Linköping University Medical Dissertation No. 1180 Linköping 2010

Collagenous colitis

The influence of inflammation and bile acids on intestinal barrier function

Andreas Münch

Gastroenterology and Hepatology unit Department of Clinical and Experimental Medicine

Faculty of Health Science Linköping University, SE-58185 Linköping

© Andreas Münch, 2010 Printed by Liu-tryck, Linköping, Sweden, 2010 ISBN: 978-91-7393-409-1 ISSN: 0345-0082

“Jedes Naturgesetz, das sich dem Beobachter offenbart, lässt auf ein höheres, noch unerkanntes schließen.“ Alexander von Humboldt (1769-1859) Dedicated to my patients

ABSTRACT Background and aims Collagenous colitis (CC) is a diarrheal disorder with an incidence rate of 5-6/100000 inhabitants, affecting mainly middle-aged women. The diagnosis is made by histology of the colonic mucosa. Classical findings are a thickened subepithelial collagenous layer and chronic inflammation in the lamina propria. In inflammatory bowel disease (IBD) the mucosal barrier function is important in pathogenesis. The main purpose of the thesis was therefore to describe the barrier function in CC. The cause of CC is uncertain but the condition seems to be associated with bile acid malabsorption. Increased faecal bile acids are known to induce diarrhea. In functional studies the influence of bile acids on mucosal permeability in biopsies of healthy human individuals and in patients with CC was investigated. Methods and patients In the first paper a single patient with intractable CC was examined before surgery, with loop-ileostomy and after bowel reconstruction. For the other studies a total of 25 patients with CC were included (20 women, 5 men, mean age 66 years). There were three groups (14 patients in clinical remission without medical treatment, 11 with active disease, and 8 of these again after 6 weeks of budesonide treatment); 17 individuals with normal histology served as controls. Endoscopic biopsies from the sigmoid colon were mounted in modified Ussing chambers and assessed for short-circuit current (Isc), transepithelial resistance (TER), and transmucosal passage of chemically killed E. coli K12 after addition of chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA). The biopsies were further investigated with confocal microscopy to assess bacterial transepithelial passage. Results: Para- and transcellular permeability was increased in active CC, but normalized with histological improvement due to faecal stream diversion. After bowel reconstruction, permeability to CrEDTA and HRP increased again. In CC, bacterial uptake in colonic biopsies was significantly higher in all groups than in controls. Despite significant alleviation of symptoms, budesonide did not normalize the increased bacterial passage. Histology was unchanged after 6 weeks of budesonide treatment. DCA augmented mucosal permeability to CrEDTA in a dose-dependent manner and even such a low dose as 100 µmol/l DCA increased bacterial uptake significantly. The combination of bile acids and E.coli K12 had additive effects on TER. 100 µmol/l CDCA and DCA increased bacterial uptake in biopsies of CC patients in remission 4-fold, but had no additive effect on biopsies from patients with active disease. Furthermore, patients in clinical remission on budesonide treatment showed no bile acid-induced effects on E.coli K12 passage. Conclusion: Collagenous colitis presents with increased para/transcellular permeability and bacterial uptake, irrespective of disease activity or budesonide treatment, signifying an underlying mucosal barrier defect. Faecal stream diversion can normalize the barrier dysfunction, but budesonide does not, despite its beneficial clinical effects which alleviate diarrhea or bowel symptoms. Bile acids in physiological concentrations have the potential to augment bacterial uptake, especially in mucosa from CC patients in remission. Budesonide treatment appears to counteract the bile acid induced mucosal impairment. These detrimental effects of bile acids on mucosal barrier function might facilitate initiation and perpetuation of mucosal inflammation in CC.

LIST OF PAPERS

I. Dynamics of mucosal permeability and inflammation in collagenous colitis before,

during and after loop-ileostomy.

Münch A, Söderholm JD, Wallon C, Öst Å, Olaison G, Ström M. Gut 2005;54:1126-28

II. Dihydroxy bile acids increase mucosal permeability and bacterial uptake in human

colonic biopsies.

Münch A, Ström M, Söderholm JD. Scand J Gastroenterol 2007;42:1-8

III. Increased transmucosal uptake of E. coli K12 in collagenous colitis persists after

budesonide treatment.

Münch A, Söderholm JD, Öst Å, Ström M. Am J Gastroenterol 2009;104(3):679-85

IV. Physiological levels of bile acids increase bacterial uptake in colonic biopsies of

collagenous colitis patients in remission.

Münch A, Söderholm JD, Carlsson AH, Magnusson KE, Öst Å, Ström M. submitted 2010

ABBREVIATIONS ASBT Apical sodium-dependent bile acid transporter AZA Azathioprine BAM Bile acid malabsorption CC Collagenous colitis CDCA Chenodeoxycholic acid CEC Colonic epithelial cells CFU Colonic forming unit CLSM Confocal laser scanning microscopy 51CrEDTA 51 Chromium-ethylene diamine tetra-acetic acid DCA Deoxycholic acid E.coli Escherichia coli ECP Eosinophil cationic protein

EGFR Epithelial growth factor receptor

FACS Fluorescence Activated Cell Sorting FAE Follicle-associated epithelium HLA Human leukocyte antigen HRP Horseradish peroxidase IBD Inflammatory bowel disease IFN γ Interferon gamma iNOS Inducible nitric oxide synthase Isc Short circuit current JAM Junctional adhesion molecules LC Lymphocytic colitis LCA Lithocholic acid MC Microscopic colitis MHC Major histocompatibility complex MLC Myosin regulatory light chain MLCK Myosin light chain kinase MMP Matrix metalloproteinase 6-MP 6-Mercaptopurine NNT Number needed to treat NSAID Non-steroidal anti-inflammatory drug PD Transepithelial potential difference PEG Polyethylene glycols 75SeHCAT 75 Selenium-labelled homocholic acid-taurine SSRI Selective serotonin reuptake inhibitors TER Transepithelial electrical resistance TIMP Tissue inhibitor of metalloproteinases TJs Tight junctions TNFα Tumour necrosis factor alpha UDCA Ursodeoxycholic acid VEGF Vascular endothelial growth factor

CONTENTS 1. INTRODUCTION 11 2. BACKGROUND TO THE STUDY 12

Collagenous colitis 12 Historical remarks/Epidemiology 12 Diagnosis: clinical, endoscopic and histological findings 13 Aetiology: mucosal and luminal factors 15 Treatment 19

Intestinal barrier function 20 Structures 20 Para-/Transcellular Permeability 23 Intestinal barrier dysfunction in IBD 25

Bile acids 27 Biochemistry/Physiology 27 The influence of bile acids on intestinal barrier function 30 3. AIMS OF THE THESIS 33 4. SUBJECTS AND METHODS 35

Patients 35 Ussing chamber 36

Permeability markers/E.coli K12 38 Bile acids 39

Histology 40 Confocal microscopy 40 Methodological considerations 41 Statistics 41

5. RESULTS 43 6. DISCUSSION 51 7. CONCLUSIONS 57 8. SVENSK SAMMANFATTNING 59 9. ACKNOWLEDGEMENTS 61 10. REFERENCES 63 Paper I Paper II Paper III Paper IV

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1. INTRODUCTION

Collagenous colitis (CC) belongs to the disease group called microscopic colitis. They are

diarrheal disorders in which the diagnosis has to be established by histology. Although

already described in 1976 by Lindström, collagenous colitis has attracted more scientific

attention only in the last decade, when firm epidemiological data from Sweden and the USA

have identified a rising incidence in the population, most certainly due to a greater awareness

of physicians to take biopsies from patients with chronic diarrhea (Wickbom, 2009; Pardi,

2007). The growing numbers of epidemiological studies have shown that CC is a disease

affecting mainly middle-aged women, although it can be seen in all ages and also in men. As

these patients frequently have concomitant autoimmune diseases such as celiac disease,

rheumatoid arthritis, diabetes mellitus or thyroid disorders, it becomes likely that a disturbed

immunological response plays a pathogenic role (Nyhlin, 2008). Furthermore, studies showed

that diverting luminal content via a loop-ileostomy can resolve intestinal inflammation.

Restoration of bowel continuity reinstalls the classical histological signs of CC, making it

evident that a luminal agent triggers the inflammatory process (Järnerot, 1995). As CC is

associated with bile acid malabsorption (Ung, 2000) it has been hypothesized that an

increased content of faecal bile acids might be of pathophysiological importance.

In classical inflammatory bowel disease (IBD) it is believed that an environmental factor

triggers an uncontrolled immunological response in the intestine of individuals who are

genetically predisposed. Hitherto no susceptibility gene has been detected in CC. In addition

to genetic, immunological and environmental factors, recent research has been examining the

role of disturbed mucosal barrier function as an important link in the pathogenesis of IBD

(Xavier, 2007). The intestinal mucosa as the main interface between the outer and inner

environment plays a crucial role in the defence from potentially harmful agents. The human

gut content is complex, consisting of more than 400-500 different bacterial species. Animal

models have shown that intestinal inflammation does not occur under sterile conditions and

the beneficial use of antibiotics in IBD further emphasizes the role that intestinal bacteria

might play in causing intestinal inflammation (Sartor, 2008). Especially newer data have

described that commensals in our own gut flora seem to have the potential to adhere and

invade the mucosa (Rhodes, 2007; Barnich, 2007).

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A broader scientific interest in different aspects of CC has emerged, in particular double-

blind, randomized trials have been conducted, looking at various medical treatment options.

Budesonide has the best documented efficacy for inducing and maintaining remission in CC

(Chande, 2009). On the other hand, cessation of budesonide treatment leads to clinical relapse

within 3 months in most patients (Miehlke, 2005).

The objective of this thesis has been to describe the mucosal barrier function in CC in

different phases of clinical activity and during budesonide therapy. Particular attention has

been paid to the passage of E.coli bacteria and the effects of bile acids on colonic mucosal

functions in biopsies, in Ussing chamber experiments.

BACKGROUND

Collagenous colitis

Historical remarks

The term collagenous colitis (CC) was first introduced by the Swedish pathologist Lindström

in 1976. He published the case report “Collagenous colitis with watery diarrhoea- a new

entity?” describing a 48-year-old woman with chronic watery diarrhea who showed no

abnormal macroscopic signs at rectoscopy, while histological examination of rectal biopsies

revealed a remarkable thickened subepithelial collagenous deposition (Lindström, 1976). He

furthermore described the clinical characteristics and showed that this condition lacks

abnormal laboratory, microbiological and endoscopic findings. He speculated that the

thickened collagenous layer formed a barrier to absorption of water and electrolytes, resulting

in diarrhea and that this disease is a seperate entity of immunological origin. In the same year

Freeman et al. published a similar case describing the same condition (Freeman, 1976).

