Application of ultra-high voltage electron microscope tomography to 3D imaging of microtubules in neurites of cultured PC12 cells.

Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three-dimensional distribution patterns of various cellular structures including microtubules in neurites. Because the penetrating power of electrons depends on the section thickness and accelerating voltage, conventional TEM, having acceleration voltages up to 200 kV, is limited to sample thicknesses of 0.2 µm or less. In this paper, we show that the ultra-high voltage electron microscope (UHVEM), employing acceleration voltages of higher than 1000 kV (1 MV), allowed distinct reconstruction of the three-dimensional array of microtubules in a 0.7-µm-thick neurite section. The detailed structure of microtubules was more clearly reconstructed from a 0.7-µm-thick section at an accelerating voltage of 1 MV compared with a 1.0 µm section at 2 MV. Furthermore, the entire distribution of each microtubule in a neurite could be reconstructed from serial-section UHVEM tomography. Application of optimised UHVEM tomography will provide new insights, bridging the gap between the structure and function of widely-distributed cellular organelles such as microtubules for neurite outgrowth. LAY DESCRIPTION: An optimal 3D visualisation of microtubule cytoskeleton using ultra-high voltage electron microscopy tomography The ultra-high voltage electron microscope (UHVEM) is able to visualise a micrometre-thick specimen at nanoscale spatial resolution because of the high-energy electron beam penetrating such a specimen. In this study, we determined the optimal conditions necessary for microtubule cytoskeleton imaging within 0.7-µm-thick section using a combination with UHVEM and electron tomography method. Our approach provides excellent 3D information about the complex arrangement of the individual microtubule filaments that make up the microtubule network.

Estimation of whole-body radiation exposure from brachytherapy for oral cancer using a Monte Carlo simulation.

Early stage oral cancer can be cured with oral brachytherapy, but whole-body radiation exposure status has not been previously studied. Recently, the International Commission on Radiological Protection Committee (ICRP) recommended the use of ICRP phantoms to estimate radiation exposure from external and internal radiation sources. In this study, we used a Monte Carlo simulation with ICRP phantoms to estimate whole-body exposure from oral brachytherapy. We used a Particle and Heavy Ion Transport code System (PHITS) to model oral brachytherapy with 192Ir hairpins and 198Au grains and to perform a Monte Carlo simulation on the ICRP adult reference computational phantoms. To confirm the simulations, we also computed local dose distributions from these small sources, and compared them with the results from Oncentra manual Low Dose Rate Treatment Planning (mLDR) software which is used in day-to-day clinical practice. We successfully obtained data on absorbed dose for each organ in males and females. Sex-averaged equivalent doses were 0.547 and 0.710 Sv with 192Ir hairpins and 198Au grains, respectively. Simulation with PHITS was reliable when compared with an alternative computational technique using mLDR software. We concluded that the absorbed dose for each organ and whole-body exposure from oral brachytherapy can be estimated with Monte Carlo simulation using PHITS on ICRP reference phantoms. Effective doses for patients with oral cancer were obtained.

Radiation therapy for primary vaginal carcinoma.

Brachytherapy plays a significant role in the management of cervical cancer, but the clinical significance of brachytherapy in the management of vaginal cancer remains to be defined. Thus, a single institutional experience in the treatment of primary invasive vaginal carcinoma was reviewed to define the role of brachytherapy. We retrospectively reviewed the charts of 36 patients with primary vaginal carcinoma who received definitive radiotherapy between 1992 and 2010. The treatment modalities included high-dose-rate intracavitary brachytherapy alone (HDR-ICBT; two patients), external beam radiation therapy alone (EBRT; 14 patients), a combination of EBRT and HDR-ICBT (10 patients), or high-dose-rate interstitial brachytherapy (HDR-ISBT; 10 patients). The median follow-up was 35.2 months. The 2-year local control rate (LCR), disease-free survival (DFS), and overall survival (OS) were 68.8%, 55.3% and 73.9%, respectively. The 2-year LCR for Stage I, II, III and IV was 100%, 87.5%, 51.5% and 0%, respectively (P = 0.007). In subgroup analysis consisting only of T2-T3 disease, the use of HDR-ISBT showed marginal significance for favorable 5-year LCR (88.9% vs 46.9%, P = 0.064). One patient each developed Grade 2 proctitis, Grade 2 cystitis, and a vaginal ulcer. We conclude that brachytherapy can play a central role in radiation therapy for primary vaginal cancer. Combining EBRT and HDR-ISBT for T2-T3 disease resulted in good local control.

