RESUMO
The effect of emetine which is a potent immunosuppresant was studied on the thymus and lymph node. The subcapsular zone of the thymus was depleted and large number of adherent cells accumulated in this thymic region. The medulla enlarged but the cortico-medullary border remained distinct. In the paracortex (T dependent area) of the lymph node many non-lymphoid pyroninophil cells appeared which is followed by an increased cell proliferation 24 hours after emetine injection. 48-60 hours after administration many macrophage-like cells appeared in the medullary sinuses. This macrophage invasion precedes the adherent cell accumulation in the subcapsular zone of the thymus suggesting a possible non-lymphoid (adherent) cell migration from the lymph node's paracortex to the thymus.
Assuntos
Emetina/farmacologia , Imunossupressores/farmacologia , Linfonodos/efeitos dos fármacos , Timo/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Linfonodos/ultraestrutura , Macrófagos/efeitos dos fármacos , Camundongos , Timo/ultraestruturaRESUMO
Emetine (33 mg/kg body weight) administered intraperitoneally blocked the immune response of mice to 10(9) sheep red blood cells (SRBC). The inhibition was almost complete when the drug was administered simultaneously or 24 hr after immunization, while partial inhibition was caused by treatment at 48 and 72 hr. Incorporation of 14C-leucine and 3H-thymidine by spleen cells isolated 4 hr after emetine injection of the mice was strongly decreased. Incorporation was approaching the control level in cells isolated 72-96 hr after emetine administration. However, the incorporation of labeled precursors was less than after SRBC treatment only, even after 72-96 hr. Emetine apparently blocked the development of immune response at an early stage and, in contrast to macromolecule synthesis, the inhibition of the antibody response was irreversible.
Assuntos
Emetina/farmacologia , Imunidade/efeitos dos fármacos , Animais , DNA/biossíntese , Eritrócitos/imunologia , Imunização , Masculino , Camundongos , Camundongos Endogâmicos , Biossíntese de ProteínasRESUMO
Emetine and chloroquine caused a dose-dependent and time-dependent inhibition of bacterial phagocytosis in rat peritoneal macrophages. The effect of emetine was found to be stronger: 50% inhibition was achieved at 5 X 10(-6) M concentration. The inhibition of phagocytosis was irreversible. Neither the binding of bacteria to the surface of phagocytes nor membrane marker 5' nucleotidase was touched by this treatment. The adherence of macrophages to plastic dishes was also impaired by the drugs but only in a smaller extent. Both emetine and chloroquine blocked the amino acid incorporation into macrophage proteins. The effect of emetine was more marked; 10(-6) M decreased the incorporation to 25% of control, while 10(-4) M chloroquine produced a similar inhibition. The block of protein synthesis was also irreversible.
Assuntos
Cloroquina/farmacologia , Emetina/farmacologia , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Animais , Radioisótopos de Carbono , Adesão Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Leucina/metabolismo , Macrófagos/imunologia , Biossíntese de Proteínas , Ratos , Ratos EndogâmicosRESUMO
Nascent Hg-DNA synthesized by the incorporation of Hg-dCTP in reversibly permeable cells of murine thymocytes has been characterized earlier. Here we describe the analysis of 5' ends of oligonucleotides isolated from thymocytes 48 hr after a single dose of emetine administration to mice. This small-molecular-weight population of nascent DNA shorter than Okazaki fragments was absent in control cells. More than 90% of the terminally 32P-labeled oligonucleotides carried a terminally phosphorylated RNA moiety at the 5' end, as demonstrated by alkaline hydrolysis. The size of the short nascent DNA fragments carrying RNA primers ranged between 9 and 50 nucleotides with an average chain length of 15 nucleotides. These oligomers are regarded as the precursors of the Okazaki fragments.
Assuntos
Replicação do DNA/efeitos dos fármacos , DNA/biossíntese , DNA/química , DNA/metabolismo , Emetina/farmacologia , Linfócitos T/metabolismo , Animais , Células Cultivadas , Cromatografia em Gel , DNA/isolamento & purificação , Eletroforese em Gel de Ágar , Masculino , Mercúrio , Camundongos , Camundongos Endogâmicos , Radioisótopos de Fósforo , Fosforilação , RNA/biossíntese , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Exponentially growing Drosophila S2 cells in suspension culture were synchronized at low- and high-resolution centrifugal elutriation, and DNA synthesis was measured by [(3)H]-thymidine incorporation throughout the S phase. At low resolution, one repair peak at the G(1)/G(0) border and two replication peaks known as early and late S subphases were observed. At high resolution, six chronologic compartments were distinguished. The distribution of these peaks indicated one repair peak at 2.05 C value, one minor replication peak at 2.43C, and four major subphases of replication corresponding to 2.64C, 2.89C, 3.32C, and 3.60C, representing 6.7%, 3.4%, 15.3%, 20.4%, 32.1%, and 22.0% of the synthetic activity, respectively. The five major peaks of cell growth with 2.32C, 2.56C, 2.85C, 3.18C, and 3.58C values consistently preceded those of replication subphases.
