Characterization of the new immunosuppressive drug undecylprodigiosin in human lymphocytes: retinoblastoma protein, cyclin-dependent kinase-2, and cyclin-dependent kinase-4 as molecular targets.

Undecylprodigiosin (UP) is the first described member of a family of related compounds showing immunosuppressive activity. We have investigated the biological effect and mechanism of action of UP in human lymphocytes. We show that UP blocks the proliferation of purified peripheral human T and B lymphocytes with an IC50 of 3 to 8 ng/ml and following stimulation by all mitogens used, with no effect on cell death. At the concentrations active on fresh lymphocytes, UP has no significant effect on the proliferation of different leukemic cell lines. UP blocks T cell activation in mid to late G1 phase and before entry into S phase, as shown by analysis of the cell cycle and of the expression of c-myc, IL-2, transferrin receptor, and B-myb. UP inhibits only partially the expression of IL-2R, suggesting that the major target of UP is localized downstream from the interaction between IL-2 and its receptor. The expression of cell cycle genes was investigated. The phosphorylation of the retinoblastoma protein was completely blocked by UP, an event alone sufficient to explain the block of S phase entry and the inhibition of proliferation. The induction of cyclin D2 and the decrease in p27 were not inhibited by UP, whereas the induction of cyclin E, cyclin A, cyclin-dependent kinase-2, and cyclin-dependent kinase-4 was strongly inhibited, potentially explaining the inhibition of retinoblastoma protein phosphorylation. These data clearly show that the site of action of UP is different from that of both cyclosporin A and rapamycin, and that this new class of compounds may, therefore, be good candidates for combined therapy.

Pattern of cytokines and pharmacomodulation in sepsis induced by cecal ligation and puncture compared with that induced by endotoxin.

The production of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 and their pharmacomodulation were evaluated in a model of polymicrobial sepsis induced in mice by cecal ligation and puncture (CLP) and were compared with the effects of endotoxin (lipopolysaccharide [LPS]) treatment. LPS levels rose as early as 1 h after CLP and increased further after 2 and 21 h. TNF-alpha was detectable in serum, spleen, liver, and lungs during the first 4 h, with a peak 2 h after CLP. IL-1 beta was measurable in serum after 24 h, and levels increased significantly in spleen and liver 4 and 8 h after CLP. IL-6 levels increased significantly in serum throughout the first 16 h after CLP. These cytokines were detectable after LPS injection, with kinetics similar to those after CLP but at a significantly higher level. To cast more light on the differences between these two animal models of septic shock, we studied the effects of different reference drugs. Pretreatment with dexamethasone (DEX); ibuprofen (IBU), an inhibitor of cyclooxygenase; and NG-nitro-L-arginine, an inhibitor of nitric oxide synthase, significantly reduced survival, while chlorpromazine (CPZ) and TNF did not affect it. Only the antibiotics and pentoxifylline significantly increased survival in mice with CLP. However, CPZ and DEX protected the mice from LPS mortality. On inhibiting TNF-alpha with DEX, CPZ, or pentoxifylline, survival was reduced, unchanged, and increased, respectively, and on increasing TNF-alpha with IBU and TNF, survival was decreased or unchanged, respectively, suggesting that the modulation of this cytokine does not play a significant role in sepsis induced by CLP, unlike treatment with LPS. The negative effects of IBU and N(G)-nitro-L-arginine suggest a protective role by prostaglandins and nitric oxide in sepsis induced by CLP.

Interleukin-10 inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin-1 beta production in the brain without affecting the activation of the hypothalamus-pituitary-adrenal axis.

Interleukin (IL) 10 inhibits endotoxin (lipopolysaccharide; LPS) induced tumor necrosis factor (TNF) production in vivo and in vitro. In turn, IL-10 is induced by LPS and acts as a negative feedback to limit TNF production. We investigated the effects of IL-10 on brain TNF and IL-1 beta production induced by a central LPS administration in mice. Because central LPS also induces peripheral TNF, we also measured the serum TNF levels. A single intracerebroventricular injection of murine recombinant IL-10 (75 ng/mouse) simultaneously with LPS (2.5 micrograms/mouse) almost completely inhibited brain TNF production. The brain IL-1 beta production was also inhibited, as was the serum concentration of the acute-phase protein serum amyloid A. On the other hand, intracerebroventricular administration of an anti-IL-10 monoclonal antibody (JES5-2A5; 60 micrograms/mouse) potentiated brain TNF and IL-1 beta production. Identical results were obtained when the serum TNF levels were measured. IL-10 did not affect the LPS-induced increase of serum corticosterone, the main endogenous inhibitor of TNF production, or the induction of IL-6. These results indicate that LPS-induced IL-10 can act as an important endogenous inhibitor of brain TNF production and suggest an anti-inflammatory role for IL-10 in the central nervous system.

