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Background and Aims Activation of platelets and leukocytes when blood comes in contact with a dialysis membrane surface leads to increased oxidative stress and release of proinflammatory cytokines, which may result in complications in dialysis patients. Within the blood vessels, nitric oxide (NO) generated by nitric oxide synthase on the endothelial cells suppresses both aggregation/activation of platelets and migration/adhesion of leukocytes. We previously showed in a rat hemodialysis model that NO (800 ppm) gas added to the dialysate can suppress blood coagulation on the surface of a dialysis membrane. The aim of the present study was to determine, using a rat hemodialysis model, the appropriate concentration of NO that would most effectively prevent blood coagulation without causing any adverse effects. Method Male Sprague Dawley rats weighing 300 to 400 g were used for the experiments. After the rats were anesthetized (isoflurane, 1.5%-3.0%), extracorporeal circulation was established by removal of blood from the carotid artery and returning it to the tail vein. During the experiment, the blood flow rate was kept at 1.0 mL/min, and the dialysate flow rate at 3 mL/min. Heparin was administered by bolus injection (0.7 unit/g-mouse) at the start of the hemodialysis, and no further heparin was added during the 4-hour hemodialysis session. A cellulose triacetate membrane and polymethylpentene membrane were used for making the small dialyzer and gas exchanger, respectively (the prototype module was manufactured by NIPRO, Co., Japan). NO (200 ppm, 400 ppm, or 800 ppm) was added to the dialysate via the gas exchanger in the NO groups, and N2 gas was added to the dialysate in the control group. During the experiment, the arterial pressure, dialyzer inlet pressure, venous pressure, activated whole blood clotting time (ACT) and methemoglobin (met-Hb) concentration were measured. After circulation, the amount of coagulated blood was measured by the hemoglobin (Hb) content and lactate dehydrogenase (LDH) activity in the eluate from the residual blood clotting in the dialyzer. Results The 4-hour hemodialysis session achievement rate was highest in the NO-800 ppm group (control, 54%; NO-200 ppm, 66%; NO-400 ppm, 77%; NO-800 ppm, 85%). The blood met-Hb content was below 10% in all the NO groups. The Hb content and LDH activity in the eluate were significantly lower in the NO groups than in the control group. These results indicate that addition of higher concentrations of NO was associated with a lower likelihood of coagulation on the dialyzer and in the dialysis circuit and a higher likelihood of smooth and successful 4-hour dialysis sessions. Addition of NO was associated with little risk of adverse effects such as met-Hb accumulation and reduction of blood pressure. Therefore, 800 ppm is considered as the most appropriate concentration of NO to be added to the dialysate for effectively inhibiting blood coagulation without causing any adverse effects. Conclusion Our experiment in a rat hemodialysis model showed that 800 ppm was the most appropriate concentration of NO to be added to the dialysate to suppress blood coagulation on the surface of the dialysis membrane without causing any adverse effects.

Background and Aims Since hemofilters used for continuous renal replacement therapy (CRRT) come in prolonged contact with blood during treatment, clotting and cell adhesion induced by contact of blood with the membrane often occur, increasing the risk to the patient and burden on the staff. Suppressed platelet adhesion and better biocompatibility have been reported with the use of a dialysis membrane whose surface has been modified with an NV polymer in patients on maintenance hemodialysis. Therefore, it may be advantageous to use the NV membrane as a hemofilter for CRRT. In the present study, we evaluated the solute removal performance change and blood cell adhesion on the NV membrane after it comes in contact with blood, to clarify the characteristics of the NV membrane for long-time use. Methods A new polysulfone membrane containing the NV polymer (SNV) and a conventional polysulfone membrane (SHG) were used for the ex vivo blood filtration experiment. To compare the solute removal performance change and blood cell adhesion after blood comes in contact with the membranes, porcine blood collected from a single animal was divided into two portions, and a 24-hour of continuous hemofiltration (CHF) experiment was performed. The circulation conditions were as follows; blood flow rate, 100 mL/min; filtrate flow rate, 20 mL/min; heparin dose adjusted as appropriate to maintain the activated clotting time from 300 to 400 sec during the experiment. We evaluated the time-courses of the transmembrane pressure (TMP) and pressure drop at the hemofilter and the change in the sieving coefficients of dextran of different molecular weights (molecular weight cutoff curve) before and after the experiment, and blood cell adhesion. The amount of blood cell adhesion was evaluated based on the hemoglobin content and lactate dehydrogenase (LDH) activity in the eluate from the residual blood clots on the hemofilter 24 hours after the CHF experiment. Results The changes in the TMP and the pressure drop were significantly lower with the use of SNV as compared to SHG (n = 9, p < 0.01). From the molecular weight cutoff curves before and after the experiment, the molecular weights when the sieving coefficient was 0.1 to 0.4, which reflect the pore size of the pores through which relatively small molecules can pass, decreased to a lower extent after blood contact with SNV as compared to SHG (n = 3, SC = 0.1-0.3, p < 0.05; SC = 0.4, p < 0.01).The hemoglobin content and LDH activity in the blood clots adhering to the membrane after the experiment were significantly lower in SNV as compared to SHG (n = 9; Hb, p < 0.01; LDH, p < 0.05), indicating that blood cell adhesion on SNV was less pronounced than that on SHG. Conclusion SNV suppressed the increase in the TMP and also the pressure drop, allowed a high rate of solute removal performance to be maintained, and suppresses blood cell adhesion to a greater extent as compared to the conventional polysulfone membrane.

