Validation of recombinant Sendai virus in a non-natural host model.

We have previously shown that recombinant Sendai virus (SeV) vector, derived from murine parainfluenza virus, is one of the most efficient vectors for airway gene transfer. We have also shown that SeV-mediated transfection on second administration, although reduced by 60% when compared with levels achieved after a single dose, is still high because of the efficient transfection achieved by SeV vector in murine airways. Here, we show that these levels further decrease on subsequent doses. In addition, we validated SeV vector repeat administration in a non-natural host model, the sheep. As part of these studies we first assessed viral stability in a Pari LC Plus nebuliser, a polyethylene catheter (PEC) and the Trudell AeroProbe. We also compared the distribution of gene expression after PEC and Trudell AeroProbe administration and quantified virus shedding after sheep transduction. In addition, we show that bronchial brushings and biopsies, collected in anaesthetized sheep, can be used to assess SeV-mediated gene expression over time. Similar to mice, gene expression in sheep was transient and had returned to baseline values by day 14. In conclusion, the SeV vector should be strongly considered for lung-related applications requiring a single administration of the vector even though it might not be suitable for diseases requiring repeat administration.

Combination therapy of an anticancer drug with the FNIII14 peptide of fibronectin effectively overcomes cell adhesion-mediated drug resistance of acute myelogenous leukemia.

We investigated whether FNIII14, a 22-mer peptide derived from fibronectin (FN) that potently impairs interaction of FN with beta1-integrin, could overcome cell adhesion-mediated drug resistance (CAM-DR) induced by very late antigen (VLA)-4-to-FN interaction in acute myelogenous leukemia (AML). Two AML cell lines, U937 cells and HL-60 cells, and fresh leukemic cells from six AML patients with high alpha4-integrin expression exhibited CAM-DR to cytosine arabinoside (Ara C) through VLA-4-to-FN interaction, while fresh leukemic cells from two AML patients with low alpha4-integrin expression did not display CAM-DR to Ara C. FNIII14 impaired VLA-4-to-FN interaction and restored sensitivity to Ara C in the CAM-DR leukemic cells. In these CAM-DR leukemic cells, upregulation of Bcl-2, which was induced through the focal adhesion kinase/Akt signal pathway upon VLA-4-to-FN interaction, was inhibited by FNIII14 treatment. In a mouse model of minimal residual disease (MRD) in bone marrow, 100% survival was achieved by combining FNIII14 with Ara C, whereas Ara C alone prolonged survival only slightly. The myelosuppression induced by Ara C was not augmented by the combination of FNIII14 in mouse experiments. Thus, the combination of anticancer drugs and FNIII14 holds promise to eradicate MRD in bone marrow after chemotherapy.

Cyclooxygenase-2 expression is related to prognosis in patients with esophageal squamous cell carcinoma.

AIM: Esophageal carcinoma is one of the most aggressive malignancies. Many studies have examined various biological factors associated with the malignant potential of esophageal carcinoma. Cyclooxygenase (COX)-2 is overexpressed in various types of human malignancies, including esophageal carcinomas. Although some groups have described COX-2 expression in esophageal adenocarcinoma, few studies have reported COX-2 expression in esophageal squamous cell carcinoma (ESCC). METHODS: We immunohistochemically investigated relationships between COX-2 overexpression in surgical specimens of primary tumors in 228 patients with ESCC. Relationships between COX-2 expression and clinicopathological factors, including prognosis, were analyzed. COX-2 expressions were classified into 4 criteria: Score 0, no staining; Score 1, <10% staining; Score 2, 10-90% staining; and Score 3, >90% staining. RESULTS: Scores of COX-2 immunoreactivity in 228 patients were as follows: Score 0, 21 of 228; Score 1, 71of 228; Score 2, 117 of 228; and Score 3, 19 of 228, respectively. COX-2 expression was significantly correlated with depth of invasion and tumor stage (p=0.03 and p=0.04, respectively). The 5-year survival rate of patients decreased significantly with increased expression of COX-2 (p=0.005). Multivariate regression analysis indicated COX-2 expression as an independent prognostic factor for ESCC. CONCLUSIONS: COX-2 overexpression was significantly correlated with depth of invasion, tumor stage and survival in ESCC. Evaluation of COX-2 expression should be useful for determining tumor properties, including prognosis, in patients with ESCC.

Randomized controlled study on preoperative chemoradiotherapy followed by surgery versus surgery alone for esophageal squamous cell cancer in a single institution.

