link1: Diabetes. 2006 May;55(5):1443-9. Links
Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives.Sapone A, de Magistris L, Pietzak M, Clemente MG, Tripathi A, Cucca F, Lampis R, Kryszak D, Cartenì M, Generoso M, Iafusco D, Prisco F, Laghi F, Riegler G, Carratu R, Counts D, Fasano A.
Mucosal Biology Research Center, University of Maryland School of Medicine, 20 Penn Street, Room 345, Baltimore, MD 21201, USA.
Zonulin, a protein that modulates intestinal permeability, is upregulated in several autoimmune diseases and is involved in the pathogenesis of autoimmune diabetes in the BB/Wor animal model of the disease. To verify the association between serum zonulin levels and in vivo intestinal permeability in patients with type 1 diabetes, both parameters were investigated in different stages of the autoimmune process. Forty-two percent (141 of 339) of the patients had abnormal serum zonulin levels, as compared with age-matched control subjects. The increased zonulin levels correlated with increased intestinal permeability in vivo and changes in claudin-1, claudin-2, and myosin IXB genes expression, while no changes were detected in ZO1 and occludin genes expression. When tested in serum samples collected during the pre-type 1 diabetes phase, elevated serum zonulin was detected in 70% of subjects and preceded by 3.5 +/- 0.9 years the onset of the disease in those patients who went on to develop type 1 diabetes. Combined, these results suggest that zonulin upregulation is associated with increased intestinal permeability in a subgroup of type 1 diabetic patients. Zonulin upregulation seems to precede the onset of the disease, providing a possible link between increased intestinal permeability, environmental exposure to non-self antigens, and the development of autoimmunity in genetically susceptible individuals.
PMID: 16644703 [PubMed - indexed for MEDLINE]
link1: Ann N Y Acad Sci. 2000;915:214-22. Links
Regulation of intercellular tight junctions by zonula occludens toxin and its eukaryotic analogue zonulin.Fasano A.
Division of Pediatric Gastroenterology and Nutrition, Gastrointestinal Pathophysiology Section, Center for Vaccine Development, Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. email@example.com
The intestinal epithelium represents the largest interface between the external environment and the internal host milieu and constitutes the major barrier through which molecules can either be absorbed or secreted. There is now substantial evidence that tight junctions (tj) play a major role in regulating epithelial permeability by influencing paracellular flow of fluid and solutes. Tj are one of the hallmarks of absorptive and secretory epithelia. Evidence now exists that tj are dynamic rather than static structures and readily adapt to a variety of developmental, physiological, and pathological circumstances. These adaptive mechanisms are still incompletely understood. Activation of PKC either by Zonula occludens toxin (Zot) or by phorbol esters increases paracellular permeability. Alteration of epithelial tj is a recently described property for infectious agents. Clostridium difficile toxin A and B and influenza and vesicular stomatitis viruses have been shown to loosen tj in tissue culture monolayers. Unlike what occurs after the Zot stimulus, these changes appear to be irreversible and are associated with destruction of the tj complex. On the basis of this observation, we postulated that Zot may mimic the effect of a functionally and immunologically related endogenous modulator of epithelial tj. We were able to identify an intestinal Zot analogue, which we named zonulin. It is conceivable that the zonulins participate in the physiological regulation of intercellular tj not only in the small intestine, but also throughout a wide range of extraintestinal epithelia as well as the ubiquitous vascular endothelium, including the blood-brain barrier. Disregulation of this hypothetical zonulin model may contribute to disease states that involve disordered intercellular communication, including developmental and intestinal disorders, tissue inflammation, malignant transformation, and metastasis.
PMID: 11193578 [PubMed - indexed for MEDLINE]
linkAffinity Purification and Partial Characterization of the Zonulin/Zonula Occludens Toxin (Zot) Receptor from Human Brain.
Structure And Function
Journal of Neurochemistry. 74(1):320, January 2000.
Lu , R.; Wang , W.; Uzzau, S. *; Vigorito, R. *; Zielke, H. R. ; Fasano, A.
The intercellular tight junctions (TJs) of endothelial cells represent the limiting structure for the permeability of the blood-brain barrier (BBB). Although the BBB has been recognized as being the interface between the bloodstream and the brain, little is known about its regulation. Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot) elaborated by Vibrio cholerae, both modulate intercellular TJs by binding to a specific surface receptor with subsequent activation of an intracellular signaling pathway involving phospholipase C and protein kinase C activation and actin polymerization. Affinity column purification revealed that human brain plasma membrane preparations contain two Zot binding proteins of ~55 and ~45 kDa. Structural and kinetic studies, including saturation and competitive assays, identified the 55-kDa protein as tubulin, whereas the 45-kDa protein represents the zonulin/Zot receptor. Biochemical characterization provided evidence that this receptor is a glycoprotein containing multiple sialic acid residues. Comparison of the N-terminal sequence of the zonulin/Zot receptor with other protein sequences by BLAST analysis revealed a striking similarity with MRP-8, a 14-kDa member of the S-100 family of calcium binding proteins. The discovery and characterization of this receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB.
