Quantitative analysis of cytokine mRNA expression in peripheral blood mononuclear cells following treatment with interleukin-2.

After activation with interleukin-2 (IL-2), peripheral blood mononuclear cells (PBMC) have been reported to induce the expression of mRNA coding various cytokines, including interleukin(IL)-1alpha, -1beta and tumor necrosis factor alpha (TNF alpha). We examined the cytokine mRNA expression of PBMC following treatment with IL-2 in vitro and in vivo by a quantitative method using the reverse transcription/polymerase chain reaction (RT-PCR). After stimulating PBMC with IL-2 in vitro, peak levels of IL-1alpha mRNA were reached between 3 h and 12 h, and thereafter declined. The IL-1beta expression increased, with levels peaking at 1-6 h and, had decreased by 96 h. The expression of TNF alpha was elevated both 1-3 h and 24-48 h after stimulation. The peak levels of IL-1alpha and -1beta mRNA and the early elevation of TNF alpha mRNA mainly accounted for the cytokine mRNA expression in adherent cells; however, the late induction of TNF alpha mRNA was observed in nonadherent cells. In patients with advanced carcinoma, the IL-1alpha and -1beta mRNA expression were elevated after IL-2 treatment for 5 consecutive days, while the expression of TNF alpha mRNA also increased. These results indicate that the quantitative RT-PCR method appears to be useful for analyzing the cytokine mRNA expression of PBMC after treatment with IL-2.

A bag carrier for continuous intravenous hyperalimentation.

We designed a new bag-carrier device system for continuous intravenous hyperalimentation. The patient carries it on his shoulder and can both walk up and down stairs and go out. The use of this device is simple and easy, and was found to increase the patient's opportunity to engage in physical activity.

Neoadjuvant chemoimmunotherapy with cisplatin and low-dose interleukin-2 for locally advanced esophageal carcinoma

After a single dose of cisplatin, the ability of peripheral blood mononuclear cells (PBMC) to generate lymphokine-activated killer (LAK) cells was significantly augmented in cancer patients. Based on this clinical finding, the patients with locally advanced esophageal carcinoma were thus treated with a combination of cisplatin and low-dose interleukin-2 (IL-2) in a neoadjuvant setting. Four patients with squamous cell carcinoma of the esophagus (T3 or T4 disease) were preoperatively treated with a regimen consisting of 50 mg/m2 cisplatin on day 1, followed by IL-2 from day 4 through day 8, when the ability of PBMC to generate LAK cells had been shown to be significantly augmented. After two to four courses of the preoperative therapy, one patient achieved a histologic CR, one showed PR and one MR. No severe toxicity was encountered. All patients thereafter underwent surgery. The median survival of these patients was 47.5 months and three of the patients had been disease free for 43 to 62 months after the initiation of the therapy. The combination of cisplatin and low-dose IL-2 administered in a neoadjuvant setting seems to result in an improved survival of locally advanced squamous cell carcinoma of the esophagus.

Apoptosis in antibody-dependent monocyte-mediated cytotoxicity with monoclonal antibody 17-1A against human colorectal carcinoma cells: enhancement with interferon gamma.

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been considered to be one of the main effector mechanisms by which unconjugated monoclonal antibody (mAb) 17-1A can exert an antitumor effect in vivo. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanism, we investigated the role of apoptosis in ADCC mediated by monocytes (ADMC) using mAb 17-1A as an antibody and the human colorectal carcinoma cell line, COLO205, as target cells in vitro. The implications of the apoptosis during ADMC was demonstrated by means of both a DNA fragmentation assay and a TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Furthermore, interferon gamma (IFN gamma) was also found to enhance the induction of apoptosis significantly. The addition of superoxide dismutase did not reduce the level of the apoptosis, although superoxide anion (O2-) was observed to be produced. However, the release of tumor necrosis factor alpha (TNF alpha) was significantly enhanced during ADMC, while, in addition, apoptosis was significantly inhibited by the addition of anti-TNF alpha antibody. These findings indicated that apoptosis might be implicated in ADMC with mAb 17-1A, which was augmented by IFN gamma, while, in addition, TNF alpha may also be one of the major mediators of apoptosis.

Surgical treatments for oesophageal cancer concomitant with gastric adenoma with severe epithelial atypia.

