Clinical significance of quantitative analysis of carcinoembryonic antigen assessed by flow cytometry in fresh human gastric cancer cells.

The expression of carcinoembryonic antigen(CEA) on tumor cells freshly excised from 51 patients with gastric cancer was studied using flow cytometry. The expression of CEA by flow cytometry was more quantitative than that by immunohistochemical staining. There was no relationship between the fluorescence intensity assessed by flow cytometry and serum CEA levels, except for patients with a high titer of serum CEA. The patients with high grade CEA expression on tumor cells by flow cytometry had poor prognoses, compared to patients with low CEA expression in undifferentiated gastric cancer. Thus, it is suggested that the quantitative CEA expression on tumor cells by flow cytometry could be a useful prognostic marker in postoperative gastric cancer patients.

Synergistic effects of tamoxifen and cepharanthine for circumventing the multidrug resistance.

We examined the synergistic effects of tamoxifen (TAM) and cepharanthine (CEP) for doxorubicin (DOX) sensitivity using MTT assay. The augmentation of DOX sensitivity by TAM and CEP was significantly correlated with the P-glycoprotein expression. The cytotoxic effect of DOX with TAM and CEP was significantly higher than that of DOX alone, or DOX with TAM, and this synergistic effect was dominant in cell lines with high expression of P-glycoprotein. It was also examined that the intracellular concentration of DOX was increased in combined exposure of TAM and CEP, compared with the exposure of TAM, because TAM and CEP promoted the influx and inhibited the efflux of DOX. Thus, TAM and CEP might be able to circumvent DOX-resistance for treatment in cancer patients.

Interleukin-4 inhibits lak and initial phase til activity via interleukin-2 receptor.

In the present study, we analyzed the proliferation and cytotoxic activities of LAK cells and initial phase TILs by stimulation with IL-4. IL-4 obviously inhibited the DNA synthesis of LAK cells and initial phase TILs at the concentration of 250 pg/ml and 25 pg/ml, respectively. Furthermore, IL-4 (25 ng/ml for LAK cells, 25 pg/ml for initial phase TILs) suppressed the cytotoxic activities against K562, KATO-III, and autologous tumor cells. The discrepancy of the concentration between the proliferation and the cytotoxicicity by IL-4 suggested different pathways in terms of the generation of LAK cells. In order to clarify the inhibitory mechanism of IL-4, we measured the expression of IL-2 receptor. IL-2 receptor alpha chain was strongly down-regulated by IL-4. Thus, IL-4 modulates the activation of LAK cells and initial phase TILs via the IL-2 receptor alpha chain.

Enhancement of tumor cell susceptibility to tumor-infiltrating lymphocytes by cisplatin.

Some means of enhancing the susceptibility of tumor cells to tumor-infiltrating lymphocytes (TIL) are required in adoptive immunotherapy. This study was designed to investigate whether or not tumor cell lysis by TIL was enhanced by treatment of the tumor cells with cisplatin, and also to clarify the mechanism of cisplatin's action on tumor cells. Autologous tumor cells and established cancer cell lines, including KATO-III and MKN-28, were used. Cytotoxic activities of TIL, the surface antigens of tumor cells, conjugation of TIL and tumor cells, and the production of TNF alpha from TIL were analyzed. Tumor cells treated with 2 micrograms/ml cisplatin for 12 h in vitro were more susceptible to bulk-cultured TIL and TIL clones. The surface antigens of tumor cells were not altered by the treatment with cisplatin. Cisplatin-treated tumor cells showed a higher binding ratio to TIL than did non-treated tumor cells. The anti-(tumor necrosis factor) (anti-TNF) or anti-TNF receptor antibody blocked the enhancement of cytotoxic activity by cisplatin. Thus, it was clarified that cisplatin enhanced the susceptibility of tumor cells to bulk-cultured TIL and TIL clones. Furthermore, the enhancement of cytotoxic activity by TIL in cisplatin-treated tumor cells was caused by a higher binding ratio to TIL and higher susceptibility to the TNF produced by TIL.

