Dextran sodium sulfate alters cytokine production in macrophages in vitro.

Macrophages have been assumed to have a crucial role in the development of inflammatory bowel disease (IBD). However, involvement of intestinal macrophages in IBD onset and functional alterations of macrophages during IBD development has not been clarified. We investigated the effect of exposure of compounds used in the induction of colitis in mice on the immune responses of peritoneal macrophages in mice. 2,4,6- trinitrobenzenesulfonic acid and oxazolone did not affect the production of interleukin (IL)-10 and IL-12 from lipopolysaccharide (LPS)-stimulated peritoneal macrophages from BALB/c mice. A significant increase in IL-10 secretion and decrease in IL-12 production from LPS-stimulated macrophages were observed upon exposure to dextran sodium sulfate (DSS). TNF-α production was enhanced significantly by exposure to DSS and LPS. The level of nitric-oxide production from macrophages was increased slightly by exposure to DSS and LPS. Expression of sphingosine kinase-1 and LIGHT (both of which are specific biomarkers of M2b macrophages) was observed in macrophages upon DSS exposure. Alteration of cytokine production in macrophages was observed upon DSS exposure in the absence of LPS stimulation. Peritoneal macrophages from C57BL/6 mice showed similar responses to peritoneal macrophages from BALB/c mice against DSS. These results suggest that DSS directs the immune response of macrophages towards the M2b phenotype.

Development of a real-time loop-mediated isothermal amplification assay for the sensitive and rapid detection of Listeria monocytogenes.

UNLABELLED: A real-time loop-mediated isothermal amplification (RealAmp) assay for the detection of Listeria was developed. The RealAmp assay, using primers specific for the hemolysin-encoding hlyA gene, was verified using Listeria monocytogenes strains (n = 58) from different regions of the world. Both the sensitivity and specificity of the RealAmp assay were high. The RealAmp assay could detect 10(3) CFU ml(-1) within 30 min. A comparative evaluation of the RealAmp assay, the API Listeria assay, and the real-time PCR assay revealed that the RealAmp assay is simpler, faster, and has a higher specificity than the other two assays. SIGNIFICANCE AND IMPACT OF THE STUDY: Conventional culture and molecular detection methods are always time consuming and require a specific laboratory infrastructure, thereby restricting their use for the rapid detection and diagnosis of pathogens. A real-time loop-mediated isothermal amplification (RealAmp) assay performed by ESEtube scanner to rapidly detect Listeria monocytogenes isolated from food was developed. The results showed that the RealAmp assay using the tube scanner was more efficient and precise than the conventional API Listeria assay and the real-time PCR assay.

Cor triatriatum sinister: an incidental finding in a patient with paroxysmal atrial fibrillation.

A 58-year-old male was referred for catheter ablation for atrial fibrillation. He was incidentally diagnosed with cor triatriatum sinister by preoperative transesophageal echocardiography and cardiovascular computed tomography. The patient has since been free from atrial fibrillation for over 24 months following successful electrical pulmonary vein isolation. The rapidly soaring number of cases undergoing catheter ablation for atrial fibrillation and imaging investigation prior to the procedure may increase the incidental detection of asymptomatic congenital heart diseases.

Less maintenance immunosuppression in lung transplantation following hematopoietic stem cell transplantation from the same living donor.

Living-donor lobar lung transplantation (LDLLT) is one of the final options for saving patients with pulmonary complications after hematopoietic stem cell transplantation (HSCT). We retrospectively investigated 19 patients who had undergone LDLLT after HSCT in Japan. Eight patients underwent LDLLT after HSCT in which one of the donors was the same living donor as in HSCT (SD group), while 11 received LDLLT from relatives who were not the HSCT donors (non-SD group). In the SD group, three patients underwent single LDLLT. The 5-year survival rate was 100% and 58% in the SD and non-SD groups, respectively. In the SD group, postoperative immunosuppression was significantly lower than in the non-SD group. Two patients died of infection and one died of post-transplant lymphoproliferative disease (PTLD) in the non-SD group, while only one patient died of PTLD 7 years after LDLLT in the SD group. Hematologic malignancy relapsed in two patients in the non-SD group. For the three single LDLLTs in the SD group, immunosuppression was carefully tapered. In our study, LDLLT involving the same donor as for HSCT appeared to have advantages related to lower immunosuppression compared to LDLLT from relatives who were not the HSCT donors.

