The effect of magnetic field during freezing and thawing of rat bone marrow-derived mesenchymal stem cells.

Previous studies showed that a programmed freezer with magnetic field can maintain a high survival rate of mesenchymal stem cells (MSCs). The purpose of this study was to evaluate the influences of magnetic field during freezing and thawing on the survival of MSCs isolated from rat bone marrow. The cells were frozen by a normal programmed freezer or a programmed freezer with magnetic field (CAS-LAB1) and cryopreserved for 7 days at -150 °C. Then, the cells were thawed in the presence or absence of magnetic field. Immediately after thawing, the number of surviving or viable cells was counted. The cell proliferation was examined after 1-week culture. Cryopreserved MSCs which were frozen by a normal freezer or a CAS freezer were transplanted into bone defects artificially made in calvaria of 4-week-old rats. Non-cryopreserved MSCs were used as a control. The rats were sacrificed at 8, 16, or 24 weeks after transplantation and the bone regeneration area was measured. Proliferation rates of MSCs after 1 week were significantly higher in the CAS-freezing-thawing group than in the CAS-freezing group. The extent of new bone formation in the CAS-freezing-thawing group tended to be larger than in CAS-freezing group 24 weeks after transplantation. These results suggest that a magnetic field enhances cell survival during thawing as well as freezing.

Cranial suture-like gap and bone regeneration after transplantation of cryopreserved MSCs by use of a programmed freezer with magnetic field in rats.

Mesenchymal stem cells (MSCs) can be used for regeneration of various organs and tissues. A previous study revealed that cryopreserved MSCs, which were frozen by a programmed freezer with a magnetic field (Cells Alive System: CAS) and cryopreserved for 7 days in a -150°C deep freezer, can maintain high survival and proliferation rates while retaining both adipogenic and osteogenic differentiation abilities. The purpose of this study was to examine MSC viability and tissue regenerative ability after long-term cryopreservation using a CAS freezer. MSCs were isolated from rat femora bone marrow and cryopreserved in a -150°C deep freezer (CAS group) or directly cryopreserved in a deep freezer (Direct group). After 3 years, the cells were thawed and the number of viable cells was counted. Cell proliferation was also examined after 14 days in culture. For histological examination, forty 4-week-old Fischer 344 male rats received bone and sagittal suture defects with a diameter of 6.0mm, and MSCs (CAS or Direct group) cryopreserved for 1 year were grafted with membranes. Non-cryopreserved MSCs (Control group) were transplanted to an additional twenty rats. The rats were sacrificed at 4, 8, 16, and 24 weeks after surgery. The parietal bones, including the sagittal suture, were observed under a light microscope and the extent of bone regeneration was measured. Our results indicate that MSCs survival and proliferation rates were significantly higher in the CAS group than in the Direct group. In the Control and CAS groups, a large amount of new bone formation and a suture-like gap was identified 24 weeks after transplantation, whereas only a small amount of new bone formation was observed in the Direct group. These results suggest that the CAS freezer is amenable to long-term cryopreservation of MSCs, which can be applied to the regeneration of various tissues, including bone tissue with suture-like gap formation.

Cryopreservation of osteoblasts by use of a programmed freezer with a magnetic field.

In order to determine a suitable condition for osteoblasts cryopreservation, murine osteoblasts were freezed by programmed freezer with a magnetic field (CAS freezer). After 7 days cryopreservation at -150°, the number of survival cells immediately after thawing and the growth rate of cultured cells for 48 hours were examined. Gene and protein expression of alkaline phosphatase (ALP), osteopontin (OPN) and bone sialoprotein (BSP) were compared between cryopreserved and non-cryopreserved groups. As a result, a plunging temperature of -30°, a hold-time at -5° for 15 minutes and a 0.1 mT of magnetic field led to the largest survival and growth rate. Moreover, there was no significant difference in ALP, OPN and BSP mRNA and protein expression between cryopreserved and control groups. From these results, it was suggested that the CAS freezer is available for osteoblast cryopreservation and bone tissue banking can be established in the future.

Characterization of amino-acid transport systems in guinea-pig intestinal brush-border membrane.

