Multicentre study to assess the performance of an artificial intelligence instrument to support qualitative diagnosis of colorectal polyps.

OBJECTIVE: Computer-aided diagnosis (CAD) using artificial intelligence (AI) is expected to support the characterisation of colorectal lesions, which is clinically relevant for efficient colorectal cancer prevention. We conducted this study to assess the diagnostic performance of commercially available CAD systems. METHODS: This was a multicentre, prospective performance evaluation study. The endoscopist diagnosed polyps using white light imaging, followed by non-magnified blue light imaging (non-mBLI) and mBLI. AI subsequently assessed the lesions using non-mBLI (non-mAI), followed by mBLI (mAI). Eventually, endoscopists made the final diagnosis by integrating the AI diagnosis (AI+endoscopist). The primary endpoint was the accuracy of the AI diagnosis of neoplastic lesions. The diagnostic performance of each modality (sensitivity, specificity and accuracy) and confidence levels were also assessed. RESULTS: Overall, 380 lesions from 139 patients were included in the analysis. The accuracy of non-mAI was 83%, 95% CI (79% to 87%), which was inferior to that of mBLI (89%, 95% CI (85% to 92%)) and mAI (89%, 95% CI (85% to 92%)). The accuracy (95% CI) of diagnosis by expert endoscopists using mAI (91%, 95% CI (87% to 94%)) was comparable to that of expert endoscopists using mBLI (91%, 95% CI (87% to 94%)) but better than that of non-expert endoscopists using mAI (83%, 95% CI (75% to 90%)). The level of confidence in making a correct diagnosis was increased when using magnification and AI. CONCLUSIONS: The diagnostic performance of mAI for differentiating colonic lesions is comparable to that of endoscopists, regardless of their experience. However, it can be affected by the use of magnification as well as the endoscopists' level of experience.

Meningeal carcinomatosis with decreased visual acuity: a case report.

Meningeal carcinomatosis is a condition in which cancer cells diffusely metastasize to the cerebral pia mater in the cerebrospinal membrane or cerebrospinal cavity. It causes a wide array of symptoms according to the site of metastasis. The prognosis is poor, especially in metastasis from solid tumors. This study reports a case of meningeal carcinomatosis caused by advanced gastric cancer, manifested by headache and vision loss. The patient was a 69-year-old man who underwent head computed tomography (CT) and magnetic resonance imaging (MRI) for persistent headaches. No abnormal findings were found; however, his vision declined, convulsions occurred, and cerebrospinal fluid (CSF) cytology showed poorly differentiated adenocarcinoma. Therefore, meningeal carcinomatosis was diagnosed. The patient died after receiving FOLFOX therapy to relieve symptoms and prolong his life. An autopsy showed no invasion of the optic nerve or surrounding tissues. As the frequency of complications of meningeal carcinomatosis in solid cancers is rare, it is crucial to actively suspect and make an early diagnosis.

Alteration in molecular properties during establishment and passaging of endometrial carcinoma patient-derived xenografts.

Patient-derived xenograft (PDX) tumor models are known to maintain the genomic and phenotypic profiles, including the histopathological structures, of the parental tumors. On the other hand, unique enrichment of single-nucleotide variants or copy number aberrations has been reported in several types of tumors. However, an understanding of endometrial carcinoma PDXs is limited. The purpose of the present study was to clarify the presence or absence of the molecular properties of endometrial carcinomas in PDXs passaged up to eight times. Established PDXs of endometrioid carcinomas maintained their histopathological characteristics, but those of carcinosarcomas predominantly consisted of sarcomatous components when compared to the parental tumors. Alterations in the proportion of cells with positive/negative immunohistochemical staining for estrogen receptor, PTEN, PAX8, and PAX2 were observed, whereas the proportions of cells with AE1/AE3, TP53, ARID1A, PMS2, and MSH6 staining were unchanged. Variants of cancer-associated genes were compared between PDXs and parental tumors. Mutations in POLE and a frameshift deletion in BRCA1 were observed in the parental tumor tissue in each of the six cases, and additional genomic alterations, which were not apparently related to histopathological and immunohistochemical alterations, were found in the PDXs of these cases. The genomic and phenotypic alterations observed between endometrial carcinoma PDXs and parental tumors were partly associated with endometrial cancer-specific characteristics related to cellular differentiation and gene mutations.

