBB, and the brain cell membrane, to trigger transport of the nanomedicine from blood into brain cells beyond the BBB. Third, the plasmid DNA must be engineered without bacterial elements, such as antibiotic resistance genes, to enable administration to humans; the plasmid DNA must also be engineered with tissue-specific gene promoters upstream of the therapeutic gene, to insure gene expression in the target organ with minimal off-target expression. Fourth, upstream manufacturing of the nanomedicine must be developed and scalable so as to meet market demand for the target disease, e.g., annual long-term treatment of 1,000 patients with an orphan disease, short term treatment of 10,000 patients with malignant glioma, or 100,000 patients with new onset Parkinson's disease. Fifth, downstream manufacturing problems, such as nanomedicine lyophilization, must be solved to ensure the nanomedicine has a commercially viable shelf-life for treatment of CNS disease in humans.
... Jde o velmi stabilní proteiny, které jsou rezistentní ke štěpení digestivními enzymy. Zvláště lektin zvaný wheat germ agglutinin (WGA) přímo stimuluje uvolnění několika prozánětlivých cytokinů z monocytů a makrofágů ve střevní sliznici a dosud neznámým mechanizmem může zvyšovat intestinální permeabilitu [12]. Inhibitory amylázy a trypsinu (ATIs -amylase/trypsin inhibitors) podobně jako lektiny fungují v zrnu jako obranný mechanizmus proti řadě infekcí a jsou rovněž rezistentní vůči proteolytickým enzymům trávicího traktu. ...
... Jde např. o lektiny či inhibitory amylázy a trypsinu, jak bylo zmíněno výše [5,12,13]. ...
Article
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Non-celiac gluten sensitivity has recently been recognized by the scientific community as a part of gluten-related disorders, and is defined as a condition with gastrointestinal and/or extra-intestinal symptoms triggered by gluten ingestion in the absence of celiac disease and wheat allergy. Currently, there is no specific serological marker and non-celiac gluten sensitivity remains a diagnosis of exclusion: testing for celiac disease and wheat allergy must be negative, symptoms must improve with a gluten-free diet, and diagnosis must be confirmed by the gluten challenge. In this article, we discuss current knowledge of pathophysiology, clinical and epidemilogical spectrum, diagnosis, and treatment of NCGS.
... While oral exposure to proteins as a general class of substances is a common and necessary nutritional process and does not normally present a health risk, a very small number of proteins have been identified in foods that are capable of causing adverse effects when consumed (Dalla Pellegrina et al., 2005;Dalla Pellegrina et al., 2009;Gabor et al., 1998;Price et al., 2014). Enteric pathogens associated with contaminated food and/or water produce a number of protein toxins that can trigger acute intestinal disease but often require the source organism to be present in order for the protein to cause effects (Aboutaleb et al., 2014;Nataro and Kaper, 1998). ...
... The dietary lectin wheat germ agglutinin (WGA) was also included in this study. WGA binds carbohydrate moieties on IECs and has been reported to cause adverse effects following oral exposure (Dalla Pellegrina et al., 2009;Pusztai et al., 1993). Two allergenic proteins present in common food sources (Ara h 2 from peanuts and bovine b-lactoglobulin from milk) that are responsible for adverse effects in sensitive individuals were also included. ...
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Human intestinal epithelial cell lines (T84, Caco-2, and HCT-8) grown on permeable Transwell™ filters serve as models of the gastrointestinal barrier. In this study, this in vitro model system was evaluated for effectiveness at distinguishing between hazardous and non-hazardous proteins. Indicators of cytotoxicity (LDH release, MTT conversion), monolayer barrier integrity ([3H]-inulin flux, horseradish peroxidase flux, trans-epithelial electrical resistance [TEER]), and inflammation (IL-8, IL-6 release) were monitored following exposure to hazardous or non-hazardous proteins. The hazardous proteins examined include streptolysin O (from Streptococcus pyogenes), Clostridium difficile Toxins A and B, heat-labile toxin from enterotoxigenic Escherichia coli, listeriolysin O (from Listeria monocytogenes), melittin (from bee venom), and mastoparan (from wasp venom). Non-hazardous proteins included bovine and porcine serum albumin, bovine fibronectin, and ribulose bisphosphate carboxylase/oxygenase (RuBisco) from spinach. Food allergenic proteins bovine milk β-lactoglobulin and peanut Ara h 2 were also tested as was the anti-nutritive food protein wheat germ agglutinin. Results demonstrated that this model system effectively distinguished between hazardous and non-hazardous proteins through combined analysis of multiple cells lines and assays. This experimental strategy may represent a useful adjunct to multi-component analysis of proteins with unknown hazard profiles.
... Furthermore, in vitro methods of enzymatic digestion introduce an important experimental variable difficult to control, and do not guarantee comparable results with what really happens in the human gut. The decision to use all the protein fractions and not only the fractions containing gliadin and glutenin is due to the possibility that other wheat proteins, such as lectins, wheat germ agglutinin or a-amylase/trypsin inhibitors, activate innate immune responses involved in the mechanisms of gluten sensitivity (Dalla Pellegrina et al., 2009;Haas et al., 1999;Junker et al., 2012). In line with this hypothesis, many investigators prefer to use the term non-celiac wheat sensitivity instead of NCGS (Carroccio, Rini, & Mansueto, 2014). ...
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Non-celiac gluten sensitivity (NCGS) is still an undefined syndrome whose triggering mechanisms remain unsettled. This study aimed to clarify how cultured peripheral blood mononucleated cells (PBMC) obtained from NCGS patients responded to contact with wheat proteins. Results demonstrated that wheat protein induced an overactivation of the proinflammatory chemokine CXCL10 in PBMC from NCGS patients, and that the overactivation level depends on the cereal source from which proteins are obtained. CXCL10 is able to decrease the transepithelial resistance of monolayers of normal colonocytes (NCM 460) by diminishing the mRNA expression of cadherin-1 (CDH1) and tight junction protein 2 (TJP2), two primary components of the tight junction strands. Thus, CXCL10 overactivation is one of the mechanisms triggered by wheat proteins in PBMC obtained from NCGS patients. This mechanism is activated to a greater extent by proteins from modern with respect to those extracted from ancient wheat genotypes.
... Many studies have addressed Caco-2 behavior when co-cultured with both non-pathogenic bacteria and peripheral blood mononuclear cells, to elucidate in particular the crosstalk with the immune system 10 . By performing different complementary laboratory assays (e.g. ...
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The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.
... An increase of IL-6 and Nfκ-B was observed while no changes were evident for TNF-α. These results, together with the observed changes in lymphocyte-granulocyte ratio and the slight increase in the splenic white pulp at the end of the treatment suggest immune response activation [32][33][34]22,25]. Upon evaluation of the histological sections of the ileum, the villi in treated intestines were found to display atrophy with respect to control intestines. ...
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Previous work showed that Tepary bean (Phaseolus acutifolius) lectins exhibit differential cytotoxic effects on cancer cell lines by apoptosis induction. In vivo studies using a Tepary bean lectin fraction (TBLF, 50 mg/kg of body weight) after colon cancer induction in rats showed that TBLF inhibited early precancerous lesions without systemic toxicity however, loss of body weight gain and activation of immune cells were observed. In order to know more about the possible adverse effects, we evaluated the administration of TBLF on digestive and immune organs. Sprague Dawley rats were administered TBLF for six weeks and allowed to recover for two weeks. Immune activation was observed through an increased lymphocyte-granulocyte ratio, an increased number of lymphoid follicles in intestinal Peyer’s patches and a slight expansion of the splenic white pulp. Atrophy was observed in small intestine villi and crypt foci of the colon without normalization after the recovery period. Pancreas histopathology showed hypertrophy after the six-week administration period, particularly vacuolation and trabecular widening; but after the two-week recovery period atrophy was observed, suggesting a partial compensatory type process. Our results show that TBLF activates the immune system and affects digestive organs through direct interaction with intestinal epithelium, and indirectly by producing pancreatic hyperfunction. Further work will focus in longer recuperation periods after TBLF treatment.
... As lectins, WGA are widely recognized as anti-nutrients in foods and bind to glycoproteins, such as human N-Acetylneuraminic acid, on the surface of cell membranes. WGA has been shown to induce the release of pro-inflammatory cytokines (TNF-α, IL-1β, IL-12 and IFN-γ) and impair the integrity of the intestinal epithelial layer [45]. However, in contrast to ATIs, no immune stimulatory activity was demonstrated for WGA in vivo. ...
Article
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Gluten-related disorders have recently been reclassified with an emerging scientific literature supporting the concept of non-celiac gluten sensitivity (NCGS). New research has specifically addressed prevalence, immune mechanisms, the recognition of non-immunoglobulin E (non-IgE) wheat allergy and overlap of NCGS with irritable bowel syndrome (IBS)-type symptoms. This review article will provide clinicians with an update that directly impacts on the management of a subgroup of their IBS patients whose symptoms are triggered by wheat ingestion.
... There are increasing doubts among the scientific community regarding whether gluten is the trigger of NCG/WS; these doubts are supported by several studies [13,[18][19][20][21][22][23][24]. It has been suggested that other molecules could determine the onset of NCG/WS [25]; possible candidates are fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) [18], wheat amylase trypsin inhibitors (ATIs) [19][20][21], wheat germ agglutinin (WGA) [26,27] and exorphins [28]. ...
Article
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Non-coeliac/non-allergic gluten/wheat sensitivity (NCG/WS) is a gluten-related disorder, the pathogenesis of which remains unclear. Recently, the involvement of an increased intestinal permeability has been recognized in the onset of this clinical condition. However, mechanisms through which it takes place are still unclear. In this review, we attempt to uncover these mechanisms by providing, for the first time, an integrated vision of recent scientific literature, resulting in a new hypothesis about the pathogenic mechanisms involved in NCG/WS. According to this, the root cause of NCG/WS is a particular dysbiotic profile characterized by decreased butyrate-producing-Firmicutes and/or Bifidobacteria, leading to low levels of intestinal butyrate. Beyond a critical threshold of the latter, a chain reaction of events and vicious circles occurs, involving other protagonists such as microbial lipopolysaccharide (LPS), intestinal alkaline phosphatase (IAP) and wheat α-amylase trypsin inhibitors (ATIs). NCG/WS is likely to be a multi-factor-onset disorder, probably transient and preventable, related to quality and balance of the diet, and not to the presence of gluten in itself. If future studies confirm our proposal, this would have important implications both for the definition of the disease, as well as for the prevention and therapeutic-nutritional management of individuals with NCG/WS.
... Being the culturing of Caco-2 cells on porous membrane an optimum in vitro model of intestinal barrier, an upgrade of this model is the coculture of Caco-2 cells with other cell types; this setup has been used in several studies to measure the interplay between different cell types (15), therefore adopted to highlight how Caco-2 response to exogenous stimuli is modified by coculture with respect to Caco-2 cells alone. For example, the Caco-2 coculture setup is common in such studies aiming to identify the complex cross talk between with the immune system due to the presence of non-pathogenic bacteria (16). ...
Article
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Colorectal cancer is one of the most frequent type of cancer, with a higher incidence in the developed countries. Colorectal cancer is usually managed with both surgeries, chemotherapy and radiotherapy. Radiotherapy has the well-known advantage of targeting the tumor, minimizing normal tissue exposure. Nevertheless, during radiation treatment, exposure of healthy tissues is of great concern, in particular because of the effects on the intestinal barrier functions and on cells belonging to the immune system. The functional role of intestinal barrier in avoiding paracellular trafficking and controlling bacterial spread from gut it is well known and it is due to the presence of tight junction complexes. However, intestinal barrier is fundamental in participating to the interplay with immune system, especially considering the gut-associated lymphoid tissue. Until few years ago, radiotherapy was considered to bear only a depressive action on the immune system. However, it is now recognized that the release of pro-inflammatory signals and phenotypic changes in tumoral cells due to ionizing radiation could trigger the immune system against the tumor. In this work, we address how intestinal barrier functions are perturbed by X-ray doses in the range 0–10 Gy, focusing on the interplay between tumoral cells and the immune system. To this aim, we adopted a coculture model in which Caco-2 cells can be grown in presence/absence of peripheral blood mononuclear cells (PBMC). We focused our attention on changes in the proliferation, trans-epithelial electrical resistance (TEER), cytokine release, and proteins of the junctional complexes. Our results indicate a high radioresistance of Caco-2 in the investigated dose range, and an increased permeability of the tumoral cell layer due to the presence of PBMC. This is found to be correlated with activation of PBMC, inhibiting the apoptotic pathway, with the enhancement of cytokine release and with variation of tight junction scaffold protein expression levels, assumed to be related to IFN-γ- and TNF-α-mediated signaling.
... Wheat is the staple food for .35% of the global population (34). Published studies have suggested that WGA increases intestinal permeability (35). In the serum of healthy individuals, Abs to WGA have been found (36). ...
Article
Plant-derived dietary lectins have been reported to be involved in the pathogenesis of several inflammatory diseases, including inflammatory bowel disease, diabetes, rheumatoid arthritis, and celiac disease, but little is known about the molecular mechanisms underlying lectin-induced inflammation. In this study, we showed that plant lectins can induce caspase-1 activation and IL-1β secretion via the NLRP3 inflammasome. Lectins were internalized and subsequently escaped from the lysosome and then translocated to the endoplasmic reticulum. Endoplasmic reticulum-loaded plant lectins then triggered Ca(2+) release and mitochondrial damage, and inhibition of Ca(2+) release and mitochondrial reactive oxygen species by chemical inhibitors significantly suppressed NLRP3 inflammasome activation. In vivo, plant lectin-induced inflammation and tissue damage also depended on the NLRP3 inflammasome. Our findings indicate that plant lectins can act as an exogenous "danger signal" that can activate the NLRP3 inflammasome and suggest that dietary lectins might promote inflammatory diseases via the NLRP3 inflammasome.
