Activation of Hepatic Stellate Cells Requires Dissociation of E-Cadherin-Containing Adherens Junctions with Hepatocytes.

Hepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC-initiating factors apart from paracrine stimuli may promote activation. The dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs was explored, establishing epithelial cadherin (E-cadherin) as an adhesion molecule linking hepatocytes and HSCs. In vivo, following carbon tetrachloride-induced liver injury, HSCs lost their spines and dissociated from adherens junctions in the early stages of injury, and were subsequently activated along with an increase in YAP/TAZ expression. After abrogation of liver injury, HSCs reconstructed their spines and adherens junctions. In vitro, reconstitution of E-cadherin-containing adherens junctions by forced E-cadherin expression quiesced HSCs and suppressed TAZ expression. Additionally, increase of TAZ expression leading to the activation of HSCs by autocrine stimulation of transforming growth factor-ß, was revealed as a mechanism of spontaneous activation. Thus, we have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechanotransduction after the loss of adherens junctions between HSCs and hepatocytes.

[A CASE OF NON-TUBERCULOUS MYCOBACTERIOSIS WITH PLEURAL EFFUSION AND THICKENING IN A PATIENT WITH AN OCCUPATIONAL HISTORY OF ASBESTOS EXPOSURE].

We report a case of a 75-year-old man with pleural effusion and an occupational history of asbestos exposure. Fluorodeoxyglucose positron emission tomography-computed tomography (FDG-PET/CT) examination revealed FDG up-takes along his pleura, leading to an initial suspicion of pleural mesothelioma. Pathological findings of a diagnostic video-associated pleural biopsy showed epithelioid cell granuloma. Repeated sputum cultures were positive for Mycobacterium intracellulare. The patient was diagnosed with pleuritis caused by non-tuberculous mycobacteria (NTM). NTM should be considered a potential cause of pleuritis.

[A Case of Anorexia Nervosa with Chewing and Spitting Improved by Treatment with Selective Serotonin Reuptake Inhibitors].

Chewing and spitting (CHSP) is the symptom of chewing and spitting out food without swallowing. CHSP is fairy common among patients with eating disorders, but no report has been published on drug treatment for it. We report a patient with anorexia nervosa showing extreme weight loss due to CHSP. After admission, CHSP was improved by treatment with Selective Serotonin Reuptake Inhibitors, leading to marked recovery of the body weight CHSP may represent a marker for illness severity, so its early treatment is critical to prevent the increasing severity of eating disorders.

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method.

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell-cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.

Morphological and microarray analyses of human hepatocytes from xenogeneic host livers.

We previously produced mice with human hepatocyte (h-hep) chimeric livers by transplanting h-heps into albumin enhancer/promoter-driven urokinase-type plasminogen activator-transgenic severe combined immunodeficient (SCID) mice with liver disease. The chimeric livers were constructed with h-heps, mouse hepatocytes, and mouse hepatic sinusoidal cells (m-HSCs). Here, we investigated the morphological features of the chimeric livers and the h-hep gene expression profiles in the xenogeneic animal body. To do so, we performed immunohistochemistry, morphometric analyses, and electron microscopic observations on chimeric mouse livers, and used microarray analyses to compare gene expression patterns in hepatocytes derived from chimeric mouse hepatocytes (c-heps) and h-heps. Morphometric analysis revealed that the ratio of hepatocytes to m-HSCs in the chimeric mouse livers were twofold higher than those in the SCID mouse livers, corresponding to twin-cell plates in the chimeric mouse liver. The h-heps in the chimeric mouse did not show hypoxia even in the twin-cell plate structure, probably because of low oxygen consumption by the h-heps relative to the mouse hepatocytes (m-heps). Immunohistochemical and electron microscopic examinations revealed that the sinusoids in the chimeric mouse livers were normally constructed with h-heps and m-HSCs. However, a number of microvilli projected into the intercellular clefts on the lateral aspects of the hepatocytes, features typical of a growth phase. Microarray profiles indicated that ∼82% of 16 605 probes were within a twofold range difference between h-heps and c-heps. Cluster and principal component analyses showed that the gene expression patterns of c-heps were extremely similar to those of h-heps. In conclusion, the chimeric mouse livers were normally reconstructed with h-heps and m-HSCs, and expressed most human genes at levels similar to those in human livers, although the chimeric livers showed morphological characteristics typical of growth.

Impact of JSS-PCS on the In-Hospital Workflow and Outcomes of Reperfusion Therapy for Acute Ischemic Stroke: Cases of a Metropolitan Secondary Emergency Facility.

