Induction of CD4+ regulatory T cells by 12-O-tetradecanoylphorbol 13-acetate in mice. Part 1: The strain difference in this induction.

12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of signal transduction, induced antigen-nonspecific regulatory T (Tr) cells for delayed-type hypersensitivity (DTH) in the spleen. A marked strain difference in the induction of Tr cells was observed when the study was performed by using the several strains of mice at 6-8 weeks old. TPA painting induced CD4+Tr cells in C3H/He (H-2k), C3H/HeN (H-2k), DBA/2 (H-2d) and AKR/N (H-2k) mice, but not in C57BL/6 (H-2b), C57BL/10 (H-2b), BALB/c (H-2d) and A/J (H-2a). Regulatory cells were also induced by incubating spleen cells from unprimed mice with TPA in vitro and were seemed to act by the production of soluble factor(s). A downregulatory activity of the soluble factor(s) was abrogated by SXC-1 (anti-IL-10 monoclonal antibody), but not by SXC-2 (anti-IL-10 monoclonal antibody) and anti-IL-4. For purification of the factor(s), we established the T cell hybridoma 4C5-1 by the fusion of spleen cells from TPA-treated C3H/He mice with AKR thymoma Bw5147 cells. The 4C5-1 cells secrete the factor(s) which can inhibit DTH response. The inhibitory activity of the factor(s) could be neutralized by SXC-1, but not by SXC-2, anti-IL-4, anti-IL-6 and anti-TGF-beta antibodies. The factor(s) could not affect the proliferation and IFN-gamma production of alpha s1-casein-specific 3D20 Th1 cells. The factor(s) termed DIF (DTH Inhibitory Factor) may be a novel cytokine, since they have reduced the footpad swelling response by local injection, and have no immune crossreactivity with the DTH regulatory cytokines and no inhibitory activity for in vitro Th1 response.

Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photoirradiation.

For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.

Automatic system for the assay of guanidino compounds to assess uremic status.

An automated system for HPLC-fluorometry of serum guanidino compounds was constructed. This system accomplished simultaneous removal of protein and uremic fluorescences, abundant in the sera of uremic patients, which interfere with the fluorometric assay. This system was applied to the detailed elucidation of the behavior of guanidinosuccinic acid and methylguanidine during and after hemodialysis therapy (HD). The uremic patients who are capable of excreting urine even under hemodialysis therapy showed low serum guanidinosuccinic acid and methylguanidine levels. The prolongation of the interval between HD for one of the patients capable of excreting urine was examined. The levels of guanidinosuccinic acid and methylguanidine did not significantly increase and no hazardous effect was observed by 2 d of prolongation.

Sensitive determination of melatonin by precolumn derivatization and reversed-phase high-performance liquid chromatography.

A sensitive determination method for melatonin was developed. Melatonin was derivatized under alkaline conditions in the presence of hydrogen peroxide. The resultant fluorophore was excited at 247 nm and the emission wavelength was 384 nm. The Stokes shift was 137 nm, which was longer than that of melatonin itself (lambda ex 280 nm, lambda em 330 nm). The melatonin derivative was separated by reversed-phase HPLC in about 15 min and the calibration curve was linear from 500 amol to 5 pmol (r > 0.999) with the detection limit of 500 amol (S/N = 5). The sensitivity of this method was about ten times higher than that of previous methods. Both the day-to-day precision and within-day precision were about 5%, and the derivative of melatonin in the aqueous solution was stable for more than 10 days. This method was successfully applied to the determination of melatonin in rat pineal gland.

Determination of N1-methylnicotinamide by HPLC postcolumn photoirradiation.

A method for the determination of N1-Methylnicotinamide (NMNA) was developed by the fluorescence formed by UV irradiation after separation by HPLC with a mobile phase containing hydrogen peroxide and 18-crown-6. This method was applied to human urine and serum.

Automatic system for the assay of guanidino compounds to assess uremic status and effect of hemodialysis.