Treatment with vonoprazan for 3 weeks is not inferior to 8 weeks for the management of gastric ESD: a multicenter noninferiority randomized study.

BACKGROUND: Vonoprazan, a potassium-competitive acid blocker (VPZ), significantly reduces postoperative bleeding after gastric ESD; however, there is no consensus on the appropriate treatment duration. We conducted a randomized controlled study to demonstrate that the 3-week administration of VPZ is not inferior to the 8-week administration for ulcer healing. METHODS: This is a prospective, open-label multicenter randomized controlled trial. Patients aged 20-85 years undergoing gastric ESD were included in this study. The key exclusion criteria were patients with bleeding tendencies and those taking NSAIDs, steroids, PPIs, or VPZ medications. Eligible patients were randomly assigned to the VPZ 3w or 8w treatment group. The primary endpoint was the proportion of patients with complete closure of the post-ESD wound at 24 weeks after ESD. The key secondary endpoints included the proportion of patients with complete closure of the post-ESD wound at 8 weeks and the proportion of bleeding or perforation more than 3 weeks after ESD. RESULTS: From May 2018 to October 2020, 234 patients were included. The proportion of patients with complete ulcer closure was significantly lower in the 3w group than in the 8w group (70.8% vs. 90.6%) at 8 weeks post-treatment. The complete closure rates at 24 weeks in the 3w and 8w groups were 99.1% and 99.2%, respectively. The absolute difference in the closure rate at 24 weeks was - 0.059% [95% confidence interval (CI) -3.4% to 3.2], and the lower limit of the 95% CI exceeded -10%, the preset threshold. None of the patients developed delayed bleeding 3 weeks after ESD. CONCLUSION: This multicenter randomized study demonstrated that 3 weeks of treatment with VPZ is sufficient for ulcer healing. Trial registry number. UMIN000031564.

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.

Involvement of neutrophils in rat livers by low-dose thioacetamide administration.

The administration with high dose (close to LD50) of thioacetamide (TAA), a hepatotoxicant used widely to induce experimental liver lesions, develops hepatocellular necrosis and subsequent inflammation (mainly M1-/M2-macrophages without neutrophil infiltration) in rats. We analyzed rat livers treated with a low dose TAA (50 mg/kg/body weight) at 6, 12, 18, 24 and 48 hr. The lesions in the affected centrilobular areas consisted of slight hepatocyte degeneration at 12 hr, and inflammatory cell infiltration at 18 and 24 hr; the lesions recovered until 48 hr. Translocation of intranuclei to cytoplasm of HMGB1, a representative molecule of damage-associated molecular patterns, was seen in some hepatocytes mainly at 6, 12, and 18 hr. As an interesting finding, at 12 hr, myeloperoxidase-positive neutrophil infiltration was observed in the affected centrilobular area. Additionally, CD68 M1-/CD163 M2-macrophages increased consistently at 12 to 48 hr. CXCL1, a chemokine for induction of neutrophils, began to increase at 6 hr and gradually increased at 12, 18 and 24 hr, apparently corresponding to the appearance of neutrophils. Collectively, the present findings at the low dose TAA indicated that along with M1-/M2-macrophages, neutrophils were characteristically seen, which might be elicited by cytoplasmic translocation of HMGB1 from nuclei. These finding would be useful for evaluation of hepatotoxicity at the early stages.

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.

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.

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).

