Takehiko Tachibana

Role of the regional lymph node in cancer metastasis

At an early phase of tumor growh, T-cell responses, i.e. the proliferation of T cells and the generation of cytotoxic T cells or killer-augmenting T cells are induced in the regional lymph node depending upon the immunological properties of the tumor cells. A small number of tumor cells seems to be rejected in the regional lymph node in situ. A progressive tumor induces suppressor activity in the regional node. Suppressor cells facilitate tumor growth and lead to lymphatic metastasis. Thus, the regional lymph node operates only as a temporary barrier to tumor growth. Experimental studies have demonstrated that cradication of the suppressor cells and effective immunization, or both, lead to tumor rejection by augmenting the immunological activity of the regional lymph node.

Antigenic heterogeneity of the reticular meshwork in the white pulp of mouse spleen

SummaryMonoclonal antibodies against cellular components of reticular meshworks were produced by immunizing rats with heterogeneous stromal-cell population of mouse spleen. Immunohistochemical screening selected two antibodies, WP-1 and RPSC-2. WP-1 proved to immunostain the meshwork of the B area densely, leaving the marginal zone unstained; it also reacted sparsely with the meshwork of the T-cell region. In contrast, RPSC-2 selectively immunostained the meshwork of the T region. Immuno-electron microscopy clearly visualized, for both antibodies, reaction products being deposited along the cytomembrane of the fibroblastic reticulum cells, along their abundant cytoplasmic processes that were densely intertwined with lymphocytes. Double immunostaining with RPSC-2 followed by WP-1 clearly divided the white pulp into the T and the B domains. The meshwork in the T-cell region proved to be immunostainable with both WP-1 and RPSC-2. Thus, the fibroblastic reticulum cells of the T-and the B-cell areas, while indistinguishable by routine microscopy, are at least partially heterogeneous.

A simple and improved method to generate human hybridomas.

By improving our previous method for production of human hybridomas, we developed a simple and remarkably efficient method for production of human hybridomas. We reformed our previous method in the following three points: (1) we added irradiated (30 Gy) myeloma cells as feeder cells to culture of fusion cells; (2) ouabain concentration in the selective medium was reduced from 2 x 10(-6) M to 1 x 10(-6) M; (3) selective medium (GIT-HAT-OL) was added to the culture after overnight cultivation of fusion cells. Consequently, we obtained higher fusion frequency (1/700 vs. 1/5500) compared with our previous method. By our present method, only so small number of human B cells (EBV-LCL) is required, so that the time necessary to establish human hybridomas is reduced.

Distribution of Cylindrical Inclusion, Amorphous Inclusion and Capsid Proteins of Watermelon Mosaic Virus 2 in Systemically Infected Pumpkin Leaves

Summary Monoclonal antibodies (MAbs) were prepared against the cylindrical inclusion protein (CIP), amorphous inclusion protein (AIP) and capsid protein (CP) of watermelon mosaic virus 2 (WMV2). Using the MAbs, CIP, AIP and CP were detected, roughly quantitatively, in WMV2-infected pumpkin leaves showing various symptoms by using electroblot-ELISA. From symptomless leaves and most dark green areas in the mosaic pattern no protein or small amounts of the three proteins were detected, but from most yellow areas in the mosaic almost equal amounts of each protein were detected in abundance. Leaves showing mild vein-yellowing and vein-clearing (respectively, the first and second leaves of the plants tested) contained AIP and CP in large amounts, but little CIP. On the other hand, expanding leaves contained CIP and AIP in large quantities, but CP in traces only. Therefore the distributions of CIP, AIP and CP in pumpkin plants were very uneven, but correlated with symptoms. In addition, the ratio of the concentrations of CIP, AIP and CP varied from tissue to tissue.

Spontaneous interferon production and growth of lymphoblastoid cells in serum-free medium

Abstract Numerous lymphoblastoid cell lines were established from human adult peripheral blood and cord blood lymphocytes, using Epstein Barr virus, and most cell lines from cord blood lymphocytes spontaneously produced abundant interferon without induction with Sendai virus, whereas lymphoblastoid cells from adult peripheral blood lymphocytes did not. These potential cells grow well in a newly developed serum-free culture medium based on Dulbeccos modified Eagle medium supplemented with non-essential amino acid, vitamins, nucleic acid derivatives, metal compounds, human transferrin, insulin and bovine or human serum albumin (Chon Fr.V). In serum-free medium, as well as in serum-containing conventional medium (RPMI-1640), the cells could also spontaneously produce interferon. The cells in the serum-free, culture could produce about 10 000 U/ml of interferon every day, harvesting the culture fluid and refeeding the cells with the fresh medium at the saturation cell density (107 cells/ml). The interferon proved to be α-type interferon on the basis of its physico-chemical and antigenic properties.

