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Ankylosing Spondylitis (AS) is the major subtype and a main outcome of an inter related group of inflammatory rheumatic disease (spondyloarthropathy) now named spondyloarthritides (SpA) (Braun and Sieper, 2007) showing familial aggregation, arthritis of sacroiliac and peripheral joints with enthesopathy, high association with HLA B27 and absence of rheumatoid factor (Brown et al.,1997; Said-Nahal et al., 2000). Others included in this family are undifferentiated spondyloarthritides (U-SpA), some forms of psoriatic arthritis, reactive arthritis (ReA), and arthritis associated with inflammatory bowel disease (IBD) (Yu et al., 2008). Many of the rheumatic diseases exact etiology and triggering factors are still not known, but a relationship between inflammatory gut lesions and SpA has been demonstrated by many authors (Mielants et al.,1996;Mielants et al., 2005; Praet et al., 2012 )
The prevalence of SpA including AS in inflammatory bowel disease (Crohn’s disease and ulcerative colitis) is high (Mielants and Vey, 2008). Prevalence rates have been described as 10-15% for sacroilitis and of 7-12% for spondylitis, although the figures are probably higher. Some 10% of patients with IBD attending a gastroenterology unit fulfilled the criteria for AS and an additonal 18% of patients had asymptomatic sacroilitis detected by conventional X- ray (Mielants and Veys, 2008).
Although only a small proportion of patients with SpA develop overt IBD, up to two thirds have evidence of subclinical gut inflammation. Ileocolonoscopic studies in patientswith SpA have revealed inflammatory gut lesions in 65% of patients with ReA and 57% of patients with AS (Stebbings et al.,2010 ) In studies performed in Belgium and Scandinavia macroscopic and microscopic changes have been identified in patients with SpA in up to 50% (Braun and Sieper, 2007).
Association between HLA-B27 and AS was reported nearly 30years ago .The frequency of HLA-B27 in AS patients ranges from 81 to 96 % while its frequency among the healthy populations is between 4 and 8% (Ebringer et al., 2006).
Gut plays an important role in the pathogenesis of many disorders that fit the concept of the SpA (Narsimulu et al., 2004). A hypothesis has been made that molecular mimicry between enteric bacteria and HLA-B27 plays a pathogenic part in HLA-B27 associated SpA . Researchers have demonstrated cross reactivity between Klebsiella pneumoniae,Yersinia enterocolitica, Shigella flexneri, Shigella sonnie, Salmonella typhimurium, and HLA-B27 (Ringrose, 1999).
The possible interaction between bacteria and HLA B27 has a crucial role in models of pathogenesis of SpA (Braun and Sieper, 2007). Ebringer et al. (2006) postulated that antibody to Klebsiella produced in the gut lymphoid tissues binds to HLA-B27 positive cells especially in entheses of the sacroiliac joints and spinal vertebrae. This causes activation of the complement cascade which leads to cell destruction and inflammation. As the inflammation subsides, a healing process takes place and new bones develops (Schett and Rudwaleit, 2010). Repeated episodes of this ‘Klebsiella reactive arthritis’ eventually produces the clinical syndrome of AS which is due to further bone formation thereby causing fusion of bones (Rashid and Ebringer, 2012).
Associations between inflammatory gut lesions caused by Salmonella,Shigella, Yersinia,Campylobacterand ReA are well established (Keat, 1983; Mielants and Veys, 1984; Clain, 1986).About 10%-20% of HLA B27 positive patients with reactive arthritis develop the full clinical picture of AS after 10-20 years (Braun and Sieper, 2007).
The observation that ReA often followed an enteric infection led to speculation that AS might also be triggered by a gut infection. Serological cross reactivity between HLA-B27 and antigens of Klebsiella pneumoniae has been established. This prompted several groups to study the fecal flora of individuals with SpA (Smith et al., 1997)
The presence and maintenance of these cross-reactive, nonpathogenic bacteria in the bowel flora of patients with AS has been found in a study. This finding suggests that the infectious process in this disordermay be different from other seronegative arthropathies, such as reactive arthritis, in that an acute episode of infection may not occur. The continued isolation of cross-reactive organisms over a period of 12 months would tend to argue for possibility of a subtle, ongoing ‘pathogenic’ process (Predergast et al.,1984)
Of the cross reactive micro-organisms, only Klebsiella could be consistently isolated from fecal cultures obtained from British patients with AS(Ebringer, 1989). In a study of 63 AS patients by Ebringer’s group an increased recovery of Klebsiella could be obtained during the active phase of the disease (Ebringer et al., 1977). A second study showed that AS patients with inactive disease and Klebsiellaculture positive subsequently followed by a relapse (Ebringer et al.,1978).
