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.

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.

Cretinism with combined hormone deficiency caused by a mutation in the PIT1 gene.

Cretinism is marked by irreversible mental and growth retardation. We describe here an entirely new case of cretinism showing combined pituitary hormone deficiencies of thyrotropin, growth hormone and prolactin that appears to be caused by homozygosity for a nonsense mutation in the gene for the pituitary specific transcription activator, Pit-1/GHF-1 (designated PIT1 in humans for pituitary specific factor 1). This is the first report in humans of a defect in a transcription activator causing deficiency of multiple target genes.

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.

Unique transcriptome, pathways, and networks in the human endometrial fibroblast response to progesterone in endometriosis.

Eutopic endometrium in endometriosis has molecular evidence of resistance to progesterone (P(4)) and activation of the PKA pathway in the stromal compartment. To investigate global and temporal responses of eutopic endometrium to P(4), we compared early (6-h), intermediate (48-h), and late (14-Day) transcriptomes, signaling pathways, and networks of human endometrial stromal fibroblasts (hESF) from women with endometriosis (hESF(endo)) with hESF from women without endometriosis (hESF(nonendo)). Endometrial biopsy samples were obtained from subjects with and without mild peritoneal endometriosis (n = 4 per group), and hESF were isolated and treated with P(4) (1 µM) plus estradiol (E(2)) (10 nM), E(2) alone (10 nM), or vehicle for up to 14 days. Total RNA was subjected to microarray analysis using a Gene 1.0 ST (Affymetrix) platform and analyzed by using bioinformatic algorithms, and data were validated by quantitative real-time PCR and ELISA. Results revealed unique kinetic expression of specific genes and unique pathways, distinct biological and molecular processes, and signaling pathways and networks during the early, intermediate, and late responses to P(4) in both hESF(nonendo) and hESF(endo), although a blunted response to P(4) was observed in the latter. The normal response of hESF to P(4) involves a tightly regulated kinetic cascade involving key components in the P(4) receptor and MAPK signaling pathways that results in inhibition of E(2)-mediated proliferation and eventual differentiation to the decidual phenotype, but this was not established in the hESF(endo) early response to P(4). The abnormal response of this cell type to P(4) may contribute to compromised embryonic implantation and infertility in women with endometriosis.

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.

Association of functional polymorphisms related to the transcriptional level of FOXP3 with prognosis of autoimmune thyroid diseases.

The severity of Hashimoto's disease (HD) and intractability (or inducibility to remission) of Graves' disease (GD) varies among patients. Forkhead box P3 (FoxP3) is a crucial regulatory factor for the development and function of regulatory T (T(reg) ) cells, and deficiency of the FoxP3 gene (FOXP3) suppresses the regulatory function of T(reg) cells. To clarify the association of the functional polymorphisms of the FOXP3 with the prognosis of GD and HD, we genotyped -3499A/G, -3279C/A and -2383C/T polymorphisms in FOXP3 gene obtained from 38 patients with severe HD, 40 patients with mild HD, 65 patients with intractable GD, in whom remission was difficult to induce, 44 patients with GD in remission and 71 healthy volunteers. The -3279CA genotype was more frequent in patients with GD in remission than in patients with intractable GD, and the -3279AA genotype, which correlates to defective transcription of FOXP3, was absent in patients with GD in remission. The -2383CC genotype was more frequent in patients with severe HD than in those with mild HD. In conclusion, the -3279A/C polymorphism is related to the development and intractability of GD and the -2383CC genotype to the severity of HD.

Dehydriding reaction of AlH3: in situ microscopic observations combined with thermal and surface analyses.

The dehydriding reaction of single-phase alpha- AlH3 was investigated by in situ microscopic observations combined with thermal and surface analyses. Before the dehydriding reaction, primary AlH3 particles of size 100 nm-1 microm were thought to be covered by an oxide layer with a thickness of less than 5 nm. Both the precipitation/grain-growth of metallic Al of size 1-50 nm and an increase in 'boundary space' were clearly observed inside the particles, while the morphologies of the particles covered by the layer did not change during the dehydriding reaction. This preliminary report provides fundamental information for a further study of AlH3 as a possible hydrogen storage material.

Development of ductal carcinoma of the pancreas during follow-up of branch duct intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND: Synchronous occurrence of intraductal papillary mucinous neoplasm (IPMN) and ductal carcinoma of the pancreas has been reported. Branch duct IPMNs with lower likelihood of malignancy are not submitted to resection but are followed-up, so ductal carcinoma may develop during the follow-up. The development of ductal carcinoma of the pancreas during follow-up of branch duct IPMNs was investigated. METHODS: 60 patients with branch duct IPMN who had an intraductal tumour of <10 mm on imaging examinations and a negative result for malignancy on cytological examination of the pancreatic juice were investigated. They were followed-up mainly by ultrasonography (US), and additionally by endoscopic ultrasonography (EUS), CT, magnetic resonance cholangiopancreatography (MRCP) or endoscopic retrograde cholangiopancreatography (ERCP) with cytological examination of the pancreatic juice for an average period of 87 months. RESULTS: Ductal carcinoma of the pancreas distinct from IPMN developed in 5 of 60 (8%) branch duct IPMNs during follow-up. The 5-year rate of development of ductal carcinoma was 6.9% (95% CI 0.4% to 13.4%), the incidence of ductal carcinoma was 1.1% (95% CI 0.1% to 2.2%) per year and the standardised incidence ratio of development of ductal carcinoma was 26 (95% CI 3 to 48). Patients >70 years old developed ductal carcinoma significantly more frequently than those under 69. Four of five ductal carcinomas identified during follow-up were resectable. Cancer developed in IPMN in 2 of 60 (3%) branch duct IPMNs during follow-up. CONCLUSIONS: During follow-up of branch duct IPMNs, ductal carcinoma of the pancreas not infrequently developed distinct from IPMN. In the follow-up of IPMN, special attention should be paid to the development of ductal carcinoma of the pancreas.

