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T Tokunaga

Publications and source records attributed to T Tokunaga.

At least 19 recordsLinked to original sources
PubMed1992

Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.

Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.

Animals↗
PubMed1992

Human soluble IL-6 receptor: its detection and enhanced release by HIV infection.

By using a fluorescence sandwich ELISA for the quantification of soluble human IL-6R, normal human PBMC were found to be induced to release IL-6R into culture supernatant by stimulation with PHA. Furthermore, certain promonocyte cell lines and human T-cell leukemia virus I (HTLV-I)-positive cell lines produced sIL-6R into culture supernatants constitutively. However, this was not found with HTLV-I negative T cell lines and Burkitt's B cell line. In addition, generation of supernatant IL-6R of the promonocyte cell line was significantly increased 27-fold after PMA treatment and sevenfold after infection with HIV. The released IL-6R molecules were characterized as an apparent m.w. of 50 to 55 kDa by both size-exclusion HPLC and immunoprecipitation of the soluble protein with IL-6R-specific mAb followed by SDS-PAGE analysis. Furthermore, increased levels of serum IL-6R were detected in blood donors seropositive for HIV. Moreover, the released IL-6R could bind efficiently to purified rIL-6 on solid phase and suppressed the proliferative responses of PBMC. These results suggest that the release of soluble IL-6R might be linked to regulatory functions of immune responses induced by IL-6 stimulation during normal and human retrovirus-infected cell growth and differentiation.

Enzyme-Linked Immunosorbent Assay↗
PubMed1992

Changes of host cell infiltration into Meth A fibrosarcoma tumor during the course of regression induced by injections of a BCG nucleic acid fraction.

MY-1, which consists of DNA and RNA extracted and purified from Mycobacterium bovis strain BCG, causes the regression of various experimental syngeneic tumors when injected intratumorally. In order to identify the host cells involved in the antitumor mechanism(s) of MY-1, we examined Meth A tumors inoculated intradermally to BALB/c mice, which were given multiple injections of MY-1 following tumor inoculation. Histological and immunohistochemical examinations were performed at several time points. On day 4 after inoculation, the MY-1-treated tumors were heavily infiltrated with a heterogeneous population of mononuclear cells with low density nuclei. The MY-1-injected tumors contained asialo-GM1-positive cells and Mac-1-positive cells, which indicated that the infiltrating mononuclear cells were natural killer cells and macrophages. On day 14 after inoculation, the tumors were infiltrated with a large number of L3T4-positive cells and fewer Lyt-2-positive cells, both of which were more abundant in the MY-1-treated tumors than in the control tumors. The observed sequence of host cell infiltration corresponded well with our previous studies which have indicated that the antitumor mechanism of MY-1 is divided into two phases, i.e. the early phase when natural killer cells and macrophages inhibit tumor growth, and the late phase when L3T4-positive cells act to induce tumor regression via a delayed-type hypersensitivity against tumor cells.

Animals↗
PubMed1992

Production of a monoclonal antibody that defines the alpha-subunit of the feline IL-2 receptor.

A mAb, termed 9F23, to feline Con A-stimulated PBMC was prepared to characterize feline IL-2R. 9F23 was identified by FACS studies, which showed that the antigen was expressed at a high density on Con A-induced feline T cell blasts while 9F23 binding was not detected on nonactivated PBMC or the Crandell feline kidney cell line CRFK. Chemical crosslinking of 125I-IL-2 to membrane IL-2Rs on Con A-stimulated feline PBMC under the low-affinity condition resulted in detection of a major 65-kDa band. 9F23 specifically immunoprecipitated the IL-2.IL-2R alpha complex in a cell extract; in contrast, neither anti-human IL-2R alpha H48 nor anti-mouse IL-2R alpha 7D4 reacted with the complex. Moreover, immunoprecipitation with 9F23 of the extract from surface-iodinated Con A-stimulated PBMC showed a major 50-55 kDa band. Furthermore, 9F23 had no effect on either IL-2-driven proliferation of the Con A-stimulated PBMC or IL-2 binding. Finally, the expression of feline IL-2R alpha on Con A-stimulated PBMC was up-regulated by addition of exogenous IL-2. Thus, 9F23 defines an epitope different from the IL-2 binding site on the alpha-subunit of feline IL-2R.

