Chronic ethanol consumption enhances inducible endothelium-dependent hyperpolarizing factor-mediated relaxation in the rat artery.

The inducible endothelium-dependent hyperpolarizing factor (iEDHF) pathway is activated as a compensatory response to adverse changes in the body. It causes vasorelaxation and maintains circulatory homeostasis in the organs. Small to moderate quantities of ethanol enhance vascular relaxation. However, its mechanism and the involvement of the iEDHF pathway in this process are unknown. Therefore, we studied iEDHF-mediated, acetylcholine-induced, endothelium-dependent relaxation in the superior mesenteric arteries (SMAs) of rats chronically fed ethanol. Rats were administered a standard diet (S-Control group), Lieber's control diet (L-Control group), or Lieber's ethanol diet (EtOH group). SMA relaxation was assessed by isometric tension measurements. Arachidonate 15-lipoxygenase (ALOX15) and soluble epoxide hydrolase (sEH) were determined by immunoblot. Acetylcholine-induced, endothelium-dependent relaxation was significantly greater in the EtOH than the control groups. These differences persisted after PGI2 and NO blockade. Thus, the increase in acetylcholine-induced relaxation was EDHF-mediated. In the EtOH group, however, it was prevented by iEDHF inhibitors. ALOX15 and sEH protein expression levels were higher in the EtOH than the L-Control group. The increase in acetylcholine-induced relaxation by chronic ethanol consumption was mediated by the iEDHF pathway. This mechanism may compensate for the blood pressure elevation induced by ethanol. This study suggests that iEDHF is induced during proper drinking and may help prevent the onset of cardiovascular conditions.

Relationship Between EGFR Expression in Oral Cancer Cell Lines and Cetuximab Antibody-dependent Cell-mediated Cytotoxicity.

BACKGROUND/AIM: Cetuximab treatment targets the epidermal growth factor receptor (EGFR) overexpressed in oral cancer. This study aimed to investigate the anti-tumour activity of cetuximab against oral cancer cell lines with respect to antibody-dependent cell-mediated cytotoxicity (ADCC), and determine the correlation between ADCC and EGFR expression. MATERIALS AND METHODS: EGFR expression in oral cancer cells was measured by quantitative flow cytometric analysis and immunohistochemistry. ADCC activity was measured by 4-h calcein release assays. RESULTS: Cetuximab-mediated ADCC against oral cancer cells was detectable at a concentration of 0.1 µg/ml. A high correlation was observed between the number of EGFR molecules on the surface of oral cancer cells and ADCC (correlation coefficient: 0.847; p=0.032). CONCLUSION: ADCC is an important mechanism underlying the therapeutic effect of cetuximab, and EGFR expression in tumour cells might serve as a predictive marker to evaluate the effect of cetuximab treatment.

Enhancement of Tissue Factor Expression in Monocyte-Derived Dendritic Cells by Pentraxin 3 and Its Modulation by C1 Esterase Inhibitor.

BACKGROUND: We have previously shown that human monocyte-derived dendritic cells (moDCs) may participate in immune system-mediated hypercoagulable state through enhanced tissue factor (TF) expression and that the complement system may be involved in this process. OBJECTIVES: The aim of this study was to explore the role of pentraxin 3 (PTX3) and the complement system in enhanced TF expression in moDCs. METHODS: moDCs were generated from isolated human monocytes. PTX3 levels in whole human blood supplemented with moDCs were determined after lipopolysaccharide (LPS) stimulation. PTX3 release by the generated moDCs upon LPS stimulation was also assessed. The effect of PTX3 on whole blood coagulation was investigated using thromboelastometric analysis. TF expression in stationary moDCs treated with LPS and/or PTX3 was determined by measuring TF activity. The effect of complement inhibitors on TF activity in moDCs treated with LPS and/or PTX3 under low-shear conditions was evaluated. RESULTS: PTX3 levels were higher in whole blood supplemented with moDCs than in the presence of monocytes and were further elevated by LPS stimulation. PTX3 release from generated moDCs was also increased by LPS stimulation. PTX3 reduced whole blood coagulation time in a dose-dependent manner. However, PTX3 did not increase TF expression in stationary moDCs. Under low-shear conditions, PTX3 increased TF expression in moDCs. C1 esterase inhibitor (C1-inh) suppressed this effect. CONCLUSIONS: PTX3 might have a thrombophilic activity and enhance TF expression in moDCs under low-shear conditions. Furthermore, suppression of moDC-associated hypercoagulability by C1-inh might be partly ascribed to its inhibitory effect on PTX3.

