A lethal case of massive hemorrhage after percutaneous liver biopsy in a patient with thrombasthenia.

Percutaneous needle liver biopsy is an important procedure in the diagnosis of and assessment of the severity of liver diseases. Although liver biopsy is considered to be a relatively safe procedure, there are occasional cases of death due to massive bleeding after liver biopsy. Thrombasthenia is a disease in which bleeding occurs in the mucosa and skin due to platelet dysfunction. A 60-year-old female was admitted for a liver biopsy for further investigation after an abnormal liver function test. She was diagnosed with thrombasthenia and was being treated with oral tranexamic acid and carbazochrome. Blood tests showed little decrease of platelet count and no abnormalities of blood coagulability. Approximately ten hours after the liver biopsy, the patient complained of nausea and lightheadedness, followed by decreased blood pressure and decreased consciousness. An emergent abdominal CT scan showed a large amount of blood in the abdominal cavity. The patient died despite multidisciplinary treatment, and a forensic autopsy was performed. At internal examination, approximately 2,620 mL of dark red blood was accumulated in the abdominal cavity. A puncture wound led 1.8 cm into the liver from the surface of the liver, and no major vascular damage was observed. The cause of death was considered to be blood loss due to bleeding from the puncture wound. Even if the platelet count is normal, such as in a case of thrombasthenia, the risk of bleeding should not be underestimated. Careful attention should be paid when performing liver biopsy in a patient with risk factors.

Cadavers found outdoor in whom fungal growth was observed on the body surface: Consideration of the role of mycology in forensic medicine.

In forensic case work, fungal growth is occasionally observed on decomposed cadavers, however, the isolation of these organisms is not always carried out routinely. The usefulness of investigating fungi isolated from corpses in forensic settings, has been reported, such as providing trace evidence, estimating the time since death, and ascertaining the time of deposition. A 32-year-old male missing for 4 months, was found in a car in a forest far from his home and had died from blood loss, having been stabbed with a kitchen knife in the left side of his chest. Whitish or white greenish colonies ranging 5 cm to 8 cm in size were observed over his whole body. Penicillium commune and Cladosporium cladosporioides were isolated from the colonies. A 49-year-old male missing for one month, and was found dead at 500 m elevation in the mountains. An autopsy was conducted, but the cause of death could not be determined. Dark greenish or whitish colonies were found on his face and neck, and Epicoccum nigrum, Mucor sp.. Cladosporium sp., and Pestalotiopsis sp. were isolated. Penicillium sp. and Cladosporium sp. are major indoor fungi, and Epicoccum sp. and Pestalotiopsis sp. are plant pathogens found in outdoor environments. There was no discrepancy between the police statement and findings of the corpse such as place discovered, types of isolated fungi, and estimated time since death. Identifying fungi on cadavers may aid in forensic casework, and further studies are needed to develop for the use of fungi as a forensic tool.

Plasma membrane damage triggered by benzalkonium chloride and cetylpyridinium chloride induces G0/G1 cell cycle arrest via Cdc6 reduction in human lung epithelial cells.

Quaternary ammonium compounds, including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC), are widely used as disinfectants. Increased use of inhalable products containing BAC or CPC has raised concerns for lung toxicity. This study sought to elucidate the microstructure of plasma membrane damage caused by BAC and CPC and the subsequent mechanism by which the damage is mediated, as assessed using two human pulmonary epithelial cell lines (A549 and BEAS-2B). Scanning electron microscopic observation showed that exposure to BAC or CPC for 3 hr reduced the length and density of microvilli on the plasma membrane in A549 cells. Analysis of cell cycle distribution following plasma membrane damage revealed that BAC and CPC promote G0/G1 cell cycle arrest in both cell lines. The protein levels of Cdc6, an essential regulator of DNA replication during G1/S transition, are decreased significantly and dose dependently by BAC or CPC exposure. CPC and BAC decreased the Cdc6 levels that had been increased by a PI3K agonist in A549 cells, and levels of phosphorylated AKT were reduced in response to BAC or CPC. Conversely, exposure to equivalent concentrations of pyridinium chloride (lacking a hydrocarbon tail) induce no changes. These results suggest that plasma membrane damage triggered by BAC or CPC causes Cdc6-dependent G0/G1 cell cycle arrest in pulmonary cells. These effects are attributable to the long alkyl chains of BAC and CPC. The reduction of Cdc6 following plasma membrane damage may be caused, at least in part, by diminished signaling via the PI3K/AKT pathway.

