The anti-adherence effect of heparin: A visual analysis

The various intrinsic bladder mucosal defenses against bacterial invasion, including the anti-adherence action of the surface mucopolysaccharide layer, have been documented in recent years. Studies have suggested Heparin, a mucopolysaccharide, may have anti-adherence action. We used scanning electron microscopy to visually evaluate the effects of intravesical HCl, Heparin and Protamine on the interaction between E. coli and rat bladders. Our findings concurred with previous statistical analyses using radio-isotope labelled bacteria, suggesting a beneficial anti-adherence effect of Heparin which can be negated by Protamine. We also noted a white coating on the bacteria in bladders treated with Heparin. We suggest this may represent minute amounts of Heparin coating the bacteria, thus decreasing bacterial adherence to bladder mucosa. Analogues of Heparin, without anti-coagulant properties, may be useful in prophylaxis against urinary tract infections.     

The effects of heparin on the adherence of five species of urinary tract pathogens to urinary bladder mucosa

Summary Previous studies performed in our laboratory have indicated that the primary antibacterial defense mechanism of the rabbit urinary bladder is the antiadsorptive action of the surface mucopolysaccharide. Removal of this layer with an acid rinse increases bacterial adherence up to 100 fold. Exogenous mucopolysaccharide (heparin) has been shown to restore Escherichia coli adherence to control levels. To determine whether this antiadherence action of heparin is species specific, we compared the adherence of 5 common urinary tract pathogens (Escherichia coli, Klebsiella ozonae, Proteus mirabilis, Pseudomonas aeruginosa, and Streptococcus fecalis) to both mucin intact and mucin deficient rabbit bladders with and without prior heparin exposure. Bacteria were radiolabeled by addition of ³H-adenine to the culture broth so that the number of bacteria adhering to the bladder could be determined using liquid scintillation spectrophometry. Results were as follows: 1) Acid removal of the mucin layer significantly increased the adherence approximately 10 fold for all 5 species tested. 2) Briefly exposing the mucin deficient bladders to heparin decreased the adherence of all species tested except Pseudomonas to mucin intact control levels. 3) Heparin treatment of mucin intact bladders slightly decreased adherence of all species except Pseudomonas below mucin intact controls, however, results were not statistically significant. 4) The magnitude of Klebsiella adherence was nearly 20 fold greater than all other species tested. While this non-species specific adherence inhibition of heparin may prove useful in the clinical setting, it appears to be less effective against Pseudomonas.This work was supported in part by grants from the Veterans Administration, NIH Grant#RO-1-AM-6508-01, and the McCabe fund.     

Prevention of human bladder tumor cell implantation in an in vitro assay

The high recurrence rate of bladder tumors can be reduced by prevention of tumor cell reimplantation on denuded urothelium following transurethral resection. This can be achieved by intravesical chemotherapy immediately after the resection of the bladder tumors. We have demonstrated, in an in-vitro system, the process of human bladder tumor cell implantation on a naturally produced extracellular matrix (ECM) which simulates the exposed bladder basement membrane and submucosa. Using this model we examined the efficacy of various cytotoxic agents in preventing tumor cell adhesion to the ECM. Human bladder tumor cell implantation was prevented following exposure of the cells to distilled water, epodyl or mitomycin C, and significantly reduced following one hour incubation in cisplatinum and doxorubicin. The maximal effect for each of these cytotoxic agents was reached within 30 to 60 minutes of treatment. Mitomycin C reached maximal effect within 10 minutes. In contrast, thiotepa did not cause a significant reduction in cell adherence to ECM as compared to untreated control cells.     

Heparin--examination of its antibacterial adsorption properties

One of the primary antibacterial defense mechanisms of the bladder is the action of the luminal mucopolysaccharide layer against adsorption of inoculated bacteria. Previous studies have shown that local instillation of the mucopolysaccharide heparin can prevent bacterial adsorption on the bladder mucosa denuded of this "antiadherence factor." To determine whether this action was due to the mucopolysaccharide composition of heparin, or rather to its anticoagulant property, protamine sulfate (a basic protein with anticoagulant properties) was tested for antiadsorptive efficacy. Protamine offered no protection against bacterial adherence in the rabbit model. It appears that heparin's protective effect is unrelated to its action as an anticoagulant.     

