• The active motif of Zot, AT1002, increases ZO-1 and Myosin 1 Beta serine phosphorylation, their interaction, and intestinal permeability

      Thakar, Manjusha; Goldblum, Simeon E.; Not, Tarcisio; Fasano, Alessio (2006)
      Background: As we have previously reported, Zonula occludens toxin (Zot) elaborated by Vibrio cholerae anchors to the bacterial outer membrane and undergoes a Vibrio-specific cleavage at amino acid residue 288, with subsequent release of its C-terminal fragment in the intestinal micromilieau. The N-terminus of the cleaved Zot fragment contains a conserved 6-mer protease activated receptor (PAR)-activating peptide (AP) motif. We synthesized the 6-mer and named it AT1002. Aims: 1.To determine whether AT1002 modulates tj both in vivo and in vitro. 2. To establish whether AT1002 signaling affect ZO-1 phosphorylation. 3. To study ZO-1 interaction with partner and scaffolding proteins in presence of AT1002. Methods: 1. Transepithelial electrical resistance (TEER) was monitored either in presence or absence of AT1002 added to the mucosal aspect of rat small intestine mounted in Ussing chambers. 2. The in vivo intestinal permeability of mouse intestine was studied by dual sugar test with HPLC technique. 3. Phosphorylation of ZO-1 induced by AT1002 was analyzed by Western immunoblotting. 4. The effect of AT1002 on protein-protein interaction of ZO-1 with partner proteins and scaffold proteins were investigated by co-immunoprecipitation analysis. Conclusions: 1.AT1002 caused the tight junctions disassembly as shown by decrease in TEER in rat tissues mounted in Ussing chambers. 2.AT1002 was biologically active both in vivo and in vitro as established by experiments performed on intestinal tissues in Ussing chambers and dual sugar test permeability test. 3.AT1002 induced ZO-1 serine phosphorylation that temporarily preceded tight junction disassembly. 4.AT1002 increased serine phosphoralytion of myosin 1 beta and increased association of myosin 1 beta with ZO-1. 5. 6. AT1002 didn’t alter the association of ZO-1 and ZO-2.
    • Expression of Tight Junction Proteins in Diseases With Compromised Intestinal Barrier Function

      Tripathi, Amit; Clemente, Maria Grazia; Sapone, Anna; Musu, Maria Paola; De Virgiliis, Stefano; Fasano, Alessio (2006)
    • Gliadin Induces Zonulin Release, Occludin Down-Regulation, and Tight Junctions Disassembly in a Rat Animal Model

      Drago, Sandro; Di Pierro, Mariarosaria; Margaretten, Klara; Thakar, Manjusha; Fasano, Alessio (2001)
    • Immunohistochemical expression of junctional proteins and intestinal permeability are altered in Hepatitis C virus (HCV) related chronic hepatitis (HCV-CH)

      Sapone, Anna; Martorelli, Luigi; Russo, Maria Itria; Generoso, Maddalena; Esposito, Pasquale; de Magistris, Laura; Ferraraccio, Franca; Esposito, Pasquale; Carteni, Maria; Fasano, Alessio (2006)
      Background: Altered intestinal permeability values (IP) and intestinal ultra-structural abnormalities have been shown in cirrhotic patients as well as other liver diseases. Aims: To study the IP, serum levels of the intestinal tight junction (tj) modulator zonulin (Z), and the immunohystochemical [immunohistochemical] expression of junctional proteins occludin, claudin, desmoglein and intracellular actin in duodenal mucosa of HCV-CH patients. Methods: The Intestinal Permeability was evaluated by the Lactulose/Mannitol (LA/MA) test. Serum Zonulin levels were determined by sandwich-ELISA. Distal duodenum biopsies were obtained by EGDS and processed for immunohystochemistry [immunohistochemistry]. Staining was obtained with antibodies specific for occludin, claudin-4, desmoglein-3, and citoskeleton of actin. Conclusions: The increased Intestinal Permeability was detected in 32% of the HCV-CH patients studied and was associated to elevated serum Zonulin levels values. These changes were associated to modification of tj proteins expression and localization. Our data suggest that in a subgroup of HCV-CH patients the up-regulation of intestinal permeability-zonulin dependent may lead to increased non-self antigens access that may influence the course of the liver disease.
    • Inhibition of the zonulin pathway blocks the progression from pre-clinical autoimmunity to Type 1 diabetes in BB/wor rats

