Microassay for measurement of binding of radiolabelled ligands to cell surface molecules

An improved technique for measuring the binding of radiolabelled ligands to cell surface molecules has been developed by modification of a procedure using centrifugation through a water-immiscible oil to separate free and cell-bound ligand. It maximises the percentage of ligand bound since cell-bound and free ligand can be separated easily and reproducibly even when very small reaction volumes are used. This permits low levels of ligand radiolabelling and relatively low numbers of cells to be used.     

IL—1对输精管结扎大鼠睾丸间质细胞功能影响的体外研究

本文观察ＩＬ－１对正常和输精管结扎大鼠睾丸间质细胞（Ｌｅｙｄｉｇｃｅｌｌｓ，莱迪希细胞）细胞功能的影响，结果表明：在人绒毛膜促性腺激素（ｈｕｎａｎｃｈｏｒｉｏｍｉｃｇｏｎａｄｏｔｒｏｐｉｎｈＣＧ）的作用下，正常和输精管结扎大鼠睾丸Ｌｅｙｄｉｇ细胞培养上清中睾酮和ｃＡＭＰ的含量均明显高于相应的对照孔，且两组比较无明显差异，说明输精管结扎后睾丸Ｌｅｙｄｉｇ细胞对垂体激素的反应性并无改变，两组细胞受ＩＬ     

Relationship of free and bound testosterone to sexual behavior in old rhesus males

Total serum testosterone concentrations, percentage of total serum testosterone bound to testosterone-binding globulin (TeBG), and estimates of free testosterone concentrations were determined in old and young male rhesus macaques. Also the sexual performance of the old (20 years and older) males was studied. The two groups did not differ in either the mean level of total testosterone or the index of free testosterone but the old males had a significantly higher percentage of testosterone bound to TeBG than did the young (10 years old) ones. We found significant negative correlations between the percentage of testosterone binding and sexual behavior in the old males. The percentage of bound testosterone was negatively correlated with the rates of contacting females, mounting, and intromission, and with the percentages of tests during which intromission and ejaculation occurred. Neither the total serum testosterone level nor the index of free testosterone correlated with the level of sexual performance.     

Organon OD 14 (tibolone) and menopausal dynamic hormone profiles

Hormonal profiles were studied in 15 post-menopausal women, 7 of whom had been treated with Organon OD 14 (Tibolone) and 8 with placebo tablets for 3 yr. In the Tibolone-treated group, the sex hormone binding globulin (SHBG) levels were significantly lower, while the estimated free testosterone levels, the testosterone/SHBG ratio and the thyroid-stimulating hormone (TSH) response to thyrotrophin-releasing hormone (TRH) were significantly higher than in the placebo group. Prolactin and triiodothyronine (T3) concentrations were lower in the actively treated group, although the differences were not statistically significant. No significant differences were observed with respect to thyroxine (T4), TSH, basal cortisol or cortisol response to synacthen.     

Plasminogen-mediated group A streptococcal adherence to and pericellular invasion of human pharyngeal cells

Alpha-enolase (SEN) is a strong plasminogen-binding protein on the surface of group A streptococci (GAS). By flow cytometry and immunofluorescence analyses and using human enolase-specific antibody, human pharyngeal cells (Detroit 562) also were found to express enolase on their surface. Detroit 562 cells preferentially bound to Lys-plasminogen and this binding was inhibited in the presence of a lysine analog, epsilon-aminocaproic acid and by carboxypeptidase-B treatment suggesting that the C-terminal lysine residue of the putative pharyngeal cell receptor(s) may play an important role in plasminogen-binding. The increased plasminogen-binding in the presence of free enolase indicated the presence of an enolase/SEN-specific receptor on the pharyngeal cell surface. GAS, when precoated with Lys-plasminogen, adhered to pharyngeal cells significantly more in numbers than when precoated with fibronectin or laminin. Similarly, GAS adhered also significantly more in numbers to pharyngeal cells which were precoated with Lys-plasminogen. GAS adhered similarly in high numbers when incubated with pharyngeal cells in the presence of soluble plasminogen. The de novo pharyngeal cell-bound protease activity, created as a result of activation of bound plasminogen by t-PA, indicated its potential role in pericellular fibrinolytic activity. Further GAS with tPA-activated plasminogen bound on their surface penetrated through Transwell-grown pharyngeal cells in significantly higher numbers. Together, the results presented in this study highlight a novel function of plasminogen in streptococcal adherence to pharyngeal cells and a newly discovered streptococcal ability to pericellularly invade pharyngeal cells as a result of tPA/endogenous plasminogen activator-mediated proteolytic activity.     