Clinical effects of chemoimmunotherapy for patients with peritoneal carcinomatosis.

The present study was undertaken to determine whether chemoimmunotherapy using activated killer cells is better than chemotherapy alone for cancer patients with peritoneal carcinomatosis. Thirty-one cancer patients received adoptive immunotherapy by activated killer cells and chemotherapy by anticancer drugs selected by a chemosensitivity test (chemoimmunotherapy group), and another 31 cancer patients received chemotherapy (chemotherapy group). The regimen of chemotherapy was determined by the results of a chemosensitivity test in both groups. The clinical effects including response rate and survival were assessed. Five patients (16.1%) achieved complete response (CR), and 17 patients (54.8%) partial response (PR) in the chemoimmunotherapy group (response rate: 22/31 patients = 71.0%), whereas 4 patients (12.9%) achieved CR, and 5 patients (16.1%) PR in the chemotherapy group (response rate: 9/31 patients = 29.0%). The response rate was higher in chemoimmunotherapy group than in chemotherapy group (p<0.05). However, no difference was observed in survival between the two groups. Therefore, it is necessary to develop methods to induce more potent killer cells for adoptive immunotherapy.

Cisplatin modulates tumor cell susceptibility to tumor-infiltrating lymphocytes in vivo.

We investigated the in vivo augmentation of susceptibility of tumor cells to tumor-infiltrating lymphocytes (TILs) with cisplatin (CDDP). TILs showed cytotoxicity against autologous and established tumor cells. Pretreatment of tumor cells with CDDP 2 mu g/ml for 12 h enhanced the susceptibility of tumor cells to TILs in vitro. TILs and autologous tumor cells were obtained from malignant ascites of patients, before and after the intraperitoneal administration of CDDP. TILs had higher cytotoxicity against autologous tumor cells of CDDP treated as compared to untreated control tumor cells, providing direct evidence of in vivo immunomodulatory effect of CDDP in cancer patients.

Autologous mixed lymphocyte reaction and postoperative prognosis in patients with gastric carcinoma.

The autologous mixed lymphocyte reaction (AMLR) represents a self recognitive response, which is very important in the immunoregulatory network system. We investigated whether the AMLR activity of patients with gastric carcinoma could reflect the postoperative prognosis to clarify the significance of autoreactivity in anti-tumor immune system in cancer patients. The AMLR activity was suppressed both in the peripheral blood and in the spleen of patients with gastric carcinoma. The patients were divided into two groups; high responder and low responder group. The former consisted of patients whose AMLR activity was extremely suppressed, and the latter of patients whose AMLR activity was mildly suppressed. The survival rate and disease-free survival rate were generally higher in the high responder group than in the low responder group, especially in the spleen. Moreover, none of the patients in the high responder group for the AMLR activity in the spleen died within three years. These results indicated that the AMLR activity could reflect the prognosis of patients who received conventional curative operation. Therefore, it was suggested that the AMLR might be a useful parameter of postoperative prognosis in gastric cancer patients and that autoreactive T cells might play a pivotal role in auto-specific immunological control of tumor growth and metastases.

Etoposide enhances the antitumor effects of cisplatin in gastric cancer cells.

We studied the combination effect of cisplatin(CDDP) plus etoposide(VP-16) in an established gastric cancer cell line, KATO-III, and also highly purified fresh human tumor cells obtained from 55 gastric cancer patients, using MTT assay. The synergistic effects of CDDP plus VP-16 were shown by both the fractional product method and median effect plot analysis in KATO-III cells, and by fractional product method in fresh human gastric cancer cells. The combination with CDDP and VP-16 showed the synergistic antitumor effects in not only KATO-III cells, but also fresh human gastric cancer cells. The antitumor effects of CDDP were enhanced by early exposure of VP-16 in KATO-III cells. The combination effects of CDDP and VP-16 were more potent in poorly differentiated gastric cancer, compared with well-differentiated cell types. Thus, it is suggested that the combination of CDDP plus VP-16 is useful in the anticancer chemotherapy of gastric cancer patients.

Augmentation of lymphokine-activated killer cell activity by lentinan.

