Involvement of the Thalamus, Hippocampus, and Brainstem in Hypsarrhythmia of West Syndrome: Simultaneous Recordings of Electroencephalography and fMRI Study.

BACKGROUND AND PURPOSE: West syndrome is a developmental and epileptic encephalopathy characterized by epileptic spasms, neurodevelopmental regression, and a specific EEG pattern called hypsarrhythmia. Our aim was to investigate the brain activities related to hypsarrhythmia at onset and focal epileptiform discharges in the remote period in children with West syndrome using simultaneous electroencephalography and fMRI recordings. MATERIALS AND METHODS: Fourteen children with West syndrome underwent simultaneous electroencephalography and fMRI at the onset of West syndrome. Statistically significant blood oxygen level-dependent responses related to hypsarrhythmia were analyzed using an event-related design of 4 hemodynamic response functions with peaks at 3, 5, 7, and 9 seconds after the onset of each event. Six of 14 children had focal epileptiform discharges after treatment and underwent simultaneous electroencephalography and fMRI from 12 to 25 months of age. RESULTS: At onset, positive blood oxygen level-dependent responses were seen in the brainstem (14/14 patients), thalami (13/14), basal ganglia (13/14), and hippocampi (13/14), in addition to multiple cerebral cortices. Group analysis using hemodynamic response functions with peaks at 3, 5, and 7 seconds showed positive blood oxygen level-dependent responses in the brainstem, thalamus, and hippocampus, while positive blood oxygen level-dependent responses in multiple cerebral cortices were seen using hemodynamic response functions with peaks at 5 and 7 seconds. In the remote period, 3 of 6 children had focal epileptiform discharge-related positive blood oxygen level-dependent responses in the thalamus, hippocampus, and brainstem. CONCLUSIONS: Positive blood oxygen level-dependent responses with hypsarrhythmia appeared in the brainstem, thalamus, and hippocampus on earlier hemodynamic response functions than the cerebral cortices, suggesting the propagation of epileptogenic activities from the deep brain structures to the neocortices. Activation of the hippocampus, thalamus, and brainstem was still seen in half of the patients with focal epileptiform discharges after adrenocorticotropic hormone therapy.

Longitudinal Findings of MRI and PET in West Syndrome with Subtle Focal Cortical Dysplasia.

BACKGROUND AND PURPOSE: Despite the development of neuroimaging, identification of focal cortical dysplasia remains challenging. The purpose of this study was to show the longitudinal changes of MR imaging and FDG-PET in patients with West syndrome and subtle focal cortical dysplasia. MATERIALS AND METHODS: Among 52 consecutive patients with West syndrome, 4 were diagnosed with subtle focal cortical dysplasia on 3T MR imaging. MR imaging and PET findings were evaluated longitudinally at onset and at 12 and 24 months of age. RESULTS: At the onset of West syndrome, MR imaging demonstrated focal signal abnormalities of the subcortical white matter in 2 patients. In the other 2 patients, focal subcortical high-intensity signals became visible on follow-up T2WI as myelination progressed. PET at onset showed focal cortical hypometabolism in 3 patients, with 1 of these patients also having focal hypermetabolism and 1 having normal findings. On PET at 24 months, hypometabolism persisted in 2 patients and disappeared in 1, and hypermetabolism disappeared in 1. In 1 patient with normal MR imaging and PET findings at onset, focal hyperintensity and hypometabolism first appeared at 24 months of age. The findings on MR imaging and PET in these patients evolved differently with brain maturation and the clinical course. CONCLUSIONS: Subtle focal cortical dysplasia can be undetectable on MR imaging at the onset of West syndrome and is not always accompanied by hypometabolism or hypermetabolism on PET. Longitudinal MR imaging and PET studies may be useful for detecting such lesions. Even in West syndrome with a congenital structural abnormality, PET findings evolve differently with brain maturation and the clinical condition.

Differential regulation of carcinoembryonic antigen and biliary glycoprotein by gamma-interferon.

