Guidelines from the Japanese Society of Echocardiography: Guidance for the management and maintenance of echocardiography equipment.

Echocardiography plays a pivotal role as an imaging modality in modern cardiology practice. Information derived from echocardiography is definitely helpful for patient care. The Japanese Society of Echocardiography has promoted echocardiography in routine clinical and research use. One of the missions of the Society is to provide information that is useful for high-quality examinations. To ensure this, we believe that equipment in good condition and a comfortable environment are important for both patient and examiner. Here, the Guideline Preparation Committee of the Japanese Society of Echocardiography has established brief guidance for the routine use of echocardiography equipment.

Biomatrices and biomaterials for future developments of bioprinting and biofabrication.

The next step beyond conventional scaffold-based tissue engineering is cell-based direct biofabrication techniques. In industrial processes, various three-dimensional (3D) prototype models have been fabricated using several different rapid prototyping methods, such as stereo-lithography, 3D printing and laser sintering, as well as others, in which a variety of chemical materials are utilized. However, with direct cell-based biofabrication, only biocompatible materials can be used, and the manufacturing process must be performed under biocompatible and physiological conditions. We have developed a direct 3D cell printing system using inkjet and gelation techniques with inkjet droplets, and found that it had good potential to construct 3D structures with multiple types of cells. With this system, we have used alginate and fibrin hydrogel materials, each of which has advantages and disadvantages. Herein, we discuss the roles of hydrogel for biofabrication and show that further developments in biofabrication technology with biomatrices will play a major part, as will developments in manufacturing technology. It is important to explore suitable biomatrices as the next key step in biofabrication techniques.

Is driving a car a risk for Legionnaires' disease?

Legionnaires' disease (LD) is a major cause of severe community-acquired pneumonia but the source and mode of transmission are not always apparent, especially in sporadic cases. We hypothesized that LD can be acquired from the air-conditioning systems of motor cars. Swabs were taken from the evaporator compartments of the air-conditioning system of scrapped cars. Healthy subjects who were mainly employees of regional transportation companies were tested for antibody to Legionella pneumophila serogroups 1-6; they also completed a questionnaire. Legionella species were detected in 11/22 scrapped cars by the loop-mediated isothermal amplification method. The prevalence of microplate agglutination titres > or =1:32 was significantly higher in subjects who sometimes used car air-conditioning systems. Although we did not prove a direct link between Legionella spp. in the car evaporator and LD, our findings point to a potential risk of car air-conditioning systems in LD, which needs further investigation.

Biochemical characterization of plasma-derived tissue factor pathway inhibitor: post-translational modification of free, full-length form with particular reference to the sugar chain.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a physiological protease inhibitor that inhibits the initial reactions of the extrinsic blood coagulation pathway. Most TFPI in human plasma is associated with lipoproteins; however, the most functionally active form is thought to be the free, full-length form (f-pTFPI). Cell culture derived TFPI and recombinant TFPI (rTFPI) exhibit variations in their respective anticoagulant activity, which may be caused by post-translational modifications, such as the frequent differences in sugar chain structures among recombinant proteins. Sugar chain structures in rTFPI expressed in Chinese hamster ovary (CHO) cells have been reported previously, but those of plasma TFPI have not been. OBJECTIVES: To purify f-pTFPI and analyze the sugar chain structures. RESULTS AND CONCLUSION: f-pTFPI was purified to homogeneity from blood plasma using a combination of anion-exchange, heparin affinity, immunoaffinity, and reversed-phase chromatographies, resulting in a yield of 76%. f-pTFPI showed a partially phosphorylated glycoprotein comprising a total of 276 amino acids by peptide mapping. The sugar chain structures were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion of the pyridylamino sugar chains and the following results were obtained. (Sialyl) Galbeta1-3GalNAc was linked to Thr(175), partially to Thr(14) and Ser(174); sialyl complex-type sugar chains to Asn(117) and Asn(167), whereas Asn(228) was not glycosylated. Neuraminidase-resistant acidic sugar chains including sulfated sugar chains were not observed significantly. The protease inhibitory activities of f-pTFPI towards activated factor (F) X and tissue factor-activated FVII complex were identical to those of full-length rTFPI expressed in CHO cells.

A femtosecond laser pacemaker for heart muscle cells.