Disease control using low-dose-rate brachytherapy is unaffected by comorbid severity in oral cancer patients.

OBJECTIVE: The aim of this study was to evaluate the outcome and complications of low-dose-rate brachytherapy (LDR-BT) for oral cancer according to comorbidity. METHODS: The records of a total of 180 patients who received LDR-BT for T1-2N0M0 oral cancers between January 2005 and December 2007 were analysed. The comorbidities of the patients were retrospectively graded according to the Adult Comorbidity Evaluation-27, and the relationships between the comorbidity grades and survival, disease control and the incidence of complications were analysed. RESULTS: The 2 year overall survival rates of patients with no comorbidity, Grade 1, Grade 2 and Grade 3 comorbidity were 87%, 85%, 76% and 65%, respectively, and the reduction in the survival rate according to comorbid severity was significant in a univariate analysis (p = 0.032) but not in a multivariate analysis including other clinical factors. Cause-specific survival, locoregional control and local control were not related to the comorbidity grade, or any other clinical factors. Grade 2 or 3 complications developed in 27% of the patients. The incidence of complications was unrelated to the comorbidity grade. CONCLUSION: The disease control of oral cancer and the incidence of complications after LDR-BT were not related to comorbid severity. LDR-BT is a useful and safe treatment for patients regardless of the presence of severe comorbidity.

A novel approach to anticancer therapies: peroxisome proliferator activator-receptor-gamma as a new target therapy in the treatment of human urological cancer.

Peroxisome proliferator activator-receptor (PPAR)-gamma is a ligand-activated transcriptional factor belonging to a steroid receptor superfamily. PPAR-gamma plays a role in both adipocyte differentiation and carcinogenesis. Up-date, PPAR-gamma is expressed in various cancer tissues, and PPAR-gamma ligand induces growth arrest of these cancer cells. In this study, we examined the expression of PPAR-gamma in human urological cancer (including renal cell carcinoma, bladder tumor, prostate cancer and testicular cancer) by RT-PCR and immunohistochemistry, and we also examined the effect of PPAR-gamma ligand in these cells by MTT assay, flow cytometry and hoechest staining. PPAR-gamma expression was significantly more extensive and intense in malignant tissues than in normal tissues. PPAR-gamma ligand induced the reduction of malignant cell viability through early apoptosis. These results demonstrated that generated PPAR-gamma in urological cancer cells may play an important role in carcinogensis and become a new target therapy in the treatment of urological cancer.

The role of cysteinyl-LT(1)receptor (CysLT(1)R) in renal ischemia-reperfusion injury.

The pathogenesis of ischemia-reperfusion (I/R) injury is known to involve cytokines and particularly surface adhesion molecules, the expression of which initiates the attachment of inflammatory cells. Renal I/R injury, a clinically important problem, is an invariable consequence of renal transplantation. The problem begins at the onset of acute tubular necrosis (ATN), when the transplantation includes a long ischemic interval or by use of a cardiac arrest donor's kidney. The cysteinyl leukotriene-1 (CysLT(1)), a potent lipid mediator in allergic disease, acts through the CysLT(1)R receptor. We researched the expression of CysLT(1)R in rat renal I/R injury as well as correlations with the degree of ATN. The right kidney was harvested and the left renal artery and vein were clamped at laparotomy. The kidney was reperfused after 90 minutes of ischemia; rats were sacrificed at 0, 3, 5, 12, and 24 hours after reperfusion. CysLT(1)R expression was analyzed by immunohistochemistry. CysLT(1)R expression was observed only in endothelial cells of a normal kidney. CysLT(1)R expression was most intense on endothelial cells at 3 hours after reperfusion, and CysLT(1)R expression on endothelial cells gradually became weaker. Twelve hours after reperfusion, ATN extended throughout the ischemic kidney. Renal I/R injury gradually progressed at time after reperfusion. Several hours after the maximal CysLT(1)R expression, we observed the maximum renal I/R injury.