Carcinoembryonic antigen (CEA), biliary glycoprotein (BGP), and non-specific cross-reacting antigen (NCA) are three closely related cell surface glycoproteins induced by gamma-interferon (IFN-gamma) in colonic epithelial cells. Maximal induction of CEA by IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the colon carcinoma cell line HT-29 occurs at 5-6 days with maximal secreted levels at 14 ng/ml for IFN-gamma and 20 ng/ml for TNF-alpha. Cell viability was reduced to 67% of controls for TNF-alpha and to 36% for IFN-gamma. Dose-response curves showed maximal induction of CEA at 500 units/ml for TNF-alpha and at 200 units/ml for IFN-gamma. Combinations of the two lymphokines revealed that the CEA induction effects were additive and the cytotoxicity effects were synergistic. Northern blot analysis of HT-29 cells treated with IFN-gamma and probed with specific probes for BGP, CEA, and NCA showed a 2-fold increase in mRNA level for BGP, and a greater than 10-fold induction for CEA and NCA. Similar results were obtained for the SW403 cell line, but in the case of the LS174T cell line, CEA mRNA levels remained constant before and after IFN-gamma treatment, while BGP and NCA mRNA levels increased by 2-5-fold. Polymerase chain reaction analysis of the four alternatively spliced transcripts of BGP revealed no differential induction of one transcript over another by IFN-gamma. A comparison of the kinetics of induction of the mRNA levels for BGP and CEA by IFN-gamma in the HT29 cell line revealed a half-time of < 6 h for BGP and 48 h for CEA. The induction of CEA mRNA was completely inhibited with either cycloheximide (protein synthesis inhibitor) or actinomycin D (RNA synthesis inhibitor), but the induction of BGP mRNA was superinduced by cycloheximide. The difference in the kinetics of induction and effect of cycloheximide on CEA and BGP mRNAs suggest that the two genes are regulated differently in the same cell line. We conclude that the regulation occurs mainly at the posttranscriptional level for CEA and involves mRNA stability. BGP regulation may be more complex, involving transcriptional and posttranscriptional regulation, and more closely resembles the regulation of MHC class II mRNA by IFN-gamma in epithelial cells. The mRNA stability effects may be mediated by the dramatically different sequences present in the 3'-untranslated regions of CEA and BGP.

3T3 fibroblasts release a low-molecular-weight inhibitory peptide of lymphocyte DNA synthesis into serum-free culture medium.

3T3 fibroblasts release a novel factor into serum-free culture medium, which strongly suppressed concanavalin A-induced thymocyte DNA synthesis. This activity was highly purified by gel filtration, ion exchange and thin-layer chromatography and was characterized as a 1 kDa heat-stable peptide. Although this peptide suppressed lymphocyte DNA synthesis when added relatively early after lectin-stimulation, the cell viability was not changed significantly. The peptide considerably repressed DNA synthesis of some mammalian fibroblast cells, but malignant-transformed cells were not affected.

T cell growth factors from adult T cell leukemia virus-transformed cell lines.

Some characteristics of T cell growth factors derived from adult T cell leukemia virus (ATLV)-transformed cell lines, MT 1 and MT 2 were analyzed. MT 1 cells release significant interleukin 2 (IL 2) activity into the culture medium, which showed the same elution pattern of gel filtration and isoelectric focusing of IL 2 from lectin-stimulated normal human lymphocytes. This activity was also detected in the cell extract of MT 1. In contrast, MT 2 cell line did not produce IL 2 activity, but non-IL 2 type growth factor was observed. The significance of these factors from MT cell lines is discussed from the viewpoint of 'autokine' in ATLV-transformed cells.

Large-molecular-weight thymocyte-activating factors in extracellular matrix of SV40-transformed human embryo fibroblasts.

It has been reported that the major thymocyte-activating factors at 10-15 kDa exist in the culture medium of SV40-transformed human embryo fibroblasts. However, these factors could not be detected in the extracellular matrix (ECM) from the same transformed cells, but the 30-40 kDa factors were found. They showed a similar lectin requirement, charge heterogeneity and Concanavalin A-binding property as compared with the factors reported previously. They were separated from fibroblast DNA synthetic activities in ECM and did not contain interleukin 2 activity. A possible relationship between 30-40 kDa factors in ECM and 10-15 kDa factors in the culture medium is discussed.

Modulation of early mRNA synthesis in lectin-induced lymphocyte activation--an association with a possible factor.

Two phases of early mRNA synthesis were observed in lectin-induced lymphocyte activation. At 0-2 h, lectin-treated lymphocytes showed a considerable decrease in nuclear RNA polymerase II activity as compared with that of untreated cells and, at later periods, a reverse increase was observed. Such modulation of mRNA synthesis is not due to the change of RNA polymerase II, but a possible factor in nuclei. This factor represses RNA chain initiation and stimulates chromatin-dependent RNA elongation catalyzed by RNA polymerase II. It was characterized to be a 10-30 kDa sugar-containing molecule and the physiological significance of this factor is discussed from the viewpoint of the transcriptional regulation.