Inhibition of interleukin-2/p55 receptor subunit interaction by complementary peptides.

Complementary peptides to interleukin-2 (IL-2) sequences important for receptor binding were tested for their ability to mimic natural receptors and act as inhibitors of the IL-2/p55 receptor subunit interaction. Peptides hydropathically complementary to IL-2 sequences 15-27 and 40-54 were synthesized in a linear and in a multimeric form and then characterized first by solid-phase binding assays for their ability to interact with IL-2. Binding between the multimeric complementary peptides and biotinylated IL-2 was specific, saturable, and inhibited by linear as well as multimeric complementary peptides. Saturable interactions, characterized by dissociation constants in the micromolar range, occurred also between IL-2 immobilized on microtiter plates and biotinylated linear and multimeric complementary peptides. Peptides corresponding to the IL-2 target sequences were able to interfere with this interaction, as well as full-length IL-2. Peptide recognition was sequence dependent, since scrambling of complementary peptide sequences or IL-2 target peptide sequences abolished binding. Multimeric complementary peptides after immobilization on solid supports proved useful also for affinity purifications of recombinant IL-2 or IL-2 fragments corresponding to the target sites, directly from crude mixtures, in high yield and with high recovery. Complementary peptides to IL-2 sequence 15-27, but not to IL-2 sequence 40-54, in the linear or in the multimeric form, even if with different potency, interfered with the IL-2/p55 receptor subunit interaction in vitro, thus suggesting a possible role of this IL-2 site in receptor recognition.

Mechanisms of interleukin-2-induced hydrothoraxy in mice: protective effect of endotoxin tolerance and dexamethasone and possible role of reactive oxygen intermediates.

Interleukin (IL)-2 is known to induce vascular leak syndrome (VLS), which was suggested to be mediated by immune system-derived cytokines, including tumor necrosis factor (TNF). To characterize the role of TNF in IL-2 toxicity in C3H/HeN mice, we used two approaches to downregulate TNF production in vivo: treatment with dexamethasone (DEX) and induction of endotoxin (lipopolysaccharide) (LPS) tolerance by a 4-day pretreatment with LPS (35 micrograms/mouse/day). Mice were then treated with IL-2 for 5 days (1.8 x 10(5) IU/mouse, twice daily). Both DEX and LPS tolerance blocked development of hydrothorax in IL-2-treated mice and inhibited TNF induction. DEX and LPS tolerance also ameliorated IL-2 toxicity in terms of decrease in food intake and inhibited the increase of the acute-phase protein, serum amyloid A (SAA). The IL-2 activation of splenic natural killer (NK) cell activity was also diminished by DEX and, to a lesser extent, by LPS-tolerance. Treatment with IL-2 also caused induction of the superoxide-generating enzyme xanthine oxidase (XO) in tissues and serum and induced bacterial translocation in the mesenteric lymph nodes (MLN). These data suggest that multiple mechanisms, including NK cell activity, cytokines, and reactive oxygen intermediates, might be important in the vascular toxicity of IL-2.

Immunodepressive activity of FCE 23762 on humoral and cell-mediated immune responses in normal mice: comparison with doxorubicin.

FCE 23762 (3' desamino-3'[2(s)methoxyl-4-morpholinyl]doxorubicin) is a new doxorubicin (Dx) derivative that has been selected for clinical testing for its favourable antitumor characteristics, which include efficacy on Dx-resistant tumors. Immunosuppression is an undesirable side-effect of anti-cancer chemotherapy and the therapeutic efficacy of Dx is probably also related to its low immunotoxicity. It was, thus, of interest to compare the effects of FCE 23762 and its parental drug on the immune responses. Both compounds were injected i.v. into healthy mice at equitoxic doses and according to different treatment schedules. Single doses of FCE 23762 and Dx, given concomitant or after the antigen, suppressed at the same degree and dose-dependently the primary anti-SRBC antibody response. Following a multiple treatment schedule after the antigen, FCE 23762 was less suppressive than Dx on both primary and secondary antibody production. Differently from Dx, that was completely inactive, FCE 23762 moderately inhibited DTH reaction to SRBC, only at the highest single dose tested or for repeated administrations given simultaneously or after priming. Both drugs were totally ineffective in delaying skin allograft rejection. Since spleen cellularity and ex vivo lymphocyte proliferation to Con A and LPS were similarly impaired by the two drugs, the differentiated immunodepressive activity of FCE 23762 and Dx cannot be merely associated to their cytotoxic and antiproliferative action. The hypothesis of a selective effect on different regulatory cell subsets and/or immune mechanisms is discussed.