... 27 In another study wheat germ agglutinin (WGA) contaminated with 1 ng mL −1 LPS was tested on CD33+ cells to appreciate the effect of endotoxin on the observed biological effects. 28 They concluded that endotoxin contamination at 1 ng mL −1 did not significantly influence the effect rendered by WGA. The low levels of endotoxins detected in our AXH samples support our suggested conclusion that the immunomodulatory effects observed in our research can be attributed to the effect of the AXH molecules rather than the trace amounts of endotoxins present in the samples. ...
Article
The use of plant derived polysaccharides as health promoters has gained immense interest in the past few years. Arabinoxylan (AX) is the predominant non-starch polysaccharide in cereals and grasses including wheat. The current research aimed to investigate the structure-function relationship of arabinoxylan hydrolyzates (AXH), obtained by the enzymatic hydrolysis of AX using xylanase and arabinofuranosidase as immunomodulators in two colon cancer cell lines: Caco-2 and HT-29. Fine structural details had a strong correlation with the immunological properties of the wheat AXH. As a general trend, as the presence of arabinose substitution increased in the AXH, the production of proinflammatory cytokines, IL-8 and TNF-α, decreased in both cell lines. Thus, AXH with a higher degree of arabinose substitution might be better adept in lowering inflammation in colon cancer cells.
... As far as the toxicity and immunogenicity concerns associated with most of the plant lectins, WGA the most studied plant lectin, however, has been shown to be toxic at micromolar concentrations, but at nanomolar concentrations, it shows less toxicity and anti-tumor effect on the gastrointestinal epithelium (Dalla Pellegrina et al., 2009). The cytotoxicity effects of the WGA, is depended on the internalization events which may differ among the cell lines used in the study, for example, in K562 cell lines its toxicity was observed to be minimum (Dalla Pellegrina et al., 2004). ...
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Lectins are proteins/glycoproteins that exhibit distinct sugar specificities and interact with cell surface molecules and have therefore being used as potential tools for drug delivery systems. In the present study we purified different sugar specific lectins by affinity chromatography. A glucose/mannose-specific lectin (DLL-I) as well as galactose-specific lectin (DLL-II) [from the seeds of Dolichos lablab], Lactose-Specific Lectin (LSL) [from the fresh water mussel Lamellidens corrianus] and Wheat Germ Agglutinin (WGA) [from wheat germ seeds] were affinity purified. Purified DLL-I migrated as a pentamer (α-1, α-2, α-3, α-4 in the range of 12-20 kDa and β subunit), DLL-II migrated as a dimer (α and β, 31 kDa and 29 kDa), LSL migrated as three subunit protein in SDS-PAGE (29 kDa, 28 kDa and 26 kDa) and WGA migrated as single subunit (18 kDa).Nanoparticles of these lectins were prepared and characterized for the size and shape by Transmission Electron Microscopy, Scanning Electron Microscopy and Atomic Force Microscopy and. The DLL-I, DLL-II, LSL and WGA nanoparticles sizes are in the range 90±20 nm, 150±20 nm, 145±10 nm and 75±15 nm respectively.
... Further, the in vitro experimental platform employs human cells rather than non-human mammalian systems and may therefore be more representative of the effects to occur in humans (Warren et al., 2015). Finally, in vitro human polarized IEC monolayers are of particular interest in the hazard assessment process for proteins because very few proteins result in adverse effects following oral exposure, but for those that do, the intestinal epithelium is often a target for damage (Dalla Pellegrina et al., 2005;Dalla Pellegrina et al., 2009;Gabor et al., 1998;Price et al., 2014;Lafont et al., 1988;Ramadass et al., 2010;Rossi et al., 1984;Weinman et al., 1989). ...
... Although Bin vitro^and Bin vivoŝ tudies demonstrated that ATIs react as an adjuvant in innate response of monocytes, macrophages, and dendritic cells activation through TLR4 complex [47, 48•], it remains to be established the role of ATIs in NCGS pathogenesis. WGA, which has epithelial-damaging and immune effects at very low doses at least in vitro [49,50], might also contribute to both intestinal and extraintestinal manifestations of NCGS but Bin vivo^effects are still questioned. ...
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Purpose of review: A new syndrome responding to gluten-free diet and defined non-celiac gluten sensitivity entered the spectrum of gluten-related disorders, together with celiac disease and wheat allergy. However, its definition, prevalence, diagnosis, pathogenesis, treatment, and follow up are still controversial. The purpose of the review is to summarize the evidence and problems emerging from the current literature. Recent findings: Direct implication of gluten in the onset of symptoms is often unproved as a low fermentable oligo-, di- and mono-saccharides and polyols diet or other components of cereals as wheat amylase trypsin inhibitor could be similarly involved. To date, no specific biomarkers or histological abnormalities confirm diagnosis, and only the self-reported response to gluten-free diet as well as a positive double blind placebo-gluten challenge characterizes these non-celiac, non-wheat allergic patients. Critical revision of published studies can offer practical indications in approaching this clinical topic and useful suggestions to standardize scientific researches.
... The binding of the targeting molecules to epithelial cells was investigated using Caco-2 cells, which exhibit a small intestinal epithelial phenotype and which have been widely used for investigating the potential of lectins as targeting molecules [27][28][29]. FITC-labeled NA bound to Caco-2 cells in a dose dependent manner (Fig. 1B) as analyzed by flow cytometry. In these experiments, biotinylated WGA and AAL revealed an even higher fluorescence intensity compared with NA, which might be explained by signal enhancement via the FITC-streptavidin-biotin complex in comparison to NA being FITC labeled alone. ...
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To improve current mucosal allergen immunotherapy Vibrio cholerae neuraminidase (NA) was evaluated as a novel epithelial targeting molecule for functionalization of allergen-loaded, poly(D,L-lactide-co-glycolide) (PLGA) microparticles (MPs) and compared to the previously described epithelial targeting lectins wheat germ agglutinin (WGA) and Aleuria aurantia lectin (AAL). All targeters revealed binding to Caco-2 cells, but only NA had high binding specificity to α-L fucose and monosialoganglioside-1. An increased transepithelial uptake was found for NA-MPs in a M-cell co-culture model. NA and NA-MPs induced high levels of IFN-γ and IL10 in naive mouse splenocytes and CCL20 expression in Caco-2. Repeated oral gavage of NA-MPs resulted in a modulated, allergen-specific immune response. In conclusion, NA has enhanced M-cell specificity compared to the other targeters. NA functionalized MPs induce a Th1 and T-regulatory driven immune response and avoid allergy effector cell activation. Therefore, it is a promising novel, orally applied formula for allergy therapy.
... Current data has shown that these lectins can increase intestinal permeability, through mechanisms still unknown. 31 ...
... Preliminary studies demonstrate that they can inhibit repair of gut epithelial cells and also stimulate the synthesis of proinflammatory cytokines leading to gastrointestinal symptoms [47,48]. ...
Article
Purpose of review: Gluten-free diets (GFDs) have seen a disproportional rise in use and popularity relative to the prevalence of established gluten-related disorders such as coeliac disease or immunoglobulin E wheat allergy. This entity has been termed noncoeliac gluten sensitivity (NCGS). This review aims to provide a current perspective on the emerging evidence for and against NCGS, along with the associated need for a GFD. Recent findings: NCGS and the benefits of a GFD are reported amongst patients with irritable bowel syndrome, inflammatory bowel disease, and nonintestinal disorders such as neuropsychiatric diseases and fibromyalgia. However, no reliable biomarkers currently exist to diagnose NCGS and hence confirmatory testing can only be performed using double-blind placebo-controlled gluten-based challenges. Unfortunately, such tests are not available in routine clinical practice. Furthermore, recent novel studies have highlighted the role of other gluten-based components in contributing to the symptoms of self-reported NCGS. These include fermentable oligo, di, mono-saccharides and polyols, amylase trypsin inhibitors, and wheat germ agglutinins. Therefore, NCGS is now seen as a spectrum encompassing several biological responses and terms such as 'noncoeliac wheat sensitivity' have been suggested as a wider label to define the condition. Summary: Despite the rising use of a GFD further studies are required to clearly establish the extent and exclusivity of gluten in NCGS.
... Preliminary studies demonstrate that they can inhibit repair of gut epithelial cells and also stimulate the synthesis of proinflammatory cytokines leading to gastrointestinal symptoms. 95,96 Therefore, given the current uncertainties regarding which gluten-based constituent is triggering symptoms, it is of some investigators' opinion that patients (particularly those who are AGA negative) should be informed that owing to the absence of diagnostic biomarkers, their condition can be considered as "self-reported" NCGS or noncoeliac wheat sensitivity, or "patients who avoid wheat and/or gluten". 41,97 When available, DBPC rechallenges should use specifically isolated gluten-based constituents, and ideally present them in capsulated form to help prevent any potential ambiguity. ...
Article
The past 5 years have seen an increase in the use of a gluten-free diet outside a diagnosis of coeliac disease or IgE-mediated wheat allergy. This trend has led to the identification of a new clinical entity termed noncoeliac gluten sensitivity (NCGS). In this Review, we discuss the evidence for NCGS as demonstrated by the results of double-blind, placebo-controlled dietary rechallenge studies. Furthermore, the characteristic phenotype of individuals with NCGS is described as well as the symptom manifestations commonly reported after gluten exposure, which include intestinal symptoms consistent with IBS, and extraintestinal symptoms such as neurological dysfunction, psychological disturbances, fibromyalgia and skin rash. Moreover, emerging evidence suggests that NCGS can be associated with organic gastrointestinal pathologies, such as IBD, in which its presence might be a reflection of severe or stricturing disease. However, NCGS is not without its controversies and uncertainties, in particular pertaining to whether it is gluten or nongluten components of the grain evoking symptoms; evidence suggests that fermentable carbohydrates, amylase trypsin inhibitors and wheat-germ agglutinin can also be responsible culprits. Finally, we discuss the novel techniques that might help diagnose NCGS in the future.
... In fact, oligosaccharides like fructans, contained in wheat and related grains, have been proven able to exert an osmotic effect in the intestinal lumen and increase gas production from bacterial fermentation [70,71] . Other plant proteins contained in wheat, such as lectins, agglutinins and amylasetrypsin inhibitors, may have a role in the development of symptoms after the ingestion of cereals by triggering the innate immune response [72][73][74] . For these reasons, and given the scattered data regarding the pathogenesis of NCGS, it has been suggested that the "non-celiac wheat sensitivity" definition may be more appropriate [75,76] . ...
Article
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Cereal crops and cereal consumption have had a vital role in Mankind's history. In the recent years gluten ingestion has been linked with a range of clinical disorders. Gluten-related disorders have gradually emerged as an epidemiologically relevant phenomenon with an estimated global prevalence around 5%. Celiac disease, wheat allergy and non-celiac gluten sensitivity represent different gluten-related disorders. Similar clinical manifestations can be observed in these disorders, yet there are peculiar pathogenetic pathways involved in their development. Celiac disease and wheat allergy have been extensively studied, while non-celiac gluten sensitivity is a relatively novel clinical entity, believed to be closely related to other gastrointestinal functional syndromes. The diagnosis of celiac disease and wheat allergy is based on a combination of findings from the patient's clinical history and specific tests, including serology and duodenal biopsies in case of celiac disease, or laboratory and functional assays for wheat allergy. On the other hand, non-celiac gluten sensitivity is still mainly a diagnosis of exclusion, in the absence of clear-cut diagnostic criteria. A multimodal pragmatic approach combining findings from the clinical history, symptoms, serological and histological tests is required in order to reach an accurate diagnosis. A thorough knowledge of the differences and overlap in clinical presentation among gluten-related disorders, and between them and other gastrointestinal disorders, will help clinicians in the process of differential diagnosis.
... This has led to the concept of NCGS being challenged although this study actually demonstrated a nocebo response to low-dose gluten, high-dose gluten, or whey protein which may be as a consequence of an anticipatory effect in view of the crossover design of the study rather than dismiss the effects of gluten (52) . Moreover, it has also been suggested that other proteins contained in wheat, such as lectins, agglutinins and amylase-trypsin inhibitors can trigger the innate immune response and therefore lead to the development of symptoms after ingestion of wheat (53)(54)(55) . For these reasons, it has been advised that in clinical practice it may be more appropriate to describe patients with NCGS using the preface 'selfreported', or alternatively use the term non-coeliac wheat sensitivity (56,57) . ...
Article
Mankind has existed for 2·5 million years but only in the last 10 000 years have we been exposed to wheat. Wheat was first cultivated in the Fertile Crescent (South Western Asia) with a farming expansion that lasted from about 9000BC to 4000BC. Thus it could be considered that wheat (and gluten) is a novel introduction to man's diet! Prior to 1939 the rationing system had already been devised. This led to an imperative to try to increase agricultural production. Thus it was agreed in 1941 that there was a need to establish a Nutrition Society. The very roots of the society were geared towards necessarily increasing the production of wheat. This goal was achieved and by the end of the 20th century, global wheat output had expanded 5-fold. Perhaps as a result the epidemiology of coeliac disease (CD) or gluten sensitive enteropathy has changed. CD is a state of heightened immunological responsiveness to ingested gluten in genetically susceptible individuals. CD now affects 1 % or more of all adults, for which the treatment is a strict lifelong gluten-free diet. However, there is a growing body of evidence to show that a far greater proportion of individuals without coeliac disease are taking a gluten-free diet of their own volition. This clinical entity has been termed non-coeliac gluten sensitivity (NCGS), although the condition is fraught with complexities due to overlap with other gluten-based constituents that can also trigger similar clinical symptoms. This review will explore the relationship between gluten, the rising prevalence of modern coeliac disease, and the new entity of NCGS along with its associated uncertainties.
... 31 Wheat germ agglutinin has also been shown to exert immune-mediated effects, which potentially lead to gastrointestinal symptoms. 32,33 Some investigators have proposed that a more appropriate term for NCGS might be nonceliac wheat sensitivity, 34 as it is a more inclusive term that might account for other components in wheat besides gluten that could contribute to symptoms. 35,36 In addition, a low-FODMAP diet has been shown to improve gastrointestinal symptoms in patients with functional bowel disorders. ...