In 1980 the term “microscopic colitis” (MC) was introduced by Read et al. (Read, 1980)

which, in 1989 was renamed to “lymphocytic colitis” (LC) when Lazenby et al. showed that

the main feature of this diarrheal disease was an increase content of intra-epithelial

lymphocytes (IEL) (Lazenby, 1989). As CC and LC share similar clinical findings and were

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found to show no endoscopic abnormality, a French and American research team proclaimed

in 1993 that these diseases should be combined under the common term “microscopic colitis”

(Levison, 1993; Flejou, 1993).

Particularly in the last decade, there has been increasing scientific focus in this field and the

number of original papers has increased exponentially reaching more than 900 publications in

January 2010. Despite the increasing interest in this field, the cause of MC and the

relationship between the two forms are still unknown.

Epidemiology

Epidemiological studies on CC have been conducted in different countries, but most of the

work with the longest follow-up has been conducted in the USA and Sweden.

In the observation period between 1984 and 2008, epidemiological data have been collected

in Olmsted County, USA and in Örebro, Sweden and both places have reported gradually

increasing incidence rates. Today it is believed that the incidence rate lies at 5.8 per 100 000

inhabitants (Wickbom, 2009) and the prevalence was reported to be 39 per 100 000

inhabitants for CC in the year 2001 (Pardi, 2007). It is most likely that the rise in incidence is

an effect of greater awareness of clinicians and pathologists when diagnosing this condition.

In all epidemiological studies, CC seems to affect mainly middle-aged women, though the

disease can occur in all ages, even in children (Benchimol, 2007). In the Örebro cohort the

mean age at diagnosis was 65 (range 53-74) years and the female:male ratio was 7:1 (Tysk,

2008).

Diagnosis: Clinical findings

The predominant symptom of CC is chronic, non-bloody, watery diarrhea (Bohr, 1996).

Abdominal pain is common, even during clinical remission (Nyhlin, 2008). Furthermore CC

is often associated with weight loss, fatigue, nausea and urgency, leading to faecal

incontinence which seriously impairs the health-related quality of life of these patients

(Hjortswang, 2005; Madisch, 2005). In one Swedish study, patients with CC were asked to

record their bowel movements in diaries and to fill out various health-related quality of life

questionnaires, making it possible to define clinical criteria for disease activity. The patients

witnessed a deterioration in their quality of life when a mean ≥ 3 stools/day or a mean ≥1

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watery stool/day in a one week registration was noted as a sign of disease activity (relapse)

(Hjortswang, 2009).

The onset of CC can be a sudden necessitating exclusion of an infectious cause, but in most

cases symptoms evolve gradually (Bohr, 1996). The course is usually chronic, intermittent,

but spontaneous remissions can occur. The risk of colorectal cancer is not increased (Chan,

1999).

CC patients often have a concomitant autoimmune co-morbidity, most commonly thyroid

disorders, celiac disease, diabetes mellitus and rheumatoid arthritis (Bohr, 1996, Kao, 2009,

Jobse, 2009). The association with other autoimmune diseases suggests an underlying

autoimmune process in CC, but hitherto no specific autoantibody or marker has been detected.

Routine blood samples are non-diagnostic and non-invasive screening for patients with CC is

not yet possible.

Endoscopic findings

Initially, CC was defined as presenting with a normal endoscopic picture but recent reports

describe abnormalities in the colon, e.g. mucosal tears as longitudinal lesions (Wickbom,

2006). Greater friability of the colon also seems to give a higher risk of post-endoscopic

perforations (Bohr, 2005; Allende, 2008).

Histology

As the diagnosis of CC is based on typical histopathological findings, it is essential that

patients with chronic diarrhea are assigned to a colonoscopy for biopsy taking. The diagnostic

features of CC are a thickening of the subepithelial collagenous layer ≥10µm in well-

orientated sections, in contrast to a normal basal membrane of <3 µm and furthermore a

chronic mononuclear inflammation in the lamina propria, epithelial cell damage and

occasionally an increased number of intra-epithelial lymphocytes, as presented in Fig.1. In

uncertain cases the use of tenascin immunostaining has been recommended (Müller, 2001).

The distribution of the typical histological findings in CC can be patchy in the colon and are

most prominent in the ascending and transverse colon and can be absent in the sigmoid colon

or rectum (Tanaka, 1992). Flexible sigmoidoscopy with multiple biopsy specimens from the

left colon is not sufficient to exclude CC when based on the presence of a thickened

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collagenous band alone. Yantiss et al. have proposed an optimal approach to obtain mucosal

biopsies for assessment of IBD of the gastrointestinal tract. To detect CC they recommend

taking two or more biopsies each from the right, transverse, descending and sigmoid colon

and additional sampling of endoscopically visible abnormalities (Yantiss, 2009).

Figure 1: A: Human colonicbiopsy showing normal histology.

B: Human colonic biopsyshowing typical findings of collagenous colitis-Increased subepithelialcollagen layer, inflammation in lamina propria and epithelial cell damage with intra-epithelial lymphocytes. Staining with trichrom.

Source=http://www.flickr.com/photos/euthman/2800899442/

A

B

Aetiology

The cause of CC is not known but it is believed that a luminal agent triggers an uncontrolled

intestinal inflammation in predisposed individuals.

That a luminal agent is a precondition in the pathogenesis of CC is best demonstrated by

diversion of intestinal content via a loop-ileostomy, leading to clinical and histopathological

remission. After operative rearrangement of the intestinal continuity the clinical symptoms

and the classical histological findings of CC resume (Järnerot, 1995). Current knowledge of

CC pathogenesis is best divided into mucosal and luminal factors.

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Mucosal factors

The mucosal inflammation in the epithelium is characterized by mainly CD8+ T lymphocytes

that carry the α/β form of the T-cell receptor. In the lamina propria there are mainly CD 4+ T-

lymphocytes (Mosnier, 1996). In a more recent study it could be demonstrated that the

increased CD 4+ T and CD8+ T cell infiltration in colonic mucosa displayed a suppressed

activation, but on the other hand the increased infiltration of eosinophils were functionally

activated in active CC (Wagner, 2009).

The thickened subepithelial collagenous layer stains intensely for collagen types I, III, VI and

particularly for tenascin. This histological finding is potentially reversible, but it is believed

that the characteristic linear deposition of extracellular matrix relies on a restricted matrix

metalloproteinase (MMP-1) RNA and increased tissue inhibitor of metalloproteinases (TIMP)

expression, leading to an imbalance of fibrogenesis and fibrolysis in CC (Günther, 1999).

Furthermore it has been suggested that vascular endothelial growth factor (VEGF) might play

a role in the accumulation of immature subepithelial matrix (Griga, 2004). By using a

colonoscope-based, segmental perfusion technique it could be shown that VEGF was

increased in the perfusate and was reduced by steroid therapy, giving rise to the hypothesis

that VEGF could be involved in the inflammatory reaction and affect mucosal permeability

(Taha, 2004).

CC demonstrates a Th1 mucosal cytokine profile with interferon gamma (IFN γ) as the

predominantly upregulated cytokine. Mucosal mRNA levels of interleukin (IL) 15, tumour

necrosis factor alpha (TNFα) and inducible nitric oxide synthase (iNOS) were also increased

(Tagkalidis, 2007).

Cytokines are known to alter tight junction permeability, especially IFN γ (Sugi, 2001) and

TNFα (Schmitz, 1999). Tagkalidis et al. found a reduction of E-cadherin and ZO-1 expression

induced by IFNγ in CC as a sign of alteration in epithelial barrier function. That paracellular

permeability is impaired in CC is corroborated by a study by Bürgel et al. showing diminished

expression of occludin and claudin 4 which are important tight junction proteins and these

findings correlated with reduced epithelial resistance, reflecting mucosal barrier dysfunction.

Furthermore the same group used Ussing chamber technique to describe the diarrheal

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mechanism in CC as being a reduced Na+ and Cl- absorption accompanied by a secretory

component of active chloride secretion (Bürgel, 2002).

Increased expression of iNOS correlated with luminal nitric oxide (NO) concentrations and

clinical activity measured as frequency of daily bowel movements (Olesen, 2003). In colonic

biopsies of CC patients, NFЌB is activated and recruited to the iNOS promoter in vivo via an

IKKβ mediated pathway (Andresen, 2005).

Faecal markers such as eosinophil protein X, myeloperoxidase and tryptase, can be increased

in the stool of CC patients (Lettesjö, 2006). The findings on faecal calprotectin as a marker

for intestinal inflammation are contradictory and can not be recommended as a diagnostic tool

(Wildt, 2007).

Genetics

A familial occurrence of CC has been reported, but the role of genetic factors remains unclear

(Abdo, 2001; Järnerot, 2001). Human leukocyte antigen (HLA) studies have demonstrated an

association between CC and HLA-DQ2 or DQ1/3 and a higher frequency of HLA-DR3DQ2

haplotype and TNFα polymorphism in CC, compared with controls (Fine, 2000; Koskela,

2008). In contrast to Crohn`s disease, functional polymorphism in the NOD2/CARD15 gene

has not been detected (Madisch, 2007) but on the other hand polymorphism of the matrix

metalloproteinase-9 gene does appear to be associated with CC (Madisch, 2006).

Luminal factors

Drug-induced CC

Several drugs have been suspected of playing a causal role in inducing microscopic colitis and

anecdotal clinical observations have been published since the early 1990s. In a systematic

review of the literature, Beaugerie and Pardi have listed 8 drugs (acarbose, aspirin,

cyclo3Fort, lansoprazole, nonsteroidal anti-inflammatory drugs, ranitidine, sertraline and

ticlopidine) as highly likely to cause LC or CC. The median interval between drug intake and

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onset of diarrhea is around 4 days and after drug withdrawal diarrhea stopped after about 5

days (median) (Beaugerie, 2005).

In a case control study the risk associated with use of non-steroidal anti-inflammatory drugs

NSAIDs in CC was confirmed; on the other hand selective serotonin reuptake inhibitors

(SSRI) caused diarrhea but not necessarily MC (Fernandez-Banares, 2007). In all patients

with chronic diarrhea a thorough drug history should be taken and cessation of suspected

drugs should be tried.

Infection

As CC can present with a sudden onset an infectious cause has been suspected. The

association of CC and Clostridium difficile infection has been discussed and presented in case

reports. The symptoms persisted after treatment of C. difficile and it was suggested that C.

difficile may be a noxious stimulant that could catalyse a chain of events resulting in CC

(Erim, 2003). Furthermore antibodies to Yersinia were more common in CC patients than in

controls, which led to the speculation that a previous Yersinia infection could have triggered

CC (Bohr, 2002). In most CC cases, despite its sudden onset, stool cultures remain negative.

Bile acids

Bile acid malabsorption (BAM) can coexist with CC, leading to more frequent bowel

movements and looser stool consistency. By using 75 Selenium-labelled homocholic acid-

taurine (SeHCAT), concurrent bile acid malabsorption (BAM) was found in up to 44% of

patients with CC. Bile acid binding treatment has been shown to be effective in CC, especially

when BAM is concomitant (Ung, 2000). The same research group studied the long term

course (mean 4.2 years) in CC, BAM and bile acid sequestrants on histopathology and clinical

features and found: 1. BAM seems to be a long-standing finding in a considerable number of

patients with CC. 2. Patients on bile acids binders had no significant change in histopathology

despite they have good effect on the symptoms. 3. Furthermore, in conclusion BAM and CC

seem to be associated although presumably independent diseases.