An automated HPLC system coupled with fluorometry was established for the sensitive, rapid, and accurate assay of serum guanidines. Naturally fluorescent materials characteristic of the sera of uremic patients (uremic fluorescences) which interfere with the assay were removed simultaneously with deproteinization. Application of this method revealed that the uremic patients who are capable of excreting urine under hemodialysis therapy show low serum guanidinosuccinic acid levels. The interval between hemodialysis sessions in one of these patients was prolonged while monitoring guanidinosuccinic acid level using the present method without any hazardous effect.

Determination of disodium cromoglycate in human urine by high-performance liquid chromatography with post-column photoirradiation-fluorescence detection.

For the determination of disodium cromoglycate in urine, a fluorimetric method using HPLC post-column photoirradiation has been developed. The mobile phase consisted of a 35 mmol l-1 phosphate buffer (pH 8)-methanol (7 + 3, %v/v) containing 75 mmol l-1 hydrogen peroxide and 20 mmol l-1 18-crown-6. The 18-crown-6 was used for separation adjustment of the disodium cromoglycate in the urine sample. Photoirradiation was carried out in tubing wound around a germicidal light in a reactor equipped with an air-cooling fan. The fluorescence was monitored with excitation at 325 nm and emission at 448 nm. The calibration graph for disodium cromoglycate was linear over the range 38-2340 ng ml-1 using an injection volume of 100 microliters. The pretreatment of the urine samples consisted of diluting and filtering steps. The mean recovery of disodium cromoglycate from urine was 99.1 +/- 2.4% (n = 6).

Fluorimetric determination of nicorandil in human plasma by a high-performance liquid chromatographic-postcolumn ultraviolet irradiation [corrected] system equipped with on-line back-pressure tubing.

For the determination of nicorandil in plasma, a fluorometric technique using HPLC-postcolumn UV radiation [corrected] has been developed. The chromatographic system consisted of a single pump, photoreactor and on-line back-pressure tubing. The system was suitable for the separation of nicorandil under the present reaction conditions. The calibration graph was linear over the range 6.5-1170 ng ml-1 using an injected volume of 100 microliters. The pretreatment of the plasma samples consisted only of deproteinizing steps by adding perchloric acid. The mean recovery from plasma was 90.2%.

Cellular induction mechanism of CD8+ suppressor T cells by DMBA and TPA: formation of CD4+ suppressor-inducer T cells.

We investigated the cellular induction mechanism of antigen-nonspecific CD8+ T suppressor cells which suppressed delayed-type hypersensitivity to sheep red blood cells, by treating BALB/c mice with 7,12-dimethylbenz[a]anthracene (DMBA), a tumor initiator, and 12-O-tetradecanoylphorbol 13-acetate (TPA), a tumor promoter. Macrophages were activated by painting with 400 nmol of DMBA on mice. And the macrophages plus TPA induced CD4+ T suppressor inducer cells in the mice spleens. These cells were efficient at inducing CD8+ T suppressor-effector cells. When 8 nmol of TPA was painted daily on mice for 3 days following the treatment with 400 nmol of DMBA, or when spleen cells from mice pretreated with 400 nmol of DMBA were cultured in 32 nmol/5 ml of TPA for 3 days, inducer cells were formed in the spleen. Both T suppressor-inducer cells and macrophages from mice treated with DMBA were shown to act with the formation of soluble factors, which may be different from IL-10.

Synthesis and antiulcer activity of N-substituted N'-[3-[3-(piperidinomethyl)phenoxy]propyl]ureas: histamine H2-receptor antagonists with a potent mucosal protective activity.

As an aim toward developing new antiulcer agents, new N-substituted N'-[3-[3-(piperidinomethyl)phenoxy]propyl]ureas were synthesized and evaluated for histamine H2-receptor antagonistic, gastric antisecretory, and gastric mucosal protective activities. A QSAR study showed that the most favorable N-substituents were electron-donating straight-chain alkyl groups of short length such as ethyl group from the viewpoint of dual action, i.e., gastric antisecretory and mucosal protective actions. Among the ureas studied, compounds 4, 5, and 8-10 were selected as candidates for further study.

