Fumarate hydratase enzyme activity in lymphoblastoid cells and fibroblasts of individuals in families with hereditary leiomyomatosis and renal cell cancer.

BACKGROUND: Hereditary leiomyomatosis and renal cell cancer (HLRCC) is the autosomal dominant heritable syndrome with predisposition to development of renal cell carcinoma and smooth muscle tumours of the skin and uterus. OBJECTIVE: To measure the fumarate hydratase (FH) enzyme activity in lymphoblastoid cell lines and fibroblast cell lines of individuals with HLRCC and other familial renal cancer syndromes. METHODS: FH enzyme activity was determined in the whole cell, cytosolic, and mitochondrial fractions in 50 lymphoblastoid and 16 fibroblast cell lines including cell lines from individuals with HLRCC with 16 different mutations. RESULTS: Lymphoblastoid cell lines (n = 20) and fibroblast cell lines (n = 11) from individuals with HLRCC had lower FH enzyme activity than cells from normal controls (p<0.05). The enzyme activity in lymphoblastoid cell lines from three individuals with mutations in R190 was not significantly different from individuals with other missense mutations. The cytosolic and mitochondrial FH activity of cell lines from individuals with HLRCC was reduced compared with those from control cell lines (p<0.05). There was no significant difference in enzyme activity between control cell lines (n = 4) and cell lines from affected individuals with other hereditary renal cancer syndromes (n = 22). CONCLUSIONS: FH enzyme activity testing provides a useful diagnostic method for confirmation of clinical diagnosis and screening of at-risk family members.

J3-crystallin of the jellyfish lens: similarity to saposins.

J3-crystallin, one of the three major eye-lens proteins of the cubomedusan jellyfish (Tripedalia cystophora), shows similarity to vertebrate saposins, which are multifunctional proteins that bridge lysosomal hydrolases to lipids and activate enzyme activity. Sequence alignment of deduced J3-crystallin indicates two saposin-like motifs arranged in tandem, each containing six cysteines characteristic of this protein family. The J3-crystallin cDNA encodes a putative precursor analogous to vertebrate prosaposins. The J3-crystallin gene has seven exons, with exons 2-4 encoding the protein. Exon 3 encodes a circularly permutated saposin motif, called a swaposin, found in plant aspartic proteases. J3-crystallin RNA was found in the cubomedusan lens, statocyst, in bands radiating from the pigmented region of the ocellus, in the tentacle tip by in situ hybridization, and in the embryo and larva by reverse transcription-PCR. Our data suggest a crystallin role for the multifunctional saposin protein family in the jellyfish lens. This finding extends the gene sharing evolutionary strategy for lens crystallins to the cnidarians and indicates that the putative primordial saposin/swaposin J3-crystallin reflects both the chaperone and enzyme connections of the vertebrate crystallins.

Demethylation, reactivation, and destabilization of human fragile X full-mutation alleles in mouse embryocarcinoma cells.

The major causes of fragile X syndrome are mutational expansion of the CGG repeat in the FMR1 gene, hypermethylation, and transcriptional silencing. Most fragile X embryos develop somatic mosaicism of disease-causing "full" expansions of different lengths. Homogeneity of the mosaic patterns among multiple tissues in the same individual indicates that these previously unstable expansions acquire mitotic stability early in fetal life. Since mitotic stability is found strictly associated with hypermethylation in adult tissues, current theory has fixed the time of instability to developmental stages when fully expanded CGG repeats exist in an unmethylated state. We used murine embryocarcinoma (EC) cells (PC13) as a model system of pluripotent embryonic cells. Hypermethylated and unmethylated full expansions on human fragile X chromosomes were transferred from murine A9 hybrids into EC cells, by means of microcell fusion. As demonstrated in the present study for the first time, even full expansion alleles that were fully methylated and stable in the donors' fibroblasts and in A9 became demethylated, reactivated, and destabilized in undifferentiated EC hybrids. When destabilized expansions were reintroduced from EC cells into A9, instability was reversed to stability. Our results strongly support the idea that fully expanded alleles are initially unstable and unmethylated in the human embryo and gain stability upon genetic or epigenetic change of the embryonic cells.

Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca(2+) receptor critical for dimerization. Implications for function of monomeric Ca(2+) receptor.

We analyzed the effect of substituting serine for each of the 19 cysteine residues within the amino-terminal extracellular domain of the human Ca(2+) receptor on cell surface expression and receptor dimerization. C129S, C131S, C437S, C449S, and C482S were similar to wild type receptor; the other 14 cysteine to serine mutants were retained intracellularly. Four of these, C60S, C101S, C358S and C395S, were unable to dimerize. A C129S/C131S double mutant failed to dimerize but was unique in that the monomeric form expressed at the cell surface. Substitution of a cysteine for serine 132 within the C129S/C131S mutant restored receptor dimerization. Mutation of residues Cys-129, Cys-131, and Ser-132, singly and in various combinations caused a left shift in Ca(2+) response compared with wild type receptor. These results identify cysteines 129 and 131 as critical in formation of intermolecular disulfide bond(s) responsible for receptor dimerization. In a "venus flytrap" model of the receptor extracellular domain, Cys-129 and Cys-131 are located within a region protruding from one lobe of the flytrap. We suggest that this region represents a dimer interface for the receptor and that mutation of residues within the interface causes important changes in Ca(2+) response of the receptor.

Analysis of cell cycle-related Ki-67 and p120 expression by flow cytometric BrdUrd-Hoechst/7AAD and immunolabeling technique.

Flow cytometric multiparameter analysis of two proliferation associated antigens, Ki-67 and p120, was combined with cell cycle kinetic analysis, achieved by continuous labeling with 5-Bromodeoxyuridine (BrdUrd), followed by staining with Hoechst 33258 and 7-Aminoactinomycin D (7AAD). Exponential and plateau phase monolayer cultures of the human bladder carcinoma cell line J82 were examined. Resting cells, characterized by their absent BrdUrd incorporation, showed no reactivity with the MIB1 antibody, which was used for the detection of the Ki-67 antigen. Proliferating cells revealed a cell cycle phase dependent Ki-67 staining intensity, which was partially related to the time period spent in G1 after mitosis. In contrast to the Ki-67 antigen expression, no decrease in p120 immunofluorescence staining intensity of non-cycling cells could be observed. We could demonstrate that a dissection of the history of cell replication, obtained by the BrdUrd/Hoechst technique combined with a simultaneous immunofluorescence staining reveals detailed information, on a single cell level, about time dependent expression of proliferation associated antigens in all cell cycle compartments.

Indocyanine green: intracellular uptake and phototherapeutic effects in vitro.

Indocyanine green (ICG; absorption peak in human plasma 805 nm) was investigated for ICG-mediated phototherapy in vitro. The cellular uptake of ICG (1 microM-50 microM) into HaCaT keratinocytes after an incubation period of 24 h increased up to an intracellular ICG concentration of 12.1 +/- 1.3 nmol per 10(6) cells. To examine dose dependent phototoxic effects in vitro, keratinocytes were incubated with 0 microM-50 microM ICG for 24 h and irradiated by a diode laser (805 nm) with different energy densities (0, 12, 24, 48 J cm-2). All applied ICG concentrations except for 5 microM yielded a cell killing effect in combination with irradiation depending significantly on ICG concentration and light dose. Cell viability for dark control and cells incubated with 50 microM ICG and irradiated with 48 J cm-2 was 0.82 +/- 0.15 and 0.07 +/- 0.02, respectively. Sodium azide (100 mM), a quencher of reactive oxygen species, inhibited significantly the cell killing using 50 microM ICG and 24 J cm-2. Taken together, photoactivation of ICG by irradiation with a diode laser was shown to induce effectively cell killing of HaCaT keratinocytes. Moreover, this effect was inhibited by sodium azide, thus irradiation of ICG might induce a photodynamic reaction.

Ophthalmic fluorometholone-gentamicin versus ophthalmic betamethasone-gentamicin following cataract surgery.