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Treatment

By taking a thorough history, the excessive use of dietary products or concomitant drugs

which can lead to chronic diarrhea should be excluded.

In patients with mild symptoms, a trial of loperamide or cholestyramine can be tested.

In minor uncontrolled studies, bismuth subsalicylate (Fine, 1999), prednisolon (Munck, 2003)

and mesalamine (Calabrese, 2007) demonstrated a favourable clinical response but sample

sizes were too small to give a general recommendation. On the other hand treatment, with

Boswellia serrata extract (Madisch, 2007) and probiotics (Wildt, 2006) failed to show

efficacy.

Budesonide has the best documented efficacy in significantly alleviating symptoms and

improving quality of life. In a Cochrane meta-analysis, budensoide was described as being

effective and well tolerated for inducing and maintaining clinical and histological responses in

patients with CC (Chande, 2009). A total of 94 patients were enrolled in three trial studies on

budesonide (9 mg daily or in a tapering schedule for 6 to 8 weeks) (Miehlke, 2002; Baert,

2002; Bonderup, 2003). Clinical remission occurred in 81% of patients given budesonide,

compared with 17% of patients who received placebo (p<0.00001). The pooled odds ratio for

clinical remission to treatment with budesonide was 12.32 (95% CI 5.53 to 27.46), with a

“number needed to treat” (NNT) = 2. A statistically significant histological response followed

treatment in all three trials studying budesonide therapy. Budesonide induction therapy has

also been shown to improve quality of life (Madisch, 2005).

On the other hand, after withholding of short-term budesonide treatment, relapse rates lied

around 61% in a follow-up period and the median time until recurrence of symptoms was 2

weeks (range 1-104 weeks) (Miehlke, 2005).

In treatment studies for maintaining remission, 80 patients who had responded to open-label

budesonide were enrolled in two trials studying budesonide (6 mg daily for 6 months)

(Bonderup, 2009; Miehlke, 2008). Clinical response was maintained in 83% of those given

budesonide, compared with 28% of patients given placebo (p=0.0002). The pooled odds ratio

for maintenance of clinical response to treatment with budesonide was 8.40 (95% CI 2.73 to

25.81), with a “number needed to treat” (NNT) = 2. Histological response was maintained in

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48% of patients given budesonide, compared with 15% given placebo (p= 0.002) (Chande,

2009).

In severe cases of CC who are steroid dependent or refractory, immunomodulating therapy

with azathioprine (AZA) or 6-mercaptopurine (6-MP) can be initiated. In a small group of

patients (N=9) AZA or 6-MP gave a response rate of 89% and a steroid sparing effect (Pardi,

2001). In a retrospective study, beneficial effects of oral low-dose methotrexate was observed

in CC patients (Riddell, 2007).

Medical therapy in CC has become so effective that surgical treatment is very seldom needed

nowadays, but an ileostomy is still an option in patients with severe and therapy resistant

illness.

Intestinal barrier function

Structures

The intestinal tract represents the body’s most important interface between internal and

external environment. The intestinal epithelium is a single-cell layer serving as a highly

selective barrier. Its role is dual, by permitting absorption of vital nutrients, electrolytes and

water on the one hand while on the other, maintaining an effective defence against

intraluminal toxins, antigens and enteric flora. The barrier is built up of a complex interaction

between several components including the unstirred water layer, mucosal surface

hydrophobicity, mucus layer containing immunoglobulins/defensins, the epithelium (cells

held together by tight junctions) and immune cells in the lamina propria that all have different

barrier-protecting properties (Fig.2).

The magnitude of transepithelial permeation of molecules gives information on mucosal

barrier integrity in health and disease. Intestinal permeability is strictly regulated and several

factors participate in this process. Not all aspects of barrier function can be discussed in this

chapter but the main focus lies on the structural components of the intestinal barrier,

especially those that are investigated with endoscopic biopsies in the modified Ussing

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chamber. Furthermore, a basic understanding of intestinal barrier dysfunction in IBD will be

highlighted.

LumenLumen

MucusMucus

EpitheliumEpithelium

Lamina Lamina propriapropria

Enteric nervous systemEnteric nervous system

Capillaries/EndotheliumCapillaries/Endothelium

Figure 2: A simplified view of the different structures comprising the intestinal barrier

function. In the lumen gastric acid, pancreatic juices, and bile take part in barrier function by

degradation of bacteria and antigens; pathogenic bacteria are kept under control by the normal

gut flora. The mucus acts as a physical barrier and contains defensins and secreted

immunoglobulins, primarily IgA. The epithelium constitutes the principal barrier to

permeation, through which molecules can pass either transcellularly (transcytosis) or

paracellularly via the junctional complexes, including tight junctions. The lamina propria

contains various cells such as myofibroblasts or cells of the innate and acquired immunity

which interact with enterocytes. Furthermore the enteric nervous system communicates with

immune cells and neural impulses influence the mucosal barrier function. Through the

endothelium of the capillaries the mucosa has contact with the circulation.

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Mucus layer

Goblet cells in the gastrointestinal tract produce a mucous gel coat that serves as a lubricant

and provides non-specific protection against chemical digestion and adhesion of bacteria.

The hydrophobic character of gastrointestinal mucus relies on a layer of surface active

phospholipids that line the top of the mucus covering the epithelium. The phospholipid layer

protects against luminal acidity by repelling the diffusing hydrogen ions. Not only in the

gastric mucosa is surface hydrophobicity high, but also in the colon. In a study where

detergents were applied to remove the phospholipid layer in rat colon, an increased mucosal

permeability to macromolecules and toxins was found (Lugea, 2000).

As already studied in the 1960s, mucus seems to envelop particles so that they do not come

into contact with epithelial cells (Florey 1962). Furthermore it protects against pathogens by

acting as a physical barrier, having binding sites for bacterial adhesins, maintaining high

concentrations of secreted immunoglobulins, primarily IgA and defensins, and also acts as a

free radical scavanger (Cross, 1984; Forstner, 1994).

Mucus is secreted continuously, nearly 10 litres daily, which is digested and mostly recycled.

The rest is shed in faeces. The thickness of the mucus layer (approx. 110-160 µm) is

determined by the balance between the rate of secretion and rate of degradation and shedding.

In an animal model it was recently demonstrated that the colonic mucus consists of two

layers, the inner layer being densely packed and devoid of bacteria. Proteomics revealed that

the gel-forming mucin Muc2 was the major structural component (Johansson, 2008).

Toxic and irritating substances can greatly stimulate mucus secretion, increasing the thickness

of the mucus layer while efficiently and rapidly moving the irritants away from the

epithelium.

The epithelial layer

The gastrointestinal epithelium is a single-cell layer that acts as a selectively permeable

barrier. It undergoes perpetual self-renewal originating from a limited pool of pluripotent stem

cells situated at or near the base of intestinal crypts (Karam, 1999). The epithelium faces the

complex task of permitting absorption of nutrients, electrolytes and water, while also

protecting the internal environment from potentially toxic products.

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There are two major routes for epithelial permeation: paracellular and transcellular (Spring,

1998). The paracellular transit is the key regulator of intestinal permeability and is formed by

a complex protein-protein network, also called the junctional complex, that mechanically

links adjacent cells and seals the intercellular space. The protein network connecting epithelial

cells forms three adhesive complexes: desmosomes, adherens junction and tight junctions

(TJs), the latter being most critical for paracellular permeability. Small hydrophilic

compounds succeed in passing through the cell via passive diffusion or via aqueous pores,

whereas larger molecules tend to pass via the paracellular route.

The transcellular pathways are only briefly mentioned as they are not further discussed.

Paracellular permeability/Tight junctions

Tight junctions are the apically-most adhesive junctional complexes in mammalian epithelial

cells. They form a continuous belt-like ring around epithelial cells at the border between the

apical and lateral membrane regions (Farquhar, 1963). They act as a dynamic gateway, able to

change in size under various condition to facilitate or hinder passage of different products. TJs

structures can be altered by osmotic load or hypertonic solution reflected by changes in

transepithelial resistance and an increased paracellular uptake of macromolecules (Madara,

1983).

TJs are a multiprotein complex build-up of four unique families of transmembrane proteins:

occludin, claudin, junctional adhesion molecules (JAM) and tricellulin.

Occludin and at least 20 members of the claudin family have different barrier-sealing

properties which are variable among cell types in terms of electrical resistance, solute and

water flux, and charge selectivity (Mitic, 2000). In the tight junctions, permeation is also

regulated by size and charge selectivity, whereby hydrophilic, positively charged molecules

and ions pass more easily. Of the junctional adhesion molecule protein family, mainly JAM-1

seems to play a major role in intestinal homeostasis by regulating epithelial permeability,

inflammation and proliferation (Laukoetter, 2007).

At points where three cells meet, tricellulin forms a central tube in a tricellular junction,

allowing passage of solutes. Tricellulin is expressed in large amounts in epithelium-derived

tissue and when tricellulin expression is suppressed, the epithelial barrier function will be

compromised (Ikenouchi, 2005).

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Furthermore, intracellular proteins such as zonula occludens (ZO) family members and

cingulin link these molecules to the actin cytoskeleton, which provides the cell with structural

integrity. The cytoskeleton includes three types of proteins filament: actin, microtubules, and

intermediate filaments that extend throughout the cytosol and make contact with the cell to

cell outer surface. Hence, the cytoskeleton is also essential for the paracellular pathway and a

critical structure for maintaining intestinal barrier function (Fig.3).

The intimate relationship between the tight junctions and the cytoskeleton is also

demonstrated by the observation that phosphorylation of the myosin regulatory light chain

(MLC) is involved in tight junction regulation. The myosin ATPase-mediated contraction of

the perijunctional actomyosin ring subsequently leads to physical tension on the TJs (Turner,

1997). Furthermore it has been shown that proinflammatory cytokines, like interferon gamma

and tumour necrosis factor alpha, influence paracellular permeability by either inducing

endocytosis of epithelial TJ proteins (Utech, 2005) or by downregulating the expression of the

tight junction strand protein occludin (Mankertz, 2000).

Figure 3: Schematic representation of the basic structural transmembranecomponents of tight junctions.

The main transmembrane proteins are occludin, claudin, junctional adhesion molecules (JAM) and tricellulin. ZO-1 or ZO-2 is important for clustering of claudins and occludin, resulting in the formation of tight junctional strands. The ZOsand cingulin can provide a direct link to the actin cytoskeleton.

Image adopted from Niessen, 2007.