Consideration of the above & other similar findings led Vaughan (1990)to conclude in a recent review ‘The probability that AS is due to a peculiar immunologic relationship between the patient & his enteric organisms & that is determined by his HLA-B27 molecules seems great.’
The information on possible source of immune dysregulation in AS is less clear. However enhanced gastrointestinal permeability, the so called leaky gut might be the possible source (Fasano, 2007).
Paneth cells (PCs), the intestinal secretory cells located at the bottom of the intestinal crypts, are well known to play an important role in innate host defence against intestinal micro-organisms. AS patients display a normal number of PC with significant over-expression of PC derived anti- microbial peptides as well as pro-inflammatory cytokines, such as IL-23.This causes pathogenesis of chronic inflammatory gut disorder. Once the disease is manifested, a deficiency in defensin could be the result of loss of epithelial integrity, resulting in bacterial invasion (Cicca et al., 2010).
Both HLA-B27 and gut inflammation play a pivotal role in development of AS. The main etiopathogenetic process is triggered by a combined genetic and environmental (mainly microbial) factors (Rashid and Ebringer, 2011).
The terminal ileum and the large bowel are hospitable places for microbial proliferation because bowel motility is slow here. Lesions of Crohn’s disease usually located where there are microbial residents (ileum and colon). Whereas ulcerative colitis is limited to the large bowel (Harry et al., 2008).The gut microflora(normal flora/microbiota) present in the intestinal lumen differs significantly from the gut flora attached and embedded in the mucosal layer (Sekirov et al., 2010).It has also been reported that the mucosa-associated bacterial communities in the colon are significantly different in composition from those in feces (Zoetendal et al., 2002).
Biopsies unlike feces,provide samples collected from regions of the intestinal tract where inflammation occurs (Rodrigo et al., 2008).Therefore, the study was designed to isolate different aerobic enteric bacteria in colonoscopy biopsy material of patients with AS.
1.2 Hypothesis
Enteric aerobic bacteria plays a role in pathogenesis of HLA-B27 positive Ankylosing Spondylitis patients.
1.3 Objectives
General Objectives:
To see enteric aerobic bacteria from colonic biopsy material among patients with Ankylosing Spondylitis.
Specific Objectives:
- To isolate enteric aerobic bacteria from colonic biopsy material among patients with Ankylosing Spondylitis and normal healthy controls.
- To observe the difference in bacterial isolates between patients with Ankylosing Spondylitis and normal healthy controls.
- To observe the difference in bacterial pattern between superficial microflora and mucosal bacteria.
- To observe any difference in bacterial load between Ankylosing Spondylitis patients and normal healthy controls.
- To determine the rate of bacterial isolates among HLA-B27 positive patients.
Review of Literature
2.1 AnkylosingSpondylitis
Ankylosing means fusing together and spondylitis means inflammation of the bones in the spine (from Greek ankylos, stiff; spondylos, vertebrae).AS is a chronic inflammatory disease which was previously known as Bekhterev's disease, Bekhterev syndrome, or Marie-Strümpell disease, (Braun and Sieper, 2002). It is the major subtype and a mainoutcome of an inter-related group of rheumatic diseasesnow named spondyloarthritidesshowing familial aggregation, arthritis of sacroiliac and peripheral joints with enthesopathy, high association with HLA B27 and absence of rheumatoid factor (Brown et al.,1997; Said-Nahal et al., 2000). Others included in this family are undifferentiated spondyloarthritides (U-SpA), some forms of psoriatic arthritis, reactive arthritis (ReA), and arthritis associated with inflammatory bowel disease (IBD).The subgroups are geneticallylinked—the strongest known contributing factor is theMHC class I molecule HLA B27 ( Yu et al., 2008).
2.1.1 History
AS has been suggested as the first recognized disease, having been distinguished from rheumatoid arthritis by Galen as early as the second century A.D. However, skeletal evidence of the disease (ossification of joints and entheses primarily of the axial skeleton, known as "bamboo spine") was first discovered in an archaeological dig that unearthed the skeletal remains of a 5000-year–old Egyptian mummy with evidence of bamboo spine (Braun and Sieper, 2002).
It was not until the late nineteenth century, however, when the neurophysiologistVladimir Bekhterev of Russia in 1893, Adolph Strümpell of Germany in 1897, and Pierre Marie of France in 1898were the first to give adequate descriptions which permitted an accurate diagnosis of AS prior to severe spinal deformity. For this reason, AS is also known as Bekhterev Disease or Marie–Strümpell Disease (Braun and Sieper, 2002).
