Beneficial effects of suplatast tosilate (IPD-1151T) in a rat cystitis model induced by intravesical hydrochloric acid

OBJECTIVE: To examine the effects of suplatast tosilate (IPD-1151T), a Th2 cytokine inhibitor recently recognized to improve the symptoms in patients with interstitial cystitis (IC), in a rat model of HCl-induced chronic cystitis, to elucidate the possible mechanisms by which the drug improves the symptoms of IC. MATERIALS AND METHODS: Chronic cystitis was induced by intravesical instillation of 0.2 mL of 0.4 m HCl in female adult rats. After a once-daily oral administration of IPD-1151T (0.1-100 mg/kg) or prednisolone (5 mg/kg) for 7 days, cystometry was performed under urethane anaesthesia. The bladder from HCl-induced cystitis rats was also assessed histopathologically. RESULTS: On cystometrography there was frequent voiding in cystitis rats. Administration of IPD-1151T for 7 days after intravesical HCl instillation dose-dependently increased the micturition volume and intercontraction intervals. Treatment with prednisolone had similar therapeutic effects. Histological analyses in the bladder from cystitis rats revealed oedema and infiltration of inflammatory cells such as mast cells and eosinophils in the lamina propria and the transitional epithelial thickening. These histological changes and the number of mast cells and eosinophils were reduced by administration of IPD-1151T or prednisolone. CONCLUSION: The present results indicate that IPD-1151T improves bladder function and pathological changes in HCl-induced cystitis rats, as previously observed in patients with IC. The rat cystitis model induced by HCl could provide useful information for studying proposed therapies for IC which might involve T cell-dependent inflammatory responses as one of its potential pathophysiologies.     

Acute stress and intravesical corticotropin-releasing hormone induces mast cell dependent vascular endothelial growth factor release from mouse bladder explants

PURPOSE: Corticotropin-releasing hormone is typically released from the hypothalamus but it has proinflammatory effects outside of the brain, possibly through the activation of mast cells. These cells express corticotropin-releasing hormone receptors with selective secretion of vascular endothelial growth factor, which may be involved in the pathogenesis of painful bladder syndrome/interstitial cystitis. This condition is characterized by bladder inflammation and worsened by stress. We investigated the effect of intravesical corticotropin-releasing hormone and acute restraint stress on vascular endothelial growth factor release from mouse bladder explants and the role of mast cells. MATERIALS AND METHODS: The bladder of C57BL/6 mast cell deficient (W/W(v)) and normal congenic (+/+) female mice (Jackson Laboratories, Bar Harbor, Maine) at ages 10 to 12 weeks was catheterized using anesthesia. After emptying urine 1) normal saline or corticotropin-releasing hormone was introduced for 45 minutes, urine was collected and the mice were allowed to recover for 4 hours before sacrifice or 2) the mice were stressed by placing them in a restrainer for 4 hours before sacrifice and urine was collected 2 hours after stress. The bladder was removed 4 hours after stress and processed for corticotropin-releasing hormone immunohistochemical staining. In other experiments the bladder was removed, minced into 1 mm(2) pieces and cultured with or without corticotropin-releasing hormone overnight. Urine and medium were frozen for histamine, interleukin-6, tumor necrosis factor-alpha and vascular endothelial growth factor assay. RESULTS: Corticotropin-releasing hormone (100 nM) or acute restraint stress (4 hours) increased histamine release in urine and vascular endothelial growth factor release in medium without increasing interleukin-6 or tumor necrosis factor-alpha in the bladder explants of C57BL/6 or +/+ but not W/W(v) mice. No vascular endothelial growth factor, interleukin-6 or tumor necrosis factor-alpha was detected in urine before or after stimulation. Corticotropin-releasing hormone immunoreactivity was present in control bladders but it increased dramatically in the bladder of stressed mice. CONCLUSIONS: Intravesical corticotropin-releasing hormone and acute restraint stress induced mast cell dependent vascular endothelial growth factor release from bladder explants. These findings suggest that stress, corticotropin-releasing hormone, mast cells and vascular endothelial growth factor might participate in the pathogenesis of painful bladder syndrome/interstitial cystitis, which is worsened by stress, and provide for new therapeutic targets.     

