Integrated microbiome and metabolome analysis reveals novel urinary microenvironmental signatures in interstitial cystitis/bladder pain syndrome patients.

BACKGROUND: The pathogenesis of interstitial cystitis/bladder pain syndrome (IC/BPS) has not been elucidated, but urinary microorganisms and metabolites have been shown to be closely associated with the inflammatory response of IC/BPS. Nevertheless, the exact mechanisms related to this response have not been clarified. METHODS: 16S rRNA sequencing and untargeted metabolomics techniques were used to analyse the urinary microbial and metabolite profiles of 30 IC/BPS patients and 30 healthy controls, and correlation analyses were performed to explore the mechanisms by which they might be involved in the inflammatory response of IC/BPS. RESULTS: Twenty-eight differential genera, such as Lactobacillus and Sphingomonas, were identified. A total of 44 differential metabolites such as 1,3,7-trimethyluric acid and theophylline were screened. The abundance of Lactobacillus and Escherichia-Shigella was significantly higher in the urine of female IC/BPS patients and healthy controls compared to males, while Bacteroides and Acinetobacter were lower than in males. The results of the Pearson correlation analysis suggested that differential microorganisms may influence the composition of metabolites. The Lactobacillus genus may be a protective bacterium against IC/BPS, whereas Sphingomonas may be a pathogenic factor. The differential metabolite theophylline, as an anti-inflammatory substance, may downregulate the inflammatory response of IC/BPS. CONCLUSIONS: This study revealed microbial and metabolite profiles in the urine of IC/BPS patients versus healthy controls in both males and females. We also found some microorganisms and metabolites closely related to the inflammatory response of IC/BPS, which provided directions for future aetiological and therapeutic research.

Usefulness of Urinary Biomarkers for Assessing Bladder Condition and Histopathology in Patients with Interstitial Cystitis/Bladder Pain Syndrome.

This study investigated the usefulness of urinary biomarkers for assessing bladder condition and histopathology in patients with interstitial cystitis/bladder pain syndrome (IC/BPS). We retrospectively enrolled 315 patients (267 women and 48 men) diagnosed with IC/BPS and 30 controls. Data on clinical and urodynamic characteristics (visual analog scale (VAS) score and bladder capacity) and cystoscopic hydrodistention findings (Hunner's lesion, glomerulation grade, and maximal bladder capacity (MBC)) were recorded. Urine samples were utilized to assay inflammatory, neurogenic, and oxidative stress biomarkers, including interleukin (IL)-8, C-X-C motif chemokine ligand 10 (CXCL10), monocyte chemoattractant protein-1 (MCP-1), brain-derived neurotrophic factor (BDNF), eotaxin, IL-6, macrophage inflammatory protein 1 beta (MIP-1ß), regulated on activation, normal T cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and 8-isoproatane, and total antioxidant capacity. Further, specific histopathological findings were identified via bladder biopsy. The associations between urinary biomarker levels and bladder conditions and histopathological findings were evaluated. The results reveal that patients with IC/BPS had significantly higher urinary MCP-1, eotaxin, TNF-α, PGE2, 8-OHdG, and 8-isoprostane levels than controls. Patients with Hunner's IC (HIC) had significantly higher IL-8, CXCL10, BDNF, eotaxin, IL-6, MIP-1ß, and RANTES levels than those with non-Hunner's IC (NHIC). Patients with NHIC who had an MBC of ≤760 mL had significantly high urinary CXCL10, MCP-1, eotaxin, IL-6, MIP-1ß, RANTES, PGE2, and 8-isoprostane levels and total antioxidant capacity. Patients with NHIC who had a higher glomerulation grade had significantly high urinary MCP-1, IL-6, RANTES, 8-OHdG, and 8-isoprostane levels. A significant association was observed between urinary biomarkers and glomerulation grade, MBC, VAS score, and bladder sensation. However, bladder-specific histopathological findings were not well correlated with urinary biomarker levels. The urinary biomarker levels can be useful for identifying HIC and different NHIC subtypes. Higher urinary inflammatory and oxidative stress biomarker levels are associated with IC/BPS. Most urinary biomarkers are not correlated with specific bladder histopathological findings; nevertheless, they are more important in the assessment of bladder condition than bladder histopathology.

