Expression, localization, and thyrotropin regulation of cathepsin D in human thyroid tissues.

Enzymatic activity and isoform expression of cathepsin D (cath D) were studied in 107 cytosols from various human thyroid tissues including 21 normal tissues, 12 cold benign nodules, 17 toxic adenomas, 22 samples from Graves' disease patients, and 35 thyroid carcinomas. Cath D assay was optimized for human thyroid tissues. We found that mean cath D specific activities, expressed as units per milligrams protein minus thyroglobulin, were higher in carcinomas (P = 0.0001), toxic adenomas (P = 0.0001), and specimens from Graves' disease patients (P = 0.0001) than in normal thyroid tissues. Mean cath D activity in carcinomas was also significantly different from that in cold benign nodules (P < 0.001) and Graves' disease tissues (P < 0.05) but not from that of toxic adenomas. To determine possible mechanisms by which the observed increase in cath D activity might be regulated, we used Western blotting to measure relative amounts of cath D isoforms in the various thyroid tissues. We found that the 31-kDa major processing form of cath D was significantly increased in carcinomas and toxic adenomas compared with normal tissues (P < 0.01), cold benign nodules (P < 0.05), and Graves' disease tissues (P < 0.05). A positive correlation of cath D activity with relative expression of the 31-kDa form (r = 0.67, P = 0.0001) was observed in 104 thyroid cytosols. These data demonstrate a deregulation at the protein level, with resulting increases in cath D activity. Immunogold labeling of cath D showed particle concentration in lysosomes or phagosomes in both normal follicles and papillary carcinoma cells, indicating that cath D localization was not altered by malignant transformation in human thyroid cells. TSH induced cath D synthesis and secretion in extracellular fluid of normal human thyroid cells in primary culture; TSH had little effect on intracellular cath D level. In conclusion, TSH-induced cath D synthesis may explain high cath D levels in Graves' disease tissues and toxic adenomas, because these tissues possess a permanently stimulated cAMP transduction pathway. Furthermore, the overexpression of cath D in thyroid carcinomas in comparison with normal controls adds further arguments for the potential role of cath D in tumor growth and metastasis.

Effect of bromolevamisole and other imidazo [2,1-b] thiazole derivatives on adenylate cyclase activity.

We studied the effect of bromolevamisole (BL) and other imidazo [2,1-b] thiazole derivatives--bromodexamisole (BD) and levamisole (LV)--on adenylate cyclase (AC) activity. BL and BD both inhibited forskolin-activated human thyroid AC, while LV had no effect. This inhibition was non-stereospecific and the IC50 values, as measured with 1 mM ATP and 40 microM forskolin, were 0.95 and 0.80 mM for BL and BD, respectively. In contrast, human thyroid alkaline phosphatase (ALP) inhibition was stereospecific, with IC50 values of 0.0012 mM for BL and 0.9 mM for BD. LV was a 10-fold weaker inhibitor of ALP than BL. These results show that ALP inhibition is not correlated with forskolin-activated AC inhibition. Furthermore, in the presence of a competitive inhibitor of GTP (0.1 mM guanosine 5'-O-(2-thiodiphosphate), BL retained its antagonizing effect on forskolin-activated AC which suggests a direct action on the catalytic subunit. The inhibition was of the mixed type, indicating a complex interaction between BL and AC. Glucagon-activated AC activity in rat liver membranes was also inhibited by BL, although to a slightly lesser degree than thyroid stimulating hormone (TSH)-activated AC from human thyroid for a given BL concentration. In cultured human thyroid cells, BL (0.25 mM) induced a potent decrease in cAMP accumulation after 2 hr of stimulation by TSH. Taken together, these results show that BL inhibits AC and that this inhibition is not organ-specific.

Cathepsin D in normal and neoplastic thyroid tissues.