Lymphokine-activated killer (LAK) activity stimulated by interleukin 2 (IL-2) and/or lentinan was examined in the peripheral blood of 9 healthy subjects and 7 cancer patients. After 4 and 8 days culture, LAK killer activity stimulated by IL-2 and lentinan against autologous tumor and K562 cells was greater than that stimulated by IL-2 alone. The optimal concentration of lentinan for the generation of killer cells ranged from 25-500 ng/ml, a level which can be achieved in vivo by the administration of clinical doses of this agent. The expression of CD25 antigen, the alpha chain of the IL-2 receptor on the activated killer cells was increased by lentinan. Thus it was shown that LAK cells stimulated with IL-2 plus lentinan had strong cytotoxicity and might be useful as effector cells for adoptive immunotherapy.

Ubenimex treatment enhances the susceptibility of gastric cancer cell lines to lymphokine-activated killer cells.

We investigated the direct effects of ubenimex on the modification of gastric carcinoma cell lines' susceptibility to killer cells, and the mechanism of its action. The susceptibility of both MKN-45 cells and KATO-III cells to LAK cells was enhanced by treatment with ubenimex for 48 h (p < 0.05), and the optimal concentration for this effect was 10 micrograms/ml. The susceptibility of ubenimex treated KATO-III cells to CD3+ LAK cells, especially to those also expressing CD8, was enhanced. DNA synthesis of tumor cells was not impaired by treatment with ubenimex at all concentrations tested. The binding rate of LAK cells and ubenimex-treated KATO-III cells was similar to that between LAK cells and untreated KATO-III cells. Moreover, no alterations in the expression of any antigen related to mononuclear cell-binding to tumor cells were induced by ubenimex. Lysis or the inhibition of DNA synthesis of tumor cells by LAK cell supernatant was enhanced by ubenimex. These results suggested that the mechanism responsible for the augmentation of tumor cell susceptibility by ubenimex may be a result of the alteration of their sensitivity to some Iytic factors released by LAK cells. Thus ubenimex shows not only an indirect host-mediated anti-tumor activity but also a direct effect on tumor cells, modifying their susceptibility to killer cells, and this may explain why ubenimex shows beneficial clinical effects as an adjuvant treatment.

In vitro augmentation of cytotoxic activity of peripheral blood lymphocytes and spleen cells of cancer patients by ubenimex.

Ubenimex is used for the immunotherapy of malignant diseases as a biological response modifier (BRM) and shows beneficial effects as an adjuvant treatment. In the present study, the in vitro effects of ubenimex on the cytotoxic activity of peripheral blood lymphocytes (PBL) and spleen cells of cancer patients and the mechanism of killer cell activation were investigated. Cytotoxic activity against K562, KATO-III and autologous tumor cells was augmented by in vitro sensitization with ubenimex (p < 0.05). The optimal concentration of ubenimex for induction of cytotoxic activity was 1 micrograms/ml, similar to serum levels after clinical oral administration. The major population of killer cells activated by ubenimex recognizing K562 was CD16+, and those recognizing KATO-III were mainly CDA+ or CD8(5) cels and CD16+ NK cells, while CDA5 or CD8+T cells comprised the majority of killer cells which showed autologous tumor-killing activity. Augmentation of the cytotoxic activity of mononuclear cells by ubenimex was blocked by both anti-IL-1 beta Ab and anti-IL-2 AB. However, the expression of IL-2 receptor (p55, p75) on effector cells was not altered. Ubenimex augmented not only NK activity but also autologous tumor killing activity of PBL and spleen cells via macrophage activation. These activities of ubenimex may be clinically beneficial as an adjuvant treatment.

Non-T cell disturbance causes the suppression of the autologous mixed lymphocyte reaction in patients with gastric carcinoma.

