Difference in the Mechanism of Iron Overload-Enhanced Acute Hepatotoxicity Induced by Thioacetamide and Carbon Tetrachloride in Rats.

Iron overload has been recognized as a risk factor for liver disease; however, little is known about its pathological role in the modification of liver injury. The purpose of this study is to investigate the influence of iron overload on liver injury induced by two hepatotoxicants with different pathogenesis in rats. Rats were fed a control (Cont), 0.8% high-iron (0.8% Fe), or 1% high-iron diet (1% Fe) for 4 weeks and were then administered with saline, thioacetamide (TAA), or carbon tetrachloride (CCl4). Hepatic and systemic iron overload were seen in the 0.8% and 1% Fe groups. Twenty-four hours after administration, hepatocellular necrosis induced by TAA and hepatocellular necrosis, degeneration, and vacuolation induced by CCl4, as well as serum transaminase values, were exacerbated in the 0.8% and 1% Fe groups compared to the Cont group. On the other hand, microvesicular vacuolation induced by CCl4 was decreased in 0.8% and 1% Fe groups. Hepatocellular DNA damage was increased by iron overload in both models, whereas a synergistic effect of oxidative stress by excess iron and hepatotoxicant was only present in the CCl4 model. The data showed that dietary iron overload exacerbates TAA- and CCl4-induced acute liver injury with different mechanisms.

Deficiency of interleukin-19 exacerbates acute lung injury induced by intratracheal treatment of hydrochloric acid.

Interleukin (IL-19) belongs to the IL-10 family of cytokines and plays diverse roles in inflammation, cell development, viral responses, and lipid metabolism. Acute lung injury (ALI) is a severe respiratory condition associated with various diseases, including severe pneumonia, sepsis, and trauma, lacking established treatments. However, the role of IL-19 in acute inflammation of the lungs is unknown. We reported the impact of IL-19 functional deficiency in mice crossed with an ALI model using HCl. Lungs damages, neutrophil infiltration, and pulmonary edema induced by HCl were significantly worse in IL-19 knockout (KO) mice than in wild-type (WT) mice. mRNA expression levels of C-X-C motif chemokine ligand 1 (CXCL1) and IL-6 in the lungs were significantly higher in IL-19 KO mice than in WT mice. Little apoptosis was detected in lung injury in WT mice, whereas apoptosis was observed in exacerbated area of lung injury in IL-19 KO mice. These results are the first to show that IL-19 is involved in acute inflammation of the lungs, suggesting a novel molecular mechanism in acute respiratory failures. If it can be shown that neutrophils have IL-19 receptors and that IL-19 acts directly on them, it would be a novel drug target.

The effect of lipopolysaccharide on liver homeostasis and diseases based on the mutual interaction of macrophages, autophagy, and damage-associated molecular patterns in male F344/DuCrlCrlj rats.

Lipopolysaccharide (LPS) has dose-dependent biphasic functions (cell protective versus cell toxic). To clarify the different effects of LPS on liver homeostasis or liver diseases, comparisons were made between low and high doses of LPS, in terms of the mutual relation of hepatic macrophages, autophagy, and damage-associated molecular patterns (DAMPs) in male F344/DuCrlCrlj rats. Rats injected with low dose (0.1 mg/kg) or high dose (2.0 mg/kg) of LPS were examined at 6, 10, and 24 hours following single injections. Histologically, focal hepatocellular necrosis was occasionally present in high-dose animals, whereas there were no significant changes in low-dose animals. In low-dose animals, Kupffer cells reacting to CD163 and CD204 were hypertrophic and regarded as M2 macrophages, which promote resolution of inflammation and tissue repair, whereas in high-dose animals, infiltration of M1 macrophages expressing CD68 and major histocompatibility complex class II, which enhance cell injury, was seen. Hepatocytes with high-mobility-group box-1 (HMGB1) (one of DAMPs)-positive cytoplasmic granules appeared more frequently in high-dose animals than in low-dose animals, indicating the translocation of nuclear HMGB1 into the cytoplasm. However, although light-chain 3 beta-positive autophagosomes in hepatocytes increased in both doses, abnormally vacuolated autophagosomes were only seen in injured hepatocytes in the high-dose group, indicating possible extracellular release of HMGB1, which might result in cell injury and inflammation. These findings suggested that low-dose LPS induced a favorable mutual relationship among hepatic macrophages, autophagy, and DAMPs leading to cytoprotection of hepatocytes, whereas failures of the relationship in high-dose LPS caused hepatocyte injury.

