Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 3

Particle Transcytosis Across the Human Intestinal Epithelium

ELISABET GULLBERG

Model Development and Target Identification for Improved Drug Delivery

ISSN 1651-6192ISBN 91-554-6145-Xurn:nbn:se:uu:diva-4780

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2005

Till pappa

List of Papers

The thesis is based on the following papers, which will be referred to by their Roman numerals:

I Gullberg, E., Leonard, M., Karlsson, J., Hopkins, A.M., Brayden, D., Baird, AW., Artursson, P. Expression of specific markers and particle transport in a new human intestinal M-cell model. Biochem Biophys. Res. Commun. (2000) 279, 808-813. Reprinted by permission. © 2000 Elsevier Science.

II Gullberg, E., Velin Keita, Å., Salim, SY., Andersson, M., Cald-well, K., Söderholm, JD., Artursson, P. Increased CD9 and 1-integrin expression in human follicle associated epithelium is linked with selective transport into human Peyer´s patches.Submitted

III Gullberg, E., Ragnarsson, EGE., des Rieux, A., Artursson, P. TNF- , TGF- 1 and LT 1/ 2 induces particle uptake and aug-ments B-cell-induced particle transcytosis in intestinal epithelial cells. Manuscript

IV Ragnarsson, EGE., Gullberg, E., Schoultz, I., Magnusson, K.-E., Söderholm, J., Artursson, P. Yersinia Pseudotuberculosis induces

1-integrin expression and particle transcytosis in human intesti-nal epithelial Caco-2 cells. Manuscript

Contents

1. Introduction.................................................................................................91.1. The intestine ........................................................................................9

1.1.1. M-cells .......................................................................................111.2. Mechanisms of intestinal uptake and transport .................................12

1.2.1. Mechanisms of endocytic uptake...............................................131.2.2. Transcytosis ...............................................................................14

1.3. Factors influencing intestinal particle uptake and transport..............151.3.1. Properties of the intestinal barrier..............................................151.3.2. Properties of the administered particle ......................................17

1.4. Methods of studying intestinal particle uptake and transport............191.4.1. In vivo studies ............................................................................201.4.2. In vitro studies on intestinal tissue.............................................201.4.3. Cell culture models ....................................................................20

1.5. Concluding remarks ..........................................................................21

2. Aims of the thesis......................................................................................22

3. Methods ....................................................................................................233.1. Establishment of a model FAE..........................................................23

3.1.1. Choice of Caco-2 cell clone/line................................................243.1.2. Choice of B-cells .......................................................................243.1.3. Choice of co-culture conditions.................................................25

3.2. Particle uptake and transport studies .................................................253.2.1. Cell culture experiments ............................................................253.2.2. Ussing chamber studies on intestinal tissue...............................253.2.3. Analysis of particle uptake ........................................................263.2.4. Analysis of particle transport.....................................................263.2.5. Assessment of integrity and viability.........................................26

3.3. Gene expression studies ....................................................................283.3.1. cDNA-array analysis .................................................................283.3.2. Interpretation..............................................................................28

3.4. Protein expression studies .................................................................293.4.1. Cell-based ELISA......................................................................303.4.2. Immunofluorescence..................................................................30

4. Results and discussion ..............................................................................314.1. Characteristics of the model FAE in comparison with human FAE .31

4.1.1. Characteristics of the induced particle transport........................324.2. Identification of target structures in human FAE ..............................33

4.2.1. Cell surface carbohydrates.........................................................334.2.2. Cell adhesion proteins................................................................33

4.3. Exploiting identified targets in human FAE......................................374.3.1. Transport of RGD-coated particles across the model FAE .......384.3.2. Selective transport of RGD-coated particles into human Peyer´s patches ....................................................................................38

4.4. Soluble factors as mediators of particle uptake and transport ...........394.4.1. Gene expression profile of Raji B-cells that induce epithelial particle transport ..................................................................................394.4.2. Effects of B-cell-secreted proteins on cell monolayer integrity and particle uptake...............................................................................404.4.3. Effects of B-cell-secreted proteins on particle transport............42

4.5. Influence of enteric bacteria on particle uptake and transport...........444.5.1. Effects of Yersinia Pseudotuberculosis on cell monolayer integrity and particle uptake ................................................................454.5.2. Effects of Yersinia Pseudotuberculosis on particle transport ....46

5. Summary and conclusions ........................................................................48

Acknowledgements.......................................................................................50

References.....................................................................................................52

Abbreviations

AP Alkaline Phosphatase BMP Bone morphogenic protein cDNA Complementary DNA CK cytokeratin ELISA Enzyme-linked immunosorbent assay FAE Follicle-associated epithelium FCS Fetal Calf Serum HBSS Hank’s Balanced Salt Solution IFN- Interferon gamma IL Interleukin IRF-1 Interferon regulatory factor 1 LT Lymphotoxin Papp Apparent permeability coefficient RANTES T-cell expressed and secreted RT-PCR Reverse transcription polymerase chain reaction SD Standard deviation TDGF Thyroid derived growth factor TER Transepithelial Electrical Resistance TGF- Transforming growth factor beta TNF- Tumor Necrosis factor alpha VE Villus epithelium

9

1. Introduction

Oral delivery of proteins using micro-particulate carriers has been under investigation for several decades, with emphasis on the formulation of anti-gens for mucosal vaccination.1, 2 The development of new peptide- and nu-cleic acid-based drugs has attracted further interest in particulate carriers for drug delivery across the intestinal epithelium.3

Drugs encapsulated in micro-particles have a low bioavailability.4 There-fore, the most appropriate, and important, application for these carrier sys-tems is the administration of mucosal vaccines. Indeed, oral delivery of anti-gens is by far the most effective method of inducing gastrointestinal mucosal immune responses.2 Furthermore, the benefits of needleless immunization in third world countries would be substantial.5

However, it is being increasingly questioned whether encapsulated anti-gens can be taken up in sufficient quantities to achieve adequate immune responses.6 While early studies in rodents and rabbits showed that up to 30% of orally administered particles were taken up across the intestinal epithe-lium,7, 8 9 more recent studies indicate that the uptake is typically less than 1% of the given dose.6 Although little is known of the extent of particle up-take in the human intestine, the moderate effects on the immune response after particulate delivery of antigens in man10-12 suggest that improvements in the efficacy of particle uptake are required before subunit oral vaccination becomes a feasible option.

Strategies for improvements to the uptake process include targeting to immune inductive sites, such as the M-cells of Peyer’s patches, in the intes-tine. However, little is known of human M-cells, and the identification of targets on these cells would significantly advance the development of effec-tive oral vaccines. In addition, adequate model systems for studying mecha-nisms of particle transcytosis and predicting particle uptake in the human intestine are required. In this thesis, a model of the human FAE was estab-lished for studies of intestinal particle transport and new targets for imroved uptake of particulate carriers were identified in human FAE.

1.1. The intestine The intestinal wall consists of three distinct layers: the muscularis mucosa, the lamina propria and a single layer of epithelial cells, which is constantly

10

being renewed (Fig. 1).13 In the small intestine, the surface area is enlarged by the presence of folds and villi and this part of the intestine is also consid-ered most important for nutrient and drug absorption in the gastrointestinal tract.14 The epithelium covering the villi consists of several different types of cells: absorptive cells, mucus-secreting goblet cells, Paneth cells secreting anti-microbial peptides, and endocrine cells. The majority (about 85%) of the cells in this epithelium are absorptive.13

The intestine undergoes continuous antigenic challenge and numerous lymphoid cells maintain the mucosal barrier to luminal pathogens. T-lymphocytes can be found within the epithelium, and the lamina propria contains numerous macrophages, dendritic cells, T-cells and antibody-secreting plasma cells.15

Figure 1. Schematic illustration of the structure of the intestinal wall, with villus, follicle-associated epithelium (FAE) and resident immune cells. For clarity, nerves and blood and lymphoid vessels have been omitted. GC: germinal center. IEL: Intra-epithelial lymphocytes.

Interspersed between the villi, within the lamina propria, isolated lymphoid follicles (ILF) or aggregates of these (Peyer´s patches) can be found, espe-

11

cially in the distal part of the small intestine (Fig. 1).16, 17 The follicular area contains numerous B-cells, T-cells, dendritic cells and macrophages and this area plays an important role in the mucosal defense against ingested patho-gens.15 A single layer of epithelial cells covering each follicle forms a dome between the villi (Fig. 1). This follicle-associated epithelium (FAE) differs from the villus epithelium (VE) in that it contains specialized M-cells, with an increased capacity to absorb proteins and particulate antigens.18

1.1.1. M-cells

Figure 2. Schematic drawing of a differentiated M-cell with resident lymphocytes in the intraepithelial pocket, surrounded by absorptive enterocytes. Adapted from19

M-cells are epithelial cells, with tight junctions that define an apical and basolateral domain, but their morphology is quite different from that of ab-sorptive enterocytes (Fig 2).20, 21 The apical membrane typically has micro-folds, or ruffles, rather than microvilli and the glycocalyx is thinner than that of absorptive cells.22 Lymphocytes reside in a pocket, or invagination, of the basal plasma membrane, which results in close contact between lymphocytes and antigens transported across the thin apical membrane of the M-cell.18

It is a matter of debate whether M-cells constitute a cell lineage of their own, whether they are generated from the same pluripotent stem cells as absorp-tive enterocytes, Goblet and Paneth cells,13, 23 or whether they even form directly from absorptive enterocytes. Several studies support the latter two hypotheses, showing that absorptive cells can differentiate into M-cell-like cells upon stimulation with lymphocytes or bacteria in vivo.20, 21, 24-26 Other studies propose that M-cells form after stimulation of lymphocyte-derived factors in specific FAE-associated crypts and therefore cannot be derived from absorptive enterocytes.27 In a recent study, however, M-cells were

12

found in the absorptive VE,28 which further supports the notion of a common precursor for all epithelial cells.

1.2. Mechanisms of intestinal uptake and transport The intestinal epithelium is primarily designed to absorb nutrients from the gut luminal contents. Microvilli protruding from the apical membrane of the absorptive cells extend the area available for absorption14 and several routes of uptake exist (Fig. 3). Small, amphiphatic molecules can easily be ab-sorbed by partioning into the lipid bilayers and crossing the epithelial cell layer by passive diffusion. Hydrophilic molecules may cross the epithelial cell layer by the paracellular route, if small enough, or by random uptake of fluids. In addition, numerous transporters are available for facilitated uptake of sugars, amino acids, oligopeptides, organic acids, vitamins,14 co-factors29

and nucleosides.30

Figure 3. Mechanisms of intestinal uptake and transport. Solutes may pass the bar-rier by passive diffusion through (1) the cell, or (2) the paracellular space. In addi-tion, there are multiple carrier-mediated mechanisms for uptake of nutrients (3). Particles and macromolecules may be transcytosed in endosomal vesicles (4). Sol-utes can also be actively transported back into the lumen after uptake (5).

While these mechanisms effectively mediate the absorption of nutrients, the entry of undigested macromolecules and pathogens into the body is pre-vented by several barriers. Mucus covers large parts of the luminal surface, the surface of the absorptive cells is covered by a glycoprotein coat (the gly-cocalyx), and the apical membranes are associated with high enzymatic ac-tivity.13 Moreover, tight junctions between the cells impedes the passage of larger molecules, and unwanted substances may be transported back into the intestinal lumen by epithelial efflux proteins.31

These barriers are of course major obstacles to the successful delivery of peptides and proteins across the intestinal wall after oral administration. However, larger peptides, proteins or particles may be absorbed in small

13

quantities by endocytic processes in absorptive cells.32 Moreover, the FAE can be considered a selective break in the intestinal barrier to macromole-cules, with lower enzymatic activity than in the VE,33, 34 no or less surface mucus, a thin glycoprotein coat35 and an increased endocytic capacity. 36

1.2.1. Mechanisms of endocytic uptake To date, four mechanisms of endocytic uptake have been described in epithelial cells: clathrin-mediated endocytosis, phagocytosis, macropinocy-tosis and caveolin-mediated endocytosis.18, 37 In addition, evidence of yet another clathrin-independent endocytic pathway exists, but further elucida-tion of this pathway is required.37 Both clathrin-mediated endocytosis and phagocytosis are receptor-mediated processes.38 The clathrin-coated vesicles may internalize both proteins and viruses but seldom become larger than 150 nm in diameter.39 In contrast, during phagocytosis, bacteria and particles up to several m in size may be internalized (Fig. 4).40, 41 Macropinocytosis is also an active, actin-dependent process, through which large volumes of fluid can be internalized.42 The process is in many ways similar to phagocy-tosis, but it is not receptor-mediated.43 Caveolae, flask-shaped invaginations of about 50 nm in diameter, have so far only been found at the basolateral membrane of epithelial cells and are probably of less interest for drug deliv-ery purposes.44 However, the relative contribution of these different path-ways to macromolecular uptake may differ considerably among epithelial cells in different stages of differentiation and under the influence of external stimuli.38

Figure 4. Endocytic mechanisms of uptake in intestinal epithelial cells

Absorptive cells Absorptive cells are capable of taking up many different proteins through clathrin-mediated endocytosis.32 Less commonly, they are also capable of taking up particles and bacteria through phagocytosis,45, 46 a process that is typically triggered by soluble factors secreted by the invading bacterium.47

14

M-cellsM-cells are capable of clathrin-mediated endocytosis,48, 49 macropinocytosis of proteins50 and phagocytosis of bacteria51, 52 viruses20, 53-59 and particles.55, 60

In contrast to absorptive cells, actin-dependent uptake is common in M-cells.61

1.2.2. Transcytosis Unless the formulation has been developed to target the epithelial cells themselves, the administered macromolecules or particulate drug-delivery systems have to be transcytosed (transported across the cell), in addition to being endocytosed (taken up), in order to exert their effects.

