Tatsunori Takayasu

Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice.

To clarify interleukin (IL)‐6 roles in wound healing, we prepared skin excisions in wild‐type (WT) and IL‐6‐deficient BALB/c [knockout (KO)] mice. In WT mice, the wound area was reduced to 50% of original size at 6 days after injury. Microscopically, leukocyte infiltration was evident at wound sites. Furthermore, the re‐epithelialization rate was ∼80% at 6 days after injury with increases in angiogenesis and hydroxyproline contents. The gene expression of IL‐1, chemokines, adhesion molecules, transforming growth factor‐β1, and vascular endothelial growth factor was enhanced at the wound sites. In contrast, the enhanced expression of these genes was significantly reduced in KO mice. Moreover, in KO mice, the reduction of wound area was delayed with attenuated leukocyte infiltration, re‐epithelialization, angiogenesis, and collagen accumulation. Finally, the administration of a neutralizing anti‐IL‐6 monoclonal antibody significantly delayed wound closure in WT mice. These observations suggest that IL‐6 has crucial roles in wound healing, probably by regulating leukocyte infiltration, angiogenesis, and collagen accumulation.

The Essential Involvement of Cross-Talk between IFN-γ and TGF-β in the Skin Wound-Healing Process

Several lines of in vitro evidence suggest the potential role of IFN-γ in angiogenesis and collagen deposition, two crucial steps in the wound healing process. In this report, we examined the role of IFN-γ in the skin wound healing process utilizing WT and IFN-γ KO mice. In WT mice, excisional wounding induced IFN-γ mRNA and protein expression by infiltrating macrophages and T cells, with a concomitant enhancement of IL-12 and IL-18 gene expression. Compared with WT mice, IFN-γ KO mice exhibited an accelerated wound healing as evidenced by rapid wound closure and granulation tissue formation. Moreover, IFN-γ KO mice exhibited enhanced angiogenesis with augmented vascular endothelial growth factor mRNA expression in wound sites, compared with WT mice, despite a reduction in the infiltrating neutrophils, macrophages, and T cells. IFN-γ KO mice also exhibited accelerated collagen deposition with enhanced production of TGF-β1 protein in wound sites, compared with WT mice. Furthermore, the absence of IFN-γ augmented the TGF-β1-mediated signaling pathway, as evidenced by increases in the levels of total and phosphorylated Smad2 and a reciprocal decrease in the levels of Smad7. These results demonstrate that there is crosstalk between the IFN-γ/Stat1 and TGF-β1/Smad signaling pathways in the wound healing process.

Muscarinic Receptor-mediated Dual Regulation of ADP-ribosyl Cyclase in NG108-15 Neuronal Cell Membranes

Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma × glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30–52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation.

The pathogenic roles of tumor necrosis factor receptor p55 in acetaminophen‐induced liver injury in mice

Acetaminophen (APAP) causes a massive production of intrahepatic tumor necrosis factor α (TNF‐α). However, it still remains elusive regarding the roles of TNF‐α in APAP‐induced liver injury. Hence, we examined pathogenic roles of the TNF‐α–TNF receptor with a molecular weight of 55 kDa (TNF‐Rp55) axis in APAP‐induced hepatotoxicity using TNF‐Rp55‐deficient [TNF‐Rp55‐knockout (KO)] mice. When wild‐type (WT) BALB/c and TNF‐Rp55‐KO mice were intraperitoneally injected with a lethal dose of APAP (750 mg/kg), the mortality of TNF‐Rp55‐KO mice was marginally but significantly reduced compared with WT mice. Upon treatment with a nonlethal dose (600 mg/kg), WT mice exhibited an increase in serum transaminase levels. Histopathologically, centrilobular hepatic necrosis with leukocyte infiltration was evident at 10 and 24 h after APAP challenge. Moreover, mRNA expression of adhesion molecules, several chemokines, interferon‐γ (IFN‐γ), and inducible nitric oxide synthase (iNOS) was enhanced in the liver. On the contrary, serum transaminase elevation and histopathological changes were attenuated in TNF‐Rp55‐KO mice injected with APAP (600 mg/kg). The gene expression of all molecules except for IFN‐γ and iNOS was significantly attenuated in TNF‐Rp55‐KO mice. Moreover, anti‐TNF‐α neutralizing antibodies alleviated liver injury when administered at 2 or 8 h after but not at 1 h before APAP challenge to WT mice. Collectively, the TNF‐α–TNF‐Rp55 axis has pathogenic roles in APAP‐induced liver failure.