Macrophage pathology in hepatotoxicity.

The liver is the most important organ that metabolizes and detoxifies chemicals taken into the body. Therefore, there is always a risk of liver damage owing to the toxic effects of chemicals. The mechanisms of hepatotoxicity have been studied extensively and deeply based on toxic effects of chemicals themselves. However, it is important to note that liver damage is variously modified by the patho-biological reactions evoked mainly via macrophages. Macrophages appearing in hepatotoxicity are evaluated by the M1/M2 polarization; M1 macrophages promote tissue injury/inflammation, whereas M2 macrophages show anti-inflammatory action including reparative fibrosis. The "portal vein-liver barrier" regulated by Kupffer cells and dendritic cells in and around the Glisson's sheath may be related to the initiation of hepatotoxicity. In addition, Kupffer cells exhibit the two-sides of functions (that is, M1 or M2 macrophage-like functions), depending on microenvironmental conditions which may be raised in part by gut microbiota-derived lipopolysaccharide. Furthermore, damage-associated molecular patterns (DAMPs) (in particular, HMGB1) and autophagy (which degrades DAMPs) also play roles in the polarity of M1/M2 macrophages. The mutual relation of "DAMPs (HMGB-1)-autophagy-M1/M2 macrophage polarization" as the patho-biological reaction should be taken into consideration in hepatotoxicity evaluation.

Analyses of damage-associated molecular patterns, particularly biglycan, in cisplatin-induced rat progressive renal fibrosis.

Damage-associated molecular patterns (DAMPs) and their receptors (TLR-2 and -4) may play important roles in renal fibrosis, of which the pathogenesis is complicated. We used rat renal lesions induced by a single intraperitoneal injection of cisplatin at 6 mg/kg body weight; consisting of tissue damage of renal tubules on days 1 and 3, further damage and regeneration with inflammation mainly on days 5 and 7, and interstitial fibrosis on days 9, 12, 15, and 20. Microarray analyses on days 5 (the commencement of inflammation) and 9 (the commencement of interstitial fibrosis) showed that DAMPs increased by more than two-fold relative to control included common extra-cellular matrix (ECM) components such as laminin (Lamc2) and fibronectin, and heat shock protein family, as well as fibrinogen, although it was limited analysis; Lamc2, an element of basement membrane, may be regarded as an indicator for damaged renal tubules. In the real-time RT-PCR analyses, TLR-2 significantly increased transiently on day 1, whereas TLR-4 significantly increased on days 9 and 15, almost in agreement with the increased biglycan (a small leucine-rich proteoglycan as ubiquitous ECM component). As M1/M2 macrophages participated in renal lesions, such as inflammation and fibrosis, presumably, TLR-4, which may be expressed in immune cells, could play crucial roles in the formation of renal lesions in association with biglycan.

Possible Cytoprotection of Low Dose Lipopolysaccharide in Rat Thioacetamide-Induced Liver Lesions, Focusing on the Analyses of Hepatic Macrophages and Autophagy.

Lipopolysaccharide (LPS) may influence hepatic macrophages and autophagy. We evaluated the potential participation of macrophages and autophagosomes in thioacetamide (TAA)-induced rat liver injury under pretreatment of a low dose LPS (0.1 mg/kg BW, intraperitoneally; nonhepatotoxic dose). F344 rats were pretreated with LPS (LPS + TAA) or saline (TAA alone) at 24 hours before TAA injection (100 mg/kg BW, intraperitoneally); rats were examined on Days 0 (controls), 1, 2, and 3 after TAA injection. Data were compared between TAA alone and LPS + TAA rats. LPS pretreatment significantly reduced TAA-induced hepatic lesion (centrilobular necrosis with inflammation) on Days 1 and 2, being reflected by declined hepatic enzyme values and decreased number of apoptotic cells. LC3B-immunoreacting autophagosomes (as cytoplasmic fine granules) were significantly increased on Days 1 and 2 in hepatocytes of LPS + TAA rats. In LPS + TAA rats, hepatic macrophages reacting to CD68, CD163, and MHC class II mainly on Day 2 and mRNA levels of macrophage-related factors (MCP-1, IL-1ß, and IL-4) on Day 1 were significantly decreased. Collectively, the low-dose LPS pretreatment might act as cytoprotection against TAA-induced hepatotoxicity through increased autophagosomes and decreased hepatic macrophages, although the dose/time-dependent cytoprotection of LPS should be further investigated at molecular levels.