25

Transcellular permeability

A controlled protein uptake via the transcytotic route is physiological and essential for antigen

surveillance in the gastrointestinal tract (Ponda, 2005). The transcellular pathway allows

many molecules to enter the cell from the luminal side and exit on the basolateral side and is

also important for the regulation of intestinal permeability. There are active mediated uptake

mechanisms for sugars, amino acids and vitamins, while larger peptides, proteins and particles

are transported through the cell by endocytosis. Endocytosis in epithelial cells can occur along

different routes, depending on the nature of the substance. There are highly specific receptor-

bound processes via the clathrin-mediated endocytosis (Liu, 2001) or more unselected uptake

of luminal antigens via phagocytosis or macropinocytosis (Conner, 2003). Most of what is

internalized is recycled to the apical membrane but the remaining proteins are degraded by

lysosomal enzymes. This process is believed to play a role in induction of oral tolerance

(Zimmer, 2000).

In in-vitro studies, horseradish peroxidise (HRP) is used as a trancellular marker and is known

to be taken up in endosomes in human colonocytes (Wallon, 2005). In one animal study it was

seen that increased intestinal permeation of HRP was associated with increases in the number

and size of the epithelial endosomes (Santos, 2001). Furthermore, epithelium under metabolic

stress increases its endocytotic activity which can result in a microtubule-, microfilament-

dependent internalization and transcytosis of bacteria (Nazli, 2006).

Intestinal barrier dysfunction in IBD

Mucosal barrier function has been extensivly examined in ulcerative colitis and Crohn`s

disease. These inflammatory bowel diseases (IBD) are of polygenetic origin characterized by

an exaggerated inflammatory response to the microbial flora inhabiting the lumen of the gut.

Microscopic colitis and IBD are clearly different entities but in rare cases, however, a double

diagnosis was made or progression of CC to genuine ulcerative colitis was observed (Geboes,

2008).

Accumulating evidence underscores the important role that the epithelium plays in both

pathogenesis and pathophysiology of IBD. Early studies suggested that functional

modification in the barrier function (increased permeability) also described with the term

26

“leaky gut” could be found not only in patients with IBD but also in some first-degree

relatives (Hollander, 1999; Söderholm, 1999). In in vivo and in vitro studies abnormal

permeability refers to a measurable increase in flux of markers across the intestinal

epithelium, whereby several mechanisms contribute to this defect.

The rate of movement is regulated primarily by the functional state of the tight junctions

controlling paracellular passage. In IBD, altered tight junction structures have been shown to

contribute to impaired epithelial barrier function (Schmitz, 1999). In Crohn`s disease an

upregulation of pore-forming claudin 2 and downregulation of sealing claudins 5 and 8 were

found (Zeissig, 2007). Furthermore, TJs are influenced by proinflammatory cytokines such as

TNF-α and IFN-γ, which increase in the IBD mucosa (Niessner, 1995). Both TNF-α and IFN-

γ have been shown to impair epithelial barrier function in cell line experiments and also

modify mucosal morphology and TJ protein rearrangement (Amasheh, 2009).

In addition to TJ changes, other changes also play a role in IBD barrier dysfunction, such as

increased transcytosis and induction of epithelial cell apoptosis and lesions. There is

increasing evidence that antigens can be taken up to a significant extent via the transcellular

route by endocytotic uptake and transcytosis. This could be identified by electron microscopy

studies in Crohn`s disease, but the transport mechanisms are still not known (Schürmann,

1999; Söderholm, 2004).

In recent years the role of luminal bacteria in the pathogenesis of IBD has attracted increased

attention as intestinal bacteria are essential for the development of mucosal inflammation as

demonstrated in numerous animal models of IBD (Barnich, 2007). Patients with IBD have

greater numbers of mucosa-associated bacteria than control patients (Swidsinski, 2002) and a

high prevalence of adherent-invasive Escherichia coli was found in the ileal mucosa in

Crohn's disease (Darfeuille-Michaud, 2004). Crohn`s disease presents initially with small

lesions at the specialized follicle-associated epithelium (FAE) that lines the Peyer`s patches in

the terminal ileum. One study demonstrated that the barrier dysfunction was localized to the

FAE of Crohn`s patients showing increased transcellular uptake of non-pathogenic bacteria

(Keita, 2008).

Little is known about mucosal barrier function in CC. In a recent study it was demonstrated

that active CC reduced E-cadherin and ZO-1 expression induced by IFNγ, signifying

modification of epithelial barrier function (Tagkalidis, 2007). That mucosal barrier function is

27

impaired in CC was further corroborated in a study by Bürgel et al. showing diminished

expression of occludin and claudin 4 which are important tight junction proteins. These

findings correlated with decreased epithelial resistance reflecting increased paracellular

permeability (Bürgel, 2002).

Bile acids Biochemistry

The common bile acids are synthesized from cholesterol in the liver and contain a saturated

ring system and a five-carbon side chain terminating in a carboxyl group. In all bile acids the

ring system is the same, though, the number and position of hydroxyl groups and the presence

or absence of conjugation to amino acids bring about important differences in the structure

and consequent physical properties. Subtle changes, such as the addition of one hydroxyl

group at position 3, 7 or 12 or the change from α- to β- configuration of the hydroxyl group

may give very different crystalline packing, solubility and behaviour in the aqueous systems

(Fig. 4 b). The α-hydroxyl groups all lie on one side of the ring and give the molecule

amphipathic character with polar and a nonpolar face responsible for its solublizing

properties. The hydroxyl group’s location, orientation and hydrophilic properties are given in

Table 1.

Bile acid pos. 3 pos. 7 pos.12

CA α OH α OH α OH hydrophilic

UDCA α OH β OH H

CDCA α OH α OH H

DCA α OH H α OH

LCA α OH H H lipophilic

Table 1: Common bile acids showing their position of the hydroxy groups, α- to β- configuration and hydrophobicity.

28

The naturally occurring bile acids in humans are cholic acid (CA), chenodeoxycholic acid

(CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA) and in a minor proportion also

ursodeoxycholic acid (UDCA) (Fig. 4 a). The primary bile acids CA and CDCA are formed in

the liver and before excretion from the hepatocytes they are conjugated with an amino acid,

taurine or glycine, by linkage to the carboxyl group of the side chain. Thus, conjugation

makes it impermeable for membranes. The secondary bile acids DCA and LCA are formed by

bacterial 7α-dehydroxylation from the primary bile acids in the intestine and, furthermore

deconjugation takes place resulting in major changes in hydrophobicity (Cabral, 2001).

Figure 4: Biochemistry of the common bile acids.

The primary bile acids cholic acid (CA), chenodeoxycholic acid (CDCA) are synthesized

from cholesterol and conjugated with an amino acid, taurine or glycine, by linkage to the

carboxyl group of the side chain. Furthermore hydroxyl groups are added to positions 3, 7 and

12 (b). In the intestine the secondary bile acids DCA and LCA are formed by bacterial 7α-

dehydroxylation and deconjugation (a). Scheme of a micelle formed by phospholipids in

aqueous solution (c).

29

Physiology

Bile acids are hydrophobic derivates of cholesterol that play an important role in the digestion

and absorption of fats. They are synthesized in the liver, stored in the gallblader, and secreted

into the intestine as conjugated bile acids linked to glycine and taurine. Bile acids serve many

important physiological functions, including cholesterol homeostasis, lipid and vitamin

absorption and excretion of drugs (Vlahcevic, 1999). Typically, after secretion into the

intestine, bile acids are efficiently reabsorbed via the apical sodium-dependent bile acid

transporter (ASBT) in the terminal ileum forming the enterohepatic circulation, although a

small percentage (~5%) is known to escape into the colon. In a steady state this faecal loss

equates approximately to the daily synthesis. Our knowledge of faecal bile acids is based

mainly on qualitative and quantitative analyses using gas-liquid chromatography-mass

spectrometry (Setchell, 1988). Quantitative determination of faecal bile acid excretion

provides important information about bile acid kinetics, whereas qualitative analysis gives us

insight into intraluminal events involving bacteria and bile acid interaction.

In general, total faecal bile acid excretion in healthy adults has been quoted to an average

range of 200-300 mg/day, mainly in unconjugated form owing to deconjugation during

passage through the small intestine and colon. The inter- and intra-individual range can differ

greatly from day to day, measurement mainly reflecting the influence that diet has on faecal

bile acid excretion. Numerous analyses have revealed a tremendous complexity and

composition of > 40 different bile acids found in faeces. Lithocholic and deoxycholic acids

are quantitatively the major bile acids, accounting for about 30-55% of all faecal bile acids

excreted. The proportions of chenodeoxycholic and cholic acids are generally low in healthy

humans. Bile acids are bound to dietary residue and intestinal microorganisms but, in the

colon, passive absorption has been demonstrated, contributing significantly to the

conservation of the bile acid pool in the healthy state. This is also demonstrated by the

presence of numerous unconjugated and secondary bile acids in peripheral blood. Our

knowledge of faecal bile acid composition in humans is based on faecal samples that have

been excreted and have passed the whole colon. Recently, in an interesting study, Hamilton et

al. looked at the concentrations and spectrum of bile acids in the human caecum. They found

that 90% of bile acids were unconjugated and dehydroxylation of bile acids was nearly

complete in the right colon. The total 3-hydroxyl bile acid concentration was 0.6±0.3mM,

thereof deoxycholic 34±16%, lithocholic 26±6%, cholic 6±9% and chenodeoxycholic acid

7±8% (Hamilton, 2007).

30

Various factors can influence bile acid excretion, the most crucial in the conservation of the

bile acid pool being the active transport of bile acids in the terminal ileum. Resection or

dysfunction due to inflammation in this region will seriously compromise the integrity of the

enterohepatic circulation. At normal intraluminal pH, conjugated bile acids will be present

principally in ionized form with high water solubility by virtue of forming micelles. Ionized

conjugated bile acids are favoured by active transport processes and a decrease in intraluminal

pH can influence bile acid uptake. The intestinal microflora metabolizes bile acids by a

number of reactions, mainly hydrolysis of the amide bond of the conjugates and 7α-

dehydroxylation. Changes in the microflora of the gut can alter both the quantitative and

qualitative patterns of faecal bile acids. Furthermore, conditions with decreased transit time or

diarrhea can lead to an excessive spillage of primary bile acids into the colon (Setchell, 1988).

The influence of bile acids on intestinal barrier function

The role that bile acids might have in the carcinogenesis of colon cancer has been vigorously

investigated but the focus of this chapter is to describe the toxicity of bile acids to the colonic

mucosa and effects on barrier function.

In perfusion studies in animals and humans, bile acids induced marked morphological

changes in the colonic mucosa, often associated with changes in fluid and electrolyte

secretion (Mekhjian, 1971; Chadwick, 1979).

Animal studies showed that bile acids with two hydroxyl groups in the alpha configuration

(CDCA and DCA) in concentrations between 1-8 mM gave a dose-related increase in

paracellular mucosal permeability and damaged the mucosa (epitheliolysis) as demonstrated

by light and electron microscopy (Camilleri, 1980; Goerg, 1982). The potency of several bile

acids as inducers of these changes appears to be related to their surface properties as

determined by critical micelle concentration and thereby loss of surface epithelium is directly

related to their detergent activity (Gullikson, 1977).