Objective: Protected code stroke has been widely introduced in the emergency medical system for acute stroke in the current coronavirus disease 2019 (COVID-19) pandemic. This study aims to confirm the effects of protected code stroke formulated by the Japan Stroke Society (JSS-PCS) on the quality and outcomes of reperfusion therapy for acute ischemic stroke (AIS), followed by evaluating its validity. Methods: The subjects were 109 consecutive patients with AIS who underwent reperfusion therapy between January 2016 and July 2021, excluding in-hospital onset cases. Patients were classified according to the treatment date into the pre-COVID-19 (n = 82) and the with-COVID-19 (n = 27) groups. JSS-PCS was applied to all patients in the latter group. Statistical comparisons were made between groups on time indicators for initial treatment (onset-to-door time, door-to-imaging time [DTI], door-to-needle time [DTN], door-to-puncture time [DTP], door-to-reperfusion time, and puncture-to-reperfusion time [PTR]). The time indicator transition over the entire period was also evaluated by subgroup analysis. Subsequently, the outcomes at discharge were statistically compared between the two periods, followed by a subgroup comparison. Finally, univariate and multivariate analyses examined whether the application of JSS-PCS affected clinical outcomes. Results: Slight delays were revealed in DTI, DTN, DTP, and PTR in the with-COVID-19 group with no statistical significance. The time indicators were delayed once entering the period of the COVID-19 pandemic and then shortened again. The outcomes at discharge tended to worsen slightly in the with-COVID-19 group with no significance. Subgroup analysis depicted a transient deterioration of outcomes early in the pandemic. Applying JSS-PCS did not significantly affect clinical outcomes in univariate and multivariate analyses. Conclusion: Regarding reperfusion therapy at our facility, the introduction and application of JSS-PCS during the COVID-19 pandemic significantly affected neither time indicators nor outcomes. Infection control should be a top priority in the first medical practice for AIS in today's world, where COVID-19 shows no signs of termination.

[Anatomists in the anatomical theater of Tartu (Dorpat) University].

In 1632, King Gustav II Adolphus of Sweden founded Academia Gustaviana,the predecessor of the present Tartu University in Estonia. After the reopening of the University in 1802, the development of the Faculty of Medicine started. The number of outstanding anatomists; Burdach, von Baer, Reichert, Bidder, Reissner, Kupffer, and Rauber made various discoveries at the Anatomical Theater (Theatrum Anatomicum). The present paper acquaints readers with profiles of these anatomists and their main contributions, and attempts to consider reasons of a quick development of Tartu University during rather a short period in the 19th century.

Adverse Events of Concurrent Radiotherapy and ALK Inhibitors for Brain Metastases of ALK-Rearranged Lung Adenocarcinoma.

BACKGROUND: We investigated acute adverse events in patients with brain metastases (BMs) of anaplastic lymphoma kinase-rearranged (ALKr) non-small cell lung cancer (NSCLC) treated with both cranial radiotherapy and tyrosine kinase inhibitors (TKIs) of ALK. PATIENTS AND METHODS: Acute AEs were retrospectively investigated in patients with BMs of ALKr-NSCLC who received both whole-brain radiotherapy (WBRT) and ALK-TKI. For comparison, they were also assessed in patients with epidermal growth factor receptor (EGFR)-mutated NSCLC and wild-type with neither ALK rearrangement nor EGFR mutation treated with WBRT. RESULTS: Two ALKr cases were consequently eligible. Grade 3 otitis media unexpectedly occurred in both cases, while there was one case out of 11 and one case out of 18 of grade 2 otitis media among the EGFR-mutated cases and wild-type cases (p=0.013), respectively. CONCLUSION: Concurrent treatment with WBRT and ALK-TKI may be associated with acute severe ear toxicity in patients with BMs of ALKr-NSCLC.

Elastic fiber system evaluated in the digestive organ of rats.