The various surgical treatments for oesophageal cancer concomitant with gastric adenoma with severe epithelial atypia (SEA) are discussed by presenting three such cases. As gastric adenoma with SEA had been considered to be a precancerous condition but not normally an indication for gastrectomy, we devised an operative strategy of laparotomy and thoracotomy. For Case 1, with early oesophageal cancer and gastric adenoma with SEA in the body of stomach, a laparotomy and local resection of the gastric lesion were carried out prior to thoracotomy in order to determine the safest reconstructive method after measuring the length of the well-nourished gastric tube. In Case 2, with advanced oesophageal cancer, a thoracotomy was first performed to assess the curability of surgery for the oesophageal cancer and a gastric adenoma was removed. In contrast, for Case 3 with advanced oesophageal cancer, and in whom post-operative survival was deemed to be short, a gastric adenoma was not resected. The most appropriate operative methods should thus be decided after careful consideration of the stage of the oesophageal cancer and characteristics of the gastric adenoma.

Microsatellite instability in multiple gastric cancers.

To better elucidate the role of genetic instability in the development of gastric cancer, microsatellite alterations were examined in a total of 30 gastric cancers that developed in 14 Japanese patients with multiple gastric cancers, which are considered to have possibly occurred under the same genetic background and in the same microenvironment of the stomach. Microsatellite instability (MSI) in multiple gastric cancers was recognized in 11 out of 14 cases (78.5%) and in 16 out of 30 cancers (53.3%). Eight out of 11 cases showing MSI exhibited a heterogeneity of microsatellite alterations. The incidence of microsatellite instability in the multiple gastric cancers cases was significantly higher than that in the solitary gastric cancer cases reported previously (20.8%: 5 out of 24 cases). These results suggested that (1) genetic instability plays a more important role in the development of multiple gastric cancers than in that of solitary gastric cancer and (2) the heterogeneity of MSI in multiple gastric cancers may not be a rare event, although the significance of the heterogeneity could not be clarified.

Immunohistochemical features of HLA-DR antigen expression and lymphoid infiltrates in gastric carcinoma after low-dose interleukin-2 and mitomycin C.

We immunohistochemically evaluated lymphoid cell infiltration and HLA-DR antigen expression in gastric tumor tissue obtained from advanced gastric cancer patients 1 day after the completion of the treatment with mitomycin C (MMC) 12 mg/m2 i.v. on day 1 and recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Then the results were compared with those in 11 patients pretreated with MMC alone, 5 treated with IL-2 alone, and 24 untreated patients. Widespread lymphoid infiltration was observed in 17% of untreated tumors, 27% of MMC-pretreated tumors, and 40% of tumors treated with IL-2 alone. However, 71% of carcinomas pretreated with MMC plus IL-2 exhibited widespread infiltration. The frequency of cases with high-grade infiltration of CD4+ cells was significantly higher in either group of patients treated with MMC alone or MMC plus IL-2. Because the CD8+ cell infiltration was not significantly altered, the ratio of CD4+ to CD8+ cells estimated as being > 1 was more frequently noted in patients given MMC alone or MMC plus IL-2, as compared with untreated control. Furthermore, 86% of tumors pretreated with MMC plus IL-2 exhibited positive HLA-DR antigen expression, whereas 29% of untreated carcinomas did so. MMC or IL-2 alone did not significantly increase HLA-DR expression. These results indicate that the combination of low-dose of IL-2 with MMC enhances the intensity of lymphoid cell infiltration in tumors, with the predominance of CD4+ cells, and HLA-DR antigen expression on tumor cells in patients with advanced gastric carcinoma.

The heterogeneity of microsatellite instability in multiple gastric cancers.

To obtain a better understanding of the role of genetic instability in developing gastric cancer, it is of great interest to examine microsatellite alterations in synchronous multiple gastric cancers that are thought may have the same genetic background and the same microenvironment of the stomach. We report our experience with two patients with synchronous multiple gastric cancers; patient 1 showed two carcinomas in the stomach, whereas patient 2 showed two carcinomas and two adenomas in the stomach. We examined the DNAs from the two cases for microsatellite instability and expected that the status of microsatellite instability in each tumor from the same stomach would be the same. However, patient 2 revealed heterogeneity in the microsatellite instability, i.e., an early cancer that showed some apparent alterations, whereas the other advanced cancer and two adenomas did not. On the other hand, neither of the two carcinomas in patient 1 showed microsatellite instability. To our knowledge, there has been no previous report of microsatellite instability in multiple gastric cancers. In this report, we describe a case that revealed such a heterogeneity of the microsatellite instability, in which the carcinogenic process of each tumor may undergo different genetic alterations even under the same genetic conditions and background.

