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CHAPTER 1 GENERAL INTRODUCTION

Abstract 1.1 1.2 1.3 1.4 Disease definition Disease characteristics Epidemiology Aetiology 1.4.1 Environmental factors 1.4.2 Immunologic factors 1.4.3 Gender 1.4.4 Genetic factors 1.5 Susceptibility genes for Inflammatory Bowel Diseases 1.5.1 Candidate Gene Association Studies 1.5.2 Genome-wide scanning and linkage studies 1.6 Pharmacogenetics

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Abstract
The aetiology and pathogenesis of the two main types of inflammatory bowel diseases, Crohns disease and ulcerative colitis, are not yet fully known. Several environmental, microbial, immunologic, genetic and lifestyle factors have been suggested to play a role in the initiation of the disease. A feasible concept is that in genetically susceptible individuals a viral infection, respectively bacterial or dietary antigens would induce an abnormal, but nonspecific mucosal immune response. This mucosal inflammation causing local and systemic changes could again induce an increased sensitivity to other antigens and physiologic and nonphysiologic factors such as smoking or oral contraceptives. As impaired immunologic functions play a central role in the inflammation of the gastrointestinal tract and the extraintestinal manifestations seen in IBD, genetic studies largely focus on genes coding for proteins having a regulatory role in the mucosal immunity. However, to date no overall and uniform agents responsible for the initiation of the disease in any of the entities could be identified. Both Crohns disease and ulcerative colitis are very likely polygenic and multifactorial diseases. Data is accumulating that these inconsistencies are partly due to the phenomenon that both diseases consist of more characteristic subgroups. Furthermore, there are indicators suggesting that the genetic background of these subgroups is different, which could also imply differences in the pathogenic mechanisms and different aetiological agents. The distinct prevalence of CD and UC in different ethnicities, colours the picture further. Therefore, there is a necessity of using welldefined study groups when analysing disease course, extraintestinal manifestations, genetic background, pathologic factors or therapeutic effects. Recently a new userfriendly classification, the Vienna classification of Crohns disease has been introduced. For ulcerative colitis a classification by disease extension, age of onset, and continous or relapsing course seems approriate. The application of these categories helps to a better understanding of factors influencing the disease pattern, such as the role of gender. Similarly to other diseases where the immune response has a pivotal role, data is accumulating that the disease susceptibility is different in men and women. These differences might include distinct disease course or diverse frequency and severity of certain extraintestinal manifestations. As an example, men were found to have at greater risk for bone loss in IBD. The reason for this is not yet known, consideration have to given to genetic, hormonal and maybe lifestyle dissimilarities. On the whole, the pathology of Crohns disease and ulcerative colitis is complex, the disease itself can manifest divergently. Studies using well defined homogenous patients groups may help to clarify the correlation between the disease presentation and its genetic background. In this chapter a review of the current knowledge on the epidemiology, aetiology, clinical and immunogenetic characteristics of inflammatory bowel diseases is described.
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GENERAL INTRODUCTION 1.1 Disease definition


Inflammatory bowel disease (IBD) is a term for a group of chronic inflammatory disorders of unknown origin involving various sites of the gastrointestinal (GI) tract. IBD may be divided into two major groups based on differences in the disease pattern: chronic non-specific ulcerative colitis (UC) and Crohn's disease (CD). Crohn's disease earlier also referred to as regional enteritis, granulomatous ileitis or ileocolitis is a non-specific chronic transmural inflammatory disease that most commonly affects the distal ileum and colon but may occur in any part of the GI tract. The first description of the disease was published in 1913 from Dalziel (1). Crohn, Ginzberg, and Oppenheimer re-discovered the disease and identified ileitis terminalis in 1932 as a distinct disease entity (2), whereas Lockhart-Mummery and Morson evidenced the existence of Crohn colitis in 1960 (3). The other most common disease entity, ulcerative colitis is a chronic, inflammatory and ulcerative disease arising in the colonic mucosa, characterised most often by bloody diarrhoea. Bargen has described already in 1924 the clinical and experimental characteristics of the disease (4), and he and his co-workers published a review in 1950 on the disease, based on the clinical history of 2000 patients (5). Early in the course of the inflammatory bowel diseases endoscopic examination and histological evaluation from biopsy specimens are the most effective techniques for differentiating the two disease entities, whereas radiological evaluation of the large and the small bowel could provide further essential information in the diagnosis of IBD. Crohn's disease is characterised by chronic inflammation extending through all layers of the intestinal wall usually involving the mesentery as well as the regional lymph nodes. The typical microscopical findings are fibrosis, cryptitis, crypt abscesses and focal aphthous ulcers located over nodules of lymphoid tissue. The most helpful histological feature distinguishing CD from other forms of IBD are non-caseating granulomas with multinucleated giant cells. However, in the histological slides granulomas can be absent in up to 50% of the patients. Other routine diagnostic techniques are upper and lower endoscopy. Endoscopic examinations typically show segments of diseased bowel sharply demarcated from the adjacent skip areas with a macroscopically normal bowel. The appearance of the mucosa depends on the severity and stage of the disease, but in contrast to the mucosa seen in UC it is often relatively normal. Typical picture shows aphtous and pinpoint lesions, whereas the cobblestone pattern is characteristic presentation in more advanced cases in the diseased section of bowel. This phenomenon appears due to the submucosal inflammation that causes
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a coarse irregularity of the mucosal surface, as well as due to the presence of patchy, longitudinal or transverse ulcers snail trails with intervening mucosal oedema. The consequent irregular thickening and fibrosis may lead to stricture formation. Withal deep sinus tracts, fistulas, strictures and pseudopolyps are also common features seen in Crohn's disease. In CD patients with upper gastrointestinal symptoms upper GI is necessary as the radiological findings mucosal stiffening and infiltration might mimic an infiltrative tumour. Colonic Crohn's disease usually presents typical changing that discriminate it from ulcerative colitis. These features comprise rectal sparing, the presence of fistulas, skip lesions and small ulcerations occurring on irregular nodules that often extend into longitudinal ulcers and transverse fissures. The role of the traditional x-ray investigations is restricted in the diagnosis of CD. However, air double-contrast barium enema and enteroclysis can be essential, especially when confirming an ileal and/or jejuneal involvement. In the presence of small bowel disease superficial aphthous and linear ulcers are seen. In more severe cases a "string sign" may be found this indicates marked ileal strictures and a separation of bowel loops. Traditional barium enema x-ray can also reveal characteristic changes due to a reflux of barium into the terminal ileum. The picture might show irregularity, nodularity, stiffness and thickening of the bowel wall. Ultrasound techniques and computed tomography (CT) are most often used to detect fistulas and extramural complications such, abscesses, inflammatory masses and could help to distinguish thickened matted loops, while leukocyte scintigraphy may further assist in the differential diagnosis, especially in active disease. Although a diagnosis of IBD as well as the specific disease entity generally precedes surgical interventions, an operation can also provide information on the disease. In the abdomen of CD patients thickened and lymphedematous mesentery may be seen. Mesenteric fat usually extends onto the serosal surface of the bowel, while mesenteric lymph nodes often enlarge. Ulcerative colitis is a chronic inflammatory disease of the colon, characterised by continuous localisation and by superficial ulcerations of the mucosa. Histological findings are marked by neutrophilic infiltration of the submucosa, the surface mucosal cells and most characteristically by crypt abscesses formed by neutrophilic infiltration of the crypt epithelium. Chronic inflammation results in a loss of surface epithelial cells, incidentally also in a loss of crypt epithelium and goblet cells. Repetitive cycles of inflammation are marked by distorted crypt architecture, crypt atrophy and mild submucosal fibrosis. In long-standing UC the surface epithelium may present with premalignant changes such as dysplasia. Submucosal oedema is characteristic, however unlike in Crohn's disease, deeper layers of the bowel beneath the submucosa are usually not involved. Total colonoscopy is the most sensitive and widely used method in the diagnosis of UC. that is also helpful in determining the severity and extent of disease. The
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endoscopic picture is characterised by a hyperaemic, friable, ulcerated, and usually haemorrhagic colonic mucosa. The bowel inflammation is uniform and continuous, extends proximally, usually involving the rectum and sometimes a few centimetres of the terminal ileum backwash ileitis. Findings often include an exudate consisting of mucus, blood, and pus. Some degree of friability or granularity usually persists during asymptomatic intervals as well. Recurrent inflammation may lead to the loss of the normal haustral pattern and the formation of inflammatory pseudopolyps. Due to the chronic inflammation fibrosis and longitudinal retraction might develop resulting in shortening of the colon. Thus the bowel has a symmetric, ahaustral, tubular appearance with a decreased mucosal and lost of vascular pattern. The typical picture of an air-contrast barium enema examination X-ray shows irregular bowel contours, ulcerations and polypoid defects. In the chronic stage of the disease further characteristic features can be appealing: the shortening of the bowel, depression of the flexures, narrowing of the bowel lumen, and rigidity.

1.2 Disease characteristics


Disease course and localisation The disease course of both Crohn's disease and ulcerative colitis vary. There are large individual differences and changes in the disease severity, presentation and activity. Both UC and CD are chronic diseases usually characterised by repeated periods of remission with only minimal symptoms and exacerbations of the disease with sometimes need of hospital admission. Ulcerative colitis typically presents with abdominal pain, bloody diarrhoea, and often fever and weight loss. The number of stools in a day and the quantity of blood and pus in the stool usually characterise the disease severity well. Thus in the milder form of UC there may be one or two semiformed stools per day, containing little blood, and probably no pus. In contrast, in the severe form of the disease patients could have even up to 15-20 times diarrhoeal stool in a day, containing blood and usually pus. Somewhat contrary to the extended forms of UC the most common complaint of patients with ulcerative proctitis is tenesmus. These patients generally present with constipation and not diarrhoea, often have rectal bleeding or discharge pus and/or mucus also independently from stool. Most patients with ulcerative colitis will have a relapse within 1 year of the first attack. In contrast to Crohn's disease, bowel inflammation is contiguous and usually gives a good idea on the severity of disease. The inflammation almost always involves the rectum, but does not exceed the colon. Ulcerative colitis is localised to the rectum in 40-50 % of the patients, while an inflammation of the procto-sigmoid colon is found in about 60-70 % of the cases. The clinically more severe forms of UC usually involve the left colon side (10-20%), colon transversum (5-10%) or the whole colon pancolitis (5-10%).

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As UC generally starts in the rectum, the definition of disease localisation and that of ulcerative proctitis the form of UC not exceeding the rectum should not be definitely set until it was followed for a minimum of 6 months. However, even after a half year may ulcerative proctitis in 20-30 percent of the cases extend proximally. Patients with localised ulcerative proctitis have the best prognosis, although ulcerative proctitis and those cases undergoing late proximal spread are frequently therapy resistant. In ulcerative proctitis the disease is usually mild, with minimal systemic or extracolonic manifestations. Severe systemic manifestations, toxic complications, and malignant degeneration are unlikely, surgery is rarely required, and life expectancy is normal. Generally seen the extent of ulcerative colitis at the diagnosis well predicts the disease severity. More extensive disease localisation is generally associated with more severe disease course and more intestinal complications respectively systemic manifestations. Thus on the whole nearly third of UC patients with extensive colitis will require surgery, while this number is lower int those with nonextensive disease. The most common clinical features of Crohn's disease are fever, abdominal pain, diarrhoea, and generalised fatigability and weight loss. Diarrhoea is often moderate; rectal bleeding and discharge of mucus and pus are far less frequent than in ulcerative colitis. Similarly to UC, Crohn's disease is characterised by intermittent exacerbations intervened by spontaneous or medical therapy induced remissions. Approximately 70-100 percent of CD patients ultimately require surgery. However, in contrast to UC operative intervention is not curative in CD; the disease is likely to recur after the resection of the whole involved bowel section. Furthermore, while normally the disease never extends into new areas of small bowel beyond its initial distribution at first diagnosis, after a surgical intervention it does often affect areas neighbouring the resected part. Crohn's disease can involve any part of the gastrointestinal tract. The frequency of specific disease localisations in CD varies in distinct studies. Most investigations reporting on the distribution of CD find 30-35% of cases involving only the small intestine (usually the terminal ileum); about 40-45% has ileocolonic involvement with a predilection for the right side of the colon and the terminal ileum; and about 20-30% comprising exclusively the colon. Involvement of the stomach, duodenum, oesophagus, buccal mucus or gingiva is present altogether in about 25% of the CD patients. Some investigators suggest that Crohn's disease of the perianal region is a distinct disease entity. Perianal region is found to be affected in about 25-33% of all CD cases, although in approximately 20-50% of CD patients with colonic localisation the rectum is spared. Patients' complaints and the clinical presentation often reflect the anatomic localisation of Crohn's disease. Furthermore localisation might also predict which complication develops (6)
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On the other hand there is also some data that the presence of extraintestinal symptoms or that of perianal complications may indicate a higher risks of relapse, thus a possibly more severe course of disease (7, 8) It has also been found that the necessity for a second surgery is higher among those patients presenting previously with fistulas and/or perforation (9). Complications The complications of IBD may be classified as local or systemic, the former being direct consequences of the inflammation or its extension. Local complications include fistulas, fissures, abscesses, strictures, bleeding, perforation, toxic dilation and in a long-standing disease the development of carcinoma. Although these complications might occur in both entities of IBD, there are characteristic differences between the typical local complications of two diseases. In ulcerative colitis one of the most common local complications is bleeding, however its extent largely varies and intractable bleeding requireing surgery is rare. Another characteristic and severe complication is toxic megacolon. The localised ileus, dilatation, peritonitis and subsequent perforation are consequences of a thin bowel wall denuded of mucosa, the transmural extension of the ulceration, and an inflammation extended to the serosa. Among the severe complications transmigration peritonitides, perforation peritonitides and toxic megacolon arising in approximately 23% of the patients are the most frequent. Therapy-resistant intestinal haemorrhages are infrequent, evolving in about 1% of the patients. Ulcerative colitis usually does not present with fistulas or with major perirectal complications (10-12). The serosal inflammation in Crohn's disease might lead to an adherence of distinct small intestinal loops most often in the right lower quadrant, forming a palpable mass. Further characteristic complications in CD fistula and abscess formation are consequences of the transmural inflammatory process that involves serosa and mesentery. Fistulas may appear between adherent structures including loops of intestine or colon as well as organs such as bladder or vagina. They may either lead to the skin or end blindly in the peritoneum or retroperitoneum. In a significant number of patients, perirectal disease often referred to as a distinct type of disease within CD might be present. The recurrent inflammation might result in scarring perirectal disease that is characterised by rectal fissures and fistulas and perirectal abscesses. Due to the thickened and leathery bowel walls stenosis with some degree of intestinal obstruction is a frequent complication that occurs in 20-30% of patients during the course of the disease. Since in CD the bowel wall is greatly thickened, free intestinal perforation is infrequent, and dilatation of the colon is less common than in UC. Likewise, rectal and intestinal bleeding is not as typical as in UC and when present, often indicates the coexistence of rectal involvement. The most frequent severe complications in CD are the acute or chronic mechanical ileus presents in about 20% of the patients (10).
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The incidence of colon carcinoma is higher in inflammatory bowel diseases than in the general population of the same ethnicity. The cumulative risk of cancer begins to rise 10 years after the disease is diagnosed, with an overall incidence of 0.5 to 1 percent per year. The incidence of cancer varies among patient populations, thus UC patients with a disease limited to the rectum have no increased risk of cancer, while patients with an extensive mucosal involvement have the highest risk. Earlier studies have found that the incidence of bowel malignancy is lower in CD than in UC (13, 14), however recently it is suggested that the long-term risk of colorectal cancer in CD patients with a colonic location is equal to that of UC patients with the same extension (15). Furthermore, CD patients with a longstanding disease of the small bowel are also at an increased risk of small bowel cancer (13). Extraintestinal manifestations Several extraintestinal symptoms and signs that occur both in CD and UC can be associated with IBD. Their exact aetiology is not known. Extraintestinal manifestations can be categorised as: a.) Complications that usually parallel the activity of the intestinal disease; b.) Extraintestinal manifestations running an independent course; and c.) Complications that relate directly to the disrupted bowel physiology. Complications that usually parallel the activity of the intestinal disease possibly represent acute immunologic or microbiologic concomitants of the bowel inflammation (16-18). According to some data these manifestations can be present in up to 5060% of all IBD patients, and are usually more common when disease is localised to the small bowel (19-21). However, there are also some manifestations that are more frequent with colonic involvement. Peripheral monoor polyarticular and often-migratory arthritis and arthralgia are typical examples for this (22). Further extraintestinal features occurring most commonly in active disease are episcleritis, iritis, aphthous stomatitis, erythema nodosum, and pyoderma gangrenosum. This parallelism could explain the improvement of the CD associated skin diseases pyoderma gangrenosum and psoriasis of patients treated with infliximab (23). Hepatosteatosis a condition that usually also resolves with the remission of IBD is probably caused by malnutrition and a concomitant steroid therapy. Extraintestinal manifestations being often present in IBD patients, but running an independent course are mainly disorders with a genetic predisposing factor. Uveitis and primary sclerosing cholangitis (PSC) belong to this group as well as ankylosing spondylitis and sacroiliitis; these latter being strongly associated with HLA-B27, whether or not IBD is present. The course of pseudotumorous chronic pancreatitis lately considered as ulcerative colitis associated extraintestinal manifestation is probably also independent from the activity of IBD (24). Among the extraintestinal manifestations discussed so far arthritis and arthralgia are most commonly detected, however their origin being a side effect of steroids, are age associated changes or truly IBD associated
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alterations is difficult to clear. Other frequently seen disorders such as primary sclerosing cholangitis, ankylosing spondylitis, iritis/uveitis, pyoderma gangrenosum, and erythema nodosum are present in approximately 6% of the patients. There seems to be both a gender and an IBD entity affinity for these features. A recent study suggests that iritis/uveitis and erythema nodosum is somewhat more frequent in women, while PSC and ankylosing spondylitis in men. Simultaneously, iritis/uveitis and PSC are more common in UC, while ankylosing spondylitis and pyoderma gangrenosum in CD (25). Extraintestinal manifestations categorised in the third group are classified as being directly related to disrupted bowel physiology, however their exact aetiology is often not known, and an influence of other affecting factors is presumptive. As an example, so far no distinct factor related to IBD is found that could be consequently associated with the quantity of bone loss in these patients. Other extraintestinal complications correlated with disrupted bowel physiology are kidney stones. Their development is explained by disorders of uric acid metabolism, impaired urinary dilution and alkalinization, dehydration, and an excessive dietary oxalate absorption. Further complications found in IBD patients include malabsorption, thus gallstones related to impaired ileal reabsorption of bile salts in extensive ileal disease. As in many other long-standing inflammatory and suppurative diseases secondary amyloidosis can evolve in IBD, preferably in CD (26). As for haematological complications hypercoagulability and thromboembolics may occur most often in patients with severe disease activity. Altered levels of clotting factors and platelet abnormalities explain this phenomenon. Liver diseases linked to UC or CD are not unusual either. Thus cholangiocarcinoma has an increased incidence in patients with chronic UC, especially in patients with sclerosing cholangitis, an autoimmune disease with a higher frequency in the UC than in the normal population. Autoimmune chronic active hepatitis and pericholangitis may also be found in IBD, and both diseases might progress to cirrhosis (16). Actually the most common complications that relate directly to disrupted bowel physiology in IBD are rather unspecific ones. Among those are weight loss and malabsorptions syndrome this latter being especially characteristic in those CD patients with diffuse ileal disease or after ileal resection. In addition in the distinct group of childhood IBD growth retardation might be observed. Among the several extraintestinal manifestations and diseases that often accompany IBD, in this study we had a closer look on the reduced bone mass of UC and CD patients. Osteoporosis Osteoporosis and osteopenia occur when bone resorption exceed bone formation. Osteoporosis is defined as a T-score value < -2.5, where T score is the number of standard deviations between the patient's bone mineral density and the peak bone mass in the young, adult population. Osteopenia is defined as a T-score between 17