The intracellular effects of focused near-infrared femtosecond laser irradiation are shown to cause contraction in cultured neonatal rat cardiomyocytes. By periodic exposure to femtosecond laser pulse-trains, periodic contraction cycles in cardiomyocytes could be triggered, depleted, and synchronized with the laser periodicity. This was observed in isolated cells, and in small groups of cardiomyocytes with the laser acting as pacemaker for the entire group. A window for this effect was found to occur between 15 and 30 mW average power for an 80 fs, 82 MHz pulse train of 780 nm, using 8 ms exposures applied periodically at 1 to 2 Hz. At power levels below this power window, laser-induced cardiomyocyte contraction was not observed, while above this power window, cells typically responded by a high calcium elevation and contracted without subsequent relaxation. This laser-cell interaction allows the laser irradiation to act as a pacemaker, and can be used to trigger contraction in dormant cells as well as synchronize or destabilize contraction in spontaneously contracting cardiomyocytes. By increasing laser power above the window available for laser-cell synchronization, we also demonstrate the use of cardiomyocytes as optically-triggered actuators. To our knowledge, this is the first demonstration of remote optical control of cardiomyocytes without requiring exogenous photosensitive compounds.

Slow Ca(2+) wave stimulation using low repetition rate femtosecond pulsed irradiation.

We demonstrated stimulation of Ca(2+) in living cells by near-infrared laser pulses operated at sub-MHz repetition rates. HeLa cells were exposed to focused 780 nm femtosecond pulses, generated by a titanium-sapphire laser and adjusted by an electro-optical modulator. We found that the laser-induced Ca(2+) waves could be generated over three orders of magnitude in repetition rates, with required laser pulse energy varying by less than one order of magnitude. Ca(2+) wave speed and gradients were reduced with repetition rate, which allows the technique to be used to modulate the strength and speed of laser-induced effects. By lowering the repetition rate, we found that the laser-induced Ca(2+) release is partially mediated by reactive oxygen species (ROS). Inhibition of ROS was successful only at low repetition rates, with the implication that ROS scavengers may in general be depleted in experiments using high repetition rate laser irradiation.

Successful treatment of recurrent intracardiac thrombus in Behçet's disease with immunosuppressive therapy.

Behçet's disease (BD) is a chronic multisystem inflammatory disorder characterized by recurrent oral and genital ulcers, skin eruptions and uveitis. Neurological, gastrointestinal, and musculoskeletal systems are also involved. Although venous and arterial vasculitis occur in up to one-third of patients, intracardiac thrombus is a very rare complication. We herein report the case of a 46-year-old man with BD who presented with a large right atrial thrombus. Within a month after surgical removal, the thrombus recurred and was successfully treated with immunosuppressants that included prednisolone and cyclophosphamide.

A novel therapeutic approach combining human plasma-derived Factors VIIa and X for haemophiliacs with inhibitors: evidence of a higher thrombin generation rate in vitro and more sustained haemostatic activity in vivo than obtained with Factor VIIa alone.

BACKGROUND AND OBJECTIVES: Therapy with recombinant Factor VIIa (rFVIIa) for haemophiliacs with inhibitors still has some unresolved problems, such as the requirement for frequent infusions of rFVIIa every 2-3 h to sustain haemostatic activity for an extended time-period and that the therapeutic dose of rFVIIa is not always predictable. In the present study, we searched for an effective combination of plasma-derived FVIIa with other blood coagulation factors, and demonstrated that a therapeutic approach combining plasma-derived FVIIa and Factor X (FX) was more useful for treating haemophiliacs with inhibitors than FVIIa alone. MATERIALS AND METHODS: The haemostatic effects of FVIIa and FX were evaluated in vitro and in vivo. In in vitro experiments we assessed the following: the ability to enhance the thrombin generation rate in a reconstituted blood coagulation model without Factor VIII (FVIII) or Factor IX (FIX); the ability to correct the activated partial prothrombin time (APTT) of FVIII-depleted plasma or FIX-depleted plasma; and the ability to correct the clotting time of haemophilia-like whole blood using thromboelastography (TEG). In in vivo experiments, the haemostatic activity of the combination treatment of FVIIa and FX was determined by measuring the bleeding time and TEG using a monkey haemophilia B model produced by the injection of anti-human FIX polyclonal antibodies. The degree of thrombogenicity of the combination was evaluated using the rabbit stasis model. RESULTS: The addition of FX to FVIIa dramatically enhanced the thrombin generation rate in the reconstituted blood coagulation model and corrected the prolonged APTTs of FVIII- and FIX-depleted plasmas to levels achieved by the replacement therapies. In contrast, the addition of prothrombin to FVIIa did not show such enhancing activity. Furthermore, FVIIa-induced whole blood clotting times in the FVIII- and FIX-inhibited states were also shortened by the addition of FX in a concentration-dependent manner. Finally, the co-administration of FVIIa (80 microg/kg) and FX (800 microg/kg) in a monkey haemophilia B model resulted in a more robust and persistent haemostatic effect on the secondary bleeding time and whole-blood clotting time of TEG than that of FVIIa alone. The results of rabbit stasis tests for evaluating the risk of thrombogenicity showed that the combination of FVIIa and FX was less thrombogenic than FEIBA. CONCLUSIONS: The present study demonstrated that the combination of FVIIa and FX appeared to have a higher and more sustainable haemostatic potential than FVIIa alone, and less thrombogenicity than FEIBA. A therapeutic approach combining FVIIa and FX could be a promising and novel approach to compensate for the disadvantages of rFVIIa and FEIBA for haemophiliacs with inhibitors.

