Immune regulation of the L5178Y murine tumor-dormant state. I. In vivo and in vitro effects of prostaglandin E2 and indomethacin on tumor cell growth.

Immunization and intraperitoneal challenge of DBA/2 mice with L5178Y lymphoma cells results in the suppression and maintenance of the L5178Y cells in a tumor-dormant state in the peritoneal cavity for many months. Cell-mediated immune responses involving lymphocytes and macrophages are involved in maintenance of the tumor-dormant state. Macrophages that have increased immunosuppressive activity and that produce increased amounts of PGE2 appear in the peritoneal cavity of tumor-dormant mice before the breakdown of the tumor-dormant state and formation of ascitic tumors. We report here that the tumor-dormant state can be terminated with formation of ascitic tumors by treatment of tumor-dormant mice with PGE2. Treatment with indomethacin results in inhibition of tumor cell growth and elimination of all recoverable tumor cells. Cultures of peritoneal cells (PC) from mice harboring L5178Y cells in a tumor-dormant state were used to analyze the PGE2 and indomethacin effects. Tumor cells did not grow out in the high-cell density PC cultures prepared from many tumor-dormant mice, but addition of PGE2 to these cultures resulted in tumor cell growth. The tumor cell growth that did occur in the PC cultures from some tumor-dormant mice was associated with PGE2 production by the associated host cells, and the addition of indomethacin to these cultures inhibited both PGE2 synthesis and tumor cell growth. Removal of plastic-adherent cells from the PC cultures eliminated the restraint on tumor cell growth. These experiments suggest that L5178Y tumor cells are maintained in a tumor-dormant state by host peritoneal cells, which are under PGE2 regulation.

Organisms can essentially be classified according to two codon patterns.

We recently classified 23 bacteria into two types based on their complete genomes; "S-type" as represented by Staphylococcus aureus and "E-type" as represented by Escherichia coli. Classification was characterized by concentrations of Arg, Ala or Lys in the amino acid composition calculated from the complete genome. Based on these previous classifications, not only prokaryotic but also eukaryotic genome structures were investigated by amino acid compositions and nucleotide contents. Organisms consisting of 112 bacteria, 15 archaea and 18 eukaryotes were classified into two major groups by cluster analysis using GC contents at the three codon positions calculated from complete genomes. The 145 organisms were classified into "AT-type" and "GC-type" represented by high A or T (low G or C) and high G or C (low A or T) contents, respectively, at every third codon position. Reciprocal changes between G or C and A or T contents at the third codon position occurred almost synchronously in every codon among the organisms. Correlations between amino acid concentrations (Ala, Ile and Lys) and the nucleotide contents at the codon position were obtained in both "AT-type" and "GC-type" organisms, but with different regression coefficients. In certain correlations of amino acid concentrations with GC contents, eukaryotes, archaea and bacteria showed different behaviors; thus these kingdoms evolved differently. All organisms are basically classifiable into two groups having characteristic codon patterns; organisms with low GC and high AT contents at the third codon position and their derivatives, and organisms with an inverse relationship.

Codon evolution is governed by linear formulas.

When nucleotide (G, C, T and A) contents were plotted against each nucleotide, their relationships were clearly expressed by a linear formula, y = alphax + beta in the coding and non-coding regions. This linear relationship was obtained from the complete single-stranded DNA. Similarly, nucleotide contents at all three codon positions were expressed by linear regression lines based on the content of each nucleotide. In addition, 64 codon usages were also expressed by linear formulas against nucleotide content. Thus, the nucleotide content not only in coding sequence but also in non-coding sequence can be expressed by a linear formula, y = alphax + beta, in 145 organisms (112 bacteria, 15 archaea and 18 eukaryotes). Based on these results, the ratio of C/T, G/T, C/A or G/A one can essentially estimate all four nucleotide contents in the complete single-stranded DNA, and the determination of any ratio of two kinds of nucleotides can essentially estimate four nucleotide contents, nucleotide contents at the three different codon positions and codon distributions at 64 codons in the coding region. The maximum and minimum values of G content were approximately 0.35 and approximately 0.15, respectively, among various organisms examined. Codon evolution occurs according to linear formulas between these two values.

