Extrahepatic neoplasms metastatic to the liver histologically are often indistinguishable from hepatocellular carcinoma (HCC). The differential diagnosis between HCC and metastatic liver tumours can be even more difficult in ultrasound guided fine-needle biopsies. Purpose of the present study was to investigate the utility of immunohistochemical staining with polyclonal anticarcinoembryonic antigen (pCEA) antibody and of in situ hybridization (ISH) revealing human albumin mRNA, with emphasis on tissues obtained via fine-needle procedure. Cases consisted of 52 primary HCC; 2 HCC metastatic to vertebral bones; 18 tumours metastatic to the liver; 24 non-hepatocellular tumours metastatic to the skin, lymph nodes and brain; 2 immature teratomas with areas of hepatoid differentiation. Forty-seven HCC (90%) and 7 liver metastases (38%) were obtained by ultrasound guided fine-needle biopsies (21 g needle was used). All the remaining cases were surgical specimens. All the cases were studied with immunohistochemistry for pCEA and ISH using a cRNA probe for human albumin mRNA. The immunohistochemical staining using pCEA showed a canalicular type of positivity in 37 cases of HCC (71%), in one HCC metastatic to vertebral bone and in the hepatoid areas of one immature teratoma. No canalicular type of positivity was obtained in non-hepatocellular neoplasms metastatic to the skin, brain, lymph-nodes and liver. Albumin mRNA was detected in 51 (98%) primary HCC, in both HCC bone metastases, and in the hepatoid areas of both immature teratomas. No positivity was obtained in non-hepatocellular tumours. The data here obtained indicate that immunostaining with pCEA and ISH revealing human albumin mRNA are markers of hepatocellular differentiation and confirm their diagnostic utility. Detection of albumin mRNA showed a higher sensitivity. In addition the cRNA probe here used seems more sensitive that the oligonucleotide probes employed in previous studies.

The Milan hypertensive strain of rat (MHS) displays abnormalities in both renal function and adrenocortical activity. While the pressor role of the former has been studied in detail, the role of the latter has not yet been clearly evaluated. In the present study, glucocorticoid receptor (GR) binding characteristics in liver cytosol from adult MHS and Milan normotensive controls (MNS) have been investigated. Dexamethasone, aldosterone and corticosterone were bound with lower affinity to cytosol of MHS rats compared with that of MNS rats. This pattern of binding could explain the raised plasma corticosterone concentrations and adrenocortical hypertrophy previously noted in MHS. The coding sequence of MHS and MNS GR genes have been determined. The MHS gene differed in four respects from that of MNS: three silent point mutations and a polymorphic microsatellite region in exon 2. The latter polymorphism has been used in cosegregation studies of F2 hybrids of MHS x MNS. The MHS GR genotype was associated with hypercalciuria and lower blood pressure in female rats and lower body weight in male rats. Although the effect on blood pressure is small, it is consistent with the affinity data. MHS GR genotype cosegregated with lower blood pressure in F2 rats and displayed a lower affinity in binding studies. In conclusion, GR polymorphism may be responsible for differences of adrenocortical function between MHS and MNS. This may lead to a reduction in the blood pressure difference between the two strains.