Differential effects of class I antiarrhythmic drugs on the guinea pig pulmonary vein myocardium: Inhibition of automatic activity correlates with blockade of a diastolic sodium current component.

The effects of class I antiarrhythmic drugs on the automaticity of isolated guinea pig pulmonary vein myocardia were investigated using microelectrode and voltage clamp methods. All of the drugs examined reduced the maximum rate of rise of automatic action potentials. The firing frequency and rate of diastolic depolarization were decreased by aprindine, flecainide and propafenone, but not by cibenzoline, disopyramide and pilsicainide, which correlated with blockade of the sodium current component induced by ramp depolarization mimicking the diastolic depolarization. In conclusion, class I antiarrhythmic drugs which block the diastolic sodium current component inhibit the automaticity of the pulmonary vein myocardium.

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.

Diffuse leiomyomatosis with circumferential thickening of the gastrointestinal wall, resembling human diffuse leiomyomatosis, in a young miniature dachshund.

Leiomyoma is the most common mesenchymal tumor in the gastrointestinal (GI) tract. Leiomyomas usually have a single or multinodular mass of various sizes, and affected animals can develop alimentary symptoms depending on the location and size. A 3-year old female miniature dachshund died after a history of refractory rectal prolapse, esophagectasis and aspiration pneumonia. At necropsy, the GI wall at the gastroesophageal and anorectal junctions was circumferentially thickened. Histologically, both GI lesions were composed of bundles of well-differentiated smooth muscles without mass formation or invasive growth. The neoplastic cells had little cellular atypia and low proliferative activity, and were positive for α-smooth muscle actin. The lesions were diagnosed as diffuse leiomyomatosis with circumferential thickening of the GI wall and has not been described in the veterinary literature.

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.

A novel splicing variant of small nucleolar RNA host gene 4 is a podocyte-selective non-coding RNA upregulated in response to puromycin aminonucleoside-induced podocyte injury.

Podocytes are terminally differentiated cells that function as the glomerular filtration barrier in the kidney, and podocyte injury leads to serious proteinuria and podocyte leakage into urine. Recent studies have demonstrated that the number of urinary podocytes is correlated with the progression of glomerular diseases. Therefore, urinary podocytes may serve as an indicator of podocyte injury. In this study, to explore podocyte injury-related genes, we performed comprehensive transcriptome analysis of primary rat podocytes cultured in the presence or absence of puromycin aminonucleoside (PAN), an agent commonly used to induce podocyte injury. RNA-seq revealed that a transcript containing the intronic sequence of small nucleolar RNA host gene 4 (Snhg4) was expressed in podocytes and upregulated by PAN. RT-qPCR analysis demonstrated that this transcript, but not Snhg4, was selectively expressed in podocytes. Therefore, we designated the novel transcript Snhg4-pod. 5'- and 3'-RACE experiments revealed that Snhg4-pod is a novel splice variant of Snhg4 lacking a poly(A) tail. PAN induced Snhg4-pod expression in podocytes in a dose-dependent manner along with their mitochondria-mediated apoptotic cell death. Further, Snhg4-pod was detected in urinary sediments from PAN-induced nephrotic rats. Our findings suggest that Snhg4-pod may serve as a novel marker for the diagnosis of glomerular injury.

Immunohistochemical characterization of myofibroblasts appearing in isoproterenol-induced rat myocardial fibrosis.

