Randomized, double-blind, placebo-controlled clinical trial of hepatocyte growth factor plasmid for critical limb ischemia.

Hepatocyte growth factor (HGF) is a potent angiogenic factor. The efficacy and safety of intramuscular injection of a naked plasmid encoding human HGF gene (beperminogene perplasmid, Collategene) was investigated in patients with critical limb ischemia (CLI) in a multicenter, randomized, double-blind, placebo-controlled trial. The randomization ratio for plasmid to placebo was 2:1. Injection sites were selected in each patient limb based on angiographic findings. Placebo or plasmid was injected on days 0 and 28. Evaluation of efficacy was carried out after 12 weeks. The primary end point was the improvement of rest pain in patients without ulcers (Rutherford 4) or the reduction of ulcer size in patients with ulcer(s) (Rutherford 5). Secondary end points were ankle-brachial pressure index, amputation, and quality of life (QOL). Forty-four patients were treated, and we performed interim analysis of efficacy in 40 patients. The overall improvement rate of the primary end point was 70.4% (19/27) in HGF group and 30.8% (4/13) in placebo group, showing a significant difference (P=0.014). In Rutherford 5 patients, HGF achieved a significantly higher improvement rate (100% [11/11]) than placebo (40% [2/5]; P=0.018). HGF plasmid also improved QOL. There were no major safety problems. HGF gene therapy is safe and effective for CLI.

The education system to master endovascular aortic repair in Japan - the Japanese Committee for Stentgraft Management.

OBJECTIVE: The Japanese Committee for Stentgraft Management (JACSM) was established with the aim of ensuring the safe and proper reach of commercial stent grafts following their regulatory approval. This study examines the validity of the practice standards developed by JACSM. METHODS: JACSM comprises 10 associations related to endovascular treatment. Based on the practice standards developed by JACSM, the status of practising institutions, practising surgeons, supervising surgeons and the results of follow-up surveys were analysed. RESULTS: In the 2.5 years following the establishment of JACSM, 298 institutions have fulfilled the practice standards. The number of practising surgeons reached 493, and the number of supervising surgeons reached 177. There were 3089 registered cases up to June 2009. The present study analysed 1570 cases registered in the 2 years from July 2006 to June 2008. The hospital mortality rate was low (0.4%) in the follow-up surveys. CONCLUSIONS: Early results following the introduction of stent grafts were generally good. The procedure spread safely without the learning curve seen in the initial stages following introduction of new medical materials, indicating that the practice standards were appropriate.

Long-term outcome of 6-month maintenance chemotherapy for acute lymphoblastic leukemia in children.

In the treatment of childhood acute lymphoblastic leukemia (ALL), excess shortening of maintenance therapy resulted in high relapse rate, as shown by our previous trial, TCCSG L92-13, in which maintenance therapy was terminated at 1 year from initiation of treatment. In this study, we aimed to confirm the long-term outcome of L92-13, and to identify who can or cannot be cured by shorter duration of maintenance therapy. To obtain sentinel cytogenetics information that had been missed before, we performed genetic analysis with genomic microarray and target intron-capture sequencing from diagnostic bone marrow smear. Disease-free survival (DFS) at 10 years from the end of therapy was 66.0±2.8%. Females (n=138) had better DFS (74.6±3.7%) than males (n=142, 57.5±4.2%, P=0.002). Patients with TCF3-PBX1 (n=11) and ETV6-RUNX1 (n=16) had excellent DFS (90.9±8.7% and 93.8±6.1%, respectively), whereas high hyperdiploidy (n=23) was the most unfavorable subgroup, with 56.6±10.3% of DFS. Short duration of therapy can cure more than half of pediatric ALL, especially females, TCF3-PBX1 and ETV6-RUNX1. Our retrospective observations suggest a gender/karyotype inhomogeneity on the impact of brief therapy.

Effect of low-density lipoprotein apheresis on patients with peripheral arterial disease. Peripheral Arterial Disease LDL Apheresis Multicenter Study (P-LAS).

