RESUMO
The interaction of lac operator DNA with lac repressor (LacI) is a classic example of a genetic regulatory switch. To dissect the role of stoichiometry, subunit association, and effects of DNA length in positioning this switch, we have determined binding isotherms for the interaction of LacI with a high affinity (Osym) operator on linearized plasmid (2500 bp) DNA over a wide range of macromolecular concentrations (10(-14) to 10(-8) M). Binding data were analyzed using a thermodynamic model involving four equilibria: dissociation of tetramers (T) into dimers (D), and binding of operator-containing plasmid DNA (O) to dimers and tetramers to form three distinct complexes, DO, TO, and TO2. Over the range of concentrations of repressor, operator, and salt (0.075 M K+ to 0.40 M K+) investigated, we find no evidence for any significant thermodynamic effect of LacI dimers. Instead, all isotherms can be interpreted in terms of just two equilibria, involving only T and the TO and TO2 complexes. As a reference binding equilibrium, which we propose must approximate the DO binding interaction, we compare the plasmid Osym results with our extensive studies of the binding of a 40 bp Osym DNA fragment to LacI. On this basis, we obtain a lower bound on the LacI dimer-tetramer equilibrium constant and values of the equilibrium constants for formation of TO and TO2 complexes. At a salt concentration of 0.40 M, the Osym plasmid binding data are consistent with a model with two independent and identical binding sites for operator per LacI tetramer, in which the binding to a site on the tetramer is only slightly more favorable than the reference binding interaction. Increasingly large deviations from the independent-site model are observed as the salt concentration is reduced; binding of a second operator to from TO2 becomes strongly disfavored relative to formation of TO at low salt concentrations (0.075 to 0.125 M). In addition, binding of both the first and second plasmid operator DNA molecules to the tetramer becomes increasingly more favorable than the reference binding interaction as [K+] is reduced from 0.40 M to 0.125 M. At 0.075 M K+, however, the strength of binding of the second plasmid operator DNA to the LacI tetramer is dramatically reduced; this interaction is much less favorable than binding the first plasmid operator DNA, and becomes much less favorable than the reference binding interaction. We propose that these differences arise from changes in the nature of the TO and TO2 complexes with decreasing salt concentration. At low salt concentration, we suggest the hypothesis that flanking non-operator sequences bind non-specifically (coulombically) by local wrapping, and that distant regions of non-operator DNA occupy the second operator-binding site by looping. We propose that wrapping stabilizes both 1:1 and 2:1 complexes at low salt concentration, and that looping stabilizes the 1:1 complex but competitively destabilizes the 2:1 TO2 complex at low salt concentration. These effects must play a role in adjusting the stability and structure of the LacI-lac operator repression complex as the cytoplasmic [K+] varies in response to changes in extracellular osmolarity.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Óperon Lac , Regiões Operadoras Genéticas , Plasmídeos/genética , Proteínas Repressoras/metabolismo , Escherichia coli/metabolismo , Repressores Lac , Modelos Químicos , Ligação Proteica , Conformação Proteica , TitulometriaRESUMO
What are the thermodynamic consequences of the stepwise conversion of a highly specific (consensus) protein-DNA interface to one that is nonspecific? How do the magnitudes of key favorable contributions to complex stability (burial of hydrophobic surfaces and reduction of DNA phosphate charge density) change as the DNA sequence of the specific site is detuned? To address these questions we investigated the binding of lac repressor (LacI) to a series of 40 bp fragments carrying symmetric (consensus) and variant operator sequences over a range of temperatures and salt concentrations. Variant DNA sites contained symmetrical single and double base-pair substitutions at positions 4 and/or 5 [sequence: see text] in each 10 bp half site of the symmetric lac operator (Osym). Non-specific interactions were examined using a 40 bp non-operator DNA fragment. Disruption of the consensus interface by a single symmetrical substitution reduces the observed equilibrium association constant (K(obs)) for Osym by three to four orders of