Study of cysteinyl leukotriene-1 receptor in rat renal ischemia-reperfusion injury.

Renal ischemia-reperfusion (I/R) injury is a major cause of renal transplant dysfunction. Recent studies of I/R injury have focused on the function of neutrophils, the mechanisms of action of inflammatory cytokines, and oxygen free radicals, as well as other mediators. However, few reports address the cysteinyl leukotriene-1 receptor (CysLT1R), an important mediator of bronchial asthma in human beings. We examined the expression of CysLT1R in rat renal I/R injury. At laparotomy, the right kidney was harvested and the left renal artery and vein were clamped. The kidney was reperfused after 90 minutes of ischemia, and the rats were killed after 0, 3, 5, 12, or 24 hours. Expression of CysLT1R analyzed at immunohistochemistry was observed only in endothelial cells in nonischemic kidney. At 0 to 3 hours after reperfusion, CysLT1R expression on endothelial cells gradually became stronger, being most intense at 3 hours after reperfusion. Twelve hours after reperfusion, necrosis extended throughout the ischemic kidney; nearly all of the tubular epithelial cells were destroyed. At 3 to 12 hours after reperfusion, CysLT1R expression gradually became weaker on endothelial cells. At 24 hours after reperfusion, CysLT1R expression was almost at the level of that in nonischemic kidney. Expression of CysLT1R was noted in a rat model of renal I/R injury. Several hours after the maximal CysLT1R expression, we observed the maximum renal I/R injury. These results may suggest a relationship between the CysLT1R and renal I/R injury.

A novel approach to successful ABO-incompatible high-titer renal transplantation.

BACKGROUND: Currently the long-term outcome among recipients of ABO-incompatible renal transplantations is excellent in Japan. However, previous reports have documented poor outcomes in patients with high (> 1:256) anti-A/B antibody titers pretreatment. The immunosuppressive protocol for ABO-incompatible high-titer renal transplantation has remained a medical challenge. METHODS: We treated 3 patients with high (> 1:512) anti-A/B antibody titers prior to ABO-incompatible renal transplantation. Our immunosuppressive protocol was initiated 1 month prior to surgery and included mycophenolate mofetil (1 g/d) and low-dose steroid (methylprednisolone [8 mg/d]). Two doses of the anti-CD20 antibody rituximab, (150 mg/m2) were administered 2 weeks before and on the day of transplantation. We performed antibody removal with 6 to 8 sessions of plasmapheresis (plasma exchange or double-filtration plasmapheresis) before transplantation. Splenectomy was also performed on the day of transplantation. Postoperative immunosuppression followed the same regimen as ABO-compatible cases, in which calcineurin inhibitors were initiated 3 days before transplantation combined with 2 doses of basiliximab. RESULT: With this protocol, the anti-A/B antibody was reduced to below 1:16 in all cases. All 3 patients underwent successful transplantation with a mean current serum creatinine of 1.32 mg/dL (range, 1.22-1.50 mg/dL). There were no episodes of antibody-mediated rejection. No serious complications or side effects were encountered. CONCLUSIONS: A preconditioning protocol consisting of rituximab infusions, splenectomy, plasmapheresis, and pharmacologic immunosuppression enabled ABO-incompatible renal transplantation in patients with high (> 1:512) anti-A/B antibody titer.

A retrospective analysis of the use of brachytherapy in relation to early stage squamous cell carcinoma of the oropharynx and its relationship to second primary respiratory and upper digestive tract cancers.