AIM: The effectiveness of low-density lipoprotein (LDL) apheresis for patients with peripheral arterial disease (PAD) was investigated to confirm a hypothesis based on subjective evidence that the amelioration of blood rheology would be the most contributing factor for improvement in clinical symptoms. Evaluation of the severity of intermittent claudication is difficult because of the lack of an accurate parameter to assess muscle ischemia during exercise, thus we objectively evaluated by non-invasive near-infrared spectroscopy (NIRS) on a treadmill in this study. METHODS: Thirty-one patients with PAD were evaluated for hemostatic function and physiological parameters such as ankle-brachial pressure index (ABI), maximum tolerated walking distance (MTWD) and recovery time (RT) or recovery ability index (RAI) on NIRS. Laboratory tests included plasma assays of total cholesterol, LDL-cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, and fibrinogen. The change in red-cell filtration rate was evaluated for the improvement of microcirculation. Statistical analysis was performed using the paired Student's t-test with Bonferroni's correction. RESULTS: A significant improvement in ABI and MTWD was observed after average 9.6+/-0.8 sessions of LDL apheresis treatment and the amelioration of microcirculation in ischemic muscle was objectively evaluated as significant improvement in RAI on NIRS. Rest pain was improved in all 5 patients with Fontaine's classification III or IV. A severe ulcer refractory to usual medications was dramatically diminished in the area by 10 sessions of LDL apheresis and fully healed 5 months after the final LDL apheresis treatment followed by medication. No angiographical change was observed in the arterial occlusive lesions in any patients. CONCLUSIONS: The effectiveness of LDL apheresis on the improvement in physiological parameters such as ABI, MTWD and clinical symptoms in patients with PAD was confirmed. The severity of intermittent claudication was objectively evaluated using non-invasive NIRS. The RT or RAI was useful parameter to evaluate the improvement in the ischemic symptoms of the extremities.

Characterization of pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia with kinase fusions in Japan.

Recent studies revealed that a substantial proportion of patients with high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harbor fusions involving tyrosine kinase and cytokine receptors, such as ABL1, PDGFRB, JAK2 and CRLF2, which are targeted by tyrosine kinase inhibitors (TKIs). In the present study, transcriptome analysis or multiplex reverse transcriptase-PCR analysis of 373 BCP-ALL patients without recurrent genetic abnormalities identified 29 patients with kinase fusions. Clinically, male predominance (male/female: 22/7), older age at onset (mean age at onset: 8.8 years) and a high white blood cell count at diagnosis (mean: 94 200/µl) reflected the predominance of National Cancer Institute high-risk (NCI-HR) patients (NCI-standard risk/HR: 8/21). Genetic analysis identified three patients with ABL1 rearrangements, eight with PDGFRB rearrangements, two with JAK2 rearrangements, three with IgH-EPOR and one with NCOR1-LYN. Of the 14 patients with CRLF2 rearrangements, two harbored IgH-EPOR and PDGFRB rearrangements. IKZF1 deletion was present in 16 of the 22 patients. The 5-year event-free and overall survival rates were 48.6±9.7% and 73.5±8.6%, respectively. The outcome was not satisfactory without sophisticated minimal residual disease-based stratification. Furthermore, the efficacy of TKIs combined with conventional chemotherapy without allogeneic hematopoietic stem cell transplantation in this cohort should be determined.

A Drosophila homolog of human proto-oncogene ret transiently expressed in embryonic neuronal precursor cells including neuroblasts and CNS cells.

We have identified a Drosophila gene encoding a putative receptor tyrosine kinase by screening a genomic DNA library with a DNA probe for a Drosophila homolog of fibroblast growth factor receptors. The newly isolated gene codes for a transmembrane protein most similar in sequence to a mammalian proto-oncogene ret; thus, the gene was termed Dret. Dret mRNA is transcribed in very small amounts in the embryonic, larval, and pupal stages. Whole mount in situ hybridization experiments revealed that the mRNA is transiently expressed in neuroblasts in early embryos. In late embryos, Dret mRNA was detected in subpopulations of differentiating CNS and PNS cells. In addition, Dret expression was affected in neurogenic mutants. These results suggest that Dret can be considered as a functional homolog of mammalian ret and should play important roles in neurogenesis.