Testosterone binding sites in the rat thymus during late embryonal and postnatal period.

Using immunoperoxidase staining with anti-testosterone Ab, has been shown that the cells binding testosterone were present in the rat thymus and that these cells are localized in the outer thymic cortex as well as in cortico-medullary region and medulla. Immunoperoxidase staining with that Ab at electron microscopy level showed that thymocytes as well as thymic epithelial cells bind this hormone. Combined immunoperoxidase staining with anti-testosterone Ab and immunofluorescence method with mAbs specific for thymocytes or thymic epithelial cells, revealed that thymocytes binding this hormone are localized mainly in the outer cortex, while thymic epithelial cells binding testosterone were found in cortico-medullary region and medulla. These testosterone binding cells were found, for the first time, in the thymus of 18-day-old fetus. It has also been shown that their density increased markedly by the day 3 of postnatal life and continued to increase up to the adult stage of organ development. These results indicate that testosterone can influence upon function of specific thymic epithelial cells, localized in the corticomedullary region and medulla. Thus, the results also suggest that this hormone can modulate T cell proliferation and/or differentiation, not only directly acting on the T cells localized in the outer thymic cortex, but also indirectly modulating function of the thymic epithelial cells that bind this hormone.     

Immune recognition of a molecule naturally presented as a monomeric or an oligomeric structure: the model of the human chorionic gonadotropin alpha subunit.

The immune recognition of a molecule naturally presented as a monomeric or an oligomeric structure is analyzed using the human chorionic gonadotropin alpha subunit (hCG-alpha) as a model. Indeed, hCG-alpha circulates as either a free subunit or combined to the beta subunit (hCG-beta) to form the dimeric hCG hormone. A T cell study was performed in BALB/c (H-2d) mice which were found to be high responders to hCG-alpha. Mice were immunized with the free hCG-alpha or the dimeric hCG alpha/beta, and their lymph node cells were challenged in vitro with either alpha subunits from different species, hCG or peptides spanning the entire primary structure of hCG-alpha. Proliferation and IL-2 assays demonstrated that hCG-alpha-primed lymph node cells responded equally well to hCG-alpha and hCG alpha/beta, suggesting that both the free and combined hCG-alpha subunits are processed in a similar way. Among the various synthetic peptides used, only those mimicking the hCG-alpha(59-92) C-terminus portion were able to stimulate hCG-alpha-primed lymph node cells, demonstrating that this region contains immunodominant T cell recognition site(s). The hCG-alpha(23-43) and (32-59) peptides, although incapable of stimulating T cells primed with hCG-alpha, elicited a T cell response when used as immunogens. These regions encompassed cryptic epitopes which were not generated during hCG-alpha processing in H-2d mice. The T cell epitopes of hCG-alpha above described as immunodominant or cryptic on the free alpha subunit, had similar characteristics when the alpha/beta dimer was used as the immunogen. In contrast, T cells primed with peptides mimicking immunodominant sites recognized differently the hCG-alpha and the hCG alpha/beta antigens. Moreover, the analysis of the B cell response to all the immunogenic hCG-alpha peptides indicated that they bear B and T cell epitopes as well. Antibodies elicited against the hCG-alpha(59-92) or (32-59) peptide were capable of recognizing the alpha subunit in its free form but not in the alpha/beta hCG dimer. Such study deserves attention for the comprehensive mechanisms of the immune response to hCG as well as for the design of anti-hCG vaccines.     

PMN binding to P-selectin is inhibited by sulfatide.

