Study comparing the double kissing (DK) crush with classical crush for the treatment of coronary bifurcation lesions: the DKCRUSH-1 Bifurcation Study with drug-eluting stents.

BACKGROUND: Classical crush has a lower rate of final kissing balloon inflation (FKBI) immediately after percutaneous coronary intervention (PCI). The double kissing (DK) crush technique has the potential to increase the FKBI rate, and no prospective studies on the comparison of classical with DK crush techniques have been reported. MATERIALS AND METHODS: Three hundred and eleven patients with true bifurcation lesions were randomly divided into classical (n = 156) and DK crush (n = 155) groups. Clinical and angiographic details at follow-up at 8 months were indexed. The primary end point was major adverse cardiac events (MACE) including myocardial infarction, cardiac death and target lesion revascularization (TLR) at 8 months. RESULTS: FKBI was 76% in the classical crush group and 100% in the DK group (P < 0.001). The incidence of stent thrombosis was 3.2% in the classical crush group (5.1% in without- and 1.7% in with-FKBI) and 1.3% in the DK crush group. Cumulative 8 month MACE was 24.4% in the classical crush group and 11.4% in the DK crush group (P = 0.02). The TLR-free survival rate was 75.4% in the classical crush group and 89.5% in the DK crush group (P = 0.002). CONCLUSIONS: DK crush technique has the potential of increasing FKBI rate and reducing stent thrombosis, with a further reduction of TLR and cumulative MACE rate at 8 months.

Time-dependent morphologic characteristics in angiographic chronic total coronary occlusions.

Intravascular ultrasound analysis of 70 chronic total occlusions (CTOs), conducted either before intervention or following dilation of a 1.5-mm balloon, showed that older CTOs have more complex plaque composition including a larger calcific burden. This may explain the adverse revascularization profile of older CTOs.

Detection of active tuberculosis by an MPB-64 transdermal patch: a field study.

The mycobacterial antigen MPB-64 was formulated for delivery in a transdermal patch and used as a diagnostic skin test reagent to detect active tuberculosis (TB) in patients attending a clinic in Manila, The Philippines. The MPB-64 Transdermal Patch was applied to 62 patients, 49 with sputum-positive active disease and 13 who had completed TB chemotherapy, and to 28 non-TB but tuberculin-positive controls. The results were read at 72 h. The sensitivity of the Transdermal Patch was 87.8%, with an efficacy of 92.9% and a specificity of 100%. The 13 TB patients who had completed 6 months of TB chemotherapy showed different reactions to the MPB64 patch test: those who had completed chemotherapy < 4 months before testing were positive; 50% of patients who completed chemotherapy 5 months previously were positive; and those who had completed chemotherapy 7 and 8 months before were negative. All the non-TB controls with positive tuberculin tests were negative to the MPB-64 Transdermal Patch, even at the highest protein dose tested. This test may be a useful method to distinguish active TB patients from TB-infected but asymptomatic individuals. Moreover, the MPB64 Transdermal Patch may be useful to monitor successful chemotherapy.

[Increased incidence of coronary artery disease and cardiac death in elderly diabetic nephropathy patients undergoing chronic hemodialysis therapy].

OBJECTIVES: The incidence of coronary artery disease and cardiac death was investigated in elderly diabetic patients undergoing chronic hemodialysis therapy. METHODS: Three hundred thirty-five patients who began hemodialysis therapy since 1992 were followed up by echocardiography and treadmill exercise testing. Coronary angiography was also performed in patients with angina pectoris. Angina pectoris was defined as clinical symptoms > Canadian Cardiovascular Society classification II, and asynergy findings by echocardiography or ST depression > 0.1 mV during the treadmill exercise test. Coronary artery stenosis was defined as narrowing > or = 75%. Patients were divided into 4 groups: diabetic nephropathy (DN) > or = 65 years old (Group O/DN, n = 56), DN < 65 years old (Group Y/DN, n = 84), non-DN > or = 65 years old (Group O/non-DN, n = 76) and non-DN < 65 years old (Group Y/non-DN, n = 119). RESULTS: Between 1992 and 1998, there were 137 patients with angina pectoris (40.9%), 79 with coronary artery stenosis (23.6%) and 37 with cardiac death (11.0%). Cumulative incidences of angina pectoris, coronary artery stenosis and cardiac death were significantly higher in the following order of groups; O/DN > Y/DN > O/non-DN > Y/non-DN. Five-year cumulative incidences of angina pectoris, coronary artery stenosis and cardiac death in Groups O/DN vs Y/non-DN were 72.2% vs 38.6%, 53.7% vs 12.2% and 50.6% vs 3.5%, respectively. Relative risks of aging and diabetic nephropathy for angina pectoris, coronary artery stenosis and cardiac death were 3.8, 7.9 and 22.4, respectively (p < 0.0001). CONCLUSIONS: Aging and the presence of diabetes are strong risk factors for coronary artery disease and cardiac death in hemodialysis patients. Therefore, diagnosis and treatment of coronary artery disease should be achieved at the early stage of hemodialysis therapy.

