Accumulation of cell cycle regulatory proteins, p21 and p27, induced after hyperthermia in human glioma cells.

It was investigated whether there was a relationship between p53 p21 and p27 induction pathways in the cellular response of glioma cells to hyperthermia. Two glioma cell lines were employed. A-172 cells had the wild-type of p53, and U251 cells had the mutant-type of p53. An adenovirus harbouring wild-type p53 was also used for the overexpression. The protein induction by hyperthermia was monitored by Western blot analysis. In U251 cells, the expression of wild-type p53 and hyperthermia had an additional cytotoxic effect, but did not affect A-172 cells. Significant p21 accumulation by hyperthermia was recognized in A-172 cells, and was also recognized in p53-transduced U251 cells. On the other hand, the accumulation of p27 by hyperthermia was not seen in A-172 or U251 cells, and the exogenous expression of p53 did not affect the accumulation of p27 by hyperthermia in U251 cells. These findings suggest that the p53-p21 pathway is involved in the signal transduction after hyperthermia, rather than the p27 pathway.

Anti-tumor promoting activity of bufadienolides from Kalanchoe pinnata and K. daigremontiana x tubiflora.

Five bufadienolides (1-5) isolated from the leaves of Kalanchoe pinnata and K. daigremontiana x tubiflora (Crassulaceae) were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All bufadienolides showed inhibitory activity, and bryophyllin A (1) exhibited the most marked inhibition (IC50 = 0.4 microM) among the tested compounds. Bryophyllin C (2), a reduction analogue of 1, and bersaldegenin-3-acetate (3) lacking the orthoacetate moiety were less active. These results strongly suggest that bufadienolides are potential cancer chemopreventive agents.

Effect of Zena F-III, a liquid nutritive and tonic drug, on the neurochemical changes elicited by physical fatigue in mice.

The effects of a liquid nutritive and tonic drug (NTD) on the neurochemical changes elicited by physical fatigue in mice were investigated in terms of the calcium-dependent dopamine synthesizing function of the brain. In this study, Zena F-III (Taisho Pharmaceutical Co., Ltd., Japan), one of the most popular NTDs in Japan, containing 15 crude drug extracts together with taurine, caffeine, and vitamins, and formulated based on the precepts of traditional Chinese medicine, was used. Male mice were forced to walk for 0-6 h at a speed of 3 m/min using a programmed motor-driven wheel cage. The serum and brain calcium levels in the mice were significantly increased following forced walking. The increase in brain calcium level began later and was more gradual than that in the serum calcium level, and reached its maximum value following forced walking for 3 h. The neostriatal dopamine level was also significantly increased, and locomotor activity significantly decreased following forced walking for 3 h. Prior oral administration of F-III (10 ml/kg) attenuated the increases in the serum and brain calcium levels, the increase in the brain dopamine levels, and the decrease in locomotor activity induced by forced walking. Taking into consideration these findings with our previous reports, it is suggested that physical fatigue leads to an increase in dopamine synthesis in the brain through a calcium/calmodulin-dependent system, thereby inducing behavioral changes, and that F-III inhibits this pathway and may alleviate overwork-induced physical fatigue.

New insecticidal bufadienolide, bryophyllin C, from Kalanchoe pinnata.

Two insecticidal bufadienolides (1 and 2) were isolated from a methanol extract of the leaves of Kalanchoe pinnata by bioassay-guided fractionation. Compound 1 was identified as known bryophyllin A (bryotoxin C). The structure of new bufadienolide 2, named bryophyllin C, was determined by spectroscopic methods and the chemical transformation of 1. Compounds 1 and 2 showed strong insecticidal activity against third instar larvae of the silkworm (Bombyx mori), their LD50 values being evaluated as 3 and 5 microg/g of diet, respectively.

Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebal cortex plasma membranes.

We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 +/- 1,469.2 microM-min in sham-treated (n = 8) to 569.0 +/- 707.8 microM-min in CSD-treated (n = 13) rats (p <0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down-regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post-CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down-regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down-regulation of these transporters may contribute to ischemic tolerance induced by CSD.