Stability of exosomes in the postmortem serum and preliminary study on exosomal miRNA expression profiling in serum from myocardial infarction cadavers.

Exosome-encapsulated miRNAs could potentially be sensitive biomarkers of human diseases. Since a lipid bilayer membrane surrounds exosomes, the exosomal miRNA may stably exist in body fluids with diseases as well as biological fluids. Therefore, exosomal miRNA may be helpful for autopsy diagnosis. Assuming cadaver blood would be most useful, we initially examined serum exosome stability with regard to storage temperatures and periods. Characteristic analyses of the exosome revealed that exosomes and the content, miRNA, were stably preserved until at least three days when stored at below 20 °C. Subsequently, exosomal miRNA expression profiling was performed on the serum of acute myocardial infarction (AMI, 4 cases) autopsy bodies and on hemorrhagic shock bodies used as the control (CT, 3 cases). Results showed that significant twofold up- and downregulations of expression of 18 and 16 miRNAs were detectable in AMI as compared to the CT, respectively. miR-126-3p, which has been reported to be increased in serum of AMI patients and a mouse model, was one of the significantly upregulated miRNAs. Furthermore, dysregulation of exosomal miRNAs, such as miR-145-5p, miR-143-3p, and miR-222-3p, which are involved in cardioprotection, may be associated with AMI pathogenesis. These findings provide a novel perspective on the potential role of exosomal miRNA in determining the cause of death.

An Autopsy Case of Spontaneous Coronary Artery Dissection With Eosinophilic Coronary Periarteritis and Degeneration of Medial Smooth Muscles.

ABSTRACT: A 45-year-old woman with no known medical history died suddenly shortly after complaining of anterior chest discomfort. The autopsy revealed a dissection at the anterior descending branch of the left coronary artery, and eosinophilic adventitial inflammation was observed both in the right coronary artery and in the vicinity of the dissection. Furthermore, there was degeneration of the tunica media in the right coronary artery, and this was thought to be a predissection lesion. In the degenerated area of the tunica media, probable apoptosis of smooth muscle cells was noted, suggesting that the degeneration was not due only to the effect of eosinophilic lytic enzymes. These findings also indicated that eosinophilic infiltration preceded the dissection. Eosinophilic infiltration around the coronary arteries is occasionally observed in cases of sudden death, but although it might be associated with the death, the pathological mechanism is yet to be elucidated. Eosinophilic periarteritis has also been observed around the site of spontaneous coronary artery dissection, although a causal relationship is unproven. The histopathology of this case indicated that the eosinophilic infiltration preceded the dissection. Detailed pathological findings are presented, together with a review of the literature.

Scavenger receptor MARCO contributes to cellular internalization of exosomes by dynamin-dependent endocytosis and macropinocytosis.

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor class-A protein that is expressed on the cell surface of macrophages. MARCO mediates binding and ingestion of unopsonized environmental particles, including nano-sized materials. Exosomes are cell-derived, nano-sized vesicles (40-150 nm) that can contain lipids, RNA, DNA, and various proteins. Exosomes play an essential role in cell-to-cell communication via body fluids. However, mechanisms for the recognition and internalization of exosomes by recipient cells remain poorly characterized. In this study, cellular association of serum-derived fluorescent exosomes and 20-nm fluorescent nanoparticles (positive control) was compared between MARCO-expressing (CHO-MARCO) and control (CHO-CT) CHO-K1 cells to examine whether MARCO expression by recipient cells mediates the cellular uptake of exosomes and environmental nanoparticles. Fluorescence microscopic studies and quantitative analyses revealed that the cellular associations of both exosomes and 20-nm nanoparticles were greater in CHO-MARCO cells than in CHO-CT cells. Exosomes and nanoparticles colocalized with green fluorescent protein (GFP)-MARCO in cells transfected with GFP-MARCO-encoding constructs . Furthermore, inhibitory studies showed that actin reorganization and dynamin are involved in the MARCO-mediated cellular internalization of exosomes. These results indicated that MARCO plays a role in the uptake of exosomes.

