RESUMO
This study aimed to evaluate the possibility of reducing boar taint in boars (Piétrain×Hybrid) by addition of different feed ingredients (raw potato starch (RPS) 10%, raw potato starch 10%+wheat bran 5% (RPS+WB), lupins 10%, inulin 5%, clinoptilolite 1%) to a standard diet over a period of 4-6 weeks before slaughter. Control boars (CBOAR) as well as barrows were fed the standard diet. Efficacy of the different feed ingredients was evaluated by different boar taint detection methods: hot iron method, consumer panel, expert panel and laboratory analysis. According to all detection methods, clear differences were noticeable between boars and barrows. No differences in boar taint incidence were found between the boars on the different dietary treatments as assessed by consumers, experts, hot iron method or the concentration of skatole in fat. A significant effect on indole level was found, but no further differentiation could be made. The concentration of backfat androstenone was significantly higher for the inulin and control boar group compared to the lupin group. In conclusion, none of the feeding strategies tested in this study reduced boar taint in boars at the given percentages.
RESUMO
Leukemic cells exert immunosuppressive effects that interfere with dendritic cell (DC) function and hamper effective antileukemic immune responses. Here, we sought to enhance the immunogenicity of leukemic cells by loading them with the double-stranded (ds) RNA Toll-like receptor 3 (TLR3) ligand polyriboinosinic polyribocytidylic acid (poly(I:C)), mimicking viral infection of the tumor cells. Given the responsiveness of DC to TLR ligands, we hypothesized that the uptake of poly(I:C)-loaded leukemic cells by immature DC (iDC) would lead to DC activation. Primary acute myeloid leukemia (AML) cells and AML cell lines markedly responded to poly(I:C) electroporation by apoptosis, upregulation of TLR3 expression, enhanced expression of major histocompatibility complex (MHC) and costimulatory molecules and by production of type I interferons (IFN). Upon phagocytosis of poly(I:C)-electroporated AML cells, DC maturation and activation were induced as judged by an increased expression of MHC and costimulatory molecules, production of proinflammatory cytokines and an increase of T helper 1 (T(H)1)-polarizing capacity. These immune effects were suboptimal when AML cells were passively pulsed with poly(I:C), indicating the superiority of poly(I:C) transfection over pulsing. Our results demonstrate that poly(I:C) electroporation is a promising strategy to increase the immunogenicity of AML cells and to convert iDC into activated mature DC following the phagocytosis of AML cells.
Assuntos
Células Dendríticas/imunologia , Leucemia Mieloide/genética , RNA de Cadeia Dupla/genética , Linfócitos T/imunologia , Receptor 3 Toll-Like/metabolismo , Transfecção , Doença Aguda , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Eletroporação , Citometria de Fluxo , Humanos , Interferon Tipo I/imunologia , Interferon gama/imunologia , Leucemia Mieloide/imunologia , Leucemia Mieloide/patologia , Ativação Linfocitária , Poli I-C/metabolismo , Células Th1/imunologia , Receptor 3 Toll-Like/genéticaRESUMO
The advent of new cell-based immunotherapies for leukemia offers treatment possibilities for certain leukemia subgroups. The wider acceptability of these new technologies in clinical practice will depend on its impact on survival and costs. Due to the small patient groups who have received it, these aspects have remained understudied. This non-randomized single-center study evaluated medical costs and survival for acute myeloid leukemia between 2005 and 2010 in 50 patients: patients treated with induction and consolidation chemotherapy (ICT) alone; patients treated with ICT plus allogeneic hematopoietic stem cell transplantation (HCT), which is the current preferred post-remission therapy in patients with intermediate- and poor-risk AML with few co-morbidities, and patients treated with ICT plus immunotherapy using autologous dendritic cells (DC) engineered to express the Wilms' tumor protein (WT1). Total costs including post- consolidation costs on medical care at the hematology ward and outpatient clinic, pharmaceutical prescriptions, intensive care ward, laboratory tests and medical imaging were analyzed. Survival was markedly better in HCT and DC. HCT and DC were more costly than ICT. The median total costs for HCT and DC were similar. These results need to be confirmed to enable more thorough cost-effectiveness analyses, based on observations from multicenter, randomized clinical trials and preferably using quality-adjusted life-years as an outcome measure.
