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1.
Poult Sci ; 99(8): 3987-3996, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32731986

RESUMO

Bacillus subtilis subsp. subtilis American Type Culture Collection deposit number PTA-125135 has recently been studied by our laboratory as a potential probiotic strain for avian species. The objective of the present study was to evaluate growth performance and feed efficiency in broiler chickens in response to a dose titration of the Bacillus strain in feed. In addition to a nonsupplemented control, Bacillus spores were supplemented into broiler chicken diets at 4 levels, which were 8.1 × 104, 1.6 × 105, 2.4 × 105, and 3.2 × 105 CFU per g of feed. The titration was applied to two different dietary regimes of standard or low metabolizable energy (ME), which differed in ME by 22, 56, and 110 kcal/kg in starter, grower, and finisher dietary phases, respectively. All diets contained 249 g per metric ton of a previously patented synbiotic feed additive. Performance data were collected at day 14, 26, and 40 of age, and the effects of Bacillus and ME treatments were evaluated by factorial ANOVA. Treatment group means were further examined for significant (P < 0.05) pairwise differences among treatments and for significant (P < 0.05) linear and quadratic effects. At day 14 of age, significant linear effects for decreased feed conversion ratio (FCR) with higher CFU of Bacillus supplementation were observed within the standard ME diet. At day 26, a linear trend was observed for increased mortality with increased dose within the standard ME diet only. Bacillus supplementation at day 26 also significantly affected FCR and mortality-adjusted FCR, where supplementation with 3.2 × 105 CFU per g feed produced lower FCR and mortality-adjusted FCR than supplementation with 1.6 × 105 CFU per g feed. We conclude from linear effects related to feed efficiency observed at day 14 and from the significant separation of Bacillus treatment means within the titrated range of supplementation at day 26 that further evaluation for effects on performance should be made of doses at 2.4 × 105, 3.2 × 105, and greater CFU per g in feed.


Assuntos
Bacillus , Galinhas , Dieta , Metabolismo Energético , Probióticos , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Galinhas/microbiologia , Dieta/veterinária , Suplementos Nutricionais , Estados Unidos
2.
Lett Appl Microbiol ; 71(5): 550-556, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32780870

RESUMO

A series of experiments was conducted to identify the molecular species responsible for surface active emulsification (surfactant) bioactivity in Bacillus subtilis subsp. subtilis strain ATCC PTA-125135, and to describe culture conditions to support the enriched production of said bioactivity in cultured plaque of the strain. The assay for methylene blue active substances (MBAS) was found to be suitable for describing surfactant activity, where a solvent-extracted molecular fraction from the biofilm was found to retain surfactant activity and positively quantified as MBAS. Furthermore, an HPLC-refined protein fraction was found to quantify as MBAS with approximately 1·36-fold or greater surfactant activity per mol than sodium dodecyl sulphate, and a proteomic analysis of solvent extracted residues confirmed that biofilm surface layer protein BslA was a primary constituent of extracted residues. Surfactant bioactivity, quantified as MBAS, was enriched in cultured plaque by the supplementation of culture media with calcium chloride or calcium nitrate.


Assuntos
Bacillus/metabolismo , Biofilmes , Cálcio/metabolismo , Azul de Metileno/metabolismo , Tensoativos/metabolismo , Meios de Cultura/metabolismo , Proteômica , Dodecilsulfato de Sódio/metabolismo
3.
Poult Sci ; 96(8): 2595-2600, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28431095

RESUMO

Avi-Lution® is a defined, patented, synbiotic product containing Saccharomyces cerevisiae, Enterococcus faecium, and Bacillus spp. Broiler chickens (n = 1,250) were experimentally treated as uninoculated controls (uCon), inoculated controls (iCon) with Clostridium perfringens, or inoculated and treated with bacitracin methylene disalicylate (BMD) at 55 mg/kg as an infected/treated control or Avi-Lution® at 1.0 (AvL1) or 2.0 (AvL2) g/kg in feed for 42 d. Each treatment was applied to 10 replicate pens of 25 straight-run, newly hatched chicks. Pens treated with AvL1, AvL2, or BMD showed improved growth, feed efficiency, or mortality from necrotic enteritis compared with iCon pens at d 14, 28, and 42. No differences in these measurements, however, were observed between pens treated with AvL1 and AvL2, which suggests that Avi-Lution® was effective at 1.0 g/kg in feed. Despite improved performance, BMD, AvL1, and AvL2 treatments did not decrease the severity of intestinal lesion scores through 42 d of age compared with the infected control. These results demonstrate that Avi-Lution® improved growth performance and feed conversion rates in broilers challenged with Clostridium perfringens despite no difference in severity of intestinal lesion scores.