We investigated the accessory function of non-T cells to autoreactive T cells in autologous mixed lymphocyte reaction (AMLR) and clarified the cause of the suppression of autoreactivity in patients with gastric carcinoma. The response of T cells in the AMLR in gastric cancer patients was significantly suppressed compared with that in controls. In patients in whom the AMLR of the spleen was suppressed more than that of the peripheral blood, the degree of stimulation of non-T cells from the spleen was remarkably suppressed, on the other hand, in patients in whom AMLR of the peripheral blood was suppressed more than the spleen, the degree of stimulation from the peripheral blood was remarkably suppressed. The expression of HLA-DR antigens on non-T cells of gastric cancer patients was lower than that of controls. AMLR was considerably decreased in controls by the treatment non-T cells with anti-HLA-DR MoAb, but not in cancer patients. Treatment of non-T cells from the spleen of gastric cancer patients with IFN-gamma remarkably improved T cell proliferation in the AMLR. IFN-gamma also enhanced the expression of HLA-DR antigens on non-T cells. The disturbance of non-T cells was not biased to a specific population. These disturbances of non-T cells suppressed the AMLR independently of stage status. Therefore, the immunological abnormality of non-T cells manifested by reduced accessory function to autoreactive T cells may cause impaired immunological surveillance against tumors and permit cancer cell growth.

Polysaccharide preparation PSK augments the proliferation and cytotoxicity of tumor-infiltrating lymphocytes in vitro.

We have investigated whether or not polysaccharide preparation PSK directly augments the proliferation and cytotoxicity of tumor-infiltrating lymphocytes (TILs). TILs were separated from 10 patients with gastrointestinal cancer (5 gastric cancers, 3 colon cancers and 2 pancreatic cancers). TILs were cultured with IL-2 and PSK for 7 days. The DNA synthesis of TILs was augmented by incubation with 100 micrograms/ml of PSK, which was similar to serum level with oral administration of PSK in cancer patients. The effect of PSK in DNA synthesis was also found by elimination of non-T cells. Furthermore, we established TIL clones and examined the effect of PSK on TILs clones. The DNA synthesis was augmented by PSK in CD4 positive and CD8 positive TIL clones without non-T cells, suggesting that PSK acts directly on TILs. We examined the cytotoxic activities of TILs by the 4-h and 16-h 51Cr release assay. PSK did not affect the cytotoxic activity of TILs against autologous tumor cells and KATO-III cells in the 4h 51Cr release assay, whereas PSK induced high lysability of TILs against autologous tumor cells in the 16-h 51Cr release assay. We studied the ability of PSK to induce cytokines from TILs using a double chamber plate. The DNA synthesis of tumor cells was more suppressed by the mixed-tumor cell culture supernatants of TILs cultured with PSK, compared to that of TILs cultured without PSK. It is suggesting that PSK induced long term killing activity of TILs by induction of cytotoxic cytokines. Thus, PSK augmented the proliferative response of TILs without interaction of T cells and non-T cells and induced cytotoxic cytokines of TILs.

Multidisciplinary treatment for gastric cancer patients by chemoimmunotherapy.

BACKGROUND/AIMS: Gastric cancer is a virulent disease with a poor prognosis despite multidisciplinary treatment. The present study was designed to clarify the clinical effects of chemoimmunotherapy for patients with advanced gastric cancer. METHODOLOGY: The enrolled gastric cancer patients had distant metastases including liver (n = 2) and peritoneal dissemination (n = 21). The patients had received the chemotherapy according to the results of chemosensitivity test and adoptive immunotherapy by activated killer cells. RESULTS: There were no severe toxicities, except fever and mild myelo-suppression. Four patients had complete response (17.4%) and 10 patients had partial response (43.5%). The performance status was improved in responders (p < 0.01, from 2.6 +/- 0.5 to 1.4 +/- 0.7); however, this was not changed in non-responders (from 2.2 +/- 0.9 to 2.0 +/- 1.2). The survival of responders was longer than that of non-responders (p < 0.05, 198 +/- 69 days vs. 104 +/- 68 days). CONCLUSIONS: It was clarified that responders by chemoimmunotherapy had a good quality of life and longer survival.

Neutrophil functions and cytokine production in patients with gastric cancer.