Improvement of interstitial cystitis symptoms and problems that developed during treatment with oral IPD-1151T

PURPOSE: We examined the efficacy of Suplatast Tosilatedouble dagger (IPD-1151T), a new immunoregulator that suppresses helper T cell mediated allergic responses, including IgE production and eosinophilic inflammation for treating patients with interstitial cystitis. MATERIALS AND METHODS: A total of 14 women (average age 43.7 years) with interstitial cystitis, which was nonulcerative in 13 and ulcerative in 1, were treated with 300 mg. IPD-1151T orally daily for 12 months. All patients received laboratory assessments, including hematology (eosinophils and CD20 positive cells) and serum chemistry (IgE, and interleukin-4 (IL-4) and 5, and immunohistochemical analyses of urine leukocytes (CD45RO positive cells as a T cell marker) before treatment. These parameters were also measured 4 and 12 months after continuous treatment. The voiding chart, and interstitial cystitis symptom and problem indexes were evaluated before and after IPD-1151T treatment. RESULTS: IPD-1151T treatment for 1 year resulted in a significantly increased bladder capacity and decreased symptoms, such as urinary urgency, frequency and lower abdominal pain, in patients with nonulcerative interstitial cystitis. These effects also correlated with a reduction in blood eosinophils, CD20 positive cells and IgE, and urine CD45RO positive memory T cells. No major side effects were observed. CONCLUSIONS: Our study suggests that immunological responses are involved in the development of interstitial cystitis symptoms. IPD-1151T could be a new oral agent for treatment of voiding symptoms and bladder pain in patients with interstitial cystitis.     

Intravesical sodium hyaluronate inhibits the rat urinary mast cell mediator increase triggered by acute immobilization stress.

PURPOSE: Mast cell activation and stress have been suggested as factors in the pathogenesis of interstitial cystitis, a painful disorder of the bladder that is diagnosed more frequently in women and characterized by increased urgency and frequency with absent infection. Intravesical sodium hyaluronate has been used to treat interstitial cystitis due to its possible replenishment of bladder glycosaminoglycans. We investigated the effect of sodium hyaluronate on the activation of bladder mast cell and release of proinflammatory mediators in the urine induced by acute immobilization stress in rats. MATERIALS AND METHODS: Using anesthesia a catheter was inserted in the bladder of 170 gm. female Sprague-Dawley rats. After emptying post-void residual urine a solution of normal saline, 0.08% or 0.4% sodium hyaluronate was introduced for 30 minutes. Each rat was allowed to recover from anesthesia and stressed for 30 minutes by confining it in a clear acrylic plastic immobilizer, while urine was continuously collected. Urinary histamine, rat mast cell protease-I and interleukin (IL)-6 were then determined. At the end of the experiments each rat was sacrificed by CO2 asphyxiation, and the bladder was removed and fixed with 4% paraformaldehyde. Frozen sections were stained with acidified toluidine blue, and the mast cell number and degree of activation were determined by granule extrusion and reduced cellular staining. RESULTS: Mean bladder mast cell activation plus or minus standard deviation in 6 control rats was 30.4% +/- 3.7% but it increased to 76.2% +/- 6.1% in 6 stressed animals (p = 0.0001). Intravesical administration of 0.4% sodium hyaluronate for 30 minutes in 6 rats before stress reduced mean bladder mast cell activation by 69.7% to 23.1% +/- 6.1% compared with stressed controls (p = 0.0003). However, compared to itself before stress there was no significant difference, indicating complete inhibition in 6 rats. Intravesical 0.08% sodium hyaluronate had a weaker inhibitory effect in 6 rats, decreasing mean degranulation by 22.5% to 59.1% +/- 7.6% (p = 0.02). In 6 rats stress increased the total mean amount of urinary rat mast cell protease-I by 271% from 0.14 +/- 0.09 to 0.52 +/- 0.17 ng. (p = 0.008). Pretreatment with 0.4% sodium hyaluronate reduced mean rat mast cell protease-I 80.8% compared with stressed controls (p = 0.008) and prevented any increase in response to stress in the same group of 8 rats with a mean pre-stress and post-stress level of 0.09 +/- 0.04 and 0.1 +/- 0.04 ng., respectively (p = 0.8). Acute stress increased mean urinary histamine in 6 rats 40.2% from 137.3 +/- 29.7 before to 193.7 +/- 7.6 ng./ml. after stress (p = 0.004). Pretreatment with 0.4% sodium hyaluronate reduced mean histamine 7.1% compared with stressed controls but completely prevented any increase in the same group of 8 rats, in which it was 174.5 +/- 23.1 before and remained 179.4 +/- 9.9 ng./ml. after stress (p = 0.75). Acute stress in 7 rats also increased the mean amount of IL-6 released in the urine by 31.5% from 775.9 +/- 69.2 to 1,021.1 +/- 93.3 pg./ml. (p = 0.007). Pretreatment with 0.4% sodium hyaluronate in 9 rats reduced mean IL-6 17% compared with stressed controls but again prevented any increase from baseline, since the value was 898.6 +/- 299.3 before and 824.4 +/- 196.4 pg./ml. after stress (p = 0.03). CONCLUSIONS: Immobilization stress induces bladder mast cell activation and the secretion of proinflammatory mediators, which are inhibited by sodium hyaluronate. Intravesical sodium hyaluronate may be a useful therapeutic option for interstitial cystitis, especially in patients with bladder mastocytosis who have symptom exacerbation with stress.     

