Chapter 9 - Springer Link

Chapter 9 Claudin-4: Functional Studies Beyond the Tight Junction Holly A. Eckelhoefer, Thejani E. Rajapaksa, Jing Wang, Mary Hamer, Nancy C. Appleby, Jun Ling, and David D. Lo Abstract Claudin-4 is an unusual member of the claudin family; in addition to its role in epithelial tight junction barrier function, it is a receptor for the Clostridium perfringens enterotoxin. We have also found that claudin-4 is regulated in mucosal epithelium M cells, both in increased expression of the protein and in redistribution into endocytosis vesicles. Our ongoing studies are studying the potential for developing ligands specific to claudin-4 for targeted delivery of cargo such as proteins and poly(dl-lactide-co-glycolide) nanoparticles to mucosal M cells. Methods for the study of claudin-4 movement within epithelial cells, and delivery of nanoparticles through targeted binding of claudin-4 are described. Key words: Tight junction, Mucosal immunity, M cell, PLGA, Nanoparticles, Drug delivery, Vaccine

1. Introduction The tight junction protein claudin-4 is one of the unusual members of the claudin gene family, sitting at a distinct locus with the closely related claudin 3, away from other family members, in both the mouse and human genome. Claudins 3 and 4 have similar c-terminal cytoplasmic tails, again distinct from other family members, and their second external domains bind to the Clostridium perfringens enterotoxin (1, 2). Thus, despite the observations that claudin-4 participates in epithelial tight junction barrier function, and indeed plays a major role in establishing transepithelial electrical resistance (3), this protein has distinct features that point to additional functions. This has been borne out in our studies identifying claudin-4 as a protein associated with the unique particle capture functions of M cells in mucosal Kursad Turksen (ed.), Claudins: Methods and Protocols, Methods in Molecular Biology, vol. 762, DOI 10.1007/978-1-61779-185-7_9, © Springer Science+Business Media, LLC 2011

115

116

H.A. Eckelhoefer et al.

epithelium (4–6). M cells are specialized epithelial cells that capture microparticles and antigens and transport them across the epithelium (“transcytosis”) to underlying immune cells (e.g., dendritic cells) for stimulation of mucosal immunity (7). While we do not yet know exactly how claudin-4 participates in M cell transcytosis, it appears to be a structural component of transcytosis vesicles. Thus, M cell delivery can be targeted by ligands with affinity for the exposed domain of claudin-4. The methods described here provide details on how we are able to work with claudin-4 both in vitro in studies on its cell biology and in vivo in studies on its role in M cell transcytosis. 1.1. Claudin-4: Cell Biology Studies Using GFP-Tagged Claudin-4

In order to visualize claudin-4 in live cells, we tagged claudin-4 with green fluorescence protein (GFP). However, since the C-terminal domain of claudin-4 has a PDZ domain which binds to PDZ proteins such as ZO-1, we fused GFP to the N-terminal to avoid interference with normal claudin-4 function. A common cell line used for studying epithelial cell tight junctions is Caco-2, or the subclone Caco-2 BBe (8); it is not as easily transfected as other cell lines, so we also generated stable integrants with specific expression levels of GFP-claudin-4, using serial cell sorting.

2. Materials 2.1. Cell Biology Studies on Claudin-4

1. Caco-2 BBe cell line was obtained from ATCC. 2. Advanced Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Biowest), 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 1 M), and pen/strep/glutamine (Invitrogen) were used for cell culture. Geneticin (Invitrogen) is added to the cell culture medium to maintain the stable transfected cell line. 3. Opti-MeM I reduced serum medium (Invitrogen) and lipofectamine 2000 (Invitrogen) were used for cell transfection. 4. Trypsin-EDTA (Invitrogen) and EDTA (Amresco). 5. The primary antibody mouse anti-ZO-1 and the secondary antibody goat anti-mouse Alexa647 were purchased from Invitrogen. Casein (Thermo Scientific), Tween-20 (Fisher), and 4% paraformaldehyde (Electron microscopy sciences). 6. The FACS machine used for cell sort is a FACSAria (BectonDickinson).