Rapidly reversible resistance to immune lysis of L5178Y lymphoma cells in a tumor-dormant state in DBA/2 mice.

L5178Y cells in a tumor-dormant state in DBA/2 mice escape lysis by cytolytic T-lymphocytes and activated macrophages. In the present report these tumor cells were found to be less susceptible to lysis by anti-H-2, anti-Thy 1.2, and anti-AKR antibodies plus complement than was the same tumor cell population removed from tumor-dormant mice and allowed to proliferate in a nonimmune environment in vivo or in vitro. This rapidly reversible resistance to immune lysis may be due to arrest of the tumor cells in the cell cycle during the tumor-dormant state.

Alteration of immunogenicity of xenogenized tumor cells in syngeneic rats by the immune responses to virus-associated antigens produced on immunizing cells.

A strong transplantation resistance to a fibrosarcoma (KMT-17) is induced in syngeneic Wistar King Aptekman/Hok rats after a single s.c. immunization with Friend virus-infected (xenogenized) viable KMT-17 cells. The resistance induced by the repeated immunizations with irradiated Friend virus-infected KMT-17 cells, however, was unexpectedly weaker when compared with that induced by irradiated KMT-17 cells. The immunogenicity of tumor-associated antigen on xenogenized Friend virus-infected KMT-17 cells was correlated with their shortened survival time in the peritoneal cavity after i.p. inoculation, especially in rats preimmunized with xenogenized tumor cells. Furthermore, xenogenized tumor cells producing a medium amount of virus-associated antigen (VAA) but not those producing a large amont of VAA showed an augmented immunogenicity even in normal rats. On the other hand, the tumor-associated antigen immunogenicity was augmented by immunization with xenogenized tumor cells which expressed a relatively small amount of VAA in rats presensitized with VAA. These findings indicate that the immunogenicity of xenogenized tumor cells is augmented by the middle-grade immune responses to VAA produced on xenogenized tumor cells.

Enhanced suppressor macrophage activity associated with termination of the L5178Y cell tumor-dormant state in DBA/2 mice.

Both cytolytic T-lymphocytes and cytolytic macrophages have been implicated in the long-term maintenance of L5178Y cells in a tumor-dormant state in DBA/2 mice. Eventually, however, the tumor-dormant state terminates, and all mice develop ascitic tumors. In an evaluation of the mechanisms involved in termination of the tumor-dormant state, we detected in the peritoneal cavity of many tumor-dormant mice macrophages with increased capacity to suppress the in vitro generation of a secondary anti-L5178Y cell cytolytic T-lymphocyte response. The incidence of macrophage-mediated immunosuppressive activity in individual tumor-dormant mice was related directly to the number of tumor cells in the peritoneal cavity of those mice. Furthermore, in tumor-dormant mice harboring fewer than 5 X 10(4) L5178Y cells, the detection of macrophage-mediated immunosuppressive activity was a prognostic indicator of termination of the tumor-dormant state and development of an ascitic tumor. These data suggest that peritoneal macrophage-mediated immunosuppressive activity, through inhibition of cytolytic T-lymphocyte generation in vivo, contributes to the termination of the tumor-dormant state and development of ascitic tumors.

Maintenance and cure of the L5178Y murine tumor-dormant state by interleukin 2: in vivo and in vitro effects.