Assuntos
Replicação do DNA , Drosophila/genética , Fase S/genética , Animais , Contagem de Células , Divisão Celular , Linhagem Celular , Separação Celular/métodos , DNA/genética , DNA/metabolismo , Drosophila/citologia , Drosophila/metabolismo , Timidina/metabolismo , TrítioRESUMO
The kinetics of DNA synthesis and the DNA pattern of isolated peripheral blood mononuclear cells from control subjects and lymphoma patients prior to drug treatment were studied as a possible tool in the early diagnosis of lymphoma. Thymidine and [H3]-dTTP incorporation represented the measure of replicative DNA synthesis in permeable murine thymocytes and HT-168 human melanoma cells as described earlier. The kinetic parameters of nucleotide incorporation were compared with the ploidity parameters of the Feulgen-stained smears examined by the DNASK TV-cytophotometric system. For better evaluation and visualization of the S-phase population, the method of silver impregnation of the Nucleolar-Organizer-Region in combination with the Feulgen technique was used. Significant differences were observed among the five determined parameters of healthy and lymphoma patients. Our results allow to explain some of the facts related to T-cell function deficiency in lymphoma.
Assuntos
DNA de Neoplasias/sangue , DNA/sangue , Doença de Hodgkin/sangue , Leucócitos Mononucleares/química , Linfoma não Hodgkin/sangue , Adolescente , Adulto , Idoso , Animais , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , DNA de Neoplasias/biossíntese , Feminino , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Nucleotídeos/metabolismo , Ratos , Ratos Wistar , Valores de Referência , Espectrofotometria/métodosRESUMO
Among the human tonsillar cells, 28.0 +/- 3.1 and 45.14 +/- 2.16% were found to be E and EAC rosette forming cells, resp. In the course of the separation procedure with nylon cotton, in the authors' modification, the cells failing to form rosettes were almost entirely adhering irreversibly to the nylon wool. The 3H-thymidine incorporation in these cells was very intensive, and we supposed them to be T and B lymphoblasts. On the basis of E and EAC rosette formation, the percentage of T and B lymphocytes in human tonsils--as reported by various authors--is different. It may be supposed that methods employed by these authors did not demonstrate the equal extent of transformed forms of lymphocytes, i.e. lymphoblasts, whose amount may be influenced by the functional condition of tonsils.
Assuntos
Linfócitos B/citologia , Tonsila Palatina/citologia , Linfócitos T/citologia , Linfócitos B/imunologia , Separação Celular , Sobrevivência Celular , Criança , Pré-Escolar , Humanos , Contagem de Leucócitos , Ativação Linfocitária , Formação de Roseta , Linfócitos T/imunologiaRESUMO
The role of endothelium in vasodilatation has only emerged in the last ten years. It was observed that many endogenous substances from endothelial cells triggered the release of a substance which was named endothelium-derived relaxing factor (EDRF). Later has been showed that NO accounted for most if not all of the biological activity of EDRF. The endothelial synthesis of NO originates from L-arginine and could be blocked by the methyl analogue (e.g. NG-mono-methyl-L-arginine). Beside endothelial cells NO could be identified in several mammalian tissues including brain, hepatocytes, lung and macrophages. NO mediated the control of vascular tone and blood pressure via vascular smooth muscle cells which exert relaxation and constriction of blood vessels. It is considered NO represents signal for the guanylate cyclase system which regulates the intracellular concentration of Ca2+ ions. It is well known that the concentration of Ca2+ ions play discern direct role in the relaxation and contraction of smooth muscle, respectively.
Assuntos
Óxido Nítrico/metabolismo , Endotélio/metabolismo , Radicais Livres/metabolismo , Humanos , Relaxantes Musculares Centrais/metabolismo , Músculo Liso/fisiologia , Vasodilatadores/metabolismoRESUMO
A large body of the evidence is available to the causative relationship between the elevated blood plasma concentrations of LDL and the atherogenesis. The oxid-LDL (modified LDL) is internalized more rapidly by the macrophages, and there is now substantial evidence that the modified LDL is actually present in atherosclerotic lesions. Recently it has been proved that the endothel cells and monocyta/macrophages generate nitric oxide (NO) from arginine, and that the LDL inhibits the formation of NO in endothel cells. The authors found that the human LDL in vitro exerts an inhibitory effect on the formation of NO in murine PED (peritoneal exudate cells) and synchronously severalfold increasing of the arginase activity in the culture media. Both effects of LDL proved to be dose dependent and the oxid-LDL has been found to be more effective. The increased activity of arginase provides a very likely explanation for the reducing of NO production in macrophage treated by LDL. The reducing or blocking of NO-formation causes a local vasocontraction which induces clinical symptoms.