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1 and -2.5 (27). When using T-score values it is difficult to distinguish between the bone loss caused by an older age and bone loss which is a complication of a disease. Therefore, in IBD patients an evaluation with Z-score values the number of standard deviations between the patients bone density and the age matched reference mean value is more reliable (28). Osteoporosis and osteopenia are more common among IBD patients than in the normal population (29). The prevalence of low bone mass in inflammatory bowel diseases ranges from 31% to 59% (30, 31), also an increased number of osteoporosis related fractures are observed in these patients (32). The reduced bone mineral density and the increased risk of osteoporosis is more understandable among those with a disease onset before the age of 20 years an age when peak bone mass is attained (33). However, so far no unequivocal features were identified as crucial causative factors explaining the increased bone loss in UC and CD patients. Risk factors for the decreased bone mineral density in IBD might include the causes of idiopathic osteoporosis, as well as specific factors connected to the core disease, including corticosteroid treatment, small bowel disease or resection. Low bone mineral density has been reported to be more common in patients with Crohn's disease - especially in those ones with small intestinal disease and resection rather than in ulcerative colitis (28, 29, 34, 35). However, this finding is not universal and might, as an example, just be a consequence of a CD patients' cohort with an earlier onset of bowel disease (36-39). Another often investigated factor is the steroid intake. A usage of corticosteroids contributes to the risk of osteoporosis (40), however low bone mass can be observed in IBD patients independently of its appliance (29, 39). Thus a high prevalence of low bone mineral density was found in patients with newly diagnosed Crohn's disease and among IBD patients with a low risk for metabolic bone disease (39). A recent study suggested that a link between steroid intake and BMD might be a secondary effect of other factors. Thus after multivariate analysis Z-score at the femoral neck was found to be significantly associated with disease type and body mass index (BMI), while at the spine only BMI. This group found no significant relationship between low BMD and calcium intake or disease site, length or severity (28). The distinct findings at femoral neck and spine draw further attention. Accordingly, while steroid induced bone disease affects vertebrae to a greater extent; numerous studies have reported a reduced hipbone mineral density in IBD when compared to the frequency of vertebral or forearm osteopenia (34, 38, 39). These results raise the possibility that other factors, present already before the onset of the disease contribute to the low bone mass and the enhanced bone loss in IBD patients. These factors could include the intestinal inflammation and the disrupted bowel function itself. In inflammatory disorders such as IBD the overregulation of proinflammatory cytokines could pose an additional risk for bone loss. Thus exposure to interleukin-6 within the bone marrow environment drives the differentiation of osteocytes to osteoclasts. Subsequently an exposure to
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interleukin-1 and tumour necrosis factor activates these osteoclasts in the bone resulting in bone resorption (41). This mechanism could be further influenced by other factors such as oestrogen and other sexual steroids (42-44). Recent studies have demonstrated that estrogens reduce the expression of signalling type IL-1 receptor (IL-1RI) in osteoclasts, while it increases the expression of decoy type IL-1 receptor (IL-1RII), altogether decreasing the IL-1 bio- responsiveness of these cells (45). This regulatory effect of oestrogen was similar in cells of both from male and female donors. On the other hand, the steady-state IL-1RII/IL-1RI ratios in osteoclast-like cells of men were found to be lower than that of early postmenopausal women, suggesting that similar IL-1 levels might have a stronger bone resorptive effect in men than in women. This seems to be verified by a finding where male sex was reported to be an independent risk factor for osteoporosis in Crohn's disease (40). Genetic association studies represent another approach in finding predisposing factors for osteoporosis. Family, twin and epidemiological studies indicate that in the collective population genetic factors play an important role in determining peak bone mass (46-48). Assessment of bone turnover markers also suggests a genetic influence in the regulation of bone metabolism thus in the rate of bone loss (49). Beside a polygenic predisposition bone mineral density is modified by the effect of environmental and lifestyle factors, such as smoking, diet, sport and medication. Therefore specific susceptibility genes likely define smaller groups of patients, which phenomenon would explain the inconsistencies found in candidate gene studies (50-55). The cytokines interleukin-6 (IL-6), tumour necrosis factor (TNF) as well as the interleukin-1 (IL-1) family play an important role in the regulation of bone formation (41), therefore genes involved in the control of their activity might influence the susceptibility to osteoporosis. Indeed, allelic variations at the interleukin-1 receptor antagonist gene (IL1RN) were found to be associated with early postmenopausal bone loss at the spine (56), while another group found a linkage between the presence of bone erosions in rheumatoid arthritis and the IL1 gene cluster (57). Another data have shown that distinct IL-6 and TNF gene polymorphisms are risk factors for the development of low bone mass (58, 59). Interleukin-1 and tumour necrosis factor also play a significant role in the pathogenesis of IBD (60, 61), while higher IL-6 levels were inspected among those IBD patients with osteoporosis mass when compared to those without (62). Furthermore allelic variations in the IL1 and TNF gene are associated with a higher risk for CD and UC (63-65). Therefore genes defining these cytokines provide good candidates for studies on the risk factors for bone loss in IBD. We found that carriers of allele 2 at the NcoI gene polymorphism in the TNF beta gene as well as carriers of allele 2 at the +3953 position of the interleukin-1 beta gene (IL1B) present significantly more frequently with low bone mass, than non-carriers (66, 67). This corresponds the results of another group focusing on the timely changes in bone mass in IBD patients. They demonstrated that non-carriers of the 240 base-pair allele of the interleukin-1
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receptor antagonist gene as well as carriers of the 130 base pair allele at the IL-6 gene are have a higher risk of bone loss than the other IBD patients (68). In summary, the predisposing factors for osteoporosis in IBD are not independent, which explains the discrepancies reported in the literature. Thus so far there are several general and IBD related osteoporosis risk factors suspected to contribute to the increased bone loss in inflammatory bowel diseases, but results on the degree of their role are controversial. Further studies are necessary to verify the value of genetic markers on their role to predict osteopenia on the long term at the time of the diagnosis of the disease. Classification and disease activity Both Crohn's disease and ulcerative colitis can present with widely different symptoms, localisation and complications. Moreover, these differences not only vary between individuals, but might also change in the course of time frequently even periodically. Inhomogeneity of patient groups may explain inconsistencies between the results of different studies as well as clinical trials. Therefore setting up characteristic features describing the severity and course of these diseases has been long an important aim of IBD research. Classification makes the comparison of studies performed in different populations, or in the same population at different times feasible. The characterisation has developed in two directions. One type of them is the activity index. It describes the actual severity of the disease at the time of the measurements. The activity index could be best used to compare disease severity in the same individual at different time points. It can be applied to register flare ups, their length, severity or frequency, thus for the check up of the therapeutic efficacy of different medications and methods. Similarly, in case of comparing dynamic variables, such as blood-chemical parameters, the usage of a reference indicating the actual severity is essential. The most widely employed activity index for adult patients with Crohn's disease is the Best index (69) or Crohn's disease activity index (CDAI), while for ulcerative colitis an index set by Truelove and Witts in 1955 (70). Interestingly, the activity suggested by clinical and laboratory data do not always confirm those shown by endoscopic and histological findings. Although these activity indexes are widely accepted and used by gastroenterologists, they include subjective elements hence some caution is recommended in their application. Although epidemiological studies suggest distinct incidence for both forms of IBD in different populations and ethnicities, here is a lack of specific data on possible dissimilarities in disease course and severity. Activity score measured during some time of the disease does provide sufficient information on the severity and characteristics of the long-term disease. Therefore, other classifications providing this type of information are also necessary. In ulcerative colitis the extension and severity of the disease at the first presentation was usually found to be well correlated with the further disease severity, course and mortality (71-73). Thus clinically more severe forms are
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mainly those with left-sided and extensive colitis. Moreover, some data have suggested that the presence of certain extraintestinal manifestations and complications could also be associated with the localisation of UC (6, 74). Probably therefore, classification of colitis ulcerosa is usually performed by disease localisation. Most commonly UC patients are divided into subgroups of proctitis, left sided colitis and extensive colitis. Others prefer to give more accurate terms on the localisation, and refer to categories such as proctitis, proctosigmoiditis, left sided colitis, involvement of the colon transversum and pancolitis. We suppose that both pancolitis and the colitis involving colon transversum are referred to as extensive colitis by the previous classification whereas procto-sigmoiditis to left-sided colitis. Therefore studies using these terminologies could be well compared; however, some inconsistencies can probably not be avoided. Most often UC patients with a disease affecting the distal colon alone have a mild disease course including diarrhoea with 3-5 movements a day. Patients with moderate disease usually present with a disease affecting the third to half of the total colon, while patients with severe disease present almost always with total colitis. As for endoscopical findings other classifications such as the grade of mucosal fragility and the tendency for spontaneous bleeding are also used to determine disease severity. The non-contiguous location of CD, the large number of possibly affected sites in the gastrointestinal tract and the wide variety of presentation make it more difficult to provide an easily applicable and proficient classification for Crohn's disease. Localisation of Crohn's disease influences the therapeutic measurements taken as well as the prognosis of the disease (20). Furthermore there is some data suggesting that the localisation is associated with the presence of certain complications and extraintestinal manifestations (6, 75-77). On the other hand, epidemiological evidence suggests that there may be at least two distinct clinical manifestations of CD. Based on the disease behaviour these have been designated to fistulising-penetrating and non-perforating forms of Crohn's disease (9). Patients with the fistulising-penetrating form of disease present with fistulas and/or abscesses, and perforation is a typical complication among them. This form is characterised by a more aggressive course of disease. Patients have more acute exacerbations, irrespectively of the anatomic localisation. Whereas patients with non-perforating form of disease were found to have a more indolent course and present typically with obstruction and bleeding. Moreover, there is some evidence that the host immune response in these two groups of patients differs (78). However, recently the non-perforating group was further divided in two patients' groups (79, 80). The first group referred to as having stricturing disease is defined as presenting with constant luminal narrowing without a sign for penetrating disease at any time in the course of disease. While patients in the second group those without stricturing or penetrating symptoms are defined as having inflammatory disease behaviour. Certainly, disease behaviour for
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example the presence of fistula or a stricture influences the therapeutic indications, the disease management and some data suggest that they might define distinct genetic groups (81-85). Therefore, homogenous patient subgroups defined by this classification provide more reliable and comparable data from clinical and etiologic studies. The first attempts to classify CD patients were proposed by Farmer and co-workers and was based on the anatomic localisation of the disease. These localisations have been linked to serological markers, indications for surgery and medical therapy (86). Sachar and co-workers set the first broad classification referred to as Rome classification - in 1992 (79). They suggested that all cases be described primarily by the anatomical localisation gastroduodenitis, jejunitis, ileitis, colitis, perianal disease and anatomical extent localised or diffuse. Further modifiers as disease behaviour primarily fibrostenotic, primarily inflammatory or primarily fistulising and operative history were also propounded. As the definition of anatomical localisation should be in most cases described with more of these identifiers, and the attached characteristics further increase the number of possible subgroups, the usage of the complete classification is not easy, and most studies do not have enough subjects to allow its full application. Although localisation and disease behaviour have been frequently used often with the application of modification the Rome classification has not been extensively adopted in its original form. Therefore, based on this classification, another more simple classification containing a smaller number of subgroups was introduced in 1998 in Vienna (80). Three variables were set to describe Crohn's disease. The first, the age at the diagnosis of the disease, was aimed to represent the genetic component; the second is localisation by definition including four exclusive categories as terminal ileal, colonic, ileocolonic involvement, and a disease affecting the upper GI tract as well. The third category is disease behaviour that provides information on the biology of the disease. On the whole this latter classification is based on largely similar features; it presents more strictly defined categories than the Rome classification. Although the number of subgroups is crucially smaller in this classification, than in the previous one, still in case of smaller study populations only one or two variables are recommended to use. The phenotypic classification and activity measurement of Crohn's disease and ulcerative colitis could help to define eligibility criteria for clinical trials. Standardised subgroups could help to identify the possible associations with environmental factors, genetic and biological markers. This would further improve the understanding of alterations between distinct ethnicities. Comparison of populations from different studies is more reliable by using homogenous and well-defined patients' groups. Therefore, a classification of Crohn's disease and ulcerative colitis based on an identification of molecular mechanisms of pathogenesis holds the promise of tailoring medical therapies to individual patients.