Pentosanpolysulfate inhibits mast cell histamine secretion and intracellular calcium ion levels: an alternative explanation of its beneficial effect in interstitial cystitis

PURPOSE: Mast cells are ubiquitous cells derived from the bone marrow and are responsible for allergic reactions as they release numerous vasodilatory, nociceptive and pro-inflammatory molecules in response to immunoglobulin E (IgE) and specific antigen. Mast cell secretion is also triggered by a number of peptides, such as bradykinin and substance P, and may also be involved in the development of inflammatory responses. An example is interstitial cystitis, which is a sterile painful bladder disorder that has been associated with a defective glycosaminoglycan bladder mucosal layer and an increased number of activated mast cells. Pentosanpolysulfate is a synthetic, sulfated polysaccharide that has been approved for the treatment of interstitial cystitis on the premise that it may replenish the defective glycosaminoglycan layer. We hypothesize that pentosanpolysulfate may also have an additional or alternate action on bladder mast cells. We report that pentosanpolysulfate has a powerful dose dependent inhibitory effect on mast cell release of histamine induced by the mast cell secretagogue compound 48/80. MATERIALS AND METHODS: Inhibition of mast cell secretion was documented by light and electron microscopy and extended to stimulation by substance P or IgE and antigen. RESULTS: The inhibition was more potent than that seen with the clinically available mast cell stabilizer disodium cromoglycate (cromolyn). Maximal inhibition by pentosanpolysulfate was apparent within 1 minute, was unaffected by the length of pre-incubation and persisted after the drug was washed off. In contrast, the effect of cromolyn was limited by rapid tachyphylaxis. In addition, while cromolyn has no effect on mucosal or rat basophilic leukemia cells, pentosanpolysulfate inhibited histamine secretion from both. Confocal microscopy using a calcium indicator dye showed that pentosanpolysulfate decreased intracellular calcium ion levels. CONCLUSIONS: Pentosanpolysulfate appears to be a potent inhibitor of allergic and nonimmune mast cell stimulation, which is an alternative explanation of its benefit in interstitial cystitis.     

Mast cell involvement in interstitial cystitis

A prospective study was designed to examine the relationship of mast cells, and eosinophilic leukocyte density and mediator levels to clinical and histological parameters of interstitial cystitis. Interstitial cystitis and control patients underwent bladder biopsy with histological examination, and quantification of intact and degranulated mast cell and eosinophilic leukocyte density. In addition, bladder tissue histamine content, urinary prostaglandin E2 excretion rates, and serum and urinary major basic protein levels were determined. A strong relationship among detrusor mast cell density, especially degranulated, and degree of epithelial loss, submucosal inflammation, epithelial ulceration, urinary pyuria and response to treatment was noted. Bladder tissue histamine content and urinary prostaglandin E2 excretion were increased in the interstitial cystitis patients. Eosinophil density in bladder biopsies was low uniformly, and interstitial cystitis and control patients showed no statistical difference. In addition, serum and urinary major basic protein levels were below the accepted normal lower limits for this protein. Therefore, our study demonstrates a relationship between the mast cell and the inflammatory process of interstitial cystitis. No similar relationship was noted for the eosinophil.     