BACKGROUND: Cathepsin D is a widely distributed lysosomal acidic endopeptidase. It is an estrogen-regulated protein that is a prognostic factor in breast cancer. The aim of this study was to measure cathepsin D concentrations in thyroid tissues and to correlate these concentrations with clinical and pathologic parameters. METHODS: Cathepsin D and thyroglobulin concentrations were measured in the cytosol of normal thyroid tissues (n = 14), benign nodules (n = 6), and thyroid carcinomas (n = 32) with an immunoradiometric assay. Statistical analysis was based on the Kruskal-Wallis and Wilcoxon tests and on the Spearman rank correlation coefficient. RESULTS: The mean level of cathepsin D, expressed as picomoles per milligram protein minus thyroglobulin, was higher in the 32 carcinomas, 29.1 +/- 15.5, than in the 14 normal thyroid tissues, 8.4 +/- 2.5 (p < 0.001) or in the 6 benign nodules, 11.2 +/- 7.3 (p = 0.003). Cathepsin D concentrations correlated with tumor size; Spearman rank correlation coefficient was rs = 0.44 (p = 0.012). No significant difference was found regarding histologic type. Cathepsin D concentrations were inversely correlated with the thyroglobulin level in the tumor; Spearman rank correlation coefficient was rs = -0.60 (p < 0.001). CONCLUSIONS: Cathepsin D concentration is higher in thyroid carcinoma than in normal thyroid tissue. Increased cathepsin D concentrations correlate with thyroid tumor size but not with histologic type. Further studies should be done to confirm the potential prognostic value of cathepsin D in patients with thyroid carcinomas.

[A comparative study of the Doppler methods for evaluation of the degree of carotid artery stenosis. Continuous, pulsed, color Doppler]. / Etude comparative des méthodes Doppler pour l'évaluation du degré des sténoses carotidiennes. Doppler continu, pulsé, couleur.

OBJECTIVE: Compare various Doppler methods for the quantification of the degree of stenosis on 85 patients. METHOD: the following parameters were measured: maximal velocity (Vmax) inside the stenosis (PW), grades of spectral disturbances at the outlet of the stenosis (PW-CW), index of spectral disturbance (STI) at the outlet of the stenosis (PW-CW), ratio of velocities I(IC/CC) in internal and common carotid (PW), ratio of vessel cross section and residual lumen area (% STEN) by color Doppler. The reference method was the Grades of spectral disturbance and the index of stenosis measured post stenosis. (Method validated against angiography and pieces of endarterectomy.) The following comparisons were done; grades and STI by CW against grades and STI by PW, Vmax (PW) against grade and STI, % STEN (color) against grade and STI, % STEN (color) against Vmax (PW), I(IC/CC) (PW) against grade and STI, I(IC/CC) (PW) against Vmax. CONCLUSION: (a) grades or stenosis index : showed the best reproductibility; (b) a high correlation was found between the grades or stenosis index post stenosis measured by CW or PW; (c) Vmax was not proportional to the stenosis degree and showed large fluctuations for the same stenosis degree; (d) the I(IC/CC) showed large fluctuations for the same stenosis degree, the correlation was poor for this velocity ratio. Both Vmax and I(IC/CC) allow to detect only 2 groups of stenosis > 75% or > 90% in area; (e) color doppler over-estimate stenosis degree by approximately 20% but was more accurate and reproducible than Vmax. An appropriate procedure was designed to avoid this over estimation.

[Effects of nifedipine on carotid blood flow and baroreflex response in essential arterial hypertension. Preliminary results]. / Effet de la nifédipine sur le débit carotidien et la réponse baroréflexe, au cours de l'hypertension artérielle essentielle. Résultats préliminaires.

The objective of this prospective study was to evaluate the effect of nifedipine administered at usual daily doses of 30 to 40 mg on the carotid flow in arterial hypertension. The study included 15 patients (8 men and 7 women), 50 to 79 (mean 59.5) years old suffering from long-standing, fixed essential hypertension becoming instable under central antihypertensive drug therapy. For calculating the carotid blood flow, vascular echotomography combined with Doppler ultrasonography and spectral analysis (Duplex probe) determining the vascular section and flow velocity were used. Arterial pressure using a mercury tonometer, flow velocity, common carotid artery diameter, carotid blood flow, Pourcelot's index, parietal tension and heart rate were measured before treatment and at the 8th day of nifedipine administration. It could be shown that the drug produced a significantly (p less than 0.001) increased carotid blood flow, in spite of a marked (p less than 0.001) decrease in systolic (p less than 0.001) and diastolic (p less than 0.005) blood pressure. The increase in carotid blood flow was directly related to the increase in flow velocity (p less than 0.001) and in the diameter of common carotid artery (p less than 0.01) and was associated with a significant decrease in the Pourcelot's index. Analysis of two groups of patients isolated from the total group according to the elevation of carotid blood flow, showed that the degree of hypotensive effect of nifedipine is negatively correlated with the baroreflex response determined by the variation of parietal tension.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative measurement of progesterone receptors in breast cancer by biochemical and immunoenzymatic assays.