      Fasano, Alessio (2005)
      Background: We have previously studied that zonulin, a protein involved in tight junctions modulation, is up regulated in BB/wor rats and is responsible for the increased intestinal permeability (IP) typical of this animal model of autoimmunity. We have also demonstrated that by blocking the zonulin pathway before the onset of autoimmunity, the incidence of Type 1 Diabetes (T1D) can be prevented in 75% of the animals. Aim : To determine whether the progression of T1D can be blocked, even though the autoimmune process is already established. Methods: 30 BB/ Wor rats were randomized after immune seroconversion (average age 55 days) in a treatment group (N=20) that received autoclaved water supplemented with 3mg /ml of zonulin receptor blocker AT1001 and HCO3- 1.5g/100ml to buffer gastric acidity, and a placebo group (N=10) that received only water with HCO3-. Serum zonulin and autoantibody levels were monitored at the beginning of the study and at its endpoint. Water intake was monitored daily, weight gain and serum glucose levels were checked weekly. Rats with fasting blood glucose  250 mg/dl were considered diabetic and were sacrificed within 24 hours of reaching the diabetic status. Results: Six out of 10 (60%) untreated rats developed T1D, while only 7/20 (35%) of the AT1001-treated animal progressed to T1D. The average age of onset of T1D was 85.410.4 in the placebo group and 86.010.3 in the treated group. AT1001 treatment did not change the serum zonulin levels between beginning (average age 55 days) and the endpoint of the experiment (average age 100 days) of the experiments. Conversely, the anti-glutamic acid decarboxylase (GAD) antibodies were significantly reduced in AT1001-treated rats that did not develop T1D (0.870.35) compared to the treated animals with the disease developed (1.870.59; p<0.05) Conclusions: The blockage of the zonulin pathway in BB/wor rats at their preclinical autoimmune stage significantly reduced the progression to T1D. This decreased incidence of T1D was associated to a significant reduction of the anti-GAD antibodies following AT1001 treatment.
    • Larazotide Acetate (AT-1001) Inhibits the Intestinal Permeability-Inflammatory Loop Caused by Gliadin and Cytokines

      Durai, Malarvizhi; Kitchens, Kelly Marie; Somerville, Robert; Gopalakrishnan, Shobha; Tamiz, Amir P.; Pandey, Niranjan; Fasano, Alessio; Murray, Joseph A., M.D.; Verdu, Elena; Alkan, Sefik S. (2009-04)
      Altered intestinal permeability and dysfunctional tight junctions (TJ) have been implicated in several autoimmune diseases including celiac disease (CeD). Elevated intestinal permeability leads to additional gluten exposure resulting in a sustained inflammatory loop which has a pivotal role in the pathogenesis of CeD. In this study, we investigated the effect of larazotide acetate (AT-1001), an 8-mer tight junction modulator peptide, on the permeability caused by factors secreted from pepsintrypsin digested gliadin (PT-gliadin) stimulated human PBMC or recombinant cytokines. Treatment of human PBMC with PT-gliadin increased the production of several pro-inflammatory cytokines including TNF-α, IFN-γ, and IL-1β. Basolateral application of this supernatant resulted in an increase in Lucifer Yellow (LY) permeability. We show that larazotide acetate inhibited PT-gliadin activated PBMC supernatants or mixture of proinflammatory cytokines (TNF-α, IFN-γ, and IL-1β)- induced increase in LY permeability in Caco-2 assay. Larazotide acetate also inhibited the transport of immune reactive 9-mer, 13-mer gliadin peptides across a Caco-2 monolayer. Finally, in a double transgenic (HCD4+/HLA/DQ8+) mouse CeD model, oral administration of larazotide acetate prior to oral gliadin treatment blocked intestinal changes such as macrophage accumulation, and intestinal permeability. These results suggest that larazotide acetate can effectively disrupt the pathogenic intestinal “permeability-inflammatory loop” by inhibition of gliadin peptide transport and inhibition of cytokine induced permeability.
    • The mechanism of tight junction disassembly by the ZOT-derived tight junction modulating peptide AT1002