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1.3 Epidemiology
The epidemiology of the two most common entities of inflammatory bowel diseases ulcerative colitis and Crohn's disease share many features. There are significant geographical, ethnical and cultural factors influencing the prevalence of both entities. IBD is more common in the developed world than in the developing world. Both UC and CD are more common in urban than in rural areas (87-90). They are both more frequent in whites than in blacks or in Asians, while Jews have an incidence three to six times greater than that of non-Jews. Furthermore, in populations where the prevalence of CD or UC is high, the prevalence is usually also high for the other form. The incidence and prevalence of the two entities differ slightly, UC being more common; however this difference is getting smaller. Earlier 4-5 times more UC cases were found than CD, now this rate is 1.5-2 to 1. (91, 92) As for the actual figures epidemiological studies of different geographical areas have shown an incidence for UC between 0.5-24.5 in 100,000 inhabitants. In Western Europe and in the United States the incidence of UC is approximately 6 to 8 cases per 100,000 population, while the prevalence is estimated approximately 70 to 150 cases per 100,000 inhabitants. On the other hand studies on the frequency of CD in several populations suggested an incidence rate between 0.1-11 cases in 100,000 people; with estimates of 2 cases per 100,000 in Western Europe and the United States. Simultaneously the prevalence in this region is estimated at a number of 20 to 40 per 100,000 inhabitants (91, 92, 93). Despite the relatively modest overall difference in incidence rates in the western world, there seems to be a considerable variation in the incidence of both diseases across Europe as well as the United States (92, 94, 95). Thus in these regions several studies reported a north-south incidence gradient, with IBD more common in the north than in the south. In Western Europe rates in the north were 40 percent higher for UC and 80 percent higher for CD than in the south. These differences could not be explained by national differences in cigarette smoking and education (92). Beside the large multicenter investigation of the European collaborative study group local epidemiological results support the existence of geographical differences in CD and UC. Epidemiological studies in Spain have shown an incidence of 2/100,000 for ulcerative colitis while 0.9/100,000 for Crohn's disease for the period of time of 1979-1988 (96), while a Danish study based on data from the Danish National Registry of Patients have found an incidence of 13.2/100,000 for ulcerative colitis and an incidence of 4.6/100,000 for Crohn's disease in the period of 1981-1992 (97). Other studies from Scandinavian countries showed an incidence for UC 12 per 100,000, while for 7 per 100,000 inhabitants for CD in a study performed in the Uppsala Health Region in Sweden (98), whereas in Stockholm region the standardised incidence with 4.9/100,000 in the time period of 1985-1989 was lower for CD (99). In Iceland, where the incidence for UC was found to be the highest in Europe, the
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mean annual incidence for the 5-year period of 1990-1994 was registrated as 16.5/100000 for UC whereas 5.5/100000 for CD (100). Outside Europe high incidence rates and dynamic epidemiology of IBD indicate the presence of important environmental risk factors in Manitoba. In this Canadian county the annual incidence rate between 1989 and 1994 was 14.6/100,000 for Crohn's disease, whereas 14.3/100,000 for ulcerative colitis. In the last year of the study period the prevalence of Crohn's disease was 198.5/100,000, while 169.7/100,000 for ulcerative colitis (101). Several studies suggest a favourable effect of nonWestern lifestyle. As an example, among Bedouin Arabs of southern Israel the prevalence rates of ulcerative colitis were 6 per 100,000 in men, while 13.7/100,000 in women. No male patient with Crohn's disease was found; the prevalence rate was 6.8/100,000 for women while 3.2/100,000 for the whole population (102). There are also changes in time in the incidence of IBD between different parts of the world. Whereas in the Western countries the incidence was increasing for both entities in the period 1950-1970, recent epidemiological studies suggested that the last 20-30 years this elevation has stopped or at least has slowed down (97). However, these findings could not be overall confirmed. For instance a prospective nation-wide study from Iceland found a continually rising incidence for both CD and UC in the period of 1950-1994 (100). Similarly a Swedish study following the incidence of CD between 1955 and 1989 described continuously increasing rates (99). At the same time, in several countries where IBD was earlier not common, such as in Japan or in India, the incidence of the disease is found to be increasing (103, 104). The increase in the incidence of ulcerative colitis preceded the increase in the incidence of Crohn's disease by about 10-15 years (95). Recently in Western Europe and North America, the incidence and prevalence of CD have been found to increase five times faster than those of ulcerative colitis. Not just the incidence, but also the disease presentation seems to change during the last decades. Some data supports that the localisation of CD and UC is getting more distal, thus the incidence of colonic CD and that of ulcerative proctitis is growing (98, 105). Other features of epidemiological studies are the age at the disease onset and the frequency of the IBD related death. Most cases of CD begin at an age earlier than 30 years, with the peak incidence among those aged 14 to 24 year. Ulcerative colitis, like Crohn's disease, may afflict people at any age, but the age-onset curve shows a bimodal distribution, with a major peak at ages 15 to 30 and a second smaller peak at ages 50 to 70. Crohn's disease is usually found to be more common in women, while ulcerative colitis in men, especially among those with an onset at older ages (92, 97, 102). Mortality rates of IBD have greatly improved during the last decades: recent North American and Northern European mortality rates are not increased compared with the general population (106-109). This improvement is probably a consequence of better therapeutic possibilities and the earlier identification of the
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patients as well as the development of severe complications of disease. The most common IBD related death causes are in ulcerative colitis fulminant colitis and carcinoma, while in CD septic complications. Differences found in the incidence and disease pattern between countries and regions could be due to distinct study designs and to divergent habits in consulting physicians in distinct populations. However the European collaborative study on IBD working with strictly described and applied criteria confirmed the geographical differences within Western Europe described by earlier studies. On the other hand, they did not find a significant variance in the severity of complaints in those registrated IBD cases between the different populations. Thus it is possible that the diversity seen in the frequency and pattern of IBD between distinct ethnicities might represent real differences reflecting environmental factors, lifestyle or genetic susceptibility. The growing incidence of IBD in countries such as in Japan and India where the Western- lifestyle is getting more widespread as well as an experience that immigrants appear to acquire the rates of their adopted country (103, 104, 110) would favour a role for environmental factors. On the other side, results suggesting that among black people in the USA IBD presentation, severity and frequency is different from the ones among whites (111-113), and the findings of higher rates of IBD in the Indian immigrant than in the indigenous population in Leicester as well as racial differences in the prevalence of ulcerative colitis and Crohn's disease in Singapore (114, 115) accent the role of a genetic background. Probably the answer to the question is not an eitheror one. It may be that in the evolution of different phenotypes of CD and UC several factors such as diet, climate, economic wealth, microorganisms and genetic susceptibility play simultaneously significant role. Therefore etiological studies have to be performed on different populations and ethnicities; likewise, when investigating the role of genetic and environmental factors in the development of IBD the disease course and ethnicity of study populations have to be well characterised.