2.1.2 Epidemiology
Ankylosing Spondylitis is a disease that affects youngpeople, who generally present at around 26 years of age. Men are more often affected than are women, with a ratio of roughly 2 to 1. About 80% of patients develop the firstsymptoms at an age younger than 30 years, and less than5% of patientspresent at older than 45 years. There is arough correlation between the prevalence of HLA B27 and the incidence and prevalence of this disease in a specific population.HLA B27 is most prevalent innorthern countries and some tribes (with up to 50% ofcases), and is highest in Eskimo populations and HaidaIndians. Overall, the prevalence of AS is between 0.1% and 1.4%, with most of these datacoming from Europe. Theincidence of AS is between 0.5 and 14 per 100,000 people per year in studies from differentcountries(Braun and Sieper, 2007). In a study done in Bangladesh AS was the commonest seronegative disease (28.89% of all inflammatory arthritis) (Hasan et al., 2009).
2.1.3 Pathophysiology
In patients with AS inflammation occurs at the site where certain ligaments or tendons attach to the bone. This area of the body is known as entheses. The inflammation is followed by some erosion (wearing away) of the bone at the site of the attachment. This is known as enthesopathy. As the inflammation subsides, a healing process takes place and new bone develops. Movement becomes restricted where bone replaces the elastic tissue of ligaments or tendons. Repetition of this inflammatory process leads to further bone formation and the individual bones which make up the backbone (vertebrae) fuse together. The pelvis is most commonly affected first. The lower back, chest wall and neck may also become involved at different times (Schett and Rudwaleit, 2010).
Fig:2.1 Ankylosing process (Ankylosing _ process.jpg)
2.2 Theories proposed to explain the link with HLA
Human Leukocyte Antigen (HLA) B27 (subtypes B*2701-2759) is a class I surface antigen encoded by the B locus in the major histocompatibility complex (MHC) on chromosome 6 and presents antigenic peptides (derived from self and non-self antigens) to T cells. HLA-B27 is strongly associated with AS, and other associated inflammatory diseases referred to collectively as SpA (Khan, 2010).
The prevalence of HLA-B27 varies markedly in the general population. For example, about 8% of Caucasians, 4% of North Africans, 2-9% of Chinese, and 0.1-0.5% of persons of Japanese descent possess this gene. In northern Scandinavia (Lapland), 24% of people are HLA-B27 positive, while 1.8% have associated Ankylosing Spondylitis.
HLA-B27 consists of a heavy chain having three α domains,which noncovalently binds short peptides and β2-microglobulin. There are 24 HLA-B27subtypes currently recognized. The structural patternsare consistent with B2705 being the ancestral allele andthe other types being generated by small mutations. B2705 is the dominant subtype and is associated withAS across broad ethnic and geographic boundaries. Ofthe subtypes studied to date, it appears that B2706 andB2709 do not confer susceptibility to AS. Although theHLA-B27 has remained a center of extensive research,the mechanism whereby HLA-B27 confers susceptibilityto AS is not well defined.
Several different hypotheses have been proposed.
1)The principle of molecular mimicry is still proposed as a possible mechanism in B27-related pathogenesis. This postulates that the antibodies directed against foreign antigens arising during a bacterial infection are cross-reactive with HLA-B27.
2)In the arthritogenic peptide theory, HLA-B27 binds unique peptides of microbial or self-origin and presents them to CD8+ Tcells . These peptides usually have an anchor arginine residue at their second position and the side chain of arginine is bound in the B pocket of HLA-B27. It was recently reported that CD4+ T cells may be involved in class I–restricted immune recognition.Consequently AS could involve an HLA-B27-restricted CD8+ T-cell or CD4+ T-cell response to microbial or self-peptides.
3)There has been considerable interest in aberrant processing or folding of the heavy chain of HLA-B27. Under normal circumstances cell surface HLA-B27 consists of a heavy chain bound to β2m and peptide. This complex is formed in the endoplasmic reticulum Heavy chain folding of HLA-B27 appears to be slower compared with other HLA alleles, however, possibly because of specific amino acid residues in the B pocket. This misfolded heavy chain is usually removed in endoplasmic reticulum, but in the event of insufficient or unavailable chaperone, peptide, or β2m, misfolded heavy chains are increased. This may increase expression of the protein BiP and generate an unfolded protein response in the endoplasmic reticulum, leading to activation of nuclear factor-Kb (Tsai, 2010).