Sudden unexpected infantile death due to undiagnosed ventricular septal defect-associated heart failure with single coronary artery.

Ventricular septal defect (VSD) generally has a good prognosis unless complicated by heart failure (HF). We report a case of sudden infant death because of clinically undiagnosed VSD in a seemingly healthy 16-day-old boy. Although a cardiac murmur was auscultated at birth, detailed clinical examination was not performed. Medicolegal autopsy revealed a perimembranous large VSD with a single coronary artery. The infant was diagnosed to have had HF based on the increased weight of the heart and extremely high serum brain natriuretic peptide levels. Histological examination revealed the degeneration of cardiomyocytes. The large VSD was thought to be the major cause of HF, although single coronary artery-associated cardiomyopathy might have also partially contributed to it. The decline in the physiological neonatal pulmonary resistance, which occurs over the first 1 or 2 weeks following birth, led to the acute progression of HF, resulting in circulatory collapse and sudden death. Detailed clinical examination should be performed for neonates with cardiac murmur to prevent avoidable death.

Experimental hypercoagulable state induced by tissue factor expression in monocyte-derived dendritic cells and its modulation by C1 inhibitor.

The crosstalk between immune and coagulation systems plays pivotal roles in host defense, which may involve monocyte-derived dendritic cells (moDCs). Our objectives were to elucidate the role of moDCs in coagulation under inflammatory conditions and the involvement of the complement system. We assessed the effects of lipopolysaccharide (LPS)-stimulated moDCs on coagulation using whole blood thromboelastometry in the presence of complement inhibitors. The sum of clotting time and clot formation time (CT plus CFT) in whole blood thromboelastometry was significantly more reduced in the presence of moDCs than in the absence of monocytes or moDCs and in the presence of monocytes, indicating a more potent coagulability of moDCs. The mRNA expression of coagulation-related proteins in moDCs was analyzed by quantitative PCR, which showed an increase only in the mRNA levels of tissue factor (TF). TF protein expression was assessed by western blot analysis and an activity assay, revealing higher TF expression in moDCs than that in monocytes. The in vitro moDC-associated hypercoagulable state was suppressed by a TF-neutralizing antibody, whereas LPS enhanced the in vitro hypercoagulation further. C1 inhibitor suppressed the in vitro LPS-enhanced whole blood hypercoagulability in the presence of moDCs and the increased TF expression in moDCs. These results suggest a significant role of moDCs and the complement system through TF expression in a hypercoagulable state under inflammatory conditions and demonstrate the suppressive effects of C1 inhibitor on moDC-associated hypercoagulation.

Acute ethanol intoxication suppresses pentraxin 3 expression in a mouse sepsis model involving cecal ligation and puncture.

Acute ethanol intoxication impairs immunological reactions and increases the risk of sepsis; however, the underlying mechanism remains unclear. Pentraxin (PTX) 3 is a humoral pattern recognition receptor whose levels rapidly increase in response to inflammation. PTX3 production is triggered by tumor necrosis factor (TNF)-α and is mediated by c-Jun N-terminal kinase (JNK). As PTX3 exerts protective effects against sepsis as well as acute lung injury, we investigated whether acute ethanol exposure exacerbates sepsis by altering PTX3 expression. Sepsis was induced in C57/BL6 mice by cecal ligation and puncture (CLP) after ethanol/saline administration. Survival rates were significantly lower in ethanol-treated than in saline-treated mice. Increased vascular permeability and attenuation of PTX3 expression were observed in the lungs of ethanol-treated mice 4 h after CLP. Concomitant with a delayed increase of plasma TNF-α in ethanol-treated mice, plasma PTX3 was also suppressed in the early phase of sepsis. Although TNF-α level in ethanol-treated mice exceeded that in saline-treated mice 16 h after CLP, PTX3 levels were still suppressed in the former group. JNK phosphorylation in lung tissue was suppressed in both groups 4 and 16 h after CLP. Furthermore, JNK phosphorylation in ethanol-treated human umbilical vein endothelial cells was suppressed even in the presence of exogenous TNF-α, resulting in inhibition of PTX3 mRNA and protein expression. Our results suggest that ethanol suppresses de novo PTX3 synthesis via two mechanisms - i.e., suppression of TNF-α production and inhibition of JNK phosphorylation. PTX3 suppression may therefore contribute to exacerbation of sepsis in acute ethanol intoxication.