1.4 Aetiology
The exact aetiology of Crohn's disease and ulcerative colitis is not yet known. Evidence is accumulating that these disorders have a multifactorial origin consisting probably both of hereditary and environmental components. Moreover, evidence is accumulating that either Crohn's disease or ulcerative colitis are single disease entities. Particularly Crohn's disease was hypothesised to present a syndrome of functional and morphological lesions, which reflect a response pattern of the gastrointestinal tract on several non-specific agents. While the causes of IBD remain unknown, certain characteristics of the two disease entities have suggested several features of possible importance. These include abreast genetic, immunologic and environmental factors such as infectious agents or diet. Next to these putative factors local changes such as mucus abnormalities,
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increased intestinal permeability, and vascular abnormalities are also observed. Thus an unregulated intestinal immune response found in IBD patients might develop as a reaction on a dietary, pharmaceutical, infectious or other environmental agent in genetically predisposed individuals. 1.4.1 Environmental factors There are several findings supporting the hypothesis that environmental factors play an important role in the expression of inflammatory bowel diseases. Such supportive observations are the increased incidence of the disease over a short time span, the higher incidence seen in urban when compared to rural areas, as well as the significantly lower concordance rates in dizygotic versus monozygotic twin pairs are all observations. Smoking One of the mostly investigated environmental factors is smoking. Interestingly, smoking diversely influences the two disease entities. In Crohn's disease smoking aggravates the disease symptoms in dose-dependent effect. It increases the needed steroid and immunosuppressive drug quantity, as well as the risk of relapse and raises the possibility of a surgical intervention. Among these patients smoking cessation favourably influences disease course (116). The observed adverse effects of smoking on the course of CD may be due to modification in the microcirculation in the intestine, a hypothesis that would be in accord with the vascular theory of the disease that was based on the discovery of vascular lesions within the affected regions. Interestingly, a recent study even suggests that those CD patients who quit smoking for more than one year have a more benign disease course than if they have never smoked (117). This could mean that in these patients smoking had been significant etiologic factor, while the contribution of other environmental and genetic factors might have had smaller magnitude. In contrast, the rate of smokers is lower among patients with ulcerative colitis than in the general population and quitting smoking has been observed to elevate the risk for the development of ulcerative colitis (118, 119). There is also evidence that smoking favourably influences the course of ulcerative colitis. Lower rates for hospitalisation and surgical intervention as well as later age of disease onset have been observed among smoking UC patients as compared to non-smokers and especially to ex-smokers (120-122). Similar trends are also observed in families with more than one affected member. In families with only Crohn's disease the rate of smokers among affected individuals is 64 percent compared to 31 percent in families having only cases of UC (123). On the other side, the prevalence of smokers was found significantly higher in CD patients with a family history for IBD than in healthy controls and in familial UC patients (124). Interestingly, the association between smoking and CD could not be confirmed in Jewish patients living in Israel. The explanation for this finding
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might be related to the higher genetic predisposition to IBD in Jewish people (125). While on the whole smoking is an established risk factor for CD, no association with passive smoking has been detected (126). Although the role of nicotine in IBD is not fully understood, transdermal nicotine patches proved to be useful in mild to moderate ulcerative colitis (127). As nicotine increases the colonic mucus production a possible explanation on its beneficial action is in agreement with the mucus theory of the aetiology of UC. Other groups found that Transdermal nicotine has no effect on mucin gene transcription. They explained its beneficial effects by the decrease of IL-8 expression (128). Further possible mechanisms include a role for diminishing the intestinal permeability or the reduction of prostaglandin-E production caused by nicotine as well as the inhibition of proinflammatory cytokines like interleukin-1, tumour necrosis factor alpha and interleukin-6 or a shift towards T-suppressor over against T-helperinducer cells (129-131). Oral contraceptives Several studies have described a weak association between oral contraceptive usage and both Crohn's disease and ulcerative colitis. Furthermore some studies found that the elevated risk for Crohn's disease falls when the use of oral contraceptives was stopped (132, 133). An explanation for these findings might be that oral contraceptives known to increase the risk of thromboembolic disorders are able to cause mucosal alterations resembling the ones seen in IBD (134, 135). However, data on their being a risk factor for IBD is conflicting, and recent studies and meta-analyses considering smoking as a co-factor - could not confirm the role of oral contraceptives in the activity of Crohn's disease and ulcerative colitis (136, 137). Dietary factors Several observations denote a role for diet in the aetiology of IBD. The rising incidence of inflammatory bowel disease since the Second World War coincides with profound changes of the dietary pattern. Moreover, increasing incidence of the disease has been observed in several populations such as the Japanese and the Indian as Western style diet became prevalent. Further supporting the role of dietary factors in the development of IBD, a difference in dietary habits was detected in Jews living in Israel. High-risk Ashkenazi Jews were found to eat more fat and meat than the low-risk non-Ashkenazi Israeli population that consumed more carbohydrate and fibre (138). These dietary differences might also explain the lower CD incidence rates in Israeli than in American Jews. Actually, dietary antigens are the majority of non-bacterial and non-self antigens that are present in the gut, and antibodies against some dietary factors have been reported both in CD and UC. Furthermore, in Crohn's disease, elemental diets and total parenteral nutrition often induce or prolong remission (139). This phenomenon was explained by the reduction of antigenic load on activated
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immune cells in the intestine, and thus by the down-regulation hyperreactive CD4 cells. Therefore, numerous case-control studies have been performed investigating various alimentary components to identify a causative substance. Refined sugar Several studies in different populations have suggested that a high intake of refined carbohydrate is associated with Crohn's disease, while generally they did not see an influence on the development of ulcerative colitis. (140-142) Other studies have found similar risk for high sucrose consumption on UC and CD. These studies detected no effect in association with lactose consumption while they have revealed a negative association between fructose intake and a risk for IBD (143). A possible explanation for these findings could be related to the presumptive trigger role of certain bacteria in Crohn's disease. Microbes in the bowel flora depend on dietary starch for their growth, thus an increased nutriment supply might elevate their potential for acting across the gut mucosa or for enhancing the inflammatory symptoms (144). However some studies suggest that high sugar consumption in Crohn's disease is a secondary phenomenon beginning after the disease onset (145). Indeed, two randomised controlled trials showed no significant changes in disease activity in patients on low refined sugar content diet (146, 147). Fatty acids Beside refined sugar another nutritional component studied in great extent in association with inflammatory bowel diseases are fatty acids. A population based study has shown that the increasing incidence of Crohn disease observed in Japan was strongly correlated with increased consumption of animal fat and protein, n-6 polyunsaturated fatty acids and the ratio of n-6 to n-3 fatty acid intake (148). Another Japanese study has suggested that margarine or chemically modified fat may play an etiological role in the development of ulcerative colitis (149). Again others emphasised the role of high animal fat and cholesterol intake as a factor responsible for an increased risk for UC (143), while a recently published retrospective study in newly diagnosed ulcerative colitis patients suggested that particularly high intakes of mono- and polyunsaturated fat are associated with an elevated risk to develop UC (150). Furthermore, Heckers and co-workers have found higher trans-hexadecenoate and trans-octadecenoate concentrations in subcutaneous adipose tissues of Crohn's disease patients when compared to controls. Since these fatty acids cannot be synthesised by man, it indicates a higher intake or uptake of chemically processed fat by CD patients (151). Moreover, in animal experiments dietary oleic acid added to elemental diet caused a reversal of the favourable effect of an elemental diet on the intestinal immune system. In contrast, supplemental dietary fish oil apparently tends to prevent relapse of Crohn's disease (152). In another study in which pigs were fed with
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chemically processed partially hydrogenated fats a bowel histologically similar to that of in Crohn's disease was seen (153). In accordance with these findings a recent randomised controlled clinical trial indicated that an immunomodulating formula containing n-3 fatty acids might play a favourable role in the treatment of CD. The study has namely found that supplementation with n-3 fatty acids and antioxidants significantly change the eicosanoid precursor profile, which may lead to the production of eicosanoids with attenuated proinflammatory activity (154). An explanation for these findings may lay in the intestinal immune function modulating role of dietary fat. Long-chain fatty acids and lipoproteins significantly stimulate lymphocyte function and the migration of T lymphocytes to Peyer's patches. Fat absorption also increases cytokine release from intestinal epithelial cells. On the other hand, unsaturated fatty acids have a suppressive effect on cell-mediated immunity (152). As dietary fat intake is closely associated with the immunologic function of the intestinal mucosa, careful manipulation of dietary fat can be important in management of this disease. Fibre intake Epidemiological studies have found a decreased relative risk of Crohn's disease for subjects who had a high intake of fibre (141). Other studies focusing on the dietary habits of IBD patients showed a negative association between vegetable consumption and the risk of Crohn's disease (143). On the other hand, some earlier case-control studies have shown negative results on the role of fibre consumption in the development of inflammatory bowel diseases (141, 155). These latter results indicate that fibre consumption might not have a general direct protective role in IBD, or that the simultaneous intake of possibly harmful factors when consuming certain high fibre containing foods, has to be taken into consideration (156). However a recent clinical study has indicated that consumption of Plantago ovata seeds containing dietary fibre might be as effective in maintaining remission in ulcerative colitis as mesalamine (157). Similarly, another recent study has found that germinated barley foodstuff has effectively suppressed dextran sulphate sodium induced experimental colitis in rats. The possible effect was reasoned by its high activity to increase short chain fatty acid levels in the gut lumen (158). Glutamine Glutamine-supplemented foods in patients with catabolic conditions have been shown to prevent deterioration of gut permeability, protect against the development of intestinal mucosal atrophy, and improve nitrogen balance. Furthermore, animal models of inflammatory bowel disease suggested that glutamine-enriched enteral diets may lead to less severe intestinal damage, less weight loss, improved nitrogen balance, and reduced disease activity (159, 160). However, other animal studies have found that excess glutamine has a deleterious
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effect on ulcers in trinitrobenzenesulfonic acid-induced colitis (161). Moreover, recent findings of a randomised clinical trial on paediatric IBD patients suggest that a glutamine-enriched polymeric diet offers no advantage over a standard lowglutamine polymeric diet in the treatment of active Crohn's disease moreover, it appears to be less effective in improving the Crohn Disease Activity Index (162). Other dietary factors Several further dietary factors have been investigated for their role in the pathogenesis of Crohn's disease and ulcerative colitis but results are rather inconsistent. A recent case-control study in newly diagnosed cases of Crohn's disease and ulcerative colitis showed numerous risk factors when compared to population controls. A positive association with cola drinks, chewing gum and chocolate consumption as well as a negative association with citrus fruit consumption and the development of both Crohn's disease and ulcerative colitis were found. Additionally, with Crohn's disease there was a positive association seen with chewing gum consumption (163). Another study in a Hindu population detected that patients with Crohn's disease eat less spicy but with more flour than controls. Patients with ulcerative colitis were on the other hand found to have lower milk consumption than controls (140). A Swedish study recorded an increased relative risk of both Crohn's disease and ulcerative colitis associated with the consumption of fast foods. However this latter observation could be also explained by the high sugar and fat while low fibre content of these nutriments (141). Among several other nutritional components a recent study has suggested that even high intakes of vitamin B6 may enhance the risk of developing UC (150), while others found a positive association between retinol intake and the development of IBD (143). One less specific, but in a way more objective environmental factor that has been associated with the induction and/or exacerbation of disease activity in patients with Crohn's disease and ulcerative colitis are ingested ultrafine particles. Based on a Western diet, recent data suggest that more than 10 ultrafine particles are ingested per person every day. These microparticles have been earlier considered inactive although they adsorb endogenous constituents of the intestinal lumen and are taken up by human intestinal lymphoid aggregates. Recent data demonstrated that ultrafine dietary particles are not immunologic inert and may be important adjuncts in overcoming normal gut cell hypo-responsiveness to endogenous luminal molecules. This may be particularly relevant to patients with inflammatory bowel disease where there is abnormal intestinal permeability (164). Another recently proposed possible mechanism by which nutrition could influence the development and progress of IBD is endogenous antisecretory factor (AF) production. Although the role of antisecretory factor in human intestinal diseases is not known, it has been shown to reduce the incidence of diarrhoeal disease in several animal species. In IBD patients consumption of hydrothermally
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processed cereals induced endogenous antisecretory factor production, and this increased activity was associated with a clinical improvement of the disease (165). On the whole, dietary components can strongly influence the effect of each other. Not studied constituents often concomitant the studied dietary element in a given population, while not in another one. Thus nutritional items and the alteration of traditional eating habits may be true risk factors for the development of IBD or just the expression of a modern life-style involving other at the present still unknown risk factors. Infectious agents The chronic inflammatory nature of Crohn's disease and ulcerative colitis has prompted a continuing search for an infectious cause. Several features suggested the possible involvement of infectious agents. The most common localisation of IBD is equivalent with the place where the bacterium concentration is the largest in acute infectious colitis, moreover Crohn's colitis and ileocolitis improves on antibiotics. Certain seasonal and geographical aggregation is also observed in the disease (166). Furthermore, recurring respiratory tract infection and diarrhoea at early childhood ages was found to be more common in patients with IBD (167). Among many suspected infectious agents Mycobacteria and measles are the ones most extensively investigated so far. Mycobacteria The similarities between Crohn's disease, intestinal tuberculosis and Jones' disease a disease caused by Mycobacterium paratuberculosis in bovines have led many researchers to suspect Mycobacterial aetiology of CD. Mycobacteria most often Mycobacterium paratuberculosis have occasionally been isolated or was demonstrated by polymerase chain reaction techniques (PCR) from affected tissues but these results were inconsistent (168, 169). Furthermore, studies using direct culture have demonstrated that some species of Mycobacteria are commonly found even in healthy individuals (170). Also the large sensitivity of PCR makes the method prone to contamination and false positive results. Although the immunohistochemistry techniques avoid many of the pitfalls of the direct culture, they proved to lack the sensitivity and specificity to show an association between pathogenic Mycobacteria and CD. Other investigators have performed serological studies in an attempt to demonstrate humoral and/or cellular response to M. paratuberculosis and other pathogenic Mycobacterium species in patients with Crohn's disease. A recent study using PCR, ELISA, and IFN-gamma tests suggested that M. avium subsp. paratuberculosis infect at least a subset of IBD patients (171). Another group found that sera from CD patients reacted to a M. paratuberculosis-recombinant antigens with the same high frequency as the sera from patients that were exposed to mycobacterial antigens (172). However, on the whole, probably also due to the ubiquitous nature of
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Mycobacteria and the cross-reactivity among species even these results are inconclusive (173, 174). Furthermore the question arise whether in the presence of Mycobacteria this infection is primary contributing to the etiopathogenesis of IBD or just secondary due to the disregulated mucosal immune functions in IBD. As for the chemotherapeutic trials, only few studies have been performed and most of these were not longer than twelve months. These studies including ones where combined antituberculotic therapy was applied did not prove to be effective in Crohn's disease (175). In summary, despite a great deal of research, the question if Mycobacteria play a role in the pathogenesis of Crohn's disease remains to be answered. Measles A viral origin of IBD was first suggested by Glahn and Pappenheimer in 1925 after finding intranuclear inclusions in the histologic samples of UC patients (176). Later a hypothesis identifying granulomatous vasculitis as the cause of Crohn's disease has (177) induced the research for possible causative viral agents. Although the granulomatous vasculitis theory is debated (178) measles virus known to infect human endothelial cells and persisting in humans is ever since a candidate for epidemiological and pathophysiologic studies on IBD. The group of Wakefield and co-workers has found evidence by immunhistochemical, electron microscopic and in situ hybridisation methods for the presence of a persisting measles infection in patients with Crohn's disease (179). However, other groups using similar methods as well as PCR techniques could not confirm these results (180-182). Similarly to the in situ experiments serological studies performed in IBD patients concerning measles virus brought contradictory results. Balzola and co-workers detected IgM antibody in majority of Crohn's disease patients as compared to the low prevalence in controls (183). On the other hand, studies in French families with high frequency of Crohn's disease could not confirm these findings (184, 185). Another group has not seen differences in the IgM levels between CD patients and controls; furthermore they did not measure measles virus IgM levels in any CD patient suggestive of acute infection (186). Conflicting data were also obtained from epidemiological studies. Data from studies in the Swedish population indicated that an exposure of mothers to measles virus in utero is a risk factor of Crohn's disease in their children (187). While a Canadian group could not confirm these findings, they have suggested that measles infection at early ages increase the risk for developing Crohn's disease and ulcerative colitis. (188, 189) Studies from the United Kingdom on the other hand have identified measles vaccination as a risk factor both for ulcerative colitis and Crohn's disease (190). The measleshypothesis of Crohn's disease supposes that early in life due to a perinatal infection an immuntolerance develops. This would at a later time deteriorate due to a predisposing genetic background or other factors such as smoking or reinfection by different tribes. However, neither of the indicative results could
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widely confirmed by other groups (191). The explanation for these contradictory results may lay in methodological problems, in the heterogeneous aetiology of Crohn's disease and the different populations examined. A higher susceptibility to measles virus of CD patients found might be as well simply a reflection of an immunologic alteration making these patients prone to Crohn's disease too. Other infectious agents Besides measles and Mycobacteria the pathogenic role of several other microorganisms were considered in Crohn's disease, ulcerative colitis or in both disease entities. In the last years several bacterium - Escherichia coli, Proteus, Yersinia, Campylobacter, Clostridium, Chlamydia, Pseudomonas, Shigella, Aerobacter, Bacteroides, Lysteria monocytogenes (192-199), different gramnegative rods (200), Streptococcal antigens (196) and virus (rotavirus, Norwalk, herpes virus) have been suggested to play a role in the pathogenesis and course of IBD (201-203). The explanation for this lies in data suggesting that the dysregulated immune response seen in IBD may be a reaction on luminal and/or enteric bacterial antigens. Thus colonic bacterial proteins would be targets of the disease-associated immune response, where own antibodies would detect a recurrent protein epitope expressed by colonic bacteria. This hypothesis is also supported by murine models of IBD caused by organisms such as Citrobacter rodentium and Helicobacter hepaticus (204). Animal models suggest a cooperation between host factors and microbial agents. Experiments have shown that IBD in rodent models involving conventionally housed animals could be prevented by raising the animals in germfree environment (205). As an example T cell receptor-alpha deficient mice maintained germfree or colonised with a limited number of intestinal bacteria fails to develop IBD-like lesions, whereas in the presence of a single microbial species, Cryptosporidium parvum, the disease evolves (206). Epidemiological and laboratory inspections supported the findings of animal models; however, on the whole, the evidence for a specific microbial aetiology for ulcerative colitis is less convincing than for Crohn's disease. The incidence of either form of IBD has been observed to increase after Shigella, Salmonella or Yersinia epidemics. Also numerous studies suggest that several bacterial (Clostridium difficile, Salmonella, Campylobacter, E. coli) and viral (influenza, mycoplasma) infections can trigger flare-ups or cause worsening in the course of IBD (207-209). Furthermore, bacterial endotoxins or lipopolysaccharides have been detected in the plasma of IBD patients (210). Interestingly, some studies found that IBD patients, especially those with Crohn's disease are less likely to be H. pylori infected than the general population. This difference was also present among those not taking Sulphasalazine (211), suggesting a specific microbial selection in these individuals. As for the specific candidate bacteria French study examined mesenteric lymph nodes and intestines of 21 patients from families with a high frequency of CD using immunhistochemistry techniques. Seventy-five percent of the specimens of
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CD patients were positively labelled with an antibody to Lysteria while none of those of the controls. The same study has found that fifty-seven percent of the CD cases contained E. coli, while 44% streptococcal antigen (212). Others detected that E. coli strains isolated from the ileal mucosa of patients with Crohn's disease adhere to differentiated intestinal cells and may disrupt the intestinal barrier by synthesising an alpha-haemolysin. (213). However, as Lysteria and E. coli are common contaminating micro-organisms, a non-specific secondary superinfection cannot be excluded. Indeed, more recent studies using immunohistochemistry techniques did not verify the presence of L. monocytogenes, and K pneumoniae in bowel section of patients with CD and UC. Although E coli antigens were found in ulcers, but not in granulomas, giant cells, or germinal centres, it is suggestive of a secondary infection in these lesions. (214) In agreement with this, other groups detected Lysteria monocytogenes DNA and E. coli strains possessing adherence factors at similar rates both in patients with IBD and in controls (215, 216). Another study investigating the possible techniques Salmonella typhimurium and Yersinia pseudotubercolosis use in order to avoid an anti-bacterial inflammatory response mechanism have found that following host cell infection they target the eukaryotic signal transduction pathways to block NF-kappaB activation (217). Other possible pathogenic factors are sulphate reducing bacteria. In the colon of patients with ulcerative colitis the hydrogen sulphide produced by these bacteria was found to be increased (218). As sulphides inhibit short chain acyl-CoA dehydrogenase, and the oxidation of n-butyrate governs the epithelial barrier function of colonocytes (219), an overgrowth of sulphate reducing bacteria might lead to mucosal damage in the bowel. A recent study found that all patients with active ulcerative colitis carried sulphate reducing bacteria (SRB), and the total viable counts were significantly related to the clinical severity grade. However counts and carriage rates of SRB in faeces of patients with ulcerative colitis was not significantly different from those in controls Therefore evidence for hydrogen sulphide as a metabolic toxin in ulcerative colitis further remains circumstantial. (220). Other results showing the presence of peptidoglycan-polysaccharides consistently in CD patients throughout the gut wall suggest that not only intact organisms but also their cell wall fragments can serve as immune stimulants (221). Somewhat in consistence with this idea, a recent study confirmed the presence of bacterial 16S rRNA gene segments in human intestinal lymph follicles in IBD patients. However, only for some of the found rRNA segments was possible to identify the respective bacteria: staphylococcus species, Streptococcus sanguis and Paracoccus marcusii. No known bacterial DNA data could be referred to the other segments showing 100% identity. The question however is again whether the bacteria found inside the lymph follicle is a primary stimulus in IBD (222). This diversity in possible causative microbial agents might be due to the non specificity of the actual provoking factor. Some researchers suggest that in Crohns disease the phagocytic activity of monocytes and natural killer (NK) cells
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NK is impaired and therefore patients experience a re-infection with most probably viral antigens and develop antigen retention in the gut lumen. The virus in this case would not have to be a very specific one, similarly to hepatitis or to commoncold, a number of viruses being susceptible to the intestinal epithelial cells could cause similar symptoms (223). Recent results showing an association between Crohns disease and a mutation a of gene coding a nuclear factor activating protein offer further explanations to the diversity of bacterial antigens indicated to play a role in the pathogenesis of IBD. Thus in the presence of the wildtype protein differential regulation by lipopolysaccharid from different bacteria have been observed. However, in the presence of the protein coded by the gene including the mutation a deficient immune answer has been observed (224, 225). In summary, to date no bacterial, fungal, or viral agents have been verified to be involved as a primer etiologic factor in the pathogenesis of UC and CD (185, 196). Whereas the primary role of these agents could not be confirmed, evidence exists for their acting as co-factors (226). Recent data suggest that even nonpathogenic bacteria of the intestinal flora might initiate the disease in susceptible hosts. Thus in genetically susceptible individuals the inflammatory and pathologic mechanisms such as increased mucosal permeability and cytokine activation initiated by a microbe or an infection might persist and end up in a chronic bowel inflammation (227). Intestinal flora The large number of infectious agents suggested to play a role in the development of IBD may partly be explained by alterations caused by these micro-organisms in the normal intestinal flora. Thus extensive experimental and clinical data suggest that normal luminal bacteria or bacterial products play a significant role in the initiation and perpetuation of chronic intestinal inflammation in IBD (228). Experiments have shown that there is an exaggerated mucosal immune response particularly in active CD but also in UC directed against cytoplasmic proteins of bacteria within the intestinal lumen; this implies that in relapse of IBD there is a breakdown of tolerance to the normal commensal flora of the gut. (229). Whether the bacterial flora contributes to the pathogenesis of IBD by increased penetration in mucus, increased adherence to epithelial cells, or invasion of the epithelium is not yet exactly known. The initiating agent might be customarily a part of the normal flora or a nonpathogenic microbe producing an inflammatory response only in the susceptible hosts. Individuals with an increased intestinal permeability might represent for instance a group of susceptible hosts. Simultaneously, genetically determined defects such as an abnormal ratio of interleukin-1 to interleukin-1 receptor antagonist, a selective activation of TH1 lymphocytes or an altered NF kappa B activation pathway may offer an explanation for the chronic inflammation (230). The substantial role of intestinal flora in the pathogenesis of IBD is confirmed by
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some animal models. As an example the evolution of enterocolitis in HLA-B27 transgenic rats is dependent upon the presence of intestinal bacteria (231). Assessment of the cecal microflora in these animals showed a rise in numbers of Escherichia coli and Enterococcus spp. corresponding to the presence and severity of IBD (232). In addition human studies has shown that the intestinal microflora of CD patients consist increased counts of both anaerobes and aerobes; among the obligate anaerobes there are more gram-positive coccoid rods and gram-negative rods than in the flora of healthy controls (233). Qualitative differences have also been detected: E. coli strains isolated from IBD patients showed increased adherent properties as well as increased production of enterotoxins (234). However other studies did not support the role of E. coli finding strains possessing adherence factors at similar rates both in patients with IBD and in controls (215). Thus Schultsz and co-workers compared the distribution of bacteria in the mucosa of rectal biopsy specimens of IBD patients and that of controls and found that the intestinal mucus in IBD patients is less protective against the endogenous microflora. This resulted in an increased association of luminal bacteria with the mucus layer. The bacteria were localised within the mucus layer but did not adhere to the epithelial cells and were not present within the lamina propria.(235). This latter finding might also give some explanation for the large inconsistencies of studies looking for a causative bacterial agent in IBD patients. The beneficial effects of antibiotic therapy in Crohn's disease as well as results with exogenous probiotics in the treatment in pouchitis (236-238) further support the key importance of intestinal milieu in the pathogenesis of IBD. Moreover administration of lactobacilli was found to lead to a normalisation of intestinal flora, reducing the number of colonic mucosal adherent and translocated bacteria has been beneficial in human IBD and in murine colitis (210). Miscellaneous environmental factors Beside the above discussed environmental factors numerous other ones have been suspected to play a role in the etiopathogenesis and/or exacerbation of IBD. Although clinicians often find that psychological and physiological stress unfavourably influence the course of IBD, there is only a low number of casecontrol studies investigating this subject and the results are not fully consistent. However, not only large prospective studies in IBD patients suggested a role for major stress events in disease activity (239), but also animal observations. Thus, after chronic stress situations ulcerative colitislike lesions were seen in gibbons (240). A recent study might offer an explanation to the partly contradictory results on the role of stress. It has suggested that while short term stress does not trigger exacerbation in ulcerative colitis, longterm perceived stress increases the risk of exacerbation over a period of months to years (241). Curiously there seems to be a difference between the two most common IBD disease entities. Published data indicate that Crohn's disease, unlike ulcerative colitis, may be statistically
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associated with lifetime psychiatric disorders. (242). Results from behavioural oriented observations are also supported by a study, which found that colitis in mice can be reactivated after resolution by stress this effect requiring sensitised CD4+ lymphocytes (243). Accordingly, human studies indicated that psychological stress leads to a decrease both in the number of NK cells and in the degree of their activity (244). The explanation for findings showing a lower risk of IBD among those performing more physical activity might also lay in a positive action on the immune system (245) Anyhow, further studies serving a better understanding of the bi-directional interactions between the brain and the gut will be probably give also an adequate comprehension on the role of stressful events in IBD. Another hypothesis for the development of IBD based on the similarities of CD lesions and the ones caused by materials such as chalk has suggested that toothpaste plays a role in the pathogenesis of CD (246). Again other researchers proposed that UC is caused by a reactive xenobiotic metabolite that is excreted in the bile and activated during its passage through the colon (247). In concordance with this idea antibiotics therapy in inadequate doses has been suggested to increase in a paradox way proinflammatory activity that in genetically susceptible individuals might lead to the development of IBD (248). Further factors that have been suspected to play a role in the pathogenesis in IBD include blood transfusions, contact with pets, tonsillectomy, appendectomy, breast feeding, perinatal events and good domestic hygiene (129). The findings are mostly explained by an unobtrusive or late infection causing a failure in the immune response, or just contrary that the finding itself is an expression of an altered mucosal immune response. Thus the sheltered child hypothesis suggest that the contact with common agents happens at a later age, causing a disturbed mucosal immune response. Supportive of this theory the incidence of IBD was detected to be higher in developed countries and in urban areas (92). On the whole the sheltered child hypothesis seemed to be confirmed for patients with Crohns disease, but not with ulcerative colitis (249). Simultaneously, the association with appendectomy was only significant for patients with ulcerative colitis. Thus appendectomy was found to be protective in UC a significantly lower percentage of UC patients underwent earlier an appendectomy, and among those who did recurrence rate was smaller and disease less extensive (250). On the other hand in Crohns disease it was identified as more frequent, however not a risk factor (251, 252). An explanation for these findings could be that the resection of appendix, an important part of the gut associated lymphoid system, might influence the balance between ileocolonic helper and suppressor functions (253) In summary, there are numerous environmental factors presumed to play the role in the pathogenesis of inflammatory bowel diseases. However, besides smoking none of them has been unequivocally proven to be causal for IBD. Hence it is

