anti-tumor effect of AlkB homolog 3 knockdown in hormone- independent prostate cancer cells.

Castrate resistant prostate cancer (CRPC) is a disease that is resistant to both hormone therapy and chemotherapy. At present, no curative therapy for CRPC has been established. Therefore, it is necessary to determine a novel molecular target for the development of therapeutic agents. We previously reported that AlkB homolog 3 (ALKBH3) is highly expressed in prostate cancer but not in benign prostatic hyperplasia or in normal prostate epithelium and that the expression levels of ALKBH3 protein are significantly correlated with the hormone-independent state of prostate cancer. Moreover, ALKBH3 regulates the invasion of prostate cancer cells via the regulation of matrix metalloproteinase 9. Here, we show that ALKBH3 gene silencing markedly induces apoptosis in hormone-independent prostate cancer cell line DU145 but not in the normal prostate epithelial cell line PNT2. Moreover, the in vivo tumorigenicity of DU145 cells was significantly inhibited by the administration of ALKBH3 siRNA. Furthermore, the anchorage-independent growth of DU145 cells was inhibited by ALKBH3 knockdown and promoted by ALKBH3 overexpression, significantly. ALKBH3 shRNA-expressing prostate cancer cells formed significantly smaller tumors than those of control shRNA transfectants in an in vivo xenograft model. These findings suggest that ALKBH3 is a promising target molecule for the development of CRPC therapeutic agents.

NEMO/IKKgamma regulates an early NF-kappaB-independent cell-death checkpoint during TNF signaling.

TNF receptor 1 (TNFR1) ligation can result in cell survival or cell death. What determines which of the two opposing responses is triggered is not fully understood. The current model suggests that it is the activation of the NF-kappaB pathway and its induction of prosurvival genes, or the lack thereof, which determines the outcome. NF-kappaB essential modifier (NEMO)/IkappaB kinase-gamma (IKKgamma)-deficient cells are highly sensitive to apoptosis, and as NEMO is essential for NF-kappaB activation, it has been assumed that this is due to the lack of NF-kappaB. This study demonstrates that this assumption was incorrect and that NEMO has another antiapoptotic function that is independent of its role in the NF-kappaB pathway. NEMO prevents receptor interacting protein-1 (RIP1) from engaging CASPASE-8 before NF-kappaB-mediated induction of antiapoptotic genes. Without NEMO, RIP1 associates with CASPASE-8 resulting in rapid tumor necrosis factor (TNF)-induced apoptosis. These results suggest that there are two cell-death checkpoints following TNF stimulation: an early transcription-independent checkpoint whereby NEMO restrains RIP1 from activating the caspase cascade, followed by a later checkpoint dependent on NF-kappaB-mediated transcription of prosurvival genes.

In vivo segmental motion of the cervical spine in rheumatoid arthritis patients with atlantoaxial subluxation.

OBJECTIVE: The dynamic mechanism underlying cervical spine involvement in rheumatoid arthritis (RA) remains unidentified. The purpose of the current study was to determine the in vivo cervical segmental motion in RA patients with atlantoaxial subluxation (AAS) using a patient-based three-dimensional magnetic resonance imaging (MRI) computer model. METHODS: Healthy volunteers and RA patients with AAS (all females, n=10) underwent MRI examination of the cervical spine. Each vertebral body from the occipital bone (Oc) to the first thoracic vertebra (T1) was reconstructed from slices of T2-weighted sagittal MR images in the neutral, flexion, and extension positions. Using volume merge methods, each reconstructed vertebral body was virtually rotated and translated. Rotational segmental and translational segmental motions were obtained in three major planes. RESULTS: Overall, the axial translational motions in the RA group were lower than those in the healthy volunteers; however the axial translational motion at only C1-C2 during flexion was at the same level as that in the healthy volunteers and was greater on the bottom side than that at other intervertebral levels. The frontal rotational motions at C1-C2 during extension were greater in the RA patients than those in the healthy volunteers (p<0.05). CONCLUSION: The atlantoaxial joints in the RA patients with AAS showed great frontal rotational motion during extension and great axial translation on the bottom side during flexion. The current noninvasive MRI-based method could be useful in evaluating the 3-D dynamic mechanism underlying cervical involvement in RA in vivo.