A novel human monoclonal antibody directed to a tumor-associated antigen.

Twelve human monoclonal antibodies (HuMAb) were established by the fusion of (mouse × human) heteromyeloma cells with B‐lymphoblastoid cells derived from the regional lymph nodes of three patients with squamous cell carcinoma of the lung. They were tested for reactivity to two kinds of proteins (purified protein derivatives and bovine serum albumin) by ELISA, Sq‐19 (squamous cell carcinoma) culture cells by indirect membrane immunofluorescence tests, and Sq‐19 tumor xenograft by immunohistological study. Among them, one HuMAb 904F (IgM, λ) was selected. In indirect membrane immunofluorescence tests, this 904F antibody reacted with various kinds of cell lines, e.g. lung cancer, esophageal cancer, endometrial cancer, and stomach cancer. It did not react with malignant hematopoietic and diploid fibroblast cell lines. Immunohistologically, it stained the tumor nests of squamous cell carcinoma, adenocarcinoma, and large cell carcinoma of the lung. It also stained those of esophagus and colon, but not those of small cell carcinoma of lung, or stomach. On frozen‐section specimens of normal tissues from various organs, it showed only limited areas of positive staining. Limited positive findings were observed at a reticular zone of the adrenal gland, at the esophagus as weak staining, and at islets of the pancreas as very weak staining. Western blotting analysis demonstrated that it recognized a 54 kDa trypsin‐sensitive molecule which is expressed on the surface of tumor cells. These results suggest the 904F monoclonal antibody detects a novel tumor‐associated antigen which is recognized by the human immune system.

Structural polymorphism of mouse complement C2 detected by microscale peptide mapping: Linkage to H-2

Complement C2 was isolated from 17 mouse strains by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined for structural polymorphism by using micro-peptide mapping. By comparing the peptide maps of tryptic digests of C2 from various strains, two allotypic variations were detected. B 10 and 14 other mouse strains demonstrated C2.1 type, while a wild mouse line (M.Mol-Ohm) and one BIO congenic strain, B10.MOL.OHM, which carries the H-2 derived from M.Mol-Ohm, demonstrated C2.2 type. (B10 × Bl0.MOL.OHM)F1 demonstrated codominantly expressed C2 type (C2.1.2). Desialation of mouse C2 did not abolish the observed variation of mouse C2. It is concluded that an H-2-linked codominant locus controls the structure of mouse complement C2, further confirming the extensive homology of the major histocompatibility complex among higher vertebrate species.

Active recombinant C3a of human anaphylatoxin produced in Escherichia coli

DNA sequence coding for the complete human C3a with 77 amino acids was divided into three portions, synthesized separately and constructed for expression in Escherichia coli. High expression of the recombinant C3a was achieved by an expression system using T7 polymerase. Purified recombinant C3a showed the same activities of ileum contraction and platelet aggregation of guinea pig as C3a purified from human serum.

Elevated Ley antigen expression on T-lymphocytes in schizophrenic patients

SummaryLey is a carbohydrate determinant of membrane glycoconjugates and is expressed in some tumor and embryonic cells. On T lymphocytes, it is known that human immunodeficiency virus (HIV)-infected human lymphocyte T-cell lines express elevated Ley antigen and AIDS patients show the highest Ley expression in T lymphocytes at lower CD4/CD8 ratio. Later, a comparative elevation of Ley expression on T-cell subsets has been noticed to be mainly present in patients with viral diseases, such as acute and chronic hepatitis, implying an association of the highest Ley expression with viral infection. We found that Ley antigen was most expressed in both CD4+ and CD8+ subsets of peripheral T lymphocytes in hospitalized schizophrenic patients. On the other hand, atypical lymphocytes with stimulated morphology are known to appear in the blood circulation of schizophrenic patients. Similar atypical lymphocytes have also been described in viral and autoimmune diseases. Two possibilities have been discussed: viral association in the pathology in some schizophrenic patients; and immunological abnormalities including environmental effects under hospitalization on immune status, since normal controls (staff in psychiatric hospitals) showed higher Ley expression than normal controls under non-psychiatric circumstances.

Evidence of Lymphocyte Abnormality in Schizophrenia

Recent interest in the lymphocytes of schizophrenics began with Kamp’s preliminary report (1962). He found that the lymphocyte nucleus of chronic schizophrenics stained deeply and that the chromatin density varied. Pennington (1963) soon confirmed this finding, but another study was not able to find cell differences between schizophrenics and normals (Bucci and Johnson, 1964). Gershon and Lonigro (1964) found a quantitative cell difference between normals and schizophrenics but also with other psychiatric patients and concluded that the results were caused by “hospitalization.”