Time course analysis of large-scale gene expression in incised muscle using correspondence analysis.

Studying the time course of gene expression in injured skeletal muscle would help to estimate the timing of injuries. In this study, we investigated large-scale gene expression in incision-injured mouse skeletal muscle by DNA microarray using correspondence analysis (CA). Biceps femoris muscle samples were collected 6, 12, and 24 hours after injury, and RNA was extracted and prepared for microarray analysis. On a 2-dimensional plot by CA, the genes (row score coordinate) located farther from each time series (column score coordinate) had more upregulation at particular times. Each gene was situated in 6 subdivided triangular areas according to the magnitude of the relationship of the fold change (FC) value at each time point compared to the control. In each area, genes for which the ratios of two particular FC values were close to 1 were distributed along the two border lines. There was a tendency for genes whose FC values were almost equal to be distributed near the intersection of these 6 areas. Therefore, the gene marker candidates for estimation of the timing of injuries were detectable according to the location on the CA plot. Moreover, gene sets created by a specific gene and its surrounding genes were composed of genes that showed similar or identical fluctuation patterns to the specific gene. In various analyses on these sets, significant gene ontology term and pathway activity may reflect changes in specific genes. In conclusion, analyses of gene sets based on CA plots is effective for investigation of the time-dependent fluctuation in gene expression after injury.

Relevance of autophagy markers to cytotoxicity of zinc compounds in macrophages.

Autophagy molecules such as microtubule-associated protein light chain 3 (LC3) and p62/SQSTM1 have been used as biomarkers of protective or conversely adverse effects of exposure to toxicants. In the present study we show changes in LC3-II (a lipidated form of LC3-I) and p62 levels in response to zinc compounds and some other toxicants in J774.1 murine macrophages. The cytotoxicity of either ZnO or ZnSO4 largely depended on the concentration of FBS or albumin in the culture medium. Accordingly, these authophagy markers were more remarkably increased when the cells were exposed to ZnO or ZnSO4 in the absence of FBS. We next addressed lysosomal function impairment and changes in LC3-II and p62 levels following exposure to TiO2, ZnO, and ZnSO4. Lysosomal pH was quickly decreased by autolysosome inhibitors such as bafilomycin A1 and chloroquine, while TiO2, ZnO and ZnSO4 did not decrease lysosomal pH. However, the amounts of LC3-II and p62 and the LC3-II/LC3-I ratio were increased either by the lysosomal inhibitors and the Zn compounds. LC3-II and p62 levels were increased after exposure to arsenite and lipopolysaccharide (LPS). The p62 and phospho-p62 levels were also increased by either ZnSO4 and bafilomycin A1 in HEK293 cells stably expressing RFP-LC3. The current observations suggest that LC3-II and p62 levels were increased as consequences of early effects of toxicants without changing lysosomal pH.

Benzalkonium chloride and cetylpyridinium chloride induce apoptosis in human lung epithelial cells and alter surface activity of pulmonary surfactant monolayers.

Quaternary ammonium compounds (e.g., benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC)) constitute a group of cationic surfactants are widely used for personal hygiene and medical care despite the potential pulmonary toxicity. To examine whether BAC and CPC aerosols deposited in the alveolar region alter pulmonary function, we studied the effects on pulmonary surfactant using two-step in vitro models; cytotoxicity using A549 alveolar epithelial cell and changes in surface activity of the pulmonary surfactant monolayer using both Surfacten® and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Cell viability was decreased with BAC and CPC dose-dependently. A comparison of cytotoxicity among BAC homologues with different length of alkyl chain showed that C16-BAC, which has the longest alkyl chain, was more cytotoxic than C12- or C14-BAC. Caspase-3/7 activity and cleaved form of caspase-3 and PARP were increased in BAC- and CPC-exposed cells. The elevated caspase-3/7 activity and their cleaved active forms were abolished by caspase-3-inhibitor. Furthermore, we examined the features of the surface pressure/trough area (π-A) isotherm by the Langmuir-Wilhelmy method and atomic force microscopy (AFM) images of lipid monolayers on a subphase containing BAC, CPC, or pyridinium chloride (PC, as a control). The π-A isotherms showed that addition of BAC or CPC yielded dose-dependent increases in surface pressure without compression, indicating that BAC and CPC expand the isotherm to larger areas at lower pressure. The collapse pressure diminished with increasing concentration of CPC. Topographic images indicated that BAC and CPC resulted in smaller condensed lipid domains compared to the control. Conversely, PC without hydrocarbon tail group, showed no cytotoxicity and did not change the isotherms and AFM images. These results indicate that BAC and CPC cause cell death via caspase-3-dependent apoptotic pathway in A549 cells and alter the alveolar surfactant activity. These effects can be attributed to the long alkyl chain of BAC and CPC.

