HLA-G-mediated NK cell senescence promotes vascular remodeling: implications for reproduction.

The uterus in early pregnancy is a non-lymphoid organ that is enriched in natural killer (NK) cells. Studies to address the role of these abundant human NK cells at the maternal/fetal interface have focused on their response to the major histocompatibility complex (MHC) molecules on fetal trophoblast cells that they contact. The interaction of maternal NK cell receptors belonging to the killer cell immunoglobulin-like receptor (KIR) family with trophoblast MHC class I molecules in pregnancy can regulate NK cell activation for secretion of pro-angiogenic factors that promote placental development. This review will cover the role of KIR at the maternal/fetal interface and focus on KIR2DL4, a KIR family member that is uniquely poised to play a role in pregnancy due to the restricted expression of its ligand, human leukocyte antigen (HLA)-G, by fetal trophoblast cells early in pregnancy. The pathways by which KIR2DL4-HLA-G interactions induce the cellular senescence of NK cells and the role of the resulting senescence-associated secretory phenotype (SASP) in vascular remodeling will be discussed in the context of reproduction.

Dienogest improves human leucocyte antigen-DR underexpression and reduces tumour necrosis factor-α production in peritoneal fluid cells from women with endometriosis.

OBJECTIVE: To determine the immunological effect of dienogest (DNG), an oral anti-endometriosis drug, on peritoneal fluid (PF) macrophages collected from women with endometriosis. Although it has been suggested that DNG has direct effects on endometriotic cells, including decreased cell proliferation and decreased anti-inflammatory cytokine production, the effects of DNG on PF cells are unclear. STUDY DESIGN: The effects of DNG on PF cells from 34 women with endometriosis and 22 women without endometriosis (controls) were investigated. Expression of human leucocyte antigen (HLA)-DR in PF macrophages, obtained from the peritoneal cavity during laparoscopic surgery, was determined by flow cytometry. HLA-DR expression was measured again after PF cells had been cultured for 72 h in a humidified atmosphere at 37 °C in 5% CO2-95% air with or without DNG. After 72 h of incubation, the concentration of pro-inflammatory tumour necrosis factor (TNF)-α in the media was measured by enzyme-linked immunosorbent assay. RESULTS: HLA-DR expression was lower in PF macrophages from women with endometriosis compared with controls. However, after DNG treatment, HLA-DR expression in PF macrophages from women with endometriosis was increased to the same level as in controls. The TNF-α concentration in the media was decreased by DNG. CONCLUSIONS: DNG can restore the antigen-presenting ability of PF macrophages by increased HLA-DR expression, and may have an anti-inflammatory effect on PF macrophages in women with endometriosis.

Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting 19-nortestosterone residue in animal tissues.

This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and ß-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 µg/kg in beef, and 2 µg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.

Production and characterisation of monoclonal antibodies against 19-Nortestosterone.

OBJECTIVE: To produce anti-19-Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. METHODS: Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate (CAAP) method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff). RESULTS: Five hybridoma cell lines, named NT-1, NT-2, NT-3, NT-4, and NT-5, were identified and their corresponding mAbs were of the IgG(1) isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7 × 10(9) L/mol. The titers and IC(50) values of purified ascite fluids were in the range of (0.64-2.56) × 10(5) and (0.55-1.0) ng/mL, respectively. Of all the cross-reacting steroids, (-NT was the most reactive with the mAbs at 62% with NT-1 mAb and 64% with NT-2 mAb. Negligible cross-reactivity (<0.01%) with other steroids was observed. CONCLUSION: The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.

Development of a solid-phase extraction-enzyme-linked immunosorbent assay for the determination of 17beta-19-nortestosterone levels in antifatigue functional foods.