[Treatment strategy and outcomes of invading apical lung cancer].

Invading apical lung cancers are generally the non-small-cell lung cancers (NSCLCs) which involve the apex of the chest wall. These tumors should be classified into 2 types based on the main location of tumor because of the difference of involved surrounding structures ; (1) the superior sulcus tumor origi nally termed Pancoast tumor which involves posterior region of the apex and (2) the anterior apical tumor which involves anterior region of the apex. Previously, these NSCLCs were considered to be inoperable showing a dismal prognosis. With the development of combined modality therapies for locally advanced NSCLCs, the prognosis of invading apical NSCLCs has been improved, especially since intro duction of the neoadjuvant chemoradiotherapy. Surgical resection for invading apical NSCLCs is 1 of challenging procedures for thoracic surgeons. The point is the anatomical complication of the small apex surrounding vital structures. Several approaches have been developed such as the posterior Paul-son's approach or anterior Masaoka's approach. In particular, the approach from anterior chest has been modified or devised to achieve safe and complete resection of tumors invading anterior structures like subclavian vessels. In this article, we reviewed our 13 cases of invading apical NSCLCs, especially from the view point of surgical approach. Thoracic surgeons should understand the properties of each approach and master them for complete resection avoiding serious complications.

[Bronchoplasty and pulmonary arterioplasty for treatment of lung cancer].

OBJECTIVE: We evaluated the outcomes of bronchoplasty (BP) and pulmonary arterioplasty (PAP) for treatment of patients with lung cancer. SUBJECTS: We studied 33 patients who underwent BP and/or PAP over the previous 7 years at our institution. METHODS: A telescope procedure was utilized for anastomosis of a tubular excision performed in the bronchus. One patient received induction chemoradiotherapy, in whom the anastomosis section was covered with an intercostal muscle flap to assure anastomosis completion, then PAP was performed under heparinization. RESULTS: BP including a sleeve resection was performed in 15 patients, while that with a wedge resection and partial side wall resection was performed in 7 and 1 patient, respectively. PAP was performed in 18 patients, and a combination of BP and PAP was used in 8. The seam was incomplete in 2 patients and stenosis was recognized in the anastomosis section in 1. No local recurrence at the anastomosis site was seen in any of the 33 cases. The survival rate for patients who underwent BP was similar to that of those who underwent a standard resection for primary lung cancer. CONCLUSION: Our results indicate that BP and PAP are safe and useful surgical procedures for patients with lung cancer.

Cardiomyocytes can be generated from marrow stromal cells in vitro.

We have isolated a cardiomyogenic cell line (CMG) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneously beating cells were repeatedly screened. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in approximately 30% of the cells; they connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. They expressed atrial natriuretic peptide and brain natriuretic peptide and were stained with anti-myosin, anti-desmin, and anti-actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials, such as sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain, and alpha-actin, indicated that their muscle phenotype was similar to that of fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.

[Surgical technique of displacement and retraction of the heart with a pigtail shaped hook and Tentacles Heart Positioner].

We describe a novel heart retracting system with Tentacles Heart Positioner (Sumitomo Bakelite, Tokyo) during off-pump coronary artery bypass grafting (OPCAB). The heart retracting system is composed of a pigtail shaped hook attached to a flexible Universal Stabilizer Arm (Estech, Minneapolis). After Tentacles suction device is applied on the surface of the heart, the retracting system is fixed on the sternal retracter so that the hook can hang and support the retracting tubes of the sucker. By regulating the shape of the Universal Stabilizer Arm and the position of the hook, the retracting system can lift the heart sufficiently and maintain the optimal position of the heart during OPCAB procedure. Together with Tentacles Heart Positioner, the heart retracting system provides excellent exposure of target coronary arteries with minimal effect on hemodynamics.

