Increased SLAMF7high monocytes in myelofibrosis patients harboring JAK2V617F provide a therapeutic target of elotuzumab.

Monocyte-derived fibrocytes recently garnered attention because the novel pathogenesis of myelofibrosis (MF), and suppression of fibrocyte differentiation by serum amyloid P remarkably improved MF. We previously revealed that human fibrocytes highly expressed signaling lymphocytic activation molecule F7 (SLAMF7) compared with macrophages and that SLAMF7high monocytes in the peripheral blood (PB) of MF patients were significantly elevated relative to those in healthy controls (HCs). In this study, we evaluated SLAMF7high monocyte percentage in the PB of HCs, myeloproliferative neoplasm (MPN) patients with MF, and MPN patients without MF by using a cross-sectional approach. We found that MPN patients with MF who harbored JAK2V617F had a significantly elevated SLAMF7high monocyte percentage, which correlated positively with the JAK2V617F allele burden. In addition, the serum concentration of interleukin-1ra (IL-1ra) was significantly correlated with the SLAMF7high monocyte percentage and JAK2V617F allele burden. These findings suggest that both SLAMF7high monocytes and IL-1ra could be useful noninvasive markers of MF onset. Furthermore, the JAK2V617F allele burden of SLAMF7high monocytes was significantly higher than that of SLAMF7low monocytes and could be a potential target of elotuzumab (Elo), an anti-SLAMF7 antibody used for treating multiple myeloma. Elo independently inhibited differentiation of fibrocytes derived not only from HCs but also from MF patients in vitro. Elo also ameliorated MF and splenomegaly induced by romiplostim administration in humanized NOG mice. In conclusion, an increase of SLAMF7high monocytes with higher JAK2V617F allele burden was associated with the onset of MF in MPN patients harboring JAK2V617F, and Elo could be a therapeutic agent for MPN patients with MF who harbor JAK2V617F.

Application of a Waveguide-Mode Sensor to Blood Testing for Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus and Treponema pallidum Infection.

Testing for blood-transmitted infectious agents is an important aspect of safe medical treatment. During emergencies, such as significant earthquakes, many patients need surgical treatment and/or blood transfusion. Because a waveguide mode (WM) sensor can be used as a portable, on-site blood testing device in emergency settings, we have previously developed WM sensors for detection of antibodies against hepatitis B virus and hepatitis C virus and for forward ABO and Rh(D) and reverse ABO blood typing. In this study, we compared signal enhancement methods using secondary antibodies conjugated with peroxidase, a fluorescent dye, and gold nanoparticles, and found that the peroxidase reaction method offers superior sensitivity while gold nanoparticles provide the most rapid detection of anti-HBs antibody. Next, we examined whether we could apply a WM sensor with signal enhancement with peroxidase or gold nanoparticles to detection of antibodies against hepatitis C virus, human immunodeficiency virus and Treponema pallidum, and HBs antigen in plasma. We showed that a WM sensor can detect significant signals of these infectious agents within 30 min. Therefore, a portable device utilizing a WM sensor can be used for on-site blood testing of infectious agents in emergency settings.

Combined treatment with benzo[a]pyrene and 1α,25-dihydroxyvitamin D3 induces expression of plasminogen activator inhibitor 1 in monocyte/macrophage-derived cells.

Benzo[a]pyrene (BaP) is an environmental pollutant found in cigarette smoke and is implicated as a causative agent of tobacco-related diseases, such as arteriosclerosis. In contrast, vitamin D signaling, which is principally mediated by conversion of vitamin D to the active form, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], decreases cardiovascular disease risk. However, combined treatment with BaP and 1,25(OH)2D3 enhances BaP toxicity, including BaP-DNA adduct formation. We further investigated the cross-talk between BaP and 1,25(OH)2D3 signaling pathways, and found that combined treatment with these compounds induces mRNA and protein expression of plasminogen activator inhibitor 1 (PAI-1) in monocyte/macrophage-derived THP-1 and U937 cells. Protein synthesis inhibitor treatment did not inhibit induction of the PAI-1 gene (SERPINE1) in these cells. BaP plus 1,25(OH)2D3 induced differentiation markers, inhibited cellular proliferation, and induced apoptosis and oxidative stress in these cells. Reactive oxygen species scavenger treatment suppressed apoptosis but not SERPINE1 induction in cells treated with BaP plus 1,25(OH)2D3. Thus, combined treatment with BaP and 1,25(OH)2D3 induced SERPINE1 mRNA expression in these cells through a mechanism that does not require de novo protein synthesis or reactive oxygen species production. These findings suggest that induction of the proinflammatory factor PAI-1 plays a role in BaP toxicity. Interestingly, PAI-1 knockdown decreased expression of the cell surface antigen CD14, a monocytic differentiation marker, in THP-1 cells treated with BaP plus 1,25(OH)2D3. PAI-1 induction may also be related to a function of monocytes/macrophages in response to xenobiotic and vitamin D signaling.