Immune cell profiles of patients with interstitial cystitis/bladder pain syndrome.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a disorder characterized by bladder pain upon filling which severely affects quality of life. Clinical presentation can vary. Local inflammatory events typify the clinical presentation of IC/BPS patients with Hunner lesions (IC/BPS-HL). It has previously been proposed that B cells are more prevalent in HL, but understanding their exact role in this environment requires a more complete immunological profile of HL. We characterized immunological dysfunction specifically in HL using immunohistochemistry. We detected significantly more plasma cells (50× increase, p < 0.0001), B cells (28× increase, p < 0.0001), T cells (3× increase, p < 0.0001), monocytes/macrophages (6× increase, p < 0.0001), granulocytes (4× increase, p < 0.0001), and natural killer cells (2× increase, p = 0.0249) in IC/BPS patients with HL than in unaffected controls (UC). Patients with IC/BPS-HL also had significantly elevated urinary levels of IL-6 (p = 0.0054), TNF-α (p = 0.0064) and IL-13 (p = 0.0304) compared to patients with IC/BPS without HL (IC/BPS-NHL). In contrast, IL-12p70 levels were significantly lower in the patients with HL than in those without these lesions (p = 0.0422). Different cytokines were elevated in the urine of IC/BPS patients with and without HL, indicating that different disease processes are active in IC/BPS patients with and without HL. Elevated levels of CD138+, CD20+, and CD3+ cells in HL are consistent B and T-cell involvement in disease processes within HL.

The bacterial microbiota of Hunner lesion interstitial cystitis/bladder pain syndrome.

OBJECTIVE: To undertake the first comprehensive evaluation of the urinary microbiota associated with Hunner lesion (HL) interstitial cystitis/bladder pain syndrome (IC/BPS). Despite no previous identification of a distinct IC/BPS microbial urotype, HL IC/BPS, an inflammatory subtype of IC/BPS, was hypothesized most likely to be associated with a specific bacterial species or microbial pattern. PARTICIPANTS AND METHODS: The bacterial microbiota of midstream urine specimens from HL IC/BPS and age- and gender-matched IC/BPS patients without HL (non-HL IC/BPS) were examined using the pan-bacterial domain clinical-level molecular diagnostic Pacific Biosciences full-length 16S gene sequencing protocol, informatics pipeline and database. We characterized the differential presence, abundances, and diversity of species, as well as gender-specific differences between and among HL and non-HL IC/BPS patients. RESULTS: A total of 59 patients with IC/BPS were enrolled (29 HL, 30 non-HL; 43 women, 16 men) from a single centre and the microbiota in midstream urine specimens was available for comparison. The species abundance differentiation between the HL and non-HL groups (12 species) was not significantly different after Bonferroni adjustments for multiple comparisons. Similarly, the nine differentiating species noted between female HL and non-HL patients were not significantly different after similar statistical correction. However, four species abundances (out of the 10 species differences identified prior to correction) remained significantly different between male HL and non-HL subjects: Negativicoccus succinivorans, Porphyromonas somerae, Mobiluncus curtisii and Corynebacterium renale. Shannon diversity metrics showed significantly higher diversity among HL male patients than HL female patients (P = 0.045), but no significant diversity differences between HL and non-HL patients overall. CONCLUSIONS: We were not able to identify a unique pathogenic urinary microbiota that differentiates all HL from all non-HL IC/BPS. It is likely that the male-specific differences resulted from colonization/contamination remote from the bladder. We were not able to show that bacteria play an important role in patients with HL IC/BPS.

Diagnostic Utility of Serum and Urinary Metabolite Analysis in Patients with Interstitial Cystitis/Painful Bladder Syndrome.