Genetic variation of the glucocorticoid receptor (GR) locus is associated with differences in blood pressure. To define the intermediate phenotypes associated with this variation, we investigated the biochemical and clinical significance of a BclI restriction fragment length polymorphism of the GR locus in 64 normal male volunteers. Blood samples were genotyped as either AA (homozygous large allele; n = 6), Aa (heterozygous; n = 51), or aa (homozygous small allele, n = 7). Four primary glucocorticoid variables were measured including GR binding characteristics and glucocorticoid-sensitive lysozyme release of leukocytes in vitro and the blanching response of forearm skin to budesonide. A large number of secondary variables (urinary and plasma steroid measurements, blood pressure and indices of body fat metabolism, and routine biochemical and hematological measurements) were also considered. In vivo sensitivity to budesonide was greater in AA than aa individuals (mean +/- SE EC50 values: 13 +/- 5 and 42 +/- 10 ng; P < 0.01). In contrast, leukocytes of AA subjects tended to have lower affinity and reduced sensitivity for dexamethasone, although these effects were not statistically significant. Based on urinary steroid measurements, 11 beta-hydroxysteroid dehydrogenase activity [ratio of tetrahydrocortisol (THF) to tetrahydrocortisone (THE) metabolites] was not affected by genotype. The relative activities of 5 alpha- and 5 beta-reductase activity (allo-THF/THF + THE) appeared lower in AA than aa subjects (0.22 +/- 0.04 cf. 0.33 +/- 0.06; P < 0.005) but were not judged to be significantly different when corrected for multiple comparisons. Single and multivariate analyses were carried out to determine which variables influence GR binding characteristics and glucocorticoid responsiveness and to see whether cardiovascular risk factors (blood pressure and body fat) were influenced by glucocorticoid-dependent functions. Only 15-20% of the variations in the dissociation constant (Kd) and maximum binding capacity (Bmax) were influenced by other variables; plasma cholesterol was the most important for affinity and plasma sodium concentration for binding capacity. Multivariate analysis showed that several factors including GR genotype and urinary cortisol account for 10% of the variation of in vivo responses to glucocorticoid hormones; plasma calcium concentration was the only variable that contributed to in vitro sensitivity of leukocytes to dexamethasone. Glucocorticoid-dependent responses were of negligible importance in determining blood pressure or percentage body fat within the narrow physiological ranges of the present study. We conclude that GR genotype affects steroid sensitivity in a tissue-specific manner because of altered GR function or possibly because of linkage to a locus that controls hormone access to the receptor by influencing steroid metabolism.

Familial hypercholesterolaemia (FH) is an inherited autosomal codominant disorder caused by many different mutations in the low-density lipoprotein receptor (LDLR) gene. The one described most frequently in patients with FH from England, arises from a G-->A transition at the first nucleotide of codon 80, resulting in the substitution of lysine for glutamic acid at residue 80 of the mature protein, FH E80K. We describe a simple method to detect this mutation in genomic DNA using the polymerase chain reaction (PCR). A 69 base pair (bp) fragment of exon 3 of the LDLR gene is amplified using a mutagenic upstream PCR primer. This substitutes a T for an A residue in the amplified product, 2 bp upstream from the mutant site, generating a restriction site for the endonuclease Taq I, in normal, but not in mutant DNA. Following digestion of amplified DNA with Taq I, normal but not mutant DNA is cut into two fragments of 29 and 40 bp, which are readily identified by polyacrylamide gel electrophoresis. Using this method, 410 patients with clinically diagnosed FH, attending lipid clinics in Edinburgh (72), Newport (158), Walsall (30) and Southampton (150), were screened for the mutation. Five individuals tested positive as heterozygotes, one from Edinburgh, three from Newport and one from Southampton. This finding was confirmed by DNA sequence analysis. We conclude that FH due to this mutation occurs in individuals throughout Great Britain and that it can be detected accurately using this simple technique. DNA from these and other individuals previously identified to be heterozygous for FH E80K, was then studied using PCR of highly informative microsatellite markers flanking the LDLR gene. Sixteen of 17 apparently unrelated individuals heterozygous for FH E80K also were heterozygous for an identical size (239 nucleotide) allele, of polymorphic microsatellite D19S394, located approximately 250 kb away from the LDLR gene. This supports the hypothesis that FH E80K in these 16 individuals arose from a single ancestor less than 1000 years ago.