Oral benzo[a]pyrene: understanding pharmacokinetics, detoxication, and consequences--Cyp1 knockout mouse lines as a paradigm.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH); this ubiquitous environmental carcinogenic agent is found in tobacco smoke, charcoal-grilled foods, and PAH-contaminated surfaces of roofs, playgrounds, and highways. Cytochrome P450 1 wild-type, Cyp1a2(-/-), Cyp1b1(-/-), or Cyp1a2/1b1(-/-) knockouts, and mice with Cyp1a1 expression deleted in hepatocytes can ingest large oral BaP doses (125 mg/kg/d) without apparent toxicity. Cyp1a1(-/-) and Cyp1a1/1a2(-/-) knockouts and mice with Cyp1a1 expression deleted in gastrointestinal (GI) tract epithelial cells develop immunotoxicity and die within 32 days, indicating that GI tract inducible CYP1A1 is absolutely required for detoxication of oral BaP. Cyp1a1/1b1(-/-) and Cyp1a1/1a2/1b1(-/-) mice are rescued from immunosuppression and early death due to absent metabolic activation of BaP by CYP1B1 in immune cells. Ten-fold lower oral BaP doses result in adenocarcinoma of the proximal small intestine (PSI) in Cyp1a1(-/-) mice; Cyp1a1/1b1(-/-) double-knockout mice show no PSI cancer but develop squamous cell carcinoma of the preputial gland duct (PGD). BaP-metabolizing CYP1B1 in the PSI and CYP3A59 in the PGD are the most likely candidates to participate in tumor initiation in the epithelial cells of these two tissues; oncogenes and tumor-suppressor genes upregulated and downregulated during tumorigenesis are completely different between these tissues. This "oral BaP Cyp1" mouse paradigm represents a powerful teaching tool, showing that gene-environment interactions depend on route-of-administration: the same oral, but not intraperitoneal, BaP exposure leads to dramatic differences in target-organ toxicity and tumor type as a function of dose and Cyp1 genotype.

Differential expression and function of alternative splicing variants of human liver X receptor α.

The liver X receptor α (LXRα) is a nuclear receptor that is involved in regulation of lipid metabolism, cellular proliferation and apoptosis, and immunity. In this report, we characterize three human LXRα isoforms with variation in the ligand-binding domain (LBD). While examining the expression of LXRα3, which lacks 60 amino acids within the LBD, we identified two novel transcripts that encode LXRα-LBD variants (LXRα4 and LXRα5). LXRα4 has an insertion of 64 amino acids in helix 4/5, and LXRα5 lacks the C-terminal helices 7 to 12 due to a termination codon in an additional exon that encodes an intron in the LXRα1 mRNA. LXRα3, LXRα4, and LXRα5 were expressed at lower levels compared with LXRα1 in many human tissues and cell lines. We also observed weak expression of LXRα3 and LXRα4 in several tissues of mice. LXR ligand treatment induced differential regulation of LXRα isoform mRNA expression in a cell type-dependent manner. Whereas LXRα3 had no effect, LXRα4 has weak transactivation, retinoid X receptor (RXR) heterodimerization, and coactivator recruitment activities. LXRα5 interacted with a corepressor in a ligand-independent manner and inhibited LXRα1 transactivation and target gene expression when overexpressed. Combination of LXRα5 cotransfection and LXRα antagonist treatment produced additive effects on the inhibition of ligand-dependent LXRα1 activation. We constructed structural models of the LXRα4-LBD and its complexes with ligand, RXR-LBD, and coactivator peptide. The models showed that the insertion in the LBD can be predicted to disrupt RXR heterodimerization. Regulation of LXRα pre-mRNA splicing may be involved in the pathogenesis of LXRα-related diseases.