Canine induced pluripotent stem cell maintenance under feeder-free and chemically-defined conditions.

Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.

Appearance of Heterogeneous Macrophages During Development of Isoproterenol-Induced Rat Myocardial Fibrosis.

Macrophages appearing in lesions are polarized toward M1 (for inflammation) and M2 (for anti-inflammation/fibrosis) types. We analyzed immunophenotypes of macrophages appearing in myocardial lesion in rats injected once with isoproterenol (10 mg/kg body weight). Inflammation following myocardial necrosis on day 1 was seen with a peak on days 3 and 5, and thereafter, reparative fibrosis developed on days 7 to 28. CD68+ M1 macrophages were seen in the early stages of injury and inflammatory on days 1 to 7, and thereafter, CD163+ M2 macrophages increased in the late stages of fibrosis on days 7 to 28. There was the polarization of M1 and M2 macrophages. The kinetics of macrophages reacting to Iba-1 and Galectin-3 was similar to that of M1 macrophages, indicating that Iba1- and Gal-3-positive macrophages might have functions of M1 type. Double immunofluorescence revealed that CD204- and MHC class II-positive macrophages are polarized toward M1 and M2 types, respectively. CCR2 messenger RNA expression is transiently elevated on day 1. Since CCR2 is a marker of blood monocytes, M1 macrophages might be recruited from blood monocytes. Collectively, macrophages expressing heterogeneous immunophenotypes participate in myocardial fibrosis. These findings would be useful for understanding the pathogenesis of myocardial fibrosis and analyzing myocardial toxicity.

Characterization of Immature Myofibroblasts of Stellate Cell or Mesenchymal Cell Origin in D-Galactosamine-Induced Liver Injury in Rats.

Lesions of D-galactosamine (D-GalN)-induced hepatotoxicity resemble those of human acute viral hepatitis. This study investigated hepatic mesenchymal cells including hepatic stellate cells (HSCs) and myofibroblasts in D-GalN-induced hepatotoxicity. Rats, injected with D-GalN (800 mg/kg body weight, once, intraperitoneally) were examined on post single injection (PSI) at 8 hours and days 1 to 5. Lesions consisting of hepatocyte necrosis and reparative fibrosis were present diffusely or focally within the hepatic lobules on PSI days 1 and 2, and then the injury recovered on PSI days 3 and 5. Myofibroblasts expressing vimentin, desmin, and α-smooth muscle actin (α-SMA) were present in the lesions. Double immunofluorescence showed that myofibroblasts reacted simultaneously to vimentin/α-SMA, desmin/α-SMA, and desmin/vimentin; furthermore, myofibroblasts reacting to vimentin, desmin, and α-SMA also co-expressed glial fibrillary acidic protein (GFAP), a marker of HSCs. Additionally, GFAP-expressing myofibroblasts reacted to nestin and A3 (both are markers of immature mesenchymal cells). Cells reacting to Thy-1, a marker for immature mesenchymal cells, also appeared in fibrotic lesions. In agreement with the myofibroblastic appearance, mRNAs of fibrosis-related factors (TGF-ß1, PDGF-ß, TNF-α, Timp2, and Mmp2) increased mainly on PSI days 1 and 2. Myofibroblasts with expression of various cytoskeletal proteins were present in diffuse or focal hepatic lesions, and they might be derived partly from immature HSCs and from immature mesenchymal cells.

Analyses of hemorrhagic diathesis in high-iron diet-fed rats.

Iron overload has been well recognized to cause oxidant-mediated cellular/tissue injury; however, little is known about the effects of iron overload on the blood coagulation system. We encountered an unexpected bleeding tendency in rats fed a high-iron diet in a set of studies using iron-modified diets. In this study, we investigated the mechanism of hemorrhagic diathesis induced by dietary iron overload in rats. Six-week-old F344/DuCrlCrlj male rats were fed a standard (containing 0.02% iron) or a high-iron diet (containing 1% iron) for 6 weeks and were then sampled for hematological, blood biochemical, coagulation, and pathological examinations. Serum and liver iron levels increased in rats fed the high-iron diet (Fe group) and serum transferrin was almost saturated with iron. However, serum transaminase levels did not increase. Moreover, plasma prothrombin time and activated partial thromboplastin time were significantly prolonged, regardless of the presence of hemorrhage. The activity of clotting factors II and VII (vitamin K-dependent coagulation factors) decreased significantly, whereas that of factor VIII was unaltered. Blood platelet levels were not influenced by dietary iron overload, suggesting that the bleeding tendency in iron-overloaded rats is caused by secondary hemostasis impairment. In addition, hemorrhage was observed in multiple organs in rats fed diets containing more than 0.8% iron. Our results suggest that iron overload can increase the susceptibility of coagulation abnormalities caused by latent vitamin K insufficiency.