Article
Full-text available
Gluten-related diseases such as celiac disease and gluten ataxia are rare conditions, affecting less than 1% of the population in the United States. Despite the rarity of these diseases, there have been significant increases in the adoption of a gluten-free lifestyle and the consumption of gluten-free foods in the United States over the last 3 decades. More than $15.5 billion were spent on retail sales of gluten-free foods in 2016. The gluten-free diet is driven by multiple factors, including social and traditional media coverage, aggressive consumer-directed marketing by manufacturers and retail outlets, and reports in the medical literature and mainstream press of the clinical benefits of gluten avoidance. Individuals may restrict gluten from their diets for a variety of reasons, such as improvement of gastrointestinal and nongastrointestinal symptoms, as well as a perception that gluten is potentially harmful and, thus, restriction represents a healthy lifestyle. Emerging evidence shows that gluten avoidance may be beneficial for some patients with gastrointestinal symptoms, such as those commonly encountered with irritable bowel syndrome. However, high-qualityevidence supporting gluten avoidance for physical symptoms or diseases other than those specifically known to be caused by immune-mediated responses to gluten is neither robust nor convincing. In fact, gluten avoidance may be associated with adverse effects in patients without proven gluten-related diseases. This article provides insight regarding gluten avoidance patterns and effects on patients without gluten-related diseases, and highlights concerns surrounding gluten avoidance in the absence of a gluten-mediated immunologic disease.
... Phytic acid complexes minerals important for human nutrition thereby limiting their bioavailability (Demir & Elgün, 2014;Gupta, Gangoliya, & Singh, 2015;Zajdel, Wilczok, Węglarz, & Dzierżewicz, 2013). Agglutinins, sugar binding proteins which increase intestinal permeability and might damage the gut lining, are also found in WG (Pellegrina et al., 2009;de Punder & Pruimboom, 2013). However, these anti-nutrients can be drastically reduced by WG stabilization (Matucci et al., 2004;de Punder & Pruimboom, 2013). ...
Article
Background Wheat germ is a precious by-product deriving from the milling industry, as it is a natural concentrated source of essential amino and fatty acids, minerals, vitamins, tocopherols, and phytosterols. However, the presence of high enzymatic activities together with a high content of unsaturated oil, induce a fast decrease in the nutritional value of wheat germ during storage and, consequently, strongly limit product's shelf-life. Scope and approach In recent decades, flour blends from raw or/and processed wheat germ received great interest from nutritional and technological perspectives. Nevertheless, the quality of the end-product strongly depended on the supplementation level, as well as the type and the severity of separation and stabilization techniques that wheat germ went through. Hence, in this review, the newest advances in wheat germ pre-handling approaches and food applications are discussed to provide relevant and updated information about its worthiness to be a part of the human diet. Key findings and conclusions To fully valorize and preserve the nutritious potential of wheat germ, effective pre-treatments of separation and stabilization are needed to guarantee its stability and suitability to meet food quality and safety standards. Such an underutilized ingredient might be a valuable fortifying component for a spectrum of foodstuffs.
... Prior reports on fermentation of wheat germ assumed that WGA is denatured during baking [45]; however, comparison of the thermal stability of WGA [21] with the crumb temperature during baking [57] suggests that WGA may not be fully denatured in all products. The impact of WGA on human health and specifically to NCWS remains controversial because adverse effects were observed in vitro [27][28][29], but suitable in vivo studies remain elusive [33,58]. Therefore, the reduction of the WGA content by sourdough fermentation reported in the present study is likely to add one piece to the large but rather incomplete puzzle that relates wheat components and sourdough fermentation to the (in)tolerance of wheat products in NCWS and irritable bowel syndrome. ...
Article
Full-text available
Non Celiac Wheat Sensitivity (NCWS) is an intolerance to wheat products and individuals with NCWS often adhere to a gluten free diet. However, gluten free diets are often associated with a reduced sensory and nutritional quality. Wheat Germ Agglutinin (WGA) is one of the wheat components linked to NCWS. This study explored the fate of WGA during sourdough fermentation. To assess the role of thiol-exchange reactions and proteolysis, sourdoughs were fermented with Fructilactobacillus sanfranciscensis DSM20451, F. sanfranciscensis DSM20451ΔgshR, which lacks glutathione reductase activity, or Latilactobacillus sakei TMW1.22, with or without addition of fungal protease. The conversion of WGA was determined by size exclusion chromatography of fluorescence-labeled WGA, and by enzyme-linked immunosorbent assay (ELISA). Commercial whole wheat flour contained 6.6 ± 0.7 μg WGA/g. After fermentation with L. sakei TMW1.22 and F. sanfranciscensis DSM20451, the WGA content was reduced (p < 0.05) to 2.7 ± 0.4 and 4.3 ± 0.3 μg WGA/g, respectively, while the WGA content remained unchanged in chemically acidified controls or in doughs fermented with F. sanfranciscensis DSM20451ΔgshR. Protease addition did not affect the WGA content. In conclusion, the fate of WGA during sourdough fermentation relates to thiol-exchange reactions but not to proteolytic degradation.
... For example, amylase trypsin inhibitors (ATIs), pest resistance molecules contained in the endosperm of wheat and related cereals, have been identified as strong activators of innate immune responses in human and murine macrophages, monocytes, and dendritic cells, eliciting the release of proinflammatory cytokines via the activation of toll-like receptor 4 (TLR4) [11]. In the same way, wheat germ agglutinins (WGA) have been shown to promote the release of pro-inflammatory cytokines, thus impairing the integrity of the intestinal epithelial layer [12]. Wheat contains also fructans-belonging to the category of FODMAPs-whose content varies according to the final product [13]. ...
Article
Full-text available
At least 40% of all the gastroenterological outpatient visits are due to functional gastrointestinal disorders (FGIDs), among which irritable bowel syndrome (IBS) is the most common, accounting for a worldwide prevalence of about 12% [...]
... Interestingly, it is well known that, beyond gluten proteins, other ingredients contained in wheat are able to trigger symptom appearance in patients affected by NCGS. Among these there are the Amylase-trypsin inhibitors, that activate the innate immune system on myeloid cells via Toll-like receptors 4 (TLR4), Wheat germ agglutinin, a lectin that protects wheat from insects, yeast and bacteria, and fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs) [56,61]. For this reason, some researchers proposed the term non-celiac wheat sensitivity (NCWS) instead of NCGS [62], as a more inclusive term, taking into account of the other substances present in the wheat, besides gluten, eliciting the activation of the immune system and consequently also the appearance of symptoms [62,63]. ...
Article
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A lifelong adherence to a gluten-free (GF) diet is currently the only treatment for Celiac disease (CD), an autoimmune disorder that arises after gluten ingestion in individuals who are genetically predisposed. The gluten intake exerts toxic effects through several pathways involving gut barrier integrity, intestinal microbiota composition and immune system stimulation. However, despite the great benefit of GF diet for CD patients, its use has been debated. Indeed, individuals who adopt this diet regime may be at risk of nutrient deficiencies. Emerging evidence supports a beneficial effect of a GF diet also for other pathological conditions, including gluten-related disorders (GRD) often associated to CD, such as Non celiac gluten sensitivity (NCGS) and Dermatitis Herpetiforme (DH) as well as Irritable bowel syndrome (IBS) and Diabetes. This suggests a pathogenic role of gluten in these conditions. Despite the growing popularity of GF diet among consumers, to date, there are limited evidences supporting its use for the management of non-celiac diseases. Therefore, in this review, we discuss whether the GF diet could really improve the general quality of life of patients with GRD and non-GRD conditions, keeping in mind its sensorial limitations and nutritional inadequacies. In addition, we discuss the current motivations, leading to the use of a GF diet, despite the inferior quality of GF products respect to those containing gluten.
... IL-8 was examined as a factor affecting intestinal barrier function, and the increased IL-8 secretion may be involved in the TEER decrease [237]. Similar results were reported by the authors of [238]. Consequently, exposure to certain mycotoxins, particularly DON, may cause damage to the intestinal integrity and lead to various chronic intestinal inflammatory diseases, such as inflammatory bowel disease [195]. ...
Article
Full-text available
The determination of mycotoxins content in food is not sufficient for the prediction of their potential in vivo cytotoxicity because it does not reflect their bioavailability and mutual interactions within complex matrices, which may significantly alter the toxic effects. Moreover, many mycotoxins undergo biotransformation and metabolization during the intestinal absorption process. Biotransformation is predominantly the conversion of mycotoxins meditated by cytochrome P450 and other enzymes. This should transform the toxins to nontoxic metabolites but it may possibly result in unexpectedly high toxicity. Therefore, the verification of biotransformation and bioavailability provides valuable information to correctly interpret occurrence data and biomonitoring results. Among all of the methods available, the in vitro models using monolayer formed by epithelial cells from the human colon (Caco-2 cell) have been extensively used for evaluating the permeability, bioavailability, intestinal transport, and metabolism of toxic and biologically active compounds. Here, the strengths and limitations of both in vivo and in vitro techniques used to determine bioavailability are reviewed, along with current detailed data about biotransformation of mycotoxins. Furthermore, the molecular mechanism of mycotoxin effects is also discussed regarding the disorder of intestinal barrier integrity induced by mycotoxins.
... 27 In another study wheat germ agglutinin (WGA) contaminated with 1 ng mL −1 LPS was tested on CD33+ cells to appreciate the effect of endotoxin on the observed biological effects. 28 They concluded that endotoxin contamination at 1 ng mL −1 did not significantly influence the effect rendered by WGA. The low levels of endotoxins detected in our AXH samples support our suggested conclusion that the immunomodulatory effects observed in our research can be attributed to the effect of the AXH molecules rather than the trace amounts of endotoxins present in the samples. ...
Article
The use of plant derived polysaccharides as health promoters has gained immense interest in the past few years. Arabinoxylan (AX) is the predominant non-starch polysaccharide in cereals and grasses including wheat. The current research aimed to investigate the structure–function relationship of arabinoxylan hydrolyzates (AXH), obtained by the enzymatic hydrolysis of AX using xylanase and arabinofuranosidase as immunomodulators in two colon cancer cell lines: Caco-2 and HT-29. Fine structural details had a strong correlation with the immunological properties of the wheat AXH. As a general trend, as the presence of arabinose substitution increased in the AXH, the production of proinflammatory cytokines, IL-8 and TNF-α, decreased in both cell lines. Thus, AXH with a higher degree of arabinose substitution might be better adept in lowering inflammation in colon cancer cells.
... Co-culture models of Caco-2 cells with other cell lines have been proposed, including studies on their interactions with the immune system (10). These latter have been mainly focused on the response of Caco-2 to exogenous stimuli and how this is modified in presence of cells of the immune system (11,12). In our previous work (13) we adopted a co-culture model of Caco-2 cells and PBMCs from healthy donors. ...
Article
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In vitro co-culture models between tumor cells and peripheral blood mononuclear cells (PBMCs) allow studying the interplay between these cell populations, potentially gaining insight into the in vivo response of the immune system to the presence of the tumor, as well as to possible other agents as radiation used for therapeutic purposes. However, great care is needed in the experimental optimization of models and choice of conditions, as some setups might offer a limited possibility to capture subtle immune perturbations. A co-culture model of PBMCs from healthy donors and colorectal adenocarcinoma Caco-2 cells was successfully adopted in a previous work to measure effects on Caco-2 and modulation of signaling when these latter are irradiated. We here tested if the same experimental setting allows to measure perturbations to the main PBMC subsets: we performed immunophenotyping by means of flow cytometry and quantified helper and cytotoxic T cells, NK cells, and B cells, when PBMCs are cultured alone (control), in presence of non-irradiated Caco-2 cells or when these latter are exposed to a 10 Gy X-ray dose from a conventional radiotherapy accelerator. To measure a baseline response in all experimental conditions, PBMCs were not further stimulated, but only followed in their time-evolution up to 72 h post-irradiation of Caco-2 and assembly of the co-culture. In this time interval PBMCs maintain a high viability (measured via the MTT assay). Caco-2 viability (MTT) is slightly affected by the presence of PBMCs and by the high radiation dose, confirming their radioresistance. Immunophenotyping results indicate a large inter-individual variability for different population subsets already at the control level. We analyzed relative population changes and we detected only a small but significant perturbation to cytotoxic T cells. We conclude that this model, as it is, is not adequate for the measurements of subtler immune perturbations (if any, not washed-out by inter-individual differences). For this purpose, the model needs to be modified and further optimized e.g., including a pre-treatment strategy for PBMCs. We also performed a pooled analysis of all experimental observations with principal component analysis, suggesting the potential of this tool to identify subpopulations of similarly-responding donors.
... The inverse relation between whole grain intake and inflammation had been consistently reported in many observational studies, [17,41,42] but its interpretation remained controversial. First of all, it was reported that gluten and wheat germ agglutinin (WGA) had strong effect in activating pro-inflammatory immune system by increasing intestinal permeability, [43,44] and inducing specific T-cell, [45] but whole grain products contained phytochemicals, like polyphenols, that can exert anti-inflammatory effects by possibly offsetting the effect of gluten and WGA. [46] Secondly, whole grain were known to a source of short-chain fatty acids (SCFA) metabolized by gut microbiota, [46] which had linked to secretion of gut hormones, [47] immune homeostasis, [48] and eventually led to whole grain-duced improvements on low-grade systematic inflammation. ...