Vitamin D receptor activation enhances benzo[a]pyrene metabolism via CYP1A1 expression in macrophages.

Benzo[a]pyrene (BaP) activates the aryl hydrocarbon (AHR) and induces the expression of genes involved in xenobiotic metabolism, including CYP1A1. CYP1A1 is involved not only in BaP detoxification but also in metabolic activation, which results in DNA adduct formation. Vitamin D receptor (VDR) belongs to the NR1I subfamily of the nuclear receptor superfamily, which also regulates expression of xenobiotic metabolism genes. We investigated the cross-talk between AHR and VDR signaling pathways and found that 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a potent physiological VDR agonist, enhanced BaP-induced transcription of CYP1A1 in human monocytic U937 cells and THP-1 cells, breast cancer cells, and kidney epithelium-derived cells. 1,25(OH)(2)D(3) alone did not induce CYP1A1, and 1,25(OH)(2)D(3) plus BaP did not increase CYP1A2 or CYP1B1 mRNA expression in U937 cells. The combination of 1,25(OH)(2)D(3) and BaP increased CYP1A1 protein levels, BaP hydroxylation activity, and BaP-DNA adduct formation in U937 cells and THP-1 cells more effectively than BaP alone. The combined effect of 1,25(OH)(2)D(3) and BaP on CYP1A1 mRNA expression in U937 cells and/or THP-1 cells was inhibited by VDR knockdown, VDR antagonists, and α-naphthoflavone, an AHR antagonist. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that VDR directly bound to an everted repeat (ER) 8 motif in the human CYP1A1 promoter. Thus, CYP1A1 is a novel VDR target gene involved in xenobiotic metabolism. Induction of CYP1A1 by the activation of VDR and AHR may contribute to BaP-mediated toxicity and the physiological function of this enzyme.

Oral benzo[a]pyrene administration attenuates dextran sulfate sodium-induced colitis in mice.

Benzo[a]pyrene (BaP) is an environmental pollutant produced by combustion processes and is present in grilled foods as well as in tobacco smoke. BaP acts as an agonist for the aryl hydrocarbon receptor (AHR), and is metabolized by AHR-inducing enzymes. BaP metabolism can result in either detoxification or metabolic activation, the latter leads to an increased risk of disease, particularly lung cancer and cardiovascular disease, in a context-dependent manner. Although AHR activation has been thought to protect against inflammatory bowel disease, it remains unknown whether BaP exerts a protective or deleterious effect on colitis. In this study, we examined the effect of oral BaP administration on colitis induced by dextran sulfate sodium (DSS) in mice, an animal model of inflammatory bowel disease. BaP administration attenuated weight loss, shortening of the colon, disease activity index scores, and histological damage in DSS-induced colitis mice. BaP also suppressed colonic expression of inflammation-associated genes and plasma interleukin-6 secretion induced by DSS treatment. BaP-DNA adduct formation, a marker of BaP metabolic activation, was not enhanced in the colon after DSS treatment. Thus, oral BaP exerts an anti-inflammatory effect on DSS-induced colitis, without the toxicity associated with metabolic activation. The results provide insights into the disease-specific roles of BaP.

Liver X receptors regulate natural killer T cell population and antitumor activity in the liver of mice.