[Endovascular stent grafting for thoracic aortic disease in elderly patients].

Despite advances in operative technique and management having improved the clinical outcomes of conventional open surgical replacement for thoracic aortic aneurysms, it remains an invasive procedure especially for aged patients. Over the past 10 years, minimally invasive endovascular surgery using a stent graft, has made significant advances for the treatment of aneurysms. For 10 years from 1995, 476 patients of thoracic aortic aneurysms were treated with the endovascular technique using the stent graft in our hospital. Exclusion of the aneurysms without endoleak were achieved within 2 weeks postoperatively in over 95%. Also in elderly patients (46/476), same good results came out. Endovascular stent grafting shows potential as a safe and useful treatment for thoracic aortic aneurysms, but further investigation should attempt to determine its efficacy over a longer postoperative period.

[Autologous fibrin sealant preparing system in coronary artery bypass grafting].

Autologous fibrin sealant (AFS) which is not based on the conventional method of co-administering fibrinogen, thrombin and aprotinin was prepared by Vivostat system, and was used in coronary artery bypass grafting (CABG). The purpose of this study was to investigate the safety and efficacy of the AFS prepared by the Vivostat system. In 6 of 68 cases of CABG, normal AFS was not prepared due to device failures. AFS was prepared and sprayed in 62 cases. There were the total of 230 anastomosis sprayed AFS and the bleeding could not seen in 225 anastomosis. Surgical hemostatic procedures (4 cases) were or other sealant usage (1 case) was performed 5 bleeding anastomosis sites. The rate of hemostasis at the anastomosis using AFS was 97.8%. This study was conducted in patients undergoing CABG. In this group of patients, a number of commercial available fibrin sealant products are routinely used. The usefulness of Vivostat as medical device to prepare and administer AFS was confirmed in this study.

Ischemic thresholds of cerebral protein synthesis and energy state following middle cerebral artery occlusion in rat.

The ischemic threshold of protein synthesis and energy state was determined 1, 6, and 12 h after middle cerebral artery (MCA) occlusion in rats. Local blood flow and amino acid incorporation were measured by double tracer autoradiography, and local ATP content by substrate-induced bioluminescence. The various images were evaluated at the striatal level in cerebral cortex by scanning with a microdensitometer with 75 microns resolution. Each 75 x 75 microns digitized image pixel was then converted into the appropriate units of either protein synthesis, ATP content, or blood flow. The ischemic threshold was defined as the flow rate at which 50% of pixels exhibited complete metabolic suppression. One hour after MCA occlusion, the threshold of protein synthesis was 55.3 +/- 12.0 ml 100 g-1 min-1 and that of energy failure was 18.5 +/- 9.8 ml 100 g-1 min-1. After 6 and 12 h of MCA occlusion, the threshold of protein synthesis did not change (52.0 +/- 9.6 and 56.0 +/- 6.5 ml 100 g-1 min-1, respectively) but the threshold of energy failure increased significantly at 12 h following MCA occlusion to 31.9 +/- 9.7 ml 100 g-1 min-1 (p less than 0.05 compared to 1 h ATP threshold value; all values are mean +/- SD). In focal cerebral ischemia, therefore, the threshold of energy failure gradually approached that of protein synthesis. Our results suggest that with increasing duration of ischemia, survival of brain tissue is determined by the high threshold of persisting inhibition of protein synthesis and not by the much lower one of acute energy failure.(ABSTRACT TRUNCATED AT 250 WORDS)

The gene encoding dipeptidyl aminopeptidase BI from Pseudomonas sp. WO24: cloning, sequencing and expression in Escherichia coli.