Risk factors for de novo acute cardiac events in patients initiating hemodialysis with no previous cardiac symptom.

An initial major adverse cardiac event (MACE) is an important predictor of future cardiovascular events in patients with chronic kidney disease (CKD). We sought to identify factors influencing occurrence of initial MACE in new maintenance hemodialysis patients without previous cardiac symptoms during the predialysis phase of CKD. Among 112 participating patients with no predialysis cardiac history, 57 underwent coronary angiography, whereas the other 55 underwent stress thallium-201 single-photon emission computed tomography within 1 month of beginning hemodialysis to detect asymptomatic coronary artery disease (CAD). In subsequent follow-up for a median of 24 months, subjects experiencing an initial MACE were compared with those who did not have such an event based on several clinical parameters at the end of predialysis phase. Asymptomatic CAD was present in 47 patients (42%), who had a higher cumulative MACE rate, than subjects without CAD (49 vs 3%, P<0.001). Multivariate Cox's regression analysis showed that three variables independently predicted initial MACE: asymptomatic CAD (hazard ratio or HR, 611.31; 95% confidence interval or CI, 14.07-26549.23; P<0.001), diabetes (HR, 20.41; 95% CI, 2.07-200.00; P=0.010), and each 1 mg/l increment in C-reactive protein (CRP) (HR, 1.94; 95% CI, 1.27-2.94; P=0.002). In conclusion, detection of asymptomatic CAD, presence of diabetes, or elevated CRP at the end of the predialysis phase were significantly associated with occurrence of an initial MACE in CKD patients starting hemodialysis who had no CAD symptoms.

Diagnostic utility of waveform analysis of compound muscle action potentials for carpal tunnel syndrome.

PURPOSE: To determine the diagnostic utility of waveform analysis of compound muscle action potentials (CMAP) for carpal tunnel syndrome (CTS). METHODS: A total of 131 hands in 71 patients diagnosed with CTS (grouped according to severity) and 80 hands in 44 normal subjects were evaluated using nerve conduction test through the carpal tunnel combined with waveform analysis of CMAP. RESULTS: Compared to normal subjects, the sensory nerve conduction velocity and mean frequency of the CMAP waveform were significantly reduced in patients with CTS. Compared with distal motor latency and sensory nerve conduction velocity, the mean frequency of the CMAP decreased significantly with increasing clinical severity. CONCLUSION: This study suggests that waveform analysis of CMAP is of diagnostic value in CTS, and is also of value in objective evaluation of postoperative recovery of carpal median nerve dysfunction.

Central role of mitochondria and p53 in Fas-mediated apoptosis of rheumatoid synovial fibroblasts.

OBJECTIVE: Fas-mediated apoptosis is preferentially observed in synoviocytes of patients with rheumatoid arthritis (RA) and is associated with the pathophysiological process of RA. To clarify the molecular mechanisms of Fas-mediated apoptosis of RA synoviocytes, we investigated the role of the mitochondrial pathway and tumour suppressor p53 in this process. METHODS: Cultured synovial fibroblasts were prepared from RA patients. After treatment of RA synovial fibroblasts with anti-Fas monoclonal antibody, the expression levels of activated caspase-9 and -3, Bid cleavage, cytochrome c release and phosphorylation of p53 at Ser15 were assessed using immunoblot analysis. The mitochondrial membrane potential (DeltaPsim) was evaluated with a fluorescence-based detection assay. Apoptotic cells were determined by a DNA fragmentation assay in the presence or absence of caspase inhibitors. Expression of p53-regulated apoptosis-inducing protein 1 (p53AIP1) was measured by real-time PCR. RA synovial fibroblasts stably transfected with a dominant-negative (DN) p53 were prepared in order to investigate the role of p53 during Fas-induced apoptosis. RESULTS: Fas ligation induced Bid cleavage, loss of DeltaPsim, cytochrome c release to the cytosol and activation of caspase-9 and -3 in RA synovial fibroblasts. Treatment with a caspase-9-specific inhibitor almost completely inhibited Fas-mediated apoptosis. Moreover, p53 activation after Fas ligation was evidenced by its phosphorylation at Ser15 and up-regulation of the p53 target gene p53AIP1. Fas-mediated apoptosis was significantly suppressed by anti-sense p53 oligonucleotides and by p53DN. CONCLUSION: Our findings strongly suggest the involvement of mitochondria and p53 in Fas-mediated apoptosis of RA synovial fibroblasts.