A prospective, randomised, investigator-masked, parallel-group study was performed to compare fluorometholone-gentamicin eye drops and ointment with betamethasone-gentamicin eyedrops and ointment in the control of ocular inflammation after cataract surgery. Seventy patients (35 in each treatment group) of both sexes undergoing cataract-lens implant surgery for visually disabling cataract were enrolled in the study. The demographic and baseline parameters on day I, the day after surgery, were similar in the two study groups. After treatment, on day 3 and day 6 post-operatively, the reduction in cells in the anterior chamber and conjunctival hyperaemia were similar in the two study groups. Both treatments were equally well-tolerated. Ophthalmic fluorometholone-gentamicin was as effective as ophthalmic betamethasone-gentamicin in the control of ocular inflammation after cataract surgery.

Influence of a haematoporphyrin derivative on the protoporphyrin IX synthesis and photodynamic effect after 5-aminolaevulinic acid sensitization in human colon carcinoma cells.

Haematoporphyrin derivatives (HPDs) are potent sensitizers in photodynamic therapy (PDT), associated with prolonged skin photosensitivity. 5-Aminolaevulinic acid (5-ALA), a natural precusor of haem, is converted intracellularly into the photosensitive agent protoporphyrin IX (PPIX), causing direct cytotoxicity after laser light irradiation but limited skin photosensitivity over 1-2 days and higher tumour selectivity. Unfortunately, the use of 5-ALA in PDT has been shown to cause only superficial tissue necrosis. Therefore, a combination of HPD and 5-ALA could be of great clinical value in the treatment of tumours if a synergistic effect of both sensitizers on tumour cell necrosis with less skin photosensitivity could be demonstrated. Human colon adenocarcinoma cells (HT-29) were cultured with either HPD or 5-ALA alone, simultaneously for 24 h with 5-ALA and HPD or in succession with 5-ALA (18 h) followed by HPD (6 h at different concentrations. Intracellular PPIX concentrations were determined by high-performance thin-layer chromatography. Furthermore, PDT was performed with an incoherent light source (lambda = 580-740 nm) using a light dose of 30 J cm(-2) and an output power of 40 mW cm(-2). The intracellular PPIX concentration correlated well with 5-ALA drug dose and incubation time and was highest after single 5-ALA sensitization. In the presence of HPD, either simultaneously or sequentially, PPIX decreased significantly. The PDT effect after simultaneous incubation with both sensitizers for 24 h was not superior to incubation with HPD alone. If 5-ALA incubation (18 h) was followed by HPD (6 h) cytotoxicity after PDT was higher than with either single drug. 5-ALA (80 microg ml(-1)) led to a decrease in tumour cell viability by 40%. A similar effect could be observed when 5-ALA and HPD were sequentially combined allowing for a reduction of the 5-ALA dose from 80 microg ml(-1) in the absence of HPD to 60 microg ml(-1) and 5 microg ml(-1) together with 0.5 microg ml(-1) and 2 microg ml(-1) HPD respectively. We speculate that the enhanced PDT effect after the combined administration of 5-ALA and HPD to cultures of colon carcinoma cells should be even more impressive in the tumour in vivo, since HPD primarily targets the tumour microvasculature and secondarily tumour cells.

Wavelength dependent photodynamic effects on chemically induced rat bladder tumors following intravesical instillation of 5-aminolevulinic acid.

PURPOSE: The aim of the study was to determine photodynamic effects in chemically induced rat bladder tumors after intravesical instillations of 5-aminolevulinic acid (5-ALA) and PDT-dependence on laser wavelength and light dose. MATERIAL AND METHODS: Following intravesical instillation of a 5-ALA solution (40 mg 5-ALA in isotonic saline and sodium-bicarbonate) rat bladder tumors have been treated with integral irradiation using laser light at wavelengths of lambda = 630 nm and lambda = 635 nm in a light dose range of 15 to 100 J/cm2. RESULTS: A significantly higher amount of necrosis including damage of normal urothelium was obtained in rat bladder tumors using laser light at lambda = 635 nm. CONCLUSIONS: For 5-ALA assisted photodynamic treatment of superficial bladder cancer in patients the irradiation wavelength of lambda = 635 nm is recommended.