Absorptive epithelial cells have both an apical and basolateral sorting compartment for endosomes, and these share a common recycling compart-ment (Fig. 5).62 These compartments seem to be used during both clathrin-mediated endocytosis and phagocytosis.38

Figure 5. The endosomal sorting compartments of the absorptive enterocyte. Endocytosed materials can follow several routes after uptake, including (1) apical recycling with/without cytoplasmic release, (2) lysosomal degradation or (3) transfer from the common recycling compartment to the basolateral sorting compartment for subsequent release from basolateral vesicles. AEE: Apical early endosome; BEE: basolateral early endosome; LE: late endosome; CRC: common recycling compart-ment; Lys: Lysosome. Adapted from 38, 41, 62

Vesicles that bud into the apical cell membrane have several possible fates. They can be recycled back to the apical membrane, with or without their contents (Fig. 5). The protein, virus or particle bound to the internalized

15

receptor is often released into the cytoplasm upon acidification of the vesi-cle, while the receptor is delivered back to the cell membrane. Although released from the vesicle, large molecules like peptides and proteins diffuse slowly through the cytoplasm and may be degraded on their way to the baso-lateral membrane.

The created vesicle may also merge with the common recycling com-partment. From there, the protein, virus or particle is often directed into a pathway leading to degradation in lysosomes. The vesicle may also merge with a vesicle from the basolateral sorting compartment and be released at the basolateral membrane, although this is a much less common process. 37,

38, 62

Thus, transport of intact proteins or carrier-systems across absorptive epithelial cells is not easily achieved, but certainly not impossible. Studies have shown that modification of endosomal sorting mechanisms, in order to decrease apical recycling of vesicles, may increase the transcytosis of pro-teins across epithelial cells.63

Although it is known that bacteria and particles are readily transported across M-cells, the fate of vesicles incorporated into them and the details of the M-cell endosomal sorting machinery have not been well studied.18 The M-cell apical cytoplasm contains numerous endosomal tubules and vesi-cles.48, 50 Although lysosomal markers have been found on these vesicles,64

studies have shown that M-cells have fewer lysosomes than epithelial cells,65

and that proteins can cross M-cells without being degraded.48

1.3. Factors influencing intestinal particle uptake and transportThe extent of particle uptake and transport across the intestinal epithelium after oral administration is dependent on a multitude of factors. The factors preceeding interactions with the intestinal barrier include the physical and chemical stability of the particle in the gastrointestinal tract, the intestinal transit time, food intake and interaction with gut luminal contents.66 The effects of various properties of the epithelial barrier and the administered particle on particle transport are discussed below.

1.3.1. Properties of the intestinal barrier The macroscopic structure of the intestine and its absorptive function seems well preserved during life.67 However, variations in the number of Peyer´s patches may potentially affect the extent of particle uptake, as will intestinal disease. Moreover, the intestinal epithelium undergoes constant renewal and

16

its composition may be influenced by luminal stimuli and secretions from cells of the underlying mucosal tissue. 26, 68, 69

Size and number of Peyer´s patches and M-cells The Peyer´s patches develop before birth and increase in number through childhood to adolescence, when hundreds of patches can be found along the intestine.16 The number of patches then decreases with age, but several tens of patches may be found even in old age.16, 17 However, the size and distribu-tion of patches in different individuals varies considerably,17 and this may potentially influence the extent of particle uptake. It is not yet known whether the number of M-cells in each patch also varies with age, but during adulthood they comprise less than 10% of the FAE.70 It is known that the number of M-cells per patch varies along the intestine, but the function and cause of this variation are not known.71

Cytokines responsible for the formation of M-cells have not yet been identified, although it has been speculated that molecules required for forma-tion of Peyer´s patches, e.g. lymphotoxin (LT) / could also play a role in M-cell formation. Recently, it was shown that the size of the FAE and numbers of M-cells are reduced upon neutralization of LT -receptor signal-ling.68 Thus, LT / may play a role in the induction of the M-cell pheno-type. However, recently, M-cells have also been found in the mouse VE28

and, interestingly, in mice deficient in LT and lacking Peyer´s patches. This indicates that the Peyer´s patch environment, or LT, is not the sole de-terminant for induction of the M-cell phenotype, although it may provide important conditions for it.

Effects of luminal antigen stimulation In 1987, Smith et al. reported that the number of M-cells in mouse FAE in-creased after transfer of pathogen-free mice to normal housing conditions.74

Since then, there have been several reports of de novo M-cell formation and increased uptake of particles after inoculation of mouse or rabbit intestinal loops with bacteria.25, 26, 75, 76 In some cases, the new M-cells were observed as early as one hour after the inoculation.76 In a recent study of the rabbit intestine by Gebert et al., however, these findings were challenged. The au-thors found that the increased uptake of particles seen after inoculation with bacteria depended on an increased transport capacity of M-cells already pre-sent in the FAE.77 Considering the changes in epithelial morphology that must take place during the transition from absorptive cell to M-cell, this explanation for the increased particle uptake seems more conceivable. Either way, bacterial stimulation leads to increased transcytosis of particles, al-though the mechanism for this increase is not known.

In general, bacterial binding to and infection of the intestinal epithelium always leads to production of a cascade of pro-inflammatory cytokines, which recruits mononuclear cells to the site of infection.47 Pro-inflammatory

17

cytokine production by these cells, in turn, is likely to result in increased transcellular and paracellular permeability to macromolecules and particles. For instance, tumor necrosis factor (TNF)- has been reported to induce endocytosis of HRP in intestinal epithelial cells in culture.78, 79 Moreover, it was recently shown that increased transcytosis of bacteria in the human FAE could be correlated to TNF- mRNA levels in the underlying mucosal tis-sue.79

Intestinal disease Intestinal diseases can affect particle uptake in different ways. Dysfunctions in intestinal motility can prolong intestinal transit times, which may affect particle uptake. Inflammation due to food allergy and inflammatory diseases are known to increase intestinal tight junction permeability to macromole-cules.80, 81 There is also evidence of an increase in M-cell numbers during inflammation.82, 83

1.3.2. Properties of the administered particle As described in earlier sections, a particulate carrier has to transverse several barriers in order to be taken up across the intestinal wall. Thus, the surface properties and size of the particle highly influence the extent of uptake. Unfortunately, studies in this area have varied greatly in experimental setup (see section 1.4). It is therefore difficult to compare results with regard to particle properties, but some general conclusions may be drawn.

SizeStudies of polystyrene and poly-lactid-co-glycolidic acid (PLGA) particles, both in cell culture8, 84, 85 and in animals85, 86 in vivo, have shown that particle uptake decreases with particle size. M-cells may take up particles up to 10 µm in size,87, 88 while particles up to 2 um have been reported to be taken up by absorptive enterocytes.85 In addition, the mucus layer may be a significant barrier for particle uptake across the absorptive epithelium. Studies have shown that the transport of small particles (about 300 nm) through mucus is significantly reduced compared with that of larger particles.89, 90

Surface properties As with lower molecular weight drugs, a specific balance between hydro-phobic and hydrophilic surface properties seems to be important for the op-timal uptake and transport particles.91 Studies of various polymers used for encapsulation of proteins show that hydrophilicity is important for passage through the mucus.92, 93 If the goal is to achieve uptake into the Peyer´s patches, the hydrophilic properties should be of less importance, because of the thinner mucus layer in this area. Hydrophobic particles, such as plain polystyrene, also seem to adhere more strongly to M-cells than to entero-

18

cytes.55, 87 However, polymeric PLGA particles have been reported to associ-ate with both FAE and VE,94 although there are contrasting reports on the preferred route of uptake.85, 95

There are no systematic reports of uptake and transport of other hydro-phobic delivery systems, such as liposomes and Iscoms™, across the intesti-nal wall. However, liposomes have been reported to be taken up by M-cells.96 Iscoms seem to be taken up across both FAE and VE, possibly be-cause of their small size (40 nm).97, 98

Since the intestinal cell surface is negatively charged,99 positively charged particles would be expected to interact more strongly with it than un-ionized or negatively charged particles. In line with this theory, plain polystyrene particles have been shown to be more effectively taken up across the intesti-nal epithelium than negatively charged particles.100

Although surface hydrophobicity and charge of the particle are important factors to consider, adsorption of gut luminal contents to the particle, which can totally change its surface properties, should also be taken into account. Adsorption of proteins can lead to increased degradation of the carrier sys-tem,66 but may also improve uptake of particles across the epithelium through opsonization.101

TargetingA more specific way of improving particle uptake and transport across the intestinal epithelium is to target specific surface receptors on epithelial cells by attaching ligands to the particle surface. One example of this process is the improvement of nanoparticle uptake across cultured epithelial cells with vitamin B12.102 Lectins have also been widely used to target both absorptive epithelial cells and M-cells.4 Although these strategies have been successful in improving uptake of peptides and proteins across absorptive epithelial cells, the mucus layer remains an effective barrier to particles. Thus, target-ing the formulation to M-cells and the FAE seems to be the most effective strategy for improving uptake and transport of oral vaccine carriers.

Targeting of mouse M-cells by attaching various ligands, including Ulex Europeaus Agglutinin 1 (UEA-1),103, 104 IgA and IgG,105, 106 to particles and targeting rat M-cells by attaching Invasin107 to particles have resulted in in-creased adherence and transcytosis of the particles across M-cells. Thus, targeting seems a feasible strategy for improving particle uptake into Peyer´s patches, although the size of the particulate carrier and accessibility of the receptor seem critical for the outcome.49, 108

19

1.4. Methods of studying intestinal particle uptake and transportThe uptake of particles has been studied in various animal models and in cell culture. In most animal studies, fluorescent latex (polystyrene) or PLGA particles have been used, but chitosan109, 110 and starch111 microparticles have also been studied. The experimental setup varies greatly, which makes it difficult to draw conclusions regarding the extent of particle uptake. In par-ticular, different doses have been given and many of the detection techniques used are not optimal for identifying particles within intestinal tissue. For example, many studies based the analysis of particle uptake on sectioning of the intestine after particle administration. This introduces considerable bias, since particles adsorbed to the luminal surface of the epithelium can be smeared over the tissue when sectioning. Even more questionable are studies where particle uptake has been assessed by determining the concentration of a fluorescent or radioactive probe in lysed intestinal tissue. Confocal micros-copy of whole tissue, flow cytometry or post-experimental detection of par-ticles using fluorescent antibodies are examples of techniques where particle uptake can be assessed with more certainty.

Regardless of the methods used, it remains difficult to identify the route of intestinal particle uptake. UEA-1 is currently employed as an M-cell marker in the mouse intestine112, 113 and Vimentin and Cytokeratin 18 are used as M-cell markers in the rabbit and pig intestine, respectively (Table 1).114, 115 However, there are no reliable M-cell markers for humans,116 which significantly hampers progress in this field.

Table 1 Markers of M-cells in various speciesa

Type of marker

Human Mouse Rat Rabbit Pig

Lectin SLAA?117**

UEA-1112,

113

EEA118

Intermediate filaments

- CK8119 Vimentin115 CK18114

Cytoskeletalproteins

F-actin120

-actinin120

Surface re-ceptors

ICAM-1121* 1-integrin122*

Enzymes Low AP123 Low AP34 Low AP34 Low AP124

Others ? IgA/IgG-adherence19

IgA/IgG-adherence19

IgA/IgG-adherence19

alectins that only stain M-cells in the caecal patch have been omitted *colon, ** negative results also obtained116, AP: alkaline phosphatase

20

In addition, M-cells of isolated lymphoid follicles have been little studied.125,

126 Therefore, one aim of this thesis was to identify M-cell or FAE markers in the human intestine. A promising approach seems to be identification of markers directly correlated with M-cell function, e.g. expression of bacterial receptors or actin-dependent endocytosis. For example, a recent study in the mouse, identified M-cells by increased F-actin and -actinin staining.120

1.4.1. In vivo studies The use of live animals in the study of particle uptake has several advan-tages. First of all, the intestinal epithelium is studied in its normal state. Sec-ondly, investigations of continuous or repeated administration can be per-formed. Thirdly, if the particles are fed orally, the influence of both gastroin-testinal transit and the epithelial barrier on particle uptake can be studied. One disadvantage of both in vivo and in vitro studies using whole tissue is that it is difficult to study isolated mechanisms of particle uptake and trans-port across the epithelium. Moreover, differences in number and size of Peyer´s patches in different animals compared with humans may signifi-cantly influence the results. Peyer’s patches are much larger in humans than in mice15, 16 and, in contrast to rodents, we have thousands of single lym-phoid follicles scattered throughout the intestinal mucosa15, 127 which may lead to underestimation of particle uptake. Rabbits, on the other hand, have both more Peyer´s patches and more M-cells per Peyer´s patch than hu-mans,71 which of course may lead to overestimation of uptake.

