Decreased expression of a glycoprotein component of bladder surface mucin (GP1) in interstitial cystitis.

Interstitial cystitis is a disease of unknown etiology characterized by unremitting urinary frequency, urgency and suprapubic pain. Recently, a change in urothelial permeability has been identified in interstitial cystitis patients that is presumably mediated by aberrations in bladder surface mucin. For this study we evaluated qualitative changes in a previously defined glycoprotein component of this layer (GP1) as it occurs in interstitial cystitis patients and normal controls. Paraffinized bladder biopsies were obtained from 23 interstitial cystitis patients (all meeting National Institutes of Health inclusion criteria) and 11 normal controls. All biopsy tissue was stained with hematoxylin and eosin, and periodic acid, Schiff reaction. The tissues were examined immunohistochemically for GP1 using an anti-GP1 serum. Periodic acid, Schiff staining clearly identified bladder surface proteoglycans in all specimens. Moderate GP1 reactivity was noted in all normal control specimens. Alternatively, GP1 expression was absent in 35% of the interstitial cystitis patient biopsies and decreased in 61%. These data demonstrate qualitative GP1 changes in a majority of interstitial cystitis patients. It is unknown whether these differences have an impact on the pathogenesis of interstitial cystitis. However, our findings suggest that the absence or decreased expression of GP1 in interstitial cystitis bladder biopsies may serve as a marker to characterize the disease further in conjunction with clinical findings.

Urinary tract glycoprotein: distribution and antigenic specificity.

It has previously been demonstrated that the urinary tract contains a unique glycoprotein (GP1) that appears to serve a function in the clearance of pathogenic bacteria. We prepared a panel of monoclonal antibodies (mAbs) to human GP1 and examined its antigenic specificity and distribution in the human urinary tract. This was also observed in relation to another glycoprotein associated with infection, Tamm-Horsfall protein (THP), as well as to URO-5, a protein used in identifying the lower urinary tract. In enzyme-linked immunoadsorbent assays (ELISA), all of the GP1 mAbs reacted with GP1 prepared from both human urine and rabbit bladder mucosa. No immunoreactivity was observed with either THP mAbs or URO-5 mAbs. Western-blot analyses using GP1 mAbs with human urine GP1 preparations detected a single band of approximately 50-52 kDa. Human urinary tract tissues were also examined by immunohistochemical techniques using the GP1 mAbs, commercial THP, and URO-5 mAbs. In paraffin-embedded bladder sections, only GP1 mAbs reacted with the urothelium. Selective staining of distal collecting tubules, renal pelvis, and ureters was demonstrated for GP1 mAbs. Proximal convoluted tubules, loops of Henle, and Bowman's capsule failed to stain for GP1. In the same tissues, THP mAbs reacted with the thick and thin segments of medullary loops of nephrons (Henle's loops). URO-5 mAbs stained fresh frozen sections of bladder urothelium, distal collecting tubules, and portions of Henle's loops; however, they failed to stain paraffin-embedded tissue sections. These GP1 mAbs provide a new tool for biochemical and histochemical investigations of the mucin layer in both healthy and diseased urinary tract tissue.

Soluble Fc gamma RIII (CD16) and immunoglobulin G levels in seminal plasma of men with immunological infertility.

Receptors for the Fc region of the immunoglobulin G (IgG) (Fc gamma R) have been recognized as a link between humoral and cellular immune responses. A soluble form of Fc gamma RIII (CD16) has been found in seminal plasma (SP), which may modulate immunosuppression of antisperm immune responses in the male and female reproductive tracts. SP from some individuals apparently have lower levels of Fc gamma RIII, but it is not known whether the diminished activities are due to low receptor concentration or steric interference from IgG. To test the hypothesis that different levels are due to steric interference, relative levels of Fc gamma RIII were measured in SP using monoclonal antibody 3G8 in an amplified enzyme-linked immunosorbent assay (ELISA) system. Men who were positive for antisperm antibodies (ASA) by Sperm Mar and direct immunobead assay (N = 26) and negative for ASA (N = 26) were tested. Individuals who were ASA positive had lower detectable levels than those who were ASA negative (t = 1.99, P = 0.05). Therefore, variation in Fc gamma RIII levels may be due to steric interference from IgG. IgG subclass concentrations in SP of both groups were determined using an ELISA method and compared to Fc gamma RIII levels. Slight correlations were seen for IgG1 (r2 = 0.237, P < 0.001), IgG2 (r2 = 0.204, P < 0.001), and total IgG (r2 = 0.299, P < 0.001) in relation to Fc gamma RIII levels in ASA-negative SP specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of an antibacterial glycoprotein in a spectrum of transitional gel carcinomas.

