Intraocular granuloma: a Schistosoma mansoni model of ocular inflammation.

An experimental uveitis model was developed in New Zealand rabbits by an intraocular injection of Schistosoma mansoni eggs. An inflammatory response was clinically apparent after 5 days and histologically was characterized by an eosinophilic infiltrate into the vitreous and choroid. The chorioretinitis that developed resulted in the disruption of the photoreceptor layer. After 30 days, eggs were enveloped by a granulomatous host response similar to that observed in animals infected systemically with Schistosoma mansoni. Reduction (immunomodulation) of granuloma size and cellularity compared with controls was observed in paraffin sections of eyes challenged (100 eggs) 4 weeks after a priming injection (500 eggs) in the contralateral eye. The granulomatous response was not evident when heat-killed eggs were injected intraocularly. Extracts made from viable eggs also induced an intense vitreous infiltrate 12 hr after injection. Serum collected from rabbits injected with 500 or more eggs showed antibody (7s) reactivity for 125I-labeled bovine S antigen, as demonstrated by immunoprecipitation with Staphylococcus aureus (Pansorbin). This model is useful for analyzing immunologic parameters involved in ocular granulomatous and parasitic diseases, humoral and cellular responses mediating autosensitization to retinal or other ocular antigens, and possible for screening chemotherapeutic agents for immunomodulation of potentially injurious host inflammatory responses.

Fc and C3b receptors on cultured retinoblastoma cells.

Fc and C3b receptors were identified on cultured retinoblastoma cells. Labeled receptor protein bound to affinity gels prepared with IgG, Aggregated IgG, and Fc but not to control gels prepared from Fab'2 or Sepharose-4B alone. Eluted Fc receptors was partially characterized by polyacrylamide gel electrophoresis. Molecular weight of the isolated receptor or its subunit was approximately 4.3 X 10(4) daltons. Cultured retinoblastoma cells were found to rosette with human indicator erythrocytes specific for C3b and Fc receptors. This study indicates that considerable reactivity between retinoblastoma patients' sera and cultured tumor cells may be mediated by these receptors as well as by reactivity toward retinoblastoma antigens.

Binding of retinoblastoma and normal sera to retinoblastoma-derived cultured cells.

We have observed increased binding of retinoblastoma patients' sera to a retinoblastoma-derived cultured cell line (Y-79). This reactivity was mediated by the serum IgG fraction and was directed toward different tumor or target cell (Y-79, Molt, Raji, and fibroblasts) cultured in media containing fetal calf serum. Normal pooled serum IgG fractions did not demonstrate any similar binding. When target cells were cultured in media containing human serum instead of fetal calf serum, a considerable reduction in retinoblastoma sera binding activity was observed. Reactivity against target retinoblastoma cells could be reduced but not entirely eliminated by quantitative absorption with nonretinoblastoma (Molt) cells grown in media with fetal calf serum. Retinoblastoma and normal sera binding to autologous fibroblasts, nonautologous fibroblasts, and cultured melanoma cells was also minimal. These findings suggest that residual binding activity in the sera tested may be directed against retinoblastoma tumor antigens. The fetal calf serum component responsible for reactivity with certain retinoblastoma sera was shown by immunoprecipitation, competitive inhibition, and gel electrophoresis to be bovine serum albumin.

Characterization of retinoblastoma immune complexes.

Immune complexes from retinoblastoma sera were characterized with molecular sieve chromatography, affinity chromatography, and polyacrylamide gel electrophoresis (PAGE). Retinoblastoma patients' sera had two well-defined peaks of immune complex activity after molecular sieve chromatography. These protein fractions had a molecular weight of approximately 1.6 x 10(5) and 2.0 x 10(6) daltons. Affinity chromatography with Sepharose 4B-protein A and analytical PAGE demonstrated that IgG was the predominant immunoglobulin in these immune compelxes. Immune complexes also had affinity for Sepharose-concanavalin A, indicating the glycoprotein nature of the antigen component.