17beta-19-nortestosterone (17beta-NT) has been illegally used in antifatigue functional foods to promote muscle growth and improve endurance. A rapid and sensitive solid-phase extraction-enzyme-linked immunosorbent assay (SPE-ELISA) method was developed and successfully applied to analyze the levels of 17beta-NT in antifatigue functional foods. A polyclonal antibody against 17beta-NT was produced from rabbits immunized with the 17beta-NT-BSA conjugate, and a competitive direct enzyme-linked immunosorbent assay was developed for the rapid detection of 17beta-NT. The concentration causing 50% inhibition (IC(50)) and the limit of detection (LOD) were found to be 0.08 and 0.0055 ng/mL, respectively; this was better than methods previously reported that had a LOD of 2.4 ng/mL. C(18) cartridges were investigated for use in removing the effects of matrix in foods, and the sample purification protocol was optimized. Using the developed SPE-ELISA method, recoveries of functional food samples were obtained in the range of 71% to 91.5%. Moreover, 2 kinds of antifatigue functional foods were analyzed using the established ELISA and HPLC methods. The correlation coefficient of the results obtained using the 2 methods was greater than 0.98. Thus, the preliminary evaluation of the SPE-ELISA method proved that it is a specific, sensitive, and precise tool that can be used for the practical detection of 17beta-NT in various antifatigue functional food samples.

Simultaneous determination of nandrolone, testosterone, and methyltestosterone by multi-immunoaffinity column and capillary electrophoresis.

A multitarget antibody immunoaffinity column was proposed for the purification and enrichment of nandrolone, testosterone, and methyltestosterone from urine. Nandrolone-3-site substituted antigen was designed and synthesized and the polyclonal antibody was prepared with immunizing rabbits. The stationary phase of the immunoaffinity column was synthesized by covalently bonding the antibodies specific to nandrolone, testosterone, and methyltestosterone onto CNBr-actived Sepharose 4B. The analytes of interest were extracted with a methanol/water mixture in one step. The immunoaffinity column showed high affinity and high selectivity to a class of structurally related compounds. The elution was then transferred to a micellar electrokinetic CE system with a running buffer of sodium borate and sodium cholate for separation and determination. Recoveries of the three steroids from complex matrix were 88-94% with RSD values less than 5.2%. Optimization of the immunoaffinity column purification was achieved and the feasibility of the technique for the analysis of steroid hormone was discussed. The results indicated that the combination of multi-immunoaffinity column and CE was an effective technique, which was rapid, simple, and sensitive for the assay of steroids.

Time-resolved fluoroimmunoassay for 19-nortestosterone residues in aquaculture tissues.

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of 19-nortestosterone (17beta-NT) residues in aquaculture tissues. The limit of detection (LOD) was determined to be 0.08 ng g-1 and the limit of quantification (LOQ) was less than 0.8 ng g-1. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by liquid chromatography tandem mass spectrometry (LC/MS/MS). This proposed technique could be applied to routine residue analysis.

A rapid and sensitive 384-well microtitre format chemiluminescent enzyme immunoassay for 19-nortestosterone.

We developed a competitive chemiluminescent (CL) enzyme immunoassay for rapid, sensitive analysis of 19-nortestosterone (19-NT) in bovine urine. Anti-19-NT polyclonal antibodies were raised in rabbits using a 19-NT-hemisuccinate derivative conjugated with ovalbumin; the derivative was also conjugated with horseradish peroxidase (HRP) as a label. Antibodies were immobilized on 384-well black polystyrene microtitre plates and HRP-labelled 19-NT activity was measured using an efficient chemiluminescent substrate (SuperSignal ELISA Femto) after 3 min incubation. Emitted light was recorded using a conventional, photomultiplier-tube-based microtitre plate reader or a sensitive back-illuminated, cooled CCD camera. The developed method fulfils all the requirements of precision (intra- and inter-assay CV < 10%) and accuracy (mean recovery 94-112%), with a detection limit of 0.03 ppb (1.1 x 10(-9) mol/L) in a urine matrix. Chemiluminescence enhances detectability of the HRP-labelled tracer (thus lowering the limit of detection with respect to colorimetry) and reduces analysis time. The 384-well microtitre plate cuts the sample/reagent volume (20 microL), a five-fold reduction with respect to the conventional 96-well microtitre plate. The developed method is suitable for high-throughput screening of 19-NT in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays.

Comparison of the efficiences of enzymatic and chemical hydrolysis of (nortestosterone and diethylstilboestrol) glucuronides in bovine urine.