Aims: The aims of the present study were to evaluate the changes in the enterobacterial flora and evaluate the hydrogen gas-producing ability of the enterobacterial flora in a mouse model of severe acute lung injury (ALI mouse model). Methods: The ALI mouse model was prepared by administering lipopolysaccharide (LPS) after activation of natural killer cells with α-galactosylceramide. The exhaled hydrogen concentrations of the mice were measured. The oxidative stress of the lung was evaluated by measuring the myeloperoxidase (MPO) activity. At the same time, feces were collected from the cecum of the mice and the hydrogen gas-producing ability was measured. Metagenomic analysis was also carried out to evaluate the changes in the enterobacterial flora. Results: The MPO activity in the lung of the ALI mouse model was significantly increased as compared to that of the control (n=25; P Conclusion: The ALI mouse model showed an increased hydrogen gas-producing ability of their enterobacterial flora, along with a change in the composition of the enterobacterial flora, which may protect organs against severe inflammation.

The purpose of the present study was to evaluate the adhesiveness of blood cells and the solute removal performance change of modified polysulfone membranes which have increased polyvinylpyrrolidone (PVP) coverage over their surface. Continuous hemofiltration (CHF) experiments for 24 h were carried out using an ex vivo hemofilter evaluation system to compare a modified polysulfone hemofilter (SHG) with the conventional polysulfone hemofilter (SH). The 25 and 50 % cutoff values of the sieving coefficient of dextran after CHF and the protein concentration in the filtrate was higher in SHG, indicating that less fouling occurred in the SHG membrane. Adhesion of blood cells after 24 h of CHF was significantly higher in the case of SH than in the case of SHG. Blood cell adhesion and membrane fouling were reduced with the use of a polysulfone membrane modified with increased PVP coverage over the surface.

Whole-lung lavage (WLL) remains the standard therapy for pulmonary alveolar proteinosis (PAP), a process in which accumulated surfactants are washed out of the lung with 0.5–2.0 l of saline aliquots for 10–30 wash cycles. The method has been established empirically. In contrast, the kinetics of protein transfer into the lavage fluid has not been fully evaluated either theoretically or practically. Seventeen lungs from patients with autoimmune PAP underwent WLL. We made accurate timetables for each stage of WLL, namely, instilling, retaining, draining, and preparing. Subsequently, we measured the volumes of both instilled saline and drained lavage fluid, as well as the concentrations of proteins in the drained lavage fluid. We also proposed a mathematical model of protein transfer into the lavage fluid in which time is a single variable as the protein moves in response to the simple diffusion. The measured concentrations of IgG, transferrin, albumin, and β2-microglobulin closely matched the corresponding theoretical values calculated through differential equations. Coefficients for transfer of β2-microglobulin from the blood to the lavage fluid were two orders of magnitude higher than those of IgG, transferrin, and albumin. Simulations using the mathematical model showed that the cumulative amount of eliminated protein was not affected by the duration of each cycle but dependent mostly on the total time of lavage and partially on the volume instilled. Although physicians have paid little attention to the transfer of substances from the lung to lavage fluid, WLL seems to be a procedure that follows a diffusion-based mathematical model.

Background: Improvements in the biocompatibility of dialysis membranes have reduced biological responses elicited by blood-membrane interactions. In this article, recent technological developments in dialysis membranes with regard to biocompatibility and recent progress in the evaluation of the biocompatibility of dialysis membranes are reviewed. Summary: The focus of investigation into dialysis membranes in recent years has focused on not only membrane materials, but also their surface textures, which have been changed, for example, by coating with vitamin E or by changing the amount and type of hydrophilizing agents used. Research and development is directed at altering the chemical and physical properties of membrane surfaces to suppress biological responses that are particularly elicited as a result of platelet activation. To develop membranes with excellent biocompatibility, biocompatibility should be evaluated on a like-for-like basis under conditions that are similar to those in clinical settings. Evaluation using actual dialyzers can be performed using porcine blood, platelet-rich plasma isolated from porcine blood (and platelet-rich plasma with leukocytes), or suspension of neutrophils isolated from porcine blood or cultured human monocytes. Key Messages: Highly biocompatible dialysis membranes can be developed when the overall correlations among biological reactions are examined by integrating all data on biological responses elicited by blood-membrane interactions or mutual interactions among blood cells.

