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Adult mammalian hearts are not regenerative. However, recent studies have evidenced that hypoxia enhances their regeneration. Islet1 (isl1) is known as a cardiac progenitor marker, which is quiescent in adult mammal hearts. In Xenopus hearts, transcriptional activation of isl1 was shown during cardiac regeneration of froglets at 3 months after metamorphosis. In this study, we examined transcriptional regulation of isl1 focusing on hypoxia-inducible factor 1α (hif1α) in Xenopus heart. We found that hif1α expression was increased in response to cardiac injury and overexpression of hif1α upregulated mRNA expression of isl1. Multiple conservation analysis including 9 species revealed that 8 multiple conserved regions (MCRs) were present upstream of isl1. DNA sequence analysis using JASPAR showed hif1α binding motifs in MCRs. By luciferase reporter assay and chromatin immunoprecipitation analysis, we found that hif1α directly bound to hif1α motifs in the most distant MCR8 and showed a specific transcriptional activity on the MCR8. In the luciferase assay using constructs carrying MCR8 without a responsive motif of hif1α, the reporter activity was lost. Pharmacologically inhibition of hif1α affected isl1 transcription and downstream events including cardiac phenotypes, suggesting functional defects of islet1. Contrarily in murine hearts, transcription of isl1 was unresponsive even after cryoinjury to adult hearts while hif1α mRNA was induced. In comparative analysis of multiple alignment, hif1α elements present in MCR8 of Xenopus or zebrafish were found to be disrupted as species are evolutionarily distant from Xenopus and zebrafish. Our results suggested an altered switch of isl1 transcription between mammals and Xenopus laevis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The patient was a 72-year-old woman who had previously undergone treatment for femoral chondrosarcoma (histologically rated as myxofibrosarcoma). She suddenly developed left homonymous hemianopsia and was diagnosed with cerebral embolism. Because she had atrial fibrillation, we treated her for cardiogenic cerebral embolism. About 3 months later, however, she developed left hemiplegia, and head magnetic resonance imaging revealed multiple tumorous lesions affecting the previously detected infracted area and several new areas. We assumed that a tumor embolus had caused cerebral embolism, which resulted in growth of the tumor from the embolus and formation of a metastatic brain tumor. The metastatic foci formed from the tumor embolus were visualized by diagnostic imaging, and histological examination of the resected tumor confirmed that the brain tumor had occluded the brain vessel (tumorigenic cerebral embolism). No such case has been reported to date, and this case seems to be important.

Even when we successfully perform a total extirpation of glioblastoma macroscopically, we often encounter tumor recurrence. We examined seven autopsy brains, focusing on tumor cell infiltration in the peripheral zone of a tumor, and compared our findings with the MR images. There has so far been no report regarding mapping of tumor cell infiltration and DNA histogram by flow cytometry, comparing the neuroimaging findings with the autopsy brain findings. The autopsy brain was cut in 10-mm-thick slices, in parallel with the OM line. Tissue samples were obtained from several parts in the peripheral zone (the outer area adjacent to the tumor edge as defined by postcontrast MRI) and then were examined by H&E, GFAP, and VEGF staining. We defined three infiltrating patterns based on number of infiltrated cells as follows: A zone, 100%-60% of the cells infiltrated tumor cells compared with tumor cell density of the tumor mass; B zone, 60%-20%; C zone, 20%-0%. In the autopsy brain, the tumor was easily identified macroscopically. We found that (1) the tumor cells infiltrated the peritumoral area; and (2) tumor cell infiltration was detected over an area measuring from 6 to 14 mm from the tumor border in the A zone. When performing surgery on glioblastoma, a macroscopic total extirpation of the tumor as defined by the contrast-enhanced area in MRI is therefore considered to be insufficient for successfully reducing tumor recurrence.

Temozolomide (TMZ) has been accepted as a standard antitumor drug for glioma worldwide. Regarding its mechanism of action, there are quite a few analyses. In the present study, we investigated the cell-killing effect and mechanism of action of TMZ with flow cytometry using glioblastoma cell lines. Each cell line was divided into three groups: a control group, a low-dose TMZ group, and a high-dose TMZ group. On day 1, TMZ was added to each cell line. Then, we counted the numbers of cells on days 2, 3, 4, and 5; in U87MG, we counted the number of cells on days 8 and 9. Simultaneously, we performed flow cytometric analysis with single- and double-staining methods. Although results varied slightly depending on the cell line, with flow cytometric analysis we identified the G(0)G(1)-, S-phase block on days 2 through 4, at the beginning of TMZ administration. After that we identified the deviation of the G(2)M block over days 3 to 5. Dominant morphological changes observed in U87MG were confined to the nuclei, with positive TUNEL staining. Early S-phase block and then a G(2)M block were observed; consecutively, we could analyze these blocks with a double-staining method more clearly. The flow cytometric method is very effective in the analysis of the antitumor mechanism of each agent. On the basis of our analysis, more effective combined chemotherapy may be expected.