1.4.2. In vitro studies on intestinal tissue In these studies, particles are injected into the intestinal lumen through everted sacs or isolated loops of the intestine.76, 122, 128 The advantage of these systems is that whole tissue, containing both FAE and absorptive epithelium, can be studied. A disadvantage is that only animal tissue can be used. Ex-cised intestinal tissue may also be mounted in Ussing chambers for studies of uptake and transport. In this case, human biopsies and surgical specimens can be used. Nonetheless, blood flow and nerve signalling are not main-tained in any of these systems, and the viability of the excised tissues de-creases within hours of isolation.129

1.4.3. Cell culture models Epithelial cells grown on filters have been widely used to investigate the uptake and transport of particles.84, 85, 102, 130-132 Epithelial cell monolayers are well-suited to kinetic studies and studies of isolated particle-epithelial inter-actions. The drawbacks of these simplified systems are, of course, that they are not in contact with the immune cells, luminal stimuli and nerve signals

21

that modulate their function in vivo. Moreover, most cultured epithelial cells employed for these studies are absorptive cells, with low phagocytic capac-ity, whereas in vivo, the FAE and M-cells of the Peyer´s patches play an important role in particle uptake and transport. Unfortunately, attempts to isolate FAE and M-cells for subsequent culture in vitro have not been suc-cessful to date. However, in a seminal paper by Kerneis et al., it was shown that co-culture of Peyer´s patch lymphocytes and intestinal epithelial cells may trigger conversion of the epithelial cells to M-cells.24 Because of the difficulties of reproducing and setting up this model system, it had not been employed in studies of particulate carriers at the onset of this thesis work. Therefore, the first aim of this thesis was to set up a simplified version of this model.

1.5. Concluding remarks Progress in optimizing the delivery of particulate carriers across the intesti-nal mucosa has been thwarted by our limited understanding of how particles are taken across the human intestinal epithelium. Furthermore, human FAE and M-cells have been sparsely studied with regard to markers and apical receptors that could be utilized for targeting.

22

2. Aims of the thesis

The overall aim of the thesis was to study particle transcytosis across the human intestinal epithelium, in particular the FAE of Peyer´s patches. The specific aims of the thesis were:

To develop an in vitro model of the human FAE for the study of par-ticle uptake and transport (I and III)

To identify target structures on human intestinal FAE, for improved uptake and transport of particles into Peyer´s patches (I and II)

To study factors influencing particle uptake and transport across the intestinal epithelium (III and IV)

23

3. Methods

In this chapter, methods central to the thesis work are presented and dis-cussed. Descriptions of materials and methods not included here, can be found in the individual papers.

3.1. Establishment of a model FAE As mentioned in the introduction, in 1997,24 Kerneís et al. established that Peyer´s patch lymphocytes can trigger conversion of intestinal epithelial cells to M-cells. In their study, Caco-2 cells were grown upside-down on filters to allow addition of Peyer´s patch lymphocytes or B-cells to the filter insert. This resulted in migration of lymphocytes into the epithelial cell monolayer during induction of M-cell properties.

Initial studies in our laboratory with the Kerneís setup resulted in poor monolayer integrity. We therefore chose to culture the Caco-2 cells on the filter insert right side up and add the lymphocytes to the basolateral medium (Fig. 6). As discussed in the results section, this setup resulted in no lympho-cytes entering the epithelial cell monolayer. Thus, we established a physi-cally separated co-culture, which was a distinct advantage compared with the original model when performing gene expression studies.

Figure 6. The established model FAE and Caco-2 control epithelium

24

3.1.1. Choice of Caco-2 cell clone/line Initially, a Caco-2 cell line already available at our laboratory was used for the establishment of the model FAE. However, after initial problems with reproducibility, the performance of alternative Caco-2 cell lines and clones was studied (paper III). Only three of the eight studied clones/cell lines (in-cluding the previously used cell line; in-house control) displayed signifi-cantly enhanced transcytosis compared with controls (Fig. 7). The Caco-2 cells obtained from ECACC and the Clone 1 Caco-2 cells were chosen for further studies. The ECACC cell line was used in paper I. However, since these cells lost their ability to transform into a model FAE only a few pas-sages after initial thawing, the Clone 1 Caco-2 cells were used for the subse-quent studies. This clone displayed increased transcytosis of particles over at least 30 passages (paper III).

Figure 7. Particle transport in different Caco-2 cell lines and clones after co-culture with Raji B-cells.

3.1.2. Choice of B-cells Kerneís et al. showed that B-cells were more important than T-cells for the induction of M-cell properties.24 Since we wanted a human model and weekly isolation of Peyer´s patch lymphocytes did not seem practical, we chose to co-culture the Caco-2 cells with the human B-cell-line Raji. A search of the literature on B-cell lines revealed that the Raji B-cell pheno-type had much in common with germinal center B-cells,133, 134 i.e. a B-cell phenotype present in the intestinal lymphoid follicles. Furthermore, Raji cells secrete LT, a cytokine known to be important for the formation of Peyer´s patches.72, 73, 135

25

3.1.3. Choice of co-culture conditions Initial studies revealed that 4 days of co-culture with Raji cells was needed to induce particle transcytosis and that no more than 6 days of co-culture should be used in order to maintain the integrity of the epithelial monolayer. However, subsequent studies revealed that the time for co-culture required adjustment according to the Caco-2 clone used.

3.2. Particle uptake and transport studies As model particles, we chose fluorescent polystyrene (latex) particles be-cause of the ease of detection and the availability of different, well defined sizes. Furthermore, the fluorophore was incorporated into these particles and not attached to the cell surface, which circumvented problems of detached fluorophores and reduced possible influences of the fluorophore on cell sur-face properties.

3.2.1. Cell culture experiments Particles with a carboxylate surface modification were chosen to avoid ad-sorption to the negatively charged plastic during the transport experiments. However, it was necessary to add 1% fetal calf serum (FCS) to the HBSS buffer during the experiments to avoid adsorption to the wells or sample vials. The presence of FCS probably resulted in opsonization of the particles to some extent, which would have affected the absolute numbers of particles transported. However, the conditions used for the transport experiments were identical for the model FAE and the control Caco-2 epithelium and thus should not have affected the relative amounts of particles transported. Ad-sorption of Pluronic and PEG to the particles, which rendered them inert to opsonization, resulted in lower transport of particles across the cell monolayers than observed with ordinary latex particles in 1% FCS. How-ever, the relative increase in transport across the model FAE compared with the Caco-2 epithelium was the same.

3.2.2. Ussing chamber studies on intestinal tissue After identification in a dissection microscope, regions of VE and FAE were cut out and mounted in modified Ussing chambers (Harvard apparatus Inc., Holliston, MA, USA136). The FAE segments were carefully adjusted so that the patches covered the entire exposed tissue surface area of 5 mm2. The segments were continuously oxygenated by circulating 95% O2 / 5% CO2 gas and equilibrated for 40 min to achieve steady-state conditions. BSA was used to block particle adsorption to the diffusion chambers and the same

26

concentration of particles/cell surface area was used as in the cell culture experiments.

3.2.3. Analysis of particle uptake Uptake studies were complicated due to the hydrophobicity of the model particles. Even at donor concentrations a thousand times below those used in the transport studies, loosely adherent particles could not be completely re-moved from the cell surface. Thus, results from cell monolayer lysates were quite difficult to interpret. Therefore, confocal microscopy was used to as-sess levels of particle uptake in the cell monolayers with more certainty (pa-per III).

3.2.4. Analysis of particle transport In order to distinguish between aggregated, fragmented and whole particles and to assess differences in particle size, the particle transport was analyzed in a Fluorescence Activated Cell Scan. Thus, detection was based on both fluorescence intensity and size.

3.2.5. Assessment of integrity and viability Assessment of integrity and viability of the epithelium during transport stud-ies was essential to these studies. Several methods were used to determine the potential influence of tight junction leakage and cell viability on particle transport during cell culture and Ussing chamber experiments.

Cell culture experiments The transepithelial electrical resistance (TER) of the cell monolayers was measured before and after the transport experiments. In addition, the perme-ability to the paracellular marker mannitol was measured after the transport experiments. The model FAE had on average a significantly lower TER and an increased permeability to mannitol (Fig. 8). However, the absolute values of the resistance and permeability to mannitol where well within previously assessed values for cell monolayers with uncompromised integrity in our laboratory. Morevover, the transepithelial resistance and mannitol perme-ability of individual filters were not correlated with the particle transport (Fig. 8). Indeed, in paper III, it was shown that the decrease in TER was the same in non-functional model FAE monolayers and model FAE monolayers that transported particles.

In order to further investigate the viability of the cell monolayers after trans-port experiments, the cells were stained with propidium iodide. Propidium iodide cannot penetrate live cells, but stains the nuclei of dead cells. There

27

was no significant increase in the number of dead cells in the model FAE cell monolayers compared with the Caco-2 control monolayers.

Figure 8. Integrity of the model FAE and Caco-2 cell monolayers. The mean per-meability to Mannitol (A) and the mean TER(B), was changed in the model FAE compared with the Caco-2 controls. However, the integrity of individual filters could not be correlated to their particle transport (C,D). n=90 (TER) and 40 (Mannitol)

In conclusion, the co-culture of Caco-2 cells with B-cells seemed to induce moderate effects on tight junction permeability to small molecules. How-ever, these effects were not associated with decreased cell viability and can-not have affected the transport of particles across the cell monolayers. Indeed, the transport of particles across the cell monolayers was inhibited at 4ºC (paper I). By confocal microscopy we also identified particles inside the cells, but not between them (Fig. 9).

Figure 9. Confocal microscopy of particle uptake in the model FAE. Cells are stained for F-actin. Particles are clearly visible as bright dots inside the cell monolayer.

28

Ussing chamber studies on intestinal tissue As described previously,129 the integrity and viability of the intestinal seg-ments was continuosly monitored during the transport experiments by meas-uring the transmucosal resistance, the resting potential and the short-circuit current across the segment.

3.3. Gene expression studies The aim of studying gene expression in the Caco-2 and Raji cells was to identify gene expression patterns associated with a certain function, e.g. cell adhesion or secretion of cytokines. However, the genes involved in these functions were not known. Therefore, we chose to use cDNA arrays, where the expression of a large number of genes could be compared at the same time. In order to reduce the time for analysis and interpretation, cDNA ar-rays representing a limited number of genes were chosen for these studies.

3.3.1. cDNA-array analysis For analysis of gene expression, messenger RNA or total RNA must be ex-tracted from the cells under study (Fig.10). Subsequently, complementary DNA (cDNA) is produced by reverse transcription. During this process, tracer nucleotides are incorporated for subsequent detection.

We used Clontech cDNA arrays, where 150-200 nucleotide polymerase chain reaction (PCR)-products from each gene were spotted in duplicates onto a nylon membrane. In order to increase the specificity of binding, the primer mix used for generation of cDNA from our cells only amplified tran-scripts from genes present on the array. After addition of our 32P-labelled cDNA to the arrays and subsequent washing, we detected spots where tran-scripts had hybridized by phospho-imaging. The intensity of each spot was thus related to the number of transcripts that had hybridized.

Differences in gene expression were analyzed by Atlas Image (Clontech) by filtering out signals lower than the average background intensity. To compare two or more arrays, the signal intensities of all genes on the arrays were normalized (detailed in paper II).

3.3.2. Interpretation Analysis of the gene expression of hundreds of genes is quite complicated. Not all genes that are expressed will be detected by the array, and some genes that are not expressed will be falsely detected. These problems can be partly resolved by setting strict criteria for the analysis and confirming the

29

Figure 10. Analysis of gene expression by cDNA-array. Adapted from.137

results with those obtained using other methods, such as reverse transcription (RT)-PCR techniques. It must also be borne in mind that the expression of genes may not always be correlated with the expression of proteins. In this study, we chose to confirm only the expression of the molecules selected for further study (CD9, 1-integrin and CD4) with RT-PCR (data not shown).

When comparing Caco-2 cell controls and the model FAE, only small changes in gene expression were detected. As discussed in the results chap-ter, only some of the Caco-2 cells were converted into cells with M-cell properties with regard to down-regulation of microvilli and adherence and uptake of particles. Thus, considering that changes in gene expression affect-ing 10% of the cell monolayer population may be difficult to detect, it is not surprising that only small differences in gene expression were found.

3.4. Protein expression studies Quantification of proteins in solution may be achieved by using ELISA. But when analyzing expression of membrane proteins, one often has to rely on relative, semiquantitative methods such as Western blotting. However, Western blotting requires solubilization and lysis of the cells and does not give information regarding the distribution of the protein in the cell mem-

30

brane. This may be solved by isolation of apical cell membranes prior to quantification. However, during these studies, a faster alternative method, cell-based ELISA, was used to assess the relative expression of membrane proteins in the model FAE and Caco-2 cell monolayers. Immunofluores-cence was used to measure relative expression of membrane proteins in tis-sue sections. Advantages, disadvantages and possible biases of interpretation are discussed below.

3.4.1. Cell-based ELISA The assay was initially described for the ICAM-1 protein.138 Briefly, cell monolayers were washed and incubated with primary antibodies, washed again and fixed in glutaraldehyde. After washing, the cell monolayers were incubated with a secondary antibody conjugated to horseradish peroxidase. The secondary antibody was then detected by conversion of HRP substrate. The relative amount of expressed membrane protein was assessed by meas-uring the total amount of protein in each cell monolayer. An advantage of this method is that both the expression of a protein and its location can be studied at the same time. A disadvantage is that there is often a high back-ground signal, since the antibodies may adhere nonspecifically to the cell membrane. Thus, the specificity of the detection system has always to be tested by immunofluorescence microscopy.

3.4.2. Immunofluorescence Quantification of protein expression by immunofluorescence is quite contro-versial, since the interpretation may be subjective and thus differ between individuals. Moreover, given the nature of tissue, sections prepared on dif-ferent occasions, from different parts of the intestine and different individu-als, may differ in antigen reactivity.