Assuntos
Custos de Cuidados de Saúde , Leucemia Mieloide Aguda/economia , Leucemia Mieloide Aguda/terapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bélgica , Quimioterapia de Consolidação/economia , Análise Custo-Benefício , Transplante de Células-Tronco Hematopoéticas/economia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imunoterapia/economia , Quimioterapia de Indução/economia , Leucemia Mieloide Aguda/mortalidade , Pessoa de Meia-Idade , Taxa de Sobrevida , Transplante Homólogo , Adulto JovemRESUMO
Electroporation of mRNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. However, many more cell types and applications might benefit from an efficient mRNA-based gene transfer method. In this study, we investigated the potential of mRNA-based gene transfer to induce short-term transgene expression in adult stem cells and activated T cells, based on electroporation with mRNA encoding the enhanced green fluorescent protein. The results show efficient transgene expression in CD34-positive hematopoietic progenitor cells (35%), in in vitro cultured mesenchymal cells (90%) and in PHA-stimulated T cells (50%). Next to presentation of gene transfer results, potential applications of mRNA-based gene transfer in stem cells and T cells are discussed.
Assuntos
Eletroporação/normas , Técnicas de Transferência de Genes/normas , Células-Tronco Hematopoéticas/metabolismo , RNA Mensageiro/genética , Linfócitos T/metabolismo , Transgenes/fisiologia , Adulto , Antígenos CD34/metabolismo , Herpesvirus Humano 8/genética , Humanos , Ativação Linfocitária , Mesoderma/metabolismo , Fito-Hemaglutininas/farmacologiaRESUMO
We investigated the cell kinetic effects of retinoic acid (RA) and the functional interaction between RA and TGF-beta on normal human bone marrow progenitor cells (CD34+). Cell cycle progression throughout the first three consecutive cell cycles and alterations in cell kinetic responses were measured using the BrdU-Hoechst quenching technique. RA stimulates the IL-3-induced growth by additionally recruiting quiescent stem and progenitor cells out of the G0/G1-phase and by increasing the cell cycle traverse rate. In contrast, TGF-beta addition resulted in a significant decrease in the number of proliferating cells. Simultaneous addition of RA and TGF-beta resulted in a stronger inhibition compared to addition of TGF-beta alone. Preincubation experiments further showed that RA is capable of sensitizing the progenitors to the inhibitory action of TGF-beta: the inhibitory effect of TGF-beta was significantly increased when cells were pretreated with RA. These data show that, in combination with IL-3, RA additionally stimulates quiescent bone marrow progenitors in a simultaneous way, and that it increases sensitivity of the progenitors to the inhibitory action of TGF-beta. The combination of RA and TGF-beta on normal and leukemic hematopoiesis has to be further investigated, since this combination may possibly provide additional therapeutic benefit.
Assuntos
Antineoplásicos/farmacologia , Células da Medula Óssea , Medula Óssea/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia , Antígenos CD34/análise , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Interações Medicamentosas , Etídio , Citometria de Fluxo/métodos , Fase G1/efeitos dos fármacos , Humanos , Interleucina-3/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Sensibilidade e EspecificidadeRESUMO
Genetically modified dendritic cells (DC) are increasingly used in vitro to activate cytotoxic T lymphocyte (CTL) immune responses. Because T cell activation protocols consist of multiple restimulation cycles of peripheral blood lymphocytes with antigen-loaded mature DC, continuous generation of DC is needed throughout the experiment. Therefore, cryopreservation of DC loaded with antigen is a valuable alternative for weekly generation and modification of DC. Recently, we described an antigen loading method for DC based on electroporation of defined tumor antigen mRNA. In this study, we demonstrate that mRNA-electroporated DC can efficiently be prepared for cryopreservation. Using an optimized maturation and freezing protocol after mRNA electroporation, we obtained high transgene-expressing viable mature DC. In addition, we showed that these modified cryopreserved DC retain stimulatory capacity in an influenza model system. Therefore, cryopreservation of mature mRNA-electroporated DC is a useful method for continuous availability of antigen-loaded DC throughout T cell activation experiments.