In a more recent study, moderate concentrations of bile acids induced increased permeability

in vivo in rat colon by mechanisms involving muscarinic and nicotinic receptors as a link

between the central nervous system and colonic mucosal barrier function (Sun, 2004).

Looking at more physiological concentrations of bile acids Mühlbauer et al. investigated the

31

molecular mechanism of bile acid-induced gene and cytokine expression in colonic epithelial

cells (CECs). They demonstrated that DCA can induce IL-8 gene expression via the NF-B

signal transduction pathway in primary colonic epithelial cells, suggesting that bile acids can

trigger a proinflammatory reaction (Mühlbauer, 2004). In a further study it was shown that

physiological concentrations of bile acids inhibited recovery of ischaemic-injured porcine

ileum, thereby implying that DCA was deleterious to mucosal barrier function due to

increased paracellular permeability (Campbell, 2004). Investigations into the effects that more

physiological concentrations of bile acids might have on barrier function, especially in human

tissue, are lacking. As CC is associated with bile acid malabsorption, presents with diarrhea

and is driven by an intestinal inflammation, the question arises to what extent bile acids

influence the barrier function in this condition?

32

33

3. AIMS OF THE THESIS As patients with inflammatory bowel disease are described as having a “leaky gut” the major

aim of this thesis was to describe barrier function in colonic biopsy material from patients

with collagenous colitis (CC), by using the Ussing chamber technique.

Furthermore, CC is associated with bile acid malabsorption, implying higher faecal bile acid

concentrations in the colon. We speculated that bile acids might affect barrier function in CC.

The specific aims of the papers are as follows:

I. to describe mucosal permeability and histological features in a single patient

with active CC, before and during faecal diversion via loop-ileostomy and after

bowel reconstruction;

II. to elucidate the effects of µM concentrations of bile acids on mucosal barrier

function in biopsies from healthy individuals with normal histology;

III. to analyse mucosal barrier function in patients with CC in clinical remission,

with active disease and during budesonide treatment.

IV. to determinate whether physiological concentrations of bile acids further

exacerbate the impaired barrier function in CC.

34

35

4. SUBJECTS AND METHODS Patients In the first paper we examined a single female patient (age 59 years) with intractable CC who

had not responded to various medical treatment options. A loop-ileostomy was performed and

she agreed to undergo repeated biopsy taking before, during faecal stream diversion and after

bowel reconstruction for functional and histological examinations.

In the second study, patients planned to be examined with endoscopy at the University

Hospital in Linköping, Sweden, and in whom we suspected a normal histology in the sigmoid

colon agreed to provide us with biopsies for research purposes. Indications for colonoscopy

were mainly screening for malignancy because of occult blood in faeces, constipation, or

previously radiographically verified polyps outside the sigmoid colon.

17 patients were included: 12 women mean age 62 years (range 38-78) and 5 men mean age

60 years (range 44-73). The patients were divided into two subgroups: Group A: 9 patients for

electrophysiological and permeability measurements, group B: 8 patients for analysis of

bacterial uptake. A further criterion for inclusion was the absence of NSAID or steroid

medication.

In the other two studies a total of 25 patients (20 women, 5 men, mean age 66 years) with CC

were included from December 2005 to April 2008. There were three groups: 14 patients in

clinical remission without medical treatment, 11 with active disease, and 8 of these were

studied again after 6 weeks of budesonide treatment. The subjects of the second study with

normal histology served as controls when comparing electrophysiological parameters and

bacterial uptake. All patients were asked to register their bowel movements during one week

on a diary chart, and thereafter undergo sigmoidoscopy where biopsies were taken from the

mid-part of the sigmoid colon. Stools were collected during a 24- hour period prior to

endoscopy, to measure stool weight and faecal calprotectin levels. Stool cultures were

performed to rule out ongoing Campylobacter, Salmonella, Shigella, Yersinia and Clostridium

infection. Routine blood samples (blood count, creatinine, CRP) were taken to detect other

possible infectious conditions. From the diary chart the mean stool frequency and quantity of

watery stools per day/week was calculated and served as reference to classify the patients into

groups of remission or active disease (relapse), according to the score by Hjortswang et al.

36

(Hjortswang, 2009). Active disease (relapse) was defined as a mean of 3 stools/day or a

mean of 1 watery stool/day over a one-week registration. The consistency of the stool was

classified in arbitrary order (1 = watery, 2 = soft, 3 = normal). 8 patients with active disease

were treated with budesonide (Entocort) 9 mg o.d. for 4 weeks and further 6 mg o.d. for 2

weeks. After 6 weeks of treatment, all patients attained clinical remission, stool collection was

repeated and the patients were re-examined with sigmoidoscopy and biopsies taken for

histology and Ussing chamber analysis. None of the patients took NSAID or other

immunomodulating agents.

All patients gave their informed consent and the studies was approved by the Ethics

Committee, Faculty of Health Sciences, Linköping, Sweden.

Ussing chamber

The “Ussing Chamber” is named after its inventor, Hans Ussing, a Danish physiologist

(Ussing, 1951). Designed initially to study vectorial ion transport through frog skin, it has

emerged to become a widely used instrument within pharmaceutical research for studies of

drug absorption (Hillgren, 1994). It has also been increasingly applied to the study of

pathophysiological processes in the intestinal mucosa of animals and humans (Stack, 1995;

Biljsma, 1995). The initial methology was rather complicated and the technique has since

been modified and simplified (Grass, 1988).

The modified Ussing chamber, which has been extensively used by our group and in these

experiments, consists of two half chambers and the endoscopically taken biopsy is mounted

between the halves, as shown in Fig. 5/6. The two compartments, one on either side of the

tissue, are filled with buffer and continuously oxygenated (95% O2, 5% CO2). The gas flow

keeps the buffer in motion, reducing the thickness of the unstirred water layer (Karlsson,

1992). A heat block keeps the solution at 37oC. The marker solutions are applied to the

mucosal or serosal compartment and withdrawn from either side for analysis. The system is

furthermore equipped with a pair of Ag/AgCl- electrodes with agar-salt bridges and a pair of

current-giving platinum electrodes to enable monitoring of electrophysiological parameters.

37

Epithelium displays two features that distinguish it from other tissue: polarity and tightness.

Polarity or the transepithelial potential difference (PD) is generated by the sum of ions and

proteins that are asymmetrically distributed either to the apical or basolateral membrane. It

reflects the electrogenic pump activity (mainly Na+/K+-ATPase) in the membrane but also

passive ion flow through channels (Armstrong, 1987).

In order to measure short-circuit current (Isc) the epithelium is short circuited by injecting a

current that is adjusted by a feedback amplifier to keep PD = 0 mV. The amount of current

needed for this reflects the summation of all active ion pump activity.

Furthermore the integrity of the tissue is determined by the formation and permeability of the

tight junction, an assembly of proteins responsible for the “tightness” between epithelial cells.

Tightness can be measured electrically by the transepithelial resistance (TER) and represents

the passive flow of ions via the paracellular pathway. Resistance is calculated by applying

Ohm`s law: TER = PD/I.

UssingUssing ChamberChamber

Figure 5: Schematic illustration of the modified Ussing chamber

The biopsy, taken by endoscopy, is mounted between the two half-chambers and continuously

oxygenated. One pair of Ag/AgCl-electrodes is used to measure the potential difference (Pd)

and another pair of platinum electrodes supplies current to the system (I) which allows

calculation of the transepithelial resistance (TER). Buffer solution is given into both

compartments and different markers can be added.

38

Figure 6: Photodocumentation of mounting an endoscopically taken biopsy in the modified Ussingchamber with an exposed tissue area of 1.76 mm2. A system (right side) contains 6 Ussing chambers.

Permeability markers

To study the mucosal barrier function, various markers such as C-mannitol, FITC-dextran and

polyethylene glycols (PEG) of varying size have been tested in Ussing chamber experiments.

We chose to use 51Cr-EDTA and the 45 kD protein antigen horseradish peroxidase (HRP)

which are widely used for permeability studies.

As a paracellular marker we chose to apply the inert probe 51Cr-EDTA (MW 384D; Perkin

Elmer, Boston, Mass., USA (3.25 µM)). EDTA binds strongly to the radioactive Cr, which

ensures that the Cr passage is equal to the passage of EDTA, and no Ca2+ can be bind to

EDTA to give detergent effects.

As a transcellular marker we applied HRP (Typ VI, 10 µM), which is known to be taken up

through the epithelial cell via macropinocytosis (Schürmann, 1999).

HRP and 51Cr-EDTA were added to the mucosal side and serosal samples were collected at 0,

30, 60, 90 and 120 min after start. An aliquot from each sample was saved for HRP analysis

and the remainder was placed in a gamma-counter for 51Cr-EDTA measurements. For HRP

analysis we used the QuantaBlu fluorigenic peroxidase substrate Kit (Pierce, Rockford; Ill.

39

USA). Permeability was calculated during the 30-90 min period for both markers. 51Cr-

EDTA permeability was expressed as Papp (apparent permeability coefficient; cm/s x 10-6),

and HRP permeability presented as transmucosal flux (pmol/h/cm2).

E.coli K-12

As commensals are increasingly known to play a pathogenetic role in IBD we wanted to study

the transmucosal passage of non-pathogenic bacteria. In papers II, III and IV all patients were

investigated for uptake of chemically killed, fluorescein conjugated E.coli K-12 BioParticles

(Molecular Probes, Leiden, The Netherlands). These bacteria are killed with

paraformaldehyde, which stops their reproduction but retains antigenicity and has previously

been used for phagocytosis studies (Wan, 1993). A concentration corresponding to 1.0 x 10

CFU/ml was added to the mucosal compartment as previously described (Keita, 2006). After

2 hours the whole content of the serosal compartment was analysed at 488 nm in a fluorimeter

(Cary Eclipse, Varian) where 1 unit corresponds to 3 x 103 CFU/ml, assessed by FACS

analysis.

Bile acids

We chose to apply CDCA and DCA in our experiments because they represent a primary and

a secondary bile acid and have been used frequently in many previous studies. Furthermore

they are known to be most abundant in the large intestine, mainly in a non-conjugated status.

Sodium-chenodeoxycholate (3α, 7α- dihydroxyl-5β-cholan-24-oic acid, >97%, Sigma) and

sodium-deoxycholic acid (3α, 12α- dihydroxyl-5β-cholan-24-oic acid, >99%, Sigma, St

Louis, Mo, USA) were diluted with mannitol Krebs to obtain concentrations of 100, 500,

1000 µmol/l. After 40 min equilibration, CDCA and DCA in mannitol Krebs were added to

the mucosal compartment.

