Vol. 18, no. 4, 799·804 (2005)
INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY
INTERSTITIAL CYSTITIS AND INFECTIOUS AGENTS D. FIORITI, M. PENTA, M. MISCHITELLI,A.M. DEGENER',A. PIERANGELP, V. GENTILE2, R. NICOSIA, C. GALLINELLI, F. CHIARINI and V. PIETROPAOLO Department ofPublic Health Sciences, 'Department ofExperimental Medicine and Pathology, 'Department ofUrology, "La Sapienza" University, Rome, Italy Received February 2, 2005 - Accepted October 7, 2005 Interstitial cystitis (IC) is a syndrome consisting of severe refractory bladder symptoms of unknown etiology. The disease tends to affect Caucasian women with a mean age of 40 years, with 25% of patients under the age of 30. Few population based epidemiological studies of IC have been performed. We analyzed a case of interstitial cystitis in a 42-year-old non-smoker woman. In two biopsy samples the presence of viral DNA of human polyomavirus BK (BKV), human herpes virus type 1 and type 2 (HHV1 and HHV-2), adenovirus, human papillomavirus (HPV) and bacterial DNA (Chlamydia trachomatis and Mycoplasma genitalium) were evaluated by means of polymerase chain reaction (PCR). Both samples resulted positive only for BKV and HPV DNA. HPV genotyping revealed the presence of HPV66 that is associated with a high risk of cancer development. Thus the finding of a viral co-infection could support the hypothesis of the multi-factorial origin of this pathology. diagnosed with "early non-ulcerative" IC and an estimated 5% to 10% of IC patients are believed to have the "classic ulcerative" IC, more severe, as demonstrated by the presence of Hunner's ulcers and glomerulations during cystoscopy. Although the first IC case may have been recognized as early as the mid 1800's, it wasn't until 1987 that the US National Institutes of Health convened the first meeting to discuss IC and in 1988 established the first definition and research criteria for IC as a severe and potentially debilitating disease (3). In 1975, the first epidemiological study on IC was conducted in Finland by Oravisto (4). Ho (2) has confirmed many of Oravisto's findings, generating much excitement among the US research community. They found that: the average age of onset for IC is 40 years, with 25% of patients under the age of 30; a late deterioration of symptoms is unusual; up to 50% of patients
Interstitial cystitis is a chronic, and often severe, inflammation of the bladder wall, the cause of which is unknown. Primary symptoms are urinary frequency, urgency, and, for some, severe lower abdominal or perineal pain. However, IC patients rarely have positive tests for infection in standard urinalysis and urine cultures (1). Unfortunately, there is no definitive diagnostic test for IC. The diagnosis is usually based upon a patient's symptoms, a cystoscopic examination of the bladder under anesthesia and exclusion of other bladder diseases. During cystoscopy urologists carefully examines the bladder wall for signs ofIC, including small petechial hemorrhages (known as glomerulations) and/or larger Hunner's ulcers. IC patients were classified in two distinct categories; Ho, Koziol and Parsons (2) in fact, classify "early non-ulcerative" IC and "classic ulcerative" IC. The great majority of patients are
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experience spontaneous rermssrons probably unrelated to treatment, with a duration ranging from 1 to 80 months; patients with IC are 10 to 12 times more likely than controls to report childhood bladder problems; patients with IC are twice as likely as controls to report a history of urinary tract infection; however, over half of all IC patients report fewer than one such infection per year before the onset of IC; 50% of IC patients have pain while riding in cars; 63% of IC patients are unable to work full time; IC patients have suicidal thoughts 3-4 times above the national average; the quality of life of IC patients is worse than patients experiencing chronic renal failure and undergoing dialysis. According to the National Institutes of