Pancreatic ribonuclease: enzymic and physiological properties of a cross-linked dimer.

Monomeric ribonuclease A has very low activity toward typically double-stranded RNA's; the dimeric form of ribonuclease A obtained by cross linking the enzyme by dimethyl suberimidate has more than 78 times the activity of the monomer toward polyadenylate . polyuridylate and 440 times the activity of the monomer toward the double-stranded RNA of a virus from Penicillium chrysogenum. The half-life of the dimer in the bloodstream of the rat is 12 times that of the mononmer.

Involvement of histamine in growth of mouse and rat tumors: antitumoral properties of monofluoromethylhistidine, an enzyme-activated irreversible inhibitor of histidine decarboxylase.

The present study suggests that newly synthesized histamine is involved in the development of some animal tumors (e.g., Lewis lung carcinoma in mice and Morris hepatoma in rats). A marked induction of histidine decarboxylase (HDC) and an increase in the histamine concentration were observed in the tumors approximately 1 week after inoculation, and there were parallel increases in ornithine decarboxylase activity and the concentrations of polyamines. The H2 receptor antagonist, cimetidine, significantly reduced tumor growth in the animal models while the H1 receptor antagonist, dexchlorpheniramine, had no effect, suggesting that histamine could act via H2 receptor sites. Extensive depletion of tumor histamine induced by local injection of Compound 48/80 did not result in a significant cytostatic effect. Monofluoromethylhistidine (MFMH), an enzyme-activated irreversible inhibitor of HDC, retarded the growth of hepatoma tissue culture cells grown in culture, and when infused s.c. at 60 mg/kg/day it greatly inhibited the development of tumors induced i.m. by hepatoma tissue culture cells in Buffalo rats. MFMH also had pronounced antitumoral effects on EMT6 sarcomas and Lewis lung carcinomas in mice, which were associated with inhibition of HDC and depletion of the histamine content of the tumors. These cytostatic effects were clearly enhanced when MFMH was combined in therapy with the specific ornithine decarboxylase inhibitor, DL-alpha-difluoromethylornithine. The antitumoral effects of the combination were associated with marked decreases in the tumor histamine and putrescine contents. It is proposed that nascent histamine, like newly synthesized putrescine and spermidine, plays a role in the rapid proliferation of animal tumors. Inhibition of HDC by essentially nontoxic drugs such as MFMH could represent a novel approach to the control of neoplastic growth.

Enhanced urinary excretion of N1-acetylspermidine and the presence of tumors.

We have studied the urinary excretion of free and acetylated polyamines in hepatoma-bearing Buffalo rats during the period of linear growth of the tumor mass. The excretion of nonconjugated polyamines was unchanged. N1-Acetylspermidine excretion did not parallel the linear increase in tumor mass but increased exponentially. Enhancement of N8-acetylspermidine excretion above control levels was observed only at a time when the average tumor mass was 35 +/- 9 (S.D.) g. shortly before the period when necrosis is usually observed. These data taken together with the analysis of urinary acetylpolyamines in rats bearing mammary tumors and in two melanoma patients show that the determination of the N1-acetylspermidine/N8-acetylspermidine ratio in urine may be of only limited value as an indicator for the presence of tumors.

Effects of alpha-difluoromethylornithine alone and combined with adriamycin or vindesine on L1210 leukemia in mice, EMT6 solid tumors in mice, and solid tumors induced by injection of hepatoma tissue culture cells in rats.

The effects of alpha-difluoromethylornithine (DFMO; RMI 71782) in combination with vindesine or Adriamycin were investigated in three different animal tumor models. When given in a concentration of 2% in drinking water to C57BL/6 X DBA/2 F1 mice inoculated i.p. with L1210 leukemia cells, DFMO prolonged the survival time 1,2-fold. Treatment with vindesine (0.1 mg/kg/week i.p. or Adriamycin (2.5 mg/kg/week i.p.) increased the mean survival time 1.4- and 2.3-fold, respectively. DFMO with vindesine doubled survival time, while DFMO with Adriamycin increased it 3.5-fold and yielded 30% long-term survivors. The growth of solid tumors induced in Buffalo rats by i.m. injection of hepatoma tissue culture cells was inhibited 65% after 2 weeks of DFMO treatment. Similar inhibition of growth could be achieved by weekly i.p. injections of vindesine (0.2 mg/kg) or Adriamycin (2.5 mg/kg). When the same doses of these drugs were administered in combination with DFMO, the growth of this hepatoma was completely arrested. Combined treatment of BALB/c mice bearing s.c. solid EMT6 tumors with DFMO and adriamycin or vindesine also resulted in enhanced inhibition of tumor growth compared to single-drug therapy. These results indicate that combination of DFMO with vindesine or Adriamycin is an effective approach to the treatment of several animal cancers.