As detailed in paper II, these biases were circumvented by comparing the relative fluorescence intensities between epithelia within the same tissue section rather than between individuals. Moreover, controls of background fluorescence were always obtained for consecutive sections present on the same slide as the samples. One might argue that the different epithelia had different antigen reactivity, due to a different location within the tissue sec-tion. However, the same secondary antibody was used to detect the CD9 and

1-integrin expression. If antigen reactivity was nonspecific, one would have expected the same pattern of expression for both antigens, and this was not the case.

31

4. Results and discussion

4.1. Characteristics of the model FAE in comparison with human FAE In the first study, the new experimental setup of the Caco-2/Raji B-cell co-culture was characterized, with regard to M-cell properties.

In co-culture with Raji B-cells, the Caco-2 cells developed an M-cell like morphology, with areas containing few, disorganized or no micro-villi (Fig. 11A), as compared with the corresponding mono-cultures of Caco-2 cells.

Figure 11. Morphology and expression of surface markers in Caco-2 cells co-cultured with Raji B-cells compared with the mono-cultured controls. (A) transmis-sion electron micrographs, (B) SLAA.-expression and alkaline phosphatase activity (B) of the co-cultured Caco-2 cells compared with controls.

As has been reported for M-cells in vivo, we observed a significant decrease in the apical activity of alkaline phosphatase (AP)123 and increased expres-sion of the Sialyl Lewis A Antigen (SLAA)117 in the co-cultures (Fig. 11B). Furthermore, the capacity for particle transport across the Caco-2 cell monolayers increased after co-culture (Table 2), without affecting cell monolayer integrity (see methods).

32

Table 2. Transport of 0.5 um particles across Caco-2 cell monolayers after co-culture with B- or T-cells compared with controls

na Median particle transport/h

Interquartile range

Caco-2 + B-cellsb 12 2190*** 215-4395

Caco-2 + T-cells 12 117 35-200

Caco-2 12 55 65-215 a Data collected from 2-3 separate experimentsb Particle transport significantly different from Caco-2/T-cell co-cultures and Caco-2 monocultures.*** = p<0.001

When studying the cell monolayers after transport experiments, it was noted that the particles remaining in the cell monolayer were not evenly distrib-uted. The alterations to the microvilli were also fragmented, which indicated that only a sub-population of the cells in each monolayer was fully trans-formed and capable of binding and transporting particles. This, however, relates well to the human intestine, where M-cells constitute less than 10% of the cells of the FAE.70 Thus, the co-cultured Caco-2 cell monolayer best fit the description of a “model FAE”.

Staining with a B-cell-specific antibody (anti-CD19) showed that no di-rect contact occurred between the Raji- and Caco-2 cells in the co-cultures (see paper I). This suggests that the morphological changes and increased transport capacity were dependent on soluble mediators, rather than cell-cell contact. The effects of cytokines and growth factors secreted by Raji B-cells on induction of particle transport was further investigated in paper III.

4.1.1. Characteristics of the induced particle transport The induced particle transport was abolished at 4°C and was saturable, which indicates that the particles were actively transported (Fig. 12).

We also found that transport of particles across the model FAE was size-dependent, which is consistent with in vivo studies.85, 139 However, particle transport in the Caco-2 cell mono-cultures remained at the same level re-gardless of particle size (paper I).

Independently of particle size, the fraction of particles transported/cm2

across the cell monolayers in our model was less than 1‰ of the added dose. A direct comparison with the fraction of particles transported in vivo is diffi-cult, given the number of parameters which contribute to particle transloca-tion. However, considering the limited particle uptake reported in some ani-

33

mal studies,55 it is fully possible that the transport of latex particles across the FAE in vivo is as limited as demonstrated in our study.

Figure 12. Influence of temperature and dose on particle transport across the model FAE and Caco-2 cell monolayers. (A) The transport of particles decreases signifi-cantly at 4ºC (n= 15) (B) The amount of particles added does not influence the num-ber of transported particles at concentrations above 1x108 particles/filter (n=12).

4.2. Identification of target structures in human FAE 4.2.1. Cell surface carbohydrates In paper I, the adherence of a number of M-cell-associated lectins to the model FAE and Caco-2 epithelium was studied. As mentioned above, the surface expression of SLAA was increased by almost 20% in the model FAE compared with the Caco-2 epithelium. In addition, binding of the rabbit cae-cal M-cell markers140 Wheat Germ Agglutinin (WGA) and Peanut Aggluti-nin (PNA) increased. The binding of UEA-1, specific for mouse and rabbit M-cells, did not increase, which is consistent with earlier studies in human tissue.118, 141 The WGA and PNA lectins are known to adhere to both absorp-tive enterocytes and M-cells in the human intestine.142, 143 Furthermore, when studying human intestinal biopsies, we found that SLAA intensely stained all epithelial cells of the VE (Fig. 13A, unpublished data). Therefore, these lectins were not further considered for targeting to the FAE.

4.2.2. Cell adhesion proteins Cell adhesion proteins specific to human M-cells or FAE have not, to date, been reported. In 1998, Clark et al. demonstrated that, in contrast to absorp-tive cells, mouse M-cells expressed 1-integrin on their apical surfaces.122

During characterization of the model FAE with cell ELISA, we identified an increase in surface expression of, among others, 1-integrin (unpublished data, fig.13B). In paper II, we investigated whether this pertained to human

34

intestinal tissue. We also employed cDNA arrays to identify additional cell adhesion proteins with specific expression in human FAE.

Figure 13. (A) SLAA staining of human intestinal villus epithelium. LP; lamina propria. Magnification 16X. (B) Surface expression of cell adhesion proteins in the model FAE compared with the Caco-2 control epithelium (n=4-6). n.d.; not de-tected, b; basolateral expression

cDNA screening for identification of new protein targets Initial attempts to isolate FAE from intestinal specimens failed because of extensive contamination with VE cells during detachment of the FAE from the underlying tissue. Therefore, we used our recently established model of the human FAE for initial exploration of membrane proteins that could be specifically expressed in human FAE tissue. Since we were interested in cell adhesion proteins, a specific cell adhesion/cell interaction cDNA array was used.

There were differences between the model FAE and Caco-2 epithelium in the expression of 18 (7%) of the 256 genes represented on the array. These genes code for proteins involved in cell adhesion, epithelial cell polarity and differentiation, actin dynamics and extracellular matrix remodeling (Fig. 14). In particular, the expression of the cell adhesion molecules CD9 and CD4,

5-integrin (ITGA5) and 8-integrin (ITGA8) were induced. CD9 and CD4 were chosen for further study, since they exhibited the most pronounced upregulation in expression. Both of these proteins have also been reported to act as co-receptors for viruses and bacteria. CD9 is a tetraspan protein that interacts with integrins in the cell membrane144, 145 and CD4 is a well-known co-receptor for HIV.146

35

Figure 14. Genes with changed expression in the model FAE compared with the Caco-2 epithelium. Gene names correspond to NCBI nomenclature and have been grouped according to reported functions. Shades of grey represent the ratio of gene expression in the model FAE compared with the Caco-2 epithelium (see legend).

Interestingly, we did not see a significant increase in 1-integrin gene ex-pression, although previous results suggested an increased apical expression of this protein (Fig.12). The distribution of 1-integrin in the model FAE was further investigated by confocal microscopy, together with that of CD9 and CD4.

Expression of potential target proteins in the model FAE Confocal microscopy studies revealed that while the protein expression of

1-integrin was evenly distributed in the Caco-2 cells, it was highly ex-pressed in the apical membrane of the model FAE (paper II).

This changed distribution may provide an explanation for the absence of increase in gene expression. The expression of CD9 was also clearly en-hanced in the model FAE compared with the Caco-2 epithelium. We were unable to find any expression of CD4 in either the model FAE or the Caco-2 epithelium (Fig. 15). Thus, it is possible that existing CD4 transcripts are not transcribed to protein in all cell types or that the cDNA array falsely detected an increase in gene expression. The expression of CD9 and 1-integrin was further analysed in human Peyer´s patches.

36

Expression of potential target proteins in human FAE Surgical specimens from the terminal ileum were obtained from 8 patients undergoing colonic surgery (see paper II). Figure 16 summarizes the levels of expression for CD9 and 1-integrin in the epithelium of these specimens. The expression levels of CD9 in the FAE were 2- to 3-fold higher than in the VE in all patients. The villi adjacent to the dome (VED) were also CD9-positive in several patients, while villi situated further away from the dome displayed no or low expression.

CD9 was expressed in both the apical and basolateral membranes of the epithelial cells. In addition, CD9 was expressed on cells in the lamina pro-pria in all patients and in the lymphoid follicles of some. This is in agree-ment with a previous study of CD9 expression in the VE of the human small intestine.147

The expression of 1-integrin was also enhanced in the FAE but, in con-trast to CD9, there was no difference in the expression between the villus adjacent to the dome and villi situated further away (Fig. 16). In all patients,

1-integrin lined the basal part of the villus and the FAE and was strongly expressed on cells in the lamina propria. This expression pattern compares well with previous studies on 1-integrin expression in human intestinal villi.148 In the FAE, 1-integrin was expressed all around the basolateral face of the epithelial cells, but was also found on the apical side of the cells. The resolution and the detection technique used did not allow discrimination of absorptive cells and M-cells. However, apical staining of 1-integrin was occasionally found where CD20-positive cells infiltrated the epithelium. Since CD20-positive cells have been found in M-cell pockets,123, 149 these cells could have been M-cells. However, 1-integrin may also be located in all cells of the FAE and the apical expression of this protein may be gov-erned by factors in the surrounding milieu, rather than a specific genotype.

In this context, the localized expression of CD9 in human FAE is interesting, especially since a recent study showed that the gene expression of yet an-other member of the tetraspan protein family, TM4SF3, was increased in human FAE.150 The tetraspan proteins are known to modulate both the loca-tion and the conformation of integrins,144, 145, 151, 152 through binding and in-teraction within tetraspanin webs in the cell membrane. Thus, CD9 and other tetraspanins may possibly play a role in the location of integrins on the api-cal surface of the FAE.

Furthermore, it was recently reported that cross-linking of a specific CD9 epitope induced activation of 1-integrins in a variety of cells.153 If this is true in human intestine, this finding could be utilized to enhance adherence to and binding of surface-exposed 1-integrins in the FAE.

37

Figure 16. Expression of (A) CD9 and (B) 1-integrin in human Peyer´s patches. Top panels display immunofluorescence of CD9 and 1-integrin in one of the tissue specimens. Graphs show the relative ecpression in each individual. Lines cross at average fluorescence intensity and bars show 95% confidence intervals (n=6-8).

4.3. Exploiting identified targets in human FAE In paper II, we also investigated whether the increased apical expression of CD9 and 1-integrin in the human FAE could be exploited for enhanced delivery of particles to the Peyer´s patches. The enhanced expression of CD9 seemed promising. However, to date, no ligands have been reported for hu-man CD9 and it was judged beyond the scope of this study to identify such ligands. Therefore, we studied the uptake and transport of integrin-adherent particles, coated with the peptide motif RGD. Many bacteria are known to use both 1-integrin and other integrins for adhesion122, 154, 155 and adhere selectively to the M-cells of the FAE.156 The RGD sequence is known to compete with Yersinia Invasin A in binding to 1-integrin,157 but also recog-nizes several other -integrin heterodimers.158

38

4.3.1. Transport of RGD-coated particles across the model FAE The targeting strategy was first validated in the cell cultures. We found that the number of transported RGD-coated particles was 50-fold higher than the number of transported non-coated RGD-negative particles in our model FAE (Fig. 17). There was no significant increase in transport of RGD-coated par-ticles in the Caco-2 control epithelium.

Figure 17. Transport of RGD-coated (+RGD) and non-coated (-RGD) particles across model FAE and human intestinal FAE, compared with Caco-2 and villus epithelium respectively. Left graph shows mean transport ± SD for each group (n=6). Right panel displays confocal microscopy sections of particle uptake in hu-man Peyer´s patches, 45 minutes after addition of particles (yellow). Cells are stained for F-actin (red). Stitched lines delineate the basal border of the epithelium. The RGD-coated particles were readily taken up across the FAE, but not the VE. Non-coated particles were not taken up across either epithelium.

4.3.2. Selective transport of RGD-coated particles into human Peyer´s patches The RGD-coated particles were also readily taken up and transported across human FAE mounted in Ussing chambers. Optical sectioning of whole tissue specimens in a confocal microscope showed uptake of RGD-coated particles as early as 15 minutes after application to the FAE. In the corresponding villus specimens, only a few particles were associated with the luminal cell surface. After 45 minutes, the RGD-coated particles had crossed the FAE and were found inside the dome. No particles were transported across the VE, but occasional particles were found within the epithelium, seemingly between cells. We could not detect any uptake of the non-coated, RGD-negative particles in either FAE or villus tissue (Fig. 17).

Thus, the increased uptake into Peyer´s patches was not merely an effect of the increased accessibility of the FAE epithelial surface, but also suggests that the increased uptake was mediated by RGD-attachment to cell surface

39

integrins. Further studies are needed to elucidate whether integrin subunits other than 1 are involved.

4.4. Soluble factors as mediators of particle uptake and transportAlthough the model FAE was useful for studies of particle transport and identification of new target structures, as in other laboratories,132 the re-sponse to Raji B-cell stimulation was variable.