Article
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Backgrounds: Observational studies had suggested an inverse association between whole grain consumption and concentration of inflammatory markers, but evidence from interventional studies was inconsistent. Therefore, we conducted a meta-analysis of randomized trials to have a better understanding of this issue. Methods: This study has been registered in PROSPERO (ID: CRD42018096533). We searched PubMed, Web of Science, Embase, Medline, and Cochrane Library for articles focusing on the topic from inception to 1 January, 2018. Summary standardized mean difference (SMD) and 95% confidence interval (CI) were calculated by using either random effect model or fixed effect model according to the heterogeneity of included studies. Subgroup analysis was also performed. Results: Totally 9 randomized trials included 838 participants were identified. In a pooled analysis of all studies, consumption of whole grains had an inverse association with inflammatory markers (SMD 0.16, 95% CI, 0.02-0.30), including C-reactive protein (CRP), Interleukin-6 (IL-6), Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β). Specific analyses for CRP and IL-6 yielded that whole grain diet was related with a significant decrease in the concentration of CRP (SMD 0.29, 95% CI, 0.08-0.50) and IL-6 (SMD 0.19, 95% CI, 0.03-0.36). Conclusions: The evidence suggested that citizens could benefit from increased whole grain intake for reducing systemic inflammation. Further well-designed studies are required to investigate the mechanism under the appearance.
... While the diet's role in celiac disease is well understood, the mechanism behind any utility in IBD is less clear. Potential mechanisms for benefit in IBD may be similar to those proposed for non-celiac gluten sensitivity, such as immune system activation by amylase-trypsin inhibitors and/or wheat germ agglutinin [29][30][31]. There is also the hypothesis that gliadin interferes with cellular tight junction proteins, thus leading to increased intestinal permeability, bacterial translocation, and activation of the innate immune response [32]. ...
Article
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Purpose of review Patients with inflammatory bowel disease (IBD) frequently look to diet to improve symptoms. Although regularly asked for dietary guidance, gastroenterologists are often unprepared to provide evidence-based recommendations. This review will summarize popular diets claiming benefits in IBD, as well as current data evaluating their efficacies. Recent findings The Specific Carbohydrate Diet has demonstrated symptom improvement and even mucosal healing; however, large trials and prospective data are lacking. The low FODMAP diet has shown benefit for functional symptoms in IBD, yet efficacy regarding inflammation is lacking. Large studies for the gluten-free diet yielded mixed results in IBD outcomes, while suggesting a negative impact on psychological well-being. Data on an “anti-inflammatory” diet were positive but remain severely limited. A currently planned large trial for the Mediterranean diet in IBD may provide much needed clinical data. Summary We provide an overview of frequently utilized diets in IBD. The body of evidence does not currently support clear dietary recommendations in IBD, as larger, prospective studies are needed.
... How this diet may benefit IBD patients is less clear [99]. A possible mechanism may involve the inactivation of the immune system by amylase-trypsin inhibitors (proteins found in wheat and commercial gluten) and/or wheat germ agglutinin, as in NGCS [139][140][141], but gliadin might also increase intestinal permeability, translocation of bacteria and immune response interfering with epithelial tight junctions [142]. Further, the gluten-free diet also involves low FODMAPs consumption, with the consequent possible benefits already outlined for that approach [139,140]. ...
Article
Full-text available
Inflammatory bowel disease (IBD) is a chronic relapsing–remitting systemic disease of the gastrointestinal tract, characterized by an inflammatory process that requires lifelong treatment. The underlying causes of IBD are still unclear, as this heterogeneous disorder results from a complex interplay between genetic variability, the host immune system and environmental factors. The current knowledge recognizes diet as a risk factor for the development of IBD and attributes a substantial pathogenic role to the intestinal dysbiosis inducing an aberrant mucosal immune response in genetically predisposed individuals. This review focused on the clinical evidence available that considers the impact of some nutrients on IBD onset and the role of different diets in the management of IBD and their effects on the gut microbiota composition. The effects of the Specific Carbohydrate Diet, low fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAP) diet, gluten free diet, anti-inflammatory diet and Mediterranean diet are investigated with regard to their impact on microbiota and on the evolution of the disease. At present, no clear indications toward a specific diet are available but the assessment of dysbiosis prior to the recommendation of a specific diet should become a standard clinical approach in order to achieve a personalized therapy.
... (Grant, Ewen et al., 1989;Pan et al., 2013;Pan, Zhao et al., 2018b;Zhao et al., 2014), affecting cellular morphology, metabolism and proliferation (Pan et al., 2017). Several studies reported that the supplementation of SBA in intestinal epithelial cells decreased the TEER value, hence affecting membrane permeability (Dalla Pellegrina et al., 2009Pan et al., 2013). Similarly, we observed significantly decrease in TEER value in the positive control group, that is, IPEC-J2 cells supplemented with 0.5 mg/ml SBA without pre-treatment with GalNAc. ...
Article
Soya bean agglutinin (SBA) is a glycoprotein and the main anti‐nutritional component in most soya bean feedstuffs. It is mainly a non‐fibre carbohydrate‐based protein and represents about 10% of soya bean‐based anti‐nutritional effects. In this study, we sought to determine the effects of N‐Acetyl‐D‐galactosamine (GalNAc or D‐GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC‐J2) cells. The IPEC‐J2 cells were pre‐cultured with 0, 0.125 × 10−4, 0.25 × 10−4, 0.5 × 10−4, 1.0 × 10−4 and 2.0 × 10−4 mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre‐incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans‐epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre‐treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin‐3 were lower in the SBA‐treated groups without pre‐treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin‐3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre‐treated groups, occludin and claudin‐3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC‐J2s.
... We have developed a repeated exposure protocol to investigate a polarized human intestinal epithelial barrier in vitro method for protein hazard characterization using proteins known to produce adverse effects following oral exposure often resulting in damage to the intestinal epithelium (Dalla Pellegrina et al., 2009;Lafont et al., 1988;Lorenzsonn and Olsen, 1982;Nusrat et al., 2001;Pusztai et al., 1993;Ramadass et al., 2010;Rossi et al., 1984;Weinman et al., 1989). Studies to date have demonstrated that the addition of hazardous proteins to human IEC monolayers for 24, 48 and 72 h results in compromised monolayer integrity and in some cases overt cytotoxicity, while the addition of non-hazardous proteins has little effect on the IEC monolayers. ...
Article
Full-text available
Recent studies suggest human-derived intestinal epithelial cell (IEC) lines cultured as polarized monolayers on permeable Transwell® filters are effective at differentiating between hazardous and non-hazardous proteins following a single exposure. In this study, IEC polarized monolayers were subjected to hazardous or non-hazardous proteins in nine exposures over 30 days and compared to a single exposure of the same protein. The objective was to evaluate whether repeated exposures to a protein differently alter barrier integrity or compromise cell viability compared to single exposures. Proteins tested included Clostridium difficile toxin A, Streptolysin O, Wheat Germ Agglutinin, Phaseolus vulgaris Hemagglutinin-E, bovine serum albumin, porcine serum albumin, and fibronectin. Evidence of diminished barrier integrity and/or cell viability following exposure to hazardous proteins was more pronounced in magnitude when IECs were subjected to multiple rather than single exposures. In some cases, an effect on IEC monolayers was observed only with repeated exposures. In general, IEC responses to non-hazardous proteins following either single or repeated exposures were minimal. Results from these studies support the utility of using cultured human IEC polarized monolayers to differentiate between hazardous and non-hazardous proteins and suggest that repeated exposures may reveal a greater magnitude of response when compared to single exposures.
... Murine peritoneal macrophages responded with high proinflammatory cytokine secretion to WGA stimulation in vitro (TNF-α, IL-1β, IL-12, and IFN-γ) [30]. Similarly, stimulation of isolated human PBMC with minimal amounts of WGA elicited the release of proinflammatory cytokines [31]. These results indicate that WGA have a high potential to initiate or maintain inflammatory responses. ...
Article
Several disorders related to the ingestion of gluten are well recognized despite overlapping clinical presentations: celiac disease, an autoimmune enteropathy triggered by gluten ingestions in susceptible individuals, allergy to wheat, and more recently non-celiac gluten sensitivity (NCGS). While celiac disease and wheat allergy are well-known disorders with a clear-cut diagnosis based on clinical tests and biological parameters, NCGS is a more difficult diagnosis, especially in children with functional gastrointestinal (GI) complaints. NCGS is considered a syndrome of intestinal but also extraintestinal symptoms occurring within hours, but sometimes even after several days of gluten ingestion. In children, the leading symptoms of NCGS are abdominal pain and diarrhea, while extraintestinal symptoms are rare, in contrast to adult patients. No precise diagnostic test nor specific biomarkers exist, except a rather cumbersome three-phase gluten-exposure, gluten-free diet, followed by a blinded placebo-controlled gluten challenge with crossover to provoke symptoms elicited by gluten in a reproducible manner that disappear on gluten-free alimentation. Recent data indicate that the peptide part of wheat proteins is not necessarily the sole trigger of clinical symptoms. Mono- or oligosaccharides, such as fructan and other constituents of wheat, were able to provoke GI symptoms in clinical trials. These new findings indicate that the term gluten sensitivity is probably too restrictive. The incidence of NCGS was reported in the range of 1-10% in the general population and to increase steadily; however, most data are based on patients' self-reported gluten intolerance or avoidance without a medically confirmed diagnosis. Treatment consists of gluten avoidance for at least several weeks or months. Patients with NCGS require regular reassessment for gluten tolerance allowing with time the reintroduction of increasing amounts of gluten.
... WGAs [19], similar to ATIs, serve as protective proteins as they are resistant to heat and proteolysis. WGAs have shown to promote the production of pro-inflammatory cytokines, which affect the integrity of the intestinal epithelium [21]. Finally, FODMAP-containing foods include such components as oligosaccharides, disaccharides, monosaccharides, and sugar alcohols. ...
Article
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It is unclear whether patients with non-celiac gluten sensitivity (NCGS) can tolerate gluten. We have evaluated the changes of both gastrointestinal symptoms and quality of life for NCGS patients after the re-introduction of dietary gluten. Twenty-two NCGS patients reporting functional gastroenterological symptoms and on gluten-free diet (GFD) for the previous three weeks were exposed to incremental gluten-containing diets. Three groups were compared at baseline (immediately after 3-weeks on GFD) and immediately after the return of symptomatology: (i) a group tolerating a low-gluten diet (3.5 g gluten/day, week 1, n = 8), (ii) a group tolerating a mid-gluten diet (8 g gluten/day, week 2, n = 6), and (iii) a group tolerating a high-gluten diet (13 g gluten/day, week 3, n = 8). Their gastrointestinal symptoms and quality of life were assessed at baseline and post-intervention. The most common symptoms were: constipation (46%), abdominal pain (50%) and dyspepsia (38%). A decrease in several short form health survey (SF-36) sub-scores (all p < 0.03) after gluten re-introduction was only observed in the group tolerating the low-gluten diet; the same group showed a lower post-intervention role-emotional SF-36 score (p = 0.01). Most gastrointestinal symptoms remained similar after gluten re-introduction. However, a decrease in the general perception of well-being was only found after gluten re-introduction in the group tolerating a low-gluten diet (p = 0.01); the same was true when comparing the post-intervention general well-being perception among the three groups (p = 0.050). In conclusion, dissimilar responses from patients with NCGS were observed after the re-introduction of gluten, with gluten at a low dosage affecting the quality of life and general well-being of a group of patients, whereas others tolerate even higher doses of dietary gluten.
Chapter
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Italy, due to its orography, is characterized by agricultural activities in Park areas and with high environmental value. In these areas, agriculture is identified above all in the production of "niche", which enjoy a rare reputation and often constitute one of the main economic resources for territory. Verifying the relationship within economic, social and environmental sustainability, is the research objective, realized on three products chosen for their typical assets: Provola delle Madonie (Sicily), Colline Teatine Olive Oil (Abruzzo) and Monreale’s Bread (Sicily). The research activities is carried out using different research methods from quantitative and empirical ones, to qualitative ones: on-site information research conducted by other scientific subjects such as universities and statistical data, focus groups with local actors, entrepreneurs and local institutions, interviews. The entire production chain was analyzed to verify its economic, environmental and social sustainability (in terms of safeguarding employment, maintaining local skills, safeguarding identity and heritage of local knowledge and traditions) and the importance from the cultural point of view as an element of territorial identity. The collaboration between public actors and local productive forces is the way to ensure concrete strategies to give value to rural territories, characterized by natural, environmental and cultural emergencies better expressed in traditional quality productions. The research carried out, lead us to affirm that the three products investigated are, undeniably, classifiable as high-quality products, with still very strong potentials and territorial value, although with many light and darkness linked to different factors, first of all the production chain and the market.
Article
The terpyridine appended glucosyl-conjugate ligand L and its complexes of Zn ²⁺ (1) and Cu ²⁺ (2) were synthesized and characterized by spectroscopy techniques. The changes in the absorption and fluorescence spectra of L in presence of Zn ²⁺ and Cu ²⁺ is supportive of interaction of L through terpyridyl metal binding core which was further confirmed by ¹ H NMR. The coordination and structural features emerged from the spectroscopy were used in order to optimize the structures of 1 and 2 by DFT computational calculations. The isolated complex 2 shows binding with ct-DNA through minor groove binding and intercalation modes and increases the thermal stability of ct-DNA by ∼20 °C. Due to the presence of Cu ²⁺ in 2, this complex showed cleavage of the plasmid pBR322 through hydroxy radical formation. The presence of glucosyl moiety at both the ends of the complex 1 interacts with the WGA and imparts dendrimeric-like micro structures due to the protein aggregation as demonstrated by SEM.
Chapter
In a clinical context, the question of what to eat is one of the most commonly asked by patients and also, at least as inflammatory bowel disease (IBD) is concerned, among the most difficult to answer by physicians. In published guidelines for management of IBD, dietary advice plays only a minor part, while public guidance, as provided by professional bodies, varies and is often based on consensus rather than on evidence [1]. In the last few years, excellent reviews of the epidemiological, clinical, and experimental works addressing the role of nutrition in IBD have been published [2–8]. The aim of this chapter is to revise the emerging knowledge, introduce some ideas and new data from recent research, and explain the authors’ point of view.