Generation of Footprint-Free Canine Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus Vector.

Using auto-erasable Sendai virus vector, we generated ciPSC line. After several passages, virus was not present in ciPSCs by RT-PCR. ciPSCs from canine PBMCs had pluripotent state, differentiated all three germ layers in vitro, and had normal 78 XX karyotype. These results proved that PBMCs were one of the good cell sources to generate ciPSC lines from companion and patient dogs.

Participation of Somatic Stem Cells, Recognized by a Unique A3 Antibody, in Mucosal Epithelial Regeneration in Dextran Sulfate Sodium (DSS)-Induced Rat Colonic Lesions.

A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma cells, recognizes somatic stem cells in rats. We analyzed the distribution of A3-positive cells in dextran sulfate sodium (DSS)-induced colonic lesions consisting of regenerating mucosa and fibrosis. Male 6-week-old F344 rats were administered 5% DSS in drinking water for 5 to 7 days, and lesions at recovery stage were also examined. In untreated control adult colons, A3-positive cells are localized around the crypts where stem cell niche is formed. Histopathologically, in colons of DSS-administered rats, mucosal atrophy, inflammatory cell infiltration, and fibrosis were observed in the lamina propria; thereafter, mucosal epithelia were desquamated, and crypts were decreased gradually with decrease in surrounding A3-positive cells. At the early recovery stage, crypts showed regeneration with reappearance of A3-positive cells. Interestingly, A3-positive cells aggregated in desquamated mucosa surface of fibrosis. Aggregated A3-positive cells coexpressed with vimentin, Thy-1, and partly CK19 but did not react simultaneously with α-SMA. Likely, aggregated A3-positive cells may be rescue cells with nature of both mesenchymal and epithelial cells to maintain self-renewal after injury in the colon. A3 antibody would become a useful tool to investigate the participation of stem cells in rat colonic lesions.

Characterization of Macrophages and Myofibroblasts Appearing in Dibutyltin Dichloride-Induced Rat Pancreatic Fibrosis.

Macrophages and myofibroblasts are important in fibrogenesis. The cellular characteristics in pancreatic fibrosis remain to be investigated. Pancreatic fibrosis was induced in F344 rats by a single intravenous injection of dibutyltin dichloride. Histopathologically, the induced pancreatic fibrosis was divided into 3 grades (1+, 2+, and 3+), based on collagen deposition. Immunohistochemically, CD68-expressing M1 macrophages increased with grade and CD163-expressing M2 macrophages also increased later than M1 macrophage appearance. Double immunofluorescence showed that there were macrophages coexpressing CD68 and CD163, suggesting a possible shift from M1 to M2 types; similarly, increased major histocompatibility complex class II- and CD204-expressing macrophages were polarized toward M1 and M2 types, respectively. These findings indicated the participation of M1- and M2-polarized macrophages. Mesenchymal cells staining positive for vimentin, desmin, and α-smooth muscle actin (α-SMA) increased with grade. There were mesenchymal cells coexpressing vimentin/α-SMA, desmin/α-SMA, and glial fibrillary acidic protein (GFAP)/α-SMA; Thy-1-expressing immature mesenchymal cells also increased in fibrotic lesions. Because α-SMA expression is a reliable marker for myofibroblasts, α-SMA-expressing pancreatic myofibroblasts might be originated from GFAP-expressing pancreatic stellate cells or Thy-1-expressing immature mesenchymal cells; the myofibroblasts could simultaneously express cytoskeletal proteins such as vimentin and desmin. The present findings would provide useful information for analyses based on features of macrophages and myofibroblasts in chemically induced pancreatic fibrosis.

Acetaminophen-Induced Rat Hepatotoxicity Based on M1/M2-Macrophage Polarization, in Possible Relation to Damage-Associated Molecular Patterns and Autophagy.