Assuntos
Bacitracina/administração & dosagem , Galinhas , Infecções por Clostridium/veterinária , Clostridium perfringens/fisiologia , Doenças das Aves Domésticas/prevenção & controle , Salicilatos/administração & dosagem , Simbióticos/administração & dosagem , Ração Animal/análise , Animais , Bacillus/química , Bacitracina/farmacologia , Galinhas/crescimento & desenvolvimento , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/efeitos dos fármacos , Dieta/veterinária , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana , Enterococcus faecium/química , Masculino , Distribuição Aleatória , Saccharomyces cerevisiae/química
4.
J Dairy Sci ; 99(11): 9040-9050, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27614838

RESUMO

Peripheral blood mononuclear cells (PBMC) and mesenteric node lymphocytes (MNL) were obtained from 30 calves that were assigned randomly at birth to 1 of 6 treatment groups with 5 calves per treatment in a 14-d study: (1) colostrum-deprived (CD), no vitamins; (2) colostrum-replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D3; (5) CR, vitamin E; (6) CR, vitamins A, D3, E. Calves were injected with appropriate vitamin supplements and fed pasteurized whole milk (CD calves) or fractionated colostrum replacer (CR calves) at birth. Thereafter, all calves were fed pasteurized whole milk fortified with vitamins according to treatment group. Calves were orally inoculated with 108 cfu of Mycobacterium avium ssp. paratuberculosis (MAP) on d 1 and 3. The PBMC and MNL harvested on d 13 were analyzed by flow cytometry as fresh cells, after 3-d culture with phytohemagglutinin (PHA), and after 6-d culture with a whole-cell sonicate of MAP (MPS). Peripheral γδ T cells were a predominant lymphocyte subset in neonatal calves, with a decreased percentage noted in CD calves compared with CR calves. As well, CD25 expression was higher in γδ T cells compared with other cell subsets, regardless of treatment group. Stimulation of PBMC with PHA resulted in increased CD4+ and CD8+ subsets, whereas MNL response was dominated by expansion of B-cell subpopulations. Stimulation with PHA and MPS decreased the relative abundance of PBMC γδ T cells, but MNL γδ T cells increased upon stimulation with MPS. These results identify γδ T cells as key early responders to intracellular infection in neonatal calves and suggest that colostrum may be an important mediator of this response.


Assuntos
Doenças dos Bovinos/imunologia , Colostro/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/imunologia , Linfócitos T/imunologia , Ração Animal/análise , Animais , Animais Recém-Nascidos , Linfócitos B/imunologia , Bovinos , Doenças dos Bovinos/microbiologia , Colecalciferol/administração & dosagem , Colostro/química , Dieta/veterinária , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Leucócitos Mononucleares/microbiologia , Leite/química , Leite/microbiologia , Pasteurização , Fito-Hemaglutininas/química , Receptores de Antígenos de Linfócitos T gama-delta , Fator de Necrose Tumoral alfa/metabolismo , Vitamina A/administração & dosagem , Vitamina E/administração & dosagem
5.
J Dairy Sci ; 99(4): 2884-2895, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26805975