Animals↗
PubMed1992

Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells.

Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.

Animals↗
PubMed1992

DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth.

The nucleic acid fraction from cells of 6 species of bacterium and 2 kinds of vertebrate, calf and salmon, was extracted and purified by the same procedures as described previously. When the spleen cells from BALB/c mice were incubated with the nucleic acid fraction from either of the bacteria, natural killer (NK) activity of the cells was remarkably elevated and the cells produced factors to activate macrophages and to inhibit viral growth. It was shown that the factor to activate macrophages was interferon (IFN)-gamma and that to inhibit viral growth was IFN-alpha/beta. On the other hand, the nucleic acid fraction from either of the vertebrate cells did not show such activities. Pretreatment of the bacterial nucleic acid fraction with DNase, but not with RNase, abrogated completely the biological activities. The activities of the bacterial nucleic acid were not influenced by the presence of polymyxin B, an inhibitor of lipopolysaccharide (LPS), and the spleen cells from not only BALB/c mice but also LPS-insensitive C3H/HeJ mice were activated, indicating that the activities of the fraction were not ascribed to LPS contaminated possibly into the fraction, but to DNA itself. Intralesional injection with the bacterial DNA fraction caused regression of mouse IMC tumors, but the injection with the vertebrate DNA fraction did not. These findings prompted us to examine the biological activities of DNA samples from a variety of animals and plants, which were provided from other laboratories or purchased from manufacturers. All of the DNA samples from cells of 5 kinds of bacterium, 2 of virus and 4 of invertebrate augmented NK activity and induced IFN, more or less, in mouse spleen calls, while the DNA from 10 kinds of vertebrate, including 3 of fish and 5 of mammal, showed no such activities. The DNA from 2 species of plants, were also inactive. Possible mechanisms to explain the different biological activities of DNA from different cell sources were discussed based on our previous finding that the particular palindromic sequences with a G-C motif(s) are required for induction of IFNs and activation of NK cells with synthetic 30-mer oligonucleotides.

Adjuvants, Immunologic↗
PubMed1992

Antitumor activity of synthetic oligonucleotides with sequences from cDNA encoding proteins of Mycobacterium bovis BCG.

Thirteen kinds of 45-mer or 30-mer synthetic oligonucleotides with sequences randomly selected from the cDNA encoding three kinds of protein of Mycobacterium bovis BCG were tested for their antitumor activity in a murine tumor system. Six out of the 13 single-stranded oligonucleotides which contained one or more hexameric palindromic sequences showed strong antitumor activity while the others without palindromic structure did not. Namely, repeated intralesional injections of 100 micrograms of the 6 oligonucleotides caused regression of the established tumor but the other 7 were ineffective. When tumor cells were mixed with 100 micrograms of an effective oligonucleotide and injected into mice, tumor growth was markedly suppressed. These results suggested that palindromic structure is essential for the antitumor activity of the synthetic oligonucleotides.

Animals↗
PubMed1992

Soluble interleukin-6 receptor is released from receptor-bearing cell lines in vitro.

Soluble interleukin-6 receptor (sIL-6R) was found to be spontaneously released from human myeloma cell line U266 cells into culture supernatant, and was quantitatively measured with a fluorescence sandwich enzyme-linked immunosorbent assay employing antibodies specific to IL-6R. The supernatant IL-6R was generated only from IL-6R-positive cell lines; myeloma cell lines RPMI8226 and PRMI1788, and myelomonocytic cell lines U937, THP-1, and HL-60. In contrast, it was not released from the IL-6R-negative cells; T cell line Molt-4 and Burkitt lymphoma cell line Raji. SDS-PAGE analysis of the soluble IL-6R from U266 cells suggested a molecular weight of approximately 50-55 kDa, 25-30 kDa smaller than the mature cell surface receptor. These results suggest that the generation of soluble IL-6R may be a maker of myeloma cells and myelomonocytic cells.