The aim of paper III was to identify the important factors for induction of particle transcytosis in intestinal epithelial cells, in order to improve the reli-ability of the model in future investigations of particulate carrier systems. We had previously optimized the Caco-2 cell line to be used for the co-culture studies (see methods). However, the influence of the Raji B-cells on model variability had not been assessed. We therefore sought to identify molecules secreted by Raji B-cells that were involved in the induction of transcytosis in epithelial cells.

4.4.1. Gene expression profile of Raji B-cells that induce epithelial particle transport Since the importance of the various secreted factors in the induction of trans-cytosis was unknown, their expression kinetics during co-culture was diffi-cult to assess. Thus, we chose to study Raji B-cell expression at the end of co-culture, when particle transcytosis was operational.

The expression of 277 genes present on the Clontech™ Cyto-kine/Receptor array was studied in:

1. Raji cells that induced epithelial particle transcytosis (Raji trp+)2. Raji cells that did not induce transcytosis (Raji trp-)3. Raji cells that had not been co-cultured with Caco-2 cells (Raji 0).

We found that 24 genes were highly expressed in the Raji trp+ cells compared with the Raji trp- and Raji 0 cells (Paper III)

Almost 40 %, or 9 of the 24 induced genes, were derived from the TNF su-perfamily of cytokines and receptors. Several growth factors were also in-duced, including TDGF1 and several from the transforming growth factor (TGF- ) superfamily (BMP1, TGF- , BMP7). The chemokine RANTES and interferon-regulatory factor IRF-1 were also among the genes with the larg-est change in expression.

40

RANTES, TNF- , TGF- have been reported to induce integrin and matrix metalloproteinase expression,159-161 which is in accordance with our previous findings with the model FAE (Fig.14). LT- and LT- are both important for the formation of Peyer´s patches68, 162 and IFN- has been reported to induce macropinocytosis in epithelial cells.163 Thus, these proteins were selected for further study.

4.4.2. Effects of B-cell-secreted proteins on cell monolayer integrity and particle uptake As detailed in paper III, the selected proteins were added to the basolateral medium of Caco-2/Raji co-cultures or Caco-2 controls at the onset of co-culture. F-actin-staining of cells after cytokine/growth factor treatment and transport experiments revealed that they grew as tight monolayers. In some cases, imperfections were found in the monolayers and also occasionally in cultures unexposed to cytokines. However, only IFN- had significantly increased cell monolayer imperfections compared with controls. The effects of the various cytokines on particle uptake and cell monolayer structure are summarized in Table 3.

Stimulation with 10 and 25 ng/ml TNF- resulted in an increased number of particles inside the Caco-2 but not the model FAE monolayers after 1 hour (Table 3, Fig 18). In contrast, stimulation with LT 1/ 2 resulted in an in-creased uptake of particles in the model FAE monolayers (Table 3, Fig. 18), while the number of particles inside the Caco-2 monolayers did not increase regardless of dose.

Following TGF- 1 stimulation, the particle uptake was dramatically in-creased in certain areas of the cell monolayers. However, the effect was very variable and in many cases the internalized particles were located to double layers of cells created by this growth factor (Fig. 18).

IFN- seemed to affect the integrity of the cell monolayers even at low doses (Table 3, Fig. 18). A significant increase in the number of monolayer imper-fections was readily visible at 1 U/ml; at 10 U/ml, several holes were found in both model FAE and Caco-2 monolayers.

41

Table 3. Summary of effects seen on the model FAE and Caco-2 epithelium after cytokine/growth factor stimulation

Celltype Intracellular particles Paracellular particles

Monolayer imperfections

Additive Model FAE

Caco-2 Model FAE

Caco-2 Model FAE

Caco-2

TNF(ng/ml)

Median Range(IQR)b

Median Range(IQR) b

Median Median

Ctrl151025

2426422469

14-4315-634-1062-17721-109

7846369*26*

0-134-1102-15039-10614-35

00100

01120

11100

10011

Lt 1/ 2(ng/ml)Ctrl120100200

33229*16*28**

1-911-479-408-3925-43

82116539

1-374-447-502-2310-71

00000

00000

13112

10210

TGF-(ng/ml)Ctrl101525

922-9

6-1214-137-5-33

131001614

4-445-23614-317-31

00-1

1100

11-0

1110

IFN-(ng/ml)Ctrl0,1110

53110

3-132-54-150-3

73120*

5-106-116-410

0000

0001

114*7*

103*10*

aImages of approximately 400 cells were collected in predetermined areas of the cell monolayers. The numbers presented here reflect median and interquartile range of particles found in each area (400 cells) after analysis of cell monolayers from two separate experiments (n=6). bIQR=Interquartile range * Significant change compared with unexposed model FAE and Caco-2 cell monolayers.

42

Figure 18. Confocal microscopy images of F-actin-stained model FAE and Caco-2 cell monolayers stimulated with different cytokines/growth factors. The monolayers were fixed one hour after addition of particles to the apical cell surface. (A) controls, (B) TNF- -treated (10 ng/ml), (C) Lt 1/b -treated (100 ng/ml), (D), TGF- 1-treated (10 ng/ml and (E) IFN- -treated (1U/ml). Arrows point to extruded cells or holes in the cell monolayers and arrowheads point to internalized particles.

4.4.3. Effects of B-cell-secreted proteins on particle transport Addition of either TNF- , LT 1/ 2, or TGF- 1 to Caco-2 cells in co-culture with Raji B-cells augmented the induction of transcytosis in the epithelial cells without changing the TER of the cell monolayers (Fig. 19).

However, only LT 1/ 2 and TNF- induced transcytosis across Caco-2 cells alone, and only at very high concentrations (200 and 25 ng/ml, respec-tively). Mono-cultured TNF- -stimulated Caco-2 cells also exhibited a moderate decrease in TER. Addition of RANTES had no effect on either Caco-2 cells alone or those stimulated by Raji cells, while low concentra-tions of IFN- had a slight effect on Caco-2 cells in co-cultures, associated with a reduction in TER.

Further studies revealed that the induction of transcytosis by LT 1/ 2 was dose-dependent (Fig. 19), while the induction by TNF- seemed independ-ent of concentration between 1 and 10 ng/ml. TGF- 1 also induced transcy-tosis at 15 ng/ml. Interestingly, this effect was abolished at 25 ng/ml. This growth factor also induced a moderate decrease in TER in the Caco-2 but not the model FAE monolayers (Fig. 19).

43

Figure 19. Effect of the selected cytokines and growth factors on induction of parti-cle transport and TER in the Model FAE and Caco-2 epithelium. Each graph is rep-resentative of 3 experiments. Bars show mean ± SD for each concentration (n=3-5).

44

Although TNF- and TGF- 1 had a direct effect on particle uptake in mono-cultured Caco-2 cells, additional factors present in the co-cultures were ob-viously needed for the transport of particles across cells. These factors could very well be a combination of the cytokines/growth factor used in the study and this prospect is currently under investigation.

However, the complexity of the system may make the specific signalling events leading to induction of particle transcytosis difficult to sort out. The kinetics of cytokine and growth factor expression during the time of co-culture are especially likely to affect the outcome. An alternative would be to control the function and reproducibility of the model FAE by adding defined amounts of one, or a few, of the identified cytokines during Raji B-cell stimulation.

Thus, the findings presented in paper III show promise for the establish-ment of a more reproducible model FAE, based on addition of either TNF-

LT 1/ 2 and/or TGF- 1. The effects of these additives on FAE and M-cell properties other than particle transcytosis await further investigation.

4.5. Influence of enteric bacteria on particle uptake and transportAs discussed in chapter 1, there are several reports in the literature of in-creased capacity for particle transcytosis following stimulation of the mouse, rat and rabbit FAE with non-enteric, non-invasive bacteria.25, 76, 77 The rea-sons for the increased transcytosis are unclear, although evidence exists of both increased M-cell numbers25, 75, 76 and an increased capacity for particle transcytosis by individual M-cells.77

In paper IV, we wanted to investigate if bacteria could induce increased ca-pacity for particle transcytosis also in absorptive epithelial cells. A recent study actually identified M-cells also in the villus epithelium and proposed that enteric pathogens may be reponsible for their formation28

As mentioned above, studies of M-cells in mice have indicated that 1-integrins are apically expressed on M-cells and that they are important for bacterial uptake.(Clark et al, 1998) Therefore, we chose to study YersiniaPseudotuberculosis, an enteric, gram-negative bacterium that selectively adheres to 1-integrin, via two of its surface proteins, Invasin154 and YadA.157 In the mouse, Yersinia has been shown to exclusively invade M-cells,164 unless Invasin is repressed.165 In this case, YadA mediates binding and uptake across the absorptive villus epithelium. Two Yersinia strains were studied; one expressing neither YadA or Invasin (YadA- /Inv-) and one that expressed only Invasin (YadA- /Inv+).

45

4.5.1. Effects of Yersinia Pseudotuberculosis on cell monolayer integrity and particle uptake Following exposure to bacteria, the TER-values of the cell monolayers were approximately 270 ohms/cm2. TER-values of around 200 ohms/cm2 has pre-viously been shown to represent Caco-2 cell monolayers with an uncom-promised integrity. The integrity of the cell monolayers was also assessed by confocal scanning laser microscopy after the experiments. We found a con-fluent, tight layer of epithelial cells both in monolayers exposed (inv+ and inv-) and unexposed to bacteria (Fig. 20).

Figure 20. Localization of Y. pseudotuberculosis and particles in Caco-2 cells by confocal microscopy. The Caco-2 cells are stained for F-actin (blue), gfp-expressing bacteria (green), and fluorescently labeled particles (red). The large arrow point to particles and bacteria located on the same cell. Small arrows point to internalized particles. Magnification 190x.

In the unexposed and inv- exposed cell monolayers, the particles were ran-domly distributed on the cell surface. We could mot detect any bound inv- bacteria. In contrast, in monolayers exposed to inv+ bacteria, numerous bac-teria were found on the apical cell surface and bacteria and particles were often located on the same cell (Fig 20, top panels).

Optical cross-sections of the cell monoalyers revealed that particles were only taken up in inv+ exposed cells (Fig. 20, bottom panels). It was not pos-sible to detect bacteria (inv+ or inv-) intracellularly or on the basolateral side of the cells.

46

4.5.2. Effects of Yersinia Pseudotuberculosis on particle transportAs shown in figure 21, the invasin-expressing Yersinia strain (inv+) had a significant influence on the transport of particles across the cell monolayers. After 2 hours, the transport had increased 77 times compared with cells un-exposed to bacteria. In contrast, the transport in cells exposed to the invasin-deficient strain (inv-) was comparable to the transport in unexposed cells. Additional experiments showed that the particle transport decreased when the particle size increased. However, the volume of transported 500 nm par-ticles was about twice the volume of transported 200 nm particles. As ex-pected from the confocal microscopy studies, the Inv+ induced particle transport was reduced to the same levels as unexposed controls at 4ºC (paper IV).

The induction of particle transport seems dependent on the interaction be-tween invasin and its receptor. However, the mechanisms involved remain unclear. Yersinia spp are known to induce their own uptake in epithelial cells, through high-affinity binding to 1-integrins on the cell surface.166

1-integrins are usually not expressed in the apical membrane of differenti-ated absorptive cells.148 However, in paper IV it was shown that both Y. pseudotuberculosis inv+ and inv- induce expression of 1-integrin on the apical cell surface of Caco-2 cells. Furthermore, it was shown that inv+ bac-teria adhered to the surface of the Caco-2 cell monolayers, while inv- bacte-ria did not. Interestingly, this binding did not result in uptake and transport of the bacteria.

Since the possibility of particles entering the cells in the same endocytic vesicles as the bacteria was excluded, the increased transcytosis must depend on modulation of the cells own endocytic machinery, up to two hours after removal of Yersinia from the cell surface. Invasin-positive Yersinia havebeen reported to activate MAP kinases, among them p38 MAPK, while inva-sin-deficient Yersinia cannot.167 Since activation of p38 MAPK may trigger endocytosis,168 this may be a mechanism whereby Yersinia stimulates parti-cle uptake, also in Caco-2 cells. Further studies are underway to elucidate the mechanisms involved in this induction.

47

Figure 21. Transport of particles in Caco-2 cells exposed to Y. pseudotuberculosis inv + and inv-. Alexa 488-labeled (a) 200 nm or (b) 500 nm particles were added to the apical side of the cell monolayers after an initial exposure to bacteria for 2 hours (106 bacteria/ml). Samples were withdrawn at 30, 60 and 120 minutes and analyzed by flow cytometry. The bars show mean values ± standard deviation (n=3-4).

48

5. Summary and conclusions

In this thesis, various aspects of particle transcytosis across the human intes-tinal epithelium were studied. A model of the human FAE was established in order to study factors influencing particle uptake and transport. This model was also successfully used to identify proteins with increased surface ex-pression in human FAE. Furthermore, an Ussing chamber technique was employed to investigate the effect of integrin targeting on particle transport across the human intestinal epithelium.

During the establishment of the model FAE, it was found that the morpho-logical changes and increased transport capacity associated with M-cells was dependent on soluble factors, rather than on cell-to-cell contact. Subsequent studies on secreted proteins responsible for the transformation revealed that not only cytokines associated with formation of Peyer´s patches (LT and TNF- ), but interestingly also a growth factor (TGF- )could influence transcytosis of particles across the intestinal epithelium. Recent studies have indicated that TNF- may affect the transcellular uptake of macromolecules across the intestinal epithelium.79 Thus, it would be in-teresting to extend the present studies by investigating the effects of TNF- ,LT 1/ 2 and TGF- 1 on macromolecule and particle transcytosis in human FAE and VE.