2.2. Surface Plasmon Resonance

1. Biacore X100. 2. Sensorchip CM5 (Biacore Cat #BR-1000-12): Stored at 4°C, equilibrate to RT before use.

9 Claudin-4: Functional Studies Beyond the Tight Junction

117

3. Amine coupling kit (#BR-1000-50): EDC and NHS are aliquoted and stored at −20°C. Ethanolamine is stored at 4°C. 4. Coupling buffers: 10 mM acetate buffer, pH 4.0, 4.5 (Cat# BR-1003-49 to 50), stored at 4°C. 5. Running buffer: HBS-EP + buffer (10× stock: 0.1 M HEPES, 1.5 M NaCl, 30 mM EDTA, and 0.5% surfactant P20) (Biacore Cat #BR-1008-26). Stored at RT, dilute to 1× before use. Use the exact same aliquot of buffer for sample preparation as is run through the instrument. 6. Regeneration solution: 2 M NaCl. 7. Sample tube & cap specific for instrument rack (Cat# BR-1002-87, BR-1004-11). 8. Microcon centrifuge tube: Millipore (Cat# 42412 for 100kD MWCO), used for buffer exchange. 9. Ligand: GST-Cldn4 R4 recombinant protein expressed and purified from bacteria (BL21), exchanged to HBS-EP + buffer for immobilization. Only GST protein is used as the control (Genscript Cat#Z02039-1). 10. Analyte: (a) Recombinant HA proteins containing Cldn4 Ecl2-binding sequences; (b) C-CPE protein (His tagged aa 184-319) used as the positive control. All proteins need to be buffer exchanged to HBS-EP + buffer using the Microcon tubes. ●●

2.3. PLGA Nanoparticles

All solutions need to be filtered using 0.22-mm filter before use. Small volumes (e.g., ligand and analyte) can be filtered through UltraFree-MC centrifugal units (Millipore, Cat#UF30GVNB).

1. The poly(dl-lactide-co-glycolide) (PLGA 85:15, MW 50,000–75,000) and poly(vinyl alcohol) (PVA, MW 30,000– 70,000, 87–90% hydrolyzed) were obtained from Sigma–Aldrich. 2. HEPES (1 M), phosphate-buffered saline (PBS, 1×), and sodium dodecyl sulfate solution (SDS, 10%), F-12 Kaighn’s medium, and geneticin were purchased from Invitrogen. 3. Methylene chloride optima®, PBS (10× ready concentrate pouches), and sodium hydroxide (certified ASC) were obtained from Fisher Scientific. 4. Rhodamine 6 G was obtained from Fluka® Analytical and 16% paraformaldehyde was obtained from Electron Microscopy Sciences. 5. Prolong Gold anti-fade reagent with DAPI and 0.2 mm 505/515 (yellow-green) Neutravidin FluoSpheres® were purchased from Molecular Probes.

118


3. Method 3.1. Design DNA Construct and Transfection

We added the GFP coding sequence to the N-terminal of claudin-4 to make our fusion GFP-CLDN4 construct. The primers used for GFP and CLDN4 cloning were GFP 5¢-CCCTACCCAAGCTTGATAATATGGCCACCACC-3¢ (For), 5¢-CGCGGATCCTGCTGACTTGTACAGCTCATCCAT3¢(Rev), CLDN4 5¢-ATATAAACGCGGATCCATGGCCTCCATGGGG-3¢ (For) 5¢-CCGGAATTCTAACACGTAGTTGCTGG-3¢ (Rev). The GFP-CLDN4 was then ligated into the pcDNA43.1 (+) plasmid, which has a CMV promoter in the sequence. After the construct was established, we transfected Caco2-BBe with the construct. Transfection of Caco-2BBe cells is often not very efficient, so we have used a variety of methods to develop transfected cell lines. 1. Caco2-BBe cells were passaged into a six-well plate 2 days before transfection. On the day of transfection, the cells were around 90% confluent. 2. Plasmid transfection was accomplished as per the manufacturer’s data sheet. Briefly, perform the following: (a) Dilute 4 mg DNA in 250 ml Opti-MEM I reduced serum medium and mix gently. (b) Mix 12 ml of lipofectamine 2000 in 250 ml of Opti-MEM I reduced serum medium. Incubate for 5 min at room temperature. (c) After 5-min incubation, combine the diluted DNA with the diluted lipofectamine 2000, mix gently by pipetting, and incubate at room temperature for 20 min. (d) Add 500 ml of complexes to the well containing cells and 2 ml of Opti-MEM I reduced serum medium. Mix gently by rocking the plate back and forth. (e) Incubate the cells at 37°C in a CO2 chamber for 24 h before subjecting to geneticin selection (600 mg/ml). Change the medium after 4 h and then every 2 days.