Population-based mapping of pulmonary adenoma susceptibility 1 locus.

Pulmonary adenoma susceptibility 1 (Pas1), the major locus affecting inherited predisposition to lung tumor development in mice, maps near the Kras2 gene. We previously reported a significant association between a KRAS2/RsaI polymorphism and the risk and prognosis of lung adenocarcinoma (ADCA) in the Italian population. In the present case-control study, we examined 269 lung ADCA patients, 121 squamous cell lung carcinoma patients, and 632 healthy individuals (general population controls) in the Japanese population with genetic markers spanning approximately 1200 kb in the KRAS2 region. Allele-specific oligonucleotide hybridization revealed the same KRAS2/RsaI polymorphism associated with risk and prognosis as in Italian lung ADCA patients; the polymorphism was significantly associated with clinical stage (P < 0.001) and survival rate (log rank = 0.0014), confirming the mapping of PAS1 and pointing to the role of this locus in human lung cancer.

Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer.

Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta-estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala-Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4-hydroxylase activity among four different variant forms of CYP1B1. Thus, inter-individual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers.

Nucleocytoplasmic shuttling of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that acts in concert with the AhR nuclear translocator (ARNT), and alters gene expression in response to environmental contaminants such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). We have previously shown that AhR contains both a nuclear localization signal (NLS), AhR(13-39), and a nuclear export signal (NES), AhR(55-75), in its NH(2)-terminal region. In this study, we obtained direct evidence for the nucleocytoplasmic shuttling of AhR and show the biological significance of the shuttling in terms of the transcriptional activation of its target gene, CYP1A1. When AhR(13-75) fused with glutathione S-transferase (GST)-green fluorescent protein (GFP) was microinjected into the nucleus of a polykaryotic of BHK21 cell, the GST-AhR(13-75)-GFP migrated from one nucleus to the other. This event, nucleocytoplasmic shuttling, was completely inhibited in the presence of leptomycin B (LMB). The interaction between chromosome region maintenance 1 (CRM1) and endogenous AhR was shown by immunoprecipitation with antibodies to AhR followed by immunoblot analysis with antibodies to CRM1. The inhibition of the nuclear export of AhR by LMB repressed the transcriptional activation of the CYP1A1 gene. The findings suggest that nuclear-cytoplasmic shuttling of AhR is essential for the inducible expression of the CYP1A1 protein.

Expression of androgen receptor in mouse eye tissues.

PURPOSE: To test the possibility that androgen directly affects the corneal cells, the possible occurrence of androgen receptor (AR) in the cornea and other eye tissues of mice was examined. METHODS: To examine the occurrence of AR protein in the mouse eye tissues, an immunocytochemical method was used. To examine the occurrence of AR mRNA in the cornea and lens, reverse transcription-polymerase chain reaction (RT-PCR) was used. RESULTS: Immunocytochemical examination revealed that antigenicity for AR antibody exists in cell nuclei of cornea, lens, iris, and ciliary body of both male and female mice. RT-PCR revealed that mRNA of AR occurs in the cornea and lens of both male and female mice. CONCLUSIONS: It is concluded that AR occurs in cells of cornea, lens, iris, and ciliary body of the mouse eye. Androgen may affect cells in these tissues directly through interaction with AR.

CYP1A1.

CYP1A1 plays an important role in the metabolism of polycyclic hydrocarbons that occur in the environment and several studies suggest that the genetic polymorphism of the gene may play a role in the predisposition to cancer. In order to evaluate the function of CYP1A1 in vivo as a host factor determinant of environmentally-caused cancers in humans, additional investigations are needed involving not only molecular epidemiological approaches in different ethnic populations but also more direct approaches such as the use of gene-targeted mice as a model system.

