Isolation of human monoclonal antibodies that bind to two different antigens and are encoded by germline VH and VL genes.

This paper reports isolation of two monoclonal antibodies (mAbs) that bind to both a membrane protein and a cytoplasmic protein. Most Abs established as markers for autoimmune disease bind to cytoplasmic or nuclear substances. However, it remains unknown how these Abs are produced. On the other hand, there were examples where clones originally isolated as Abs that bind to membrane proteins also showed binding activity to cytoplasmic or nuclear substances. Based on these results, the following hypothesis has been proposed. The Abs that had been originally produced against a membrane protein showed cross-reactivity against cytoplasmic or nuclear substances. In the present study we reported isolation of Abs that bound to both a membrane protein, CADM1, and a cytoplasmic protein, α-actinin-4. The method adopted in the present study could be generally applicable to isolation of Abs showing such dual specificity. Firstly, we constructed a huge human Ab library using various organs including naïve B-cell-rich organs such as bone marrow and umbilical cords. Then, we developed a comprehensive screening method for isolation of Abs that bound to cell surface antigens. Through extensive screenings with many kinds of cell we newly obtained a library composed of around 4000 independent clones that bind to membrane proteins. We screened this library with α-actinin-4 and succeeded in isolating two Abs. They bound to α-actinin-4 and a membrane protein CADM1. Furthermore, they are encoded by naïve heavy and light chain variable genes (VH & VL). These results suggested that cross-reactive Abs to both a membrane protein and a cytoplasmic protein could be present in germline repertoire of Ab in humans. This methodology adopted in the present study could be applied to isolation of cross-reactive Abs possibly involved in autoimmune diseases.

Web survey-based selection of controls for epidemiological analyses of a multi-prefectural outbreak of enterohaemorrhagic Escherichia coli O157 in Japan associated with consumption of self-grilled beef hanging tender.

An outbreak of enterohaemorrhagic Escherichia coli O157 occurred in multiple prefectures of Japan in November 2009. We conducted two case-control studies with trace-back and trace-forward investigations to determine the source. The case definition was met by 21 individuals; 14 (66.7%) were hospitalised, but no haemolytic uraemic syndrome, acute encephalopathy or deaths occurred. Median age was 23 (range 12-48) years and 14 cases were male (66.7%). No significant associations with food were found in a case-control study by local public health centres, but our matched case-control study using Internet surveys found that beef hanging tender (or hanger steak), derived from the diaphragm of the cattle, was significantly associated with illness (odds ratio = 15.77; 95% confidence interval, 2.00-124.11). Pulsed-field gel electrophoresis analysis of isolates from patients and the suspected food showed five different patterns: two in faecal and food samples, and another three in patient faecal samples only, although there were epidemiological links to the meat consumed at the restaurants. Trace-back investigation implicated a common food processing company from outside Japan. Examination of the logistics of the meat processing company suggested that contamination did not occur in Japan. We concluded that the source of the outbreak was imported hanging tender. This investigation revealed that Internet surveys could be useful for outbreak investigations.

Real space observation of current-induced magnetic domain wall displacement in Co/Ni nano-wire by photoemission electron microscopy.

Current-induced magnetic domain wall (DW) displacement in a Co/Ni nano-wire with perpendicular magnetic anisotropy was investigated in real space by photoemission electron microscopy (PEEM) for the first time. DW velocity determined from the PEEM observation was 40 m s(-1) for the current density of 2.5 × 10(12) A m(-2), which was consistent with the result obtained by the electrical measurement used in our previous reports.

Observation of the intrinsic pinning of a magnetic domain wall in a ferromagnetic nanowire.

