Autoimmune murine thyroiditis. Relationship of humoral and cellular immunity to thyroiditis in high and low responder mice

Good responder (C57Br/cd) and poor responder (C57BL/10) mice were immunized with mouse thyroid extract in Freund's complete adjuvant. The good responder mice first showed antibody to thyroglobulin on the seventh day while the poor responder animals had slightly lower titers and antibody did not appear until day 12. The first signs of thyroid infiltration appeared on day 4 in the responder strain, and severe lesions were seen between the third and fifth week. In contrast, the poor responder mice showed almost no evidence of infiltration. The macrophage disappearance reaction, a measure of cell-mediated immunity, was similar in the responder and nonresponder strains.     

MicroRNAs May Mediate the Down-Regulation of Neurokinin-1 Receptor in Chronic Bladder Pain Syndrome

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B₁ receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor.Bladder pain syndrome (BPS) should be diagnosed on the basis of symptoms of pain associated with the urinary bladder, accompanied by at least one other symptom, such as day-time and/or night-time urinary frequency after exclusion of confounding symptoms, according to the European Society for the Study of Interstitial Cystitis proposal and European Association of Urology guidelines.¹ The term “interstitial cystitis” (IC) was not omitted to avoid problems of reimbursement, disability benefits and so forth. The European Society for the Study of Interstitial Cystitis proposal coined the term BPS, however considered the use of painful bladder syndrome and IC in parallel acceptable. Overall, this disease, which has a significant impact on social and psychological well-being, affects approximately 1 million patients in the United States alone,² with at least 230 confirmed cases per 100,000 females.³ The etiology of BPS is unknown, and its treatment is largely empirical. A multitude of pathogenetic mechanisms have been postulated ranging from neuroinflammatory to autoimmune or possibly infectious or toxic agents, but an inflammatory component is commonly thought to be involved.⁴ Epithelial damage has often been invoked: the mucinous layer of the healthy bladder is often compromised in patients with BPS/IC, as well as in some animal models.^5,6 An initiating event (toxin) may lead to increased urothelial permeability, which in turn leads to nerve sensitization and possibly up-regulation of neurotransmitter release (tachykinins, glutamate, calcitonin gene-related peptide).⁷ Sensory nerves secrete inflammatory mediators such as substance P (SP), a nociceptive neurotransmitter in the central and peripheral nervous system. It is not clear whether there are differences in urinary SP between IC patients and controls, although several studies demonstrated increased amounts of the released SP in the urine of BPS/IC patients, and there is a suggestion that increased SP-positive nerve fibers may be present in IC patients.^8–10Activated G protein-coupled receptors are implicated in inflammatory conditions, including BPS/IC and prostatitis.¹¹ Recent results suggest that neurogenic inflammation may be involved in the pathogenesis of BPS/IC in the animal model.¹² However to date this has not been clearly shown in human tissue. Tachykinins (TK), including SP, mediate neurogenic inflammation and smooth muscle contraction in the genitourinary tract through stimulation of neurokinin (NK)1 and NK2 receptors (NK1R and NK2R).¹³ NK1 receptor-expressing sensory neurons are found in the muscle layer of bladder (detrusor), within and just below the urothelium, and around blood vessels, being more abundant in the bladder base than in the dome.¹⁴ In the urinary system, TKs are thought to be responsible for overactivity, hypersensitivity, inflammation, and changes in urothelial permeability.¹⁵ Experiments with NK1R knockout mice demonstrated an obligatory requirement of NK1R in cystitis and participation of these receptors in mast cell degranulation and inflammation.¹² It has previously been shown, using semiquantitative methods, that the NK1R mRNA is increased in bladder biopsies from patients with BPS/IC.¹⁶ In addition, NK2 receptors, which play a pivotal role in the modulation of motor and sensory functions, have been localized in human bladder detrusor muscle.¹⁷ In human urothelium, NK2R expression has not been detected, which is in contrast to animal studies, where NK2 receptors are present in rat urothelium and have a role initiating micturition.^18,19 The apparent species-specific differences in expression patterns of TK receptors warrant caution in interpreting the data obtained from animal models of BPS/IC, and prompted us to investigate the expression and localization of these receptors in healthy and diseased human bladders.Changes in bladder function are often concomitant with structural alterations in smooth muscle and urothelial cells, which manifest themselves in changes of gene expression.²⁰ Such changes are indicative of the pathophysiological processes taking place during the progression of a disease; therefore some of the causative factors of BPS/IC might be elucidated by uncovering a link between the expression of proteins, mediating neurogenic inflammation, bladder contractility and epithelial permeability. Although there are several studies examining gene expression changes during experimentally induced cystitis in animals,^21–25 human data are scarce. In human BPS patients, the molecular markers for bladder permeability and proteoglycan core proteins have been shown to be down-regulated,^26,27 and there was an increased permeability and decreased tight junction formation of bladder epithelial cell monolayers grown from biopsies in patients with BPS/IC, as compared with cells from normal controls.²⁸Recently, microRNAs (miRNAs) have been defined as an important class of gene expression regulators, involved in the processes of inflammation and cancer.^29,30 MiRNAs are noncoding single-stranded RNAs of about 22 nucleotides that regulate the expression of mRNAs, inhibiting the protein production by base-pairing with complementary sequences.³¹ Studies in psoriasis, atopic eczema, and inflammatory bowel disease have shown an involvement of miRNAs in the pathogenesis of these inflammatory diseases.^32,33We collected biopsy material from 28 BPS patients with a high degree of continuous pain and 8 controls and quantified the disease-related changes in the expression levels of the genes involved in the regulation of epithelial permeability, bladder contractility, and neurogenic inflammation. We identified and validated several miRNA species, significantly up-regulated in BPS patients. Our findings suggest a causative role of miRNAs in the regulation of gene expression in BPS, and possible mechanisms of their induction and targeting.     

