Asymmetric profiles of infection and innate immunological responses in human iPS cell-derived small intestinal epithelial-like cell monolayers following infection with mammalian reovirus.

The intestinal mucosa plays an important role as an immune barrier due to its continual exposure to invading pathogens, including viruses. It is thus highly important to evaluate virus infection profiles in the intestinal mucosa for prevention of virus infection and development of antivirus medicines; however, only a few enterocyte lines are available as in vitro intestinal models for the evaluation of virus infection. In this study, we evaluated profiles of infection and innate immune responses following infection with a mammalian orthoreovirus (hereafter reovirus), which has often been used as a tractable model for studies of viral pathogenesis, in human iPS cell-derived small intestinal epithelial-like cell (hiPS-SIEC) monolayers and cells of a human colon adenocarcinoma cell line, Caco-2. The levels of reovirus infection were similar between hiPS-SIEC and Caco-2 cell monolayers, which are often used as an intestinal model, after apical and basolateral infection. In hiPS-SIEC monolayers, more efficient replication of the virus genome was observed following basolateral infection than apical infection, while apical infection resulted in higher levels of virus protein expression and progeny virus production than basolateral infection. Reovirus significantly induced innate immune responses, including expression of type I and III interferons (IFNs), in hiPS-SIEC monolayers more efficiently than Caco-2 cells. Higher levels of type I and III interferon (IFN) expression were found in hiPS-SIEC monolayers following apical infection than basolateral infection. These results suggested that hiPS-SIECs are a promising in vitro model for the evaluation of virus infection.

miR-27b-mediated suppression of aquaporin-11 expression in hepatocytes reduces HCV genomic RNA levels but not viral titers.

BACKGROUND: MicroRNAs (miRNAs) have gained much attention as cellular factors regulating hepatitis C virus (HCV) infection. miR-27b has been shown to regulate HCV infection in the hepatocytes via various mechanisms that have not been fully elucidated. In this study, therefore, we examined the mechanisms of miR-27b-mediated regulation of HCV infection. METHODS: In silico screening analysis, transfection with miR-27b mimic, and a cell-based reporter assay were performed to identify miR-27b target genes. Cell cultured-derived HCV (HCVcc) was added to Huh7.5.1 cells knocked down for aquaporin (AQP) 11 (AQP11) and overexpressing AQP11. HCV replication levels were evaluated by real-time RT-PCR analysis of HCVcc genome. RESULTS: Infection of Huh7.5.1 cells with HCVcc resulted in significant elevation in miR-27b expression levels. In silico analysis revealed that AQP11, which is an AQP family member and is mainly localized in the endoplasmic reticulum (ER), was a candidate for a target gene of miR-27b. Transfection of a miR-27b mimic significantly reduced AQP11 expression, but a cell-based reporter assay demonstrated that miR-27b did not suppress the expression of a reporter gene containing the 3'-untranslated region of the AQP11 gene, suggesting that miR-27b indirectly suppressed AQP11 expression. AQP11 expression levels were significantly reduced by infection with HCVcc in Huh7.5.1 cells. Knockdown and over-expression of AQP11 significantly reduced and increased HCVcc genome levels in the cells following infection, respectively, however, AQP11 knockdown did not show significant effects on the HCVcc titers in the culture supernatants. CONCLUSIONS: These results indicated that HCV infection induced a miR-27b-mediated reduction in AQP11 expression, leading to a modest reduction in HCV genome levels in the cells, not HCV titers in the culture supernatants.

[Anesthetic management using the arterial pressure-based cardiac output monitor and a central venous oximetry catheter for tricuspid valve replacement in a patient receiving hemodialysis].

A 64-year-old man receiving hemodialysis underwent a tricuspid valve replacement. We used an arterial pressure-based cardiac output (APCO) monitor and a central venous oximetry catheter instead of pulmonary artery catheter because the enlargement of the right ventricle caused by tricuspid valve regurgitation might make it difficult to insert the pulmonary artery catheter. Aortic calcification was so severe that an arterial cannula was sewn into the right subclavian artery. Therefore, we had to insert an arterial catheter into the shunt placed on his left upper limb, and connected it to the APCO monitor FloTrac (Edwards Life-sciences, U.S.A.). Anesthesia was induced with intravenous fentanyl, midazolam and rocuronium, and maintained with fentanyl and propofol. A triple lumen PreSep central venous oximetry catheter (Edwards Lifesciences, USA) was inserted via his right internal jugular vein. Under cardiopulmonary bypass beating tricuspid valve replacement was performed. Although a large amount of catecholamine was needed for weaning from cardiopulmonary bypass, it was performed referring to the cardiac index of APCO monitor. The operation was finished successfully. We concluded that cardiac function should be evaluated with the relative change of cardiac index from APCO monitor when the absolute value was uncertain from patient's pathology (e.g., valvular disease).