We studied the antitumor effects of recombinant human interleukin 2 (HuIL-2) in DBA/2 mice which harbor L5178Y lymphoma cells in a tumor-dormant state in the peritoneal cavity and in peritoneal cell (PC) cultures prepared from such mice. Intraperitoneal injection of 10,000 to 100,000 units of HuIL-2/day for 10 days eliminated tumor cells from 30-45% of the mice, whereas no mice became tumor-free in the phosphate-buffered saline-treated control group. In vitro, as little as 10 units/well HuIL-2 failed to stimulate cytotoxic activity in PC from normal mice. HuIL-2 directly stimulated antitumor cytotoxic activity in nonadherent but not in adherent PC cultures from tumor-dormant mice; however, treatment of whole PC from tumor-dormant mice with HuIL-2 resulted in the development of antitumor cytotoxic activity in the adherent PC population which were derived from such cultures. This suggests that the HuIL-2-treated nonadherent PC contributed to the cytotoxic activation of the adherent PC. Flow cytometric analysis of the PC from tumor-dormant mice revealed a polyclonal expansion of T-lymphocytes. Lyt-1+, Lyt-2+, and L3T4+ lymphocytes were all required for HuIL-2 to induce antitumor cytotoxic activity.

Effects of dietary vitamin B-2 and vitamin E on the delta9-desaturase and catalase activities in the rat liver microsomes.

The effects of dietary vitamin B-2 and vitamin E on delta9-desaturation of stearoyl-CoA, catalase, glutathione peroxidase, superoxide dismutase and electron transport components in rat liver microsomes have been investigated. delta9-desaturase activities were decreased on diets deficient of vitamin B-2, E and supplemented with E. Among the peroxide-scavenging enzymes, only the catalase activity in microsomes correlates significantly with delta9-desaturase activity. In vitro addition of bovine catalase had no effect on microsomal delta9-desaturase activity on control diet. However, it enhanced the delta9-desaturation in microsomes on vitamin B-2-deficient diet which contained low catalase and high superoxide dismutase activities, compared to those in microsomes of control diet. It is suggested that the hydrogen peroxide-generating and -decomposing systems may play an important role on the delta9-desaturase activity in microsomes.

Metabolism of platelet-activating factor in primary cultured adult rat hepatocytes by a new pathway involving phospholipase C and alkyl monooxygenase.

The metabolic fate of 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether) upon interaction with primary cultured adult rat hepatocytes was investigated. [3H]PAF-acether was transformed time-dependently into [3H]lyso-PAF-acether, 1-O-[3H]alkylglycerol and finally converted to 3H-labeled fatty aldehyde. 1-O-[3H]Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) was formed after a long incubation time and with a smaller amount compared with that formed in platelets and neutrophils. When lipids from cells, cell surfaces and incubation medium were analyzed separately, most of the transformed products of [3H]PAF-acether remained in the cells. When 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine was incubated with hepatocytes, it was mainly converted into 1-O-[3H]alkylglycerol. 3H-labeled fatty aldehyde and [3H]alkylacyl-GPC were also found. Hepatocytes metabolized slowly from 1-O-[1-14C]hexadecylglycerol to 3H-labeled fatty aldehyde and 3H-labeled phospholipid. These findings suggest that cultured hepatocytes mainly catabolize exogeneous PAF-acether by removing the acetyl residue and the polar head group and, finally, by cleaving an ether bond. The deacetylation-reacylation step, which is important in platelets and neutrophils, was not shown to be a main metabolic pathway of PAF-acether in cultured hepatocytes.

Possible involvement of NADPH-cytochrome P450 reductase and cytochrome b5 on beta-ketostearoyl-CoA reduction in microsomal fatty acid chain elongation supported by NADPH.

The chain elongation products from [2-14C]malonyl-CoA and palmitoytl-CoA by rat liver microsomes initiated by NADPH were found mainly to be 18:1 and 18:0. When anti-NADPH-cytochrome P450 reductase or anti-cytochrome b5 immunoglobulin G were included in this system, not only was the overall chain elongation significantly suppressed, but also a new radioactive product, which was identified as 2-heptadecanone derived from an intermediate beta-ketostearate, is accumulated depending upon the amounts of IgG added. These results suggest that NADPH-cytochrome P450 reductase and cytochrome b5 participate in the conversion from beta-ketostearoyl-CoA to beta-hydroxystearoyl-CoA, which is the first reductive step of the microsomal chain elongating system.

