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.

Carbon nanotube enhanced label-free immunosensor for amperometric determination of oocyte maturation-inducing hormone in fish.

Maintaining high-quality fish eggs stably and efficiently is important for aquaculture. We developed a label-free immunosensor system for measuring 17,20ß-dihydroxy-4-pregnen-3-one (DHP). DHP is suddenly secreted before ovulation as a maturation-inducing hormone in fish, and therefore, DHP levels are an indicator for predicting ovulation. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry (CV). For biomolecular immobilization on the surface of sensing electrode, Au electrode, we used self-assembled monolayers of thiol-containing compounds to fix anti-DHP immunoglobulin. In addition, we used a single-walled carbon nanotube to improve sensitivity. Using this electrode, we were able to determine the CV signal change caused by the antigen-antibody complex. The proposed immunosensor system showed a linear correlation (correlation coefficient: 0.9827) between the anodic peak current of the CV and the DHP level in range from 15.6 to 50,000 pg ml(-1). The sensor system was then applied to monitor DHP of goldfish (Carassius auratus). Blood plasma of fish was collected every 3 h after administering a DHP inducer. In the measurement, the anodic peak current of the CV showed distinct changes depending on DHP levels in the blood plasma. A good relationship was observed between DHP levels determined by our proposed system and the conventional method (correlation coefficient: 0.9351).

Monoclonal antibodies to progesterone: characterization and selection for enzyme immunoassay in bovine milk.

Thirty-one stabile murine monoclonal antibody (MAb) producing cell lines to progesterone were generated by using a short and a long immunization protocol. Long-term immunization with high doses of 11alpha-hydroxyprogesterone-hemisuccinate-bovine serum albumin (11alpha-OH-P-HS-BSA) antigen led to very good antibody response in Balb/c mice. The donor mouse produced antiserum with a high titre of 1/250,000. Eleven MAbs were selected for further characterization since they showed high sensitivities (<35 pg/well to inhibit 50% of the tracer) in bridge homologous enzyme immunoassay (EIA). The results were compared to the donor mouse polyclonal antiserum. The MAbs and the donor mouse antiserum were generally found to be highly specific, when tested with 30 different steroids. Employing MAb 9C11, with affinity constant, K(alpha), to 11alpha-OH-P-HS of 1.1 x 10(10) M(-1), a bridge heterologous microtitre plate EIA for milk progesterone was developed, using the second-antibody coating technique and horseradish peroxidase (HRP) as an enzyme label. The assay is simple and convenient to use, as it permits direct addition of undiluted milk samples, at the same time maintaining high sensitivity, high precision, and a wide range of optical density (OD) values. The major advantage of the assay developed, compared to previously published direct addition milk progesterone immunoassays, is that progesterone concentrations, measured by the EIA, were not influenced by changing milk fat concentrations, even when milk samples containing up to 10% of milk fat were used for analysis.

Reproductive steroids during maturation in a primitive teleost, the Pacific herring (Clupea harengus pallasi).

Concentrations of reproductive steroids were measured in the plasma of captive Pacific herring, Clupea harengus pallasi, (1) prior to ovulation and milt production, (2) during the periovulatory and newly milt-producing period, (3) during a "ripe" holding period after ovulation and during milt production, and (4) before and after spawning. 17alpha,20beta-Dihydroxyprogesterone (17,20beta-P), despite being present only in low concentrations in the unconjugated (free) form (<10 ng/ml), is likely to be the maturation inducing steroid in females and was associated with the initiation of milt production in males since its levels are elevated coincidentally with these events. Glucuronated 17,20beta-P, free 17alpha-hydroxyprogesterone (17-P), and free and glucuronated 3alpha, 17alpha-dihydroxy-5beta-pregnan-20-one (3alpha,17-P-5beta) were present in high concentrations (140-250 ng/ml) in periovulatory females and newly milt-producing males. This steroid pattern suggests that the low levels of 17,20beta-P are due to glucuronation and competitive conversion of its precursor, 17-P, to free and glucuronated 3alpha, 17-P-5beta. Glucuronated testosterone was the principal steroid in preovulatory and premilt-producing fish (200-350 ng/ml), coincident with similar levels of glucuronated 11-ketotestosterone in males. After ovulation females did not spawn synchronously until 2 months later, which may be partially due to reduced environmental cues in the captive situation, while male fish released milt sporadically throughout the ripe holding period. Steroidal indicators of readiness to spawn in females or males were not detected. Rather, levels of all steroids gradually decreased in ripe holding fish (<30 ng/ml) to reach even lower levels (<1 ng/ml) after spawning. We suggest that "runniness" of gametes is a distinctive characteristic of females that are ready to spawn, but that this may result from relaxation of sphincter muscles rather than being an additional maturational step.