BACKGROUND/AIMS: One of the most important factors in the prevention of postoperative infection is the patient's own capacity to protect against infection. Neutrophils play a major role in this protection through phagocytosis and superoxide generation. Inflammatory cytokines are suitable for estimating the degree of surgical stress. The present study was designed to elucidate whether neutrophil functions are impaired in gastric cancer patients, and are related with cytokine production after surgery. METHODOLOGY: Phagocytosis and superoxide generation by neutrophils was studied in 84 patients with gastric cancer by flow cytometry. IL-6, IL-8 and tumor necrosis factor alpha were studied in 18 patients with gastric cancer by enzyme-linked immunosolubent assay. RESULTS: In gastric cancer patients phagocytosis was not impaired, whereas superoxide generation was lower than benign diseases and it was inhibited relative to the clinical stage. Moreover, superoxide generation was correlated with the nutritional parameters and was more suppressed in 7 patients who suffered from postoperative infection than in 40 patients whose postoperative course were uneventful. The fluctuation of superoxide generation correlated well with the serum cytokine levels in the postoperative course and its correlation was clarified in vitro. Nine patients with gastric cancer received intravenous hyperalimentation, and their superoxide generation was increased. CONCLUSIONS: Superoxide generation by neutrophils was suppressed in gastric cancer patients and it is suggested that nutritional support prevents postoperative infection via the augmentation of superoxide generation.

P-glycoprotein expression and chemosensitivity in highly purified fresh human gastrointestinal cancer cells.

BACKGROUND/AIMS: Colorectal cancer is one of the tumors most refractory to treatment by chemotherapy. One of the major problems associated with cancer chemotherapy is drug-resistance of tumor cells, and resistance to doxorubicin (DOX) is mainly due to the effect of P-glycoprotein. We have tried to prove the correlation between P-glycoprotein expression and DOX-sensitivity in highly purified fresh human colorectal cancer and, moreover, to prove the differentiation of P-glycoprotein expression between the different kinds of cancers, including gastric cancer. METHODOLOGY: The present study was designed to quantify P-glycoprotein expression by flow cytometry, and DOX-sensitivity by MTT assay in highly purified fresh human tumor cells obtained from 29 cancer patients including 13 colorectal cancers and 16 gastric cancers. RESULTS: DOX-sensitivity decreased in proportion to P-glycoprotein expression in colorectal cancer. P-glycoprotein expression in colorectal cancer was higher than that in gastric cancer. Particularly, P-glycoprotein expression in colorectal cancer in the DOX low-sensitivity group was higher than in the DOX high-sensitivity group. CONCLUSIONS: The chemotherapeutic management of patients with colorectal cancer might be more effective if we can circumvent the effect of P-glycoprotein.

[Isolation rate of E. coli from surgical infections and their susceptibilities].

Escherichia coli isolated from surgical infections during the period from July 1983 to June 1995 were investigated in a multicenter study involving 19 hospitals in Japan, and the following results were obtained. 1. Although the isolation rate of E. coli was not high from postoperative infections, it was most frequently isolated from primary infections throughout the study period. E. coli, Klebsiella spp. and anaerobic bacteria were predominant from fresh infections. From the cases that had previous antibiotics treatment, Enterococcus spp. were the most predominant isolates followed by MRSA and Pseudomonas spp. in this order. 2. Against E. coli, cefozopran, carumonam and aztreonam had the strongest activity, followed by cefmenoxime, imipenem, latamoxef, gentamicin and ofloxacin. Recently, we have noticed that antibiotic resistant E. coli strains particularly against cefazolin are increasing year by year.

Clinical effect of immunochemotherapy for a patient with advanced gallbladder cancer: report of a case.