[Functions of large granular lymphocytes--a case of large granular lymphocytosis with characteristic cell surface antigens].

A 58-year-old male visited the hematological clinic of Surugadai Nihon University Hospital, Tokyo, complaining of numbness around both elbows. The peripheral leukocyte count was 12,400/microliters, and large granular lymphocytes (LGL) occupied 79% of the leukocytes. The cell surface antigen studied by flow cytometry were the positive CD 2, 3, 5, 8, 11, and Leu-7, and the negative CD1, 4, 10, 16 (Leu-11), 19, 20, and OKTIa1. IgG-FCR checked by mean of the EA-rosette formation was positive. The LGL showed the negative NK cell activity and the positive ADCC and LAK cell activities. It was interesting that LGL was negative for CD16 (Leu-11) while they had ADCC activity. Since the rearrangement of the receptor gene in T-cells was demonstrated by the southern blot analysis, the proliferation of LGL was considered to be a clonal one. LGL did not inhibit the colony formation of granulocyte and erythrocyte precursors in the plasma clot culture. It was thus considered that this might partially explain the fact that the patient's neutrophil, Hb and platelet levels remained normal.

Involvement of cytochrome b5 in the oxidative desaturation of linoleic acid to gamma-linolenic acid in rat liver microsomes.

The effects of antibodies against microsomal electron-transport components on the in vitro activity of delta6-desaturation of linoleic acid to gamma-linolenic acid have been studied in intact microsomal membranes of rat liver. Reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (0.87 mM) served as electron donors, and effectively prompted the delta6-desaturase activities with yields of about 1.1 to 1.3 nmol per mg of protein in 10 min. Of the two antibodies studied under the same in vitro conditions, i.e., rabbit antisera preparations against rat liver microsomal hydrophilic parts of cytochrome b5 and NADPH-cytochrome c reductase, only the antibody against cytochrome b5 demonstrated a marked ability to inhibit the delta6-desaturase activity. This evidence supports a participation of cytochrome b5 in the delta6-desaturation of linoleic acid and suggests a pathway analogous to the delta9-desaturation of stearyl-CoA.

[Clinical evaluation of Dumon stent for tracheobronchial stenosis due to advanced esophageal carcinoma].

We inserted Dumon stent to 13 patients with tracheobronchial stenosis due to advanced or recurrent esophageal carcinoma. Severe dyspnea was improved in 11 patients except for 2 patients with bilateral recurrent nerve palsy. 3 cases who had radiation therapy or chemotherapy lived over 150 days. We inserted esophageal stent in 4 cases. 2 patients died due to hemoptysis after 156 days and 35 days. We conclude that Dumon stent is one of the useful treatments in order to improve quality of life and prognosis of advanced or recurrent esophageal carcinoma.

[Histological effect and prognosis after preoperative chemotherapy in esophageal cancer].

Preoperative chemotherapy for esophageal cancer was performed and the toxic effects, post-operative complications, histological effects and survival time were evaluated. Toxic effects and post-operative complications were rare. The histological effect was evaluated according to the Guidelines for Clinical and pathologic studies on carcinoma of the esophagus by the Japanese Society for Esophageal Disease. A histological effect of more than Grade II was seen from 25 to 42.1%. A significant difference was seen in the Kaplan-Meier survival curves between the response group and the no response group (p < 0.05). If we perform preoperative chemotherapy for esophageal cancer only to responsive cases determined by chemosensitivity tests, the prognosis of advanced esophageal cancer may be improved.

[Surgical analysis for lung cancer with scalene node metastasis].

In this study, we evaluated 15 surgically treated cases of lung cancer with scalene node metastasis. Eleven males and 4 females with ages ranging from 41 years to 78 years (average 59.1 years) were examined. Seven cases was in the right upper lobe, 3 in the left upper lobe, 2 in the right lower lobe, 2 in the left lower lobe, 1 in the middle lobe. Adenocarcinomas were found in 9 cases squamous cell carcinomas were in 4 cases and large cell carcinomas were in 2 cases. Two cases was in T 1, 7 in T 2, 2 in T 3, 4 in T 4. In all cases, lobectomy was performed. Five year survival rate was 14.3% as determined by the Kaplan-Meier method. Average survival period was 26.6 months. Three cases survived over 3 years including one case survived over 10 years.