BACKGROUND Most but not all data from different ethnic groups fit the Global Lung Function Initiative (GLI) spirometric reference model. This study investigates to what extent discrepancies are caused by secular changes in body proportions. METHODS FEV 1 and FVC from 20,336 healthy Japanese subjects (13,492 women) aged 17 to 95 years were compared with GLI-2012 reference values for Europeans. Data on the sitting height/standing height ratio (Cormic index) in 17-year-old students, collected from 1949 to 2012 in successive birth cohorts, were used to assess secular changes in body frame. The cohort-specific Cormic index was used to assess how variation in body frame affected pulmonary function. RESULTS FEV 1 and FVC were lower than GLI-2012 reference values, with values progressively falling until age 35 to 40 years and then rising to European levels in the elderly. The Cormic index rose until 1942, then fell, with a nadir in the 1970s, before rising again until 1995. Nearly one-half of the spirometric variability from predicted values could be explained by differences in the Cormic index between birth cohorts. CONCLUSIONS In low-income countries, improving health conditions are likely to drive increases in height and changes in relative leg length similar to those observed in Japan and, thus, to a change in body frame. This implies that height-based prediction equations for such populations will need to be periodically updated.

The purpose of the present study was to establish a continuous hemofiltration model using porcine blood to compare filter life. Continuous hemofiltration (CHF) experiments were performed using an in vitro hemofilter evaluation system utilizing porcine blood containing trisodium citrate in addition to nafamostat mesilate as anticoagulants. The lifetime of the hemofilter was evaluated using the transmembrane pressure and the pressure drop across the hemofilter at varying trisodium citrate concentrations. The porcine blood used in this experiment was considered to be in a slightly hypercoagulable state because of the continuous contact with non-biological materials and calcium inflow from substitution fluid. Blood containing 7 or 8 mM of trisodium citrate and nafamostat mesilate could be effectively used to compare the lifetimes of hemofilters utilized under the same conditions. In this CHF model using porcine blood, the plugging of the hollow fibers occurred shortly after the plugging of the membrane pores. In conclusion, a CHF model using porcine blood can be established by adjusting the concentration of trisodium citrate added to the blood.

Background Although the benefits of ex vivo lung perfusion (EVLP) have been globally advocated, the potentially deleterious effects of applying EVLP, in particular activation of proinflammatory cascades and alteration of metabolic profiles, are rarely discussed. This study examined proinflammatory events and metabolic profiles in lung grafts on EVLP and tested whether preconditioning lung grafts with inhaled hydrogen, a potent, cytoprotective gaseous signaling molecule, would alter the lungs' response to EVLP. Methods Rat heart-lung blocks were mounted on an acellular normothermic EVLP system for 4 hr and ventilated with air or air supplemented with 2% hydrogen. Arterial and airway pressures were monitored continuously; perfusate was sampled hourly to examine oxygenation. After EVLP, the lung grafts were transplanted orthotopically into syngeneic rats, and lung function was examined. Results Placing lung grafts on EVLP resulted in significant upregulation of the messenger RNAs for several proinflammatory cytokines, higher glucose consumption, and increased lactate production. Hydrogen administration attenuated proinflammatory changes during EVLP through upregulation of the heme oxygenase-1. Hydrogen administration also promoted mitochondrial biogenesis and significantly decreased lactate production. Additionally, in the hydrogen-treated lungs, the expression of hypoxia-inducible factor-1 was significantly attenuated during EVLP. These effects were maintained throughout EVLP and led to better posttransplant lung graft function in the recipients of hydrogen-treated lungs. Conclusions Lung grafts on EVLP exhibited prominent proinflammatory changes and compromised metabolic profiles. Preconditioning lung grafts using inhaled hydrogen attenuated these proinflammatory changes, promoted mitochondrial biogenesis in the lungs throughout the procedure, and resulted in better posttransplant graft function.

Infliximab (IFX), a known monoclonal antibody against tumor necrosis factor-α (TNF-α), is used to treat Kawasaki disease (KD) patients with intravenous immunoglobulin (IVIG) resistance. The transcriptional modulation of inflammation following IFX therapy has not been reported in KD patients. We investigated the transcript abundance profiles in whole blood obtained from eight IVIG-resistant KD subjects treated with IFX therapy using microarray platforms and compared them with those in initially IVIG-responsive subjects. A pathway analysis was performed using WikiPathways to search for the biological pathways of the transcript profiles. Four transcripts changed by IFX therapy were subsequently validated using quantitative real-time polymerase chain reaction. The pathway analysis showed the reduced abundance of transcripts in the nucleotide-binding oligomerization domain, matrix metalloproteinase (MMP), and inflammatory cytokine pathways and the increased abundance of transcripts in the T-cell receptor, apoptosis, TGF-β, and interleukin-2 pathways. Additionally, the levels of four transcripts (peptidase inhibitor-3, MMP-8, chemokine receptor-2, and pentraxin-3) related to KD vasculitis and IVIG resistance decreased after IFX therapy. The administration of IFX was associated with both the signaling pathways of KD inflammation and several transcripts related to IVIG resistance factors. These findings provide strong theoretical support for the use of IFX in KD patients with IVIG resistance.

