Mizuho Nosaka

Absence of IFN-γ accelerates thrombus resolution through enhanced MMP-9 and VEGF expression in mice

Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism, a leading cause of death in individuals with DVT. Several lines of evidence indicate proinflammatory cytokines such as TNF-α are involved in thrombus formation and resolution, but the roles of IFN-γ remain unclear. To address this issue, we performed ligation of the inferior vena cava to induce DVT in WT and IFN-γ-deficient (Ifng-/-) mice. In WT mice, intrathrombotic IFN-γ levels were elevated progressively as the postligation interval was extended. Thrombus size was substantially smaller at 10 and 14 days in Ifng-/- mice than in WT mice. Intrathrombotic collagen content was remarkably reduced at more than 10 days after the ligation in Ifng-/- mice compared with WT mice. The expression and activity of MMP-9, but not MMP-2, was higher at the late phase in Ifng-/- mice than in WT mice. Moreover, intrathrombotic recanalization was increased in Ifng-/- mice, with enhanced Vegf gene expression, compared with that in WT mice. Activation of the IFN-γ/Stat1 signal pathway suppressed PMA-induced Mmp9 and Vegf gene expression in peritoneal macrophages. Furthermore, administration of anti-IFN-γ mAbs accelerated thrombus resolution in WT mice. Collectively, these findings indicate that IFN-γ can have detrimental roles in thrombus resolution and may be a good molecular target for the acceleration of thrombus resolution in individuals with DVT.

Critical Role of TNF-α-Induced Macrophage VEGF and iNOS Production in the Experimental Corneal Neovascularization

PURPOSE We evaluated the roles of tumor necrosis factor (TNF)-α in alkali-induced corneal neovascularization (CNV). METHODS CNV was induced by alkali injury and compared in wild-type (WT) BALB/c mice, and TNF receptor 1-deficient (TNF-Rp55 KO) counterparts, or in mice treated with TNF-α antagonist and recombinant TNF-α. Angiogenic factor expression and leukocyte accumulation in the early phase after injury were quantified by real-time PCR and immunohistochemical analysis, respectively. RESULTS Alkali injury augmented the intraocular mRNA expression of TNF-α and its receptor, together with a transient macrophage and neutrophil infiltration. Compared to WT mice, TNF-Rp55 KO mice exhibited reduced CNV. Intraocular F4/80-positive macrophages and Ly-6G-positive neutrophils infiltration did not change in KO mice compared to WT mice after the injury. Alkali injury induced a massively increased intraocular mRNA expression of angiogenic factors, including vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), interleukin (IL)-6, E-selectin, and intercellular adhesion molecule (ICAM)-1 in WT mice, whereas these increments were retarded severely in KO mice. Immunofluorescence analysis demonstrated that F4/80-positive cells expressed VEGF and iNOS. Moreover, TNF-α enhanced VEGF and iNOS expression by peritoneal macrophage from WT, but not KO mice. Topical application of TNF-α antagonist reduced CNV, while topical application of recombinant TNF-α enhanced it. CONCLUSIONS TNF-Rp55-KO mice exhibited impaired alkali-induced CNV through reduced intracorneal infiltrating macrophage VEGF and iNOS expression.

Time-dependent organic changes of intravenous thrombi in stasis-induced deep vein thrombosis model and its application to thrombus age determination

Using histochemical and immunohistochemical techniques, we examined the intrathrombotic collagen contents and the appearance of hemosiderin-positive cells, neovessels, and myofibroblasts in a stasis-induced venous thrombosis model. The intrathrombotic collagen deposition area occupied about 20% at 5 days, and exceeded 80% at 21 days after ligation of the inferior vena cava (IVC). Hemosiderin-positive cells in the thrombus first appeared at 3 days in only one of the five samples, and positive cells were constantly detected in all thrombi at 5 days or later. CD31-positive neovessels in the thrombus first appeared at 5 days in one of five samples and were detected in all samples after 10 days. At 7 days, alphaSMA-positive myofibroblasts at the periphery of the thrombus first appeared in three of five samples, and were detected and enhanced time-dependently in all samples after 10 days. These observations demonstrated that these markers would be applicable for thrombus age determination.