Assuntos
Criopreservação , Células Dendríticas , Técnicas de Transferência de Genes , RNA Mensageiro , Apresentação de Antígeno , Células Dendríticas/imunologia , Eletroporação , Humanos , Imunoterapia , Células K562 , Ativação Linfocitária , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Linfócitos T/imunologiaRESUMO
In this study, analogues of olomoucine, a previously described plant cytokinin analogue with cyclin-dependent kinase (CDK) inhibitory activity, were investigated for effect on CDK1 and CDK2 and for effect on cell proliferation. Eight new compounds exhibit stronger inhibitory activity on CDK1 and CDK2 and on cell proliferation than olomoucine. Some active compounds showed low inhibition of proliferation of normal myeloid growth. Improvement of inhibitory activity of known compounds with a C6-benzylamino group was brought about by substitution with one hydroxyl. Also, new C2 substituents associated with inhibitory activity on CDK and on cell proliferation are described. There was a significant correlation between effect on CDK and antiproliferative effect on the KG1 and Molt3 cell lines and on primary human lymphocytes, strongly suggesting that at least part of the antiproliferative effect of cytokinin analogues was due to inhibition of CDK activity. Cytokinin analogues induced apoptosis in a time- and concentration-dependent manner and changes in cell cycle distribution. The antiproliferative and pro-apoptotic effects of plant cytokinin analogues suggest that they are a new class of cytostatic agents and that they may find an application in the chemotherapy of cancer.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Citocininas/farmacologia , Inibidores Enzimáticos/farmacologia , Leucemia/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Purinas/farmacologia , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , DNA de Neoplasias/efeitos dos fármacos , Relação Dose-Resposta a Droga , Cinetina , Leucemia/enzimologia , Plantas , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
Important functional differences exist between primitive CD34++ CD38- hematopoietic progenitor cells derived from human fetal liver (FL) and adult bone marrow (ABM). FL progenitors are known to have higher proliferative capacities and lower cytokine requirements than their ABM counterparts. In this study, we isolated FL and ABM CD34++ CD38- cells and used a two-stage culture system to investigate the effects of transforming growth factor-beta (TGF-beta) and blocking anti-TGF-beta antibodies (anti-TGF-beta) on these cells. First, we demonstrate that FL progenitors are significantly less sensitive to the inhibitory effects of TGF-beta than ABM cells. Second, whereas ABM cells are significantly stimulated by anti-TGF-beta, only very limited effects are seen on FL cells. Third, we show that the effect of anti-TGF-beta is mainly situated at the level of the initial cell cycles of very primitive progenitor cells with a high proliferation potential. Fourth, we demonstrate that blocking the effects of endogenous TGF-beta reduces the growth factor requirements of ABM cells in order to proliferate and differentiate. Based on these data, we hypothesize that at least part of the functional differences that exist between adult and fetal stem cells can be accounted for by a developmental different responsiveness to TGF-beta.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Adulto , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Feto/citologia , Feto/fisiologia , Células-Tronco Hematopoéticas/citologia , Humanos , GravidezRESUMO
The immediate cell kinetic response of highly purified human bone marrow progenitor cells (CD34+ sorted fraction) to the inhibitory effects of transforming growth factor-beta (TGF-beta) was studied using the BrdU-Hoechst flow-cytometric technique. The progenitor cells were stimulated with either interleukin-3 (IL-3) alone or with IL-3 in combination with IL-1, stem cell factor (SCF), or IL-6, and the inhibitory action of TGF-beta was evaluated in each phase of the first three consecutive cell cycles. Semisolid methylcellulose cultures were also performed to compare these initial events to the effects observed after 7, 14, and 21 days of incubation. Within the CD34+ compartment, the progenitor cells can be discriminated on a functional basis, i.e., in terms of TGF-beta sensitivity. Very primitive progenitors, recruited out of the G0 phase by IL-3 plus an early-acting factor (IL-1, SCF) are, upon addition of TGF-beta, arrested specifically in the G1 phase of the second cell cycle. In the clonogenic assays, the increased colony formation due to IL-1 or SCF was completely abolished by the counteracting effect of TGF-beta that diminished colony output back to the level of TGF-beta-plus-IL-3 supplemented colony growth. Addition of TGF-beta to CD34+ progenitors responding to IL-3 alone resulted in an overall retardation, but without an apparent specific accumulation of cells in any of the cell cycles. Finally, within the CD34+ compartment, there exists a subset of IL-3-responsive, but TGF-beta-insensitive, progenitor cells that were, upon addition of TGF-beta, not arrested at all. In conclusion, our results demonstrate that TGF-beta exerts different cell kinetic effects on CD34+ progenitor cell growth depending on the applied stimulus.
