Tatsuya Kondo

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.

Neuronal localization of fibroblast growth factor-9 immunoreactivity in human and rat brain

Fibroblast growth factor-9 (FGF-9) is a relatively new member of the FGF family isolated from the conditioned medium of a human glioblastoma cell line as a secreting-type factor that exhibits a growth-stimulating effect on cultured glial cells. In order to elucidate the roles of FGF-9 in the central nervous system, we investigated in detail the distribution of FGF-9 proteins in the normal human and rat brains by immunohistochemistry using two different antibodies specific to FGF-9. In both human and rat, a strong expression of FGF-9 immunoreactivity was localized mainly in neurons throughout the normal brain. Immunoreactive glial cells were rarely encountered. In the human brain, strong and uniform immunoreactivity was observed in neurons of cerebral cortex, hippocampus, substantia nigra, motor nuclei of the brainstem, and Purkinje cell layer. A detailed mapping in the rat brain showed a distribution of FGF-9 immunoreactivity in a widespread population of neurons, though the intensity varied between different locations and even among the same nucleus. The most prominent expression in rat was observed in neurons of the mitral cell layer of the olfactory bulb, red nucleus, mesencephalic trigeminal nucleus, motor trigeminal nucleus, facial nucleus, reticular nucleus and Purkinje cell layer. These findings suggest that FGF-9 plays an important role in the central nervous system and may have a potential function closely connected to neurons in the normal brain.

Chromosome studies in 70 brain tumors with special attention to sex chromosome loss and single autosomal trisomy

Chromosome analysis was performed on 70 brain tumors. Thirty-six tumors showed clonal karyotypes characterized by many autosomal abnormalities; 20 meningiomas revealed monosomy 22 as a consistent abnormality, and 12 gliomas showed various abnormalities frequently involving chromosomes 3, 7, and 22. Of the remaining 34 tumors, 24 had normal karyotypes and 10 had clonal cells with loss and/or an extra sex chromosome with single trisomy of chromosomes 3, 6, 7, or 14. Sex chromosome aneuploidy was mostly due to loss of the Y or an X chromosome and was observed in 25 tumors, usually together with autosomal abnormalities. In these tumors the average frequency of cells with sex chromosome aneuploidy was 52%, with a range from 12% to 100%. Loss of the Y was found significantly more frequently in tumors of aged patients. Chromosome analysis in materials subcultured for a long period showed a tendency for cellular selection in which clonal cells with many autosomal abnormalities disappeared rapidly and karyotypes having loss or an extra sex chromosome and/or trisomy 7 were present in an increasing proportion with advance of cell generations in vitro. We infer that the cells having loss of one sex chromosome or trisomy 7 have a proliferative advantage. And that cells bearing only these abnormalities may exist in normal brain tissue more abundantly than in any other body tissue. The possibility of tissue-specific aneuploid mosaicism in the normal tissue would allow an alternative interpretation for simple autosomal trisomy in solid tumors.

Primary ductal adenocarcinoma of the lacrimal gland

The morphological similarity of salivary and lachrymal gland tumors is known. Many clinicopathological studies and characteristics of salivary duct carcinoma, which bears histological similarlties to mammary duct carcinoma, have been recently reported; however, only one case of lacrimal duct carcinoma is reported. A second case of lacrimal duct carcinoma is presented. A 67‐year‐old male with a painlass mass in the right upper eyelid underwent total removal of the tumor mass. Microscopic examination of the tumor mass revealed ductal adenocarcinoma of the lacrimal gland, which was the equivalent of salivary duct carcinoma. The Immunohistological studies of the lacriml duct carcinoma showed similar results to those reported for salivary duct carcinoma. The recurrent tumor in the subdural spaces was removed 2 years after the initial surgery and the patient is followed as an outpatient.

Expression and Growth Stimulatory Effect of Fibroblast Growth Factor 9 in Human Brain Tumors

OBJECTIVE Fibroblast growth factor 9 (FGF-9) is a relatively new member of the FGF family isolated from the conditioned medium of a human glioblastoma cell line as a secreting type factor that exhibits a growth-stimulating effect on primary glial cells. To elucidate the roles of FGF-9 in human brain tumors, the expression and biological activities of FGF-9 were studied using culture cells and surgically obtained tumor specimens. METHODS Measurement of FGF-9 and basic FGF in conditioned media of cell cultures was performed by using a sandwich enzyme immunoassay. The mitogenic effect of FGF-9 was evaluated by cell growth studies. FGF-9 expression in vivo was demonstrated by immunohistochemistry. RESULTS One of 4 glioma cell lines and 4 of 16 human meningiomas examined actually secreted detectable amounts of FGF-9 proteins. In comparison, basic FGF production was detected from 3 of 4 glioma cell lines and 11 of 16 human meningiomas. Similarly to basic FGF, recombinant human FGF-9 significantly stimulated the in vitro cell proliferation in three of four glioma cell lines investigated in a dose-dependent manner. A time course growth study using U87 MG cells revealed an accelerated growth stimulation by FGF-9 after Day 4. The growth stimulatory activity was also shown in three of four human meningiomas studied. Moderate to strong immunoreactivity for FGF-9 was observed in 40 (82%) of 49 human brain tumors examined irrespective of origin, tumor type, grade of malignancy, or whether initial or recurrent. In contrast, strong immunostaining was localized in neurons in the normal human cerebral cortex. CONCLUSION The present findings suggest that FGF-9 may be involved in the biology of human brain tumors with a possible importance in tumor cell growth. Whether the growth factor is more generally involved in oncogenesis of human tumors awaits further investigation.