The mucin lining of the bladder is thought to serve as a primary defense mechanism against bacterial colonization, and has recently been implicated in the urothelial resistance to carcinogenic insult. We have isolated a unique glycoprotein fraction (GP1) of this lining from the normal rabbit bladder which may have a function in preventing bacterial adherence and colonization in the urinary tract. This glycoprotein has been shown to bind to a wide range of uropathic bacteria. The present study examines changes in the bladder's antibacterial defense mechanisms as measured by GP1 expression in the neoplastic state. Using an anti-GP1 serum, immunohistochemical staining was performed on 20 paraffinized and fresh frozen transitional cell carcinomas ranging from low grade, superficial tumors to high grade, invasive tumors. The presence of GP1 was seen throughout the mucosal layer in normal specimens with increased amounts noted towards the mucosal surface. Progressively decreased expression was noted with increasing grades of all transitional carcinoma specimens. Mucosal field changes in GP1 expression were not noted in any of the patients. Intestinal mucosal controls failed to detect the presence of GP1. These studies suggest that the expression of GP1 decreases with tumor dedifferentiation and that bladder tumorogenesis may serve a role in handicapping the bladder's primary antibacterial defense mechanism.

Antisperm antibodies in seminal plasma of spinal cord-injured men.

Central to the problem of reproductive rehabilitation of spinal cord-injured men treated by assisted ejaculatory techniques is the consistent observation of deficient semen quality. Most studies have reported asthenospermia despite the presence of normal sperm concentration in most men undergoing these procedures. To date little attention has been given to the incidence and relevance of sperm autoimmunity in this group. In 9 anejaculatory spinal cord-injured men, electroejaculation was performed. Antegrade ejaculates were obtained in 7 men and analyzed. Mean sperm antegrade concentration was 74.4 +/- 113 x 10(6)/mL with a mean motile sperm concentration of 28.6 +/- 54.0 x 10(6)/mL. Enzyme-linked immunosorbent assay (ELISA)-determined antisperm antibody response was positive in the seminal plasma of 5 of 7 patients. Because of the disproportionately high incidence of an immunologic factor in men with neurogenic infertility, sperm autoimmunity should be considered among the important causes underlying their seminal dysfunction.

The production of antibodies to pentosanpolysulfate (ELMIRON, SP-54).

Pentosanpolysulfate (PPS) represents the product obtained after sulfation of xylan and is composed of beta 1----4-D-xylopyranose residues sulfated at C2 and C3. Studies have shown that this compound can often be effective in relieving the symptoms of interstitial cystitis (IC). In order to elucidate the mode of action of PPS in IC, a sensitive and reliable assay was needed. To this end we prepared an immunogenic form of PPS by coupling it to methylated bovine serum albumin (MBSA). This complex was used to immunize NZW rabbits (1 mg, IM). Four of five animals responded with anti-PPS antibodies, three of which had high titer (greater than 1/2000) as measured by an enzyme-linked immunosorbent assay (ELISA). All sera were routinely absorbed with an MBSA-Sepharose immunoadsorbent to remove anti-MBSA antibodies. ELISA inhibition tests were used to determine the sensitivity and specificity of the sera. At least 50 ng/ml of PPS could be routinely detected by this assay. A number of naturally occurring proteoglycans, polysaccharides, monosaccharides and disaccharides were examined for reactivity with the antibodies but only heparin was an effective inhibitor. Absorption with heparin immunoadsorbents reduced, but did not eliminate, the ability of heparin to inhibit anti-PPS binding. This activity could be destroyed by treatment with heparinase without affecting PPS inhibition. Normal urine did not affect the ELISA or ELISA inhibition tests and thus allowed the determination of PPS levels in IC patient urines. Initial analysis of seven IC patients receiving oral PPS revealed urine concentration of 0.8-16.0 micrograms/ml. No inhibition could be detected in pre-treatment urine samples.

Antisera to a rabbit urinary tract antigen also react with human bladder and kidney tissue.

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.

Systemic sperm autoimmunity in spinal-cord injured men.

Historically, spinal-cord injured men have been considered virtually sterile because of ejaculatory dysfunction commonly resulting from their injury. Assisted ejaculatory techniques, however, have overcome the problem of sperm transport and have allowed both the establishment of pregnancy through artificial insemination and the assessment of their semen quality. Most studies have noted the presence of asthenozoospermia in the setting of normal sperm concentration following electroejaculation or vibratory stimulated ejaculation. Thus far, little attention has been given to the basis for the frequent finding of asthenozoospermia, and the possibility of sperm autoimmunity in this group has not been adequately studied. In nine spinal-cord injured men, reproductive evaluation was performed consisting of hormonal measurements, testicular biopsy, and indirect immunobead tests for sperm autoimmunity. A mean sperm concentration was 144 +/- 185 x 10(6)/ml. However, the mean motile concentration was 33 +/- 62 x 10(6)/ml. Indirect serum immunobead showed positive IgG or IgA titers in 3 of 8 patients. Because of the disproportionately high incidence of an immunologic factor in spinal-cord injured men compared to able-bodied infertile men, sperm autoimmunity should be considered among the important causes underlying seminal dysfunction following spinal cord injury.

Distribution of rabbit mucosal glycoprotein throughout urinary tract.