Aberrant Expression of Histo-blood Group A Type 3 Antigens in Vascular Endothelial Cells in Inflammatory Sites

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1–4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.

Exaggerated arsenic nephrotoxicity in female mice through estrogen-dependent impairments in the autophagic flux

Gender is one of the essential factors in the development of various diseases and poisoning. Therefore, we herein examined gender differences in sodium arsenite (NaAsO2)-induced acute renal dysfunction. When male and female BALB/c mice were subcutaneously injected with NaAsO2 (12.5mg/kg), serum and urinary markers for proximal tubular injury were significantly higher in female mice than in male ones. NaAsO2-induced histopathological alterations were consistently more evident in females than in males. Ovariectomy, but not orchiectomy significantly attenuated NaAsO2-induced renal injury. These results imply that the hypersusceptibility of female mice is attributed to estrogen signals. NaAsO2 suppressed the autophagic flux in tubular cells through the activation of ERK. Enhancements in the activation of ERK were significantly greater in females than in males, with the eventual accumulation of LC3-II and P62 in the kidneys, implying that the autophagic flux is impaired in females. The IL-6/STAT3 signaling pathway had protective roles in NaAsO2-induced nephrotoxicity through the suppression of ERK activation. Despite the absence of differences in intrarenal IL-6 expression between male and female mice, STAT3 was less activated with enhanced SOCS3 expression in females than in males. An in vitro study using mProx24 cells revealed that the estrogen treatment induced SOCS3 expression, and eventually suppressed the autophagic flux, as evidenced by greater increases in the accumulation of LC3-II and p62 with ERK activation, which was canceled by the knockdown of Socs3. Collectively, these results indicate that estrogen has a negative impact on the development of NaAsO2 nephrotoxicity through its suppression of the autophagic flux.

AG490, a Jak2 inhibitor, suppressed the progression of murine ovarian cancer.

Ovarian cancer is the major cause of cancer death among female genital malignancies, and requires developing novel therapeutic measures. Immune escape and acquisition of tolerance by tumor cells are essential for cancer growth and progression. An immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) overexpression in tumors is essential for host immune tolerance. Janus-activated kinase-signal transducer and activator of transcription (JAK-STAT) pathway is involved in various kinds of tumor biology. Thus, we examined the effects of STAT1 inhibition by AG490 (a JAK2 inhibitor) on ovarian cancer progression in mice. In vitro study, IFN-γ treatment up-regulated Ido mRNA expression with STAT1 activation in OV2944-HM-1 cells, whereas AG490 treatment significantly inhibited this effect with the suppression of STAT1 phosphorylation. In vivo model, OV2944-HM-1 cells were intraperitoneally/subcutaneously transplanted into syngeneic immunocompetent female mice. AG490 treatment significantly suppressed subcutaneous tumor growth, compared with control. Consistently, in mice intraperitoneally inoculated HM-1 cells, the same treatment significantly improved survival rate with the reduced number of intraperitoneal tumors. Actually, intratumoral IDO expression was significantly suppressed with the reduction of STAT1 activation in AG490-treated mice. Moreover, in tumor microenvironment of mice treated with AG490, the accumulation of anti-tumor leukocytes such as CD8(+) T-cells, M1 macrophages, and NK cells was apparently exaggerated with the reciprocal reduction of regulatory T cells. Furthermore, intratumoral expression of anti-tumor cytokines such as IL-1α, IL-1β and IL-12 expression was significantly enhanced in mice treated with AG490. Collectively, JAK/STAT signal pathways may be good molecular target for immunotherapy of ovarian cancer.