RESUMO

Thirty Holstein calves were obtained from 2 dairy farms in central Iowa at birth and randomly assigned to 1 of 6 treatment groups: (1) colostrum deprived (CD), no vitamins; (2) colostrum replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D3; (5) CR, vitamin E; and (6) CR, vitamins A, D3, E, with 5 calves per treatment in a 14-d study. Calves were fed pasteurized whole milk (CD) or fractionated colostrum replacer (CR) at birth (d 0) and injected with vitamins according to treatment group. From d 1 through d 14 of the study, all calves were fed pasteurized whole milk (PWM) supplemented with vitamins as assigned. All calves were inoculated with Mycobacterium avium ssp. paratuberculosis on d 1 and 3 of age. Calves fed CR acquired IgG1 and haptoglobin in serum within 24 h of birth, whereas CD calves did not. The CR-fed calves were 2.5 times less likely to develop scours, and CR calves supplemented with vitamins D3 and E also demonstrated a decreased incidence of scours. Serum vitamin levels of A, D, and E increased within treatment group by d 7 and 14 of the study. Interestingly, synergistic effects of supplemental vitamins A, D3, and E on serum 25-(OH)-vitamin D were observed at d 7, resulting in higher levels than in calves administered vitamin D only. Further, vitamin D3 deficiency was observed in CD and CR calves fed a basal diet of pasteurized whole milk and no supplemental vitamins. Colonization of tissues with Mycobacterium avium ssp. paratuberculosis was negligible and was not affected by colostrum feeding or vitamin supplementation. Results demonstrated passive transfer of haptoglobin to neonatal calves, and potential health benefits of supplemental vitamins D3 and E to calves fed pasteurized whole milk.


Assuntos
Ração Animal/normas , Doenças dos Bovinos/prevenção & controle , Colostro/metabolismo , Dieta/veterinária , Haptoglobinas/metabolismo , Paratuberculose/prevenção & controle , Vitaminas/farmacologia , Animais , Animais Recém-Nascidos , Bovinos , Doenças dos Bovinos/patologia , Feminino , Haptoglobinas/análise , Imunoglobulina G/sangue , Mycobacterium avium subsp. paratuberculosis/fisiologia , Paratuberculose/patologia , Distribuição Aleatória
6.
J Dairy Sci ; 97(6): 3715-27, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24704235

RESUMO

To observe the effects of supplemental dietary d-α-tocopherol in relation to dietary energy on growth and immune status in dairy calves, 32 newborn Holstein bull calves were assigned to 1 of 4 treatments for 5 wk in a 2 × 2 factorial, randomized complete block, split-plot design. Calves received moderate growth (MG) or low growth (LG) all-milk dietary treatments, formulated to support daily gains of 0.5 or 0.25 kg/d, respectively, per the dietary energy recommendation for milk-fed calves according to the National Research Council's Nutrient Requirements of Dairy Cattle. Calves in both groups were either injected i.m. with Vital E-A+D (injectable solution of vitamins E, A, and D) on d 1 and supplemented with Emcelle Tocopherol (micellized vitamin E) via milk daily (MG-S and LG-S), or were not supplemented (MG-C and LG-C) during the study period. Total weight gain of MG calves was greater than that of LG calves and tended to be greater in MG-S calves than in MG-C calves. Calves receiving vitamin supplementation demonstrated greater concentrations of plasma α-tocopherol, retinol, and 25-(OH)-vitamin D than did control calves, whereas MG calves demonstrated a lower concentration of plasma α-tocopherol than did LG calves. The apparent increased utilization of α-tocopherol by MG calves was accompanied by a rise in serum haptoglobin, a positive acute-phase protein and indicator of inflammation, especially in MG-C calves. Serum amyloid A, also a positive acute-phase protein, was not different among groups, but was elevated from baseline in all groups during wk 1 through 3. Plasma IgG1 concentrations were higher in MG-S and LG-S calves than in their nonsupplemented dietary counterparts, whereas plasma IgG2, IgA, and IgM concentrations were not different among groups. In summary, dietary supplementation of d-α-tocopherol improved plasma α-tocopherol status and tended to increase growth in calves fed for 0.5 kg of average daily gain. Vitamin supplementation ameliorated the rise of serum haptoglobin associated with acute inflammation in MG calves, and may have improved passive transfer of maternal antibody. These results indicate a role for α-tocopherol in prevention of proinflammatory state associated with greater dietary energy and onset of infectious disease.