magnitude; double symmetrical substitutions approach the six orders in magnitude difference between specific and non-specific binding to a 40 bp fragment. At these adjacent positions in the consensus site, the free energy effects of multiple substitutions are non-additive: the first reduces /deltaG(obs)o/ by 3 to 5 kcal mol(-1), approximately halfway to the non-specific level, whereas the second is less deleterious, reducing /deltaG(obs)o/ by less than 3 kcal mol(-1). Variant-specific dependences of K(obs) on temperature and salt concentration characterize these LacI-operator interactions. In general, binding constants and standard free energies of binding both exhibit characteristic extrema near 290 K. As a consequence, both the enthalpic and entropic contributions to stability of Osym and variant complexes change from positive (i.e. entropy driven) at lower temperatures to negative (i.e. enthalpy driven) at higher temperatures, indicating that the heat capacity change upon binding, deltaC(obs)o, is large and negative. In general, /deltaC(obs)o/ decreases as the specificity and stability of the variant complex decreases. Stabilities of complexes of LacI with Osym and all variant operators are strongly [salt]-dependent. Binding constants for the variant complexes exhibit a power-dependence on [salt] that is larger in magnitude (i.e. more negative) than for Osym, but no obvious trend relates changes in contributions from the polyelectrolyte effect and the observed reductions in stability (delta deltaG(obs)o). These variant-specific thermodynamic signatures provide novel insights into the consequences of converting a consensus interface to a less specific one; such insights are not obtained from comparisons at the level of delta deltaG(obs)o. We propose that this variant-specific behavior arises from a strong effect of operator sequence on the extent of induced conformational changes in the protein (and possibly also in the DNA site) which accompany binding.
Assuntos
Proteínas de Bactérias/metabolismo , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Ligação Competitiva , DNA/química , Proteínas de Ligação a DNA/química , Eletrólitos , Óperon Lac , Repressores Lac , Modelos Químicos , Nucleoproteínas/química , Ligação Proteica , Dobramento de Proteína , Proteínas Repressoras/química , TermodinâmicaRESUMO
In our studies of lac repressor tetramer (T)-lac operator (O) interactions, we observed that the presence of extended regions of non-operator DNA flanking a single lac operator sequence embedded in plasmid DNA produced large and unusual cooperative and anticooperative effects on binding constants (Kobs) and their salt concentration dependences for the formation of 1:1 (TO) and especially 1:2 (TO2) complexes. To explore the origin of this striking behavior we report and analyze binding data on 1:1 (TO) and 1:2 (TO2) complexes between repressor and a single O(sym) operator embedded in 40 bp, 101 bp, and 2514 bp DNA, over very wide ranges of [salt]. We find large interrelated effects of flanking DNA length and [salt] on binding constants (K(TO)obs, K(TO2)obs) and on their [salt]-derivatives, and quantify these effects in terms of the free energy contributions of two wrapping modes, designated local and global. Both local and global wrapping of flanking DNA occur to an increasing extent as [salt] decreases. Global wrapping of plasmid-length DNA is extraordinarily dependent on [salt]. We propose that global wrapping is driven at low salt concentration by the polyelectrolyte effect, and involves a very large number (>/similar 20) of coulombic interactions between DNA phosphates and positively charged groups on lac repressor. Coulombic interactions in the global wrap must involve both the core and the second DNA-binding domain of lac repressor, and result in a complex which is looped by DNA wrapping. The non-coulombic contribution to the free energy of global wrapping is highly unfavorable ( approximately +30-50 kcal mol(-1)), which presumably results from a significant extent of DNA distortion and/or entropic constraints. We propose a structural model for global wrapping, and consider its implications for looping of intervening non-operator DNA in forming a complex between a tetrameric repressor (LacI) and one multi-operator DNA molecule in vivo and in vitro. The existence of DNA wrapping in LacI-DNA interactions motivates the proposal that most if not all DNA binding proteins may have evolved the capability to wrap and thereby organize flanking regions of DNA.