Spinal reconstruction for symptomatic thoracic haemangioma using a titanium cage.

Most vertebral haemangiomas are asymptomatic. A case of spinal reconstruction for symptomatic extraosseous thoracic haemangioma using a titanium cage is reported. Radiographs of the T11 vertebra demonstrated characteristic vertical striations. Magnetic resonance imaging and computed tomography showed spinal cord compression by extraosseous tumour extension. Several tumour feeding vessels were shown by angiography. Through a transpedicular biopsy, a histological diagnosis of cavernous haemangioma was made. Embolisation of feeding vessels was performed using coils before surgery. Laminectomy and subtotal vertebrectomy were performed by a single posterior approach. Rigid stabilisation of the spine was achieved with pedicle screw systems and a cage filled with an autogenous bone graft. Five months postoperatively, stabilisation of the spine was established without loosening of the cage or pedicle screws. Clinical symptoms were improved.

Independent predictors of restenosis after percutaneous coronary revascularization in haemodialysis patients.

BACKGROUND: Percutaneous balloon angioplasty has become a well-established and routine procedure for coronary revascularization of haemodialysis patients with coronary artery disease. However, the incidence of restenosis after balloon angioplasty is significantly higher in haemodialysis patients than in the general population. We performed a retrospective study comparing balloon angioplasty with coronary stenting in haemodialysis patients. We evaluated the long-term clinical and angiographic outcome after successful percutaneous coronary revascularization in haemodialysis patients. METHODS: A total of 103 consecutive haemodialysis patients (123 lesions) underwent procedurally and clinically successful percutaneous revascularization. Patients were treated with three different strategies: (i) balloon angioplasty in 55 patients (69 lesions); (ii) coronary stenting with balloon angioplasty in 23 patients (25 lesions); and (iii) coronary stenting with rotational atherectomy in 25 patients (29 lesions) who had severely calcified stenotic coronaries. RESULTS: The rates of in-hospital mortality were similar in the three groups. The 1-year incidence of overall events and major adverse cardiac events (MACE) were significantly higher in the balloon group than in the stent with/without rotational atherectomy groups (75% vs 36 and 28%, P<0.01; 71% vs 32 and 28%, P<0.01). Use of coronary stenting (relative risk=0.006, P<0.001) and the presence of calcified coronary lesion (relative risk=68.2, P<0.001) were independent predictors of the 1-year MACE-free survival after percutaneous revascularization. The 3-year MACE-free survival rate was significantly lower in the balloon group than in the stent with/without rotational atherectomy groups (11% vs 33 and 47%, P<0.005 and P<0.001). CONCLUSIONS: This study shows that coronary stenting reduces the incidence of MACE in haemodialysis patients with/without calcified coronary lesions. Moreover, coronary stenting reduces the restenosis rate of both complex and restenotic lesions, and rotational atherectomy prior to coronary stenting reduces the restenosis rate of the severely calcified coronary lesions. These results suggest that coronary stenting with/without rotational atherectomy has led to an improved long-term outcome in the haemodialysis patients with coronary artery disease.

Haematopoietic progenitor cells from the common marmoset as targets of gene transduction by retroviral and adenoviral vectors.

To establish a new non-human primate model for human cytokine and gene therapy, we characterized lymphocytes and haematopoietic progenitor cells of the small New World monkey, the common marmoset. We first assessed the reactions of marmoset bone marrow (BM) and peripheral blood (PB) cells to mouse anti-human monoclonal antibodies (mAbs) for the purpose of isolating marmoset lymphocytes and haematopoietic progenitor cells. Both cell fractions stained with CD4 and CD8 mAbs were identified as lymphocytes by cell proliferation assay and morphological examination. Myeloid-specific mAbs such as CD14 and CD33 did not react with marmoset BM and PB cells. No available CD34 and c-kit mAbs could be used to purify the marmoset haematopoietic progenitor cells. Furthermore, we studied the in vitro transduction of the bacterial beta-galactosidase (LacZ) gene into CFU-GM derived from marmoset BM using retroviral and adenoviral vectors. The transduction efficiency was increased by using a mixed culture system consisting of marmoset BM stromal cells and retroviral producer cells. It was also possible to transduce LacZ gene into marmoset haematopoietic progenitor cells with adenoviral vectors as well as retroviral vectors. The percentage of adenovirally transduced LacZ-positive clusters was 15% at day 4 (multiplicity of infection=200), but only 1-2% at day 14. The differential use of viral vector systems is to be recommended in targeting different diseases. Our results suggested that marmoset BM progenitor cells were available to examine the transduction efficiency of various viral vectors in vitro.