YKL‐40 and mast cells are associated with detrusor fibrosis in patients diagnosed with bladder pain syndrome/interstitial cystitis according to the 2008 criteria of the European Society for the Study of Interstitial Cystitis

Richter B, Roslind A, Hesse U, Nordling J, Johansen J S, Horn T & Hansen A B
(2010) Histopathology 57 , 371–383
YKL‐40 and mast cells are associated with detrusor fibrosis in patients diagnosed with bladder pain syndrome/interstitial cystitis according to the 2008 criteria of the European Society for the Study of Interstitial Cystitis Aims: Bladder pain syndrome/interstitial cystitis (BPS/IC), diagnosed according to the new 2008 criteria of the European Society for the Study of Interstitial Cystitis (ESSIC), may lead to detrusor fibrosis. In some inflammatory diseases, fibrosis is related to YKL‐40. The aims were to examine YKL‐40 antigenic expression in bladder tissue and levels in serum and urine in BPS/IC and to evaluate whether YKL‐40 could be a non‐invasive, prognostic biomarker for bladder fibrogenesis and treatment intensity. Methods and results: Immunohistochemistry, immunoelectron microscopy and enzyme‐linked immunosorbent assay (ELISA) analyses in 45 patients showed YKL‐40 expression in detrusor mast cell granules and submucosal macrophages, and elevated YKL‐40 levels in serum and urine compared to healthy individuals (median 72 versus 7 μg/l, P < 0.001). Clinicopathological parameters showed associations of detrusor fibrosis with YKL‐40‐positive cells (P = 0.001), mast cells (P = 0.014) and urine YKL‐40 (P = 0.009). Bladder capacity correlated inversely with YKL‐40‐positive cells (P < 0.001) and mast cells (P = 0.029). Treatment intensity was not associated with YKL‐40. Conclusion: Serum and urine levels of YKL‐40 may be used as non‐invasive biomarkers in BPS/IC for the evaluation of bladder fibrogenesis.     