The spin transfer torque is essential for electrical magnetization switching. When a magnetic domain wall is driven by an electric current through an adiabatic spin torque, the theory predicts a threshold current even for a perfect wire without any extrinsic pinning. The experimental confirmation of this 'intrinsic pinning', however, has long been missing. Here, we give evidence that this intrinsic pinning determines the threshold, and thus that the adiabatic spin torque dominates the domain wall motion in a perpendicularly magnetized Co/Ni nanowire. The intrinsic nature manifests itself both in the field-independent threshold current and in the presence of its minimum on tuning the wire width. The demonstrated domain wall motion purely due to the adiabatic spin torque will serve to achieve robust operation and low energy consumption in spintronic devices.

Transient decreases in Bispectral Index without associated changes in the level of consciousness during photic stimulation in an epileptic patient.

This case report describes a patient with a history of epileptic seizures who showed unusual decreases in the Bispectral Index (BIS) attributable to the induction of abnormal slow electroencephalographic (EEG) waves by photic stimulation, without any associated decrease in his level of consciousness. After starting anticonvulsive therapy, photic stimulation no longer induced abnormal EEG activity nor decreased BIS values. These findings suggest that BIS values may not accurately reflect a patient's actual level of consciousness in the presence of epilepsy-related abnormal EEG activity and that the BIS monitor may be able to track such EEG changes.

[Aortic regurgitation caused by rupture of a fibrous strand; report of a case].

Rupture of a fibrous strand of a tricuspid aortic valve is extremely rare. We describe a patient with aortic regurgitation due to spontaneous rupture of the fibrous strands that suspend the aortic valve leaflets. The fibrous strands were recognized between the left and right coronary cusps, and between the left coronary and non-coronary cusps during the operation. The fibrous strand between the left coronary and non-coronary cusps had been ruptured, and thus the left coronary and non-coronary cusps were prolapsed into the left ventricle. Since a degenerative change was observed histologically in the resected specimen, these fibrous strands were considered to be an embryonic remnant of the aortic valve.

System design of a bioartificial liver with a high performance hemodialyzer as an immunoisolator using a mathematical kinetic model.

We propose a new bioartificial liver (BAL) system equipped with a high performance hemodialyzer to act as an immunoisolation device. We discuss the design of the BAL system using a mathematical kinetic model with the experimentally obtained mass-transfer performances of various hemodialyzers. The mass transfer resistances of the hemodialyzers did not adversely influence the ammonia-removal and bioactive-substance-supply performances of the BAL system. A suitable hemodialyzer for the BAL system is available even at present using an engineering design. The remaining problems to be overcome before realizing clinical use of the BAL system are to increase the rate constant of the first order reaction of the BAL for ammonia metabolism and to develop a new method of blood access that can be used safely with long term reliability at a high blood flow rate (ca. 556 ml/min).

Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.

Vascular endothelial growth factor (VEGF) expression regulates angiogenesis accompanying tumor growth in a peritoneal disseminated tumor model.

Quantitative analysis of the process of tumor angiogenesis was performed in a new animal model of tumor microcirculation, in which colon carcinoma cells were inoculated into the peritoneal cavity of rats. Time-dependent changes in the microvascular architecture of mesenteric microvessels of tumor-bearing rats were visualized using an intravital microscope. Simultaneously, the expression of vascular endothelial growth factor (VEGF) by the tumor cells and VEGF secretion into ascites were analyzed. The results showed that VEGF increases microvascular permeability and stimulates the growth of microvessels into the tumor and that the spatial and temporal concentration of VEGF is strongly correlated. Such a correlation was stronger in the early angiogenic stages of tumor growth than in the subsequently occurring multiple metastatic stage, when VEGF was still observed at a high level in tumor surroundings. Thus, VEGF is suggested to be primarily involved in the pathophysiological control of angiogenesis accompanying tumor progression.

Somatosensory nociceptive mechanical stimulation modulates systemic and mesenteric microvascular hemodynamics in anesthetized rats.