According to our previous reports, the intraperiodontal elastic fiber system comprises oxytalan fibers, whereas all types of elastic system fibers are present in the gingiva. Much remains to be elucidated regarding the topographic development of the elastic fiber system that constitutes the walls of the digestive organs. This study aimed to examine the topographic development of the elastic fiber system in the periodontal tissue, oral cavity and digestive tract of rats at light- and electron microscopic levels. At embryonic day 20, in situ hybridization revealed the mRNA expression of tropoelastin in the putative gingival lamina propria but not in the dental follicle. At the postnatal stage, the masticatory mucous membrane of the gingiva and hard palate comprised three different types of elastic system fibers (oxytalan, elaunin and elastic fibers). Conversely, the elastic fiber system comprised elaunin and elastic fibers in other oral mucosae and the lining mucosae of digestive tract organs (the esophagus, stomach and small intestine). The findings of our study suggest that the elastic fiber system is mainly related to tissue resistance in the periodontal ligament and tissue elasticity in the oral mucosae without masticatory mucosae and the overlying mucosa of digestive tracts and both functions in the gingiva and hard palate, respectively. The appearance of elaunin fibers in the periodontium of rats aged 14 weeks suggests the expression of tropoelastin induced by mechanical stressors such as mastication. The intraperiodontal difference in the distribution of elaunin fibers suggests heterogeneity among fibroblasts constituting the periodontium.

Roles for Cell-Cell Adhesion and Contact in Obesity-Induced Hepatic Myeloid Cell Accumulation and Glucose Intolerance.

Obesity promotes infiltration of inflammatory cells into various tissues, leading to parenchymal and stromal cell interaction and development of cellular and organ dysfunction. Liver sinusoidal endothelial cells (LSECs) are the first cells that contact portal blood cells and substances in the liver, but their functions in the development of obesity-associated glucose metabolism remain unclear. Here, we find that LSECs are involved in obesity-associated accumulation of myeloid cells via VLA-4-dependent cell-cell adhesion. VLA-4 blockade in mice fed a high-fat diet attenuated myeloid cell accumulation in the liver to improve hepatic inflammation and systemic glucose intolerance. Ex vivo studies further show that cell-cell contact between intrahepatic leukocytes and parenchymal hepatocytes induces gluconeogenesis via a Notch-dependent pathway. These findings suggest that cell-cell interaction between parenchymal and stromal cells regulates hepatic glucose metabolism and offers potential strategies for treatment or prevention of obesity-associated glucose intolerance.

Development of the Heart Endocardium at an Early Stage of Chick Embryos Evaluated at Light- and Electron-Microscopic Levels.

Development of the endocardium in the heart of 4 to 4·1/2-day-incubated chick embryos was observed light and electron microscopically, and these results were evaluated by immunohistochemistry for desmin, FLK1 (VEGFR-2) or CD31, and by in situ hybridization assays for flk1-mRNA expression. At this developmental stage, the atrium and the ventricle were already discriminated by formation of the atrio-ventricular junction. The cardiac wall consisted of three layers; the inner endocardium, the middle myocardium, and the outer epicardium. The developing endocardium was seen as a chain of single-layered endocardial cells. Along its inner surface, numerous clusters of blood corpuscles were distributed, which seemed to contain some undifferentiated endocardial cells estimated from their characteristic ultrastructure and histological topography. Several blood corpuscles were in directly contact with the myocardium at the missing portions of the developing endocardial cell-chains. Differentiating endocardial cells individually showed roundish, small and large crescent, or flat in shapes. Such a prominent change of cell shapes appeared to be in parallel with their secretory activity during the transformation from the undifferentiated cells to the endocardial cells. Furthermore, immunohistochemistry for FLK1 or CD31, and in situ hybridization assays for flk1-mRNA labeled the cells composing developing endocardial cell-chains. Though these expressional analyses could not document clearly the transition of precursor cells into endocardial cells, the present study provided for the first time some important information regarding the morphological transition process toward endocardial cells at ultrastructural levels. Anat Rec, 299:1080-1089, 2016. © 2016 Wiley Periodicals, Inc.

Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source.

BACKGROUND: We previously reported that hepatocytes can be differentiated from embryonic stem (ES) cells by way of embryoid body (EB) formation and are transplantable into the mouse liver. However, the transplantation of EB-derived cells frequently resulted in teratoma formation in the recipient liver. In the present study, we eliminated the tumorigenic cells from EB outgrowths and examined the effects of enriched ES-cell-derived hepatocyte transplantation into an injured liver. METHODS: On day 15 in culture, the EBs were partially disaggregated and subcultured. Hepatocytes in the subcultured cells were examined by the expression of hepatocyte markers. Undifferentiated cells contaminating in the EB-derived cells were eliminated by Percoll discontinuous gradient centrifugation. Furthermore, undifferentiated cells, endothelial cells, and macrophages were eliminated by magnetic cell sorting using platelet/endothelial cell adhesion molecule (PECAM)-1 and Mac-1 antibodies. These enriched ES-cell-derived hepatocytes were then transplanted into the injured mouse liver. RESULTS: Percoll centrifugation and PECAM-1 antibodies eliminated the undifferentiated cells expressing Oct-3/4 from the EB-derived cells. ES-cell-derived hepatocytes showed expression of liver-related genes, synthesis of urea and glycogen, and structural characteristics during subculture. A transplantation study showed that the enriched ES-cell-derived hepatocytes integrated into the injured mouse liver and produced no teratomas. When the ES-cell-derived hepatocytes were transplanted into a CCl4-injured liver, the liver function was subsequently improved. CONCLUSIONS: Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity.

