The induction of apoptosis by methotrexate in activated lymphocytes as indicated by fluorescence hyperpolarization: a possible model for predicting methotrexate therapy for rheumatoid arthritis patients.
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The objectives of this study were to test the in vitro response of healthy non-activated, activated, and rheumatoid arthritis (RA) lymphocytes to methotrexate (MTX), and design an in vitro model for predicting the efficiency of MTX treatment for RA patients. Considering the RA profile of clonal-expanded CD4(+) T cells, phytohemagglutinin-activated mononuclear cells taken from healthy donors were incubated with different concentrations of MTX. The MTX-immunosuppressive effect was tested by fluorescence intensity measurements, including PI assay and annexin V assay. For simple detection, we used the Individual Cell Scanner (IC-S), which enables the measurement of early events in individual cells. Healthy mononuclear cells (MNC), and MNC derived from RA patients, were tested by the IC-S while utilizing fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) as an established marker of activation or suppression. In healthy activated MNC, we found that MTX, through its early incubation period, interferes with the activation signal obtained by PHA and exerts an apoptotic signal, which is noted by increases in the FP. Comparing our model to six long-standing RA patients and five newly-diagnosed patients revealed significant differences in the FP measurements, including fluorescence depolarization as an early established measurement of lymphocyte activation, and hyperpolarization as a measurement of an early immunosuppressive effect. We conclude that MTX, an effective therapy for RA patients, could easily be tested by fluorescence polarization measurements of FDA before (or during) clinical use in order to predict its efficiency on a specific RA patient. Moreover, the FP measurements can be used for the diagnosis, and making timing and dosage decisions. (+info)
Antimicrobial activity of SMAP-29 against the Bacteroides fragilis group and clostridia.
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OBJECTIVES: The cathelicidin-derived peptide SMAP-29 exerts rapid and broad-spectrum antimicrobial activity against aerobic bacteria and fungi. In this study, the effects of the peptide against the Bacteroides fragilis group, including antibiotic-resistant isolates, Clostridium perfringens and Clostridium difficile reference and clinical isolates, were investigated. METHODS: The microbicidal activity of SMAP-29 against eight reference and 100 clinical anaerobic strains from a national collection was assessed using a microdilution susceptibility assay, and by determining the killing kinetics on selected strains. The killing mechanism was investigated further by means of a two-colour fluorescent permeabilization assay, and by scanning electron microscopy (SEM). RESULTS: The Bacteroides fragilis group, Clostridium reference strains and most clinical isolates were inhibited in vitro by 1-2 microM (3.2-6.4 mg/L) SMAP-29, and killed by 1.5- to 2-fold higher peptide concentrations. The anaerobic bacterial cells were 90%-100% permeabilized within 2 h of exposure to bactericidal concentrations of the peptide. The SEM images of bacteria exposed to SMAP-29 provide morphological evidence that the envelope is an important target of the bactericidal activity of this peptide. These results are consistent with earlier studies indicating that SMAP-29 kills aerobic bacteria with a membranolytic mechanism, and suggest that both aerobic and anaerobic bacteria share surface features that are targeted by this peptide. CONCLUSIONS: These studies demonstrate that the spectrum of antibacterial activity of SMAP-29 includes the B. fragilis group and Clostridium species, and encourage further investigations of the therapeutic potential of this peptide. (+info)
Study on interactions of endocrine disruptors with estrogen receptor-beta using fluorescence polarization.
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In this study, we developed a rapid, simple and homogeneous human recombinant estrogen receptor-beta (hrER-beta) binding assay method using fluorescence polarization (FP) by applying a fluorescent ligand, fluorescein-labeled estradiol (F-E2). A Scatchard plot and a Hill plot analysis of the saturation binding assay using F-E2 and hrER-beta were studied. F-E2 showed a high affinity for hrER-beta, the dissociation constant was 5.53 nM, indicating that F-E2 is applicable to the hrER-beta binding assay. Competitive binding assays using F-E2, in which the FP values decreased upon the addition of compounds (competitors) were carried out to evaluate the binding characteristics of compounds with and without biological activities to hrER-beta. Twenty-one compounds, such as hormones, pharmaceuticals, industrial chemicals and phytoestrogens, were examined. The obtained sigmoidal inhibition curves were transformed into pseudo-Hill plots and the concentrations at 50% inhibition (IC50) and Hill coefficients, the degree of cooperativity in ER-ligand binding, were obtained from the regression equations. Antagonists exhibited larger Hill coefficients than agonists, showing the correlation between the Hill coefficients and the estrogenic/antiestrogenic activities. (+info)
Fluorescence polarization assay for diagnosis of human brucellosis.
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Fluorescence polarization immunoassay (FPA) uses molecular rotational properties to measure antibody binding to antigen directly. The potential use of this method was assessed in comparison to a competitive enzyme immunoassay (CELISA) and conventional serological tests for the diagnosis of brucellosis on a total of 587 human sera. Based on 340 sera from asymptomatic blood donors with no evidence of brucellosis, the specificity of the FPA was 97.9 % using a cut-off value of 72 mP. Sera from Brucella-infected patients (11 Brucella melitensis, 32 Brucella abortus, 32 Brucella suis and one Brucella sp.) yielded a sensitivity estimate of 96.1 %. In tests on 84 sera from suspected brucellosis patients, the FPA detected 80 cases. Of 87 sera from patients with probable infection, 15 were detected by both CELISA and FPA, three by CELISA only and four by FPA only. The discrepancies in both groups involved sera with low, declining titres. The FPA uses a sample of 40 micro l serum, takes about 5 min to complete and has been demonstrated to be accurate for the detection of antibodies to B. abortus, B. melitensis and B. suis and for identifying patients suffering relapses. Because of the ease of the procedure, it could be readily adopted for use in clinical laboratories and blood banks. (+info)
Cross-reactivity of anti-digoxin antibodies with digitoxin depends on tracer structure.