Pancreaticoduodenectomy under epidural anesthesia without endotracheal intubation for the elderly.

To evaluate the efficacy of a single application of epidural anesthesia without endotracheal intubation for pancreaticoduodenectomy (PD), the data on 30 patients who underwent PD were analyzed for their operative morbidity and mortality. These patients were classified into two groups according to the type of anesthesia performed: 15 received epidural anesthesia alone (Group I) and 15, general anesthesia under endotracheal intubation (Group II). The clinical characteristics of the patients in both groups were comparable at the time of operation, except that Group I included a significantly larger proportion of elderly patients than Group II (p < 0.05). Postoperative pulmonary complications (PPCs) occurred in 5 (33.3%) of the Group II patients, especially in elderly patients who underwent lengthy operations, whereas no such complications occurred in the Group I patients (p < 0.05), even in elderly patients with a long operating time. The curability of the malignant tumors and the incidences of other complications were not significantly different between the two groups. These findings suggest that a single application of epidural anesthesia is effective in preventing PPCs when performing a time-consuming PD, especially in elderly patients.

Lymphokine-activated killer cell function of lymphocytes from regional lymph nodes in patients with gastric carcinoma.

Lymphokine-activated killer (LAK) cells generated by culture of regional lymph node cells (LNC) with interleukin 2 (IL 2) for 4 and 11 days were examined for their functional capabilities in comparison with those of peripheral blood mononuclear cells (PBM) in 25 patients with gastric carcinoma. The cytotoxic activity of LAK cells induced from LNC for 4-day culture with IL 2 was significantly lower than that from PBM. However, the LNC-LAK cytotoxicity was markedly increased up to almost the same level as that of PBM after 11-day culture. The production of interferon-gamma (INF-gamma) and tumor necrosis factor-alpha (TNF-alpha) from nonadherent LAK cells in LNC was also significantly reduced as compared to that from PBM 4 days after culture, when stimulated with or without tumor target, Raji cells. After 11-day culture with IL 2, however, the levels of these cytokines produced by LNC-LAK cells either with or without stimulation by tumor target were comparable to those by PBM-LAK cells, although the release of these cytokines was markedly reduced when compared to that after 4-day culture. Phenotypic analysis revealed decreased proportion of cells mediating NK activity in LNC before and 4 days after culture. CD56+ and CD57+ cells in LNC were increased after 11-day culture, although the percentages of these cells were still low as compared to those in PBM. The proportions of OKIa1+ and CD25+ cells were uniformly increased after 4 and 11-day culture in both cell populations. Changes in subpopulations of CD4+ and CD8+ cells in LNC were not apparently different from PBM. These results indicated the differential LAK cell function of cells from regional lymph nodes from PBM in patients with gastric carcinoma.

Minimal increase in serum interleukin-6 levels during laparoscopic cholecystectomy.

The chronologic changes in the serum levels of interleukin-6 (IL-6), a mediator for acute-phase inflammation, were compared between laparoscopic cholecystectomy (LC) and open cholecystectomy (OC), since these two types of operations were considered to be a unique model for examining the role of local tissue injury in postoperative inflammatory reactions. The increase in the serum IL-6 level during LC was found to be significantly smaller than that during OC and resulted in a smaller extent of postoperative elevations for C-reactive protein. These results suggest that laparoscopic surgery associated with minimal tissue injury can help limit an increase in the serum IL-6 level during surgery, thus contributing to a reduction in surgical stress.

Production of interleukin-6 at operative wound sites in surgical patients.