Chapter
The ingestion of the wheat products causes inconvenience or even major disturbances for a limited segment of the population because of wheat intolerance or celiac disease. Unfortunately, because of the marketing interests, a crusade has tried to convince the people that tolerate the wheat products to adhere to avoid to wheat products and to consume expensive substitutes with marked inferior nutritious quality.
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Noncoeliac gluten sensitivity (NCGS) has gained attention as an emerging clinical entity. Data regarding the epidemiology, pathogenesis, and management of NCGS are scattered in view of the diagnostic uncertainty surrounding the disorder. We aim to provide a current perspective of NCGS and its associated controversies. NCGS consists of a spectrum of intestinal and extraintestinal symptoms related to the ingestion of gluten-containing food, yet in the absence of coeliac disease or wheat allergy. To date, no specific biomarker exists for NCGS, thereby leaving the diagnosis to be confirmed by dietary elimination followed by double-blind placebo-controlled gluten-based rechallenges. Unfortunately, this technique is cumbersome, not readily-available in routine clinical practise, and can still leave the diagnosis of NCGS open to debate as to whether the effects demonstrated can be specifically attributed to the gluten-protein per se or rather coexisting nongluten components, such as fermentable carbohydrates and amylase-trypsin inhibitors. Physicians are increasingly being posed with the dilemma of patients presenting with self-reported NCGS. However, this appears to be the tip of the iceberg and future studies are in need of delineating which gluten-based component is responsible for each individual patient's complaint.
Article
While it is well documented and widely appreciated that ingestion of wheat (and less so rye and barley) is associated with gastrointestinal symptoms such as bloating or abdominal pain, the component of wheat to which such an effect is attributed is less well established. Key Messages: Wheat is a complex of proteins (80% gluten, 20% metabolic proteins), carbohydrates (starch, non-starch polysaccharides, fructans), lipids and other components. The majority of attention has focused on gluten as the culprit in triggering symptoms, but re-challenge studies have nearly all used wheat flour-related products (such as bread) as the stimulus. When carbohydrate-deplete gluten was used as the challenge agent, gluten-specific feelings of depression and not gut symptoms were observed in those who fulfilled strict criteria of 'non-coeliac gluten sensitivity', thereby underlining the complexity of cereals and of undertaking research in this area. Candidate components other than gluten include poorly absorbed oligosaccharides (mainly fructans), non-gluten wheat proteins such as amylase-trypsin inhibitors or wheat germ agglutinin, and exorphins released during the digestion of gluten. Specific biological and/or clinical effects associated with gluten-free diets or wheat ingestion need to be carefully dissected before attribution to gluten can be claimed. Currently, coeliac disease is the only common condition that has been unequivocally linked to gluten. Inaccurate attribution will be associated with suboptimal therapeutic advice and at least partly underlies the current gluten-free epidemic gripping the Western world. © 2015 S. Karger AG, Basel.
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The consumption of wheat-based products is high in most West- ern countries (e.g. Europe, United States) and is increasing in East- ern countries as a consequence of a shift toward a Western lifestyle. Historically, Wheat-Related Disorders (WRD) referred only to wheat allergy and celiac disease. In recent years, a growing number of sub- jects worldwide have reported intestinal and extraintestinal symp- toms, without the diagnostic features of celiac disease or wheat al- lergy. In these subjects, symptoms of a wheat-related disorder may originate from non-celiac gluten sensitivity, the non-gluten antibody target protein serpins, purinins, α-amylase/protease inhibitors, glob- ulins, farinins, wheat exorphin sensitivity, wheat germ agglutinin sen- sitivity, wheat amylase-trypsin inhibitor sensitivity, and/or FODMAP (fermentable oligo-, di-, mono- saccharides and polyols) sensitivity. This article reviews the pertinent literature and presents three pediat- ric cases of wheat-related disorders that present with varied clinical presentations: liver failure, type 1 diabetes mellitus and a conjuncti- val tumor believed to be Kaposi’s sarcoma. All conditions responded rapidly to a wheat-free diet. A wheat-related disorder (with or without celiac disease) may affect any organ or system including cardio- vascular disease, neurological diseases, connective tissue diseas- es, allergies, in ammatory bowel disease, nephritis and others. While there is increasing awareness that wheat-related disorders can manifest with extra-intestinal symptoms, clinicians still rely too heavily on the presence of gastrointestinal symptoms to suspect a wheat-related disorder. These three cases showed wheat-related pathogenesis affecting diverse organs, such as the liver, pancre- as, or eye, without marked gastrointestinal symptoms. Therefore, in patients with unexplained symptoms, who do not improve with stan- dard therapies, it may be prudent to screen for serological indicators of a WRD. Clinical awareness of the diversity of presentations of a WRD may lead to earlier suspicion, investigation and a reduction in co-morbidity.
Article
Wheat-related disorders have become a growing area of clinical and scientific interest and can be categorized broadly as: autoimmune-mediated; allergic; and non-autoimmune/non-allergic conditions. Non-celiac gluten sensitivity (NCGS) and non-celiac wheat sensitivity (NCWS) present on this spectrum as disorders associated with adverse gastrointestinal and extra-intestinal manifestations following exposure to gluten and/or other wheat-related constituents. NCGS/NCWS is increasingly considered in patients with unexplained symptoms after the exclusions of celiac disease and wheat allergy. As objective diagnostic data and specific biomarkers are lacking, response to a gluten-free/wheat-free diet can confirm the presence of NCGS/NCWS. An association with irritable bowel syndrome has been detected, and the effects of other food components, such as fermentable oligosaccharides, disaccharides, monosaccharides, and polyols, may contribute. Our organization and synthesis of extant knowledge pertaining to wheat-related disorders may advance current practice and research efforts toward an improved understanding of NCGS/NCWS as an evolving clinical entity.
Article
The Tepary bean (Phaseolus acutifolius) lectin fraction (TBLF) exhibits differential cytotoxicity on colon cancer cells and inhibition of early tumorigenesis in the colon (50 mg/kg, three times per week, for 6 weeks). TBLF showed low toxicity with the ability to activate the immune system; however, some adverse effects are the loss in body weight gain, intestinal atrophy, and pancreatic hyperplasia. After a recovery period of 2 weeks after treatment, reversion of pancreatic hyperplasia but no recovery of intestinal atrophy was observed. As TBLF has shown anticancer effects on the colon, it is important to characterize the adverse effects and how they can be reversed. Sprague Dawley rats were administered with TBLF (50 mg/kg) for 6 weeks, three times per week, and then allowed to recover for 6 weeks post-treatment. After TBLF administration, small intestine atrophy, villus atrophy, and cryptic hyperplasia were confirmed, as well as increased intestinal mucus production, increased permeability and a decrease in the apparent ileal digestibility of crude proteins. The colon showed damage in the simple prismatic tissue and decreased crypt depth, and changes in microbiota and a decrease in the apparent fecal digestibility of crude protein were determined. Our results show that the adverse effects provoked by TBLF were partially reversed after 6 weeks of recovery post-treatment, suggesting that increasing the recovery period it could be possible to reverse all adverse effects observed.
Technical Report
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Die Diagnostik getreideassoziierter Nahrungsmittelunverträglichkeiten wie Zöliakie, IgE-vermittelter Weizenallergie oder Weizensensitivität stellt in der täglichen Praxis aufgrund überlappender Beschwerdebilder eine besondere Herausforderung dar. Bei vielen Betroffenen stehen dabei Symptome eines Reizdarmsyndroms so stark im Vordergrund, dass nicht selten die korrekte Diagnose über Jahre verzögert wird. Eine valide Abgrenzung der Ursachen sowie eine exakte Identifizierung der die Unverträglichkeit auslösenden Getreidebestandteile sind daher Voraussetzung für ein erfolgreiches Therapieregime.
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Incorporation of N-acetylglucosamine-specific agglutinins from wheat germ (Triticum aestivum; WGA), thorn apple (Datura stramonium) or nettle (Urtica dioica) rhizomes in the diet at the level of 7 g/kg reduced the apparent digestibility and utilization of dietary proteins and the growth of rats, with WGA being the most damaging. As a result of their binding and endocytosis by the epithelial cells of the small intestine, all three lectins were growth factors for the gut and interfered with its metabolism and function to varying degrees. WGA was particularly effective; it induced extensive polyamine-dependent hyperplastic and hypertrophic growth of the small bowel by increasing its content of proteins, RNA and DNA. Furthermore, an appreciable portion of the endocytosed WGA was transported across the gut wall into the systemic circulation, where it was deposited in the walls of the blood and lymphatic vessels. WGA also induced the hypertrophic growth of the pancreas and caused thymus atrophy. Although the transfer of the gene of WGA into crop plants has been advocated to increase their insect resistance, as the presence of this lectin in the diet may harm higher animals at the concentrations required to be effective against most pests, its use in plants as natural insecticide is not without health risks for man.
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The in vitro production of interleukin-1beta (IL-1beta), IL-6, and the IL-1 receptor antagonist (IL-1ra) in whole blood upon stimulation with different bacterial strains was measured to study the possible relationship between disease severity and the cytokine-inducing capacities of these strains. Escherichia coli, Neisseria meningitidis, Neisseria gonorrhoeae, Bacteroides fragilis, Capnocytophaga canimorsus, Staphylococcus aureus, Enterococcus faecalis, Streptococcus pneumoniae, and Streptococcus pyogenes induced the cytokines IL-1beta, IL-6, and IL-1ra. Gram-negative bacteria induced significantly higher levels of proinflammatory cytokine production than gram-positive bacteria. These differences were less pronounced for the anti-inflammatory cytokine IL-1ra. In addition, blood was stimulated with E. coli killed by different antibiotics to study the effect of the antibiotics on the cytokine-inducing capacity of the bacterial culture. E. coli treated with cefuroxime and gentamicin induced higher levels of IL-1beta and IL-6 production but levels of IL-1ra production similar to that of heat-killed E. coli. In contrast, ciprofloxacin- and imipenem-cilastatin-mediated killing showed a decreased or similar level of induction of cytokine production as compared to that by heat-killed E. coli; polymyxin B decreased the level of production of the cytokines.
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Based on the observation that pathogen-derived lectins play an important role in cell adhesion and invasion, we examined the possible role of host carbohydrate-bearing molecules in inducing the secretion of IL-12, a crucial proinflammatory cytokine. The ability of 12 plant lectins to recognize and stimulate naive murine mononuclear cells in vitro has been characterized in this study. Mitogenic lectins (comprising Con A, PHA, PSA, and LCA) were found to induce the secretion of multiple cytokines in vitro, including IL-2, interferon (IFN)-gamma, and IL-12. Of interest, WGA, a nonmitogenic lectin unable to promote IL-2 secretion, was found to induce IL-12 and IFN-gamma production in a T and B cell-independent fashion. The functional properties of WGA were inhibited by N-acetylneuraminic acid and N,N'-diacetylchitobiose. WGA therefore represents a potentially useful tool for the study of membrane glycoproteins involved in the early proinflammatory response characteristic of innate immunity.
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Wheat germ agglutinin (WGA), a lectin with specificity for N-acetylglucosamine and sialic acid, was investigated with respect to its ability to activate the NADPH-oxidase of in vivo-exudated neutrophils (obtained from a skin chamber), and the activity was compared to that of peripheral blood neutrophils. The exudate cells responded to WGA, by both releasing reactive oxygen species into the extracellular milieu and producing oxygen metabolites intracellularly. The peripheral blood cells were unresponsive. To mimic the in vivo-exuded neutrophils with regards to receptor exposure, peripheral blood neutrophils were induced to mobilize their granules and vesicles to varying degrees (in vitro priming), prior to challenge with WGA. The oxidative response to WGA increased with increasing levels of granule mobilization, and the receptor(s) could be shown to reside in the secretory vesicles and/or the gelatinase granules in resting neutrophils. Several WGA-binding glycoproteins were detected in subcellular fractions containing these organelles. The extra- and intracellular NADPH-oxidase responses showed differences in sialic acid dependency, indicating that these two responses are mediated by different receptor structures.
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The CD14 receptor is a pattern recognition molecule in the innate immune response against microorganisms and other exogenous and endogenous stress factors. The most important CD14 signalling co-receptor is toll-like receptor 4 (TLR4), which activates, among others, the nuclear factor kappaB (NF-kappaB) inflammatory pathway. Besides its role in innate immunity and host defence, the proinflammatory cytokines expressed upon TLR4/NF-kappaB pathway activation exert proatherogenic effects. The CD14 C(-260)T promoter and TLR4 Asp299Gly functional polymorphisms have been recently implicated in the development of cardiovascular events, suggesting that the genetically determined inflammatory response against pathogens or their antigens may have a major role in atherogenesis and subsequent acute events. Is the association of these polymorphisms with cardiovascular disease more evidence for the implication of infection, especially by Gram negative bacteria, in the development of acute coronary events? This article reviews the molecular basis, biological functions, and clinical implications of the CD14/TLR4 polymorphisms in the development of cardiovascular events.
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Here we describe the protocols for negative or reverse detection of proteins, nucleic acids and lipopolysaccharides separated in polyacrylamide electrophoresis gels. These protocols are based on the selective synthesis and precipitation of a white imidazole-zinc complex in the gel, which is absent from those zones where biomolecules are located. These methods are highly sensitive (1-10 ng of biomolecules per band), very cheap as they use inexpensive, common laboratory reagents (imidazole and a Zn II salt), rapid (less than 20 min after gel washing), robust and simple (two steps). Reverse-stained biomolecules are reversibly fixed in the gel. After brief incubation in a zinc chelating agent, biomolecules can be recovered from the gel with the same efficiency as from unstained gels. In consequence, they are procedures of choice for micropreparative applications. References covering typical applications are included.
