Generation of a sensitive TNFR2-specific murine assays system.

Tumor necrosis factor (TNF)/TNF receptors (TNFR1/TNFR2) are considered to be potential drug targets to treat refractory diseases, including autoimmune diseases and malignant tumors. However, their specific functions, especially in the case of TNFR2, are poorly understood. In this study, we constructed a mouse TNFR2 (mTNFR2)-mediated biological assay system that shows no effects of mouse TNFR1 (mTNFR1) in order to screen mTNFR2-selective stimulating agents. Mouse TNFR1(-/-)R2(-/-) preadipocytes were transfected with the gene encoding the mTNFR2/mouse Fas (mFas) chimeric receptor in which the extracellular and transmembrane domains of mTNFR2 were fused to the intracellular domain of mFas. Our results demonstrated that this cell line exhibits highly sensitive mTNFR2-mediated cytotoxic effects. We propose that this mTNFR2-mediated biological assay system would be a useful tool to screen for mTNFR2-selective stimulating agents.

Superselective intra-arterial cisplatin infusion and concomitant radiotherapy for maxillary sinus cancer.

BACKGROUND: The purpose of this study was to evaluate the efficacy of superselective cisplatin infusion with concomitant radiotherapy (RADPLAT) for previously untreated patients with the squamous cell carcinoma of maxillary sinus (SCC-MS). METHODS: Between 1999 and 2010, 54 patients were given superselective intra-arterial infusions of cisplatin (100-120 mg m(-2) per week) with simultaneous intra-venous infusions of thiosulfate to neutralise cisplatin toxicity and conventional radiotherapy (65-70 Gy). RESULTS: One patient (1.9%) was diagnosed with T2, 14 (25.9%) with T3, 27 (50%) with T4a, and 12 (22.2%) with T4b disease. Lymph-node involvement was present in 12 patients (22.2%). During the median follow-up period of 6.4 years, the 5-year local progression-free and overall survival rates were 65.8 and 67.9% for all patients, respectively. No patient died as a result of treatment toxicity or experienced a cerebrovascular accident. Osteonecrosis (n=5), brain necrosis (n=1), and ocular/visual problems (n=14) were observed as late adverse reactions. CONCLUSION: We have shown excellent overall survival and local progression-free rate in SCC-MS patients treated by RADPLAT with acceptable rates of acute and late toxicity. A multi-institutional trial is needed to prove that this strategy is a feasible and effective approach for the treatment of SCC-MS.

Targeted insertion of a variable region gene into the immunoglobulin heavy chain locus.

A mutant mouse strain has been generated in which a rearranged immunoglobulin heavy (H) chain variable (V) region gene is placed into the heavy chain locus in its natural position, replacing the JH elements. In homozygous mutant mice, essentially all B cells in the spleen express the transgenic VH region in their antibodies. The proper location of the transgene relative to the constant region genes allows it to participate in isotype switching and undergo somatic hypermutation. Immunoglobulin transgenic mice generated in this fashion by gene targeting should prove useful for the exploration of immunoregulatory mechanisms.

DNA damage-induced apoptosis and Ice gene induction in mitogenically activated T lymphocytes require IRF-1.

Lymphocytes are highly sensitive to DNA damage-induced apoptosis. In thymocytes, the tumor suppressor p53 has been shown to be required for this type of apoptosis. However an as yet unknown, p53-independent pathway(s) appears to mediate the same event in mitogenically activated mature T lymphocytes. By using mice with a null mutation in the IRF-1 gene, we revealed that DNA damage-induced apoptosis in the latter cell type is dependent on the anti-oncogenic transcription factor interferon regulatory factor-1 (IRF-1). Thus two different anti-oncogenic transcription factors, p53 and IRF-1, are required for distinct apoptotic pathways in T lymphocytes. Furthermore, we found that mitogen induction of the interleukin-1 beta-converting enzyme (Ice) gene, a mammalian homolog of the Caenorhabditis elegans cell death gene ced-3, is also IRF-1-dependent. An IRF-1 binding sequence was identified in the 5' flanking region of the Ice gene. In addition, ectopic overexpression of IRF-1 results in the activation of the endogenous Ice gene and enhances the sensitivity of cells to radiation-induced apoptosis. Thus, induction of Ice gene may be involved in IRF-1 dependent DNA damage-induced apoptosis in activated mature T lymphocytes.

