Yasuhiko Kimoto

Expression of heavy-chain constant region of immunoglobulin and T-cell receptor gene transcripts in human non-hematopoietic tumor cell lines

Expression of gene transcripts for immunoglobulins and a T‐cell receptor was investigated in non‐hematopoietic tumor cell lines using the highly sensitive RT‐nested PCR method. These proteins are reported to be produced and secreted or expressed in malignancies originating from hematopoietic organs only. Originally designed PCR primers for different exons coding for the heavy‐chain constant regions of IgM, IgD, IgG3, IgG1, IgE, and IgA and the T‐cell receptor‐α were used. All gene transcripts were detected in the 5 investigated cancer cell lines without exception. The results suggest that even non‐hematopoietic cancer cells transcribe immunoglobulin and T‐cell receptor genes and may produce the corresponding proteins. Genes Chromosomes Cancer 22:83–86, 1998.

Immunohistochemical Detection of P-glycoprotein in Breast Cancer and Its Significance as a Prognostic Factor.

Overexpression of P-glycoprotein (Pgp) in tumors is one of the major mechanisms which mediates the multidrug resistance (MDR) phenotype. To evaluate the prognostic significance of Pgp in breast cancer, Pgp expression was examined in paraffin-embedded tissue sections of 94 breast cancer specimens by immunohistochemistry. Tissue specimens were obtained by mastectomy without preoperative chemotherapy. UIC2 monoclonal antibody which recognizes an extracellular epitope of human Pgp was employed. Of the 94 breast cancer specimens, 35 (37.2%) were positive for Pgp expression. Pgp expression had no correlation with menopausal or hormone receptor status, axillary lymph node involvement or tumor size. However, a significant correlation was observed between Pgp expression and disease relapse (p = 0.0322). Pgp-positive patients showed a significantly shorter disease-free survival period than Pgp-negative patients by the Kaplan-Meier method (p = 0.0433). These results suggest that immunohistochemical detection of Pgp in breast cancer tissue may have prognostic value after radical operation.

Killing of human tumor cell lines by human monocytes and murine monoclonal antibodies

We evaluated the capacity of freshly isolated blood monocytes to mediate antibody-dependent cellular-mediated cytotoxicity (ADCC) in cooperation with murine anti-tumor monoclonal antibodies (MAbs). Blood monocytes isolated from most donors by adherence selection to fibronectin-coated plastic surfaces and subsequently depleted of natural killer/killer (NK/K) cells exhibited significant ADCC activity against tumor cell lines in combination with an IgG3 antitumor MAb (BR55-2). However, significant variation in ADCC competence was observed among donors. Culture parameters influencing monocyte ADCC activity were evaluated and optimized. The influence of MAb isotype on ADCC capacity of anti-tumor MAbs was also evaluated using anti-tumor class-switch variant hybridoma proteins and a panel of anti-tumor MAbs. MAbs of the IgG2a and IgG3 subclasses exhibited high ADCC potential, whereas MAbs of the IgG2b subclass exhibited no ADCC activity. One of two IgG1 MAbs tested exhibited high ADCC activity with monocyte effectors. The role of monocytes or macrophages in tumor remission of cancer patients undergoing MAb immunotherapy is not known. However, correlative studies of monocyte ADCC capacity and responsiveness of cancer patients to MAb immunotherapy may help to establish the role of these effectors in MAb-mediated tumor remissions.

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies

Clinical effects and side effects were studied in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of normal donors by means of initial stimulation with pokeweed mitogen (PWM). Six of 15 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastasis, pleural effusion and primary tumor. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Generally toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently except one patient who showed preshock like symptom accompanied with leukocytopenia and continuous hypotension immediately after infusion but was finally rescued. In the patients who received more than 1011 of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies appeared in the patients. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells.

Apoptosis as a Mechanism of Lectin-Dependent Monocyte-Mediated Cytotoxicity

In this study we investigated the mechanisms of cytotoxicity mediated by pokeweed mitogen (PWM)-activated human peripheral blood monocytes. By using DNA electrophoresis and propidium iodide (PI)-DNA staining flow cytometry, we demonstrated that apoptotic cell death of target U937 cells and Raji cells was induced in lectin (PWM)-dependent monocyte-mediated cytotoxicity (LDMC). The LDMC-mediated DNA fragmentation in U937 cells and Raji cells was induced in lectin (PWM)-dependent monocyte mediated cytotoxicity(LDMC). The LDMC-mediated DNA fragmentation in U937 cells was completely inhibited by anti-TNF alpha monoclonal antibody (mAb), but not by the addition of monosaccharide (N-acetylglucosamine, GlcNAc, a sugar specifically recognized by PWM and a lectin-like receptor on monocytes). In contrast, GlcNAc inhibited the DNA fragmentation in Raji cells induced by LDMC which the anti-TNF alpha mAb had no effect. PWM was found to stimulate the production of nitric oxide (NO) from monocytes. The NO-production was enhanced in the presence of target Raji cells, while the enhancement was abolished by the treatment with GlcNAc. By flow cytometry, we found that PWM bound to tumour cells as well as monocytes, and inhibited the expression of HLA-DR antigen on tumour cells. These results suggest that the presence of lectin molecules on the surface of monocytes and tumour cells may bring the two cells together, thus facilitating the induction of apoptosis in target cells by triggering the production of cytolytic factors (TNF and NO) and the modification of target cell surface antigen (HLA-DR).

