Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus-infected mice.

UNLABELLED: Essentials H1N1 Influenza A virus (IAV) infection is a hemostatic challenge for the lung. Tissue factor (TF) on lung epithelial cells maintains lung hemostasis after IAV infection. Reduced TF-dependent activation of coagulation leads to alveolar hemorrhage. Anticoagulation might increase the risk for hemorrhages into the lung during severe IAV infection. SUMMARY: Background Influenza A virus (IAV) infection is a common respiratory tract infection that causes considerable morbidity and mortality worldwide. Objective To investigate the effect of genetic deficiency of tissue factor (TF) in a mouse model of IAV infection. Methods Wild-type mice, low-TF (LTF) mice and mice with the TF gene deleted in different cell types were infected with a mouse-adapted A/Puerto Rico/8/34 H1N1 strain of IAV. TF expression was measured in the lungs, and bronchoalveolar lavage fluid (BALF) was collected to measure extracellular vesicle TF, activation of coagulation, alveolar hemorrhage, and inflammation. Results IAV infection of wild-type mice increased lung TF expression, activation of coagulation and inflammation in BALF, but also led to alveolar hemorrhage. LTF mice and mice with selective deficiency of TF in lung epithelial cells had low basal levels of TF and failed to increase TF expression after infection; these two strains of mice had more alveolar hemorrhage and death than controls. In contrast, deletion of TF in either myeloid cells or endothelial cells and hematopoietic cells did not increase alveolar hemorrhage or death after IAV infection. These results indicate that TF expression in the lung, particularly in epithelial cells, is required to maintain alveolar hemostasis after IAV infection. Conclusion Our study indicates that TF-dependent activation of coagulation is required to limit alveolar hemorrhage and death after IAV infection.

Analysis and measurement of dielectrophoretic manipulation of particles and lymphocytes using rail-type electrodes.

A particle manipulation and sorting device using the dielectrophoretic (DEP) force is described in this study. The device consists of "ladder-type", "flip-type" and "oblique rail-type" electrode regions. The ladder-type and rail-type electrodes can generate a DEP force distribution that captures the particles, the DEP force of which is negative, in the area located at the center of the electrodes. The ladder-type electrode can align the particles with equal spacing in the streamwise direction. Using the flip-type electrode, which pushes the particles away, in combination with these electrodes, the direction of the particle and timing can be selected with high accuracy, reliability, and response. In the first half of this study, a numerical simulation is carried out to calculate the particle motion and evaluate the performance of the ladder-type electrode. Several models are used to investigate the influences of the non-uniformity of the electric field and the electric interaction of the surface charges and polarizations. Experiments are then carried out to demonstrate the motions of the particles and the sorting reliability. The trajectories and the probability density functions of the particles at the inlet and outlet of the electrode region showed that by using these electrodes the particles can be aligned, sorted, and guided accurately.

Expression of p53 synergistically augments caspases-mediated apoptosis induced by replication-competent adenoviruses in pancreatic carcinoma cells.

We examined cytotoxicity of replication-competent type 5 adenoviruses (Ad5) in human pancreatic carcinoma cells with a p53-defective genotype. The replication-competent Ad5 of which E1A gene was activated by exogenous transcriptional regulatory sequences, derived from the midkine and survivin genes, achieved cytotoxicity to the pancreatic carcinoma. These cells were susceptible to replication-incompetent Ad5 expressing the wild-type p53 gene. We also produced the replication-competent Ad5 bearing the same exogenous regulatory sequences and the type 35 Ad-derived fiber-knob region, and showed that the cytotoxicity was comparable to that of the replication-competent Ad5 prototype. We then investigated possible combinatory effects of the fiber-modified replication-competent Ad and Ad5 expressing the wild-type p53 gene, both of which did not interfere respective infections. The combination produced synergistic cytotoxic effects with enhanced cleavages of caspase-3 and PARP molecules, and with increased sub-G1 fractions and annexin V-positive populations although the viral production of the replication-competent Ad was rather suppressed by expressed p53. Pancreatic cells infected with both Ad showed increase of p53 and decrease of MDM2 and p21 levels, compared with those infected with Ad expressing the p53 gene. These data collectively indicated that replication-competent Ad augmented susceptibility of pancreatic cells to apoptosis through upregulated p53 expression.

Administration of DNA Encoding the Interleukin-27 Gene Augments Antitumour Responses through Non-adaptive Immunity.