Inactivation of antiadherence effect of bladder surface glycosaminoglycans as possible mechanism for carcinogenesis

We have shown that the urinary bladder secretes and binds to its surface a glycosaminoglycan layer whose nonspecific antiadherence effect protects the bladder from infection and perhaps from stone formation. If bladder cancer is caused by agents present in the urine, as is widely believed, this mechanism may also protect against carcinogenesis. We performed the current study to determine whether suspected carcinogens or cocarcinogens in the urine gain access to the transitional cells by impairing or inactivating the surface antiadherence effect. Using an in vivo method to quantitate bacterial adherence to the rabbit bladder, we compared adherence in control and glycosaminoglycan-deficient bladders to adherence in bladders treated with one of several suspected urinary carcinogens. There were statistically significant differences between adherence in control bladders and adherence in bladders treated with the tryptophan metabolites 3-hydroxykynurenine and 3-hydroxyanthranilic acid, sodium cyclamate, and sodium saccharin. These data indicate that perhaps certain suspected urinary bladder carcinogens inactivate the anti-adherence effect of the glycosaminoglycan layer at the bladder surface and thereby penetrate to the transitional cells to exert their tumorigenic effects; or they may serve as cocarcinogens that inactivate the glycosaminoglycan barrier and permit other urinary carcinogens to transform the transitional cells.     

The protective effect of heparin in experimental bladder infection.

The surface mucopolysaccharide layer of the urinary bladder has been shown to interfere with bacterial attachment, and we therefore consider it to be the primary mechanism of combatting bacterial colonization and subsequent infection. Experimentally, hydrochloric acid alters this layer in such a way as to permit massive bacterial adsorption to the vesical mucosa. Heparin, an acid mucopolysaccharide, was instilled into rabbit bladders that had their naturally occurring mucopolysaccharide altered by hydrochloric acid. Heparin was shown to inhibit the attachment of inoculated Escherichia coli to these acid-treated bladder mucosas, and the degree of protection afforded was similar to that of the mucopolysaccharide layer of the intact urothelium.     

Heparin as antibacterial agent in rabbit bladder.

Previous studies performed in our laboratory indicated that the primary antibacterial defense mechanism of the rabbit bladder is the antiadsorptive action of the surface mucopolysaccharide. The increased bacterial adsorption that occurs when the bladder is denuded of this layer was prevented by the instillation of heparin. Additional studies showed that the protective effect of heparin is inhibited by protamine, a further indication that the bladder's "antiadherence factor" is a mucopolysaccharide. Small amounts of heparin, applied directly to the mucoprotein-deficient bladder or to the surface of the inoculated bacteria, produced a statistically significant reduction in bacterial adsorption.     

Effect of vesical overdistention on bladder mucin

Histochemical staining of bladder tissue demonstrates a discrete layer of mucopolysaccharide (mucin) on the surface of human and rabbit bladders. Studies have shown that an intact mucin layer may be important in helping the bladder to resist bacterial infections. This report correlates vesical overdistention with destruction of the mucin layer in the rabbit bladder. These findings suggest that vesical overdistention may predispose to urinary tract infection because of bladder mucin disruption.     

Sucralfate inhibition of tumor cell implantation in the murine urinary bladder

Superficial transitional cell carcinoma of the bladder has a 50 to 70% recurrence rate. Recurrences may be related to tumor cell implantation during transurethral resection which disrupts the glycosaminoglycan layer of the bladder. This study demonstrated decreased tumor formation in murine bladders which were pre-treated with sucralfate, a synthetic glycosaminoglycan, prior to tumor cell instillation. These results suggest a possible role for sucralfate as a glycosaminoglycan supplement.     