Catalytic properties of polymorphic human cytochrome P450 1B1 variants.

Four polymorphic human cytochrome P450 (CYP) 1B1 allelic variants, namely Arg48,Ala119,Leu432,Asn453, Arg48,Ser119,Leu432,Asn453, Arg48, Ala119,Val432,Asn-453 and Arg48,Ser119,Val432,Asn453, were expressed in Escherichia coli together with human NADPH-P450 reductase and the recombinant proteins (in bacterial membranes) were used to assess whether CYP1B1 polymorphisms affect catalytic activities towards a variety of P450 substrates, including diverse procarcinogens and steroid hormones. Activities for activation of 19 procarcinogens to DNA-damaging products by these four CYP1B1 variants in a Salmonella typhimurium NM2009 umu response system were found to be essentially similar, except that a Arg48, Ser119,Leu432,Asn453 variant was slightly more active (1.2- to 1.5-fold) than the other three CYP1B1 enzymes in catalyzing activation of (+)- and (-)-benzo[a]pyrene-7, 8-diols, 7,12-dimethylbenz[a]anthracene-3,4-diol, benzo[g]chrysene-11,12-diol, benzo[b]fluoranthene-9,10-diol, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminofluorene. Kinetic analysis of 17beta-estradiol hydroxylation showed that V(max) values for 4-hydroxylation ranged between 0.9 and 1.5 nmol/min/nmol P450 for 4-hydroxylation and 0.3 and 0.6 nmol/min/nmol P450 for 2-hydroxylation in these CYP1B1 variants, with K(m) values ranging from 1 to 9 microM. Interestingly, the ratio of product formation of 4-hydroxyestradiol to 2-hydroxyestradiol was higher for the Val432 variants of CYP1B1 variants than the Leu432 variants of the enzyme. The same trend was noted in the ratio of estrone 4-hydroxylation to estrone 2-hydroxylation catalyzed by CYP1B1 variants. Mutation in the CYP1B1 genes also affected the K(m) and V(max) values in the 6beta-hydroxylation of testosterone and 6beta- and 16alpha-hydroxylation of progesterone. These results indicate that the polymorphisms in the human CYP1B1 gene cause some alterations in catalytic function towards procarcinogens and steroid hormones and thus may make some contribution to susceptibilities of individuals towards mammary and lung cancers in humans.

Immunoregulatory roles of IL-10 in innate immunity: IL-10 inhibits macrophage production of IFN-gamma-inducing factors but enhances NK cell production of IFN-gamma.

In our study of the immunoregulatory roles of IL-10 in innate immunity, nonantigenic phagocytosable chitin particles were administered i.v. to IL-10-deficient (knockout (KO)) mice or KO mice pretreated with anti-NK1.1 or anti-IFN-gamma Abs. The results established that chitin treatment of KO mice increased superoxide anion release from alveolar macrophages (Mphi) to a level much higher than that in wild-type (WT) mice. The results also suggested that the NK cell is the source of IFN-gamma that is primarily responsible for this alveolar Mphi priming. To further study the roles of IL-10-inhibiting chitin-induced IFN-gamma production, we used spleen cell cultures. The experiments showed that IL-12, IL-18, and TNF-alpha, which were produced by chitin-stimulated Mphi, contributed to the IFN-gamma-inducing activity of chitin. Our results established that exogenous IL-10 inhibited chitin-induced IFN-gamma production in spleen cell cultures from both KO and WT mice. Exogenous IL-10 also inhibited IL-12 and TNF-alpha production by chitin-stimulated Mphi. Exogenous IL-10 decreased IL-12- or IL-18-induced IFN-gamma levels in KO but not in WT NK cell cultures. However, exogenous IL-10 enhanced IFN-gamma levels when NK cells were stimulated simultaneously with both IL-12 and IL-18 in KO and WT cultures. Our in vitro data indicate that IL-10 has differential effects on chitin-induced IFN-gamma production. However, the inhibitory effects of endogenous IL-10 appear to be dominant in the chitin-induced alveolar Mphi priming response in vivo.

MPB64 mycobacterial antigen: a new skin-test reagent through patch method for rapid diagnosis of active tuberculosis.