Relevance of hemolysis-induced tissue factor expression on monocytes in soft clot formation in alcohol-containing blood.

The fluidity of cadaveric blood is an important characteristic in the post-mortem examination of cases of asphyxial death. Although it is empirically known that soft blood clots are present in cadaveric blood containing alcohol, the relationship between such clots and blood alcohol is unclear. We addressed this issue through in vitro studies using blood collected from healthy volunteers. Assessment of global hemostasis by rotational thromboelastometry revealed that ethanol treatment enhanced the procoagulant activity of whole blood. However, ethanol inhibited epinephrine-induced platelet aggregation, whereas plasma levels of von Willebrand factor and the activity of coagulation factors VIII and IX were unaffected. In contrast, tissue factor (TF) activity was higher in plasma obtained from ethanol-treated whole blood than that in plasma from untreated blood. Ethanol induced hemolysis of red blood cells, and the consequent hemoglobin (Hb) release promoted de novo synthesis of TF in isolated monocytes, as determined by real-time reverse transcription PCR, western blotting, and flow cytometry. However, ethanol itself did not induce TF expression in monocytes. Given that TF activates the extrinsic coagulation pathway and amplifies hemostatic reactions, Hb-induced TF expression in monocytes might contribute to soft blood clot formation.

[Two autopsy cases in which previous surgery facilitated the positive identification of decomposed bodies]. / Dve pitvy, ve kterých predchozí operace usnadnila ztotoznení posmrtne dekomponovaných tel.

The positive identification of decomposed corpses is often difficult. We describe two autopsy cases in which medical materials, which had been implanted during previous surgical treatments, facilitated positive identification. The discovery of decomposed corpses is increasingly common in Japan, due to the increasing number of lonely deaths. Implanted medical materials and devices may be a useful tool for personal identification in the near future.

Sudden Death Due to Bilateral Pulmonary Thromboembolism Following Laparoscopic Cholecystectomy.

Cases of sudden death due to pulmonary thromboembolism (PTE) following laparoscopic surgery are very rare. The risk factors for PTE include sex, operation duration, age, obesity, and underlying diseases. The development of thromboprophylaxis according to specific risk factors has contributed to the decrease in postoperative mortality. Here, we describe the case of a 50-year-old patient with sudden death due to PTE at 24 hours after laparoscopic cholecystectomy. The origin of the thrombi were bilateral deep vein thromboses in both the lower extremities. No severe risk factors for PTE were detected in the patient, and pneumatic compression devices were used during the surgery for thromboprophylaxis. We believe that the accumulation of minor risk factors may have contributed to the onset of PTE. Hence, a more cautious assessment of the risk factors for PTE prior to surgery is required in such cases.

Therapeutic approaches for treating hemophilia A using embryonic stem cells.

