Cloning of regions required for contact hemolysis and entry into LLC-MK2 cells from Shigella sonnei form I plasmid: virF is a positive regulator gene for these phenotypes.

Two distinct regions required for both contact hemolysis and entry into LLC-MK2 cells were cloned into Escherichia coli from the Shigella sonnei form I plasmid, pSS120. The first region was cloned into an E. coli HB101 strain containing noninvasive Tn1 insertion mutants of the form I plasmid, and expression of ipa (invasion plasmid antigen) gene products was restored. The plasmid carrying the first region was then transformed into E. coli lacking the form I plasmid, and additional DNA fragments from the form I plasmid were cloned into the same recipient on compatible vectors. Five of these double transformants were found to be positive for contact hemolysis activity. Restriction analysis of these five clones indicated that the previously reported ipa locus and the invA locus were present on the second plasmid region. Only the strains carrying both of these regions were active in contact hemolysis and cell invasion assays. Several proteins, including the a, b, c, and d proteins encoded by the ipa genes, were detected in the double transformants by Western blot (immunoblot) analysis with serum of a monkey convalescing from shigellosis. A positive regulator was suggested to exist in the first region, since the amounts of most of these proteins were simultaneously increased in the presence of this region. Subcloning and nucleotide sequencing indicated that this positive regulator gene was virF. Product analysis of the virF gene with minicells showed that two peptides (30 and 21 kilodaltons) were synthesized and that at least the 30-kilodalton protein was essential for regulation of the ipa genes.     

Successful catheter ablation for incessant ventricular tachycardia in a patient with hypertrophic cardiomyopathy.

A 35-year-old man was referred to Nihon University Hospital because of repetitive ventricular tachycardia (VT) at 180-200 beats/min. QRS morphology of the VT was right bundle branch block with a northwest axis. Transthoracic echocardiography showed hypertrophic cardiomyopathy. Coronary angiography was normal and left ventriculography showed neither obstruction in the left ventricle (LV) nor any pressure gradients within the LV or between the LV and aorta. Hemodynamic deterioration occurred during VT. Intracardiac mapping showed that the VT originated from the posteroseptal portion of the LV near the apex and Purkinje potentials that preceded the onset of the QRS complex by 58-70 ms were documented. Radiofrequency ablation at these sites terminated the VT, which has not recurred for 25 months.     

Cancer detection using a PET tracer, 11C-glycylsarcosine, targeted to H+/peptide transporter.

H+/peptide transporter, PEPT1, is functionally expressed in some human cancer cell lines and might be a candidate molecular target for detection of cancers in vivo using PET. The aim of the present study was to establish a novel tumor-imaging technology using a PET tracer targeted to H+/peptide transporter(s). We also compared the tracer with 18F-FDG, focusing on the specificity of their accumulation between tumor and inflammatory tissues. METHODS: A dipeptide PET tracer, 11C-glycylsarcosine (11C-Gly-Sar), was injected intravenously into athymic mice transplanted with human pancreatic, prostate, and gastric cancer cells. The distribution patterns of 11C-Gly-Sar and 18F-FDG in the tumor-bearing mice, and in mice with inflammatory tissue, were assessed by imaging with a positron planar imaging system (PPIS). Tissue distributions of tracer radioactivity were also measured. The expression levels of PEPT1 and PEPT2 (PEPTs) proteins in tumor xenografts and inflammatory tissue were examined by immunohistochemical analysis. The messenger RNA expression levels of PEPTs in 58 available cancer cell lines were quantified by means of real-time polymerase chain reaction. RESULTS: All 3 tumor xenografts were well visualized with the PPIS after injection of 11C-Gly-Sar. Expression of PEPTs in those xenografts was confirmed by immunohistochemical analysis. Tumor-to-blood concentration ratios of 11C-Gly-Sar increased in a time-dependent manner and were much higher than unity. Most of the radioactivity found in the tumor tissue was recovered as the intact tracer. These results indicated that 11C-Gly-Sar was taken up by the PEPTs in tumor xenografts. It is noteworthy that 11C-Gly-Sar was minimally present in inflammatory tissues that expressed no PEPT1 or PEPT2 protein, whereas 18F-FDG was highly accumulated, with the values of the selectivity index being >25.1 and 0.72 for 11C-Gly-Sar and 18F-FDG, respectively. The mRNAs of PEPT1 and PEPT2 were expressed in 27.6% and 93.1%, respectively, of the cancer cell lines examined in the present study. CONCLUSION: The present study indicates that 11C-Gly-Sar is a promising tumor-imaging agent and is superior to 18F-FDG for distinguishing between tumors and inflammatory tissue. Because PEPTs were ubiquitously expressed in various types of tumor cells examined, 11C-Gly-Sar could be useful for the detection of many types of cancers.     

Risk factors for invasive aspergillosis in living donor liver transplant recipients.

Invasive aspergillosis (IA) is a severe complication of liver transplantation. Risk factors for IA after deceased donor liver transplantation (DDLT) have been presented in several reports, but are not well established for living donor liver transplant recipients. Here, a retrospective case-control study was performed. Five cases with IA were investigated after living donor liver transplantation (LDLT) between January 1999 and December 2002 at Kyoto University Hospital. For comparison, living donor liver transplant recipients without IA were taken as controls. These patients had undergone LDLT 1 month before or after each IA case and had the same survival times as the latter. We evaluated the clinical and laboratory findings for both groups up until their demise. Patients with IA after LDLT had a very poor prognosis. By univariate analysis, risk factors for IA were preoperative intensive care unit stay (P = 0.02) and preoperative steroid administration (P = 0.02). Preoperative steroid administration for fulminant hepatitis possibly predisposed to the development of IA after LDLT.     