(C) 2000 International Society for Neurochemistry
link1: J Pharm Sci. 2003 Feb;92(2):414-23. Links
Zonula occludens toxin increases the permeability of molecular weight markers and chemotherapeutic agents across the bovine brain microvessel endothelial cells.Karyekar CS, Fasano A, Raje S, Lu R, Dowling TC, Eddington ND.
Pharmacokinetics Biopharmaceutics Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 100 Penn Street, Baltimore, Maryland 21201, USA.
The purpose of this study was to examine the ability of Zonula occludens toxin (Zot) to reversibly open tight junctions in bovine brain microvessel endothelial cells (BBMECs) to enhance drug delivery via the paracellular pathway. Transport across BBMEC monolayers was examined for molecular weight markers and chemotherapeutic agents ([(14)C]sucrose, [(14)C]inulin, [(3)H]propranolol, [(3)H]doxorubicin, and [(14)C]paclitaxel) with Zot (0.0-4.0 microg/mL). TEER of monolayers was measured to assess effect and reversibility of Zot. Cell viability of BBMEC in the presence of Zot was assessed by trypan blue exclusion staining. Apparent permeability (P(app)), enhancement ratio (R), and percent increase in transport determined were statistically compared by ANOVA. A significant increase (p < 0.05) in P(app) was observed for the transport of [(14)C]sucrose, [(14)C]inulin, [(3)H]doxorubicin, and [(14)C]paclitaxel at a 4.0 microg/mL concentration of Zot. A significant concentration-dependent decrease in TEER was observed on treatment with Zot with rapid reversal to baseline after removal. Zot (4 micro/ml) was found to be nontoxic to the BBMECs after 2 hours incubation. In conclusion, Zot increased paracellular transport across the BBMEC in a reversible, concentration-dependent manner. Modulation of paracellular transport with Zot may be used to increase the brain permeability of potent central nervous system-active drugs, including anticancer agents. Copyright 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:414-423, 2003
PMID: 12532391 [PubMed - indexed for MEDLINE]
Related ArticlesEnhanced permeability of molecular weight markers and poorly bioavailable compounds across Caco-2 cell monolayers using the absorption enhancer, zonula occludens toxin. [Pharm Res. 2002] Enhancing the permeation of marker compounds and enaminone anticonvulsants across Caco-2 monolayers by modulating tight junctions using zonula occludens toxin. [Eur J Pharm Biopharm. 2001] Pluronic P85 increases permeability of a broad spectrum of drugs in polarized BBMEC and Caco-2 cell monolayers. [Pharm Res. 1999] Effect of the six-mer synthetic peptide (AT1002) fragment of zonula occludens toxin on the intestinal absorption of cyclosporin A. [Int J Pharm. 2008] The effect of beta-turn structure on the permeation of peptides across monolayers of bovine brain microvessel endothelial cells. [Pharm Res. 1997] » See all Related Articles...
link1: Gastroenterology. 2008 Jul;135(1):194-204.e3. Epub 2008 Mar 21. Links
Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3.Lammers KM, Lu R, Brownley J, Lu B, Gerard C, Thomas K, Rallabhandi P, Shea-Donohue T, Tamiz A, Alkan S, Netzel-Arnett S, Antalis T, Vogel SN, Fasano A.