Hemophilia A is an X-linked rescessive bleeding disorder that results from F8 gene aberrations. Previously, we established embryonic stem (ES) cells (tet-226aa/N6-Ainv18) that secrete human factor VIII (hFVIII) by introducing the human F8 gene in mouse Ainv18 ES cells. Here, we explored the potential of cell transplantation therapy for hemophilia A using the ES cells. Transplant tet-226aa/N6-Ainv18 ES cells were injected into the spleens of severe combined immunodeficiency (SCID) mice, carbon tetrachloride (CCl4)-pretreated wild-type mice, and CCl4-pretreated hemophilia A mice. F8 expression was induced by doxycycline in drinking water, and hFVIII-antigen production was assessed in all cell transplantation experiments. Injecting the ES cells into SCID mice resulted in an enhanced expression of the hFVIII antigen; however, teratoma generation was confirmed in the spleen. Transplantation of ES cells into wild-type mice after CCl4-induced liver injury facilitated survival and engraftment of transplanted cells without teratoma formation, resulting in hFVIII production in the plasma. Although CCl4 was lethal to most hemophilia A mice, therapeutic levels of FVIII activity, as well as the hFVIII antigen, were detected in surviving hemophilia A mice after cell transplantation. Immunolocalization results for hFVIII suggested that transplanted ES cells might be engrafted at the periportal area in the liver. Although the development of a safer induction method for liver regeneration is required, our results suggested the potential for developing an effective ES-cell transplantation therapeutic model for treating hemophilia A in the future.

An autopsy case of complete adipocere formation.

A car containing a male corpse with complete adipocere formation was found at the bottom of a lake. The deceased had presumably driven into the lake 7 years earlier. The surface of the deceased was unusually hard and firm like a gypsum board, and the entire internal viscera had turned into adipocere. Since the time required for adipocerous changes depends largely on environmental conditions, we considered the key conditions, namely, water temperature, pH, and oxygen content. In our case, cold, acidic water may have delayed adipocere formation, thus necessitating a long period of time for completeness. On the other hand, anoxic conditions and the peculiar environment of a lake bottom presumably contributed to complete adipocere formation.

Potential Biomarker Peptides Associated with Acute Alcohol-Induced Reduction of Blood Pressure.

The purpose of this study was to explore the peptides that are related to acute reduction of blood pressure after alcohol drinking. Venous blood was collected from male healthy volunteers before and after drinking white wine (3 ml/kg weight) containing 13% of ethanol. Peptidome analysis for serum samples was performed using a new target plate, BLOTCHIP®. Alcohol caused significant decreases in systolic and diastolic blood pressure levels at 45 min. The peptidome analysis showed that the levels of three peptides of m/z 1467, 2380 and 2662 changed significantly after drinking. The m/z 1467 and 2662 peptides were identified to be fragments of fibrinogen alpha chain, and the m/z 2380 peptide was identified to be a fragment of complement C4. The intensities of the m/z 2380 and m/z 1467 peptides before drinking were associated with % decreases in systolic and diastolic blood pressure levels at 45 min after drinking compared with the levels before drinking, while there were no significant correlations between the intensity of the m/z 2662 peptide and % decreases in systolic and diastolic blood pressure levels after drinking. The m/z 1467 and 2380 peptides are suggested to be markers for acute reduction of blood pressure after drinking alcohol.

sHLA-G and sHLA-I levels in follicular fluid are not associated with successful implantation.

In the field of in vitro fertilization (IVF), useful markers for the prediction of successful implantation for oocyte or embryo selection are essential. It has been reported that sHLA-G (sHLA-G1/HLA-G5) could be detected in the supernatant of the fertilized embryo and in follicular fluid samples (FFs), and that the presence of sHLA-G was related to successful implantation. If sHLA-G could be used as a marker of oocyte selection from multiple FFs, oocytes could be selected without physical contact, thus reducing the likelihood of damage. To investigate the potential for sHLA-G as a marker of oocyte selection from multiple FFs in one patient, protein levels of total protein, sHLA-G, and sHLA-I (sHLA-A, B, and C) were examined in FFs. The variation among multiple FFs in total protein level and sHLA-G level was not related to successful pregnancy. The average sHLA-I levels did not differ in the successful implantation and unsuccessful implantation groups, indicating that sHLA-I levels were not related to successful pregnancy. Furthermore, sHLA-G in FFs was not detected by western blotting, despite being detected by ELISA, while sHLA-I was detected by both ELISA and western blot. These data suggest that sHLA-G in FF might not be a useful marker for oocyte selection as measurements of sHLA-G were inconsistent and there was no association with successful pregnancy. Further, more rigorously tested ELISA systems for detecting sHLA-G in body fluids are necessary before the utility of sHLA-G for diagnosis can be established.