The effects of somatosensory nociceptive pinch stimulation of the hindpaw on mesenteric microvascular hemodynamics and systemic circulatory parameters were investigated in anesthetized rats using an intravital microscope-television system. Blood flow velocity in the terminal (18-40 microm in diameter) or precapillary (10-20 microm in diameter) arterioles of the mesentery was monitored by the dual sensor method developed by the authors. In the proximal terminal arterioles, blood flow velocity decreased substantially along with arteriolar constriction induced by pinching of the hindpaw for 30 s. In the distal terminal arterioles and precapillary arterioles, blood flow velocity increased after pinching. In the proximal terminal arterioles, the decrease of velocity in response to reflex vasoconstriction was abolished by intravenous injection of an alpha-blocker (phentolamine, 10 mg/kg). The increase in mesenteric precapillary arteriolar blood flow velocity (43+/-9%, p < 0.01) associated with the increase in mean arterial pressure (MAP) (22+/-1%, p < 0.01) was observed within a few seconds after the onset of the stimulation, and then the response in blood flow velocity returned to the baseline ahead of MAP response recovery after the end of the stimulus. These responses were diminished by alpha-adrenergic receptor blockade. The heart rate (HR) increase (4+/-1%, p < 0.01) induced by pinching was abolished by beta-adrenergicreceptor blockade (propranolol, 3 mg/kg, i.v.). There was a strong correlation between the increase in MAP and the decrease in renal blood flow measured by laser Doppler flowmeter (r = 0.87-0.98). Pinch stimulation of the rat hindpaw evoked changes in mesenteric arteriolar blood flow velocity that were mediated via the somato-sympathetic reflex vasoconstriction and the pressor response.

Effects of intra-arterial administration of prostaglandin E1 on rat cremaster muscle microcirculation.

In plastic and reconstructive surgery, postoperative drug therapy with prostaglandin E1 (PGE1) has been widely used to prevent flap necrosis and to extend flap surviving area. In the present study, we assessed the effects of intra-arterial administration of PGE1 on microcirculation. The left cremaster muscle of male Wistar rats were used to measure microcirculatory hemodynamic parameters. The values of internal vessel diameter and erythrocyte velocity were measured by using the confocal laser-scanning microscope (CLSM) system and fluorescent dyes. The blood flow rate was calculated from measured internal vessel diameter and erythrocyte velocity. Internal vessel diameter and flowing erythrocytes were satisfactorily visualized by using the CLSM system. The blood flow rate of arteriole, venule and capillary were dose dependently increased by the intra-arterial administration of PGE1 from the minimum dose of 0.01 ng/kg/min. It is suggested that the intra-arterial administration of PGE1 is a more effective way of postoperative drug therapy than intravenous injection for flap surgery.

Insulin increases blood flow rate in the microvasculature of cremaster muscle of the anesthetized rats.

The hemodynamic actions of insulin in skeletal muscle microvasculature are not yet well elucidated. In the present study, we investigated the effects of systemic insulin injection on arteriole and capillary diameter and blood flow rate in rat cremaster muscle, using intravital real-time confocal laser-scanning microscope system in combination with selective fluorescent labeling. Subcutaneous insulin injecbon (1 U/kg) significantly increased serum insulin levels at 15 minutes as compared with saline injection. At 15 and 30 minutes after insulin injection, blood glucose levels were significantly lower compared to saline injected controls. Arteriolar diameter was significantly increased at 15 and 30 minutes by insulin. Arteriolar erythrocyte flow velocity was significantly increased at 15 and 30 minutes. In addition, capillary erythrocyte flow velocity was increased at 15 and 30 minutes. These results demonstrated that calculated blood flow rates in capillary and arteriole increased after insulin injection.

The effects of 30% and 60% xenon inhalation on pial vessel diameter and intracranial pressure in rabbits.