The purpose of the present study was to compare the clinical results between preoperative chemoradiotherapy followed by surgery (CRT group) and surgery alone (Surgery group) by a randomized controlled study. Twenty-two patients were assigned to the CRT group and 23 to the Surgery group. A total radiation dose of 40 Gy was applied and in the same period, intravenous chemotherapy was performed using cisplatin (7 mg over 2 h) and 5-fluorouracil (5-FU; 350 mg over 24 h). Surgical treatment was performed in 20 patients in the CRT group except for two patients with bone metastasis after CRT. According to histological effects of primary tumors, the number of patient with Grades 1, 2 and 3 was 11, 7 and 3, respectively. Frequency of lymphatic and venous invasion was significantly lower in the CRT group than in the Surgery group. The 5-year survival rate was 57% in the CRT group and 41% in the Surgery group (P = 0.58). According to the histological effect in the CRT group, 5-year survival was 30% for Grade 1, 83% for Grade 2 and 100% for Grade 3 (P = 0.0069). This randomized trial did not demonstrate a statistically significant survival difference between the CRT group and the Surgery group.

Expression of osteopontin in oesophageal squamous cell carcinoma.

Osteopontin is a multifunctional 34 kDa extracellular matrix protein with a cell-binding domain. It is involved cell adhesion and cell migration and is therefore considered to influence tumorigenesis and/or metastasis. The purpose of the present study was to evaluate the clinical significance of Osteopontin expression in oesophageal squamous cell carcinoma (ESCC). In the present study, we immunohistochemically investigated the relationship between Osteopontin expression and clinicopathological factors including prognosis in surgical specimens of primary tumours in 175 patients with ESCC. Osteopontin was expressed in 48% of 175 patients. Osteopontin expression was significantly correlated with lymph node metastasis, lymphatic invasion, and stage (P=0.0015, 0.037 and 0.033, respectively). Tumours with expressing Osteopontin exhibited more lymph node metastasis, lymphatic invasion and advanced stage than the tumour with negative Osteopontin expression. Five-year survival rate was better in patients with negative Osteopontin expression than in those with positive Osteopontin expression (P=0.035). However, multivariate analysis revealed that Osteopontin expression was not an independent prognostic factor. As our findings suggest that Osteopontin may play an important role in progress of ESCC, the evaluation of Osteopontin expression is useful for predicting the malignant properties of ESCC.

The role of salvage surgery for recurrence of esophageal squamous cell cancer.

AIM: A consensus treatment strategy for recurrent esophageal squamous cell cancer (ESCC) has not been established. The purpose of the present study was to analyse the mode of recurrence, and evaluate the role of surgical salvage treatment in recurrence of ESCC. METHODS: Recurrence was detected in 131 of 367 consecutive patients with ESCC. We retrospectively analysed the mode of recurrence and treatment for recurrence. Recurrence was divided into four types; lymph node, hematogeneous, mixed and local. Treatments were classified into four groups; chemotherapy alone (C group), radiation therapy +/- chemotherapy (R group), surgery +/- other therapy (S group), and no therapy (N group). RESULTS: Of the 131 recurrences, the number of patients with lymph node, hematogeneous, mixed and local recurrence was 43, 44, 40 and 4, respectively. The number of patients in the C, R, S, N groups was 35, 35, 24 and 37, respectively. Of the 24 patients who received surgical treatment for recurrence, the number of patients with lymph node, hematogeneous, mixed and local recurrence was 11, 6, 6 and 1, respectively. The number of lesions in hematogeneous recurrence was 2 or less. The survival rate from recurrence to death in the C, R, S and N groups was 0, 3.9, 6.7 and 0%, respectively. A statistically significant difference was found in these groups (p < 0.0001). CONCLUSIONS: Salvage surgery is one of the useful treatment tools for resectable metastatic lesions. In such cases, the number of lesions, recurrent sites and effectiveness of chemotherapy and/or radiotherapy should be carefully evaluated.

Effect of tolerance induction to immunodominant T-cell epitopes of Sendai virus on gene expression following repeat administration to lung.