Antitumor properties of (2R,5R)-6-heptyne-2,5-diamine, a new potent enzyme-activated irreversible inhibitor of ornithine decarboxylase, in rodents.

(2R,5R)-6-Heptyne-2,5-diamine hydrochloride (MDL 72175) is a new, potent, and selective inhibitor of mammalian ornithine decarboxylase. MDL 72175 given p.o. in drinking fluid reduced by 80% the growth of EMT6 sarcoma in mice and of HTC hepatoma in rats. It prolonged the survival of mice bearing L1210 or P388 leukemias and inhibited the development of Lewis lung carcinoma in mice at doses 10- to 20-fold lower than those of alpha-difluoromethylornithine, the most widely used irreversible inhibitor of ornithine decarboxylase. MDL 72175 depleted putrescine and spermidine levels in the tumors to the same extent as did alpha-difluoromethylornithine. In the EMT6 sarcoma, MDL 72175 achieved at low doses a greater maximal antitumor effect than did alpha-difluoromethylornithine. In combination therapy, MDL 72175 plus Adriamycin gave at least additive antitumor effects on solid tumors and experimental leukemias in animals. The combination MDL 72175 plus methylglyoxal bis(guanylhydrazone) also gave additive antitumor effects on P388 leukemia, associated with an increased uptake of methylglyoxal bis(guanylhydrazone); in contrast, antagonistic effects were observed with this combination on EMT6 tumors in mice. Since MDL 72175 did not present toxicity at effective antitumor doses, this new ornithine decarboxylase inhibitor can be considered as a promising antitumor drug.

Characterization of surface markers of continuously growing murine resident macrophages.

Conditions have been described which allow an in vitro indefinite multiplication of differentiated murine macrophages (Lombard et al: Biol Cell 53, 219, 1985). R. and MAY-1 cell lines, which were obtained, respectively, from mouse (Balb/c) spleen and resident peritoneal macrophages, have been further characterized. They present at their surface, besides the Mac-1 antigen and Fc-receptor, a mannose receptor which was characterized for its binding properties. This receptor is responsive for a specific phagocytosis of mannosylated particles, i.e., mannosylated latex beads or oil droplets containing mannosylated bovine serum albumin. Moreover, R and MAY-1 cells present an ectoenzyme profile (NAD+ glycohydrolase and nucleotide pyrophosphatase) similar to those of the corresponding resident macrophages.

Establishment and characterization of long-term cultured cell lines of murine resident macrophages.

Murine resident macrophages can proliferate in vitro when they are grown in coculture on a layer of mesothelial or endothelial type feeder cells. Resident macrophages were obtained from lung explants of C57Bl/6 lpr/lpr mice and from spleen explants or peritoneal washing of Balb/c mice; the cells were seeded without further washing. After 3-4 weeks of culture, the macrophages began to proliferate on a confluent layer of feeder cells. The macrophages then could be collected in the fluid phase and reseeded for permanent culture after generation of a new feeder layer. These cells were characterized as macrophages by the following criteria: 1) their morphology, ultrastructure, and adherence properties; 2) more than 90% of the macrophages phagocytized yeasts compared with less than 1% of the feeder cells; 3) the presence of functional Fc and mannose receptors, nonspecific cytoplasmic esterases, and membrane ectoenzymes such as nicotinamide adenine dinucleotide (NAD) glycohydrolase and nucleotide pyrophosphatase; 4) by cytofluorographic phenotype analysis with monoclonal antibodies, characterizing a normal macrophage population (MAC1+, Fcrec+, H-2K+, THY1-, LYT2-, L3T4-). 5) by functional studies proving that the expanded macrophages could function as accessory cells in the induction of lymphocyte proliferation in response to concanavalin A (Con A), that they generated reactive oxygen radicals and that they were cytotoxic for tumor cells. During coculture, growth or activating factors such as macrophage colony-stimulating factor or gamma-interferon were released in the medium. Long-term cultured macrophages had chromosomal abnormalities. Our study suggests that tissue macrophages can proliferate in vitro and hence that it is possible to establish long-term cultured cell lines of macrophages of defined and reproducible characteristics.