The purpose of this study was to retrospectively evaluate brachytherapy for early stage squamous cell carcinoma of the oropharynx (SCO) in relation to second primary respiratory and upper digestive tract cancers (RUDT). Between 1976 and 2001, 111 previously untreated patients with stage I or II SCO were treated with Au-198 seed brachytherapy alone (36 cases) or Au-198 seed brachytherapy plus external irradiation (75 cases). Of the 111 patients, 28 patients had stage I disease and 83 patients had stage II disease. Each patient was evaluated for therapeutic efficacy, post-treatment quality of life (QOL) and a second cancer. The 5-year and 10-year cause-specific actuarial survival rates for stage I and II SCO were 87% and 86%, respectively. We found that the 5-year and 10-year survival rates for all SCOs combined with second primary RUDT cancers were 71% and 45%, respectively. 51 second primary RUDT cancers occurred successively in 41 patients following treatment for early stage oropharyngeal cancer and this was the sole prognostic factor by the multivariate analysis. Au-198 seed brachytherapy with or without ipsilateral external irradiation of up to 30 Gy was associated with fewer late complications in the oral cavity and salivary gland. We concluded that our treatment policy of brachytherapy with or without external irradiation for patients with early stage SCO was effective and acceptable from the standpoint of tumour control and post-treatment QOL.

The risk of radiation-induced cancer in patients with squamous cell carcinoma of the head and neck and its results of treatment.

The purpose of this study was to determine the incidence and the results of treatment of cancer induced by radiotherapy for early stage (stage I and II) squamous cell carcinoma of the head and neck (SCH). The clinical records of 355 patients with early stage malignant lymphoma of the head and neck region treated by radiotherapy were reviewed, and then the records of 1358 patients with early stage SCH (oral cavity, 956; larynx, 154; oropharynx, 110; maxillary sinus, 86; lip, 20; epipharynx, 17; hypopharynx, 15) who underwent radiotherapy were reviewed. The disease-specific 10-year survival rate of the patients with 355 malignant lymphoma was 61%, and 5 cases of radiation-induced cancer occurred more than 8 years after irradiation. The crude incidence of radiation-induced cancer in the malignant lymphoma patients was 1.4%, and the 10-year probability by the actuarial life table method was 0.8%. The 10-year survival rate of the early stage SCH patients was 71%. The crude incidence of a second cancer in a previously irradiated field after an 8-year latent period (SCI) in the SCH patients was 1.8% (25/1358), and the 10-year probability was 1.6%. 12 SCIs were treated by surgery and 8 of those 12 patients (67%) resulted in success, whereas treatment by radiation resulted in failure in every other case. The risk of SCIs in the SCH group was higher than in the early stage malignant lymphoma group, although the difference was not statistically significant. The possibility of radiation-induced cancer in SCH is small, and the advantage of radiation therapy compares favourably with the risks of other treatments.

Human interleukin-10 transduced fetal liver stem cells prolong survival of mouse skin and heart allografts.

Interleukin (IL)-10 regulates immune responses, acting as a suppressive cytokine by inhibiting the synthesis of Th1 cytokines, such as IL-2 and interferon (IFN)-gamma. It also strongly down-regulates major histocompatibility complex (MHC) class II determinants on antigen presenting cells (APC). On the other hand, long-term tolerance is well correlated with the persistence of a peripheral microchimerism. In this study, we investigated the synergistic effect of human IL-10 (huIL-10) and hematopoietic microchimerism for the induction of long-term tolerance. Irradiated Balb/c mice (H-2d) were used as recipients (fetal liver stem cells [FLSC], skin and heart) and C57BL/6 (H-2b) mice were used as donors of FLSC, skin and heart. Recipients were simultaneously transplanted with the heart, the skin and with huIL-10 gene-transduced FLSC. Microchimerism was checked using fluorescent flow cytometry, huIL10 production using enzyme-linked immunosorbent assay (ELISA), and graft survival was evaluated by daily observation. Significant level of huIL10 (up to 900 pg/mL) was detected for more than 2 weeks in the serum of mice that underwent transplantation. Four weeks after the FLSC injection, microchimerism was identified in the recipient lymphoid organs (spleen, thymus, and bone marrow) by the presence of donor cells (H-2b). Finally, in the group of mice treated with huIL-10 gene-transduced FLSC, skin allografts survived for 18.9 +/- 1.8 days compared with 9.5 and 9.6 days in the groups of mice treated with nontransduced FLSC or huIL-10 alone, respectively. The same pattern for heart allograft survival was observed. HuIL-10 transduction of donor hematopoietic stem cells resulted in production of huIL-10, cell engraftment, and chimerism. Although full tolerance was not obtained, specific and highly significant (P < .001) prolongation of the survival of donor heart allografts with (more than 2-fold compared with nontreated groups) was observed. The infiltration of the transplanted heart and its late rejection demonstrate that stem cells transduced with huIL-10 gene induce "prope" tolerance in this model.

