Splanchnic sympathectomy prevents translocation and spreading of E coli but not S aureus in liver cirrhosis.

INTRODUCTION: Spontaneous bacterial peritonitis (SBP) is mainly caused by bacterial translocation of enteric Gram-negative bacteria, predominantly Escherichia coli. The sympathetic nervous system (SNS) is activated in advanced cirrhosis, particularly in the splanchic circulation, and exerts potent immunosuppressive actions. However, the role of splanchnic SNS activity in bacterial translocation and bacterial spreading in cirrhosis remains unclear. METHODS: E coli or Stapylococcus aureus (10(6) CFU) were given intraperitoneally. After 6 h, mesenteric lymph nodes (MLN), liver, spleen, lung and peripheral blood were harvested from ascitic cirrhotic rats (LC) and healthy controls with and without splanchnic sympathectomy (SE). The bacterial tissue burden was determined by standard microbiological culture techniques. In vitro phagocytic activity of peritoneal polymorphonuclear leucocytes was assessed by FACS analysis. RESULTS: Under basal conditions SE reduced bacterial translocation to MLN in LC rats from 45% to 17%. LC rats had a marked increase in bacteraemia after E coli and S aureus challenge and an increased incidence and degree of E coli translocation to MLN, liver, spleen and lung compared with control rats. SE prevented bacteraemia in LC rats after E coli but not after S aureus challenge. Prior SE abolished the difference in incidence as well as the bacterial tissue burden in each organ after E coli application in LC rats, being no longer significantly different from control rats with or without SE. The protective effects of SE against E coli were associated with a greater influx of mononuclear cells into the peritoneal cavity and increased phagocytic activity of peritoneal polymorphonuclear leucocytes. CONCLUSIONS: In cirrhosis with bacterial peritonitis, hyperactivity of the splanchnic sympathetic nervous system contributes to the translocation of E coli but not S aureus to MLN and extraintestinal sites. This indicates a key role for sympathetic drive in the impairment in host defence against Gram-negative bacteria in cirrhosis.

In vivo clonogenic tumor cell kinetics following 1,3-bis(2-chloroethyl)-1-nitrosourea brain tumor therapy.

An in vitro colony formation assay was modified to determine the effects of in vivo 1,3-bis(2-chloroethyl)-1-nitrosourea therapy on tumor cell kill and subsequent clonogenic cell kinetics. The measured surviving fraction must be multiplied by the relative total number of tumor cells for each posttreatment interval in order to eliminate inaccuracies caused by dead cell removal in vivo and the lysis of damaged cells by the disaggregation procedure. The assumptions, limitations, and applications of the technique are discussed. 1,3-Bis(2-chloroethyl)-1-nitrosourea doses of 0.25, 0.50, and 1.00 X dose lethal to 10% of animals resulted in approximately at 1-, 2-, and 3-log cell kill, respectively. Significant proliferation of surviving clonogenic cells was observed after a latency period of approximately 2 days, and the rate of tumor regrowth was dose dependent. The cell-doubling times following treatment with 0.25, 0.50, and 1.00 X doses lethal to 10% of animals were 15, 21, and 38 hr, respectively. The interval to complete repopulation of the clonogenic pool corresponds to the observed increase in animal life-span for the 2 larger doses and further validates the assay as a true measure of in vivo chemotherapeutic efficacy.

In vitro evaluation of in vivo brain tumor chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea.

An in vitro colony formation assay was used to determine the efficacy of in vitro therapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on a rat brain tumor. The fraction of clonogenic cells surviving in vivo therapy was determined by a comparison between the in vitro colony-forming capacity of cells derived from previously treated and untreated tumors. With this intracerebral solid tumor a direct correlation was found between the surviving fraction of cells and animal survival, implying that the in vitro assay system is a reliable test of therapeutic effect. The BCNU dose-response curve was exponential up to a dose of 0.75 times the LD10 dose with little additional cell kill noted at higher drug levels. This plateau does not appear to represent a resistant subpopulation of cells, since retreatment of tumors derived from cells surviving an LD10 dose were as sensitive to BCNU as those with no prior drug exposure. Instead, it may represent, at least in part, failure of the drug to reach and/or enter cells in all parts of solid tumors. On the average BCNU doses of 0.75 times the LD10 dose or greater resulted in slightly more than a 3-log cell kill and doubled the life-span for our tumor-bearing animals. The finding that an increase in animal life-span requires at least a 1-log tumor cell kill indicates that survival studies with intracranial tumor models may be insensitive to single courses of many chemotherapeutic agents with modest but significant antitumor activity.

Brain-tumor therapy. Quantitative analysis using a model system.