Identification and characterization of a group 2 conifer pollen allergen from Chamaecyparis obtusa, a homologue of Cry j 2 from Cryptomeria japonica.

BACKGROUND: Not only Cryptomeria japonica (Japanese cedar) pollen but also that of Chamaecyparis obtusa (Japanese cypress) induces the allergic symptoms of Japanese cedar pollinosis. However, allergens from C. obtusa pollen have not been as well characterized as those from C. japonica pollen. OBJECTIVE: We sought to identify and characterize a homologue of the second major allergen of C. japonica pollen, Cry j 2, from the pollen of C. obtusa. METHODS: An allergen homologous to Cry j 2 was identified in C. obtusa pollen extract by immunoblot analysis, probed with anti-Cry j 2 monoclonal antibodies and purified by a series of column chromatographic steps. RESULTS: The allergen isolated from the extract showed a slightly diffuse band of 45 kDa and closely spaced double-bands of 42 and 45 kDa on SDS-PAGE, under reducing and non-reducing conditions, respectively; the bands were approximately 5-7 kDa larger than those of Cry j 2. In 24 of 30 residues, the N-terminal amino acid sequence of the allergen was identical with corresponding sequence in Cry j 2. Most patients with pollinosis who were IgE antibody-positive to Cry j 2 were shown to be IgE antibody-positive to this allergen, and the IgE antibody levels to both allergens were highly correlated. CONCLUSION: The results indicate that the allergen isolated from C. obtusa pollen in this study is a homologue of Cry j 2. The allergen was designated as Cha o 2 according to the WHO/IUIS Allergen Nomenclature Subcommittee recommendation.

[Change in skin reactivity to common allergens in allergic patients over a 30-year period. Association with aeroallergen load].

Five-year changes in the percentage of positive intradermal test to allergens in patients with bronchial asthma (BA) and allergic rhinitis (AR) over a period of 30 years from 1969 to 1998 were analyzed, with consideration of their association with the yearly changes in the levels of airborne pollens and molds over 30 years. Allergens used throughout the period were house dust, pollens from various plants (Japanese cedar, pine, oak, Japanese alder, Zelkova, orchard grass, ragweed, mugwort and Japanese hop) and molds (Alternaria, Cladosporium, Aspergillus, Penicillium and Candida). Overall percentages to house dust were higher in BA patients than in AR patients, but those to pollens were distinctly higher in the latter. Those to molds were higher in BA patients, except that those to Alternaria were about the same in both BA and AR patients. Therefore, the clinical expression of allergy may differ according to the type of allergen to which subjects are sensitized. Over the course of 30 years, the increase in the percentage of positive intradermal test to all of the allergens used was noted in both BA and AR patients, although this did not always reflect the aeroallergen load. This includes the two extremes to pollens from Japanese cedar and pine, either of which is the most common source of allergen in Japan. Significant increases in the percentages to Japanese cedar pollen (from 12.5% to 54.4% in BA patients, and from 35.1% to 81.5% in AR patients) appeared to be roughly correlated to marked increases in pollen count. In contrast, that to pine pollen was kept low at about 2%, because of their weak allergenicity, but abruptly increased to 5-6% in the last decade, despite the almost invariable pollen count over the last 30 years. Therefore, the increased sensitivity to aeroallergens may also be associated with other factors besides their load in the air.

Effect of histamine H2-receptor antagonist on the phosphorus-binding abilities of calcium carbonate and calcium lactate in hemodialysis patients.