Study of peroxisome proliferator-activated receptor (PPAR)-gamma in renal ischemia-reperfusion injury.

Recent studies of ischemia-reperfusion (I/R) injury have focused on the function of neutrophils, the action mechanism of inflammatory cytokines. However, few reports have addressed peroxisome proliferator-activated receptor (PPAR)-gamma. PPAR-gamma is a ligand-activated transcriptional factor belonging to the steroid receptor superfamily. It plays a role in both adipocyte differentiation and tumorigenesis. We researched the expression of PPAR-gamma in renal I/R injury of the rat. Male Lewis rats were used. The right kidney was harvested and the left renal artery and vein were clamped at 90 minutes of ischemic time. Rats were killed at 0, 1.5, 3, 5, and 12 hours after reperfusion. PPAR-gamma expression was studied by immunohistostaining. PPAR-gamma expression was observed only on mesangial and endothelial cells of normal kidney. From 1.5 to 3 hours after reperfusion, PPAR-gamma expression gradually became stronger on mesangial and endothelial cells. PPAR-gamma expression was most intense on mesangial cells and endothelial cells at 3 hours after reperfusion. Twelve hours after reperfusion, necrosis extended throughout the ischemic kidney and nearly all the tubular epithelial cells were destroyed, but 12 hours after reperfusion PPAR-gamma expression gradually became weaker on mesangial and endothelial cells. PPAR-gamma was expressed in the rat model having renal I/R injury. Several hours after maximal of PPAR-gamma expression, maximal renal I/R injury was observed. These results may indicate a relationship between PPAR-gamma expression and renal I/R injury.

Cyclooxygenase-1 and -2 in human testicular tumours.

In this study, we investigated the expression of cyclooxygenase (COX)-1 and -2 in human testicular cancer (TC) and normal testis (NT) tissues, as well as the effects of COX ligands on viability and proliferation. Tumour specimens were obtained from 72 patients with TC and 20 patients with NT. RT-PCR and immunohistochemical methods were used to determine COX expression. While COX expression was not noted in any of the NT tissues, a marked expression was observed in the TC samples. The extent and intensity of immunoreactive COX-1 and -2 polypeptides in the TC tissues was statistically greater than the expression in the NT tissues. The synthetic COX inhibitors inhibited the growth of the TC cells. Both COX-1 and COX-2 are induced in testicular cancer, and these results indicate that both COX-1 and COX-2 are essential for the growth of TC cells.

Thymidine phosphorylase levels as a prognostic factor in renal cell carcinoma.

OBJECTIVE: To investigate the relationship between thymidine phosphorylase (TP), a vascular growth factor, and established prognostic factors for renal cell carcinoma (RCC), e.g. histological grade or Tumour-Node-Metastasis (TNM) classification. PATIENTS AND METHODS: TP levels were measured in RCC tissue (tumour TP) and in adjacent non-neoplastic kidney tissue (normal tissue TP), using a sandwich-type enzyme-linked immunosorbent assay. The 59 patients, diagnosed with organ-confined RCC before surgery and who had undergone radical nephrectomy, were divided into two groups according to their prognosis after surgery. Group 1 (nine patients) had a poor prognosis and group 2 (50) had no evidence of disease within a 65-month follow-up. The relationships among TP level, TNM classification, histological subtypes, V factor and prognosis, and of tumour TP to normal tissue TP levels were investigated. Multiple regression analysis was used to determine the importance of factors associated with increased TP levels. RESULTS: Normal tissue TP levels correlated with histological grade (r = 0.31, P < 0.01); in patients with venous invasion or with a poor prognosis, the levels were significantly higher than in those without (P < 0.05 and < 0.001, respectively). The normal tissue TP levels were also significantly higher in the non-clear cell than in the clear cell subtype. Multiple regression analysis showed that the independent factor associated with elevated normal tissue TP levels was histological grade (R2 = 0.189, P < 0.01). There was no correlation between tumour TP and other factors. CONCLUSION: Normal tissue TP levels in localized hypervascular RCC were associated with histological grade. These data suggest that normal tissue TP levels could be a prognostic factor.