Inhaled nitric oxide (NO) has been reported to decrease the infarct size in cardiac ischemia-reperfusion (I/R) injury. However, reactive nitrogen species (RNS) produced by NO cause myocardial dysfunction and injury. Because H2is reported to eliminate peroxynitrite, it was expected to reduce the adverse effects of NO. In mice, left anterior descending coronary artery ligation for 60 min followed by reperfusion was performed with inhaled NO [80 parts per million (ppm)], H2(2%), or NO + H2, starting 5 min before reperfusion for 35 min. After 24 h, left ventricular function, infarct size, and area at risk (AAR) were assessed. Oxidative stress associated with reactive oxygen species (ROS) was evaluated by staining for 8-hydroxy-2′-deoxyguanosine and 4-hydroxy-2-nonenal, that associated with RNS by staining for nitrotyrosine, and neutrophil infiltration by staining for granulocyte receptor-1. The infarct size/AAR decreased with breathing NO or H2alone. NO inhalation plus H2reduced the infarct size/AAR, with significant interaction between the two, reducing ROS and neutrophil infiltration, and improved the cardiac function to normal levels. Although nitrotyrosine staining was prominent after NO inhalation alone, it was eliminated after breathing a mixture of H2with NO. Preconditioning with NO significantly reduced the infarct size/AAR, but not preconditioning with H2. In conclusion, breathing NO + H2during I/R reduced the infarct size and maintained cardiac function, and reduced the generation of myocardial nitrotyrosine associated with NO inhalation. Administration of NO + H2gases for inhalation may be useful for planned coronary interventions or for the treatment of I/R injury.

Background: X-rays are not thought to cause electromagnetic interference (EMI) in implantable cardiac pacemakers. However, x-ray radiation during computed tomography (CT) scanning has been reported to cause EMI in some implantable cardiac pacemakers. The objectives of this study were to identify the location within the pacemakers where x-ray radiation causes EMI and to investigate the association of EMI with the x-ray radiation conditions. Methods: We verified the location where x-ray radiation caused EMI using a CT scanner and conventional radiographic x-ray equipment. An inhibition test and an asynchronous test were performed using five types of implantable cardiac pacemakers. Results: X-ray radiation inhibited the pacing pulses of four types of implantable cardiac pacemakers when the body of each implantable cardiac pacemaker, containing a complementary metal-oxide semiconductor (CMOS), was scanned using a CT scanner. We confirmed that x-ray-induced EMI depends on the x-ray radiation conditions, that is, the tube voltage, tube current, x-ray dose, and direction of x-ray radiation, as well as the sensing thresholds of the implantable cardiac pacemakers. Conclusions: X-ray radiation caused EMI in some implantable cardiac pacemakers, probably because the CMOS component was irradiated. The occurrence of EMI depended on the pacemaker model, sensing threshold of the pacemaker, and x-ray radiation conditions. (PACE 2010; 33:1174–1181)

Endotoxin-retentive filters are used to purify the dialysate used for hemodialysis, but endotoxin fragments whose molecular weight is below the molecular weight cut-off point of the filter may pass through the filter. The objective of the present study was to determine whether lipid A, an endotoxin fragment, is sufficiently removed by an endotoxin-retentive filter. A filtration test for lipid A was carried out using an endotoxin-retentive filter. The filtrate was tested for the presence of lipid A by a biochemical analysis: the Limulus lysate test and a bioassay: the lymphocyte stimulation test. The endotoxin-retentive filter removed lipid A to below the limit of detection by the Limulus lysate test. However, the filtrate stimulated lymphocytes, suggesting that a small amount of lipid A whose concentration was below the limit of detection by the Limulus lysate test had passed through the filter. In conclusion, the endotoxin-retentive filter was capable of removing lipid A to a concentration below the limit of detection by the Limulus lysate test but the filtrate still stimulated lymphocytes.

Intravenous immunoglobulin (IVIG) treatment-resistant patients are high risk of developing coronary artery lesions with Kawasaki disease. The IVIG-responsive (Group A; n = 6) and IVIG-resistant patients (Group B) were predicted before starting the initial treatment using the Egami scoring system and randomly allocated as a single-IVIG treatment group (group B1; n = 6) or as a IVIG-plus-methylprednisolone (IVMP) combined therapy group (group B2; n = 5). We investigated the transcript abundance in the leukocytes of those patients using a microarray analysis. Five patients in group A and one patient in group B1 responded to initial IVIG treatment. All group B2 patients responded to IVIG-plus-IVMP combined therapy. Before performing these treatments, those transcripts related to IVIG resistance and to the development of coronary artery lesions, such as IL1R, IL18R, oncostatin M, suppressor of cytokine signaling-3, S100A12 protein, carcinoembryonic antigen-related cell adhesion molecule-1, matrix metallopeptidase-9, and polycythemia rubra vera-1, were more abundant in group B patients in comparison with group A patients. Moreover, those transcripts in group B2 patients were more profoundly and broadly suppressed than group B1 patients after treatment. This study elucidated the molecular mechanism of the effectiveness of IVIG-plus-IVMP combined therapy.