The measurement of progesterone receptors (PR) by enzyme immunoassay (Abbott Laboratories, EIA monoclonal) and biochemical assay using a tritiated ligand (promegestone, R5020) was studied and compared by using the statistical method of Passing and Bablok. In order to improve the reliability of the biochemical method, data were analysed using a hyperbolic model which avoids the need to determine non-specific binding experimentally. The comparison of hyperbolic (Y) and Scatchard (X) plots gave a regression curve of Y = 0.93 x + 1.34 fmol/mg of protein. Cytosols from 70 human breast cancers homogenized in the absence of KCl were assayed for PR by both the EIA (Y) and biochemical (X) methods. The linear regression obtained gave Y = 1.21 X + 1.97 fmol/mg of protein. In the presence of 0.4 M KCl-Tris buffer, the corresponding result for 80 human breast cancers was Y = 3.11 X + 1.91 fmol/mg of protein. The slopes of the two regression lines obtained in the presence or absence of KCl were significantly different. Results of the biochemical and EIA methods were similar in the absence of KCl, whereas EIA gave higher values when KCl was used. This discrepancy probably stem from the methodological differences between the two methods: the biochemical assay measures active steroid binding sites while EIA measures antigenic activity. The authors conclude that clinical studies are required before using high-salt extraction buffer in routine PR determination by the EIA method; this will result in improvements in the determination of the hormone dependence and/or the prognosis of human breast cancers.

[Immunoenzymatic assays of c-erbB-2 oncoprotein and epidermal growth factor receptor in breast cancer: correlation with clinical and biological parameters]. / Dosages immunoenzymatiques de l'oncoprotéine c-erbB-2 et du récepteur du facteur de croissance de l'épiderme dans le cancer du sein: corrélation avec les paramètres cliniques et biologiques.

Two new immunoenzymatic assays for c-erbB-2 oncoprotein and epidermal growth factor receptor (EGF-R) (Oncogene Science) in human breast cancer were validated. Correlations between these assays and some clinical and biological parameters were also studied. The repeatability and reproducibility of standard curves for the two methods gave a coefficient of variation (CV) of less than 4% and about 10% respectively. The accuracy of c-erbB-2 oncoprotein and EGF-R assays was examined by using dilution and recovery tests throughout the standard curves. The linear relations between theoretical and measured values, for these tests, had slopes close to 1 and an intercept near 0. The median value for EGF-R, measured on solubilized membranes of 290 primary tumors, was 0.12 fmol/micrograms protein, the mean value was 0.37 (range 0 to 35.7). For c-erbB-2 oncoprotein, the median value, measured using the same population, was 2.75 human neu unit/micrograms protein, the mean value was 7.85 (range 1 to 125). There was an inverse relationship between EGF-R values and those for the estrogen receptor (ER), progesterone receptor and pS2 protein as well as menopausal status. C-erbB-2 oncoprotein concentrations were positively correlated with ER, pS2 protein and cathepsin D. Furthermore, a significant positive correlation was observed between EGF-R levels and c-erbB-2 oncoprotein levels. In conclusion, immunoenzymatic assays of EGF-R and c-erbB-2 oncoprotein are easy to use, sensitive and reliable. The accurate standardisation of immunoenzymatic assays could contribute to the clinical use of EGF-R and c-erbB-2 oncoprotein as prognostic factors in breast cancer.

[Evolution of salivary estradiol levels during the spontaneous menstrual cycle. Correlation between saliva and plasma]. / Evolution des taux d'estradiol salivaire au cours du cycle menstruel spontané. Corrélation salive-plasma.