卡介苗素膀胱灌注预防膀胱癌术后复发的临床研究

目的探讨卡介苗素膀胱内灌注预防膀胱癌术后复发的疗效、安全性．方法47例膀胱癌术后患者分2组。分别应用卡介苗素和卡介苗定期行膀胱内灌注，随访10～32个月，了解灌注后肿瘤复发情况及并发症。并于灌注前、后检测2组尿液中IL－2、IL－6、IL－8的变化情况．结果卡介苗膀胱灌注组肿瘤复发4例（17．4％）。副反应发生18例（78．3％），卡介苗素膀胱灌注组肿瘤复发5例（20．8％）。副反应发生率11例（45．8％），2组尿液中IL－2、IL－6、IL－8值灌注后高于灌注前（P〈0．05）．两组肿瘤复发率、IL-2、IL-6、IL-8值灌注前后比较无显著差异，副反应卡介苗组明显高于卡介苗素组（P〈0．05）．结论卡介苗素膀胱灌注预防膀胱癌术后复发的有效率与卡介苗相同，但不良反应明显减少。患者耐受性好，因此卡介苗素可成为膀胱浅表移行上皮细胞癌临床治疗和预防复发的一种有效药物．     

Histamine and mucosal mast cells in interstitial cystitis

Interstitial cystitis (IC) is a chronic inflammatory disorder of the urinary bladder of unknown aetiology and pathogenesis. The classic form (Hunner's ulcer) is characterized by high mast cell numbers in the detrusor muscle and an expansion of mucosal mast cells in the lamina propria and also in the epithelium. Such cells can be recovered in bladder washings and in the urine in single cell suspensions. We have counted the mast cells and measured the histamine in bladder washings from 16 patients with classic IC and from a control group of 15 patients with so-called early, non-ulcerative IC. The bladder washings from all patients with classic IC contained well preserved mast cells (median 2.16, range 0.5–8.6×10³ cells/I) and histamine (median 14.3, range 6–66 ng/l), while only occasional mast cells and traces of histamine were found in washings from patients with non-ulcerative IC. The histamine content was strongly correlated to the number of mast cells (r=0.87). The mean histamine content per mast cell was estimated at 7.6±0.65 (SEM) pg/cell. The high histamine content per mast cell in relation to previously published data (2.8–4.6 pg/cell) can be attributed to the mild and rapid handling of the specimens.     

Highly specific detection of muscarinic M3 receptor,G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA)

Purpose

Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis – BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples.

Conditioned medium derived from mesenchymal stem cells culture as a intravesical therapy for cystitis interstitials

The treatment of Interstinal Cystitisis (IC) is still challenge for urologist. Available therapies do not result in long-term control of symptoms and do not provide pain relive to patients. Unique abilities of mesenchymal stem cells (MSC) could be used to develop new treatment approaches for Interstitial Cystitis. Conditioned Medium (CM) derived from MSC culture is rich in plenty of growth factors, cytokines and trophic agents which were widely reported to enhance regeneration of urinary bladder in different conditions. This ready mixture of growth factors could be used to develop intravesical therapy for patients with IC. MSC-CM has anti-apoptotic, anti-inflammatory, supportive, angiogenic, immunosuppressive and immunomodulative properties and seems to be ideal substance to prevent IC recurrence and to create favorable environment for regeneration of damaged bladder wall.     

Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette–Guérin (BCG) in patients with superficial bladder cancer

Few studies have analysed the antibody response during intravesical BCG immunotherapy for superficial bladder cancer. We have examined the evolution in serum antibody response against several heat shock proteins (hsp), including the recombinant mycobacterial hsp65 and the native protein P64 from BCG, GroEL from Escherichia coli (hsp60 family), recombinant mycobacterial hsp70 and the E. coli DnaK (hsp70 family), against purified protein derivative of tuberculin (PPD) and the AG85 complex of Mycobacterium bovis BCG, as well as against tetanus toxoid in 42 patients with a superficial bladder tumour, 28 treated with six intravesical BCG instillations and 14 patients used as controls. We also analysed the lymphoproliferative response of peripheral blood mononuclear cells against PPD in this population. Data of antibody responses at 6 weeks post BCG were available in all 28 patients, and at 4 month follow up in 17 patients. All patients who demonstrated a significant increase in IgG antibodies against PPD at 4 months follow up had a significant increase already at 6 weeks of follow up. In contrast, IgG antibodies against hsp increased significantly from 6 weeks to 4 months post-treatment. A significant increase in IgG antibodies against PPD, hsp65, P64, GroEL, and hsp70 at 4 months follow up was observed in 10/17, 8/17, 10/17, 4/17 and 8/17 patients. Native P64 protein elicited a higher antibody response than recombinant mycobacterial hsp65. No increase in antibody response was observed against Dnak from E. coli, against AG85 or tetanus toxoid after BCG therapy. An increase in IgG antibodies against P64 at 4 months follow up compared with pretreatment values was found to be a significant predictor of tumour recurrence (P< 0.01). Further studies with a larger number of patients are needed to confirm the value of the antibody response against P64 as a clinical independent prognostic factor.     