We have isolated the dipeptidyl aminopeptidase BI (DAP BI) gene from the plasmid library of Pseudomonas sp. WO24 chromosomal DNA by the enzymatic plate assay using a chromogenic substrate. The DAP BI gene, designated dap b1, was further subcloned and sequenced. Sequence analysis of an approx. 3-kb fragment revealed an open reading frame of 2169 nucleotides, which was assigned to the dap b1 gene by N-terminal and internal amino acid sequences. The predicted amino acid sequence of DAP BI containing a serine protease Gly-X-Ser-X-Gly consensus motif displays extensive homologies to the several proteases belonging to the prolyl oligopeptidase family, a novel serine protease family possessing the catalytic triad with a specific array of Ser, Asp and His in this order, which is the hallmark of the member of this family including DAP IV. The dap b1 gene was expressed in Escherichia coli and the expressed enzyme was purified about 230-fold with 2.6% recovery from the cell-free extracts. The enzymatic properties such as molecular mass, substrate specificity and effect of inhibitor were similar to the native enzyme from Pseudomonas sp. WO24.

Structure and expression of hedgehog, a Drosophila segment-polarity gene required for cell-cell communication.

The complete nucleotide sequence of the coding region of hedgehog (hh), a segment-polarity gene in Drosophila melanogaster, was determined. The gene was found to include three exons which would encode a 421- (or 471-) amino acid (aa) polypeptide with a long hydrophobic stretch. The hh mRNA was about 2.3 kb long and expressed throughout development. The hh expression in an embryo occurred in stripes, while that in imaginal discs occurred in the posterior compartment. As a whole, the spatial expression pattern of hh mRNA was very similar to that of engrailed (en), a homeobox gene required for the formation of the anterior-posterior compartment boundary. Unlike en, no hh expression was observed in the central nervous system.

Haemostatic clot formation at anastomosis of synthetic venous graft in defibrinogenated dogs: a scanning electron microscopic study.

A segment of the inferior vena cava was replaced by an expanded polytetrafluoroethylene graft in 13 dogs. Five of them served as a control group, while the other 8 were moderately or severely defibrinogenated with subcutaneous batroxobin. Plasma fibrinogen decreased to extremely low values throughout the experiment in the defibrinogenated dogs except in the moderately treated group in which it temporarily rose to 0.72-0.87 g/l on the first postoperative day. Scanning electron microscopic observations of the haemostatic clot formed at the anastomoses of the graft revealed no significant morphological differences in platelet adhesion and/or aggregation between the three groups. These findings confirmed that platelets play a key role in primary haemostasis during defibrinogenation. The fibrin network was slightly diminished and only short fibrin filaments could be seen in the moderately and severely defibrinogenated groups respectively. These differences in composition of the clots are discussed in relation to their haemostatic capacity.

Respiratory involvement and immune status in yusho patients.

Clinical and experimental studies on respiratory involvement and alterations in immune status were carried out. Respiratory distress occurring in these patients has improved gradually for 14 years but still remains. Copious expectoration at an early stage of the disease may be related to the fact that a number of discrete polychlorinated biphenyls (PCBs) are distributed throughout the lung parenchyma. For accumulation in the bronchial mucosa, structural requirements and specific dose dependence of PCBs have been clearly shown; however, pathological and physiological studies have indicated that respiratory involvement in yusho is mainly small airway disease that may be caused by involvement of cellular component (Clara cells) in bronchioles and/or associated infection. Respiratory distress is often exacerbated by viral or bacterial infection. Changes in the immune status in PCB and polychlorinated dibenzofuran (PCDF) poisoning are as follows: IgA and IgM in the serum are decreased at an early stage of the disease and then return to normal; suppression of cellular immunity was reported in Taiwanese patients and some may remain in the later stages of the disease, as shown in our patients. PCDFs now appear to be the main causal agents in yusho. Rats given PCDFs showed necrosis of the Clara cells in bronchioles and marked thymus atrophy, while few such changes were noted in rats given PCBs. Therefore, further examination is needed for the difference of the toxic effects between two compounds.