OBJECTIVE: To identify the potential biomarkers of interstitial cystitis/painful bladder syndrome (IC), a chronic syndrome of bladder-centric pain with unknown etiology that has an adverse impact on quality of life, we analyzed the urine and serum metabolomes of a cohort of IC patients and non-disease controls (NC). METHODS: Home collection of serum and urine samples was obtained from 19 IC and 20 NC females in the Veterans Affairs (VA) Health Care System. IC was diagnosed independently by thorough review of medical records using established criteria. Biostatistics and bioinformatics analyses, including univariate analysis, unsupervised clustering, random forest analysis, and metabolite set enrichment analysis (MSEA), were then utilized to identify potential IC biomarkers. RESULTS: Metabolomics profiling revealed distinct expression patterns between NC and IC. Random forest analysis of urine samples suggested discriminators specific to IC; these include phenylalanine, purine, 5-oxoproline, and 5-hydroxyindoleacetic acid. When these urinary metabolomics-based analytes were combined into a single model, the AUC was 0.92, suggesting strong potential clinical value as a diagnostic signature. Serum-based metabolomics did not provide potential IC discriminators. CONCLUSION: Analysis of serum and urine revealed that women with IC have distinct metabolomes, highlighting key metabolic pathways that may provide insight into the pathophysiology of IC. The findings from this pilot study suggest that integrated analyses of urinary metabolites, purine, phenylalanine, 5-oxoproline, and 5-HIAA, can lead to promising IC biomarkers for pathophysiology of IC. Validation of these results using a larger dataset is currently underway.

Urine biomarkers in ESSIC type 2 interstitial cystitis/bladder pain syndrome and overactive bladder with developing a novel diagnostic algorithm.

This study aimed to investigate the diagnostic values of urine cytokines in interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder (OAB) patients, and to develop a novel diagnostic algorithm. Urine samples were collected from 40 IC/BPS, 40 OAB patients, and 30 controls. Commercially available multiplex immunoassays were used to analyze 31 targeted cytokines. Urine cytokine profiles were significantly different among study groups and controls. MIP-1ß showed the highest sensitivity (92.2%) for identifying diseased study patients from controls. The cytokines with high diagnostic values for distinguishing between IC and OAB included IL-10, RANTES, eotaxin, CXCL10, IL-12p70, NGF, IL-6, IL-17A, MCP-1, and IL-1RA. The diagnostic algorithm was subsequently developed according to the diagnostic values obtained. MIP-1ß was selected for the initial screening test to diagnose diseased patients and controls with diagnostic rates of 81.6% and 68.4%, respectively. As confirmation tests for IC/BPS, the diagnostic rates of eotaxin, CXCL10, and RANTES were 73.3%, 72.7%, and 69.7%, respectively. As the confirmation test for OAB, the diagnostic rate of IL-10 was 60%. Urine cytokine profiles of IC/BPS and OAB patients differed from those of controls and might be useful as biomarkers for diagnosis. A novel pilot diagnostic algorithm was developed based on these profiles.

Cultivable Bacteria in Urine of Women With Interstitial Cystitis: (Not) What We Expected.

OBJECTIVE: Multiple studies show cultivatable bacteria in urine of most women. The existence of these bacteria challenges interstitial cystitis (IC)/painful bladder syndrome (PBS) diagnosis, which presumes a sterile bladder. The aims of this study were (1) to compare the female bladder microbiomes in women with IC/PBS and unaffected controls and (2) to correlate baseline bladder microbiome composition with symptoms. METHODS: This cross-sectional study enrolled 49 IC/PBS and 40 controls. All provided catheterized urine samples and completed validated questionnaires. A subset of the IC/PBS cohort provided voided and catheterized urine samples. All samples from both cohorts were assessed by the expanded quantitative urine culture (EQUC) protocol; a subset was assessed by 16S rRNA gene sequencing. RESULTS: Of the IC/PBS cohort, 49.0% (24/49) were EQUC positive; in these EQUC-positive samples, the most common urotypes were Lactobacillus (45.8%) and Streptococcus (33.3%). Of the controls, 40.0% were EQUC positive; of these EQUC-positive samples, the most common urotype was Lactobacillus (50.0%). The urotype distribution was significantly different (P < 0.05), as 16% of the IC/PBS cohort, but 0% of controls, were Streptococcus urotype (P < 0.01). Symptom-free IC/PBS participants were less likely to be EQUC positive (12.5%) than IC/PBS participants with moderate or severe symptoms (68.8% and 46.2%) and the control cohort (60%; P < 0.05). CONCLUSION: Lactobacillus was the most common urotype. However, the presence of Lactobacillus did not differ between cohorts, and it did not impact IC/PBS symptom severity. Bacteria were not isolated from most participants with active IC/PBS symptoms. These findings suggest that bacteria may not be an etiology for IC/PBS.