A decrease of RNA synthesis during macrophage activation with lipopolysaccharide--an association with novel regulating factors.

RNA synthesis during macrophage activation with lipopolysaccharide (LPS) was analyzed in mice. The RNA synthesis of macrophages was considerably increased early after LPS stimulation, but drastically decreased at a later period. The decrease of RNA synthesis was observed in mouse strains responding to LPS such as BALB/c, ATL and ddY, but not in nonresponder strain, C3H/HeJ. Furthermore, the decrease of RNA synthesis occurred in both alpha-amanitin resistant and sensitive RNA polymerase reactions. However, RNA polymerases in nuclei were not changed significantly in LPS-treated macrophages, but the factors affecting RNA polymerase reactions were drastically changed. Amongst these factors, the stimulating factors were decreased and a novel repressing factor was remarkably increased. The factors were partially characterized and their significance in macrophage activation is discussed.

Heterogeneous macrophage-activating factors from extracellular matrix of human embryo fibroblasts.

Previously, the author found a low-molecular-weight stimulating activity for macrophages in the extracellular matrix of a human embryo fibroblast cell strain. It showed stimulatory effects on the attachment ability of macrophages and their production of superoxide anion (-O2) and [3H]UTP incorporation (Zool. Sci. 3, (1986) 621-626). When the author further analyzed this activity, it was found to be associated with two different factors which could be separated by ion exchange chromatography. Their molecular weights were estimated to be about 5 kDa and 10 kDa by gel filtration chromatography; they are designated fibroblast-derived macrophage activating factor I and II (FMAF I and II). They were sensitive to trypsin, but resistant to heat and did not show significant colony stimulating factor and interferon activities. In addition, FMAF II exhibited much higher activity for granulocyte RNA synthesis than FMAF I. The properties of these factors are compared with other factors reported previously and the biological significance of the factors is discussed.

Dual functions of DNA synthesis inhibitory peptide in fetal calf serum.

A novel factor in fetal calf serum was found which showed the dual inhibitory effects on DNA synthesis of murine lymphocytes and some species of bacteria. The factor was purified by gel filtration, ion exchange and thin-layer chromatography and characterized to be a 1 kDa basic peptide. In addition, this factor is not nonspecific to inhibitors, thymidine or polyamines. The modes of action and the spectrum of target cells were analyzed and its biological significance is discussed.

A human embryo fibroblast-derived peptide suppresses the zymosan-induced biochemical activation in human polymorphonuclear leukocytes.

Previously, a low-molecular-weight peptide was found in the serum-free culture media conditioned by a human embryo fibroblast cell strain (YH-1), which showed inhibitory effects on the proliferation and functions of B and T lymphocytes (Okai et al. (1987) Zool. Sci. 4, 99-105). In this paper, the inhibitory effect of this peptide on the functional activation in human polymorphonuclear leukocytes (PMNs) is documented. This peptide suppressed the opsonized zymosan-induced generation of chemiluminescence and superoxide anion and concomitant RNA synthesis. However, this peptide did not show significant cytotoxic effects on human PMNs as judged by a cytoplasmic enzyme-releasing assay. These results indicate that a fibroblast-derived peptide reported previously has a suppressive activity on the functional activation of human PMNs. The immunological significance of this finding is discussed.

Different inducing activities for cytotoxic T cells by heterogeneous thymocyte-activating factors from SV40-transformed human embryo fibroblasts.

Thymocyte-activating factors are produced by human embryo fibroblasts and their production is enhanced by SV40-induced transformation (Okai, Gotoh and Yamashita, Immununol. Lett. (1985), 9, 153-159). The activities for thymocyte DNA synthesis in the culture medium from SV40-transformed cells are separated into the two fractions by a DEAE Sephadex A-25 column chromatography eluting at 150 and 500 mM NaCl. Each fraction contains the heterogeneous molecular weight activities as judged by a Sephadex G-100 column chromatography. In the fraction eluted at 150 mM NaCl, the inducing activities for cytotoxic T cells by the factors are dependent upon their molecular weights; the larger factors exhibit much higher cytotoxic activity than that of the smaller factors. In contrast, the heterogeneous molecular weight factors eluted at 500 mM NaCl do not show the remarkable difference of their biological activities. In addition, the inducing activities by thymocyte-activating factors are also dependent upon their charge properties. These different inducing activities for cytotoxic T cells by thymocyte-activating factors is discussed from the aspect of the immunological regulation.