Electrophysiologic monitoring of the effects of soluble virulence factors produced by Escherichia coli infection in urine.

OBJECTIVES: The presence of soluble virulence factors in urine infected by Escherichia coli has been postulated by recent studies in a rabbit bladder model. These substances may enhance bacterial adherence by damaging the glycosaminoglycan (GAG) layer that normally blocks both bacterial adherence and diffusion of solutes to the level of the epithelial membrane. We evaluated the effects of E. coli-infected urine on New Zealand white rabbit bladder mucosal permeability by measuring conductance (g) and current (I) and then calculating resistance (R) using the formula R = I divided by g. METHODS: Forty-five rabbit bladders were prepared and mounted in Ussing chambers. The group was divided equally into three smaller cohorts (n = 15). The first cohort (NL) was exposed to uninfected sterile-filtered urine, the second cohort (PS) was a positive control exposed to uninfected sterile-filtered urine containing protamine sulfate, and the final cohort (INFX) was exposed to supernatants of E. coli-infected (more than 10(7) colony-forming units) sterile-filtered urine. The average time of exposure was 160 minutes, and the previously mentioned electrophysiologic parameters were measured and recorded. RESULTS: The results of the PS group showed statistically significant differences in conductance, current, and resistance compared with the NL group. The decline in resistance accompanied by elevation in both current and conductance was an indirect indication that the GAG layer was functionally disturbed with a secondary increase in mucosal permeability. In a similar fashion, the INFX group also showed a statistically significant rise in conductance as well as a decline in resistance. CONCLUSIONS: These data support the concept that E. coli-infected urine contains soluble factors that can damage the GAG layer. Although these substances appear to be less potent than quaternary or polyamines such as protamine sulfate, their mechanism of action appears similar and may enhance bacterial virulence.

Intestinal de-epithelialization and augmentation cystoplasty: an animal model.

OBJECTIVES: An animal model of augmentation cystoplasty was developed in New Zealand rabbits to study the effects of intestinal de-epithelialization on subsequent re-epithelialization by bladder urothelium. METHODS: Twenty-four rabbits underwent augmentation cystoplasty using intestinal segments that were either treated with protamine sulfate and urea solution or else anastomosed with an intact epithelium. Half of the rabbits receiving the de-epithelialized intestinal segments were subjected to glycosaminoglycan replacement therapy by administration of intravesical heparin. Experimental and control rabbits were sacrificed at 1-, 2-, and 3-month intervals. RESULTS: Histologic examination of the augmented sections showed small areas of urothelium growing over the intestinal epithelium (approximately 15%). The heparin-treated group demonstrated the greatest amount of re-epithelialization. There was no obvious histologic difference in the amount of collagen present in the augmented tissues in any of the experimental groups. CONCLUSIONS: In a preliminary study, New Zealand rabbits appear to be satisfactory as an experimental animal for studying the augmentation cystoplasty procedure and for the development of therapeutic interventions for enhancing epithelial growth. Protamine and urea will de-epithelialize the bowel and heparin may promote epithelialization of augmented intestinal segment by transitional epithelium.

Dynamic bis-intercalation of a homodimeric thiazole orange dye in DNA: evidence of intercalator creeping.

We have used one and two dimensional exchange 1H NMR spectroscopy to characterize the dynamics of the binding of a homodimeric thiazole orange dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis- 4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinol inium tetraiodide (TOTO), to double stranded DNA (dsDNA). The double stranded oligonucleotides used were d-(CGCTAGCG)2 (1) and d(CGCTAGCTAGCG)2 (2). TOTO binds preferentially to the (5'-CTAG-3')2 sites and forms mixtures of 1:1 and 1:2 dsDNA-TOTO complexes with 2 in ratios dependent on the relative amount of TOTO and the oligonucleotide in the sample. The dynamic exchange between preferential binding sites in the case of a 2:1 1-TOTO mixture is an intermolecular exchange process between two binding sites on different oligonucleotides. In the case of the 1:1 2-TOTO complex an intramolecular exchange process occur between two different binding sites on the same strand. Both processes were studied. The results demonstrate the ability of TOTO to migrate along a dsDNA strand in an intramolecular exchange process. The migration process ("creeping") along the DNA strand is 6 times faster than the rate of intermolecular exchange between sites in two different oligonucleotides.