Interleukin-5 and interleukin-10, as important mediators of vascular permeability, contribute to the development of various pathologic effusions. However, little is known regarding the involvement of these two cytokines in the formation of cysts associated with central nervous system (CNS) tumors. Twenty-eight patients with various cystic CNS tumors were investigated for expression of interleukin-5 and interleukin-10 in cyst fluid and their matched cytokine receptors in tumor tissue. Interleukin-5 and interleukin-10 were detected in cyst fluid, and interleukin-5 concentration was significantly correlated with interleukin-10 concentration (r=0.508, p=0.006). Moreover, both receptors were also detectable in the tumor tissue specimens and high levels of expression were also found in perivascular cells. Therefore, the local production of interleukin-5 and interleukin-10 might be implicated in some types of cyst formation.

We removed 12 intraorbital tumors (5 schwannomas, 3 meningiomas, 2 cavernomas, 1 pleomorphic adenoma, and 1 neuroblastoma) using the frontozygomatic approach. No patients died. Postoperatively, 1 patient developed transient ptosis, and 3 patients had mild enophthalmos. Two patients with a meningioma developed transient worsening of their visual acuity and visual field. The frontozygomatic approach for surgical treatment of intraorbital tumors provides a wide visual field exposing the entire optic nerve. This approach is indicated for large intraorbital tumors, tumors affecting the optic nerve or orbital apex, intraorbital tumors that have extended into the intracranial cavity, and intracranial tumors that have extended into the orbit. The operative procedure for intraorbital tumor is determined by the location of the lesion and by the direction of its growth. The procedure is applicable to all intraorbital tumors. It reduces discomfort for surgeons while providing a relatively wide surgical field.

There have been few reports on investigation of the cell kinetics of brain tumors using a laser scanning cytometer (LSC). We compared DNA analysis using a flow cytometer (FCM) with that by LSC using established cell lines of brain tumors, and we demonstrated the similarity of both analytical results. The DNA-index (DI) and proliferating index (PI) of various brain tumors were determined, and the results showed that these indices reflected the malignancy of the tumors. The DI values were higher for astrocytoma grade IV than for astrocytoma grade II, frequently showing an aneuploid pattern. The determination of DNA-ploidy using LSC, together with cryopathological diagnosis, was considered to be useful for deciding the area to be resected for glioma perioperatively and to be worth applying clinically.

Cyclins are key components of the cell cycle progression machinery. Immunohistochemical detection of cyclins in relation to cell position, DNA content, by multiparameter flow cytometry has provided new cell cycle studies. T98G, U373MG, and U251MG were scheduled expression of cyclin A, B1 which are maximal in late S and G2M phase cells. T98G was scheduled expression of cyclin E. being maximal in G1 and S phase cells, but U251MG, U373MG were unscheduled expression of cyclin E. The expression of cyclin D1 were not expressed a pattern. These result may mean that cyclin E in an important protein of cell cycle machinery of malignant brain tumor.

Meningioma is one of the popular benign brain tumors. However, the recurrence of this tumor is not infrequently encountered. In an attempt to establish the useful adjuvant therapy for the recurrent meningioma, in vitro chemosensitivity study for meningioma was conducted. Among various chemotherapeutic agents tested here, cisplatin showed highest cytotoxicity on cultured meningioma cells. In conclusion, cisplatin may be useful in adjuvant chemotherapy for the recurrent meningioma.

An unusual case of cervical astrocytoma due to hematomyelia was reported. A 68-year-old man was admitted to our institute because of sudden onset of paraplegia and urinary and bowel disturbance. CT and plain X ray on admission showed no abnormal findings, but emergency MRI revealed a high signal intensity area in Th 8-9 level on T1 weighted image. Preoperative diagnosis was idiopathic hematomyelia and emergency laminectomy was performed. After the dural incision from Th 8-10, the intramedullary hematoma was totally removed, but at the cephalic portion within the hematoma cavity an abnormal tumorous tissue was detected. It was noticed with the bleeding sauce of hematomyelia, and it, too, was totally removed. The microscopic finding concerning the surgical specimen was fibrillary astrocytoma (grade 2), and its vascular component was relatively rich.