Identification of colony-forming activities of various hemopoietic cells in the serum-free culture medium of a human embryo fibroblast cell strain.

Ralph et al. reported that human cell lines for the production of colony stimulating factor cannot secrete the factor in serum-free media (Blood 68, 633-639, 1986). However, we found various colony-forming activities to be present in the serum-free culture medium of a human embryo fibroblast cell strain (YH-1). When a soy bean trypsin inhibitor was added to the serum-free media, semisolid cultures of mouse and human bone marrow cells exhibited a considerable degree of colony formation by macrophages and to a lesser extent by other cells. A difference in apparent molecular weights in macrophage colony-forming activities by YH-1 cells from serum-free and serum-containing cultures was observed. The physiological significance of these findings is discussed in terms of regulation of hemopoietic cell differentiation by human embryo fibroblasts.

Thymocyte activating factors from SV40-transformed human embryo fibroblasts.

A novel factor was found in the medium conditioned by SV40-transformed human embryo fibroblasts, which stimulate concanavalin A-induced thymocyte DNA synthetic response. This activity was estimated to be 10-15 kD and divided into two activities by ion exchange chromatography. One of them is a protein molecule and the other is a glycoprotein. In addition, these activities are not derived from the growth factors reported previously such as interleukin 2 (Morgan, R., Ruscetti, F. and Gallo, R. C. (1976) Science 193, 1007-1008) and transforming growth factor (De Larco, J. E. and Todaro, G. J. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4001-4005).

Effects of a purified cecropin D from a Chinese silk moth on growth, function and differentiation of murine hemopoietic cells.

The effects of cecropin D, a small basic peptide isolated from a Chinese oak silk moth, on the functions or differentiation of mammalian hemopoietic cells are described in the present paper. This peptide suppressed lectin-induced DNA synthesis of murine splenocytes in a dose-dependent manner without any significant cytotoxic effects. It also exhibited inhibitory effects on antibody production in lipopolysaccharide-stimulated lymphocytes and on colony formation of hemopoietic progenitor cells in plasma clots culture. These results indicate that cecropin D can regulate growth, function and differentiation of murine hemopoietic cells. The biological significance of this finding is discussed from the comparative immunological point of view.

Potent suppressing activity of the non-polyphenolic fraction of green tea (Camellia sinensis) against genotoxin-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002)--association with pheophytins a and b.

Antigenotoxic and antimutagenic activities of green tea extract and tea-derived polyphenols have been studied using in vitro and in vivo experiments. However, antigenotoxic substances in the non-polyphenolic fraction of green tea have been poorly elucidated. In the present study, the effect of the non-polyphenolic fraction of green tea on genotoxin-induced umu C gene expression was analyzed using a tester bacteria, and potent antigenotoxic substances in the non-polyphenolic fraction were identified. The non-polyphenolic fraction of green tea showed strong suppressive activities against umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1) or mitomycin C (MMC) in the presence or absence of S9 metabolizing enzyme mixture. The non-polyphenolic fraction of green tea exhibited major two-color bands in a silica gel TLC and they were identified as chlorophyll-related compounds, pheophytins a and b, judged by their specific colors, Rf values in silica gel TLC and absorption spectra. These pigments showed significant suppressive activities against umu C gene expression in tester bacteria induced by Trp-P- and MMC in a dose-dependent manner. These results suggest that the non-polyphenolic fraction of green tea contains pheophytins a and b as potent antigenotoxic substances.

Potent suppressive activity of pheophytin a and b from the non-polyphenolic fraction of green tea (Camellia sinensis) against tumor promotion in mouse skin.

