Veer P. Bhavanandan

A comparison of multiple urine markers for interstitial cystitis.

PURPOSE We measured several urine markers in 24-hour specimens from patients with interstitial cystitis and healthy controls. For each marker we determined whether the urine level was significantly different in interstitial cystitis and control cases, and whether the marker level correlated with the symptom score. MATERIALS AND METHODS Study participants included 36 female patients with interstitial cystitis and 36 age matched female volunteers. Multiple urine aliquots were obtained to measure the various markers. RESULTS Certain markers were significantly increased in interstitial cystitis, including anti-proliferative factor, epidermal growth factor, insulin-like growth factor (IGF) binding protein-3 and interleukin (IL)-6. Markers significantly decreased in interstitial cystitis were heparin-binding epidermal growth factor-like growth factor, cyclic guanosine monophosphate and methylhistamine. Other markers were not significantly different in the interstitial cystitis and control groups, including total glycosaminoglycans, epitectin, hyaluronic acid, IL-8, IL-1 and nitrates plus nitrites. IGF-1 was undetectable in 24-hour urine samples but spot voided samples from the same interstitial cystitis population had IGF-1 levels similar to previously reported levels. The only significant association of marker with symptom score was a positive correlation of IL-6 with nocturia. For all markers the conclusions were the same whether the marker was normalized to creatinine or to 24 hours. CONCLUSIONS This study confirmed several previously reported urine alterations in interstitial cystitis, including increased anti-proliferative factor, epidermal growth factor, IGF binding protein-3 and IL-6, and decreased heparin-binding epidermal growth factor-like growth factor and cyclic guanosine monophosphate. Of all markers studied anti-proliferative factor had the least overlap in the interstitial cystitis and control groups, and so it is the most likely candidate to become a diagnostic test.

Studies on glycoproteins XI. The O-glycosidic linkage of N-acetylgalactosamine to seryl and threonyl residues in ovine submaxillary gland glycoprotein

Ovine submaxillary glycoprotein (OSM) was digested exhaustively with pronase and the sialoglycopeptides separated by gel filtration. By subsequent neuraminidase (EC 3.2.1.18) treatment and further purification, glycopeptides of average molecular weight 660 and containing 50% of the total galactosamine of OSM were obtained. Serine and threonine were the only amino acids with a functional group in the side chain present in a concentration high enough to accommodate all hexosamine residues. Treatment of the glycopeptides with 0.5 N NaOH at 0° or 22° resulted in the release of N-acetylgalactosamine coincident with the loss of an equimolecular amount of hydroxyamino acids. Most likely the carbohydrate was released by the mechanism of β-carbonyl elimination. Some evidence for the formation of an unsaturated compound, presumably α-aminoacrylic acid, was afforded spectrophotometrically. It is concluded that in OSM at least 50% of the prosthetic groups are joined glycosidically to the hydroxyl groups of serine and threonine. When native OSM was treated at pH 8.0 and 42° for 120 h, about one-third of the prosthetic groups was released. After this treatment the isolated, non-dialyzable residual glycoprotein reacted strongly positive in the Warren test. The mechanism of the reaction is discussed.

Plasmodium falciparum-Infected Erythrocytes Adhere Both in the Intervillous Space and on the Villous Surface of Human Placenta by Binding to the Low-Sulfated Chondroitin Sulfate Proteoglycan Receptor

In pregnant women infected with Plasmodium falciparum, the parasite-infected red blood cells (IRBCs) sequester in the placenta through chondroitin 4-sulfate (C4S)-mediated adherence. The pattern of IRBC adherence in P. falciparum-infected placenta has been controversial. Moreover, the identity of the chondroitin sulfate proteoglycan (CSPG) receptor, that mediates IRBC adherence, and its location in the placenta have not been established. This study, using immunohistochemical techniques, clearly shows, for the first time, that the low-sulfated CSPGs of the placenta are localized predominantly in the intervillous space. Ex vivo IRBC adherence analyses demonstrate that the IRBCs are adhered to the CSPG receptors in the placenta in a C4S-dependent manner. This IRBC binding pattern was similar to that observed in P. falciparum-infected placentas. These data and the results of dual-fluorescence staining of the endogenous RBCs and syncytiotrophoblasts, and co-localization of CSPG and IRBC adherence unequivocally establish that the low-sulfated CSPGs are the major natural receptors for IRBC adherence in the placenta. Further, it was found that IRBCs adhere mainly in the intervillous space and also at significant levels to the syncytiotrophoblasts. Finally, the ex vivo IRBC adherence method described herein provides a reliable procedure for future studies for the assessment of the efficacy of C4S inhibitors and adhesion inhibitory antibodies.

