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The mucin layer covering the bladder transitional cell mucosa appears to function as a primary defense mechanism against bacterial infection. We have previously prepared a glycoprotein fraction (GP1) from the urinary bladder mucosa of NZW rabbits and raised murine antisera against it. These antisera react with bladder, ureter and kidney tissue from rabbits, rats, guinea pigs, and hamsters. We now show that a similar substance occurs in human kidneys and bladder. In order to remove antibodies reactive with the Tamm-Horsfall protein (THP), the antisera were initially absorbed with an immunoadsorbent composed of purified human THP covalently bound to Sepharose CL-4B gel. Using an enzyme linked immunosorbent assay (ELISA) it could be shown that the absorbed antisera did not react with THP but retained a high titer in binding to GP1. Immunohistochemical procedures involving avidin-biotin-immunoperoxidase staining demonstrated that the absorbed anti-GP1 reacted well with six human urinary bladder biopsy specimens and two kidney autopsy specimens while normal murine sera showed little or no binding. Although this reactivity was not as strong as that found with homologous tissue (rabbit) these studies suggest that GP1, an antigen common to several animal species, is also related to a human urinary tract component.

Histochemical staining of bladder tissue has demonstrated a discrete layer of mucopolysaccharide (mucin) at the surface of rabbit and human bladders. This mucin is disrupted by acid treatment and is probably resynthesized by the transitional cells in less than twenty-four hours and replaced by forty-eight hours. Physiologic data indicate that bladder mucose can resist bacterial attachment, a function that is also disrupted by acid and recovers in less than twenty-four hours. These findings suggest that the surface mucopolysaccharide inhibits bacterial binding and may be the primary antibacterial defense of the urinary tract.

We have previously shown that the transitional cells lining the urinary bladder are capable of producing glycosaminoglycan (GAG). By use of a quantitative in vivo method of measuring bacterial adherence, we demonstrated that bacterial adherence to the mucosal cells is diminished in the presence of this GAG, rises when it is removed (by acid), and returns to normal when the GAG is resynthesized (in less than 24 h). We also found that this much layer could be removed (with a corresponding rise in bacterial adherence) and that addition of exogenous GAG (heparin) to the bladder prevented the expected rise in bacterial adherence. This study analyzed in depth the manner by which heparin prevents the rise in adherence seen when the mucin is removed. Pretreatment of bacteria with heparin had no effect on adherence, whereas pretreatment of the bladder with heparin inhibited adherence. To corroborate our impression that the heparin was coating the transitional cells, [3H]heparin was added to bladders after removal of mucin. Autoradiography revealed the heparin to be adherent to the surface of the transitional cells.

To implicate further the surface mucoprotein as the factor responsible for preventing bacterial attachment to the bladder mucosa, rabbit urothelium was severely disrupted and allowed to regenerate for varying periods of time. Quantitative measurements of the attachment of 14C-labeled Escherichia coli were correlated with the histologic presence of the surface mucoprotein. Control levels of bacterial attachment were observed only when the PAS-positive layer had regenerated, further supporting the role of this layer in the bladder's antibacterial defense.

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.

14C-labeled Escherichia coli was introduced into acid-treated and intact rabbit bladders in vivo. Live, dead, and metabolically inhibited bacteria displayed similar interaction with the urothelium with and without intact surface mucoprotein. Disruption of the mucoprotein layer by acid reduced the defense potential in each group to a similar degree. Neither active metabolism nor bacterial cell death seems to enhance bacterial adherence to bladder mucosa.

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.

Pre-existing bacteriuria of 2 to 3 weeks' duration in the rabbit had no effect on either the histological integrity of the sialomucin layer (anti-adherence factor) of the bladder mucosa or the protective effect of this layer against super-infection.

The virulence of urease-producing bacteria depends on the ability of urease to degrade urea into ammonia and thereby to alkalinize the urine. Infections caused by urease-producing organisms such as Proteus mirabilis are particularly difficult to manage clinically. We have shown that the layer of glycosaminoglycans at the bladder surface protects against infection by blocking the adherence of bacteria to the epithelium. To determine whether urease-producing urinary pathogens owe their virulence in part to an ability to inactivate the protective effect of the glycosaminoglycan layer, we tested the ability of ammonium chloride to alter bacterial adherence to the normal vesical mucosa. We used an in vivo adherence assay that we have described previously in rabbits. Control animals received sodium chloride adjusted to the same pH as the ammonium chloride. We found that 0.25 M ammonium chloride significantly increases bacterial adherence to normal vesical mucosa as compared to adherence in controls receiving 0.25 M sodium chloride (p less than 0.05). These data suggest that urease plays a hitherto undescribed role in bacterial virulence by altering the antiadherence activity of the glycosaminoglycan layer present at the transitional cell surface.