Coronary artery disease as a definitive risk factor of short-term outcome after starting hemodialysis in diabetic renal failure patients.

Cardiovascular disease is a major cause of morbidity and mortality in patients with diabetes who are receiving dialysis. It is known that cardiac mortality is high in the first year of hemodialysis (HD). The purpose of this study was to clarify the prevalence of coronary artery disease (CAD) in patients with diabetes at the initiation of HD, and to investigate the relationship between short-term outcomes after starting hemodialysis and CAD. We performed coronary angiography (CAG), whether or not patient had angina, within one month of initiation of maintenance HD in 17 of 34 consecutive unselected diabetic patients. We studied the two-year survival rate of those with CAD. Eleven patients (65%) were classified CAD-positive. Nine (82%) of these 11 had multivessel disease. Clinical and hematologic factors did not differ significantly between the groups with and without CAD. During the two-year follow-up period, 28 (82%) of 34 patients survived. In patients with CAD the two-year survival rate was 60%. None of the patients without CAD died within 2 years after starting HD. These results suggest that the presence of CAD at the initiation of HD may play a critical role in short-term outcomes after starting HD. We believe that an evaluation of CAD should be performed at the initiation of HD.

Estimation of cervical cord dysfunction by somatosensory evoked potentials.

The purpose of this study was to examine the relationship of abnormal short-latency somatosensory evoked potentials (SSEPs) recorded by a noncephalic reference montage with clinical variables in cervical myelopathy patients and to reexamine the diagnostic utility of SSEPs in such patients. We studied cervical SSEPs elicited by stimulating the median and ulnar nerves in 87 patients. Our grade classification of spinal N13, which is based on the normal limits of latencies or amplitudes, corresponded well with the clinical variables and is of value when trying to localize the cervical lesion segmentally. The N9-P14 interpeak latency in response to ulnar nerve stimulation correlated well with lower extremity function (r = -0.440, P <0.0001). We suggest a combined assessment of N13 amplitude, and N9-N13 and N9-P14 interpeak latencies to estimate dorsal column and dorsal horn function separately in patients with cervical myelopathy.

Progress reports on immune gene therapy for stage IV renal cell cancer using lethally irradiated granulocyte-macrophage colony-stimulating factor-transduced autologous renal cancer cells.

There is no effective treatment for patients with stage IV renal cell cancer (RCC), although the introduction of new therapy is imminent. Cancer gene therapy is currently considered to be one of the most promising therapeutic modalities in the field of cancer treatment. Based on the results of animal studies, vaccination using autologous granulocyte-macrophage colony-stimulating factor-transduced renal cancer cells appears promising. Before initiating a clinical study using an ex vivo gene-transduced autologous cell vaccine-based immunogene therapy for RCC in Japan, in 1992 we initially planned a Japanese version of a clinical protocol in collaboration with a US group. In 1993, the original protocol was refined. We performed five preclinical qualification studies using RCC nephrectomy specimens from patients in 1997, and the results showed that preparation of RCC cells for autologous vaccines at the Clinical Cell Technology Facility, Research Hospital of the Institute of Medical Science, University of Tokyo, was feasible. Subsequently in August 1998, the Ministry of Health and Welfare and the Ministry of Education, Science, Culture, and Sport approved our clinical protocol. We have recruited two patients with stage IV RCC to our study so far. Here we report the background to the initiation of cancer gene therapy in Japan.

TCR-Vbeta repertoire analysis with RT-PCR was useful for the early detection of pulmonary relapsed T-cell lymphoma after autologous peripheral blood stem cell transplantation.