Residues of 19-nortestosterone (19-NT) and diethylstilboestrol (DES) are excreted in bovine urine, mainly conjugated to glucuronic acid. Prior to quantification, urine must be deconjugated, which is commonly performed by enzymatic or chemical hydrolysis. The efficiencies of two enzymatic and two chemical deconjugation methods were studied. The range of efficiencies obtained for DES were 51.8% (beta-glucuronidase, incubation at 37 degrees C overnight) and 2.7% (methanolic HCl), respectively. Similarly, efficiencies for NT ranged from 43.1% (beta-glucuronidase, incubation at 55 degrees C for 2 h) to 12.7% (methanolic HCl). The results highlight that within control laboratories significant underestimation of drug residue content in samples may occur, due to poor deconjugation.

Development of a competitive enzyme immunoassay for 17 alpha-19-nortestosterone.

Because 17 beta-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17 alpha-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17 alpha-19-nortestosterone-3-carboxy-methyloxime-bovine serum albumin (17 alpha-19-NT-3-CMO-BSA), the competitive incubation of 17 alpha-19-NT and the 17 alpha-19-nortestosterone-3-CMO-horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17 alpha-19-nortestosterone was used to produce an antibody with selective affinity for the 17 alpha-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B0 50% of +/- 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples.

Immunoaffinity pre-column for selective on-line sample pre-treatment in high-performance liquid chromatography determination of 19-nortestosterone.

A liquid chromatographic column-switching system for automated sample pretreatment and determination of the anabolic hormone beta-19-nortestosterone (beta-19-NT) and its metabolite alpha-19-nortestosterone (19-norepitestosterone) in calf urine is described. The system consists of an immunoaffinity pre-column (immuno pre-column) packed with Sepharose-immobilized polyclonal antibodies against beta-19-NT, a second pre-column packed with C18 bonded silica and an analytical C18 column. Urine (25 ml) is directly loaded on the immuno pre-column, where the analytes of interest are trapped by the immobilized antibodies. Next the analytes are desorbed selectively with a solution containing an excess of the cross-reacting steroid hormone norgestrel and transferred, via the second pre-column, to the analytical column. The recovery of beta-19-NT in spiked urine samples was over 95%. The detection limit was 50 ng/l for a 25-ml urine injection. The system showed no loss of analytical performance over a 6-month period, during which about 100 samples were analysed with the same immuno pre-column. The general applicability of this sample pretreatment method is discussed.

[Bovine serum albumin conjugates of the new progestagen dienogest, synthesis and immunogenic properties]. / Rinderserumalbuminkonjugate des neuen Progestagens Dienogest--Syntheseund immunogene Eigenschaften.

In order to develop a radioimmunoassay for the new progestagen dienogest (STS 557, 17 alpha-cyanomethyl-17 beta-hydroxy-estra-4,9-dien-3-one), bovine serum albumin (BSA) conjugates of STS 557-3-carboxymethyloxime and of STS 557-11-hemisuccinate were synthesized as antigens for the production of antisera. It was proved that an excess of isobutylchlorocarbonate in the coupling reaction using the "mixed anhydride method" results in an acylation of free NH2-groups in the BSA. By the immunization of rabbits with the STS 557-antigen-antisera of high specificity and affinity to STS 557 were produced. Endogenous steroids show no cross reaction with the STS 557-antisera. Steroids with a 17 alpha-CH2CN-group, being obtained by chemical synthesis or microbial transformation, compete with STS 557 for the binding positions of the antibodies to a different extent.

19-nortestosterone, a model for the use of anabolic steroid conjugates in raising antibodies for radioimmunoassay.

1. The raising of antibodies in Soay sheep to an immunogenic conjugate of 19-nortestosterone-3-(O-carboxymethyl)oxime with bovine serum albumin is described. 2. High titre plasma so obtained can be used for the radioimmunoassay of 19-nortestosterone. 3. Cross-reactivities of the antibody with endogenous steroids is confined to testosterone (20-25%) and 17beta-estradiol (1-2%). Cross-reactivities with a range of other 17beta-hydroxy synthetic anabolic steroids and with some potential 17beta-hydroxy metabolites of 19-nortestosterone vary between 2-3% for 5beta-estrane-3alpha,17beta-diol and 26% for 1-dehydrotestosterone. There is limited cross-reactivity (ca. 1-2%) with anabolic steroids with 17beta-hydroxy, 17alpha-methyl substituents. 4. 19-Nortestosterone (phenyl propionate) administered to a horse (1 mg/kg, intra-muscular) gives urinary metabolites readily detectable by radioimmunoassay, for more than 4 days.