Assuntos
Proteínas de Bactérias/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli , Óperon Lac , Conformação de Ácido Nucleico , Proteínas Repressoras/metabolismo , Sítios de Ligação , Repressores Lac , Modelos Moleculares , Potássio/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , TermodinâmicaRESUMO
When incubated for 1.0 hr with neutrophils isolated from bovine peripheral blood, hydrocortisone (0.05, 0.5, or 5.0 micrograms/ml) had no significant effect on their ability to ingest Staphylococcus aureus, to reduce nitroblue tetrazolium (NBT) or iodinate protein, or to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) against chicken erythrocytes. Random migration was significantly enhanced by the highest concentration of hydrocortisone (HC) but was unaffected by the lower concentrations. Resting bovine monocytes were incubated for 24 hr in medium with or without added hydrocortisone (0.05, 0.5, or 5.0 micrograms/ml). Purified bovine neutrophils were then incubated for 1.0 hr with the resulting monocyte supernatant (MS). The MS from untreated monocytes significantly enhanced neutrophil ADCC but did not affect the other neutrophil functions evaluated. Supernatants from monocytes incubated with 0.5 or 5.0 micrograms/ml hydrocortisone (HC-treated MS) failed to enhance neutrophil ADCC but did enhance neutrophil random migration under agarose. The other parameters of neutrophil function were unaffected. Production of the factors in MS was reduced by inhibition of protein synthesis in monocytes. Their activity was reduced by exposure of MS to proteolytic enzymes, suggesting that the monocyte factors were polypeptides or proteins. Protein synthesis by the neutrophil was not necessary for its response to the monocyte-produced ADCC-enhancing factor but was necessary for its response to the HC-induced monocyte factor. The results suggest that the antiinflammatory effects of glucocorticoids may be partially mediated by factors released by monocytes.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Hidrocortisona/farmacologia , Monócitos/efeitos dos fármacos , Neutrófilos/fisiologia , Proteínas/fisiologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Atividade Bactericida do Sangue , Movimento Celular/efeitos dos fármacos , Temperatura Alta , Humanos , Monocinas , Oxirredução , Puromicina/farmacologiaRESUMO
The activity of four recombinant human cytokines on porcine neutrophils was evaluated. Porcine neutrophils were treated with varying doses of recombinant human tumour necrosis factor-alpha (rHu-TNF), interferon-gamma (rHu-IFN), interleukin-8 (rHu-lL-8), or granulocyte-macrophage colony-stimulating factor (rHu-GM-CSF). The function of treated neutrophils was compared with that of non-treated controls in the following assays: antibody-independent neutrophil cytotoxicity (AINC), antibody-dependent cell-mediated cytotoxicity (ADCC), iodination, Staphylococcus aureus ingestion, cytochrome C reduction, random migration, and chemotaxis. Treatment with rHu-TNF produced significant (P < 0.05) depression of neutrophil random migration (2.5, 25, and 250 ng ml-1 rHu-TNF) and iodination (250 ng ml-1) and a near significant (P = 0.08) depression in ADCC (250 ng ml-1). Treatment with 25,000 U ml-1 of rHu-IFN caused a significant increase in AINC. At lower doses of rHu-IFN, there was a trend (0.05 < P < or = 0.08) toward depression of AINC (250 U ml-1) and ADCC (25 U ml-1) and enhancement of iodination (250 U ml-1). Treatment with 50 ng ml-1 of rHu-IL-8 caused a near significant increase (P = 0.06) in AINC. There were no significant differences noted when porcine neutrophils were treated with rHu-GM-CSF (2.5-2500 U ml-1). No synergism was noted between rHu-TNF and rHu-IFN.
Assuntos
Citocinas/imunologia , Neutrófilos/imunologia , Suínos/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Movimento Celular , Quimiotaxia de Leucócito/imunologia , Grupo dos Citocromos c/metabolismo , Citotoxicidade Imunológica/imunologia , Fagocitose/imunologia , Proteínas Recombinantes/imunologiaRESUMO
The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.