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The effect of formyl chemotactic peptide (fCTP, fMet-Leu-Phe), beta-amyloid peptides (beta-AP, 1-42, 1-16 and 25-35), and bradykinin (BK) on functional activity of phagocytic cells has been investigated. Wheat germ agglutinin (WGA) was also used as a model membrane binding agent of polypeptide nature. Murine monocyte-macrophage cell line J774.2 and normal human blood polymorphonuclear (PMN) cells were used as target phagocytic cells. Their activity was quantitatively estimated by measuring phagocytosis of killed yeast cells. Beta-AP (1-41) maximally stimulated phagocytosis at 0.1 microg/ml, BK--at 1.0 microg/ml, and fCTP--at 2.0 microg/ml. Beta-AP (1-16) and beta-AP (25-35) were inactive in used test-systems. Phagocytosis-inducing activity of beta-AP (1-42) and BK reached maximal levels in 2 h and decreased after 4-6 h of incubation. Phagocytosis numbers were compared with the indicators of phagocytic cell activation, such as absorption of neutral red dye, glucose utilization, production of super-oxide anion (NBT-test) and nitrite accumulation (indicator of NO production). NBT-test, which may be related to the killing ability of phagocytic cells towards the ingested objects, was positive only in stimulated PMN leukocytes, while the nitrite accumulation was detected only in stimulated macrophages. Nitrite accumulation in macrophages was markedly induced by lipopolysaccharide and to a lower extent by 0.5 microg/ml beta-AP (1-42). In high dose (5.0 microg/ml) beta-AP suppressed nitrite accumulation in macrophages stimulated by lipopolysaccharide. Other studied peptides were inactive in inducing nitrite accumulation. Transforming growth factor type beta suppressed phagocytic activity of PMN cells activated by beta-AP or WGA. The anti-inflammatory drugs (indomethacin and L-lysine aescinate) inhibited beta-AP (1-42)-induced phagocytosis. The interrelations between the regulatory pathways of BK, beta-AP and fCTP are discussed.
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Interleukin-1 (IL-1) is a polypeptide that is produced following infection, injury, or antigenic challenge. Although the macrophage is a primary source of IL-1, epidermal, epithelial, lymphoid, and vascular tissues synthesize IL-1. When IL-1 gains access to the circulation, it induces a broad spectrum of systemic changes in neurologic, metabolic, hematologic, and endocrinologic systems. However, because IL-1 lacks a signal peptide, a considerable amount of the IL-1 that is synthesized may remain associated with the cell, particularly as part of the plasma membrane; moreover, membrane-associated IL-1 is biologically active, especially in its ability to participate in lymphocyte activation and mesenchymal tissue remodeling. There are two gene products coding for IL-1:IL-1-beta and IL-1-alpha. The spectrum of biologic activities of IL-1 are induced by both forms. IL-1 activates lymphocytes and plays an important role in the initiation of the immune response. Receptors for IL-1 recognize both forms, but receptors are scare and their affinities often do not match the potency of the biologic response. The most consistent property of IL-1 is up-regulation of cellular metabolism and increased expression of several genes coding for biologically active molecules. IL-1 is a highly inflammatory molecule and stimulates the production of arachidonic acid metabolites. IL-1 also acts synergistically with other cytokines, particularly tumor necrosis factor. The multitude of biologic responses to IL-1 is an example of the rapid adaptive changes that take place to increase the host's defensive mechanisms.
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Flow cytometry is increasingly used for cytokine detection where it serves to complement ELISA (enzyme-linked immunosorbent assay) and ELISPOT assays. Since it is possible to stain both extracellular epitopes and intracellular cytokines on the same cells, this is a powerful technique for analysing cytokine expression in defined cell populations. However unstimulated cells do not express cytokines. Thus, appropriate stimulation is a prerequisite for studying cytokine expression. Here phorbol 12-myristate 13-acetate (PMA)/ionomycin in vitro stimulation has been applied. In order to accumulate the cytokines within the cells, protein secretion needs to be inhibited, by the addition of reagents that inhibit protein secretion during the stimulation. The two most widely used reagents are monensin and brefeldin A (BFA). These reagents differ somewhat in their mode of action, which might explain their different effects. Monensin is an inhibitor of trans-Golgi function, while BFA inhibits protein transport between the endoplasmic reticulum (ER) and the Golgi. CD69, a very early activation marker on lymphocytes and neutrophils, was monitored in order to measure the efficacy of the protein secretion inhibition. Here we report that: (a) BFA, but not Monensin, is able to completely block extracellular CD69 expression on mice splenocytes after in vitro stimulation with PMA/ionomycin; (b) Monensin is more toxic than BFA and increases the relative amount of CD4+ cells due to a more profound increase in dead cells in the CD4− population; (c) CD69 is a useful marker when setting up intracellular staining of cytokines for flow cytometry.
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When injected intravenously into humans and animals, interleukin-1β (IL-1β) is perhaps the most potent of the endogenous pyrogens. However, IL-1β is initially synthesized as a relatively inactive precursor molecule (proIL-1β) which lacks a signal peptide and hence remains inside the cell. To be active as a fever-producing molecule, proIL-1β must first be processed to an active mature molecule and secreted. Although several enzymes associated with inflammatory tissues are capable of processing proIL-1β into an active molecule in the extracellular compartment, the IL-1β converting enzyme (ICE, also called caspase-1) cuts intracellular proIL-1β after the aspartic acid residue in position 116, resulting in a highly active mature IL-1β that is secreted into the extracellular space. IL-18 is also initially synthesized as an inactive precursor molecule (proIL-18) lacking a signal peptide. IL-18 is a member of the IL-1 family, and like IL-1β, proIL-18 is cleaved by ICE to yield an active molecule. However, unlike IL-1β, IL-18 is not an endogenous pyrogen following intraperitoneal injection into mice. Nevertheless, IL-18 may contribute to inflammation and fever because IL-18 is a potent inducer of tumor necrosis factor, chemokines, and interferon-γ production.
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Cadherin-mediated cell-cell adhesion is perturbed in protein tyrosine kinase (PTK)-transformed cells. While cadherins themselves appear to be poor PTK substrates, their cytoplasmic binding partners, the Arm catenins, are excellent PTK substrates and therefore good candidates for mediating PTK-induced changes in cadherin behavior. These proteins, p120ctn, β-catenin and plakoglobin, bind to the cytoplasmic region of classical cadherins and function to modulate adhesion and/or bridge cadherins to the actin cytoskeleton. In addition, as demonstrated recently for β-catenin, these proteins also have crucial signaling roles that may or may not be related to their effects on cell-cell adhesion. Tyrosine phosphorylation of cadherin complexes is well documented and widely believed to modulate cell adhesiveness. The data to date, however, is largely correlative and the mechanism of action remains unresolved. In this review, we discuss the current literature and suggest models whereby tyrosine phosphorylation of Arm catenins contribute to regulation or perturbation of cadherin function.
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It is now generally accepted that macromolecules, including food proteins, can and do cross the mature small intestinal epithelium and reach the systemic circulation. In most instances the extent of this absorption is small and nutritionally not significant. However, from the points of view of immune reactions, hormonal or toxic effects or as a potential delivery route for orally taken macromolecular drugs, the systemic absorption of even small amounts of intact proteins and other macromolecules may have far-reaching consequences. There are a number of possible routes of uptake from the small intestine. These include transport through the membranous epithelium of Peyer's patches, where antigen sampling by and presentation to the gut immune system occurs. At higher luminal concentrations the importance of the more general transepithelial absorption routes may increase. Thus, the extent of uptake by passive transport through aqueous pores, lipid bilayers of membranes and/or paracellular/intercellular routes may become appreciable, particularly in the damaged mucosa. Moreover, and much more importantly, transport by receptor-mediated endocytosis also increases, particularly when either the receptors or the ligands are lectins. Because of their resistance to gut proteolysis, binding to brush-border membranes and interference with the limitation on antigen uptake normally imposed by the local immune system, appreciable amounts of lectins and/or toxins of the general structure of A (toxin)-B(lectin), either free or included in liposomes, may be taken up by and transported through the epithelial cells of the small intestine. Thus oral drug applications based on lectins either as receptors or ligands may now be ready for experimental exploration.
Article
To further investigate the interaction between human mononuclear leucocytes [peripheral blood mononuclear cells (PBMC)] and enterocytes, the effect of a confluent layer of differentiated CACO-2 cells on cytokine kinetics during challenge with bacteria in a compartmentalized coculture model was investigated. Nonpathogenic Escherichia coli were added either to the apical or the basolateral compartment of this transwell cell culture system, the latter of which contained human leucocytes. The synthesis of tumour necrosis factor (TNF-alpha) and interleukin (IL)-12 was significantly suppressed by CACO-2 cells when leucocytes were stimulated directly with bacteria. This suppression was not paralleled by changes in the production of IL-10, IL-6 and transforming growth factor (TGF)-beta. When the bacteria were applied apically to the CACO-2 cell layer, the production of TNF-alpha, IL-12, IL-1beta, IL-8, IL-6, IL-10, TGF-beta and interferon-gamma was pronouncedly lower as compared to the bacterial stimulation of leucocytes beneath the CACO-2 cells. In the latter experiments, IL-6, IL-8 and TNF-alpha were the cytokines being mostly induced by apical addition of E. coli. Quantitative mRNA expression analysis revealed that IL-8 gene expression was equally induced in both CACO-2 and PBMC after apical stimulation with bacteria. Of note, bacteria-stimulated CACO-2 cells produced little or no cytokines in the absence of leucocytes, supporting the concept of leucocyte-epithelial cell cross-talk in modulating cytokine responses in the gut mucosa.
Article
Wheat germ agglutinin (WGA) is low mitogenic or nonmitogenic for human T lymphocytes and inhibits phytohemagglutinin (PHA)-induced mitotic response of the lymphocytes. In this study, the effect of WGA was analyzed in terms of interleukin 2 (IL2) production, expression of IL2 receptor, and IL2 responsiveness of the T lymphocytes. WGA as well as PHA could induce IL2 mRNA and IL2 production and also elevate cytoplasmic free Ca2+ concentration. The IL2 production was reduced by inhibitors of calmodulin and protein kinase C. The IL2 receptor (Tac) expression was induced at about 20% of the lymphocytes by WGA and the expression induced by PHA was not blocked by the addition of WGA. The lymphocytes precultured with WGA for 3 days could proliferate by the addition of IL2 after removal of WGA. The IL2-dependent proliferation of PHA-blasts was blocked by the addition of WGA. These results indicate that WGA inhibits T lymphocyte proliferation by inhibiting the responsiveness of the lymphocytes to IL2 but not by interfering with IL2 production and IL2 receptor expression.
Article
Wheat germ agglutinin (WGA) inhibits proliferation of human peripheral blood mononuclear cells (PBMC) induced by mitogens and antigens. We investigated the mechanism by which WGA inhibits PHA-induced human lymphocyte proliferation with regard to the interleukin pathway. Our data revealed that although PBMC-proliferation was markedly suppressed by WGA, levels of IL 2 activity in WGA-inhibited cultures were not reduced, but instead were increased, suggesting failure to utilize IL 2. Furthermore, the addition of exogenous IL 2 failed to overcome the suppression. Consistent with these observations, culturing PBMC with PHA plus WGA markedly decreased the number of high-affinity IL 2 receptor per cell, as determined by binding of purified [3H]IL 2, relative to cultures containing PHA alone. WGA immobilized on support beads bound detergent-solubilized IL 2 receptors from PHA-activated T cells, but did not bind human IL 2. However, WGA did not competitively block the binding of [3H]IL 2 to PHA-induced lymphoblasts. These results suggest that WGA inhibits lymphocyte proliferation by binding to and decreasing the number of high-affinity IL 2 receptors displayed on T cells, without impairing IL 2 production.
Article
Wheat germ agglutinin (WGA), previously regarded strictly as a nonmitogenic or anti-mitogenic lectin, can under appropriate conditions markedly stimulate in vitro synthesis and secretion of immunoglobulin (Ig) by human B lymphocytes. Stimulation of Ig production by WGA is 1) confined to a narrow lectin dose range (2 to 10 micrograms/ml; 2) abrogated by the simple sugar N-acetyl-D-glucosamine but not by a variety of other monosaccharides; 3) effective only after early additions of WGA within the initial 72 hr of 12-day cultures; 4) detected in the presence of B and T cells but not B cells alone; and 5) polyisotypic in nature, as indicated by augmented synthetic rates of Ig in each of 3 major classes (IgG, IgA, and IgM). With few exceptions, WGA produces equivalent or greater rates of Ig production as obtained in cultures activated with pokeweed mitogen (PWM), a well-recognized T-dependent polyclonal activator of human B cells. Furthermore, periperal blood lymphocytes from select individuals that respond weakly to PWM are markedly stimulated with WGA. In contrast to these stimulatory effects of WGA on Ig production by lymphocytes exposed to low lectin concentrations, addition of WGA in amounts greater than 15 micrograms/ml to PWM-stimulated human lymphocyte cultures produces marked suppression of the expected level of Ig synthesis. These data indicate that varying doses of WGA can produce contrasting stimulatory and inhibitory effects on human B cell metabolism.