Relationship of Bladder Pain With Clinical and Urinary Markers of Neuroinflammation in Women With Urinary Urgency Without Urinary Incontinence.

PURPOSE: The pathogenesis of bladder pain is poorly understood. Our hypothesis is that in women with urinary urgency without incontinence, bladder pain is associated with the presence of neurogenic inflammation in the bladder wall and neuroinflammatory biomarkers in the urine. METHODS: We conducted a prospective cross-sectional study of women with urinary urgency without incontinence. Urinary symptoms were measured using Female Genitourinary Pain Index. Neuropathic pain, a clinical biomarker of neuroinflammation, was measured using the PainDETECT questionnaire. Inflammatory neuropeptides measured in the urine included nerve growth factor (NGF), brain-derived neurotrophic factor, vascular endothelial growth factor, and osteopontin. Neuropathic pain scores and urinary neuropeptide levels were compared between patients with and without bladder pain using univariable and multivariable analyses. RESULTS: In 101 women with urinary urgency without incontinence, 62 (61%) were in the bladder pain group (visual analog scale score, ≤ 3), whereas 39 (39%) were in the no bladder pain group. Urinary symptom scores (5.0 ± 3.1 versus 3.5 ± 2.4, P < 0.001) and neuropathic pain scores (13.3 ± 8.6 vs 5.1 ± 4.8, P < 0.001) were significantly higher for the bladder pain group than for the no bladder pain group. On multivariable analysis after controlling for age, body mass index, and severity of urinary urgency, bladder pain score was significantly associated with elevated urinary levels of vascular endothelial growth factor (P = 0.04) and osteopontin (P = 0.02), whereas the neuropathic pain score was significantly associated with an increased NGF level (P = 0.03). CONCLUSIONS: In women with urinary urgency without incontinence, bladder pain is associated with the presence of clinical and urinary biomarkers of neuroinflammation.

Repeated intravesical injections of platelet-rich plasma improve symptoms and alter urinary functional proteins in patients with refractory interstitial cystitis.

Repeated intravesical injections of autologous platelet-rich plasma (PRP) have been shown to improve symptoms in patients with interstitial cystitis/bladder pain syndrome (IC/BPS); however, there is a paucity of objective evidence of the effectiveness of this therapy. In this study, we investigated the changes in urinary markers after PRP treatment. Forty patients with IC/BPS who were refractory to conventional therapy received four injections of PRP at monthly intervals; 10 mL PRP solution with 2.5 times the peripheral blood platelet concentration was used. Urine levels of thirteen functional proteins, growth factors, and cytokines were assessed at baseline and at the 4th PRP injection. The clinical parameters included visual analog scale (VAS) pain score, daily urinary frequency, nocturia episodes, functional bladder capacity, and global response assessment (GRA). The GRA and symptom score significantly decreased post-treatment. In patients with GRA ≥ 2, the success rates at 1 month and at 3 months after the 4th PRP injection were 70.6% and 76.7%, respectively. The VAS pain score, frequency, and nocturia showed a significant decrease (all p < 0.05). Urinary levels of nerve growth factor, matrix metalloproteinase-13, and vascular endothelial growth factor significantly decreased post-treatment (p = 0.043, p = 0.02, and p = 0.000, respectively); platelet-derived growth factor-AB showed a significant increase (p = 0.004) at the 4th PRP treatment compared with baseline. In this study, repeated intravesical PRP injections provided significant symptom improvement in IC/BPS patients with concomitant changes in the related biomarker levels.Trial registration: ClinicalTrial.gov: NCT03104361; IRB: TCGH 105-48-A.

Analysis of viruses present in urine from patients with interstitial cystitis.

The question of whether some cases of interstitial cystitis may have an infectious etiology has been debated for some time. Previous studies have looked for the presence of certain specific viruses, but generally did not use the types of sensitive and unbiased approaches that are currently available. As part of the MAPP (Multidisciplinary Approach to the Study of Chronic Pelvic Pain) Research Network, we examined urine specimens from interstitial cystitis patients who provided specimens over time and also reported various symptoms at the time of urine collection. We first performed next-generation sequencing to look for the presence of viruses in urines, and detected two human polyomaviruses that are known to be excreted into urine, BKPyV and JCPyV. We were especially interested in BKPyV because it is a known cause of another bladder disease, hemorrhagic cystitis, in bone marrow transplant recipients. Further analysis of individual samples indicates a trend toward higher excretion of polyomaviruses in patients experiencing increased symptoms.