Cell Line↗
PubMed1992

Oligonucleotide sequences required for natural killer cell activation.

Based on the previous finding that certain 30-mer single-stranded oligodeoxyribonucleotides (oligonucleotides) having particular 6-mer palindromic sequences could induce interferon-alpha and -gamma, and enhance natural killer activity, the present study was carried out to clarify the entire relationship between the activity and the sequence of 30-mer oligonucleotides. The results indicated that the activity depended critically on the presence of particular palindromic sequences including the 5'-CG-3' motif(s). The size and the number of palindromes as well as the extra-palindromic sequences also influenced the activity. An oligonucleotide with a 10-mer palindrome and extra-palindromic oligoguanylate sequences showed the strongest activity among the oligonucleotides tested.

Animals↗
PubMed1992

Contralateral development of acute subdural hematoma following surgery for chronic subdural hematoma--case report.

An unusual case of acute subdural hematoma developed after drainage of chronic subdural hematoma in a 71-year-old male. The acute subdural hematoma was located over a membranous layer in the subdural space similar to the outer membrane of the chronic subdural hematoma. Intraoperatively, bleeding from the bridging vein and oozing from the superior sagittal sinus were observed. The membranous layer probably separated from the dura mater following decompression after drainage of the contralateral hematoma, and this separation then damaged the bridging vein and superior sagittal sinus, resulting in the acute subdural hematoma.

Acute Disease↗
PubMed1992

[Brain metastasis of lung cancer with Eaton-Lambert syndrome--case report].

A case of brain metastasis of lung cancer with Eaton-Lambert syndrome (ELS) is reported. A 45-year-old male was admitted to the Department of Surgery in Kurume University Hospital on November 13, 1985, complaining of general fatigue. On admission, neurological examination revealed diplopia and fatigue of the extremities. The electromyogram (EMG) showed the waning phenomenon in low frequency repetitive stimulation (2Hz) and the waxing phenomenon in high frequency repetitive stimulation (10Hz, 20Hz). His clinical symptoms, radiological findings and EMG findings demonstrated lung cancer with ELS. Left pulmonary lobectomy with lymphnode dissection of the anterior mediastinum and pulmonary hilus was performed on December 4. Intraoperatively, the tumor was strongly adherent to a medium lymphnode. The patient experienced complete relief symptoms due to ELS. Histological examination disclosed a small cell carcinoma without lymphnode metastasis. He was discharged without any neurological deficits following chemotherapy on February 27, 1986. He was readmitted to the Department of Neurosurgery on August 29, 1986, because of the development of nausea and vomiting. Neurological examination demonstrated no abnormalities except for choked disc in the bilateral ocular fundi. The computed tomography scan revealed a metastatic brain tumor with a mural nodule and cyst. The tumor was totally removed on September 2. Histological examination revealed a typical appearance of small cell carcinoma. He followed a satisfactory postoperative course. He was discharged following radiation therapy on November 2, 1986, and was followed as an outpatient. He has no problem in daily life since then. Though the patient had an expanding metastatic brain tumor from lung cancer after the first operation, he experienced no symptoms due to ELS.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Neoplasms↗
PubMed1992

Evidence for postjunctional release of ATP evoked by stimulation of muscarinic receptors in ileal longitudinal muscles of guinea pig.