3.2. FACS Selection

1. The cells were cultured until 100% confluent in the full medium with 600 mg/ml geneticin in two 75-cm2 flasks. 2. The medium was removed from the flasks and the cells were washed twice with PBS. To detach the cells, 1 ml of


119

t rypsin-EDTA was added into each flask and incubated at 37°C for 5 min. 3. The trypsinization was quenched with 5 ml sorting buffer (PBS 500 ml, EDTA 1 mM, HEPEs 25 mM, and 1%FBS), the cells were then transferred into a separate, 15-ml cornical tube, and spun at 1,000 rpm for 5 min. 4. After aspirating the supernatant, the cells were resuspended by pipetting in 1 ml sorting buffer and spun again at 1,000 rpm for 5 min. 5. The supernatant was again aspirated and resuspended by pipetting in 1 ml sorting buffer. The cells were then passed through the cell strainer and combined in a new 15-ml tube. 6. The cells were counted and then diluted with the sorting buffer at a concentration of 10 million/ml. 7. The cells were sorted using the FACSAria. The non-transfected Caco2-BBe cells were used as a negative control. A higher GFP expression may cause cell death, so we sorted for the medium expression of GFP cells (Fig. 1). 8. The sorted cells were spun down at 1,000 rpm for 5 min. The supernatant was aspirated and the cells were washed with 1 ml full medium twice before the sorted cells were seeded back to the flask and incubated at 37°C.

Fig. 1. Selection of Caco2-BBe cells expressing GFP-CLDN4 by FACS. Figure (a) demonstrates the expression of GFP in BBe control cells (green), GFP-CLDN4 transfected cells before sorting (red ), and GFP-CLDN4 transfected cells after two rounds of sorting (blue). It is clear that after two rounds of sorting, the transfected cells significantly increased their GFPCLDN4 expression. Figure (b) shows the gate of GFP expression. In order to avoid any toxic effects induced by overexpression of GFP, we sorted for medium GFP-expressing cells.

120


Fig. 2. Immunohistochemistry. Three-dimensional confocal image of stable GFP-CLDN4 transfected cells. Stable GFPCLDN4 transfected Caco2-BBe after three rounds of sorting was used for immunostaining. Green indicates GFP, red indicates ZO-1, and blue indicates nuclei. In the GFP-CLDN4 transfected cells, GFP-CLDN4 can not only go to the tight junction where it co-localizes with ZO-1 (red) but can also be found at the apical membrane (arrow ).

9. The cells were cultured with geneticin (600 mg/ml). When they were confluent, steps 1–9 were repeated twice. 10. After sorting the transfected cells, the GFP expression of the transfected cells was significantly shifted to the right (Fig. 1). We also performed histocytochemistry to further confirm the expression pattern of GFP-CLDN4 in the cells (Fig. 2). The sorted cells were then stored in liquid nitrogen (see Note 1). 3.3. Histocyto chemistry

1. After the cells were sorted by FACS and cultured in the 75-cm2 flask until confluent, 1 × 105 cells were passaged into the chamber slides and cultured with geneticin for 48 h before staining. During staining, the medium was removed and washed twice with PBS.


