Cinnamyl Schiff bases: synthesis, cytotoxic effects and antifungal activity of clinical interest.

The aim of this study was to synthesize and investigate the in vitro antifungal properties of 23 cinnamyl Schiff bases. In addition, cytotoxic effects of such cinnamyl Schiff bases against human lung, kidney or red blood cells were also checked. The compounds were synthesized in a single-step, 2 min of reaction under microwave irradiation produced up to 97% yield. Six of the 23 cinnamyl Schiff bases possessed antifungal activities against strains of Candida, Aspergillus, Fonsecaea and, particularly, Cryptococcus species. Indeed, cinnamyl Schiff bases 1 and 23 exhibited minimum inhibitory concentration (MIC) values more than twofold lower than fluconazole (FCZ) against all the Cryptococcus neoformans strains (MIC = 1·33, 1·4 and 5·2 µg ml-1 , respectively) and Cryptococcus gattii strains (MIC = 5·3, 2·8 and 9·2 µg ml-1 , respectively) (12 strains of each species) while cinnamyl Schiff base 11 was as potent as FCZ against all strains from both Cryptococcus species. No significant cytotoxic effects were observed for Schiff bases against human lung, kidney or red blood cells, all presenting selective indexes higher than 10. In conclusion, this study revealed cinnamyl Schiff bases, especially 1 and 23, as new lead anticryptococcal agents for the discovery of novel antifungal drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: The occurrence and severity of fungal infections have increased in recent decades due to resistance to available antifungal drugs and the appearance of new emerging pathogens. Thus, the search for new antifungal agents is mandatory. From a series of 23 cinnamyl Schiff bases, two compounds (1 and 23) were interrogated as new anticryptococcal agents without significant cytotoxicity against human lung, kidney or red blood cells. In turns, these new Schiff bases are lead compounds for the discovery of novel antifungal drugs.

2-deoxy-D-glucose-induced metabolic stress enhances resistance to Listeria monocytogenes infection in mice.

Exposure to different forms of psychological and physiological stress can elicit a host stress response, which alters normal parameters of neuroendocrine homeostasis. The present study evaluated the influence of the metabolic stressor 2-deoxy-D-glucose (2-DG; a glucose analog, which when administered to rodents, induces acute periods of metabolic stress) on the capacity of mice to resist infection with the facultative intracellular bacterial pathogen Listeria monocytogenes. Female BDF1 mice were injected with 2-DG (500 mg/kg b. wt.) once every 48 h prior to, concurrent with, or after the onset of a sublethal dose of virulent L. monocytogenes. Kinetics of bacterial growth in mice were not altered if 2-DG was applied concurrently or after the start of the infection. In contrast, mice exposed to 2-DG prior to infection demonstrated an enhanced resistance to the listeria challenge. The enhanced bacterial clearance in vivo could not be explained by 2-DG exerting a toxic effect on the listeria, based on the results of two experiments. First, 2-DG did not inhibit listeria replication in trypticase soy broth. Second, replication of L. monocytogenes was not inhibited in bone marrow-derived macrophage cultures exposed to 2-DG. Production of neopterin and lysozyme, indicators of macrophage activation, were enhanced following exposure to 2-DG, which correlated with the increased resistance to L. monocytogenes. These results support the contention that the host response to 2-DG-induced metabolic stress can influence the capacity of the immune system to resist infection by certain classes of microbial pathogens.

Distinctive immunomodulating properties and interactivity with model membranes and cells of two homologous muramyl dipeptide derivatives differing by their lipophilicity.

We have studied and compared the immunomodulating activities of two muramyl dipeptide (MDP) derivatives: beta-heptylglycoside-MDP (C7H15MDP) and beta-hexadecylglycoside-MDP (C16H33MDP). The amphiphilic derivative C7H15MDP has been found to be a more effective stimulator of T lymphocyte proliferation and allospecific cytotoxic T cell generation in a mixed lymphocyte culture, and a more effective activator of interleukin-1 (IL-1) and tumour necrosis factor (TNF) production by murine peritoneal macrophages, in comparison with MDP and C16H33MDP used in equimolar concentrations. C7H15MDP also stimulated cytotoxicity of natural killer (NK) cells: On the contrary, its lipophilic homologue C16H33MDP did not show such activities in vitro except for its influence on IL-1 and TNF production. We have found significant differences in the interaction of these two 14C-labelled MDP derivatives with model membranes and in the uptake of these preparations by erythroleukemia K562 cells. We consider the hydrolipophilic balance of the above preparations to be the main cause of their different interactions with membranes and their uptake by cells and, as a result, their opposite immunomodulating activities in vitro.