Sudden Death Caused by Rupture of Spontaneous Ductus Arteriosus Aneurysm in an Adult.

A 55-year-old man complained of sudden onset of severe neck pain. This was followed by prompt loss of consciousness and death. Autopsy revealed rupture of a saccular aneurysm, which was considered to have resulted from enlargement of the remaining ductal tissue, and was located on the medial aspect of the uppermost portion of the descending aorta. Dense blood extravasation was noted in the posterior mediastinum and extending to the strap muscles of the neck and larynx. Histological examination of the rupture site revealed disappearance of the medial elastic fibers and thickened intima covered with dense fibrous tissue. Spontaneous ductus arteriosus aneurysm in adults is a rare finding, but widespread use of imaging technologies has revealed that it develops more frequently than previously recognized. Fatal complications may occur even when the aneurysm is relatively small. Therefore, pathologists should be aware of this aneurysm as a potential cause of sudden death.

Cellular uptake of paraquat determines subsequent toxicity including mitochondrial damage in lung epithelial cells.

Paraquat (PQ) is one of the commonly used herbicides in the world, despite its high toxicity. The ingestion of PQ accidentally or intentionally causes severe damage in diverse organs including the lung. Pulmonary fibrosis triggered by PQ accumulation in the lung epithelial cells is one of the major causes of death. This study investigated the intracellular accumulation of PQ, reactive oxygen species (ROS) generation and mitochondrial injury using two lung epithelial cell lines A549 and BEAS-2B (BEAS). Although A549 exhibit greater resistance to oxidative stress than BEAS, a cytotoxicity assay for PQ demonstrated that EC50 for lethality in A549 was 7 times lower than that in BEAS. When exposed to PQ at a concentration around EC50 for lethality, the amount of ROS generated in A549 was as low as that in BEAS. Conversely, the cellular concentration of PQ in A549 after exposure was higher than that in BEAS, which suggests a distinct difference in the susceptibility to PQ between these cell lines. After a 16 h exposure to PQ, mitochondrial membrane potential (MMP) decreased in A549, but decreased only slightly in BEAS even following a 30 h exposure. PQ-exposed A549 reduced an accumulation of PTEN-induced kinase 1 (PINK1), which works in degradation of damaged mitochondria, following the decrease of MMP, whereas PQ did not decline the PINK1 in BEAS. These results suggest that mitochondrial dysfunction due to cellular accumulation of PQ might contribute to the PQ-provoked toxicity more than the ROS generation in the lung epithelial cells.

Differential Regulation of IL-1ß and IL-6 Release in Murine Macrophages.

Asbestos and silica (exogenous danger) and adenosine triphosphate (ATP, endogenous danger-signaling molecule) synergistically increase IL-1ß release from endotoxin-primed macrophage, which is mediated by NOD-like receptor protein 3 (NLRP3) inflammasome. However, the conversion of pro-IL-1ß to its active form seems to depend on the macrophage cell types. In the present study, bone marrow-derived macrophages (BMM) and three murine macrophage cell lines, J774.1, J774A.1, and RAW264.7 were exposed to ATP or fibrous titanium dioxide (FTiO2) in the presence or absence of lipopolysaccharide (LPS), and the concentrations of IL-1ß and IL-6 in both cell lysates and in the culture media were measured by immunoblotting to differentiate active form of IL-1ß from pro-IL-1ß. IL-1ß release was synergistically increased when the cells were exposed to both LPS and ATP or FTiO2, while IL-6 was readily released by LPS alone. IL-1ß released into the culture medium was pro-IL-1ß in J774.1 and RAW264.7, and most of the pro-IL-1ß remained inside the cells. In contrast, the active form of IL-1ß was released together with pro-IL-1ß from J774A.1 and BMM after the co-stimulation. J774A.1 and BMM express apoptosis-associated speck-like protein contains a carboxyl-terminal CARD (ASC) while J774.1 and RAW264.7 do not or only faintly express ASC, and accordingly, caspase-1, which converts pro-IL-1ß to its active form, is activated only in J774A.1 and BMM. Collectively, the canonical inflammasome pathway is not activated in J774.1 and RAW264.7, and the apparent synergistical increase of IL-1ß in the culture medium mostly reflects the leakage of pro-IL-1ß from these cells.

Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway.

The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2-3 µm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.

Inflammatory responses to neutral fat and fatty acids in multiple organs in a rat model of fat embolism syndrome.

Fat embolism syndrome (FES) is a common complication of long bone fractures. FES is rare but with significant morbidity and occasional fatalities. Studies of animal models of FES are numerous; however, few studies compare inflammatory reactions in multiple organs. The present study investigated the effect of neutral fat and fatty acids, which cause changes in multiple organs and induce FES. Using rats we evaluated the ratio of lung-to-body weight and conducted histological analyses and quantitative analysis of inflammatory cytokine mRNAs in the lungs following intravenous administration of neutral fat or fatty acids. Neutral fat increased the ratio of lung-to-body weight, and neutral fat formed emboli in lung capillaries. The levels of interleukin-1 beta (IL-1ß), IL-6 and tumor necrosis factor-alpha (TNF-α) in the lungs increased after injection of neutral fat and oleic acid. Analysis of the histologic changes revealed that the highest numbers of fat droplets, occluding the capillaries of the lungs, kidney, heart, and brain formed 12h after the injection of neutral fat and fat droplets gradually diminished 48h later. Fat droplets were not detected in any organs after the injection of oleic acid. IL-1ß and TNF-α levels in the lungs were elevated 9-24h after the injection of neutral fat, although IL-6 levels peaked at 6h. After injection of oleic acid, peak levels of IL-1ß, IL-6, and TNF-α were detected at 6h, and IL-6 again increased in all organs and plasma at 15h. Neutral fat, but not fatty acids, formed emboli in the capillaries of multiple organs. These findings suggest that neutral fat increased inflammatory cytokine levels by forming emboli in organ capillaries, particularly in the lungs, while oleic acid augmented inflammatory cytokine levels by stimulating endothelial cells of multiple organs.

The role of Rho-kinases in IL-1ß release through phagocytosis of fibrous particles in human monocytes.

Long fibers, such as asbestos and carbon nanotubes (CNTs), are more potent activators of inflammatory and genotoxicity than short or tangled fibers. Fibrous particles trigger interleukin (IL)-1ß secretion and cause inflammatory diseases through NLRP3 inflammasomes in phagocytotic cells. However, the mechanism involved in fibrous particle-induced inflammation has not been well documented. In this study, we focused on GTPase effector Rho-kinases (ROCK1, and 2), which are known to be involved in a wide range of cellular functions such as adhesion, regulation of cytoskeleton, and phagocytosis. We examined whether ROCKs are associated with multi-walled CNT (MWCNT)- or asbestos-induced IL-1ß secretion in human monocytic THP-1 cells using a selective inhibitor and small interfering RNA. THP-1 cells were differentiated to macrophages by PMA and were exposed to MWCNTs, crocidolite asbestos or lipopolysaccharide (LPS) in the presence or absence of Y27632 (ROCK inhibitor) or Z-YVAD (caspase-1 inhibitor). Exposure of the cells to MWCNTs or asbestos provoked IL-1ß secretion, but this secretion was suppressed by both Y27632 and Z-YVAD, whereas LPS-induced IL-1ß secretion was inhibited only by Z-YVAD and not by Y27632. siRNA designed for knockdown of both ROCK1 and ROCK2 suppressed MWCNT- and asbestos-induced IL-1ß secretion, but did not change LPS-induced IL-1ß secretion. Moreover, Y27632 suppressed pro-IL-1ß protein levels and the release of activated-cathepsin B and activated-caspase-1 induced by MWCNTs or asbestos. In contrast, LPS-induced pro-IL-1ß protein was not suppressed by Y27632. These results suggest that ROCKs are involved in fibrous particle-induced inflammasome responses in THP-1 cells.

Human organic cation transporter 2 (hOCT2): Inhibitor studies using S2-hOCT2 cells.