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possible that these agents are nonspecific, activating a specific immunanswer in predisposed individuals. 1.4.2 Immunologic factors Numerous observations support the idea that immune mechanisms are involved in the pathogenesis of inflammatory bowel diseases. Extraintestinal manifestations such as arthritis and dermatological disorders often accompany these disorders and may represent autoimmune phenomena. Besides, immunosuppressive therapeutic agents glucocorticoids, azathioprine, and cyclosporine are beneficial in both Crohns disease and ulcerative colitis. The increased frequency of childhood upper respiratory tract infections detected in patients with CD, could also mean that in these patients express symptomatic infections that in normal individuals remain symptomless (129). Patients with IBD may have humoral antibodies to colon cells and to foreign proteins such as cow's milk protein. In addition, IBD has been described in association with agammaglobulinemia as well as IgA deficiency. Abnormalities of cell-mediated immunity that have been reported in relation to IBD include cutaneous anergy, diminished responsiveness to various mitogenic stimuli, and a decreased number of peripheral T cells. Since many of these changes may revert to normal when the disease is quiescent, they are probably secondary phenomena. Animal models can provide another evidence for an immunologic pathway in the disease evolution of IBD. Thus an evolution of spontaneous colitis is experienced in transgenic rat expressing human HLAB27, in mouse being deficient in interleukin 2, as well as in cotton-top tamarin (254). Presumably an inadequate or prolonged activation of the intestinal immune system plays an important role in the pathophysiology of the chronic mucosal inflammation seen in IBD. This idea is supported by the increased levels of inflammatory mediators in the inflamed mucosa, by the experimental inflammation in animal models, and by clinical trials inhibiting inflammatory mediators (255). Normally, the gastrointestinal mucosa is exposed to several infectious and food antigens. The mucosal epithelium of the alimentary tract represents a crucial barrier to a broad spectrum of noxious and immunogenic substances within the intestinal lumen. Under physiologic conditions temporary damage of the epithelial surface mucosa may be caused by proteases, residential flora, dietary compounds or other factors. The rapid reestablishment of the intestinal mucosal surface barriers integrity observed in healthy individuals seems to fail in inflammatory bowel diseases. The exact reasons for this are not yet known. A comprehension of the immunologic functions in the bowel can lead us to a better understanding of the malfunction of the process. The earliest lesion in CD is the aphthous ulcer that overlies a lymphoid follicle. The distribution of these findings is significant as it refers to a possible relationship with specialised antigen presenting M cells.

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The mucosal immune system being functionally independent of the systemic immune system is located in the intestinal mucosa. It is functionally divided into two sites. The first is the inductive site, where foreign antigens are selectively taken up for initiation of immune responses by the gut-associated lymphoid tissue (GALT). The second, the effector site, consists of IgA producing plasma cells, T and B lymphocytes, macrophages and other antigen presenting cells of the lamina propria (256). IgA secretion is an important component of gastrointestinal protective mechanisms. Antigens complexed with secretory IgA in the mucus are largely degraded, thus do not penetrate the mucosa. Therefore it is interesting that in contrast to normal subjects where IgA is the main immunoglobulin in the intestine, patients with active IBD produce high concentrations of IgG from intestinal lymphocytes (229). Luminal antigens are preferentially taken up by specialised antigen sampling M cells (membrane cells). Specialised M cells overlying the lymphoid follicles in Peyers patches are important sites where antigens, e.g. bacteria and bacterial cell wall products gain access to the lamina propria. These sites also serve as a means of transporting luminal antigens to the extracellular space. Besides, Mcells also directly present antigens to the neighbouring lymphocytes. Likewise, luminal antigens transported into Peyers patches are retained by follicular dendritic cells and are presented to T and B cells under the influence of CD4+ regulatory T cells. Effector T cells in the lamina propria appear to have a helper (Th2) bias, while intraepithelial lymphocytes cytotoxic (257, 258). The CD4+ Tlymphocytes recognise the antigenpeptide/MHC II complex, while CD8+ Tlymphocytes the antigenpeptid MHC I complex. After being taken up, nonself antigen peptides are presented together with MHC II molecules, while endogenous peptides with MHC I molecules. In this context it is interesting that contrary to healthy individuals in IBD patients there is a significant MHC II expression in the colon epithelium (259). As one of the main tasks of MHC II is the antigen presentation, this high expression in the colon directs to a locally activated immune system. Upon longstanding specific stimulation and activation neutrophils and macrophages synthesise and secrete proinflammatory mediators, like cytokines. Viral, bacterial, dietary or other type of antigens could all activate mucosal macrophages to release proinflammatory cytokines such as IL1, TNF. This initiates the inflammatory cascade thus enhances chemotaxis and permeability and activates further immunocytes. These would again release the immunoregulatory cytokines IL2, IL6 and IFN which promote lymphocyte proliferation and differentiation. Thus the process sustains an active inflammation leading to tissue destruction in the gastrointestinal mucosa. Disturbed balances between proinflammatory and contrainflammatory cytokines promote long standing active inflammation and subsequent tissue injury (260). However, it is not yet fully known which factor is defected in the mucosal immuneresponse in CD and UC. Experimental results indicate several candidates. As an example there is some evidence that the non-specific killing
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system, especially the monocyte-macrophage system is impaired in Crohns disease (223). This could also offer an explanation for granuloma evolution in CD. Thus the reported decreased phagocytic activity of monocytes could drive to antigen retention in the macrophages consequently induce the development of granulomas. Other results suggest that one of the critical agents in the local immune response of inflammatory bowel diseases are the CD4+ T cells. CD4+ T cells are classified as Th1 (Thelper 1) and Th2 (Thelper 2) on the basis of their function. Their distinct task is also reflected in their different cytokine production profiles. Th1 type lymphocytes produce mainly interleukin2 (IL2), interleukin12 (IL12), interferon (IFN) and tumour necrosis factor and act in cellmediated immune answer. On the other hand Th2 type lymphocytes produce interleukin4 (IL4), interleukin5 (IL5), interleukin10 (IL10), interleukin6 (IL6) and interleukin13 (IL13) and primarily contribute to the humoral immunity. These subsets regulate each other reciprocally through key cytokines. Thus IFN produced by Th1 cells suppresses the expansion of Th2 answer, while IL4, IL 10 and IL13 secreted by Th2 cells inhibit the Th1 responses (261). Although in human the division of Th1 and Th2 cell subsets is not as strict as in mouse, it provides a good model to understand the pathology of IBD in humans. Thus in the pathogenesis of the two most common forms of IBD ulcerative colitis is thought have a Th2 pattern, while Crohns disease rather a Th1. Hence Tlymphocytes from the bowel of CD patients produce after in vitro polyclonal stimulation predominantly IFN and TNF. Consistently, compared to healthy controls, cells of CD patients produced lesser amounts of Th2 cytokines such as IL4 and IL5 (262-264). A similar phenomenon could be observed on the mRNA level in CD patients: significantly elevated IFN values in the lamina propria were detected (265). On the other side, T lymphocytes from resected bowel of UC patients were found to have normal IFN, but elevated IL5 production (263). Experimental models of IBD further support the idea that CD4+ lymphocytes have a crucial role in the pathomechanism of these diseases. As an example, transfer of the CD54RBHigh subpopulation of CD4+ spleen cells from Balb/c mouse leads in C.B17 SCID mouse to a chronic colitis resembling to Crohns disease (266). Evidence is accumulating for the existence of another group of specialised T lymphocytes in the mucosal immune system. These lymphocytes are responsible for suppressing a pathologic immune response against dietary antigens and the normal intestinal flora (267). Although the initiation factor and process for their maturation is not fully known, some data suggest that the location could be the environment of the Mcells (268). Thus the decreased resistance and tolerance against luminal antigens observed in Crohns disease and ulcerative colitis might be explained partly by Tcell dysfunction.

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Recent results show a significant role for mucosal immune cells from inflamed gut also in the frequent onset of arthritis in IBD patients. Thus these have found that these leukocyte populations bind avidly to synovial vessels (269). Recent results showing a significant association between variations in the NOD2 gene and Crohns disease, point to the role of immune response regulation in the pathogenesis of IBD (224, 225). Thus NOD2 itself is a cytosolic protein whose expression is restricted to monocytes (270). Its function includes recognising bacterial components, such as lipopolysaccharides and activate nuclear factor B (NFB) (270). NFB is a transcription factor, playing a pivotal role in the induction of cytokine synthesis and in the regulation of many other inflammation associated genes, thus its altered activation could lead to an exaggerated inflammatory response by the adaptive immune system. A related possibility that besides NFB, NOD2 mediates induction of cytokines as interleukin10, that can downregulate inflammatory response (271). Alterations in the NFB levels could be observed in inflammatory bowel disease. Ths immunohistochemical techniques and nuclear extracts from biopsies taken from inflamed and non-inflamed areas of the bowel of IBD patients show a different degree of activation of NF-B (272). A correlation between the number of cells showing NF-B activation and the degree of mucosal activation has been observed (273). In vitro studies comparing the levels of NF-B in monocytes from active CD or UC patients with normal controls found similar results (274). The activity of NF-B is regulated by multiple factors (275). The most important way of regulation is based on the subcellular location of NF-B, namely being inactive when it is in the cytoplasm and active inside the nucleus. The retention of these molecules in the cytoplasm is achieved by means of the formation of a complex with inhibitory proteins, the I-B. These proteins can be found in both the cytoplasm and the nucleus. In response to various agents, I-B is phosphorilated (276), which signals for its ubiquitation and rapid degradation, thus allowing nuclear traslocation of NFkB. One of the most interesting aspects of NF B is the variety and nature of the inducers that lead to its activation, which include bacterial products, viruses and viral products, parasites, inflammatory cytokines, T and B cell mitogens, drugs and reactive oxigen metabolites. The chromosomal location of the genes that code for the NFB complex and IB is: NFKB1 (for p50) on 4q23; NFKB3 (for p65) on 11q12 and NFKBI (for IB) on 14q13. Cytokines Beside a pathologically altered Tcell distribution or function and deteriorated regulatory mechanisms changes in the cytokine profile might have a pathognostic value in UC and CD. Cytokines are soluble, nonantigen specific proteins and glucoproteins with a strong biological effect playing a key regulatory role in the
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immune responses. They are produced by leukocytes and a number of other cell types principally by cells of the immune and hematopoietic system. Currently there are more than 100 cytokines known forming a complex interactive regulatory network. Cytokines induce the production of adhesion molecules and other inflammatory mediators, thus they can induce prolong but also terminate inflammation. (277). Beside their role in the immunologic reactions cytokines have an influence virtually on almost all other type of physiologic processes as well. Beside general symptoms as fever, loss of weight some functions are distinguished in inflammatory bowel diseases. As an example cytokines affect bone remodelling by regulating the differentiation as well as the activity of osteoclasts and osteoblasts. Furthermore, they are involved in the cartilage metabolism and destruction as well as in the development of vascular lesions. The proinflammatory cytokines interleukin1, tumour necrosis factor and interleukin6 play a central regulatory role in the inflammatory process in both the systemic and the localised inflammatory reactions. They are such as the chemokines predominantly monocyte/macrophage derived. Interleukin-1 The term interleukin1 involves two polypeptides (IL1 and IL1) possessing a broad range of physiologic functions. IL1 plays a role in the regulation of inflammatory, metabolic, haematopoietic and immunologic mechanisms. In healthy state, with exception of few specific cell types, no significant amounts of mRNA coding for IL1 are observed (278). IL1 is the prototype of pro inflammatory cytokines, as it induces the expression of several genes and the synthesis of numerous proteins, which are responsible for inflammatory changes. IL1 is produced by several cell types, including macrophages, neutrophils and endothelial cells. Its effect naturally depend also on the organ and tissue, thus the surrounding of its pace of production. The immunologic properties of interleukin 1 include T and Bcell activation, induction of natural killer cell activity and initiation of the expression of several lymphokine genes (279). Furthermore some data suggest that IL1 inhibits the antibody binding to the CD4 antigens, which could also have relevance in IBD. Furthermore, the nuclear factor kappaB (NF B) can be induced by IL1 (278). This feature is important in the context of recent findings of an association between frameshift mutations in the NOD2 gene and Crohns disease. Since wildtype NOD2 protein activates NFB, while this induction was deficient in mutant NOD2 (224, 225). Although IL1 levels are usually low in the healthy, higher circulating levels are detected in women following ovulation as well as in physiological stress situations such as strenuous exercise (279). Elevated levels of interleukin1 beta have been reported in several diseases and pathologic conditions. Beside inflammatory bowel diseases these include autoimmune diseases like rheumatoid arthritis, sarcoidosis and Kawasaki disease or newly diagnosed IDDM.
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Furthermore among others, higher IL1 levels are detected in infectious diseases including HIV and AIDS, tuberculosis, chronic hepatitis B, in obstructive jaundice, alcoholic cirrhosis, in patients with periodontitis and in thermal injury (278, 280) Multiple mechanisms are working in the body against an overproduction of this central proinflammatory cytokine. Both interleukin1 and interleukin1 can bind on IL1 receptors; however signal transduction is not mediated by the soluble IL1 receptor type II. Further natural control mechanisms include the IL 1 converting enzyme, and the interleukin1 receptor antagonist. This latter is produced by actually the same cell types as IL1, and has been shown to inhibit its biologic activities both in vitro and in vivo (281, 282). Interleukin1 receptor antagonist (IL1ra), however does not block all activities of IL1. As an example, it does block the effect that IL1 exerts on synovial cells, fibroblasts and chondrocytes, and has been reported to reduce the inflammation and tissue destruction in rabbit model of colitis (282). However in vitro it does not inhibit the proliferation of Tcells and T cell responses to antigens, influenced by IL1 (283). The biological capacity of the IL1 and IL1ra are not equal. Thus 50% inhibition of the biological effects of IL1 requires 10 to 500fold excess amounts of IL1ra (283). This disparity might give an explanation for findings in the inflammatory mucosa of IBD patients showing upregulation both in the IL1 and IL1ra levels. Generally, interleukin-1 is a central element in the regulation of inflammation both systemically and at the mucosal level of the gastrointestinal tract. There is also ample evidence that in IBD interleukin1 beta respectively an imbalance of IL1 and IL1ra plays an important role. Thus enhanced circulating and mucosal levels of IL-1 have been reported in patients with IBD in comparison to healthy controls (60, 284-286), but this imbalance was not found in patients with selflimiting colitis (287). Supporting the results of human studies elevated levels of IL1 have been reported in the gut tissue of animal models of intestinal inflammation (285). Not just on the protein levels were these differences verified. Higher IL-1 mRNA steady state expressions were found in biopsy specimens from patients with inflammatory bowel disease than in the ones of normal intestine (288). Even in the gut lavage of IBD patients were IL-1 levels increased; moreover these levels have correlated with the activity of disease and were predictive for relapse (289). Furthermore, a mucosal imbalance of IL-1 and IL-1ra ratio was described in IBD (287); later it was found that the levels of IL1ra were significantly higher in IBD patients compared to healthy controls both in serum and in intestinal biopsy specimens (290). Critically seen these findings could just refer to a chronic inflammatory process in the bowel. However, more interestingly, there were also differences found in IL1 levels between patients with distinct disease behaviour. Thus non-perforating form of CD was associated with a higher expression of IL-1 mRNA in intestinal tissue when compared to
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intestinal specimens of patients with the perforating form of CD or other controls (78). Our studies have further established these results. Thus we found that CD patients having the nonperforating form of the disease are more frequently associated with an IL1 genotype that was correlated to a higher production of IL1 (291). Other studies have correlated diminished IL1ra/IL1 protein ratio with the clinical severity of Crohns disease (287). As IL1 was found to came from the lamina propria, while IL1ra largely form the epithelial fraction, this disparate contribution was accounted for the difference in IL1ra/IL1 ratios. Another, although an indirect point, to the role of IL1 in the pathomechanism of IBD, is that corticosteroids effective therapeutic tools in IBD inhibit IL1 gene expression. Beside its central role in regulating the inflammatory processes, its other effects might affect the course, severity or behaviour of IBD. As an example, higher levels of interleukin1 in Crohns disease, causing a greater induction of fibroblast activity (292, 293) would lead to the development of the stricturing form of CD (291). Similarly, the effect of IL1 on synovial cells and cartilage could offer an explanation for the arthritis often accompanying IBD. Thus IL1 induces the collagenase production in synovial cells, and increases the release of the proteoglycanases and proteases from chondrocytes. This would be in consistence with the observations that arthritis belongs to those extraintestinal manifestations that parallel the activity of IBD. Another in IBD possibly important feature of interleukin1 beta is that it is a major modulator of extracellular matrix catabolism and bone resorption. Thus IL1 was described to enhance bone resorption by stimulating osteoclast development and activity (294), and was found to decrease bone collagen and noncollagen protein synthesis (295). This is in consistence with other results showing greater spinal bone loss among premenopausal women with higher stimulated production of IL1 (296). Furthermore, another group found a significantly higher secretion of IL1 by cultured peripheral blood mononuclear cells in osteoporotic patients when compared to healthy subjects (297). In accordance with these results the naturally occurring antagonist of IL1 interleukin1 receptor antagonist was shown to stimulate bone formation and decrease the number of osteoclasts on the endocortical surfaces (298). Therefore we could hypothesise that the elevated levels of IL1 and/or a dysbalance between the IL1 and IL1ra production seen in IBD patients could effect in increased bone loss. This would be further supported by previous studies, which showed increased rates of bone resorption, but not bone formation markers in inflammatory bowel diseases (38, 39). However, the effect of interleukin1 beta and its natural antagonist on the bone metabolism is further modified by other cytokine and noncytokine factors, such as estrogens and interleukin6. As earlier suggested, estrogens can further modify the role of IL1 on bone turnover in both genders (42-44). Thus estrogens reduce