Mucosal Biology Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
BACKGROUND & AIMS: Celiac disease is an immune-mediated enteropathy triggered by gliadin, a component of the grain protein gluten. Gliadin induces an MyD88-dependent zonulin release that leads to increased intestinal permeability, a postulated early element in the pathogenesis of celiac disease. We aimed to establish the molecular basis of gliadin interaction with intestinal mucosa leading to intestinal barrier impairment. METHODS: Alpha-gliadin affinity column was loaded with intestinal mucosal membrane lysates to identify the putative gliadin-binding moiety. In vitro experiments with chemokine receptor CXCR3 transfectants were performed to confirm binding of gliadin and/or 26 overlapping 20mer alpha-gliadin synthetic peptides to the receptor. CXCR3 protein and gene expression were studied in intestinal epithelial cell lines and human biopsy specimens. Gliadin-CXCR3 interaction was further analyzed by immunofluorescence microscopy, laser capture microscopy, real-time reverse-transcription polymerase chain reaction, and immunoprecipitation/Western blot analysis. Ex vivo experiments were performed using C57BL/6 wild-type and CXCR3(-/-) mouse small intestines to measure intestinal permeability and zonulin release. RESULTS: Affinity column and colocalization experiments showed that gliadin binds to CXCR3 and that at least 2 alpha-gliadin 20mer synthetic peptides are involved in this binding. CXCR3 is expressed in mouse and human intestinal epithelia and lamina propria. Mucosal CXCR3 expression was elevated in active celiac disease but returned to baseline levels following implementation of a gluten-free diet. Gliadin induced physical association between CXCR3 and MyD88 in enterocytes. Gliadin increased zonulin release and intestinal permeability in wild-type but not CXCR3(-/-) mouse small intestine. CONCLUSIONS: Gliadin binds to CXCR3 and leads to MyD88-dependent zonulin release and increased intestinal permeability.
PMID: 18485912 [PubMed - indexed for MEDLINE]
1: Mult Scler. 2008 Sep;14(8):1036-43. Epub 2008 Aug 13. Links
Multiple sclerosis: chemokine receptor expression on circulating lymphocytes in correlation with radiographic measures of tissue injury.Fox RJ, Kivisakk P, Fisher E, Tucky B, Lee JC, Rudick RA, Ransohoff RM.
Department of Neurology, Mellen Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. firstname.lastname@example.org
BACKGROUND: Leukocytes expressing inflammatory chemokine receptors (CKRs), most consistently CCR2, CCR5, and CXCR3, have been identified in multiple sclerosis (MS) tissue lesions and provide attractive therapeutic targets. Our previous studies found large inter-individual differences in expression of these CKRs but stable levels over time within subjects. This observation suggests a CKR "set-point" within individuals, which might relate to inflammatory injury in MS. We evaluated the correlation between CKR levels and magnetic resonance imaging (MRI) measures of disease activity. METHODS: Fifty-five relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients were prospectively followed with annual CKR and MRI studies. Multiparameter flow cytometry was used to determine CCR2, CCR5, and CXCR3 expression on CD4 and CD8 cells. Simultaneous cranial MRIs were performed, and quantitative measures of T2, T1, and gadolinium lesions, brain parenchymal fraction (BPF), and whole brain and fractionated magnetization transfer ratio (MTR) were performed using automated software. Spearman's rank correlations evaluated the relationship between CKR levels and MRI measures. RESULTS: Significant correlations were observed between CXCR3 expression on CD8 cells and measures of new (T1) and total (T1, T2) lesion volumes, lesion MTR, and BPF; higher levels of CXCR3 expression were correlated with greater injury on MRI (|r| = 0.27-0.42). In contrast, CD4 cell CKR expression was only minimally correlated with MRI measures. CONCLUSIONS: Over 2 years, we observed significant correlations between the percent of CD8 cells expressing CXCR3 and MRI measures of MS inflammatory activity and tissue destruction. These observations are consistent with a pathogenic role for cytotoxic T cells in MS brain and have significant implications regarding T-cell targeted therapeutic strategies.
PMID: 18701575 [PubMed - in process]
link1: J Pharmacol Exp Ther. 2006 Sep;318(3):1153-62. Epub 2006 Jun 2. Links
Periplocoside E inhibits experimental allergic encephalomyelitis by suppressing interleukin 12-dependent CCR5 expression and interferon-gamma-dependent CXCR3 expression in T lymphocytes.Zhu YN, Zhong XG, Feng JQ, Yang YF, Fu YF, Ni J, Liu QF, Tang W, Zhao WM, Zuo JP.
Laboratory of Immunopharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People's Republic of China.
Periplocoside E (PSE) was found to inhibit primary T-cell activation in our previous study. Now we examined the effect and mechanisms of PSE on the central nervous system (CNS) demyelination in experimental allergic encephalomyelitis (EAE). C57BL/6 mice immunized with myelin oligodendrocyte glyco-protein (MOG) were treated with PSE following immunization and continued throughout the study. The effect on the progression of EAE and other relevant parameters were assessed. PSE reduced the incidence and severity of EAE. Spinal cord histopathology analysis showed that the therapeutic effect of PSE was associated with reduced mononuclear cell infiltration and CNS inflammation. As reverse transcription-polymerase chain reaction analysis showed, PSE decreased the CD4(+), CD8(+), and CD11b(+) cell infiltration. T cells from lymph nodes of MOG-immunized mice expressed enhanced levels of CCR5 and CXCR3 mRNA compared with T cells from normal mice. However, CCR5 and CXCR3 expressions were suppressed in T cells from PSE-treated mice. In vitro study also showed PSE inhibited interferon (IFN)-gamma-dependent CXCR3 expression in T cells through suppressing T-cell receptor (TCR) ligation-induced IFN-gamma production, whereas it inhibited interleukin (IL)-12-dependent CCR5 expression through suppressing IL-12 reactivity in TCR-triggered T cells. As a result, the initial influx of T cells into CNS was inhibited in PSE-treated mice. The consequent activation of macrophages/microglia cells was inhibited in spinal cord from PSE-treated mice as determination of chemokine expressions (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10). Consistently, the secondary influx of CD4(+), CD8(+), and CD11b(+) cells was decreased in spinal cords from PSE-treated mice. These findings suggest the potential therapeutic effect of PSE on multiple sclerosis.