Overdose of acetaminophen (APAP), an antipyretic drug, is an important cause of liver injury. However, the mechanism in the rat model remains undetermined. We analyzed APAP-induced hepatotoxicity using rats based on M1/M2-macrophage functions in relation to damage-associated molecular patterns (DAMPs) and autophagy. Liver samples from six-week-old rats injected with APAP (1000 mg/kg BW, ip, once) after 15 h fasting were collected at hour 10, and on days 1, 2, 3, and 5. Liver lesions consisting of coagulation necrosis and inflammation were seen in the affected centrilobular area on days 1 and 2, and then, recovered with reparative fibrosis by day 5. Liver exudative enzymes increased transiently on day 1. CD68+ M1-macrophages increased significantly on days 1 and 2 with increased mRNAs of M1-related cytokines such as IFN-g and TNF-α, whereas CD163+ M2-macrophages appeared later on days 2 and 3. Macrophages reacting to MHC class II and Iba1 showed M1-type polarization, and CD204+ macrophages tended to be polarized toward M2-type. At hour 10, interestingly, HMGB1 (representative DAMPs) and its related signals, TLR-9 and MyD88, as well as LC3B+ autophagosomes began to increase. Collectively, the pathogenesis of rat APAP hepatotoxicity, which is the first, detailed report for a rat model, might be influenced by macrophage functions of M1 type for tissue injury/inflammation and M2-type for anti-inflammatory/fibrosis; particularly, M1-type may function in relation to DAMPs and autophagy. Understanding the interplayed mechanisms would provide new insight into hepato-pathogenesis and contribute to the possible development of therapeutic strategies.

Participation of Somatic Stem Cells, Labeled by a Unique Antibody (A3) Recognizing both N-glycan and Peptide, to Hair Follicle Cycle and Cutaneous Wound Healing in Rats.

A monoclonal antibody (A3) was generated by using rat malignant fibrous histiocytoma (MFH) cells as the antigen. Generally, MFH is considered to be a sarcoma derived from undifferentiated mesenchymal cells. Molecular biological analyses using the lysate of rat MFH cells revealed that A3 is a conformation specific antibody recognizing both N-glycan and peptide. A3-labeled cells in bone marrow were regarded as somatic stem cells, because the cells partly coexpressed CD90 and CD105 (both immature mesenchymal markers). In the hair follicle cycle, particularly the anagen, the immature epithelial cells (suprabasal cells) near the bulge and some immature mesenchymal cells in the disassembling dermal papilla and regenerating connective tissue sheath/hair papilla reacted to A3. In the cutaneous wound-healing process, A3-labeled epithelial cells participated in re-epithelialization in the wound bed, and apparently, the labeled cells were derived from the hair bulge; in addition, A3-labeled immature mesenchymal cells in the connective tissue sheath of hair follicles at the wound edge showed the expansion of the A3 immunolabeling. A3-labeled immature epithelial and mesenchymal cells contributed to morphogenesis in the hair cycle and tissue repair after a cutaneous wound. A3 could become a unique antibody to identify somatic stem cells capable of differentiating both epithelial and mesenchymal cells in rat tissues.

Effects of dexamethasone on hepatic macrophages in normal livers and thioacetamide-induced acute liver lesions in rats.

Resident and infiltrative macrophages play important roles in the development of pathological lesions. M1/M2 macrophage polarization with respective CD68 and CD163 expression remains unclear in chemically induced liver injury. This study was aimed at investigating the influence of macrophages on normal and chemically induced liver injury. For this, dexamethasone (DX), an immunosuppressive drug, was administered in normal rats and thioacetamide (TAA)-treated rats. Liver samples were collected and analyzed with immunohistochemical methods. Repeated injections of DX (0.5 or 1.0 mg/kg BW) for 3, 7 and 11 days reduced the number of CD163 positive hepatic resident macrophages (Kupffer cells) in normal livers, while increasing AST and ALT levels. In TAA (300 mg/kg BW)-treated rats injected with DX (0.5 mg/kg BW) pretreatment, the number of M1 and M2 macrophages showed a significant decrease compared with that of TAA-treated rats without DX treatment. Additionally, reparative fibrosis resulting from hepatocyte injury induced by TAA injection was suppressed by DX pretreatment. Our data suggested that macrophages could influence not only normal hepatic homeostasis (reflected by AST and ALT levels) but also chemically induced hepatic lesion development (reduced reparative fibrosis).