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the expression of signalling type IL1 receptor (IL1RI) in osteoclasts, while it increases the expression of decoy type IL1 receptor (IL1RII), altogether decreasing the IL1 bio- responsiveness of these cells (34). Not just interleukin1, but generally intestinal cytokines are regarded as key components in the pathogenesis of IBD. Cytokines interact in the regulation of their expression. This interaction can occur at both at transcriptional and post transcriptional level and can present in the form of autoinduction, stimulation of another cytokine or inhibition of cytokine expression. Therefore, an altered expression in the intestinal mucosa of many cytokines can be supposed, and extensive investigations indeed verified distinct changes in the cytokine profile in IBD patients. Among others interleukin1, interleukin2, interleukin4, interleukin5, interleukin6, interleukin8, interleukin10, interleukin11, interleukin12, tumour necrosis factor and interferon have been suggested to play a role in the pathogenesis of either CD or UC (299, 300). Therefore, the question arises, which factor and which cytokine could have an initiator role. As the proinflammatory cytokines, interleukin1, and tumour necrosis factor are central features of the inflammatory cascades, they make ideal candidates. This hypothesis was further confirmed by the favourable results of clinical trials using neutralising antibodies against TNF in severe forms of Crohns disease (301). Furthermore, similarly to IL1, an increased activation of TNF could offer an explanation for extraintestinal manifestation observed in IBD such as osteoporosis and arthritis. Thus TNF and IL6 stimulate cartilage destruction as well as osteoclast development and activity consequently boosting bone resorption (41, 302, 303). Furthermore genetic variants associated with higher production of the respective cytokine have been linked with a larger prevalence of osteoporosis in the studied populations (58, 59). Therefore IL1 and TNF have been favourite aspirants in studies using candidate genes for trying to define the genetic background for groups and subgroups of IBD patients. In summary, although there are mucosal and immunologic changes being more characteristic either for ulcerative colitis or Crohns disease, to date no immunologic alterations specific for either of the entities have been verified. Concerning the disease course and characteristics there are distinct subgroups both in Crohns disease and ulcerative colitis. Data is accumulating that these subgroups have also different genetic background, which could also indicate a disturbance at various points in the inflammatory cascade. Therefore performing immunologic investigations on well defined subgroups may be necessary for concordant immunologic results. 1.4.3 Gender Several studies have shown in high-incidence areas a female preponderance at a young age (20-40 years) for Crohn's disease, thus the incidence and prevalence is 20-30% higher in women than in men. At the same time, especially at older ages
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there are somewhat more men with ulcerative colitis, than women (95). Furthermore data from Scandinavian countries suggested that also age specific incidence rates were greater for males with ulcerative colitis and females for Crohn's disease (97, 98). Prevalence rates for both ulcerative colitis and Crohns disease are low in the Bedouin Arabs of southern Israel, and interestingly both entities of IBD were found to be more common in women. Moreover, have there was no male patient with Crohn's disease found (102). However, some other studies found equal gender frequencies, or sometimes even marginally significant male predominance in Crohn's disease (20, 304). Remarkably most of these studies were based on populations where differences to the typical Westernlifestyle and/or diet might be detected offereing a possible explanation for these differences. As an example, in Greece and in the neighbouring Mediterranean countries such as Italy and Spain, women tend to smoke less frequently, and take less oral anticontraceptives, which might be the case as well in Korea. (20, 305308). Not just the prevalence, but also the disease course has been suggested to be associated with the gender of the patients. Thus some investigators have found female sex as a risk factor for relapse in Crohn's disease (309-311), but not every study could confirm their results (312, 313). Inconsistencies when observing association between disease course and gender might also be due to a distinct age of disease onset in different studies. Thus another study in childhood CD recognised again a difference in the severity of the disease between the genders (77). Others seen different disease localisation in women with CD than in men, thus in women the disease involved the colon more often than in men (20) While again others found ileocecal resections and extraintestinal manifestations more frequent in female CD patients (311). Whether these differences are due to genetic or environmental factors is still unclear. A larger prevalence of Crohn's disease in Turner syndrome (314), the reported higher transmission rate from mothers to children than from fathers to children (315) would suggest a genetic background. The observed more severe disease course in smoking women in comparison with men (316) and the missing female dominance in the frequency of CD in studies from Southern Europe could on the other hand support the cooperation of genetic and/or other intrinsic factors as well as environmental features (20, 305-308). Actually the most likely mechanisms include several issues such as gender (related differences in the immune responsiveness, sex steroid effects, and gender-related genetic factors. As an example, estrogens were demonstrated to decrease in certain cell types the bio-responsiveness on interleukin-1, a major proinflammatory cytokine playing a significant role in the pathology of IBD (45). It is in consistence with earlier results indicating that plasma IL-1beta binding capacity differs between men and women, and a main contributing factor is the soluble interleukin1 receptor II (317). Nevertheless, there is a sex bias present in other syndromes where impaired immune mechanisms play a significant role in the disease expansion. In several
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autoimmune diseases differences in the disease prevalence as well as in the prognosis and clinical course have been seen (318, 319). On the whole, in the United States about 80 percent of those suffering from autoimmune diseases are women. Multiple sclerosis, rheumatoid arthritis and myasthenia gravis are all more prevalent in the female than in the male population. At least one part of the explanation for these findings is that women produce a more vigorous immune response to infection. Thus when challenged by an antigen, men tend to have more like a Th-2 immune response, while women more like a Th-1 (320). These findings together with those results suggesting a Th-1 immunologic pattern in CD, while Th-2 in UC, are actually in consistence with the gender bias seen in CD and UC. As for the role of hormones, estrogens, progesterone, cortical and even testosterone can induce anti-inflammatory mechanisms. On the other hand, sex steroids dominating in women might also contribute the onset of the disease. For example in Myasthenia gravis there is a characteristic peak of disease onset in young women in their second and third decades, thus when the concentration of these hormones are highest (321). The average age of onset of multiple sclerosis is also different between the two genders, men getting the disease at a later age than women (322, 323). Furthermore females were found to be more frequently IL1RN*2 carriers than males (324) Thus a parallel could be drawn with ulcerative colitis, where the incidence in older men is higher when compared to women. On the basis of these results we could also hypothesise that the contradictory results of studies investigating the role of oral anti-contraceptives in IBD were partly due to this two-sided effect of female sex hormones. Further, we could speculate that as pills with lower hormone concentration and different composition became widespread, pathogenic effects previously seen with "heavier" pills have changed or disappeared. On the whole, sex-related hormones and genetic factors might modulate the immune system and thus modify the probability for the development and progression of the disease, in a combination with genetic and environmental factors. 1.4.4 Genetic factors There is increasing evidence that a genetic predisposition is present in inflammatory bowel diseases (325-329). Furthermore, data is accumulating that this predisposition influences not only the development of the disease, but also the disease extent, extraintestinal manifestations and possibly the disease course (81, 82). Information suggesting that genetic background plays a role in the development of Crohns disease and ulcerative colitis include the results of epidemiological, family and twin studies as well as complex segregation analyses. As discussed above the frequency of both ulcerative colitis and Crohns disease differs among diverse ethnic groups. Thus the prevalence is the highest among Whites, while lower in Blacks and even smaller in Asians (91). There are also significant differences within the white population: the occurrence of IBD is
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highest in Ashkenazi Jews (330), while lower in the in the compound Jewish population living in Israel (331, 332). Furthermore differences were found within the Jewish population living in the United States. Thus the disease was more prevalent among those originated from middle Europe than among those from Poland and Russia suggesting that Jewish patients with inflammatory bowel disease probably represent a non-random genetically predisposed subset of the Jewish population (333). Interestingly, a recent multicenter study including centres from northern as well as from southern Europe indicated substantial differences within Europe, however not in a northsouth axis manner as anticipated (92). Whereas the epidemiological clustering of cases could be due to an effect of either genetic factors and/or common environmental influences, family, and especially twin studies provide clearer evidence for the role of hereditary factors in IBD. Familial aggregation of IBD has been verified by several studies. These include investigations comparing the frequency of familial and sporadic IBD cases, while another approach is to inspect the prevalence of the disease in the relatives of IBD patients. Some investigators estimate that 25% (327, 334-336) while again others found that between 1030% (326, 337, 338) of individuals with CD or UC will have at least one first-degree relative affected. The population relative risk calculated from the prevalence of IBD in the relatives of CD and UC patients and in the background population is around 1015, being somewhat higher in the relatives of CD than that of UC patients (327, 328, 338). Again, in Jewish patients the familial occurrence was found to be significantly higher, than in nonJewish patients living in the same geographical area (339). Although the relatives of patients with both CD and UC tend to have the same disease entity, there is no specificity for a given form of IBD within one family. Even a monozygotic twin pair has been discovered to present with discordant disease entities (340). Concordance rates for CDCD versus CDUC respectively for UCUC versus UCCD were detected to be about 7290% (82, 123, 336, 338). Familial aggregation of several diseases could occur partly due to common environmental and lifestyle factors. However, this does not seem to be the case in IBD, as the prevalence in spouses was generally not elevated (334, 341, 342). On the other hand, in the geographical area of Northern France and Belgium 28 couples with IBD had been registrated in which either both partners got the disease after cohabitation or one of them had IBD before cohabitation and the other experienced first symptoms afterwards (343). This finding together with another report on three close collage friends getting the disease (344) might refer to the role of the environment in the expression of the disease. However, as such reports are rare, and patients numbers included are low, aggregation of diseased individuals could be accidental. Twin studies are those genetic epidemiological studies that make the most sensitive differentiation between genetic and epidemiological factors. Monozygotic twins share 100% of their genes, while dizygotic twins like other
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siblings share 50% of their genes. As normally twins live in the same environment during their childhood, higher concordance rates for monozygotic twin pairs in comparison to dizygotic twin pairs would point to a more explicit role for genetic factors in the disease etiology. Simultaneously, as twins may share environmental factors more extensively than siblings, higher concordance rates in dizygotic twins versus ordinary siblings imply a greater role for environmental than for genetic factors. Twin studies on IBD showed a significantly greater monozygotic twin concordance (40-60%) compared to dizygotic concordance (2-14%) in Crohns disease, combined with comparable DZ and sibling concordance. Simultaneously, in ulcerative colitis the respective values for monozygotic twin concordance (6-17%) and dizygotic concordance (0-5%) indicate a lesser role for genetic factors when compared to CD (325, 345-347). Unfortunately, due to the limited number of candidates, there are relatively few twin studies performed. However in order to delineate the extent of environmental effects an exciting opportunity would be to examine those monozygotic IBD twin pairs and their environment that grew up apart. Another interesting point is the finding of several studies that children of patients with IBD have an earlier age of disease onset, however not significantly more severe disease than their affected parents (123, 338, 348, 349). In all of these studies a difference of about 16 years could be observed. However the median age of the parents was 3338 years, which is higher than reported in the sporadic cases. Recently a multicenter study including 111 CD families with 62 affected child-parent pairs found a significant correlation between age of onset and the calendaryear of birth, indicating an overall population trend over the last decades (350). Therefore, the original findings could be probably explained by a selection bias, thus patients with a higher age of onset and/or a less severe disease course would have more offspring. Besides, there are also timely changes in the general IBD population in respect to the age of disease onset, and disease course. Thus the phenomenon above is presumably not genetic anticipation, but an outcome of contribution of environmental factors. We should also raise the question whether genetic susceptibility for specific disease course and presentation exist. In respect to this subject family studies are somewhat inconsistent. Thus LennardJones and coworkers examining 213 family members with IBD belonging to 67 families found no significant similarities as to clinical appearance and course within families (123). Another group studying patients with CD found no concordance for disease behaviour. On the other hand this latter group reported a concordanec for localisation, and within generations also for the age of onset. These findings were most explicit between siblings (338). Again others detected greaterthanexpected concordance for site and clinical type of Crohn's disease within individual families (82). Comparable results were shown by Jewell and coworkers where parentchild repectively sibling pairs were found to be concordant for disease type, extent and extraintestinal manifestations (81). In addition to the family studies, case control
49

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studies indicated that there might be distinct genetic groups both in CD and UC (84, 351). Furthermore, statistical clustering of unselected population of CD patients confirmed the presence of well defined subgroups (352). The reason for the lack of agreement of family studies regarding disease presentation might be that several genes contribute to the expression and the presentation of IBD. Thus some of these genes would be clear etiologic factors, necessary to the disease manifestation in certain groups of the patients. These genes would be the ones shared by affeced family members in a larger amount. Beside these the presence of genetic variations at other points of the genome would affect the disease course and presentation by influencing regulatory mechanisms such as cytokine production. These genes not being necessary for the manifestation of the disease are probably only present in a smaller number of the affected family members. These genes however would be overrepresentated in those IBD population and family subgroups that present with the respective disease appearance or behaviour. In summary, these results indicate that in these multifactorial diseases genetic factors exert a major pathogenic role, whereas there seems to be a difference in the extent of genetic influence between UC and CD, indicating a lesser role in patients with ulcerative colits. Genetic epidemiologic studies, seggregation analyses and recently the reults of the genomewide linkage studies point to a polygenic etiology, with minor genes excerting incomplett penetrance.

1.5 Susceptible genes for Inflammatory Bowel Diseases


The findings of genetic epidemiologic stuides together with the results of seggregation analysis and genomewide linkage studies indicate that IBD represents a complex, non-Mendelian genetic disorder, defining distinct subgroups in both common disease entities. Seggregation analysis has postulated that multiple susceptibility genes contribute to IBD, with some of them common to CD and UC. The presence of shared susceptibility genes between CD and UC is suggested by the relatively modest cross-disease relative risks of 3.85 and 1.72, given a CD and UC proband, respectively. It is postulated that opposed to Mendelian disorders where a single, critical mutation is sufficient, in IBD any single mutation/polymorphism is insufficient to cause disease. Recent results on genes found to be associated with Crohns disease seem to somewhat controvert this idea, however, none of the polymorphisms have been found to be associated with the whole CD population, and the subgroup of those patients possessing the mutation was not clinically characterised (224, 225). Furthermore, these patients could definitely have other, still noncharacterised mutations. The idea that defects at distinct genes could result in a disease with similar presentations, identified as one disease is not unusual. Having a closer look on the divergent disease presentation and course of patients a diversity in the genetic causative background is through plausible.
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Furthermore, genetic engineering in mice of a broad array of different genes (interleukin-10, interleukin-2/R, T-cell receptor, tumour necrosis factor (TNF), multidrug resistance gene, N-cadherin, HLA-B27) can result in a stereotypic, IBDlike picture. Genetic heterogeneity may similarly exist in humans, where different subsets of genes in different patients result in similar disease expression. Moreover, host-responses are affected by environmental factors, including specific characteristics of intraluminal bacteria. Again, seen in other diseases, there are large interindividual differences as to the extent of the influence of a specific environmental factor. Actually the presumption that in Crohns disease and maybe in ulcerative colitis similar phenotypes would appear as an effect of different mutations or distinct environmental factors is not unusual. A somewhat similar phenomenon is experienced in diabetes mellitus and even more expressively in most of the hereditary metabolic diseases, such as the Hurler disease (353). The recent finding suggesting that multiple genetic variations in the NOD2 gene and probably in its surrounding could separately contribute to the evolution of Crohns disease would support this idea (224, 225). Furthermore, the assumed gender differences in Crohns disease that recently receive a growing attention could be partly reasoned by a multifactorial inheritance. As an example in hypertrophic pylorus stenosis the genetic threshold is higher in women, resulting in larger number of men presenting with the disease (353). With respect to Crohns disease the genetic or maybe the environmental threshold in women would be higher for some while lower for other genes than in men, eventuating in differences in the typical disease presentation. Whether these variances are due to the differences in the hormonal regulation, immune functions or the X chromosome is still not known. As to the inconsistencies of family studies with respect to concordance in disease presenation the phenomenon of phenotype differences within the same family is wellknown even for disorders with major dominant genes (353): the effect of major genes can be influenced by environmental factors and genetic components resulting in an altered gene expression. Compared to Mendelian diseases, in which coding region mutations with obvious effects on protein function predominate, the nature of specific risk alleles will be fundamentally different for complex genetic disorders. Thus in the pathogenesis of complex genetic disorders alterations in noncoding regulatory regions with subtle effects on protein expression may constitute a significant component. Further, until specific risk alleles within disease genes are firmly established for IBD, the precise relationship between familial and the more common sporadic cases cannot be defined. Approaches to Identifying Disease-Associated Genes There are two different techniques used to identify genes associated with a certain disease. Genomewide linkage studies are trying to identify chromosomal regions of excess sharing within families with multiple affected members. These
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Chapter 1