Highly expressed in kidney and located on the basolateral membrane, human organic cation transporter 2 (hOCT2) can transport various compounds (i.e. drugs and toxins) into the proximal tubular cell. Using cultured proximal tubule cells stably expressing hOCT2 (i.e. S2-hOCT2 cells), we sought to probe different compound classes (e.g. analgesics, anti-depressants, anti-psychotics, disinfectant, herbicides, insecticides, local anesthetic, muscarinic acetylcholine receptor antagonist, sedatives, steroid hormone, stimulants and toxins) for their ability to inhibit (14)C-TEA uptake, a prototypical OCT2 substrate. Aconitine, amitriptyline, atropine, chlorpyrifos, diazepam, fenitrothion, haloperidol, lidocaine, malathion, mianserin, nicotine and triazolam significantly inhibited (14)C-TEA uptake; IC50 values were 59.2, 2.4, 2.0, 20.7, 32.3, 13.2, 32.5, 104.6, 71.1, 17.7, 52.8 and 65.5µM, respectively. In addition, aconitine, amitriptyline, atropine, chlorpyrifos, fenitrothion, haloperidol, lidocaine, and nicotine displayed competitive inhibition with Ki values of 145.6, 2.5, 2.4, 24.8, 16.9, 51.6, 86.8 and 57.7µM, respectively. These in vitro data support the notion that compounds pertaining to a wide variety of different drug classes have the potential to decrease renal clearance of drugs transported via hOCT2. Consequently, these data warrant additional studies to probe hOCT2 and its role to influence drug pharmacokinetics.

Sulfide induces apoptosis and Rho kinase-dependent cell blebbing in Jurkat cells.

Hydrogen sulfide (H2S) is a toxic gaseous substance, and accidental exposure to high concentrations of H2S has been reported to be lethal to humans. Inhaled and absorbed H2S is partially dissolved within the circulation and causes toxic effects on lymphocytes. However, the mechanisms involved in H2S toxicity have not been well documented. In this study, we examined the cellular uptake and injury of sulfide-exposed human T lymphocytes (Jurkat). Cells were exposed to a H2S donor, sodium hydroxysulfide (NaHS), at pH 6.0, 7.0, or 8.0 for 1 h at 37 °C in a sealed conical tube to avoid the loss of dissolved H2S gas. Cytotoxicity and cellular sulfide concentrations increased dramatically as the pH of the NaHS solution decreased. Sulfide enhanced the cleavage of caspase-3 and poly (ADP-ribose) polymerase and induced early cellular apoptosis. A pan-caspase inhibitor reduced sulfide-induced apoptosis. These results indicate that sulfide induces pH-dependent and caspase-dependent apoptosis. We also found that blebbing of the plasma membrane occurred in sulfide-exposed cells. Both ROCK-1 and ROCK-2 (Rho kinases) were activated by sulfide, and sulfide-induced cell blebbing was suppressed by a ROCK inhibitor, suggesting that a Rho pathway is involved in sulfide-induced blebbing in lymphocytes.

Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 cells.

The toxicity of carbon nanotubes (CNTs), a highly promising nanomaterial, is similar to that of asbestos because both types of particles have a fibrous shape and are biopersistent. Here, we investigated the characteristics of macrophage receptor with collagenous structure (MARCO), a membrane receptor expressed on macrophages that recognizes environmental or unopsonized particles, and we assessed whether and how MARCO was involved in cellular uptake of multi-walled CNTs (MWCNTs). MARCO-transfected Chinese hamster ovary (CHO-K1) cells took up polystyrene beads irrespective of the particle size (20nm-1µm). In the culture of MARCO-transfected CHO-K1 cells dendritic structures were observed on the bottom of culture dishes, and the edges of these dendritic structures were continually renewed as the cell body migrated along the dendritic structures. MWCNTs were first tethered to the dendritic structures and then taken up by the cell body. MWCNTs appeared to be taken up via membrane ruffling like macropinocytosis, rather than phagocytosis. The cytotoxic EC(50) value of MWCNTs in MARCO-transfected CHO-K1 cells was calculated to be 6.1µg/mL and transmission electron microscopic observation indicated that the toxicity of MWCNTs may be due to the incomplete inclusion of MWCNTs by the membrane structure.