Highly metastatic cells, especially in the lungs, are known to be resistant to nitric oxide (NO)-mediated cytotoxicity, compared with poorly or non-metastatic cells. However, the precise mechanisms connecting NO and metastasis remain to be determined. To clarify the role of NO in the characteristic changes in NO-resistant cells in response to inflammatory cytokines, we used Lewis lung tumor (LLT) cells, which are known to be highly metastatic NO-resistant cells, and determined the changes in cell deformability and the gene expression profile after the cells were stimulated using cytokine mixture or an NO donor. Both exogenous NO and endogenous NO via inducible NO synthase produced by cytokines decreased cell deformability by enhancing actin polymerization. The expression of several genes associated with actin polymerization was changed so as to increase actin filaments in the cells by enhancing actin polymerization and by suppressing actin depolymerization, actin filament severing, and barbed-end actin filament capping. In conclusion, inflammatory cytokine stimulation reduces deformability of LLT cells and enhances actin polymerization which is mainly controlled by the same genes induced by NO.

The aim of this study was to investigate the response of auto-CPAP devices to respiratory events (apnea, hypopnea, flow-limitation and snoring) on the same condition using a physiological upper airway model. The hypothesis of this study is that collapsibility of the flow-limiting collapsible segment of the airway is influenced by the upstream airway resistance. Five auto-CPAP devices, AutoSet T, AutoSet Spirit, Goodnight 420E, PV10i and REMstar Auto were evaluated. Apnea: all the devices increased the auto-CPAP level, while AutoSet T and AutoSet Spirit did not respond to apnea for 30s. Hypopnea: all the devices except the AutoSet T and Goodnight 420E increased pressure. Flow-limitation: all the devices except the PV10i and REMstar Auto increased pressure. Snoring: the snoring sounds disappeared when REMstar Auto and PV10i were used, and the Goodnight 420E lowered the level of snoring. In conclusion, the response of auto-CPAP devices to respiratory events differed. Collapsible upper airway model with upstream resistance is useful for the first-step assessment of auto-CPAP devices.

We conducted a Phase I trial of irinotecan (CPT-11), a topoisomerase I inhibitor, combined with amrubicin, a topoisomerase II inhibitor. The aim was to determine the maximum tolerated dose (MTD) of amrubicin combined with a fixed dose of CPT-11 as well as the dose-limiting toxicities (DLT) of this combination in patients with advanced non-small cell lung cancer. Eleven patients with stage IIIB or IV disease were treated at 3-week intervals with amrubicin (5-min intravenous injection on days 1–3) plus 60 mg/m2 of CPT-11 (90-min intravenous infusion on days 1 and 8). The starting dose of amrubicin was 25 mg/m2, and it was escalated in 5 mg/m2 increments until the maximum tolerated dose was reached. The 30 mg/m2 of amrubicin dose was one dose level above the MTD, since three of the five patients experienced DLT during the first cycle of treatment at this dose level. Diarrhea and leukopenia were the DLT, while thrombocytopenia was only a moderate problem. Amrubicin did not affect the pharmacokinetics of CPT-11, SN-38 or SN-38 glucuronide. Except for one patient, the biliary index on day-1 correlated well with the percentage decrease of neutrophils in a sigmoid E max model. There were five partial responses among 11 patients for an overall response rate of 45%. The combination of amrubicin and CPT-11 seems to be active against non-small cell lung cancer with acceptable toxicity. The recommended dose for Phase II studies is 60 mg/m2 of CPT-11 (days 1 and 8) and 25 mg/m2 of amrubicin (days 1–3) administered every 21 days.

Gefitinib is a synthetic, oral anilinoquinazoline specifically designed to inhibit the epidermal growth factor receptor tyrosine kinase, and is the first targeted drug to demonstrate reproducible activity in non-small cell lung cancer patients who do not respond to platinum-based chemotherapy. In this report, we present two cases of an interaction between gefitinib and warfarin which has not been reported previously. Because of the potentially serious consequences of this interaction, close monitoring of the International Normalized Ratio and warfarin dosage adjustment are recommended for patients receiving warfarin together with gefitinib.

Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) is up-regulated in response to inflammatory stimuli. To evaluate the extent to which local pleural inflammation involves additional site in the pleural cavity and elsewhere, we investigated the time course of the levels of iNOS and its product in the inflammatory and other sites, and compared those with a level of COX-2 in rat carrageenin-induced pleurisy. The exudate and plasma NOx levels rose, reaching peaks at 9 and 14 h, respectively. Both COX-2 and iNOS became detectable in exudate leukocytes, their levels reaching peaks at 3 and 9 h after irritation, respectively. COX-2 was detectable mainly in neutrophils, but iNOS was detectable in both neutrophils and mononuclear leukocytes. Furthermore, iNOS became detectable in neutrophils and mononuclear leukocytes in enlarged parathymic lymph nodes from 3 h in addition to those in peripheral blood and Kupffer cells from 3 to 14 h, respectively. The gene product is also detectable in thymic large dendritic cells of pleurisy-induced rats as well as normal control rats. COX-2 became detectable in stellar dendritic cells of the enlarged draining lymph nodes from 14 h. Thus, these gene products were induced in the immediate proximity of regional lymph nodes, and even at a considerable distance of liver by the local inflammatory stimulus. Although their expression pattern was quite different from each other, these gene products were detectable in phagocytic cells.

Previous studies have indicated that NO plays a crucial role in the metastasis of tumor cells and that tumor cells produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS). Since the deformability of tumor cells is an important factor governing their metastatic potential, in this study we investigated the regulation of tumor cell deformability by NO. Lewis lung tumor cells (3LL cells) were also incubated with a cytokine mixture (IL-1 beta, IFN gamma, and TNF alpha). The nitrite/nitrate content of the supernatant was then measured by the Griess method, and iNOS expression was evaluated by RT-PCR in vitro. Nitrite/nitrate was produced in response to administration of the cytokine mixture, and iNOS mRNA was expressed in the cytokine-treated cells. The deformability of the 3LL cells was evaluated by measuring the peak pressure generated during their passage through a microfilter at a constant flow rate. Both the cytokine mixture and NO donor (NOC 18) significantly increased the filtration pressure, and the staining of the cells with rhodamine-phalloidin revealed assembly of F-actin in the cell membrane. In conclusion, NO plays a role in the decreased deformability of tumor cells, suggesting that NO is one of the factors that regulates metastasis.

Although hemodynamic-based functional brain imaging techniques are powerful tools to explore the brain functions noninvasively, hemodynamic-based signal is strongly affected by spatial configuration of microvessels. Understanding the quantitative relations between microvascular structure and functional activity is therefore significant to make a valid signal interpretation for the imaging techniques. In the present study, we evaluated depth profiles of microvascular distributions in rat somatosensory subfields (barrel field, forelimb region, trunk region and hindlimb region) and characterized depth variations in microvascular structures, such as locations, lengths and directions of microvessels, throughout the cortical layers (I–VI). To obtain the accurate microvascular structure, we made a customized casting method by using confocal laser scanning microscope. We observed that microvascular distribution successively varied throughout the cortical layers (I–VI) and that the maximum number density of microvessels was consistently found in middle layers (III–V). In addition, superficial layers had relatively long microvessels, almost perpendicular to the cortical surface, whereas middle layers had short microvessels propagating in all directions. These regional differences in microvascular structures were closely related to the somatosensory subfields, e.g., barrel field was the greatest number density of microvessels among the investigated subfields. Based on these observations, we compared microvascular profiles with previously reported distribution patterns of tissue partial pressure of oxygen (pO2). The results showed that tissue pO2 was correlated with microvascular distribution in some but not all of the subfields. This finding shows that detailed microvascular profiles are helpful to investigate causal relationships between microvascular structure and functional activities in cerebral cortex.

We measured and compared the oxygen partial pressure (pO2) profiles in experimental tumors after irradiation with carbon ions and with X-rays. The NFSa fibrosarcomas grown in the hind legs of C3H male mice received isoeffect single doses of carbon ions or X-rays. Coaxial oxygen microelectrodes of high spatial resolution were inserted into the tumor with 20 micrometer steps by a computerized micromanipulator. The number of pO2 peaks that reached 15 mmHg were at least 0.45 per 3,000 micrometer in unirradiated tumors and significantly increased to 1.55 per 3,000 micrometer as early as day 1 of carbon-ion irradiation (p