UNLABELLED: Xenon may increase cerebral blood flow and intracranial pressure (ICP). To evaluate the effects of xenon on brain circulation, we measured pial vessel diameter changes, CO(2) reactivity, and ICP during xenon inhalation in rabbits. Minimum alveolar anesthetic concentration (MAC) for xenon was established in rabbits (n = 6). By using a cranial window model, pial vessel diameters were measured at 30% and 60% xenon inhalation and in time control groups (n = 15). ICP, mean arterial blood pressure, and heart rate were recorded during 30% and 60% xenon inhalation (n = 5). Pial vessel diameters were measured during hypocapnia and hypercapnia conditions in 60% Xenon and Control groups (n = 14). MAC for xenon was 85%. Xenon (0.35 and 0.7 MAC) dilated the arterioles (10% and 18%, respectively) and venules (2% and 4%, respectively) (P < 0.05). Dilation of arterioles was more prominent than that of venules. ICP, mean arterial blood pressure, and heart rate did not change during xenon inhalation. No difference in CO(2) reactivity was observed between Xenon and Control groups (P = 0.79). Sixty percent xenon (0.7 MAC) dilated brain vessels, but venule changes were small. Xenon did not increase ICP and preserved CO(2) reactivity of the brain vessels. IMPLICATIONS: Xenon might increase cerebral blood flow; however, 0.7 minimum alveolar anesthetic concentration xenon preserved both low intracranial pressure and CO(2) reactivity of the cerebral vessels in the normal rabbit.

Novel cell immobilization method utilizing centrifugal force to achieve high-density hepatocyte culture in porous scaffold.

Cell seeding is one of the key procedures in the construction of tissue-engineered organs. In our previous efforts to create a bioartificial liver, high-density cultures of hepatocytes (>1 x 10(7) cells/1 cm(3)-substrate) and long-term maintenance of metabolic function were achieved with a packed-bed reactor utilizing porous poly(vinyl formal) (PVF) resin as a scaffold. However, a low seeding efficiency of about 30% remains a major obstacle to the scaleup of the reactor. In the present study, a new cell seeding method, centrifugal cell immobilization (CCI), which is based on alternating centrifugation and resuspension, was used to achieve high-density seeding and improve the seeding efficiency. Using the CCI method, the maximum density of the immobilized hepatocytes reached 3.8 x 10(7) cells/1 cm(3)-PVF, and the seeding efficiency was improved to about 43% after a relatively short immobilization process (about 15 min). Moreover, further improvement of the seeding efficiency was obtained by serial immobilization procedures. Thus, we concluded that this method is useful and effective for seeding cells into 3-dimensional scaffolds.

Improvement of metabolic performance of cultured hepatocytes by high oxygen tension in the atmosphere.

Maintaining metabolic functions of cultured hepatocytes at higher levels is an essential requirement for the development of a bioartificial liver. We investigated the effect of oxygen tension (10--40%) of the medium on immobilization efficiency and metabolic functions of cultured hepatocytes obtained from a rat for up to 4 days. Immobilization efficiencies of cultures in 10% oxygen showed a significantly lower value from those for the other conditions. The ammonium metabolic rate and the albumin secretion rate were significantly improved with an increase of dissolved oxygen tension for up to 2 days. These values remained similar in the later stage of the culture. The urea secretion rate showed similar values in all conditions. In conclusion, higher oxygen tension improved immobilization efficiency and metabolic functions of cultured rat hepatocytes in the earlier stage of culture for up to 2 days.

Effect of shear stress on platelet adhesion to expanded polytetrafluoroethylene, a silicone sheet, and an endothelial cell monolayer.