Opposite roles of neutrophils and macrophages in the pathogenesis of acetaminophen-induced acute liver injury

Neutrophils and macrophages infiltrate after acetaminophen (APAP)‐induced liver injury starts to develop. However, their precise roles still remain elusive. In untreated and control IgG‐treated wild‐type (WT) mice, intraperitoneal APAP administration (750 mg/kg) caused liver injury including centrilobular hepatic necrosis and infiltration of neutrophils and macrophages, with about 50% mortality within 48 h after the injection. APAP injection markedly augmented intrahepatic gene expression of inducible nitric oxide synthase (iNOS) and heme oxygenase (HO)‐1. Moreover, neutrophils expressed iNOS, which is presumed to be an aggravating molecule for APAP‐induced liver injury, while HO‐1 was mainly expressed by macrophages. All anti‐granulocyte antibody‐treated neutropenic WT and most CXC chemokine receptor 2 (CXCR2)‐deficient mice survived the same dose of APAP, with reduced neutrophil infiltration and iNOS expression, indicating the pathogenic roles of neutrophils in APAP‐induced liver injury. However, APAP caused more exaggerated liver injury in CXCR2‐deficient mice with reduced macrophage infiltration and HO‐1 gene expression, compared with neutropenic WT mice. An HO‐1 inhibitor, tin‐protoporphyrin‐IX, significantly increased APAP‐induced mortality, implicating HO‐1 as a protective molecule for APAP‐induced liver injury. Thus, CXCR2 may regulate the infiltration of both iNOS‐expressing neutrophils and HO‐1‐expressing macrophages, and the balance between these two molecules may determine the outcome of APAP‐induced liver injury.

Simultaneous determination of local anesthetics including ester-type anesthetics in human plasma and urine by gas chromatography–mass spectrometry with solid-phase extraction

The present study describes the simultaneous determination of seven different kinds of local anesthetics and one metabolite by GC-MS with solid-state extraction: Mepivacaine, propitocaine, lidocaine, procaine (an ester-type local anesthetics), cocaine, tetracaine (an ester-type local anesthetics), dibucaine (Dib) and monoethylglycinexylidide (a metabolite of lidocaine) were clearly separated from each other and simultaneously determined by GC-MS using a DB-1 open tubular column. Their recoveries ranged from 73-95% at the target concentrations of 1.00, 10.0 and 100 microg/ml in plasma, urine and water. Coefficients of variation of the recoveries ranged from 2.3-13.1% at these concentrations. The quantitation limits of the method were approximately 100 ng/ml for monoethylglycinexylidide, propitocaine, procaine, cocaine, tetracaine and dibucaine, and 50 ng/ml for lidocaine and mepivacaine. This method was applied to specimens of patients who had been treated with drip infusion of lidocaine, and revealed that simultaneous determination of lidocaine and monoethylglycinexylidide in the blood and urine was possible.

Reduced acetaminophen-induced liver injury in mice by genetic disruption of IL-1 receptor antagonist

Acetaminophen (APAP) induced increases in intrahepatic expression of interleukin (IL)-1α, IL-1β, and IL-1 receptor antagonist (IL-1ra), when administered intraperitoneally. These observations prompted us to define the pathophysiological roles of IL-1ra in APAP-induced liver injury. Compared with wild-type (WT) mouse-derived hepatocytes, IL-1ra-deficient (IL-1ra KO)-derived hepatocytes exhibited more resistance against APAP but not APAP-derived major toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Moreover, the amounts of a major APAP adduct (selenium-binding protein), an indicator of NAPQI generation from APAP, was significantly lower in IL-1ra KO mice than WT mice with depressed intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, the enzymes crucially involved in NAPQI generation from APAP. These observations would indicate that IL-1ra deficiency impaired APAP metabolism. IL-1α and IL-1β were expressed to similar extents in livers of untreated IL-1ra KO and WT mice. By contrast, the intranuclear amount of p65 of NF-κB, which can suppress the gene expression of CYP1A2, CYP2E1, and CYP3A11, was higher in untreated IL-1ra KO than WT mice. Moreover, when mice were intraperitoneally administered APAP (200 mg/kg), IL-1ra KO mice exhibited attenuated APAP-induced liver injury as evidenced by reductions in serum alanine transferase levels and histopathological changes such as centrilobular necrosis, hemorrhages, and leukocyte infiltration. Finally, when given 12 h before APAP challenge, IL-1α repressed the intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, eventually reducing APAP-induced liver injury, along with reduction in APAP adducts. Collectively, NF-κB was activated without any stimuli by the genetic disruption of IL-1ra, and suppressed cytochrome P450 enzyme expression, thereby reducing APAP-induced liver injury.