Loading therapy consisting of the ACNU regimen was instituted as postoperative anticancer chemotherapy for malignant gliomas. The efficacy rate of the regimen was 25% at a dose of 3 mg/kg. A high incidence of hematological changes, such as leukopenia (neutropenia) and thrombocytopenia, were observed after chemotherapy. The former could be prevented by the administration of G-CSF, but platelet infusions were necessary in some patients for amelioration of thrombocytopenia. Gastroenterological symptoms, such as nausea and vomiting, were also frequently noted. Granisetron (Kytril), which is a recently developed selective competitive inhibitor of the 5-HT3 receptor, was used for the treatment of these adverse effects, and was found to be clinically effective.

A 39-year old woman presented with hallucinatory paranoid state, particularly with erotomania, around September, 1988 (at the age of 39), and was hospitalized in a mental hospital for 9 days from May 1, 1989, to receive major tranquilizer therapy. At admission, the leukocyte count was 10,400/mm3 showing a mild leukocytosis, and there was temporary adynamia in the upper extremities. Thereafter, mild leukocytosis persisted intermittently. On May 12, 1989, the patient visited the Department of Neuropsychiatry, Kansai Medical University, and clinical examinations revealed mental symptoms including insomnia and erotomania, delusion of reference and auditory hallucination without persecutory taint. She showed clear consciousness and well understanding. Characteristically, her expression and behavior were smooth and emotional communication was available. There were neither alterations in her basic mood, nor flaccid association of idea. No abnormalities were seen in the hair and skin, and buffalo hump was not observed. Blood examination revealed a leukocyte count of 10,700/mm3, suggesting a mild leukocytosis. According to the patient, the menses have been regular. Although major tranquilizer therapy has been maintained, she gradually developed emotional instability, and tended to show fatigue and regressive changes in her personality. She was hospitalized in a mental hospital from October 25, 1989 to July 24, 1991. Since 1990, when she was in the hospital, she gradually developed obesity, hypertension, acne, and diabetes mellitus.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic effects of interferons (IFNs) on brain tumors have often been reported in the literature, however, so far as we know, there are no reports of the study on the antineoplastic effect of IFNs (alpha, beta, and gamma) labelled with fluorescein isothiocyanate (FITC) using flow cytometry (FCM). Three established glioma cell lines and 11 cultured cells of brain tumor from surgical specimens were exposed to IFN-alpha, beta, and gamma at the concentrations of 10(2)-10(5) IU/ml for 24 h, respectively. Using FCM, the viability of the cells was evaluated with fluorescein diacetate stain and the cell cycle was analyzed from the DNA-histogram with propidium iodide stain. Furthermore, FITC-labelled IFN-alpha, beta and gamma were also contacted with each cell to calculate respective positive cells. The viability decreased about 60% on day 1 and day 3, indicating the effect of IFN-alpha and beta on U373MG cells and on some cultured glioma cells from surgical materials, whereas, IFN-gamma had no effects. Antineoplastic effect of each IFN well correlated with FITC-positive rates, demonstrating S phase block in the cell cycle. IFN-gamma had no antineoplastic effects, whereas IFN-alpha and beta showed antineoplastic effects, which fact suggested that IFN-gamma receptor be different from those of IFN-alpha and beta. The method of FITC-labelling for IFNs with the aid of FCM has the advantages as follows: 1) Antineoplasticity of IFN can be simply evaluated with FCM; 2) It is easy to analyze the action mechanism of IFN; 3) Information on the receptor is obtainable; and 4) Sensitivity can be evaluated prior to administration of IFN, suggesting possibilities of clinical application of this method.

For the purpose of FCM, we have developed a new method of sterile sorting of NK cells by negative selection using FITC-labelled monoclonal antibodies. In this method, over 95% lymphocytes with large granular lymphocytes (NK cells) were obtained by exclusion of Leu-1 and HLA-DR positive cells from human peripheral blood. From the results of cytotoxicity test of NK cells against 4 types of glioma cell lines and 6 kinds of cultured cells from surgical materials, the antineoplastic effect was recognized in 4 out of 6 surgical materials. Administration of NK cells through an Ommaya tube in 2 brain tumor patients caused no side effects. This technique is clinically applicable because 1) NK cells are easy to obtain, 2) NK cells show antineoplastic effect on brain tumors, and 3) the sensitivity can be evaluated in individual patients.