RDP58 inhibits T cell-mediated bladder inflammation in an autoimmune cystitis model

Interstitial cystitis (IC) is a chronic inflammatory condition of the urinary bladder with a strong autoimmune component. Currently, the major challenge in IC treatment is the development of effective therapies. RDP58 is a novel d-amino acid decapeptide with potent immunosuppressive activity. In this study, we investigated whether RDP58 was effective as an intravesical agent for treating bladder autoimmune inflammation in a transgenic mouse model (URO-OVA mice). URO-OVA mice were adoptively transferred with syngeneic activated splenocytes of OT-I mice transgenic for the OVA-specific CD8(+) TCR for cystitis induction and treated intravesically with RDP58 at days 0 and 3. Compared with controls, the RDP58-treated bladders showed markedly reduced histopathology and expressions of mRNAs and proteins of TNF-alpha, NGF and substance P. To determine whether the inhibition of bladder inflammation by RDP58 was due to the interference with effector T cells, we treated the cells with RDP58 in vitro. Cells treated with RDP58 showed reduced production of TNF-alpha and IFN-gamma as well as apoptotic death. Collectively, these results indicate that RDP58 is effective for treating T cell-mediated experimental autoimmune cystitis and may serve as a useful intravesical agent for the treatment of autoimmune-associated bladder inflammation such as IC.     

Activation of bladder mast cells in interstitial cystitis.

Interstitial cystitis (IC) is a sterile, inflammatory bladder condition characterized by urinary frequency and urgency, as well as burning and suprapubic pain, which occurs more frequently in women who may suffer for years before diagnosis. An increased number of mast cells have been associated with IC, but the published reports are inconclusive and often conflicting. Human bladder biopsies were analysed blindly for the degree of activation of mast cells in control and IC patients. It was found that mast cells from IC patients averaged as high as 34 cells/mm2 as compared to less than 16/mm2 in controls. Electron microscopy revealed that over 90% of mast cells from IC patients were activated to various degrees. It is concluded that mast cell activation is a pathologic characteristic for IC.     

Analysis of the immunologic mechanism of intravesical bacillus Calmette-Guerin therapy for superficial bladder tumors: distribution and function of immune cells.

Intravesical bacillus Calmette-Guerin (BCG) administration has been used as an adjuvant therapy after transurethral resection for superficial bladder cancer, but the exact mechanisms of its antitumor activity are not yet known. The aim of this study was to characterize the immunologic aspects of antitumor activity of BCG using an animal model. C3H/He inbred mice and murine bladder tumor cell line, MBT-2 were used. The changes in immune cells such as helper T cells, suppressor T cells, macrophages and natural killer cells in the bladder and spleen were analysed by immunohistochemical method in intravesical BCG instilled in normal bladder, MBT-2 implanted after electrocauterization of the bladder mucosa and MBT-2 implanted and intravesical BCG treated group. The changes in natural killer cell activity of the splenocytes and peritoneal lymphocytes were evaluated using 51chromium release assay at regular time intervals following intraperitoneal BCG instillation. The prophylactic anticancer effect was evaluated by observing the tumor growth in the intravesically BCG treated group after intravesical MBT-2 implantation. In immunohistochemical examination, a remarkable infiltration of macrophage and helper T cell was observed in the lamina propria of the bladder, and the helper and suppressor T cells ratio (Th/Ts ratio) was increased after intravesical BCG therapy. In 51chromium release assay, enhanced natural killer cell activity of the splenocytes and peritoneal lymphocytes was observed after intraperitoneal BCG inoculation. The growth of implanted tumor was suppressed following intravesical instillation of BCG. These results suggest that the antitumor activity of BCG is not related to the simple inflammatory reaction but to the local and systemic immune response in which helper T lymphocytes and mononuclear cells play an important role.     