The nuclear receptors liver X receptor α (LXRα) and LXRß are lipid sensors that regulate lipid metabolism and immunity. Natural killer T (NKT) cells, a T cell subset expressing surface markers of both natural killer cells and T lymphocytes and involved in antitumor immunity, are another abundant immune cell type in the liver. The potential function of the metabolic regulators LXRα/ß in hepatic NKT cells remains unknown. In this study, we examined the role of LXRα and LXRß in NKT cells using mice deficient for LXRα and/or LXRß, and found that hepatic invariant NKT (iNKT) cells are drastically decreased in LXRα/ß-KO mice. Cytokine production stimulated by the iNKT cell activator α-galactosylceramide was impaired in LXRα/ß-KO hepatic mononuclear cells and in LXRα/ß-KO mice. iNKT cell-mediated antitumor effect was also disturbed in LXRα/ß-KO mice. LXRα/ß-KO mice transplanted with wild-type bone marrow showed decreased iNKT cells in the liver and spleen. The thymus of LXRα/ß-KO mice showed a decreased population of iNKT cells. In conclusion, LXRα and LXRß are essential for NKT cell-mediated immunity, such as cytokine production and hepatic antitumor activity, and are involved in NKT cell development in immune tissues, such as the thymus.

Oral benzo[a]pyrene-induced cancer: two distinct types in different target organs depend on the mouse Cyp1 genotype.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1 and CYP1B1) can both detoxify PAHs and activate them to cancer-causing reactive intermediates. Following high dosage of oral BaP (125 mg/kg/day), ablation of the mouse Cyp1a1 gene causes immunosuppression and death within ∼28 days, whereas Cyp1(+/+) wild-type mice remain healthy for >12 months on this regimen. In this study, male Cyp1(+/+) wild-type, Cyp1a1(-/-) and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) double-knockout mice received a lower dose (12.5 mg/kg/day) of oral BaP. Tissues from 16 different organs-including proximal small intestine (PSI), liver and preputial gland duct (PGD)-were evaluated; microarray cDNA expression and >30 mRNA levels were measured. Cyp1a1(-/-) mice revealed markedly increased CYP1B1 mRNA levels in the PSI, and between 8 and 12 weeks developed unique PSI adenomas and adenocarcinomas. Cyp1a1/1b1(-/-) mice showed no PSI tumors but instead developed squamous cell carcinoma of the PGD. Cyp1(+/+) and Cyp1b1(-/-) mice remained healthy with no remarkable abnormalities in any tissue examined. PSI adenocarcinomas exhibited striking upregulation of the Xist gene, suggesting epigenetic silencing of specific genes on the Y-chromosome; the Rab30 oncogene was upregulated; the Nr0b2 tumor suppressor gene was downregulated; paradoxical overexpression of numerous immunoglobulin kappa- and heavy-chain variable genes was found-although the adenocarcinoma showed no immunohistochemical evidence of being lymphatic in origin. This oral BaP mouse paradigm represents an example of "gene-environment interactions" in which the same exposure of carcinogen results in altered target organ and tumor type, as a function of just 1 or 2 globally absent genes.

Knock-in mouse lines expressing either mitochondrial or microsomal CYP1A1: differing responses to dietary benzo[a]pyrene as proof of principle.

In the past, CYP1A1 protein was known to be located in the endoplasmic reticulum (ER; microsomes). More recently, CYP1A1 was shown also to be targeted to the inner mitochondrial membrane; mitochondrial import is dependent on NH(2)-terminal processing that exposes a cryptic targeting signal. It is interesting that microsomal and mitochondrial CYP1A1 enzymes exhibit different substrate specificities, electron donors, and inducer properties. To understand the physiological functions of microsomal versus mitochondrial CYP1A1, we have generated three knock-in lines by altering the CYP1A1 NH(2) terminus. Cyp1a1(mtt/mtt) mice encode an NH(2)-terminal 31-amino acid-truncated protein, deleting the ER-targeting signal and exposing the cryptic mitochondrial-targeting signal. Cyp1a1(mtp/mtp) mice encode a protein carrying L7N and L17N mutations; this mutant lacks the signal recognition particle (SRP)-binding site and subsequent ER-targeting, but requires proteolysis by a cytosolic peptidase for mitochondrial import. Cyp1a1(mc/mc) mice encode a microsomal protein having R34D and K39I mutations, which abolish the mitochondrial targeting signal. After dioxin or beta-naphthoflavone treatment of these mouse lines, the CYP1A1 protein was shown to be located in the mitochondria of the Cyp1a1(mtp/mtp) and Cyp1a1(mtt/mtt) lines and in microsomes of the Cyp1a1(mc/mc) line. To test for differences in function, we compared the response to dietary benzo[a]pyrene (BaP). After 18 days of daily oral BaP, wild-type and Cyp1a1(mc/mc) mice were completely protected, whereas Cyp1a1(-/-) and Cyp1a1(mtp/mtp) mice showed striking toxicity and compensatory up-regulation of CYP1A2 and CYP1B1 mRNA in several tissues. Our data support the likelihood that it is the microsomal rather than mitochondrial CYP1A1 enzyme that protects against oral BaP toxicity.