间质性膀胱炎大鼠膀胱ICAM-1与功能膀胱容量的关系

目的探索间质性膀胱炎(IC)大鼠膀胱组织细胞间粘附分子(ICAM-1)水平与膀胱容量之间的关系。方法雌性SD大鼠分为正常对照、IC模型和透明质酸(HA)治疗3组,每组10只。以腹腔注射环磷酰胺联合膀胱灌注鱼精蛋白和脂多糖制作IC模型(IC模型组),模型鼠膀胱灌注HA进行治疗者为HA组。检测各组的平均排尿量和24h总尿量,以及膀胱组织肥大细胞浸润数、ICAM-1和肿瘤坏死因子α(TNF-α)水平。结果 IC模型组的平均排尿量显著小于正常大鼠(1.10mL vs.1.88mL,P0.001)和HA治疗组(1.30mL vs.1.10mL,P=0.009),而3组的24h总尿量无显著差异。IC模型大鼠无论是肥大细胞浸润数目还是ICAM-1、TNF-α含量,均较正常对照有极显著升高(P均0.001)。该3项指标在HA组有显著减少。相关分析显示,平均尿量与ICAM-1水平呈显著负相关(r=-0.725,P0.001),而与肥大细胞浸润数和组织TNF-α含量无显著相关性。结论 SD大鼠IC模型膀胱组织ICAM-1水平与功能性膀胱容量之间存在密切相关性,降低ICAM-1可有效增加功能性膀胱容量。ICAM-1有望成为IC治疗的观测指标。     

Stimulated release of urine histamine in interstitial cystitis.

The diagnosis of interstitial cystitis is primarily made based on clinical and cystoscopic findings with exclusion of other bladder diseases. Despite all of the efforts at definitive identification, interstitial cystitis lacks universal objective findings. Mast cell activation with associated histamine release has been postulated as an etiological factor leading to the symptom complex associated with interstitial cystitis. To investigate this hypothesis, a 3-step controlled prospective study was conducted. In step 1 reliability of urine histamine assay was critically examined, and the assay was established to be simple, reliable and valid. In step 2 random spot urine histamine levels (basal state) were measured in 25 noninterstitial cystitis and 15 interstitial cystitis patients (22.1 +/- 0.95 ng./ml. versus 19.2 +/- 1.19 ng./ml.). There was no significant difference in the random urine histamine levels between the 2 groups (p greater than 0.05). In step 3 urine histamine levels were measured before and after hydrodistention (acute stimulation) in 7 noninterstitial cystitis controls and 6 newly diagnosed interstitial cystitis patients under general anesthesia. The urine histamine-to-creatinine ratio was used to correct for the dilutional effect of normal saline used during hydrodistention. The urine histamine-to-creatinine ratios of the control group showed no significant difference before and after hydrodistention. However, the difference in the urine histamine-to-creatinine ratios of the interstitial cystitis group compared to the controls before and after hydrodistention was highly significant (p less than 0.001). Although measurement of random spot urine histamine alone (basal state) was not found useful to make the diagnosis of interstitial cystitis, measurement of urine histamine before and immediately after hydrodistention (acute stimulation) may become an important objective parameter to assist in the diagnosis of interstitial cystitis.     

Histamine content and mast cell count of detrusor muscle in patients with interstitial cystitis and other types of chronic cystitis

The quantitative mast cell count in the detrusor muscle, the histamine content and the degree of collagen staining material in the bladder wall have been evaluated in order to elucidate their value in distinguishing between patients with interstitial cystitis and other types of chronic cystitis. The number of mast cells in the detrusor muscle was statistically significantly increased in patients with interstitial cystitis compared with the control group (P less than 0.0001). With a proposed level of greater than 20 mast cells/sq mm of muscle tissue the diagnostic specificity was 88% and the diagnostic sensitivity 95%. The histamine content in the bladder wall was significantly increased in patients with interstitial cystitis (P less than 0.05) but not useful as a diagnostic test. The amount of collagen staining material was significantly increased in the intra- and inter-fascicular muscle tissue of the bladder in patients with interstitial cystitis (P less than 0.0005, P less than 0.001) and might be used as a support for the histological diagnosis, even in patients with uncontracted bladders.     