We compared glucocorticoid receptor binding characteristics and glucocorticoid responsiveness of human mononuclear leukocytes (HML) from hypertensive patients and matched normotensive volunteers. We also considered associations of these variables with plasma renin activity, aldosterone, cortisol, corticotropin, and electrolyte concentrations. We calculated binding affinity (Kd; nmol/L) and capacity (Bmax; sites/cell) for dexamethasone and cortisol from homologous and heterologous competition curves for specific [3H]dexamethasone binding sites on HML isolated from the blood of normotensive volunteers and subjects with essential hypertension. Glucocorticoid responsiveness of HML was evaluated as IC50 values (nmol/L) for dexamethasone and cortisol for the inhibition of lysozyme release. We measured plasma hormones by radioimmunoassay. Kd values (mean+/-SE) for cortisol in HML of hypertensive patients were higher than in control subjects (24.6+/-2.4 versus 17.5+/-1.7 nmol/L, P<.04). Binding capacity (4978+/-391 versus 4131+/-321 sites/cell), Kd values for dexamethasone (6.7+/-0.5 versus 5.7+/-0.3 nmol/L), and IC50 values for dexamethasone (3.4+/-0.3 versus 3.1+/-0.2 nmol/L) and cortisol (12.2+/-1.6 versus 9.5+/-0.3 nmol/L) were not significantly different. Patients with renin values less than 0.13 ng angiotensin I/L per second were markedly less sensitive to cortisol than those with higher values. Both Kd (30.3+/-2.5 versus 19.2+/-2.4 nmol/L) and IC50 values (15.5+/-1.8 versus 8.9+/-1.2 nmol/L) for cortisol were significantly higher in patients with lower renin values (P<.03). Other variables, including plasma hormone and electrolyte values and binding characteristics for dexamethasone, were not different. These data suggest that cortisol binding to glucocorticoid receptor is slightly impaired in patients with essential hypertension. In vivo, this could lead to inappropriate binding of cortisol to mineralocorticoid receptors. Hence, decreased sensitivity to cortisol is associated with renin suppression. This hypothesis is supported by evidence of hypertension and low renin activity, which others have described in patients with primary glucocorticoid resistance due to mutations of the glucocorticoid receptor.

Carbenoxolone potentiates the mineralocorticoid activity of endogenous glucocorticoid hormones by inhibiting the enzyme 11 beta-hydroxysteroid dehydrogenase, which converts cortisol and corticosterone to inactive 11-oxo-derivatives. We addressed the question of whether glucocorticoid activity is also affected by carbenoxolone. Using a rat model involving low dose corticosterone treatment, we found that carbenoxolone neither potentiated nor inhibited the modest increases in blood pressure or reductions in weight gain caused by steroid treatment. Other indices of glucocorticoid activity including white blood cell number, thymus weight, and down regulation of the glucocorticoid receptor were unaffected. In vitro studies with liver and kidney cytosol preparations indicated that carbenoxolone did compete for 3H-dexamethasone binding sites. Carbenoxolone was 5-10 times more effective than glycyrrhetinic acid, 20-30 thousand times less effective than dexamethasone, and is therefore, approximately 1000 times less effective than corticosterone. Analysis of dexamethasone-binding curves indicated a single class of receptor. We conclude that carbenoxolone at the dose tested does not have intrinsic glucocorticoid activity in vivo, nor does it modulate the activities of corticosterone. Carbenoxolone binds weakly to the glucocorticoid receptor. It is not clear whether this weak affinity accounts for some or any of the direct in vitro effects of high concentrations of carbenoxolone that others have described.

HistopathologyVolume 28, Issue 6 p. 554-555 Mammary adenohibernoma S. DAMIANI, S. DAMIANI Institute of Anatomic Pathology, University of Bologna, Bologna, ItalySearch for more papers by this authorM. PANARELLI, M. PANARELLI Institute of Anatomic Pathology, University of Bologna, Bologna, ItalySearch for more papers by this author S. DAMIANI, S. DAMIANI Institute of Anatomic Pathology, University of Bologna, Bologna, ItalySearch for more papers by this authorM. PANARELLI, M. PANARELLI Institute of Anatomic Pathology, University of Bologna, Bologna, ItalySearch for more papers by this author First published: June 1996 https://doi.org/10.1046/j.1365-2559.1996.d01-474.xCitations: 9 Dr Stefania Damiani Istituto di Anatomia Patologica, Policlinico S. Orsola, via Massarenti 9, 40137, Bologna, Italy. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume28, Issue6June 1996Pages 554-555 RelatedInformation