The origin of the ATP release evoked by muscarinic agonists and veratridine from longitudinal muscles of the guinea pig was assessed with muscarinic antagonists. Acetylcholine (ACh) (1 microM) and bethanechol (10 microM) produced an immediate and marked ATP release, which was almost completely blocked by atropine (0.3 microM) and by the M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (1 microM); release was only slightly affected by tetrodotoxin (0.6 microM). The bethanechol-evoked release of ATP was partly, but not significantly, inhibited by pirenzepine, a M1 muscarinic antagonist. Veratridine, an ACh releaser, also elicited a delayed ATP release, in a concentration-related manner. This ATP release was greatly antagonized by atropine and by Ca++ removal from the medium, implying mediation by endogenous ACh released after depolarization. In contrast, electrically evoked ACh release was enhanced by atropine and 4-DAMP. Bethanechol, unlike veratridine, failed to elicit measurable ACh release from the tissue. The contraction evoked by bethanechol was notably inhibited by atropine and 4-DAMP, but not by pirenzepine and AFDX-116, a M2 muscarinic antagonist, at a concentration of 0.3 microM. These findings suggest strongly that ATP is postjunctionally released from the ileal smooth muscles after stimulation of postsynaptic muscarinic receptors, presumably M3 receptors.

Acetylcholine↗
PubMed1992

[Emergency manual aspiration for severe hypertensive cerebellar hemorrhage].

We treated 16 patients with hypertensive cerebellar hemorrhage and coma, or deep coma. Their ages ranged from 44 to 79 years (mean age: 66.3 years). Manual aspiration was performed in 9 patients and suboccipital craniectomy was performed in 7 patients. The difference in outcome between the manual aspiration group and the suboccipital craniectomy group with severe hypertensive cerebellar hemorrhage was then evaluated. Manual aspiration was performed for 4 patients in coma, and 5 in deep coma, with a mean age of 69.0 years. The average size of the hematoma was 48.7mm and the mean volume was 31. 7ml on CT scan. The mean interval from admission to operation was about 60 minutes. The mean aspiration rate was 79.6% and 7 patients (77.8%) had a good response to drainage. The suboccipital craniectomy patients included 5 in coma, and 2 in deep coma, with a mean age of 63.4 years. The average size of the hematoma was 51.1 mm and the mean volume was 33.1 ml on CT scan. The mean interval from admission to operation was about 112 minutes and the mean evacuation rate was 86.4%. The results were as follows: 1) After manual aspiration, 5 patients (55.6%) had a good outcome and 2 patients (40%) with deep coma showed good recovery. In contrast, after suboccipital craniectomy only 2 patients (28.6%) had a good outcome and all of the deep coma patients showed poor recovery. 2) The outcome may be most strongly influenced by the duration from admission to operation. 3) All patients with a hematoma volume of over 30 ml had a poor outcome.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

[MRI findings of closed head injury in children; with special reference to the effect of central shearing force].

It is considered that shearing effect as introduced by Holbourn may produce central concussion, diffuse brain swelling and diffuse axonal injury according to its grade of force. MRI was performed in 38 children who had been admitted to our hospital during the previous 1 year for the treatment of closed head injury of varying severity. In 8 out of 38 cases, abnormal high signal intensity was observed in the medial and para-medial brain parenchyma on MRI. All of these 8 cases suffered from head trauma caused by motor vehicle accidents. They included 2 cases of cerebral concussion, 1 of diffuse brain swelling, and 5 cases of diffuse axonal injury. In 2 cases of cerebral concussion, MRI (T2 weighted) revealed only localized high intensity in the corpus callosum, while CT showed normal and subarachnoid hemorrhage only at the interposium. These two children had been unconscious for periods of 20 to 30 minutes. In one case of diffuse brain swelling, MRI (T2W) showed a slightly obscure border between gray and white matter due to generally increased intensity. In 5 cases of diffuse axonal injury, most of these cases manifested lesions at the corpus callosum, deep white matter, periventricular gray matter, pons, midbrain and the cerebellum as demonstrated by high signal intensity on MRI (T2W) while CT in the acute stage showed small hemorrhage at the corpus callosum, corticomedullary junction and mid-brain and in the ventricles. Among these, two cases also demonstrated subdural hematoma and cortical contusional hemorrhage. At 3-4 weeks after injury, the area of high intensity previously demonstrated in the deep white matter and the corpus callosum on MRI (T2W) was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Axons↗
PubMed1992

[Multiple intracranial aneurysms following radiation therapy for pituitary adenoma; a case report].