Chlorophyll-related compounds pheophytin a and b have been recently identified as antigenotoxic substances in the non-polyphenolic fraction of green tea (Camellia sinensis), which suppressed umu C gene expression in tester bacteria induced by various genotoxins (Okai and Higashi-Okai, Cancer Lett. 118 (1997) 117-123). In the present study, the authors analyzed in vivo and in vitro effects of pheophytin a and b from the non-polyphenolic fraction of green tea on tumor promotion in mouse skin as follows. (1) When pheophytin a and b from green tea were topically applied prior to each treatment with a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on BALB/c mouse skin initiated by 7,12 dimethylbenz[a]anthracene (DMBA), they caused suppression in a dose-dependent fashion against skin tumorigenesis. (2) Pheophytin a and b exhibited significant suppressions against TPA-induced inflammatory reaction, such as edema formation, in BALB/c mouse ear skin in a dose-dependent manner. (3) Pheophytin a and b from green tea showed inhibitory effects against early induction of ornithine decarboxylase (ODC) in BALB/c mouse skin fibroblasts caused by TPA. These results suggest that pheophytin a and b from the non-polyphenolic fraction have potent suppressive activities against tumor promotion in mouse skin.

All-trans beta-carotene enhances mitogenic responses and ornithine decarboxylase activity of BALB/c 3T3 fibroblast cells induced by tumor promoter and fetal bovine serum but suppresses mutagen-dependent umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002).

Although previous epidemiological studies have indicated that beta-carotene is an important agent for the chemical prevention against carcinogenesis, a recent prospective study has strikingly suggested that supplementation with beta-carotene significantly increased the incidence of some types of cancer (The alpha-Tocopherol and beta-Carotene Cancer Prevention Study Group, New Engl. J. Med., 330 (1994) 1031-1035). To analyze the discrepancy of this problem, the authors analyze the effects of beta-carotene on biochemical and biological events associated with carcinogenesis by in vitro experiments. (1) All-trans beta-carotene enhanced the proliferation and DNA synthesis of BALB/c 3T3 cells induced by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and fetal bovine serum, although beta-carotene itself did not show mitogenic activity. (2) All-trans beta-carotene caused a remarkable stimulation for the early induction of ornithine decarboxylase (ODC) activity after the stimulation of TPA and fetal bovine serum. (3) All-trans beta-carotene exhibited significant antimutagenic activity which suppresses umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by a typical mutagen, 2-aminoanthracene (2-AA). These experimental results suggest that all-trans beta-carotene might cause beneficial and harmful effects on different phases of carcinogenesis.

Suppressive effects of the extracts of Japanese edible seaweeds on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) and tumor promotor-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells.

Some of epidemiological data indicated that ubiquitous consumption of seaweeds in Japan may be a possible protective factor against some types of tumor. To analyse this problem, the authors studied the antimutagenic and antitumor promotion activities in methanol-soluble extracts of typical edible seaweeds which showed suppressive effects on 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1)-induced umu C gene expression in SOS response of Salmonella typhimurium (TA 1535/pSK 1002) and 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells. Although eight varieties of edible seaweeds including chlorophyta, Phaenophyta and Rhodophyta showed significant antimutagenic and antipromotion activities, they expressed the activities different from each other. Among these seaweeds, Enteromorpha prolifera ('Sujiaonori' in Japanese) and Porphyra tenera ('Asakusanori') showed relatively strong suppressive activities in both antimutagenic and antipromotion assays compared with other seaweeds. These seaweeds contained considerable amounts of beta-carotene as a possible active principle with anticarcinogenic activity. This compound was partially associated with the antimutagenic activity in the seaweed extract, but did not contribute to the antipromotion activity of seaweed extract under our experimental conditions. These results strongly suggest that Japanese edible seaweeds have possible antimutagenic and antipromotion activities probably associated with antitumor activity.

Identification of antimutagenic substances in an extract of edible red alga, Porphyra tenera (Asakusa-nori)

Recently, a relatively strong antimutagenic activity has been detected in the extract of Porphyra tenera (Asakusa-nori in Japanese) which showed a suppressive effect on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002 (Okai et al. (1994) Cancer Lett., 87, 25-32). In the present paper, we analyzed the active principles for the antimutagenic activity in an extract of Porphyra tenera and detected three color spots on a silica gel TLC plate which indicated very similar Rf values and absorbance spectra of standard pigments such as beta-carotene, chlorophyll a and lutein. The seaweed pigments recovered from preparative silica gel TLC corresponding to beta-carotene, chlorophyll a and lutein exhibited significant suppressive activities against mutagen-induced umu C gene expression and combined treatment with these pigments showed an additive effect compared with single treatment with each pigment. Furthermore, the standard pigments prepared from other biological sources also exhibited similar anti-mutagenic activities. The significance of this finding is discussed from the protective role of seaweed pigments against mutagenesis probably associated with carcinogenesis.