Hirofumi Naganuma

Trastuzumab (Herceptin) Enhances Class I-Restricted Antigen Presentation Recognized by HER-2/neu-Specific T Cytotoxic Lymphocytes

Purpose: Numerous examples from animal models and clinical trials showed that HER-2-derived peptides are naturally processed as a CTL epitope and can be recognized by tumor-specific CTLs in several tumors with HER-2 overexpression. The humanized anti-HER-2 monoclonal antibody, Herceptin, has been designed to specifically antagonize the HER-2 function by directing against the extracellular domain of the HER-2 protein. One of the actions of Herceptin includes the internalization and degradation of HER-2, which might increase the amount of HER-2-derived peptides available for loading to MHC class I. Experimental Design: In the present study, we investigated how Herceptin treatment of HER-2-overexpressing targets affects lysis by HER-2-specific CTLs. Results: We showed that Herceptin sensitized HER-2-overexpressing tumors to lysis by HLA-A2-restricted or HLA-A24-restricted CTLs, without any effect of the expression of MHC class I, costimulatory molecules, adhesion molecules, or TAP-1 on the targets. Furthermore, the enhancement of cytolytic activity with Herceptin was inhibited by addition of a specific proteasome inhibitor, lactacystin. Conclusions: These results suggested that Herceptin treatment might enhance the class I-restricted presentation of endogenous HER-2 antigen via the proteasome step, resulting in higher susceptibility of HER-2-overexpressing tumors to lysis by the HER-2-specific CTLs.

Correlation of thrombospondin‐1 and transforming growth factor‐β expression with malignancy of glioma

The expression of thrombospondin‐1 (TSP‐1) and its role in gliomas have not been well examined. In the present study TSP‐1 expression in a panel of malignant glioma cell lines and the expression of TSP‐1 and transforming growth factor (TGF‐β) proteins in low‐grade and malignant glioma tissues were investigated. Reverse transcription‐polymerase chain reaction analysis showed that nine of nine malignant glioma cell lines expressed TSP‐1 mRNA, and seven of nine glioma lines expressed TSP‐2 mRNA. Production and secretion of TSP‐1 were examined in the T98G glioblastoma cell line by western blot analysis. Total TSP‐1 protein content in the supernatant was 10 times higher than that in the cell lysate. Secretion of TSP‐1 was examined in these glioma cell lines by western blot analysis. All glioma lines secreted significant levels of TSP‐1. Bioassay showed that all tumor lines had the capacity to activate latent TGF‐β. Localization of TSP‐1, TGF‐β1, ‐β2, and ‐β3 was examined immunohistochemically in surgically resected glioma tissues, including 11 glioblastomas, six anaplastic astrocytomas, and eight astrocytomas. Most glioblastomas expressed high levels of both TSP‐1 and TGF‐β. Anaplastic astrocytomas expressed moderate levels of TSP‐1 and TGF‐β. Most malignant gliomas expressed various levels of TGF‐β1, ‐β2, and ‐β3. The expression of both proteins, however, was weak in low‐grade gliomas. Normal brain tissues around the tumors were negatively or very weakly positively stained for TSP‐1 and TGF‐β. These results indicate that most malignant glioma cells express TSP‐1 in vitro and in vivo, and the expression of TSP‐1 and TGF‐βin vivo correlates with the histologic malignancy of glioma. Overexpression of both TSP‐1 and TGF‐β may increase the biologic malignancy of malignant gliomas, through generating the active form of TGF‐β in tumor tissues.

Spontaneous resolution of chronic subdural hematomas.

Four patients with chronic subdural hematomas, all of which resolved spontaneously, were followed from the time of injury to resolution of the chronic subdural hematoma. Periodic computed tomographic (CT) scans showed spontaneous resolution 78, 174, 231, and 326 days after the development of the chronic subdural hematoma, respectively. Features of the CT scans and a possible mechanism of spontaneous resolution are discussed.