We visualized in real-time platelets adhering to the surface of three representative biomaterials, by using an apparatus consisting of a modified cone and plate rheometer combined with an upright epifluorescence microscope under two shear flows (0.1 and 5.0 dyne/cm2). The materials were expanded polytetrafluoroethylene (ePTFE), silicone sheet, and a monolayer of bovine endothelial cells (ECs) formed on glass, all of which are opaque materials used for artificial blood vessels and medical devices. According to quantitative analysis, the monolayer of ECs formed on glass had better blood compatibility than did either the ePTFE or the silicone sheet under shear flow conditions. Under a shear flow condition of 0.1 dyne/cm2, platelet adhesion was silicone sheet > ePTFE. In contrast, under a shear flow condition of 5.0 dyne/cm2, ePTFE > silicone sheet. These results indicate that the intensity of shear stress could modify the order of hemocompatibility of the materials. Therefore, direct observation of platelet adhesion under shear flow conditions is indispensable for testing and screening biomaterials and for providing a precise quantitative evaluation of platelet adhesion.

New type of matrix support for bone marrow cell cultures: in vitro culture and in vivo transplantation experiments.

A new type of bone marrow cell culture system was developed by using a highly porous substrate matrix, i.e., porous polyvinyl formal (PVF) resin. Murine bone marrow (BM) cells were cultured without the use of exogenous growth factors in a three-dimensional matrix support made of collagen coated porous PVF resin. To examine the optimal conditions for highest stromal cell density, short-term and long-term in vitro culture experiments using PVF were performed. In the short-term culture experiments, it was found that cubes of PVF (10 x 10 x 2 mm and 130 microm in pore size) coated with type I collagen with a seeding density of 2x10(7) BM cells offered the most appropriate culture conditions. In the long-term cultures, BM cells in PVF maintained their viability for up to 6 weeks. In another series of re-inoculation experiments, freshly isolated BM cells were inoculated onto the already developed stromal layer. In this study, a higher cell density of the stromal layer was obtained in the PVF culture compared with those in the control dish culture. Based upon the results of in vitro experiments, in vivo transplantation studies were also performed. Histologic examinations of the subcutaneously transplanted PVF with stroma revealed host derived hematopoiesis inside the PVF matrix. Moreover, survival of approximately 15% of the transplanted BM cells that were cultured in PVF were confirmed in X-ray irradiated recipients. From these results, it is suggested that PVF resin is a promising three-dimensional substrate for BM cell culture and that it can maintain hematopoietic stem cells or progenitor cells after transplantation.

[Coronary arterial revascularization in dialysis patients].

It is recently reported that among chronic renal dialysis patients myocardial infarction accounts 7.4% of all the cause of death in Japan. In order to improve the mortality of dialysis patients the treatment of ischemic heart disease (IHD) is a major problem. We review previous reports and discuss about the treatment of IHD in dialysis patients. The collected data from the previous reports written about CABG in dialysis patients shows that the mortality of CABG is 7.8% (30/387), the probability of perioperative myocardial infarction (PMI) 8.6% (21/243), brain infarction 1.6% (4/243) and major infection 5.8% (14/243). 3-year and 5-year mortality rate after CABG is reported to be 69-80% and 48-56%, respectively. The result of CABG is excellent and shows that this procedure improves the mortality of dialysis patients with IHD. The procedural mortality of PTCA is 5.6% (8/143), the probability of PMI is 7% (10/143). Many reports shows the high probability of the recurrence of chest pain (60-86%). This procedure when performed in dialysis patients is incomplete because of the calcification of the coronary artery. Even among the dialysis patients it is preferable to perform CABG positively.

Relationships between age of onset, antisocial history and general psychopathological traits in Japanese alcoholics.

This study investigated the relationships of age of onset, antisocial history and general psychopathological traits measured by the Minnesota Multiphasic Personality Inventory (MMPI) in Japanese alcoholics (n = 84). A 2 (earlier vs later onset) x 2 (antisocial vs non-antisocial) multivariate analysis of covariance showed that age of onset had a significant correlation with some subscales of the MMPI such as L (lie), Sc (schizophrenia), and Si (social introversion), whereas history of antisocial behavior had no significant correlation with any MMPI clinical subscales. This result indicated that age of onset was a more significant variable than was antisocial history with regard to the current general psychopathological traits on MMPI in Japanese alcoholics.