A vast amount of evidence suggests a role also for luminal antigens in the transcytosis of particles across the FAE.25, 26, 74, 75, 77, 169 The results presented here suggest that in addition, interactions between the intestinal epithelium and bacteria may influence the transcytosis of particles across absorptive epithelial cells. These findings could possibly be exploited for improved delivery of particulate carrier systems. However, previous use of viral and bacterial vectors has raised several safety issues, especially with regard to unwanted immune responses.170 Therefore, the fact that particle transport was induced without uptake of the pathogen is promising. Further studies are underway to investigate if fixed bacteria or isolated invasin exerts similar effects on Caco-2 cell monolayers as live bacteria.

Although bacteria interact with the villus epithelium, the binding and uptake of many pathogens - at least in various animal models - is mainly restricted to the FAE.171 This specific interaction is almost certainly due to the low

49

accessibility of cell adhesion molecules on the absorptive epithelium. The discovery that RGD, an integrin-adherent peptide, mediates selective uptake into Peyer´s patches shows that cell adhesion molecules are readily accessi-ble for targeting in human FAE. Studies are currently underway to identify the integrin receptors responsible for the RGD-induced uptake, since this concept could be exploited for improved uptake of oral vaccine formula-tions.

However, the crucial question remains whether the uptake of vaccine parti-cles, even after targeting, is sufficient to initiate an immune response. After local administration to the FAE, we found between 5 and 10 particles inside each dome. During similar experiments in the mouse intestine, using parti-cles known to initiate an immune response after oral administration, only one to three particles were taken up in a small proportion of the studied domes.128 Thus, it is possible that the uptake of RGD-coated particles will be sufficient to initiate an immune response after oral administration. However, the influence of gastrointestinal passage on particle uptake needs further investigation.

In conclusion, this thesis has provided a platform for further investigation of particle uptake and transport across the human intestinal epithelium. The model FAE has already been used to study the uptake of particulate carriers across the human FAE132 and other studies are underway. Furthermore, the identification of targets for delivery of particulate carriers into human Peyer´s patches, gives new hope for the development of subunit oral vac-cines.

50

Acknowledgements

The studies in this thesis were carried out at the Department of Pharmacy, Uppsala University. I wish to express my sincere gratitude to:

My supervisor, professor Per Artursson, for your creativity, for sharing your broad knowledge with me and for letting me try my own crazy ideas.

Professor Göran Alderborn for providing excellent research facilities and creating a friendly atmosphere at the department.

Dr Johan Söderholm (co-author) for your mentorship and for bringing enthu-siasm to my research project when I needed it the most. I am glad this thesis work took time, so that we could collaborate.

Eva Ragnarsson (co-author) for lots of interesting research discussions and for a helping hand during the finalization of this thesis.

All my other co-authors for your contributions to this thesis and for making research much more fun. Special thanks to the Linköping “gang”: Åsa Keita, Sa’ad Salim and Ida Schoultz, for always helping me out when I needed it and for good times at meetings and trips. I look forward to working more with you! Thank you also to Professor Karl-Eric Magnusson for helpful dis-cussions regarding the manuscripts. Dr Johan Karlsson, for valuable input to my research. My Irish collegues: Dr Maurice Leonard, Dr Alan Baird (I will never forget your hospitality during my Dublin visit), Dr David Brayden and Dr Ann Hopkins. Anne des Rieux for fruitful collaboration and many inter-esting research discussions. Dr. Margaretha Andersson and Professor Karin Caldwell for your interest in my research and for your expertise in surface chemistry.

Dr. Johan Karlsson, Dr. Bertil Abrahamsson, Dr. Anna-Lena Ungell, Dr. Mårten Hammar and all former members of my ”reference group” at Astra-Zeneca, Mölndal for your valuable input to my research.

“My” master thesis students, Åsa Schoug and Lotta Svensson, for your con-tributions to this thesis.

51

My room mates: Sofia Berggren, Dr Karin Östh and Gunilla Englund for your pleasant company and for putting up with my bright ideas.

Dr. Lucia Lazorova for help with all kinds of practical matters and discus-sions of research during these years. Johan Gråsjö for help with statistical problems. Dr Göran Ocklind for teaching me immunofluorescence micros-copy and helping me out with various computer problems. Du är en klippa!

All past and present members of the cell group for fun times and for being there during both ups and downs. Special thanks to Dr Staffan Tavelin for fruitful and interesting research discussions and to Dr Kicki Bergström for your positive attitude and “party-mind”.

Professor Ingvar Sjöholm for taking time to dicuss research with me and Linda Stertman for solving acute problems during the past hectic months.

Eva Nises-Ahlgren, Christin Magnusson, Harriet Pettersson, Ulla Wästberg-Galik, Eva Lide, Lotta Wahlberg and Birgitta Rylén for help and support with practical matters.

All past and present members of the department of Pharmacy for fun trips, nice breaks, sports activities, parties and for making the department such a nice place to work.

AstraZeneca, The Swedish Academy of Pharmaceutical Sciences, CD Carls-sons stiftelse and IF: s stiftelse, for financial support.

Alla gamla och nya medlemmar i Västgöta Nations Damkör/Upptakt för massa roligt under dom här åren. Kommer snart tillbaks till er.

Mina vänner, för roliga middagar och fester, för att ni finns och ger mig per-spektiv på livet. Lovar att bli mer kontaktbar nu.

Hela min fantastiska släkt, för att ni finns. Tack speciellt till Katti och Matti-as och Peter och Carola för inkvartering under Göteborgsbesöken.

Mina svärföräldrar, Anna-Greta och Lars-Eric, för ert stöd och för hjälp med bl. a. barnpassning under det sista året.

Mamma och Per, för att ni finns och för att ni alltid trott på mig.

Alice, min älskling. Tror inte jag skulle blivit färdig om det inte var för dig.

Mats, för ditt fantastiska stöd och uppmuntran under de här åren. Älskar dig.

52

References

1. O'Hagan DT, Rappuoli R. Novel approaches to vaccine delivery. Pharm Res 2004;21:1519-30.

2. Foster N, Hirst BH. Exploiting receptor biology for oral vaccination with biodegradable particulates. Adv Drug Deliv Rev 2005;57:431-50.

3. Gander B. Trends in particulate antigen and DNA delivery systems for vaccines. Adv Drug Deliv Rev 2005;57:321-3.

4. Russell-Jones GJ. Use of targeting agents to increase uptake and localiza-tion of drugs to the intestinal epithelium. J Drug Target 2004;12:113-23.

5. Friede M, Aguado MT. Need for new vaccine formulations and potential of particulate antigen and DNA delivery systems. Adv Drug Deliv Rev 2005;57:325-31.

6. Brayden DJ. Oral vaccination in man using antigens in particles: current status. Eur J Pharm Sci 2001;14:183-9.

7. Lewis DA, Field WN, Hayes K, Alpar HO. The use of albumin micro-spheres in the treatment of carrageenan-induced inflammation in the rat. J Pharm Pharmacol 1992;44:271-4.

8. Jani P, Halbert GW, Langridge J, Florence AT. Nanoparticle uptake by the rat gastrointestinal mucosa: quantitation and particle size dependency. J Pharm Pharmacol 1990;42:821-6.

9. Desai M, Labhasetwar V, Amidon GL, Levy RJ. Gastrointestinal uptake of biodegradable microparticles effect of paticle size. Pharmaceutical Re-search 1996;13:1838-1845.

10. Stertman L. Starch microparticles as an oral vaccine adjuvant with empha-sis on the differentiation of the immune response. Acta Universitatis Upsa-liensis, 2004.

11. Tacket CO, Reid RH, Boedeker EC, Losonsky G, Nataro JP, Bhagat H, Edelman R. Enteral immunization and challenge of volunteers given en-terotoxigenic E. coli CFA/II encapsulated in biodegradable microspheres. Vaccine 1994;12:1270-4.

12. Katz DE, DeLorimier AJ, Wolf MK, Hall ER, Cassels FJ, van Hamont JE, Newcomer RL, Davachi MA, Taylor DN, McQueen CE. Oral immuniza-tion of adult volunteers with microencapsulated enterotoxigenic Es-cherichia coli (ETEC) CS6 antigen. Vaccine 2003;21:341-6.

13. Madara JL, Trier JS. Functional morphology of the mucosa of the small intestine. In: Johnson LR, ed. Physiology of the intestinal tract. New York: Raven press, 1994:1577-1622.

14. Daugherty AL, Mrsny RJ. Transcellular uptake mechanisms of the intesti-nal epithelial barrier Part one. Pharm. Sci. Technol. Today 1999;4:144-151.

53

15. MacDonald TT. The mucosal immune system. Parasite Immunol 2003;25:235-46.

16. Cornes JS. Peyer's patches in the human gut. Proc R Soc Med 1965;58:716. 17. Van Kruiningen HJ, West AB, Freda BJ, Holmes KA. Distribution of

Peyer's patches in the distal ileum. Inflamm Bowel Dis 2002;8:180-5. 18. Kraehenbuhl J-P, Neutra MR. Epithelial M cells: Differentiation and func-

tion. Annu. Rev. Cell Dev. Biol 2000;16:301-332. 19. Weltzin R, Lucia-Jandris P, Michetti P, Fields BN, Kraehenbuhl JP, Neutra

MR. Binding and transepithelial transport of immunoglobulins by intestinal M cells: demonstration using monoclonal IgA antibodies against enteric vi-ral proteins. J Cell Biol 1989;108:1673-85.

20. Bye WA, Allan CH, Trier JS. Structure, distribution, and origin of M-cells in the Peyer's patches of mouse ileum. Gastroenterology 1984;86:789-801.

21. Owen RL, Jones AL. Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles. Gastroen-terology 1974;66:189-203.

22. Gebert A, Rothkotter HJ, Pabst R. M cells in Peyer's patches of the intes-tine. Int Rev Cytol 1996;167:91-159.

23. Karam SM. Lineage commitment and maturation of epitehlial cells in the gut. Frontiers in Bioscience 1999;4:286-298.

24. Kerneís S, Bogdanova A, Kraehenbuhl J-P, Pringault E. Conversion by Peyer's patch lymphocytes of human enterocytes into M cells that transport bacteria. Science 1997;277:949-952.

25. Meynell HM, Thomas NW, James PS, Holland J, Taussig M, Nicoletti C. Up-regulation of microsphere transport across the follicle-associated epi-thelium of Peyer's patch by exposure to Streptococcus pneumoniae R36a.FASEB Journal 1999;13:611-619.

26. Savidge TC, Smith MW, James PS. Salmonella-induced M-cell formation in germ-free mouse Peyer's patch tissue. American Journal of Pathology 1991;139:177-184.

27. Gebert A, Fassbender S, Werner K, Weissferdt A. The development of M cells in Peyer's patches is restricted to specialized dome-associated crypts. Am J Pathol 1999;154:1573-82.

28. Jang MH, Kweon MN, Iwatani K, Yamamoto M, Terahara K, Sasakawa C, Suzuki T, Nochi T, Yokota Y, Rennert PD, Hiroi T, Tamagawa H, Iijima H, Kunisawa J, Yuki Y, Kiyono H. Intestinal villous M cells: an antigen en-try site in the mucosal epithelium. Proc Natl Acad Sci U S A 2004;101:6110-5.

29. Tsuji A, Tamai I. Carrier-mediated intestinal transport of drugs. Pharm Res 1996;13:963-77.

30. Griffith DA, Jarvis SM. Nucleoside and nucleobase transport systems of mammalian cells. Biochim Biophys Acta 1996;1286:153-81.

31. Borst P, Elferink RO. Mammalian ABC transporters in health and disease. Annu Rev Biochem 2002;71:537-92.

32. Swaan PW. Recent advances in intestinal macromolecular drug delivery via receptor-mediated transport pathways. Pharm Res 1998;15:826-34.

54

33. Savidge TC, Smith MW. Evidence that membranous (M) cell genesis is immuno-regulated. Adv Exp Med Biol 1995;371A:239-41.

34. Owen RL, Bhalla DK. Cytochemical analysis of alkaline phosphatase and esterase activities and of lectin-binding and anionic sites in rat and mouse Peyer's patch M cells. Am J Anat 1983;168:199-212.

35. Neutra MR, Mantis NJ, Frey A, Giannasca PJ. The composition and func-tion of M cell apical membranes: implications for microbial pathogenesis. Semin Immunol 1999;11:171-81.

36. Owen RL. Uptake and transport of intestinal macromolecules and microor-ganisms by M cells in Peyer's patches--a personal and historical perspec-tive. Semin Immunol 1999;11:157-63.

37. Apodaca G. Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton. Traffic 2001;2:149-59.

38. Marsh M. In: Marsh M, ed. endocytosis. New York: Oxford University press, 2001.

39. Liu S-H, Mallet WG, Brodskt FM. Clathrin-mediated endocytosis. In: Marsh M, ed. Endocytosis. New York: Oxford University Press, 2001:1-25.

40. Stossel TP. The early history of phagocytosis. In: Gordon S, ed. Phagocyto-sis: the host. Volume 5. Stamford, Connecticut: JAI Press Inc., 1999:3-.

41. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature 2003;422:37-44.