Langerhans cell histiocytosis of the urinary bladder in a patient with bladder cancer previously treated with intravesical Bacillus Calmette–Guérin therapy

We report an extremely rare case of Langerhans cell histiocytosis (LCH) of the urinary bladder. A 68-year-old man presented with gross hematuria. Cystoscopy showed multiple papillary tumors in the urinary bladder, and transurethral resection was performed. Pathological diagnosis was high-grade papillary urothelial carcinoma with lamina propria invasion. The patient received six treatments with intravesical Bacillus Calmette–Guérin (BCG) therapy. Seven months after surgery, follow-up cystoscopy showed three elevated lesions in the urinary bladder, two of which were identified histologically as recurrent urothelial carcinoma. Microscopic examination of the lesion at the anterior wall revealed diffuse infiltration of medium to large histiocytoid cells in the lamina propria, many of which had distorted nuclei and nuclear grooves. Dense eosinophilic infiltration was also observed. Immunohistochemically, the histiocytoid cells were diffusely positive for S-100 and CD1a, but negative for cytokeratin AE1/AE3 and melanosome-associated antigen recognized by HMB-45. Based on the histological and immunohistochemical features, we diagnosed the lesion as LCH of the urinary bladder. There was no evidence of recurrence of either bladder cancer or LCH after an 18-month follow-up. To avoid misdiagnosis, urologists and pathologists should be aware that LCH may develop in the urinary bladder after intravesical BCG therapy for bladder cancer.     

Luteal support with micronized progesterone following in-vitro fertilization using a down-regulation protocol with gonadotrophin-releasing hormone agonist: a comparative study between vaginal and oral administration.

This study aimed to compare the efficacy of micronized progesterone administered as luteal support following ovulation induction for in-vitro fertilization (IVF)- embryo transfer in cycles using gonadotrophin-releasing hormone agonist, either orally (200 mgx4/day) or vaginally (100 mgx2/day) and to characterize the luteal phase hormonal profile during such treatments. A total of 64 high responder patients requiring intracytoplasmic sperm injection due to male factor infertility were prospectively randomized into two treatment groups. Patients treated orally or vaginally were comparable in age (31.9 +/- 6.1 versus 30.6 +/- 5.2; mean +/- SD), number of oocytes retrieved (17 +/- 8.2 versus 18 +/- 7.0), and number of embryos transferred (3.1 +/- 1.2 versus 2.7 +/- 0.9) per cycle. Following low dose vaginal treatment, a significantly higher implantation rate (30.7 versus 10.7%, P < 0.01), but similar clinical pregnancy rate (47.0 versus 33.3%) and ongoing pregnancy rate (41.1 versus 20.0%) was observed, compared with oral treatment. In conception cycles, luteal serum progesterone and oestrogen concentrations did not differ between the treatment groups. In non-conception cycles, late luteal progesterone concentrations were significantly lower following vaginal treatment. As low dose micronized progesterone administered vaginally is simple, easy and well tolerated, it could be recommended as the method of choice for luteal support, especially for high responder patients at risk for ovarian hyperstimulation syndrome.     

Anidulafungin for the treatment of candidaemia/invasive candidiasis in selected critically ill patients