MR detection of iliac bone marrow involvement by malignant lymphoma with various MR sequences including diffusion-weighted echo-planar imaging.

OBJECTIVE: To assess the diagnostic accuracy of MR imaging for detecting bone marrow infiltration by malignant lymphoma. PATIENTS AND DESIGN: Fifty-three patients with malignant lymphoma underwent MR imaging and bone marrow biopsy. In 80 iliac crests of the 53 patients (13 positive specimens in 9 patients and 67 negative specimens in 44 patients), biopsy results and the signal intensity characteristics were compared. MR sequences included T1-weighted SE, T2-weighted FSE with fat suppression, FSE STIR, and diffusion-weighted EPI with fat suppression at 1.5 T. RESULTS AND CONCLUSIONS: To detect lymphoma infiltration, T1-weighted SE had the highest sensitivity (92%) and diffusion-weighted EPI with fat suppression and FSE STIR had the highest specificity (92.5% and 92%, respectively). A combination of T1-weighted SE and FSE STIR yielded the highest sensitivity and specificity (85% and 97%, respectively). A combination of T1-weighted SE and FSE STIR sequences seems to be the current choice of imaging protocol for detecting bone marrow infiltration by malignant lymphoma.

Sonographic appearances of malignant lymphoma of the salivary glands.

PURPOSE: We undertook this retrospective study to describe the sonographic findings in patients with malignant lymphoma of the major salivary glands. METHODS: We reviewed the sonograms and medical records of 7 patients with histologically proven lymphoma of the parotid (3 patients) or submandibular glands (4 patients). RESULTS: Primary lymphoma was found in 1 parotid gland and 2 submandibular glands. The remaining 4 cases were secondary lymphomas. One patient had been diagnosed with Sjögren's syndrome and had been followed up with sonography. In parotid glands, both parenchymal and intraparotid nodal lymphomas were found. All submandibular gland tumors were parenchymal. Intraparotid nodal involvement appeared as multiple small nodules with relatively smooth margins, whereas the parenchymal parotid and submandibular gland lymphomas were larger (25 to 45 mm in longitudinal diameter) and showed various degrees of margin irregularity. All tumors were hypoechoic relative to the normal parenchyma. The primary parotid lymphoma and intraparotid nodal lymphomas had a homogeneous echotexture; the secondary parotid lymphomas and submandibular gland lymphomas were heterogeneous. One submandibular gland lymphoma showed intratumoral echogenic stripes. Neither calcification nor cystic degeneration was observed within the lesions. CONCLUSIONS: Lymphomas of the salivary glands present a variety of sonographic appearances, ranging from multiple small, hypoechoic nodules to an irregularly shaped heterogeneous mass without cystic areas or calcifications.

Association of plasma free-3-methoxy-4-hydroxyphenyl (ethylene)glycol, natural killer cell activity and delirium in postoperative patients.

We measured and compared levels of plasma free 3-methoxy-4-hydroxyphenyl (ethylene)glycol (pMHPG), a major metabolite of noradrenaline, and natural killer (NK) cell activity in 26 patients prior to their undergoing an operation for cardiovascular diseases; 11 of whom expressed delirium and 15 who did not. In conclusion, we found that pMHPG levels before an operation were higher in patients with postoperative delirium than in the patients without, while NK cell activity showed no difference between the two groups. It is possible that hyperactivity of noradrenargic neurons is connected with the development of postoperative delirium. Furthermore, we considered that measurement of pMHPG level before operation might be a useful tool to predict the occurrence of postoperative delirium.