40

Histology

All biopsies were examined by the same pathologist (Åke Öst). Two biopsies from the

sigmoid colon were taken at each investigation and stained with haematoxylin-eosin (HE) and

van Gieson. The degree of surface epithelial cell degeneration was assessed in arbitrary units

(0=none, 1=mild, 2=moderate, 3=severe). The thickness of the collagenous band was

measured in five different areas and the mean value was determined. Immunohistochemical

staining for CD3 was also performed according to routine procedures. The number of intra-

epithelial lymphocytes (IEL/100 enterocytes; mean value of three counts) was assessed. The

infiltration of mononuclear cells (lymphocytes and plasma cells) in the lamina propria was

defined in arbitrary units (0=none, 1=mild, 2=moderate, 3=severe) (Geboes, 2000).

Confocal laser scanning microscopy

From 2 patients in the second and fourth paper, six extra biopsies were processed for confocal

laser scanning microscopy to study the passage routes. E.coli K-12 and 100 or 1000 µmol/l of

CDCA or DCA were added to the mucosal side and after 15 min the tissues were rinsed in

phosphate-buffered saline (PBS) and then carefully removed to be mounted in OCT

Compound (Miles Inc., Ind., USA). The biopsies were stored at –72° C. The tissue blocks

were subsequently cryosectioned (6 μm thickness) onto glass slides using a Leica CM3050

microtome (Sollentuna, Sweden). The slides were air-dried overnight, fixed in ice-cold

acetone for 30 min, and stored at 40C until further use. The sections were then incubated for

10 min with Alexa Fluor 581 conjugated phalloidin (Molecular Probes, Leiden, The

Netherlands). The slides were thoroughly washed with PBS (5 times). A drop of mounting

medium (Dako Cytomation, CA, USA) was added. Prolong Gold with DAPI was used as

mounting medium to achieve a parallel nuclear and chromosome stain. In experiments where

rhodamine conjugated dextran (10.000 MW) (Invitrogen) was used it was added in the Ussing

chambers at the same time as the bacteria.

The slides were examined in a Nikon Eclipse E600W confocal laser-scanning microscope

(Nikon, NY, USA) using Nikon EZ-C1 software, with a 60x oil-immersion objective. An ion

laser permitted simultaneous excitation wavelengths of 488 nm for fluorescein-labelled E. coli

and 594 nm for Alexa-labelled phalloidin.

41

Methodological considerations

The taking of human colonic biopsies cannot be standardized; the biopsies may vary in size

and thickness. This leads to a scattering of results due to variability of the examined tissue.

To reduce inter-individual differences in biopsy taking, this task was performed mainly by

one doctor (Magnus Ström), as mounting the biopsies in the Ussing chamber was done by

Andreas Münch. To avoid systematic repetition and unconscious mounting of the largest

biopsies first, the order of placement of the Ussing chambers in the system was randomly

changed. To further reduce biological variability, multiple biopsies were examined.

In CC the typical histological findings are patchy throughout the colon but more present in the

right side of the colon. In this region the concentration of bile acids is greater, declining on

their way through the colon due to passive absorption. In the studies biopsies were taken from

the sigmoid colon, due mainly to practical reasons and to reduce discomfort for the patients.

To what extent results could differ between the right and sigmoid colon is not known but

should be considered.

Furthermore, to extrapolate findings derived from in-vitro experiments into the in-vivo

situation should be undertaken with caution. The complexity of the biological circumstance

can not be reproduced by the Ussing chamber, which has obvious limitations such as the lack

of circulation and nervous control, making viability crucial for the specimens. Nevertheless it

was found that colonic biopsies had good viability and could be used to study transmucosal

uptake of various molecules for 160 min with stable levels of ATP and lactate (Wallon, 2005).

Biopsies that did not fulfil the viability criteria (PD > -0.5 mV) at the beginning of the

experiment were excluded.

Statistics

In all papers the data were presented as mean/SEM, median and 25th-75th percentiles. As our

results in humans are not normally distributed we used non-parametric methods for the

permeability calculations. Comparisons between groups were initially done with Kruskal-

Wallis test and further analysed with the Mann-Whitney test. For the comparison of patients

before and after budesonide treatment, Wilcoxon`s-matched pairs signed rank test was

applied. Spearman`s test was used for correlation between histological findings and bacterial

uptake. The two-sided p-value <0.05 was considered significant.

42

43

5. RESULTS

Detailed descriptions of the results obtained are given in the respective papers. This section

will only highlight the main findings.

Paper 1 In paper I we described a patient with intractable collagenous colitis who was treated with a

temporary loop-ileostomy. She was followed clinically, histopathologically, and functionally

by measuring mucosal permeability before, with ileostomy, and after bowel reconstruction.

The changes in histological findings at different time-points are given in Table 2.

Table 2. Time schedule of sigmoid histology mean (range) of 3-5 counts.

Before surgery

2 months with diverting stoma

4 months with diverting stoma

7 months after bowel reconstruction

Epithelial cell degeneration (au 0-3)

2

1

0

1

Collagenous band thickness (µm) 30 (22-38) 25 (2-50) <10 <10

Intraepithelial lymphocytes (number per 100 enterocytes)

17 (13-23) 8 (5—11) 14 (11-17) 12 (9-16)

Density of inflammatory (mononuclear cells) in lamina propria (au 0-3)

2 1 1 2

au = arbitrary units no=0, slight=1, moderate=2 and heavy=3

In the Ussing chamber experiments, Cr-EDTA and HRP permeability was substantially

increased before surgery, when the patient had active colitis. Permeability decreased at 2

months and normalized after 4 months when compared with the control group. Seven months

after bowel reconstruction, colonic mucosa permeability increased again to a level above the

95th percentile for the controls (Fig.7). Electrophysiological measurements (Pd) were stable,

44

indicating viability of all specimens. The results indicate that faecal stream diversion leads not

only to histological remission but also restores barrier function.

Figure 7. Permeability of Cr-EDTA and HRP in the sigmoid colon of 19 healthy controls and

at different stages of disease in the patient with CC (3-5 biopsies of the sigmoid colon studied

at each time point). Permeability is expressed as the apparent permeability coefficient (Papp).

Bars indicate mean values with SEM. The dashed line indicates 95th percentile of controls.

Paper 2 In this paper Ussing chamber experiments were performed in biopsies taken from patients

with normal histology. Nine patients were included for electrophysiological measurement (Pd,

Isc and TER) and analysis for Cr-EDTA and HRP permeability while adding increasing

concentrations of bile acids. Bacterial uptake with addition of CDCA and DCA was

investigated separately in 8 patients, in these patients an analysis of bacterial effects on TER

was also carried out. A total of 204 biopsies were investigated and 14 were excluded because

of Pd above -0.5 mV indicating loss of viability.

Both bile acids caused increased bacterial uptake. Significant differences were present

between controls and 500 µmol/l (p=0.01) and 1000 µmol/l (p=0.04) CDCA. With DCA,

bacterial uptake increased significantly already with 100 µmol/l (p=0.03) (Fig.8).

45

CDCA DCA

% p

assa

ge

% p

assa

ge-1

0

1

2

3

4

5

6

Control 100 µM 500 µM 1000 µM-1

0

1

2

3

4

5

6

Control 100 µM 500 µM 1000 µM

* * * *

Figure 8: Uptake of E. coli bacteria during 120 min exposure to 0/100/500/1000 µmol/l CDCA and DCA. Values are given as % passage of mucosal concentration. * = p<0.05 compared to controls.

The results showed that E.coli K12 can per se decrease TER. When combining100 µmol/l of

CDCA or DCA with bacteria, we observed stronger effects on TER and especially CDCA

seems to augment this effect, though not reaching significance compared with control biopsies

(p=0.06) (Fig. 9).

Figure 9: Changes in transepithelial resistance (TER) during luminal exposure to E-

coli (bac) and/or 100 µmol/l CDCA (bile).

TER

-10

-8

-6

-4

-2

0

min

Ohm

cm2

No bac/No bile

No bac/100µM CDCA

bac/No bile

bac/100µM CDCA

0 30 60 90 120

46

When using confocal microscopy, fluorescent E.coli K12 bacteria were detectable in the

lamina propria after 15 min when the biopsies had been exposed to 1000 µmol/l CDCA or

DCA. E.coli bacteria adhered to the epithelium and were found to cross the cell layer mainly

via the paracellular route (Figs 10/11).

Figure 10: Confocal microscopy of colonic biopsies after exposure to 1000 µmol/l CDCA for 15 min. Overview showing fluorescent E.coli bacteria in the epithelium (fine arrow) and translocation of bacteria into lamina propria (thick arrow).

Figure 11: Magnified view showing adhesion and initiated paracellular uptake of fluorescent E.coli in colonic epithelium after 15 min exposure to 1000 µmol/l CDCA .

47

Paper 3

The main finding of the third paper was the significantly increased uptake of E.coli bacteria in

the Ussing chamber in all groups of CC patients, compared with controls. Active disease also

showed significantly increased uptake compared with patients in remission (p=0.03). After 6

weeks of budesonide treatment the passage of E.coli K12 decreased numerically, though not

significantly compared with active disease and the values did not normalize (Fig. 12).

Figure 12: Uptake of E.coli bacteria during 120 min in Ussing chamber. Comparison in

controls vs. patients with CC in remission or active disease, and after budesonide treatment.

Values are given as units and IQR. One unit denotes to 3 x 103 CFU/ml.

On commencing the experiments (time 0) TER in the active disease group was significantly

increased compared with controls; 47 (38-53) Ωcm2 versus 34 (27-37) Ωcm2 (p=0.005). After

mucosal exposure to E.coli K12, TER decreased significantly more in active disease

compared with remission and controls. TER did not change after budesonide treatment

(Fig.13).

p=0.004

p=0.03

p=0.001

p=0.006

48

Figure 13: Change in electrical resistance during 120 min exposure to E.coli K12 in Ussing

chamber. Comparison between controls and patients with CC in remission or active disease,

and after budesonide treatment.

After addition of E.coli K12 in the mucosal compartment, the change in short-circuit current

was significantly altered in active disease, compared with controls. The change in Isc (Δ 0-

120min) by E.coli stimulation normalized after budesonide treatment (Fig. 14).

Figure 14: Change in short-circuit current (Isc) during 120 min in Ussing chamber and after

adding of E.coli.

p=0.03

p=0.01

p=0.001

p=0.01

49

Paper 4

In paper 4 the biopsies of CC patients in all groups (remission, active disease and after

budesonide treatment) were stimulated with either 100 µmol/l CDCA or 100 µmol/l DCA.

The most interesting result is the 4-fold increase in E.coli uptake in biopsies of patients in

clinical remission, due to addition of bile acids. In patients with active disease, no further

increase in bacterial passage was induced by bile acids and this was also the case in

individuals undergoing budesonide treatment (Fig. 15).

Figure 15: Uptake of E.coli bacteria during 120 min in Ussing chamber with or without

adding 100 µmol/l CDCA or DCA to the mucosal side of colonic biopsies. Comparison in

controls and in groups of patients with CC in remission, with active disease and during

budesonide treatment. In remission, addition of bile acids increased bacterial uptake

significantly in colonic biopsies (♦ p=<0.01). In healthy controls 100 µmol/L DCA increased

bacterial passage (* p= <0.05). Values are given as units and range on a logarithmic scale.

