Roberto Salerno

Expression and Function of Gonadotropin-releasing Hormone (GnRH) Receptor in Human Olfactory GnRH-secreting Neurons AN AUTOCRINE GnRH LOOP UNDERLIES NEURONAL MIGRATION

Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmanns syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3–4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.

Luminescent immunoassay (LIA) of cortisol—I. Synthesis and evaluation of two chemiluminescent labels of cortisol

Abstract Two cortisol-chemiluminescent marker conjugates, cortisol-21-hemisuccinate-aminobutyl ethyl-iso-luminol (C-21-HS-ABEI) and cortisol-21-hemisuccinate-aminobutyl-isoluminol (C-21-HS-ABI), were synthesized and evaluated as potential labels for the development of an immunoassay for cortisol based on chemiluminescence. Both conjugates were able to compete with tritiated cortisol for the binding sites of an antibody raised against a cortisol-21-hemisuccinate-BSA and they showed higher affinity than unaltered cortisol. These conjugates produced light upon oxidation by a hydrogen peroxide-microperoxidase system. The chemiluminescent reaction was optimized in terms of pH, concentration of oxidant and enzyme. Under optimal conditions both conjugates were detectable at femtomolar levels. The detection limits of C-21-HS-ABEI and C-21-HS-ABI were respectively 0.2 fmol and 1.0 fmol. These results indicated that immunoassay procedures for cortisol based on monitoring chemiluminescence can be developed with these labels.

Des (1-3) IGF-I-stimulated growth of human stromal BPH cells is inhibited by a vitamin D3 analogue

Prostate growth and differentiation is under the control of androgens not only during fetal life and childhood but also in adulthood, and it has been proposed that increased prostatic concentration of androgens, or increased androgen responsiveness, causes benign prostatic hyperplasia (BPH). However, different androgen ablation strategies such as treatment with GnRH agonists and finasteride resulted in a modest decrease of the hyperplastic prostate volume. In the last few years it became evident that both androgen-dependent and androgen-independent growth factors promote prostate enlargement by inducing cell proliferation or reducing apoptosis. Therefore, new therapeutic strategies, aimed at reducing intraprostatic growth factor signaling, are under investigation. In this study, we report further evidence that a non hypercalcemic-analogue of vitamin D(3), analogue (V) decreases growth factor-induced human BPH cell proliferation and survival. We found that Des (1-3) insulin-like growth factor [Des (1-3) IGF-I], an IGF-I analogue, which does not bind to IGF-binding proteins, is a potent mitogen for BPH stromal cells via a dual mechanism: stimulation of cell growth and inhibition of apoptosis. Similar results were previously reported for another growth factor for BPH cells, keratinocyte growth factor (KGF). Accordingly, we speculate that both KGF and IGF might be involved in the pathogenesis of BPH. We also found analogue (V) not only inhibits the mitogenic activity of growth factors on BPH cells, but even decreased the basal expression of bcl-2, and induced apoptosis. Therefore, vitamin D(3) analogues might be considered for the medical treatment of BPH.

Evaluation of different progesterone-isoluminol conjugates for chemiluminescence immunoassay

The effect of varying the length of the alkyl bridge linking the chemiluminescent label isoluminol to progesterone on the light yield and binding affinity of progesterone chemiluminescent-marker conjugates was investigated. For this purpose five different derivatives of isoluminol, aminoethyl isoluminol (AEI), aminoethylethyl isoluminol (AEEI), aminobutyl isoluminol (ABI), aminobutyl-ethyl isoluminol (ABEI) and aminoethyl-ethyl isoluminol (AHEI) were covalently linked through a peptide bond to progesterone-11 alpha -hemisuccinate (P-11-HS). The resulting progesterone chemiluminescent-marker conjugates were then evaluated as potential labels for the development of an immunoassay based on monitoring chemiluminescence. These conjugates were able to compete with tritiated progesterone for the binding sites of an antibody raised against progesterone-11 alpha-HS bovine serum albumin, and they showed higher affinity than unaltered progesterone. These conjugates produced light upon oxidation by a hydrogen peroxide-microperoxidase system. The chemiluminescent reaction was optimized in terms of pH, concentration of oxidant, catalyst and conjugate design. Under optimal conditions, all the conjugates were detectable at femtomolar levels. The lowest detection limit was obtained using P-11-HS-ABEI (0.1 fmol). These results indicated that immunoassay techniques based on chemiluminescence can be developed with these labels.

Luminescent immunoassay (lia) of cortisol—2. development and validation of the immunoassay monitored by chemiluminescence

Abstract An immunoassay procedure for determination of cortisol in human plasma and in urine is described, which utilizes chcmiluminescence as the end point. The assay utilizes cortisol-21-hemisuccinate-aminobutyl-ethyl-isoluminol (C-21-HS-ABEI) as the chemiluminescent marker, and dextran coated charcoal is used for separation of bound and free forms of the ligand. The chemiluminescent assay for cortisol was validated in terms of sensitivity, specificity, linear range and precision. An assay procedure for measuring of plasma and urinary free cortisol was established and assay results were compared with radioimmunoassay, using the same antiserum with tritiated cortisol as the label. The two methods agreed well ( r = 0.95 for plasma cortisol and r = 0.97 for urinary free cortisol). The proposed method takes full advantage of specificity and sensitivity that result from the use of an antibody, without employing an isotopically labelled ligand.