Ganglioside Composition and Its Relation to Clinical Data in Brain Tumors

The ganglioside composition of 15 cases of meningioma, 15 cases of astrocytoma, 5 cases of neurinoma, 4 cases of ependymoma, 3 cases of metastatic brain tumor and 1 case each of mixed glioma, oligodendroglioma, medulloblastoma, embryonal carcinoma, and cultured glioma cell line were analyzed by thin-layer chromatography. The GM2, GD3, and GD2 content of the tumors was determined using specific monoclonal antibodies (MAb). Cases were grouped according to the difference in ganglioside pattern and various clinical features. In meningiomas and astrocytomas, GM3 and GD3 were the major gangliosides. The tumor content of the rather simple gangliosides (GM3, GM2, GD3, GD2) increased or was almost equal to that of normal tissue (leptomeninges tissue in the case of meningiomas, and brain tissue in the case of astrocytomas), while the tumor content of complex gangliosides (GM1, GD1a, GT1a, GT1b) decreased as compared with normal tissue. The GM3 content of meningiomas increased in middle-aged patients, who comprised the majority of the patients with these tumors. The GD2 content decreased in middle-aged patients with initial symptoms of meningioma within a year. The GM3 content of astrocytomas decreased in patients who underwent radiotherapy. The amount of GM3 and GD3 increased in small tumors. GM3 may be related to the early proliferative stage. The ganglioside patterns of brain tumors are shown in this study to differ according to clinical features and also to be changeable in their clinical courses.

Outcomes of 24 patients with subarachnoid hemorrhage aged 80 years or older in a single center.

OBJECTIVE In the developed countries, elderly population is rapidly increasing, but outcomes of elderly patients with subarachnoid hemorrhage (SAH) remain unclear. PATIENTS AND METHODS We retrospectively reviewed the medical records of non-traumatic SAH patients aged 80 years or older, who were hospitalized in a single center between 2000 and 2005. RESULTS There were 24 patients (80-92 years old and 83% female), representing 8.8% of all non-traumatic SAHs (n=272). Of those, six patients received an intervention (five clipping and one endovascular coiling) and the remaining 18 patients were managed conservatively. The patients who received an intervention were younger and had a better consciousness at presentation. Early mortality rate within 30 days after SAH was higher in the conservative group (61% [11/18] and 17% [1/6], p=0.155). At 6 months, mortality rate was significantly higher in the conservative group (83% [15/18] and 33% [2/6], p=0.038), and independence rate was higher in the intervention group (33% [2/6] and 0% [0/18], p=0.054). Logistic regression analysis showed that age and degree of consciousness on admission were significant predictor of outcome in 4 weeks, and that receiving intervention was significant predictor of outcome in 6 months. CONCLUSION In elderly SAH patients with good clinical condition at presentation, an active intervention may improve the outcome.

Large cysts of the pineal gland: report of two cases.

Two cases of large, nontumorous cysts of the pineal gland are reported, the histopathology of which was confirmed using surgically resected specimens. Both patients were middle-aged, and the pineal cysts were found incidentally. The histopathological findings in the two cases were strikingly similar to each other and were characterized by the following points: 1) the cyst wall typically consisted of three layers, namely, collagenous fibers, glia-like cells, and normal pineal cells; 2) the cyst wall was relatively thin, approximately 100 to 300 microns in thickness; and 3) deposits of calcification almost always existed on the side of the layer of collagenous fiber. Because these findings are clearly different from those previously reported, large benign cysts of the pineal gland, or at least some of them, may constitute a new pathological entity. It is important to consider this possible pathological diagnosis when dealing with pineal cysts.

Stimulation of Thrombopoiesis in Mice by Fibroblast Growth Factor 9

Fibroblast growth factor 9 (FGF-9), a novel member of the FGF family, was found to have thrombopoietic activity in vitro and in vivo. In an in vitro megakaryocyte colony-stimulating factor assay, anti-mouse interleukin-6 (IL-6) monoclonal antibody neutralized FGF-9 activity. This suggests that the activity may be exerted via IL-6 induction. BALB/c mice that received subcutaneous FGF-9 injections of 4 to 100 micrograms/day for 2 weeks showed a dose-dependent transient increase in peripheral platelet counts 10 to 12 days after the first treatment. Histologic studies showed a marked increase in megakaryocytes in bone marrow and extramedullary hematopoiesis in the spleen and the liver. Examination of changes in the DNA content of bone marrow megakaryocytes revealed that the ploidy distribution underwent a marked shift 3 days after FGF-9 injection, with a large increase in the 2N megakaryocyte population. The major modal ploidy shifted from the normal 16N to 2N. The number of megakaryocyte progenitor cells in FGF-9-treated mice increased up to 1.5-fold in the bone marrow and 10-fold in the spleen on day 6. These results indicate that FGF-9 acts on the in vivo proliferation of megakaryocytes.