[A simplified method in determination of blood 17-hydroxyprogesterone by radioimmunoanalysis]. / Método simplificado para medir 17-OH progesterona plasmática por radioinmunoanálisis.

A practical method to measure 17-OHP, in peripheral blood by radioimmunoanalysis using a highly specific antiserum, is described. The method usefulness described in dependability studies present a sensitivity of the pattern curve of 10 picograms; precision shows a variation rate intranalysis < or = 11.9% and interanalysis of < or = 10.0%. Accuracy was > or = 95% and specificity is demonstrated by antiserum characterization with other steroids. Measuring 17-OHP4 in ng/ml in plasma of women under different physiological conditions, it was found that the levels of this hormone on day -5 of proliferative phase were 0.31 +/- 0.24 nanograms (ng)/ml, and at day +7 of secretory phase of 1.47 +/- 0.65 ng/ml. In patients with stimulation with ACTH show as to the basal sample an increase (double) of levels at 30 minutes, that keeps increasing until 90 minutes with certain trend to diminish at 120 minutes. This study shows that it is possible, to measure 17-OHP4 in human plasma with good dependability degree and easy handling reducing, in addition, operative cost.

Autoimmune progesterone dermatitis: absence of contact sensitivity to glucocorticoids, oestrogen and 17-alpha-OH-progesterone.

Autoimmune progesterone dermatitis is a rare condition, characterized by recurrent premenstrual exacerbations of a dermatosis, in which sensitivity to progesterone can be demonstrated. The sensitizing mechanism is unknown. The aim of this study was to test the hypothesis that cross-sensitivity between steroid groups could induce allergy to endogenous progesterone in these patients. 5 patients with autoimmune progesterone dermatitis and 1 with oestrogen-sensitive dermatitis have been patch tested with a corticosteroid series, conjugated oestrogen 1% in petrolatum (pet.), and 17-alpha-OH-progesterone 2% pet. There were no immediate or delayed reactions at 2 and 4 days to any steroid group. We have therefore been unable to demonstrate steroid cross-sensitivity, or a use for 17-alpha-OH-progesterone in the investigation of oestrogen - and progesterone-sensitive dermatoses.

Monoclonal antibodies against progesterone: effect of steroid-carrier coupling position on antibody specificity.

Monoclonal anti-progesterone antibodies were raised by immunizing mice with progesterone coupled through either the C3, C6 or C11 positions to protein carrier (bovine serum albumin, BSA). The specificity of four antibodies for a range of steroids related to progesterone, some carrying substitutions at various ring positions, was studied by competitive inhibition in an ELISA system. The results demonstrated that the ring coupling position has a determining effect on the cross-reactivity of the antibodies obtained. The patterns of cross-reaction were interpreted in the light of the structure of the combining site of an anti-progesterone antibody (DB3) recently determined by X-ray crystallography, and inferences drawn about the orientation of steroid in the combining sites of the antibodies studied. Specifically, in two antibodies raised against progesterone-11-BSA, the orientation of steroid resembled that of the progesterone-DB3 complex, with positions C11 and C3 exposed and C6 and C20 buried; an antibody raised against progesterone-6-BSA bound steroid in an apparently similar disposition, except that C6 was exposed and C11 buried; finally, in an antibody raised against progesterone-3-BSA, all steroid positions other than C3 were apparently buried in the steroid-antibody complex.

Analysis of blood spot 17 alpha-hydroxyprogesterone concentration in premature infants--proposal for cut-off limits in screening for congenital adrenal hyperplasia.

Blood Spot 17 alpha-hydroxyprogesterone (17-OHP) concentrations in neonates, especially in premature babies, were determined in relation to 1) the gestational age at birth, 2) the equivalent age of gestation at blood sampling and 3) the birth weight. The 17-OHP concentrations were found to be higher with prematurity. Accordingly, the cut-off limit in screening for congenital adrenal hyperplasia (CAH) in premature infants is proposed as 20 ng/ml. Ideal cut-off limits were set by the equivalent age of gestation at blood sampling. Cut-off limits on the basis of gestational age at birth and birth weight are also suggested, where the sampling age is not so advanced. The rate of false positivity in premature infants can be reduced by this method.

Molecular characterization of monoclonal anti-steroid antibodies: primary structures of the variable regions of seven antibodies specific for 17 alpha-hydroxyprogesterone or 11-deoxycortisol and their pH-reactivity profiles.