Assuntos
Bovinos/fisiologia , Ingestão de Energia , Haptoglobinas/metabolismo , Imunidade Inata/efeitos dos fármacos , Proteína Amiloide A Sérica/metabolismo , alfa-Tocoferol/metabolismo , Ração Animal/análise , Animais , Análise Química do Sangue/veterinária , Bovinos/crescimento & desenvolvimento , Bovinos/imunologia , Dieta/veterinária , Suplementos Nutricionais/análise , Feminino , Ganho de Peso/efeitos dos fármacos , alfa-Tocoferol/administração & dosagem
7.
J Thromb Haemost ; 3(2): 312-20, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15670038

RESUMO

OBJECTIVE: To investigate the effects of abciximab, eptifibatide and no GPIIb-IIIa antagonist (control) on soluble CD40 ligand (sCD40L) and the formation of leukocyte-platelet aggregates (LPA) in 98 ACS patients undergoing percutaneous coronary intervention (PCI). BACKGROUND: sCD40L and LPA are increased in patients with ACS. METHODS: sCD40L was measured by enzyme-linked immunosorbent assay (ELISA) and LPA by whole blood flow cytometry. RESULTS: There were no baseline differences between the three groups in sCD40L and LPA. At the end of PCI, sCD40L was unchanged in the controls, decreased by 30% (P < 0.001) in the abciximab group and by 11% (P < 0.02) in the eptifibatide group. Eighteen to 24 h after PCI, sCD40L was unchanged in the controls, reduced 30% (P < 0.001) in the abciximab-treated group and 9% (P < 0.01) in the eptifibatide-treated group. At the end of PCI, circulating monocyte-platelet aggregates (MPA) were reduced by 12% (P = NS) in the abciximab-treated group, 13% in the eptifibatide-treated group (P = NS), but slightly increased in the controls (P = NS). Eighteen to 24 h after PCI, MPA were reduced by 41% (P < 0.001) compared to baseline in the abciximab-treated group, by 23% (P = NS) in the eptifibatide-treated group, and 15% (P = NS) in the controls. In contrast to control patients presenting while on clopidogrel, control patients presenting not on clopidogrel demonstrated a reduction in sCD40L and LPA 18-24 h post-PCI (P = NS). At low receptor occupancy, GPIIb-IIIa antagonists did not augment the release of sCD40L or the number of circulating LPA. CONCLUSIONS: GPIIb-IIIa antagonists reduce circulating sCD40L and LPA formation in patients with ACS undergoing PCI. At low receptor occupancy, GPIIb-IIIa antagonists do not activate platelets.


Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/patologia , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Ticlopidina/análogos & derivados , Abciximab , Doença Aguda , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Plaquetas/patologia , Ligante de CD40/sangue , Clopidogrel , Doença das Coronárias/complicações , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos Fab das Imunoglobulinas/farmacologia , Inflamação/tratamento farmacológico , Leucócitos/patologia , Masculino , Pessoa de Meia-Idade , Adesividade Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Ticlopidina/administração & dosagem , Ticlopidina/farmacologia
8.
Am Heart J ; 142(5): 790-8, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11685164