SETTING: A collaborative study between the Japan BCG Laboratory, Tokyo, Japan, and the Infectious Disease Section, Philippine General Hospital, Manila, the Philippines. Tuberculosis patients from four clinics in the vicinity of Manila, Our Lady of Grace Parish, Sto. Niño de Tondo Parish, the Canossa Health and Social Center, and the Health Care Development Center, were examined. OBJECTIVE: To develop a new, simple and rapid diagnostic method for active tuberculosis. Subjects were tested for skin reaction to a special antigen, MPB64, by the patch test method instead of intradermal injection of purified protein derivative (PPD). DESIGN: Fifty-three active tuberculosis patients and 43 healthy PPD-positive controls were tested to determine whether or not the reaction to MPB64 was positive only in active tuberculosis patients. RESULTS: Fifty-two of the 53 active tuberculosis patients showed a positive reaction to MPB64, while none of the 43 PPD-positive controls did. The specificity of MPB64 to active tuberculosis was 100%, and the sensitivity was 98.1%. The efficacy of the test was 98.9%. CONCLUSION: The patch test with MPB64 is a promising method for the diagnosis of active tuberculosis, distinguishing tuberculous patients from those who are infected but have not developed the disease, and also from BCG-vaccinated individuals. This new skin test is a subject for further evaluation and it is important to compare the results with PPD Mantoux.

Nuclear localization and export signals of the human aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the Ahr nuclear translocator (hypoxia-inducible factor 1beta). We found in this study that Ahr contains both nuclear localization and export signals in the NH2-terminal region. A fusion protein composed of beta-galactosidase and full-length Ahr translocates from the cytoplasm to the nucleus in a ligand-dependent manner. However, a fusion protein lacking the PAS (Per-Ahr nuclear translocator-Sim homology) domain of the Ahr showed strong nuclear localization activity irrespective of the presence or absence of ligand. A minimum bipartite Ahr nuclear localization signal (NLS) consisting of amino acid residues 13-39 was identified by microinjection of fused proteins with glutathione S-transferase-green fluorescent protein. A NLS having mutations in bipartite basic amino acids lost nuclear translocation activity completely, which may explain the reduced binding activity to the NLS receptor, PTAC58. A 21-amino acid peptide (residues 55-75) containing the Ahr nuclear export signal is sufficient to direct nuclear export of a microinjected complex of glutathione S-transferase-Ahr-green fluorescent protein. These findings strongly suggest that Ahr act as a ligand- and signal-dependent nucleocytoplasmic shuttling protein.

A nuclear localization signal of human aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor 1beta is a novel bipartite type recognized by the two components of nuclear pore-targeting complex.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a component of the transcription factors, aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1, which transactivate their target genes, such as CYP1A1 and erythropoietin, in response to xenobiotic aromatic hydrocarbons and to low O2 concentration, respectively. Since ARNT was isolated as a factor required for the nuclear translocation of AhR from the cytoplasm in response to xenobiotics, the subcellular localization of ARNT has been of great interest. In this investigation, we analyzed the subcellular distribution of ARNT using transient expression of a fusion gene with beta-galactosidase and microinjection of recombinant proteins containing various fragments of ARNT in the linker region of glutathione S-transferase/green fluorescent protein. We found a clear nuclear localization of ARNT in the absence of exogenous ligands to AhR, and identified the nuclear localization signal (NLS) of amino acid residues 39-61. The characterized NLS consists of 23 amino acids, and can be classified as a novel variant of the bipartite type on the basis of having two separate regions responsible for efficient nuclear translocation activity, but considerable deviation of the sequence from the consensus of the classical bipartite type NLSs. Like the well characterized NLS of the SV40 T-antigen, this variant bipartite type of ARNT NLS was also mediated by the two components of nuclear pore targeting complex, PTAC58 and PTAC97, to target to the nuclear rim in an in vitro nuclear transport assay.

Prognostic significance of germ line polymorphisms of the CYP1A1 and glutathione S-transferase genes in patients with non-small cell lung cancer.