The variable region nucleotide sequences of seven monoclonal anti-steroid antibodies that are specific for the closely related progesterone derivative, 11-deoxycortisol or 17 alpha-hydroxyprogesterone (17-OHP), have been determined by genomic cloning and DNA-sequencing or by direct mRNA-sequencing. As for their heavy chain variable regions, the nucleotide sequences of the SCET.M8.1 (SCET) and OHP 4B2.2.1 (4B2) antibodies were classified into the VH-9 family, while OHP 7D7.2.3 (7D7), 1E9.3.1 (1E9), 57.G6.1 (57) and 138.H8.1 (138) used VH-3 family genes. OHP 101.B11.1 (101) used a gene of the VH-1 family. For their light chain variable regions, SCET and 57 used VK-28 group genes, while 4B2, 7D7, 1E9, 101 and 138 antibodies used genes of the VK-21 subgroups (21A, 21B or 21C). All of the antibodies used different combinations of genes in the VH families and VK groups or subgroups. This indicates that the antibody response against the steroid hapten, 17-OHP, is fairly polyclonal, and several VH/VL combinations show high affinity for progesterone-related steroids. Although the primary structures of hypervariable loop regions of the mAbs were relatively diverse, generally, hydrophobic and aromatic amino acids were rich in these regions. Moreover, the length of heavy chain CDR3 was constant in all the antibodies investigated in this paper as well as the previously reported anti-progesterone monoclonal antibodies (mAbs). This suggests that the length of VH CDR3 in these mAbs has a considerable influence on the formation of antigen-combining pockets. The pH-reactivity profiles for the anti-17-OHP mAbs indicated that the the steroid-mAb binding was independent of pH between pH 4 and 11 in most of the mAbs. The results suggest that the steroid-mAb interactions are not largely affected by the electrostatic environments near the combining sites of these mAbs. Taken together, these data imply that the shape of hydrophobic depressions in the combining sites is important for the binding of relatively large, hydrophobic and rigid haptens like steroids.

Direct solid-phase time-resolved fluoroimmunoassay of 17 alpha-hydroxyprogesterone in serum and dried blood spots on filter paper.

We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (17OHP) in serum and blood spots on filter paper. We used 17OHP-3-carboxymethyloxime (17OHP3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of 17OHP, for a very high specific activity. The assay is based on competition between labeled 17OHP3CMO and 17OHP in blood specimens for polyclonal rabbit anti-17OHP antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace 17OHP from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of 17OHP (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.

Erythema multiforme associated with autoreactivity to 17 alpha-hydroxyprogesterone.

We report a case of a 23-year-old woman who was afflicted with disseminated skin erythema multiforme-like eruptions that started at the menarche, relapsed at the premenstrual periods, dramatically spread during two pregnancies and cleared after abortion; the skin lesions responded dramatically to thalidomide treatment. A high-affinity binding factor to 17 alpha-hydroxyprogesterone (17-OHP) was found in the serum of this patient. Her lymphocytes did not proliferate in vitro after exposure to exogenous 17-OHP but showed significant chromatin activation. There was a decreased expression of HLA antigens at the surface of the patient's blood lymphocytes. This is a unique well-documented case of erythema multiforme most possibly due to autoreactivity to 17-OHP; the precise mechanism(s) of this autoreactivity has not been established.

[Enzyme immunoassay of 17-hydroxyprogesterone in dried blood spot on filter paper using specific antibody for 17-hydroxyprogesterone].

Specific antiserum for 17-hydroxyprogesterone (17-OH-P) was prepared by immunizing 7 alpha-(2-carboxyethylthio)-17-OH-P conjugated bovine serum albumin (BSA) in rabbits. Using this antiserum, 17-OH-P enzyme immunoassay for dried blood spots on filter paper was established. As a label, alkaline phosphatase was coupled covalently with 7 alpha-carboxy-methylthio-17-OH-P by carbodiimide method. B/F separation was carried out by the addition of anti-rabbit IgG goat antiserum. All specimens used were punched out with a paper puncher of 3mm diameter. The assay sensitivity was 2pg/tube, which was estimated by two standard deviation at zero concentrations of the calibration curve. Cross reactivities of this antibody were as follows: 11-deoxycortisol (8.21%), 17-OH-pregnenolone (3.33%), progesterone (1.67%), 11-deoxycorticosterone (0.31%), cortisol (0.16%), pregnenolone-3-sulfate Na salt (0.03%), dehydroepiandrosterone (DHEA) (less than 0.03%), 16 alpha-OH-DHEA (less than 0.03%), DHEA-3-glucuronide (less than 0.03%), DHEA-3-sulfate Na salt (less than 0.03%), pregnenolone (less than 0.02%). Intra- and inter-assay coefficient of variations were 4-14% and 9-18%, respectively. In normal babies, 17-OH-P concentrations measured directly (without sample extraction) were below 23pg/disk (n=204). The histogram of 17-OH-P level in normal babies obtained by the direct method was distributed lower than that obtained by the enzyme immunoassay system (range: 4-79pg/disk, n=268) which used antibody raised against 17-OH-P-3-O-carboxymethyloxime conjugated BSA (Enzaplate, Sapporo Diagnostic Laboratory).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of 11 alpha-hydroxyprogesterone haptens.