We previously reported the results of quantitative and histochemical studies implicating the surface mucin of the bladder mucosa as an important antibacterial defense mechanism, which functions by preventing bacteria from adhering to the bladder wall. We call the mucin "anti-adherence factor" and we feel this is a previously undocumented role for mucin as a type of host antibacterial defense. These experiments were conduced with Escherichia coli. In an effort to determine whether the anti-adherence ability of the vesical mucin was a generalized phenomenon, we repeated these studies using unrelated bacterial species, including E coli, Klebsiella pneumoniae, and Staphylococcus aureus. The ability of the vesical mucosa to resist bacterial adherence to its surface was found to be independent of the bacterial species that was investigated.

In order for micro-organisms to infect the urinary tract they must adhere to the vesical mucosa so that they remain after voiding. Experiments in our laboratory demonstrated that the bladder has a self-protective mechanism capable of preventing bacterial adherence to the surface. Histochemical staining demonstrated that this substance is a mucopolysaccharide. Treatment of the mucosa with acid disrupted the protective layer and resulted in a 20 to 100-fold increase in bacterial adherence. Within 24 hours this layer reappeared and binding was again reduced to normal. This phenomenon was not related to microbial species or metabolic activity.

We have previusly provided physiologic and histochemical data implicating the bladder surface mucin layer as an important new antibacterial defense mechanism. This mucin or its contents seems to act as an "antiadherence factor", inhibiting bacterial adherence to the bladder mucosa and thereby facilitating the removal of bacteria by the voiding process. The present study was designed to investigate three mechanisms by which the mucin might repel bacterial attachment. Our data suggest that neither IgA nor a chelating agent are anti-adherence factors. We did find, however, that pH had a significant effect on the adherence of bacteria to mucosal cells stripped of their mucin layer. This result suggest that electrochemical charge is important in bacterial adherence. We beleive that the mucin layer both provides an electrochemical coat on the bladder surface that is a poor substrate for bacterial adherence and blocks the receptor sites of the transitional cells to which the microbes might adhere.

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.

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.

We have developed a model in rabbits for quantitatively determining the attachment of microbial organisms to the vesical mucosa. Our data suggest that the bladder has a primary antibacterial defense mechanism that serves to interrupt microbial attachment. This mechanism seems to be secretory, since after destruction with acid, the resistance to microbial binding returns in less than 24 hr.

Hormonal manipulation has been shown to result in less efficient bladder clearance of bacteria. We describe the use of a double antibody technique to semiquantitatively demonstrate the diminution of bladder glycoproteins from the transitional epithelium in oophorectomized rabbits. Rabbit bladder glycoprotein was isolated and used to immunize Swiss-Webster mice. Bladders of normal and oophorectomized rabbits were sequentially stained with mouse antirabbit sera and fluoresceinated goat antimouse antibody. A significant loss of bladder epithelial glycoprotein was evident in oophorectomized rabbits as compared to controls. This study, utilizing a semiquantitative immunologic staining technique, suggests an intact glycoprotein layer is important in the bladder defense mechanism.

The mucopolysaccharide (MPS) layer of the bladder has been implicated as a bacterial anti-adherence factor in previous experimentation. Production of MPS is known to be related to hormones in other systems of the body and probably to the urinary system. Due to the known rising incidence of urinary tract infection in most menopausal females, an experimental model using oophorectomized rabbits was designed. Oophorectomized and control rabbits were observed for their ability to clear their bladder of experimentally induced Escherichia coli 04 infections. There was a 2-fold increase in the mean number of days for clearance of bacteria among oophorectomized rabbits versus control rabbits. Twice as many rabbits cleared their infection within 1 week in the control population versus the oophorectomized population. To further examine the relationship of oophorectomy to urinary tract infection, a group of 1030 female canines in an animal veterinary practice was studied. It was found that the incidence of infection was higher in the spayed population than in the non-spayed group. The non-spayed group seem to have an age dependent history of urinary infection whereas ovariohysterectomy appeared to abolish this relationship to age.

The mucin layer of the bladder covering the transitional epithelium is thought to be an anti-adherence substance for bacteria. We have previously demonstrated that removal of this layer results in increased bacterial colonization. In this present communication we report our attempts to isolate and characterize the components of the mucin layer. Bladders were removed from female NZW rabbits and the mucosal layer was extracted with 0.1 M NaCl. Fractional centrifugation and column chromatography on AcA 34 and AcA 22 resulted in the separation of a partially purified glycoprotein, containing 30% carbohydrate, migrating as a single peak (4.5S) in the analytical ultracentrifuge. It was immunogenic in mice and the antisera were used to develop both a radioimmunoassay and an enzyme linked immunosorbent assay (ELISA). The antigenic activity of the glycoprotein was destroyed by protease or extremes of pH, but not by several glycosidases. In addition the murine antisera could not be inhibited by a panel of naturally occurring glycoproteins or glycosaminoglycans.