A case of lupus cystitis diagnosed from urinary symptoms as the sole initial complaint.

Cystitis related with systemic lupus erythematosis (SLE) is termed as lupus cystitis. It is a subtype of SLE in which gastrointestinal and bladder symptoms are prominent and it usually manifests with other symptoms of SLE. We present a case in a 37-year-old woman whose sole complaint was the severe urinary symptoms. A biopsy of the bladder showed inflammation in the submucosa and the finding of vasculitis. An autoimmune disease was suspected from the blood serum sample. A further laboratory study was performed and she was diagnosed with SLE and lupus cystitis. Prompt therapy with corticosteroids resulted in the improvement of the severe bladder symptoms.

Dynamic observation of oxygenation-induced contraction of and transient fiber-network formation-disassembly in cultured human brain microvascular endothelial cells.

Oxygenation-induced contraction of nonconfluent cultured human brain microvascular endothelial cells (HBECs, n = 30) was examined by video-enhanced contrast-differential interferential contrast microscopy. After administering a continuous gentle blow of pure oxygen gas to the surface of the medium just above the flattened HBEC, the plasma membrane exhibited tensioning and wrinkling, resulting in a strong contraction of the cell body by 14 +/- 7% (P < 0.001). When the cell stopped contracting, transient formation of a fiber network starting from certain spots (possibly adhesion plaques, though these were not visible in the majority of cases) and expanding to the whole cell was observed. The occurrence of fiber network formation was statistically significant (26 of 30 separate cells, P < 0.05). After cessation of oxygen delivery, the observed network of fibers broke up rapidly (in a period of 3.3 +/- 1.2 seconds) into small particles of <0.5 microm in diameter, which subsequently fused into the cellular structure. The HBEC completely recovered the control appearance. The sequential process was completed within 30 seconds and was reproduced in individual cells each time that oxygen gas was supplied. The authors conclude that the HBEC strongly contracts in response to a transient oxygenation stimulus, followed by rapid formation/disassembly of a network structure.

[Empyema of the ureteral stump with multiple stones after nephrectomy].

A 64-year-old woman, who had undergone right nephrectomy because of right incomplete double pyeloureter and dysplastic kidney with a ureteral stone at the age of 25, presented with a chief complaint of repeated urinary tract infection associated with right lower abdominal pain. A diagnosis of empyema of the ureteral stump with multiple stones was made based on X-ray findings and cystoscopy. In December 1999, the right residual ureter and ureteral stones were removed. Histopathologically, non-specific inflammatory change and fibrosis of the wall were observed. The ureter including the stones should have been resected at the previous nephrectomy.

[Fibrous pseudotumor of the tunica vaginalis testis: a case report].

A case of fibrous pseudotumor originating from the tunica vaginalis testis is reported. A 42-year-old man was referred to our hospital with the complaint of painless nodules along his right epididymis. There was no evidence of specific history. Ultrasonography showed an isoechoic small mass attached to the right testis. Inflammatory granulomas of the right epididymis were suspected, and an operation was carried out. The operative appearance indicated tumors of various sizes originating from the tunica vaginalis testis. Tumors were resected with the tunica vaginalis testis. Pathological diagnosis of the tumor was a fibrous pseudotumor. We reviewed and discussed 33 cases of intrascrotal fibrous pseudotumor, including our case, in the Japanese literature.

Apoptotic response through a high mobility box 1 protein-dependent mechanism in LPS/GalN-induced mouse liver failure and glycyrrhizin-mediated inhibition.