DNA-mediated immunization of a tumour antigen is a possible immunotherapy for cancer, and interleukin (IL)-27 has diverse functions in adaptive immunity. In this study, we examined whether IL-27 DNA administration enhanced antitumour effects in mice vaccinated with DNA encoding a putative tumour antigen, ß-galactosidase (ß-gal). An intramuscular injection of cardiotoxin before DNA administration facilitated the exogenous gene expression. In mice received ß-gal and IL-27 DNA, growth of ß-gal-positive P815 tumours was retarded and survival of the mice was prolonged. Development of ß-gal-positive Colon 26 tumours was suppressed by vaccination of ß-gal DNA and further inhibited by additional IL-27 DNA administration or IL-12 family cytokines. Nevertheless, a population of ß-gal-specific CD8(+) T cells did not increase, and production of anti-ß-gal antibody was not enhanced by IL-27 DNA administration. Spleen cells from mice bearing IL-27-expressing Colon 26 tumours showed greater YAC-1-targeted cytotoxicity although CD3(-)/DX5(+) natural killer (NK) cell numbers remained unchanged. Recombinant IL-27 enhanced YAC-1-targeted cytotoxicity of IL-2-primed splenic NK cells and augmented a phosphorylation of signal transducer and activator of transcription 3 and an expression of perforin. These data collectively indicate that IL-27 DNA administration activates NK cells and augments vaccination effects of DNA encoding a tumour antigen through non-adaptive immune responses.

Nutritional management of anastomotic leakage after colorectal cancer surgery using elemental diet jelly.

BACKGROUND/AIMS: Anastomotic leakage is major complication of colorectal surgery. Total parenteral nutrition (TPN) and fasting are conservative treatments for leakage in the absence of peritonitis in Japan. Elemental diet (ED) jelly is a completely digested formula and is easily absorbed without secretion of digestive juices. The purpose of this study was to assess the safety of ED jelly in management of anastomotic leakage. METHODOLOGY: Six hundred and two patients who underwent elective surgery for left side colorectal cancer from January 2008 to December 2011 were included in the study. Pelvic drainage was performed for all patients. Sixty-three (10.5%) patients were diagnosed with an anastomotic leakage, and of these, 31 (5.2%) without diverting stoma were enrolled in this study. RESULTS: Sixteen patients received TPN (TPN group) and 15 patients received ED jelly (ED group). The duration of intravenous infusion was significantly shorter in the ED group than in the TPN group (15 days versus 25 days, P= 0.008). In the TPN group, catheter infection was occurred in 2 patients who required re-insertion of the catheter. CONCLUSION: Conservative management of anastomotic leakage after colorectal surgery with ED jelly appears to be a safe and useful approach.

Zoledronic acid induces apoptosis and S-phase arrest in mesothelioma through inhibiting Rab family proteins and topoisomerase II actions.

Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.

Combination of adenoviruses expressing melanoma differentiation-associated gene-7 and chemotherapeutic agents produces enhanced cytotoxicity on esophageal carcinoma.

We examined the combinatory antitumor effects of adenoviruses expressing human mda-7/IL-24 gene (Ad-mda-7) and chemotherapeutic agents on nine kinds of human esophageal carcinoma cells. All the carcinoma cells expressed the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) receptor complexes, IL-20R2 and either IL-20R1 or IL-22R1, and were susceptible to Ad-mda-7, whereas fibroblasts were positive only for IL-20R2 gene and resistant to Ad-mda-7-mediated cytotoxicity. Sensitivity of these esophageal carcinoma cells to Ad-mda-7 was however lower than that to Ad expressing the wild-type p53 gene. We thereby investigated a possible combination of Ad-mda-7 and anticancer agents and found that Ad-mda-7 with 5-fluorouracil (5-FU), cisplatin, mitomycin C or etoposide produced greater cytotoxic effects than those by Ad-mda-7 or the agent alone. Half-maximal inhibitory concentration values of the agents in respective cells were decreased by the combination with Ad-mda-7. Cell cycle analyses showed that Ad-mda-7 and 5-FU increased G2/M-phase and S-phase populations, respectively, and the combination augmented sub-G1 populations. Ad-mda-7-treated cells showed cleavages of caspase-8, -9 and -3 and poly (ADP-ribose) polymerase, but the cleavage levels were not different from those of the combination-treated cells. Ad-mda-7 treatments upregulated Akt phosphorylation but suppressed IκB-α levels, whereas 5-FU treatments induced phosphorylation of p53 and extracellular signal-regulated protein kinases 1 and 2. Molecular changes caused by the combination were similar to those by Ad-mda-7 treatments, but the Ad-mda-7-mediated upregulation of Akt phosphorylation decreased with the combination. These data collectively suggest that Ad-mda-7 induced apoptosis despite Akt activation and that the combinatory antitumor effects with 5-FU were produced partly by downregulating the Ad-mda-7-induced Akt activation.