Diffusion Tensor Imaging MRI With Spin-Echo Sequence and Long-Duration Measurement for Evaluation of Renal Fibrosis in a Rat Fibrosis Model.

BACKGROUND: Renal fibrosis (RF) is a well-known marker for chronic kidney disease (CKD) progression, including chronic renal injury after renal transplantation. However, invasive biopsy is an available examination for evaluation of RF. Diffusion MRI was once recognized as a promising option for RF. However, it is now controversial for RF evaluation in a unilateral ureteral obstruction (UUO) model. METHODS: To seek an optimal imaging method applicable for RF in UUO model kidneys, we attempted a series of MRI methods, including proton density-weighted imaging, T1-weighted imaging, T2-weighted imaging, T2*-weighted imaging, diffusion-weighted imaging, and diffusion tensor imaging (DTI). RESULTS: We identified DTI MRI by spin-echo sequence plus a special kidney attachment as the best option for evaluation of renal UUO fibrosis, compared with normal kidney on the opposite side. To confirm these results, we applied this technique to a rat UUO therapeutic model with the anti-fibrotic reagent Fasudil. Fractional anisotropy values calculated from DTI MRI showed statistically significant linear correlation with the RF area measured by use of Sirius red or Masson trichrome staining of the positive area [cortex (r = 0.6397, P = .0283) and outer stripe of the outer medulla (r = 0.7810, P = .0039)]. CONCLUSIONS: By use of the DTI MRI with spin-echo sequence, it may be possible to accurately evaluate RF in CKD.

Calmodulin kinases II and IV and calcineurin are involved in leukemia inhibitory factor-induced cardiac hypertrophy in rats.

We recently reported that leukemia inhibitory factor (LIF) enhances Ca(2+)](i) through an increase in L-type Ca(2+) current (I(Ca,L)) in adult cardiomyocytes. The aim of this study was to investigate whether LIF activates Ca(2+)-dependent signaling molecules, such as calcineurin and calmodulin kinases II and IV (CaMKII and CaMKIV), and, if so, whether these Ca(2+)-mediated signaling events contribute to LIF-mediated cardiac hypertrophy. We first confirmed that LIF increased I(Ca,L) and [Ca(2+)](i) in primary cultured rat neonatal cardiomyocytes. Calcineurin, CaMKII, and CaMKIV activities increased at 2 minutes and peaked by 1.6-, 2.2-, and 2.2-fold, respectively, at 15 minutes. Nicardipine or verapamil fully inhibited these activities. Autophosphorylation of CaMKII was also observed to parallel the timing of CaMKII activity, and this phosphorylation was blocked by nicardipine, verapamil, or EGTA. LIF treatment led to a 3-fold increase in nuclear factor of activated T cell-luciferase activity. To confirm that inositol triphosphate (IP(3))-induced Ca(2+) release from sarcoplasmic reticulum was not involved in this process, IP(3) content and phosphorylation of phospholipase Cgamma were investigated. LIF did not increase IP(3) content or phosphorylate phospholipase Cgamma. KN62 (an inhibitor of CaMKII and CaMKIV) attenuated c-fos, brain natriuretic peptide, alpha-skeletal actin, and atrial natriuretic peptide expression. KN62 suppressed the LIF-induced increase in [(3)H]phenylalanine uptake and cell size. Cyclosporin A and FK506 slightly attenuated brain natriuretic peptide but did not affect c-fos or atrial natriuretic peptide expression. Cyclosporin A significantly reduced the LIF-induced increase in [(3)H]phenylalanine uptake. These findings indicated that LIF activated CaMKII, CaMKIV, and calcineurin through an increase in I:(Ca,L) and [Ca(2+)](i) and that CaMKII, CaMKIV, and calcineurin are critically involved in LIF-induced cardiac hypertrophy.