Induction of CD8+ suppressor T cells by treatment with DMBA and TPA in vitro.

Antigen-nonspecific CD8+ T suppressor cells, which suppressed delayed-type hypersensitivity (DTH) against sheep red blood cells in BALB/c mice, were induced by incubating spleen cells from mice treated with 7,12-dimethylbenz[a]anthracene (DMBA), a tumor initiator, with 12-O-tetradecanoylphorbol 13-acetate (TPA), a tumor promoter. The optimal condition was incubation in 3.2 x 10(-8) mol/5 ml of TPA for 4 days. It was shown that induction of the suppressor cells required macrophages from mice treated with DMBA. These data were consistent with the results of previous work, in which CD8+ suppressor cells were induced by painting BALB/c mice with TPA following DMBA treatment. DTH was suppressed in the culture supernatants of spleen cells from mice treated with DMBA and TPA; the suppression was genetically unrestricted. The suppressor factor was resistant to trypsin and sensitive to heating at 56 degrees C for 30 min and had affinity for the macrophages.

Fluorometric determination of urinary kynurenic acid by flow injection analysis equipped with a "bypass line".

A flow injection analysis involving a photochemical reaction and fluorometric detection has been developed for the determination of urinary kynurenic acid. Kynurenic acid was found to fluoresce on irradiation with ultraviolet light at pH 7.2 in the presence of hydrogen peroxide. This method was applied to flow injection analysis using a new procedure involving a "bypass line" for the simultaneous determination of urinary kynurenic acid and background fluorescence. The calibration graph showed linearity over the range of 0.20 to 120 pmol. For pretreatment of urinary kynurenic acid, a PRE-SEP C18 cartridge was used. The mean recovery of kynurenic acid from urine was 94.5%. The content of urinary kynurenic acid was 13.0 +/- 2.68 mumol/day. There was good correlation (r = 0.9729) between values determined by flow injection analysis and high-performance liquid chromatography.

Fluorimetric determination of indomethacin in human serum by high-performance liquid chromatography coupled with post-column photochemical reaction with hydrogen peroxide.

A high-performance liquid chromatographic method involving post-column photochemical reaction and fluorimetric detection has been developed for the determination of indomethacin in serum. The bioanalytical set-up consisted of single-pump system for post-column photochemical reaction. The mobile phase consisted of 0.07 M phosphate buffer (pH 6.6)-acetonitrile (65:35, v/v) containing 180 mM hydrogen peroxide. The column effluent was irradiated with UV light to give fluorescence. The fluorimetric detection was monitored with excitation at 358 nm and emission at 462 nm. The calibration curve was linear over the range 0.05-30 micrograms/ml by injecting a volume of 10 microliters of indomethacin solution. The detection limit (signal-to-noise ratio = 5) for indomethacin was 10 ng/ml using a 100-microliters aliquot of deproteinized serum. The mean recovery from serum was 94.3%.

Fluorimetric determination of kynurenine in human serum by high-performance liquid chromatography coupled with post-column photochemical reaction with hydrogen peroxide.

A high-performance liquid chromatographic method involving post-column photochemical reaction and fluorimetric detection has been developed for the determination of kynurenine in serum. Kynurenine was separated on a column of Capcell Pak C18 (resistant to pH 10). The mobile phase consisted of 0.05 M Na2B4O7-0.1 M KH2PO4 buffer (pH 8.5)-ethanol (97:3, v/v) containing 60 mM hydrogen peroxide. The post-column reagent, containing 60% (v/v) ethanol, was mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The recovery of kynurenine was 95.9 +/- 5.0% (n = 6). The method allows the determination of as little as 2 pmol of kynurenine.