The measurement of steroid concentrations is recommended in the exploration of the different endocrine functions. The salivary concentrations are the reflect of the free biologically active fraction in the plasma. In this study we have used a sensitive and specific method that can be routinely applied in salivary extradiol (SE2) RIA, using a 3 CMO 125I histamine derivative as a tracer. This study related to 12 normal spontaneous menstrual cycles and two abnormal spontaneous cycles. Saliva samples were collected daily throughout the menstrual cycle. Plasma samples were collected simultaneously to compare the salivary and plasma patterns. In the follicular phase SE2 was low: M +/- SD = 9.4 +/- 2.4 pmol/l. During the periovulatory period, the mean level of SE2 the day of the preovulatory peak was 27.6 +/- 8.6 pmol/l. In the luteal phase the SE2 levels fluctuated in a serrated manner with a mean of 11.7 +/- 3.4 pmol/l. The difference between the different phases is significant at the probability threshold 0.01. We have compared the salivary and plasma patterns during the different phases. At the beginning the salivary/plasma ratio was 3.4 +/- 0.9%. During the periovulatory period this ratio was only 2.5 +/- 0.6% and the end of the cycle 2.8 +/- 1%. The statistic study showed that these differences were significant at the probability threshold 0.01.

[Detection and quantification of carotid atheroma. Respective value of vascular ultrasonography, spectrum analysis and digital intravenous angiography]. / Dépistage et quantification de l'athérome carotidien. Intérêts respectifs de l'échotomographie vasculaire, de l'analyse spectrale et de l'angiographie numérisée intraveineuse.

This retrospective study involved 37 subjects who were examined by means of intravenous digitalised angiography, vascular ultrasonography and spectral analysis of the Doppler signal. The last two examinations were found to give the most precise images and functional information. The concordances between angiography and ultrasonography on the one hand and spectral analysis on the other were 82 and 90 per cent. The authors have established a protocol in order to limit and systematize these examinations during screening for carotid atheroma: the risk factors are weighted; a score is obtained by summation; depending on the value of this score and the patient's age, the investigation procedure is pursued or not. The sequence always consists of ultrasonography followed by spectral analysis when plaque is detected and by angiography when the plaque is stenotic or ulcerated. In symptomatic patients, ultrasonography and digitalised angiography are performed routinely.

One-step chemiluminescent immunoassay of free thyroxin with acridinium-ester-labeled thyroxin evaluated and compared with a two-step radioimmunoassay.

This new one-step chemiluminescent immunoassay of free thyroxin (FT4) involving a thyroxin-immunoglobulin conjugate labeled with acridinium ester (Magic Lite System; Ciba Corning Diagnostics Corp., Medfield, MA) is rapid (one 1-h incubation), requires two calibrators per run, and takes 10 s per sample for the quantification step. Analytical performances were excellent: within- and between-run CVs of less than 10% in the working range, no significant effect of hemolysis, bilirubin, or lipemia, and no significant interaction between the conjugate and the thyroxin-binding proteins. Magic Lite results (y) correlated well with those obtained by the Sclavo (x) two-step radioimmunoassay (Sclavo, Siena, Italy): y = 1.35x + 1.32 (r = 0.94, n = 267, P less than 0.001, Sxy = 6.29). Clinical sensitivities (diagnostic efficiencies) for hypothyroidism and hyperthyroidism were 0.91 and 0.98 for normal interval limits of 12 and 21.5 pmol/L (95% confidence interval). Magic Lite results in situations where patient therapy, treatment, or unusual conditions can result in a lack of correlation between the clinical status and the FT4 values were qualitatively the same as those obtained by the Sclavo assay.

A computer-aided system for the diagnosis of hepato-biliary diseases. A comparison with the performance of physicians.