2.3 Ankylosing Spondylitis and gut infection
Although ReA, another B27-related SpA, has a clear relationto antecedent infection, this is less clear for AS. B27-transgenic rats raised in a germ-free environment do notdevelop inflammatory pathology in the gut or the joints,and induction of arthritis following reintroduction ofcommensal gut flora supports the notion that such organismsplay an important role in the pathogenesis of B27-associated gut and joint inflammation (Rashid and Ebringer, 2011).
2.3.1 Link between HLA-B27 and gut-mediated Arthritic diseases
The association of HLA-B27 with AS is amongst the strongest genetic link with any common disease which has been encountered in the field of rheumatology (Thomas and Brown, 2010). This genetic bond was discovered in early 1970s, where more than 95% of patients with AS have been found to possess HLA-B27 alleles, whilst the frequency of this gene in the general population was below 10% (Brewerton et al., 1973; Schlosstein et al.,1973). Other diseases in the SpA group have lower but different degrees of associations with this allelotype, depending on the clinical presentation and pathological location of the disease (Braun and Sieper, 2010). For example, the frequency of this allelotype in patients with IBD/CD without associated arthritis is comparable to those of the normal population but increases to 40-60% in those patients with spondylitis/sacroiliitis. The frequency of HLA-B27 in patients with ReA/Reiter’s syndrome and U-SpA is ranging between 30 to 90% and 50 to 70%, respectively, meanwhile its frequency in patients with psoriatic arthritis (PsA) with or without peripheral arthritis is around 20%, but it is increased to up to 60% in patients with associated sacroiliitis. From these data results, it appears that a spondyloarthropathic patient presenting with spinal involvement had a higher chance of possessing HLA-B27 genes than those with peripheral joints involvement only (Rashid and Ebringer, 2010).
2.3.2 Gut inflammation and interrelation between different disease
entities of Spondyloarthiritides
Many data support the existence of an inter-connection or inter-relation between different disease entities in the SpA group. For example:
1). The prevalence of AS in patients with UC and CD was found to be reaching 2.6% and 6%, respectively, giving an overall 3.7% prevalence in patients with IBD (Palm et al., 2002). It has also been reported that AS is frequently associated with IBD, with 5-10% of cases having clinical IBD and approximately 70% of cases having subclinical bowel inflammation (Thomas and Brown, 2010), and this link was emphasized previously through an analytical review (Ebringer et al., 2007). Furthermore, HLA-B27 positive patients with IBD were shown to have higher chances of developing AS when compared to those without IBD (Wright, 1978).
2). Many studies have shown macroscopical and/or microscopical features of gut mucosal inflammation, especially in patients with IBD, ReA, AS, and U-SpA (Mielants et al., 2005). In one particular study, gut changes varying from an acute to asymptomatic chronic intestinal inflammation have been observed in about 60% of patients with SpA (Demetter et al., 2002), and more interestingly, those patients who showed signs of articular remissions were preceded by disappearance of the gut inflammation, which supports the concept that the gut is involved in the pathogenesis of SpA.
3). Another fundamental support for the role of gut and the intestinal flora in the development of SpA in relation to the presence of HLA-B27 genes is the absence of arthritis and colitis in germ-free HLA-B27-transgenic rats and the induction of these features when the rats were relocated to non germ-free environment (Hammer et al., 1990).
4). Twenty to 30 percent of patients with unclassified HLA-B27-positive inflammatory rheumatic diseases (Sany et al., 1980) or oligoarthritis have been shown to develop into one form of the definite spondyloarthropathic group such as IBD, ReA, or AS (Schattenkirchner and Kruger, 1987).
5). It has also been stated that more than half of patients with U-SpA will develop AS over a certain period of time (Mau et al., 1988). In a later study, however, no significant differences were observed in some clinico-radiological or genetic features of AS and U-SpA among the Middle Eastern and South Asian populations (Uppal et al., 2006).
It appears from these results that both HLA-B27 and gut inflammation play a pivotal role in the development of SpA, especially AS and CD, and that the main etiopathogenetic process is triggered by a combined genetic and environmental (mainly microbial) factors (Rashid and Ebringer, 2011).
2.4 Ankylosing Spondylitis and microbial link
The first evidence of the epidemiological link between microbes and SpA was detected in the early Twentieth Century, where a triad of urethritis, conjunctivitis, and arthritis, being termed as Reiter’s syndrome, was found to follow a dysenteric or venereal infection (Calin, 1998). Reiter’s syndrome was later recognized as a form of ReA and since then each of the triggering bacterial agents including Yersinia, Campylobacter, Shigella, and Salmonella enterogenic bacteria as well as Chlamydia urogenital pathogens has been found to have an approximately equal role in the development of this disease (Leirisalo-Repo, 2005).