Party size and diet of syntopic atelids (Ateles chamek and Lagothrix cana) in Southwestern Brazilian Amazonia.

Syntopic Alouatta seniculus, Ateles chamek and Lagothrix cana (Atelidae) were studied in southwestern Amazonia. Primate populations were first surveyed, and then the party size, diet and vertical spacing were monitored over a 5-month period. Atelids accounted for more than half the survey sightings and Lagothrix was the most abundant. Party sizes recorded for both Alouatta and Lagothrix during monitoring were significantly larger than those recorded during surveys, but no such difference was found for Ateles. Monitored parties were significantly larger in Lagothrix in comparison with either Alouatta or Ateles, as were groups of Ateles in comparison with Alouatta. Mean party size in Ateles decreased progressively during the course of the study, from 8.9 +/- 3.4 in June to 3.9 +/- 2.3 in October. Moraceae was the most important dietary resource for Ateles and Lagothrix, in terms of both feeding records and number of species exploited. There was considerable overlap in the plant taxa exploited, but some notable differences, such as the exclusive use of Hymenaea courbaril (Caesalpinaceae) by Lagothrix and of Euterpe precatoria (Arecaceae) by Ateles. As at other sites in the region, Ateles occupied significantly higher forest strata in comparison with Lagothrix. Despite the preliminary nature of the study, the results indicate a number of ecological differences between species that undoubtedly play an important role in niche separation.

The 2.0-A crystal structure of tachylectin 5A provides evidence for the common origin of the innate immunity and the blood coagulation systems.

Because invertebrates lack an adaptive immune system, they had to evolve effective intrinsic defense strategies against a variety of microbial pathogens. This ancient form of host defense, the innate immunity, is present in all multicellular organisms including humans. The innate immune system of the Japanese horseshoe crab Tachypleus tridentatus, serving as a model organism, includes a hemolymph coagulation system, which participates both in defense against microbes and in hemostasis. Early work on the evolution of vertebrate fibrinogen suggested a common origin of the arthropod hemolymph coagulation and the vertebrate blood coagulation systems. However, this conjecture could not be verified by comparing the structures of coagulogen, the clotting protein of the horseshoe crab, and of mammalian fibrinogen. Here we report the crystal structure of tachylectin 5A (TL5A), a nonself-recognizing lectin from the hemolymph plasma of T. tridentatus. TL5A shares not only a common fold but also related functional sites with the gamma fragment of mammalian fibrinogen. Our observations provide the first structural evidence of a common ancestor for the innate immunity and the blood coagulation systems.

Characterization of anti-myocardial autoantibodies in Japanese patients with dilated cardiomyopathy.

Few previous reports have comprehensively screened all the anti-myocardial autoantibodies (AMCA) in relation to other clinical profiles in patients with idiopathic dilated cardiomyopathy (IDC), so the present study used both immunohistochemistry (FITC) and immunoblotting (IB) for screening patients with IDC in order to characterize the clinical significance of AMCA. Sera were collected from 100 patients with IDC and age-matched 100 healthy control subjects (CTL). For FITC, an unfixed frozen section of human myocardium was used for the standard indirect immunofluorescence; for IB, total cardiac homogenates of the same myocardium were blotted to serum at 2 sets of dilution (1:200 and 1:10,000). The positive rates of AMCA detection for each method were as follows (IDC vs CTL); 39% vs 6% for FITC, 38% vs 4% for IB (1:200), and 10% vs 0% for IB (1:10,000). Fifty-nine patients with IDC and 8 CTL were positive for AMCA by either method, and 18 patients with IDC and 2 CTL were positive for AMCA by both methods. IB-positivity at 1:200 was an independent predictor by multiple logistic regression analysis of non-sustained ventricular tachycardias as well as left ventricular end-diastolic diameter and plasma norepinephrine concentration.

Differentiation between high-grade glioma and metastatic brain tumor using single-voxel proton MR spectroscopy.