Chronic kidney disease after 5/6 nephrectomy disturbs the intestinal microbiota and alters intestinal motility.

Organ-organ crosstalk is involved in homeostasis. Gastrointestinal symptoms are common in patients with renal failure. The aim of this study was to elucidate the relationship between gastrointestinal motility and gastrointestinal symptoms in chronic kidney disease. We performed studies in C57BL/6 mice with chronic kidney disease after 5/6 nephrectomy. Gastrointestinal motility was evaluated by assessing the ex vivo responses of ileum and distal colon strips to electrical field stimulation. Feces were collected from mice, and the composition of the gut microbiota was analyzed using 16S ribosomal RNA sequencing. Mice with chronic kidney disease after 5/6 nephrectomy showed a decreased amount of stool, and this constipation was correlated with a suppressed contraction response in ileum motility and decreased relaxation response in distal colon motility. Spermine, one of the uremic toxins, inhibited the contraction response in ileum motility, but four types of uremic toxins showed no effect on the relaxation response in distal colon motility. The 5/6 nephrectomy procedure disturbed the balance of the gut microbiota in the mice. The motility dysregulation and constipation were resolved by antibiotic treatments. The expression levels of interleukin 6, tumor necrosis factor-α, and iNOS in 5/6 nephrectomy mice were increased in the distal colon but not in the ileum. In addition, macrophage infiltration in 5/6 nephrectomy mice was increased in the distal colon but not in the ileum. We found that 5/6 nephrectomy altered gastrointestinal motility and caused constipation by changing the gut microbiota and causing colonic inflammation. These findings indicate that renal failure was remarkably associated with gastrointestinal dysregulation.

Hepatic Myoepithelial Carcinoma in a Dog: Immunohistochemical Comparison With Other Canine Hepatic Carcinomas.

An 11-year-old female miniature Dachshund dog presented with a solid, soft, gray mass on the hepatic lateral left lobe. Histologically, the mass consisted of neoplastic proliferation of cells with round nuclei and eosinophilic and vacuolated cytoplasm arranged in alveolar, trabecular, and solid patterns. Immunohistochemically, the neoplastic cells were positive for pancytokeratin (CK AE1/AE3), CK5, CK14, vimentin, Sox9, and myoepithelial markers (α-smooth muscle actin, p63, and calponin). The morphological and immunohistochemical findings indicated a diagnosis of myoepithelial carcinoma. We conducted immunohistochemical studies on other representative canine hepatic tumors. Although the myoepithelial phenotype was not observed in the hepatocellular carcinoma, some tumor cells in cholangiocarcinoma showed immunohistochemical features of myoepithelium, suggesting that some neoplastic cells in cholangiocarcinoma may have the potential to differentiate into myoepithelial cells. To our knowledge, this is the first report in veterinary medicine of a hepatic carcinoma with a myoepithelial phenotype.

The localization and distribution of cells labeled by a somatic stem cell-recognizing antibody (A3) in rat colon development; possible presence of a new cell type forming the intestinal stem cell niche.

A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma (MFH)-derived cloned cells, recognizes somatic stem cells (bone-marrow/hair follicle stem cells). We investigated the distribution of cells immunoreactive to A3 in the developing rat intestine (particularly, the colon), focusing on the ontogenic kinetics of A3-positive cells. In the rat intestine, A3 labeled spindle-shaped stromal cells localized in the submucosa and labeled endothelial cells of capillaries in the lamina propria forming villi in the early development stage. With development progression, A3-positive cells were exclusively localized around the crypts of the colon. Double immunofluorescence revealed that A3-positive cells around the crypts reacted to vimentin (for mesenchymal cells) and Thy-1 (for mesenchymal stromal cells) but not to α-SMA (for mesenchymal myofibroblastic cells) or CD34 (for hematopoietic stem cells), indicating that A3-positive cells around the crypts may have characteristics of immature mesenchymal cells. In addition, A3 labeled a few epithelial cells at the base of colon crypts. Furthermore, immunoelectron microscopy revealed that A3-positive cells lay inside myofibroblasts adjacent to the epithelium of the crypts. A3-positive cells were regarded as a new type of immature mesenchymal cells around the crypts. Collectively, A3-positive cells might take part in the stem cell niche in the colon, which is formed through epithelial-mesenchymal interaction.

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death.

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death.