The amino-acid transport systems have been characterized in brush-border membrane vesicles prepared from guinea-pig small intestine. Uptake of all amino acids tested was measured at the initial velocity for 5 s. L-Proline, alpha-(methylamino)isobutyrate, glycine, L-alanine and L-methionine were transported dependent solely on an Na+ gradient from the outside to the inside of the vesicles, and L-cysteine, L-phenylalanine and L-leucine were transported dependent largely on the Na+ gradient with a small fraction of Na+-independent transport. The transport of L-aspartic acid and L-lysine was independent of the Na+ gradient and L-lysine transport was somewhat inhibited by the presence of cations, including Na+, K+ and Li+. A cross-inhibition study of the uptake of these amino acids in the brush border of guinea-pig intestine revealed the presence of at least three Na+-dependent and three Na+-independent carrier-mediated systems. One Na+-dependent system interacted mainly with imino acid. Another Na+-dependent system interacted with neutral amino acids, while a third system was selective for glycine. One Na+-independent system is for acidic amino acids, another is responsible for neutral amino acids and a third for cationic amino acids. These transport systems of amino acids in guinea-pig small intestine are compared with those in rabbit and mouse intestine.

Degradation of vitronectin by matrix metalloproteinases-1, -2, -3, -7 and -9.

The susceptibility of vitronectin (Vn) purified from human plasma to digestion by matrix metalloproteinases (MMPs) was examined. MMP-2, -3, -7 and -9 except for MMP-1 degraded Vn into multiple fragments. MMP-7 showed the highest activity to the substrate among these MMPs, digesting 8-, 30- and 44-fold more preferentially than MMP-2, -3, and -9, respectively. These data suggest that MMP-2, -3, -7 and -9 may be responsible for the pathological degradation and/or normal turnover of Vn.

Increased expression of type VI collagen genes in drug-induced gingival enlargement.

Fibrotic gingival enlargements induced by phenytoin or nifedipine were examined with special reference to type VI collagen expression. Immunolocalization studies showed abnormal accumulation of type VI collagen around the collagen fiber bundles in the fibrotic gingival enlargements. Examination of total RNA extracted from fibroblasts and tissues of enlarged gingivae demonstrated increased type VI collagen steady-state mRNA levels. These results suggest that excessive deposition of type VI collagen in drug-induced gingival enlargement is attributed to increased expression of the collagen genes.

Identification of creatine as a cofactor of thiamin-diphosphate kinase.

Thiamin-diphosphate (TDP) kinase which catalyzes thiamin triphosphate formation from TDP requires a low-molecular-mass cofactor in addition to ATP and Mg2+. The cofactor was isolated in a crystalline form from pig skeletal muscle and identified as creatine by proton NMR, mass spectrometry, infrared spectrometry and elemental analysis. The isolated cofactor and authentic creatine supported the same activity of partially purified TDP kinase at identical molar concentrations. Neither creatine phosphate nor creatinine showed activity as a cofactor. This is the first report showing evidence of the existence of a creatine-dependent enzyme.

Flow velocity of central retinal artery and retrobulbar vessels during cardiovascular operations.

OBJECTIVE: Both blood flow monitoring and pressure monitoring are necessary to avoid inadequate cerebral perfusion during cardiovascular operations. Inasmuch as transcranial Doppler ultrasonography does not provide a consistently good signal, especially during cardiopulmonary bypass, we examined the blood flow through the central retinal artery, which has proved to reflect an obstruction of the carotid artery. METHOD: Twenty-eight consecutive cases were examined with a 5 or 7.5 MHz conventional echocardiographic probe. Correlation between the maximal velocity at the central retinal artery and systolic blood pressure was examined. The blood flow of the central retinal artery and retrobulbar vessels was examined during selective or retrograde cerebral perfusion or intraaortic balloon pumping. RESULTS: Blood flow could be clearly visualized but disappeared below a certain pressure in every case. With data from 478 measuring points, systolic blood pressure correlated with maximal velocity (r = 0.6902, p < 0.0001). The blood pressure-axis intercept, known as "critical closing pressure," was 35.8 +/- 14.8 mm Hg, varying among individuals and bilateral eyes. Pulsatility index increased after cardiopulmonary bypass (1.095 +/- 0.245 to 1.525 +/- 0.268, p < 0.0001). Patency of the circle of Willis was confirmed by the blood flow during anastomosis of the ipsilateral artery. During retrograde cerebral perfusion, blood flow was detectable at the retrobulbar vessels. During intraaortic balloon pumping, the central retinal artery flow was augmented on inflation of the balloon. CONCLUSION: Orbital vessel monitoring provides the critical closing pressure of the central retinal artery and confirms patency of the circle of Willis. The eye can be "an acoustic window" into intracranial blood flow during cardiovascular surgery.