      Tripathi, Amit; Rai, Usha; Goldblum, Simeon E.; Thakar, Manjusha; Watts, Tammara; De Leo, Luigina; Di Toro, Nicola; Not, Tarcisio; Hollenberg, Morley D., 1942-; Fasano, Alessio (2009)
    • New insights on celiac disease pathogenesis: gliadin effects on Zonulin release and intestinal permeability

      Drago, Sandro; Di Pierro, Mariarosaria; Margaretten, Klara; Thakar, Manjusha; Fasano, Alessio (2002)
    • Proteinase-Activated Receptor 2 (PAR-2) Involvement In The Zot/Zonulin-Mediated Regulation Of Intestinal Tight Junctions

      Clemente, Maria Grazia; Vogel, Stefanie N.; Hollenberg, Morley D., 1942-; Fasano, Alessio (2004-05)
    • Role of Zonulin-Dependent Increased Gut Permeability in the Pathogenesis of Type I Diabetes in Humans

      Sapone, Anna; de Magistris, Laura; Iafusco, Dario; Prisco, Franco; Carratu, Romano; Bizzarri, Barbara; Clemente, Maria Grazia; Musu, Maria Paola; Lampis, Rosanna; Cucca, Francesco; et al. (2003)
    • Salmonella typhi induces increased mucosal permeability and elicits a strong pro-inflammatory response ameliorated by vaccine candidates in vitro

      Fiorentino, Maria R., Ph.D.; Lammers, Karen M.; Dyson, Tristan; Vogel, Stefanie N.; Fasano, Alessio (2010)
    • Seric Zonulin, a Modulator of Intestinal Tight Junctions Permeability, is Increased in Different Autoimmune Disorders

      Clemente, Maria Grazia; Musu, Maria Paola; Counts, Debra; De Virgiliis, Stefano; Fasano, Alessio (2002)
    • Seric Zonulin, A Modulator of Tight Junctions Permeability, Is Increased in Different Autoimmune Disorders

      Clemente, Maria Grazia; Musu, Maria Paola; Counts, Debra; De Virgiliis, Stefano; Fasano, Alessio (2002)
    • Serum zonulin and intestinal permeability before and after a gluten-containing meal in both Type 1 diabetes and in their relatives

      Sapone, Anna; Fasano, Alessio (2005)
      Background: The trigger of the autoimmune destruction of pancreatic beta cells in Type 1 diabetes (T1D) is unclear. One theory is that antigens absorbed through the gut may be involved. We have recently described a protein, zonulin, that opens intestinal tight junctions and allows paracellular absorption of macromolecules. We have reported that zonulin is increased in the serum of a subset of patients with T1D and first degree relatives. Aim: The objective of this study was to determine if dysregulation of the zonulin pathway, both at baseline and following a gluten-containing meal, is linked to increased intestinal permeability (IP) and is involved in the pathogenesis of T1D. Methods: After obtaining informed consent, blood was obtained from children with T1D, parents, and siblings. Zonulin was measured in the serum of children with T1D (n=11) and their first degree relatives (n=19), by sandwich ELISA. IP was determined by HPLC measurement of both serum (at baseline and every hour up to 5 hours) and urine (5 hours collection) lactulose (LA) and mannitol (MA) after subjects ingested the sugars test solution. Results were expressed as LA/MA ratio. Results: There was a significant increase in zonulin (53%) and IP (47 %) in subjects compared to the negative cutoff. There was a significant relationship between elevation of zonulin and increased IP (p=0.026) and between LA/MA ratio in post-prandial serum samples and LA/MA ratio in urine (p=0.01). Although there was no change in mean zonulin levels with a meal, all subjects for whom IP data is available had elevation of zonulin at baseline and post-meal, and all had an increase in IP 1-hour post-meal. The 1 h post-prandial IP increase was statistically higher in T1D subjects (5.6 folds) as compared to their relatives (2.7 folds, p<0.01). There was no relationship between serum glucose and zonulin levels. Conclusions: Zonulin is significantly correlated with increased IP in children with T1D and their first degree relatives. These data suggest that increased serum zonulin is indicative of increased gut leakiness in children with T1D. Studies of the temporal relationship of an increase in serum zonulin and the development of diabetes autoantibodies are planned to suggest a causative relationship in a subset of genetically susceptible individuals.