42. Lewis W. Pinocytosis. Johns Hopkins Hosp. Bull. 1931:17. 43. Maniak M. Macropinocytosis. In: Marsh M, ed. Endocytosis. New York:

Oxford University Press, 2001:78-93. 44. Torgersen ML, Skretting G, van Deurs B, Sandvig K. Internalization of

cholera toxin by different endocytic mechanisms. J Cell Sci 2001;114:3737-47.

45. Lu L, Walker WA. Pathologic and physiologic interactions of bacteria with the gastrointestinal epithelium. Am J Clin Nutr 2001;73:1124S-1130S.

46. Florence AT. The oral absorption of micro- and nanoparticulates: neither exceptional nor unusual. Pharm Res 1997;14:259-66.

47. Viswanathan VK, Sharma R, Hecht G. Microbes and their products--physiological effects upon mammalian mucosa. Adv Drug Deliv Rev 2004;56:727-62.

48. Neutra MR, Phillips TL, Mayer EL, Fishkind DJ. Transport of membrane-bound macromolecules by M cells in follicle-associated epithelium of rab-bit Peyer's patch. Cell Tissue Res 1987;247:537-46.

49. Frey a, Giannasca KT, Weltzin R, Giannasca PJ, Reggio H, Lencer, w. I., Neutra MR. Role of the glycocalyx in regulating access of micropartcles to apical plasma membranes of intestinal epithelial cells: implications for mi-crobial attachment and oral vaccine targeting. Journal of Experimental Medicine 1996;184:1045-1059.

50. Owen RL. Sequential uptake of horseradish peroxidase by lymphoid folli-cle epithelium of Peyer's patches in the normal unobstructed mouse intes-tine: an ultrastructural study. Gastroenterology 1977;72:440-451.

51. Owen RL, Pierce NF, Apple RT, Cray WC, Jr. M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for

55

antigen sampling and for microbial transepithelial migration. J Infect Dis 1986;153:1108-18.

52. Yamamoto T, Kamano T, Uchimura M, Iwanaga M, Yokota T. Vibrio cholerae O1 adherence to villi and lymphoid follicle epithelium: in vitro model using formalin-treated human small intestine and correlation be-tween adherence and cell-associated hemagglutinin levels. Infect Immun 1988;56:3241-50.

53. Wolf JL, Rubin DH, Finberg R, Kauffman RS, Sharpe AH, Trier JS, Fields BN. Intestinal M cells: a pathway for entry of reovirus into the host. Sci-ence 1981;212:471-2.

54. Woode GN, Pohlenz JF, Gourley NE, Fagerland JA. Astrovirus and Breda virus infections of dome cell epithelium of bovine ileum. J Clin Microbiol 1984;19:623-30.

55. Jepson MA, Simmons NL, Savidge TC, James PS, Hirst BH. Selective binding and transcytosis of latex microspheres by rabbit intestinal M cells. Cell and Tissue Research 1993;271:399-405.

56. Pappo J, Owen RL. Absence of secretory component expression by epithe-lial cells overlying rabbit gut-associated lymphoid tissue. Gastroenterology 1988;95:1173-7.

57. Pappo J, Steger HJ, Owen RL. Differential adherence of epithelium overly-ing gut-associated lymphoid tissue. An ultrastructural study. Lab Invest 1988;58:692-7.

58. Pappo J, Ermak TH, Steger HJ. Monoclonal antibody-directed targeting of fluorescent polystyrene microspheres to Peyer's patch M cells. Immunology 1991;73:277-80.

59. Pappo J, Mahlman RT. Follicle epithelial M cells are a source of inter-leukin-1 in Peyer's patches. Immunology 1993;78:505-7.

60. LeFevre ME, Vanderhoff JW, Laissue JA, Joel DD. Accumulation of 2-micron latex particles in mouse Peyer's patches during chronic latex feed-ing. Experientia 1978;34:120-2.

61. Neutra MR. M-cells and microbial pathogens. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant L, eds. Infections of the gastrointestinal tract. New York: Raven press, 1995:163-178.

62. Mostov KE, Verges M, Altschuler Y. Membrane traffic in polarized epithe-lial cells. Curr Opin Cell Biol 2000;12:483-90.

63. Spiro DJ, Boll W, Kirchhausen T, Wessling-Resnick M. Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro. Mol Biol Cell 1996;7:355-67.

64. Allan C, Mendrick D, Trier JS. Rat intestinal M cells contain acidic en-dosomal-lysosomal compartments and express class II major histocompa-bility complex determinants. Gastroenterology 1993;104:698-708.

65. Owen RL, Apple RT, Bhalla DK. Morphometric and cytochemical analysis of lysosomes in rat Peyer's patch follicle epithelium: their reduction in vol-ume fraction and acid phosphatase content in M cells compared to adjacent enterocytes. Anat Rec 1986;216:521-7.

66. Florence AT, Hussain N. Transcytosis of nanoparticle and dendrimer deliv-ery systems: evolving vistas. Adv Drug Deliv Rev 2001;50 Suppl 1:S69-89.

56

67. Holt PR, Balint JA. Effects of aging on intestinal lipid absorption. Am J Physiol 1993;264:G1-6.

68. Debard N, Sierro F, Browning J, Kraehenbuhl JP. Effect of mature lym-phocytes and lymphotoxin on the development of the follicle-associated epithelium and M cells in mouse Peyer's patches. Gastroenterology 2001;120:1173-82.

69. Kerneís S, Pringault E. Plasticity of the gastrointestinal epithelium: The M cell paradigm and oppurtunism of pathogenic microorganisms. Seminars in Immunology 1999;11:205-215.

70. Owen RL, Ermak, T. H. Structural specializations for antigen uptake and processing in the digestive tract. Springer Seminars in Immunopathology 1990;12:139-152.

71. Gebert A, Rothkötter H-J, Pabst R. M cells in Peyer's patches of the intes-tine. International Review of Cytology 1996;167:91-159.

72. De Togni P, Goellner J, Ruddle NH, Streeter P, Fick A, Mariathasan S, Smith SC, Carlson R, Shornick LP, Strauss-Schoenberger J, Russell JH, Karr R, Chaplin DD. Abnormal development of peripheral lymphoid or-gans in mice deficient in lymphotoxin. Science 1994;264:703-707.

73. Koni PA, Sacca R, Lawton P, Browning JL, Ruddle NH, Flavell RA. Dis-tinct roles in lymphoid organogenesis for lymphotoxins alpha and beta re-vealed in lymphotoxin beta-deficient mice. Immunity 1997;6:491-500.

74. Smith MW, James PS, Tivey DR. M cell numbers increase after transfer of SPF mice to a normal house enviroment. American Journal of Pathology 1987;128:385-389.

75. Borghesi C, Regoli M, Bertelli E, Nicoletti C. Modifications of the follicle-associated epithelium by short-term exposure to a non-intestinal bacterium. J Pathol 1996;180:326-32.

76. Borghesi C, Taussig MJ, Nicoletti C. Rapid appearance of M cells after microbial challenge is restricted at the periphery of the follicle-associated epithelium of Peyer's patch. Lab Invest 1999;79:1393-401.

77. Gebert A, Steinmetz I, Fassbender S, Wendlandt KH. Antigen transport into Peyer's patches: increased uptake by constant numbers of M cells. Am J Pathol 2004;164:65-72.

78. Clark EC, Patel SD, Chadwick PR, Warhurst G, Curry A, Carlson GL. Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate. Gut 2003;52:224-30.

79. Soderholm JD, Streutker C, Yang PC, Paterson C, Singh PK, McKay DM, Sherman PM, Croitoru K, Perdue MH. Increased epithelial uptake of pro-tein antigens in the ileum of Crohn's disease mediated by tumour necrosis factor alpha. Gut 2004;53:1817-24.

80. Soderholm JD, Peterson KH, Olaison G, Franzen LE, Westrom B, Magnus-son KE, Sjodahl R. Epithelial permeability to proteins in the noninflamed ileum of Crohn's disease? Gastroenterology 1999;117:65-72.

81. Soderholm JD, Olaison G, Peterson KH, Franzen LE, Lindmark T, Wiren M, Tagesson C, Sjodahl R. Augmented increase in tight junction perme-

57

ability by luminal stimuli in the non-inflamed ileum of Crohn's disease. Gut 2002;50:307-13.

82. Lugering A, Floer M, Lugering N, Cichon C, Schmidt MA, Domschke W, Kucharzik T. Characterization of M cell formation and associated mononu-clear cells during indomethacin-induced intestinal inflammation. Clin Exp Immunol 2004;136:232-8.

83. Kucharzik T, Lugering A, Lugering N, Rautenberg K, Linnepe M, Cichon C, Reichelt R, Stoll R, Schmidt MA, Domschke W. Characterization of M cell development during indomethacin-induced ileitis in rats. Aliment Pharmacol Ther 2000;14:247-56.

84. Desai MP, Labhasetwar V, Walter E, Levy RJ, Amidon GL. The mecha-nism of uptake of biodegradable microparticles in Caco-2 cells is size de-pendent. Pharm Res 1997;14:1568-73.

85. McClean S, Prosser E, Meehan E, O'Malley D, Clarke N, Ramtoola Z, Brayden D. Binding and uptake of biodegradable poly-DL-lactide micro-and nanoparticles in intestinal epithelia. European Journal of Pharmaceuti-cal Sciences 1998;6:153-163.

86. Jani P, McCarthy DE, Florence AT. Nanosphere and microsphere uptake via Peyer´s patches: observation of the rate of uptake in the rat after a sin-gle oral dose. Int J Pharm 1992;86:239-246.

87. Eldridge JH, Hammond CJ, Meulbroek JA, Staas JK, Gilley RM, Tice TR. Controlled vaccine release in the gut-associated lymphoid tissues. I. Orally administered biodegradable microspheres target the Peyer´s patches. J Con-trol Rel 1990;11:205-214.

88. Tabata Y, Inoue Y, Ikada Y. Size effect on systemic and mucosal immune responses induced by oral administration of biodegradable microspheres. Vaccine 1996;14:1677-85.

89. Sanders NN, De Smedt SC, Demeester J. The physical properties of biogels and their permeability for macromolecular drugs and colloidal drug carri-ers. J Pharm Sci 2000;89:835-49.

90. Norris DA, Sinko PJ. The role of surface hydrophobicity in the transport of polystyrene microspheres through Caco-2 cell monolayers and intestinal mucin. Proc. Int Symp. Control. Rel. Bioact. Mater 1997;24:17-18.

91. Norris DA, Puri N, Sinko PJ. The effect of physical barriers and properties on the oral absorption of particulates. Advanced Drug Delivery Reviews 1998;34:135-154.

92. Norris DA, Sinko PJ. Effect of size, surface charge, and hydrophobicity on the translocation of polystyrene microspheres through gastrointestinal mucin. J Appl Poly Sci 1997;63:1481-1492.

93. Andrianov AK, Payne LG. Polymeric carriers for oral uptake of micropar-ticulates. Adv Drug Deliv Rev 1998;34:155-170.

94. Jepson MA, Simmons NL, O'Hagan DT, Hirst BH. Comparison of Poly(dl-Lactide-co-glycolide) and Polystyrene Microsphere Targeting to Intestinal M Cells. J Drug Target 2003;11:269-72.

95. Damgé C, Aprahamian M, Marchais H, Benoit JP, Pinget M. Intestinal absorption of PLGA microspheres in the rat. Journal of Anatomy 1996;189:491-501.

58

96. Childers NK, Denys FR, McGee NF, Michalek SM. Ultrastructural study of liposome uptake by M cells of rat Peyer's patch: an oral vaccine system for delivery of purified antigen. Reg Immunol 1990;3:8-16.

97. Claassen I, Osterhaus A, Boersma W, Schellekens M, Claassen E. Fluores-cent labelling of virus, bacteria and iscoms: in vivo systemic and mucosal localisation patterns. Adv Exp Med Biol 1995;371B:1485-9.

98. Lazorová L, Artursson P, Engström Å, Sjölander a. Transport of an influ-enza virus vaccine formulation (iscom) in caco-2 cells. American Journal of Physiology 1996;270:G554-G564.

99. Andrianov AK, Payne LG. Polymeric carriers for oral uptake of micropar-ticulates. Adv Drug Deliv Rev 1998;34:155-170.

100. Florence AT, Hillery AM, Hussain N, Jani PU. Factors affecting oral up-take and translocation of polystyrene nanoparticles: histological and ana-lytical evidence. Journal of Drug Targeting 1995;3:65-70.

101. Carron E, Hall A. Phagocytosis. In: Marsh M, ed. Endocytosis. New York: Oxford University Press, 2001:58-77.

102. Russell-Jones GJ, Arthur L, Walker H. Vitamin B12-mediated transport of nanoparticles across Caco-2 cells. Int J Pharm 1999;179:247-55.

103. Clark MA, Blair H, Liang L, Brey RN, Brayden D, Hirst BH. Targeting polymerised liposome vaccine carriers to intestinal M cells. Vaccine 2001;20:208-17.

104. Foster N, Clark A, Jepson MA, Hirst BH. Ulex europeaus 1 lectin targets microspheres to mouse Peyer’s patch M-cells in vivo. Vaccine 1998;16:536-541.

105. Smith MW, Thomas NW, Jenkins PG, Miller NG, Cremaschi D, Porta C. Selective transport of microparticles across Peyer's patch follicle-associated M cells from mice and rats. Exp Physiol 1995;80:735-43.

106. Porta C, James PS, Phillips AD, Savidge TC, Smith MW, Cremaschi D. Confocal analysis of fluorescent bead uptake by mouse Peyer's patch folli-cle-associated M cells. Exp Physiol 1992;77:929-32.