Health (NIH), IC affects about 700,000 people in the United States, 90% of whom are women. These data were confirmed by Clemens et al. (5). Increasing awareness ofthe disease is helping to speed diagnosis and treatment. The etiology ofIC is currently unknown. One hypothesis under study is that IC could be due to an autoimmune response following a bladder infection (6). Several possible causes of IC have been proposed including infections, changes in neuronal function, autoimmune reactivity, hypersensitivity response with mast cell accumulation and release, and defects in the urothelial cell permeability barrier (6-11). Many patients with IC present with varying degrees of bladder inflammation, which appear to correlate with the severity of the symptoms (11). Increasing evidence indicates that IC is associated with bladder mast cell activation, in particular, the activation of these cells initiates activation of monocytes and/or macrophages and their resulting products such as IL6 may modulate bladder inflammation (12), although it may be in a secondary role rather than as a direct cause ofIC. The mast cells and numerous secretory cytokines such as IL-lP, IL-6, proteases, arachidonic acid metabolites and TNF-alpha mediate the bladder inflammation and the response to pathogens and trauma and then they are an integral parts of bladder cancer processes (13). IL-6 is the primary cytokine elevated in the urine of interstitial cystitis patients (11). Mast cells express adhesion molecules themselves (14-15), and can also regulate the expression of cellular adhesion molecules (CAMs), thus augmenting the inflammatory responses by
increasing leukocyte binding (16-17). Histamine, a major secretory product of mast cells by a process called degranulation, can cause localized pain and irritation in tissues where the mast cells are present. Various authors demonstrated that the level of histamine in bladder tissues of patients with IC was higher and that the mast cells were more degranulated or activated in IC than in other conditions, clearly demonstrating local irritation of bladder tissues (12-13, 16-17). Some studies (18-20) suggest that the cooperative action between histamine and bacterial components lead to amplified inflammatory responses; in fact, histamine amplifies endothelial cell responsiveness to both Gramnegative and Gram-positive cell wall components and the synergy between histamine and bacterial pathogens is associated with enhanced expression of toll-like receptors (TLR). The mechanism by which histamine enhances sensitivity to the bacterial component is unknown. Moreover research continues to evaluate the role of the mast cell in IC. The possibility that infectious agents could playa role in the etiology of interstitial cystitis has been investigated for several years but the origin of IC remains an open question (21-26). Some authors have suggested that IC could be the consequence of the irritating effect of some compounds present in the urine (22), but a particular molecule has not yet been identified in patients with IC. Other authors are beginning to explore the possibility that heredity may playa role in some cases of IC (23). As already mentioned, viruses may be involved in the pathogenesis of interstitial cystitis (IC). Adenoviruses and BKV can infect bladder epithelial cells, thus becoming the causative agents for hemorrhagic cystitis, especially in immunocompromised patients such as transplanted individuals. However some authors reported that adenovirus do not playa major pathogenetic role in interstitial cystitis, whereas HHV-l and -2 are commonly recognized for their capability to cause chronic and latent infections within the urogenital tract (27). IC is a poorly understood disease with unknown causes. Although no bacteria or viruses (pathogens) have been found in the urine of IC sufferers, an unidentified infectious agent may be a hypothetical cause. Others believe that IC occurs with ischemia (tissue death) or a deficiency of glycoaminoglycan
Int. J. Immunopathol. Pharmacol.