Diagnosis of germinal neoplasm in the thalamus and basal ganglia

Germinal neoplasms originating in the thalamus and basal ganglia were histologically verified by stereotactic biopsies in five cases and by other methods in three cases. Immunoperoxidase staining was performed on the tumors using antibodies against human chorionic gonadotropin and placental alkaline phosphatase. The presence of human chorionic gonadotropin was demonstrated in one germinoma and two mixed tumors, but not in three germinomas. Placental alkaline phosphatase was demonstrated to be present in four germinomas and one mixed tumor. Stereotactic biopsy specimens can be studied immunohistochemically, and the placental isoenzyme of alkaline phosphatase appears to be a new tumor marker for germinoma.

Germ cell tumor in the basal ganglia: Immunohistochemical demonstration of α-fetoprotein, human chorionic gonadotropin, and carcinoembryonic antigen

A case of germ cell tumor in the basal ganglia and its vicinity was successfully treated surgically and by radiation. alpha-Fetoprotein, human chorionic gonadotropin, and carcinoembryonic antigen were found in the tumor tissue, using the immunoperoxidase method. The presence of three immunohistochemically demonstrated tumor markers in a single germ cell tumor is extremely unusual. These results strongly suggest a cell differentiation or transformation in germ cell tumors.

Pathobiology of intracranial germ-cell tumors: immunochemical, immunohistochemical, and electron microscopic investigations.

SummaryIn an attempt to clarify the pathobiology of intracranial germ-cell tumors, we studied 56 patients with these tumors, both clinically and pathologically. Our evaluations included immunochemical, immunohistochemical, and electron microscopic observations. Thirty tumors originated in the pineal region, ten in the suprasellar region, nine in the basal ganglia, and two in other regions. Five had double sites of origin. Histologically, there were 39 germinomas, six teratomas, four embryonal carcinomas, two choriocarcinomas and five mixed tumors. Among tumors tested immunochemically, serum carcinoembryonic antigen (CEA) was positive in 3.4%, serum alpha-fetoprotein (AFP) in 25.8%, and serum human chorionic gonadotropin (HCG) in 32.1%. In CSF, CEA was positive in 14.3%, AFP in 26.3%, and HCG in 75%. One third of germinomas were positive for serum HCG, and 88.9% for CSF HCG. One half of teratomas were positive for CEA of CSF. In immunohistochemical testing, the positive rates for CEA, AFP, HCG, and placental alkaline phosphatase (PLAP) were 26.9%, 11.5%, 24.1%, and 55.6%, respectively. Most teratomas were positive for CEA and the reactions were prominant in gland-like structures. HCG-positive syncytial cells were found in three germinomas and two embryonal carcinomas as well as in choriocarcinomas. The majority of germinomas were positive for PLAP and the reactive sites were tumor cell membranes and cytoplasm. On electron microscopy, germinomas were least differentiated, followed in order by embryonal carcinomas, choriocarcinomas, and teratomas. Further clinical and pathological studies will be necessary for a better understanding of the biology of these tumors.

Suppression of TGF-β1 in Human Gliomas by Retroviral Gene Transfection Enhances Susceptibility to LAK Cells

Human glioma cell line, Onda 10 produces TGF-β1. TGF-β1 has a biological role for the immunosuppression of the host. We have investigated whether suppression of TGF-β1 on human glioma cell enhanced the susceptibility to lymphokine-activated killer (LAK) cells. In vitro, susceptibility to LAK cells on Onda 10 cell is augmented by retroviral gene transfection with antisense TGF-β1. Nude mice bearing Onda 10 cells transduced with antisense TGF-β1 gene has a longer life span compared to mice carrying that of sense TGF-β1 gene or vector alone. The cytotoxic activity of LAK cells induced from spleen cells of mice carrying antisense TGF-β1 gene transduced cells is higher against Onda 10 cell than that of LAK cells from mice carrying vector alone transduced cells. Also, antisense TGF-β1 gene transduced cells are much more sensitive to LAK cells compared to Onda 10. These suggest that the augmented host systemic immunity in mice is one of the mechanisms of the reduced tumorigenicity of antisense TGF-β1 gene transduced cells and that the increased systemic immunity could be ascribed to the increased immunogenicity of the tumor cells. The gene therapy for malignant glioma with antisense TGF-β1 gene is expected to be promising.