[A mild variant case of maple syrup urine disease].

We reported a mild variant case of maple syrup urine disease. He was unable to walk alone at 2 years of age and developed seizures and intermittent ataxia at 5 years of age. Activity of 1-14C-leucine decarboxylase in fibroblasts revealed 40% of normal activity in the boy and 90% in the mother. MRI showed hypo-myelination of white matter and mild atrophy of brain stem and cerebellum. Dietary treatment was not effective for ataxia and brain atrophy.

[A case of mediastinal schwannoma originating from the intrathoracic vagal nerve].

A 46-years-old man was admitted because of an abnormal shadow on X-ray. The operation underwent under the diagnosis of benign tumor in the antero-superior mediastinum. At the operation, the tumor arise from the right intrathoracic vagal nerve and it was resected with the transection of vagal nerve. The histological diagnosis was schwannoma. Postoperative course was uneventful except the slight hoarseness. Intrathoracic vagal tumor is rare and we have found 19 reported cases among Japanese literatures. This case is considered to be the 20th case originated from the intrathoracic vagal nerve in Japan.

[Surgery for intrathoracic recurrence of non-small cell lung cancer].

In this study, we defined a solitary lung nodle in the same histology which could be traced its' origin from carcinoma in situ or was found over than two years' follow up as a second primary lung cancer. These cases were excluded. Eighteen cases underwent second surgery for intrathoracic recurrence. Fourteen cases were male and four cases were female. Their ages ranged from 23 to 75 (average 59.6) years. The histology were adenocarcinoma in 9 cases, squamouscarcinoma in 7, adenosquamous carcinoma in 1, large cell carcinoma in 1. The initial surgical procedures were lobectomy in 17, partial resection in 1. The initial stage were I in 13, II in 2, IIIA in 1. Pulmonary recurrence were found in 10, bronchial stump recurrence were found in 4, pulmonary hilus lymph node recurrence were found in 2, mediastinal lymph node recurrence were found in 2, pulmonary stump recurrence was found in 1. The second surgical procedures were completion pneumonectomy in 7, completion lobectomy in 1, lobectomy with segmentectomy in 1, segmentectomy or partial resection in 7, mediastinal dissection in 2. The overall 5-year survival rate of the patients with recurrence after reoperation was 31.8%. An aggressive surgical approach for recurrent lung cancer should be recommended.

[Clinical study on reoperation for recurrent pulmonary metastasis].

Ninety seven patients with metastatic lung tumors were treated surgically in our department. Second pulmonary resections were performed in 13 patients. They consists of 9 males and 4 females, their age ranged from 12 to 75 years old (average 54.4 years old). Tumors originate from sarcoma in 5 cases, laryngeal cancer, colorectal cancer, renal cell cancer in 2 cases respectively, oral cavity cancer and transitional cell cancer in 1 case respectively. No second pulmonary resection was performed in patients with metastatic lung tumors originating from lung cancer or breast cancer, because they metastatize not only lung but also general organs. No second pulmonary resection was required in patients with testicular tumor and choriocarcinoma, because chemotherapy take good effect on them.

[A case of primary pulmonary cryptococcosis].

A 53-year-old male was admitted because of an abnormal chest roentgenogram showing a solitary tumor shadow in the right upper lung field. Laboratory data was normal. Although examination with the bronchoscope and sputum cytology showed no abnormal findings, a partial resection of the right upper lobe was performed. Microscopic examination of the lesion was cryptococcosis. As another focus was not found by systemic examination, the lesion was disclosed primary pulmonary cryptococcosis. Cryptococcosis is rare but we must think of it when we meet a solitary tumor shadow on the chest roentgenogram.