Analysis of interleukin 6 (IL-6)/IL-6 receptor system using monoclonal anti-IL-6 antibodies.

To investigate the IL-6/IL-6 receptor system, we obtained five murine monoclonal antibodies against human IL-6, which neutralize its biological activity. We classified them into two groups (Type I mAb and Type II mAb) according to the epitopes they recognized. These two types of antibodies showed no difference in the manner in which they neutralized IL-6 activity, but they differed in the way they inhibited the binding of IL-6 to its receptor. While Type I mAb inhibited IL-6 binding to its receptor completely, Type II mAb inhibited only partially, even at a concn of Type II mAb sufficient to neutralize biological activity. Scatchard plot analysis revealed that in the case of the human myeloma cell line, the U266 cell, which showed two-phase binding of IL-6 to its receptor, the high affinity binding disappeared and the affinity of the low affinity binding decreased in the presence of Type II mAb. These results suggest that Type I mAb neutralizes IL-6 activity by the blocking of IL-6 binding to its receptor directly. In contrast, Type II mAb neutralizes IL-6 activity not by direct blocking of IL-6 binding to its receptor, but by modulating the binding affinity of IL-6 to its receptor, that is, inhibiting the formation of high affinity binding in IL-6 receptor system. This also indicates that the formation of high affinity may be necessary to transduce the IL-6 signal.

Biotinylation of human interleukin-2 for flow cytometry analysis of interleukin-2 receptors.

We designed a method to analyze receptors for interleukin-2 (IL-2R) using biotinylated IL-2 (b-IL-2). To optimize the condition of biotinylation of IL-2 for flow cytometry, the degree of biotinylation was controlled by monitoring the relative biotin contents in b-IL-2 with a newly developed ELISA. The b-IL-2 prepared by incubating 150 micrograms IL-2 in 150-300 micrograms/ml N-hydroxysuccinimidyl biotin retained biological activity and was appropriate for flow cytometry analysis. Positive fluorescence appeared in the IL-2R-bearing cell lines but not in those without IL-2R. This binding was inhibited by preincubation of the cells with unlabelled IL-2. The b-IL-2 bound to both low and high affinity IL-2Rs, but the binding to the latter was more intense. The advantage of this method is that expression of IL-2Rs of these two categories of affinity can be separately monitored.

A new method of gene transfer into hematopoietic progenitors using liquid culture with interleukin-3 and interleukin-6.

The technique of gene transfer into hematopoietic cells is expected to offer a new form of therapeutics. As a result of studies on a gene-delivery system using granulocyte-macrophage colony-forming units (CFU-GM), a type of hematopoietic progenitor, we have established a technique for efficient gene transfer into CFU-GM. DGL, a retrovirus vector containing the SV40 promoter and the neomycin resistance gene, was constructed and found to transfer genes effectively into murine CFU-GM, which subsequently expressed the neomycin resistance gene. After gene transfer of murine non-adherent bone marrow cells precultured in liquid culture with recombinant murine IL-3 (rmIL-3) and recombinant human IL-6 (rhIL-6) for 6 days, gene transferred CFU-GM in bone marrow cells were able to proliferate 5-10-fold and the ratio of gene transferred CFU-GM to total CFU-GM reached 70-100% from less than 1% in the liquid culture with rmIL-3, rhIL-6 and neomycin for 6 days. Using this protocol, we have been able to obtain large amounts of highly concentrated gene-transferred CFU-GM for fundamental research on CFU-GM gene-delivery systems.

Comparison of [(18)F]FDG PET and (201)Tl SPECT in evaluation of pulmonary nodules.