Antisense-mediated reduction in thrombospondin-1 expression reduces cell motility in malignant glioma cells

Thrombospondin‐1 (TSP‐1) is a multifunctional matrix protein implicated in cancer cell adhesion, migration, invasion, inhibition of angiogenesis and activation of latent transforming growth factor‐β. The involvement of TSP‐1 in the motility of malignant glioma cells was investigated by transfection of TSP‐1 complementary deoxyribonucleic acid (cDNA) sense and antisense expression vectors into the glioblastoma cell line T98G‐G7 that secretes high amounts of TSP‐1. TSP‐1 production in the 3 antisense cDNA‐transfected clones was significantly reduced to 51%, 43% and 47% compared to the host T98G‐G7 cells. Motility of the 3 clones was evaluated by invasion assay and compared to the motility of host T98G‐G7 cells and 2 sense‐transfected clones. Migration of cells was significantly reduced in the 3 antisense‐transfected clones with reduced TSP‐1 production to 56%, 61% and 43% compared to the host T98G‐G7 cells. The host T98G‐G7 and another TSP‐1‐secreting A172 and YMG5 glioblastoma cells were also treated with a synthetic peptide, WSHWSPWSSCSVTCG, which includes 3 consecutive sequences of the adhesion sites in the TSP‐1 molecule and with a control peptide. The synthetic peptide significantly inhibited the migration of T98G‐G7 and A172 cells in a dose‐related manner. Maximum inhibition of migration was achieved by 100 μg/ml of the peptide and the reduction of cell motility compared to untreated cells was 34.6 % and 53.9 %, respectively. On the other hand, the inhibition of migration by the peptide was minimal in YMG5 cells, which secretes a smaller amount of TSP‐1 than T98G‐G7 and A172 cells. These results suggest that TSP‐1 secreted by malignant glioma cells is involved in the motility of glioma cells.

Down-regulation of transforming growth factor-β and interleukin-10 secretion from malignant glioma cells by cytokines and anticancer drugs

The effect of treatment with interleukin-1β (IL-1β), interferon-γ (IFN-γ), vincristine, and etoposide was evaluated on the secretion of transforming growth factor-β (TGF-β) and IL-10 and the expression of major histocompatibility complex (MHC) class I, intercellular adhesion molecule-1 (ICAM-1), and CD80 molecules by malignant glioma cells. Five malignant glioma cell lines were treated with IL-1β, IFN-γ, and/or anticancer agents (vincristine and etoposide). Combined treatment with IL-1β and IFN-γ caused greater inhibition of TGF-β secretion compared to treatment with IFN-γ, and almost the same levels of inhibition as treatment with vincristine and etoposide. The greatest inhibition of TGF-β secretion was achieved by treatment with all agents. Low levels of IL-10 secretion were determined in two out of five malignant glioma cell lines. This IL-10 secretion was inhibited by treatment with IL-1β, IFN-γ, vincristine, and/or etoposide. Treatment with both cytokines and anticancer agents increased the expression of MHC class I and ICAM-1 in all tumor cell lines. The mean increase of expression of MHC class I was 50% and that of ICAM-1 was 12-fold. No tumor cell lines expressed CD80 molecules on the cell surface, and no treatment caused CD80 expression. These results suggest that TGF-β and IL-10 secretion by malignant glioma cells can be suppressed by treatment with a combination of IL-1β, IFN-γ, vincristine, and etoposide, and the treatment up-regulates MHC class I and ICAM-1 expression on tumor cells. These results have implications for immunotherapy and chemotherapy in patients with malignant tumors.

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor-β (TGF-β), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF-β secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF-β activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF-β in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF-β secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF-β and activate latent TGF-β evenafter large dose irradiation, despite the inhibition oftumor growth.