High flux circularly polarized gamma beam factory: coupling a Fabry-Perot optical cavity with an electron storage ring.

We report and discuss high-flux generation of circularly polarized γ-rays by means of Compton scattering. The γ-ray beam results from the collision of an external-cavity-enhanced infrared laser beam and a low emittance relativistic electron beam. By operating a non-planar bow-tie high-finesse optical Fabry-Perot cavity coupled to a storage ring, we have recorded a flux of up to (3.5 ± 0.3) × 108 photons per second with a mean measured energy of 24 MeV. The γ-ray flux has been sustained for several hours. In particular, we were able to measure a record value of up to 400 γ-rays per collision in a full bandwidth. Moreover, the impact of Compton scattering on the electron beam dynamics could be observed resulting in a reduction of the electron beam lifetime correlated to the laser power stored in the Fabry-Perot cavity. We demonstrate that the electron beam lifetime provides an independent and consistent determination of the γ-ray flux. Furthermore, a reduction of the γ-ray flux due to intrabeam scattering has clearly been identified. These results, obtained on an accelerator test facility, warrant potential scaling and revealed both expected and yet unobserved effects. They set the baseline for further scaling of the future Compton sources under development around the world.

The synthesis of a pyridyl analog of adenosylcobalamin and its coenzymic function in the diol dehydratase reaction.

A novel analog of adenosylcobalamin in which 5,6-dimethylbenzimidazole and D-ribose moieties of the nucleotide loop are replaced by pyridine and the trimethylene group, respectively, was synthesized and examined for coenzymic function. The coordination of pyridine to the cobalt atom in this analog was stronger than that of 5,6-dimethylbenzimidazole in the corresponding homolog. The adenosyl form of pyridyl analog served as partially active coenzyme for diol dehydratase. The kcat/Km values calculated from the initial velocity indicate that this analog is a better coenzyme than the 5,6-dimethylbenzimidazolyl or imidazolyl counterpart. However, the reaction with the pyridyl analog as coenzyme was accompanied with a concomitant inactivation during catalysis, with a kcat/Kinact value 50-100 times lower than that for adenosylcobalamin or the 5,6-dimethylbenzimidazolyl analog. Therefore, it can be concluded that the 5,6-dimethylbenzimidazole moiety of adenosylcobalamin is important for continuous progress of a catalytic cycle by protecting the reactive intermediates from side reactions.

Specificity for various imino-acid-residues of a proline-specific dipeptidylcarboxypeptidase from a Streptomyces species.

A proline-specific dipeptidylcarboxypeptidase, which removes diproline from the C-terminus of the proline-containing peptides, such as Boc-Pro-Pro-Pro-Pro and Leu-Pro-Pro-Pro-Pro-Pro, has recently been purified from a Streptomyces sp. The specificity of the enzyme for various imino acid-containing synthetic peptide substrates was further studied. The peptides with proline, hydroxyproline, or dehydroproline at the P2' position were found to be good substrates, while those with pipecolic acid, D-proline or other usual amino acids at the P2' position were scarcely hydrolyzed. The peptides with proline, dehydroproline, pipecolic acid, or N-methyl-alanine at the P1' position were well-hydrolyzed, while those with hydroxyproline or D-proline at the P1' position were not hydrolyzed. Utilizing this high specificity for imino acids, Boc-Pro-Pro-Pro-Pro was synthesized by the enzyme using Boc-Pro-Pro as the acidic component and Pro-Pro as the basic component.

Autonomic and antiarrhythmic drug modulation of ST segment elevation in patients with Brugada syndrome.