A prospective, multicentre, phase Illb study with an exploratory, open-label design was conducted to evaluate efficacy and safety of anidulafungin for the treatment of candidaemia/invasive candidiasis (C/IC) in specific ICU patient populations. Adult ICU patients with confirmed C/IC meeting ≥l of the following criteria were enrolled: post-abdominal surgery, solid tumour, renal/hepatic insufficiency, solid organ transplant, neutropaenia, and age ≥65 years. Patients received anidulafungin (200 mg on day 1, 100 mg/day thereafter) for 10- 42 days, optionally followed by oral voriconazole/fluconazole. The primary efficacy endpoint was global (clinical and microbiological) response at the end of all therapy (EOT). Secondary endpoints included global response at the end of intravenous therapy (EOIVT) and at 2 and 6 weeks post-EOT, survival at day 90, and incidence of adverse events (AEs). The primary efficacy analysis was performed in the modified intent-to-treat (MITT) population, excluding unknown/missing responses. The safety and MITT populations consisted of 216 and 170 patients, respectively. The most common pathogens were Candida albicans (55.9%), C. glabrata (14.7%) and C. parapsilosis (10.0%). Global success was 69.5% (107/154; 95% CI, 61.6-76.6) at EOT, 70.7% (111/157) at EOIVT, 60.2% (77/128) at 2 weeks post-EOT, and 50.5% (55/109) at 6 weeks post-EOT. When unknown/missing responses were included as failures, the respective success rates were 62.9%, 65.3%, 45.3% and 32.4%. Survival at day 90 was 53.8%. Treatment-related AEs occurred in 33/216 (15.3%) patients, four (1.9%) of whom had serious AEs. Anidulafungin was effective, safe and well tolerated for the treatment of C/IC in selected groups of ICU patients.     

Expression and function of bradykinin B1 and B2 receptors in normal and inflamed rat urinary bladder urothelium

The bladder urothelium exhibits dynamic sensory properties that adapt to changes in the local environment. These studies investigated the localization and function of bradykinin receptor subtypes B1 and B2 in the normal and inflamed (cyclophosphamide (CYP)-induced cystitis) bladder urothelium and their contribution to lower urinary tract function in the rat. Our findings indicate that the bradykinin 2 receptor (B2R) but not the bradykinin 1 receptor (B1R) is expressed in control bladder urothelium. B2R immunoreactivity was localized throughout the bladder, including the urothelium and detrusor smooth muscle. Bradykinin-evoked activation of this receptor elevated intracellular calcium  (EC₅₀= 8.4 n m )  in a concentration-related manner and evoked ATP release from control cultured rat urothelial cells. In contrast, B1R mRNA was not detected in control rat urinary bladder; however, following acute (24 h) and chronic (8 day) CYP-induced cystitis in the rat, B1R mRNA was detected throughout the bladder. Functional B1Rs were demonstrated by evoking ATP release and increases in [Ca²⁺]_i in CYP (24 h)-treated cultured rat urothelial cells with a selective B1 receptor agonist (des-Arg⁹-bradykinin). Cystometry performed on control anaesthetized rats revealed that intravesical instillation of bradykinin activated the micturition pathway. Attenuation of this response by the P2 receptor antagonist PPADS suggests that bradykinin-induced micturition facilitation may be due in part to increased purinergic responsiveness. CYP (24 h)-treated rats demonstrated bladder hyperactivity that was significantly reduced by intravesical administration of either B1 (des-Arg¹⁰-Hoe-140) or B2 (Hoe-140) receptor antagonists. These studies demonstrate that urothelial expression of bradykinin receptors is plastic and is altered by pathology.     

Local immunostimulation induced by intravesical administration of autologous interferon-gamma-activated macrophages in patients with superficial bladder cancer

We conducted a phase I/II clinical trial of the safety and efficacy of intravesical administration of autologous IFN-gamma-activated macrophages (MAK) in patients with superficial bladder cancer. Monocyte-derived MAK cells were prepared in vitro and patients received six instillations of 1.4 x 10(8) to 2.5 x 10(8) cells, once a week, for five consecutive weeks. Treatment was well tolerated, with seven grade 1 and five Grade 2 protocol-related adverse effects. Nine out of 17 included patients had no recurrences during the year following the first instillation of MAK. The aim of the present study was to search for immune parameters related to local immunostimulation induced by MAK. Monitoring of the patients showed that urinary IL-8, GM-CSF and, to a lesser extent, IL-18 were increased following MAK instillations, with inter-individual differences. The urinary IL-8 level was about 10-fold higher than that observed for other cytokines, and its biological activity was reflected by a concomitant increase of urinary elastase, indicating neutrophil activation and degranulation. We also showed that nine out of 12 patients investigated presented an increase of urinary neopterin, a marker of IFN-gamma-activated macrophages, 7 days after MAK instillation, while serum neopterin levels were almost stable. These results are in line with persistence of activated macrophages in the bladder wall after infusions. Moreover, there was evidence of macrophages in urine smears 2 months after the sixth MAK instillation, and the score of macrophages correlated with the quantity of neutrophils in the urine. Overall, this study provides evidence of a local immunostimulation induced by this novel and safe immunotherapeutic approach of MAK instillations in patients with superficial bladder cancer.     