The purpose of this study was to clarify the efficacy of single-voxel proton magnetic resonance spectroscopy (MRS) in differentiating high-grade glioma from metastasis. Thirty-one high-grade gliomas (11 anaplastic gliomas and 20 glioblastomas) and 25 metastases were studied. Proton MRS was performed using point-resolved spectroscopy with echo times (TEs) of both 136 and 30 ms. The peaks for lipid were evaluated at short TE, and those for N-acetyl-aspartate (NAA), creatine (Cr), and choline-containing compounds (Cho) were assessed at long TE. All the tumors exhibited a strong Cho peak at long TE. Twenty-one of 25 metastases showed no definite Cr peak. The remaining 4 metastases showed NAA and Cr peaks; however, the presence of NAA and relatively high NAA/Cr ratio (1.58+/-0.56) indicated normal brain contamination. All the gliomas, except for a single glioblastoma, showed a Cr peak with (n=16) or without (n=14) NAA. At short TE all metastases and glioblastomas showed definite lipid or lipid/lactate mixture, but anaplastic gliomas showed no definite lipid signal. Intratumoral Cr suggests glioma. Absence of Cr indicates metastasis. Definite lipid signal indicates cellular necrosis in glioblastoma and metastasis, and no lipid signal may exclude metastases.

Induction of acquired factor IX inhibitors in cynomolgus monkey (Macaca fascicularis): a new primate model of hemophilia B.

Inherited hemophilia dog and other transient hemophilic animal models have been used for evaluation of hemostatic agents for use in treatment of hemophilia. We established the first nonhuman primate hemophilic model by immunizing cynomolgus monkeys with human FIX (hFIX) in adjuvants. FIX activities of all three hFIX-immunized monkeys decreased transiently to less than 10% in accordance with prolongation of activated partial thromboplastin time (APTT). Forty micrograms of human factor VIIa (hFVIIa) per kilogram body weight (that was reported to be clinically effective) was administered to the monkey with the highest inhibitor titer to evaluate its usefulness as a hemophilia inhibitor model. Results of thromboelastography (TEG) after the injection demonstrated that the hemostatic effect of FVIIa in this model would be similar to that in hemophiliacs with inhibitors. The antibodies purified from the monkey's plasma by hFIX-immobilized gel were composed of two types: Ca(2+)-dependent and -independent antibodies, with features of IgG(1) and IgG(4). Both types of antibodies reacted to cynomolgus FIX, and only Ca(2+)-dependent antibodies also expressed inhibitory activity against cynomolgus FIX. Immunoblotting analyses of Ca(2+)-dependent antibodies using hFIX and its derivatives suggested that they recognized the Ca(2+)-dependent conformation related to the gamma-carboxyglutamic acid (Gla) domain. Comparison of FIX cDNA from human, cynomolgus monkey, and other species, and the results of immunization of various animals (goats, beagle dogs, rabbits, and rats) with hFIX in adjuvants strongly suggested that the development of acquired FIX inhibitors in the monkeys might be due to high cross-reactivity of the antibodies to molecular mimic antigens, hFIX, and cynomolgus FIX.

Comparison of image reconstruction algorithms in myocardial perfusion scintigraphy.

The purpose of this study was to compare the clinical utility of two image reconstruction algorithms in myocardial perfusion SPECT (single-photon emission computed tomography): filtered back-projection (FBP) and ordered subset expectation maximization (OSEM). A rest/stress one-day protocol with 99mTc-MIBI or tetrofosmin was performed on 102 consecutive patients who underwent coronary angiography. After SPECT data acquisition, images were reconstructed with FBP and OSEM algorithms. We assessed diagnostic performance (sensitivity, specificity and accuracy) in detecting coronary artery stenosis and evaluated regional tracer uptake with a 4-point scoring system. Although there were no significant differences in diagnostic performance between FBP and OSEM reconstruction, the OSEM method yielded higher uptake in the RCA area than the FBP method by reducing the count-loss artifact due to hepatic uptake of the tracers. In addition, regional uptake in the LCX area was significantly lower in the OSEM image than in the FBP image; this phenomenon was observed mainly in patients with coronary stenosis and/or infarction in the LCX territory. In conclusion, OSEM and FBP offered comparable diagnostic performance in stress myocardial perfusion SPECT. The OSEM method contributed to reduction of the count-loss artifact in inferior and posterior walls and to easy recognition of hypoperfusion in the LCX area.

Factor VIIa modified in the 170 loop shows enhanced catalytic activity but does not change the zymogen-like property.