RESUMO

BACKGROUND: Plaque disruption with resultant platelet activation and leukocyte-platelet aggregation is a pathophysiologic process common to both acute coronary syndromes and percutaneous coronary interventions. Unfractionated heparin is a standard antithrombotic therapy in patients with both acute coronary syndromes and in those undergoing percutaneous coronary interventions. Low-molecular-weight heparins have been reported to cause less platelet activation than unfractionated heparin. METHODS: Monocyte-platelet aggregates, neutrophil-platelet aggregates, platelet surface P-selectin, and platelet surface glycoprotein (GP) IIIa were measured serially by whole blood flow cytometry in 40 patients with unstable angina (randomly assigned to either unfractionated heparin 70 U/kg or the low-molecular-weight heparin dalteparin 60 IU/kg) undergoing coronary intervention with planned abciximab administration (in 2, one-half-dose boluses). Assays were performed at baseline, 5 minutes after administration of either type of heparin, 10 minutes after the first bolus of abciximab, 10 minutes after second bolus of abciximab, and 8 to 10 and 16 to 24 hours after administration of either heparin. RESULTS: No significant differences in clinical outcomes were observed between patients receiving either unfractionated heparin or dalteparin. The number of circulating P-selectin-positive platelets was increased by unfractionated heparin but not dalteparin, and abciximab reversed this increase. The number of circulating P-selectin-positive platelets was reduced below baseline levels in both treatment groups 8 to 10 and 16 to 24 hours after study drug administration. At 8 to 10 and 16 to 24 hours after administration of study drug, platelet degranulation in response to iso-thrombin receptor agonist peptide 1.5 mmol/L was significantly reduced by almost 50% (compared with immediately after study drug administration). Both unfractionated heparin and dalteparin significantly increased the numbers of circulating monocyte-platelet and neutrophil-platelet aggregates, which were subsequently reduced to baseline levels after administration of the second abciximab bolus and to below baseline at both 8 to 10 and 16 to 24 hours in all patients. After both unfractionated heparin and dalteparin administration, platelet surface GP IIIa expression was significantly increased compared with baseline at both 8 to 10 and 16 to 24 hours. CONCLUSIONS: Dalteparin in combination with abciximab in patients with unstable angina undergoing coronary intervention appears to be safe. Unfractionated heparin, but not dalteparin, degranulates platelets in patients with unstable angina. Both heparins increase the number of circulating monocyte-platelet and neutrophil-platelet aggregates. Abciximab therapy during coronary interventions rapidly reduces the number of degranulated platelets and leukocyte-platelet aggregates.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD/efeitos dos fármacos , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/cirurgia , Dalteparina/farmacologia , Dalteparina/uso terapêutico , Fibrinolíticos/uso terapêutico , Heparina/uso terapêutico , Fragmentos Fab das Imunoglobulinas/farmacologia , Leucócitos/química , Selectina-P/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Abciximab , Angioplastia Coronária com Balão , Anticorpos Monoclonais/uso terapêutico , Aterectomia , Fibrinolíticos/farmacologia , Heparina/farmacologia , Humanos , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Integrina beta3 , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo
9.
J Am Coll Cardiol ; 38(4): 1002-6, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11583872

RESUMO

OBJECTIVES: We investigated whether elevated levels of circulating monocyte-platelet aggregates (MPA) can be used to identify patients with acute myocardial infarction (AMI). BACKGROUND: Commonly used blood markers of AMI reflect myocardial cell death, but do not reflect the earlier pathophysiologic processes of plaque rupture, platelet activation and resultant thrombus formation. Circulating MPA form after platelet activation. METHODS: In a single center between October 1998 and November 1999, we measured circulating MPA in a blinded fashion by whole blood flow cytometry in 211 consecutive patients who presented to the emergency department (ED) with chest pain and were admitted to rule out AMI. Acute myocardial infarction was diagnosed by a CK-MB fraction greater than three times control. RESULTS: Patients with AMI (n = 61), as compared with those without AMI (n = 150), had significantly higher numbers of circulating MPA (11.6 +/- 11.4 vs. 6.4 +/- 3.6, mean +/- SD, p < 0.0001). After controlling for age, the adjusted odds of developing AMI for patients in the 2nd, 3rd and 4th quartiles of MPA, in comparison with patients in the lowest quartile (odds ratio = 1.0), were 2.1 (95% confidence interval [CI]: 0.7, 6.8), 4.4 (95% CI: 1.5, 13.1) and 10.8 (95% CI: 3.6, 32.0), respectively. The number of circulating MPA in patients with AMI presenting within 4 h of symptom onset (14.4) was significantly greater than those presenting after 4 h (9.4) and after 8 h (7.0), (p < 0.001). Of the 61 patients with AMI, 35 (57%) had a normal creatine kinase isoenzyme ratio at the time of presentation to the ED, but had high levels of circulating MPA (13.3). CONCLUSIONS: Circulating MPA are an early marker of AMI.