The effect of histamine H2-receptor antagonist (famotidine) on the phosphorus-binding abilities of calcium carbonate and calcium lactate were examined in 13 chronic hemodialysis patients. In seven patients receiving calcium carbonate, famotidine (20 mg/d) was given because of gastroduodenal disorders, and calcium carbonate was replaced with calcium lactate as a phosphorus binder after 4 wk of treatment with famotidine. With the 4-wk administration of famotidine accompanied by calcium carbonate, the serum phosphorus level increased from 6.3+/-0.9 to 7.1+/-0.5 mg/dl (P<0.05). However, with the substitution of calcium lactate, the serum phosphorus level decreased significantly when compared to that before substitution (6.3+/-0.2 and 6.0+/-0.9 mg/dl after 4 and 8 wk of substitution, respectively), despite continued administration of famotidine. Serum calcium, creatinine, alkaline phosphatase, high sensitive parathyroid hormone, blood urea nitrogen, arterial blood pH, and bicarbonate were not significantly altered during the trial period. In six control patients treated with calcium carbonate alone, there were no statistical changes in serum calcium and phosphorus levels after substitution of calcium lactate for calcium carbonate. These results suggest that famotidine significantly affects the phosphorus-binding ability of calcium carbonate, but not that of calcium lactate. A careful observation of changes in the serum phosphorus level should be required in hemodialysis patients receiving calcium carbonate and histamine H2-receptor antagonists. Calcium lactate may be useful as a phosphorus binder in such hemodialysis patients.

Enhanced AP-1 binding in brain induced by D1 and D2 agonists in methamphetamine-sensitized rats.

The activator protein-1 (AP-1) binding activities induced by a separate challenge with SKF38393 and quinpirole after 1 weeks' abstinence from chronic methamphetamine (4 mg/kg/day, 14 days) were increased significantly in the striatum, nucleus accumbens and cingulate cortex compared with the saline-treated controls. Quinpirole-, but not SKF38393-induced AP-1 binding activities were still significantly higher after a 4-week abstinence period in the chronic methamphetamine group than in the chronic saline control group. Downward sniffing, which occurred following a quinpirole-challenge, was significantly intensified after both a 1 and 4 weeks' abstinence from chronic methamphetamine. These results indicate that chronic administration of methamphetamine induces alterations of the interactions of dopamine D1 and D2 receptors which are reflected as enhanced AP-1 binding activities.

Regulation of blood pressure with calcium-dependent dopamine synthesizing system in the brain and its related phenomena.

The effects of calcium on blood pressure regulation remain controversial. Although the mechanism by which calcium increases blood pressure when it is given intravenously and acutely has been elucidated, that by which calcium reduces blood pressure when it is supplemented chronically and slightly through daily diet is unclear. From a number of animal experiments concerning the effects of calcium on blood pressure, we believe that calcium ions have two separate roles in the regulation of blood pressure through both central and peripheral systems: (1) calcium ions reduce blood pressure through a central, calcium/calmodulin-dependent dopamine-synthesizing system and (2) calcium ions increase blood pressure through an intracellular, calcium-dependent mechanism in the peripheral vasculature. These concepts were applied to elucidate the mechanisms underlying hypertension in spontaneously hypertensive rats (SHR) and changes in blood pressure in other experimental animals, and the following conclusions were reached. The decrease of the serum calcium level in spontaneously hypertensive rats (SHR) causes a decrease in calcium/calmodulin-dependent dopamine synthesis in the brain. The subsequent low level of brain dopamine induces hypertension. The increase in susceptibility to epileptic convulsions and the occurrence of hypertension in epileptic mice (El mice) may be linked through a lowering of calcium-dependent dopamine synthesis in the brain, and epilepsy and hypertension may be associated. Exercise leads to increases in calcium-dependent dopamine synthesis in the brain, and the increased dopamine levels induce physiological changes, including a decrease in blood pressure. Cadmium which is not distinguished from calcium by calmodulin, activates calmodulin-dependent functions in the brain, and increased dopamine levels may decrease blood pressure. In this report, our studies are considered in light of reports from many other laboratories.

[One stage operation for sternal turnover with preserved rectus muscle pedicles and aortic root replacement associated with Marfan's syndrome].