Targeting of the tumor microcirculation by photodynamic therapy with a synthetic porphycene.

9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) is a chemically pure substance with fast pharmacokinetics and superior photodynamic properties in vitro as compared to Photofrin. In this study the pharmacokinetics, photodynamic efficacy and tissue localization of ATMPn were investigated in vivo. Amelanotic melanomas (A-Mel-3) were implanted in dorsal skin fold chambers fitted to Syrian Golden hamsters. Fluorescence kinetics of ATMPn (1.4 mumol kg-1 b.w.i.v.; n = 8) were monitored by intravital microscopy. Quantitative measurements of fluorescence intensity were carried out by digital image analysis. For tumor growth studies 1.4 mumol kg-1 was injected 24 h (n = 3), 3 h (n = 3), 1 min (n = 6) and 2.8 mumol kg-1 1 min (n = 6) before PDT (Laser (630 nm) or lamp (600-750 nm), 100 mW cm-2, 100 J cm-2). Tumor volume was measured for 28 d. Solid tumors (n = 3) were excised 1 min after injection of ATMPn (2.8 mumol kg-1) and cryostat sections (20 mm) were analyzed by confocal laser scanning microscopy (CLSM) for tissue localization of the dye. Maximal fluorescence (mean +/- S.E.) arose in the tumor (94 +/- 7%) and surrounding host tissue (67 +/- 5%) 30 s post injection followed by a rapid decrease. Hardly any fluorescence was detectable 12 h after administration. Only PDT 1 min after injection of ATMPn was effective yielding 3/6 complete remissions (2.8 mmol kg-1, laser) and 6/6 complete remissions (2.8 mumol kg-1, lamp), respectively. One minute after injection the dye is primarily localized in the vascular wall of normal and tumor vessels as shown by CLSM. PDT at a time, when the dye is localized primarily in the tumor microcirculation, exhibits the best tumor killing effects showing that vascular targeting is effective in treating solid malignant tumors. ATMPn in liposomes makes administration and light irradiation in one session possible due to its fast pharmacokinetics. Thus, using ATMPn as a photosensitizer may provide more flexibility to perform PDT after surgical exploration and debulking as adjuvant therapy.

Side effects of high-energy shockwaves in the human kidney: first experience with model comparing two shockwave sources.

The side effects of high-energy shockwaves (HESW) from two different sources on kidney parenchyma obtained from 10 patients treated by radical nephrectomy for renal cell carcinoma were examined. Immediately after nephrectomy, the kidneys were perfused with cold HTK solution and kept in hypothermia (8 degrees C) for a maximum of 4 hours. In five cases, the tumor-free parenchyma was treated at the upper or lower renal pole with 2000 shocks, energy output 21 kV, in an experimental electromagnetic shockwave system (Siemens Co., Erlangen). In the other five cases, the upper or lower poles were treated with 2000 shocks, energy output 24 kV, in an electrohydraulic spark gap system (MFL 5000; Dornier Medizintechnik, Germering). The resulting tissue defects were analyzed by histologic examinations. Changes after treatment with the electromagnetic system were found mainly in the tubules and midsized blood vessels in a well-defined focal area. Treatment with the electrohydraulic system was followed by tubular and glomerular lesions combined with vessel defects in a patchy pattern. The model is able to define the side effects of HESW in the human kidney and to test the side effects of different lithotripters.

Expression of proliferating cell nuclear antigen (PCNA) and Ki-67 in dysplasia in inflammatory bowel disease.