C-4 and C-6 bridged haptens of 11 alpha-hydroxyprogesterone, an anti-androgen, were respectively synthesized via 4, 5 and 5 alpha, 6 alpha epoxide ring openings using 3-mercaptopropanoic acid. Substitution of 6-bromo derivative of progesterone using the same reagent unexpectedly afforded a C-4 substituted product instead of the normal C-6 substituted product previously reported in the literature.

Influence of functional groups on homologous antibody affinity of 11 alpha-hydroxyprogesterone derivatives. I. Synthesis and affinity evaluation.

Syntheses and cross-reactivities with progesterone toward the same specific antibody are reported for a series of amides of 11 alpha-hydroxyprogesterone 11-hemisuccinate. Some hypotheses are made regarding the effects of the chemical structure of the substituents on the immunological properties of derivatives.

Production and characterization of monoclonal antibodies to 17 alpha-hydroxyprogesterone.

Hybridoma clones producing antibodies to 17 alpha-hydroxyprogesterone (17-OHP) were established by using a 17-OHP-bovine serum albumin conjugate as an immunogen. Six representative IgG-class monoclonal antibodies of high affinity (10(8)-10(9) M-1) showed differential reactivities with several structurally related steroids. Two enzyme immunoassay (EIA) systems (fluorescence EIA and micro-EIA) for 17-OHP using OHP 4B2.2.3, which showed the lowest cross-reactivity with other steroids, were established. The micro-EIA system was shown to be applicable to the mass-screening of congenital adrenal hyperplasia.

The production and characterisation of antisera to 17-hydroxyprogesterone.

Sheep were immunised with 11-deoxycortisol-21-hemisuccinate-bovine serum albumin (11-deoxycortisol-21-HS-BSA) or with 17-hydroxyprogesterone-7 alpha-carboxyethyl thioether-keyhole limpet haemocyanin (17-OHP-7 alpha-CETE-KLH) or with 17-OHP-3-(O-carboxymethyl)oxime-KLH (17-OHP-3-CMO-KLH). The resultant antisera were assessed using [3H]17-OHP and dextran-coated charcoal to separate the antibody bound and free fractions. All sheep produced antisera with an apparent affinity constant of from 1.4 to 6.6 X 10(9) 1/mol. Those raised against 11-deoxycortisol-21-HS-BSA had titres ranging from 1:12,000 to 1:78,000 but showed significant cross-reactivity with many of the steroids tested. Sheep immunised with 17-OHP-7 alpha-CETE-KLH had antisera titres of from 1:102,000 to 1:180,000 and only 17-hydroxypregnenolone cross-reacted significantly (10-20%). The best antisera were raised in sheep immunised with 17-OHP-3-CMO-KLH. Titres ranged from 1:168,000 to 1:390,000 and there were about 8 g/l of specific antibodies which cross-reacted 5.7% or less with 17-hydroxypregnenolone, and less than 0.5% with progesterone, 11-deoxycortisol and the other steroids studied. The antisera to 17-OHP-3-CMO-KLH were further assessed using [125I]17-OHP; titres ranged from 1:5,700,000 to 1:18,000,000 with affinity constants of from 1.67 to 2.5 X 10(10) 1/mol. They showed minimal or no cross-reactivity with the steroids studied. Reimmunisation after an 8-month interval produced antisera with a higher affinity constant and even lower cross-reactivity with other steroids.

Possible synthesis of steroid-protein for immunization with a fixed narrow range of the hapten-protein ratio.

A comparison of two methods for synthesis of a steroid-protein conjugate (progesterone-11 alpha-hemisuccinate-BSA) has been made. Under the conditions of the method described, using tetrahydrofurane as a medium for the coupling reaction, stable intermediate products were obtained. The resulting conjugate had a narrow range of the hapten-protein ratio (18-20 steroid molecules per molecule of BSA), very good solubility in water and almost no unreacted BSA in the sample. Antisera raised against this conjugate, applied in low dose (50 micrograms), had quite satisfactory characteristics concerning their titre and specificity. The tests for recovery, sensitivity and the range of measurement established the possibility of using such antisera for the radioimmunoassay of progesterone.

Mathematical analysis of antiserum titre and affinity in rabbits injected with 11 alpha OH-progesterone-hemisuccinate-BSA.

A mathematical procedure is presented here to determine the behaviour of antiserum titre in rabbits, after repeated injections of 11 alpha OH-Progesterone-hemisuccinate-BSA. By means of methods recently applied to the time series, the peaks of maximum response were determined. A common behaviour of rabbits was revealed for the delay times in the response after each booster injection. The titre and affinity of antiserum were determined by means of a special method for mathematical treatment of data in RIA analysis, which gives directly such parameters with a statistical interpretation.