Screening and determination of methamphetamine and amphetamine in the blood, urine and stomach contents in emergency medical care and autopsy cases

Methamphetamine (MA) and amphetamine (AMP) were screened and their levels were determined using the Toxi-Lab thin-layer chromatography system and gas chromatography-mass spectrometry, respectively, in the blood, urine and stomach contents from 211 emergency medical care and 417 autopsy cases. MA and AMP were detected in 5 emergency medical cases, and the blood MA and AMP concentrations ranged from 0.697-0.041 micromol/100 g and from 0.0944-0.0003 micromol/100 g, respectively. MA and AMP were detected in 19 autopsy cases, in which blood MA and AMP concentration ranged from 14.3-0.123 micromol/100 g and from 0.256-0.0017 micromol/100 g, respectively. The autopsy cases included 5 cases of sudden death with blood MA concentration of less than 3 micromol/100 g. MA and AMP screening and determination in emergency medical care and autopsy cases provide useful information and are indispenable in clarifying the dimensions of MA abuse in Japan.

Use of REMEDi HS in Emergency Toxicology for a Rapid Estimate of Drug Concentrations in Urine, Serum, and Gastric Samples

The REMEDi HS is a broad spectrum drug identification system, designed for emergency toxicology screening and forensic applications. The total analysis time is about 20 min. The current library has 555 drugs and metabolites. The system has a software routine that uses an internal standard (IS) to perform quantitative analysis for target compounds when calibrators are available; further, response factors (RF) are supplied for a rapid estimate of drug concentrations when calibrators are unavailable. In the present study, The concentrations of six drugs (bromisovalum, ephedrine, hydroxyzine, diphenhydramine, ranitidine, and lidocaine) and a metabolite of lidocaine (glycinexylidide) were determined using both methods. The slopes of the regression lines between the rapid estimate method and the IS method were generally within 20% of unity, in agreement with the manufacturers claim. Semiquantitative estimates based on RF also showed good agreement with results obtained using multipoint calibration. These estimates were sufficient for clinical differentiation of routine and toxic levels. Our study demonstrated that the REMEDi HS is particularly useful for a rapid estimate of drug concentrations in the samples from emergency cases when calibrators are not readily available. Our study also showed that this system can be used for the therapeutic monitoring of ranitidine, bromisovalum, lidocaine, and diphenhydrmine.

Postmortem degradation of administered ethanol-d6 and production of endogenous ethanol: experimental studies using rats and rabbits

Deuterium-labeled ethanol-d6 was employed to study the metabolism and postmortem change of ethanol in putrefied organ tissues. First, 4 ml/kg body weight of 25% (w/v) solution of ethanol-d6 was administered orally to each of 15 rats. The heart blood and organs were collected 15-90 min after the administration and the ethanol-d6 was analyzed by head space gas chromatography/mass spectrometry. The ethanol-d6 concentration in the organ tissues reached its maximum at 15 min after the administration and then gradually declined, showing the same pattern as human ethanol metabolism. Ethanol-d6 (3 ml of the same solution/kg body weight) was injected into the vein of a rabbits ear (total of 12 rabbits). The rabbit was killed with carbon monoxide 30 min after the administration and the carcass was allowed to stand for 1-4 days at 30 degrees C in a moist chamber. The concentration of ethanol-d6 decreased moderately. Postmortem ethanol and 1-propanol concentrations, in contrast, showed marked increases 2.5 days and more after sacrifice in line with the degree of putrefaction of each organ tissue including skeletal muscle. This suggests the postmortem activation of micro-organism activity. These results indicate that ethanol concentrations in cadaver tissues must be carefully assessed with due consideration of postmortem degradation and production.