A 40-year-old man with Marfan's syndrome had annulo aortic ectasia with Sellers grade 4 aortic valve regurgitation and Wada grade 3 pectus excavatum. Simultaneous operation was successfully performed by aortic valve composite graft insertion and sternal turnover with the rectus muscle pedicles. Following a midline skin incision, the cost-sterno complex (plastron) was dissected together with the bilateral rectus muscle pedicles, and the sternum was divided transversely through the second intercostal space. The plastron with muscle pedicles was retracted away from the anterior chest toward the abdomen and was covered by the moistened sternal bag made of polyethylene to prevent dryness and contamination during the composite graft insertion. The aortic root was replaced with a composite graft consisting of a 25 mm SJM valve and a 26 mm Hemashield graft. A short interposed 10 mm Hemashield graft was inserted between the ostia of the left coronary artery and the composite graft. The right coronary artery was reimplanted in the aortic conduit using the button technique with a doughnut pledget. This one stage method offered excellent operative exposure and enabled us to prevent possible necrosis of the sternum, infection of the mediastinal sinus, and postoperative cardiac failure resulting from chest wall compression. In this procedure, active usage of the rapid autologous transfusion system effectively reduced the total amount of blood transfusion.

A novel method for chemo-enzymatic synthesis of elicitor-active chitosan oligomers and partially N-deacetylated chitin oligomers using N-acylated chitotrioses as substrates in a lysozyme-catalyzed transglycosylation reaction system.

N,N',N"-Tri(monochloro)acetylchitotriose prepared by N-monochloroacetylation of chitotriose trihydrochloride was successfully polymerized into higher-molecular-weight oligomers by a lysozyme-catalyzed transglycosylation reaction, and a following base-catalyzed N-demonochloroacetylation gave a chitosan oligomer mixture mainly composed of oligomers with dp > 6. Partially N-deacetylated chitin oligomers (DAC oligomers) with dp 4-12 were synthesized by the enzyme reaction using N,N',N"-tri(monochloro)acetylchitotriose and N,N',N"-triacetylchitotriose (chitin trimer) as initial substrates followed by N-demonochloroacetylation. The structures of synthetic oligomers were analyzed by 1H NMR spectroscopy, enzymatic hydrolysis and nitrous acid deamination-NaBH4 reduction treatment. The dp of synthetic oligomers was measured by MALDI TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) using per-N-acetylated derivatives. The synthetic chitosan and DAC oligomers were strong elicitors for phytoalexin induction in Pisum sativum and Phaseolus vulgaris. This chemo-enzymatic method utilizing N-acylated chitotrioses as substrates is a novel approach to the synthesis of high-molecular-weight chitosan oligomers and DAC oligomers of biological importance.

Hypertension in epileptic mice: a phenomenon related to reduction of Ca(2+)-dependent catecholamine synthesis in the brain.

The possible complication of hypertension and epilepsy was investigated through the response in epileptic El mice. The systolic blood pressure in El mice (male, 8 weeks of age) and that in normal ddY mice (the parent strain of El mice) were compared by a tail-cuff method, using a programmed sphygmomanometer. The systolic blood pressure in El mice (120.5 +/- 5.6 mm Hg) was 28% (P < 0.01) higher than that in ddY mice (93.9 +/- 5.3 mm Hg). The higher systolic blood pressure in El mice was lowered by the acute intracerebroventricular administration of CaCl2 (10 mumol/kg, 30 min before measurement) or dopamine (30 nmol/mouse, 15 min before measurement), and was also improved by the chronic oral supplementation with 1.2% calcium (Ca2+) solution. Combining these results with those in our previous reports, where it is stated that lowering of Ca(2+)-calmodulin-dependent catecholamine synthesis increases the susceptibility to epileptic convulsions, we suggest that the increase in susceptibility to epileptic convulsion and occurrence of hypertension in El mice may be linked and that the two diseases may be associated.

[Release of chemical mediators in the conjunctival lavage fluids after eye provocation with allergen or compound 48/80].