screenings use anonymous informative markers throughout the genome to search for the chromosomal location of susceptibility genes. This technique is hypothesis free, thus there is no need for a previous presumption on the pathogenic mechanisms involved or a model of disease inheritance. A drawback of these studies that they require a large number of study subjects, in this case families. As either Crohns disease or UC are very common diseases and familial aggregation is only found in about 10 percent of the patients, families from different ethnicities have to be considered together. However, several studies indicated that both diseases comprise different entities, which might have different genetic background (81, 84, 351). Knowing the rather large variations in the prevalence of IBD in different populations, diversity in the genetic background based partly on ethnicity can not be excluded (112, 113). Furthermore, an identified chromosomal region still contains a large number of genes, from which the ones causatively associated with the disease have to be further detected. Candidate gene studies on the other hand, test specific risk alleles or mutations within candidate genes, and determine if there is an increased prevalence in patients compared to control population. Traditionally these genes have been selected on the basis of their putative functions such as immune regulation while now these include positional candidate genes that lie in areas of linkage. The obvious disadvantage for these types of studies is the large number of possible candidate genes. On the other hand, these studies are feasible to perform on somewhat smaller, but well defined, ethnically homogenous patients subgroups. Anyhow recent achievements (224) point to the fact that future research will be most effective when combining these approaches. Thus candidate genes within chromosomal regions of linkage for the disease entity or for a specific course will provide aspirants for further analysis. 1.5.1 Candidate Gene Association Studies Considering the central role of immune mechanisms in the pathogenesis of both ulcerative colitis and Crohns disease genes participating in the regulation of the immune and inflammatory response provide ideal candidates for etiologic investigations. Therefore many association studies have been concentrating on the role of HLA and cytokine gene polymorphisms. HLA genes The major histocompatibility complex (MHC) in men called the human leukocyte antigen (HLA ) complex located on the short arm of chromosome 6 is crucial in antigen presentation, thus in the immune response of the individual. While Class I molecules (HLAA, HLAB, HLAC) present endogenously synthesised peptides, class II molecules (HLADP, HLADQ, HLADR) show peptides derived from the environment to CD+ cells. These HLA molecules are highly polymorphic, while also showing typical patterns in distinct ethnicities. Therefore it is actually not surprising that data analysing HLA class I and II
52

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antigens in relation to IBD in relatively small groups of patients are largely inconsistent. On the whole, these studies have been more heterogeneous for Crohns disease, suggesting that HLA antigens might have a larger impact on the evolvement of ulcerative colitis. Considering the current theory on the pathogenesis of IBD, HLA class II genes could be regarded as better candidates for association studies. However, Japanese studies found an association with HLAB52, a subtype of HLAB5, in patients with UC (354-356), and by pointing out that a linkage with A24Bw52DR2DPw9 haplotype exist, suggested that the HLAB locus itself plays an important role (357). Other groups described associations usually with HLADR types and subtypes. Thus HLADRB1*1502, a subtype of DR15 (HLADR2) was suggested to be primarily associated with UC and its progression (356). Some studies of USA and Western Europe have reported increased prevalence of HLADR2 as well (83, 358, 359). Beside others not confirming an association with this subtype, two studies reported a decreased prevalence of HLADR2 (329, 360-363). Some groups have suggested additional protective alleles with decreased frequency in UC patients, such as DRB1*04 (329, 358, 364, 365). Studies on HLA class II genes in Crohns disease have revealed positive respectively negative association with a large number of alleles, but none of these results is widely confirmed (366). Later a metaanalysis performed suggested that both ulcerative colitis and Crohn's disease were associated with specific HLA class II phenotypes. Thus in this meta-analysis a positive association with DR2, DR9, and DRB1*0103 while a negative with DR4 for ulcerative colitis was reported. Furthermore the group has also identified positive association with DR7, DRB3*0301 and DQ4 while negative association with DR2 and DR3 for CD (367). Recently, a study in Japanese CD patients indicated again another allele, the HLA-DRB1*0405 to contribute to the susceptibility to CD (368). Recent results provided evidence to the idea that the large number of HLA alleles suggested to take part in the pathogenesis of IBD is partly a consequence of ethnic differences. Thus beside identifying DR3 haplotype as being protective against Crohns disease, they found that an association with DR15, normally present in UC patients, was observed in the Jewish, but not in the non-Jewish CD group (369). Other findings suggesting that an interaction between DR and DQ determine the extent of disease risk (370) are very much in accordance with the detected minor effects of the HLA genes as well as their functional interaction. Although HLA genes do not seem to have a major effect on the evolution of inflammatory bowel diseases, association might be stronger or only apparent in specific disease subgroups. This could indicate an effect in influencing the disease course either by HLA genes or by genes in linkage disequilibrium with them. One of the first studies indicating the necessity of dividing patients into subgroups demonstrated a difference in the genetic background between pANCA positive and negative UC patients (371), however these results could not be later confirmed (372). Others found HLADRB1*0103 to be correlated with extensive
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ulcerative colitis (65, 329, 373), while an association between HLADR3, DQ2 was only recognised in females with distal disease. Masuda and co-workers found that in the Japanese population DRB1*1502 was significantly more frequent in intractable UC patients, whereas DRB1*1501 in cases with a more benign disease course (374). These results have been confirmed in Dutch patients (83) as well as in an English patients population among those who underwent colectomy for extensive disease (65). Furthermore, association have also been detected with IBD subgroups with distinct arthritic manifestations. Thus in the subgroup with dominantly large joint acute arthritis individuals with HLAB27, B*35 were more frequently found than in the normal population, while among those with small joint chronic polyarthritis carriers of HLAB*44 were overrepresented (351). These results altogether suggest that HLA genes might have a minor role in the etiology of IBD, influencing the disease course and onset in ethnically matched subgroups with specific disease presentation. Clustering of nonMHC susceptibility loci between different autoimmune diseases such as Crohn's disease, multiple sclerosis, psoriasis, asthma, and type 1 diabetes has been observed. This clustering of loci supports the hypothesis that distinct autoimmune disorders may have some common susceptibility genes (375). However, in contrast to other immune disorders such as celiac sprue and insulin-dependent diabetes mellitus, the role of the major histocompatibility complex (MHC) was not found to be dominant. This suggests that nonMHC genes might have a stronger pathogenic role in IBD. Beside HLA genes, genes encoding cytokines are in the limelight of candidate gene studies. Indeed, stable interindividual variations in the production rates of several cytokines have been reported. These differences or some of them might be related to genetic variations in the regions of cytokine genes that influence their transcription, mRNA stabilisation, translation or receptor binding (376, 377). Moreover, alterations in the genes of other, regulatory proteins influencing cytokine gene expression could also affect the rates of protein production. In inflammatory bowel diseases a dysregulated immune response could be observed both on systemic and mucosal level (284, 286). Due to the longstanding inflammation notable changes in the levels of several cytokines are detected (60). Therefore cytokine gene polymorphisms provide ideal candidates for association studies for Crohns disease and ulcerative colitis. As a central proinflammatory cytokine interleukin-1 and its natural occurring antagonist, the interleukin1 receptor antagonist plays an elemental role in the initiation, regulation and termination of the inflammatory process. As IL1 levels were found to be elevated and the IL1/IL-1ra ratio dysregulated in patients with IBD (60, 284, 286, 287, 290), furthermore there seemed to be genetically determined differences in the production of these proteins (377), the IL-1 family became a preferred candidate for association studies.

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IL1 gene family The IL1 genes, IL1A, IL1B and IL1RN, are located in close proximity to each other within 430 kb on chromosome 2q12-21(378) (Figure 1).

IL1 gene family

40-110 kb

220-320 kb

IL1A

IL1B

IL1RA

Chromosome 2 q12-21

Figure 1. The loci of IL1 and (Il1RA) IL1RN genes are closely linked in chromosome 2 Various sites of single nucleotide polymorphisms (SNPs) have been described in the IL1 genes. When we started our studies four SNPs had been described in the IL1B gene. The restriction enzyme TaqI cleaves a327 bp product at position +3953 of the IL1B gene into two fragments of 211 bp and 116 bp, this constitutes allele 1. A C T substitution at this position avoids the action of the TaqI restriction enzyme and let the 327 bp product intact (allele 2). This polymorphism seems to be correlated with IL-1 secretion in vitro (379). Another C T substitution at the 511 position in the promoter region of the IL1B gene results in two RFLP (381) (Figure 2.). Genetic heterogeneity in the IL1RN is caused by a variable number of tandem repeats (VNTR) of 86 bp in intron 2 (381). Later other SNPs have been described in IL1RN gene at the positions +1731, +1821, +1868, +1887, +8006, +8061, +9589, (382), and +1934 (383) but these alleles are in linkage disequilibrium with the VNTR polymorphisms (383-385). Some reports have found higher production of IL-1ra in the plasma and by the monocytes of patients with the rare allele 2 of the VNTR in intron 2 (386, 387), but carriage of IL1RN*2 was associated with decreased production of total IL-1ra protein in cultured PBMCs from both UC patients and controls (388).

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Location of the polymorphisms in the IL1B gene

Promoter region

Exon 5

TATA box Position 31 Position 511

Position +3953

Figure 2. Several gene polymorphisms in the IL1B gene There are inconsistencies in studies associating the presence of specific alleles in the IL1 gene with the production of IL1 and IL1ra (290, 377, 379, 388-391). These differences could be partly explained by different methodology applied in the experiments. On the other hand genetic polymorphisms might differently influence the basal and activated levels of interleukins in distinct cell types. Besides cytokine production is regulated by complex factors, such as the local environment of the specific cells, thus divergent IL1 production rates might be observed in the bowel and in the plasma. This could be in vivo further affected by the extent of IL1 receptor density or downregulation. Additionally, linkage disequilibrium between distinct genetic polymorphisms in the IL1 gene couldfalsely suggest a geneproduction effect. However, on the whole these data suggest that genetic polymorphims in the IL1 gene have a regulatory effect on basal and activated IL-1 production. At present, however, it appears that these polymorphisms do not regulate directly the secretion of IL-1 and can be used as disease markers rather than functionally relevant. Very recently, after completing the chapters of this thesis a functionally relevant gene polymorphism has been found in the promoter region of IL1B. It is in a TATA-box region that markedly affects DNA-protein interactions in vitro (392). Fortunately for the studies reported in this thesis this polymorphisms, which is located in the -31 position of IL1B gene, is in tight linkage disequilibrium with another polymorphism in the promoter region at -511 position studied by us. The IL1B-511T (allele 1) variant is in positive linkage disequilibrium with the IL1B-31C allele (393, 394), while the greater DNA binding of the IL1B-31T-bearing oligonucleotide represents the wild-type IL1B511C (allele 2).
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Interleukin1 plays a central role in the inflammatory mechanisms, as well as in the regulation of the connective tissue differentiation and in distinct hormonal and neurological functions. Furthermore large interindividual variations were found in the production of IL1 in healthy populations, and these variations have been associated with distinct genetic polymorphisms in the IL1 gene. Therefore several candidate gene studies investigated the possible association between this gene and various diseases with unknown etiology. Recent studies suggest that polymorphisms of the IL1 gene complex may be significant severity factors in a number of multifactorial, often chronic inflammatory disorders. As an example a specific IL1 genotype that was associated with high levels of IL-1 production has been linked to the severity of periodontitis in non-smokers (395, 396). Furthermore, antibody responses to periodontal microbiota were also found to be different between specific IL1 subgroups (397). On the other hand in smokers disease severity was not correlated with genotype these patients possessing this other already well-known periodontitis risk factor (395, 396). Bearing in mind the osteoclast and chondrocyte activating functions of IL-1, other studies investigated arthritis and bone loss in relation to the IL1 gene. Two English groups have found that IL1B polymorphisms are associated with the severity of rheumatoid arthritis (398, 399). Likewise, Cox and co-workers showed a linkage between the presence of bone erosions in rheumatoid arthritis and the IL1 gene cluster, while this linkage was not present in the non-erosive form of disease (57). Other groups presented similar results investigating the etiology of osteoporosis. Keen and coworkers suggested that allelic variations at the IL1RN gene are associated with early postmenopausal bone loss of the spine (56). Members of the IL1 family play an important role in the regulation of bone formation, and there is evidence that IL1B genotype influences the course of IBD (63, 291). Therefore genes involved in the control of their activity provide as well candidate loci for an increased bone loss in IBD. Thus our results indicating that carriers of IL1B511*2 (Figure 3.) have a higher risk to present with low bone mass in IBD would confirm that the inflammatory process alters the regulation of bone resorption by a long-term upregulation of IL-1. Furthermore, in our healthy control population no association between bone mass and IL1B polymorphisms was seen, which is in consistance with the findings of Langdahl and coworkers. They reported that in their healthy controls respectively in the population of patients with osteoporosis, but not with IBD polymorphisms in the IL-1beta gene are not associated with osteoporotic fractures or alterations in bone mass or bone turnover (383). On the other side IL-1ra VNTR polymorphism were significantly more frequent in the gruop of English osteoporotic patients (56.2%) compared with age-matched normal controls (43.3%) The relative risk of osteoporotic fractures was increased, while BMD of the lumbar spine was reduced in these individuals. The difference in bone mass between the genetic subgroups tended to increase with increasing age, which finding would explain the lack of this association in our relatively young IBD patients and age matched controls.
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1 .1 (1 0 6 b p , 1 9 9 b p )

1 .2

2 .2 (3 0 5 b p )

Figure 3. Bi-allelic gene polymorphism at position -511 of IL1B gene Most of the studies investigating the effect of different members of the IL1 gene on distinct diseases suggest that genetic variations influences the course, but not the onset of the disorders. As an example the presence of diabetic nephropathy in insulin-dependent diabetes mellitus, but not IDDM itself was found to be associated with the IL1 gene (401, 402). The carriage of IL1RN*2 allele was linked to a significantly poorer longterm outcome of another kidney disorder IgA nephropathy (403). Findings suggesting a more severe course in acute pancreatitis (404) and in preeclampsia (405) among those being carriers of specific IL1RN genotypes might be explained by minor antagonistic effect on the proinflammatory IL-1beta. While other results showing higher prevalence of liver cirrhosis among those alcoholic patients being carriers of IL1B511*2 (406) probably account for the fibroblast activating capacity of IL1beta. Another effect, concerning the regulatory role of IL1beta in the central nervous system, may explain the association between the IL1 gene cluster and the onset and severity of psychiatric diseases such as schizophrenia (407) or Alzheimer disease (408). Interestingly in another disorder of the neurological system, in multiple sclerosis gender seems to contribute to the disease development among carriers of IL1RN*2. Thus in the MS cohort, females were found to be more frequently IL1RN*2 carriers than males, and among IL1RN*2 carriers the relative frequency of women with MS was higher than that of control women (324). These latter findings could be of large interest in respect to IBD, hence evidence is

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accumulating that gender plays an important role in the disease course of IBD, however the extent and exact significance is not yet understood. Recently published studies has shown example for an interaction between the interleukin-1 gene cluster, the presence of an infectious agent and additional host factors towards progression to disease. Thus the IL1B31T allele was found to be associated with a greater vulnerability to persistent Helicobacter pylori infection, and this vulnerability is modified by smoking. (394). Another study indicated that this allele is linked to an increased risk of both hypochlorhydria induced by H. pylori and gastric cancer. (393). Also other results indicated that IL1 genotypes play a significant role in the outcome of infections. As an example those HIV patients being homozygous for IL1RN*2 had a lower median level of HIV RNA than carriers of IL1RN*1 (409). On the basis of these studies we could hypothesise that IL1 gene influences the severity and course of different diseases by controlling the production of interleukin1beta and interleukin1 receptor antagonist, whereas additional genetic, environmental and lifestyle factors or infections would determine if any of these diseases develops. Similarly to the diseases above, several studies have found association between the IL1 gene cluster and the presence or course of inflammatory bowel diseases. An association between the allele 2 of the IL1RN gene VNTR polymorphism in intron 2 and ulcerative colitis was first reported in 1994 (410), and has subsequently been investigated both in Europe and in the US. While these findings were later confirmed in UC patients with extensive disease in another British population (65), they could not be confirmed in Dutch patients; however, a trend in patients with more severe UC was detected (372). On the other hand neither in a Southern German nor in our Hungarian population studied could an association be identified between IL1RN gene polymorphism (Figure 4.) and ulcerative colitis or pancolitis. (410, 411). An explanation might lie in the divergent UC severity thus in the population studied by us there were only 2 patients needing colectomy. This hypothesis is supported by the findings of Heresbach and coworkers reporting a higher frequency of IL1RN*2 only in the surgically treated patients (412). A recent study involved 320 patients with ulcerative colitis and 827 ethnically matched controls and at the same time it has performed a metaanalyses on IL1RN*2 and ulcerative colitis. They detected only a minor association in the undivided UC group as well as in those with extensive colitis. It suggests that beside the small genetic risk due to the IL1RN gene additional genetic or environmental factors facilitate the evolvement of ulcerative colitis (413). As the final outcome of interleukin-1 effects depends both on the IL1beta and the IL1ra levels, simultaneous consideration of genetic polymorphisms encoding these proteins might be important. Bioque and coworkers found that among IBD patients noncarriers of IL1B*2 (Figure 5.) were more often present in the subgroup of patients carrying the IL1RN*2. By contrast, no association of these alleles was detected in the group of healthy controls.
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IL 1 R N g e n e , V N T R p o ly m o r p h ism in in tr o n 2

bp

A lle le (b p )