Urine cytokines as biomarkers for diagnosing interstitial cystitis/bladder pain syndrome and mapping its clinical characteristics.

The objective of the present study was to investigate the diagnostic values of urine cytokines in patients with interstitial cystitis/bladder pain syndrome (IC/BPS) and to identify their correlations with clinical characteristics. Urine samples were collected from 127 patients with IC/BPS [European Society for the Study of Interstitial Cystitis (ESSIC) types 1 and 2] and 28 controls. Commercially available multiplex immunoassays (MILLIPLEX map kits) were used to analyze 31 targeted cytokines. Cytokine levels between patients with IC/BPS and controls were analyzed using ANOVA. Receiver-operating characteristic curves of each cytokine to distinguish IC/BPS from controls were generated for calculation of the area under the curve. Patients with IC/BPS had urine cytokine profiles that differed from those of controls. Between patients with ESSIC type 1 and 2 IC/BPS, urine cytokine profiles were also different. Among cytokines with high diagnostic values (i.e., area under the curve > 0.7) with respect to distinguish patients with ESSIC type 2 IC/BPS from controls, regulated upon activation, normal T cell expressed and presumably secreted (RANTES), macrophage inflammatory protein (MIP)-1ß, and IL-8 were of higher sensitivity, whereas macrophage chemoattractant protein (MCP)-1, chemokine (C-X-C motif) ligand 10 (CXCL10), and eotaxin-1 were of higher specificity. In multivariate logistic regression models controlling for age, sex, body mass index, and diabetes mellitus, the urine cytokines with high diagnostic values (MCP-1, RANTES, CXCL10, IL-7, and eotaxin-1) remained statistically significant in differentiating IC/BPS and controls. MCP-1, CXCL10, eotaxin-1, and RANTES were positively correlated with glomerulation grade and negatively correlated with maximal bladder capacity. In conclusion, patients with IC/BPS had urine cytokine profiles that clearly differed from those of controls. Urine cytokines might be useful as biomarkers for diagnosing IC/BPS and mapping its clinical characteristics.

Urine Gene Expression Profiles in Bladder Pain Syndrome Patients Treated with Triamcinolone.

BACKGROUND: The pathogenesis of bladder pain syndrome (BPS) remains incompletely defined, and there is no standard treatment for BPS as yet. OBJECTIVE: To gain detailed insight into the disease pathobiology of BPS through comparative gene expression analysis of urine from BPS patients versus control individuals and, furthermore, to determine the efficacy of triamcinolone treatment in BPS patients in terms of the gene expression profiles in urine. DESIGN, SETTING, AND PARTICIPANTS: A prospective pilot study including 21 urine samples from patients with Hunner's lesions (n=6) and controls (n=9) between January and August 2017. INTERVENTION: Triamcinolone treatment of BPS patients. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Urine samples from BPS patients were collected before (pretreatment group) and 2 wk after triamcinolone treatment (post-treatment group). Gene expression of urine sediment was analyzed using RNA sequencing. Pathways and biological processes in which differentially expressed genes are involved were analyzed. RESULTS AND LIMITATIONS: A total of 3745 genes were found to be differentially expressed between the three groups tested. Gene expression differences between controls and BPS samples (630 differentially expressed genes) were more pronounced than the differences between pre- and post-treatment BPS samples (197 differentially expressed genes). Gen Set Enrichment Analysis showed that differentially expressed genes in BPS patients (pretreatment), compared with controls, were enriched for some functional gene networks associated with several metabolic processes and ribosome biogenesis. The limited number of patients included may not accurately represent the BPS population. CONCLUSIONS: Gene expression profiles of urine sediment are able to discriminate between BPS and control patients. Moreover, we show that triamcinolone induces changes in urine gene expression profiles. PATIENT SUMMARY: In this report, we looked at gene expression profiles of urine sediment from patients with Hunner's lesions, before and after triamcinolone treatment, and control individuals. We found that urine gene expression profiles are able to discriminate Hunner's lesions patients from controls. Furthermore, we report, for the first time, that triamcinolone treatment of patients with Hunner's lesions induces changes in bladder gene expression profiles that can be observed in urine samples.