Effect of space flight on cytokine production and other immunologic parameters of rhesus monkeys.

During a recent flight of a Russian satellite (Cosmos #2229), initial experiments examining the effects of space flight on immunologic responses of rhesus monkeys were performed to gain insight into the effect of space flight on resistance to infection. Experiments were performed on tissue samples taken from the monkeys before and immediately after flight. Additional samples were obtained approximately 1 month after flight for a postflight restraint study. Two types of experiments were carried out throughout this study. The first experiment determined the ability of leukocytes to produce interleukin-1 and to express interleukin-2 receptors. The second experiment examined the responsiveness of rhesus bone marrow cells to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Human reagents that cross-reacted with monkey tissue were utilized for the bulk of the studies. Results from both studies indicated that there were changes in immunologic function attributable to space flight. Interleukin-1 production and the expression of interleukin-2 receptors was decreased after space flight. Bone marrow cells from flight monkeys showed a significant decrease in their response to GM-CSF compared with the response of bone marrow cells from nonflight control monkeys. These results suggest that the rhesus monkey may be a useful surrogate for humans in future studies that examine the effect of space flight on immune response, particularly when conditions do not readily permit human study.

Countermeasures for ameliorating in-flight immune dysfunction.

We analyzed immune disturbances caused by the influence of spaceflight factors and proposed approaches for their correction. First, we determined the significant resistance of humoral immunity to the action of spaceflight factors, for example, B cell and immunoglobulin content, B cell reactivity to lipopolysaccharide, and distribution of B cell clones to trinitrophenyl (TNP group). A brief reversible increase of IgA and/or IgG concentration was the most characteristic shift observed. However, we noted prominent changes of T cells and natural killer (NK) cells, such as decreases in the content and phytohemagglutinin (PHA) reactivity of T lymphocytes, reduced T helper activity, and a reduction in NK cell cytotoxic activity in humans and animals during prolonged, spaceflights. Rat bone marrow cells showed a decreased response to macrophage colony-stimulating factor. Studies with rat lymph node cells showed that microgravity and spaceflight stressors may influence lymphocytes in a tissue-specific manner. In experiments on isolated cells, human lymphocytes produced interferon more intensively in a reduced-gravity (microgravity) environment than under terrestrial conditions. However, lymphocyte activation by concanavalin A was sharply suppressed. Under microgravity conditions the transfer of activating signal to protein kinase C was strongly reduced. Thus, in-flight disturbances of T and NK cell functions are quite significant and could cause some diseases. However, to date the in-flight immune dysfunctions have not been excessive. Probably, we are mainly dealing with a complex of changes of immunity in space that is similar to that in the presence of acute stress. Therefore, it is reasonable to consider stress-related immunotherapy approaches in the practice of space medicine. The most effective methods and approaches of modern immunotherapy, such as artificial vaccines, monoclonal antibodies and immunoconjugates, recombinant cytokines, or immunomodulators etc, are also reviewed.

Effect of spaceflight on lymphocyte proliferation and interleukin-2 production.

Studies of peripheral blood lymphocytes from astronauts indicate that microgravity depresses T-cell responsiveness. However, this effect has not been examined in cells of peripheral lymphatic tissue, where most lymphocytes are located. In this study, inguinal lymph node lymphocytes from rats flown on the COSMOS 2044 mission were tested for proliferation and interleukin-2 (IL-2) production. Cells cultured with mitogenic lectins, phorbol ester, and calcium ionophore, or T-cell mitogen and lymphokine, were assayed for DNA synthesis by [3H]thymidine incorporation. Lymphocytes incubated with a T-cell mitogen alone also were tested for IL-2 production. Proliferation of lymphocytes from flight rats was not significantly different from controls for any of the mitogens tested. Furthermore, lymph node lymphocytes from flight rats was not significantly different from controls for any of the mitogens tested. Furthermore, lymph node lymphocytes from control and flown rats produced similar amounts of IL-2. Thus microgravity may act on lymphocytes in a tissue-specific manner, a new finding that could impact on the evaluation of spaceflight effects on immunocompetence.