[Evaluation of urine specific gravity as an index of hypotension after spinal anesthesia for cesarean section].

BACKGROUND: Although most cesarean sections are done under spinal anesthesia, we often experience severe hypotension. Fluid resuscitation is usually carried out for prevention of hypotension, but it is difficult to assess the suitable infusion volume. We examined whether the urine specific gravity can predict hypotension after spinal anesthesia for cesarean section. METHODS: Ninety nine patients (ASA 1 or 2) undergoing elective cesarean section were recruited. After dural puncture, we collected the cerebrospinal fluid and injected 2 ml of hyperbaric 0.5% bupivacaine. Thereafter urethral catheters were inserted, and then we collected the urine sample. The specific gravity of each sample was measured by using refractometer after the operation. RESULTS: There was a good correlation between the urinary output and the urine specific gravity. The minimum systolic blood pressure until delivery, the total dose of ephedrine, and the maximum sensory block level showed a significant, but not particularly strong correlation with the urine specific gravity. CONCLUSIONS: We concluded that it was difficult to predict hypotension by using urine specific gravity because the correlation was too weak.

A cell leakproof PLGA-collagen hybrid scaffold for cartilage tissue engineering.

A cell leakproof porous poly(DL-lactic-co-glycolic acid) (PLGA)-collagen hybrid scaffold was prepared by wrapping the surfaces of a collagen sponge except the top surface for cell seeding with a bi-layered PLGA mesh. The PLGA-collagen hybrid scaffold had a structure consisting of a central collagen sponge formed inside a bi-layered PLGA mesh cup. The hybrid scaffold showed high mechanical strength. The cell seeding efficiency was 90.0% when human mesenchymal stem cells (MSCs) were seeded in the hybrid scaffold. The central collagen sponge provided enough space for cell loading and supported cell adhesion, while the bi-layered PLGA mesh cup protected against cell leakage and provided high mechanical strength for the collagen sponge to maintain its shape during cell culture. The MSCs in the hybrid scaffolds showed round cell morphology after 4 weeks culture in chondrogenic induction medium. Immunostaining demonstrated that type II collagen and cartilaginous proteoglycan were detected in the extracellular matrices. Gene expression analyses by real-time PCR showed that the genes encoding type II collagen, aggrecan, and SOX9 were upregulated. These results indicated that the MSCs differentiated and formed cartilage-like tissue when being cultured in the cell leakproof PLGA-collagen hybrid scaffold. The cell leakproof PLGA-collagen hybrid scaffolds should be useful for applications in cartilage tissue engineering.

[Case of pulmonary edema and transient heart failure during difficult airway management].

Many kinds of side effects are likely to occur while managing difficult airway. This article describes a case of a man who fell into pulmonary edema and heart failure during the difficult airway management. He was to undergo arthroscopic surgery on his knee. At first, we planed spinal anesthesia but he took an antiplatelet drug (aspirin). Since we wanted to avoid the epidural hematoma, we chose general anesthesia. After induction of general anesthesia, positive pressure ventilation via face mask was possible, but laryngeal mask ventilation was impossible. Although tracheal intubation was attempted, we recognized that he had difficult airway. After some intubation trials, we decided to quit the operation and awoke the patients. Spontaneous breathing appeared soon, but the oxygenation was getting worse. We performed fiberoptic nasal intubation under spontaneous breathing. Although his blood pressure was quite high during intubation, after intubation the vasopressor was needed to maintain blood pressure. Ventilation with 100% O2 could not maintain the oxygenation well. Chest X-ray revealed the pulmonary edema. We presumed that hypertension associated with airway stimulation had caused acute pulmonary edema and heart failure resulting from diastolic dysfunction induced by increased catecholamine.