(GAG) in the epithelium (17). Therefore, research continues to evaluate the role of several factors that cause this inflammatory condition. MATERIALS AND METHODS A 42-year-old non-smoker woman presented persistent symptoms correlated to urinary tract infections for approximately 18 months. Antibiotic treatment was based on the patient symptoms: pain and pressure in pelvic situ, dysuria, and increased urinary frequency. Urine chemicalphysical analysis was negative for white blood cells and bacteria. Urine cultural analysis showed no bacterial growth after 48 hours. Moreover the symptoms of the patient worsened before menstruation and after sexual intercourse; finally, when the patient needed to urinate every 20 minutes at work and 10 times during the night, leaving her exhausted and depressed in the morning. The patient did not have back pain, nausea, vomiting, fever or sexually transmitted diseases. The diagnosis of interstitial cystitis is routinely based on the techniques necessary to elicit the criteria established at the conferences of the National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases (NIDDK). Essentially, this requires a thorough patient history, urinary-dynamic evaluation and cystoscopy carried out under anesthesia with hydrodistention of the bladder and bladder biopsy (28). In this case the diagnosis of interstitial cystitis was performed as exclusion diagnosis of urinary tract infections, bladder cancer and other pelvic disorders. During cystoscopy, that showed increased or prominent vasculature and submucosal haemorrhage, two bioptic bladder samples were taken. For PCR analysis samples were incubated in lysis buffer (0.5 M TRIS, 0.02 M EDTA, 0.01 M NaC!, 1% SDS) with 200 ug/ml proteinase K at 55°C for 24 h. Digestion was followed by phenol-chloroform extraction and ethanol precipitation. One ug of totally purified DNA was used for each PCR assay. Beta-globin PCR was performed on extracted DNA to assess the efficacy of nucleic acid extraction (29). The PCR products were analysed on 2% agarose gel by ethidium bromide staining and the positive samples were utilised for further PCR. The assays included positive (purified viral DNA) and negative (all the PCR components except the template) controls to exclude false-positive and false-negative results. DNA detection ofAdenovirus, HHV-I and HHV-2, HPY, BKV, C. trachomatis, M. genitalium was performed by PCR assay using specific target DNA sequences showed in Table I. The treatment was based on traditional antibiotics and on biophysical techniques used to control IC symptoms
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include bladder retraining electrical nerve stimulation (TENS), stress reduction with biofeedback, diet modification and exercise; then local medication with dimethyl sulfoxide (DMSO, Rimso-50®) was instilled (intra-vescical) through the urethra and directly into the bladder via a catheter.
RESULTS Our results indicated that the bioptic samples were suitable for PCR analysis and resulted positive only for BKV and HPV DNA. HPV genotyping was performed by sequencing the PCR product and revealed the presence ofHPV 66 DNA. Munoz (30) has recently classified the HPV-66 as probable highrisk genotype capable of causing progression to cancer in the uterine cervix. DNA of human herpes virus type 1 and type 2, adenovirus, C. trachomatis and M genitalium was not detected. DISCUSSION Although others studies demonstrated the presence ofBKV DNA in bladder mucosa (31), BKV was never before associated with interstitial cystitis. Since chronic pathologies often have multiple etiologies, the finding ofBKV and HPV DNA in both samples of the same patient suggests their possible involvement in the disease. Although until today the etiology of IC has not been assessed, current evidence indicates that the majority of IC patients have a small toxic factor (APF) in their urine that may playa role in the pathogenesis of this disorder by inhibiting the proliferation of bladder epithelial cells. Whether the APF is encoded by the eukaryotic cells themselves or induced by an unidentified intracellular microorganism is still not determined (32). Therefore, this case may be of some interest to better understand the etiology of interstitial cystitis; in fact, the finding of two different viral agents could support the hypothesis of a multi-factorial infectious origin of this pathology. In this case we could say that viruses could be involved in etiology ofIC even if the relation between these viruses and involved cells in bladder inflammation was not examined in our study. Reports of other cases could contribute to clarify the still open question on the IC etiology.
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Microorganism
Amplified region
PCRprodnct length
HPV
Ll protein
450bp
ADV
Hexon
308 bp
HHV-I/HHV-2
DNA polimerase
179 bp
HHV-l/HHV-2
GlycoproteinB
149 bp
BKV (est)
Regulatory region
748 bp
BKV (int)
Regulatoryregion
354 bp
Clamy
Criptic plasmid
201 bp
Myc
Adhesin gene
281 bp
ACKNOWLEDGEMENTS This work was partially supported by Italian MURST grants. REFERENCES 1. 2.
3.
4. 5.
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Table I: Primers utilized in PCR ADV= Adenovirus; HHV-l/HHV-2= Herpes simplex virus 1/2; HPV= Human papillomavirus; BKV= Human polyomavirus BK, Clamy= Clamydia trachomatis; Myc=M. genitalium.
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