To compare the spatial heterogeneity of brain tissue partial pressure of oxygen (pO(2)) among local brain regions, we focused on functional and anatomical variations in rat somatosensory cortex. Tissue pO(2) was measured by using an oxygen microelectrode with high spatio-temporal resolution, and investigated in three somatosensory areas including hindlimb (HL), forelimb (FL), and trunk region (Tr). Their anatomical structures were determined with histological techniques (Nissl stain). In addition to the measurement of baseline tissue pO(2), we examined temporal shifts in tissue pO(2) distribution elicited by functional stimulation using the brushing stimulation to the hindlimb, forelimb, and trunk regions of the body. We observed that average tissue pO(2) in the Tr (14+/-10 Torr) was significantly lower than those in the HL (25+/-13 Torr) and FL (24+/-13 Torr). Such regional differences in tissue pO(2) were closely related to the cytoarchitectonic variations among these three areas. In addition, the functional stimulation enlarged the regional differences in the pO(2) depending on each somatosensory area; the pO(2) in the HL increased by 3.6+/-2.9% after the stimulation to hindlimb, whereas that in the Tr decreased by -2.9+/-2.5% after the stimulation to trunk region. Such dual responses of tissue pO(2) (i.e. increase or decrease) after the functional stimulation to the corresponding body regions may provide a criterion to clinically predict regions susceptible to tissue hypoxia, because considerable decrease in tissue pO(2) occurred in the Tr showing the lowest baseline pO(2).

Cross-sectional oxygen distribution in microvessels in most previous studies has been assumed to be homogeneous. Recent studies using phosphorescence quenching microscopy or microspectrophotometry showed a decline in oxygen profile near the arterial wall. In this study we performed theoretical analysis of intravascular P(O(2)) and S(O(2)) profiles in arterioles by using a radial diffusion model with a constant oxygen efflux from the vascular lumen, taking intravascular flow distribution into account. Theoretical calculations indicated that radial oxygen diffusion and a laminar flow pattern would create inhomogeneous intravascular oxygen profile with a decline toward the arterial wall. As mean blood flow velocity became lower, the difference between the centerline oxygen level and the inner surface level became larger. In conclusion, it is suggested that oxygen efflux from the vascular lumen and less convective supply near the vascular wall create a decline in P(O(2)) as well as S(O(2)) toward the arterial wall.

Background Previous studies have shown that lipopolysaccharide-induced inflammation in the lung results in tyrosine nitration. The objective of this study was to evaluate the contribution of myeloperoxidase and peroxynitrite pathway to the tyrosine nitration in lipopolysaccharide-administered lungs of rats that were otherwise untreated or leukocyte-depleted by cyclophosphamide or received inhaled nitric oxide (NO). Methods The authors analyzed the immunoreactivity of inducible nitric oxide synthase (iNOS), nitrotyrosine (a product of the myeloperoxidase or peroxynitrite pathway), and chlorotyrosine (a byproduct of the myeloperoxidase pathway) by use of specific antibodies. The number of neutrophils in bronchoalveolar lavage fluid (BALF) and levels of myeloperoxidase activity in lung homogenates were also measured. Results Lipopolysaccharide enhanced the immunoreactivity of iNOS, nitrotyrosine, and chlorotyrosine in alveolar wall cells, alveolar macrophages, and neutrophils. Leukocyte depletion by cyclophosphamide and inhibition of leukocyte accumulation in the lungs by NO inhalation did not eliminate the increase in iNOS immunoreactivity in alveolar macrophages after lipopolysaccharide treatment, but nitrotyrosine and chlorotyrosine were not produced in these cells. Tyrosine nitration in response to lipopolysaccharide was associated with increases in neutrophil count in BALF and in myeloperoxidase activity in lung homogenates, whereas NO inhalation suppressed the neutrophil count in BALF and reduced tyrosine nitration and chlorination. Conclusions These findings suggest that myeloperoxidase pathway has a role in tyrosine nitration in the lungs of lipopolysaccharide-treated rats, and that NO inhalation during early phase of inflammation does not increase but rather decreases tyrosine nitration and chlorination, possibly by reducing neutrophil sequestration.

Background— The proximity of capillaries, arterioles, and venules provides complex spatial relationships that lead to oxygen transfer among microvessels. Although a conceptual image of complex oxygen transfer among microvessels has been hypothesized, in vivo mapping of oxygen saturation (S o 2 ) levels in microvessels had never been performed. Methods and Results— The oxygen profile of the arterioles and venules of the rat cremaster muscle during normoxia and hypoxia was visualized by preparing pseudo-color images of S o 2 levels based on microspectrophotometry data obtained by using 3 different optical filters and a cooled CCD camera. The S o 2 images showed lower S o 2 levels in arterioles close to their walls, and the S o 2 levels in the paired venules showed higher S o 2 levels close to the arterioles. There were capillaries that crossed the microvessels whose S o 2 levels changed as they crossed the microvessels. The S o 2 levels were lower close to the vessel wall than in the centerline level of the microvessels, and the highest S o 2 levels in venules paralleling arterioles were skewed toward the arterial side. The S o 2 images showed that the S o 2 level in arterioles decreased after crossing venules, whereas the S o 2 level in venules increased after crossing arterioles. Conclusions— Visualization of intravascular S o 2 levels suggested that oxygen is transferred between paired microvessels and between crossing microvessels in rat cremaster muscle. The possibility that oxygen is transported from some arterioles to venules and tissue through adjacent capillaries is proposed.