121

2. The cells were fixed with 1% paraformaldehyde in PBS at room temperature for 20 min and washed with 0.1% Tween in PBS (PBST) twice for 5 min. They were then permeabilized with 0.5% Tween in PBS for 10 min and then washed twice again with 0.1% Tween in PBS. 3. Before being incubated with the primary antibody (ZO-1) at room temperature for 90 min, the cells were blocked with casein plus Tween (CT) for 10 min. 4. After primary antibody incubation, the cells were washed with PBST twice for 5 min and then incubated with secondary antibody (goat anti-rabbit 568 1:1,000 in CT) for 45 min. They were then washed with PBST again and post-fixed with 4% paraformaldehyde for 15 min. The cells were then washed with PBS once, mounted with Prolong Gold anti-fade reagent with DAPI, and dried at room temperature for 24 h. 5. The cells were imaged with a BD CARV II spinning disc confocal microscope using IPLab software and analyzed with Volocity imaging processing software (Perkin–Elmer/ Improvision). 3.4. Surface Plasmon Resonance Studies on Claudin-4: Binding to the Second External Domain

This assay is designed to measure recombinant HA protein containing claudin-4-binding sequences to the claudin-4 Ecl2 region (9). Purified GST-Cldn4 R4 is used as the ligand, and GST protein only is the control, both of which are immobilized onto a CM5 sensorchip using an amine-coupling reaction.

3.4.1. Protein Expression and Purification

Claudin-4: mouse claudin-4 (cldn4, NM_00903) was subcloned into pGEX4T-2 by PCR with high-fidelity DNA polymerase Pfu (Stratagene). The primers for the R4 deletion mutant (Ecl2.CT) were F: 5¢-GGATCCTGGACCGCTCACAACG-3¢ and reverse primer 1: 5¢-CTCGAGTTACACATAGTTGCTGGCGGGG-3¢. The constructs were confirmed by DNA sequencing. GSTCldn4/pGEX4T-2 construct was transformed into Escherichia coli (BL21, pLysS) for protein expression. The soluble protein was purified by glutathione-agarose affinity chromatography (Pierce), and the co-purified GST protein was separated by gel filtration chromatography on FPLC with Superdex 200 column. For use as the analyte in Biacore assay, GST-Cldn4 was balanced to HBS-EP buffer by Microcon (Millipore) centrifugation.

3.4.2. Preparation of the Chip for Use

1. Turn on the Biacore ~1 h before use to allow temperature to stabilize to 25°C. 2. Take CM5 sensorchip out of 4°C, remove it from the sealed bag, and allow to warm to RT. 3. Disconnect the inlet tubes from the H2O storage bottle and connect to the running buffer HBS-EP + bottle. 4. Dock new CM5 chip into the instrument.

122


5. Immobilize GST and GST-R4 to the chip by diluting each to 15 mg/ml in pH 4.0 acetate buffer, and 30 mg/ml in pH 4.5 acetate buffer, respectively. GST will be immobilized to the control channel, Fc1; GST-R4 will be immobilized to the assay channel, Fc2. The immobilization is accomplished by an amine-coupling mechanism that reacts the –NH2 on the protein with the –COOH of the dextran on the chip. 6. Prepare the required amounts of EDC, NHS, and ethanolamine (85 ml, 85 ml, and 237 ml, respectively), and load all of these reagents into Biacore tubes and into the rack. 7. Use the “amine coupling” tool in surface preparation Wizard to operate the immobilization, choose Fc2 for GST-R4 and Fc1 for GST, and select the “aiming for certain RU parameter” to 3,000RU to control the amount of ligand that will be bound. The procedure will continue automatically, and the resultant immobilized RU will be shown at completion. 3.4.3. Binding Assay

1. Prepare enough of each analyte (70 ml) in HBS-EP + buffer. Normally, recombinant HA proteins are prepared at a 1–5 mM concentration range. Prepare 5 mM of C-CPE as the positive control, which should give a clear positive binding result on the sensorgram, indicating the chip is active. 2. Load all of the analyte samples, positive control, and regeneration solution into sample rack. 3. Measure the binding by using the “Binding assay” tool from the “Wizard.” Select the variable options: sample contact time 120 s, dissociation time 120 s, regeneration time 180 s, and 30 s stabilization. The procedure will run automatically using the wizard software (see Note 2). 4. Analyze the data by using the “Evaluation” software. Alignments and presentation modifications can be done very specifically, depending on the need. 5. Undock the active chip and store at 4°C in a 50-ml conical tube with ~2 ml of H2O to keep it moist. The chip can be reused until noticeable activity is decreasing in regard to binding C-CPE. 6. Shut down the instrument.