T-cell large granular lymphocytic leukemia occurring after autologous peripheral blood stem cell transplantation.

A 61-year-old man with angioimmunoblastic lymphoma in first complete remission underwent autologous peripheral blood stem cell transplantation. At 1 month post transplant, asymptomatic large granular lymphocytosis developed. The surface marker profile of the cells was CD3+CD8+CD56-CD57+. The disease course was chronic and indolent. The patient remains in complete remission from angioimmunoblastic lymphoma more than 6 months post transplant with persistent large granular lymphocytosis (lymphocyte count, 5-15 x 10(9)/l). Although post transplantation T-cell lymphoproliferative disorders have mostly occurred in allogeneic transplantation recipients and presented as aggressive lymphomas/leukemias, we suggest that chronic indolent T-cell large granular lymphocytic leukemia can occur after autologous stem cell transplantation.

Characterization of topographic EEG changes when smoking a cigarette.

The acute effects of cigarette smoking on the human electroencephalogram (EEG) were investigated by the topographic mapping technique. Twenty-six subjects participated in this study, which involved sham smoking and real smoking of preferred cigarettes. Effects of smoking were analyzed by statistical and multivariate analysis. Analysis of variance and t-test results showed a significant decrease in the theta and alpha 1 bands but a significant increase in the alpha 2, beta 1, and beta 2 bands. Factor analysis and cluster analysis showed that there were two or three independent regions on the scalp that indicate the effects of smoking on topographic EEG. A hypothesis was formed that smoking has different effects on human EEG profiles for different brain regions and that there are individual variations in the EEG responses to smoking.

Effect of calcium deficiency on the content of nonreducible crosslinks in rat mandibular bone.

The effect of calcium deficiency on the content of the nonreducible cross-linking amino acid, pyridinoline, in rat mandibular bone was studied. An apparent decrease in serum and bone calcium levels was observed beginning at four weeks after initiation of a calcium-deficient diet. The pyridinoline content in the bone of calcium-deficient animals began to increase at four weeks and thereafter increased markedly as compared to that of controls. Collagen content increased significantly six weeks after calcium deficiency. These results suggest that calcium deficiency inhibits mineralization and promotes the formation of pyridinoline in rat mandibular bone.

Selective intercostal arterial perfusion during thoracoabdominal aortic aneurysm surgery.

BACKGROUND: This clinical study evaluated changes in motor evoked potentials (MEP) elicited by direct cerebral cortical stimulation and evoked spinal cord potentials (ESCPs) elicited by direct spinal cord stimulation during selective intercostal arterial perfusion for thoracoabdominal aortic aneurysm (TAAA) repair. We also determined the efficacy of this perfusion method for prevention of paraplegia. METHODS: Two kinds of ESCPs and MEPs were monitored during the prosthetic replacement step for TAAA surgeries. We performed selective intercostal arterial perfusion from the T7 intercostal artery to the L1 intercostal artery through a small piece of Dacron graft while monitoring spinal cord potentials in five cases of TAAA. RESULTS: The MEP amplitude decreased after clamping the aorta but quickly recovered after selective perfusion of intercostal arteries. Other spinal cord potentials did not change during the reconstruction of intercostal arteries. Postoperative paraplegia or parapalesis did not occur in any of the patients. CONCLUSIONS: Monitoring of MEPs during selective intercostal arterial perfusion was a useful adjunct to prevent postoperative paraplegia in TAAA surgery.

Retained intracardiac air in open heart operations examined by transesophageal echocardiography.