A computer-aided system has been developed for the diagnosis of disease of the liver and biliary system. The program is based on the use of 30 indicants, all of which are available within six hours after patient's admission: 18 of them are clinical signs, 12 are laboratory parameters including routine liver-function tests. To date, the program concerns 52 hepato-biliary diseases. From the analysis of the 30 items collected in any patient, the diagnoses are computed according to Bayes' theorem and printed by decreasing order of probability. The performance of the program was tested using records of patients with fully proven diagnosis. The first diagnosis given by the computer was correct in 57 per cent of the cases. In 80 per cent, the right diagnosis was among the first four proposed. When the performance of the model was compared to that of physicians, the number of correct answers was roughly the same for the computer and for the specialists in hepatology; in contrast, the computer's responses were far better than those of general practitioners. These results demonstrate the efficiency of the program for the diagnosis of hepato-bilitary diseases and its potential interest for helping clinicians in decision-making.

Breast cancer estrogen receptor assays: assessment of a direct hyperbolic fitting to analyse multipoint binding data.

Analysis of breast cancer estrogen receptor multipoint binding assay is performed by fitting experimental data to a hyperbolic model derived from the law of mass action. The calculations performed on a microcomputer are carried out from the total bound and free ligand concentrations. The parameters estimated by hyperbolic fitting, receptor concentration N and constant of dissociation K, well agree with those obtained by Scatchard's transformation. N and K derived from hyperbolic analysis are much less susceptible to the influence of experimental errors. The method is more reliable at low receptor concentrations. The main advantage of the hyperbolic fitting is to simplify the technical methodology in clinical laboratory practice; there is no need to determine the non-specific bindings experimentally. Calculations can be easily automated on any laboratory microcomputer. Assays of any kind of receptor could be analysed by the hyperbolic fitting when the physical-chemical equilibrium between receptor, nonsaturable component and ligand can be approximated by a two-component model.

Estrogen receptors and cathepsin D in human thyroid tissue.

BACKGROUND: To investigate the significance of estrogen receptors (ER) in the pathogenesis of thyroid dysplasia, the authors analyzed, by analogy with breast cancers, ER and three estrogen-regulated proteins: progesterone receptor (PR), cathepsin D, and pS2 protein, in cytosols of 42 human thyroid tissues. METHODS: ER and PR were measured by an immunoenzymatic assay and cathepsin D and pS2 by an immunoradiometric assay. Tissue specimens included 7 normal tissues, 6 benign nodules, 8 toxic adenomas, 7 from patients with Graves disease, and 14 carcinomas. RESULTS: ER was present at very low concentrations, with no statistical difference between neoplastic and nonneoplastic tissues. The mean levels of cathepsin D, expressed as pmol/mg protein minus thyroglobulin, were higher in the 14 carcinomas (P = 0.0003), the 7 specimens from patients with Graves disease (P = 0.006), and the 8 toxic adenomas (P = 0.04) than in the 7 normal thyroid tissues. A significant difference also was observed between the carcinomas (P = 0.003) and six benign nodules. Compared to TNM parameters, cathepsin D concentrations correlated with tumor size: higher cathepsin D levels were found in pT4 than in pT2 and pT3 carcinomas. All the tissues tested were negative for PR and pS2 protein. CONCLUSIONS: The results clearly indicate a significant difference between neoplastic and normal thyroid tissue in terms of the amount of cathepsin D, but not that of ER. This suggests that cathepsin D probably is not regulated by estrogen but simply is a marker of protease activity during invasion by thyroid carcinomas.

Chemiluminescence immunoassay of thyrotropin with acridinium-ester-labeled antibody evaluated and compared with two other immunoassays.

A new chemiluminometric immunoassay of thyrotropin (TSH) involves antibody labeled with acridinium ester ("Magic Lite System," Ciba Corning Diagnostic Corp.). The assay is rapid, with two incubations totaling 2.5 h, requires two standards per run, and takes 10 s per sample for the quantification step. Analytical performance, within- and between-run reproducibilities, and linearity were excellent. The detection limit is 0.04 milli-int. unit/L. Results correlated well with those obtained by immunoradiometric assay (RIA-gnost hTSH, Hoechst-Behring) and immunofluorometric assay (hTSH Delfia, LKB): r = 0.975. TSH measurements in 32 euthyroid subjects ranged from 0.4 to 4.8 milli-int. units/L (mean 1.35 milli-int. units/L). TSH values for 51 hypothyroid and subclinically hypothyroid patients ranged from 2 to 65 milli-int. units/L. TSH values for 33 hyperthyroid patients (less than 0.14 milli-int. unit/L, less than 0.04 milli-int. unit/L in 16 of the 33) were clearly lower than for most untreated euthyroid subjects. For 169 other individuals whose thyroid function was being routinely assessed. TSH ranged from 0.4 to 4.8 milli-int. units/L, three had TSH less than 0.14 milli-int. unit/L, and four had TSH between 0.14 and 0.4 milli-int. unit/L. This system is as efficient and reliable for screening for thyroid function as the two comparison systems.