Determination of testosterone and its tissue metabolites (DHT and 3α-diol) in human plasma and prostatic tissue by isotopic dilution mass spectrometry

5 alpha-Dihydrotestosterone has been widely measured in human prostatic tissue using RIA since it is involved in the pathogenesis of human prostatic hyperplasia and seems to be the best index for the follow-up of patients affected by prostatic cancer under endocrine treatment. A GC-MS method for the simultaneous determination of testosterone (T), 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol) in prostatic tissue based on the isotopic dilution technique was developed. Tri-deuterated internal standards of each compound were previously synthetized in our laboratory. After extraction and purification on Sep-Pak C18 and Sephadex LH-20, T and its metabolites were measured as heptafluorobutyric ester (HFB) derivatives. Quantitative analysis was performed on a VG 7070 EQ mass spectromer equipped with a fused silica capillary column using the Selected Ion Monitoring technique. Steroid values (mean +/- SD; ng/g tissue) found in nine human hypertrophic prostates were: T: 0.71 +/- 0.43; DHT: 4.46 +/- 1.41; 3 alpha-diol: 0.34 +/- 0.23. Preliminary results obtained from the detection of the three androgens in human prostatic hyperplasia treated for 3 months with GnRH before surgery seem to indicate that DHT concentration decreases more than 10 times. Values obtained (n = 1; ng/g tissue) were: T: 0.194; DHT: 0.255; 3 alpha-diol: 0.015.

Quantitative determination of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, in human plasma, using isotope dilution mass spectrometry

An original method is described for the determination in human plasma of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, by isotope dilution mass-spectrometry using 7,7-[2H2]-4-OHA as internal standard. This compound was synthesized starting from 7,7-[2H2]-4-androstene-3,17-dione. The procedure includes an extraction step using an Extrelut 1 column and a derivatization with N,o-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The minimum detection level of the method is 0.650 pg and the coefficients of variation for the 0.5 ng/ml (plasma) and 5 ng/ml (plasma) concentrations are 3.2% (within assay) and 6.7% (between assay) and 1.86% (within assay) and 2.3% (between assay) respectively.

Measurement of testosterone and its 5-alfa-reduced metabolites in human prostatic tissue using isotope dilution mass spectrometry

5 alpha-Dihydrotestosterone (DHT) has been widely measured in human prostatic tissue using RIA since it is found to be involved in pathogenesis of human prostatic hyperplasia (BHP) and to be the best index for the follow-up of patients affected by prostatic cancer under endocrine treatment. A GC/MS method for the simultaneous determination of testosterone (T), dihydrotestosterone (DHT), 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstan-3 beta-17 beta-diol (3 beta-diol) in prostatic tissue was developed based on the isotopic dilution technique. Trideuterated internal standards of each compound were previously synthesized in our laboratory. After previous extraction and purification on Sep-Pak C-18 cartridges and Lipidex DEAP columns, T and its metabolites were measured as heptafluorobutyric esters (HFB). Quantitative analysis was performed on a VG 7070 EQ mass spectrometer equipped with a fused silica capillary column using the Selected Ion Monitoring technique. Steroid values (means +/- SD; ng/g tissue) found in 9 human hypertrophic prostates were: T, 0.71 +/- 0.43; DHT, 4.46 +/- 1.41; 3 alpha-diol, 0.34 +/- 0.23; 3 beta-diol, 0.14 +/- 0.32.

Luminescent immunoassay (LIA) methods for steroid hormones

The application of luminescence to the development of non-isotopic immunoassay methods for steroids and urinary steroid metabolites is reviewed. On-line computer analysis of the light emission is particularly useful, as it reveals the interfering effects of biological compounds on the reaction and this can improve the quality of luminescence immunoassay (LIA) methods. The main characteristics of chemiluminescent tracers are stability, safety, speed and sensitivity of detection, and reliability. Homogeneous methods, not requiring phase separation, have also been reported and validated. Heterogeneous methods which use dextran-coated charcoal or solid-phase techniques can be used for direct determination of urinary steroids in unextracted samples.

Effects of surgery and epidural or general anaesthesia on testosterone, 17-hydroxyprogesterone and cortisol plasma levels in prepubertal boys.

Testosterone (T), 17-hydroxyprogesterone (17P) and cortisol (F) plasma levels have been measured in two groups of prepubertal boys before and during surgery under general anaesthesia (Group 1) and epidural anaesthesia (Group 2) respectively. The mean plasma levels of T, 17P and F increased significantly (P less than 0.05; P less than 0.001; P less than 0.005, respectively) during surgery in Group 1; in Group 2 the plasma levels of T and F did not show any significant variation, whereas 17P significantly increased (P less than 0.05). However the mean level reached by 17P in Group 2 was significantly lower (P less than 0.005) than that observed in Group 1. No significant variation of LH and FSH plasma levels was observed in either group. Our report suggests that the modifications of T and 17P plasma levels observed during surgery under general anaesthesia (GA) are probably due to the stress induced adrenal response. This response can be inhibited or reduced by epidural anaesthesia (EA).