Using transesophageal echocardiography during open heart operations, we found another form of retained intracardiac air, "pooled air," in addition to the form of "bubbles" that had been reported by other authors. The pooled air was detected in all of 13 patients (100%); it was located at the right upper pulmonary vein in 13 (100%), left ventricular apex in 9 (69.2%), left atrium in 8 (61.5%), right coronary sinus of Valsalva in 8 (61.5%), left atrial appendage in 4 (30.8%), and left upper pulmonary vein in 3 (23.1%). The pooled air was found also in the pulmonary artery in 6 of 8 patients (75.0%) in whom the pulmonary artery was clearly visualized. In 1 patient, 5 mL of air was aspirated from the left ventricular apex, followed by a reduced size of the air on the transesophageal echocardiographic image. Because intracardiac air rapidly changes its locations and appearances, continuous monitoring is important, especially at weaning from bypass. The long-axis view of the heart is useful not only for detecting and locating the air, but also for guiding and evaluating the procedures to remove air.

Surgical repair of Wolff-Parkinson-White syndrome complicated with myocardial bridging.

Myocardial bridging causes myocardial ischemia during supraventricular tachycardia. We present a case of Wolff-Parkinson-White syndrome combined with myocardial bridging. The patient complained of angina pectoris during paroxysmal supraventricular tachycardia because of severe constriction of the left anterior descending coronary artery during systole. A myocardial scintigram revealed myocardial ischemia in the anteroseptal wall during paroxysmal supraventricular tachycardia. Myotomy to prevent myocardial bridging and interruption of the accessory conduction pathway was successfully accomplished in a one-stage operation.

Simple left atrial procedure for chronic atrial fibrillation associated with mitral valve disease.

BACKGROUND: A computerized 48-channel mapping system was used to investigate the characteristics of an atrial epicardial electrogram during chronic atrial fibrillation (AF) in patients with solitary mitral valve disease. We have devised a simple left atrial procedure to eliminate the chronic AF during a mitral valve operation. METHODS: Using this mapping system, we performed intraoperative atrial mapping in 11 patients with chronic AF associated with mitral valve disease. The AF duration ranged from 0.4 to 15 years (mean, 8.0 +/- 4.5 years). A simple surgical ablation of the AF on the left atrium only was performed during the mitral valve operations. RESULTS: The mean AF cycle length of the atria ranged from 129 to 169 milliseconds in the right atrium and from 114 to 139 milliseconds in the left atrium. The mean AF cycle length of the left atrium was shorter than that of the right atrium. Regular and repetitive activation was found in the left atria of 7 of 11 patients. The AF disappeared in all patients immediately after the operation, and 10 of these patients continued to have a sinus rhythm postoperatively (AF-free rate, 91%). CONCLUSIONS: Computerized intraoperative mapping revealed a shorter mean AF cycle length in the left atrium. A simple left atrial procedure was effective in eliminating chronic AF associated with solitary mitral valve disease.

Pooled air in open heart operations examined by transesophageal echocardiography.

BACKGROUND: The clinical significance of pooled air detected by transesophageal echocardiography during open heart operations is not clear. METHODS: Thirty-eight consecutive patients undergoing an open heart operation or an operation on the ascending aorta were divided into two groups on the basis of the absence (group 1, n = 14) or presence (group 2, n = 24) of pooled air. They were examined for intramyocardial echo contrast, ST segment elevation, conduction disturbances, and regional wall motion abnormalities. RESULTS: Echo contrast was found in no patient in group 1 and 66.7% of group 2 patients (p < 0.001). New regional wall motion abnormalities were detected in no patient in group 1 versus 33.3% of group 2 patients (p < 0.05), and ST segment elevation was seen in 33.3% of group 2 patients versus no group 1 patients (p < 0.05). Intramyocardial cardial echo contrast was newly detected after the appearance of pooled air more frequently in patients with ST segment elevation (p < 0.001). Atrioventricular block and sinus arrest appeared in 3 patients and 2 patients, respectively. Postoperative regional wall motion abnormalities were found in 25.0% of patients and were not closely related to intraoperative echo contrast findings. CONCLUSIONS: Pooled air, which is often detected in open heart operation by means of transesophageal echocardiography, is related to several cardiac events, including ST segment elevation, conduction disturbances, and regional wall motion abnormalities, although most of these are transient.