Assuntos
Antígenos CD/análise , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/citologia , Interleucina-1/farmacologia , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Antígenos CD34 , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Interações Medicamentosas , Citometria de Fluxo , Fluorescência , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Fator de Células-TroncoRESUMO
We investigated the effects of transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) on very primitive CD34++CD38- and on more mature CD34++CD38+ human hematopoietic progenitor cells by means of a two stage pre-colony-forming cell (pre-CFC) assay. The first (liquid) stage of this assay allows evaluation of the effects of TGF-beta and MIP-1 alpha on the "primary" proliferation of the progenitors under study and on the generation of "secondary" colony-forming cells (CFC, cells for which a second stage semisolid clonogenic assay was used as a read-out). TGF-beta inhibited the proliferation and CFC generation of CD34++CD38- and CD34+CD38+ cells, showing the strongest inhibitory activity on CD34++CD38- cells. MIP-1 alpha exerted a weaker inhibitory activity on CD34+2CD38- cells, whereas it enhanced the primary proliferation of CD34+CD38+ cells and generation of secondary CFC in this subpopulation. Thus, TGF-beta, and MIP-1 alpha both inhibit very primitive CD34+2)CD38- cells, but they are not equally potent. The effects of TGF-beta and MIP-1 alpha on more mature progenitor cells are more complex. Our results and data from the literature indicate that, as progenitor cells mature, they reach a "pivotal point" at a certain stage in their differentiation pathway, depending on the inhibitor, where they are no longer inhibited or where they may even be stimulated by the former inhibitor to proliferate.
Assuntos
Antígenos CD34/análise , Antígenos CD , Antígenos de Diferenciação/análise , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Proteínas Inflamatórias de Macrófagos/farmacologia , N-Glicosil Hidrolases/análise , Fator de Crescimento Transformador beta/farmacologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL4 , Humanos , Glicoproteínas de MembranaRESUMO
CD34++ CD38- and CD34+ CD38+ hematopoietic progenitor cells (HPCs) from human fetal liver (FL), cord blood (CB), and adult bone marrow (ABM) were isolated and investigated for their growth characteristics, cytokine requirements and response to two modulators of early hematopoiesis, interferon (IFN)-gamma and macrophage inflammatory protein (MIP)-1alpha. We observed first that a significantly lower percentage of CD34++ cells were CD38- in ABM than in FL and CB. Second, the functional differences between CD34++ CD38- and CD34+ CD38+ cells were less pronounced in FL and CB than in their ABM counterparts. Third, an inverse correlation was found between growth factor response and the ontogenic age of HPCs, and a direct correlation was noted between cytokine requirements and the ontogenic age of HPCs. Fourth, spontaneous colony formation in a classic semisolid culture system was reproducibly obtained only in the ontogenically earliest cells, that is, in FL but not in CB and ABM, in which no such spontaneous colony formation was observed. Fifth, the modulatory effects of IFN-gamma and MIP-1alpha were qualitatively different depending on the ontogenic age of the progenitor source: whereas IFN-gamma was only a selective inhibitor of primitive CD34++ CD38- ABM progenitor cells, it inhibited both CD34++ CD38- and CD34+ CD38+ FL and CB cells to the same extent. In contrast to the effects of MIP-1alpha on ABM, we could not find any consistently stimulatory or inhibitory effects on FL and CB progenitors. In conclusion, important functional and biologic differences exist between FL, CB, and ABM progenitor cells, and these differences could have major implications for the use of these cell populations in preparative protocols of ex vivo expansion, transplantation strategies, or gene transfer experiments.