Prevention of urinary tract infection by the exogenous glycosaminoglycan sodium pentosanpolysulfate

The transitional cells at the surface of the urinary bladder secrete and bind to their surfaces 1 or more glycosaminoglycans whose presence prevents bacterial adherence to the mucosa. Because it is believed that adherence is prerequisite to infection, we did the current studies to determine whether the antiadherence effect of natural and synthetic glycosaminoglycans prevents infection. We exposed intact rabbit bladders, mucin deficient rabbit bladders, and rabbit bladders treated with sodium pentosanpolysulfate, a similar but synthetic substitute for the surface glycosaminoglycan(s), to bacteria in vivo. We measured infection rates 48 hours after exposure. The infection rate was significantly higher in mucin deficient bladders than in controls (p less than 0.02). There was no significant difference between infection rates in controls and infection rates in bladders treated with sodium pentosanpolysulfate. These results support our impression that a bladder with an intact mucin layer is better able to resist infection than is a mucin deficient bladder. The natural surface glycosaminoglycan(s) and the synthetic substitutes that reproduce their antiadherence effect appear to be protecting factors.     

Pentosanpolysulfate coating of silicone reduces encrustation

BACKGROUND AND PURPOSE: A significant problem associated with catheterization in the urinary tract is the encrustation of the catheter materials. One approach to reducing encrustation is to alter the surface properties of the catheters. We evaluated the effectiveness of coating with pentosanpolysulfate (PPS), a semisynthetic polysaccharide similar to heparin, in reducing encrustation and the foreign-body inflammatory response to silicone stents in the bladders of male New Zealand White rabbits. MATERIALS AND METHODS: Sixteen rabbits were divided into three groups to receive placement in their bladders of uncoated (N = 7), PPS-coated (N = 7), or sham matrix-processed silicone rings (N = 2) via open cystotomy. After 50 days of maintenance on normal food and water, all rabbits were sacrificed, and the air-dried, unfixed silicone ring surfaces were examined by scanning electron microscopy. Bladders and remaining silicone rings were removed and preserved separately. Silicone rings, cleaned of all encrustation, were stained with toluidene blue to determine the presence or absence of PPS coating on the surface. RESULTS: Histologic examination revealed normal tissue in bladder sections exposed to coated silicone rings and an inflammatory response in sections from bladders having uncoated silicone rings. Coating with PPS was associated with an eightfold reduction in the amount of encrustation of silicone and a marked reduction in the inflammatory response of the bladder wall to the foreign body. CONCLUSIONS: A PPS coating may be useful in reducing the encrustation of long-term indwelling silicone stents or catheters in the human urinary tract.     

Inhibition of tumor implantation by intravesical gemcitabine in a murine model of superficial bladder cancer

PURPOSE: We determined if a single intravesical instillation of gemcitabine (2',2'-difluorodeoxycytidine) could prevent the implantation of urothelial cancer cells in the bladder wall of mice, and if 4 weekly treatments could eliminate early implanted bladder cancer in this model. MATERIALS AND METHODS: Tumor implantation and orthotopic bladder tumors were induced in mice by electrocautery of the bladder wall and subsequent instillation of MB49 bladder cancer cells. In the first experiment the tumor cell suspension was left in place for 30 minutes, immediately followed by bladder irrigation and a single intravesical instillation of 250 or 500 microg gemcitabine for 10, 30, 60 or 120 minutes. In the second experiment dwell time was 2 hours, bladders were not irrigated after tumor cell instillation and mice were treated with 4 weekly instillations starting 24 hours after tumor cell implantation. The animals were monitored for side effects and bladder cancer signs, and autopsied at the end of followup. RESULTS: A single intravesical instillation of 500 microg gemcitabine (10 mg/ml) for 30 minutes decreased tumor outgrowth significantly from 90% (control) to 30% (chi-square test p = 0.022). Gemcitabine at 250 microg and prolonged instillations of 500 microg during 60 or 120 minutes were less effective. In the second experiment a short dwell time (30 minutes) was effective at 500 microg doses, resulting in an outgrowth decrease in 89% (control) to 30% of mice, whereas longer instillations (greater than 120 minutes) resulted in significantly reduced tumor outgrowth (11% at 250 microg). The apparent loss of efficacy as a factor of time could not be fully explained. Prolonged bladder distention caused by increased bladder volume due to diuresis may have resulted in trauma and caused enhanced susceptibility to tumor implantation. In the second experiment prolonged instillations (greater than 120 minutes) at 250 microg or higher doses (500 microg) were effective with 11% and 30% outgrowth, respectively. CONCLUSIONS: If given early (within 30 minutes.) after tumor cell seeding, gemcitabine is effective for preventing tumor cell implantation and the resulting tumor outgrowth.     