L3/Lhx8 is a pivotal factor for cholinergic differentiation of murine embryonic stem cells.

L3/Lhx8 is a member of the LIM-homeobox gene family. Previously, we demonstrated that L3/Lhx8-null mice specifically lacked cholinergic neurons in the basal forebrain. In the present study, we conditionally suppressed L3/Lhx8 function during retinoic acid-induced neural differentiation of a murine embryonic stem (ES) cell line using an L3/Lhx8-targeted small interfering RNA (siRNA) produced by an H1.2 promoter-driven vector. Our culture conditions induced efficient differentiation of the ES cells into neurons and astrocytes, but far less efficient differentiation into oligodendrocytes. Suppression of L3/Lhx8 expression by siRNA led to a dramatic decrease in the number of cells positive for the cholinergic marker ChAT, and overexpression of L3/Lhx8 recovered this effect. However, no significant changes were observed in the number of Tuj1+ neurons and GABA+ cells. These results strongly suggest that L3/Lhx8 is a key factor in the cholinergic differentiation of murine ES cells and is involved in basal forebrain development.

The effects of FasL on inflammation and tumor survival are dependent on its expression levels.

The apoptosis-inducing Fas ligand (FasL) is expressed in a variety of human cancers and has been implicated in tumor immune evasion. Paradoxically, ectopic expression of FasL in experimental tumors triggers a neutrophil-mediated inflammatory response and tumor rejection. To resolve these conflicting findings, we have established B16 melanoma and P29 Lewis lung carcinoma lines expressing different levels of FasL and examined their tumorigenicity in vivo. While tumors with a high level of FasL were rapidly rejected as previously reported, those expressing a low level of FasL were not rejected but grew faster than did FasL-negative parental cells. The growth enhancement of FasL(low) tumors was not observed in T-cell-deficient nude mice, suggesting that FasL expressed in tumors at low levels counteracted against T-cell-dependent antitumor responses. In support of this notion, FasL(low) tumors were found to grow faster than parental cells in mice that had acquired tumor-specific immunity. Furthermore, histological examinations revealed apoptosis of lymphocytes in tissue sections of FasL(low) tumors. These results collectively suggest that FasL on tumors is a double-edged sword: at high levels it triggers tumor rejection whereas at low levels it facilitates tumor growth possibly by suppressing antitumor immune responses.

A novel role for Fyn: change in sphere formation ability in murine embryonic stem cells.

Fyn, a member of the Src-family protein tyrosine kinase (PTK), is an essential factor in myelination in the CNS and is involved in murine embryonic stem (ES) cell growth and differentiation. Although dysfunctions of Fyn have been comparatively studied, the gain of function by ectopic expression, especially using ES cells, has seldom been investigated. In this article, we give the first report of the involvement of Fyn alteration in the sphere formation ability of murine ES cells. First, transient transfection of Fyn hardly affected multiplication and specialization. Then, we investigated Fyn overexpression using ES cells, which stably express Fyn. As a result, altered sphere formation capability was observed in all clones stably expressing Fyn. These results may provide important information for reproduction medical treatment using ES cells.

Reductive metabolism of aromatic nitro compounds including carcinogens by rabbit liver preparations.

Reductive metabolism of aromatic nitro compounds was examined with rabbit liver preparations. Under anaerobic conditions, carcinogenic 2-nitrofluorene, 4-nitrobiphenyl, and 1-nitro-naphthalene were reduced to the corresponding hydroxylamines and amines, whereas the carcinogenic 1-nitropyrene was reduced only to the corresponding amine by liver cytosol in the presence of 2-hydroxypyrimidine, an electron donor of aldehyde oxidase. These metabolites were identified unequivocally by comparing their mass spectra and thin-layer chromatographic behaviors with those of the authentic samples. Both liver microsomes and cytosol catalyzed the reduction of these aromatic nitro compounds in varying degrees. The microsomes required reduced pyridine nucleotides for occurrence of the nitroreductase activities. In this case, reduced nicotinamide adenine dinucleotide phosphate was more effective than reduced nicotinamide adenine dinucleotide as an electron donor. The cytosol by itself exhibited some nitroreductase activities, which were markedly enhanced by addition of an electron donor of aldehyde oxidase, i.e., N1-methylnicotinamide or 2-hydroxypyrimidine. The full activities of the cytosol with the electron donor were much higher than those of the microsomes with the reduced pyridine nucleotide. Purified liver aldehyde oxidase, like the cytosol, exhibited significant nitroreductase activities in the presence of its electron donor. These results indicated that cytosolic aldehyde oxidase functions as a major enzyme responsible for the reduction of aromatic nitro compounds including carcinogens in rabbit liver.