UNLABELLED: Recent reports have indicated the value of [(18)F]FDG PET and (201)Tl SPECT in diagnosing lung cancer. In this study, we compared the diagnostic value of FDG PET and (201)Tl SPECT in the evaluation of pulmonary nodules. METHODS: Sixty-three patients with 66 pulmonary nodules suspected to be lung cancer on the basis of chest CT were examined by FDG PET and (201)Tl SPECT (early and delayed scans) within a week of each study. For semiquantitative analysis, the standardized uptake value (SUV) or the tumor-to-nontumor activity ratio (T/N) (or both) was calculated. All of these lesions were completely removed thoracoscopically or by thoracotomy and were examined histologically. RESULTS: Fifty-four nodules were histologically confirmed to be malignant tumors, and 12 were benign. Both techniques delineated focal lesions with an increase in tracer accumulation in 41 of 54 lung cancers. (201)Tl SPECT on early or delayed scans (or both) identified 4 additional lung cancers that FDG PET images did not reveal: 3 bronchioloalveolar carcinomas and a well-differentiated adenocarcinoma. FDG PET identified 3 additional lung cancers that (201)Tl SPECT images did not reveal; 2 of these lung cancers were <2 cm in diameter. The mean FDG SUV and T/N of bronchioloalveolar carcinomas (2.06 +/- 0.76 and 3.49 +/- 1.03, respectively) were significantly lower than those of poorly differentiated adenocarcinomas (5.55 +/- 2.01 [P = 0.026] and 8.23 +/- 2.16 [P = 0.01], respectively). However, no significant difference was found in (201)Tl T/N on early and delayed scans between bronchioloalveolar carcinomas (1.64 +/- 0.29 and 1.87 +/- 0.42, respectively) and poorly differentiated adenocarcinomas (1.58 +/- 0.32 and 2.76 +/- 1.36, respectively). Of the 12 benign nodules, FDG PET and (201)Tl SPECT showed false-positive results for the same 7 benign nodules (58.3%) (4 granulomas, 1 sarcoidosis, 1 inflammatory pseudotumor, and 1 aspergilloma). Negative FDG PET findings and positive (201)Tl SPECT findings were obtained only for bronchioloalveolar carcinomas or a well-differentiated adenocarcinoma but not for other histologic types of lung cancers or benign pulmonary nodules. CONCLUSION: No significant difference was found between FDG PET and (201)Tl SPECT in specificity for the differentiation of malignant and benign pulmonary nodules. The degree of differentiation of lung adenocarcinoma correlated with FDG uptake but not with (201)Tl uptake. Bronchioloalveolar carcinoma (a well-differentiated, slow-growing tumor) findings typically were positive with (201)Tl but were negative with FDG. The combination of FDG PET and (201)Tl SPECT may provide additional information regarding the tissue characterization of pulmonary nodules.

T cell-dependent antibody production by Ly-1 B cells.

Through the use of a SCID transfer system, we have demonstrated that under certain conditions, the production of Ig by Ly-1 B cells can be modulated by T cells. This modulation can take the form of enhanced isotype production or isotype-switch induction and to some extent appears to be dependent on the activation state of the T cells. Furthermore we have shown that Ly-1 B cells can mount an idiotypically restricted T cell-dependent immune response to the antigen PC-KLH. This result suggests that the previous failure to observe T cell-dependent responses by Ly-1 B cells has been due to these B cells being "blind" to the antigens used and is not due to some inherent property of these B cells. When one considers the previous reports of the substantial contribution of Ly-1 B cells to the natural serum immunoglobulin levels and the ability of T cells to affect Ig production by Ly-1 B cells documented in this report, it is clear that the interaction of T cells with the Ly-1 B-cell population is important in determining the "natural" serum Ig repertoire of the mouse.

Regulation of the interferon system, immune response and oncogenesis by the transcription factor interferon regulatory factor-1.

Interferon regulatory factor-1 (IRF-1) is a transcriptional activator which was originally identified as the regulator of the type I interferon (IFN-alpha/beta) gene expression. Subsequent studies have revealed that IRF-1 is involved in a wide spectrum of the host defense mechanisms, including the antiviral response by IFNs. IRF-1 has also been shown to regulate a variety of cytokines and their target genes, thereby contributing to the development and function of the Th1-type immune response. Furthermore, IRF-1 is a critical regulator of cell growth and death, and its inactivation accelerates cell transformation. IRF-1 may be a prototypical example of a transcription factor which can selectively modulate distinct sets of genes depending on the cell type and/or nature of the cellular stimuli, so as to evoke appropriate response in each.

FDG PET in the evaluation of the aggressiveness of pulmonary adenocarcinoma: correlation with histopathological features.