Fibrotic lesion is formed by myofibroblasts capable of producing collagens. The myofibroblasts are characterized by immunoexpressions of vimentin, desmin and α-smooth muscle actin (α-SMA) in varying degrees. The cellular characteristics remain investigated in myocardial fibrosis. We analyzed immunophenotypes of myofibroblasts appearing in isoproterenol-induced myocardial fibrosis in rats until 28 days after injection (10 mg/kg body weight); the lesions developed as interstitial edema and inflammatory cell reaction on 8 hr and days 1 and 3, and fibrosis occurred on days 1, 3, 7, 14, and 21 by gradual deposition of collagens, showing the greatest grade on day 14; the lesions gradually reduced with sporadic scar until day 28. Myofibroblasts expressing vimentin and α-SMA increased with a peak on day 3, and then, gradually decreased onwards. Interestingly, Thy-1 expressing cells appeared in the affected areas, apparently being corresponding to the grade similar to vimentin- and α-SMA-positive cells. Thy-1 is expressed in immature mesenchymal cells such as pericytes with pluripotent nature. The immunoreactivity for A3-antigen, a marker for immature mesenchymal cells, was seen in some surrounding cells. There were no cells reacting with antibodies to nestin or glial fibrillary acidic protein, although hepatic myofibroblats have been reported to react with these antibodies. Collectively, myofibroblasts appearing in rat myocardial fibrosis may have been derived from immature mesenchymal cells positive for Thy-1 or A3-antigen, with thereafter showing expressions of vimentin and α-SMA in differentiation.

Generation of Footprint-Free Canine Induced Pluripotent Stem Cells Using Auto-Erasable Sendai Virus Vector.

Canine induced pluripotent stem cells (ciPSCs) can be used in regenerative medicine. However, there are no reports on the generation of genome integration-free and completely exogenous gene-silenced (footprint free) ciPSCs that are tolerant to enzymatic single-cell passage. In this study, we reprogrammed canine embryonic fibroblasts using the auto-erasable replication-defective and persistent Sendai virus vector, SeVdp(KOSM)302L, and generated two ciPSC lines. The ciPSCs were positive for pluripotent markers, including alkaline phosphatase activity as well as OCT3/4, SOX2, and NANOG transcripts, and NANOG, stage-specific embryonic antigen-1, and partial TRA-1-60 protein expression, even after SeVdp(KOSM)302L removal. The ciPSCs were induced to differentiate into all the three germ layers as embryoid bodies in vitro and as teratomas in vivo. Furthermore, SeVdp(KOSM)302L-free ciPSCs maintained a normal karyotype even after repeated enzymatic single-cell passaging. Therefore, to our knowledge, for the first time, we demonstrated the generation of footprint-free and high-quality ciPSCs that can be passaged at the single-cell stage using enzymatic methods. Our method for generation of ciPSCs is a good step toward the development of clinical application of ciPSCs.

M1/M2-macrophage Polarization-based Hepatotoxicity in d-galactosamine-induced Acute Liver Injury in Rats.

d-galactosamine (d-GalN) is a well-known hepatotoxic agent that causes liver injury. Conversely, hepatic macrophages play a crucial role in maintaining liver tissue integrity. Macrophage functions were investigated in hepatic lesions induced by a single intraperitoneal injection of d-GalN (800 mg/kg body weight [BW]) in 6-week-old F344 rats. Blood and liver samples were examined at 8 hr and on 1, 2, 3, and 5 days postsingle injection (PSI). Hepatic lesions consisting of degeneration/sporadic foci of coagulation necrosis, inflammatory cell reaction, and reparative fibrosis were seen on PSI days 1 and 2, reflected by significantly increased serum levels of aspartate transaminase and alanine transaminase and upregulation of CD68 M1 (tumor necrosis factor-α, interleukin [IL]-6, and interferon-γ) and CD163 M2 (transforming growth factor-ß1, IL-10, monocyte chemoattractant protein-1, and IL-4) macrophage-related factors. Double immunofluorescence staining on PSI day 2 demonstrated that 82% of hepatic macrophages expressed of CD163/CD68 simultaneously; 65-75% of MHC class II macrophages showed co-expression of CD163 or CD68 and 95% CD204-expressing macrophages reacted to CD163 or CD68. These findings showed that both M1- and M2-macrophages contributed to the development of hepatic lesions induced by d-GalN and provided information about macrophage activation, indicating the importance of analysis of macrophage phenotypes for hepatotoxicity based on M1/M2-polarization.