The endothelial adhesion protein P-selectin binds to a ligand present on the surface of leukocytes. We have characterized the binding interaction between P-selectin and polymorphonuclear leukocytes (PMNs) in an in vitro assay. These studies have utilized a soluble chimeric protein termed receptor globulin (Rg), which consists of the lectin-EGF-CR-CR extracellular domains of P-selectin fused to a human immunoglobulin G Fc domain. The PMNs bound to immobilized Rg in a saturable and concentration-dependent manner. The binding was specific for the Rg, as preincubation of the cells with soluble Rg inhibited binding to immobilized Rg, and binding was dependent on the presence of free divalent cations. The PMNs expressed a ligand for both P-selectin and E-selectin but not for L-selectin. Previously it was shown that sulfatide is a ligand for P-selectin binding in transformed cells. We have demonstrated that the presence of sulfatide in the P-selectin-PMN adhesion assay inhibits binding in a dose-dependent manner.     

What about bioavailable estradiol?

Estradiol (E(2)) - similarly to testosterone - is a hormone mainly bound to SHBG and albumin in serum. Only the non SHBG-bound (free and albumin-bound hormone, i.e. bioavailable) hormone diffuses easily from circulation to tissues and is available for target cells. Bioavailable hormone measured or calculated seems to represent the best access to bioactive hormone concentration. Several studies reported that this bioavailable E(2) could be usefully measured for the understanding of chronic diseases in men or women, such as osteoporosis, cardiovascular disease and Alzheimer's disease. E(2) assays require a high sensitivity to assess low concentrations. It is currently difficult to know if bioavailable E(2) is really implicated or not in a given pathology but its interest is reported in many epidemiological studies.     

Leydig cell micronodules are a common finding in testicular biopsies from men with impaired spermatogenesis and are associated with decreased testosterone/LH ratio

To assess the biological significance of Leydig cell 'hyperplasia' in man, Leydig cell distribution, volume, and function were studied in patients with infertility or testicular cancer and in suddenly deceased controls. A total of 156 biopsies from 95 patients and 18 necropsies from 13 controls were examined using a semi-quantitative stereological method. In patients, serum concentrations of testosterone, sex hormone binding globulin (SHBG), luteinizing hormone (LH), follicle stimulating hormone (FSH), oestradiol and inhibin-B were correlated with the findings on histological examination. Leydig cell clusters of more than 15 cells in a cross-section, for which we proposed the name 'micronodules', were frequently seen in testicles exhibiting Sertoli-cell-only syndrome (SCO), a mixed pattern of impaired spermatogenesis, or complete spermatogenesis in combination with elevated FSH. Median numbers of micronodules per 1.77 mm(2) (four fields of vision) in these three histological patterns were 6, 4, and 3.5, respectively. In contrast, micronodules were only occasionally observed in testicular biopsies from patients with complete spermatogenesis and normal gonadotrophin levels (median 1), and were rare in testes from controls (median = 0, p = 0.02). The proportion of testicular tissue occupied by Leydig cells increased with decreasing spermatogenic capacity. In contrast, the total volume of Leydig cells per testis was roughly comparable irrespective of the histological pattern, with the exception of testes with bilateral micronodules, which had significantly increased Leydig cell volume compared to those without micronodules. The number of micronodules correlated positively to LH (r = 0.577, p < 0.01) and FSH (r = 0.595, p < 0.01) and the presence of micronodules was most pronounced in the hyperstimulated testes, as reflected by an increased LH/testosterone ratio. In conclusion, Leydig cell micronodules were more frequent in biopsies with impaired spermatogenesis and associated with decreased ratios of testicular hormones to gonadotrophins. The presence of micronodules thus seems to be a histological marker of testicular failure in man.     