Autologous lymphocytes prevent the death of monocytes in culture and promote, as do GM-CSF, IL-3 and M-CSF, their differentiation into macrophages.

Blood monocytes collected by apheresis from healthy donors were differentiated in vitro to macrophages which were subsequently activated with recombinant human interferon-gamma. 7 day cultures were established by seeding Ficoll-separated mononuclear cells or elutriation-purified monocytes under different culture conditions. The best macrophage yields required the seeding of mononuclear cells (instead of purified monocytes) in teflon bags with a high air-liquid surface interface. The effects of GM-CSF, IL-3 and M-CSF on the macrophage yield were then evaluated. GM-CSF increased the average yield by 3.6- and 2.3-fold when purified monocytes or total mononuclear cells were seeded respectively. The corresponding increases with IL-3 were 2.5- and 2.1-fold respectively and with M-CSF 1.2- and 1.4-fold respectively. Macrophages matured under these various conditions displayed similar CD14, CD64, CD71, HLA-DR and Max 1 antigen expression and similar in vitro anti-tumoral activity against U937 cells. Culturing in the presence of cytokines permits the large scale production of activated macrophages for adoptive immunotherapy trials.

Large scale isolation of human blood monocytes by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation for adoptive cellular immunotherapy in cancer patients.

The increasing interest in mononuclear phagocytes for adoptive cellular immunotherapy (ACI) trials in cancer patients led us to define a procedural approach to harvest reproducibly highly purified single-cell suspensions of large numbers of functional human circulating blood monocytes (Mo). A semiclosed counterflow centrifugal elutriation (CCE) system has been developed, using a new large capacity Beckman JE 5.0 rotor with one interchangeable 40 ml or 5 ml separation chamber, to purify Mo from mononuclear cell (MNC) concentrates of healthy donors and cancer patients obtained by continuous flow centrifugation leukapheresis (CFCL). This method does not require a Ficoll density gradient centrifugation step. A total of 115 leukapheresis procedures were carried out in 35 patients and in 30 healthy donors by either Cobe 2997 or Cobe Spectra, with a similar efficiency in MNC apheresis. The average yield per leukapheresis procedure was 5.6 x 10(9) MNC of purity 90-100% (25-45% Mo, 40-65% lymphocytes). The average yields per elutriation procedure (R/O fraction) were 1.1 x 10(9) cells (purity 93% Mo) using the 5 ml separation chamber, and 1.5 x 10(9) cells (purity 91%) using the 40 ml separation chamber, with a respective recovery of 82 +/- 7% and 78 +/- 8% Mo. In vitro analysis of the viability and function of the purified Mo shows that neither morphological integrity nor physiological activity was compromised by this two-step isolation procedure, which additionally provides highly purified human Mo suspensions, in a quantity suitable for ACl of cancer patients.

Frequency of human anti-FVIII antibodies in humanized SCID mice elicited by recombinant deleted factor VIII and by a plasma derived factor VIII.

SCID mice were grafted with human PBL (hu-PBL-SCID) from healthy or haemophilia A donors. Those containing human and no murine Ig in their plasma, were injected with 100 U VIII:Ag of a plasma derived (pd) FVIII or recombinant deleted Factor VIII (FVIII deltaII) and with 10 microg of tetanus toxoid as control immunogen. The frequency and the intensity of the humoral specific responses were measured in 253 mice humanized with PBL from 13 different donors. There was no significant difference in the frequency or intensity of the anti-FVIII immune responses to pd FVIII and FVIII deltaII. Neutralizing antibodies were only detected in the plasma of mice humanized with cells from haemophiliacs having FVIII inhibitors in their blood. The immune responses observed in hu-PBL-SCID mice correlated with the immune status of the corresponding human donor.

Subcellular distribution of ornithine decarboxylase in rat liver and accessibility of the enzyme to alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase.

The subcellular localisation of ornithine decarboxylase and of its synthetic irreversible inhibitor, alpha-difluoromethylornithine, was investigated in control rat livers and in livers of animals in which the enzyme was induced by partial hepatectomy or by treatment with dexamethasone. Ornithine decarboxylase activity was distributed in normal rat liver between the nuclear (40%, mainly nucleolar) and the cytosolic (43%) fractions. Cytosolic liver ornithine decarboxylase was markedly induced after partial hepatectomy or treatment with dexamethasone, whereas the enzyme associated with the nuclear fraction was not induced by these procedures. The irreversible inhibitor was found only in the cytosol fraction and was totally absent from the nuclear fraction.