Bge snail cell-line antigens: ineffectiveness as antischistosomal vaccine in mice.

Mice and rabbits were immunized with antigens derived from Bge cells, Biomphalaria glabrata hemolymph, or Schistosoma mansoni. Antisera from mice given molluscan antigens did not form immunoprecipitates with soluble antigen from adult worms, but their binding to surfaces of sporocysts, cercariae, and schistosomules suggests the presence of cross-reacting determinants. In vitro, cell-mediated immune responses to Bge antigens were not demonstrable in infected nor in immunized mice. Mice immunized with Bge cell-line antigens and challenged with S. mansoni cercariae showed no reduction in worm burden when compared with control mice.

Metacercarial cyst formation in vitro of Echinostoma paraensei.

Cercariae of Echinostoma paraensei Lie and Basch 1968 encysted normally in the presence of Biomphalaria glabrata embryo (Bge) cells in culture, partially in culture conditioned medium, and not at all in fresh culture medium alone. At the ultrastructural level the cyst is composed of 2 well defined regions. The outer cyst wall (OCW) is particulate to fibrous in nature, formed from secretory granules released from the cercarial tegument. Membranous scrolls or rodlets secreted from the subtegumental cystogenous gland cells are then added to this layer, forming the inner cyst wall (ICW). After 24 hr the cultured cyst is enclosed by a thin cellular capsule similar to that found around cysts in the snail host. The capsule also contains collagen fibers, not found elsewhere in Bge cell cultures.

In vivo quantitative NMR imaging of fruit tissues during growth using Spoiled Gradient Echo sequence.

Nondestructive studies of physiological processes in agronomic products require increasingly higher spatial and temporal resolutions. Nuclear Magnetic Resonance (NMR) imaging is a non-invasive technique providing physiological and morphological information on biological tissues. The aim of this study was to design a robust and accurate quantitative measurement method based on NMR imaging combined with contrast agent (CA) for mapping and quantifying water transport in growing cherry tomato fruits. A multiple flip-angle Spoiled Gradient Echo (SGE) imaging sequence was used to evaluate the intrinsic parameters maps M0 and T1 of the fruit tissues. Water transport and paths flow were monitored using Gd(3+)/[Fe(CN)6](3-)/D-mannitol nanoparticles as a tracer. This dynamic study was carried out using a compartmental modeling. The CA was preferentially accumulated in the surrounding tissues of columella and in the seed envelopes. The total quantities and the average volume flow of water estimated are: 198 mg, 1.76 mm(3)/h for the columella and 326 mg, 2.91 mm(3)/h for the seed envelopes. We demonstrate in this paper that the NMR imaging technique coupled with efficient and biocompatible CA in physiological medium has the potential to become a major tool in plant physiology research.

Proteoglycan core protein syndecan in bladder biopsies.

The etiology of interstitial cystitis (IC) may be related to a dysfunctional epithelium caused by an abnormal permeability barrier. The presence of deleterious urinary substances (quaternary amines) that alter an otherwise normal epithelium may also be contributory. IC disease could reflect an inability of the bladder to repair its protective surface-coat material (glycosaminoglycans and proteoglycans), which is constantly exposed to a toxic urine environment. Bladder biopsy tissue from IC patients and derived explant cells were investigated to determine if mRNA for a proteoglycan core protein could be extracted and evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Syndecan was chosen for this investigation because the available sequence information permitted PCR primers to be synthesized. The results indicated that biopsy tissue and explant cells could be utilized for the isolation of syndecan core protein mRNA. This proteoglycan was also demonstrated in mouse bladders by immunostaining and immunoblotting (but not in human tissues) using a syndecan-specific monoclonal antibody (281-2). Quantitative differences in IC tissues versus normal bladder tissue with respect to gene expression for this proteoglycan core protein can now be determined.