Article
We have used biochemical and morphological techniques to demonstrate that hepatocytes in the perfused liver bind, internalize, and degrade substantial amounts of murine epidermal growth factor (EGF) via a receptor-mediated process. Before ligand exposure, about 300,000 high-affinity receptors were detectable per cell, displayed no latency, and co-distributed with conventional plasma membrane markers. Cytochemical localization using EGF coupled to horseradish peroxidase (EGF-HRP) revealed that the receptors were distributed along the entire sinusoidal and lateral surfaces of hepatocytes. When saturating concentrations of EGF were perfused through a liver at 35 degrees C, ligand clearance was biphasic with a rapid primary phase of 20,000 molecules/min per cell that dramatically changed at 15-20 min to a slower secondary phase of 2,500 molecules/min per cell. During the primary phase of uptake, approximately 250,000 molecules of EGF and 80% of the total functional receptors were internalized into endocytic vesicles which could be separated from enzyme markers for plasma membranes and lysosomes on sucrose gradients. The ligand pathway was visualized cytochemically 2-25 min after EGF-HRP internalization and a rapid transport from endosomes at the periphery to those in the Golgi apparatus-lysosome region was observed (t 1/2 approximately equal to 7 min). However, no 125I-EGF degradation was detected for at least 20 min. Within 30 min after EGF addition, a steady state was reached which lasted up to 4 h such that (a) the rate of EGF clearance equaled the rate of ligand degradation (2,500 molecules/min per cell); (b) a constant pool of undegraded ligand was maintained in endosomes; and (c) the number of accessible (i.e., cell surface) receptors remained constant at 20% of initial values. By 4 h hepatocytes had internalized and degraded 3 and 2.3 times more EGF, respectively, than the initial number of available receptors, even in the presence of cycloheximide and without substantial loss of receptors. All of these results suggest that EGF receptors are internalized and that their rate of recycling to the surface from intracellular sites is governed by the rate of entry of ligand and/or receptor into lysosomes.
Article
Tight junction permeability control is important in a variety of physiological and pathological processes. We have investigated the role of tyrosine phosphorylation in the regulation of tight junction permeability. MDCK epithelial cells and brain endothelial cells were grown on filters and tight junction permeability was determined by transcellular electrical resistance (TER). The tyrosine phosphatase inhibitor pervanadate caused a concentration- and time-dependent decrease in TER in both MDCK and brain endothelial cells. However, as expected, pervanadate resulted in the tyrosine phosphorylation of many proteins; hence interpretation of its effects are extremely difficult. Phenylarsine oxide, a more selective tyrosine phosphatase inhibitor, caused the tyrosine phosphorylation of relatively few proteins as analyzed by immunoblotting of whole cell lysates. This inhibitor, like pervanadate, also elicited a decrease in TER in the two cell types. In the MDCK cells, the action of phenylarsine oxide could be reversed by the subsequent addition of the reducing agent 2,3-dimercaptopropanol. Immunocytochemistry revealed that phenylarsine oxide rapidly stimulated the tyrosine phosphorylation of proteins associated with intercellular junctions. Because of the known influence of the adherens junction on tight junctions, we analyzed immunoprecipitates of the E-cadherin/catenin complex from MDCK cells treated with phenylarsine oxide. This revealed an increase in the tyrosine phosphorylation of beta-catenin, but not of alpha-catenin. However, the tight junction associated protein ZO-1 was also tyrosine phosphorylated after PAO treatment. These data indicate that tight junction permeability may be regulated via mechanisms involving tyrosine phosphorylation of adherens junction and tight junction proteins.
Article
To investigate the usefulness of wheat germ agglutinin as a targeting carrier protein for an acid-labile chemotherapeutic prodrug directed against colon carcinoma cells in vitro. Cis-aconityl-linked doxorubicin-wheat germ agglutinin was prepared by a two step procedure and the conjugate-binding capacity of target- and non-target cells was assayed by flow cytometry. The antiproliferative activity of the prodrug on Caco-2 and MOLT-4 cells was determined by the XTT- and BrdU-test and compared with that of the parent drug and the lectin alone. At pH 4.0, about 50% of the conjugated doxorubicin were released within 24 h from the water soluble prodrug exhibiting a conjugation number of 24 (mol doxorubicin/mol WGA). The prodrug-binding capacity of colon carcinoma cells exceeded that of human colonocytes and lymphoblastic MOLT-4 cells 4.5-fold. Additionally, the antiproliferative effect of the conjugate on Caco-2 cells was 39% as opposed to 5% in case of MOLT-4 cells. As the unmodified carrier protein inhibited or stimulated Caco-2 cell growth in a concentration-dependent manner, the cytostatic activity of the conjugate was determined at WGA concentrations without an effect on cell-proliferation. Considering 50% release of conjugated drug at the most, the prodrug yielded 160% of the cytostatic activity of free doxorubicin. WGA-prodrug targeting offers new perspectives for site-specific, cytoinvading drug delivery in colon cancer chemotherapy.
Article
The biochemical features that distinguish human M cells from other intestinal epithelial cell types are important for understanding microbial pathogenesis and for targeting vaccines to the mucosal immune system. We applied a large panel of carbohydrate-specific monoclonal antibodies and lectins to Peyer's patch and cecum biopsy specimens from three normal individuals and a patient with inflammatory bowel disease. The results show that human M-cell glycosylation patterns are distinct from those of other species examined and that human M cells preferentially display the sialyl Lewis A antigen. This carbohydrate epitope is also present in a small subpopulation of enterocytes in the follicle-associated epithelium and in goblet cell mucins.
Article
Mucosal immunization with soluble protein Ag alone may induce Ag-specific tolerance, whereas mucosal immunization with Ag in the presence of a mucosal adjuvant may induce Ag-specific systemic and mucosal humoral and cell-mediated immune responses. The most widely used and studied mucosal adjuvant is cholera toxin (CT). Although the mechanism of adjuvanticity of CT is not completely understood, it is known that CT induces mucosal epithelial cells to produce the proinflammatory cytokines IL-1, IL-6, and IL-8 and up-regulates macrophage production of IL-1 and the costimulatory molecule B7.2. Because IL-1 may duplicate many of the activities of CT, we evaluated IL-1alpha and IL-1beta for their ability to serve as mucosal adjuvants when intranasally administered with soluble protein Ags. IL-1alpha and IL-1beta were as effective as CT for the induction of Ag-specific serum IgG, vaginal IgG and IgA, systemic delayed-type hypersensitivity, and lymphocyte proliferative responses when intranasally administered with soluble protein Ag. Our results indicate that IL-1alpha and IL-1beta may be useful as mucosal vaccine adjuvants. Such an adjuvant may be useful, and possibly required, for vaccine-mediated protection against pathogens that infect via the mucosal surfaces of the host such as HIV.
Article
Flow cytometry is increasingly used for cytokine detection where it serves to complement ELISA (enzyme-linked immunosorbent assay) and ELISPOT assays. Since it is possible to stain both extracellular epitopes and intracellular cytokines on the same cells, this is a powerful technique for analysing cytokine expression in defined cell populations. However unstimulated cells do not express cytokines. Thus, appropriate stimulation is a prerequisite for studying cytokine expression. Here phorbol 12-myristate 13-acetate (PMA)/ionomycin in vitro stimulation has been applied. In order to accumulate the cytokines within the cells, protein secretion needs to be inhibited, by the addition of reagents that inhibit protein secretion during the stimulation. The two most widely used reagents are monensin and brefeldin A (BFA). These reagents differ somewhat in their mode of action, which might explain their different effects. Monensin is an inhibitor of trans-Golgi function, while BFA inhibits protein transport between the endoplasmic reticulum (ER) and the Golgi. CD69, a very early activation marker on lymphocytes and neutrophils, was monitored in order to measure the efficacy of the protein secretion inhibition. Here we report that: (a) BFA, but not Monensin, is able to completely block extracellular CD69 expression on mice splenocytes after in vitro stimulation with PMA/ionomycin; (b) Monensin is more toxic than BFA and increases the relative amount of CD4+ cells due to a more profound increase in dead cells in the CD4- population; (c) CD69 is a useful marker when setting up intracellular staining of cytokines for flow cytometry.
Article
Recent experiments by a number of workers have suggested that it may be possible to use various targeting molecules, which bind to the intestinal epithelium, to promote the uptake and transport of nanoparticles from the intestine to the circulation. We have used commercial nanoparticles to examine the effect of size, density and inhibitors on uptake of lectin-coated nanoparticles by epithelial cells. The degree of uptake was most influenced by the density of lectin on the particle, with size and type of lectin being less important. Uptake could be inhibited by the presence of specific sugars or free lectin. These studies should provide a good basis for the design of targetable biodegradable drug-loadable particles suitable for oral delivery.
Article
Previous studies found that monolayers formed from canine oxyntic epithelial cells in primary culture displayed remarkable resistance to apical acidification and both mitogenic and migratory responses to epidermal growth factor (EGF) treatment. In our present studies, we found that EGF increased transepithelial resistance (TER) but not short-circuit current in these monolayers. Parallel effects of EGF on decreasing mannitol flux and increasing TER implicate direct regulation of paracellular permeability. EGF acting at either apical and basolateral receptors rapidly increased TER, but the apical response was sustained whereas the basolateral response was transient. (125)I-labeled EGF binding revealed specific apical binding, but receptor numbers were 25-fold lower than on the basolateral surface. Both apical and basolateral EGF activated tyrosine phosphorylation of EGF receptors (EGFR), beta-catenin, and cellular substrate as evident on confocal microscopy. Although apical EGF activated a lesser degree of receptor autophosphorylation than basolateral EGF, phosphorylation of beta-catenin was equally prominent with apical and basolateral receptor activation. Together, these findings indicate that functional apical and basolateral EGFR exist on primary canine gastric epithelial cells and that these receptors regulate paracellular permeability. The sustained effect of apical EGFR activation and prominent phosphorylation of beta-catenin suggest that apical EGFR may play a key role in this regulation.
Article
The trend towards an increased consumption of minimally processed plant food results in a higher intake of non-nutritive compounds such as lectins. Lectins are typically globular proteins that are resistant to digestion in the gastrointestinal tract. They affect the integrity of the intestinal epithelium and the absorption of dietary antigens, and induce the release of allergic mediators from mast cells in vitro. Based on this information we have studied whether dietary wheat germ agglutinin (WGA) could be involved in triggering food allergies. Brown Norway rats were immunized intraperitoneally using ovalbumin (OVA; 10 microg/rat) and 10 d later treated for five consecutive days with WGA (10 mg/rat per d) administered intragastrically. Rats were then orally challenged with OVA (100 microg/rat) 1 h after the last WGA application, and blood was collected 4 h later. Immunological responses (anti-OVA immunoglobulins E and G, rat mast cell protease II, interferon-gamma and lymphocyte proliferation) were measured and lymphocyte subpopulations were determined. In immunized rats WGA treatment resulted in increased serum rat mast cell protease II concentrations (pre-challenge 0.26 (SE 0.08) microg/ml, post-challenge 0.49 (SE 0.09) microg/ml; P < 0.01) 4 h after the OVA challenge. After 5 d serum concentrations of anti-OVA immunoglobulin E were significantly increased only in the immunized controls (absorbance at 405 nm on days 14 and 19 was 0.09 (SE 0.008) and 0.24 (SE 0.046) respectively; P = 0.02), while in WGA-treated rats no significant increase was seen (0.08 (SE 0.004) and 0.15 (SE 0.037 respectively; P = 0.14). CD4+ : CD8+ T lymphocytes in the spleen was significantly increased at this time (OVA 1.1 (SD 0.2), 1.4 (sd 0.1), P < 0.05). The treatment did not impair the proliferation and interferon-gamma production of mesenteric lymphocytes. In conclusion, these data suggest that high dietary intake of lectins such as WGA may affect the allergic response towards oral antigens in the gut-associated lymphoid tissue.
Article
To examine whether the dietary lectin wheat germ agglutinin (WGA) can facilitate binding and uptake of protein drugs due to its cytoadhesive and cytoinvasive properties, conjugates were prepared by covalent coupling of fluorescein-labeled bovine serum albumin (F-BSA) to WGA using divinylsulfone for crosslinking. Increasing the molar ratio of F-BSA/WGA resulted in 2.6-8.7 times higher Caco-2 binding as compared with glycyl-F-BSA. About 75% of F-BSA-WGA were bound specifically to Caco-2 cells according to inhibition studies in presence of the complementary carbohydrate. The Caco-2 association of F-BSA-WGA was temperature-dependent indicating active uptake of membrane bound conjugate, which was confirmed by confocal microscopy. The conjugate accumulated within lysosomal compartments followed by proteolytic degradation of F-BSA-WGA 1-4 h after conjugate loading as observed by equilibrating the intracellular pH with monensin. Finally low molecular weight degradation products of the proteinaceous prodrug appear in the extracellular medium. Contrary to Caco-2 single cells, a minor part of the conjugate is degraded by brush border proteases already 30 min after exposure to Caco-2 monolayers. But most of the conjugate is taken up into differentiated cells and processed as in single cells. Though the enzymic barrier remains to be surmounted, WGA-mediated drug delivery is a promising strategy for peroral delivery of even high molecular weight drugs to overcome the mucosal barrier.
Article
An immunoenzymatic method for the quantitative determination of dietary lectin activities employing immobilized glycoproteins was studied. Lectins from wheat germ (WGA), peanut (PNA), and jack bean (ConA) were added to microtiter plates coated with ovalbumin or asialofetuin and quantified by enzyme-linked immunosorbent assay (ELISA) with lectin-specific antibodies. ELISA responses for lectin activity were dose-dependent in the concentration range 30-1000 ng/mL for WGA and 80-1000 ng/mL for both PNA and ConA. Inhibition assays carried out with different saccharides confirmed that the binding of lectins to immobilized glycoproteins was specific. The proposed method is specific and sensitive, allowing the quantitative determination of lectin activities on raw samples by simple dilution of the extracts. Examples of application to wheat germ and roasted peanut extracts are reported.
Article
Bioactive molecules that can gain access to body tissues through the gastrointestinal tract may interact with immune regulatory circuits and effector functions. Among these are plant lectins, such as wheat germ (WG) agglutinin, which constitute common components of the human diet and target the immune system on a daily basis. Dietary bioactive molecules might be considered as immunomodulatory signals. To investigate the possible effects on the immune system of the long-term absence of such signals, two groups of rats were fed on a diet containing or deprived of WG. The WG-deprived diet induced a state of functional unresponsiveness in lymphocytes from primary and secondary lymphoid organs, as evaluated by in vitro stimulation with T cell mitogen phytohemoagglutinin (PHA) and B cell mitogen lypopolysaccarides (LPS). The unresponsive state of the immune cells could be reversed by injection of antigen emulsified in oil with inactivated mycobacteria (complete Freund's adjuvant, CFA) Dietary signals can thus interact with the immune system possibly influencing its shaping during ontogenesis.