Ovarian carcinoma with fistula formation to the sigmoid colon and ileum: report of a case.

We describe herein an extremely rare case of clear cell type ovarian carcinoma resulting in fistula formation into the colon and intestine. The patient was a 61-year-old woman in whom a large tumor with extravasation from the sigmoid colon was found by barium enema examination. The tumor was preoperatively diagnosed as left ovarian cancer by angiography which showed the tumor feeder arising from the left ovarian and uterine arteries.

Enhancing effect of pokeweed mitogen on the proliferation and the cytotoxicity of lymphokine-activated killer cells.

In order to obtain more potent lymphokine‐activated killer (LAK) cells for use in adoptive immunotherapy, pokeweed mitogen (PWM) was added to the culture medium for the initial 24–48 h of culturing. The proliferation rate of PWM‐stimulated LAK cells reached about 1000‐fold after 3‐week culture. This rate was nearly the same as that of LAK cells stimulated by 10 ng/ml of OKT3, the mouse anti‐CD3 monoclonal antibody. However, the cytotoxicity of PWM‐stimulated LAK cells was significantly more potent than that of OKT3‐stimulated LAK cells. Phenotypic analysis revealed that PWM‐stimulated LAK cells were CD3+CD56+‐dominant while OKT3‐stimulated LAK cells were CD3+CD56‐‐dominant. About half of CD3+CD56+ PWM‐stimulated LAK cells was CD8+. These results suggest that more efficient adoptive immunotherapy is possible by using high‐dose PWM‐stimulated LAK cells with more potent cytotoxicity. Interleukin‐1β and tumor necrosis factor a were significantly increased in the culture media after 24‐h incubation with 1 μg/ml of PWM. Secretion of interferon‐γ was not enhanced by this concentration of PWM within 24 h. Therefore, PWM is considered to activate monocytes or macrophages to produce these cytokines in advance, influencing the proliferation and the cytotoxicity of LAK cells.

Polyethylene glycol-modified pokeweed mitogen (PWM) as a potential non-immunogenic stimulator of lymphokine-activated killer cells

Among the various plant lectins, pokeweed mitoge (PWM) is most effective in enhancing the cytotoxicity of human lymphokine-activated killer (LAK) cells. However, the use of PWM in adoptive immunotherapy has been limited due to the strong immune response against the protein of plant origin. Amino groups in PWM was modified with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chioro-s-triazine, activated PEG2, to form PEG-PWM conjugates. Its immunoreactivity towards anti-PWM antibodies was reduced by increasing the degree of modification of amino groups in PWM. PEG-PWM, in which 54% of amino groups in PWM was modified with activated PEG2, had a nearly complete reduction of immunoreactivity. Intraperitoneal administration of PEG-PWM to mice did not produce substantial levels of anti-PWM antibodies. Nevertheless, PEG-PWM retained the ability to induce the maximum levels of cytotoxicity of human LAK cells in vitro.

Pokeweed mitogen induces p55 interleukin-2 receptor expression on human monocytes and its effect on interleukin-2 activated monocyte cytotoxicity

Activation of human peripheral blood monocytes by pokeweed mitogen (PWM) results in the expression of interleukin 2 receptor(IL-2R) p55 chains, which are absent on resting monocytes. By dual-fluorescence flow cytometry, we found PWM induced detectable numbers of IL-2R+ cell which were further identified as LeuM3+ monocytes (22.91% of the LeuM3+ cells were IL-2R+ within 24h, and 32.17% in 48h). In addition, LPS can also induce IL-2R on 15.39% of LeuM3+ cells. On the contrary, other mitogens such as PHA or Con A, and cytokines as IFN-gamma, IL-2, M-CSF, TNF-alpha and IL-1 beta showed no influence on the IL-2R expression on monocytes. It was also noticed that PWM itself had no direct effect on HLA-DR antigen expression on LeuM3+ cells. The addition of IL-2 to PWM-pretreated IL-2R positive monocytes significantly augmented their tumoricidal activity. Thus monocytes when activated underwent a series of phenotypic and functional changes including the expression of IL-2R which may provide an important immunoregulatory pathway.

Fine structure of experimental canine gastric carcinoma, with special reference to signet ring cells

Fine structure of poorly differentiated adenocarcinoma selectively induced in the canine stomach by a low concentration of N-ethyl-N′-nitro-N-nitrosoguanidine was studied with special reference to the so-called signet ring cells. These cells were electron microscopically classified into two groups: 1) mucin-containing cells and 2) intracellular microcyst cells. The cytoplasm of mucin-containing cells was packed with fused large and low electrondense secretory granules. In another type of signet ring cells, there were intracellular microcysts with microvilli on the internal surface. A comparison of canine and human signet ring cells revealed a close resemblance in the fine structure. Canine gastric carcinoma should be a good model for human gastric carcinoma therapeutics.