The mucin layer covering the transitional epithelium of the bladder is thought to be an anti-adherence substance for bacteria. We describe the use of an immunoperoxidase staining technique to demonstrate the presence of glycoprotein lining the urothelium of both the upper and lower urinary tracts of the rabbit. Antisera against this glycoprotein (GP1) were raised in Swiss-Webster mice. The genitourinary tracts of male and female NZW rabbits were removed and sequentially treated with mouse anti-GP1 sera, biotin-labelled anti-mouse IgG, and an avidin-biotinylated horseradish peroxidase complex. The results demonstrated that an antigenically similar (or identical) glycoprotein covers the distal renal tubules and urothelium of the renal pelvis, ureters, bladder, and urethra, suggesting that it may function as an antibacterial defense mechanism throughout the urinary tract.

Antigenic similarities among rodent urinary tract glycoproteins.

Urinary glycoconjugates (glycosaminoglycans, glycoproteins, mucopolysaccharides) have been postulated as the natural defense mechanisms which prevent urinary tract infections. As a direct approach to establish the validity of this hypothesis, we have prepared a glycoprotein fraction (GP1) from rabbit bladder mucosal tissue and shown that it may be involved in the prevention of bacterial adherence. Immunohistochemical studies using fluorescence have demonstrated that murine antibodies raised to rabbit GP1 can be used as a semiquantitative index of glycoprotein production. The present investigation addresses the question of the species restriction of the glycoprotein--whether it is confined to the rabbit or whether a similar or identical substance occurs in other species. Using an immunoperoxidase staining technique, we have examined the genitourinary tracts of Sprague Dawley rats, Golden hamsters, and Hartley guinea pigs. Mouse anti-rabbit GP1 was used as the primary antibody with an avidin-biotin complexing system. All three species reacted well with the murine antiserum but were negative with normal sera. Semiquantitative estimates of the relative amounts of this material in different areas of the genitourinary tract showed that the distal renal tubules, renal pelvic mucosa, ureters and bladders were rich in this glycoprotein while urethra and vagina were not.

Urinary bladder glycoproteins of the rabbit: extraction, biochemical and immunological studies.

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.

The detection of infectious mononucleosis antibodies by radioimmunoassay and ELISA.

An infectious mononucleosis receptor (IMR) preparation has been isolated from sheep erythrocyte stroma. This material was used to detect the presence of human infectious mononucleosis (IM) antibodies with rabbit anti-human immunoglobulin antisera by radioimmunoassay and ELISA. These procedures use 10-20 ng of IMR and 5-10 microliter of human serum. A single treatment of the IMR with neuraminidase reduces its ability to bind to the IM antibodies by 80%. In addition absorption of the IM sera with sheep red cells completely removes IM specific antibodies.

The isolation of an antigen binding factor from cultured murine T-lymphocytes.

BALB/c mice were immunized with the random sequence polypeptide [Glu60 Ala40] (GA). 125I-lymphocyte suspensions were prepared from lymph nodes, radiolabelled with 125I and cultured for 18 hours. Supernatant fluids were collected and passed over an immunoabsorbant composed of [D-Glu60 D-Ala40] (D-GA) bound to Sepharose. The non-adherent fraction was then applied to GA Sepharose and the adherent material eluted with 2 M NaSCN. The eluate was relabelled with 125I, reabsorbed to GA Sepharose and eluted. This fraction bound well to GA-Sepharose (50-80%) but not to the enantiomorphic D-GA Sepharose (5-10%). This material did not react with goat antisera directed against murine IgG, IgM or IgA. It was capable of binding to rabbit antiidiotypic sera raised against BALB/c anti-GA, but not to those antibodies raised against BALB/c anti-GAT. It also showed some reaction with rabbit antiserum to a rat T cell factor (TCF). Polyacrylamide gel electrophoresis revealed a major component with an approximate molecular weight of 63,000 which was not affected by reduction and alkylation.

Preparation of an infectious mononucleosis receptor from sheep erythrocyte stroma.

A method is described for the isolation and purification of infectious mononucleosis receptor (IMR) in high yield from sheep erythrocyte stroma. The procedure involves extraction of the stroma successively with boiling acetone, 100, 75 and 50% ethanol which removes approximately 40% non-infectious mononucleosis-active material. Subsequent phenol/buffered saline extraction of the residue from the organic extraction yields a highly active IMR preparation in the aqueous phases. This material was further purified by ethanol precipitation and gel filtration chromatography. The yield was 0.2% of the stroma. Approximately 0.1 mug/ml of the purified IMR inhibits 4 agglutinating units of infectious mononucleosis (IM) serum. The material is a glycoprotein containing N-acetyl- and N-glycolylneuraminic acids, hexose, hexosamine and approximately 46% protein. It gives one band in immunoelectrophoresis with IM serum and loses IM specificity upon treatment with neuraminidase.

The occurrence of antibodies in rabbit anti-pneumococcus antisera directed against calcium dependent and independent determinants.

Two type specific acidic pol-saccharides from Pneumococci (type III and type VIII) have been used to prepare immuno-adsorbents. The polysaccharides were coupled to Sepharose 4b via a 1,6 hexanediamine spacer and used to isolate two populations of type specific antibodies from rabbit anti-pneumococcus antisera. One population requires calcium to affect interaction with the homologous polysaccharide, while the other population has no such requirement. The isolated antibodies are of the lgG class and are fully active in precipitin assays.