Spaceflight alters immune cell function and distribution.

Experiments were carried out aboard COSMOS 2044 to determine the effects of spaceflight on immunologically important cell function and distribution. Control groups included vivarium, synchronous, and antiorthostatically suspended rats. In one experiment, rat bone marrow cells were examined in Moscow, for their response to recombinant murine granulocyte/monocyte colony-stimulating factor (GM-CSF). In another experiment, rat spleen and bone marrow cells were stained in Moscow with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and shipped to the United States for analysis on a flow cytometer. Bone marrow cells from flown and suspended rats showed a decreased response to granulocyte/monocyte colony-stimulating factor compared with bone marrow cells from control rats. Of the spleen cell subpopulations examined from flown rats, only those cells expressing markers for suppressor-cytotoxic T- and helper T-cells showed an increased percentage of stained cells. Bone marrow cells showed an increase in the percentage of cells expressing markers for helper T-cells in the myelogenous population and increased percentages of anti-asialo granulocyte/monocyte-1-bearing interleukin-2 receptor-bearing pan T- and helper T-cells in the lymphocytic population. Cell populations from rats suspended antiorthostatically did not follow the same pattern of distribution of leukocytes as cell populations for flown rats. The results from COSMOS 2044 are similar, but not identical, to earlier results from COSMOS 1887 and confirm that spaceflight can have profound effects on immune system components and activities.

Effect of spaceflight on natural killer cell activity.

The effects of spaceflight on immune cell function were determined in rats flown on COSMOS 2044. Control groups included vivarium, synchronous, and antiorthostatically suspended rats. The ability of natural killer cells to lyse two different target cell lines was determined. Spleen and bone marrow cells obtained from flight rats showed significantly inhibited cytotoxicity for YAC-1 target cells compared with cells from synchronous control rats. This could have been due to exposure of the rats to microgravity. Antiorthostatic suspension did not affect the level of cytotoxicity from spleen cells of suspended rats for YAC-1 cells. On the other hand, cells from rats flown in space showed no significant differences from vivarium and synchronous control rats in cytotoxicity for K-562 target cells. Binding of natural killer cells to K-562 target cells was unaffected by spaceflight. Antiorthostatic suspension resulted in higher levels of cytotoxicity from spleen cells for 51Cr-labeled K-562 cells. The results indicate differential effects of spaceflight on function of natural killer cells. This shows that spaceflight has selective effects on the immune response.

Effects of spaceflight on levels and activity of immune cells.

Experiments were carried out on cells from rats that had been flown on Soviet Biosputnik Cosmos 1887 to explore the effects of spaceflight on immune responses. Rat bone marrow cells were examined for their response to colony stimulating factor-M. Rat spleen and bone marrow cells were stained with antibodies directed against cell surface antigenic markers. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell and interleukin-2 receptor cell surface antigens. A small increase in the percentage of cells staining positively for helper-T-cell antigens was also noted. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin.

A study of the action of immunosuppressive factors from tumour cells on lymphocytes and macrophages in vitro and on the graft-versus-host reaction in mice.

The effect of immunosuppressive factors from P815, B16, and EL-4 mouse tumour cells on the permeability of the lymphocyte membrane to RNAase (the 'membrane-toxic effect'), on the generation of tumour necrosis factor (TNF) by mouse peritoneal macrophages, and on the survival rate of mice undergoing a graft-versus-host reaction was investigated. The immunosuppressive factors were found to have a membrane-toxic effect on mouse lymphocytes. Moreover, in the presence of pancreatic RNAase there was a dose-dependent increase in the inhibitory effect of the immunosuppressive factors on concanavalin-A-induced spleen cell proliferation. The immunosuppressive factors reduced the production of muramyl-dipeptide-induced TNF by mouse peritoneal macrophages. When immunosuppressive factors from P815 cells were administered to F1 hybrid mice (CBA x C57B1/6), there was a marked decrease in the intensity of the graft-versus-host reaction induced by injection of C57B1/6 parent mouse spleen cells to the F1 hybrids, and the life span of these mice was increased. It is suggested that the membrane-toxic effect may be one mechanism by which cells in the immune system are inhibited by immunosuppressive factors from tumour cells.