Previously, cross-sectional oxygen distribution in microvessels was assumed to be homogeneous. The oxygen profile in the arterioles of rat cremaster muscle was measured using microspectrophotometry and a PO(2) microelectrode, showing a drop in SO(2) as well as PO(2) close to the vascular wall with a flat PO(2) profile in the perivascular tissue.

There are only a few reports of the use of impulse oscillation system (IOS) for the evaluation of COPD treatment. In this study, we applied IOS and spirometry to evaluate the effectiveness of fluticasone propionate and salmeterol (SFC) combined with tiotropium (TIO) in COPD patients. Following a 4-week run-in period with TIO (18 μg once daily) treatment, COPD patients were randomized to SFC (250/50 μg twice daily; SFC + TIO group, n = 25), or TIO alone (TIO group, n = 31). Pulmonary functions were recorded by IOS and spirometry before and after the study period. The SFC + TIO group showed significant improvements in inspiratory resistance at 5 Hz and resonant frequency, as well as in FVC and FEV1, after the 12-week treatment (p

It has been reported that several bronchoconstrictors generate nitric oxide (NO), counteracting bronchoconstriction, and removal of bronchial epithelia reduces NO production. However, it has not been elucidated whether neurokinin A (NKA), a potent bronchoconstrictor liberated from nerve terminals, generates NO. Specific questions in this study were (1) does NKA also generate NO, (2) does NO counteract NKA-induced bronchoconstriction, and (3) does the NO generation require bronchial epithelial cells? In an in vivo study exogenous as well as endogenous (capsaicin-induced) NKA increased airway opening pressure (Pao) and the exhaled NO level, and both were inhibited by an antagonist selective for NK2 receptor (a receptor for NKA), SR48968. The exhaled NO level became negligible with an inhibitor of NO synthase (NOS) type 1-3 (NG-nitro- l -arginine methyl ester, l -NAME) with increased Pao, but not with a NOS type 2 inhibitor. In an in vitro study, NKA increased the nitrite/nitrate level in superfused fluid of tracheal segments. Removing smooth muscle reduced nitrite/nitrate in the fluid to negligible levels, while the level was unchanged with removal of the epithelia. Pretreatment with l -NAME enhanced the tension of epithelia-removed tracheal segments. These findings indicate that (1) NKA generates NO, (2) NO counteracts NKA-induced bronchoconstriction, and (3) NKA activates NOS in the muscle layer, independently of bronchial epithelia.

There are theoretical and experimental indications that the presence of He as a balance gas markedly increase the diffusion velocity of other gases contained in a gas mixture. We allowed dogs with pulmonary vasoconstriction induced by hypoxia to inhale a mixture of 5 parts per million (ppm) of nitric oxide (NO) and O2 balanced with He (NO in He) instead of N2 (NO in N2). The dilating effect of NO in He and NO in N2 on the pulmonary artery was evaluated by determining conventional pulmonary hemodynamic parameters, mean pulmonary artery (PA) pressure (MPAP), and pulmonary vascular resistance indexed to body surface area (PVRI), pulmonary impedance ( Z), and the recently developed hemodynamic index, time-corrected wave intensity (WI). The main findings in this study were as follows: 1) hypoxia increased MPAP, PVRI, Z at 0 Hz ( Z 0), Z at the first harmonics, characteristic impedance ( Z c), the reflection coefficient (Γ), and the first peak of WI; 2) NO in N2 reduced Z 0and Γ; and 3) NO in He reduced the first peak of WI and reduced Z 0 and Γ more than NO in N2. The enhanced vasodilatory effect of NO in He might be associated with facilitated diffusion of NO diluted in the gas mixture with He. In conclusion, increased efficacy of NO in He offers the possibility to reduce the inhaled NO concentration.

In previous studies the exhaled nitric oxide (NO) level of asthma patients was investigated only in association with bronchial inflammation, and whether the degree of bronchoconstriction itself influences the exhaled NO level has never been investigated. We therefore evaluated the effect of inhalation of a bronchoconstrictor (methacholine) or a bronchodilator (salbu-tamol) on the exhaled NO level of healthy volunteers and asthma patients. The exhaled NO level of the healthy volunteers decreased after methacholine inhalation. The exhaled NO level of patients with mild or moderate persistent asthma, who had no asthma attacks on the day of measurement, increased after salbutamol inhalation, and the exhaled NO level of asthma patients during asthma attacks increased after salbutamol inhalation followed by intravenous drip infusion of aminophylline. It is suspected that large amounts of NO are trapped in the lung distal to the constricted airway, contributing little to the exhaled NO level at the mouth. However, we expect that the trapped NO is exhaled at a larger fraction after the dilatation of the constricted small airway, thereby increasing the exhaled NO level at the mouth. In conclusion, the results of this study suggest that acute changes in bronchoconstriction themselves influence the exhaled NO level independently of the change in NO synthase activity associated with airway inflammation.