Assuntos
Doenças dos Bovinos/imunologia , Leucose Enzoótica Bovina/imunologia , Vírus da Imunodeficiência Bovina/imunologia , Infecções por Lentivirus/imunologia , Infecções por Lentivirus/veterinária , Vírus da Leucemia Bovina/imunologia , Animais , Bovinos , Citotoxicidade Imunológica , Leucose Enzoótica Bovina/sangue , Leucose Enzoótica Bovina/etiologia , Imunidade Celular , Infecções por Lentivirus/sangue , Estudos Longitudinais , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Neutrófilos/imunologia , Distribuição AleatóriaRESUMO
Salmonella typhimurium infection in swine causes an enterocolitis followed by a persistent carrier state, but little is known about the mechanisms that allow this organism to colonize and persist in host tissues. Neutrophils provide a first line of defense against invading pathogens such as Salmonella typhimurium. The purpose of this study was to evaluate porcine neutrophil function after in vivo exposure to Salmonella and to determine if the immunomodulator, bacillus Calmette Guerin (BCG), exerts any effect on neutrophil function or on the colonization and persistence of S. typhimurium in the pig. Compared to negative controls, neutrophils from pigs exposed to S. typhimurium exhibited significantly decreased iodination, cytochrome-C reduction, antibody-dependent cell-mediated cytotoxicity, random migration, and chemotaxis (P less than or equal to 0.05). Neutrophil bactericidal activity against S. typhimurium was significantly enhanced. Most of the significant differences were noted in the first two days after exposure to Salmonella. Often the functional alterations were biphasic, peaking again 7-10 days after exposure. BCG alone significantly depressed random migration and cytochrome-C reduction in unstimulated neutrophils. The clinical course, colonization pattern, and persistence of Salmonella were similar between pigs receiving BCG and untreated pigs. These data suggest that S. typhimurium infection causes a depression in oxidative metabolism and motility, yet an increase in overall bactericidal activity against S. typhimurium in circulating porcine neutrophils. It also appears that BCG treatment, as reported here, does not enhance resistance of pigs to S. typhimurium colonization or reduce the number of persistent organisms in the porcine ileum.
Assuntos
Vacina BCG/administração & dosagem , Neutrófilos/imunologia , Salmonella typhimurium/imunologia , Doenças dos Suínos/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Atividade Bactericida do Sangue/imunologia , Quimiotaxia de Leucócito/imunologia , Grupo dos Citocromos c/biossíntese , Feminino , Contagem de Leucócitos , Masculino , Neutrófilos/enzimologia , Salmonelose Animal/imunologia , Suínos , Doenças dos Suínos/sangue , Doenças dos Suínos/microbiologiaRESUMO
The brain stem has long lost the designation of "no-man's land." Armed with a detailed knowledge of skull base and parenchymal neuroanatomy, coupled with the advances in intraoperative mapping and monitoring, most intrinsic brain stem cavernous malformations can be resected microsurgically. Success continues to depend on proper patient selection, optimal timing, thorough planning, meticulous technique, and completeness of the resection.
Assuntos
Tronco Encefálico/cirurgia , Cerebelo/cirurgia , Craniotomia/métodos , Hemangioma Cavernoso/cirurgia , Neoplasias Infratentoriais/cirurgia , Microcirurgia/métodos , Humanos , Bulbo/cirurgia , Mesencéfalo/cirurgia , Planejamento de Assistência ao Paciente , Seleção de Pacientes , Ponte/cirurgia , Medição de Risco/métodos , Resultado do TratamentoRESUMO
As a feed additive, ascorbic acid has been shown to have a protective effect against bacterial and viral diseases and to reduce the impact of detrimental stress in chickens. This study examined the effect of ascorbic acid treatment on in vitro heterophil function by examining random migration and phagocytosis and bacterial killing of Staphylococcus aureus. Heterophils were evaluated in broiler chickens ranging from 5 to 16 wk of age, and age differences were seen. Significant increases in bacterial killing were found in heterophils treated with ascorbic acid, and this difference tended to be greater in chickens from 5 to 10.5 wk of age. No significant differences were found in phagocytosis or random migration, but ascorbic acid tended to decrease random migration. The most significant effect on in vitro heterophil function was an increase in bacterial killing.