Thyroxine (T4) and tri-iodothyronine (T3) determinations: techniques and value in the assessment of thyroid function

Hormonal production of the thyroid gland is constituted of thyroxine or T4 (80%) and triiodothyronine or T3 (20%). In the circulation, whole T4 originates from thyroid secretion but most of T3 (80%) is produced extrathyroidally from T4 deiodination. Conversion of T4 to T3 may be influenced by various conditions and circulating T3 is a less reliable reflection of thyroid hormone production than T4. In serum most of T4 and T3 is bound to binding proteins and only 0.02% of T4 and 0.3% of T3 is free. Because of their higher diagnostic performance, free T4 (FT4) and free T3 (FT3) measurements have superseded total (free + bound) hormone determination. Total hormone measurements remain useful for research studies or in case of severe hyperthyroidism. Equilibrium dialysis/RIA is considered as the reference method for free hormone measurements. Routine clinical laboratories use automated direct two-step or one-step immunoassays with a high molecular weight ligand or labelled antibody. Free hormone measurement remains technically demanding, especially in sera from severe non-thyroid ill patients with low serum thyroxine binding capacity. Interference from anti-thyroid hormone antibodies and familial dysalbuminemic hyperthyroxinemia depends on the assay method, but is now less marked and less frequently detected. To be able to correctly interpret the results of an assay, it is necessary to assess its performance in biologically and clinically well-characterised serum samples. FT4, and FT3 measurements, if FT4 is normal and hyperthyroidism suspected, are used to confirm and assess the level of hypo and hyperthyroidism (overt or subclinical). When the thyroidal status is unstable (first months of a thyroid treatment, altered L-T4 dose, subacute thyroiditis) or when the hypothalamic-pituitary function is disturbed (central hypothyroidism), TSH determination is diagnostically misleading and only free hormone measurements are reliable for thyroid function assessment.     

Photoperiod and testosterone modulate growth and melanogenesis of s91 murine melanoma

In vivo and in vitro assays were performed with S91 murine melanoma cells aiming to investigate the effects of testosterone and photoperiod on tumor growth and melanogenesis (tyrosinase activity). In vivo assays were performed by inducing melanoma tumors in castrated mice receiving increasing concentrations of testosterone and submitted to varying photoperiod regimens. The results demonstrated that the increase of melanin content was higher in animals submitted to the longest days, thus demonstrating the importance of photoperiod length in melanin synthesis. Increase in tumor growth and protein content was observed in testosterone-treated animals submitted to 12L:12D; in testosterone-treated animals submitted to 4L:20D and 20L:4D tumor growth was significantly smaller. In S91 cultured cells, testosterone increased cell proliferation and reduced tyrosinase activity in a dose-dependent manner. Radioactive binding assays demonstrated that the hormone was acting through low affinity testosterone receptors, since the presence of aromatase inhibitor did not affect the binding assay in a statistically significant way, and all the in vitro experiments were performed in the presence of the inhibitor. Our in vivo data added to the in vitro results corroborate the hypothesis that S91 melanoma cells directly respond to testosterone and that this effect is modulated by light.     

In vivo demonstration by radioautography of binding sites for insulin in liver, kidney, and calcified tissues of the rat

An in vivo binding assay using radioautography was employed to visualize insulin receptors in rat tissues. Two and one-half minutes after the intravenous injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with lactated Ringer's solution followed by perfusion with glutaraldehyde. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. Nonspecific binding of labeled insulin was noted in the proximal convoluted tubules of the kidney cortex, prebone and adjacent bone, predentin and adjacent dentin, and enamel. Specific binding sites were observed at the periphery of hepatocytes, over osteoblasts, and in relation to the endothelial cells of fenestrated capillaries within the papillary layer of the maturation zone of the incisors.     

Soluble Interleukin 2 Receptors Released from Mitogen Stimulated Human Peripheral Blood Lymphocytes Bind Interleukin 2 and Inhibit IL2 Dependent Cell Proliferation

In this communication the binding characteristics and possible regulatory role of sIL2R were investigated. Soluble IL2R are released or secreted in high concentrations by phytohemagglutinin (PHA) stimulated human lymphoid cells. The addition of sIL2R, purified by gel filtration chromatography, to cultures of PHA stimulated lymphoblasts resulted in a dosedependent inhibition of [³H]TdR incorporation that could be overcome by the addition of exogenous IL2. Scatchard analysis of IL2 binding demonstrated that the presence of sIL2R did not inhibit ligand interaction with the high affinity IL2R. Immunoprecipitation studies utilizing [¹²⁵I]IL2 and the non-inhibitory anti-Tac protein antibody 7G7/B6 revealed that most of the ¹²⁵I-labeled IL2 migrated with a protein of approximately 45-50 kDa on SDS/PAGE. Together, these results provide evidence that the sIL2R limits the availability of free IL2 to proliferating cells and down-regulates their response without directly affecting the number or function of the cell bound high affinity IL2R.     