1. The role of genetically determined changes in adrenal steroid production, metabolism and action in the pathogenesis of cardiovascular disease in man is considered by studying three loci that are important in corticosteroid function. 2. Variation at the glucocorticoid receptor locus can be identified as a biallelic restriction fragment length polymorphism (Bcl1); subjects with contrasting genotypes show altered skin vasoconstrictor responses to topically applied budesonide without any significant change in leucocyte receptor binding characteristics. 3. In a case control study of patients with essential hypertension, we have shown evidence of reduced 11 beta-hydroxysteroid dehydrogenase activity, with an elevated ratio of cortisol to cortisone metabolites in urine. 4. The genes encoding 11 beta-hydroxylase and aldosterone synthase are highly homologous. Studies in the Milan hypertensive rat show variation at this locus, which may account for the increased steroid synthesis noted in the hypertensive strain; in man, a chimaeric gene comprising 5' regulatory regions from 11 beta-hydroxylase and 3' coding sequence from aldosterone synthase accounts for the autosomal dominant condition Dexamethasone Suppressible Hyperaldosteronism. Variation in the precise location of the crossover site between the two genes does not account for the observed phenotypic heterogeneity in this condition. 5. Measurement of basal plasma steroid levels in subjects with essential hypertension show an increased ratio of 11-deoxycortisol/cortisol, consistent with reduced activity of 11 beta-hydroxylase in the zona fasciculata. 6. In summary, three loci involved in corticosteroid synthesis, metabolism and action can independently affect cardiovascular phenotypes; their roles in determining pathophysiological changes, including hypertension, remain to be studied.

The effect of a low dose of the glucocorticoid receptor antagonist, RU486, was tested in spontaneously hypertensive (SHR) and in Sprague Dawley (SD) rats. In SD rats, RU486 (50 gmg/day, 18 days) significantly increased growth rate and thymus weight, probably by antagonising the actions of endogenous glucocorticoid hormones. Blood pressure and plasma corticosterone levels were not affected by RU486 at this dose. In leucocytes, RU486 treatment in vivo reduced the number of glucocorticoid binding sites but did not affect binding affinity. In contrast, growth rate and thymus weight were not altered in SHR when treated with the same dose of RU486 for a longer period (60 days). Blood pressure was unaffected as were leucocyte glucocorticoid receptor characteristic. Glucocorticoid receptor binding characteristics for RU486 were similar for liver cytosol of SD and SHR. We conclude that the apparent difference in sensitivity to RU486 between strains is probably not due to differences in interaction of the antagonist with the glucocorticoid receptor but may be caused by differences in pharmacokinetics of RU486 between strains.

Glucocorticoid receptor binding was compared in liver cytosol preparations from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats using homologous displacement of [3H]dexamethasone. At 5°C, there was no difference in receptor binding affinity or concentration between strains for dexamethasone, corticosterone or aldosterone. At 37°C, affinity for dexamethasone was lower than at 5°C for both rat strains and decreased with time. However, at this higher temperature, binding affinity in the SHR preparation was consistently higher than in the WKY preparation. The WKY preparation had a higher receptor concentration. The rate of dissociation of the [3H]dexamethasone-receptor complex prepared at 5°C and then incubated at 37°C was rapid but not different between strains. A possible explanation of these results is that the relationship of the heat shock proteins to the receptor heterocomplex is different between strains. Evidence exists of a genetic difference in Hsp 70 between SHR and WKY rats, although its cosegregation with blood pressure has not been established.