Randomized phase II trial comparing amrubicin with topotecan in patients with previously treated small-cell lung cancer: North Japan Lung Cancer Study Group Trial 0402.

PURPOSE: Amrubicin, a new anthracycline agent, and topotecan are both active for previously treated small-cell lung cancer (SCLC). No comparative study of these agents has been reported. This randomized phase II study was conducted to select amrubicin or topotecan for future evaluation. PATIENTS AND METHODS: Patients with SCLC previously treated with platinum-containing chemotherapy were randomly assigned to receive amrubicin (40 mg/m(2) on days 1 through 3) or topotecan (1.0 mg/m(2) on days 1 through 5). Patients were stratified by Eastern Cooperative Oncology Group performance status (0, 1, or 2) and type of relapse (chemotherapy sensitive or refractory). The primary end point was overall response rate (ORR), and secondary end points were progression-free survival (PFS), overall survival, and toxicity profile. RESULTS: From February 2004 to July 2007, 60 patients were enrolled, and 59 patients (36 patients with sensitive and 23 patients with refractory relapse) were assessable for efficacy and safety evaluation. Neutropenia was severe, and one treatment-related death owing to infection was observed in the amrubicin arm. ORRs were 38% (95% CI, 20% to 56%) for the amrubicin arm and 13% (95% CI, 1% to 25%) for the topotecan arm. In sensitive relapse, ORRs were 53% for the amrubicin arm and 21% for the topotecan arm. In refractory relapse, ORRs were 17% for the amrubicin arm and 0% for the topotecan arm. Median PFS was 3.5 months for patients in the amrubicin arm and 2.2 months for patients in the topotecan arm. Multivariate analysis revealed that amrubicin has more influence than topotecan on overall survival. CONCLUSION: Amrubicin may be superior to topotecan with acceptable toxicity for previously treated patients with SCLC. Further evaluation of amrubicin for relapsed SCLC is warranted.

[A case of malignant pleural mesothelioma with elevation of G-CSF and CYFRA in the serum and pleural fluid].

A 68-year-old man complaining of hoarseness and back pain, with no history of exposure to asbestos, was referred to our hospital in June 2002. He was admitted because his chest X-ray and CT scan showed atelectasis and a tumor-like region in the right lower lobe of the lung. Serum-CYFRA was 2.8 ng/ml, elevated slightly; however, no other tumor markers for lung cancer were elevated. A diagnosis of squamous cell lung cancer was made based on bronchial washing cytology. Persistent high fever and WBC count elevation did not respond to antibiotics, and reduced only after chemotherapy. Both serum G-CSF (217.0 pg/ml) and CYFRA in the pleural effusion (107.1 ng/ml) were elevated. The biopsy of the growing tumor in the right lateral abdominal wall revealed carcinoma with sarcomatous component or biphasic-type malignant pleural mesothelioma (MPM). In spite of chemotherapy and radiation therapy for the abdominal wall tumor, the tumor rapidly progressed and the patient died three months after admission. The findings at autopsy suggested the tumor was a sarcomatous MPM. However, immunohistochemical staining and tissue HABP staining revealed biphasic type MPM. Although CYFRA elevation in the serum and/or the pleural effusion in MPM patients has been previously reported, it has not been reported in any of the 5 MPM patients reported to have G-CSF elevation. Therefore, this is the first reported case of G-CSF-producing MPM with CYFRA elevation in both serum and the pleural effusion.

Regulation of proliferation, motility, and contractivity of cultured human endometrial stromal cells by transforming growth factor-beta isoforms.