Quantitation of human chorionic gonadotrophin by the planimetry of latex agglutination-inhibition results on microtiter plates

A new type of heavy latex particle (LP) coated with the conjugates of human chorionic gonadotrophin (hCG) and bovine serum albumin (BSA) was used as indicator for the microtiter agglutination-inhibition assay of hCG using a monoclonal antibody (McAb) to hCG. The precipitation patterns of LP were measured by an automatic planimetry system. The detection limit of this method was 4 to 8 IU/l. Human luteinizing hormone (hLH) and follicle-stimulating hormone (FSH) caused hardly any cross-reaction in the presence of 3,350 IU/l and 1,500 IU/l, respectively. This planimetric immunoassay (PMIA) method provides a potentially useful method for hormone assay.     

中青年男性血清睾酮水平变化的初步探讨

了解健康中、青年男性血清性激素水平的变化.选取126例健康男性分为20～29岁、30～39岁、40～49岁共3个年龄组.分别测定血清睾酮(T)、游离睾酮(FT)、性激素结合球蛋白(SHBG)、雌二醇(E2)、黄体生成素(LH)、卵泡刺激素(FSH),对各组测定值进行统计学分析.结果显示:血清T、FT水平在30～39岁组...     

Testosterone and alcoholic cirrhosis. Epidemiologic, pathophysiologic and therapeutic studies in men

The present review summarizes the pathogenic mechanisms leading to variation of plasma testosterone concentrations, consequences of hypoandrogenization and hyperoestrogenization, and effects of oral testosterone treatment in men with alcoholic cirrhosis. These patients have normal median plasma testosterone concentrations, but 20% have values above and 20% have values below the normal limits. The majority of patients have raised sex hormone binding globulin (SHBG) concentrations. This increase accounts for the supranormal plasma testosterone concentrations. With decreasing liver function, plasma testosterone concentrations decrease significantly. The combination of increased SHBG levels and decreasing liver function leads to low or subnormal plasma concentrations of non-protein bound and non-SHBG bound testosterone. This decrease, together with raised oestrogen concentrations, may explain the increased prevalence of gynecomastia and testicular atrophy which raises with decreasing liver function. Oral testosterone treatment of alcoholic cirrhotic men produces an increase in the plasma concentrations of testosterone, androstenedione and dihydrotestosterone, but oestrogen concentrations increase as well. Oral testosterone treatment significantly reduces the prevalence of gynecomastia, but is without significant effects on liver biochemistry, morphology, haemodynamics, and function, general well being, sexual dysfunction and survival of alcoholic cirrhotic men. A pooled estimate of the mortality risk of cirrhotic patients treated with anabolic-androgenic steroids does not disclose any significant difference compared with placebo treatment (relative risk 0.98; 95% confidence limits 0.77-1.22). Seldom, but serious, side-effects of oral testosterone treatment can not be excluded.     

Quantitative explanation for increased affinity shown by mixtures of monoclonal antibodies: Importance of a circular complex

Mixtures of some but not all monoclonal antibodies which bind to separate epitopes on human chorionic gonadotropin (hCG) show an increased affinity for the hormone. To find an explanation for the increase in affinity, we developed a mathematical model which predicts the quantities of intermediates formed when pairs of IgG1 mouse monoclonal antibodies having affinities of ～ 10⁸M^?1 for hCG are mixed with the hormone. At low antibody concentrations (i.e. < 1 nM or 0.15 μg/ml) analysis of possible antibody-hormone combinations, including linear and circular chains composed of less than 12 molecules of antibody and 12 molecules of hCG, suggests the increase in affinity is due to formation of a circular complex containing two molecules of antibody and two of hCG. Further, the model predicts that the circular complex will be the major species formed at antibody-antigen equivalence. This prediction is supported by experimental observations on the molecular weight of a new complex formed in the presence of hCG and the mixture of the monoclonal antibodies. In addition, based on experimental values of binding constants for individual antibodies to hCG, the model correctly quantifies the loss in complex observed in the presence of excess hCG antigen. At high antibody concentrations (i.e. > 10 nM or 1.5 μg/ml) the formation of linear chains of antibody-hCG pairs becomes appreciable and contributes to the increase in apparent affinity of the mixture for hCG. These results suggest that the observed affinity of complex mixtures of antibody for antigens containing multiple epitopes calculated from Scatchard plots may not be related to the affinity or avidity of any of the antibody species for a given epitope.     