Long noncoding RNA (MEG3) in urinal exosomes functions as a biomarker for the diagnosis of Hunner-type interstitial cystitis (HIC).

BACKGROUND: Toll-like receptor-7 (TLR7) is functionally involved in the pathogenesis of Hunner-type interstitial cystitis (HIC). In addition, maternally expressed gene 3 (MEG3) is implicated in many urethral diseases. In this study, we aimed to verify the hypothesis that exosomal MEG3 in urine can be used as a novel diagnostic biomarker for HIC. METHODS: Electron microscopy was utilized to observe the distribution of urinary exosomes between the case group and the control group. Receiver operating characteristic analysis was utilized to compare the diagnostic values of MEG3 and miR-19a-3p. Computational analysis and luciferase assay were conducted to identify the correlation between MEG3 and miR-19a-3p as well as between TLR7 and miR-19a-3p. In addition, real-time polymerase chain reaction and Western blot were performed to establish the signaling pathways implicated in the pathogenesis of HIC. RESULTS: When age and gender distributions are excluded, urinary exosomes were equally distributed between case and control groups. The area under the curve of MEG3 was larger than that of miR-19a-3p, indicating that MEG3 has a better value in the diagnosis of HIC. In addition, patients with HIC showed elevated MEG3 expression and inhibited miR-19a-3p expression, thus establishing a negative correlation between MEG3 and miR-19a-3p. MEG3 and TLR7 were both identified as targets of miR-19a-3p, establishing a MEG3/miR-19a-3p/TLR7 signaling pathway, in which MEG3 enhances the expression of TLR7 via inhibiting the expression of miR-19a-3p. CONCLUSION: MEG3 level was upregulated in patients with HIC. In addition, MEG3 downregulated miR-19a-3p expression while upregulating TLR7 expression. Furthermore, MEG3 contributes to the pathogenesis of HIC. Therefore, exosomal MEG3 in urine can be used as a biomarker for HIC diagnosis.

Urinary fungi associated with urinary symptom severity among women with interstitial cystitis/bladder pain syndrome (IC/BPS).

PURPOSE: To correlate the presence of fungi with symptom flares, pain and urinary severity in a prospective, longitudinal study of women with IC/BPS enrolled in the MAPP Research Network. METHODS: Flare status, pelvic pain, urinary severity, and midstream urine were collected at baseline, 6 and 12 months from female IC/BPS participants with at least one flare and age-matched participants with no reported flares. Multilocus PCR coupled with electrospray ionization/mass spectrometry was used for identification of fungal species and genus. Associations between "mycobiome" (species/genus presence, relative abundance, Shannon's/Chao1 diversity indices) and current flare status, pain, urinary severity were evaluated using generalized linear mixed models, permutational multivariate analysis of variance, Wilcoxon's rank-sum test. RESULTS: The most specific analysis detected 13 fungal species from 8 genera in 504 urine samples from 202 females. A more sensitive analysis detected 43 genera. No overall differences were observed in fungal species/genus composition or diversity by flare status or pain severity. Longitudinal analyses suggested greater fungal diversity (Chao1 Mean Ratio 3.8, 95% CI 1.3-11.2, p = 0.02) and a significantly greater likelihood of detecting any fungal species (OR = 5.26, 95% CI 1.1-25.8, p = 0.04) in high vs low urinary severity participants. Individual taxa analysis showed a trend toward increased presence and relative abundance of Candida (OR = 6.63, 95% CI 0.8-58.5, p = 0.088) and Malassezia (only identified in 'high' urinary severity phenotype) for high vs low urinary symptoms. CONCLUSION: This analysis suggests the possibility that greater urinary symptom severity is associated with the urinary mycobiome urine in some females with IC/BPS.

Effect of Water Avoidance Stress on serum and urinary NGF levels in rats: diagnostic and therapeutic implications for BPS/IC patients.