3.5. Claudin-4 Targeting In Vivo: PLGA Nanoparticles for Targeting to M Cells

Our studies have suggested that claudin-4 is a component in mucosal M cell particle uptake as part of its role in immune surveillance at the mucosal epithelium. This raised the intriguing possibility that ligands targeting claudin-4 could be used to mediate delivery of cargo such as vaccines. We have begun to test this potential using PLGA nanoparticles incorporating a recombinant protein with a claudin-4-binding peptide, confirmed by Surface Plasmon Resonance to have high affinity binding (10).

9 Claudin-4: Functional Studies Beyond the Tight Junction 3.5.1. Preparation of Claudin-4-Targeted Recombinant ProteinLoaded PLGA Nanoparticles

123

Given that the CPE30 targeting peptide can indeed mediate measurable uptake of fluorescent beads by M cells, we developed nanoparticles that could incorporate the recombinant HA-HTCPE30 fusion protein (a fusion between the extracellular domain of influenza hemagglutinin and a C-terminal CPE30 peptide). It was critical that such particles would retain the proteins long enough for delivery to the target tissue, yet display enough of the protein on the surface to enable the CPE30 targeting peptide moiety to mediate uptake by M cells. PLGA polymer nanoparticles seemed to be an appropriate choice, since they could be produced with incorporated protein, yet they are biodegradable and can release the protein over time in vivo once delivered (11). PLGA nanoparticles containing targeting (HA-HT-CPE30) and nontargeting (HA-HT) peptides were prepared from 85:15 PLGA using solvent evaporation/double emulsion (also known as water-in-oil-in water, w/o/w) method. 1. Preparation of stock solutions (a) 4% PLGA polymer (85:15) solution was prepared by adding 0.18 g of PLGA into 4.5 ml of methylene chloride in a glass beaker and stirring until dissolved (see Note 3). (b) 2% PVA solution was prepared by dissolving 0.6 g of PVA in 30 ml 10 mM HEPES and adjusting the pH to 7.5 with NaOH (see Note 4). (c) Protein solutions: HA-HT-CPE30 or HA-HT protein in HEPES buffer at 3.0–4.5 mg/ml concentration (see Note 5). (d) For labeling experiments; a 40 mg/ml Rhodamine 6 G (R6G) solution was prepared by dissolving 1 mg of R6G in 25 ml of methylene chloride (see Note 6). 2. Preparation of first w/o emulsion: The reagents listed in the table were added to a 18 × 150 mm disposable glass tube in the order listed. 4% PLGA solution

4.25 ml

Protein

0.5 ml of HA-HT-CPE30 or HA-HT

2% PVA stabilizer

0.25 ml

For labeled nanoparticles, 25 ml of 40 mg/ml R6G was added to the PLGA solution before adding the protein. The solution was emulsified by probe sonication (Branson Sonifier 450) for 20 s (duty cycle 20%, output control 3) to obtain w/o emulsion. 3. The resulting w/o emulsion was divided into two disposable glass tubes (see Note 7), and 12.5 ml of 2% PVA solution was added to each tube. The solution was emulsified by probe sonication for 30 s (duty cycle 20%, output control 3) to obtain the final w/o/w emulsion.

124


4. The final w/o/w was then combined in a 50-ml glass beaker and stirred uncovered for 20 h with a magnetic stirrer at 400 rpm at 4°C to allow solvent evaporation. 5. The solution was added to a 40-ml Oakridge tube and centrifuged at 3,800 rpm (RCF 1000 g) for 30 min. The supernatant was discarded and the pellet was resuspended gently in 20 ml of distilled water. The washing step was repeated with two 20-min and one 15-min centrifugation. The supernatant was discarded. 6. The resulting nanoparticle pellet was frozen in liquid nitrogen and lyophilized overnight at −88°C, 0.006 Torr. 7. The final product was stored at 4°C and kept dry with Dryrite calcium sulfate pellets till ready to use. 3.5.2. Nanoparticle Characterization Scanning Electron Microscopy