Alternate Occurrence of Allergic Disease and an Unusual Form of Interstitial Cystitis

Background : It has been postulated that interstitial cystitis can be induced by an allergy. This is partly based on the observation that many patients with interstitial cystitis also have allergic diseases. In this study, an allergic evaluation was conducted on patients with interstitial cystitis complicated by bronchial asthma, a typical allergic disease.
Methods : Clinical histories were obtained and biopsy specimens from the vesical walls of the study patients were examined histologically. Cutaneous tests and IgE radioallergosorbent tests (RAST) were performed. Further, intravesical provocation tests were carried out using IgE RAST-positive antigens, and histamine release assays were performed on the vesical biopsy specimens using anti-lgE antibodies.
Results : Five of 6 patients alternately exhibited symptoms of allergic disease and bladder symptoms. The eosinophil and mast cell counts in the vesical biopsy specimens of these 5 patients were increased. Furthermore, an intravesical provocation test performed using the IgE RAST-positive antigen was positive in 4 patients. The mean vesical biopsy specimen histamine release was 1 7.7% for patients with interstitial cystitis with bronchial asthma which was significantly higher than that for interstitial cystitis patients without bronchial asthma (8.9%) or the control group (4.5%). The prognosis of patients with interstitial cystitis with allergic complications was relatively good.
Conclusion : Patients with bronchial asthma exhibited hypersensitivity both generally and locally in the bladder. The alternation phenomenon was observed between the hypersensitive organs.     

Tibial nerve stimulation diminishes mast cell infiltration in the bladder wall induced by interstitial cystitis urine

OBJECTIVES: To investigate whether the urine of interstitial cystitis (IC) patients has a toxic effect on the bladder wall, as determined by mast cell infiltration, and to evaluate the preventive effect of tibial nerve electric stimulation (TNES) on bladder mastocytosis induced by IC urine. MATERIAL AND METHODS: The bladders of female rats were catheterized and instilled with IC urine (Group IC; n=10) and normal urine (Group NU; n=5) obtained from humans, saline (Group S; n=5) and protamine sulphate (Group PS; n=10) for 6 weeks. Additionally, in five rats instilled with IC urine and five instilled with PS, TNES was also performed (Groups IC + TNES and PS + TNES). RESULTS: In the lamina propria of the bladder, the mean number of mast cells per square millimetre was significantly higher in Groups IC (32.5+/-12.3) and PS (39.4+/-11.1) than in Groups S (11.9+/-4.3) and NU (13.7+/-3.5). After TNES, the corresponding values were decreased significantly to 15.3+/-5.4 and 15.3+/-4.1 in Groups IC + TNES and PS + TNES, respectively (p<0.001). A significant reduction in mast cell infiltration in the detrusor was also determined after TNES compared with the value in Group IC (4.6+/-1.6 vs 12.1+/-3.0; p<0.001). CONCLUSIONS: We demonstrated that IC urine may result in increased mast cell infiltration in the bladder wall. TNES may play a therapeutic role by diminishing the mast cell count in the bladder wall, which has a strong relationship with nociceptive neural endings.     

The role of beta-catenin signaling in the malignant potential of cystitis glandularis

PURPOSE: Chronic inflammation is a risk factor for malignant transformation in the bladder. The pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) is a mediator of such inflammation that induces nuclear localization of the adherens junction component beta-catenin. This mechanism has a key role in the initiation and progression of the premalignant lesion Barrett's metaplasia of the esophagus. Cystitis glandularis is a metaplastic lesion of the bladder urothelium occurring in the presence of chronic inflammation and in up to 13% of asymptomatic bladders. Two subtypes are described (typical and intestinal/colonic) with uncertain malignant potential. Etiologically and histologically cystitis glandularis mimics Barrett's metaplasia. We investigated the roles of beta-catenin and TNFalpha in cystitis glandularis. MATERIALS AND METHODS: Immunohistochemistry and immunofluorescence were used to demonstrate the expression and localization of E-cadherin, beta-catenin and TNFalpha in 9 sections of typical cystitis glandularis and 4 of intestinal/colonic cystitis glandularis. Appropriate controls were used for all experiments. RESULTS: Immunohistochemistry demonstrated normal membranous expression of E-cadherin and beta-catenin in all cystitis glandularis sections with increased TNFalpha expression. Immunofluorescence showed nuclear localization of beta-catenin in the intestinal/colonic subtype only, which was not observed in typical cystitis glandularis. CONCLUSIONS: The presence of nuclear beta-catenin suggests that intestinal/colonic cystitis glandularis shares the same signaling pathway with the premalignant lesion Barrett's metaplasia of the esophagus and the intestinal/colonic subtype of cystitis glandularis may have the potential to progress to malignancy. This finding has important implications for the management of this lesion.     