One unit corresponds to 3x103 CFU/ml.

♦ ♦

*

50

51

6. DISCUSSION

During the last decade, numerous, mainly epidemiological studies have given us more insight

into collagenous colitis. We have learned that this diarrheal disorder is not as uncommon as

previously predicted and that the incidence rate lies between 5-6/100000 inhabitants making it

nearly as common as Crohn`s disease (Wickbom, 2009; Pardi, 2007). Furthermore, it has been

shown that patients with CC have poorer quality of life compared with a background

population (Hjortswang, 2005). Luckily, good treatment is available and budesonide has the

best documented efficacy for inducing and maintaining clinical and histological remission

with a “number needed to treat” (NNT) of 2 as published in a Cochrane meta-analysis

(Chande, 2009).

Despite the increasing interest in clinical studies on collagenous colitis, little experimental

research has been undertaken into the pathogenesis of this disorder.

In classical inflammatory bowel disease the cause of intestinal inflammation is apparently

multifactorial (Xavier, 2007; Baumgart, 2007). It is believed that an environmental factor

triggers an uncontrolled intestinal inflammatory response in a genetically predisposed

individual. Besides genes, disturbed innate immunity and environmental factors, the intestinal

mucosa as a barrier between the inner and outer environment plays a crucial role in IBD. The

“leaky gut” theory describes a dysfunctional barrier leading to increased mucosal permeability

of potential noxious agents (Hollander, 1999; Clayburgh, 2004; Xavier, 2007). The tight

junctions (TJs) constitute the rate-limiting components of the paracellular permeability

pathway.) TJs structures and function can be modulated by pro-inflammatory cytokines such

as IFN-γ and TNF-α, which increase in the mucosa of IBD patients (Bruewer, 2006).

In recent studies the normal gut flora or commensals, especially E.coli species with the ability

to adhere and invade the mucosa, have been focused on as being possible initiators of

intestinal inflammation (Rhodes, 2007; Barnich, 2007; Sartor, 2008).

Studies on intestinal permeability in humans have previously been carried out mainly with in

vivo techniques (Bjarnason, 1995). The Ussing chamber technique for in vitro studies of

epithelial function is well established for experiments on intestinal ion transport, drug

absorption, and permeability to variously sized marker molecules and antigens, mainly in the

gut mucosa of laboratory animals (Holtug, 1996; Albin; 2001). The modified Ussing chamber

52

which was used in our experiments allows investigation of endoscopic biopsies of humans

(Wallon, 2005). The obvious advantages with this technique are the opportunity for detailed

studies on epithelial function in samples from healthy individuals or patients before and after

treatment of gut disorders.

By this approach we could for the first time describe that CC has an underlying mucosal

barrier defect, irrespective of whether the patients were in clinical remission, had active

disease or were undergoing budesonide therapy. All patient groups had significantly increased

transmucosal passage of E.coli K12 compared with controls, and confocal microscopy

revealed that the bacteria apparently crossed the epithelium via the paracellular route.

Alterations of paracellular permeability was furthermore demonstrated by a significant

reduction of TER when adding E.coli K12 in the Ussing chamber.

Information on the expression of tight junction proteins in CC is limited. Bürgel et al. found

that the decreased epithelial resistance in CC was accompanied by diminished expression of

occludin and claudin 4, while claudin -1, claudin-3, and claudin 5 remained unaltered. Despite

that the exact physiological role of these integral membrane proteins are still unclear, the

authors conclude that these changes might represent the structural correlate for the epithelial

barrier defect (Bürgel, 2002).

CC is associated with a T helper cell type 1 mucosal cytokine profile with 60-100 times

greater mRNA levels for interferon gamma (IFN γ) and tumor necrosis factor alpha (TNFα)

compared to controls (Tagkalidis, 2007). In the same study CC was associated with reduced

staining of cell junction protein E-cadherin and even complete loss of ZO-1 staining in active

disease. These TH1 cytokines are known to have critical effects on barrier function and

especially in Crohn`s disease it has been established that TNFα causes an increase in intestinal

epithelial TJ permeability (Gibson, 2004; Ma, 2004).

In other experimental studies it has been speculated that eosinophil activation and vascular

endothelial growth factor (VEGF), a potent enhancer of vascular permeability, could be

mediators of mucosal permeability in CC. Taha et al. used a unique colonoscopy-based

perfusion technique and found increased levels of eosinophil cationic protein (ECP) and

VEGF in the perfusion fluid in distal segments of the colon. As ECP and VEGF levels

correlated with increased concentrations of albumin, which can be interpreted as a sign of

mucosal leakage, the authors concluded that activated eosinophils and VEGF could alter

53

mucosal permeability (Taha, 2001; Taha, 2004). Activation of eosinophils in CC was also

confirmed in a recent study (Wagner, 2009). Furthermore, the epithelium of CC patients

shows strong immunostaining of VEGF and it has been suggested that VEGF might have an

important role in counteracting the local imbalance of fibrogenesis and fibrolysis, leading to

an accumulation of immature subepithelial matrix in CC (Griga, 2003). The thickened

subepithelial collagenous band has been described as a significant diffusion barrier, making

up one third of the resistance of the epithelium, thus reducing absorptive efficiency of ions

(Bürgel, 2002).

In our study histology had not changed at all after 6 weeks budesonide treatment whereas

other studies have reported a reduction in thickness of the collagenous layer and decrease of

inflammation grade (Baert, 2002; Bonderup, 2003; Miehlke, 2002). These differences could

have been due to differing treatment duration (6 or 8 weeks) and dose regimes (without

tapering). Another study of CC patients found that the degree of inflammation in lamina

propria could be used to predict response to medical treatment; the more intense the

inflammation, the more likely it was that the patient needed anti-inflammatory therapy (Abdo,

2002). On the other hand yet another prior study failed to show any correlation between the

thickness of the collagenous layer and stool frequency (Wang, 1987). In our studies we found

no correlation between grade of histological alterations and transmucosal bacterial uptake.

Inspired by the study by Järnerot et al., describing how faecal stream diversion leads to

induction of clinical and histopathological remission in CC, we investigated histology and

mucosal permeability in a single patient with CC before operation, with loop-ileostomy, and

after bowel reconstruction. Our findings corroborated Järnerots findings and in the period

with a diverting stoma, repeated biopsies of the sigmoid colon showed that the transcellular

and paracellular permeability decreased at the same time as the intestinal inflammation abated

and the thickened sub-epithelial collagenous layer and the epithelial degeneration

disappeared. After bowel reconstruction, however intestinal permeability increased prior to

the appearance of the thickened collagenous layer. These findings suggest that the disturbed

mucosal barrier function is triggered by a hitherto unknown noxious luminal factor and the

increased mucosal permeability precedes the histological changes in CC.

54

Studies by Ung et al. found an association between CC and bile acid malabsorption measured

with SeHCAT (Ung, 2000). A pathological outcome in the SeHCAT implies greater losses of

bile acids via the colon. Faecal concentrations of bile acids in CC patients have not been

analysed, but effective treatment with bile acid resins such as cholestyramine suggests that

faecal bile acids contribute to patient’s symptoms. Bile acids are known to induce diarrhea by

increasing Cl- secretion (Potter, 1998). In animal studies it could be shown that bile acids with

two hydroxyl groups in the alpha configuration (CDCA and DCA) in concentrations between

1-8 mM gave a dose-related increase of paracellular mucosal permeability and damaged the

mucosa as assessed by light and electron microscopy (Chadwick, 1979; Camilleri, 1980;

Goerg, 1982). Little is known however, about the effect of µM concentrations of bile acids on

mucosal permeability in the human colon. Therefore we first wanted to investigate the

functional effect of bile acids on colonic biopsy material from healthy individuals in the

Ussing chamber and speculated that they might impair barrier function. Hereby we found that

concentrations of CDCA and DCA below 1mM increased paracellular mucosal permeability

and enhanced bacterial uptake in normal human colon biopsies. However these results have to

be interpreted with caution as the Ussing chamber experiment cannot simulate the complex in

vivo situation in all aspects. The luminal content with variable pH and calcium concentrations

can influence the toxicity of bile acids (Rafter, 1991). Furthermore it is uncertain how mucus

production, as a cell surface protection, is affected in biopsies that lack nervous stimulation

and blood circulation. However, stable epithelial PD values during all our experiments

contradicted cytotoxicity after addition of 100µmol/l CDCA or DCA.

Hamilton et al. (2007) were the first to demonstrate that the total caecal concentrations of 3-

hydroxy bile acids in humans were 0.6mM ± 0.3mM. As CDCA constitutes 7±8% and DCA

34±16% of the total bile acid composition, a concentration of 100 µmol/l CDCA and DCA

lies within the physiological range. We therefore continued our Ussing chamber experiments

with only 100 µmol/l CDCA and DCA, looking at the effects bile acids might have on barrier

function in CC.

The major finding of paper IV is that physiological concentrations of bile acids increase E.coli

passage in biopsies of patients in clinical remission by 4-fold, which is highly significant. In

experimental studies in Caco-2 monolayers it could be demonstrated that tight junction

structures are modulated by µM concentrations of bile acids via epithelial growth factor

receptor (EGFR) activation (Raimondi, 2008) or generation of reactive oxygen species (Araki,

2005). Furthermore it has been demonstrated that non-pathogenic E.coli strains decrease TER

55

and alter localization of claudin-1 in T84 cells, signifying tight junction modulation (Zareie,

2005). The combination of bile acids and bacteria seems to have additional effects on

paracellular permeability (TER), as shown in paper II, and the question arises if bile acids in

concentrations found in the colon contribute to the uptake of commensal bacteria through the

mucosa in vivo. Taken together, these findings give rise to the hypothesis that faecal bile acids

in CC patients may be of pathogenic importance by affecting the mucosal barrier which could

initiate intestinal inflammation and lead to a manifest clinical relapse. Bile acid binders are

known to ameliorate diarrheal symptoms in CC, but it has not yet been investigated whether

this medication can keep patients in remission, reduce histological signs of intestinal

inflammation and improve mucosal barrier function in controlled trials. Furthermore, it could

be of interest to study the potential effects of bile acids on the mucosal barrier function in

classical IBD.

On the other hand, in active disease where bacterial uptake is already significantly increased,

no further augmentation arises with bile acid stimulation. This could imply that structures of

the mucosal barrier might already be rearranged in active disease in such degree that no

further impairment is induced by bile acids. Moreover, patients in clinical remission during

budesonide treatment had no increased passage after adding bile acids, suggesting that

steroids have protective properties on bile acid induced mucosal impairment and improve

barrier function.