2-[Fluorine-18]fluoro-2-deoxy-d-glucose (FDG) uptake within the primary lesion correlates with survival on positron emission tomography (PET) studies of patients with non-small cell lung cancer. The more metabolically active the tumour, the worse the outcome. The aim of this study was to determine whether a correlation exists between aggressiveness as determined by pathology and the findings of FDG PET in pulmonary adenocarcinoma. Thirty-five patients with 38 adenocarcinomas of the lung were studied. All patients underwent thoracotomy within 4 weeks of the FDG PET study. For semiquantitative analysis, standardized uptake values (SUVs) were calculated. Patients were classified into high SUV (> or = 4.0) and low SUV (<4.0) groups. The degree of FDG uptake (SUVs) in primary lung lesions was correlated with the histopathological features of aggressiveness (pleural involvement, vascular invasion or lymphatic permeation). The mean SUV of aggressive adenocarcinomas (4.36+/-1.94, n = 22) was higher than that of non-aggressive ones (1.53+/-0.88, n = 16) (P < 0.0001). Tumours with a high FDG uptake have a significantly higher likelihood of aggressiveness than those with a low FDG uptake (P = 0.0004). Analysis by the Kaplan-Meier methods revealed that the groups had different prognoses (log-rank test, P = 0.0099). The high SUV group had a significantly worse prognosis. In conclusion, a correlation was seen between aggressiveness as determined by pathology and glucose metabolism as measured by FDG PET in adenocarcinoma of the lung. FDG PET may be used as a non-invasive diagnostic technique in measuring aggressiveness and prognosis in patients with pulmonary adenocarcinoma.

201Tl SPET in the differential diagnosis of brain tumours.

The aims of this study were to assess the utility of 201Tl single photon emission tomography (SPET) in the differential diagnosis of brain tumours and to elucidate the relationship between 201Tl tumour uptake and degree of contrast-enhancement on magnetic resonance imaging (MRI). Early (15 min) and delayed (3 h) 201Tl SPET imaging and T1-weighted MRI were performed before and after Gd-DTPA enhancement in 101 (41 malignant and 60 benign) untreated brain tumours. The 201Tl uptake ratio (tumour-to-normal brain count ratio) for both the early and delayed SPET studies and the retention index (the ratio of delayed to early 201Tl uptake) were calculated. Malignant tumours were separated from benign tumours with 87% accuracy based on the assumption that tumours with a 201Tl retention index < 0.7 or no abnormal uptake are benign. Meningiomas and pituitary adenomas were differentiated from other benign tumours by their characteristic pattern on SPET. The degree of contrast-enhancement of the tumour on MRI was concordant with the early 201Tl uptake ratio for most histological types. However, schwannomas and cavernous haemangiomas showed a low 201Tl uptake ratio in spite of a high degree of contrast-enhancement on MRI. In conclusion, 201Tl SPET provides additional information that helps in the differential diagnosis of brain tumours.

Intense accumulation of Tc-99m MDP and Ga-67 in multiple periapical cemental dysplasia.

A case of multiple periapical cemental dysplasia is presented and intensive accumulation of Tc-99m MDP and Ga-67 is described. A 53-year-old woman was admitted with an intermittent pain and swelling of the left buccal region. The radiograph showed multiple sclerotic masses covering entire periapical regions of the teeth, in both the maxilla and mandibula. Biopsy of the maxilla facilitated a definitive diagnosis of multiple periapical cemental dysplasia.

Delayed visualization of gallbladder with in vivo labeled Tc-99m-red blood cell scintigraphy for gastrointestinal bleeding.

A case of a 78-year-old man with delayed visualization of the gallbladder during in vivo labeling for 99mTc-red blood cell scintigraphy for gastrointestinal bleeding is reported. The patient had several episodes of renal failure, melena, anemia, blood transfusion, and hemodialysis. The mechanism of this unusual phenomenon is not clear; however, excess binding of heme and 99mTc-pertechnetate caused by autohemolysis and oliguria are suggested to be the main factor in this phenomenon.

Acute subendocardial infarction with diffuse intense Tc-99m PYP uptake and minimal Tl-201 abnormality.

Tc-99m PYP scintigraphy performed on a patient with severe anterior chest pain showed diffuse intense uptake with central decreased activity corresponding to the left ventricular cavity. Tl-201 myocardial perfusion scintigraphy at rest revealed a minimal perfusion abnormality with decreased apical uptake in the lateral view. Because of these findings, diffuse subendocardial infarction was suggested.

Influenza virus A/Kumamoto haemagglutinin induces type I, III, and IV hypersensitivity in mice.