Particle Size Measurements

The morphology of the protein-loaded nanoparticles was visualized by scanning electron microscopy (SEM). A very small amount of nanoparticles was placed on a double-sided adhesive tape attached to an aluminum stub and sputter coated with gold/palladium beam for 2 min. The coated samples were imaged with Philips XL30-FEG SEM at 10 kV (Fig. 3). The particle size of the nanoparticles was measured with ImageJ software using the obtained SEM images. The diameter of

Fig. 3. SEM and size distribution of HA-HT-CPE nanoparticles showing a narrow range of particle size around 300 nm in diameter. Particle diameter was measured by ImageJ® software using SEM images obtained for three different preparations of nanoparticles. The diameter of approximately 150 particles was measured for each preparation. The frequency distribution of particle size was plotted with Prism software. X-axis represents the particle diameter in micrometer, while y-axis represents the percentage of particles at a given diameter.


125

approximately 150 nanoparticles was measured, and the size distribution was plotted using Prism software. Determination of Protein Loading

Total protein loading was estimated using BCA assay. Approximately 5–8 mg of freeze-dried nanoparticles were accurately measured, added to 2 ml of 5% SDS in 0.1 M NaOH solution, and incubated with shaking for 24 h at room temperature until a clear solution was obtained. The protein content was measured in triplicates for each sample using BCA protein assay. The protein loading (%, w/w) was expressed as the amount of protein relative to the weight of the nanoparticles assayed (12).

3.6. In Vitro Uptake of Claudin-4-Targeted Protein-Loaded PLGA Nanoparticles

In vitro, we found that the nanoparticles with the HA-HT-CPE protein were readily taken up by GFP-claudin-4 CHO transfectants, showing both the function of the targeting peptide and the accessibility of the functional targeting peptide in the nanoparticles. The GFP-claudin-4 CHO cell transfectants were selected by serial FACS selection as described above for transfected Caco2BBe cells. 1. In vitro uptake studies of R6G-labeled protein-loaded nanoparticles were performed in GFP-tagged claudin-4 transfected Chinese hamster ovary (CHO) cells (9). 2. The cells were maintained in F-12 Kaighn’s medium supplemented with 10% fetal bovine serum and 0.8 mg/ml geneticin. 3. For the confocal studies, the cells were plated on cover slides placed in six-well plates and grown at 37°C in 5% CO2 incubator for 48 h. 4. The cells were washed with PBS and the medium was replaced by 1 ml of nanoparticle solution in culture medium prewarmed to 37°C (10 mg of protein/well). The cells were incubated at 37°C in 5% CO2 incubator for 1 h. 5. Upon incubation, the cells were washed three times with PBS to remove unbound nanoparticles. 6. The cells were then fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and washed with PBS + 0.1% Tween20 for 3–5 min, two times. 7. The cover slides were mounted on glass slides with Prolong Gold anti-fade reagent with DAPI and incubated for 24 h at room temperature. 8. The cells were imaged using a BD CARV II spinning disc confocal microscope, using IPLab software (Fig. 4). 9. Histological analysis of particle uptake was performed by counting the number of particles taken up per cell in randomly selected fields of the slides for three different experiments.

126


Fig. 4. PLGA nanoparticle uptake by GFP-claudin-4 transfected CHO cells. R6G-labeled nanoparticles were given to GFP-claudin-4 transfectants (green) for 1 h, and the cells were processed for confocal microscopy. Control HA-HT nanoparticles were not taken up (top), while HA-HT-CPE/R6G particles (bottom) were found to be readily bound and ingested, with particles shown to co-localize internally with GFP-claudin-4 (yellow spots, arrows). Cell nuclei, blue.

4. Notes 1. In general, the high level expression of the GFP is stable after three or more rounds of FACS selection. 2. To better account for free GST in the GST-R4 sample, set the target RU to 3,000 for that channel, but set the Fc1 GST RU