A 56-year-old woman was admitted presenting with a sensation of abdominal fullness. She was diagnosed to have advanced gallbladder cancer with carcinomatous peritonitis, as well as lymph node and liver metastases. We obtained highly purified tumor cells and tumor-infiltrating lymphocytes (TIL) from extirpated cervical lymph nodes and peritoneal effusion, and the chemosensitivity of these cells was tested with an MTT assay. Intensive chemotherapy with cisplatin (CDDP) and 5-fluorouracil (5-FU) was then performed according to the results of the MTT assay. Thereafter, cytotoxic T-lymphocytes (CTL) were induced in mixed cultures of autologous tumor cells and peripheral blood lymphocytes, and adoptive immunotherapy was performed with TIL and CTL. The malignant ascites and metastatic lesions disappeared after the intraperitoneal administration of CDDP and the transfer of TIL and CTL, and subsequently the patient's quality of life improved. This patient could return to work; however, liver metastasis was later observed, and she died 14 months after the initial diagnosis. Combination therapy with anticancer drugs and activated killer cells was thus found to be effective in a patient with advanced gallbladder cancer.

Induction of cytotoxic T cells and their antitumor activity in mice transgenic for carcinoembryonic antigen.

In order to develop immunotherapy strategies that are based on eliciting immune responsiveness to the self-antigen, human carcinoembryonic antigen (CEA), we examined whether cytotoxic T lymphocyte (CTL) activity against CEA could be elicited in CEA-transgenic and nontransgenic mice. CEA-transgenic [C57BL/ 6-TGN(CEAGe)18FJP] and nontransgenic mice were primed with CEA-transfected syngeneic fibroblasts in combination with Corynebacterium parvum. Spleen cells from immunized mice were cultured with irradiated syngeneic MC-38 colon carcinoma cells transfected with CEA (MC-38.CEA) as stimulators prior to the measurement of CTL activity. Primed nontransgenic spleen cells showed augmented CTL activity against MC-38.CEA cells as compared with control parental MC-38 cells, nontransfected or transfected with vector only. Moreover, primed CEA transgenic spleen cells showed augmented CTL activity against MC-38.CEA cells that was similar to that observed in nontransgenic mice. All CTL clones derived from either transgenic or nontransgenic mice showed cross-reactivity with MC-38 cells expressing the CEA-related antigen, nonspecific cross-reacting antigen, but not biliary glycoprotein. CEA-specific CTL clones were not identified. Adoptive transfer of cloned CTL resulted in inhibition of MC-38.CEA but not MC-38.BGP tumor growth. Tumor cures were elicited in mice treated with a combination of cloned CTL and cyclophosphamide. Histopathological examination of CEA-expressing colons from either immunized mice or recipients of cloned CTL did not reveal any autoimmune reactions. These studies demonstrate that CTL recognizing cross-reactive class I epitopes on the CEA molecule can be induced in transgenic mice. The expression of these epitopes on tumor cells creates effective targets for CTL in vivo without inducing adverse reactions in CEA-expressing normal tissues. Since anti-CEA CTL have been generated in humans, CEA-transgenic mice may be a useful model to study vaccines that are based on CTL effector mechanisms.

Clonal and functional analysis for the augmentation of tumour-infiltrating lymphocytes by interleukin 4.

In the adoptive immunotherapy for cancer, the amounts of induced effector cells play a major role in improving therapeutic efficacy. We have already demonstrated that interleukin 4 (IL-4) augments proliferation of tumour-infiltrating lymphocytes (TILs) without altering the cytotoxic activity against autologous tumour cells. The present study is designed to investigate how IL-4 augments TILs by using established TIL clones in terms of IL-2/IL-2 receptor system. CD4+, CD8+ and CD4+ CD8+ (double positive) TIL clones were established from cancer patients. At clonal level, IL-4 augmented the proliferation of IL-2-activated TIL clones irrespective of phenotypes. In order to clarify the mechanism of IL-4 at clonal level, the blocking assay by anti-IL-2 receptor alpha and beta chain and binding assay of IL-2 on the cell surface and the measurement of the internalisation of IL-2 in the cell were performed. It was clarified that IL-4 up-regulated the IL-2 receptor and then augmented the action of IL-2 molecule on the cell surface stimulated by IL-4. Furthermore, binding IL-2 internalised rapidly into the cells. Thus, it is suggested that signal transduction is augmented and proliferation of TILs is enhanced by IL-4 via the action of IL-2/IL-2 receptor system.