CYP1A1 is responsible for the metabolic activation of benzo(a)pyrene in cigarette smoke, and high susceptibility to smoking-related lung cancer has been associated with the MspI polymorphism of the CYP1A1 gene. Individuals with a susceptible CYP1A1 genotype have been found to be at remarkably high risk when the genotype is combined with a deficient Mu-class glutathione S-transferase (GSTM1) genotype. In this study, we investigated the relationship between germ line polymorphisms of these genes and clinical characteristics or survival rates in 232 patients with non-small cell lung cancer (NSCLC). Statistical analysis revealed a significant association (P < 0.05) of the MspI polymorphism of the CYP1A1 gene with histological type, performance status (general conditions of patients), and the extent of the primary tumor (T-factor). On the other hand, the GSTM1 polymorphism was significantly associated with performance status, the extent of regional lymph node metastasis (N-factor), and the extent of distant metastasis (M-factor). NSCLC patients with at least one susceptible allele of the MspI polymorphism of the CYP1A1 gene [heterozygous genotype B or a rare homozygous genotype C; n = 131; median survival time (MST) = 24.2 months] were associated with a shortened survival compared with those with nonsusceptible homozygous alleles (genotype A; n = 101; MST = 65.2 months; P = 0.005 by log-rank test). Smokers with susceptible genotypes (n = 104; MST = 18.2 months) were markedly associated with a shortened survival compared with those with genotype A (n = 76; MST = 69.2 months; P = 0.024); such an association was not found among nonsmokers by genotypes. Genotype-dependent survival was also observed in patients at an advanced stage of disease (P = 0.010), but not in those at an early stage of disease (P = 0.382). Patients with the susceptible CYP1A1 genotype had remarkably shortened survivals when the genotype was combined with a deficient genotype GSTM1(-) (P = 0.017; degree of freedom = 3). Multivariate analysis by the Cox proportional hazards model also revealed that the CYP1A1 polymorphism was an independent prognostic factor in patients at a nonresectable advanced stage of NSCLC (P = 0.005; hazard ratio = 1.98; 95% confidence interval, 1.24-3.17).

Effective isolation of MPB64 from a large volume of culture filtrate of Mycobacterium bovis BCG Tokyo.

MPB64, a secretory protein of Mycobacterium bovis BCG Tokyo, was isolated from a culture filtrate of the bacteria in Sauton synthetic medium harvested on day 8. The protein was isolated by five steps; (i) concentration of the culture filtrate by cutting the molecules smaller than 5 kDa with the Millipore Pellicon Cassette system, (ii) affinity separation by a Phenyl Sepharose CL-4B column, (iii) separation with a DEAE-Sepharose CL-6B column with 3 M urea, (iv) separation with a Sephacryl S200HR column, and (v) separation with a DEAE-Sepharose column without urea. MPB64 in each fraction was determined by comparing the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with that of standard MPB64. The specificity of isolated MPB64 was tested by immunoblotting with anti-MPB64 antibody. The potency of isolated MPB64 in eliciting skin reaction in the BCG-sensitized guinea pigs was the same to that of standard MPB64. The method described herein is an improved one for isolating MPB64 from a large volume of culture filtrate of M. bovis BCG Tokyo. The technique should be applicable to isolation of other mycobacterial secretory proteins.

Association of CYP1A1 germ line polymorphisms with mutations of the p53 gene in lung cancer.

We reported an association of smoking-induced lung cancer susceptibility with the human cytochrome P450 1A1 (CYP1A1) polymorphisms in our previous studies. To investigate a relationship between genetically determined individual predispositions and mutations of target genes in the early stage of lung carcinogenesis, we examined p53 mutations in relation to germ line polymorphisms of the CYP1A1 and GSTM1 genes, using surgical specimens of 148 non-small cell lung cancer patients who were smokers. The frequency of p53 mutations among heavy smokers was higher than in patients who had never smoked [P < 0.01; odds ratio (OR), 3.74; 95% confidence interval (CI), 1.46-9.56]. By single-strand conformational polymorphism, aberrant migration bands of p53 gene fragments were detected in 56 cases (38%). Smokers with susceptible rare homozygous alleles of either the MspI or Ile-Val polymorphism of the CYP1A1 gene have a 4.5-fold (P < 0.005; OR, 4.48; 95% CI, 1.64-12.26) or 5.5-fold (P < 0.01; OR, 5.52; 95% CI, 1.55-19.64) higher risk of having a mutation of the p53 gene than those with nonsusceptible predominant homozygous alleles of the gene. Non-small cell lung cancer patients with a susceptible CYP1A1 genotype were at remarkably high risk of having a mutation of the p53 gene when the genotype was combined with a deficient genotype, GSTM1(-). However, there was no difference between the types of p53 mutation and genotypes of the drug-metabolizing enzymes. These results showed that CYP1A1 germ line polymorphisms, which were associated with the genetic predisposition for lung cancer, were related to cigarette smoking-associated p53 mutations.