Preliminary report on prediction of spinal cord ischemia in endovascular stent graft repair of thoracic aortic aneurysm by retrievable stent graft.

OBJECTIVE: To predict spinal cord ischemia after endovascular stent graft repair of descending thoracic aortic aneurysms, temporary interruption of the intercostal arteries (including the aneurysm) was performed by placement of a novel retrievable stent graft (Retriever) in the aorta under evoked spinal cord potential monitoring. METHODS: From February 1995 to October 1997, endovascular stent graft repair of descending thoracic aortic aneurysms was performed in 49 patients after informed consent was obtained. In 16 patients with aneurysms located in the middle and distal segment of the descending aorta, the Retriever was placed temporarily before stent graft deployment. The Retriever consisted of two units of self-expanding zigzag stents connected in tandem with stainless steel struts. Each strut was collected in a bundle fixed to a pushing rod, and the stent framework was lined with an expanded polytetrafluoroethylene sheet. The Retriever was delivered beyond the aneurysm through a sheath and was retracted into the sheath 20 minutes later. A stent graft for permanent use was deployed in patients whose predeployment test results with the Retriever were favorable. Evoked spinal cord potential was monitored throughout placement of the Retriever and stent grafting until the next day. RESULTS: The Retriever was placed in 17 aneurysms in 16 patients. There were no changes in amplitude or latency of evoked spinal cord potential records obtained before or during Retriever placement. After withdrawal of the Retriever, all aneurysms were excluded from circulation immediately after permanent stent grafting. There were no changes in evoked spinal cord potential, nor were neurologic deficits seen after stent graft deployment in any patient. CONCLUSIONS: These results suggest that predeployment testing with the Retriever under evoked spinal cord potential monitoring is promising as a predictor of spinal cord ischemia in candidates for stent graft repair of thoracic aortic aneurysms.

Effect of flow competition on internal thoracic artery graft: postoperative velocimetric and angiographic study.

OBJECTIVES: To assess the effects of competitive blood flow on internal thoracic artery grafts, we investigated postoperative flow velocity characteristics and angiographic findings of the grafts with various grades of native coronary artery stenosis. METHODS: Fifty patients who had an internal thoracic artery graft to the left anterior descending artery underwent intravascular Doppler graft velocimetry during postoperative angiography. Patients were divided into 3 groups according to the grade of native coronary stenosis: group H (28 patients), 80% stenosis or greater; group M (16 patients), 60% to 79% stenosis; and group L (6 patients), 40% to 59% stenosis. Phasic flow velocity of the grafts was measured with an intravascular Doppler ultrasound-tipped guide wire during angiography. Graft flow volume was calculated from the diameter and the average peak velocity. RESULTS: Average peak velocity (group H, 27.1 +/- 8.6 cm/s; group M, 16.9 +/- 3.9 cm/s; group L, 7.2 +/- 3.7 cm/s), distal graft diameter (group H, 2.27 +/- 0.23 mm; group M, 2. 00 +/- 0.28 mm; group L, 1.07 +/- 0.27 mm), and calculated graft flow volume (group H, 33.1 +/- 12.0 mL/min; group M, 16.2 +/- 5.8 mL/min; group L, 2.3 +/- 2.0 mL/min) significantly differed among the 3 groups. Graft flow in diastole and systole also differed among the 3 groups. CONCLUSIONS: Competitive blood flow reduces internal thoracic artery graft flow and diameter according to the grade of the native coronary artery stenosis. These data suggest that grafting the internal thoracic artery to the coronary artery with stenosis of a low grade can cause graft atrophy and failure.

Identification of a novel Drosophila gene encoding a Cdc2-related protein kinase.