Biochemical and immunochemical analysis of gangliosides of human small cell lung carcinoma: production of monoclonal antibodies against a unique marker of small cell lung carcinoma, ganglioside Fuc GM1.

Immunization of mice with a pure preparation of the ganglioside adsorbed on Salmonella typhimurium and hybridization of splenocytes with myeloma P3-X63-Ag 8.653 have resulted in hybridomas producing monoclonal antibodies against ganglioside Fuc GM1, a marker of human small cell lung carcinoma. Characterization of four hybridomal clones and data on the antigenic specificity of the monoclonal antibodies are given. All four monoclonal antibodies reacted only with Fuc GM1 in an enzyme-linked immunosorbent assay. In radioimmunodetection of the antigen on thin-layer plates, two of the four monoclonal antibodies gave cross-reactions with Fuc GD1b. The obtained monoclonal antibodies have revealed the presence of Fuc GM1 in all seven cases of small cell lung carcinoma we have studied and the absence of Fuc GM1 in the normal human lung tissue and in lung adenocarcinomas.

Tumour cell toxicity for the targets of natural killer cells.

Cells of intraperitoneally transplanted murine leukaemias and sarcomas (L-1210, EL-4, MC-11 and SA-1), were found to be toxic for target cells usually employed for assaying NK cells (K-562, EL-4, YAC-1). The 3H-uridine-ribonuclease method used in our study, in contrast to the 51Cr assay, revealed not only cytotoxicity but also reversible damage to target cell membrane (membrane toxicity). This damage became irreversible if target cells had been pretreated with actinomycin D. To exclude the possible role of an admixture of NK, macrophages and T killer cells, contaminants were removed from suspensions of peritoneal tumour cells from syngeneic mice by adherence to plastic surfaces and separation on Hypaque-Ficoll, and from tumour cells grown in vivo or in vitro and then maintained in allogeneic animals by additional treatment with anti-H-2 sera and complement. The toxic effect depended directly on the dosage of tumour cells. Unlabelled target cells inhibited tumour cell toxicity. The medium used for incubating tumour cells was not toxic for target cells.

Studies of the immunologically specific retention of antigen-activated lymphoid cells in antigen-containing lymph nodes.

Mouse spleen lymphocytes proliferating under the influence of SRBC, or educated thymocytes were labelled in vivo or in vitro with 3H-thymidine and transferred intravenously to syngeneic recipients, which had received a subcutaneous injection of the same, or a non-cross-reacting, antigen (SRBC, rat or chicken RBC, DNP-proteins) into the right front footpads. The left footpads had been injected with a control (non-cross-reacting) antigen. The right (ipsilateral) and left (contralateral) regional lymph nodes were compared on the basis of their radioactivity and radioautography. SRBC-containing lymph nodes retained specifically both viable primed spleen cells and educated thymocytes. Treatment of donor spleen cells with anti-T serum and complement prevented specific retention. Administration of anti-SRBC serum to the recipients had a similar effect. Retention occurred during 6 days after administration of the antigen to the recipients and was mediated by radioresistant host lymph node cells. It declined profoundly when host macrophages were blocked with charcoal particles. The data obtained suggest a major role of the direct contact between T cell receptors and macrophage-bound antigen in the specific lymphocyte retention.

Bone marrow suppressor B cells in vitro.

Marrow cells from intact CBA and (CBA x C57B1)F1 mice added to a culture of syngeneic splenocytes at its initiation suppressed the generation of anti-SRBC antibody-forming cells. Removal of cells bearing surface immunoglobulins and/or MBLA led to a substantial reduction in the marrow suppressive activity. Marrow cells from mice pretreated with five doses of hydroxyurea did not suppress the immune response of syngeneic splenocytes to SRBC. While the total number of cells in the marrow of such animals declined 5-fold, the relative content of Ig-positive cells was somewhat increased. The proportion of blast cells dropped from 10.3 to 2.4%. No blast cells bearing surface immunoglobulins were observed. The proportion of cells incorporating [3H]-thymidine fell 40-fold. We conclude that suppression of the immune response to SRBC in vitro is mediated by immature precursors of the B lymphocyte series which are present in the bone marrow of intact mice.