BACKGROUND: Interleukin-6 (IL-6) plays a central role in the acute phase of inflammation after surgical injury. The serum concentration of IL-6 increases during an operation. The mechanisms of this increase in the serum IL-6 level, however, has not yet been fully clarified. STUDY DESIGN: To determine the possibility of production of IL-6 at the operative wound site and its regulation by humoral factors in surgical patients, the IL-6 secretion of biopsied skin obtained from an operative wound both before and after the operation were quantitated by using organ culture techniques. RESULTS: When skin explants obtained from the uninjured skin were cultivated and the amounts of IL-6 secreted into the culture medium were measured, IL-6 secretion increased exponentially during culture, which indicated that the stress of the skin incision induced IL-6 production. The skin specimens obtained from the operative wounds postoperatively secreted a significantly larger amount of IL-6 than those obtained from uninjured skin either preoperatively or postoperatively, implying that skin at the site of the operative wound had been more sensitized to produce IL-6 because of the surgical injury. The IL-6 secretion by skin explants was significantly enhanced either by tumor necrosis factor or interleukin-1, while it was inhibited by corticosteroids. CONCLUSIONS: Interleukin-6 production at the site of the operative wound is partly responsible for the elevation of the serum IL-6 level during the operation. Organ cultures of the skin explants may provide a feasible system for research on the cytokine networks in surgical patients.

Enhanced induction of lymphokine-activated killer activity following a single dose of cisplatin in cancer patients.

The effects of intravenous cisplatin (CDDP) administration on the generation of lymphokine-activated killer (LAK) activity in peripheral blood mononuclear (PBM) cells were investigated in cancer patients. The ability of PBM to generate LAK activity was significantly augmented 3, 5 and 7 days after a single dose, 50 mg m-2, of CDDP injection when compared to that before injection. NK activity of PBM was not altered. The distribution of lymphocyte subsets exhibited no significant change following CDDP injection, except CD2+ cells. However, the ability of monocytes in PBM to produce TNF-alpha was significantly enhanced 5 days after the drug administration, although IL-1-alpha and IL-1-beta production was not augmented.

Lymphokine-activated killer cell function of peripheral blood mononuclear cells, spleen cells and regional lymph node cells in gastric cancer patients.

Lymphokine-activated killer (LAK) cells generated by culture of peripheral blood mononuclear cells (PBMC), spleen cells (SPC) and regional lymph node cells (LNC) with IL-2 for 4 days were examined for their functional capabilities in 29 patients with gastric carcinoma. The cytotoxic activity of LAK cells induced from LNC was significantly lower than that from either PBMC or SPC, although there was no difference between PBMC or SPC. The induction of mRNA of interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) and the production of these cytokines in the non-adherent LAK cells from LNC were also significantly reduced compared with those from PBMC or SPC. Further, the LAK cells from LNC secreted significantly lower levels of these cytokines when stimulated with tumour target, Raji cells, although the production of these cytokines was markedly increased by stimulation with the targets in all three cell populations. Phenotypic analysis of each cell population revealed a decreased proportion of the cells mediating natural killer (NK) activity, including CD16+, CD56+, and CD57+ cells in LNC either before or after culture, although OKIa1+ and CD25+ cells were uniformly increased in all cell populations after culture. Changes in subpopulations of CD4+ and CD8+ cells in LNC were not apparently different from PBMC or SPC. These results indicated the differential reactivity of each lymphocyte population to IL-2 and the reduced LAK cell function of LNC compared with PBMC or SPC in patients with gastric carcinoma.

The feasibility of epidural anesthesia without endotracheal intubation for abdominal surgery in patients over 80 years of age.

To evaluate the efficacy of a single application of epidural anesthesia without endotracheal intubation for elderly patients over 80 years of age, the data on 108 patients who underwent abdominal surgery were analyzed for the occurrence of postoperative complications. These patients were classified into two groups according to the type of anesthesia performed: 66 received epidural anesthesia alone (Group I) and 42, general anesthesia under endotracheal intubation (Group II). There were no lethal pulmonary complications in Group I, whereas 2 patients (4.8%) died of respiratory failure resulting from pulmonary complications in Group II. The incidence of postoperative pulmonary complications in Group I was 6.1%, which was significantly lower than the 28.6% observed in Group II (p < 0.005). The occurrence of pulmonary complications in Group I was not related to the operating time, while pulmonary complications frequently occurred in patients who underwent lengthy operations in Group II. These findings suggest that a single application of epidural anesthesia would improve the overall safety in performing abdominal surgery in elderly patients over 80 years of age.

Lymphokine-activated killer cell activity of peripheral blood, spleen, regional lymph node, and tumor infiltrating lymphocytes in gastric cancer patients.