Nerve growth factor (NGF) is thought to play a key role in chronic pain felt by bladder pain syndrome/interstitial cystitis (BPS/IC) patients by activating its high affinity receptor tropomyosin-related kinase subtype A (Trk A). Whether this pathway is also involved in the aggravation of pain sensation during stress events was here investigated. The levels of plasmatic NGF were increased in rats submitted to Water Avoidance Stress test (WAS), compared to controls. The administration of the alpha1A adrenoceptors blocker silodosin prevented the increase of plasmatic NGF. Urinary NGF levels were also moderately increased in animals submitted to WAS. WAS increased pain behaviour score, lowered abdominal mechanical pain threshold and increase voiding bladder reflex activity. These changes were prevented by the administration of TrkA antagonist GW441756. These findings prompt the use of plasmatic NGF as diagnosis tool for chronic visceral painful conditions and opens therapeutic opportunities for TrkA receptors antagonist/NGF sequestration.

Biomarkers in the diagnosis and symptom assessment of patients with bladder pain syndrome: a systematic review.

INTRODUCTION AND HYPOTHESIS: Bladder pain syndrome (BPS) is a disease of unknown etiology defined as an unpleasant sensation related to the bladder, associated with lower urinary tract symptoms of more than 6 weeks' duration, in the absence of any identifiable causes. Despite its impact on quality of life (QoL) and socioeconomic burden, there are no objective methods for the diagnosis or assessment of therapeutic response. We systematically reviewed biomarkers associated with BPS to update the current knowledge on this issue. METHODS: A systematic review of the Cochrane Library, Embase, PubMed/MEDLINE, LILACS, SCOPUS, and ClinicalTrials.gov databases was conducted following the PRISMA statement. Original articles investigating biomarkers for the diagnosis or symptom assessment of patients with BPS were assessed; no language restrictions were applied. Animal or post-mortem studies were excluded. RESULTS: Of the 478 records retrieved, 11 articles were included. MIF, NGF, Etio-S, APF, and a combined methylhistamine/Il-6 model were increased in BPS urine samples versus controls. Also increased were glyceraldehyde in stool, in addition to the expression of some genes (ARID1A, ARF, CHAT, eNOS, GLI-1, iNOS, MCP-1, NGF, WNT-8A, WNT-10A), nerve density, IL-16, VCAM-1, and ICAM-1 in bladder tissue specimens. In contrast, some fecal bacteria, expression of other genes (CHT, HB-EGF, OCT-1, SMRT-1, WNT11) in the bladder urothelium, and urinary DNA methylation in CpG-sites, MCP-3, G5P1, and HB-EGF were decreased in BPS. As none of the biomarkers was studied more than once, a Forest plot could not be constructed. Only 4 articles reported the relation of biomarkers to symptom scores. CONCLUSIONS: Potential biomarkers for BPS in urine, stool, and bladder biopsy specimens are described. Further research is needed before their use in clinical practice.

Urine nerve growth factor (NGF) level, bladder nerve staining and symptom/problem scores in patients with interstitial cystitis.

BACKGROUND: Interstitial cystitis (IC)/bladder pain syndrome (BPS) is a challenging disease, affecting thousands of people all around the world, especially women. Although there have been numerous theories regarding IC/BPS etiology, the physiopathology of the disease still remains unclear and there is a lack of certain treatment. OBJECTIVES: The aim of the study was to assess the role of nerve fibers and nerve growth factor (NGF) in the etiopathogenesis of IC/BPS symptoms by demonstrating if there is a correlation between urine NGF levels, amount of peripheral nerves in bladder mucosa and symptom severity. MATERIAL AND METHODS: A prospective clinical study was conducted with 15 IC/BPS patients and 18 controls. Urine NGF levels were measured by enzyme-linked immunosorbent assay (ELISA). Bladder punch biopsies were obtained from 15 IC/BPS patients and 9 controls. Immunohistochemistry was performed for S-100 to highlight peripheral nerve twigs in bladder mucosa. The O'Leary-Sant Interstitial Cystitis Symptom and Problem Index (OSICSPI) was used to assess symptom severity and effects of the disease on the patients' life. RESULTS: NGF normalized to urine creatinine (NGF/Cr) levels in IC/BPS patients were significantly higher than in controls, 0.34 ±0.22 and 0.09 ±0.08 pg/mL: mg/dL, respectively (p < 0.001). The mean symptom score in IC patients was 12.27 ±2.4 (median: 12) and the mean problem score was 10.9 ±2.3 (median: 12). The mean mucosal nerve (S-100 stained) area in the IC/BPS group was significantly higher than in the controls, 2.53 ±1.90 vs 1.0 ±0.70, respectively (p = 0.018). In correlation analyses, the NGF/Cr level in IC/BPS patients was found significantly correlated with the O'Leary-Sant IC Symptom and Problem Index scores independently (p = 0.001 and p = 0.028, respectively). CONCLUSIONS: NGF seems to be a promising biomarker in IC/BPS. It may help clinicians in diagnoses and patient follow-up. Thus, unnecessary, expensive and invasive tests, interventions and treatments might be avoided.