C-peptide and insulin, but not C19-steroids, support the predictive value of body mass index on leptin in serum of premenopausal women

Hyperleptinaemia is known to be positively associated with obesity in females. Therefore, circulating leptin concentrations are predicted by body mass index (BMI). Additional effects of endogenous C19-steroids, sex hormone binding globulin (SHBG), luteinizing hormone (LH), follicle stimulating hormone (FSH), C-peptide and insulin on the predictive value of BMI on serum leptin were investigated in 56 hyperandrogenaemic and/or hyperinsulinaemic and/or obese premenopausal women. Serum concentrations (after an overnight 12 h fast) of leptin, total testosterone, free testosterone, SHBG, dehydroepiandrosterone sulphate (DHEAS), LH, FSH, and oestradiol as well as serum concentrations of C- peptide and insulin prior to, and 1 h after, an oral 100 mg glucose load (1 h values) were determined by immunoassays. Subjects with regular menstrual cycles were studied in the mid-follicular phase while the remainder were studied at random. Nineteen normotestosteronaemic, normoinsulinaemic, lean and ovulatory volunteers served as controls; in order to determine the effect of different stages of the menstrual cylce, serum concentrations of leptin (and of oestradiol in 12 out of the 19 individuals) were determined at the preovulatory, the mid-luteal and the following mid-follicular phase. Significant differences between the patients versus control were not found possibly because of the heterogeneity in the patient group. Multiple regression indicated a hyperbolic correlation between BMI and leptin concentrations. As expected, BMI was the major determinant responsible for >50% (R2=0.51) of the elevation of leptin concentrations. The combination of BMI with fasting C-peptide or fasting insulin enhanced the R2 up to 0.59. The multiple regression with two explaining parameters showed a significant regression coefficient for BMI at the 0.001 level, and for fasting C- peptide and fasting insulin at the 0.01 level, which was as statistically significant as the combination of BMI with the 1 h values of C-peptide and of insulin. In contrast, total testosterone, free testosterone, SHBG, free testosterone/SHBG ratio, DHEAS and LH/FSH ratio had no effect. Similarly, models with more than two variables did not measurably improve the explained variation. In the control group, leptin concentrations were significantly higher in preovulatory and mid- luteal phases than the two mid-follicular phases (P < or = 0.05) and must be considered when determining sampling time. In conclusion, hyperandrogenaemia does not have a predictive value on leptin concentrations in premenopausal subjects but hyperinsulinaemia exerts an effect independent of obesity that is the strongest predictor for elevation of leptin concentrations. Hyperinsulinaemia might contribute to the hyperbolic correlation of circulating leptin in obese patients.      

不同剂量电离辐射所致Leydig细胞对hCG反应性的变化

本文报告了不同剂量X线所致Leydig细胞对hCG反应性的变化。幼年雄性大鼠睾丸的Leydig细胞经预培养24小时后,分别接受25,50,75,100,250mGy小剂量的X线照射(剂量率为12.5mGy/分)以及0.5,1.0,2.0,4.0,8.0Gy大剂量X线照射(剂量率0.51Gy/分)。然后加入浓度为0.025或0.05i.u./ml的hCG,继续培养48小时,观察Leydig细胞对hCG反应性的变化。结果表明,小剂量辐射在25,50mGy组引起Leydig细胞对0.05i.u./ml hCG的反应性增高,而在各小剂量组对0.025i.u./ml hCG的反应性无明显变化。大剂量辐射Leydig细胞在各剂量组对0.025和0.05i.u/ml hCG的反应性均降低,此种降低有照射剂量依赖性。对上述变化的可能机制进行了讨论。