Assuntos
Antígenos CD/sangue , Medula Óssea/imunologia , Citocinas/farmacologia , Sangue Fetal/imunologia , Células-Tronco Hematopoéticas/imunologia , Fígado/imunologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adulto , Antígenos CD34/sangue , Antígenos de Diferenciação/sangue , Medula Óssea/embriologia , Divisão Celular/imunologia , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , Ensaio de Unidades Formadoras de Colônias , Desenvolvimento Embrionário e Fetal/fisiologia , Humanos , Interferon gama/farmacologia , Fígado/embriologia , Proteínas Inflamatórias de Macrófagos/farmacologia , Glicoproteínas de Membrana , NAD+ Nucleosidase/sangueRESUMO
1 The influence of rhein anthrone on the peristaltic reflex was studied with a modified Trendelenburg technique in a range from 10(-8) M to 4 x 10(-5) M, on a normal and reversed guinea-pig ileum segment. Rhein anthrone had no significant effects on longitudinal muscle tension, intraluminal pressure or volume displacement when tested on the normal segment in doses up to 10(-5) M. When applied to the mucosal side (reversed segment), rhein anthrone produced a dose-dependent increase of longitudinal muscle tension (significant from 10(-7) M), of intraluminal pressure (significant from 3 x 10(-6) M) and of volume displacement (significant from 10(-7) M). The data show that rhein anthrone possesses in vitro activity which is dependent on contact with the mucosa. 2 The action of rhein anthrone on the reversed segment was inhibited by BW755C (a dual inhibitor of cyclo-oxygenase and lipoxygenase), by indomethacin and by SC19220 (an antagonist of prostaglandin E2 (PGE2) and PGF2 alpha). The effects remaining on longitudinal muscle tension, intraluminal pressure and volume displacement, calculated as percentage (mean +/- s.e.mean) of the initial value, were respectively: 13 +/- 8; 23 +/- 13; 112 +/- 5 for BW755C; 66 +/- 19; 51 +/- 8; 53 +/- 8 for indomethacin and 27 +/- 12; 13 +/- 7; 50 +/- 5 for SC19220. It is concluded that arachidonic acid metabolites, especially PGE2 and PGF2 alpha are involved in the effects of rhein anthrone on the reversed segment.
Assuntos
Antracenos/farmacologia , Íleo/efeitos dos fármacos , Peristaltismo/efeitos dos fármacos , Prostaglandinas/fisiologia , 4,5-Di-Hidro-1-(3-(Trifluormetil)Fenil)-1H-Pirazol-3-Amina/farmacologia , Animais , Antraquinonas/farmacologia , Ácido Dibenzo(b,f)(1,4)oxazepina-10(11H)-carboxílico, 8-cloro-, 2-acetilidrazida/farmacologia , Feminino , Cobaias , Íleo/fisiologia , Técnicas In Vitro , Indometacina/farmacologia , MasculinoRESUMO
This study shows that rhein anthrone has no laxative potency in germ-free rats because after intracaecal administration of a dose of 50 mg/kg the large intestine transit exceeded 240 min. The time course of the laxative potency of rhein anthrone injected intracaecally was evaluated after peroral inoculation of germ-free rats with the caecal contents of conventional rats. Large intestine transit was measured at consecutive periods, on days 0, 1, 2, 3 and 5 after peroral inoculation. It appeared that 1 day after peroral inoculation the laxative potency of rhein anthrone was already established (large intestinal transit < 10 min) and laxation remained on the following days (days 2, 3 and 5). We concluded that rhein anthrone is inactive in germ-free rats and acquires laxative potency after peroral inoculation of germ-free rats with caecal contents of conventional rats.