Clinical implication of orbital ultrasound monitoring during selective cerebral perfusion.

BACKGROUND: We evaluated clinical relevance of orbital ultrasound (OUS) monitoring to neurological events in aortic surgery associated with selective cerebral perfusion (SCP). METHODS: In 24 consecutive cases, blood flow was monitored at central retinal artery (CRA) and retrobulbar vessels. The threshold perfusion pressure for detecting CRA flow in the color Doppler mode (BPt) was determined in individual eyes. RESULTS: The BPt ranged from 25 to 71 mm Hg. Events (infarction, anisocoria, delirium) occurred in 8 cases. Infarction occurred in all 3 cases when retrobulbar flow was severely impaired for 40 minutes or longer, while none of the remaining 21 cases had infarction (p = 0.0005). Among the latter cases, perfusion pressure was below BPt for longer than 100 minutes in all 5 cases with events, and in 5 of 16 cases without events (p = 0.0124). No significant difference was found in age, duration of cardiopulmonary bypass, SCP, and circulatory arrest, and duration of blood pressure below 50 mm Hg. CONCLUSIONS: Sustained hypoperfusion detected with OUS monitoring is related to an occurrence of neurological events.

Use of the monoclonal antibody M30 for detecting HSG cell apoptosis.

An immunocytochemical method using a monoclonal antibody (MoAb), M30, which reacts with the product resulting from the cleavage of cytokeratin 18 by activated caspase, was applied to detect the apoptosis of human salivary gland tumor (HSG) cells induced by epigallocatechin gallate (EGCG), gallic acid (GA) and sodium ascorbate (SA). EGCG, GA and SA dose-dependently induced HSG cell death. Immunoreactive products were significantly observed in the cytoplasm of HSG cells after treatment with all these compounds. The reactions occurred with lower concentrations of these agents and after shorter treatment times, in comparison with DNA fragmentation detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. These results suggest that immunocytochemical staining with the MoAb M30 may be useful for detecting the apoptosis-inducing activities of various chemical compounds.

Immunohistochemical detection of cytokeratin 18 and its neo-epitope in human salivary glands and pleomorphic adenomas.

An immunohistochemical method using a monoclonal antibody M30 (MAb M30), which reacts with the product released by the cleavage of cytokeratin 18 (CK18) by activated caspase, was used to investigate the extent of apoptosis in human salivary glands and pleomorphic adenomas. The distribution of CK18 in the salivary glands and adenomas was also determined and compared with that of the product detected by MAb M30. CK18 was detected in the cytoplasm of serous acinar and ductal cells in normal human salivary glands. In pleomorphic adenomas, CK18 was observed mainly in the tumor cells of duct-like structures, but not in those of myxomatous or chondroid tissues. Positive MAb M30 reaction products were found in the cytoplasm of acinar cells in the restricted lobules of normal salivary glands and in the luminal contents of duct-like structures in pleomorphic adenomas. These results suggest that a mechanism which suppresses apoptosis may be linked to the growth of human pleomorphic adenomas.

Comparison studies of passive and active shunting during cross-clamping of the descending thoracic aorta.

The effects of passive (no pump) and active (using centrifugal pump) shunting on hemodynamics and metabolisms during cross-clamping of the descending thoracic aorta were compared in mongrel dogs. In Group I (control), the aorta was cross-clamped for 2 hours without shunting; in Group II (passive), a temporary shunt was placed between the left common carotid and left femoral arteries during cross-clamping; and in Group III (active), left heart by-pass, matching flow to that in group II, was performed. Hemodynamic and metabolic parameters were recorded prior to cross-clamping and following every 30 min for 4 hours. All animals in Group I suffered from hemodynamic instability, metabolic abnormal and neurologic injury and died within 12 hours. Hemodynamic changes were unstable in Group III than II, and three dogs in Group III, but none in Group II, suffered from neurologic injury. Metabolic changes between Group II and III were not significant. We conclude that passive shunting is superior to active shunting for aortic by-pass.