增强卡介苗与膀胱壁结合力的实验研究

目的为提高卡介苗的抗肿瘤作用提供实验依据. 方法家兔30只,膀胱内分别行切割伤、电灼伤、冷冻伤后随机分成5组,每组6只.A组膀胱内单纯灌注磷酸盐缓冲液;B组膀胱内灌注磷酸盐缓冲液+同位素标记后的卡介苗(3H-卡介苗);C组灌注氨基已酸+3H-卡介苗;D组灌注氨甲苯酸+ 3H-卡介苗;E组灌注肝素+3H-卡介苗.随后切取各损伤处及未损伤处膀胱壁,消化后以液体闪烁计数器测定3H-卡介苗的结合量 (单位次/min). 结果卡介苗在膀胱壁损伤处的结合量远高于未损伤处; C组、D组卡介苗的结合量(27809±6580、 28772±6058)明显高于B组 (12462±2412); 与B组比较(χ2分别为5.12、5.09,均P＜0.01),差异均有非常显著意义.而E组卡介苗结合量则明显低于B组,也远低于C组与D组,统计学检验差异亦有非常显著意义. 结论对家兔膀胱内灌注纤维蛋白溶解抑制剂可增强卡介苗与其膀胱壁的结合力,而纤溶促进剂肝素则作用相反,提示前者可提高卡介苗的抗肿瘤作用.     

The effect of nitrofurantoin on bladder tumor cell lines: in vitro growth and implantation in the cauterized mouse bladder

Nitrofurans compounds and derivatives demonstrate antineoplastic activity in vitro as well as in vivo. Nitrofurantoin caused in vitro growth inhibition of a FANFT-induced murine bladder tumor (MBT2) and a human transitional cell carcinoma cell line (GIBB) in concentrations of 125 microM, 250 microM and 500 microM. The implantation and growth of MBT2 in the cauterized mouse bladder was inhibited by 250 microM nitrofurantoin. The bladder mucosa of two groups of C3Hf/HeHa female mice was electrically cauterized. In group I, 1 X 10(6) MBT2 cells were injected into the bladders of 42 mice, while in group II 1 X 10(6) MBT2 cells in 250 microM nitrofurantoin solution were injected into the bladders of 51 mice. Positive tumor implantation was seen in 25 bladders (59.5 per cent) of group I as compared to 15 bladders (29.5 per cent) of group II. All tumors in group I were large, occupying more than 50 per cent of bladder cross sectional area with 24 per cent showing extravesical extention. Sixty-six per cent of tumors in groups II were less than 25 per cent of bladder cross sectional area and 13.4 per cent had extravesical extention.     

Prophylaxis of bladder tumor implantation Intravesical and systemic chemotherapy

Several antitumor agents have been evaluated for their effectiveness in reducing the implantation and progressive growth of transitional cell carcinoma in an animal model. This system allows the evaluation of both the topical cytotoxic action as well as the systemic toxicity of the drugs given intravesically. Systemic cyclophosphamide and intravesical epipodophyllotoxin significantly reduced the incidence of tumor cell implantation.     

Differentiation of Smooth Muscle Cells from Human Amniotic Mesenchymal Cells Implanted in the Freeze-Injured Mouse Urinary Bladder

Background

The multipotency of human amniotic mesenchymal cells (HAMCs) has been reported, but the role of HAMCs in urinary tract regeneration is unknown.

Objective

The aim of the study was to determine if cells derived from HAMCs support the structural and functional reconstruction of freeze-injured mouse bladders.

Design, setting, and participants

HAMCs were harvested from an amnion membrane, and cells were cultured for 7 d prior to injection into the freeze-injured bladder walls of nude mice.