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The most common methods for measuring digoxin concentrations in serum are immunoassays. The prerequisite for exact determination of the digoxin value is an antibody that specifically binds digoxin. Because digitoxin differs from digoxin only in the C-12 hydroxy group, it is difficult to obtain anti-digoxin antibodies that do not cross-react with this compound. During the development of a fluorescence polarization immunoassay (FPIA) for digoxin, we investigated digoxin tracers with different structures. We found that in FPIA the digitoxin cross-reactivity of an antibody could be reduced by varying the structure of the tracer molecule. (+info)
Measurement of cyclosporine by liquid chromatography and three immunoassays in blood from liver, cardiac, and renal transplant recipients.
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In an effort to replace HPLC for whole-blood determination of cyclosporine (CsA), we compared HPLC with radioimmunoassay (RIA; INCSTAR, Cyclo-Trac SP assay), fluorescence polarization immunoassay (FPIA; Abbott TDx), and in-house modified enzyme-multiplied immunoassay technique (EMIT; Syva Co.). For blood samples from 200 various transplant recipients, RIA = 1.262 (HPLC) - 8.16, r = 0.983; FPIA = 1.200 (HPLC) + 19.90, r = 0.981; and EMIT = 1.038 (HPLC) + 11.28, r = 0.985. For segregation by transplant type, RIA, FPIA, and EMIT demonstrated positive biases of 27%, 12%, and 3%, respectively, for liver transplant recipients (n = 50) when compared with HPLC. Heart transplant recipients (n = 50) gave positive bias values of 23%, 14%, and 4% for RIA, FPIA, and EMIT, respectively. Adult renal transplant recipients (n = 50) demonstrated positive bias values of 30%, 31%, and 0% for RIA, FPIA, and EMIT, respectively. For pediatric renal transplant recipients (n = 50), positive biases of 40%, 31%, and 9% were obtained for RIA, FPIA, and EMIT, respectively. We conclude that the modified EMIT represents the best replacement for HPLC. (+info)
Release of gentamicin and vancomycin from temporary human hip spacers in two-stage revision of infected arthroplasty.
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AIM: Evaluation of the delivery of gentamicin and vancomycin from polymethylmethacrylate (PMMA) spacers before and after implantation for the treatment of total hip replacement infections. METHODS: Twenty industrially produced spacers containing gentamicin (1.9%) were utilized. Vancomycin (2.5%) mixed with PMMA cement was used to fill holes drilled in the cement of 14 of the 20 spacers immediately before implantation. The spacers were removed from 20 patients 3-6 months after implantation and then immersed in phosphate buffer at 37 degrees C for 10 days. Antibiotic concentrations were determined by fluorescence polarization immunoassay. RESULTS: Gentamicin and vancomycin were still present in all the spacers removed from the patients. The release of gentamicin alone and in combination with vancomycin was in the range 0.05%-0.4% of the initial amount present, whereas the release of vancomycin was in the range 0.8%-3.3%. The release kinetics showed a similar pattern for both drugs. After a high initial release of drug, a reduced, but constant, elution was observed over the next few days. CONCLUSIONS: The delivery of gentamicin and vancomycin from PMMA cement was high initially, with sustained release over several months. Incorporation of vancomycin into the surface of the spacers permitted spacers to be prepared with multiple antibiotics present and without adversely affecting the release kinetics of the agents. The gentamicin-vancomycin combination shows potential for the treatment of infection following total hip replacement in specific patients. (+info)
Neutralization of free digoxin-like immunoreactive components of oriental medicines Dan Shen and Lu-Shen-Wan by the Fab fragment of antidigoxin antibody (Digibind).
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Dan Shen and Lu-Shen-Wan, traditional Chinese medicines used as remedies for heart diseases, demonstrate digoxin-like immunoreactivity. The digoxin-like immunoreactive components of Lu-Shen-Wan show approximately 55% protein binding, while Dan Shen demonstrates concentration-dependent protein binding (68% bound at lower concentrations but only 25% bound at higher concentrations). Because Dan Shen and Lu-Shen-Wan can cause substantial toxic effects in patients, we studied the potential use of Digibind (Fab fragment of polyclonal antidigoxin antibody; Burroughs Wellcome, Research Triangle Park, NC) for neutralizing the pharmacologically active free fractions of Dan Shen and Lu-Shen-Wan. Drug-free serum pools were supplemented with Dan Shen or Lu-Shen-Wan to achieve apparent digoxin concentrations expected in severe overdoses. Aliquots of supplemented serum pools were supplemented further with aqueous Digibind solution to achieve final Digibind concentrations between 5 and 20 microg/mL (expected in vivo range in patients overdosed with digoxin and being treated with Digibind). We observed complete removal of the free apparent digoxin in the presence of Digibind for Dan Shen and Lu-Shen-Wan. Digibind binds free digoxin-like immunoreactive components of Dan Shen and Lu-Shen-Wan in vitro. (+info)