Vitamin D3 modulates the expression of bile acid regulatory genes and represses inflammation in bile duct-ligated mice.

Vitamin D receptor (VDR), a nuclear receptor that regulates calcium homeostasis, has been found to function as a receptor for secondary bile acids. Because the in vivo role of VDR in bile acid metabolism remains unknown, we investigated the effect of VDR activation in a mouse model of cholestasis. We treated mice with 1alpha-hydroxyvitamin D(3) [1alpha(OH)D(3)] after bile duct ligation (BDL) and examined mRNA expression and cytokine levels. 1alpha(OH)D(3) treatment altered the expression of genes involved in bile acid synthesis and transport in the liver, kidney, and intestine but did not decrease bile acid levels in the plasma and liver of BDL mice. 1alpha(OH)D(3) treatment suppressed mRNA expression of proinflammatory cytokines in the liver and strongly decreased the plasma levels of proinflammatory cytokines in BDL mice. These findings indicate that 1alpha(OH)D(3) regulates a network of bile acid metabolic genes and represses proinflammatory cytokine expression in BDL mice. VDR ligands have the potential to prevent the cholestasis-induced inflammatory response.

CYP1A1 and CYP1A2 expression: comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines.

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how "human-like" can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

Diallyl Trisulfide Enhances Benzo[a]pyrene-induced CYP1A1 Expression and Metabolic Activation in Hepatic HepG2 Cells.

BACKGROUND/AIM: Benzo[a]pyrene (BaP), an environmental pollutant produced by combustion processes, induces expression of cytochrome P450 (CYP) 1A1 via the activation of aryl hydrocarbon receptor (AHR). Induced CYP1A1 is involved in BaP metabolism, resulting in either detoxification or metabolic activation in a context-dependent manner. The effect of diallyl trisulfide (DATS), a garlic-derived organosulfur compound, on BaP metabolism has not been investigated. MATERIALS AND METHODS: The combined effect of DATS and BaP on BaP metabolism in hepatocyte-derived HepG2 cells was examined. RESULTS: DATS enhanced BaP-induced CYP1A1 and CYP1B1 mRNA expression, BaP hydroxylation and BaP-DNA adduct formation. Combined treatment of BaP and DATS also increased reactive oxygen species levels. DATS enhanced BaP-induced AHR recruitment and histone H3 acetylation on the CYP1A1 promoter. CONCLUSION: DATS combined treatment enhances BaP metabolic activation through an AHR-modulating mechanism.

Basal and inducible CYP1 mRNA quantitation and protein localization throughout the mouse gastrointestinal tract.

The CYP1A1, CYP1A2, and CYP1B1 enzymes are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); metabolism of BaP by these enzymes leads to electrophilic intermediates and genotoxicity. Throughout the gastrointestinal (GI) tract, we systematically compared basal and inducible levels of the CYP1 mRNAs by Q-PCR, and localized the CYP1 proteins by immunohistochemistry. Cyp1(+/+) wild-type were compared with the Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) and Cyp1a2/1b1(-/-) double-knockout mice. Oral BaP was compared with intraperitoneal TCDD. In general, maximal CYP1A1 mRNA levels were 3-10 times greater than CYP1B1, which were 3-10 times greater than CYP1A2 mRNA levels. Highest inducible concentrations of CYP1A1 and CYP1A2 occurred in proximal small intestine, whereas the highest basal and inducible levels of CYP1B1 mRNA occurred in esophagus, forestomach, and glandular stomach. Ablation of either Cyp1a2 or Cyp1b1 gene resulted in a compensatory increase in CYP1A1 mRNA - but only in small intestine. Also in small intestine, although BaP- and TCDD-mediated CYP1A1 inductions were roughly equivalent, oral BaP-mediated CYP1A2 mRNA induction was approximately 40-fold greater than TCDD-mediated CYP1A2 induction. CYP1B1 induction by TCDD in Cyp1(+/+) and Cyp1a2(-/-) mice was 4-5 times higher than that by BaP; however, in Cyp1a1(-/-) animals CYP1B1 induction by TCDD or BaP was approximately equivalent. CYP1A1 and CYP1A2 proteins were generally localized nearer to the lumen than CYP1B1 proteins, in both squamous and glandular epithelial cells. These GI tract data suggest that the inducible CYP1A1 enzyme, both in concentration and in location, might act as a "shield" in detoxifying oral BaP and, hence, protecting the animal.