OBJECTIVES: We examined the modulatory effects of autonomic nervous system and antiarrhythmic drugs on the ST segment in patients with Brugada syndrome to gain an insight into the mechanism of ST segment elevation. BACKGROUND: Right bundle branch block, ST segment elevation and ventricular tachyarrhythmias define a distinct clinical and electrocardiographic (ECG) syndrome (Brugada syndrome). However, the mechanism of ST segment elevation and the causes of this syndrome are unknown. METHODS: The study included four patients in whom structural heart or coronary artery disease was excluded by noninvasive and invasive tests. High take-off ST segment elevation of either the coved or saddle-back type in precordial leads V1, V2 and V3 was seen in all patients. Three patients experienced recurrent episodes of syncope or aborted sudden cardiac death, and the remaining patient had palpitation. Autonomic receptor stimulation and blockade and intravenous administration of antiarrhythmic drugs were performed during sinus rhythm while the 12-lead ECG was recorded. Metaiodobenzylguanidine (MIBG) scanning and Holter monitoring were also performed. RESULTS: Beta-adrenoceptor stimulation by intravenous isoproterenol consistently reduced (> or = 0.1 mV) ST segment elevation at or 80 ms after the J point in all four patients. Selective alpha-adrenoceptor stimulation by intravenous norepinephrine in the presence of propranolol or by intravenous methoxamine consistently augmented, whereas alpha-adrenoceptor blockade reduced, ST segment elevation in three patients. Intracoronary acetylcholine or intravenous edrophonium or neostigmine augmented ST segment elevation without inducing coronary spasm in three of four patients. Class IA antiarrhythmic drugs also consistently augmented (three patients), whereas class IB drugs had no effect on (two patients) ST segment elevation. No abnormality was found on MIBG imaging or heart rate variability in three patients, suggesting that autonomic dysfunction is not a primary disease process. Class IA drugs had no effect on ST segment in three control patients, suggesting that the ST segment elevation seen in patients with Brugada syndrome in response to the drugs is not a nonspecific response. CONCLUSIONS: ST segment elevation in patients with Brugada syndrome was augmented by selective stimulation of alpha-adrenoceptors or muscarinic receptors or by class IA drugs but was mitigated by beta-adrenoceptor stimulation or alpha-adrenoceptor blockade. These responses might be explained by postulating the presence of an area of early repolarization or a local "depolarized" area in the ventricle causing ST segment elevation in this syndrome. Because only a small number of patients were studied, these possibilities need further evaluation.

Potassium channel blocker activates extracellular signal-regulated kinases through Pyk2 and epidermal growth factor receptor in rat cardiomyocytes.

OBJECTIVES: We sought to determine whether potassium (K(+)) channel blockers (KBs) can activate extracellular signal-regulated kinase (ERK) and to characterize the upstream signals leading to ERK activation in cardiomyocytes. BACKGROUND: Because KBs attenuate K(+) outward current, they may possibly prolong the duration of action potentials, leading to an increase in calcium (Ca(2+)) transient ([Ca(2+)](i)) in cardiomyocytes. Elevation of intracellular Ca(2+) levels can trigger various signaling events. Influx of Ca(2+) through L-type Ca(2+) channels after membrane depolarization induced activation of MEK and ERK through activation of Ras in neurons. Although KBs are frequently used to treat cardiac arrhythmias, their effect on signaling pathways remains unknown. METHODS: Primary cultured rat cardiomyocytes were stimulated with four different KBs-4-aminopyridine (4-AP), E-4031, tetra-ethylammonium and quinidine-and phosphorylation of ERK, proline-rich tyrosine kinase 2 (Pyk2) and epidermal growth factor receptor (EGFR) was detected. Action potentials were recorded by use of a conventional microelectrode. (Ca(2+))(i) was monitored by the fluorescent calcium indicator Fluo-4. RESULTS: E-4031, 4-AP, tetra-ethylammonium and quinidine induced phosphorylation of ERK. 4-Aminopyridine prolonged the duration of action potentials by 37% and increased (Ca(2+))(i) by 52% at 1 mmol/l. Pre-incubation of ethyleneglycoltetraacetic acid, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis and diltiazem completely blocked this phosphorylation, whereas flufenamic acid and benzamil did not. 4-Aminopyridine induced tyrosine phosphorylation of Pyk2 and EGFR, which peaked at 5 and 10 min, respectively. Cytochalasin D, AG1478 and dominant-negative EGFR strongly inhibited the phosphorylation of ERK, whereas calphostin C, calmidazolium and KN62 did not. CONCLUSIONS: These findings indicate that KBs induce ERK activation, which starts with Ca(2+) entry through the L-type Ca(2+) channel in cardiomyocytes, and that EGFR and Pyk2 are involved in this activation.