127

to 1,500. Roughly half of our GST-R4 sample is free GST, which can skew binding results when comparing Fc2 to Fc1 as the evaluation software will do. If immobilization is difficult, and the optimum RU cannot be reached, increase the problem sample concentration by 50% and repeat. 3. When dissolving PLGA polymer in methylene chloride, it is important to add the methylene chloride into the beaker first and then add PLGA. Otherwise, PLGA would get stuck on the walls of the beaker, making it harder to dissolve. In addition, glass pipettes should be used when measuring methylene chloride. 4. When making PVA stock solution, first add the buffer into the beaker and while stirring, add PVA gradually to avoid formation of clumps, which would take a longer time to dissolve. 5. When nontargeted HA-HT and claudin-4-targeted HA-HTCPE recombinant fusion proteins are incorporated into nanoparticles, they should be in a low ionic buffer such as HEPES, or PBS:HEPES 1:10, compared to full strength PBS. 6. For labeled nanoparticles, R6G dye should be dissolved in an organic solvent such as methylene chloride, ethanol, or DMSO. 7. Glass pipettes should be used when dividing the w/o emulsion into two tubes as this solution contains methylene chloride.

Acknowledgments This work was supported by a Grand Challenges in Global Health award from the Foundation for the National Institutes of Health (FNIH), and Grants R21 AI73689 and R01 AI63426 from the National Institutes of Health. References 1. Sonoda, N., Furuse, M., Sasaki, H., Yonemura, S., Katahira, J., Horiguchi, Y., Tsukita, S. (1999) Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: evidence for direct involvement of claudins in tight junction barrier. J. Cell Biol. 147, 195–204. 2. Katahira, J., Sugiyama, H., Inoue, N., Horiguchi, Y., Matsuda, M., Sugimoto, N. (1997) Clostridium perfringens enterotoxin utilizes two structurally related membrane

proteins as functional receptors in vivo. J. Biol. Chem. 272, 26652–8. 3. Colegio, O.R., Van Itallie, C., Rahner, C., Anderson, J.M. (2003) Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture. Am. J. Physiol. Cell Physiol. 284, C1346–54. 4. Lo, D., Tynan, W., Dickerson, J., Scharf, M., Cooper, J., Byrne, D., Brayden, D., Higgins, L., Evans, C., O’Mahony, D.J. (2004) Cell

128


culture modeling of specialized tissue: Identification of genes expressed specifically by Follicle Associated Epithelium of Peyer’s Patch by expression profiling of Caco-2/Raji co-cultures. Int. Immunol. 16, 91–99. 5. Clark, R.T., Hope, A., Lopez-Fraga, M., Schiller, N., Lo, D.D. (2009) Bacterial particle endocytosis by epithelial cells is selective and enhanced by tumor necrosis factor-receptor ligands. Clin. Vacc. Immunol. 16, 397–407. 6. Wang, J., Lopez-Fraga, M., Rynko, A., Lo, D.D. (2009) TNFR and LTbR agonists induce Follicle-Associated Epithelium and M cell specific genes in rat and human intestinal epithelial cells. Cytokine 47, 69–76. 7. Neutra, M.R., Pringault, E., Kraehenbuhl, J-P. (1996) Antigen sampling across epithelial barriers and induction of mucosal immune responses. Annu. Rev. Immunol. 14, 275–300. 8. Peterson, M.D., Mooseker, M.S. (1993) An in vitro model for the analysis of intestinal brush border assembly. I. Ultrastructural analysis of cell contact-induced brush border

assembly in Caco-2BBe cells. J. Cell Sci. 105, 445–460. 9. Ling, J., Liao, H., Clark, R., Wong, M. S., Lo, D. D. (2008) Structural constraints for the binding of short peptides to claudin-4 revealed by surface plasmon resonance. J. Biol. Chem. 283, 30585–30595. 10. Rajapaksa, T. E., Stover-Hamer, M., Fernandez, X., Eckelhoefer, H. A., Lo, D. D. (2009) Claudin 4-targeted protein incorporated into PLGA nanoparticles can mediate M cell targeted delivery. J. Control Release. 11. Zhao, A., Rodgers, V.G. (2006) Using TEM to couple transient protein distribution and release for PLGA microparticles for potential use as vaccine delivery vehicles. J. Control. Release. 113, 15–22. 12. Coombes, A. G., Yeh, M. K., Lavelle, E. C., Davis, S. S. (1998) The control of protein release from poly(dl-lactide co-glycolide) microparticles by variation of the external aqueous phase surfactant in the water-in oil-in water method. J. Control. Release. 52, 311–320.