Distribution of carbamazepine and its metabolites carbamazepine-10,11-epoxide and iminostilbene in body fluids and organ tissues in five autopsy cases

Carbamazepine (CMZ) and its metabolites, carbamazepine-10,11-epoxide (CMZepo) and 2,2’-iminostilbene (Imi), were simultaneously assayed by gas chromatography-mass spectrometry (GC-MS) in body fluids and organ tissues taken from victims in five autopsy cases. Clomipramine-HCl was added to each specimen as internal standard (IS); target compounds and IS were simultaneously extracted with an Extrelut NT column before analysis by GC-MS. The concentrations of CMZ were generally much higher in organ tissues than in blood and urine, and were higher in the liver than in the lung in three cases. The concentrations of metabolites CMZepo and Imi were lower than those of CMZ in all body fl uid and organ tissue specimens; the Imi concentrations in the brain were higher than CMZepo concentrations in four cases. Imi concentrations in the kidney, however, were lower than those of CMZepo in four cases. In the lung and liver, the concentrations of Imi did not show a consistent trend; those of Imi were higher or lower than those of CMZepo, but both metabolites were not at negligible levels. To our knowledge, this is the fi rst demonstration of the distribution of Imi among human body fluids and tissues.

Postmortem distribution of chlorpyrifos-methyl, fenitrothion, and their metabolites in body fluids and organ tissues of an intoxication case

We herein report a fatal intoxication case caused by the ingestion of the insecticides chlorpyrifos-methyl (CPFM) and fenitrothion (MEP). A 70-year-old man was found dead in his house and a cup containing a small amount of agricultural chemicals was on the table near his body. External and internal examinations revealed no injuries. In a gas chromatography-mass spectrometry (GC-MS) screening test, CPFM, MEP, and their metabolites, 3,5,6-trichloro-2-pyridinol (TCPY) and 3-methyl-4-nitrophenol (3MNP), respectively, were qualitatively detected in his stomach contents. The concentrations (µg/g) of CPFM, TCPY, MEP, and 3MNP in the extracts of each body fluid and organ tissue were assessed by GC-MS and were as follows: 27.8, 56.2, 17.2, and 2.82 (heart blood); 6.60, 42.9, 1.80, and 2.59 (peripheral blood); 0.0821, 45.9, 2,09, and 102 (urine); 21.4, 26.6, 76.2, and 3.83 (brain (frontal portion)); 16.1, 101, 9.67, and 1.26 (liver); 7.45, 101, 21.4, and 26.1 (right kidney); and 73,500, 9750, 232,000, and 1880 (stomach contents), respectively. Based on these results and autopsy findings, the cause of death was acute fatal intoxication by CPFM and MEP.

Fatal acute intoxication of accidentally ingested nifedipine in an infant – A case report

A fatal case of acute nifedipine intoxication in a two-year-old boy is presented. The boy accidentally orally ingested an unknown amount of his grandfathers nifedipine (40mg/tablet), mistaking it for a ramune confectionery. Despite intensive medical treatment, his death was confirmed at 31h after the accidental ingestion. The forensic autopsy revealed that there were neither pathological alterations or injuries in all of the organs. Toxicologically, nifedipine could be detected at the concentrations of 0.463, 0.669 and 13.0μg/g in cardiac blood, peripheral blood and stomach contents, respectively. These concentrations were evaluated as fatal levels, and the cause of death was diagnosed as acute nifedipine intoxication. Recently, the number of infants and children who accidentally ingest drugs in the home is increasing. This case report prompts forensic pathologists and toxicologists to emphasize that children are always exposed to the risk of accidental drug ingestion in daily life.

High concentration of methidathion detected in a fatal case of organophosphate-poisoning

We report a case of fatal intoxication caused by the ingestion of an organophosphate pesticide, methidathion (DMTP). An 80-year-old male was found dead in his bed. Forensic autopsy revealed no remarkable morphological changes. However, in a toxicological screening test, methidathion was qualitatively detected in extracts of stomach contents. Concentrations of methidathion (μg/g) in body fluids and organ tissues, determined by gas chromatography-mass spectrometry, were as follows; 66.2 in heart blood, 8.33 in peripheral blood, 8.80 in urine, 2000 in the brain (frontal lobe), 4800 in the left lung, 810 in the liver, 150 in the left kidney, and 64,000 in the stomach contents (total 1.9 g). These results strongly suggested that the victim orally ingested methidathion. Additionally, xylene was determined in body fluids and organ tissues. From the toxicological data together with autopsy findings, the cause of his death was diagnosed as acute poisoning by an emulsion of methidathion.