OBJECTIVE: To evaluate the involvement of transforming growth factor (TGF)-beta isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) on endometrial tissue remodeling during the perimenstrual period. DESIGN: The effects of TGF-beta isoforms on the cell proliferation, motility, and contractivity of cultured human endometrial stromal cells (ESCs) were investigated. SETTING: Research laboratory at a medical school. PATIENT(S): Nine endometrial specimens in the late secretory phase were used. INTERVENTION(S): Endometrial stromal cells were incubated with recombinant human recombinant TGF-beta1, TGF-beta2, and TGF-beta3. MAIN OUTCOME MEASURE(S): The cell proliferation, motility, and contractivity of ESCs were accessed by a modified methylthiazoletetrazolium assay, in vitro wound repair assay, transwell invasion assay, and collagen gel contraction assay. RESULT(S): All three isoforms of TGF-beta significantly inhibited the cell proliferation of ESCs in a dose-dependent manner. In vitro wound repair assay and transwell invasion assay demonstrated that the TGF-beta isoforms significantly inhibited the motility of ESCs. However, the TGF-beta isoforms were shown to have a clear effect on the collagen gel contractivity of ESCs. CONCLUSION(S): These results suggest that TGF-beta isoforms may promote endometrial tissue repair through the inhibition of the proliferation, expansion, and migration of ESCs, and through the stimulation of the contraction of the collagen gel matrix by these cells. Transforming growth factor-beta may be involved in the protection of the endometrium from extensive fibrosis and scarring by regulating ESC function during the perimenstrual period.

Effects of theanine on alcohol metabolism and hepatic toxicity.

We previously showed that theanine, is a major amino acid in green tea, enhanced doxorubicin (DOX) induced antitumor activity. Besides, theanine induced the elevation of glutathione (GSH) level attributable to the increase of glutamate in the liver of mice, namely theanine would reduce the adverse reaction of DOX. Consequently, theanine was thought to be effective against the tissue changes with GSH level reduction. On the other hand, it is suggested excessive uptake of alcohol causes a production of free radicals, a decrease of GSH level, and an increase in the amount of lipid peroxide (LPO) in liver, and shifting to an alcoholic liver injury. Then, aiming at the prevention and medical treatment of a hepatic toxicity by the food components with little toxicity, we have studied the effect of theanine (i.p.) on ethanol metabolism and hepatic toxicity using ethanol (p.o.) single-administered mice. On the 1st hour after ethanol administration, the ethanol concentrations in blood of the theanine combined groups decreased compared with the ethanol-alone group. The alcohol dehydrogenase and aldehyde dehydrogenase activities in the liver increased by combined theanine. Since the elevation of cytochrome P450 (CYP) 2E1 activity was controlled in the theanine-combined groups, it was considered that these disorders attributable to CYP2E1 in ethanol long-term uptake might be avoidable by theanine. Although LPO increased in 3 h after by single-administration of ethanol, the increase was controlled by theanine-administration and was improved until the normal level. In conclusion, it was indicated that theanine was effective against alcoholic liver injury.

Squamous cell carcinoma of the cervix producing granulocyte colony-stimulating factor.

BACKGROUND: Marked leukocytosis is occasionally observed in patients with a malignant nonhematopoietic tumor. Granulocyte colony-stimulating factor (G-CSF) may be responsible for this phenomenon. We report a case of G-CSF-producing squamous cell carcinoma of the cervix that showed marked leukocytosis. CASE: A 71-year-old Japanese woman was admitted for further investigation for leukocytosis. Her white blood cell (WBC) count had been gradually increasing over a period of 10 months. Laboratory data on admission revealed marked leukocytosis, with a WBC count of 30,400/microL, which consisted primarily of mature granulocytes (93%). Her serum G-CSF level was significantly elevated. However, there was no evidence of infection or hematopoietic disorders. Further examinations showed stage IIIb cervical cancer. The pathological diagnosis was squamous cell carcinoma of the nonkeratinizing type. Immunohistochemical staining of the biopsied specimens confirmed the production of G-CSF protein by the tumor cells. The patient was successfully treated by radiation therapy. Her WBC count returned to a normal level (3,700/microL). Her serum G-CSF level also decreased. The patient is alive without evidence of recurrence at 8 months after the treatment. CONCLUSION: It is suggested that the leukocytosis manifested in this patient was due to G-CSF produced by the tumor. It was possible to use the WBC count and serum G-CSF levels as additional tumor markers.

A case of mixed connective tissue disease showing a lymphoid follicle in muscle pathology.

We report a 74-year-old man suffering from mixed connective tissue disease (MCTD) accompanied by a lymphoid follicle in muscle pathology. He showed proximal muscle weakness and arthritis in finger joints. His laboratory findings were characteristic of MCTD. Prednisolone administration favorably improved his symptoms. A lymphoid follicle is a rare pathological finding in inflammatory myopathies. The immunohistochemical findings suggest that the lymphoid follicle would be involved in the specific role for the pathogenesis of MCTD.