Anti-inflammatory effect of an Escherichia coli extract in a mouse model of lipopolysaccharide-induced cystitis

The bacterial extract, Uro-Vaxom, which consists of immunostimulating components derived from 18 Escherichia coli strains, was used for the prophylaxis of recurrent cystitis. To evaluate the anti-inflammatory effect of E. coli extract, we measured the cytokine levels of bladder tissue after oral administration and analyzed bladder inflammation by histopathologic examination in a model of lipopolysaccharide (LPS)-induced cystitis in mice. After oral administering the E. coli extract for 10 days, the cytokine [interleukin-6 (IL-6), IL-10, monocyte chemoattractant protein-1, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha, IL-12p70] levels present in the bladder of female Balb/C mice were determined using a cytometric bead array. The bladder macrophage inflammatory protein-2 level was also measured using a sandwich enzyme immunoassay. After immunization with the E. coli extract, E. coli LPS was instilled into the bladders intravesically. Twenty-four hours later, the mice were sacrificed and the level of bladder inflammation was quantified using the bladder inflammatory index (BII). Significant changes in the bladder IL-6 and IFN-gamma levels were observed after the E. coli extract treatment. Secretions of the other cytokines were not stimulated by the E. coli extract. The bladder instilled with LPS had high inflammation scores for edema, leukocyte infiltration, and hemorrhage in the saline treated control mice. In contrast, the E. coli extract treated mice exhibited mild inflammation of their bladders with a significant reduction in the BII scores compared with the controls. These findings might explain the anti-inflammatory effect of the E. coli extract demonstrated in clinical studies.     

Comparison of histamine release in human skin mast cells induced by morphine, fentanyl, and oxymorphone

Human leukocyte and skin mast cell preparations were incubated with morphine sulfate in concentrations ranging from 1.5 X 10(-5) M to 4.5 X 10(-3) M. Skin mast cells also were incubated with oxymorphone and fentanyl in the same concentrations. Human leukocytes did not release histamine in response to any concentration of morphine. In skin mast cells, histamine release by morphine first was detected at 1.5 X 10(-4) M. Histamine release further increased at 5.0 X 10(-4) M with no incremental increase at higher concentrations. Oxymorphone and fentanyl failed to release histamine at any concentration. Histamine release by morphine required calcium but was not influenced by changes in the 1-4 mM range. Skin mast cell preparations were pretreated for 30 min in naloxone 5 X 10(-4) M and then morphine 5 X 10(-4) M was added for 30 min without removing naloxone. Naloxone neither released histamine nor inhibited morphine-induced histamine release. The release of histamine by morphine but not equimolar concentrations of fentanyl and oxymorphone indicates that histamine release by narcotics is not a nonspecific effect of high drug concentration. The failure of naloxone to inhibit morphine-induced histamine release suggests that histamine release by morphine is not dependent on opiate receptor binding or activation. These results indicate that this human mast cell preparation will be useful in further understanding the mechanism of histamine release induced by morphine and other agents.     

A guinea pig model for study of bladder mast cell function: histamine release and smooth muscle contraction.

To study the function of mast cells in bladder tissue, guinea pigs were sensitized with ovalbumin by intraperitoneal injections, bladder tissue strips were superfused, and tissue contractile force and histamine release were studied. Upon challenge with ovalbumin, bladder tissue contracted 64 +/- 4% (mean +/- S.E.M.) of the maximum carbachol contraction and released 14.1 +/- 1.6% of the total tissue histamine content. Incubation of sensitized bladder tissue with indomethacin led to an increased force and duration of the contraction while incubation with nordihydroguaiaretic acid combined with pyrilamine reduced histamine release and abolished the contraction. Tissue histamine content was significantly higher in the bladder neck than in the dome, and significantly elevated following sensitization. Histochemical studies of bladder tissue demonstrated mast cell degranulation in antigen challenge experiments. In addition, a group of guinea pigs were sensitized to ovalbumin through bladder instillations. With this model, study of the functional characteristics of bladder mast cells and the acute actions of mast cell products on the bladder microenvironment, should now be feasible.     