Impaired erythropoiesis in transgenic mice overexpressing a truncated erythropoietin receptor.

Erythropoietin (EPO), one of the pivotal regulators of erythrocyte production, transmits signals through the EPO receptor (EPOR). We have previously reported that human bone marrow (BM) cells express two dominant forms of the EPOR, one full-length and one truncated (EPOR-F and EPOR-T). Experiments with a cell line have shown that the EPOR-T acts as a dominant-negative regulator of EPOR-F-mediated signals. Its role in erythropoiesis in vivo, however, has yet to be clarified. Here we show the presence in mouse BM of a truncated form of the EPOR that is essentially the same as EPOR-T in humans. To investigate its role in vivo, we generated transgenic mice overexpressing mouse EPOR-T (EPOR-T-Tg mice). As a result, two independent EPOR-T-Tg lines were established. One line revealed mild anemia, but another line did not. When anemia was induced experimentally in these mice, however, both lines showed apparently poor recovery resulting in higher mortality than wild-type control mice. The impaired erythropoiesis found in these mice thus strongly suggests the EPOR-T's role as a negative regulator of erythropoiesis in vivo.

Demonstration of the stabilization technique for nonplanar optical resonant cavities utilizing polarization.

Based on our previously developed scheme to stabilize nonplanar optical resonant cavities utilizing polarization caused by a geometric phase in electromagnetic waves traveling along a twisted path, we report an application of the technique for a cavity installed in the Accelerator Test Facility, a 1.3-GeV electron beam accelerator at KEK, in which photons are generated by laser-Compton scattering. We successfully achieved a power enhancement of 1200 with 1.4% fluctuation, which means that the optical path length of the cavity has been controlled with a precision of 14 pm under an accelerator environment. In addition, polarization switching utilizing a geometric phase of the nonplanar cavity was demonstrated.

Activation mechanism of human Hageman factor-plasma kallikrein-kinin system by Vibrio vulnificus metalloprotease.

Vivrio vulnificus, an opportunistic human pathogen, secretes a metalloprotease (VVP). The VVP inoculated into a guinea pig is known to generate bradykinin through activation of the Hageman factor-plasma kallikrein-kinin system. VVP was shown to possess the ability to activate the human system through the same mechanism as that clarified in the guinea pig system, namely, VVP converted both human zymogens (Hageman factor and plasma prekallikrein) to active enzymes (activated Hageman factor and plasma kallikrein), and the then generated kallikrein liberated bradykinin from high-molecular-weight kininogen. However, in the presence of plasma alpha 2-macroglobulin (alpha 2M), the VVP action was drastically decreased. This finding suggests that the human system might be activated only at the interstitial-tissue space which contains negligible amounts of alpha 2M or in the bloodstream of the individuals whose plasma alpha 2M level is extremely reduced.

Microbial metalloproteases and pathogenesis.

Zinc metalloproteases produced by human pathogenic microorganisms show a wide variety of pathological actions. In local infections, the proteases cause necrotic or hemorrhagic tissue damage through digestion of structural components of the ground substance, and also form edematous lesions through generation of inflammatory mediators, while in systemic infections, the proteases act as a synergistic virulence factor through disordered proteolysis of many plasma proteins. Clostridial neurotoxins, Bacteroides fragilis enterotoxin and Bacillus anthracis lethal factor are also zinc metalloproteases.