We have employed IAR therapy [combination of postirradiation, chemotherapy and interferon (IFN)] for malignant glioma patients. Changes of lymphocyte fractions in patients were evaluated before and after IAR therapy, using a recently developed two-color analysis. Eight malignant glioma patients received irradiation, chemotherapy (ACNU) and immunotherapy (OK-432 and IFN-beta). Peripheral blood lymphocytes taken during hospitalization with IAR therapy (first half and latter half), and every 3 to 6 months for 2 years at the longest after IAR therapy were double-stained with FITC- and PI-labelled antibodies and two-color analysis was conducted by a FACS Analyzer. Six patients out of 8 survived for 6 months to 2 years, 2 died after 3 and 6 months, respectively. Leu-2a (suppressor/cytotoxic T), especially Leu-2a+ 15- (cytotoxic T) showed a high value. Leu-2a level decreased during treatment, and both Leu-2a+ 15- and Leu-2a+ 15+ (suppressor T) values decreased. Two thirds of the patients showing an increased Leu-2a+ 15+ level died. Leu 3a (helper/inducer T), especially Leu-3a+ 8+ (inducer T) level decreased, but Leu-3a+ 8- (helper T) level increased during treatment. The level decreased in the worse patients. Leu-3a/Leu-2a ratio was low, but it increased during treatment as compared with the results of conventional therapy. Leu-7, Leu-11a, NK activity, and gamma-IFN productivity were further studied. Treatment combined with IFN revealed an influence on the T cells resulting in an increase of helper T level and suppression of suppressor T level.

The antineoplastic effects and mechanism of IFN-alpha with a newly designed fluorescent isothiocyanate-labelled IFN-alpha were studied on three established glioma-cell lines and cultured cells of clinical brain tumors. Viability, cell cycle and cells showing positive reaction with fluorescent isothiocyanate-labelled IFN were analyzed by flow cytometry. U373MG glioma cells in contact with 10(4) IU/ml of IFN for 24 hours had a dose dependency, decreased viability, S-phase block in the cell cycle and high rate of positive reaction with fluorescent isothiocyanate-labelled IFN. Two surgical cases, except for one case, showed similar results and other cell lines or meningiomas were not affected by IFN. As the antineoplastic effects of IFN-alpha correlated with the rate of occurrence of positive cells with fluorescent isothiocyanate-labelled IFN, sensitive tumor cells may have a respective specific receptor for IFN-alpha, and, therefore, fluorescent isothiocyanate-labelled IFN-alpha might be of clinical value with regard to the problem of the receptor as well as sensitivity test.

Though we recently found some reports on sterile sorting of natural killer cells (NK cells) by flow cytometer (FCM), which manifested cytotoxicity, no reports have concerned antineoplasticity of sterile NK cells on glioma cell lines and cultured surgical materials of brain tumor so far as we know. Monocytes were sampled from the peripheral blood of healthy adults, stained with fluorescein isothiocyanate (FITC)-labeled antihuman monoclonal antibodies, and sorted by an FCM, which had been sterilized with 0.5% Hibitane alcohol solution. Over 95% of the cells obtained were NK cells, and their viability was disclosed to be 97% by the FDA staining. Using thus obtained NK cells, the antineoplastic effects were evaluated in 3 kinds of glioma cell lines and 5 surgical specimens by the microcytotoxicity test. The effects varied widely from 9 to 74% (E/T ratio 40) for glioma cell lines, and from 1 to 66% (E/T ratio 10-40) for surgical specimens. It is expected in the future to apply NK cells clinically using this sterile sorting technique.

Eleven patients with Dubin-Johnson syndrome (DJS) were studied clinicopathologically. In three patients with DJS, concomitant chronic hepatitis was observed. They showed long-standing jaundice with the symptoms of general fatigue and anorexia. Laboratory tests revealed mild hypertransaminasemia, elevated serum bilirubin (over 5.0 mg/dl) and a high level of serum Bromsulphalein (BSP) retention at 45 min (above 18%). Two patients complicated with chronic hepatitis showed a rather slow secondary rise in the BSP excretion curve in comparison with the patients who had no complication. One patient accompanied with the most severe fibrosis showed no secondary rise in the BSP excretion curve. After the treatment of the patient with phenobarbital, however, a secondary rise in the BSP excretion curve appeared and the serum BSP level was also significantly decreased at all points on the BSP excretion curve. Histological examination of the liver revealed the decreased number of the Dubin-Johnson pigments in the patients complicated with chronic hepatitis. Laparoscopically, a patient with a chronic aggressive hepatitis showed a dark gray decololization of the liver surface and another patient with a chronic aggressive hepatitis showed a black color of the liver surface with partial nodule formation.

The biochemical properties of preneoplastic antigen (PN antigen) were investigated in order to resolve inconsistencies in previous reports. First of all, epoxide hydrase was purified biochemically from the liver of phenobarbital-treated rats and anti epoxide hydrase serum was prepared in rabbits. Immunochemically, in Ouchterlony double diffusion, PN antigen had the same antigenicity as epoxide hydrase. From an immunoprecipitation study using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis, it was found that PN antigen may be composed of epoxide hydrase and at least one more protein (mol. wt. 27,000). However, the biochemical and biological properties of the protein binding to epoxide hydrase were not determined. The existence of PN antigen (epoxide hydrase and its binding protein) may be associated with microsomal membrane alterations during chemical carcinogenesis.