Plant homeodomain finger protein 11 promotes class switch recombination to IgE in murine activated B cells.

BACKGROUND: Polymorphisms of the Plant homeodomain finger protein 11 (PHF11) are strongly associated with high serum IgE levels and clinical severity of atopic patients. However, the precise mechanism has not been fully elucidated. We investigated the role of Phf11 in class switch recombination (CSR) to IgE by activated B cells. METHODS: We generated Phf11 transgenic (Lckd-Phf11-Tg) mice that express the exogenous murine Phf11 in lymphocytes under the control of distal Lck promoter. We examined IL-4-induced CSR to IgE in activated Lckd-Phf11-Tg B cells in vitro. We analyzed production of ovalbumin (OVA)-specific IgE and nose-scratching symptoms in Lckd-Phf11-Tg mice using an OVA-induced allergic rhinitis model. RESULTS: The exogenous Phf11 promoted CSR to IgG1 and IgE in activated B cells with an increase in germ line transcript (GLT) γ1 and GLT ε expression. The exogenous Phf11 augmented transcriptional activity of the GLT γ1 and GLT ε promoters through permissive histone modifications and binding of NF-κB and STAT6. Furthermore, the exogenous Phf11 bound to the GLT ε promoter with increased binding of NF-κB. Silencing of the endogenous Phf11 reduced the frequency of CSR to IgE and GLT ε expression, but not to IgG1 or GLT γ1 expression, in activated B cells. In an allergic rhinitis model, Lckd-Phf11-Tg mice showed a significant increase in the production of OVA-specific IgE and the frequency of nose scratching. CONCLUSION: Phf11 accelerates CSR to IgE in activated B cells by increasing the transcriptional activity of GLT ε promoter and contributes to the exacerbation of allergic responses. These findings provide a novel therapeutic target for allergic diseases.

Perioperative haemostatic management of haemophilic mice using normal mouse plasma.

Intense haemostatic interventions are required to avoid bleeding complications when surgical procedures are performed on haemophilia patients. The objective of this study was to establish an appropriate protocol for perioperative haemostatic management of haemophilic mice. We assessed the prophylactic haemostatic effects of normal mouse plasma (NMP) on haemophilia B (HB) mice for both a skin flap procedure and a laparotomy. When 500 µL of NMP was administered to the mice, plasma factor IX (FIX:C) levels peaked at 15.1% immediately after intravenous (IV) administration, at 6.1% 2 h after intraperitoneal (IP) administration and at 2.7% 6 h after subcutaneous administration. Administering 500 µL of NMP via IP or IV 30 min in advance enabled the skin flap procedure to be performed safely without any complications. After the laparotomy procedure, several mice in the IP administration group exhibited lethal bleeding, but all mice survived in the IV administration group. Anti-mouse FIX inhibitors did not develop, even after repetitive administrations of NMP. However, human FIX concentrates, especially plasma-derived concentrates, elicited the anti-human FIX inhibitors. The results show that administering 500 µL of NMP via IV or IP 30 min in advance enables surgical procedures to be safely performed on HB mice, and that IV administration is more desirable than IP if the procedure requires opening of the abdominal wall.

Parameter-free extraction of EMCD from an energy-filtered diffraction datacube using multivariate curve resolution.

We present a parameter-free method of extraction of the electron magnetic circular dichroism spectra from energy-filtered diffraction patterns measured on a crystalline specimen. The method is based on a multivariate curve resolution technique. The main advantage of the proposed method is that it allows extraction of the magnetic signal regardless of the symmetry and orientation of the crystal, as long as there is a sufficiently strong magnetic component of the signal in the diffraction plane. This method essentially overcomes difficulties in extraction of the EMCD signal caused by complexity of dynamical diffraction effects.

Upregulated p53 expression activates apoptotic pathways in wild-type p53-bearing mesothelioma and enhances cytotoxicity of cisplatin and pemetrexed.