OBJECTIVE: Previous studies have revealed large variations in the interobserver agreement of dysplasia grading in inflammatory bowel disease. Therefore, we investigated the diagnostic value of two novel monoclonal antibodies (MIB 1 against Ki-67 and PC 10 against PCNA) in the detection of dysplasia. METHODS: A total of 62 biopsies were investigated and histologically classified as follows: 13 probably positive for dysplasia; 15 low-grade dysplasia; five high-grade dysplasia; and 15 inflammation without dysplasia and 14 normal controls. The percentage of positive Ki-67- or PCNA-stained nuclei (= labelling index) was determined in relation to the distribution throughout the mucosa. RESULTS: In all biopsies PCNA-labelling index exceeded that of Ki-67-labelling index. In the superficial half of the crypt PCNA- and Ki-67-labelling indices in the biopsies with 'indefinite for dysplasia, probably positive' or low-grade dysplasia exceeded that of normal tissue (P < 0.001). However, an unequivocal discrimination between biopsies with 'indefinite for dysplasia, probably positive' or low-grade dysplasia and inflammation was not possible. PCNA- and Ki-67-labelling indices were significantly higher in high-grade than in low-grade dysplasia (PCNA 81.4% vs. 44.3%, Ki-67 54.8% vs 30.9%, P < 0.001). Most interestingly, labelling indices of both markers were significantly (P < 0.0001) higher in biopsies with high-grade dysplasia than with active inflammation in the superficial half of the crypt. CONCLUSION: PCNA and Ki-67 are useful adjuncts in the diagnosis of high-grade dysplasia, because high-grade dysplasia can easily be distinguished from low-grade dysplasia or active inflammation if the distribution of the positive-stained cells within the mucosa is taken into account. Lower unspecific binding and lower influence on proliferation activity by inflammation prompts us to prefer Ki-67 (MIB 1).

Cell-type-specific response to shock waves of suspended or pelleted cells as analysed by flow cytometry or electrical cell volume determination.

Shock-wave-induced cell damage of suspended or pelleted bladder cancer cells was analysed with the flow cytometric propidium iodide (PI)/fluorescein diacetate assay, and electrical volume determination using the CASY 1 analyser system and growth curves. The CASY system revealed a smaller fraction of suspended RT4 cells with impaired membrane integrity than the flow cytometric assay. No differences were found for pelleted RT4 cells and suspended J82 cells. The discrepancies of the two viability assays indicated a different response of the cell membrane to shock waves which was dependent on the exposure system and the cell type. Growth curves indicated delayed cell death for suspended RT4 cells and exclusively immediate cell death for pelleted RT4 cells and suspended J82 cells. PI positive suspended RT4 cells were morphologically intact while pelleted RT4 cells and suspended J82 cells were mainly disrupted. From these data it can be concluded that intracellular or membrane alterations seem to be correlated with the occurrence of cavitational effects while cell disruption can likewise occur by the direct impact of the shock wave.

The combined effects of high-energy shock waves and ionising radiation on a human bladder cancer cell line.

The effects of high-energy shock waves (HESW) generated by an experimental Siemens lithotripter in combination with 137Cs gamma-rays were examined in vitro. Proliferation after treatment of immobilised pellets of either single cells or multicellular spheroids of the bladder cancer cell line RT4 was determined using colony-forming assays and cell cycle analysis. Surviving and cell cycle fractions were calculated for each shock wave and radiation application mode separately, and for sequential combination in different successions for the purpose of characterizing the interaction of both treatment modalities. Combination of HESW and ionising radiation turned out to act additively or slightly supra-additively on both biologic models.

Mechanisms of shockwave action in the human kidney.

The effects on the human kidney parenchyma of high-energy shockwaves (HESW) with different energy densities were examined. Kidneys of patients treated by radical nephrectomy for renal cell carcinoma were perfused with cold HTK solution immediately after nephrectomy and kept in hypothermia (8 degrees C) for a maximum of 4 hours. The tumor-free parenchyma was treated with 2000 shocks at energy outputs of 15 kV (16 MPa, 0.15 mJ/mm2), 17 kV (32 MPa, 0.25 mJ/mm2), 19 kV (50 MPa, 0.4 mJ/mm2), and 21 kV (65 MPa, 0.6 mJ/mm2) in an experimental electromagnetic shockwave system (Siemens Co., Erlanger, Germany). Resulting tissue effects were analyzed by histologic and immunohistochemical examinations and confocal laser scanning microscopy. Different sensitivities of cell components, blood vessels, and tubules were found. Laser scanning microscopy revealed nuclear alterations in the vicinity of the focus up to a distance of approximately 10 mm. Severe histologic changes were found in a smaller zone, while immunohistochemistry studies revealed negative collagen IV staining in an area of approximately 4 x 4 mm (all distances measured within the plane perpendicular to the acoustic axis). From these results, it can be concluded that HESW directly damage the tubules and the vascular system, which might explain the clinical changes after extracorporeal shockwave lithotripsy in human patients. The extent of these effects seems to be dependent on the applied energy.