Budesonide has the best documented efficacy for inducing and maintaining remission in CC

and all studies have in common that budesonide reduces watery stools significantly and this

effect can be seen quite rapidly, within days (Chande, 2009). However, the clinical course of

CC is mainly chronic relapsing and the successful induction therapy with budesonide is

compromised by a high relapse rate in 61% of patients after 2 weeks and in 88% after 3

months (Miehlke, 2005). Relapse risk remains high even after 6 months of maintenance

treatment with budesonide (Bonderup, 2009). The mechanisms underlying frequent relapses

in CC have not yet been clarified.

In general, diarrhea is driven by osmotic forces including malabsorption, active secretion and

altered ion flux. Bürgel et al. showed that the diarrheal mechanism in CC relies predominantly

on a reduced net Na+ and Cl- absorption and is accompanied by a minor component of active

chloride secretion (Bürgel, 2002). In our study the short-circuit current (Isc) was significantly

56

reduced in patients with active disease, possibly due to reduced Na+ absorption, thus

corroborating Bürgels findings. Furthermore Sandle et al. previously found that inflamed but

structurally intact human colonic tissue exhibits only a moderate degree of electrogenic

sodium transport (Sandle, 1990). In inflamed tissue, T cell derived mucosal cytokines

mediated inactivation of Na+/K+ ATPase in mice (Musch, 2002) and in CC a Th1 cytokine

profile is predominant (Tagkalidis, 2007). Steroids are known to stimulate Na+ and fluid

absorption in the colon (Sellin, 1985) and a more recent study showed that a possible

mechanism for this could be a steroid-induced up-regulation of epithelial sodium channels

(ENaC) (Zeissig, 2007). In our study, budesonide normalized the alterations in Isc, which

were induced by E.coli K12 in biopsies from active CC patients. These data demonstrate that

budesonide exerts a primarily direct and substantial anti-diarrheal effect, while its anti-

inflammatory properties, reflected by improvement of the histological picture, appears more

gradually.

Concerning effects of steroids on barrier function it has been demonstrated that the induction

of remission in Crohn`s disease with corticosteroids is associated with an improvement in

intestinal permeability as measured by the lactulose/mannitol ratio in vivo (Wild, 2003).

Although it is well established that steroids have positive effects on intestinal permeability it

is still unclear whether this is a result of their anti-inflammatory properties, including the

ability to inhibit the expression of proinflammatory cytokines such as TNFα (Barnes, 1998) or

due to a direct TJ barrier “tightening” effect of glucocorticoids. To address this question,

Boivin et al. looked at mechanism of glucocorticoid regulation of the intestinal tight junction

barrier and found that steroids inhibit the TNFα –induced increase in myosin light chain

kinase (MLCK) protein expression, a key process mediating tight junction permeability

(Boivin, 2007).

Despite favourable results of short-term budesonide treatment our findings suggest that

increased bacterial uptake may be a genuine phenomenon in patients with CC that does not

normalize after effective clinical treatment with budesonide and one may speculate that this

ongoing barrier dysfunction has pathogenetic significance in explaining the development and

recurrence of CC.

57

7. CONCLUSIONS

- Collagenous colitis is associated with increased paracellular and transcellular

permeability, which normalizes following faecal stream diversion. Mucosal

permeability was altered prior to reappearance of the thickened collagenous layer after

restoration of bowel continuity.

- Collagenous colitis is associated with increased mucosal bacterial passage irrespective

of disease activity.

- Active CC is associated with altered tight junction reactivity.

- Dihydroxy bile acids alter gut barrier function, causing increased antigen and bacterial

uptake in normal human colonic biopsies.

- Physiological concentrations of bile acids exacerbate barrier dysfunction in colonic

biopsies from CC patients in clinical remission.

- Budesonide gives short-term clinical relief but does not restore the gut barrier

function.

- Budesonide appears to counteract the bile acid-induced mucosal impairment.

- It can be speculated that faecal bile might exacerbate and perpetuate mucosal

inflammation in CC.

58

59

8. SVENSK SAMMANFATTNING Bakgrund: Kollagen kolit (KK) är en diarrésjukdom med en incidens på 5-6/105 invånare och drabbar mest medelålders kvinnor. Diagnosen ställs histologiskt. De klassiska tecknen på kollagen kolit är ett förtjockat subepitelialt kollagenband och en kronisk inflammation i lamina proria. Vid inflammatorisk tarm sjukdom är slemhinnans barriärfunktion störd. Därför är barriärfunktionen vid KK intressant att kartlägga. Orsaken till KK är oklar men det finns en association med gallsaltmalabsorption. Det är känt att ökad mängd gallsalter i tjocktarmen leder till diarré. I funktionella studier undersöktes hur gallsalterna påverkar den mukosala permeabiliteten hos friska försökspersoner och hos patienter med KK. Metod och patienter: I det första arbetet undersöktes en kvinnlig patient med uttalad KK innan operation, efter loop-ileostomi and vid tarm rekonstruktion. I de andra studierna inkluderades sammanlagd 25 patienter med KK (20 kvinnor, 5 män, genomsnittlig ålder 66 år). De delades in i tre grupper: 14 patienter i klinik remission, 11 med aktiv sjukdom (8 av dessa igen med budesonid behandling) och 17 friska kontrollpersoner med normal histologi. Endoskopiska vävnadsprover från sigmoideum monterades i en modifierad Ussing kammare och undersöktes med elektrofysiologiska parametrar (kortslutningsström Isc, transepitelial resistans TER) och transmukosal passage av avdödade E.coli K12 bakterier efter tillägg av chenodeoxychol syra (CDCA) och deoxychol syra (DCA) i olika koncentrationer. Vävnadsproverna undersöktes också med konfokal mikroskopi för att studera passagväg genom slemhinnan. Resultat: I fallbeskrivningen visar aktiv sjukdomen tecken på ökad paracellular och transcellular permeabilitet men detta normalisera vid urkoppling av tarminnehåll via stomin. Efter återkoppling ökar den mukosala permeabiliteten för makromolekyler igen. Vid KK syns ett signifikant ökat upptag av bakterier både i remission och aktivitet samt under budesonid behandling jämfört med kontroller. Trots signifikant förbättrade symtom under budesonid behandling normalisera inte den ökade passagen av bakterier. Histologin är oförändrad efter 6 veckors behandling med budesonid. DCA påverkar den mukosala permeabiliteten dosberoende och redan 100µmol/l DCA ökar det bakteriella upptaget signifikant i biopsierna från friska försökspersoner. Kombinationen av gallsalter och E.coli K12 verkar ha additiva effekter på TER. 100µmol/l CDCA och DCA leder till en 4 faldig ökning av bakteriepassagen hos patienter i klinisk remission men har ingen effekt på biopsier från patienter med aktiv sjukdom. Ytterligare visar det sig att gallsalterna inte har någon inverkan på bakteriers upptag i biopsier från patienter som står på budesonid behandling. Slutsats: KK har en ökad permeabilitet för makromolekyler och bakterier oberoende av sjukdomsaktivitet eller budesonid behandling som ett tecken på en underliggande barriärstörning i slemhinnan. Att koppla ur tarmen via en stomi resulterar i normalisering av barriärfunktionen men så är inte fallet vid budesonid behandling trots dess goda effekt att minska symtomen. Gallsalter i fysiologiska koncentrationer har potentialen att öka bakteriers upptag framförallt i biopsier från patienter i klinisk remission. Budesonid behandling tycks motverka gallsalternas inverkan på slemhinnan. Dessa negativa effekter av gallsalter och inverkan på den mukosala barriär funktionen kan möjligtvis initiera och upprätthålla den intestinala inflammationen vid KK.

60

61

9. ACKNOWLEDGEMENTS Magnus Ström: My chief tutor! Your door was always open for me. You have been a father-

figure and role model, taking every opportunity to discuss all aspects of research and even

other matters of life. Your enthusiasm and thoroughness was inspiring.

Johan D Söderholm: My co-tutor, the rising star in barrier function research. Thank you for

introducing me to the Ussing chamber and our laboratory. It is a pleasure to work with you.

Henrik Hjortswang: My chief. Thank you for maintaining an academic atmosphere at our

Department and supporting me on my way as a PhD student and doctor. You have a great

leadership style and allow everybody to grow and develop their abilities.

Susanna Walter: Du hast mich nach Schweden geholt. Dafür sei Dir für immer gedankt. Du

bist viel mehr als eine gute Kollegin. Ich wünsche Dir viel Erfolg beim Forschen und Freude

mit der Familie. Danke für die kritische Durchsicht meines Manuskripts.

Ylva Braaf, Anders Carlsson: Thank you for your technical assistance in the lab and helping

me to understand the Ussing chamber.

Conny Wallon: Thank you for your help in the first paper and your thesis on the modified

Ussing chamber, making this method reliable and more widely accepted.

Sa`ad Salim: Your help was essential in providing good pictures with the confocal

microscopy. I wish you all the best for your future research.

Karl-Eric Magnusson: Thank you for your collaboration and help with the confocal

microscopy.

Alan Hoffmann: Mr bile acids! A real gentleman who took time to talk to me, leaving an

unforgettable impression.

62

Kjell Arne Ung: Your thesis and interest in bile acids in collagenous colitis has been crucial

for my work.

My colleagues Sven Almer, Stergios Kechagias, Rikard Svernlöv, Tilman Weisselberg,

Mathias Ekstedt, Mats Carlson, Georgios Katinius: You make work worthwhile and

joyful. Not a day has passed when I felt regret. I thank you all for our discussions and feel

proud to be part of the team.

Ann and Lotta: My ladies in the Endoscopy Department. With patience, humour and

understanding have you helped me to organize all examinations and collection of biopsies.

Britt, Berit, Lotta, Eva, Helen: Thank you for helping me with the administration, contact

with patient and for creating such a friendly atmosphere at the open ward department.

Members of the “MC klubb”: Thank you for sharing our knowledge on microscopic colitis.

I feel privileged in joining the expert team and having the opportunity to discuss research.

Mathias Haarhaus: Mein Freund und Wegbegleiter in allen Höhen und Tiefen. Wir kennen

uns durch und durch. Möge deine Forschung Dir Freude bringen und deine Familie Glück im

Leben.

Meine Kommilitonen an der Universität Witten/Herdecke: Voller Dankbarkeit blicke ich

zurück auf unser Medizinstudium. Es war ein Privileg mit und an Euch wachsen zu dürfen.

Papa: Ich danke für all deine Unterstützung und Großzügigkeit mit der Du mich auf meinem

Weg begleitet hast. Mit Respekt begegnen wir uns von Mann zu Mann.

Mama: Du hast immer an mich geglaubt und mich unterstütz. Ich weiß Deine lieben

Gedanken bei mir.

Christiane: Meine Schwester. Wir werden uns am längsten im Leben begleiten. Ich wünsche

Dir Freude in der Malerei und Glück in der Liebe.

Meine Kinder Amina, Elias, Leon und Leander: Ihr seid die wahre Freude in meinem

Leben. Euch werde ich immer in meinem Herzen tragen.

63

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