L-canavanine restores blood pressure in a rat model of endotoxic shock.

Administration of lipopolysaccharide to anaesthetised rats produced a reduction in mean arterial pressure, an increase in heart rate, and death at 4-6 h. Intravenous infusion of NG-nitro-L-arginine methyl ester (50 mg/kg), an inhibitor of constitutive and inducible nitric oxide (NO) synthase, 60 min after challenge with lipopolysaccharide, caused an immediate increase in blood pressure followed by a precipitous fall in pressure, and death. In contrast, intravenous infusion of L-canavanine (100 mg/kg), reported to be a selective inhibitor of inducible NO synthase in vitro, 60 min and 180 min after lipopolysaccharide challenge, produced an increase in mean arterial pressure and reversed the lipopolysaccharide induced hypotension. However, in lipopolysaccharide challenged animals protected from hypotension by administration of L-canavanine (60 min post challenge), intravenous infusion of NG-nitro-L-arginine methyl ester at 180 min post challenge caused an immediate rise in mean arterial pressure, followed by a rapid fall in blood pressure and heart rate, and sudden death. In contrast, a second dose of L-canavanine at 180 min post challenge maintained blood pressure for the duration of the experiment. These findings indicate that inhibition of both constitutive and inducible NO synthase during endotoxaemia is lethal. However, the use of a selective inhibitor of inducible NO synthase restores mean arterial pressure to baseline, and offers a therapeutic approach to managing hypotension in shock.

Inhibition of nitric oxide synthesis improves survival in a murine peritonitis model of sepsis that is not cured by antibiotics alone.

Inhibition of nitric oxide synthesis was investigated in a murine model of advanced sepsis in which antibiotic therapy alone did not improve survival. Seven hours after receiving a lethal intraperitoneal challenge with live Escherichia coli, mice were given either NG-monomethyl-L-arginine (L-NMMA) intravenously, imipenem-cilastatin subcutaneously or a combination of both. L-NMMA (3-300 mg/kg) or imipenem-cilastatin (10 or 50 mg/kg) given alone did not improve survival; co-administration of L-NMMA and either 10 or 50 mg imipenem-cilastatin/kg improved survival significantly. These findings suggest that nitric oxide contributes to the morbidity associated with advanced sepsis and that nitric oxide synthase inhibition may improve the efficacy of conventional antimicrobial treatment of severe infections.

Modulation of type IV collagenase and plasminogen activator in a hamster fibrosarcoma by basement membrane components and lung fibroblasts.

The effect of basement membrane components (laminin, fibronectin and type IV collagen) and lung fibroblasts on type IV collagenase and plasminogen activator activity was investigated in a primary HSV-2-induced hamster fibrosarcoma, and its in vivo derived sublines and in vitro derived clones of varying metastatic potential. Fibronectin and type IV collagen were ineffective at influencing the expression of either type IV collagenase or plasminogen activator activity. Laminin, however, at concentrations of 1-10 micrograms ml-1 added to the serum-free culture supernatants, increased the release of type IV collagenase by up to 100% for the parental cell line. Three highly metastatic sublines (two from in vivo origin and one from in vitro cloning) showed increases of up to 300%. Non-metastatic sublines (two from in vivo origin and one from in vitro cloning), however, showed no increase in type IV collagenase activity. Plasminogen activator release from either the parental line cell or its metastatic sublines and clones, was unaffected by the addition of laminin. Addition of tumour cells to lung fibroblast monolayers resulted in an increased expression of PA activity in the supernatant, whilst type IV collagenase activity was reduced.

Type IV collagenase activity of a primary HSV-2-induced hamster fibrosarcoma and its in vivo metastases and in vitro clones.

The expression of a basement membrane (BM) collagen-degrading metalloprotease (Type IV collagenase) was studied in a herpes simplex virus (HSV)-2 transformed hamster fibrosarcoma and its in vivo derived sublines and in vitro derived clones of varying metastatic potential. The primary parent tumor was shown to release more or less Type IV collagenolytic activity compared with its sublines (derived from lung nodules that developed after resection of the primary tumor). Normal baby hamster kidney and hamster embryo fibroblasts did not secrete detectable amounts of BM collagenase, whereas normal hamster lung fibroblast secreted intermediate levels of Type IV collagenase activity. The collagenase IV activity of the parent tumor and its in vivo and in vitro derived sublines was assayed in vitro and compared with the ability of the cells lines to spontaneously metastasize in vivo. No correlation between the ability to secrete type IV collagenase and metastatic propensity was detected. Although all cell lines secreted type IV collagenase, the highest activity was recorded for a nonmetastatic variant.

