Haruhiko Yagi

Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease.

Summary:  Naturally arising CD25+CD4+ regulatory T (Treg) cells, most of which are produced by the normal thymus as a functionally mature T‐cell subpopulation, play key roles in the maintenance of immunologic self‐tolerance and negative control of a variety of physiological and pathological immune responses. Natural Tregs specifically express Foxp3, a transcription factor that plays a critical role in their development and function. Complete depletion of Foxp3‐expressing natural Tregs, whether they are CD25+ or CD25–, activates even weak or rare self‐reactive T‐cell clones, inducing severe and widespread autoimmune/inflammatory diseases. Natural Tregs are highly dependent on exogenously provided interleukin (IL)‐2 for their survival in the periphery. In addition to Foxp3 and IL‐2/IL‐2 receptor, deficiency or functional alteration of other molecules, expressed by T cells or non‐T cells, may affect the development/function of Tregs or self‐reactive T cells, or both, and consequently tip the peripheral balance between the two populations toward autoimmunity. Elucidation of the molecular and cellular basis of this Treg‐mediated active maintenance of self‐tolerance will facilitate both our understanding of the pathogenetic mechanism of autoimmune disease and the development of novel methods of autoimmune disease prevention and treatment via enhancing and re‐establishing Treg‐mediated dominant control over self‐reactive T cells.

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (−)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (−)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (−)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (−)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ∼30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ∼5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of Salmonella typhimurium and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of S. typhimurium and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of S. typhimurium . The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.

Metabolism of benzo[a]pyrene: Effect of substrate concentration and 3-methylcholanthrene pretreatment on hepatic metabolism by microsomes from rats and mice

Abstract The metabolism of [ 14 C]benzo[a]pyrene by liver microsomes from normal and 3-methylcholanthrene-treated mice and rats was quantitatively analyzed by high pressure liquid chromatography. The ratio of dihydrodiols of benzo[a]pyrene to total metabolites formed was greater with microsomes from Long-Evans rats when compared to the ratio produced by microsomes for DBA/2J or C57BL/6J mice. Treatment of rats or C57BL/6J mice with 3-methylcholanthrene resulted in marked increases in the metabolism of benzo[a]pyrene to phenols, dihydrodiols, and quinones; but this effect did not occur in DBA/2J mice. The profile of benzo[a]pyrene metabolites formed by rat and mouse liver microsomes was dependent on both substrate and protein concentration. The use of incubation conditions with limiting substrate concentrations resulted in massive secondary metabolism of benzo[a]pyrene metabolites and was accompanied by the production of substantial amounts of metabolites that were no longer extractable into the organic phase.

Differences in mutagenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides

Summary Substantial differences in the mutagenic activities of the optically pure (+)- and (−)-enantiomers of the diastereomeric 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrenes were observed in strains TA98 and TA100 of Salmonella typhimurium and Chinese hamster V79 cells. In strains TA98 and TA100 (−)-7β,8α-dihydroxy-9β, 10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was the most mutagenic compound, inducing from 1.3 to 9.5 times as many mutations as the three other optically active stereoisomers. In Chinese hamster cells (+)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was the most mutagenic compound inducing from 6 to 18 times as many variant colonies as the three other isomers. These results with known metabolites of the environmental carcinogen benzo[a]pyrene represent the first report of differences in mutagenic activity among optical enantiomers.

Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in self-tolerance and autoimmune disease.

Naturally arising CD25+CD4+ regulatory T cells, which express the transcription factor Foxp3, play key roles in the maintenance of immunologic self-tolerance and negative control of a variety of physiological and pathological immune responses. The majority of them are produced by the normal thymus as a functionally mature T cell subpopulation specialized for suppressive function. Their generation is in part genetically and developmentally controlled. Genetically determined or environmentally induced abnormality in CD25+CD4+ regulatory T cell development, maintenance, and function can be a cause of autoimmune disease in humans.