We have identified a novel gene encoding a putative protein kinase from a Drosophila genomic library. The gene, about 2 kbp in length, consists of four exons and codes for a protein of 349 amino acid residues. The deduced sequence shows significant similarity to various kinases, especially to a subgroup of Ser/Thr kinases related to Cdc2 kinase; thus, the gene was termed Dcdrk (Drosophila cdc2-related kinase gene). Among the kinases examined, mammalian galactosyltransferase-associated 58 kDa protein kinase showed the highest homology (about 50% identity in the kinase domain) to Dcdrk kinase. Northern blot analysis revealed that the Dcdrk mRNA is expressed throughout development in nearly constant amounts. Moreover, a whole mount in situ hybridization experiment showed that the Dcdrk mRNA is ubiquitously distributed in almost all embryonic cells and tissues, suggesting a universal function of Dcdrk, possibly in cell cycle regulation.

Two-stage resuscitation of the cat brain after prolonged cardiac arrest.

Following prolonged cardiac arrest, reperfusion of the brain is endangered by the low blood perfusion pressure during the early resuscitation phase. In order to avoid low perfusion brain injury, a two-stage resuscitation protocol was applied to cats submitted to 30 min potassium chloride induced cardiac arrest: first, the heart was resuscitated, followed--after stabilisation of blood pressure--by recirculation of the brain. During cardiac resuscitation the brain was disconnected from the general circulation by inflating a pneumatic cuff around the neck. The results were compared with the outcome of conventional one-stage resuscitation following 15 min cardiac arrest. Cardiac resuscitation was successful in 5 out of 8 animals with 15 min and in 6 out of 13 animals with 30 min cardiac arrest. In successfully resuscitated animals of both groups, brain energy metabolism recovered to normal within 3 h although two-stage resuscitation increased brain ischemia time to 37-61 min. Two-stage resuscitation, in consequence, is a promising approach for revival of the brain after prolonged cardiac arrest.

Role of endothelin-1/endothelin receptor system in endotoxic shock rats.

Endothelin (ET)-1, a potent vasoconstrictor peptide derived from the endothelium, is markedly increased in endotoxic shock, although the pathophysiological role of ET-1 under septic conditions remains obscure. To delineate the role of ET-1 and its receptor subtype in endotoxic shock, we here attempted to determine the changes of circulating levels of ET-1 and its biosynthetic intermediate big ET-1 in endotoxic shock rats, to evaluate the gene expression of ET-1 as well as the ET-1 receptor subtypes (ETA and ETB) in the heart, lung and liver, and to study the effects of ET receptor antagonists on systemic arterial blood pressure, heart rate and survival rate. Administration of bacterial lipopolysaccharide (LPS) caused profound hypotension, increased heart rate and death, and these effects were blocked by a nonselective ETA/ETB receptor antagonist (TAK044), but not by an ETA selective antagonist (BQ123). Administration of exogenous ET-1 caused a profound pressor response in control rats, but not in the LPS-pretreated rats. Injection of LPS caused marked elevation of plasma levels of both ET-1 and big ET-1, which were not affected by treatment with either ET receptor antagonist. Administration of LPS caused up-regulation of ET-1 and ETB receptor mRNA in the heart, whereas ETA receptor mRNA was markedly down-regulated in the heart, lung and liver. These data suggest differential gene regulation of ET-1 and its receptor subtypes in various organs from endotoxic shock rats, and that nonselective ETA/ETB receptor antagonist, but not ETA receptor antagonist, ameliorates endotoxin-induced hypotension and death.

Coronary revascularization with arterial conduits collateral to the lower limb.

A 62-year-old man with left main coronary artery disease had coronary artery bypass grafting. Angiography disclosed total occlusion of the left common iliac artery. The left internal thoracic artery and the left inferior epigastric artery were well developed as collateral pathways to the left external iliac artery. With concomitant femoro-femoral crossover bypass, these two large arterial conduits were harvested and grafted to the coronary artery.

An unusual six-co-ordinate platinum(II) complex containing a neutral I2 ligand.

The present paper deals with a rare platinum(II) complex containing the kappa-I2 ligand, which is an unusual example of a six-co-ordinated octahedral platinum(II) complex.