Allergen extract was used to perform conjunctival provocation test on patients with Japanese cedar pollen conjunctivitis during the off-season, and histamine, tryptase and immunoreactive-leukotriene C4 (i-LTC4), which mainly consisted of LTE4, were measured in the lavage fluids. The results obtained in this study were as following. 1) After instillation of the allergen extract (1:20, w/v, 0.01 ml), histamine reached a maximal level of 4.63 +/- 1.67 ng/ml (mean +/- SE, n = 11) at 5 min. Tryptase appeared more slowly and the peak levels were reached at 5 min (116.6 +/- 49.6 ng/ml) or 10 min (119.4 +/- 63.8 ng/ml). In another set of the experiments, histamine peaked at 5 min (3.84 +/- 1.08 ng/ml, n = 7), and i-LTC4 reached peaks at 5 min (320 +/- 44.4 pg/ml) or 10 min (367 +/- 64.8 pg/ml). The amounts of these mediators decreased to base-line levels by 30 min. 2) The amounts of histamine retrieved from the lavage fluid were significantly correlated with those of tryptase (r = 0.819, p < 0.01, n = 11) and the weight ratio of tryptase to histamine was 41.2 +/- 10.0. These observations suggest that histamine was released from mast cells. 3) A significant correlation between the levels of histamine and i-LTC4 recovered from the lavage fluids was also observed (r = 0.736, p < 0.05, n = 7). 4) Compound 48/80 was instilled in the right eye (7.5 mg/ml, 0.01 ml) and the left eye was given allergen solution.(ABSTRACT TRUNCATED AT 250 WORDS)

[Immunological and physicochemical properties of Cry j II, the second major allergen of Japanese cedar pollen (Cryptomeria japonica)].

Cry j II, the second major allergen of Japanese cedar (sugi, Cryptomeria japonica) pollen was examined for the allergenicity by intradermal test and RAST. Nineteen of the 25 allergic patients examined, showed positive reaction to the Cry j II. Contents of Cry j II in the extracts of the pollen collected in various regions from 1977 to 1991 showed yearly variation ranging from 2.9 to 14 mg/100 g pollen, whereas the amount of Cry j I in the extract was comparatively stable at about 35 mg/100 g pollen. Physicochemical treatments of Cry j I and Cry j II suggested that specific human IgE antibodies and some mAbs bind to conformational epitopes which are denatured and destroyed by certain treatments.

Purgative activity and principals of the fruits of Rosa multiflora and R. wichuraiana.

Pseudocarps or seeds of Rosa multiflora, crude drug "Eijitsu" have been used as purgative in Japanese traditional medicine. R. wichuraiana was generally thought to be able to substitute for the plant. The n-butanol fractions of both plant seeds were tested on purgative activities with mice, and the values of the 50% effective dose (ED50) were 5.6 g/kg as the seed weight for R. multiflora and 57 g/kg as the seed weight for R. wichuraiana. From pseudocarps of R. multiflora, a new purgative compound, multinoside A acetate, was isolated, and its ED50 value was tested to be 150 mg/kg (77-291 mg/kg, 95% confidence limit). The other isolated compounds were three known quercetin glycosides, quercetin 3-O-xyloside, isoquercitrin and hyperin. From pseudocarps of R. wichuraiana, three quercetin glycosides, isoquercitrin, hyperin and quercetin 3-O-beta-D-glucuronide were isolated similarly, but no purgative components of R. multiflora were detected.

Peripheral acting muscle relaxants alter the effects of baclofen on the electrocorticograms in the rat.

The effects of intraperitoneally administered baclofen on behavior and electrocorticograms (ECoG) were studied in freely moving and paralyzed rats. In the freely moving rats, 10 mg/kg of baclofen suppressed ECoGs with behavioral sedation, and 20 mg/kg caused whole body twitching accompanied with spike discharges in low voltage slow waves background. In the freely moving rats, baclofen acted as depressant in low dose and convulsant in high dose. In the paralyzed rat, 10 mg/kg of baclofen first suppressed ECoGs. Though fast waves components recovered with slow waves background in the ECoGs of rats paralyzed with gallamine (50 mg/kg) 3 hours after the baclofen injection, fast waves activities were not observed in the ECoG of rats paralyzed with succinylcholine (50 mg/kg), d-tubocurarine (3 mg/kg) or pancuronium (1 mg/kg). Under paralysis with d-tubocurarine, baclofen suppressed ECoG first, and then spike discharges started 90 min after the injection. These results suggest that the effects of baclofen on ECoG were different depending on the muscle relaxant used.