Pulmonary recurrence of malignant lymphoma is a rare event after stem cell transplantation. We report here a 45-year-old male who was successfully diagnosed with relapsed pulmonary T-cell lymphoma using an RT-PCR method. Clonal expansion of T cells expressing identical TCR V-D-J junction size (Vbeta5-Jbeta1.5) was demonstrated in lymphocyte groups obtained from both bronchoalveolar lavage fluid at relapse, and paraffin embedded lymph node samples resected when he was first diagnosed with angioimmunoblastic T-cell lymphoma. This method provided evidence to diagnose relapsed pulmonary angioimmunoblastic T-cell lymphoma in its early phase.

[Long-term outcome of coronary events in hemodialysis patients younger and older than 65 years of age].

BACKGROUND AND AIM: It has been reported that a coronary event is the leading cause of mortality in HD patients. The aim of this study was to examine and compare prospectively the effect of aging in relation to the in-hospital and the long-term outcome in HD patients with or without revascularization therapy who had experienced a coronary event. STUDY PATIENTS AND METHODS: Seventy consecutive HD patients with coronary events (9 AMI, 48 AP, and 13 CHF) were registered in this study and 69 patients underwent CAG. Patients were classified into elderly (> or = 65, n = 33) and younger (< 65, n = 37) groups based on their ages at the time of the events. Forty-six patients (21 vs 25) underwent initial coronary revascularization therapy. We followed 70 HD patients with coronary events for a mean period of 31 +/- 21 months (range: 1 day to 77 months). RESULTS: A level of 64% of the elderly group and 41% of the younger group experienced coronary events within the first year of HD. The diseased vessels (2.2 vs 1.9 per patient) and stenotic lesions (2.8 vs 2.5 pre patients) were not significantly different between the two groups. The 70-month survival rate was significantly lower (21% vs 65%, p = 0.0423) in the elderly group than in the younger group. The complicated rate of stroke after a major event was significantly higher (14 vs 4, p = 0.0025) in the elderly group than in the younger group. Moreover 21 elderly patients (11 cardiac death, 5 stroke, 4 cancer) and 9 younger patients (8 cardiac death, 1 stroke) died during the 70-month follow-up period. CONCLUSIONS: Coronary events were most frequent in the first year of HD. Long-term survival rate was significantly lower in elderly patients than in younger patients. Cardiac death was the most common cause of death in both groups regardless of performing coronary revascularization. Death due to stroke and cancer was also more common in elderly patients.

Vaccine effect of granulocyte-macrophage colony-stimulating factor or CD80 gene-transduced murine hematopoietic tumor cells and their cooperative enhancement of antitumor immunity.

To develop immunogene therapy targeting minimal residual hematopoietic tumor cells in patients, we transduced murine GM-CSF or CD80 gene into murine WEHI 3B myelomonocytic leukemia and EL-4 thymic lymphoma cells using retroviral vectors and evaluated their effects on inducing antitumor responses in syngeneic host mice. Subcutaneously injected GM-CSF- and CD80 gene-transduced WEHI 3B (GMCSF/WEHI/3.2 or CD80/WEHI/1.8, respectively) cells lost their original tumorigenicity in immunocompetent syngeneic mice. Results from tumor inoculation experiments using athymic nude mice suggested that the rejection of GMCSF/WEHI/3.2 in immunocompetent mice depended fully on T cells and that of CD80/WEHI 1.8 depended partly on T cells and partly on NK cells. In both WEHI 3B and EL-4 models, irradiated GM-CSF gene-transduced cells provided strong immuno-protection against wild-type cells, but irradiated CD80 gene-transduced cells did not. A remarkably high cooperative effect was obtained when irradiated GMCSF/EL-4 and CD80/EL-4 were inoculated together. These results suggested that the tumor vaccine effect is efficiently enhanced by GM-CSF gene transduction and CD80 gene transduction induces some protective antitumor immunity in co-operation with GM-CSF gene transduction.

Onset of coronary artery disease prior to initiation of haemodialysis in patients with end-stage renal disease.