Glucocorticoids inhibit translation of the lysozyme gene. This effect may be the basis of an improved method of measuring glucocorticoid responsiveness in human tissues. We have compared lysozyme synthesis in various types of white blood cells and examined the specificity of inhibitory responses to various steroid hormones. The dose-related effects of the glucococorticoid receptor antagonist RU486 on dexamethasone responses were also assessed. Glucocorticoid receptor binding in mononuclear leukocytes (HML) was characterized by homologous displacement of [3H]dexamethasone and compared with the dose-related inhibitory effect of dexamethasone on lysozyme synthesis. Lysozyme activity was measured photometrically as the ability to cause lysis of Micrococcus lysodeikticus in the medium. The greatest effect of dexamethasone was observed after 72 h of culture. Qualitatively similar effects of dexamethasone were observed on cell lysozyme content and lysozyme activity in the medium, but for convenience, activity in medium, rather than cell content, was measured in subsequent assays. Lysozyme activities in various cell types prepared from the blood of healthy volunteers were ranked as follows: polymorphonuclear cells > monocytes > mononuclear cells > lymphocytes. However, dexamethasone inhibited lysozyme synthesis to a similar degree for all types. As mononuclear cells are more conveniently prepared in greater yield compared with other cells, this HML fraction formed the basis of a method of assessing glucocorticoid responsiveness and sensitivity. Lysozyme activity from HML was not significantly affected by incubation with 1 mumol/L estradiol, progesterone, dehydroepiandrosterone, or aldosterone. Dexamethasone and cortisol at 1 mumol/L both inhibited release by 45-50%. Although RU486 when added alone partially inhibited lysozyme activity, the same concentration (1 mumol/L) antagonized glucocorticoid responses and shifted the IC50 and threshold values for the effect of dexamethasone from 1.2 nmol/L to more than 1 mumol/L and from less than 1.0 to 19 nmol/L, respectively. The equilibrium dissociation constants (Kd) for dexamethasone binding to the glucocorticoid receptor ranged from 2.8-12.5 nmol/L and were positively correlated with dexamethasone IC50 values for lysozyme synthesis (r = 0.57; P = 0.002). In conclusion, the inhibition of lysozyme synthesis by dexamethasone in human mononuclear cells is a convenient and specific method of measuring responsiveness to glucocorticoids.

(1994). The glucocorticoid receptor and hypertension. Endocrine Research: Vol. 20, No. 2, pp. 101-116.

We have explored the variability of P300 event-related potentials in patients affected by Hakim-Adams syndrome, with raised or intermittent intracranial pressure, treated with surgical cerebrospinal fluid shunting. The clinical utility of P300 is confirmed in the light of the improvement of neurophysiological data after the surgical procedure, parallel with amelioration of neuropsychological performances.

This study was designed to determine plasma endothelin-1 levels in patients with essential hypertension and diabetes mellitus. Endothelin immunoreactivity was measured in normal controls (n = 30), mild-moderate essential hypertensives (n = 25), Type II diabetic normotensives (n = 25) and hypertensive patients (n = 20). In addition, in ten patients of each group we investigated the relationships of endothelin with other vasoactive hormones. Plasma endothelin concentrations were similar in healthy controls, in essential hypertensives and in diabetic patients with or without hypertension, averaging 8.23 +/- 1.68 pg/ml, 7.7 +/- 1.1 pg/ml, 5.05 +/- 0.94 pg/ml, 7.88 +/- 1.41 pg/ml, respectively. No correlations were observed between endothelin and concentrations of plasma renin activity, aldosterone, catecholamines, atrial natriuretic peptide and arginine-vasopressin. The present study suggests that Type II diabetic patients with or without essential hypertension do not have demonstrably higher values of plasma endothelin than essential hypertensives or healthy subjects.