Therapy of spontaneously metastatic HSV-2 induced hamster tumours with cortisone acetate administered with or without heparin.

Subcutaneous injection of cortisone acetate administered with or without oral heparin retarded the growth of two HSV-2 induced hamster fibrosarcomas. Histological sections showed no obvious difference between the vasculature of treated and untreated tumours although there were fewer infiltrating lymphocytes in treated tumours. Treatment was, however, found to be ineffective against metastatic development following resection of primary tumours. Natural killer cell activity was found to be greatly reduced in animals receiving heparin and/or cortisone acetate treatment and this may influence the effectiveness of treatment on the metastatic process. We conclude that treatment of tumours with cortisone plus heparin is no different from the response to cortisone used alone.

Metastatic heterogeneity in a spontaneously metastatic HSV-2 induced hamster fibrosarcoma: association of phenotypic and genotypic properties with metastatic potential.

A spontaneously metastatic tumour of hamsters and cell lines derived from its in vivo metastatic deposits, or from in vitro cloning were compared for immunobiological and genotypic variation and correlation sought with regard to metastatic potential. Cell lines were established from seven individual lung metastases following primary tumour resection of the parent (HSV-2-333-2-26) cell line. When inoculated subcutaneously, and upon resection of the subsequent tumour mass, four cell lines (MetA, MetB, MetE and MetF) demonstrated greater, one similar (MetG) and two (MetC and MetD) less metastatic capacity compared with the parent tumour line. In further studies cell lines were established in vitro by single cell cloning of the parent tumour either by limiting dilution or soft agar cloning. In vivo tumour resection experiments showed eight cell lines (S4, S7, S4A, S7A, S7B, C1.1, C1.4 and C1.5) to have an increased metastatic potential and five cell lines (S8, S9, S8B, S9D and S9E) a decreased metastatic potential compared to the parental line. Karyotypic analysis of the cells revealed that all highly metastatic cell lines were of a near diploid genotype, whilst non- and weakly metastatic cell lines, including the parental line, were aneuploid or near tetraploid. The immunobiological characteristics of these cell lines was studied. Assessment of in vivo immunogenicity showed that eight clones (MetA, MetB, MetE, MetF, S7A, C1.1, C1.4 and C1.5) were non-immunogenic whilst the parental tumour line and three clones (MetC, MetD and MetG) exhibited a strong transplantation rejection antigen; immunogenicity showed an inverse correlation with metastatic potential. Susceptibility to NK cytolysis was demonstrated for cell lines exhibiting a weak or non-metastatic/immunogenic phenotype. The origin of metastatic variants and their association with genotype and immunobiological properties is discussed.

Production of plasminogen activator by a primary HSV-2-induced hamster fibrosarcoma and its in vivo derived sublines.

The plasminogen activator (PA) content of a primary HSV-2-induced hamster fibrosarcoma and sublines derived from its in vivo metastases was investigated using the indirect 125I-labeled fibrin plate method. Fresh tissue culture lines established from primary HSV-2-333-2-26 tumors were shown to produce levels of PA similar to sublines derived from lung or kidney foci that developed after resection of primary tumors. In comparison, normal hamster embryo fibroblast (NHEF) and baby hamster kidney cells produced little or no PA, although baby hamster lung fibroblasts produced intermediate levels of PA. In addition, no correlation was found between PA levels of the sublines and their ability to metastasize from subcutaneous tumors, although tumor cell lines were shown to produce significantly more PA than normal cells.

Non-random chromosome changes in a herpes-virus-transformed Syrian hamster cell line and its metastatic derivatives.