Establishment of an Orthotopic Mouse Non-Muscle Invasive Bladder Cancer Model Expressing the Mammalian Target of Rapamycin Signaling Pathway

We established an orthotopic non-muscle invasive bladder cancer (NMIBC) mouse model expressing the mammalian target of the rapamycin (mTOR) signaling pathway. After intravesical instillation of KU-7-lucs (day 0), animals were subsequently monitored by bioluminescence imaging (BLI) on days 4, 7, 14, and 21, and performed histopathological examination. We also validated the orthotopic mouse model expressing the mTOR signaling pathway immunohistochemically. In vitro BLI photon density was correlated with KU-7-luc cell number (r² = 0.97, P < 0.01) and in vivo BLI photon densities increased steadily with time after intravesical instillation. The tumor take rate was 84.2%, formed initially on day 4 and remained NMIBC up to day 21. T1 photon densities were significantly higher than Ta (P < 0.01), and histological tumor volume was positively correlated with BLI photon density (r² = 0.87, P < 0.01). The mTOR signaling pathway-related proteins were expressed in the bladder, and were correlated with the western blot results. Our results suggest successful establishment of an orthotopic mouse NMIBC model expressing the mTOR signaling pathway using KU-7-luc cells. This model is expected to be helpful to evaluate preclinical testing of intravesical therapy based on the mTOR signaling pathway against NMIBC.     

Cannabinoid receptor 2 is increased in acutely and chronically inflamed bladder of rats

Cannabinoid receptors 1 and 2 (CB1 and CB2) are G-protein coupled receptors that are expressed throughout the body. Cannabinoid receptors are expressed in the urinary bladder and may affect bladder function. The purpose of this study was twofold: to confirm the presence of cannabinoid receptors in the bladder, the L6/S1 spinal cord, and dorsal root ganglia (DRG), and to determine the effects of acute and chronic bladder inflammation on expression of cannabinoid receptors. Acute or chronic bladder inflammation was induced in rats by intravesical administration of acrolein. Abundance of CB1 and CB2 protein and their respective mRNA was determined using immunoblotting and quantitative real-time PCR, respectively. We confirmed the presence of CB1 and CB2 receptor protein and mRNA in bladder, L6-S spinal cord, and DRG. Acute bladder inflammation induced increased expression of CB2, but not CB1, protein in the bladder detrusor. Chronic bladder inflammation increased expression of bladder CB2 protein and mRNA but not CB1 protein or mRNA. Expression of CB1 or CB2 in spinal cord or DRG was unaffected by acute or chronic bladder inflammation. CB1 and CB2 receptors are present in the bladder and its associated innervation, and CB2 receptors are up-regulated in bladder after acute or chronic inflammation. CB2 receptors may be a viable target for pharmacological treatment of bladder inflammation and associated pain.     

Mast cell activation in sterile bladder and prostate inflammation

Sterile inflammation of the bladder has often been associated with interstitial cystitis (IC), a urologic condition of unknown etiology, predominantly affecting young and middle-aged females, for which no effective therapy is known. Recent reports have indicated that IC is associated with an increased number of bladder mast cells. Here we report the case of a middle-aged man with chronic sterile hematuria, dysuria and lower abdominal pain associated with a high number of bladder and prostate mast cells. Following therapeutic transurethral resection of the trigone area, bladder neck and prostate, samples of bladder and prostate were examined with light and electron microscopy and contained many mast cells (about 150 cells/mm2) which were not degranulated. Nevertheless, mast cells contained many altered granules and urinary levels of histamine were elevated, implying secretion without exocytosis. These findings are discussed in the context of the pathophysiology of IC and possible therapeutic interventions.