Inhibitions of protein kinases and protein phosphatases have opposite effects on thyrotropin-stimulated cAMP accumulation in human thyroid cells.

We investigated the effects of inhibitions of protein phosphatases and protein kinases on thyrotropin (TSH) stimulation of cAMP accumulation in human thyroid cells. Okadaic acid (OA) and calyculin-A (CL-A), two potent inhibitors of type-1 (PP-1) and type-2A (PP-2A) protein phosphatases, had a biphasic concentration-dependent response on cAMP formation. An inhibitory effect (41.3% and 47.2% inhibition with OA and CL-A) was first observed at 1 microM OA and 10 nM CL-A, followed by a reduction of this effect with OA (24% inhibition) or by a complete reversal of inhibition with CL-A, at 10-fold higher concentrations of both products. Addition of purified PP-1 and PP-2A to crude membranes from cells preincubated with OA, reversed OA-induced adenylyl cyclase inhibition, confirming that these protein phosphatases regulate TSH-mediated cAMP production. Levels of protein incorporation of 32P were higher with 10 microM OA than with 1 microM OA and did not correlate with the biphasic effect of OA on cAMP production. These results support a dual action of protein phosphorylation in the control of adenylyl cyclase activity stimulated by TSH. H-7, an inhibitor of nucleotide- and calcium/phospholipid-dependent protein kinase (PKC), increased by 197% the stimulation of cAMP accumulation by TSH in thyroid cells. Phorbol 12-myristate 13-acetate (PMA) counteracted the effect of H-7 on cAMP levels, which suggests that PKC is involved in the action of H-7. Moreover, KT5926, an inhibitor of calcium/calmodulin-dependent protein kinase II and myosin light chain kinase, increased basal cAMP levels rather than cAMP levels stimulated by TSH. In light of these results, we suggest that phosphorylation/dephosphorylation cycles regulate basal and TSH-stimulated adenylyl cyclase activities in human thyroid.

Serum-to-urinary prostate specific antigen ratio: its impact in distinguishing prostate cancer when serum prostate specific antigen level is 4 to 10 ng./ml.

PURPOSE: Benign prostatic hyperplasia (BPH) was shown to be associated with high concentrations of urinary prostate specific antigen (PSA). We investigated the serum-to-urinary PSA ratio in patients undergoing prostate biopsy to assess its efficacy in enhancing serum PSA specificity in the detection of prostate carcinoma. MATERIALS AND METHODS: From November 1995 through January 1996 consecutive patients undergoing prostate biopsy were prospectively included in the study. Serum and urine PSA levels were measured at our laboratory with the Tandem-R assay. Samples were drawn 24 hours before prostate biopsy and at a distance from prostatic manipulation or ejaculation. RESULTS: We studied 73 patients with BPH and 57 with prostate cancer. Differences between BPH and prostate cancer were statistically significant considering serum PSA or serum-to-urinary PSA ratios. In the 50 patients with a serum PSA of 4.0 to 10.0 ng./ml. (35 with BPH and 15 with prostate cancer) the differences between prostate cancer and BPH were still significant only when considering serum-to-urinary PSA ratio. Receiver operating characteristic curves showed that serum-to-urinary PSA ratio was a better predictor of prostate cancer than serum PSA. CONCLUSIONS: Our results suggest that the serum-to-urinary PSA ratio may be useful in distinguishing BPH from prostate cancer, particularly in the diagnostic gray zone of serum PSA between 4.0 and 10.0 ng./ml.