Assuntos
Antracenos/farmacologia , Catárticos/farmacologia , Trânsito Gastrointestinal/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Vida Livre de Germes , Intestino Grosso/efeitos dos fármacos , Intestino Grosso/microbiologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Ratos WistarRESUMO
The present study was undertaken to investigate the role of prostaglandins in the shortening of transit time observed after intraduodenal administration of rhein anthrone and rhein. After intraduodenal administration of rhein anthrone (0.5-10 mg/rat), a dose-dependent acceleration of small intestinal transit was observed. The effect for rhein (1-10 mg/rat) was far less pronounced. In the same test conditions, analysis of small intestinal tissue revealed a significant increase of prostaglandin E2 (PGE2), reaching its maximum value 30 min after administration of rhein anthrone. The increase in PGE2 found 30 min after administration of rhein was not significant. The effects provoked by rhein anthrone could be largely prevented by pretreatment of the animals with indomethacin (1-3 mg rat) or cortisol (10 mg/rat). It is concluded that prostaglandins play an important role in the acceleration of the transit provoked in rats by rhein anthrone.
Assuntos
Antracenos/farmacologia , Antraquinonas/farmacologia , Dinoprostona/metabolismo , Trânsito Gastrointestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Animais , Catárticos/farmacologia , Relação Dose-Resposta a Droga , Intestino Delgado/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Extrato de Senna , Senosídeos , Fatores de TempoRESUMO
The effect of rhein and rhein anthrone on the transit and the transport of water and electrolytes in the large intestine was investigated in germ-free rats. After intracaecal administration, neither of the two compounds was found to accelerate the transit of a colour marker through the large intestine. Both drugs reduced the net absorption of sodium and chloride in the colon and enhanced net potassium secretion. Net water absorption was decreased by rhein and even reversed into net secretion by rhein anthrone. Our results show that the secretagogue activity of the compounds is not sufficient to induce laxation in germ-free rats. Furthermore rhein and rhein anthrone had no laxative properties under our experimental conditions.
Assuntos
Antracenos/farmacologia , Antraquinonas/farmacologia , Água Corporal/metabolismo , Trânsito Gastrointestinal/efeitos dos fármacos , Intestino Grosso/efeitos dos fármacos , Animais , Eletrólitos/metabolismo , Vida Livre de Germes , Absorção Intestinal/efeitos dos fármacos , Intestino Grosso/metabolismo , Masculino , RatosRESUMO
Changes in the number, maturity and function of neutrophils, concomitant changes in plasma concentrations of hormones and metabolites, and the increased susceptibility of cows to infectious diseases around parturition, led us to investigate the effect of beta-hydroxybutyric acid (BHBA), acetoacetic acid (AcAc), hydrocortisone-21-acetate (HCAc) and bovine pregnancy-associated glycoprotein (bPAG) on the proliferation of bovine bone marrow progenitor cells in methylcellulose in vitro cultures. Myeloid progenitors were stimulated with concanavalin A-stimulated leukocyte conditioned medium (LCM) and erythroid progenitors with erythropoietin in the presence of hemin. Erythroid and myeloid colonies were scored after five and seven days, respectively. BHBA and AcAc induced inhibitory effects on the proliferation of bovine bone marrow cells at concentrations of 1.0, 2.5, and 5.0 mM. HCAc significantly inhibited growth of progenitors at concentrations of 10, 20, 50, and 100 ng/ml, and bPAG at concentrations of 2400 and 3000 ng/ml. The results of this study suggest that in the cow high concentrations of BHBA, AcAc, HCAc and bPAG, which can be reached in the circulation around calving, could alter the number of circulating neutrophils after parturition. This phenomenon might contribute to the increased susceptibility of dairy cows to environmental mastitis.
Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Acetoacetatos/farmacologia , Ácido Aspártico Endopeptidases/farmacologia , Medula Óssea/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hidrocortisona/análogos & derivados , Proteínas da Gravidez/farmacologia , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias/veterinária , Concanavalina A , Feminino , Hemina , Hidrocortisona/farmacologia , Ativação Linfocitária/efeitos dos fármacos , GravidezRESUMO
In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.