Intervention

Three days prior to implantation, the posterior bladder walls were freeze injured for 30 s. The cultured HAMC-derived cells (0.5 × 10⁵ cells per 50 μl) were implanted into the injured regions. Control bladders received a cell-free injection. At 1, 2, 4, and 6 wk after the cell implantation, the experimental bladders were extirpated.

Measurements

The bladder tissues were examined by immunohistochemistry for α-smooth muscle actin (SMA). The HAMC-derived cells were detected by antihuman nuclei antibody (HuNu). Separately, bladder muscle strips were examined for contractile responses to potassium.

Results and limitations

At 1 wk after implantation, the HAMC-derived cells, which were detected by HuNu, differentiated into muscular layers composed of SMA-positive cells. From 2 to 6 wk after implantation, abundant layers of SMA-positive and HuNu-positive cells developed. In control bladders, few SMA-positive cells remained at the injured regions at 1 wk, but by 6 wk, more were present. At 1 wk, the contractile responses to potassium of the cell-implanted bladders were significantly higher than those of the control-injected ones. Control-injected bladders also recovered by 6 wk, but the rate of recovery was slower.

Conclusions

Freeze-injured mouse bladders implanted with HAMC-derived cells recovered morphology and function faster than control-injected bladders.     

Estimation of ABO(H) isoantigen expression in bladder tumors

ABO(H) isoantigen expression was estimated semiquantitatively with the red cell adherence test on single epithelial cell suspensions from 89 bladder tumor and 7 normal bladder specimens. Correlations of red cell adherence counts positive for antigen with the pathological findings of the bladder (histology and stage of tumor) were made in a blind fashion at the end of the study. The mean count positive for antigen from normal bladders was 86.0, which was significantly different (p less than 0.001) from those of muscle-invading lesions (12.6) and flat carcinoma in situ (21.6). This study shows a good correlation among the cell counts positive for antigen, histological findings and stage of disease, and provides the basis for a prospective study that may help to determine the role of ABO(H) isoantigen measurement in bladder tumor patients.     

Urothelial susceptibility to tumor cell implantation: comparison of cauterization with N-methyl-N-nitrosourea

To determine whether or not transitional cell carcinoma cells will preferentially implant and grow on an altered urothelial surface, cauterization and instillation of N-methyl-N-nitrosourea (MNU) were used to alter the murine bladder urothelium. Transplantable tumor cells (2.09 X 10(6) ) were placed into the bladders of 53 mice. Tumor cell implantation occurred in only 6 per cent of the mice with a normal bladder, whereas tumors were present in 28 per cent of mice pretreated with intravesical MNU and in 67 per cent of mice which had a portion of the bladder cauterized prior to insertion of tumor cells (p less than 0.005). This study not only establishes the optimal technique for implantation in this experimental model, but also suggests that seeding may be a contributory factor in the high recurrence rate following endoscopic treatment of bladder tumors in man.     

Enhancement of the bladder defense mechanism by an exogenous agent

The mucopolysaccharide layer produced by the transitional cells coating the bladder plays an important role in the defense mechanism of the lower urinary tract. Carbenoxolone, a drug derived from licorice, has been shown to be beneficial to patients with peptic ulcer disease by stimulating increased mucous production in the stomach. Since the bladder epithelium has an endodermal derivation, the question of whether carbenoxolone has an effect on the mucopolysaccharide layer in the bladder was put forth. Carbenoxolone was shown to provide a protective effect to laboratory-induced lower urinary tract infections in the rabbit model.     

Adherence ofCandida albicans to epithelial cells from normal and cancerous urinary bladders

Patients with malignancies are at high risk to develop infections byCandida albicans. We have compared the adherence ofC. albicans isolated from urine cultures to bladder epithelial cells obtained from healthy volunteers and patients with cancer of the bladder. The mean number ofC. albicans adhering per epithelial cell from areas infiltrated from cancer was significantly higher as compared to cells obtained from intact areas of cancerous bladders bladders and from normal bladders. The increased adherence ofC. albicans to cancerous epithelial cell suggests that malignancies are associated with alterations of the epithelial cell surface which render the cells more susceptible to colonization byC. albicans. The increased colonization may predispose these patients toC. albicans infections.