Assuntos
Monócitos/fisiologia , Infarto do Miocárdio/diagnóstico , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Creatina Quinase/sangue , Creatina Quinase Forma MB , Feminino , Citometria de Fluxo , Humanos , Isoenzimas/sangue , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/fisiopatologia , Selectina-P/análise
10.
Blood ; 98(8): 2518-25, 2001 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11588050

RESUMO

Chromosomal translocations involving the platelet-derived growth factor beta receptor (PDGFbetaR) gene have been reported in some patients with chronic myelomonocytic leukemia (CMML). The resultant fusion proteins have constitutive PDGFbetaR tyrosine kinase activity, but the partner genes previously reported (tel, Huntingtin interacting protein 1 [HIP-1], H4/D10S170) have poorly understood roles in the oncogenic activity of the fusion proteins. A novel PDGFbetaR fusion protein has been characterized in a patient with CMML and an acquired t(5;17)(q33;p13). Southern blot analysis on patient leukemia cells demonstrated involvement of the PDGFbetaR gene. Using 5' rapid amplification of complementary DNA ends-polymerase chain reaction (RACE-PCR) on patient RNA, rabaptin-5 was identified as a novel partner fused in-frame to the PDGFbetaR gene. The new fusion protein includes more than 85% of the native Rabaptin-5 fused to the transmembrane and intracellular tyrosine kinase domains of the PDGFbetaR. Transduction with a retroviral vector expressing rabaptin-5/PDGFbetaR transformed the hematopoietic cell line Ba/F3 to growth factor independence and caused a fatal myeloproliferative disease in mice. Rabaptin-5 is a well-studied protein shown to be an essential and rate-limiting component of early endosomal fusion through interaction with the Ras family GTPases Rab5 and Rab4. The fusion protein includes 3 of 4 coiled-coil domains (involved in homodimerization of native rabaptin-5), 2 caspase-3 cleavage sites, and a binding site for the tumor suppressor gene tuberin (tuberous sclerosis complex-2). Early endosomal transport is critical in regulation of various growth factor receptors, through ligand-induced clathrin-mediated endocytosis, and thus this new fusion protein links together 2 important pathways of growth regulation.


Assuntos
Leucemia Mielomonocítica Crônica/sangue , Proteínas de Membrana/genética , Proteínas de Fusão Oncogênica/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Proteínas de Transporte Vesicular , Adulto , Animais , Medula Óssea/patologia , Linhagem Celular , Clonagem Molecular , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Translocação Genética
11.
Circulation ; 104(12): 1374-9, 2001 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-11560852

RESUMO

BACKGROUND: The primary mechanism of action of glycoprotein (GP) IIb/IIIa antagonists is inhibition of the final common pathway of platelet aggregation: fibrinogen binding to the GP IIb/IIIa complex. However, it has been reported that induction of fibrinogen binding and platelet aggregation is an intrinsic prothrombotic property of low-dose GP IIb/IIIa antagonists. These apparently paradoxical results have been extensively referenced in the cardiology literature. METHODS AND RESULTS: By platelet aggregation and flow cytometry, we demonstrate that (1) dissociation of GP IIb/IIIa antagonists (abciximab, tirofiban, eptifibatide, or xemilofiban) from platelets does not result in platelet aggregation; (2) tirofiban and eptifibatide can induce a fibrinogen-binding-competent conformation of the GP IIb/IIIa receptor, but stable fibrinogen binding does not occur without fixation; (3) the slow off-rate of abciximab exposes only a small proportion of unblocked GP IIb/IIIa receptors at any time, and these also fail to stably bind fibrinogen; and (4) the GP IIb/IIIa antagonist-induced fibrinogen binding in some previously reported experiments was probably the result of artifactual thrombin generation. CONCLUSIONS: Under physiological conditions, GP IIb/IIIa antagonists currently in clinical use do not have an intrinsic activating property that results in platelet aggregation or stable fibrinogen binding to GP IIb/IIIa.