PMID: 16751252 [PubMed - indexed for MEDLINE]
Periploca sepium Bge, a traditional Chinese herb medicine, is used for treating rheumatoid arthritis in China. Followed the bioactivity-guided isolation, the most potent immunosuppressive compound, periplocoside E (PSE), a pregnane glycoside had been identified from Periploca sepium Bge. We investigated the immunosuppressive effects of PSE in vitro and in vivo. The results showed that PSE in a dose-dependent manner significantly inhibited the proliferation of splenocytes induced by concanavalin A, and mixed lymphocyte culture reaction at no cytotoxic concentrations (<5 µM). Administration of PSE suppressed a delayed type hypersensitivity reaction, and ovalbumin (OVA) induced antigen-specific immune responses in mice. In vivo treatment with PSE dose-dependently suppressed OVA-induced proliferation and cytokine (IL-2 and IFN-) production from splenocytes in vitro. Purified T cells from OVA-immunized mice with PSE treatment showed its low ability for activation by OVA plus normal antigen presenting cells stimulation again in vitro. Further studies showed PSE dose-dependently inhibited anti-CD3 induced primary T cell proliferation, activation for IL-2R (CD25) expression, and cytokine (IFN- and IL-2) production also at the transcriptional level. PSE was highly specific, significantly inhibited the activation of ERK and JNK, whereas activation of p38 was not affected in T cells stimulated with anti-CD3. These results demonstrated that PSE is an immunosuppressive compound in Periploca sepium Bge, which directly inhibit T cell activation in vitro and in vivo. This study provided evidence to understand the therapeutic effects of Periploca sepium Bge and indicated that this herb is appropriate for treatment of T cell-mediated disorders, such as autoimmune diseases.
Chemokine receptor CXCR3 is a Gαi protein-coupled receptor in the CXC chemokine receptor family. Other names for CXCR3 are G protein-coupled receptor 9 (GPR9) and CD183. There are two variants of CXCR3: CXCR3-A binds to the CXC chemokines CXCL9, CXCL10, and CXCL11 whereas CXCR3-B can also bind to CXCL4 in addition to CXCL9, CXCL10, and CXCL11.
CXCR3 is expressed primarily on activated T lymphocytes and NK cells, and some epithelial cells and some endothelial cells. CXCR3 and CCR5 are preferentially expressed on Th1 cells, whereas Th2 cells favor the expression of CCR3 and CCR4. CXCR3 ligands that attract Th1 cells can concomitantly block the migration of Th2 cells in response to CCR3 ligands, thus enhancing the polarization of effector T cell recruitment.
CXCR3 is able to regulate leukocyte trafficking. Binding of chemokines to CXCR3 induces various cellular responses, most notably integrin activation, cytoskeletal changes and chemotactic migration. CXCR3-ligand interaction attracts Th1 cells and promotes Th1 cell maturation.
As a consequence of chemokine-induced cellular desensitization (phosphorylation-dependent receptor internalization), cellular responses are typically rapid and short in duration. Cellular responsiveness is restored after dephosphorylation of intracellular receptors and subsequent recycling to the cell surface. A hallmark of CXCR3 is its prominent expression in in vitro cultured effector/memory T cells, and in T cells present in many types of inflamed tissues. In addition, CXCL9, CXCL10 and CXCL11 are commonly produced by local cells in inflammatory lesion, suggesting that CXCR3 and its chemokines participate in the recruitment of inflammatory cells. Additionally, CXCR3 has been implicated in wound healing.
CXCR3 has been implicated in the following diseases, atherosclerosis, multiple sclerosis, pulmonary fibrosis, type 1 diabetes, autoimmune myasthenia gravis, nephrotoxic nephritis, acute cardiac allograft rejection and possibly Celiac Disease. Development of agents to block CXCR3-ligand interactions may provide new ways to treat these diseases.
