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#28288389   2017/03/13 Save this To Up

Ovarian ablation for premenopausal breast cancer: A review of treatment considerations and the impact of premature menopause.

Historically, ovarian ablation (OA) was used as therapy for women with recurrent hormone-receptor-positive (HRP) premenopausal breast cancer. With the publication of the SOFT (Suppression of Ovarian Function Trial) and TEXT (Tamoxifen and Exemestane Trial) randomized trials, there is considerable interest in OA as an adjuvant treatment, either in combination with tamoxifen or an aromatase inhibitor (AI). Thus, we have reviewed current guidelines and key studies on this important topic and have highlighted the relevant biological and pharmacological aspects of the various endocrine therapies. The results of two key randomized trials addressing the use and controversies of OA in premenopausal breast cancer are discussed and recent research emphasizing the detrimental consequences of premature menopause and the cost-effectiveness of OA is presented. In low-risk patients with HRP premenopausal breast cancer, OA is not beneficial and tamoxifen remains the anti-hormone treatment of choice. In high-risk women (previous chemotherapy or women younger than 35), OA in combination with AI is more effective but is arguably not cost-effective, particularly when OA is achieved medically using a GnRH agonist/antagonist. Compared to tamoxifen alone, the SOFT trial showed a 4.5-7.7% reduction in breast cancer relapse using OA (in combination with either tamoxifen or AI) in high-risk women, though the 5-year overall survival benefit was limited (1.4-3.6%). Premature menopause is associated with long-term mortality risks and women often experience significant menopausal symptoms that impact on quality of life. These considerations should play a role in the treatment selection of those patients who may benefit from adjuvant OA.

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#27832728   2016/11/11 Save this To Up

Gonadal steroids block the calpain-1-dependent intrinsic pathway of apoptosis in an experimental rat stroke model.

Apoptosis plays an important role in the progression of the ischemic penumbra after reperfusion. Estrogen and progesterone have neuroprotective effects against ischemic brain damage, however the exact mechanisms of neuroprotection and signaling pathways is not completely understood. In this study, we investigated the possible regulatory effects of a combined steroid treatment on extrinsic and intrinsic apoptotic signaling pathways after cerebral ischemia.

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#24295179   2013/12/03 Save this To Up

Effect of different combinations of antibodies and enzyme labels on ELISA of progesterone.

In steroid immunoassays, selection of right combination of antibody and enzyme-labeled antigen determine the sensitivity and specificity of ELISA. Antibodies raised against different positions of progesterone adopting heterologous systems were reported to provide better assays for progesterone. Four different antibodies developed against progesterone-11α-hemiglutarate-BSA (P-11-HG-BSA), progesterone-11α-hemisuccinate-BSA (P-11-HS-BSA), progesterone-3-O-carboxymethyloxime-BSA (P-3-CMO-BSA), and progesterone-3-O-carboxymethyloxime-ovalbumin (P-3-CMO-ova) were tested in combination with enzyme-labeled P-11-HG, P-11-HS, progesterone-11α-carboxymethyl ether (P-11-CME), P-3-CMO, 17-hydroxyprogesterone-3-O-carboxymethyl oxime (17-P-3-CMO), and progesterone-4-carboxymethyl thioether (P-4-CMTE). These were variously labeled with penicillinase, alkaline phosphatase (ALP), and horseradish peroxidase (HRP). When antibody developed against P-11-HS-BSA was tested with P-3-CMO labeled separately with penicillinase, ALP, and HRP, the type of enzyme used had no effect on the performance of the assay. It was found that a homologous assay using P-3-CMO-ova as immunogen and P-3-CMO-HRP as label, as well as a heterologous ELISA with antibody raised against P-11-HS-BSA in combination with P-3-CMO-HRP, provided sensitive assays for progesterone. The use of 17α-hydroxy progesterone-3-O-carboxymethyl oxime-HRP with the same antibodies against P-3-CMO-BSA and P-11-HS-BSA also proved to be better than P-3-CMO-HRP. These findings implied that the sensitivity and specificity of ELISA to a great extent depended on the nature of the antibody produced, while the choice of enzyme labels could be manipulated.

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#23323985   2013/01/17 Save this To Up

Influence of different length spacers containing enzyme conjugate on functional parameters of progesterone ELISA.

In steroid enzyme immunoassay (EIA), there is an increase or decrease of labeled steroid recognition by antibody due to homologous and heterologous combinations of enzyme conjugate with immunogen that affects sensitivity of the assay. We have introduced three to 18 atomic length linkers between enzyme and steroid moieties and studied their effects on functional parameters such as sensitivity, ED(50), and specificity of progesterone enzyme immunoassays. Progesterone-3-carboxymethyloxime-bovine serum albumin (P-3-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using 17-α-hydroxy-progesterone-3-carboxymethyloxime (17-α-OH-P-3-CMO) as carboxylic derivative of 17-α-hydroxy-progesterone and horseradish peroxidase (HRP) as label. These were 17-α-OH-P-3-CMO-HRP, 17-α-OH-P-3-CMO-urea-HRP (17-α-OH-P-3-CMO-U-HRP), 17-α-OH-P-3-CMO-ehylenediamine-HRP (17-α-OH-P-3-CMO-EDA-HRP), 17-α-OH-P-3-CMO-carbohydrazide-HRP (17-α-OH-P-3-CMO-CH-HRP), and 17-α-OH-P-3-CMO-adipic acid dihydrazide-6-aminocaproic acid-HRP (17-α-OH-P-3-CMO-ADH-6ACA-HRP). The influence of different atomic length linkers on sensitivity, ED(50), and specificity were studied with reference to label without linker. The results of the present investigation revealed that the incorporation of ADH-6ACA spacer in 17-α-hydroxy-progesterone-enzyme conjugate improved the sensitivity in antigen plus bridge heterologous EIA system. The presence of spacer in enzyme conjugate improved the sensitivity and specificity (cross-reactivity) in some antigen plus bridge heterologous assay of progesterone.