Lymphokine-activated killer (LAK) cell activity of peripheral blood mononuclear cells (PBM), spleen cells (SPC), regional lymph node cells (LNC), and tumor-infiltrating lymphocytes (TIL), induced by activation with interleukin 2 (IL 2) for 4 days, was evaluated in patients with gastric carcinoma. TIL exhibited the lowest LAK activity and the cytotoxicity of LNC was significantly lower than that of either PBM or SPC. There was no difference between PBM and SPC. Then, there were significant correlations of LAK activity among PBM, SPC, and LNC, whereas poor correlations were observed in the cytotoxicity between TIL and PBM, SPC, or LNC. Phenotypic analysis of each cell population was performed before and after activation with IL 2. Before culture, the cells mediating natural killer (NK) activity such as CD16+, CD56+, and CD57+ cells were few in LNC and TIL. However, CD56+ and CD57+ cells in TIL were increased after culture. Then, CD4+Leu8+ and CD8+CD11+ cells, which identify suppressor cell function, were not elevated in LNC or TIL, as compared to that in PBM or SPC. Further, the proportions of OKIa1+ and CD25+ cells expressing T-cell activation and IL 2 receptor were uniformly increased in all cell populations after culture. These results indicate the differential reactivity of each lymphocyte population to IL 2 and fundamental dysfunction of LNC and, especially TIL, suggesting the specific influence of the local tumor environment on the lymphocyte function in the area in patients with gastric carcinoma.

Laboratory correlates of chemoimmunotherapy with low-dose recombinant interleukin-2 and mitomycin C in patients with advanced carcinoma.

Based on our clinical findings that the ability of cancer patients to generate lymphokine-activated killer (LAK) cells was remarkably augmented after mitomycin C (MMC) administration, we designed a treatment regimen that consisted of MMC 12 mg/m2, i.v. on day 1 and recombinant interleukin-2 (IL-2) 700 U/m2, i.v. every 12 hr from day 4 through day 8. Of 29 patients with advanced carcinoma treated with this regimen, 10 had a partial response (PR) and 4 had a minor response. The correlation of hematological and immunological changes associated with this treatment with the antitumor response to this therapy was investigated. Pretreatment values of total white blood cell and lymphocyte counts, and the level of increase of eosinophil counts in responder patients who showed a PR, were significantly greater than those in nonresponder patients. However, there was no correlation between clinical response and cytotoxic activities of peripheral blood mononuclear (PBM) cells, including NK and LAK activity, and the ability to generate LAK cells after the treatment. The capacity of adherent cells in PBM to produce IL-1-beta was increased after the treatment in both responders and nonresponders, whereas IL-1-alpha production was not increased. In addition, a significant increase in the ability to produce TNF-alpha was observed only in responders, indicating the correlation of TNF-alpha production with clinical response to this therapy. Since these correlations had been reported in the previous studies using IL-2, the present results suggested that the therapeutic effectiveness of this therapy against advanced carcinoma, is due to IL-2 probably augmented by its combination with MMC. In addition, these parameters might be predictive of therapeutic efficacy of this treatment.

Neuroleptic malignant syndrome occurring after an emergency operation for traumatic duodenal perforation: report of a case.

Neuroleptic malignant syndrome (NMS) is a potentially fatal complication which may develop in psychiatric patients taking neuroleptic drugs. We report herein the successful treatment of a 33-year-old schizophrenic man, prescribed neuroleptic drugs, who underwent an emergency operation for traumatic duodenal perforation with a retroperitoneal infection. Five days after the operation, he began to demonstrate clinical features consistent with NMS such as high fever, abnormalities in vital signs, leukocytosis, and an elevated serum level of creatine phosphokinase; however, these findings were first presumed to be secondary to either the preexisting tissue injuries or to postoperative complications. A definite diagnosis of NMS was thus delayed until muscle rigidity and autonomic instability became evident. After a tentative diagnosis of NMS had been made, sodium dantrolene, a drug used specifically for the treatment of NMS, was administered and the patient's condition remarkably improved. Since NMS can be induced by either interrupting the course of neuroleptic drugs or by the additional administration of sedative drugs, and since its mortality rate is high if prompt and appropriate treatment is not carried out, surgeons should bear in mind the possibility of NMS developing postoperatively in psychiatric patients.