Estrogenic regulation of HSP90 kD synthesis in rat urinary bladder.

The role of heat shock protein (HSP90 kD) has been investigated in regard to its association with steroid receptors. HSP90 kD may play a role in steroid receptor stabilization and activation. Oophorectomized Sprague-Dawley rats (n = 25) were placed into five groups and injected subcutaneously with 30 microg beta-estradiol 17-benzoate in sesame oil, with one group injected with carrier oil (control). After estrogen administration, the rats were killed, and their bladders removed for immunostaining, immunoblotting and enzyme-linked immunosorbent assay (ELISA). Immunoblot analysis demonstrated a 90-kD band in bladder homogenates, even in the absence of estrogen. However, the bands were more intense 12 and 24 h after administering estrogen. ELISA showed significant differences in HSP90 kD synthesis as early as 6 h compared to controls (P< 0.05). After 48 h the estrogen-treated rats and controls were identical. The above results were confirmed by immunostaining for HSP90 kD. HSP90 kD synthesis in the rat urinary bladder is under estrogenic regulation. These findings may be relevant in the etiology and pathobiology of interstitial cystitis and menopausal voiding dysfunctions since the bladder is enriched with estrogenic receptors and is under estrogenic influence.

Prevention of acrolein-induced bladder injury by pentosanpolysulfate.

The active metabolite of cyclophosphamide, acrolein, which is capable of damaging the transitional epithelium of the bladder, was evaluated in both in vivo and in vitro models to determine if its damaging effect could be reduced by the presence of a sulfated polysaccharide pentosampolysulfate. It was discovered that in all models pentosanpolysulfate was capable of reducing transitional cell injury due to acrolein.

Production of soluble virulence factor by Escherichia coli.

Experimental evidence suggests that adherence is a prerequisite for bacterial infection. We demonstrated that transitional cells at the surface of the bladder are coated with glycosaminoglycans (proteoglycans and mucus) whose presence efficiently decreases bacterial adherence to the mucosa. Exposure of mucus to protamine sulfate, a quaternary amine (known to form salts with glycosaminoglycans and inactivate them) significantly increases the bacterial adherence to the bladder. Investigators have primarily focused on bacterial surface factors (that is pili or fimbriae, glycocalix) in relation to the ability to adhere. We explored the hypothesis that Escherichia coli produces a soluble virulence factor that increases the infection rate in rabbits by promoting bacterial adherence to the bladder mucosa. In addition, it was proposed that this factor is a quaternary amine similar to protamine. For these studies an in vivo bacterial infection assay (which we described previously in rabbits) was used to examine E. coli metabolic products (soluble virulence factor) that could promote bacterial persistence in the bladder by perturbing mucus (glycosaminoglycans), and promote bacterial adherence and virulence. E. coli was grown in human urine and a bacterial-free supernatant was collected. Rabbit bladders were then exposed to either this supernatant or to the same human urine that was not infected with E. coli. Results show a significantly higher bacterial persistence (bacterial count) in bladders pretreated with urine containing the E. coli supernatants compared to controls pretreated with uninfected urine (p = 0.03). The molecular weight of the putative soluble virulence factor is less than 3.5 kD. (p = 0.056) based on dialysis studies and binds to heparin agarose affinity chromatography matrix, suggesting that it is cationic and capable of adhering to the highly anionic bladder mucus (glycosaminoglycans).

Elevated urinary norepinephrine in interstitial cystitis.