Immune control of neoplasia by adoptive transfer of macrophages: potentiality for antigen presentation and gene transfer.

Human macrophages could be differentiated from mononuclear precursors present in the blood circulation. After IFN-gamma activation, they became antitumoral and adhered to transformed cells. Low amounts of activated macrophages (MAK) caused regression of experimental tumors in animal models. In cancer patients, MAK were well tolerated and caused tumor necrosis but no clear therapeutic response has been reported up to now. Improvements can be expected using local treatment and more specific macrophages presenting tumor antigens. This restoration of immune recognition of growing tumors with low levels of reactants should ultimately reestablish, after exogenous stimulation, the insufficient immune response of the host against a malignant tumor. Antitumoral macrophages can also be optimized by gene transfection. Macrophages are proposed as stable and long lasting cell vectors for adoptive gene treatments.

Adoptive immunotherapy of solid tumors with activated macrophages: experimental and clinical results.

Adoptive immunotherapy in cancer has been essentially restricted to the use of lymphoid effector cells (NK, TIL, LAK) stimulated with IL-2. Differentiated macrophages represent another key effector population even more important for the immune control of cancer. We have shown that activated murine macrophages reduced primary tumors and experimental metastases. Human macrophages differentiated from circulating monocytes and activated with IFN gamma (MAK) were cytotoxic in vitro for a variety of tumor cell and caused regression of human tumors implanted in nude mice. A large scale technology has been developed for the generation of antitumor macrophages. These MAK cells (10(8) to 10(9] were injected in cancer patients in pilot clinical trials and were well tolerated. MAK treatment is technically feasible, clinically safe and presents several advantages compared to other immunotherapies.

Polyamine metabolism and polyamine excretion in normal and tumor bearing rodents.

Aminoguanidine sulfate (AG) inhibits in vivo oxidative deaminations of the polyamines and their derivatives. This compound was used to study urinary polyamine excretion by normal, and tumor bearing rodents. Of the total expendable polyamines, 64 percent were catabolized by AG-sensitive oxidases and escaped observation. Tumor bearing animals did not excrete enhanced amounts of polyamines at any stage of tumoral growth. However, treatment with adriamycin caused an increased polyamine excretion. Prolonged administration of a 2% solution of a-difluoromethylornithine (DFMO), reduced urinary polyamine excretion to the same level of about 27%, irrespective whether the animals carried a large tumor or not. Cadaverine excretion was not affected by treatment with DFMO. Based on these animal data, it appears that urinary polyamines are of restricted value in the diagnosis of tumors.

Immunotherapy of murine sarcoma by adoptive transfer of resident peritoneal macrophages proliferating in culture.

Normal resident peritoneal macrophages from BALB/c mice were continuously grown and expanded in vitro as non tumorigenic cells on a confluent layer of mesothelial cells. These peritoneal macrophages expanded in vitro (EPM) were very cytotoxic against EMT6 sarcoma, Abelson myeloma, EL4, and L929S cells in culture. This tumoricidal effect was fully expressed without further activation with bacterial lipopolysaccharides (LPS). In vivo, adoptive transfer of one million EPM to BALB/c mice bearing subcutaneous EMT6 sarcoma caused regression of the solid tumor. In contrast, macrophages produced by 10 days' culture of bone marrow stem cells, or freshly isolated from the peritoneal cavity of BALB/c mice, were not cytotoxic in vitro or in vivo. Local injection in the vicinity of the tumor as well as intravenous transplantation of EPM effectively inhibited tumor growth. This antitumoral effect was further enhanced by intraperitoneal injection of 2 micrograms LPS to the tumor bearing mice.

Antitumoral properties and reduced toxicity of LPS targeted to macrophages via normal or mannosylated liposomes.

Neo-mannosylated liposomes have been prepared by coupling a mannose derivative bearing a hydrophilic spacer arm to preformed large unilamellar liposomes containing 4-(p-maleimidophenyl) butyryl-phosphatidylethanolamine. Lipopolysaccharide (LPS) was encapsulated in normal or neo-mannosylated liposomes; the neo-mannosylated vesicles showed specificity for the in vitro activation to toxicity of macrophages only in the case of differentiated macrophages presenting mannose receptors at their surface. In vivo, LPS entrapped in neo-mannosylated vesicles showed a reduced toxicity for animals hypersensitive to LPS. Moreover targeting of LPS to tissue macrophages with neo-mannosylated liposomes induced regression of experimental solid tumors in mice (EMT6 sarcoma, 3LL carcinoma) and was effective on lung metastases.