Article
Hen eggs represent an easily available and inexpensive source of glycoproteins expressing a variety of sugars. Egg glycoproteins might therefore be exploited to purify by affinity chromatography carbohydrate-binding proteins (lectins) with different specificities. A method to generate an affinity matrix from hen eggs is described. The matrix was assayed for its ability to purify in a single step biologically active phytohemagglutinin, wheat germ agglutinin, lentil lectin, and peanut agglutinin. Milligrams of purified lectins per gram of matrix was obtained, with the only exception of peanut agglutinin that was not efficiently retained into the affinity column. Hen egg chromatography is a relatively simple, fast, and reproducible method to purify high amount of plant lectins.
Article
The relationships between degree of lectin-cell binding, cytotoxicity and cytoagglutinating activity of three Wheat Germ Agglutinin isolectins (WGA-1, WGA-2, WGA-3) against normal lymphocytes and cultured leukemic cell lines (Jurkat, MOLT-4, Raji, Daudi, K-562) were studied. All WGA-isolectins interacted in a similar degree with normal lymphocytes, while in the case of leukemic cells, the degree of isolectin-cell binding increased in the order: WGA-1< or =WGA-3<WGA-2 at isolectin concentrations 0.5 microM and higher, and WGA-3<WGA-2< or =WGA-1 at 0.25 microM isolectin concentration. The WGA interacted in higher degree with Jurkat, Raji, Daudi and K-562, followed by MOLT-4 and normal lymphocytes. The velocity of cytoagglutination in the presence of 0.25 microM WGA-isolectins increased in the order: WGA-3<WGA-2< or =WGA-1, and was better expressed in Jurkat, Raji, Daudi and K-562, followed by MOLT-4 and normal lymphocytes. The cytotoxicity of isolectins was very well expressed against Jurkat, MOLT-4, Raji and Daudi, and less expressed against K-562 and normal lymphocytes. In the case of leukemic cells, the cytotoxic effect of WGA-isolectins increased in the order: WGA-3<WGA-2=WGA-1. A very good positive correlation was determined between velocity of cytoagglutination and degree of lectin-cell binding (r=0.77, P<0.001). A good inverse correlation was found between cytotoxicity and degree of lectin-cell binding (r=-0.34, P<0.001), and poor correlation was observed between cytotoxicity and cytoagglutinating activity of WGA-isolectins (r=0.16, P<0.01). The results suggest that the WGA-isolectins, structurally distinguishable in only several amino acid sequences, interacted in different degrees with leukemic cells and manifested different cytoagglutinating and cytotoxic activity.
Article
Lectin-mediated drug delivery may become a promising strategy to improve the efficacy of poorly permeable drugs by utilising active high-capacity transport pathways of epithelial tissues. This requires the elucidation of the basic mechanisms of lectin uptake prior to their practical use. We studied the interaction between the dietary lectin wheat germ agglutinin (WGA) and Caco-2 cells (single cells and monolayers) by a newly established assay design that is able to discriminate between cellular binding and uptake as well as by confocal microscopy: (i) All binding sites available for WGA at the cell membrane were occupied within 10 min of incubation. (ii) Cytoadhesion was followed by immediate uptake. After 20 min, 60% (single cells) or 30% (monolayers) of the membrane bound lectin were internalised. However, regardless of cell arrangement, 80% of the surface bound lectin was taken up into the cells during the course of the experiment. (iii) About 50% of the internalised lectin accumulated within the lysosomes after 1 h. This was confirmed by assays in the presence of monensin, an inhibitor of endosomal acidification, and by colocalisation with lysosomal cathepsin followed by semiquantitative image analysis. Further analysis by immunocytochemistry suggested that the trans-Golgi complex and the caveoli were not involved. Due to cytoadhesion, cytoinvasion and partial lysosomal accumulation, WGA-mediated drug delivery may provide for improved intracellular availability of conjugated drugs or colloidal carrier systems.
Article
The gut associated immune system fences off potentially harmful intestinal antigens from the systemic circulation and induces systemic tolerance against luminal antigens. Intestinal immune responses against luminal antigens include IgA secretion and induction of regulatory cells. Unlike few other cytokines, lymphotoxin alpha/beta regulates the development of intestinal lymphoid organs. The embryonic development of Peyer's patches, postnatal lamina propria B cell development, and isolated lymphoid follicle development all depend on lymphotoxin beta receptor interactions. Lymphotoxin alpha/beta signalling also contributes to the development of mesenteric lymph nodes. In addition, intestinal inflammation is suppressed by inhibition of lymphotoxin beta signalling, an observation which has initiated clinical studies using this treatment principal. Intestinal follicular lymphoid organs are sites of antigen presentation. Antigen presenting cells tune the delicate balance between intestinal immune tolerance and inflammation. Therefore, gut associated lymphatic organs and factors regulating their development are critical for the prevention of adverse immune reactions to intestinal antigens. This review provides an overview on the role of lymphotoxin and the gut associated lymphatic organs in the regulation of oral tolerance and intestinal inflammation.
Article
In this study of lectin-induced apoptosis we found that wheat germ agglutinin (WGA) initiated an accelerated type of programmed cell death developing after only 30 min of incubation with tumor cells. To analyze possible mechanisms, studies were focused using the WGA lectin whose carbohydrate specificity is well defined. We found that WGA could induce apoptosis by binding to either N-acetylneuraminic acid or N-acetylglucosamine (GlcNAc) on the cell surface of normal and malignant cells. We also showed that it is unlikely that WGA triggers apoptosis by binding to the carbohydrate portion of Fas. CrmA gene transfection did not inhibit WGA-mediated apoptosis of Jurkat cells. In addition, Jurkat-R cells selected for resistance to Fas signaled apoptosis manifested high sensitivity to WGA as did Fas-negative BL6 melanoma cells. WGA-induced apoptosis is also caspase-3-independent and was found to be triggered via a mitochondrial pathway. WGA induced a loss of transmembrane potential, disruption of the inner mitochondria membrane, and release of cytochrome c and caspase-9 activation after 30 min of cell interaction. Interestingly, Bcl-2 gene transfection did not affect sensitivity of Jurkat cells to WGA. The Jurkat-R subline that has been shown to be Bax and Bak deficient and resistant to various apoptotic signals was highly sensitive to WGA-induced apoptosis. In summary, WGA triggers a unique pattern of apoptosis that is extremely fast, Fas- and caspase-3-independent, and is mediated via a mitochondrial pathway. However, its mitochondrial component is unrestrained by the loss of Bax and Bak or the upregulation of Bcl-2 expression.
Article
Wheat Germ Agglutinin (WGA) cytotoxicity has been studied using two human leukemia cell lines, Molt3 and K562, and human peripheral blood mononuclear cells (PBMC). In spite of similar binding at the cell surface, WGA was found to promote cell death to a different extent in Molt3, K562 and PBMC and to induce different death events leading to apoptosis in Molt3 and either apoptosis and necrosis in K562 cells and PBMC. In Molt3 but not in K562 cells, WGA cytotoxicity could be potentiated 66-200 fold by 50 nM monensin, a carboxylic ionophore that perturbs the intracellular trafficking of endocytosed molecules. Synergism between the cytotoxic activities of WGA and monensin was demonstrated in Molt3 cells by comparing non toxic, or slightly toxic, doses of WGA and monensin alone or in combination. These results show that the cytotoxic effect of WGA is dependent on internalisation events which may differ among the cell lines used. WGA and monensin can enter the human diet being a component of wheat germ and an antibiotic used for zootechnic reasons in the bioindustry, respectively. These data reveal the synergistic effect between two dietary molecules, otherwise per se toxic at much higher concentrations, with possible implications for human and animal health.
Article
CXCL8 (previously known as Interleukin-8), a member of the alpha-chemokine family of chemotactic cytokines, stimulates intestinal neutrophil activation and chemotaxis. As intestinal epithelial cells have been recently shown to produce CXCL8, the aim of this study was to identify functional activities of CXCL8 on intestinal epithelial cells. The expression of CXCL8 receptors CXCR1 and CXCR2 was assessed by RT-PCR and FACS analysis in human Caco-2 and HT-29 cells. The effects of CXCL8 on intestinal epithelial proliferation were assessed with colorimetric MTT assays and the effects on epithelial restitution with an in vitro migration model using Caco-2 and HT-29 cells. While the expression of both CXCR1 mRNA and protein could be demonstrated by RT-PCR and FACS analysis in human Caco-2 and HT-29 cells, no expression of CXCR2 was observed in these cell lines. Colorimetric MTT assays revealed that CXCL8 does not modulate cell proliferation in HT-29 and Caco-2 cells. In contrast, CXCL8 significantly enhanced intestinal epithelial migration in an in vitro migration model of HT-29 and Caco-2 cells. Enhancement of intestinal epithelial cell migration by CXCL8 was partially CXCR1-dependent and TGFbeta-independent. CXCL8 exerts functional effects on intestinal epithelial cells that may be relevant for intestinal inflammation and mucosal healing.
Article
Based on the fact that oligosaccharides encode biological information, the biorecognition between lectinised drug delivery systems and glycosylated structures in the intestine can be exploited for improved peroral therapy. Basic research revealed that some lectins can mediate mucoadhesion, cytoadhesion, and cytoinvasion of drugs. Entering the vesicular pathway by receptor mediated endocytosis, part of the conjugated drug is accumulated within the lysosomes. Additionally, part of the drug is supposed to be transported across the epithelium. Moreover, factors probably adversely influencing feasibility of the concept such as toxicity, immunogenicity, and intestinal stability of plant lectins are discussed. As exemplified by lectin-grafted prodrug and carrier systems, this strategy is expected to improve absorption and probably bioavailability of poorly absorbable drugs, peptides and proteins as well as therapeutic DNA.
Article
Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed also by gastrointestinal epithelial cells. WGA is currently investigated as an anti-tumor drug and as a carrier for oral drugs. Information on whether it can cross the gastrointestinal epithelium and on its possible effects on the integrity of the epithelial layer is however scanty or lacking, and herein we address these issues. Differentiated Caco2 cells have been used as a model of polarized intestinal epithelium. WGA concentration at both the apical and the basolateral side of the epithelium has been quantified using a sensitive ELISA assay (sensitivity threshold 0.84 nM). Trans epithelial electrical resistance (TEER) has been measured to evaluate the integrity of the epithelium upon treatments with WGA. (3)H-Mannitol (182.2 Da) and FITC-dextran (3000 Da) have been used to measure the permeability of the epithelium. Cell viability has been measured by the MTT, by 7-AAD uptake, and Annexin-V binding assays. Up to a concentration of 5.6 microM, approximately 0.1% of intact WGA molecules only could cross the epithelial layer. WGA perturbed the integrity of the epithelium and increased the permeability of the tissue in a dose- and time-dependent manner. WGA did not induce cell death but increased the permeability of individual cells to 7-AAD which is normally not uptaken by viable cells. These data allowed us to define a toxicity threshold for WGA on epithelial cells. WGA suitability as a carrier for oral drugs can therefore be evaluated on a rational basis.
Article
The purpose of this study was to identify one of the ligands that mediate carbohydrate-specific cytoadhesion and cytoinvasion of wheat germ agglutinin (WGA)-containing drug delivery systems. The receptor-ligand studies were performed with isolated epidermal growth factor (EGF) receptors as well as biomimetic membranes prepared from Caco-2 and A-431 cells. The binding of fluorescent labeled WGA was detected by the silver nanoparticle enhanced fluorescence technique. The binding of WGA to isolated EGF receptors is saturable and the equilibrium is reached within 1 min. The interaction between WGA and isolated EGF receptors is fully inhibited by the complementary carbohydrate and at least 85% of WGA binding to artificial Caco-2 membranes is caused by protein-carbohydrate interactions involving the tetrasialo-binding motif. The integrity and the presence of EGF-receptors in artificial Caco-2 membranes as well as their WGA-binding capacity were confirmed by immunoblot detection. The glycosylated extracellular domain III of the EGF receptor is involved in the WGA-Caco-2 cell interaction. Accordingly, receptor mediated endocytosis is the basic mechanism for internalization of WGA. As the EGF receptor is overexpressed in a high number of tumors but also occurs in non-malignant tissue at considerable density, WGA-mediated drug delivery opens exciting possibilities for specific binding and uptake of poorly absorbable drugs.
Article
In addition to its role as a mediator of innate pro-inflammatory responses following bacterial lipopolysaccharide (LPS) binding, the 55kDa glycosyl-phosphatidylinositol-linked macrophage plasma membrane glycoprotein CD14 is now also known to play a role in phagocytic clearance of apoptotic cells. Although apoptotic cell-associated ligand(s) for CD14 await definition, initial findings suggest that ligand binding occurs close to, or at the same site as, LPS binding. Significantly, in contrast to LPS clearance and in keeping with the non-phlogistic nature of apoptosis, CD14-dependent engulfment of apoptotic cells fails to elicit pro-inflammatory cytokine release from macrophages. Therefore CD14 may be regarded as an innate immune receptor both for microbial products--after binding which activates inflammatory responses--and for self components, which either fail to induce, or alternatively actively suppress, inflammatory responses. Here we review current knowledge of the structure and functions of CD14, its ligands, its possible modes of signal transduction and its place in the panoply of macrophage molecules implicated in apoptotic-cell clearance.
Handbook of Plant Lectins: Properties and Biomedical Applications
  • E J M Van Damme
  • W J Peumans
  • A Pusztai
  • S Bardocz
Van Damme, E.J.M., Peumans, W.J., Pusztai, A., Bardocz, S., 1998. Handbook of Plant Lectins: Properties and Biomedical Applications. John Wiley and Sons, West Sussex.
  • C D Pellegrina
C.D. Pellegrina et al. / Toxicology and Applied Pharmacology 237 (2009) 146–153