TwistedHelix wrote:Here's a link to my first ever post, way back in 2005 when I was a naive and virginal young man. Intestinal permeability was all the rage back then, and I had just read that the nasal mucosa is packed with neural stem cells, so I was postulating on whether fragments from those cells could survive into the gut, find their way into the bloodstream, and set off an immune response rather than a digestive one. Zonulin gets a mention, too:
Multiple sclerosis patients have peripheral blood CD45RO+ B cells and increased intestinal permeability.
Dig Dis Sci 1996 Dec;41(12):2493-8
Yacyshyn B, Meddings J, Sadowski D, Bowen-Yacyshyn MB
Department of Medicine, University of Alberta, Edmonton, Canada.
Increased intestinal permeability and the CD45RO isoform expression of the leukocyte common antigen on peripheral blood CD20+ B cells are found in Crohn's disease. Others have observed that multiple sclerosis (MS) patients may have an increased risk of coacquisition of Crohn's disease. The aim of this study was to identify an association between these diseases using peripheral blood CD45 isoform expression and intestinal permeability in MS. Lactulose/mannitol permeability and peripheral blood CD20+ B cell CD45RO expression were defined in healthy controls, MS patients, and patients coincidentally affected by MS and Crohn's or MS and ulcerative colitis (UC). Five of 20 MS patients had increased intestinal permeability, a finding not previously reported. High levels of CD45RO were found on circulating CD20+ B cells from patients with MS. This has not been reported previously in MS and is found in very few other conditions. Eight patients with coincident MS and Crohn's disease or MS and UC were studied. Coincident MS and UC patients expressed CD45RO on CD20+ B cells, a finding not identified in UC patients alone. A subgroup of MS patients has increased intestinal permeability. These patients express CD45RO CD20+ B cells, also found in Crohn's disease.
Gastrointestinal absorption of intact proteins.
Annu Rev Nutr 1988;8:329-50
School of Biomedical Sciences, University of Bradford, West Yorkshire, England.
There is now no reasonable doubt that small quantities of intact proteins do cross the gastrointestinal tract in animals and adult humans, and that this is a physiologically normal process required for antigen sampling by subepithelial immune tissue in the gut. It is too small to be nutritionally significant in terms of gross acquisition of amino-nitrogen, but since it has important implications relating to dietary composition it must receive consideration from nutritionists. The process of intact protein absorption occurs without eliciting harmful consequences for most individuals, but it appears likely that a small number of people absorbing these "normal" amounts may react idiosyncratically; also, some individuals may absorb excessive amounts, and they may suffer clinically significant consequences. Likewise, individuals with diminished absorption of intact protein may be at risk. Normal absorption probably occurs predominantly by transcellular endocytosis with some possible contribution by a route between cells; increased net entry of protein to the circulation may reflect (a) increased paracellular (intercellular) passage, (b) increased transcellular passage, and/or (c) decreased lysosomal proteolysis. Tests to distinguish among these possibilities are strongly desirable. Intact protein absorption may be involved in the pathogenesis of inflammatory bowel disease, "food allergies, " and other diseases, including even major psychiatric disorders, but the current evidence is mainly indirect and suggestive. Great caution and careful objective studies are needed to establish whether such relationships with disease do exist and to unravel the underlying basic physiological mechanisms. Now that interest has developed in the assessment of intestinal permeability to small- and medium-sized molecules, it is hoped that equally simple methods for studying macromolecular permeability will be developed and applied. Therapeutic methods for enhancing intact polypeptide absorption would be valuable for vaccine and peptide drug administration by the oral route. Therapeutic reduction of the process may be relevant in food-sensitive patients.
Antigen absorption from the small intestine and gastrointestinal disease.
Pediatr Clin North Am 1975 Nov;22(4):731-46
In this article, I have attempted to summarize the concept of intestinal permeability to antigens such as ingested food proteins, bacterial breakdown products, endotoxins, and enzymes. The mature gut retains the capacity to absorb macromolecules by a pinocytotic mechanism which is more pronounced during the neonatal period. The vast majority of individuals have no ill effects from the intestinal transport of large molecules. However, when increased quantities of toxic or antigenic macromolecules gain access to the body because of a derangement in the intraluminal digestive process or because of a defect in the mucosal barrier, antigen absorption may be altered and result in either local intestinal or systemic disorders. The speculative concepts suggesting that clinical disease states may be associated with altered mucosal permeability have been discussed.
Review article: Intestinal permeability in Crohn's disease.
Aliment Pharmacol Ther 1997 Dec;11 Suppl 3:47-53; discussion 53-6
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