Initial steroid bolus injection promotes vigorous CD8+ alloreactive responses toward early graft acceptance immediately after liver transplantation in humans.

We have found that steroid bolus withdrawal prior to graft reperfusion increased the incidence of acute cellular rejection (ACR). This study aims to clarify how initial steroid bolus (ISB) injection at reperfusion influences the kinetics of CD8(+) alloreactive immune responses immediately after living donor liver transplantation (LDLT). A total of 49 hepatitis C virus (HCV)-infected recipients were classified into 3 groups according to hierarchical clustering by preoperative CD8(+)CD45 isoforms. The naive T cell proportion was considerably higher in Group I than in Groups II and III, whereas Group II recipients had the highest effector memory (EM) T cells and Group III the highest effector T cells. The frequency of ACR was significantly higher in recipients without ISB than in those with ISB. In particular, the ACR rates were the highest in Group II without ISB. Following ISB, the proportion of effector T cells was promptly upregulated within 6 hours after graft reperfusion, simultaneously with the upregulation of CD27(-)CD28(-) subsets, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha and perforin expression, which significantly correlated with increasing interleukin (IL)-12 receptor beta 1 cells. These were then downregulated to below preoperative levels by tacrolimus (Tac) administered at 24 hours. These changes did not occur in the absence of ISB. In Group II without ISB, the downregulation of IL-12Rbeta1(+) cells was the greatest, consistent with the highest rates of ACR and mortality (60%). In conclusion, ISB must be done in place, especially in Group II with preexisting high EM T cells, to enable the development of early allograft acceptance.     

Myocardial 11C-diacylglycerol accumulation and left ventricular remodeling in patients after myocardial infarction.

Left ventricular (LV) remodeling after myocardial infarction (MI) is a maladaptive process that increases the risk of heart failure and death. The myocardial phosphoinositide cycle, which is located downstream from several neurohumoral factors, plays a crucial role in LV remodeling. Our animal studies demonstrated that 1-[1-11C]butyryl-2-palmitoyl-rac-glycerol (11C-DAG) can be used to visualize regions with an activated phosphoinositide cycle. Therefore, we examined whether myocardial 11C-DAG accumulation assessed by PET is relevant to LV enlargement and systolic dysfunction in post-MI patients. METHODS: We performed PET with 11C-DAG in 13 post-anteroseptal MI patients and 4 healthy volunteers. We placed regions of interest on the noninfarcted myocardium and calculated the myocardium-to-left atrial (LA) chamber ratio of 11C-DAG accumulation. RESULTS: The myocardium-to-LA chamber ratio of 11C-DAG was significantly higher in the post-MI patients (mean +/- SD, 1.73 +/- 0.35) compared with that of the healthy volunteers (mean +/- SD, 1.25 +/- 0.13; P < 0.05). In the post-MI patients, the myocardium-to-LA chamber ratio of (11)C-DAG was significantly correlated with the LV end-diastolic volume index (r = 0.79, P < 0.01) and the plasma concentration of brain natriuretic peptide (r = 0.85, P < 0.001) and negatively correlated with the LV ejection fraction (r = -0.69, P < 0.01). CONCLUSION: These findings suggest that the myocardial 11C-DAG accumulation assessed by PET is relevant to LV enlargement, LV systolic dysfunction, and humoral activation in post-MI patients. This new imaging strategy based on intracellular signaling may contribute to the assessment and treatment of post-MI patients.     

Distinct osteogenic mechanisms of bones of distinct origins

Mammalian bones have three distinct origins (paraxial mesoderm, lateral plate mesoderm, and neural crest) and undergo two different modes of formation (intra-membranous and endochondral). Bones derived from the paraxial mesoderm and lateral plate mesoderm mainly form through the endochondral process. During this process, hypertrophic chondrocytes play a vital role in inducing both osteogenesis and angiogenesis. One of the essential osteogenic factors secreted from hypertrophic chondrocytes is Indian hedgehog (Ihh). In contrast, bones derived from the neural crest mainly form through the intramembranous pro-cess and do not require Ihh. Thus, depending on their origin, bones have distinct signaling properties, which need to be considered in the research and application of bone biology.Presented at the 18th Annual Research Meeting of the Japanese Orthopaedic Association, Kitakyushu, Japan, October 17, 2003     

Ruptured pediatric posterior cerebral artery aneurysm 9 years after the onset of Kawasaki disease: a case report

CASE REPORT: A 12-year-old boy who had a history of Kawasaki disease 9 years ago experienced a subarachnoid hemorrhage by ruptured right posterior cerebral artery aneurysm. On day 1 operation, as the aneurysm was very fragile and bled easily, two intraoperative ruptures, including a very premature rupture, were encountered. As a result, a left hemiparesis especially severe in the left hand was caused by the right anterior thalamic infarction due to the occlusion of a thalamo-perforating artery arising near the neck of the aneurysm. DISCUSSION: The histopathological examination of the intraoperative excised aneurysmal dome disclosed the thickening of the endothelial inner due to the endothelial hypertrophy and the invasion of inflammatory cells. This finding of the aneurysm was partially mimicking the finding of the coronary artery of the patients with Kawasaki disease. The combination of cerebral aneurysm and Kawasaki disease has never been reported until now, and the etiology of the aneurysm of this patient is unclear.