600 500 400 300 200 100

3 1 4 2

(5 0 0 b p ) (4 1 0 b p ) (3 2 5 b p ) (2 4 0 b p )

1 .1

2 .2

1 .2

1 .3

2 .3

1 .4

Figure 4. VNTR gene polymorphisms in the IL1RN gene

IL1B gene, position +3953

bp
400 300 200

Allele

2 ( 249 )

(135)
100 ( 114 )

1.2

2.2

1.1

Figure 5. Bi-allelic gene polymorphism at position +3953 of IL1B gene

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These results suggest that the IL1B/IL1RN allelic cluster may participate in defining the biological basis of predisposition to chronic inflammatory bowel diseases (63). Although these findings have been confirmed on a Hungarian population (411), no such association was detected in Southern German IBD patients (414). Genetic polymorphisms in the IL1B gene might have a coregulatory effect on the cytokine production (389, 390). Therefore we have investigated whether specific associations of these genes exist in CD or UC. Furthermore we aimed to know if these associations would determine the disease course. In patients with ulcerative colitis there was a higher frequency of IL1B 511*2 among those with a diarrhoeal leading symptom. Moreover in CD patients an allelic combination of the two genes significantly separates those with non fistulising course of disease (291). Distinct prevalence rates in dissimilar ethnicities as well as an involvement of multiple genetic factors and environmental agents in the disease etiology could explain the inconsistencies of studies relating IL1 genotypes to IBD populations. Thus there might be parallels between the findings IBD and the different contribution of smoking in distinct disorders. As an example, smoking has a role in Helicobacter pylori related hypochlorhydria, while in the etiology of periodontitis genetic factors and smoking seem to act apart. Beside different dietary and lifestyle habits and divergent occurrence of infectious agents the frequency of allelic variations might differ in distinct populations. For instance whereas in individuals northern European heritage IL1B+3953*2 allele confers a susceptibility factor for localized juvenile periodontitis, taken the high frequency of the IL1B+3953*1 allele in the African-American population, this polymorphism can not provide valuable diagnostic or predictive information for the disease (415). Similarly, in Caucasians with juvenile idiopathic inflammatory myopathy the IL1RN*1 allele was found to be a risk factor, whereas in African-Americans the IL1RN*3 allele was associated with the disease (416). This suggests that distinct alleles may confer susceptibility for the same disease in different ethnic groups. Other data suggested that the frequency of the pro-inflammatory IL1B511T and IL1B+3954C alleles were significantly higher among Taiwanese compared with Caucasians. At the same time the frequency of a novel polymorphism with 3 nucleotide substitutions in the IL1RN VNTR 2-repeat allele was significantly lower in Taiwanese than in Caucasians (417). Again others found that the IL1B 3954T allele was more frequent in the SouthAfrican white IBD population than in the white healthy controls, while no such association was found in the black population (418). Other candidate genes involved in the control of the immune response Beside the genetic variations in the interleukin-1 gene family the possibility of a linkage with other cytokine gene polymorphisms have been investigated in inflammatory bowel diseases. On of them is the gene encoding the tumour necrosis factor (TNF). The explanation for this lies in the function of TNF, its
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chromosomal location as well as the alterations in its expression in IBD. Thus the TNF gene is located on chromosome 6p within the MHC region. Multiple linkage studies have demonstrated suggestive evidence for linkage in this region (419). Increased expression of TNF has been observed in IBD (60, 61) and anti-TNF antibodies constitute a major new means of treating Crohn's disease (420). Furthermore, a murine model similar to Crohn's disease resulting from deletion of an AT-rich region in the 3' untranslated region of the TNF gene increasing the expression of TNF has been reported. However, association studies in IBD patients at selected TNF variants have demonstrated conflicting results (421). Investigators from Los Angeles have found a particular haplotype TNFa2b1c2d4e1 defined by five TNF microsatellite loci (TNFa, TNFb, TNFc, TNFd, and TNFe), that was present in 24% of patients with CD,while only 4% of patients with UC and 7% of control subjects (64), however later on these results could not be replicated. Recently a Japanese group has investigated the effects of newly identified polymorphisms within the upstream promoter region of TNFA gene that may affect the transcriptional activity. They found that TNF gene polymorphisms -1031C, -863A, and - 857T are positively associated with CD. Furthermore, among the subgroups of CD small bowel disease showed the highest frequencies, suggesting that these alleles may influence not only the susceptibility to CD but also the disease location (422). Later, another Japanese group has partly confirmed these findings showing that in CD the TNFA promoter haplotypes TNFA-U03 (-1031C, -863A, -857C) was significantly increased, while TNFA-U04 (-1031C, -863C, -857C) was significantly decreased (368). Nevertheless yet no data on other ethnicity confirmed these findings. Another group investigating the effect of a single base pair polymorphism located in the promoter region of TNF gene, found that the frequency and carriership of allele TNF308*2 was significantly higher in steroid-dependent Crohns disease, and tended to be higher in colonic and in fistulising disease. Furthermore patients carrying at least one copy of allele 2 were found to produce slightly more TNF-alpha at the colonic level (423). Somewhat incocnsistently again others found no significant differences in TNFA and LT alpha haplotype frequencies between patients and controls overall. On subgroup analysis however, haplotype TNF-2 was more frequent in women with extensive colitis compared to distal colitis. This difference was even greater for the combined TNF-2-LT alpha-2 haplotype. The TNF-2 and LT alpha-2 haplotypes were associated with higher TNF production in CD patients (424). The interpretation of these studies is not easy. The diveregent associations suggested by different studies are either consequences of the presence of a complex haplotype, or they are just markers for other possibly variable genes in association with specific subgroups of IBD patients. Not just interleukin1 and tumour necrosis factopr, but several other cytokines might determine the susceptibility of IBD mucosa and facilitate tissue repair. Transforming growth factor beta (TGF) plays a central role in intestinal
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epithelial wound healing (425), and increased levels of plasma TGF levels have been associated with augmented fibrogenesis (426). Genetic polymorphisms in the gene encoding TGF were found to be associated with the active and activatable serum levels of the cytokine (427). Although the functional role of TGF induction of fibrosis respectively regulation of wound healing are important features in the disease behaviour in IBD no significant associaitons were found between polymorphisms in the TGFB1 gene and CD, UC or their subgroups (428, 429). Due to the chronic inflammation levels of several cytokines are upregulated in inflammatory bowel disases. Therefore functionally relevant polymorphisms in genes encoding these cytokines provide attractive candidates for association studies. As an example the gene encoding the proinflammatory cytokine interleukin6 maps to an area of chromosome 7 known to be significant for susceptibility to inflammatory bowel disease. Nevertheless no significant differences were apparent in the allele, genotype and carrier frequencies of a functionally relevant polymorphism in the promoter region IL6174 between IBD patients and controls (430). Another group noted a modest increase in the frequency of the IL6+4470*G allele in CD and UC patients as compared to controls. However this and another polymorphism located in the 3' flanking region of IL6 was not found to be functionally relevant (431). Therefore the weak association might only indicate linkage disequilibrium with a susceptibility gene. Another cytokine raising large interests is interleukin10. It has anti-inflammatory effects and is known to downregulate the production of Th1-derived cytokines, thus excerts a key role in the regulaiton of mucosal inflammation. The role of four gene polymorphisms in the promoter region at positions 592, 627, 1082 and 1117 have been evaulated (432, 433). Although an excess of the functionally relevant 627A allele was observed in patients with left-sided colitis compared with controls suggesting that IL-10 may influence the extent of the disease, later these results could not be replicated in a newly recruited group of patients with UC. No significant differences were apparent either in the allele frequencies or in the haplotype frequencies of any other polymorphism, suggesting that these sites do not contribute to the susceptibility or severity of IBD. Based on findings of mice colitis modells, also interleukin-4 is suggested to play a critical role development of Crohns disease. Functionally significant polymorphisms have been described at position -34 in the IL-4 gene and at codon 576 in the IL-4 receptor gene. Alleles in the IL4 and IL4 receptor gene resulting in high IL-4 transcription and enhanced signalling activity were found to be associated with Crohns disease (434). However a study carried out in a Finnish populaiton could not verify these results (435). Therefore further studies in other populations and a accurate defininition of patinets subgroups associated with these polymorphism are still necessary. Further posititve associations have been reported for polymorphisms within the intracellular adhesion molecule (ICAM1) located in the chromosome 19p13
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(436). Posititve data also exist for the genes encoding the natural resistance associated protein (NRAMP1) located in the chromosome 2q35 (437), for the gene encoding 1 and 2 kinin receptors (438). Association exsist between certain CD groups while not between UC and the genes encoding the TAP molecules (transporters associated with antigen processing) (439, 440). However, further replication studies as well as functional studies are still necessary to judge the significance of these findings. A recent study suggested an association between genetic variations in MICA (MHC Class I chain like gene A) and disease behaviour in ulcertaive colitis (441). The exact function of these polymorphisms is not yet known, but they may affect the interaction with T-cells. Beside functional candidates the definition of regions of linkage has allowed the investigation of positional candidate genes within the identified areas. One of these genes is the intestinal mucin gene (MUC3) that is located near the reported linkage on chromosome 7q (442). As previous reports indicated a possible mucin abnormality in UC patients, the role of polymorphisms of variable number of tandem repeats was analysed in Japanese and Caucasian UC patients. The rare alleles of the MUC3 gene were reported to be more frequent in UC patients of both ethnicities, suggesting that they may confer genetic predisposition to UC (442). Others have focused on the CC-chemokine receptor 5 (CCR5). The chromosomal location of the CCR5 gene on 3p21 coincides with an IBDsusceptibility locus identified by genome-wide scanning. However, there were no significant differences in the CCR5delta32 mutation frequency a mutation which coding for a nonfunctional receptor neither between IBD patients and healthy controls, nor between CD and UC patients. Similarly no correlation was detected between the CCR5delta32 genotype and the age at IBD-diagnosis, the frequency of surgical intervention, or disease localization, suggesting that the IBD susceptibility locus of disease region probably do not involve the CCR5 gene (443, 444). Another functional candidate is the vitamin D receptor gene, located on chromosome 12 in a region that has been linked to IBD by genome screening techniques. Vitamin D plays an important role in distinct immune functions susch as the inhibition of Th1 cytokine production, and the suppression of lymphocyte proliferation. The single nucleotid polymorphisms TaqI, ApaI and FokI located in the gene were typed in CD and UC patients as well as in healthy controls. Among patients with Crohns disease there were significantly more homozygotes (genotype tt) at the TaqIsite than in UC or healthy controls (445). Again further functional and repetitive studies are necessary to verify and ecxplain the association found. 1.5.2 Genome-wide scanning and linkage studies The technique of genome-wide scanning has been applied successfully in inflammatory bowel disease. A number of putative susceptibility loci have been
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identified through genomewide searches including replicated regions of linkage on chromosomes 12, 16, 6 (the HLA region), and 14. Although the role of these regions was verified in different ethnicities, various linkage studies often implicate different regions of varying effects. An example for ethnical variability is the susceptibility gene located on chromosome 16 and designated IBD1. After the first report on its relevance in IBD (446) several studies confirmed the significance of chromosome 16 region in the development of Crohn's disease and ulcerative colitis, with a greater significance in CD (447-451). However, these findings were observed in predominantly non-Jewish populations, while in those studies where Ashkenazi Jews were included for analysis, the results have been widely divergent. However, in a recent study where the Ashkenazi population was subdivided on the basis of age of onset, there was a striking increase in linkage in families where affected individuals had an age of onset < or = 21 yr (nonparametric linkage 3.02, p = 0.002). In contrast, there was no evidence of linkage in the Jewish families where all affected individuals had an age of onset > 21 yr (452). Recently three groups in different European and in a North American population have reported association between Crohns disease an insertion mutation in NOD2 gene that is located in the pericentromeric region of the chromosome 16 (224, 225, 453). However given its frequency this polymorphism is unlikely to account completely for the observed linkage at IBD1, and other variants of NOD2 may confer additional disease risk. Furthermore, other suceptibility genes might also be present in this broad region of linkage on chromosome 16. The function of NOD2 could also support its role in the pathogenesis of IBD. Thus wild type NOD2 activates NFB, making it responsive to bacterial lipopolysaccharides, while this induction is deficient in mutant NOD2. The expression of the protein coded by this gene is restricted to monocytes. Thus a deficit in sensing bacteria by the monocyte/macrophage system might result in an exaggerated inflammatory response. Beside the repeatedly confirmed linkage in the chromosome 16, several other chromosomal regions have been suggested to be relevant in the etiology of Crohns disease and ulcerative colitis. The strongest and replicated evidence was foud for a region sapnning 41 centiMorgans on the long arm of Chromosome 12 (450, 454). Furthermore other studies suggested that a linkage with 3p and 3q might exist (449, 454) both for UC and CD. This earlier suggested linkage to chromosome 3p21 was also identified in a Finnish IBD population, with lod scores peaking at D3S2432, with a maximum two-point lod score of 1.68 (435). Similarly a linkage for both entities was found for 7q, 1p and 4q (449, 450, 454). Later Hampe and collegues reporting the largest genome wide screening to date found novel regions for further investigation on chromosomes 1, 4, 6 (the HLA region), 10, 22 and X (450). Beside the already known choromosomal linkage region a recent study has identified a linkage within the X chromosome in the region to Xq21.3 around dXs1203, where the NPL-1 interval around the linkage peak comprises 19.7 cM. (455). Respecting the results of all genome wide
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searches in many of the implicated regions, evidence for linkage has been observed in both CD and UC, suggesting that there might be major pathogenic genes common to both diseases. On the other hand, data from investigators in Oxford provided first evidence for linkage alone for UC with regions in chromosomes 2 and 6, while for Crohns disease alone chromosome 16 (454). In order to identify the responsible genes in the replicated linkages of chromosomes fine mapping and/or functional candidate gene studies are further necessary. Genes identified by this way have probably major effects and would rather influence the disease onset than the disease course. Other genes influencing the disease appearance and behaviour might be associated in ethnically matched groups showing phenotypic similarity. 1.6 Pharmacogenetics Another interesting question is how the individual background defines the therapeutic effects in IBD. In the pathomechanism of IBD genes playing regulatory or effecting role in immune functions are likely significant, determining the development and/or the course of IBD. Disease course on the other hand is also influenced by the response of the patient on the medical treatment. Thus beside the indications based on the severity of disease, its course and location, there are other important hereditary factors influencing the therapeutic efficiency, such as the rate of drug metabolism. Therefore investigating genetic polymorphisms in drug-metabolising enzymes possibly playing a determinant role in the inter-individual differences in drug metabolism and effects is an important new area of IBD research. The immunosuppressive agents, azathioprine and 6-mercaptopurine are inactivated by S-methylation by thiopurine S-S-methyltransferase (TMPT). Recent studies showed that approximately 10 percent of the population is heterozygous and further 0.3 percent homozygous recessive for a defective TMPT gene. These homozygous recessive individuals could accumulate excessive thioguanine nucleotides and develop severe myelosuppression with usual drug dosages (456). Indeed, 27% of patients with CD and myelosuppression during azathioprine therapy had mutant alleles of the TPMT gene associated with enzyme deficiency. Furthermore the delay between the drug administration and the occurrence of bone marrow toxicity was markedly shorter for those patients with 2 mutant alleles, while longer in patients with 1 mutant allele. The longest average time before the occurrence of myelosuppresion was observed in those patients with normal genotype (85). Another study has found multidrug resistance gene expression significantly elevated in those IBD patients who required bowel resection for failed medical therapy. Indicating a constant effect, mucosal MDR expression was found to remain stable over the time and appeared to be independent of disease activity (457). Elucidating the molecular basis of inherited differences in drug metabolism will help to select the optimal medication and dosage for individual patients.
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On the whole, the data suggest that genetic background plays an important role in the pathogenesis of inflammatory bowel diseases. Genes would regulate the evolution and progression of the disease on different levels. Thus major susceptibility genes would determine an increased chance for a disease onset in the presence of certain environmental factors. While the presence of other genes would additively increase this chance respectively affect which entity Crohns disease or ulcerative colitis presents. Again other genes would influence the disease course including the disease behaviour, extension and the presence of extraintestinal manifestations. In addition environmental factors would control the process, contribute to the expression of the genes and to the phenotype.

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