The majority of malignant mesothelioma possesses the wild-type p53 gene with a homologous deletion of the INK4A/ARF locus containing the p14(ARF) and the p16(INK4A) genes. We examined whether forced expression of p53 inhibited growth of mesothelioma cells and produced anti-tumor effects by a combination of cisplatin (CDDP) or pemetrexed (PEM), the first-line drugs for mesothelioma treatments. Transduction of mesothelioma cells with adenoviruses bearing the p53 gene (Ad-p53) induced phosphorylation of p53, upregulated Mdm2 and p21 expression levels and decreased phosphorylation of pRb. The transduction generated cleavage of caspase-8 and -3, but not caspase-9. Cell cycle analysis showed increased G0/G1- or G2/M-phase populations and subsequently sub-G1 fractions, depending on cell types and Ad-p53 doses. Transduction with Ad-p53 suppressed viability of mesothelioma cells and augmented the growth inhibition by CDDP or PEM mostly in a synergistic manner. Intrapleural injection of Ad-p53 and systemic administration of CDDP produced anti-tumor effects in an orthotopic animal model. These data collectively suggest that Ad-p53 is a possible agent for mesothelioma in combination with the first-line chemotherapeutics.

Historical changes in epidemiology of diffuse panbronchiolitis.

BACKGROUND AND OBJECTIVE: Japanese pulmonologists, experienced in treating patients with diffuse panbronchiolitis (DPB) prior to the 1980s, have uniformly observed that new incidences of DPB are now a rare event in Japan. However, there is no epidemiological data to support this observation. We examined epidemiological trends of the number of patients with DPB in a large company. DESIGN: The computerized health records of JR East Company employees were used to identify patients with DPB and then these were followed up using the assessments of these patients in JR Tokyo General Hospital and two other JR hospitals. The whole study period was 27 years (1976-2003), although detailed analyses were carried out for three specific periods; the first was 1976-1980, the second was 1989-1993, and the third was 1999-2003. RESULTS: In the first period, 11 DPB cases (four incidence, and seven prevalence) were detected among a total of 355,572 workers. In the second period, three DPB cases (one incidence, and two prevalence) were identified from a total of 180,359 workers. In the third period, no case was found in a total of 144,485 workers. CONCLUSION: This epidemiological trend suggests that both the incidence and prevalence of DPB may have decreased.

Feasibility of cytological diagnosis of sarcoidosis with endobronchial US-guided transbronchial aspiration.

BACKGROUND: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has a high diagnostic value in sarcoidosis if the obtained histological specimen is indicative of a non-caseating epithelioid-cell granuloma. However, EBUS-TBNA in sacoidosis sometimes affords solely cytological specimens. OBJECTIVE: To investigate the relevance of EBUS-TBNA cytology specimens in diagnosing sarcoidosis. DESIGN: The study population comprised 72 patients with sarcoidosis and 116 patients who had thoracic malignancies and intrathoracic lymphadenopathy but were eventually proven to be metastasis-free (controls). The EBUS-TBNA samples obtained for these subjects were blindly evaluated for the presence of epithelioid cell clusters by 2 independent cytoscreeners and a pathologist. RESULTS: Interobserver variability in the specimen grading was minimal. The sensitivity and specificity were 65.3% and 94.0%, respectively. The sensitivity was high, at 87.5%, for the combined cytological and histological examinations. Of 7 controls whose cytological specimens showed epithelioid cell clusters, 3 were also deemed positive for sarcoidosis on histological examination, which indicated that they had sarcoid reaction to cancer. CONCLUSIONS: Cytological evaluation of the EBUS-TBNA specimens had higher sensitivity than histological evaluation alone for intrathoracic lymphadenopathy due to sarcoidosis. It should be recognized, however, that up to 6% of patients with thoracic malignancy may have sarcoid reaction in non-metastatic lymph nodes.

Differential expression and the anti-apoptotic effect of human placental neurotrophins and their receptors.

Neurotrophin (NT) is important in the survival, maintenance and differentiation of neuronal tissue, and functions in follicle maturation, tumor growth, angiogenesis and immunomodulation; however, the expression of NT and its receptors (NTR) in human placenta and their influence on fetal growth are unclear. Here we investigated the correlation of NT and NTR in human placenta with uterine environment and fetal growth. TrkB, a NTR, mRNA was expressed on decidual and villous tissue and increased with gestational age, localizing in the trophoblast layer and endothelium by immunohistochemistry. Villous TrkB mRNA was significantly increased in preeclampsia (PE) than in controls and was higher in the normotensive small for gestational age (SGA) placenta, although it was not significant. It was also significantly increased in the small twin of discordant twin pregnancies. Brain-derived neurotrophic factor (BDNF), the main ligand of TrkB, was expressed in membranous chorion and villous tissue and was significantly higher in maternal plasma in normotensive SGA and PE than in controls. TrkB mRNA expression was up-regulated on cultured villous tissue explants and on JEG-3, a choriocarcinoma cell line, by H(2)O(2) treatment. BDNF decreased apoptotic cells in H(2)O(2)-treated JEG-3, indicating that BDNF/TrkB signaling had anti-apoptotic effects against oxidative stress in JEG-3, suggesting a protective role of BDNF/TrkB in human villous tissue under unfavorable conditions in utero.