Factor VIIa (VIIa) is an unusual trypsin-type serine proteinase that appears to exist in an equilibrium between minor active and dominant zymogen-like inactive conformational states. The binding of tissue factor to VIIa is assumed to shift the equilibrium into the active state. The proteinase domain of VIIa contains a unique structure: a loop formed by a disulfide bond between Cys310 and Cys329, which is five residues longer than those of other trypsin types. To examine the functional role of the loop region, we prepared two mutants of VIIa. One of the mutants, named VII-11, had five extra corresponding residues 316-320 of VII deleted. The other mutant, VII-31, had all of the residues in its loop replaced with those of trypsin. Functional analysis of the two mutants showed that VIIa-11 (Kd = 41 nm) and VIIa-31 (Kd = 160 nm) had lower affinities for soluble tissue factor as compared with the wild-type VIIa (Kd = 11 nm). The magnitude of tissue factor-mediated acceleration of amidolytic activities of VIIa-11 (7-fold) and that of VIIa-31 (2-fold) were also smaller than that of wild-type VIIa (30-fold). In the absence of tissue factor, VIIa-31 but not VIIa-11 showed enhanced activity; the catalytic efficiencies of VIIa-31 toward various chromogenic substrates were 2-18-fold greater than those of the wild-type VIIa. Susceptibility of the alpha-amino group of Ile-153 of VIIa-31 to carbamylation was almost the same as that of wild-type VIIa, suggesting that VIIa-31 as well as wild-type VIIa exist predominantly in the zymogen-like state. Therefore, the tested modifications in the loop region had adverse effects on affinity for tissue factor, disturbed the tissue factor-induced conformational transition, and changed the catalytic efficiency of VIIa, but they did not affect the equilibrium between active and zymogen-like conformational states.

A dose-response study of anticholinesterase drugs on contractile and phosphatidylinositol responses of rat trachea.

UNLABELLED: We investigated whether anticholinesterase drugs in large doses inhibit muscarinic receptors of airway smooth muscle. In vitro measurements of isometric tension and [(3)H]inositol monophosphate (IP(1)) that formed were conducted by using rat tracheal rings or slices. Neostigmine and pyridostigmine caused muscular contraction and IP(1) accumulation in small doses (10 microM and < or = 100 microM, respectively), but they attenuated muscular contraction and IP(1) accumulation in larger doses (1000 microM). Edrophonium did not affect the smooth muscle tone and IP(1) levels. Neostigmine, pyridostigmine, and edrophonium attenuated the carbachol (5.5 microM)-induced smooth muscle contraction and IP(1) accumulation, when administered in large doses (1000 microM). The attenuation of contraction by neostigmine at large doses was not affected by methoctramine, an M(2) muscarinic receptor antagonist, but was reversed by washing with fresh Krebs-Henseleit solution. The results suggest that anticholinesterase drugs have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy. IMPLICATIONS: Neostigmine and pyridostigmine, but not edrophonium, have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy.

Fibrinogen binds to integrin alpha(5)beta(1) via the carboxyl-terminal RGD site of the Aalpha-chain.

Fibrinogen interactions with vascular endothelial cells are implicated in various physiological and pathophysiological events, including angiogenesis and wound healing. We have shown previously that integrin alpha(5)beta(1) is a fibrinogen receptor on endothelial cells [Suehiro, K., Gailit, J., and Plow, E.F. (1997) J. Biol. Chem. 272, 5360-5366]. In the present study, we have characterized fibrinogen interactions with purified alpha(5)beta(1) and have identified the recognition sequence in fibrinogen for alpha(5)beta(1). The binding of fibrinogen to immobilized alpha(5)beta(1) was selectively supported by Mn(2+). Fibrinogen bound to purified alpha(5)beta(1) in a time-dependent, specific, and saturable manner in the presence of Mn(2+), and the binding was blocked completely by Arg-Gly-Asp (RGD)-containing peptides and by anti-alpha(5) and anti-alpha(5)beta(1) monoclonal antibodies. A monoclonal antibody directed to the C-terminal RGD sequence at Aalpha572-574 significantly inhibited the binding of fibrinogen to alpha(5)beta(1), whereas monoclonal antibodies directed to either the N-terminal RGD sequence at Aalpha95-97 or the C-terminus of the gamma-chain did not. Furthermore, substituting RGE for RGD at position Aalpha95-97 in recombinant fibrinogen had a minimal effect on binding, whereas substituting RGE for RGD at position Aalpha572-574 decreased binding by 90%. These results demonstrate that the C-terminal RGD sequence at Aalpha572-574 is required for the interaction of fibrinogen with alpha(5)beta(1).