[Effect of alcohol on vascular function].

Vascular function is regulated by a balance of vasoconstriction and vasorelaxation. Disorder in this balance due to alcohol consumption causes various clinical conditions. In this review, we discuss the effects of acute and chronic ethanol consumption on vascular responses, including vasoconstriction, endothelium-dependent vasorelaxation, and nerve-mediated vasorelaxation. Acute ethanol administration induces vasoconstriction in ethanol-naive animals in vitro. Furthermore, ethanol can both potentiate and suppress agonist-induced Ca(2+)-dependent vasoconstriction. Moreover, ethanol augments Ca(2+)-independent vasoconstriction by increasing Ca2+ sensitivity. Endothelium-dependent relaxation is mediated by the nitric oxide (NO) pathway and the endothelium-derived hyperpolarizing factor (EDHF) pathway. Acute ethanol treatment inhibits both NO- and EDHF-mediated relaxation. Furthermore, acute ethanol ingestion can also potentiate and suppress calcitonin gene-related peptide (CGRP)-induced nerve-mediated relaxation. These opposing effects may be due to differences in species or vascular beds. Thus, acute ethanol treatment decreases vasorelaxation, thereby shifting the contraction-relaxation balance towards contraction. Combined, these effects are one mechanism by which acute heavy alcohol consumption causes circulatory disturbances such as vasospasms or ischemic heart disease. In contrast, chronic low-dose ethanol has no effect on vasoconstriction, whereas chronic high-dose ethanol increases vasoconstriction. Additionally, chronic ethanol intake has diminished, unchanged, and even increased effects on nerve-mediated relaxation; therefore, conclusions on these effects are not possible at present. Interestingly, chronic low-dose ethanol administration enhanced endothelium-dependent relaxation; however, higher doses inhibited these responses. Therefore, regular or light-to-moderate alcohol intake increases vasorelaxation and may suppress elevated blood pressure, whereas chronic heavy alcohol consumption may raise blood pressure, causing various clinical conditions.

The Inhibitory Effect of Ethanol on Interleukin-1 ß-Induced Suppression of Contractile Response in the Rat Superior Mesenteric Artery.

Interleukin (IL)-1 ß is a cytokine that is upregulated by the pro-inflammatory bacterial endotoxin lipopolysaccharide. This study examined the effect of ethanol on IL-1 ß-mediated suppression of phenylephrine-induced contractility and inducible nitric oxide synthase (iNOS) expression in the rat superior mesenteric artery (SMA). IL-1 ß suppressed the phenylephrine-induced contractile response, and this effect was inhibited by ethanol. The IL-1 ß-mediated effects were also blocked by cycloheximide, an inhibitor of protein synthesis, as well as AMT and 1400W, which are iNOS inhibitors, and PTIO, an NO scavenger. However, indomethacin, a cyclooxygenase (COX) inhibitor that promotes NO-independent vasodilation, did not affect IL-1 ß-mediated suppression of the contractile response. Western blot analysis revealed that iNOS levels in SMA were upregulated by IL-1 ß and inhibited by ethanol (50 and 100 mM). These results indicate that the suppression of the SMA contractile response by IL-1 ß requires iNOS activity, but not COX-2. Furthermore, these data suggest that ethanol inhibits the effects of IL-1 ß on the contractile response via inhibition of iNOS, rather than COX-2.

Asphyxial death related to postextraction hematoma in an elderly man.

We here report an autopsy case of a man in his seventies who died from asphyxia due to compression of the trachea caused by postextraction bleeding after extraction of his left mandibular third molar by a dentist in private practice. On the morning after the tooth extraction, he had complained of dyspnea and became unconscious at home. Although he was brought to the emergency room by ambulance, he died 7 days later without regaining consciousness. Autopsy examination revealed that the lingual side of the alveolar bone was fractured at the extraction socket. Moreover, subcutaneous bleeding that extended from the extraction socket to the thyrohyoid ligament in the cervical region and deviation of the epiglottis due to the bleeding were observed. Histological findings revealed liver cirrhosis; there were no significant findings in other organs. On the basis of these findings, we concluded that alveolar bone fracture occurred during the extraction and that the bleeding spread to the cervical region. Thus, the patient had died from asphyxia resulting from airway obstruction caused by cervical subcutaneous bleeding derived from postextraction bleeding. We emphasize that tooth extraction may cause fatal complications in patients with bleeding tendencies, particularly in the elderly.