Bcl6 in pulmonary epithelium coordinately controls the expression of the CC-type chemokine genes and attenuates allergic airway inflammation.

BACKGROUND: There is synteny in the CC-type chemokine gene clusters between humans (CCL2/MCP-1, CCL7MCP-3, CCL11/eotaxin, CCL8/MCP-2, CCL13/MCP-4, and CCL1/I-309) and mice (CCL2, CCL7, CCL11, CCL12/MCP-5, CCL8, and CCL1). OBJECTIVE: As many putative Bcl6/STAT-binding sequences are observed in the clusters, we examined the roles of a transcriptional repressor Bcl6 and the regional histone modification in the expression of these chemokine genes in pulmonary epithelium. METHODS: We generated transgenic (Tg) mice carrying the Bcl6 or the dominant-negative (DN)-Bcl6 gene under the control of the surfactant protein C (SPC) promoter that induces the exogenous gene expression in the distal lung epithelium. For in vitro studies, A549, alveolar type II-like epithelial cell line transfected with the SPC-DN-Bcl6 gene were stimulated with IL-4+TNF-α, and Bcl6 or STAT6 binding to and histone modification of the cluster in the transfectants were analysed by chromatin immunoprecipitation assays. Tg mice sensitized with ovalbumin (OVA) were challenged with OVA inhalation. The amounts of mRNAs in each sample were analysed by quantitative RT-PCR. RESULTS: The amount of Bcl6 bound to the cluster decreased in A549 cells stimulated with IL-4 and TNF-α, whereas STAT6 binding increased in association with regional histone H3-K9/14 acetylation and H3-K4 methylation. The expression of all chemokine genes in the gene cluster was augmented in activated A549 cells transfected with the DN-Bcl6 gene. We also induced allergic airway inflammation in Tg mice. Expression of the chemokine genes and infiltrated cell numbers in the lungs of these Tg mice with allergic airway inflammation were inversely correlated with the amount of Bcl6 in the lungs. CONCLUSION AND CLINICAL RELEVANCE: Expression of the pulmonary epithelium-derived CC-type chemokine genes in the cluster is orchestrated by the conserved machinery related to Bcl6. Thus, Bcl6 in pulmonary epithelium may be a critical regulator for pathogenesis of various pulmonary inflammatory diseases.

Prenatal diagnosis of anterior sacral meningocele.

Anterior sacral meningocele is an extremely rare condition and there has been only one previous report of a prenatal diagnosis. We report the case of a 36-year-old primigravida who was referred following detection of a huge fetal pelvic cyst on routine ultrasound examination at 19 + 4 weeks' gestation. Neither fetal ultrasound nor magnetic resonance imaging (MRI) at 20 + 5 weeks' gestation could detect communication between the cyst and the spinal cord. Because extension of the pear-shaped cyst through the pelvic diaphragm down to the perineum was reminiscent of dilated vagina and uterine cervix, a tentative diagnosis of hydrometrocolpos secondary to imperforate hymen was considered. On follow-up MRI at 33 + 5 weeks' gestation, a narrow stalk connecting the pelvic cyst and the spinal canal through the anterior sacral foramen was clearly delineated, allowing us to reach the prenatal diagnosis of anterior sacral meningocele.

Prenatal treatment of meconium peritonitis with urinary trypsin inhibitor.

We describe a case of congenital meconium peritonitis with progressive fetal ascites and polyhydramnios. Fetal ascites could be only partially reduced on paracentesis at 29 weeks' gestation, and it subsequently increased. Urinary trypsin inhibitor (UTI), a physiological anti-inflammatory substance, was administered into the fetal abdominal cavity at a second paracentesis performed at 35 weeks' gestation. There was a significant amount of fetal ascites remaining 1 day after the second paracentesis, but this completely resolved within 5 days. A healthy infant was delivered vaginally and no surgical intervention was required. The case suggests that UTI can reduce meconium-induced chemical peritonitis and thereby facilitate intrauterine remission of fetal ascites.