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#22959769   2012/10/02 Save this To Up

Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles.

The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3'-diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal.

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#22203942   2011/12/28 Save this To Up

Immunohistochemical evaluation of hormone receptors with predictive value in mammary carcinomas.

Immunohistochemical evaluation of hormone receptors (ER, PR) and correlation of immunohistochemical and morpho-clinical data.

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#21707363   2011/06/28 Save this To Up

One-step monoclonal antibody based enzyme-linked immunosorbent assay for direct determination of cortisol in serum.

Monoclonal antibodies to cortisol have obvious potential advantages as starting materials for assay systems to detect their levels in body fluids. This is very important for monitoring pituitary gland and adrenal functions. To develop a one-step competitive heterogeneous enzyme-linked immunosorbent assay (ELISA), a monoclonal anti-cortisol antibody was generated using a reasonably designed haptenic derivative. Cortisol-3-O-carboxymethyloxime was coupled to carrier protein bovine serum albumin (BSA) to enhance its immunogenicity. Spleen cells were prepared from a BALB/c mouse, which had repeatedly been immunized with a conjugate of cortisol-3-O-carboxymethyloxime-bovine serum albumin (cortisol-3-O-CMO-BSA), to be fused with SP2/0 myeloma cells. After one fusion experiment, four hybridoma clones secreting a practical antibody were established. One of the resulting monoclonal antibodies, 2C9D11B5, showed an affinity constant (Ka) of 1.4 × 10(10) M(-1) for cortisol and provided a practical calibration curve (limit of detection [LOD], 0.26 ng per assay) in this ELISA system employing cortisol-21-hemisuccinate-horseradish peroxidase (cortisol-21-HS-HRP) as a tracer. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (3.5%), cortisone (0.47%), corticosterone (<0.01%), progesterone (<0.01%), 17-hydroxyprogesterone (1.2%), 6-hydroxycortisol (7.6%), and tetrahydrocortisol (<0.01%). The intra-assay and inter-assay coefficient of variations (CVs) ranged from 4.3% to 9.2% and 3.8% to 10.4 %, respectively. The analytical recoveries were 92.3% to 116.3%. Serum cortisol levels of healthy volunteers were determined after chilled acetone, stripped to be 292.76 ± 201.38 ng/mL (n=5), which are in the reference range.

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#19330648   2009/03/30 Save this To Up

Development of an immunoassay to measure progesterone using printed biosensors, and its application to the assessment of ovarian function in the numbat (Myrmecobius fasciatus).

A biosensor system was developed to measure progesterone levels in the urine of female numbats (Myrmecobius fasciata) as an index of ovarian function. Screen printed sensors were coated with a monoclonal progesterone antibody, and incubated in a mixture of sample/standard and progesterone-3-CMO-horseradish peroxidase (HRP). The difference in potential between the working and reference electrode was measured, after exposure to an HRP substrate. EIA and biosensor standard curves showed parallelism, and the biosensor gave values similar (r = 0.83) to the conventional EIA. Progesterone concentrations at different stages of the oestrus cycle were not significantly different to those obtained by EIA.

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#19330644   2009/03/30 Save this To Up

Development of ELISA for measurement of progesterone employing 17-alpha-OH-P-HRP as enzyme label.

The present study was aimed to develop a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) to measure progesterone in human serum using a heterologous combination of immunogen and enzyme conjugate. The antiserum was raised against Progesterone-3-O-carboxymethyloxime bovine serum albumin (P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime (17-alpha-OH-P-3-O-CMO) with Horseradish Peroxidase (HRP) to form 17-alpha-OH-P-3-CMO-HRP. A Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 100microL enzyme conjugate along with 50microL of standards in the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using tetramethyl benzidine/hydrogen peroxide (TMB/H(2)O(2)) as substrate. The enzyme substrate reaction was terminated with 100microL of 0.5 M H(2)SO(4) after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The lowest detection limit of the assay was 0.2 ng/mL. Cross-reaction with analogous steroids pregnenolone and 17-alpha-OH-P were found to be 6.8 and 6.1%, respectively. For other analogous steroids, it was less than 0.1%. The intra- and inter-assay coefficient of variation ranges from 4.52-7.39% and 4.65-9.55%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.91 (n = 40).

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#18360807   2008/03/24 Save this To Up

Development of rapid and sensitive one-step direct enzyme linked immunosorbent assay for 17-alpha-OH-progesterone in serum.

Using a homologous combination of immunogen and enzyme conjugate, a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) was developed to measure 17-alpha-hydroxy-progesterone (17-alpha-OH-P) in human serum. The antiserum was raised against 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime bovine serum albumin (17-alpha-OH-P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime with horseradish peroxidase (HRP). Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 25 microL enzyme conjugate along with 50 microL of standards on the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using Tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The enzyme substrate reaction was terminated with 100 microL of 0.5 M H2SO4 after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The detection limit of the assay was 180 pg/mL. The assay was more specific as compared to most other reported immunoassays for 17-alpha-OH-P. Cross reaction with analogous C18, C19, and C21 steroids was less than 0.1% except for progesterone which showed 2.1% cross reaction. The intra- and inter-assay coefficients of variation ranges from 3.7-7.5% and 6.9-11.7%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.9 (n=30).

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