A primary subcutaneous tumour of low spontaneous metastatic capacity, produced after inoculation of Herpes-virus hominis type-2-transformed hamster fibroblasts (parent line) and two in vivo derived highly metastatic lung deposits (Met A and Met B) were karyotyped and compared after trypsin G-banding. The parent line was cytogenetically heterogeneous with a modal chromosome number of 74. However, a number of cells were of a higher ploidy level. A large variation in both numerical and structural abnormalities was observed, the chromosome rearrangements were often complex and unstable, but all the cells contained a theme of common marker chromosomes. Met A and Met B were near diploid (mean chromosome numbers 42 and 44 respectively) with a low level of tetraploid cells. They shared many chromosome rearrangements but could be readily distinguished by an additional translocation unique to Met A. Cytogenetic homogeneity within and between metastases suggested that they were of monoclonal origin and had been derived from a karyotypically similar subpopulation within the parent tumour. We were unable to detect such cells in the parent line; thus, their numbers within the parent tumour were likely to be low. Metastasis, therefore, has been highly selective, depending on the particular phenotypic properties of Met A and Met B. All cells of the parent and metastatic lines have homogeneously staining regions (HSR) and abnormalities of chromosomes 15 (C15) which may be important in tumorigenesis. In addition, Met A and Met B cells have a number of chromosome rearrangements [translocations, deletions and a double minute chromosome (DM)] not present in the parent cells. They are retained at a high frequency in the cells of Met A and Met B and thus it seems likely that the metastatic phenotype is associated with one or more of these chromosome aberrations.

Properties of a herpesvirus-transformed hamster cell line: immunogenicity of sublines of high and low metastatic potential.

The immunogenicity of a herpesvirus hominis type-2-transformed hamster cell line (HSV-2-333-2-26) of low spontaneous metastatic ability was compared with that of its two in vivo-derived sublines of increased metastatic potential. The parent (HSV-2-333-2-26) tumour was immunogenic as assessed by protection against tumour challenge afforded by implantation of irradiated tumour cells or tissue. In contrast, the two metastatic sublines, designated Met A and Met B, were non-immunogenic as defined by the above critera . However, the parent Met A and Met B tumours were shown to possess a common antigen(s), since immunization with irradiated parent tumour cells afforded protection to challenge with Met A or Met B. Immunization with the metastatic sublines, however, gave no protection to homologous or heterologous tumour challenge. Bacillus Calmette-Guérin (BCG) inoculated in admixture with irradiated tumour cells and followed 7 days later by one immunization with X-irradiated tumour cells alone, increased host immunocompetence to subsequent homologous or cross-tumour cell challenge with parent, Met A or Met B cells. The immunity raised by using BCG plus irradiated tumour cells was shown to be specific to antigens expressed on the HSV-2 parent cell line and its metastatic sublines. In addition, BCG admixed with live inocula of parent, Met A or Met B cells induced contact suppression of in vivo tumour growth of the parent cells, but not of Met A or Met B cells. It is suggested from these studies that the parent tumour possesses a tumour-specific transplantation antigen(s) ( TSTAs ) which is not functionally active on its metastatically derived sublines. Common antigens, shared between the parent and Met A and Met B cells, are detectable by cross-challenge experiments, but they themselves appear not to be immunologically offensive. The loss of immunogenicity is discussed as a possible mechanism for the in vivo selection of sublines with increased metastatic potential.

Reduced susceptibility to natural killer cell lysis of hamster tumours exhibiting high levels of spontaneous metastasis.

The natural killer (NK) cell susceptibility of hamster tumours exhibiting high and low levels of spontaneous metastasis in vivo was investigated. The parent cell line (HSV-2-333-2-26), which was weakly metastatic, and the NK-sensitive target line K562 were both sensitive to hamster natural killer cell activity. In contrast, 2 in vivo cloned sublines, designated met A and met B, exhibited a high level of spontaneous metastasis and were shown to be weakly or non-susceptible to hamster UK reactivity.

Detection and characterization of natural killer cells in Syrian golden hamsters.

Natural killer (NK) cell activity was observed in the peripheral blood and spleen of 8- to 10-week-old Syrian golden hamsters, but not in the bone marrow or thymus. Low, but significant, levels of cytotoxicity were also observed in mesenteric and axillary lymph nodes and cells harvested from the peritoneum. Cytotoxicity, in a 4-hr 51Cr-release assay, was found to be nylon wool non-adherent and was significantly reduced by treatment with trypsin or incubation at 37 degrees C for 18 hr. Natural cytotoxicity was shown to be low at 1 week of age, but increased to a maximum at 8 weeks and was maintained into old age. Correlation was observed between peripheral blood cytotoxicity and the presence of large, often granular, lymphocytes following fractionation of effector cells by Percoll discontinuous gradient separation. These findings are compared with previous studies in hamsters, where the results of longer-term cytotoxicity tests differ in some respects to those of the present study.