Elevated Urine Levels of Macrophage Migration Inhibitory Factor in Inflammatory Bladder Conditions: A Potential Biomarker for a Subgroup of Interstitial Cystitis/Bladder Pain Syndrome Patients.

OBJECTIVE: To investigate whether urinary levels of macrophage migration inhibitory factor (MIF) are elevated in interstitial cystitis/bladder pain syndrome (IC/BPS) patients with Hunner lesions and also whether urine MIF is elevated in other forms of inflammatory cystitis. METHODS: Urine samples were assayed for MIF by enzyme-linked immunosorbent assay. Urine samples from 3 female groups were examined: IC/BPS patients without (N = 55) and with Hunner lesions (N = 43), and non-IC/BPS patients (N = 100; control group; no history of IC/BPS; cancer or recent bacterial cystitis). Urine samples from 3 male groups were examined: patients with bacterial cystitis (N = 50), radiation cystitis (N = 18) and noncystitis patients (N = 119; control group; negative for bacterial cystitis). RESULTS: Urine MIF (mean MIF pg/mL ± standard error of the mean) was increased in female IC/BPS patients with Hunner lesions (2159 ± 435.3) compared with IC/BPS patients without Hunner lesions (460 ± 114.5) or non-IC/BPS patients (414 ± 47.6). Receiver operating curve analyses showed that urine MIF levels discriminated between the 2 IC groups (area under the curve = 72%; confidence interval 61%-82%). Male patients with bacterial and radiation cystitis had elevated urine MIF levels (2839 ± 757.1 and 4404 ± 1548.1, respectively) compared with noncystitis patients (681 ± 75.2). CONCLUSION: Urine MIF is elevated in IC/BPS patients with Hunner lesions and also in patients with other bladder inflammatory and painful conditions. MIF may also serve as a noninvasive biomarker to select IC/BPS patients more accurately for endoscopic evaluation and possible anti-inflammatory treatment.

A genome-scale DNA methylation study in women with interstitial cystitis/bladder pain syndrome.

AIMS: To assess the feasibility of using voided urine samples to perform a DNA methylation study in females with interstitial cystitis/bladder pain syndrome (IC/BPS) as compared to age- and race-matched controls. A unique methylation profile could lead to a non-invasive, reproducible, and objective biomarker that would aid clinicians in the diagnosis of IC/BPS. METHODS: Nineteen IC/BPS patients and 17 controls were included. IC/BPS patients had an Interstitial Cystitis Symptom Index score of >8; controls had no bladder symptoms. DNA was extracted from pelleted urine sediment. Samples with >500 ng of genomic DNA underwent quantitative DNA methylation assessment using the Illumina Infinium MethylationEPIC BeadChip. Age- and race-matching was applied prior to analysis. Linear regression models were used to compare average methylation between IC/BPS cases and controls at each cytosine guanine dinucleotide site (loci where methylation can occur). RESULTS: Sixteen participants (eight IC/BPS age- and race-matched to eight controls) had adequate DNA for methylation analysis. The median age was 43.5 years (interquartile range 33.8, 65.0), the median BMI was 27.1 (IQR 22.7, 31.4), and 14 were Caucasian (87.5%). A total of 688 417 CpG sites were analyzed. In exploratory pathway analysis utilizing the top 1000 differentially methylated CpG sites, the mitogen-activated protein kinase (MAPK) pathway was overrepresented by member genes. CONCLUSIONS: The results demonstrate the feasibility of using voided urine specimens from women with IC/BPS to perform DNA methylation assessments. Additionally, the data suggest genes within or downstream of the MAPK pathway exhibit altered methylation in IC/BPS.