Development and applications of a radioimmunoassay (RIA) for the in vitro and in vivo quantification of murine IL-1 beta.

A specific, precise, and accurate radioimmunoassay (RIA) for murine interleukin-1 beta (mIL-1 beta), with a sensitivity of 250 pg/ml, has been established. Although mIL-1 beta shares structural homology and multiple biological properties with mIL-1 alpha, this RIA did not detect mIL-1 alpha or other murine cytokines such as TNF and IL-6. Recombinant mIL-1 beta, freshly added in different concentrations to murine plasma, was recovered from 88 to 104%, and intra- and interassay coefficients of variation never exceeded the 10% value. Parallel analysis showed that murine plasma and cell or organ supernatants did not affect the test. This characteristic allowed mIL-1 beta analysis directly in the nonmanipulated biological specimens. In murine macrophage supernatants collected after 24 h of in vitro stimulation with LPS, nanogram fractions of IL-1 beta were detected by RIA. These values corresponded to approximately 50% of the total IL-1 detected by the LAF bioassay. In spleen, liver, and lung, IL-1 beta appeared at significant levels (110 ng/g of lung, 638 ng/g of spleen, and 78 ng/g of liver) as early as 1 h after LPS administration, reached the plateau 1-2 h later, and then slowly but progressively decreased. In plasma and brain, nanogram fractions of IL-1 beta were detectable by 4 h post-LPS. Thereafter, IL-1 beta levels progressively increased to reach the value of 44 ng/g in the brain and 2 ng/ml in plasma 8 h after LPS treatment.

Synthesis and immunomodulating activity of condensed N-aryl-2-cyano-3-oxo-3-pyrazolyl-propanamides.

A series of condensed N-aryl-2-cyano-3-oxo-3-pyrazolyl-propanamides were synthesized and evaluated for immunomodulating activity following intraperitoneal administration. These new molecules were found to enhance macrophage cytotoxicity and stimulate host mediated antibacterial defences in mice. The compound 3-cyano-3-(1,4-dihydro-1-phenyl-[1]-benzothiopyrano[4,3-c]pyrazol- 3-yl]-3-oxo-N-phenyl-propanamide, chosen for wider pharmacological investigation, proved effective in preventing adjuvant-induced arthritis development in rats.

Effects of the new immunoactive compound FCE 20696 on rat and murine models of autoimmune diseases.

FCE 20696 is a new synthetic immunomodulator, capable to induce suppressor cells after repeated oral administrations in mice. We have tested it by oral route in three established models of autoimmunity. Experimental Allergic Encephalomyelitis (EAE) has been induced in rats and FCE 20696 has been given for two weeks before the encephalitogenic stimulus and continued afterwards until the complete recovery. Adjuvant Arthritis (AA) has been induced in rats and FCE 20696 has been given for five weeks starting one week before the induction of the disease. Female NZB/WF1 mice were treated with FCE 20696 biweekly throughout their life, starting from nine weeks of age. In EAE, the compound at dose of 2.5 mg/kg reduced the symptoms of the disease and protected 60% of animals from becoming ill. In the AA model, FCE 20696 at 1.1 mg/kg reduced inflammatory lesions in both the injected and the controlateral legs. NZB/WF1 mice treated with 1.5 mg/kg lived 20 weeks longer than controls and proteinuria of 5 mg/ml or more was delayed by 22 weeks. This drug has a definite value in experimental autoimmunity and seems a good candidate for clinical testing.

MTT colorimetric assay for testing macrophage cytotoxic activity in vitro.

The MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) cleavage assay, originally described by Mosmann (1983, J. Immunol. Methods 65, 55) for measuring cell survival/proliferation, has been used successfully to quantitate macrophage-mediated cytotoxicity. Peritoneal macrophages, from control or pyran copolymer MVE2-treated mice and from TU5 and L929 murine cell lines, were used as effectors and targets respectively in a 48 h cytotoxicity test, at three effector: target ratios (10:1, 5:1, 2:1). The amount of MTT reduced by cells to its blue formazan derivative during an additional 4 h of culture was quantified spectrophotometrically at 570 nm using an ELISA reader. A linear relationship between the formazan generated and the number of viable TU5 and L929 cells was demonstrated, together with time-dependent growth characteristics for these cells. The formazan produced by macrophages was independent of their functional state and did not interfere with the target cell signal. MVE2-activated macrophages strongly inhibited the survival/growth of target cells in a dose-related way, whereas the cytotoxic activity of control macrophages was very low. Finally, the MTT method compared favorably with the 3H-TdR uptake method in evaluating macrophage cytotoxicity, and both of them were more sensitive than the 3H-TdR release assay. The MTT cleavage method is a useful alternative to radioisotopic methods for quantitating macrophage cytotoxicity for actively growing in vitro targets. Its main advantages are: (a) sensitivity and reproducibility; (b) elimination of the need for radioactive compounds; (c) ease with which it can be performed and quantified; (d) rapidity.