Effect of the 21-aminosteroid on nuclear factor-kappa B activation of Kupffer cells in endotoxin shock

BACKGROUND: The 21-aminosteroid (U-74389G) is a nonglucocorticoid steroid that was synthesized to inhibit lipid peroxidation without the glucocorticoid activity. We recently demonstrated that the 21-aminosteroid administered to endotoxin shock mice reduces liver injury and improves the survival rate of mice through inhibition of nuclear factor-kappa B activation in the liver. The study was undertaken to determine whether the 21-aminosteroid could suppress pro-inflammatory gene up-regulation through inhibition of nuclear factor-kappa B activation in Kupffer cells. METHODS: Kupffer cells were isolated from rats by collagenase perfusion followed by pronase digestion. After a lipopolysaccharide addition, each assay was performed for tumor necrosis factor-alpha, interleukin-6, tumor necrosis factor-alpha messenger RNA, nuclear factor-kappa B, and I kappa B proteins. RESULTS: After the lipopolysaccharide addition, Kupffer cells released both tumor necrosis factor-alpha and interleukin-6. The 21-aminosteroid treatment suppressed the release of tumor necrosis factor-alpha in a dose-dependent manner. The 21-aminosteroid also inhibited the increase of tumor necrosis factor-alpha messenger RNA expression and nuclear factor-kappa B activation in Kupffer cells 1 hour and 30 minutes, respectively, after lipopolysaccharide addition. Furthermore, the 21-aminosteroid treatment suppressed the degradation of I kappa B proteins in lipopolysaccharide-stimulated Kupffer cells. CONCLUSIONS: These results suggest that the 21-aminosteroid inhibits release of the tumor necrosis factor-alpha and interleukin-6 from lipopolysaccharide-stimulated Kupffer cells by inhibiting nuclear factor-kappa B activation. This is accomplished by inhibiting I kappa B degradation in endotoxin shock and this may prove useful for the treatment of endotoxin shock.     

Modulating bladder neuro-inflammation: RDP58, a novel anti-inflammatory peptide, decreases inflammation and nerve growth factor production in experimental cystitis

PURPOSE: In interstitial cystitis (IC) inflammation induces and perpetuates neurotrophic changes in the bladder, resulting in the symptoms of frequency, urgency and pain. RDP58 (NH2-arg-norleucine (nle)-nle-arg-nle-nle-nle-gly-tyr-CONH2) (Sangstat Corp., Fremont, California) is a novel synthetic peptide that inhibits early signal transduction pathways for the expression of inflammatory cytokines. In this study we evaluated the effects of intravesical RDP58 on an established model of cystitis. MATERIALS AND METHODS: Mice were catheterized and equal volumes of Escherichia coli lipopolysaccharide (LPS) or saline were instilled into the bladder. After 45 minutes the bladders were drained and distilled water or RDP58 (1 mg/ml) was instilled for 30 minutes. At 24 hours later the bladders were excised and cultured for analysis of tumor necrosis factor-alpha (TNF-alpha), substance P (SP) and nerve growth factor (NGF) production, as quantified by enzyme-linked immunosorbent assay. RESULTS: LPS caused severe inflammation in mouse bladders compared with controls. Exposure to LPS increased the levels of TNF-alpha, SP and NGF production compared with controls (each p <0.05). In LPS exposed mice RDP58 significantly decreased inflammatory parameters by 82% 24 hours after treatment (p <0.05). Within 4 hours RDP58 abolished TNF-alpha production and at 24 hours TNF-alpha remained undetectable. RDP58 also significantly decreased SP and NGF production in LPS exposed bladders by more than 40% and 85%, respectively (each p <0.05). CONCLUSIONS: Inflammatory models of cystitis result in increased levels of TNF-alpha, SP and NGF production in the bladder, paralleling the hypothesized neuro-inflammatory etiology of IC. RDP58 decreases inflammation and neurotrophic factors in vivo and it may potentially treat bladder disorders with an inflammatory component, such as IC.