Sendai virus (SeV) is able to transfect airway epithelial cells efficiently in vivo. However, as with other viral vectors, repeated administration leads to reduced gene expression. We have investigated the impact of inducing immunological tolerance to immunodominant T-cell epitopes on gene expression following repeated administration. Immunodominant CD4 and CD8 T-cell peptide epitopes of SeV were administered to C57BL/6 mice intranasally 10 days before the first virus administration with transmission-incompetent F-protein-deleted DeltaF/SeV-GFP. At 21 days after the first virus administration, mice were again transfected with DeltaF/SeV. To avoid interference of anti-GFP antibodies, the second transfection was carried out with DeltaF/SeV-lacZ. At 2 days after the final transfection lung beta-galactosidase expression, T-cell proliferation and antibody responses were measured. A state of 'split tolerance' was achieved with reduced T-cell proliferation, but no impact on antiviral antibody production. There was no enhancement of expression on repeat administration; instead, T-cell tolerance was, paradoxically, associated with a more profound extinction of viral expression. Multiple immune mechanisms operate to eradicate viruses from the lung, and these findings indicate that impeding the adaptive T-cell response to the immunodominant viral epitope is not sufficient to prevent the process.

Endoscopic quadrantectomy for breast cancer with sentinel lymph node navigation via a small axillary incision.

A great deal of clinical experience has firmly established the concept of the sentinel lymph node (SN) in breast cancer. SN biopsy allows treatment without axillary lymphadenectomy and has made it possible to perform a surgical intervention via just a small skin incision. In partial resection of the breast (quadrantectomy), we use a double retractor to form a workspace under the skin via a small axillary incision. Resection does not require a large incision even in cases in which the cancer lesion is located in the upper inner or lower inner quadrant of the breast, as the endoscope allows the surgeon to see the workspace formed by the double retractors.

A study of relationship between multifocal VEF responses and binocular disparity.

Multifocal visual evoked magnetic field (mfVEF) is useful for shortening the measurement time and/or improving the signal-to-noise ratio of recording, in investigating cortical activities elicited by multiple stimulus patches across the visual field. To record the whole-head mfVEF, we previously developed custom software to deliver multifocal stimuli and to analyze the acquired data, and we applied it to investigation of stereopsis. In the present study, relationship between mfVEF responses and binocular disparity was investigated. Subjects were instructed to fixate a nonius line in the center of a screen, and they were presented with rapid alteration of random-dot (RD) patches. The experiments consisted of a luminance change condition (condition L) and seven binocular disparity conditions (condition BDs) ranging from a crossed disparity of 1 deg to an uncrossed disparity of 1 deg. Each response evoked by one of the RD patches was obtained with the multifocal technique. Although all subjects showed obvious responses in the condition L, only one of the subjects showed obvious responses in condition BDs. The sources of the responses recorded in condition BDs were mainly localized around calcarine sulci according to the known retinotopic cortical representation. The responses obtained in condition BDs showed dependence on binocular disparity in the magnitude and the latency. These results would be the basis for further analysis of brain magnetic responses related to stereopsis.

The ultrasonic coagulating and cutting system injures nerve function.

BACKGROUND: Although, many surgeons have used ultrasonic coagulating and cutting systems and shears (LCS) when carrying out endoscopic thyroidectomies and parathyroidectomies, in our experience some patients had temporary paralysis of the recurrent laryngeal nerve (RLN) after these operations. We had sometimes noticed that the heat of the blade of the LCS was increased just after use; therefore, we designed an RLN model, and investigated the damage to the RLN which could be attributed to the heat of the LCS. METHODS: We investigated the effects of the heat of the LCS blade on rat femoral and sciatic nerves, using temperature measurement, histological examination and evoked electromyography. RESULTS: The temperature of the LCS blade exceeded 150 degrees C after 30 seconds when it was not used for cutting. When we used the LCS to cut rat muscle or fat tissue, the temperature of the blade exceeded 100 degrees C after 20 seconds. There was no damage to the nerve histologically when the LCS was used for less than 20 seconds at a distance of 3 mm. Electrophysiological study showed that touching the nerve with the LCS blade after only 5 seconds of use resulted in damage to the nerve. CONCLUSIONS: Our results suggest that the RLN should not be touched directly with the blade just after it has been used, and that it is possible to use the LCS at a distance of 3 mm from the RLN for less than 20 seconds at level 3. In order to maintain these distances, the RLN must be endoscopically visualized during surgery of the neck.

Endothelial cells potentiate phagocytic killing by macrophages via platelet-activating factor release.