Assuntos
Arginase/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/química , Óxido Nítrico/metabolismo , Animais , Macrófagos/enzimologia , CamundongosRESUMO
OBJECTIVE AND DESIGN: The effects of various inflammatory stimuli on the cytokine profile and phagocytic capacity of mouse and rat peritoneal macrophages were investigated in vitro. The correlations between cytokine concentrations and the expressions of NOS II and arginase were also studied. METHODS: Mice and rats were injected intraperitoneally with various inflammatory agents. Peritoneal macrophages were isolated. The levels of eight cytokines were determined in macrophage cultures by ELISA test. Phagocytic capacity of macrophages was measured by the ingestion of M. Luteus. RESULTS: The most marked changes caused by i. p. treatments were observed in the levels of IL-1 and IL-6 in mice and of IL-12 in rats. IFN-gamma level were increased mainly in rat cells while TNF-alpha production was rather enhanced in mice. Phagocytic capacity of macrophages was higher in rat samples and it increased with all treatments, except BCG, without marked differences between different treatments. CONCLUSIONS: Each inflammatory agent caused an increase in cytokine productions in both species, with marked differences among cytokines. Correlations were found in mouse between IL-6 level and NOS II expression, and IL-10 level with arginase expression. In rat macrophages, IFN-gamma, TNF-alpha and MIP-2 productions were in good correlation with NOS II expression.
Assuntos
Citocinas/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Arginase/metabolismo , Interferon gama/metabolismo , Interleucina-1/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Micrococcus luteus/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Fagocitose , Ratos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE AND DESIGN: The effects of various inflammatory stimuli on the alternative arginine metabolic pathways in mouse and rat peritoneal macrophages were investigated in vitro and compared. TREATMENTS: Mice and rats were injected i. p. with thioglycollate, carrageenan, casein, BCG and Newcastle Disease Virus (NDV) vaccines. METHODS: Peritoneal macrophages were isolated from untreated and treated animals. The activities of nitric oxide synthase (NOS) II and arginase were measured and expressions were followed by Western blotting. The uptake of arginine and nitrite formation of macrophages were also measured. RESULTS: Inflammatory stimuli increased the NO production and the expression and activity of both NOS II and arginase in mice in vitro. On the contrary, the same treatments changed the expression and activity of NOS II only, but not those of arginase in rats. The most marked effects on NO metabolism were produced by casein and NDV treatments. CONCLUSIONS: The activity and expression of NOS II and arginase can be stimulated in peritoneal macrophages in vitro by injecting inflammatory agents into the peritoneal cavity. A markedly different response in arginine metabolism was observed in mouse and rat macrophages. Casein treatment was a potent inducer for both enzymes. NDV vaccines induced mainly NOS II, while thioglycollate induced arginase.
Assuntos
Arginina/metabolismo , Inflamação , Macrófagos/efeitos dos fármacos , Animais , Arginase/metabolismo , Vacina BCG/farmacologia , Carragenina/farmacologia , Caseínas/farmacologia , Macrófagos/metabolismo , Masculino , Camundongos , Vírus da Doença de Newcastle/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Peritônio/metabolismo , Ratos , Ratos Wistar , Tioglicolatos/farmacologia , Vacinas Virais/farmacologiaRESUMO
The arginase produced by peritoneal macrophages is not synthesized de novo in short-term (3 h) cultures after harvesting the cells. In long-term cultures the arginase synthesis is restored. In contrast to arginase lysozyme is continuously synthesized in short-term cultures. These statements were proved by the following experimental results: 1. Protein synthesis inhibitor and lysosomotropic agents did not alter the arginase level. 2. Arginine and its analogue, canavanine and ornithine were not able to change the arginase activity. 3. The product of an alternative metabolic pathway of arginine, sodium nitrite, did not affect arginase activity. 4. Effectors influencing the synthesis of cyclic nucleotides (cAMP, cGMP), indomethacin, sodium nitroprusside and an analogue of cAMP had no effect on the arginase activity. 5. Arginase activity could not be significantly modified either by an in vitro Micrococcus luteus treatment or by changing the adherence period of peritoneal exudate cells. 6. When arginase was produced in murine peritoneal macrophages at various periods with medium change, the total arginase released into the media from murine and rat macrophages did not exceed the original intracellular arginase content of the adhered cells during the first 6 hours.