A report is made here on a rare case in which occurrence of multiple cerebral aneurysm was observed after radiotherapy. The case was that of a female aged 51 who was hospitalized with the chief complaint of consciousness disorder. The patient was discharged from the hospital 1 year before, after undergoing subtotal extirpation of a tumor through the transsphenoidal sinus in a case of pituitary adenoma, and post-operative radiotherapy (topical 50 Gy). Mild hyperlipidemia associated with hypothyroidism was observed by blood biochemical test during the patient's hospitalization, and multiple cerebral infarction was also observed by CT scanning and MR imaging. Therefore, conservative treatments including intensified endocrine-supplementing treatment, centering on thyroid hormone, were attempted. The patient's thyroid gland function and hyperlipidemia improved but the consciousness disorder persisted. Exacerbation of the consciousness disorder occurred suddenly 6 weeks after the patient's hospitalization. Subarachnoid hemorrhage and hydrocephalus were observed by CT scanning. Cerebral angiography detected a saccular aneurysm in the trifurcation of the right middle cerebral artery, 3 fusiform aneurysms in the periphery of the right middle cerebral artery, 2 fusiform aneurysms in the posterior cerebral artery and irregularity of wall width in the central artery including the terminal region of the internal carotid artery. These findings were not observed at the initial hospitalization and were considered to have been formed newly afterwards. Further, these findings were observed unexceptionally in all the radiated fields. The patient died 8 weeks after hospitalization, and no autopsy finding was obtained. From the above, we presumed that radiation vasculopathy caused by the radiation therapy made one year previously, had led to the formation of multiple cerebral aneurysms.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoma↗
PubMed1991

Development of a new host vector system in mycobacteria.

The hybrid plasmid pYT72/pYT92 constructed from an Escherichia coli plasmid pACYC177 and mycobacterial plasmid pMSC262 isolated from Mycobacterium scroflaceum strain W262 transformed both E. coli and BCG. Phage-sensitive mutants S-10 and S-20 isolated from BCG Tokyo strain showed higher frequency of transformation than the wild-type strain. Frequency of transformation was dependent on age of the culture and the electroporation condition used. Several deletion mutants were generated from pYT72/92 to determine the minimum region for the replication in the mycobacteria. A 2.3-kb fragment of pMSC262 was found to contain an essential region. Using this fragment and pACYC177, a small shuttle vector pYT937 containing two drug-resistance markers, kanamycin- and ampicillin-resistance, was constructed. pYT937 contains AatII, BamHI, BbvII, GsuI, HincII, PstI, ScaI and XbaI cloning sites.

Cloning, Molecular↗
PubMed1991

Granulocyte-macrophage colony-stimulating factor promotes the proliferation of human alveolar macrophages in vitro.

The effects of granulocyte-macrophage (GM-CSF) or macrophage-CSF on in vitro proliferation of human alveolar macrophages (AM) were evaluated. AM of healthy volunteers incubated with recombinant human GM-CSF revealed incorporation of [3H]thymidine in vitro. The maximum incorporation was observed at 20 U/ml of GM-CSF on day 3. The proportion of proliferating cells incubated with 20 U/ml of GM-CSF from day 3 to day 4 was 8 to 11% of the total, whereas 3 to 5% of cells proliferated without GM-CSF. The number of cell nuclei increased 1.30- to 1.68-fold in the initial 7 days during incubation with 20 U/ml of GM-CSF, whereas there was a 1.07- to 1.13-fold increase without GM-CSF. Conditioned media obtained by the incubation with human lung tissue exhibited similar effects as recombinant human GM-CSF on macrophages. The effects were completely abrogated by antibody against human GM-CSF. Immunohistochemically, GM-CSF was detected in lung cells including AM, alveolar epithelium, alveolar interstitial cells, and endothelial cells. In contrast, recombinant macrophage-CSF did not induce the proliferation of human AM, although it has been known to promote the proliferation of murine AM. These observations suggest that GM-CSF plays an important role among the regulatory factors that locally support the population of AM in human lungs.

Adult↗