The immunomodulatory function of endothelial cells (EC) includes the initiation of leukocyte margination, diapedesis, and activation through the upregulation of various cell surface-associated molecules. However, the effect that EC have on the phagocytic function of neighboring monocytes and macrophages is less well described. To address this issue, microvascular EC were cocultured with murine peritoneal macrophages, first in direct contact, then in a noncontact coculture system, and macrophage phagocytosis and phagocytic killing were assessed. The presence of increasing concentrations of EC resulted in a dose-dependent increase in macrophage phagocytic killing. This stimulatory effect was inhibited in a dose-dependent manner by the pretreatment of macrophage/EC cocultures with WEB-2086 or CV-6209, specific platelet-activating factor (PAF)-receptor antagonists, but not by anti-tumor necrosis factor-alpha, anti-interleukin (IL)-1alpha, or anti-IL-1beta. Furthermore, the effect was reproduced in the absence of EC by the exogenous administration of nanomolar concentrations of PAF. Microvascular EC potentiate macrophage phagocytic killing via the release of a soluble signal; PAF appears to be an important component of that signal.

Automated determination of 5-fluorouracil and its metabolite in urine by high-performance liquid chromatography with column switching.

We report a quantitative assay of 5-fluorouracil (FU) and its metabolite, 5-fluorodihydrouracil (FDHU) in human urine by used a column-switching high-performance liquid chromatographic method. The analyses were carried out using a molecular exclusion column for sample purification, and a cation-exchange column for separation. Each sample required only 40 min to analyze, and required no preparation other than filtration. Linearity was verified up to 1000 nmol/ml (r > 0.993). The recovery of FU was 96-101%; recovery of FDHU was 96-105%. The imprecision (RSD) for FU (10-100 nmol/ml) was < 1.5%, same-day (n = 5), and < 1.8%, day-to-day (n = 5). The imprecision (RSD) for FDHU (10-100 nmol/ml) was < 3.2%, same-day (n = 5), and < 4.0%, day-to-day (n = 5). The detection limits were, respectively, 0.1 nmol/ml. We measured FU and FDHU in urine of seven cancer patients after oral administration of FU. The cumulative quantity ratio of the FDHU and FU (FDHU/FU) excreted in their urine within 120 min after FU administration was a constant value in all seven patients. Based on these results, we believe that our method provides a useful tool for evaluating FU metabolism.

Endothelial cells potentiate oxidant-mediated Kupffer cell phagocytic killing.

Phagocytosis and killing of circulating organisms by Kupffer cells (KCs) are discrete, important components of host defense. However, the killing mechanism(s) are not fully understood, and the potential role of adjacent nonparenchymal cells such as hepatic endothelial cells has not been defined. Rat KCs -/+ an hepatic endothelial cell enriched cellular fraction (HECEF) were incubated with Candida parapsilosis and assayed for phagocytosis and phagocytic killing by validated fluorochromatic vital staining. The role of reactive oxygen metabolites in KC phagocytic functions was examined by inhibition with superoxide dismutase and/or catalase. Diphenyleneiodonium and allopurinol were used to examine the potential roles of NADPH oxidase and xanthine oxidase, respectively, in generating these toxic oxidants. Coculture with HECEF increased KC phagocytic activity (from 75% to 88%) and candidacidal activity (from 20% to 31%). Superoxide dismutase, catalase, diphenyleneiodonium, or allopurinol caused inhibition of candidacidal activity, but did not affect phagocytosis, and did not block the potentiation of phagocytosis or of killing caused by coculture with HECEF. Reactive oxygen intermediates generated by both NADPH oxidase and xanthine oxidase-dependent pathways are important in KC killing of Candida parapsilosis. In vitro, KC phagocytosis and killing are potentiated (via a non-oxidant-mediated mechanism) by coculture with a preparation of hepatic non-parenchymal cells composed primarily of endothelial cells.

Study of needlestick accidents and hepatitis C virus infection in healthcare workers by molecular evolutionary analysis.

In the five-year period between 1989 and 1993, 87 needlestick accidents occurred among healthcare workers at our hospital. Thirty-seven (43%) of these needlestick accidents involved blood contaminated with hepatitis C virus (HCV), and two of them (5.4%) led to the occurrence of hepatitis C infection. Case 1 was a 43-year-old nurse who was accidentally injured by a needle contaminated with blood from a patient who had cirrhosis and hepatocellular carcinoma due to hepatitis C. Acute hepatitis C occurred after five weeks and HCV RNA was positive after eight weeks. Case 2 was a 33-year-old nurse who was injured by a needle contaminated with blood from a patient who had chronic hepatitis C. Liver function was normal at 11 days after the accident. However, hepatitis C was diagnosed 21 months later after she had successfully given birth to her baby. The nucleotide sequence of the HCV E2/NS1 region was determined in the two patients and the needlestick victims, and phylogenetic trees were constructed by molecular evolutionary analysis. On the basis of these trees, transmission of HCV could be confirmed in both cases. This method of analysis may be useful for confirming the transmission of HCV even long after the event.