[An enzyme-linked immunosorbent assay (ELISA) for the quantitation of sugi pollen and Dermatophagoides mite allergens and its application for standardization of allergen extracts].

A simple enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of the major allergens of sugi pollen, Cry j I and of Dermatophagoides mites, Der I (Der p I/Der f I) and Der II (Der p II/Der f II) for use in the in vitro standardization of allergen extracts. Polystyrene microplates coated with a IgG fraction of rabbit antiserum were incubated first with allergen extracts and then with biotinylated antiserum IgG. The bound allergen-biotinylated antibody complex was detected with commercially available streptavidin-enzyme conjugate followed by the addition of colorimetric substrate. The assay was very sensitive (-0.2 ng/ml) and reproducible (CV% = 1.9-13.8%). The ELISA was compared with the radioimmunoassay previously described, and the results showed a very good correlation between the assays (r = 0.967-0.990). The allergen content in three sugi pollen and three house dust extracts measured by the ELISA also demonstrated a good agreement with the relative potency of these extracts as determined by the intradermal skin test. These results indicate that the ELISA could be useful in the standardization of allergen extracts.

[The effects of crude drugs using diuretic on horse kidney (Na+ + K+)-adenosine triphosphatase].

In the folk-medicine, several kinds of crude drugs are used as diuretics. Twenty three kinds of diuretic drugs were chosen, and examined for their effects on the horse kidney (Na+ + K+)-adenosine triphosphatase (ATPase), which is an intrinsic enzyme of the plasma membrane and responsible for the active transport of Na+ and K+ across the membrane. Twenty one out of twenty three kinds of ethanol extracts of diuretic drugs inhibited the kidney (Na+ + K+)-ATPase activity. The intensity of the inhibition of these drugs was compared by estimating the amounts of their ethanol extracts which inhibited the (Na+ + K+)-ATPase activity by 50% (I50, micrograms/ml). Among these drugs, Atractylodis Lanceae Rhizoma (I50 = 12.8) Atractylodis Rhizoma (I50 = 15.2), Plantaginis Semen (I50 = 16.0), Plantaginis Herba (I50 = 16.0) and Alismatis Rhizoma (I50 = 22.0), have strong inhibitory effects on the kidney (Na+ + K+)-ATPase activity. The ethanol extracts of the rhizomes of Atractylodes lancea De Candolle and Atractylodis japonica Kitamura were examined with varying concentrations of ATP and ouabain. The mode of inhibition of these two extracts on the (Na+ + K+)-ATPase activity appeared to be uncompetitive with respect to ATP as judged from Lineweaver-Burk plot. The ethanol extract of Atractylodes japonica Kitamura decreased the I50 for ouabain from 1.6 x 10(-7) to 7.0 x 10(-9) M, while that of Atractylodes lancea De Candolle did not change the I50 for ouabain.

[Combined treatment in patients with carcinoma in situ of the urinary bladder using intravesical pirarubicin, irradiation and hyperthermia].

The treatment with hyperthermia in combination with radiation and intravesical pirarubicin (THP-ADM) was preliminary investigated in 5 patients with urinary bladder carcinoma in situ. Following intravesical administration of 30 mg THP-ADM, external irradiation of 3.0 Gy was delivered to the urinary bladder. Immediately then, hyperthermia using Thermotron RF-8 was performed for 50 min (intravesical temperature: 42-43 degrees C for 35 min). After five courses of the treatment, complete response has been maintained for 6, 8, and 9 months in 3 patients. However, in a patient with complete response, urinary cytology became positive in the 6th month after the treatment. In the remaining patient treatment was interrupted after only 3 courses due to urinary irritation, urinary cytology didn't become negative. The side effects of the combined treatment were limited to the transient symptoms of bladder irritation in all patients and thermal burn in 2 patients. These preliminary results suggest that this combined treatment may represent an effective conservative therapy for patients with urinary bladder carcinoma in situ.