Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3.

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.

Enhancement of brain distribution of anticancer agents using DeltaG, the 12 kDa active fragment of ZOT.

OBJECTIVE: The objective of this study was to evaluate the ability of DeltaG, the 12 kDa active fragment of ZOT, to increase the brain distribution of MTX and paclitaxel, two commonly used anticancer agents with poor distribution into the brain. METHODS: As part of dose estimation of DeltaG, [14C]-sucrose (40 microCi/kg), a hydrophilic paracellular marker, was co-administered with DeltaG (0, 400 and 800 microg/kg) with and without protease inhibitors to male Sprague-Dawley rats (n=3 per group) via an intracarotid cannula. MTX (50 mg/kg) and [3H]-paclitaxel (120 microCi/kg) were co-administered with the effective doses of DeltaG determined from the above study via the intracarotid cannula. Animals were euthanized by carbon dioxide asphyxiation at the specified time periods and brain and plasma samples were analyzed for the respective drug. RESULTS: The brain distribution of [14C]-sucrose was significantly enhanced at both doses of DeltaG. A fold enhancement in the B/P ratios of 1.88 and 2.68 was observed at the 400 and 800 microg/kg doses respectively, when the protein was protected from metabolic degradation with PIs. DeltaG significantly increased the brain distribution of MTX at each of the doses administered, with over a seven-fold increase at the 600 microg/kg dose. [3H]-paclitaxel brain AUC(0-60 min) was significantly higher in the presence of DeltaG (800 microg/kg with PIs) with a 2.5-fold enhancement in brain exposure. CONCLUSIONS: DeltaG significantly enhances the brain distribution of MTX (hydrophilic) and paclitaxel (lipophilic) and has the potential to be further developed as adjunct therapy to increase delivery of poorly permeable chemotherapeutic and other CNS targeted compounds.

Zonula occludens toxin increases the permeability of molecular weight markers and chemotherapeutic agents across the bovine brain microvessel endothelial cells.

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.

Effect of the biologically active fragment of zonula occludens toxin, delta G, on the intestinal paracellular transport and oral absorption of mannitol.

OBJECTIVE: Many therapeutically active agents experience low bioavailability upon oral administration due to low permeability, low solubility, interaction with efflux transporters or first pass metabolism. In general, absorption enhancers are agents that can modulate the paracellular permeability of drugs, thus, potentially increasing oral bioavailability. The objective of this study was to examine the effect of the active fragment of Zonula occludens toxin (Zot), deltaG, on the transport of a paracellular marker, mannitol, using in vitro (Caco-2 cell monolayers) and in vivo (intraduodenal administration in rats) experimental methods. METHODS: The transport of [14C]mannitol with deltaG (0, 50, 80, or 100 microg/ml) was determined across Caco-2 cells. Male Sprague-Dawley rats were assigned to receive one of the following treatments: [14C] mannitol (40 microCi/kg), [14C]mannitol/deltaG (417 microg/kg), or [14C] mannitol/deltaG/Protease inhibitors (PI). RESULTS: The mean (+/-S.E.M.) apparent mannitol permeability coefficients (P(app)) observed after incubation with 0, 50, 80, and 100 microg/ml deltaG were 3.5 (+/-0.4), 4.17 (+/-0.27), 4.33 (+/-0.61), and 9.94 (+/-0.24)x10(-6) cm/s. After oral administration, C(max) (3.8 x 10(-4) vs. 4.4 x 10(-4) mM) and AUC(0-6 h) (0.096 vs. 0.088 mM min), obtained for [14C]mannitol and [14C]mannitol/deltaG, respectively, were not statistically different. However, both C(max) (7.6 x 10(-4) mM) and AUC(0-6 h) (0.25 mM min) were significantly higher for the [14C]mannitol/deltaG/PI treatment. CONCLUSIONS: The 12 kDa fragment of Zot, deltaG, enhanced the in vitro transport and oral absorption of the paracellular marker, mannitol, in the presence of protease inhibitors (PI).