A recently developed colony-formation assay has been used to evaluate in vivo 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) therapy of a transplantable rat brain-tumor model. A comparison of the in vitro colony-forming capacity of treated and untreated tumor cells permits calculation of the fraction of clonogenic tumor cells surviving in vivo therapy; The plateau that we previously observed o the BCNU dose-response curve is not the result of repair of potentially lethal damage, since no change in the 0.1% of surviving clonogenic tumor cells occurs during the first 2 to 4 days after treatment. Although reanalysis of the dose-response curve indicates that sublethal damage exists, its repair is probably minimal. The most likely explanation for the observed limitation of the BCNU effect is the drug's failure to reach all clonogenic cells. A dose of BCNU that kills more than 99.9% of clonogenic tumor cells within 30 minutes of treatment results in only a 60% decrease in tumor weight by Day 14. This disparity is explained by retarded removal of dead cells, and, along with a previously determined 90% cell-kill threshold necessary to appreciate increased animal survival, demonstrates the inherent limitations of measurements of tumor size (including brain scans and clinical patient evaluations) in evaluating the efficacy of brain-tumor therapy. Following at LD10 dose of BCNU the surviving clonogenic tumor cells increase in number after latency period of 2 to 4 days; during regrowth the cell doubling time is 40 hours. Marked variability in tumor response and regrowth was noted. The determination of information regarding disturbed tumor cell kinetics and tumor heterogeneityis essential for the proper planning of combination chemotherapy and multimodality regimens.

The distribution of nuclear DNA from human brain-tumor cells.

Flow cytometry (FCM) is a technique that measures the quantity of DNA contained in individual nuclei and records a frequency distribution of the DNA content per nucleus in the sampled cell population. Nuclei from a variety of human brain-tumor types were isolated by means of tissue grinding, purified by centrifugation through 40% sucrose (15 minutes at 4000 rpm), fixed with 10% formalin, stained with acriflavin-Feulgen, and analyzed by FCM. Profiles of DNA distribution in histologically benign tumors, such as meningiomas, pituitary adenomas, neuroblastomas, and low-grade astrocytomas, revealed a large diploid population (2C) with a few nuclei in DNA synthesis, as well as a small premitotic population (G2 cells) that contains a 4C DNA complement. In contrast, malignant gliomas, including glioblastomas, consist of more cells in DNA synthesis; these tumor cells show a highly variable distribution of ploidy consisting not only of diploid, and/or aneuploid, but also of triploid, tetraploid, and possibly octaploid populations. Also, a large variability between different regions of each tumor was always observed. In contrast, metastatic brain tumors, despite the fact that they contain a considerable number of cells undergoing DNA synthesis, demonstrate little variability within each individual tumor. The ability to rapidly characterize the cell populations of human brain tumors with FCM may enhance the effectiveness of their clinical management.

Recovery from potentially lethal damage induced by spirohydantoin mustard on 9L cells in vitro.

Spirohydantoin mustard (SHM) has been shown to cross the blood-brain barrier in dogs and has shown activity against mouse brian tumors in vivo and against 9L rat brain tumor cells in vitro. Factors influencing the recovery of monolayer 9L cells from SHM-induced potentially lethal damage were studied. The pattern of recovery was similar in exponentially growing and plateau-phase cultures. Recovery occurred in either Earle's balanced salt solution (EBSS) or depleted medium (DM), and was inhibited in fresh medium (FM). The recovery process had a half-life of approximately 4 hours and was completed within 18 hours. The enhancement of survival was dependent on drug concentration and temperature. The magnitude of recovery decreased as the temperature was lowered and was inhibited at ambient temperature, which suggested that an enzymatic repair process was responsible for recovery. The distribution of cells through the cell cycle after SHM treatment was determined using a flow cytometer. Incubation of exponentially growing and plateau-phase cells in either EBSS or DM impeded the progression of cells from G1 into S phase, but cells incubated in FM progressed into S phase by 24 hours. We conclude that the recovery occurred primarily when cells were in G1 phase and potentially lethal damage would be fixed when cells initiate DNA synthesis within 18 hours after administration of SHM. Therefore, optimal therapeutic benefit may depend on drug scheduling.

Effect of dibutyryl cAMP on cell cycle progression of rat brain tumor cells in vitro.

Autoradiographic and flow microfluorometry analyses have been applied to a study of perturbed cell kinetics in 9L rat brain tumor cells treated with dibutyryl cyclic AMP and theophylline alone and in combination in vitro. At a concentration of 1 mM each, cell growth ceased shortly after the administration of these drugs. The results indicate that cells in S and G2 phase at the time of drug administration can undergo mitosis even though a considerable prolongation of G2 phase was apparent. However, cells in G1 at the time of drug administration was arrested in that phase, whereas those cells in S or G2 were able to complete one mitosis before becoming arrested in the G1 phase. This blocking effect was reversible, and cells resumed proliferation at a normal rate shortly after the removal of these drugs.