Assuntos
Arginase/biossíntese , Precursores Enzimáticos/biossíntese , Macrófagos Peritoneais/enzimologia , Animais , Arginase/efeitos dos fármacos , Canavanina/farmacologia , Células Cultivadas , Lisossomos/efeitos dos fármacos , Masculino , Camundongos , Muramidase/biossíntese , Nucleotídeos Cíclicos/farmacologia , Ornitina/farmacologia , Inibidores de Proteases/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos WistarRESUMO
1. Murine macrophages showed a considerably higher in vitro arginase production in short time cultures than rat peritoneal cells. 2. The in vivo stimulation with casein or thioglycollate resulted in an enhanced in vitro enzyme production in mice. 3. The adherence is not the condition of the enzyme production. 4. The difference between the two species cannot be explained by the lack of bivalent ions, the absence of energy supply, proteolysis, the low number of macrophages or by the different cell types of the peritoneal exudate of mouse and rat. 5. The lysozyme production of murine and rat peritoneal macrophages was also investigated and no difference was observed between the two species.
Assuntos
Arginase/metabolismo , Ativação de Macrófagos , Macrófagos/enzimologia , Animais , Células Cultivadas , Cromatografia em Camada Fina , Cinética , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Muramidase/metabolismo , Cavidade Peritoneal/citologia , Ratos , Ratos EndogâmicosRESUMO
It was found that the supernatant of mouse PEC culture medium (MCM) (both resident and casein-elicited cells) has an inhibitory effect in vitro on the incorporation of [3H]TdR into DNA of mouse spleen cells. The inhibitory effect in the MCM appears in the first 24 hr and also reaches its maximum value within this time. The inhibitory effect of this factor could not be demonstrated in the extract of freshly harvested M phi cells. The factors responsible for inhibition proved to be heat stable at 80 degrees C for longer than 30 min. Following heat treatment, the crude extract was separated into four fractions absorbing uv light at 280 nm using Sephadex G-25 column chromatography, and the most potent biologically active inhibitory factor was eluted in the last fraction. This fraction could also be obtained with a more effective permeation volume using Trysacryl GF 05 gel chromatography, and the active B fraction from this chromatography could be separated into four subfractions by isotachophoresis (ITP). The active fraction, which was obtained by Trysacryl GF 05 gel chromatography and further separated by ITP, was found to be highly inhibitory. It contained a peptide-like substance with a molecular mass of approximately 2.0 kDa and had an anionic character at pH 4.0. The inhibitory effect of MCM cannot be influenced either by inhibitory compounds of protein synthesis or by proteolysis blocking agents. Furthermore, the inhibitory effect is shown to be reversible and is more pronounced on B cells than on T lymphocytes.
Assuntos
Divisão Celular/efeitos dos fármacos , Linfócitos/citologia , Macrófagos/fisiologia , Peptídeos/toxicidade , Proteínas/fisiologia , Animais , Células Cultivadas , Temperatura Alta , Ativação de Macrófagos , Camundongos , Peso Molecular , Monocinas , Peptídeos/isolamento & purificação , Cavidade Peritoneal/citologiaRESUMO
1. The de novo synthesis of arginase was much higher in murine than in rat peritoneal macrophages. This process was inhibited irreversibly by protein synthesis inhibitors and reversibly by glycolysis blockers. 2. Rat macrophages produce more nitric oxide (NO) than murine cells. NO production was inhibited by the inhibitors of protein synthesis or glycolysis. 3. The loading of macrophages by exogenous arginine for 24 hr in vitro resulted in the increase of arginase and nitrite in macrophages to different extents. 4. No great differences in lysozyme production was observed. 5. The proportion of arginine taken up and incorporated is contrasted in murine and rat macrophages.
Assuntos
Arginase/biossíntese , Macrófagos/metabolismo , Camundongos Endogâmicos/metabolismo , Óxido Nítrico/metabolismo , Ratos Wistar/metabolismo , Aminoácidos/metabolismo , Animais , Arginase/fisiologia , Arginina/metabolismo , Células Cultivadas , Camundongos , Cavidade Peritoneal/citologia , Ratos , Fatores de TempoRESUMO
The uptake of Leu-OMe and Leu-Leu-OMe was studied in vitro in porcine PMN cells. Both methylesters are metabolized leading to the intracellular accumulation of leucine. Part of the hydrolyzed leucine gradually filtrates back into the culture medium in a time-, temperature- and methylester substrate concentration-dependent manner. Another portion of Leu-OMe is converted to Leu-Leu dipeptide. With respect to the cellular effects of Leu-OMe treatment ultrastructural studies showed the presence of large vacuoles without significant alteration of cell viability. Increased exocytosis of lysosomal enzymes did not lead to lytic events. Changes in the plasma membrane are indicated by the observation that Leu-OMe treatment causes the loss of the chemotactic activity to formyl-Met-Leu-Phe.