Wavelength dependency of photodynamic effects after sensitization with 5-aminolevulinic acid in vitro and in vivo.

A promising new therapeutic modality for skin cancer, administration of the heme precursor 5-aminolevulinic acid followed by light irradiation, is known as photodynamic therapy. Photofrin, the only clinically approved sensitizer, has an absorption maximum at 630 nm, the wavelength used in most experimental and clinical trials with 5-aminolevulinic acid. We investigated photodynamic efficacy of irradiation with coherent light at wavelengths ranging from 622 to 649 nm in vitro and in vivo as well as the content and distribution of intracellular porphyrin after administration of 5-aminolevulinic acid. HaCaT immortalized human keratinocytes were sensitized with 30 micrograms/ml 5-aminolevulinic acid for 24 h in vitro. By cell viability determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, the best cell-killing effects were observed after irradiation at 635 nm. Using an amelanotic melanoma (A-Mel-3) grown subcutaneously in Syrian Golden hamsters, we confirmed these results in vivo: tumor growth was markedly delayed in animals treated with 100 mg/kg 5-aminolevulinic acid intravenously and irradiated with coherent light at 635 nm as compared to animals irradiated at 630 nm. This photodynamic effect is probably mediated by large amounts of the photosensitizing porphyrin, protoporphyrin IX, localized in cell membranes as visualized by confocal laser scan microscopy and as determined by high pressure liquid chromatography in vitro. The results suggest that irradiation at 635 nm with a coherent light source is more effective than irradiation at 630 nm for photodynamic therapy with 5-aminolevulinic acid.

Cellular fluorescence of the endogenous photosensitizer protoporphyrin IX following exposure to 5-aminolevulinic acid.

Supplying 5-aminolevulinic acid (ALA), a precursor in the biosynthetic pathway to heme from an external source leads to an accumulation of the endogenous fluorescent photosensitizer protoporphyrin IX (PPIX). Following instillation of ALA in the urinary bladder neoplastic tissue can be discerned by fluorescence cystoscopy or treated by illumination with light of an appropriate wavelength. In order to provide a biological rationale for the clinical findings, we have analyzed the capacity of three different cell lines to accumulate PPIX by flow cytometry. Three different urothelial cell lines, normal fibroblasts and endothelial cells were exposed to ALA under varying conditions. Urothelial cell lines J82 and RT4, derived from malignancies of the bladder displayed fluorescence intensities 9- and 16-fold, respectively, above the fluorescence level of the normal urothelial cell line HCV29. Human umbilical cord endothelial cells fluoresced moderately while the fibroblast cell line N1 exhibited a fluorescence level comparable to those of the cancer cells. Fluorescence increased with increasing cell density and was also dependent on the growth of cells as monolayers or multicellular spheroids. Increasing ALA concentrations led to saturation of fluorescence after 4 h of incubation at cell type-specific fluorescence levels obtained at different ALA concentrations. Continuous incubation in medium containing serum resulted in a linear rise of fluorescence during the first 4 h, which was followed by a saturation period (8-24 h) and a renewed rise. In the case of serum depletion, fluorescence intensities were significantly higher and increased linearly during the entire 48 h incubation period.(ABSTRACT TRUNCATED AT 250 WORDS)

Human follicular and papillary thyroid carcinoma cells interact differently with human venous endothelial cells.