Progesterone receptor antagonists with a 3-phenylquinazoline-2,4-dione/2-phenylisoquinoline-1,3-dione skeleton.

Novel non-steroidal progesterone receptor antagonists with a 3-phenylquinazoline-2,4-dione/2-phenylisoquinoline-1,3-dione skeleton were developed and their structure-activity relationships were investigated. Among the prepared compounds, 4-(4,4-diethyl-3,4-dihydro-1,3-dioxoquinolin-2(1H)-yl)benzonitrile (DEPIQ-4CN) showed the most potent activity, with IC(50) values of 74-78nM in alkaline phosphatase activity and reporter gene assays.

Cytochrome P450 1A1 (CYP1A1) protects against nonalcoholic fatty liver disease caused by Western diet containing benzo[a]pyrene in mice.

The Western diet contributes to nonalcoholic fatty liver disease (NAFLD) pathogenesis. Benzo[a]pyrene (BaP), a prototypical environmental pollutant produced by combustion processes, is present in charcoal-grilled meat. Cytochrome P450 1A1 (CYP1A1) metabolizes BaP, resulting in either detoxication or metabolic activation in a context-dependent manner. To elucidate a role of CYP1A1-BaP in NAFLD pathogenesis, we compared the effects of a Western diet, with or without oral BaP treatment, on the development of NAFLD in Cyp1a1(-/-) mice versus wild-type mice. A Western diet plus BaP induced lipid-droplet accumulation in liver of Cyp1a1(-/-) mice, but not wild-type mice. The hepatic steatosis observed in Cyp1a1(-/-) mice was associated with increased cholesterol, triglyceride and bile acid levels. Cyp1a1(-/-) mice fed Western diet plus BaP had changes in expression of genes involved in bile acid and lipid metabolism, and showed no increase in Cyp1a2 expression but did exhibit enhanced Cyp1b1 mRNA expression, as well as hepatic inflammation. Enhanced BaP metabolic activation, oxidative stress and inflammation may exacerbate metabolic dysfunction in liver of Cyp1a1(-/-) mice. Thus, Western diet plus BaP induces NAFLD and hepatic inflammation in Cyp1a1(-/-) mice in comparison to wild-type mice, indicating a protective role of CYP1A1 against NAFLD pathogenesis.

Sensitive typing of reverse ABO blood groups with a waveguide-mode sensor.

Portable, on-site blood typing methods will help provide life-saving blood transfusions to patients during an emergency or natural calamity, such as significant earthquakes. We have previously developed waveguide-mode (WM) sensors for forward ABO and Rh(D) blood typing and detection of antibodies against hepatitis B virus and hepatitis C virus. In this study, we evaluated a WM-sensor for reverse ABO blood typing. Since reverse ABO blood typing is a method for detection of antibodies against type A and type B oligosaccharide antigens on the surface of red blood cells (RBCs), we fixed a synthetic type A or type B trisaccharide antigen on the sensor chip of the WM sensor. We obtained significant changes in the reflectance spectra from a WM sensor on type A antigen with type B plasma and type O plasma and on type B antigen with type A plasma and type O plasma, and no spectrum changes on type A antigen or type B antigen with type AB plasma. Signal enhancement with the addition of a peroxidase reaction failed to increase the sensitivity for detection on oligosaccharide chips. By utilizing hemagglutination detection using regent type A and type B RBCs, we successfully determined reverse ABO blood groups with higher sensitivity compared to a method using oligosaccharide antigens. Thus, functionality of a portable device utilizing a WM sensor can be expanded to include reverse ABO blood typing and, in combination with forward ABO typing and antivirus antibody detection, may be useful for on-site blood testing in emergency settings.

Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor.

In large-scale disasters, such as huge significant earthquakes, on-site examination for blood typing and infectious disease screening will be very helpful to save lives of victims who need surgical treatment and/or blood transfusion. However, physical damage, such as building collapse, electric power failure and traffic blockage, disrupts the capacity of the medical system. Portable diagnostic devices are useful in such cases of emergency. In this study, we evaluated a waveguide-mode sensor for detection of anti-hepatitis virus antibodies. First, we examined whether we can detect antigen-antibody interaction on a sensor chip immobilized hepatitis B virus surface (HBs) antigen and hepatitis C virus (HCV) core antigen using monoclonal mouse antibodies for HBs antigen and HCV core antigen. We obtained significant changes in the reflectance spectra, which indicate specific antigen-antibody interaction for anti-HBs antibody and anti-HCV antibody. Next, we examined the effect of horseradish peroxidase-conjugated secondary antibody using aminoethyl carbazole as the peroxidase substrate and found that the colorimetric reaction increases detection sensitivity for anti-HBs antibody more than 300 times. Finally, we successfully detected anti-HBs antibody in human blood samples with an enhancing method using a peroxidase reaction. Thus, a portable device utilizing a waveguide-mode sensor may be applied to on-site blood testing in emergency settings.

The heat shock protein inhibitor KNK437 induces neurite outgrowth in PC12 cells.

The nervous system is highly sensitive to various environmental stresses, such as ischemia. Stress response mechanisms that result in neuroprotection, including the induction of heat shock proteins (HSP), are not well understood. We examined the effect of KNK437, a compound that inhibits the synthesis of inducible heat shock proteins, on neuronal differentiation in rat pheochromocytoma PC12 cells. KNK437 decreased the expression of HSP70, and induced the neurite outgrowth of PC12 cells in the absence of stress stimulation, although with lower efficacy than nerve growth factor (NGF). Neurite outgrowth stimulated by KNK437 and NGF was blocked by inhibitors of ERK mitogen-activated protein (MAP) kinase, p38 MAP kinase, and glycogen synthase kinase 3beta signaling pathways. NGF, and not KNK437, induced acetylcholine esterase (AChE) activity, a functional differentiation marker, indicating that KNK437 utilizes a mechanism distinct from that of NGF. KNK437 enhanced the activity of low dose NGF treatment on neurite outgrowth induction and ERK phosphorylation in PC12 cells, a finding that identifies KNK437 as a possible nerve regeneration agent. This compound may be a useful tool for the investigation of neuronal differentiation and neuroprotection against environmental stress.

Synthesis, Biological Activities, and X-ray Crystal Structural Analysis of 25-Hydroxy-25(or 26)-adamantyl-17-[20(22),23-diynyl]-21-norvitamin D Compounds.

Novel 19-norvitamin D analogues (ADYW1-4, 5a-d) in which an adamantyl diyne side chain is attached directly to the 17-position of the D ring are designed and stereoselectively synthesized. The adamantane ring of these analogues was expected to interfere with helix 12 (H12, activation function 2) of the vitamin D receptor (VDR) to modulate its activities. The analogue 5b binds to the VDR (7% of the natural hormone) and shows significant partial agonistic activity in transactivation assay. Compound 5b showed considerable selectivity in VDR target genes expressions in vitro, it was taken up by target cells 2-3 times more readily, and its lifetime was three times longer than the natural hormone. The X-ray crystal structure of 5b in complex with VDR reveals that the ligand binds similarly to the natural hormone, but the diyne moiety is slightly bent (angles around the diyne 5° to 8°) with respect to the original diyne vitamin D compound 6 in VDR (<1°) due to steric hindrance with helix 12.