Immunosuppressive activity of 4'-iodo-4'-deoxy-doxorubicin on humoral and cell mediated immune responses in mice: comparison with doxorubicin.

4'-Iodo-4'-deoxy-doxorubicin (I-DX) is a new halogenated doxorubicin (DX) derivative, selected for clinical testing in view of a number of pharmacotoxicological advantages vis-à-vis the parental compound. It was thus of interest to compare the immunological effects of I-DX to those of DX. The compounds were injected i.v. into mice at equal doses in terms of acute toxicity, over a variety of treatment schedules. When tested in single doses for anti SRBC primary and secondary antibody response, I-DX, unlike DX, was not immunodepressive; on the contrary it often caused a moderate amplification of the response. Following a multiple treatment schedule, I-DX was as depressive as DX only when administered after the antigen, any other treatment regimen, before or concomitant with the antigen, being uneffective. I-DX was completely uneffective and DX only slightly active in delaying skin allograft rejection and in reducing DTH reactivity to SRBC. I-DX was more inhibitory than DX for lymphoid cells in vitro, and both drugs reduced spleen cellularity without modifying the relative percentage of B and T cells after administration in vivo. However, the splenic reactivity to LPS ex vivo, unlike that to ConA, was restored earlier in I-DX than in DX-treated mice.

FCE20696, a new synthetic immunomodulator: immunopharmacological profile.

A preliminary characterization of the immunopharmacological profile of the new immunomodulating agent FCE20696 (6H,6[2-(dimethylamino)-ethoxycarbonyl]-dibenzo[b,d]pyran-HCl) was performed in mice. Single i.p. doses of this chemical, concomitantly given with the antigen, increased the antibody response and decreased the delayed hypersensitivity reaction to a suboptimal dose of SRBC, the active doses ranging from 6.25 to 50 mg/kg. No activity was observed using a full antigen dose. Macrophage cytotoxic activity was enhanced 2-4 days after a single i.p. treatment with 50 and 10 mg/kg. Spleen cell proliferation to T and B mitogens was inhibited by a single dose of 30 mg/kg given i.p. 6 days before the test, or by 10 mg/kg x 3 days ending one day before the test. Finally, generation of suppressor cells was enhanced by the compound, given p.o. biweekly for at least 7 weeks, at doses ranging from 0.1 to 10 mg/kg. Collectively taken, these data suggest that FCE20696 has a broad range of immunomodulating activities and that macrophages and suppressor cells are presumed to be the main targets of its pharmacological activity.

Monoclonal antibodies against doxorubicin.

Hybridomas which secrete monoclonal antibodies (MAbs) reacting with doxorubicin (DXR), a widely used anthracycline cytotoxic antibiotic, have been obtained by fusing NSO/P3 mouse myeloma cells with spleen cells from BALB/c mice immunized with DXR-BSA conjugate. The best producer among the several clones obtained was expanded and the secreted MAb (MAD2) purified and characterized. MAD2 cross-reacts to varying degrees with anthracycline compounds such as some DXR analogues and derivatives, but does not recognize anthracene and anthraquinone structures, with the exception of weakly reacting Mitoxantrone. MAD2 and the panel of MAbs which are at present being purified may become a tool for studying the relevance of different domains of the anthracyclin molecule in terms of biologic activity.

Cholecystokinin octapeptide in rat central nervous system: Immunocytochemical studies using a monoclonal antibody that does not react with CGRP.

The biological activity of the important neuropeptide cholecystokinin octapeptide (CCK8) resides at the C-terminus. Antibodies with C-terminal specificity have been reported to cross-react with a different neuropeptide, calcitonin gene related peptide (CGRP) and this has frustrated the interpretation of immunohistochemical studies. We describe here the properties of a monoclonal antibody to the CCK-related peptide, caerulein, that reacts with the C-terminal region of CCK8, but does not react with CGRP in radioimmunoassay or immunohistochemistry. The distribution of CCK-like activity revealed by immunohistochemistry using this antibody broadly resembles that described previously with a single major exception: in the dorsal horn of the spinal cord. The results support the suggestion that apparent CCK activity in the terminals of rat primary sensory neurones is due to cross-reactivity with CGRP.