Assuntos
Artrite Infecciosa/veterinária , Ácido Ascórbico/farmacologia , Granulócitos/fisiologia , Doenças das Aves Domésticas/sangue , Infecções Estafilocócicas/veterinária , Staphylococcus aureus , Animais , Artrite Infecciosa/sangue , Ácido Ascórbico/administração & dosagem , Quimiotaxia de Leucócito/efeitos dos fármacos , Galinhas , Aditivos Alimentares , Granulócitos/efeitos dos fármacos , Granulócitos/microbiologia , Técnicas In Vitro , Fagocitose/efeitos dos fármacos , Projetos Piloto , Doenças das Aves Domésticas/microbiologia , Infecções Estafilocócicas/sangueRESUMO
The present study was designed to determine the effect of recombinant porcine somatotropin (rpST; 5, 15, or 25 mg.pig-1.d-1 for 57 d) on a variety of immune function variables. We observed no significant effect of rpST treatment on the gross pathology of the pigs, histopathology of the immune system organs, total and differential white blood cell counts, lymphocyte blastogenic response to mitogens, or the neutrophil functions of chemotaxis, ingestion, reduction of cytochrome C, iodination, and antibody-dependent cell-mediated cytotoxicity. Those variables that were significantly affected by rpST treatment include a decreased hemoglobin and packed red blood cell volume (at some time points for all three rpST dosages), a decrease in plasma protein level at the 25-mg dose, a small increase in neutrophil random migration (at all three rpST dosages), and a decrease in IgG antibody response to tetanus toxoid at 15 d after immunization (but not at d 8, 22, or 29). Additionally, rpST treatment was associated with a decreased rate of BW gain (at 15-mg dose), increased liver and kidney weights (at all three dosage levels), and an increased incidence of renal tubular cytoplasmic vacuolation of minor severity. There were no observed differences in the overall health of the pigs due to rpST treatment, based on clinical observations as well as determination of antibody titer to, and isolation of, common swine pathogens. Therefore, there was no evidence that the observed influence of rpST treatment on immune function would be clinically relevant.
Assuntos
Hormônio do Crescimento/toxicidade , Sistema Imunitário/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Suínos/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Proteínas Sanguíneas/análise , Peso Corporal/efeitos dos fármacos , Bordetella bronchiseptica/isolamento & purificação , Feminino , Imunidade Celular/efeitos dos fármacos , Contagem de Leucócitos/veterinária , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Tamanho do Órgão/efeitos dos fármacos , Pasteurella multocida/isolamento & purificação , Proteínas Recombinantes/toxicidadeRESUMO
Recombinant human interleukin-2 (rhIL-2) was evaluated for its influence on total and differential WBC counts, lymphocyte blastogenic responsiveness to mitogens, and several measurements of neutrophil function in clinically normal and in dexamethasone-treated cattle. A single dose of rhIL-2 (2.5 X 10(7) U) given SC had no influence on the total or differential WBC count; however, it did cause an inhibition of neutrophil random migration. The other measurements of neutrophil function (Staphylococcus aureus ingestion, cytochrome C reduction, iodination, and antibody-dependent and antibody-independent cell-mediated cytotoxicity) evaluated were not significantly altered. The rhIL-2 treatment was associated with a significant (P less than 0.01) decrease in uptake of [3H]thymidine in unstimulated lymphocytes and a tendency toward enhanced blastogenesis of lymphocytes stimulated with phytohemagglutinin. This enhancement was significant (P less than 0.05) only when the results were expressed as a stimulation index. Lymphocyte responsiveness to concanavalin A and pokeweed mitogen was not significantly influenced by rhIL-2 administration. Dexamethasone (0.04 mg/kg) administered every 24 hours for 3 consecutive days altered the WBC count and several measurements of lymphocyte and neutrophil function. The administration of a single dose of rhIL-2 (2.5 X 10(7) U) 8 hours after the first dose of dexamethasone did not alter the influence of dexamethasone on any of the measurements. These results indicated that rhIL-2 has some biologic activity in cattle, but when used as administered here, did not overcome the influence of dexamethasone on the in vitro measurements of lymphocyte and neutrophil function that were evaluated.