Respiratory complications of Ehlers-Danlos syndrome type IV.

We describe a case of Ehlers-Danlos syndrome (EDS) type IV in a male in early half in his twenties, who experienced recurrent and eventually fatal pulmonary hemorrhage. EDS type IV is a rare disorder of type III collagen synthesis that is characterized by unusual facies, thin translucent skin with a venous vascular pattern, easy bruising, and hypermobility of the small joints. Autopsy findings showed hypermobility of the joints and distensibility of the skin. Microscopically, the abdominal skin showed substantially decreased dermal thickness. Moreover, the reticular dermis showed fine collagen bundles and large interstitial spaces compared with the skin from a normal control that showed large collagen bundles. Individual elastic fibers were also thicker than those observed in the skin of a normal control. The thoracic aorta showed thin adventitia and a relative increase in elastic fibers. The parenchyma of both the lungs showed markedly diffuse hemorrhage with hemosiderin-laden alveolar macrophages or old thrombi and organized thrombi in the small bronchi. Furthermore, both sections of the lung showed multiple fibrous nodules containing benign metaplastic bone. Vascular wall disruption and tearing of the vessel walls in the lung parenchyma were also observed. We concluded that EDS type IV led to the patient's death because of pulmonary hemorrhage. Because this syndrome resulted in the patient's death from arterial and bowel rupture, it is important to consider EDS as a potential cause of sudden death.

[Effect of ethanol on nerve dependent vasorelaxation and vasoconstriction].

Vascular tone is controlled by endothelium and nerves innervating blood vessels. Endothelium releases various substances such as PGI2 (Prostacyclin), NO (nitric oxide), TXA2 (thromboxane A2) and ET-1 (endothelin-1). It is well documented that ethanol affects vascular responses mediated by these substances. On the other hand, peripheral vascular tone is also regulated not only by sympathetic adrenergic nerves but also by nonadrenergic noncholinergic (NANC) nerves. NANC nerves include NO nerves and sensory nerves which release calcitonin gene-related peptide (CGRP) and nerve-mediated hyperpolarizing factor (NDHF). However, effect of ethanol on nerve-mediated vascular responses remains poorly understood. Therefore, this review summarizes the effect of ethanol on the sympathetic adrenergic nerve-mediated vasoconstriction and the sensory nerve-mediated vasorelaxation.

Expression of coagulation factors from murine induced pluripotent stem cell-derived liver cells.

A protocol to differentiate liver cells from induced pluripotent stem (iPS) cells is being established. However, the ability of these differentiated iPS cells to express liver-specific proteins, such as coagulation cascade and related factors, has yet to be assessed. This study evaluated whether liver-like populations differentiated from murine iPS cells gain the ability to produce coagulation-related factors. Following differentiation of murine iPS cells into hematopoietic-like and liver-like embryoid bodies, we assessed gene expression profiles for coagulation-related markers, including fibrinogen, factors II, V, VII, VIII, IX, X, XI, XII, and XIIIß, protein C, protein S, antithrombin, plasminogen, von Willebrand factor, and ADAMTS13 by real-time reverse transcription PCR. Liver-like embryoid bodies demonstrated strong expression levels of nearly all the coagulation-related genes assessed, compared with undifferentiated iPS cells and hematopoietic-like embryoid bodies. We also confirmed efficient translation and secretion of fibrinogen and albumin (hepatocyte-specific marker proteins) into the conditioned medium by these differentiated cells, suggesting successful differentiation of iPS cells into the liver lineage. These findings suggest that iPS cells can be differentiated into liver-like populations that express coagulation-related factors. Liver-like embryoid bodies may provide a source for cell-based therapies directed toward liver diseases, including coagulation factor deficiencies in the future.