Perturbed cell kinetics of 9L rat brain tumor cells following dianhydrogalactitol.

Kinetic changes induced by dianhydrogalactitol (DAG) in cultured 9L rat brain tumor cells were studied using conventional autoradiography and flow cytometry. Cell cycle parameters for the untreated cell line were: G1, 8.5 hours; S, 8.2 hours; G2, 3.2 hours; M, 0.5 hour; and cell cycle time, 19.5--20 hours. Treatment with 5 micrograms/ml of DAG, which caused 89.8% cell kill as measured by the colony-forming efficiency assay, resulted in the following effects: (a) undisturbed progression of G2 cells toward mitosis; (b) disturbed progression of the cells in the S--G2 boundary; (c) extreme prolongation of DNA synthesis time; and (d) temporary accumulation in G2 phase of cells that were in G1 or S phase at the time of treatment.

Development of an in vitro colony formation assay for the evaluation of in vivo chemotherapy of a rat brain tumor.

An in vitro colony formation assay for the evaluation of in vivo brain tumor therapy has been developed. When plated, disaggregated cells derived from solid tumors proliferated to form relatively homogeneous colonies after a latency period of 2 to 6 days. Increasing concentrations of fetal calf serum enhanced colony-forming efficiency (CFE) with a plateau between 7 and 16%. Supplementation with either irradiated feeder cells (10(3) to 10(5) cells per dish), or medium conditioned by 1 to 3 days of in vitro incubation with the same cell line, doubled the CFE. The density of tumor cells (untreated or previously treated with chemotherapeutic agents) did not affect the CFE when a minimum of 10(4) total cells (tumor plus feeder) were plated. Therefore, in this system the optimal experimental conditions for evaluating chemotherapy and radiotherapy require incubation of disaggregated tumor cells for 12 days in medium containing 10% of fetal calf serum and enough feeder cells to provide a minimum of 10(4) cells per dish. The CFE for untreated tumors was 18 +/- 10% (+/-S.D.), demonstrating that there is significant biological variation. The assay appeared sensitive, with reproducible results, when applied to individual chemically treated tumors. An estimate of the percentage of clonogenic cells affected by in vivo chemotherapy may be obtained by comparing the CFE of cells from treated and untreated tumors. This assay can measure up to a 5 log(10) cell kill, and it should prove to be valuable in developing more effective regimens for the treatment of solid tumors in animals and man.

Clonogenicity of multiple populations of human glioma cells in vitro sorted by DNA content.

Malignant human gliomas have a highly variable distribution of cell nuclei, consisting of diploid and/or other populations in terms of nuclear DNA content. In order to study in vitro clonogenicity of each population, dissociated or cultured human glioma cells were stained with 20 microM/ml of Hoechst 33342 dye (which stains viable DNA with minimal cell kill), and were sorted sterile into separate populations, based on specific nuclear DNA content, for clonogenicity assay. The colony-forming efficiency (CFE) of tumor cells plated immediately after disaggregation of the biopsy specimens ranged from 0.0044 to 0.149%, and the CFEs increased dramatically with successive passages (to 5 to 40%). The CFEs of the individual populations sorted according to DNA content were similar within individual tumors. These results suggest not only that malignant gliomas are composed of multiple populations in terms of DNA content, but also that each of these populations contain clonogenic cells. The morphologic structure of cells within and among colonies did not appear to relate to DNA content.

Human antibody responses to components of the monoclonal antimelanoma antibody ricin A chain immunotoxin XomaZyme-MEL.

Human antibody responses to immunotoxin components were evaluated in 21 melanoma patients who were treated with XomaZyme-MEL, a murine monoclonal antimelanoma antibody-ricin A chain conjugate. Twenty of the 21 melanoma patients produced antibodies against ricin A chain, while 15 of 21 produced antibodies reactive with the murine monoclonal antibody component. Both IgM and IgG antibody responses were produced. Immunoglobulin responses were usually detected 1 to 2 weeks following initiation of therapy, with peak levels generally attained 2 to 4 weeks posttherapy. Titers of the anti-ricin A chain antibodies were generally higher than those of the antimurine monoclonal antibodies for the dose range tested. There was no clear correlation between the dose of immunotoxin administered and the antibody titer. By use of a competitive flow cytometry assay, antiidiotype responses were demonstrated in eight of 10 melanoma patients who had antimurine antibodies. Both the kinetics of appearance and the relative titers of the antiidiotype responses generally corresponded to the antimurine responses. The development of antimmunotoxin antibodies can reduce the therapeutic potential of immunotoxins through several mechanisms. The development of antibodies in a significant number of patients suggests that optimally effective, repeated courses of therapy will require some procedure for suppressing or abrogating the response against the immunotoxin.