Assuntos
Leucina/análogos & derivados , Leucócitos Mononucleares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Exocitose/efeitos dos fármacos , Técnicas In Vitro , Leucina/análise , Leucina/metabolismo , Leucina/farmacologia , Leucócitos Mononucleares/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Neutrófilos/metabolismo , SuínosRESUMO
Human tonsillar lymphocytes separated on nylon wool and rat macrophages showed different sensitivity to deoxycholate (DOC) treatment at a low (0.24 mM, 0.01%) concentration for 3 h. The T cell-enriched fraction was stimulated more readily by PHA whereas the B-cell enriched fraction lost its adherence and a decrease of chromium binding capacity was observed after the detergent treatment. Rat peritoneal macrophages under the same conditions lost their chromium label and lysozyme content, whereas their adherence and phagocytic capacity decreased dramatically without affecting their binding capacity. Higher sensitivity to the detergent was observed in peritoneal macrophages compared to tonsillar lymphocytes when various DOC concentrations were used. These findings proved that this low concentration DOC treatment, at least in macrophages, touched mainly the adhesive proteins and the dynamics of the membrane and not its receptor-associated properties.
Assuntos
Ácido Desoxicólico/farmacologia , Macrófagos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Líquido Ascítico , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Criança , Pré-Escolar , Radioisótopos de Cromo/metabolismo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Muramidase/análise , Tonsila Palatina , Ratos , Ratos Endogâmicos , Sensibilidade e EspecificidadeRESUMO
The emetine effectively abolished the plasma cell population in the chicken's gland of Harder by day 3 of treatment. The plasma cell content regenerated by day 5 following emetine injection, possibly from a metabolically inactive, resting B cell population which was resistant to the emetine treatment. By day 7 extracellular substance in a very large quantity appeared among the plasma cells and epithelial cells which might represent a hyperactive plasma cell secretion. The changes in the circulating antibodies measured by hemagglutination well-correlated with the plasma cell content in the gland of Harder. The gland of Harder (GH) is an accessory lacrimal gland. Its main function is to lubricate the nictitating membrane and keep the surface of the eyeball wet. The presence and regulatory function of cAMP dependent histone kinase was showed. Since it has been published that in chicken the interstitium of this gland contains a proper amount of plasma cells, this observation called the attention of many investigators to study the role of GH in the immune response. The B cell maturation in the chicken GH has been studied. The surface marker studies have proved that beside the B cells the gland contains functionally adequate number of T cell which exert stimulatory effect on B cells to promote their transformation to plasma cells. In addition to T and B cells small number of macrophages also occur. In the 9 H the number of plasma cells is age dependent. At hatch only a few plasma cells occur in the interstitium of the gland but by 3 weeks of age they become predominant. Different isotypes of immunoglobulins are secreted by these plasma cells.
Assuntos
Emetina/farmacologia , Glândula de Harder/citologia , Plasmócitos/efeitos dos fármacos , Animais , Galinhas , DNA/biossíntese , Células Epiteliais , Epitélio/efeitos dos fármacos , Eritrócitos/metabolismo , Glândula de Harder/efeitos dos fármacos , Imunoglobulinas/biossíntese , Mitose/efeitos dos fármacos , Biossíntese de Proteínas , RNA/biossíntese , Ovinos/imunologiaRESUMO
Protein synthesis of nylon-wool adherent (B) and non-adherent (T) human tonsillar lymphocytes was examined in the presence of three protein fractions precipitated with ammonium sulfate from the medium of the above lymphocytes incubated in vitro for 4 hours. One fraction precipitated with ammonium sulfate at 0--30% saturation was found to inhibit amino acid incorporation into proteins mainly in unseparated cells while the fraction precipitated at 30--70% saturation decreased the rate of protein synthesis in B cells. The fraction precipitated at 70--100% saturation inhibited protein synthesis slightly in T lymphocytes. Protein synthesis in B and T as well as in unseparated cells was also examined in the presence of proteins obtained from the medium of B and T lymphocytes. It is assumed that proteins released by lymphocytes without mitogenic activation in vitro are involved in the mediation of lymphocyte interactions and may be related to lymphokines synthesized and released by activated cells.