Follicular thyroid carcinomas (FTC) characteristically spread via blood vessels, while papillary thyroid carcinomas (PTC) predominantly metastasize to lymph nodes. This different behavior of cancer cells originating from one organ was investigated by layering multicellular tumor spheroids (MCTS) consisting of various kinds of human thyroid cells onto confluent monolayers of human venous endothelial cells (HEC). The MCTS and HEC were cocultured in an incubation chamber fixed under a microscope, and the behavior of the cells was investigated. In this way significant differences between FTC, PTC, and follicular adenoma cells (FTA) were observed regarding their in vitro behavior upon interaction with HEC. FTC cells required 20 min for adhesion and another hour until they migrated out of a spheroid, whereas PTC- and FTA-MCTS were adhesive after 2 h or later, and their cells did not start migration until 5 h of incubation. Furthermore, one FTC-spheroid triggered about 100 endothelial cells to enter the replication cycle, while no spheroid consisting of either PTC or FTA cells induced more than 20 endothelial cells to start proliferation. During these processes, the cells of the MCTS and the endothelial cells contacted each other directly and remained viable. The results show that FTC cells interact faster and more intensively with human endothelial cells than PTC and FTA cells. Thus the study suggests that an enhanced capability of the FTC cells to interact with venous endothelial cells might favor the clinically observed hematogenous spreading of follicular thyroid carcinomas.

Flow cytometric analysis of cell suspensions exposed to shock waves in the presence of the radical sensitive dye hydroethidine.

The occurrence of intracellularly and extracellularly generated free radicals during shock wave exposure on an experimental Siemens lithotripter was tested with the radical sensitive dyes hydroethidine and dichlorofluorescin (DCFH). DCFH, a nonfluorescent compound, is oxidised to dichlorfluorescein (DCF) by hydrogen peroxide in the presence of peroxidase. DCF green fluorescence intensity was used for fluorescence spectrometric measurement of hydrogen peroxide generated during shock-wave treatment of cell-free dye solutions. The fluorescence intensity of ethidium, the oxidised form of hydroethidine, was used for the flow-cytometric measurement of intracellular oxidising reagents present in RT4 tumour cells during shock-wave exposure. Changes in membrane permeability, which influence the intracellular content of ethidium, were controlled by counterstaining the cells with propidium iodide, an indicator for membrane integrity. We observed no increase in intracellular ethidium fluorescence intensity after shock-wave treatment of single cell suspensions and therefore no indication for shock-wave-induced intracellular free radicals.

Fluorescence cystoscopy following intravesical instillation of 5-aminolevulinic acid: a new procedure with high sensitivity for detection of hardly visible urothelial neoplasias.

Methods have been sought for the in vivo marking of tiny papillary tumors of the bladder and flat urothelial lesions such as dysplasia or carcinoma in situ, which can easily be missed during conventional endoscopy under white light. A new procedure is reported for the fluorescence detection of urothelial dysplasia and early bladder cancer. The method is based on intravesical application of 5-aminolevulinic acid (ALA). ALA if applied exogenously induces accumulation of protoporphyrin IX (PPIX) in the urothelium of the bladder. PPIX is an intensively red fluorescing agent. The mean ratio of fluorescence intensity between urothelial cancer and normal epithelium was found to be 17:1. Fluorescence excitation was achieved by violet light from a krypton ion laser (lambda = 406.7 nm) or from a xenon arc lamp with a bandpass filter system (lambda = 375-440 nm). Both light sources proved to be of equal suitability for fluorescence excitation. Fluorescence microscopy revealed that the PPIX fluorescence is strictly limited to the urothelium. It could not be detected from the submucosa or muscle of the bladder. Bladder wall biopsies were taken from 90 patients with suspicion of bladder cancer under fluorescence view. The fluorescence detection proved to be of high sensitivity (98%). No serious side effects which would preclude further clinical testing, especially no cutaneous photoreaction, were observed. Tumor-associated fluorescence induced by topical ALA application offers new perspectives in the diagnosis and treatment of bladder cancer. In case of suspicious or positive urine cytologic findings, ALA fluorescence cystoscopy may be useful for detecting the precise site of the malignancy. The procedure might be helpful in complete resection or coagulation of urothelial neoplasms. Due to this, diminishing recurrence rates are expected. However, this hypothesis has to be studied in prospective clinical trials.