Antigen specific, suppressor cells induced by the immunomodulator FCE 20696 in aged NZB/WF1 mice.

FCE 20696 is a new synthetic immunomodulating agent, active on some manifestations of the autoimmune disease of NZB/WF1 mice, in which a disorder of T suppressor cells (SC) has been described to be of pathogenetic relevance. The ability of FCE 20969 to induce SC in NZB/WF1 mice, which are then able to specifically inhibit the production of anti autologous red blood cells antibodies (aRBC Ab), was investigated. NZB/WF1 mice were chronically treated with the compound starting from the ninth week of age, sacrificed at different times and their spleen cells transferred to 9 weeks old, syngeneic mice. In donor animals SC were induced by rat RBC, whereas in the recipients the suppressive activity of transferred cells was evaluated from the appearance of aRBC Ab induced by the same antigen. The results show that antigen specific SC were present in both FCE 20696 treated and control young animals. In older controls, SC couldn't be induced, whereas in treated animals SC were present and able to transfer suppression into the recipients. FCE 20696 was active at 1.5 mg/Kg.

Macrophage characteristics of a cell line isolated in vitro from a Moloney lymphoma.

A new cell line, LM-A, established in vitro from a murine Moloney lymphoma, is described. The line shows the cytologic, adherence, and phagocytic properties of normal macrophages. It forms specific rosettes with antibody-coated erythrocytes. The cells mediate antibody-dependent cellular cytotoxicity as assayed by release of radioactivity from 51Cr-labeled erythrocytes. The cytolytic reaction proved to be functionally distinct from the phagocytic process as demonstrated by enhanced cytolysis in the presence of iodo-acetamide, an inhibitor of phagocytosis. The line has Moloney murine leukemia virus (MLV)-related antigens as detected by membrane immunofluorescence. The LM-A line is useful for the study of macrophage functions and also provides an interesting tool for investigating the antigenic and immunogenic properties induced by MLV in a cell as relevant as the macrophage in the immune response.

Pharmacokinetic approach to the selection of dose schedules for medroxyprogesterone acetate in clinical oncology.

The pharmacokinetic and bioavailability properties of medroxyprogesterone acetate (MPA) after single PO and IM doses in man were used as a basis to predict, on a theoretical pharmacokinetic basis, the blood level profile of the drug during repeated dose administration with various dosage schedules. Because of the unusually long-lasting depot effect of IM MPA, a different build-up process of blood levels is expected during repeated IM or PO administration, and this should be taken into account when dose schedules for use in clinical oncology are selected. As regards the IM route, dose schedules based on 4 weeks' treatment with daily injections of 500-1,000 mg followed by a maintenance therapy with 1,000 mg/week are suggested, since they permit rapid achievement and maintenance of relatively high plasma levels. A similar plasma level profile can be obtained with oral MPA provided that daily doses twice as large as the IM doses are given during the first month of treatment and continued during the maintenance period. The serum levels observed in 25 patients with advanced breast cancer treated with MPA given IM or PO according to various dose schedules and recent literature data are very close to the serum level profiles predicted on a theoretical pharmacokinetic basis.

Modification of immune responsiveness in murine Schistosomiasis mansoni. I. Time course after cercarial exposure.

The immune responsiveness of mice infected with 90 cercariae of Schistosoma mansoni has been investigated. Post-infection kinetics of antibody responses, delayed hypersensitivity and mitogen responsiveness show that there are profound disturbances of these immunological parameters starting 2 weeks after the infection. Although the final outcome is immunodepression the S. mansoni infection can produce immunostimulation. We have observed differences in the kinetics of the depression of humoral antibody responses in two strains of mice. It seems that the worms and not the eggs are of major importance in determining the observed alterations of immune responsiveness.

Affinity and specificity of penicillin-antibody interaction determined by an enzyme (E. coli beta-galactosidase) immunoassay.

An enzyme immunoassay (EIA) for a penicillin derivative is described with a sensitivity at least at the nanogram level. The label, E. coli beta-galactosidase is a macromolecule of 540 000 daltons: the size of the enzyme and the ease of linking penicilloyl residues to it make it an interesting model to study the effect of the degree of haptenic substitution (DS) in the tracer on the parameters of EIA. Our results show that the affinity of the binding reaction between antibody and tracer is proportional to the DS but the sensitivity of inhibition is not affected, at least not between 1 and 10 penicilloyl residues per GZ molecule. The theoretical consequences and practical applications of multivalent tracers in EIA are discussed.