Assuntos
Células da Medula Óssea/fisiologia , Técnicas de Cultura de Células/métodos , Células Precursoras Eritroides/fisiologia , Animais , Bovinos , Sobrevivência Celular , Meios de Cultura , Leucócitos , MetilceluloseRESUMO
The defence against infection in high-yielding dairy cows is correlated with the number and function of circulating neutrophils and depends on their production in bone marrow. Therefore, the DNA content of isolated bone marrow cell suspensions from 7 calves, 7 cows and 14 humans was assayed by flow cytometry. Bovine sternal bone marrow samples were collected within 30 min of death, and human marrow samples were collected by sternal puncture and aspiration. Mononucleated cells were isolated by gradient centrifugation. In the bone marrow samples from calves and cows, 35 +/- 2.6% and 31.8 +/- 1.5% of the isolated bone marrow cells respectively were in the S/G2/M-phase. The difference between calves and cows was not significant. In the human samples, only 12 +/- 0.8% of the cells were in the S/G2/M-phase. A significant (P < 0.001) difference was observed between the two species. These results indicated that the proliferative, in activity of haematopoietic cells is significantly higher in cattle than in humans.
Assuntos
Medula Óssea/fisiologia , Bovinos/fisiologia , DNA/análise , Células-Tronco Hematopoéticas/fisiologia , Animais , Medula Óssea/química , Centrifugação com Gradiente de Concentração/veterinária , Corantes/química , Feminino , Citometria de Fluxo/veterinária , Células-Tronco Hematopoéticas/química , Humanos , Propídio/químicaRESUMO
Three potential early-age predictors of which boars are likely to develop boar taint (testes volume, skin lesions and dirtiness) were measured on 102 boars every fortnight from 10 weeks of age until slaughter. These predictors were correlated with the level of boar taint according to the hot iron method and the concentrations of skatole and androstenone as determined by chemical analysis. The chance of no/low boar taint according to the hot iron method decreased with higher testes volume (weeks 22 and 24) and increased with skin lesion score (weeks 12, 16 and 18). For the concentrations of androstenone and skatole, the strongest correlation was found with testes volume in week 12. Skin lesions in week 16 were negatively correlated with skatole levels. Dirtiness was negatively correlated with skatole concentrations (week 18) but positively correlated with androstenone concentrations (weeks 20 and 22). Testes volume has the greatest potential for predicting the likelihood of developing boar taint.
Assuntos
Contaminação de Alimentos , Carne/análise , Feromônios/análise , Sus scrofa/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Androstenos/efeitos adversos , Androstenos/análise , Criação de Animais Domésticos , Animais , Bélgica , Cruzamentos Genéticos , Contaminação de Alimentos/prevenção & controle , Inspeção de Alimentos/métodos , Temperatura Alta , Humanos , Masculino , Odorantes , Tamanho do Órgão , Feromônios/efeitos adversos , Escatol/efeitos adversos , Escatol/análise , Pele/lesões , Dermatopatias/veterinária , Gordura Subcutânea Abdominal/químicaRESUMO
Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (iDC) is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)-beta and interleukin (IL)-10 double-positive CD4(+) T cells within 1 week of autologous DC/T cell co-cultures. In iDC/T cell cultures, where antigen-specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF-beta and IL-10 secreted by CD4(+)CD25(-)FOXP3(-) T cells. In addition, the suppressive capacity of CD4(+) T cells conditioned by iDC was transferable to already primed antigen-specific CD8(+) T cell cultures. In contrast, addition of CD4(+) T cells conditioned by mDC to primed antigen-specific CD8(+) T cells resulted in enhanced CD8(+) T cell responses, notwithstanding the presence of TGF-beta(+)/IL-10(+) T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine-secreting regulatory T cells. We show that iDC-conditioned CD4(+) T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4(+) T cells. Furthermore, TGF-beta(+)/IL-10(+) T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.