Clinical significance of the NKG2D ligands, MICA/B and ULBP2 in ovarian cancer: high expression of ULBP2 is an indicator of poor prognosis

ObjectiveTo investigate the clinical significance of the expression of the NKG2D ligands MICA/B and ULBP2 in ovarian cancer.MethodsEighty-two ovarian cancer patients and six patients without ovarian cancer from Department of Obstetrics and Gynecology of Kyoto University Hospital were enrolled in this study between 1993 and 2003. Expression of MICA/B, ULBP2, and CD57 in ovarian cancer tissue and normal ovary tissue was evaluated by immunohistochemical staining, and the relationship of these results to relevant clinical patient data was analyzed. Expression of MICs, ULBP2, and HLA-class I molecules in 33 ovarian cancer cell lines and two normal ovarian epithelial cell lines, as well as levels of soluble MICs and ULBP2 in the culture supernatants, were measured.ResultsExpression of MICA/B and ULBP2 was detected in 97.6 and 82.9% of ovarian cancer cells, respectively, whereas neither was expressed on normal ovarian epithelium. The expression of MICA/B in ovarian cancer was highly correlated with that of ULBP2. Strong expression of ULBP2 in ovarian cancer cells was correlated with less intraepithelial infiltration of T cells and bad prognoses for patients, suggesting that ULBP2 expression is a prognostic indicator in ovarian cancer. The expression of NKG2D ligands did not correlate with the levels of the soluble forms of the ligands.ConclusionsHigh expression of ULBP2 is an indicator of poor prognosis in ovarian cancer and may relate to T cell dysfunction in the tumor microenvironment.

Metabolism of benzo[A]pyrene III. An evaluation of the fluorescence assay

Summary Fluorescence spectra of the twelve isomeric phenols and phenolate anions of benzo[a]pyrene have been determined. The 3- and 9-isomers had the strongest fluorescence as phenols or phenolate anions. Many of the phenolate anions have insignificant fluorescence compared to the 3-hydroxybenzo[a]pyrene anion, whereas the phenols have more comparable fluorescence. Comparison of the fluorescence in acid (i.e., phenols) and alkali (i.e., phenolate anions) produced by the phenolic products obtained from benzo[a]pyrene after incubation with hepatic microsomes or with a reconstituted system established that a substantial percentage (35–50%) of the phenolic products formed are not detected by the standard fluorimetric assay in alkali.

High mutagenicity of metabolically activated chrysene 1,2 dihydrodiol: Evidence for bay region activation of chrysene

Abstract Chrysene and the 3 metabolically possible vicinal trans dihydrodiols of chrysene were tested for mutagenicity towards S. typhimurium strain TA100 in the presence of hepatic microsomes or a highly purified hepatic microsomal monooxygenase system. The products formed during the metabolic activation of chrysene 1,2-dihydrodiol were more than 20 times as mutagenic to the bacteria than the metabolites formed from chrysene, chrysene 3,4-dihydrodiol or chrysene 5,6-dihydrodiol. When the double bond in the 3,4-position of chrysene 1,2-dihydrodiol was saturated, the resulting tetrahydrodiol could not be metabolically activated. These results, which strongly suggest that chrysene 1,2-dihydrodiol is activated by metabolism to either or both of the diastereomeric chrysene 1,2-diol-3,4-epoxides, provide additional support for the bay region theory of polycyclic hydrocarbon carcinogenicity.

A structural gap in Dpo4 supports mutagenic bypass of a major benzo[a]pyrene dG adduct in DNA through template misalignment

Erroneous replication of lesions in DNA by DNA polymerases leads to elevated mutagenesis. To understand the molecular basis of DNA damage-induced mutagenesis, we have determined the x-ray structures of the Y-family polymerase, Dpo4, in complex with a DNA substrate containing a bulky DNA lesion and incoming nucleotides. The DNA lesion is derived from an environmentally widespread carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BP). The potent carcinogen BP is metabolized to diol epoxides that form covalent adducts with cellular DNA. In the present study, the major BP diol epoxide adduct in DNA, BP-N2-deoxyguanosine (BP–dG), was placed at a template–primer junction. Three ternary complexes reveal replication blockage, extension past a mismatched lesion, and a −1 frameshift mutation. In the productive structures, the bulky adduct is flipped/looped out of the DNA helix into a structural gap between the little finger and core domains. Sequestering of the hydrophobic BP adduct in this new substrate-binding site permits the DNA to exhibit normal geometry for primer extension. Extrusion of the lesion by template misalignment allows the base 5′ to the adduct to serve as the template, resulting in a −1 frameshift. Subsequent strand realignment produces a mismatched base opposite the lesion. These structural observations, in combination with replication and mutagenesis data, suggest a model in which the additional substrate-binding site stabilizes the extrahelical nucleotide for lesion bypass and generation of base substitutions and −1 frameshift mutations.