Assuntos
Bovinos/imunologia , Dexametasona/farmacologia , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Animais , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Humanos , Tolerância Imunológica/efeitos dos fármacos , Interleucina-2/administração & dosagem , Contagem de Leucócitos/efeitos dos fármacos , Contagem de Leucócitos/veterinária , Linfócitos/fisiologia , Masculino , Neutrófilos/fisiologia , Distribuição Aleatória , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
Neutrophil function and mononuclear cell proliferative responses to mitogens were determined in healthy cattle and in cattle persistently infected with bovine viral diarrhea (BVD) virus. Uptake of [3H]thymidine by resting and mitogen-stimulated peripheral blood mononuclear cells was significantly lower in cattle persistently infected with BVD virus than in healthy cattle. Neutrophils from cattle persistently infected with BVD virus had significantly impaired capability to ingest Staphylococcus aureus, but were normal in respect to random migration under agarose, cytochrome C reduction, iodination, and antibody-dependent cell-mediated cytotoxicity. Impairment of neutrophil function in cattle persistently infected with BVD virus differs from impairment of neutrophil function reported in healthy cattle mounting an immune response to recent BVD virus infection.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/sangue , Doenças dos Bovinos/sangue , Bovinos/sangue , Ativação Linfocitária , Linfócitos/fisiologia , Neutrófilos/fisiologia , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/fisiopatologia , Feminino , MasculinoRESUMO
OBJECTIVE: To develop a model to study the kinetics and relative amounts of cytokines produced by liver cells during enteric infection. DESIGN: Salmonella enteriditis lipopolysaccharide (LPS)- or live S choleraesuis-stimulated isolated livers from clinically normal pigs and pigs with active acute phase response. ANIMALS: 7- to 14-day-old salmonellosis-free pigs, 4 to 12/group. PROCEDURE: Livers were removed and perfused with oxygenated Krebs-Henseleit solution for 30 minutes and with S choleraesuis or LPS added for 7 minutes. Livers were then perfused with 500 ml of fresh solution in a closed loop procedure for 180 minutes. Perfusate samples were collected for tumor necrosis factor-alpha (TNF alpha) and interleukin 6 (IL-6) bioassays. RESULTS: Tumor necrosis factor-alpha values remained constant during perfusion of normal livers and increased in those exposed to LPS. Interleukin 6 values increased in perfusate from normal livers from 30 to 150 minutes, then decreased. In livers from pigs with an active acute phase response, TNF alpha values were reduced; IL-6 appeared by 2 minutes and decreased after 25 minutes. CONCLUSIONS: Isolated livers could be kept viable for 3 hours, and IL-6 and TNF alpha could be measured by the bioassays used. CLINICAL RELEVANCE: Model can be used for studying and modifying the response of liver cells to infective agents.
Assuntos
Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/imunologia , Salmonella/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos , Bioensaio , Sobrevivência Celular/efeitos dos fármacos , Glucose/análise , Humanos , Técnicas In Vitro , Interleucina-6/análise , Interleucina-6/farmacologia , Cinética , Células L , L-Lactato Desidrogenase , Fígado/citologia , Fígado/efeitos dos fármacos , Camundongos , Perfusão , Potássio/análise , Suínos , Fatores de Tempo , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Twenty Holstein steers subclinically infected with coccidia were allotted to 2 groups of 10 steers each. One group received a diet containing 0.5 mg of decoquinate/kg of body weight. After 25 days on the diet, there was no difference between the groups in lymphocyte blastogenic responsiveness to mitogens; however, there were differences in neutrophil function. Lymphocytes from steers of the decoquinate-fed group had decreased random migration under agarose, enhanced cytochrome C reduction, and enhanced iodination activity. Other measures of neutrophil function evaluated (chemotactic index, Staphylococcus aureus ingestion, and antibody-dependent and -independent cell-mediated cytotoxicity) were not affected. After 30 days of decoquinate feeding, half of the cattle in each group received 5 daily IM injections of dexamethasone (0.04 mg/kg of body weight). The dexamethasone-treated steers from the group that did not have decoquinate in the diet developed clinical coccidiosis, whereas the decoquinate-treated steers remained clinically normal. Lymphocyte and neutrophil function were again evaluated for a 3-day period beginning 4 days after dexamethasone treatment was halted. Neutrophils from the steers that developed clinical coccidiosis after dexamethasone administration had significantly (P less than 0.05) inhibited random migration under agarose, cytochrome C reduction, and iodination activity, but significantly (P less than 0.01) enhanced S aureus ingestion. The feeding of decoquinate prevented the inhibition of neutrophil cytochrome C reduction and lessened the inhibition of neutrophil iodination in the dexamethasone-treated group. Dexamethasone treatment was associated with an inhibition of lymphocyte blastogenic responsiveness to phytohemagglutinin in principals as well as controls.