To determine whether the onset of coronary artery disease may precede the initiation of dialysis in patients with end-stage renal disease, we performed coronary angiography within 1 month of initiation of maintenance haemodialysis in 24 patients (age range 42-78 years; mean 63.7 +/- 11). Coronary angiography was performed regardless of the absence or presence of angina. Fifteen patients had diabetic nephropathy, and nine had non-diabetic nephropathy. Significant coronary stenosis was defined as at least 75% narrowing of the reference segment. Fifteen patients (62.5%) with a total of 49 lesions were classified as the coronary artery disease present group. Eleven of those 15 (73.3%) had multivessel disease. The average number of stenotic lesions was 3.3 per patient. The most common patterns of stenosis were complex (23 lesions; 47%), and diffuse lesions over 20 mm long (14 lesions; 29%). None of the clinical or haematological factors evaluated differed significantly between the groups with and without coronary artery disease. The prevalence of coronary artery disease was 72.7% in the symptomatic patients and 53.8% in the asymptomatic patients. The diagnosis of coronary artery disease at the start of maintenance haemodialysis based only on chest symptoms and clinical factors proved to be difficult. Coronary angiography is thus essential for evaluating coronary artery disease in uraemic patients. Many patients with end-stage renal disease had coronary artery disease prior to the start of haemodialysis.

Epidural hematoma of the lumbar spine, simulating extruded lumbar disk herniation: clinical, discographic, and enhanced magnetic resonance imaging features. A case report.

STUDY DESIGN: This case report describes a patient with epidural hematoma of the lumbar spine and progressive intermittent claudication whose imaging findings were similar to those of a patient with extruded disk herniation. OBJECTIVES: To highlight the diagnosis and cause of the lumbar epidural hematoma. SUMMARY OF BACKGROUND DATA: The characteristics of imaging of lumbar epidural hematomas have been reported. To the authors' knowledge, a case of epidural hematoma with leakage of the contrast medium into the mass at discogram has never been reported. METHODS: A case of a lumbar epidural hematoma with 10-15 minutes of intermittent claudication was described. There was no history of major trauma or coagulation disorders. Diagnosis was made using magnetic resonance imaging, myelography, discography, and computed tomography. The magnetic resonance image demonstrated a relatively large, rounded mass posterior to the S1 vertebral body with L5-S1 disc protrusion. A gadolinium-enhanced magnetic resonance image showed a rim-enhancing lesion. A discogram revealed leakage of the contrast medium into the mass. The mass was diagnosed as extruded disk herniation, and surgery was performed. RESULTS: At surgery no evidence of obvious disk herniation was detected, but the encapsulated hematoma was found. CONCLUSIONS: A case of chronic lumbar epidural hematoma in which clinical and imaging findings were similar to those of an extruded lumbar disk herniation was reported. Clinical information and results of imaging studies should be analyzed carefully because the imaging findings of contained epidural hematomas are often quite specific to this condition and can be differentiated from those of extruded disc herniations. Management of chronic lumbar epidural hematoma may be altered by a specific diagnosis.

Further investigation of some inhibitors on myogenic differentiation: mechanism of inhibition with HMBA on quail myoblasts transformed with Rous sarcoma virus.

To investigate the mechanism of myogenic differentiation, we are using quail myoblast cells (QM cells) transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV), termed QM-RSV cells. At 35.5 degrees C, a permissive temperature for RSV, QM-RSV cells repeatedly proliferate without differentiation, but, at 41 degrees C, a nonpermissive temperature, myogenic differentiation proceeds. This temperature dependency of the differentiation is derived from protein kinase activity of pp60v-src, as tyrosine dephosphorylation is necessary for myogenic differentiation of QM-RSV cells. In this study, it was demonstrated that among four fusion inhibitors, aspirin, doxorubicin, HMBA and TPA, three of the inhibitors, except for TPA, inhibited myogenin gene expression. Moreover, HMBA inhibited myoblast fusion accompanying inhibition of tyrosine dephosphorylation of certain proteins, and recovered the tyrosine kinase activity of pp60v-src to a certain extent. To study the effect of HMBA on the intracellular localization of pp60v-src, detergent-soluble and detergent-resistant fractions were prepared with Triton X-100. As a result, it was shown that pp60v-src mainly exists in detergent-resistant fraction at 35.5 degrees C. While almost all of the pp60v-src at 41 degrees C exists in detergent-soluble fraction. HMBA treatment retained pp60v-src in detergent-resistant fraction even at 41 degrees C. These results suggest that HMBA inhibits myogenic differentiation of QM-RSV cells by affecting the regulation of pp60v-src.