Assuntos
Plaquetas/efeitos dos fármacos , Fibrinogênio/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Plaquetas/citologia , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Hirudinas/farmacologia , Humanos , Agregação Plaquetária/fisiologia , Inibidores da Agregação Plaquetária/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Fixação de Tecidos
12.
Circulation ; 104(13): 1533-7, 2001 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-11571248

RESUMO

BACKGROUND: Platelet surface P-selectin is considered the "gold standard" marker of platelet activation. Degranulated, P-selectin-positive platelets, however, aggregate with leukocytes in vitro and rapidly lose surface P-selectin in vivo. METHODS AND RESULTS: Flow cytometric tracking of autologous, biotinylated platelets in baboons enabled us to directly demonstrate for the first time in vivo that (1) infused degranulated platelets very rapidly form circulating aggregates with monocytes and neutrophils, and (2) 30 minutes after infusion of the degranulated platelets, the percentage of circulating monocytes aggregated with infused platelets persist at high levels, whereas the percentage of circulating neutrophils aggregated with infused platelets and the platelet surface P-selectin of nonaggregated infused platelets return to baseline. We therefore performed 2 clinical studies in patients with acute coronary syndromes. First, after percutaneous coronary intervention (n=10), there was an increased number of circulating monocyte-platelet (and to a lesser extent, neutrophil-platelet) aggregates but not P-selectin-positive platelets. Second, of 93 patients presenting to an Emergency Department with chest pain, patients with acute myocardial infarction (AMI) (n=9) had more circulating monocyte-platelet aggregates (34.2+/-10.3% [mean+/-SEM]) than patients with no AMI (n=84, 19.3+/-1.4%, P<0.05) and normal control subjects (n=10, 11.5+/-0.8%, P<0.001). Circulating P-selectin-positive platelets, however, were not increased in chest pain patients with or without AMI. CONCLUSIONS: As demonstrated by 3 independent means (in vivo tracking of activated platelets in baboons, human coronary intervention, and human AMI), circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin.


Assuntos
Plaquetas/fisiologia , Monócitos/fisiologia , Infarto do Miocárdio/patologia , Selectina-P/metabolismo , Ativação Plaquetária/fisiologia , Doença Aguda , Animais , Biomarcadores , Plaquetas/metabolismo , Agregação Celular , Dor no Peito/diagnóstico , Dor no Peito/patologia , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Masculino , Infarto do Miocárdio/diagnóstico , Neutrófilos/fisiologia , Papio
13.
Cytometry ; 43(4): 308-13, 2001 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-11260598

RESUMO

BACKGROUND: Adherence of platelets to endothelial cells may be a significant event in the development of vascular thrombosis. Existing models, which examine platelet-endothelial cell interactions, compromise endothelial cell integrity or use radioactivity to identify platelets that adhere to endothelial cells. We report a novel method for in vitro detection of platelet-endothelial cell adhesion that allows endothelial cells to remain as an intact monolayer and for visualization of individual platelets. METHODS: Fluorescently labeled platelets were incubated with a confluent monolayer of endothelial cells. Laser scanning cytometry (LSC) identified platelets bound to endothelial cells based on their fluorescent signals. RESULTS: LSC detection of platelets reliably reproduced well-described findings of thrombin-induced platelet-endothelial cell adhesion. Results demonstrating reduced adhesion with a glycoprotein IIb-IIIa-specific blocking monoclonal antibody confirmed the specificity of the LSC detection of platelet-endothelial cell adhesion. CONCLUSIONS: LSC is a novel method for detecting platelet--endothelial cell adhesion. Its advantages over other methods are: (a) endothelial cells remain undisturbed and adherent throughout; (b) the ability to detect individual bound platelets and subpopulations; (c) the ability to store images and slides and then relocate, revisualize, and reanalyze individual cells or cell populations of interest; and (d) no radioactivity.