The complete enzymic degradation of glycopeptides containing O-seryl and O-threonyl linked carbohydrate

Abstract In recent years a considerable amount of information on the molecular structure of glycoproteins has been published. Little is known, however, on the biosynthesis of these compounds and no information is available on the enzymic breakdown of glycoproteins to their individual constituents. In the present paper the purification and properties of a β-N-acetylhexosaminidase and its action on glycopeptides containing O-seryl and O-threonyl linked N-acetylgalactosamine are described.

Structure and function of the Ca antigen

The Ca antigen, which can be detected in a wide range of malignant human tumours by means of the Cal antibody, is a glycoprotein of the mucin type. At least 95% of the carbohydrate is 0-glycosidically linked to the polypeptide which contains high proportions of glycine, serine and glutamic acid. The carbohydrate has a very simple structure: it is composed almost entirely of tetra- tri- and disaccharides having the general formula (NeuNac)n leads to [Gal leads to GalNac] alpha leads to, where n = 0, 1 or 2. In many malignant cell lines, the antigen is produced constitutively in vitro; but in one that has been examined, its synthesis can be induced by high concentrations of lactate. Evidence is presented for the view that a primary function of this glycoprotein is to shield the cells that produce it from hydrogen ion concentrations outside of the physiological range. The presence of the Ca antigen in malignant tumours may thus be a reflection of metabolic conditions that are known to be characteristics of such tumours.

INCREASED URINARY HYALURONIC ACID AND INTERSTITIAL CYSTITIS

PURPOSE We compared urinary levels of hyaluronic acid in patients who met the National Institute for Diabetes, and Digestive and Kidney Diseases criteria for interstitial cystitis and in age matched healthy female controls. MATERIALS AND METHODS Urinary hyaluronic acid was measured by solid phase radiometric assay using hyaluronic acid binding protein. Hyaluronic acid and symptom scores were compared in interstitial cystitis patients who gave multiple urine samples during treatment. Since hyaluronic acid changed with treatment in some patients, 17 samples from untreated interstitial cystitis patients were selected and compared with 17 control samples. RESULTS Mean plus or minus standard deviation urinary hyaluronic acid concentrations were similar in the 2 groups (interstitial cystitis group 574 +/- 496, controls 512 +/- 324 ng./ml., p = 0.77). When normalized to creatinine urinary hyaluronic acid was significantly higher in interstitial cystitis patients (interstitial cystitis group 674 +/- 220, controls 446 +/- 220 ng./mg. creatinine, p = 0.0019). Urinary creatinine concentrations did not differ significantly (interstitial cystitis group 842 +/- 715, controls 1,162 +/- 516 mg./l., p = 0.12). CONCLUSIONS Urinary hyaluronic acid was higher in interstitial cystitis patients than healthy controls. Since bladder hyaluronic acid is below the epithelium, this finding may indicate leakage across the epithelium into the urine in interstitial cystitis patients.

A Novel Monoclonal Antibody Specific for Sialylated MUC1 Mucin

Development of a new monoclonal antibody (mAb) MY.1E12 which reacts with sialylated MUC1 mucins is described. The mAb did not react with any component in the lysates of COS‐1 cells, whereas it bound to sialylated MUC1 mucins produced by COS‐1 cells transiently transacted with MUC1 mucin cDNA, strongly suggesting that the expression of the epitope of mAb MY.1E12 depends on the presence of the MUC1 mucin core peptide. The requirement of sialyl residues for antibody recognition was established by Western blotting analysis of extracts of various carcinoma cells and in situ desialylation. In all cases, the mAb binding of electrophoretically separated MUC1 mucin diminished after desialylation by mild acid hydrolysis. When Capan‐1 pancreatic carcinoma cells were pretreated with benzyl‐JV‐acetylgalactosaminide in culture, the MUC1 mucins produced under these conditions, which were detected by core peptide‐specific mAbs, did not react with mAb MY.1E12. These results suggest that 0‐linked carbohydrate chains are important for the mAb binding.