Assuntos
Doenças dos Bovinos/imunologia , Coccidiose/veterinária , Decoquinato/farmacologia , Dexametasona/farmacologia , Hidroxiquinolinas/farmacologia , Neutrófilos/fisiologia , Animais , Bovinos , Quimiotaxia de Leucócito/efeitos dos fármacos , Coccidiose/imunologia , Contagem de Leucócitos/veterinária , Ativação Linfocitária/efeitos dos fármacos , Masculino , Neutrófilos/imunologia , FagocitoseRESUMO
Cattle persistently infected with bovine viral diarrhea (BVD) virus have decreased neutrophil and lymphocyte functions. We reevaluated these functions and further characterized the inhibition of persistent BVD virus infection in neutrophils, using sensitive kinetic assays. In addition, the influence of in vitro incubation of neutrophils with recombinant bovine interferon gamma (rBoIFN gamma) and in vitro incubation of lymphocytes with recombinant bovine interleukin-2 was evaluated. Significant (P less than 0.05) decrease in random migration under agarose, Staphylococcus aureus ingestion, cytochrome-C reduction, iodination, antibody-independent cell-mediated cytotoxicity, oxidant production, and cytoplasmic calcium flux were observed in neutrophils from cattle persistently infected with BVD virus, compared with noninfected control cattle. Incubation of neutrophils from noninfected controls with rBoIFN gamma significantly (P less than 0.05) decreased random migration under agarose, cytochrome-C reduction, and cytoplasmic calcium flux. Neutrophils from cattle persistently infected with BVD virus also had decreased random migration under agarose after incubation with rBoIFN gamma; in addition, antibody-independent cell-mediated cytotoxicity, elastase release, and cytoplasmic calcium flux were significantly enhanced. The rBoIFN gamma induced significantly (P less than 0.05) different effects on chemotaxis, cytochrome-C reduction, iodination, and cytoplasmic calcium flux of neutrophils from infected and control cattle. The rBoIFN gamma was more effective at improving the function of neutrophils from cattle persistently infected with BVD virus, compared with neutrophils from controls. Lymphocytes from infected cattle had decreased blastogenesis in response to phytohemagglutinin, concanavalin A, and pokeweed mitogen.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Neutrófilos/imunologia , Complicações Infecciosas na Gravidez/imunologia , Animais , Cálcio/metabolismo , Bovinos , Inibição de Migração Celular , Células Cultivadas , Quimiotaxia de Leucócito , Grupo dos Citocromos c/metabolismo , Citotoxicidade Imunológica , Feminino , Interferon gama/farmacologia , Interleucina-2/farmacologia , Cinética , Contagem de Leucócitos/veterinária , Ativação Linfocitária , Masculino , Oxirredução , Elastase Pancreática/metabolismo , Gravidez , Proteínas Recombinantes/farmacologia , Staphylococcus aureus/imunologiaRESUMO
Compared with neutrophils from healthy dogs, neutrophils from 2 dogs with primary ciliary dyskinesia had increased distance of random migration, but fewer of the neutrophils migrated. The affected dogs had an increase in the numbers of Staphylococcus aureus phagocytized. Lymphocyte blastogenesis in the affected dogs in response to standard mitogens was considered to be normal.
Assuntos
Transtornos da Motilidade Ciliar/veterinária , Doenças do Cão/sangue , Neutrófilos/fisiologia , Sistema Respiratório/patologia , Animais , Movimento Celular , Cílios/patologia , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/sangue , Transtornos da Motilidade Ciliar/imunologia , Transtornos da Motilidade Ciliar/patologia , Doenças do Cão/imunologia , Doenças do Cão/patologia , Cães , Feminino , Ativação Linfocitária , Microscopia Eletrônica , Neutrófilos/imunologia , Neutrófilos/microbiologia , Fagocitose , Sistema Respiratório/ultraestrutura , Staphylococcus aureus/imunologiaRESUMO
Three dosages (0.1, 0.5, and 2.5 mg/animal, subcutaneously), of recombinant bovine interferon-gamma (rBoIFN-gamma) were evaluated for their in vivo influence on neutrophil function and lymphocyte blastogenesis in cattle. The optimal of the three dosages tested (0.5 mg/animal or 1.1 x 10(6) U/animal) was then evaluated for its influence on neutrophils and lymphocytes in both normal and dexamethasone-treated cattle. One animal, which received 2.5 mg of rBoIFN-gamma, died by 24 h after administration due to acute diffuse interstitial pneumonia with interlobular edema and emphysema. The two highest dosages used caused fever at 24 h and the highest dosage caused a decrease in lymphocyte blastogenesis at 24 h after administration. The influence of rBoIFN-gamma on neutrophil function was dose dependent and depended on the baseline values for neutrophil function. Random migration by neutrophils was consistently inhibited in animals that received 0.5 mg or more of rBoIFN-gamma. Staphylococcus aureus ingestion and antibody-dependent cell-mediated cytotoxicity by neutrophils was enhanced by rBoIFN-gamma treatment in both dexamethasone-treated cattle and in nondexamethasone-treated cattle, which had relatively low values for these parameters before treatment. Iodination by neutrophils was also enhanced by rBoIFN-gamma when either a suboptimal concentration of neutrophil stimulant was used or when the cattle were treated with dexamethasone. In summary, the rBoIFN-gamma had greater immunomodulator activity in immunosuppressed than in normal cattle. The in vivo influence of rBoIFN-gamma therefore depends on the physiologic status of the animal.