OBJECTIVES: To measure urinary catecholamines and determine the extent to which they may be elevated in urine from patients with interstitial cystitis (IC). METHODS: Random urine samples from patients with IC (n = 111) and urine from normal volunteers (n = 92) were acidified on collection (voided and catheterized specimens) and assayed for catecholamine (norepinephrine or normetanephrine) by enzyme-linked immunosorbent assay. Creatinine levels in these urine samples were also measured. RESULTS: Analysis of the data indicated that patients with IC had a higher urinary level of the neurotransmitter norepinephrine compared with the measured levels in the urine of normal volunteers (89.1 +/- 58.3 versus 54.9 +/- 37.1 microg/g creatinine, P <0.05). The metabolite normetanephrine was similar in the urine samples from these two groups. Urine from patients with bladder outlet obstruction (n = 11) did not have elevated amounts of urinary norepinephrine. The norepinephrine levels were not statistically different in the urine samples from patients with symptomatic and asymptomatic IC. The elevated urinary levels in patients with IC did not decrease after treatment with sodium pentosanpolysulfate (Elmiron), heparinoids, dimethyl sulfoxide, or combinations of these during 1 to 15 months of treatment. CONCLUSIONS: Norepinephrine was found to be elevated in the urine from patients with IC compared with urine from normal controls. This would be consistent with increased sympathetic (adrenergic) activity from the bladders of patients with IC or possibly from increased adrenal activity, since stress is associated with symptom increase in some patients with IC. Norepinephrine levels did not decrease with treatment nor did they differ between symptomatic and asymptomatic patients at the time of urine collection.

A new method for cytodestruction of bladder epithelium using protamine sulfate and urea.

Bladder epithelium relies primarily on the presence of a surface glycosaminoglycan (GAG) layer and the structural integrity of cell-cell contact to maintain impermeability to toxic urinary wastes. Previous clinical studies evaluating bladder permeability characteristics in interstitial cystitis patients had indicated that epithelial desquamation occurs after treatment with protamine sulfate (PS) followed by hypertonic urea. The following study was performed using rabbits to further investigate this finding. The urinary bladder was evaluated for optimal treatment conditions for epithelial removal. Protamine sulfate (1 to 10 mg./ml.) and urea (100 to 200 gm./ml.) were instilled into the bladder at volumes ranging from 5 to 60 ml. to that required for near maximum distention. After incubation at room temperature for 15 minutes, the bladders were fixed and evaluated histologically for epithelial removal. The maximum epithelial removal occurred when the bladders were distended, and when PS concentration was 5 to 10 mg./ml. and urea at 200 gm./l. There was greater epithelium removal after repeated treatments. Epithelial cells that were removed were not viable based on Trypan blue staining. There was no significant increase of C14 labeled urea in the plasma after 15 minutes. Rabbits that were followed for 6 weeks after treatment did not show any histological evidence of increased collagen deposition and/or fibrosis. This procedure may have important clinical value since it may remove sufficient bladder epithelium in patients with transitional cell carcinoma to have therapeutic benefit. This offers a realistic option for selective, nontoxic destruction of bladder epithelium.

Abnormal sensitivity to intravesical potassium in interstitial cystitis and radiation cystitis.

The urgency-frequency syndrome (UFS) (non-bacterial cystitis, interstitial cystitis) may well represent a heterogenous group with several etiologies. This study was based on the hypothesis that one subset of UFS patients has a leaky (to solutes) epithelium and cations such as potassium could thereby diffuse subepithelially and provoke symptoms. It was also hypothesized that normal impermeable transitional epithelium would not allow cations to diffuse across the cells during the K+ provocation test and no symptoms would be experienced. If the epithelium was permeable ("leaky"), diffusion would occur and provoke symptoms. Water or 0.4 M KCl was placed intravesically into normal volunteers and interstitial cystitis (IC) patients. Water did not provoke symptoms in either group but KCl provoked 4.5% of normals and 70% of IC patients. Differences were significant (P < 0.0001). This test provides a valuable diagnostic tool for UFS and a valuable research tool to separate epithelial permeability problems from other subsets of patients. A third group, consisting of 11 IC patients in remission on heparinoid therapy, was also tested and only 18% were provoked by KCl. Four patients with radiation cystitis were also examined and all four (100%) were provoked by the potassium.