Neuraminidase assay utilizing sialyl-oligosaccharide substrates with tritium-labeled aglycone

A new method for the radioisotopic assay of neuraminidase activity has been developed. The substrate utilized, α-d-N-acetylneuraminosyl-(2 → 3′)-lactit[3H]ol, was prepared by reduction of α-d-N-acetylneuraminosyl-(2 → 3′)-lactose with tritiated borohydride and purified by ion-exchange chromatography. After incubation with neuraminidase, the reaction mixtures were applied to small columns of AG 1-X2 (formate) in order to remove free sialic acid and unhydrolyzed substrate. The lactit[3H]ol released by neuraminidase action was then recovered by washing the columns with distilled water and quantitated by utilizing a liquid scintillation spectrometer. Studies with bacterial, avian, and mammalian neuraminidases are described.

Urinary Chondroitin Sulfates, Heparan Sulfate and Total Sulfated Glycosaminoglycans in Interstitial Cystitis

PURPOSE We compared urinary glycosaminoglycan levels in patients with interstitial cystitis and healthy controls. MATERIALS AND METHODS Total sulfated glycosaminoglycans assayed by dimethylmethylene blue binding and individual glycosaminoglycans analyzed by cellulose acetate electrophoresis were compared in patients with interstitial cystitis and healthy controls. Also, multiple urine samples were obtained from healthy female controls for 2 months to assess the relationship of urinary glycosaminoglycan and creatinine concentrations, and to determine whether glycosaminoglycan excretion changes during the menstrual cycle. RESULTS Total sulfated glycosaminoglycan and creatinine concentrations correlated well in random voided samples. Menstrual cycle day did not affect total sulfated glycosaminoglycan levels. Cellulose acetate electrophoresis revealed 3 bands corresponding to chondroitin sulfates, heparan sulfate and acidic glycoprotein. Patients with interstitial cystitis had decreased urinary concentrations of each of these individual components and total sulfated glycosaminoglycans. However, glycosaminoglycan-to-creatinine ratios were similar in interstitial cystitis and control urine. CONCLUSIONS Using these assays total and individual urinary glycosaminoglycan levels normalized to creatinine were not altered in interstitial cystitis.

A NEW DIRECT TEST OF BLADDER PERMEABILITY

PURPOSE A proposed cause of interstitial cystitis is increased bladder permeability but to our knowledge this theory has not been proved by direct testing. We developed a safe, relatively painless, direct test of bladder permeability. MATERIALS AND METHODS The original permeability test involved placing 4% lactulose and 1% rhamnose intravesically, then drawing blood to assay for these sugars. Initial feasibility studies were performed in rabbits with bladder epithelium that was intact or disrupted by a 50% acetone rinse. In humans the initial goal was to distinguish intact bladders from those known to have increased permeability. Since distention is known to increase permeability temporarily, we studied patients with interstitial cystitis immediately after distention. RESULTS Neither sugar was absorbed from intact rabbit bladders, while each was absorbed from acetone rinsed bladders at 10, 20 and 30 minutes. We used 100 ml. of solution in the initial 8 humans, including 6 with interstitial cystitis and 2 controls. At 30 minutes each sugar was absorbed from interstitial cystitis bladders but neither was absorbed from intact bladders. The test solution was then changed to 5% rhamnose. Mean rhamnose absorption plus or minus standard deviation was much greater in the 6 patients with interstitial cystitis than in 8 controls (26.3 + or - 26.1 versus 0.78 + or - 0.87 nmol. /ml. serum, p = 0.008). With 1 exception interstitial cystitis serum levels were at least 4-fold higher than the highest control level. CONCLUSIONS This new permeability test clearly distinguishes intact versus distended bladders. It may be performed to test whether bladder permeability is increased in interstitial cystitis.