Assuntos
Adjuvantes Imunológicos/farmacologia , Dexametasona/farmacologia , Interferon gama/farmacologia , Animais , Temperatura Corporal/efeitos dos fármacos , Bovinos , Contagem de Leucócitos/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Masculino , Neutrófilos/efeitos dos fármacos , Proteínas RecombinantesRESUMO
Transcription of the chicken ovalbumin gene is induced both in vivo and in vitro by four classes of steroid hormones. Recent experiments identified a steroid-dependent regulatory element (SDRE) in the 5'-flanking region of the ovalbumin gene between -900 and -521. To characterize the regulatory properties of the SDRE more precisely, additional mutations were created in this region, and fusion genes prepared by linking the ovalbumin 5'-flanking region and promoter to the chloramphenicol acetyltransferase structural gene. When the ovalbumin-chloramphenicol acetyltransferase fusion genes were transfected into steroid-responsive primary oviduct cells, mutants lacking sequences between -900 and -732 were no longer responsive to estrogen, corticosterone, progesterone, or dihydrotestosterone. The SDRE did not confer steroid-dependent expression on the heterologous thymidine kinase promoter by itself but did in conjunction with the negative regulatory element identified between -350 and -100. This suggests that the two elements act as a single functional entity and that the SDRE is not behaving as a typical steroid response element. Gel shift analyses revealed that two SDRE.protein complexes were formed when nuclear protein extracts were derived from estrogen-treated chicken oviduct but that only one complex was formed with extracts from estrogen-withdrawn oviduct or from other tissues. Neither an estrogen response element oligomer nor a glucocorticoid/progesterone response element oligomer competed for either of the DNA.protein complexes. Partially purified progesterone receptor also did not bind to the SDRE. These data indicate that induction of the ovalbumin gene by steroid hormones requires complex interactions involving both the SDRE and the negative regulatory element.
Assuntos
Corticosterona/farmacologia , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reguladores , Genes/efeitos dos fármacos , Ovalbumina/genética , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Galinhas , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Sondas de Oligonucleotídeos , Oviductos/metabolismo , Plasmídeos , TransfecçãoRESUMO
The objective of this work was to evaluate a proprietary ultrafiltered bovine whey product for its in vitro influence on the function of neutrophils from normal and dexamethasone-treated cattle, and for its in vivo influence on neutrophil function in periparturient dairy cows. The ultrafiltered bovine whey was produced by hyperimmunizing cows to various bacterial pathogens by intramammary injection, collecting and pooling the colostrum and milk for the first 3 wk after parturition, then separating and processing the whey through a filter with a nominal molecular mass cut off of 10,000 Da. In vitro treatment of neutrophils from normal calves with ultrafiltered bovine whey significantly increased neutrophil random migration, cytochrome C reduction, iodination activity, antibody-dependent cell-mediated cytotoxicity, and antibody-independent cell-mediated cytotoxicity. Cytochrome C reduction was the only neutrophil function parameter significantly enhanced by the in vitro treatment of neutrophils from dexamethasone-treated cattle with ultrafiltered bovine whey. In vivo treatment of periparturient cows with ultrafiltered bovine whey did not alter the total or differential leukocyte counts in the animals but did significantly increase the total erythrocyte counts. In vivo treatment with ultrafiltered bovine whey also significantly increased neutrophil iodination activity in the periparturient cows. Neutrophil iodination activity (a measure of the myeloperoxidase/hydrogen peroxide/halide antibacterial system) is a very potent bactericidal mechanism of neutrophils and has previously been shown to be suppressed in periparturient cows.