Young (3-week-old) spontaneously hypertensive rats (SHR) had significantly higher basal plasma corticosterone levels than WKY rats and maximum responses to ACTH were also higher. In isolated adrenocortical cells from these rats, corticosterone production was also more responsive to ACTH in SHR. There was no significant difference in aldosterone production. Mononuclear leucocytes from older (10-week-old) SHR had a higher affinity for dexamethasone but a smaller number of binding sites per cell. The SHR therefore has higher circulating glucocorticoid levels and the target cells have a higher apparent affinity for this agonist. However, the target cells also have a smaller binding capacity. The precise resultant effect of these changes on glucocorticoid activity will require additional studies on specific glucocorticoid-dependent variables.

Nine hundred and ninety patients, ages 20 years or older, undergoing non-cardiac elective surgery were prospectively studied to identify high cardiac risk preoperative factors in a case-mix population and to assess cardiological risk. The prevalence of major cardiac complications was 2.3%, including 0.8% mortality. Univariate analysis showed that: age; history of chest pain; dyspnea; hypertension; presence of systolic murmur and third sound; diastolic pressure greater than 95 mmHg; electrocardiogram left ventricular hypertrophy; cardiothoracic ratio greater than 0.5 and valvular calcifications are associated with cardiac complications (p = 0.001-0.02), with low sensitivity (range: 14-38%) and high specificity (range: 85-98%). Cardiological referral was required for 169 patients (17%) that showed a higher prevalence of cardiovascular diseases (85%) and of cardiac complications (5.3%). Cardiologists required further tests for 13 patients (7.7%) and modified therapy for 93 (55%). High cardiac risk patients are identified preoperatively in current practice and cardiological referral is frequent; further studies are mandatory to evaluate the most effective and efficacious procedures.

The association of liver cirrhosis with arterial essential hypertension has been previously described. The present study extends the previous reports by investigating the hormonal relationships that may occur in patients with established essential hypertension associated to liver cirrhosis. We studied the renin-angiotensin, the adrenergic systems and other vasoactive hormones such as arginine-vasopressin, atrial natriuretic peptide, endothelin and parathyroid hormone in cirrhotic patients with and without essential hypertension. The data suggested that the coincidence of arterial hypertension in cirrhotic patients was characterized by the following findings: a decreased renin-angiotensin activity; a reduced systemic vasodilatation; an increased peripheral pressor effect of vasoactive hormones and an increased effective blood volume.

In 45 patients with essential hypertension and 15 age-matched normotensive control subjects, the renal resistive index, as an expression of arterial impedance, was determined using Doppler ultrasound. In both kidneys the resistive index was assessed at baseline and after captopril test (50 mg orally). In the moderate and severe hypertensives, compared to mild hypertensives and control subjects, the baseline resistive index was significantly higher (P less than .05). Following captopril, the resistive index increased only in normotensives (P less than .05) and in mild hypertensives (P less than .05). Univariate and multivariate analyses show that the duration and severity of hypertension correlated with an increase of the resistive index both in basal and in dynamic conditions. Thus, the use of the resistive index, as determined by echo-Doppler, could provide useful information for the assessment of renal vascular impedance in essential hypertensive patients. This would help us detect the evolution of hypertensive disease to the higher degrees of severity that are correlated to renal arteriolar damage.

Endothelin-1, a potent vasoconstrictor peptide with 21 amino acid residues, is released by the vascular endothelium. Plasma immunoreactive endothelin levels were measured in 23 patients with cirrhosis and in 20 healthy subjects. Concentrations were significantly lower in patients with non-uraemic cirrhosis than in normal subjects (19.4 +/- 8.9 pmol/l vs. 48.8 +/- 24.8 pmol/l, p less than 0.002). Plasma renin, aldosterone, atrial natriuretic peptide, arginine-vasopressin and catecholamines did not show significant correlations with plasma endothelin-1 levels. Furthermore, there were no significant differences in plasma endothelin levels for etiology of cirrhosis, presence of ascites or varices. These data suggest that low circulating endothelin may be involved in the development or maintenance of systemic vasodilatation in cirrhosis.