Antitumoral effects of lipopolysaccharides, tumor necrosis factor, interferon and activated macrophages: synergism and tissue distribution.

Treatment of C57BL/6 mice bearing Lewis lung carcinoma or of BALB/c mice bearing EMT6 sarcoma with tumor necrosis factor (TNF), lipopolysaccharides (LPS) or interferon caused necrosis of the solid tumors and regression. Toxicity was observed in tumor-bearing animals when TNF or LPS were used at effective antitumoral doses. Similar antitumoral effects could be achieved using less than 1 million macrophages from C57BL/6, lung of from BALB/c peritoneal cavity expanded in vitro, and spontaneously fully activated to cytotoxicity during culture. This effect, observed after transfer twice a week by intravenous or peritumoral route, was not dependent on histocompatibility. Additive effects were observed after combined treatment with activated macrophages and a low dose of LPS or TNF. The biodistribution of labelled LPS and of labelled cytotoxic macrophages was studied in tumor-bearing mice. Although, as expected, LPS was concentrated essentially in the liver, a slow accumulation in the center of the tumor was observed. Macrophages injected intravenously accumulated in the lung and were then redistributed towards liver, kidney and the tumor periphery. Macrophages injected locally remained essentially in the tumor periphery with a slow redistribution in the body. The complementary localization of LPS and of cytotoxic macrophages respectively in the center and periphery of solid tumors might explain their synergism.

Study of the dependence of human monocytes and macrophages antitumoral properties upon TNF-alpha expression or release.

This study compares the antitumoral properties of isolated circulating human blood monocytes (Mo) and of mature macrophages (MO) obtained by 7 days differentiation of Mo or isolated from alveolar washing. These cells were activated to cytotoxicity in the presence of recombinant human interferon-gamma (rHuIFN-gamma). This antitumoral effect was measured at a low (1/1) effector/target ratio without pretreatment of the tumor cells. Activated Mo released tumor necrosis factor-alpha (TNF-alpha) in the culture medium where their antitumoral activity could be totally neutralized by specific anti-rHuTNF-alpha antibodies. In contrast, blood monocytes derived macrophages differentiated and activated in vitro expressed TNF-alpha on their membrane where it could be labelled and partially neutralized by anti-rHuTNF-alpha antibodies. Direct effector/target contact was required for the activity of macrophages differentiated in culture or collected from the lung cavity of healthy subjects. When these macrophages were obtained from infected patients or subjected to LPS treatment, they directly released cytotoxic amounts of TNF in the extracellular fluid after activation with IFN-gamma. Monocytes act mainly by soluble mediators (TNF-alpha being a key factor), while differentiated macrophages in the absence of endotoxin act by close cell to cell contact involving the lytic action of membranous TNF-alpha as well as some release of soluble TNF-alpha. We also present evidences (based on the use of various protease inhibitors) that the role of proteases is much less crucial in the cytotoxic action of monocytes and macrophages.

Production of human macrophages with potent antitumor properties (MAK) by culture of monocytes in the presence of GM-CSF and 1,25-dihydroxy vitamin D3.

Antitumoral macrophages (MAK) were obtained by the culture of human mononuclear cells in hydrophobic bags. From one cytapheresis, up to 10(9) mature macrophages could be purified by elutriation after one week of culture in IMDM medium in the presence of 2% human AB serum. These MAK cells were used for adoptive treatment in metastatic cancer patient with no dose-limiting toxicity. The present study aimed to improve the average MAK yield by addition of GM-CSF and of dihydroxy-cholecalciferol. The differentiated macrophages obtained presented higher antitumoral functionality in response to rh-IFN gamma than in their absence. These MAK presented all the differentiation antigens of cytotoxic macrophages compared to MAK cells differentiated in standard medium. They killed human tumor targets effectively in vitro at a low (1/1) effector/tumor ratio; furthermore, the antitumoral activity reached by MAK cells after IFN gamma activation appeared to be stabilized for several days.

Effect of alpha-monofluoromethyl histidine, an irreversible inhibitor of histidine decarboxylase, on gestation in mice.

Chronic administration of alpha-monofluoromethyl histidine, a specific inhibitor of histidine decarboxylase, to pregnant mice blocked histidine decarboxylase activity and markedly depleted histamine levels in 18-day-old foetuses and in newborn mice. Such treatment had no effect on implantation or embryonic development or parturition suggesting that de novo synthesis of histamine is not a significant factor in these processes in the mouse.