Assuntos
Endotélio Vascular/fisiologia , Citometria por Imagem/métodos , Microscopia Confocal/métodos , Adesividade Plaquetária/fisiologia , Difosfato de Adenosina/farmacologia , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Células Cultivadas , Combinação de Medicamentos , Endotélio/citologia , Endotélio/fisiologia , Endotélio Vascular/citologia , Epinefrina/farmacologia , Humanos , Adesividade Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Trombina/farmacologia
14.
Thromb Haemost ; 84(3): 492-8, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11019977

RESUMO

In addition to inhibition of platelet aggregation, GPIIb-IIIa antagonists may reduce thrombotic events via other mechanisms. In a novel whole blood flow cytometric system, we investigated the effects of GPIIb-IIIa antagonists, in the presence or absence of thrombin inhibitors, on platelet surface-bound factor V/Va and platelet surface phospholipids. Diluted venous blood was incubated with either buffer or a GPIIb-IIIa antagonist (abciximab, tirofiban, or eptifibatide). Some samples were pre-incubated with clinically relevant concentrations of unfractionated heparin (UFH), a low molecular weight heparin, a direct thrombin inhibitor, or buffer only. Platelets were then activated and labeled with mAb V237 (factor V/Va-specific) or annexin V (binds phosphatidylserine), fixed, and analyzed by flow cytometry. In the absence of thrombin inhibitors, GPIIb-IIIa antagonists (especially abciximab) significantly reduced agonist-induced platelet procoagulant activity, as determined by reduced binding of V237 and annexin V. At high pharmacologic concentrations, unfractionated heparin and enoxaparin, but not hirudin, further reduced factor V/Va binding to the surface of activated platelets in the presence of GPIIb-IIa antagonists. Agonist-induced platelet procoagulant activity was reduced in a patient with Glanzmann's thrombasthenia. We conclude that GPIIb-IIIa antagonists reduce platelet procoagulant activity in whole blood and heparin and enoxaparin augment this reduction. Fibrinogen binding to GPIIb-IIIa is important in the generation of platelet procoagulant activity.


Assuntos
Fator V/metabolismo , Fosfatidilserinas/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Tirosina/análogos & derivados , Abciximab , Anticorpos Monoclonais/farmacologia , Plaquetas/metabolismo , Colágeno/farmacologia , Relação Dose-Resposta a Droga , Eptifibatida , Citometria de Fluxo , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Recém-Nascido , Proteínas de Membrana/metabolismo , Peptídeos/farmacologia , Ligação Proteica , Trombastenia/sangue , Trombina/antagonistas & inibidores , Trombina/farmacologia , Tirofibana , Tirosina/farmacologia
15.
Methods ; 21(3): 259-70, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10873480

RESUMO

Platelet function in whole blood can be comprehensively evaluated by flow cytometry. Flow cytometry can be used to measure platelet reactivity, circulating activated platelets, platelet-platelet aggregates, leukocyte-platelet aggregates, procoagulant platelet-derived microparticles, and calcium flux. Clinical applications of whole blood flow cytometric assays of platelet function in disease states (e.g., acute coronary syndromes, angioplasty, and stroke) may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of GPIIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.


Assuntos
Plaquetas/fisiologia , Citometria de Fluxo/métodos , Anticorpos Monoclonais/imunologia , Bancos de Sangue , Plaquetas/imunologia , Plaquetas/metabolismo , Plaquetas/patologia , Coleta de Amostras Sanguíneas , Isquemia Encefálica/sangue , Isquemia Encefálica/patologia , Calibragem , Humanos , Leucócitos/metabolismo , Ativação Plaquetária/imunologia , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Deficiência do Pool Plaquetário/sangue , Deficiência do Pool Plaquetário/diagnóstico , Deficiência do Pool Plaquetário/patologia , Controle de Qualidade , Fixação de Tecidos
17.
Science ; 246(4927): 251-5, 1989 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-2799386

RESUMO

Fumigant applicators who, 6 weeks to 3 months earlier, were exposed to phosphine, a common grain fumigant, or to phosphine and other pesticides had significantly increased stable chromosome rearrangements, primarily translocations in G-banded lymphocytes. Less stable aberrations including chromatid deletions and gaps were significantly increased only during the application season, but not at this later time point. During fumigant application, measured exposure to phosphine exceeds accepted national standards. Because phosphine is also used as a dopant in the microchip industry and is generated in waste treatment, the possibility of more widespread exposure and long-term health sequelae must be considered.


Assuntos
Aberrações Cromossômicas , Praguicidas/envenenamento , Fosfinas/envenenamento , Bandeamento Cromossômico , Exposição Ambiental , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestrutura , Masculino
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