Influenza virus A/Kumamoto haemagglutinin was found to induce type I, III, and IV hypersensitivity in mice. MRL/Mp-I pr/lpr (MRL/l) mice are known to be lower responders to, and poor inducers of, interleukin-2 (IL-2). Recombinant IL-2 was found to augment the type III reaction in BALB/c mice and to suppress the reaction in MRL/I mice in vivo. Cyclophosphamide 100 mg/kg had a selective suppressive effect on the suppressor T cells responsible for type IV reaction, and subsequently the delayed type hypersensitivity (DTH) was augmented. Higher doses of cyclophosphamide suppressed both suppressor and effector T cells in DTH, and subsequently the DTH was suppressed markedly. This experimental model system employing viral haemagglutinin may provide a new screening method for the development of immunomodulators.

[Superimposition of MR angiography and three dimensional radionuclide brain perfusion image with personal computer].

To investigate the correlation of arterial obstruction with brain perfusion, MR angiography (MRA) and three dimensional (3D) radionuclide brain perfusion image were superimposed. Eleven cases with intracranial artery obstructive patients were studied. Three dimensional brain perfusion images were generated based on the ray-tracing method. Superimposition of MRA on to 3D brain perfusion image was performed on a personal computer. Reconstructing time for 3D image was about 15 minutes for each patient, Superimposing time was about 5 minutes for each image. Correlation of arterial obstruction with decrease in brain perfusion was demonstrated clearly by superimposed image. With a personal computer, it was possible to produce clinically useful synthesized images with relatively short time and conveniently.

[Secondary hepatic resections in a case of sigmoid colon cancer with multiple liver metastasis (H3) after successful continuous hepatic artery infusion chemotherapy oriented by in vitro chemosensitivity test].

A 43-year-old woman was admitted to our hospital for sigmoid colon cancer with multiple liver metastasis (H3). As preoperative CTAP (CT during arterial portography) examination showed 23 metastatic nodules in the whole liver, hepatic resections were not indicated. Angiographic findings showed that right and left hepatic arteries branched separately from the celiac artery. Sigmoid colon resection with D3 lymph node dissection and catheterization to the right hepatic artery via gastroduodenal artery were undertaken as a first operation. Continuous hepatic artery infusion chemotherapy with MMC, 5-FU oriented by in vitro chemosensitivity test (SDI test: Succinic Dehydrogenase Inhibition test) of primary tumor was performed 7 days after the first operation. After administration of MMC (40 mg) and 5-FU (16,500 mg), metastatic nodules in the right lobe almost disappeared except for the one tumor of S7, but the size and number of the nodules in the left lobes increased. At 10 months after the first operation, the left hepatic lobectomy and extirpation of only one tumor in the right lobe (S7) underwent. This case showed the usefulness of continuous hepatic artery infusion chemotherapy oriented by in vitro chemosensitivity test for multiple liver metastasis from colon cancer.

[Case of stage IVb gastric cancer in which favorable anti-tumor effect and good QOL were observed due to UFTP therapy after low-dose CDDP-tegafur therapy].

A patient with stage IVb advanced gastric cancer, who was Group 4 lymph node metastasis positive, underwent two postoperative courses of low-dose CDDP-tegafur therapy (800 mg/body/day of tegafur + 5 mg/body/5 administrations, 2 days of rest, of cisplatin). UFTP therapy (400 mg/body/day of UFT + 5 mg/body/twice weekly of cisplatin) was thereafter given on an outpatient basis. The patient has now been receiving this therapy for one year and six months. The anti-tumor effect has been maintained and the tumor has been reduced in size by 89% without any adverse reactions. A good QOL has been observed. The present therapy can be performed safely at home and appears to be a favorable treatment from the viewpoint of QOL.

[Study of complications after endoscopic sclerotherapy for esophageal varices--particularly 2 cases that changed portal circulation after sclerotherapy].

In 116 patients with esophageal varix due to liver cirrhosis who received the endoscopic sclerotherapy, the following complications were observed after the sclerotherapy: 38 ulcer formation; 19 chest pain; 16 hypotension; 15 fever; 12 pleural effusion; 6 esophageal stenosis and so on. Furthermore, two cases with marked changes of portal circulation were experienced after the sclerotherapy. The first case had severe bleeding out of duodenal varix four months after the sclerotherapy and died because of massive bleeding. The second case had hepatic encephalopathy six months after the sclerotherapy. In both cases, angiography revealed the development of collateral veins after the sclerotherapy. Because the abrupt intercept of the esophageal varix by the successful sclerotherapy causes the increase of the other collateral blood flow, the changes of portal circulation must be watched carefully afterward.