[7 years of annual mass screening for colorectal cancer in office workers--usefulness of 3-day RPHA method].

Immunological fecal occult blood test have been proved to be useful in the mass screening for colorectal cancer. Since 1986, we have started annual mass screening by the use of 3-day RPHA method and medical questionnaire for office workers over 40 yr old. During 7 yr, 261 out of 5386 examinees were testing positive for fecal occult blood, a test positive rate was 4.8% and 12 colorectal cancer (7: early, 5: advanced) were found. Among 12 cases, 10 were detected by positive results of RPHA test and 2 by questionnaires of symptoms or family history. The sensitivity and specificity of this method were calculated at 83.3% and 95.3% respectively. But as for advanced cancers, the sensitivity of this method was 100%. 75% of patients with colorectal cancer were over 55 yr old. The predictive value of positive test (PV) were much more higher in subjects with 2 or 3 times test positive (high risk group: PV = 14.3%) than in subject with only 1 time test positive (low risk group: PV = 0.6%). So it is considered to be reasonable that the high risk group (24% of total test positive) should be immediately undergone further examinations (Ba. enema or colonoscopy), and the low risk group should be undergone re-examinations of 3-day RPHA method. By this system, almost 50% of further examinations will be reduced. In conclusion, 3-day RPHA method with this further examination system is reasonable and efficient for screening of colorectal cancer in office workers, considering the high reliability in cancer detection and decreased further examinations.

Clinical application of transvenous mitral commissurotomy by a new balloon catheter.

A new balloon catheter was developed which allows mitral commissurotomy without thoracotomy. The procedure has been successful in five of the six patients with mitral stenosis so treated. In the remaining patient, the procedure could not be performed because of technical difficulties. The balloon is reinforced with a nylon micromesh and its shape changes in three stages, depending on the extent of inflation. It is inserted from the saphenous vein into the mitral orifice transseptally, fixed across the mitral orifice with partial inflation, and finally inflated to full its extent, separating the fused commissures by its expansile force. After the procedure, catheterization revealed a significant reduction in the mean diastolic pressure gradient across the mitral valve without resultant mitral regurgitation in each patient. Two-dimensional echocardiograms showed a marked to moderate degree of dilatation of the mitral orifice in each patient. All five patients are well with remarkable clinical improvements 2 to 16 months after the procedure.

[Noninvasive evaluation of cardiac involvements and left ventricular function in schoolchildren with the history of mucocutaneous lymph node syndrome].

Mucocutaneous lymph node syndrome (MCLS) in the young has been known to have coronary aneurysms, and then it has been recently suspected as a cause of premature atherosclerosis and cardiomyopathy. Thirty-three schoolchildren who suffered from MCLS were studied to evaluate cardiac involvements and left ventricular function using two-dimensional (2-D) echocardiograms and submaximal stress test. Fifteen normal schoolchildren were studied as normal control. All these MCLS children were asymptomatic and had no significant findings in routine chest X-ray and electrocardiographic examinations. According to submaximal stress test, 8 cases showed a J type ST depression of only 0.5--1.0 mm, and there were no positive cases. Using 2-D echocardiograms, the left coronary artery was detected in 85% and the right coronary in 27%. One case showed an aneurysm of the left coronary artery. However, none of them showed abnormal left ventricular wall motion or the wall motion abnormality compatible with cardiomyopathy. There were no significant differences between MCLS and normal control in ejection fraction, mean VCF, diastolic descent rate of the anterior mitral valve, D/S ratio of the left ventricular wall, and Weissler's index (PEP/ET). These findings suggested that 1) most of MCLS schoolchildren do not have obvious cardiac involvement and their left ventricular function is within normal limits, 2) because of its low sensitivity, submaximal stress test is not so useful in screening coronary lesions, and 3) the 2-D echocardiogram works not only in detecting coronary aneurysms but also in evaluating left ventricular function.