HMGB1 is a nuclear component involved in nucleosome stabilization and transcription regulation, but extracellularly it is able to serve as a potential late mediator of lethality. In the present study, we explored inflammation-promoting activity of HMGB1 and blockade of extracellular release of HMGB1 by glycyrrhizin (GL) in LPS/GalN-triggered mouse liver injury. At 1 to 10 h after LPS/GalN-treatment, mice were anesthetized to collect blood from heart puncture, and serum transaminase and HMGB1 were evaluated. Administration of LPS/GalN precipitated tissue injury associated with time-dependent alteration in HMGB1 serum levels. At 8 h nuclear immunoreactive products were remarkably reduced and extracellular HMGB1 expression was found exclusively in the pericentral foci. The treatment with GL significantly down-regulated the serum levels of ALT, AST, and HMGB1 in addition to the strong inhibition of tissue injury and extracellular immunoreactivity to HMGB1 and to acetylated-lysine. Furthermore, GL brought about a significant decrease in the number of apoptotic hepatocytes labeled with TUNEL-method. On the basis of these results, three apoptosis-associated genes were identified with microarray analysis and real-time PCR. The ChIP-assay revealed the binding of HMGB1 protein to Gsto1 promoter sequence in LPS/GalN-treated mice and the remarkable decrease in combined HMGB1 protein by GL. The current findings claim that a single injection of LPS/GalN might stimulate apoptosis of hepatocytes through the binding of HMGB1 protein to Gsto1 promoter region and that GL-treatment might prevent the apoptosis and inflammatory infiltrates caused with LPS/GalN-injection by disturbing the binding of HMGB1 protein to Gsto1 promoter sequence.

Development of the oxytalan fiber system in the periodontal space of rat incisors.

The present study clarifies developmental organization of the oxytalan fiber system in the periodontal space of both the enamel (labial) and cementum (lingual) sides of rat incisors. The number of oxytalan fibers per unit area (µm(2)) was counted in rat incisors at stages of embryonic day 20 (E20) to postnatal day 35 (P35). Oxytalan fibers in the periodontal space of the enamel side were apt to decrease in number during the postnatal period, whereas their number remained almost unchanged on the cementum side during the developmental period. When the incisor emerged through the gum at P11, thinner oxytalan fibers distributed in the apical growing periodontium of the cementum side seemed to be fused with one another to become thicker fibers as has been reported for rat molars (Inoue et al., 2012). Thus, the oxytalan fiber system in the periodontal space represented significant differences in its distributional density between the enamel and cementum sides after E23. At the stage of P35, oxytalan fibers presented significantly denser distribution in all territories of the periodontal ligament of the cementum side versus the enamel side. The present findings claim that the oxytalan fiber system might bind the tooth to the periodontal ligament and provide equilibrium of vascular system and control of blood flow in the periodontal ligament of the cementum side, while it might exclusively regulate the high level of physiologically adapted vasculature in the periodontal space of the enamel side.

Association between EGFR-TKI resistance and efficacy of radiotherapy for brain metastases from EGFR-mutant lung adenocarcinoma.

AIM: To clarify how patients with epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma with acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs) respond to radiotherapy (RT) for brain metastases. PATIENTS AND METHODS: Forty-seven patients were divided into the following three groups: a TKI-naïve group with EGFR mutation (n=11), a TKI-resistant group with EGFR mutation (n=10), and an EGFR-wild-type group (n=26). Patients received stereotactic RT (n=23) or whole-brain RT (n=24). RESULTS: The response rate for patients with TKI-resistant tumor at three months after RT tended to be lower (11%) than that of those who were TKI-naïve (82%, p=0.006) and for patients with wild-type EGFR (48%, p=0.10). On univariate analysis, central nervous system progression-free and overall survival were significantly shorter for patients with TKI-resistant tumors than for those who were TKI-naïve (p=0.018 and p=0.005, respectively). Multivariate analysis showed that TKI resistance was an independent predictor of poorer overall survival (p=0.011). CONCLUSION: Acquired resistance to TKIs appears to be associated with low efficacy of brain RT.

Drug-induced thrombotic thrombocytopenic purpura successfully treated with recombinant human soluble thrombomodulin.

A 61-year-old woman with recurrent non-small cell lung cancer presented with thrombocytopenia, microangiopathic hemolytic anemia, neurological abnormalities, renal failure and a fever that appeared during chemotherapy with gemcitabine and bevacizumab. She was diagnosed with drug-induced thrombotic thrombocytopenic purpura (TTP). After the discontinuation of chemotherapy, the administration of recombinant human soluble thrombomodulin and fresh-frozen plasma rapidly ameliorated the TTP. Hypertension preceded the onset of TTP and required the administration of quadruple therapy on admission. However, after three months, the hypertension was controllable without anti-hypertensive drugs. Twelve months later, the ninth course of vinorelbine was administered safely, preventing the patient's lung cancer from progressing.