107. Hussain N, Florence AT. Utilizing bacterial mechamisms of epithelial cell entry: invasin-induced oral uptake of latex nanoparticles. Pharmaceutical Research 1998;15:153-156.

108. Mantis N, Frey A, Neutra M. Accessibility of glycolipid and oligosaccha-ride epitopes on rabbit villus and follicle-associated epithelium. American Journal of Physiology 2000;278:G915-G923.

109. van der Lubben IM, Verhoef JC, van Aelst AC, Borchard G, Junginger HE. Chitosan microparticles for oral vaccination: preparation, characterization and preliminary in vivo uptake studies in murine Peyer's patches. Biomate-rials 2001;22:687-94.

110. Van Der Lubben IM, Konings FA, Borchard G, Verhoef JC, Junginger HE. In vivo uptake of chitosan microparticles by murine Peyer's patches: visu-alization studies using confocal laser scanning microscopy and immunohis-tochemistry. J Drug Target 2001;9:39-47.

111. Stertman L, Strindelius L, Sjoholm I. Starch microparticles as an adjuvant in immunisation: effect of route of administration on the immune response in mice. Vaccine 2004;22:2863-72.

59

112. Clark MA, Jepson MA, Simmons NL, Booth TA, Hirst BH. Differential expression of lectin-binding sites defines mouse intestinal M-cells. J Histo-chem Cytochem 1993;41:1679-87.

113. Giannasca PJ, Giannasca KT, Falk P, Gordon JI, Neutra MR. Regional differences in glycoconjugates of intestinal M cells in mice: potential tar-gets for mucosal vaccines. Am J Physiol 1994;267:G1108-21.

114. Gebert A, Rothkotter HJ, Pabst R. Cytokeratin 18 is an M-cell marker in porcine Peyer's patches. Cell Tissue Res 1994;276:213-21.

115. Jepson MA, Mason CM, Bennett MK, Simmons NL, Hirst BH. Co-expresion of vimentin and cytokeratins in M-cells of rabbit intestinal lym-phoid follicle-associated epithelium. Histochemical Journal 1992;24:33-39.

116. Wong NA, Herriot M, Rae F. An immunohistochemical study and review of potential markers of human intestinal M cells. Eur J Histochem 2003;47:143-50.

117. Giannasca P, Giannasca K, Leichtner AM, Neutra MR. Human intestinal M cells display the Sialyl Lewis A antigen. Infection and Immunity 1999;67:946-953.

118. Sharma R, van Damme EJM, Peumans WJ, Sarsfield P, Schumacher U. Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer's patches. Histochemistry and Cell Biology 1996;105:459-465.

119. Rautenberg K, Cichon C, Schmidt MA. Immunocytochemical characteriza-tion of the follicle-associated epithelium of Peyer's patches: anti-cytokeratin 8 antibody (Clone 4.1.18) as a molecular marker for rat M-cells. European Journal of Cell Biology 1996;71:361-375.

120. Clark MA, Hirst BH. Expression of junction-associated proteins differenti-ates mouse intestinal M cells from enterocytes. Histochem Cell Biol 2002;118:137-47.

121. Ueki T, Mizuno M, Uesu T, Kiso T, Tsuji T. Expression of ICAM-1 on M-cells covering isolated lymphoid follicles of the human colon. Acta Medi-cina Okayama 1995;49:145-151.

122. Clark MA, Hirst BH, Jepson MA. M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells. Infect Immun 1998;66:1237-43.

123. Farstad IN, Halstensen TS, Fausa O, Brandtzaeg P. Heterogenity of M-cell-associated B and T cells in human Peyer's patches. Immunology 1994;83:457-464.

124. Schmedtje JF. Some histochemical charcteristics of lymphoepithelial cells of the rabbit appendix. Anat. Rec. 1965;151:412-413.

125. Rosner AJ, Keren DF. Demonstration of M cells in the specialized follicle-associated epithelium overlying isolated lymphoid follicles in the gut. J Leukoc Biol 1984;35:397-404.

126. Hamada H, Hiroi T, Nishiyama Y, Takahashi H, Masunaga Y, Hachimura S, Kaminogawa S, Takahashi-Iwanaga H, Iwanaga T, Kiyono H, Yama-moto H, Ishikawa H. Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J Immunol 2002;168:57-64.

60

127. Dukes C, Bussey HJR. The number of lymphoid follicles of the human large intestine. J Pathol Bacteriol 1926;29:111-116.

128. Larhed A, Stertman L, Edvardsson E, Sjoholm I. Starch microparticles as oral vaccine adjuvant: antigen-dependent uptake in mouse intestinal mu-cosa. J Drug Target 2004;12:289-96.

129. Soderholm JD, Hedman L, Artursson P, Franzen L, Larsson J, Pantzar N, Permert J, Olaison G. Integrity and metabolism of human ileal mucosa in vitro in the Ussing chamber. Acta Physiol Scand 1998;162:47-56.

130. Delie F, Rubas W. Caco-2 monolayes as atool to examine intestinal uptake of particulates. Proceedings of the International Symposium of Controlled Release....... 1996;23:149-150.

131. Russell-Jones G, J., Veitch H, Arthur L. Lectin-mediatedtransport of nanoparticles across Caco-2 OK cells. International Journal of Pharmaceu-tics 1999;190:165-174.

132. van der Lubben IM, van Opdorp FA, Hengeveld MR, Onderwater JJ, Ko-erten HK, Verhoef JC, Borchard G, Junginger HE. Transport of chitosan microparticles for mucosal vaccine delivery in a human intestinal M-cell model. J Drug Target 2002;10:449-56.

133. Ho YS, Subhendu C, Hsu SM. Induction of differentiation of African Burkitt's lymphoma cells by phorbol ester: possible relation with early B cells. Cancer Invest 1987;5:101-7.

134. Laskov R, Lancz G, Ruddle NH, McGrath KM, Specter S, Klein T, Djeu JY, Friedman H. Production of tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) by murine pre-B and B cell lymphomas. J Immu-nol 1990;144:3424-30.

135. Körner H, Cook M, Riminton DS, Lemckert FA, Hoek RM, Ledermann B, Köntgen F, Fazekas de St Groth B, Sedgwick JD. Distinct roles for lym-photoxin- and tumor necrosis factor in organogenesis and spatial organi-zation of lymphoid tissue. European Journal of Immunology 1997;27:2600-2609.

136. Grass GM, Sweetana SA. In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell. Pharm Res 1988;5:372-6.

137. Rydell N. Development of a new oral vaccine against diphteria and the study of its immunogenicity in mouse and man. Acta Universitatis Upsa-liensis, 2004.

138. Moynagh P, Williams D, O'Neill L. Activation of NF-kB and induction of ICAM-1 expression in human glial cells by IL-1. The Journal of Immunol-ogy 1994;153:2681-2690.

139. Jani PU, Halbert GW, Langridge J, Florence AT. The uptake and transloca-tion of latex nano-spheres and microspheres after oral administration to rats. J Pharm. Pharmacol. 1989;41:809-812.

140. Jepson MA, Clark MA, Foster N, Mason CM, Bennett MK, Simmons NL, Hirst BH. Targeting to intestinal M-cells. Journal of Anatomy 1996;189:507-516.

141. Jepson MA, Clark MA. Studying M cells and their role in infection. Trends in Microbiology 1998;6:359-65.

61

142. Jacobs LR, De Fontes D, Cox KL. Cytochemical localization of small in-testinal glycoconjugates by lectin histochemistry in controls and subjects with cystic fibrosis. Dig Dis Sci 1983;28:422-8.

143. Forsberg G, Fahlgren A, Horstedt P, Hammarstrom S, Hernell O, Hammar-strom ML. Presence of bacteria and innate immunity of intestinal epithe-lium in childhood celiac disease. Am J Gastroenterol 2004;99:894-904.

144. Jones PH, Bishop LA, Watt FM. Functional significance of CD9 associa-tion with beta 1 integrins in human epidermal keratinocytes. Cell Adhes Commun 1996;4:297-305.

145. Rubinstein E, Poindessous-Jazat V, Le Naour F, Billard M, Boucheix C. CD9, but not other tetraspans, associates with the beta1 integrin precursor. Eur J Immunol 1997;27:1919-27.

146. Dalgleish AG, Beverley PC, Clapham PR, Crawford DH, Greaves MF, Weiss RA. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 1984;312:763-7.

147. Sincock PM, Mayrhofer G, Ashman LK. Localization of the transmem-brane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: comparison with CD9, CD63, and alpha5beta1 integrin. J Histo-chem Cytochem 1997;45:515-25.

148. Beaulieu J-F. Differential expression of the VLA family of integrins along the crypt-villus axis in the human small intestine. Journal of Cell Science 1992;102:427-236.

149. Yamanaka T, Straumfors A, Morton H, Fausa O, Brandtzaeg P, Farstad I. M cell pockets of human Peyer's patches are specialized extensions of ger-minal centers. Eur J Immunol 2001;31:107-17.

150. Lo D, Tynan W, Dickerson J, Scharf M, Cooper J, Byrne D, Brayden D, Higgins L, Evans C, O'Mahony DJ. Cell 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 2004;16:91-9.

151. Hemler ME. Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu Rev Cell Dev Biol 2003;19:397-422.

152. Yanez-Mo M, Tejedor R, Rousselle P, Sanchez -Madrid F. Tetraspanins in intercellular adhesion of polarized epithelial cells: spatial and functional re-lationship to integrins and cadherins. J Cell Sci 2001;114:577-87.

153. Gutierrez-Lopez MD, Ovalle S, Yanez-Mo M, Sanchez-Sanchez N, Rubin-stein E, Olmo N, Lizarbe MA, Sanchez-Madrid F, Cabanas C. A function-ally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated beta1 integrin. J Biol Chem 2003;278:208-18.

154. Isberg RR, Leong jM. Multiple 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 1990;60:861-871.

155. Kerr JR. Cell adhesion molecules in the pathogenesis of and host defence against microbial infection. Mol Pathol 1999;52:220-30.

156. Siebers A, Finlay BB. M-cells and the patogenesis of mucosal and systemic infections. Trends in Microbiology 1996;4:22-29.

62

157. Marra A, Isberg RR. invasin-dependent and invasin-independent pathways for translocation of Yersinia pseudotuberculosis across the Peyer's patch in-testinal epithelium. Infection and Immunity 1997;65:3412-3421.

158. Ruoslahti E. RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 1996;12:697-715.

159. Wahl SM, Allen JB, Weeks BS, Wong HL, Klotman PE. Transforming growth factor beta enhances integrin expression and type IV collagenase secretion in human monocytes. Proc Natl Acad Sci U S A 1993;90:4577-81.

160. Vaddi K, Newton RC. Regulation of monocyte integrin expression by beta-family chemokines. J Immunol 1994;153:4721-32.

161. Gan X, Wong B, Wright SD, Cai TQ. Production of matrix metallopro-teinase-9 in CaCo-2 cells in response to inflammatory stimuli. Journal of Interferon and Cytokine Research 2001;21:93-98.

162. Lorenz RG, Chaplin DD, McDonald KG, McDonough JS, Newberry RD. Isolated lymphoid follicle formation is inducible and dependent upon lym-photoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function. J Immunol 2003;170:5475-82.

163. Koda T, Minami H, Ogawa T, Yamano M, Takeda E. Higher concentra-tions of interferon-gamma enhances uptake and transport of dietary anti-gens by human intestinal cells: a study using cultured Caco-2 cells. J Nutr Sci Vitaminol (Tokyo) 2003;49:179-86.

164. Grassl GA, Bohn E, Muller Y, Buhler OT, Autenrieth IB. Interaction of Yersinia enterocolitica with epithelial cells: invasin beyond invasion. Int J Med Microbiol 2003;293:41-54.

165. Eitel J, Dersch P. The YadA protein of Yersinia pseudotuberculosis medi-ates high-efficiency uptake into human cells under environmental condi-tions in which invasin is repressed. Infect Immun 2002;70:4880-91.

166. Boyle EC, Finlay BB. Bacterial pathogenesis: exploiting cellular adher-ence. Curr Opin Cell Biol 2003;15:633-9.

167. Grassl GA, Kracht M, Wiedemann A, Hoffmann E, Aepfelbacher M, von Eichel-Streiber C, Bohn E, Autenrieth IB. Activation of NF-kappaB and IL-8 by Yersinia enterocolitica invasin protein is conferred by engagement of Rac1 and MAP kinase cascades. Cell Microbiol 2003;5:957-71.

168. Cavalli V, Vilbois F, Corti M, Marcote MJ, Tamura K, Karin M, Arkinstall S, Gruenberg J. The stress-induced MAP kinase p38 regulates endocytic trafficking via the GDI:Rab5 complex. Mol Cell 2001;7:421-32.

169. Yamanaka T, Helgeland L, Farstad IN, Fukushima H, Midtvedt T, Brandtzaeg P. Microbial colonization drives lymphocyte accumulation and differentiation in the follicle-associated epithelium of Peyer's patches. J Immunol 2003;170:816-22.

170. Chuah MK, Collen D, VandenDriessche T. Biosafety of adenoviral vectors. Curr Gene Ther 2003;3:527-43.

171. Clark MA, Jepson MA. Intestinal M cells and their role in bacterial infec-tion. Int J Med Microbiol 2003;293:17-39.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 3

Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy. (Prior to January, 2005, the series was published under the title "Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy".)

Distribution: publications.uu.seurn:nbn:se:uu:diva-4780

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2005