Aquaporin 9 is expressed in the epididymis of immature and mature pigs.

Aquaporins (AQPs) are channel proteins that facilitate the transepithelial and bidirectional movement of water. AQP9 is an aquaporin that is expressed in the mammalian epididymis. This water transport contributes to epididymal sperm concentration. This study aimed to examine the morphology of epididymal epithelium in piglets and boars, as well as the expression and immunolocalization of AQP9. The piglets presented an epididymal epithelium in differentiation with principal, basal and apical cells. The cellular population of the epididymal epithelium in boars consisted of principal, basal, apical, clear and narrow cells. The migratory cells known as halo cells were observed in the epididymis of both piglets and boars. AQP9 expression presented differences between piglets and boars. Moderate intensity of AQP9 immunoreaction was observed in the apical border of the epididymal epithelium of the caput and cauda regions in the piglet epididymis. A moderate-to-intense reaction for AQP9 was observed in the nuclei of epithelial cells of the three epididymal regions in the boar epididymis. The region of the cauda epididymis showed reactivity for AQP9 also in the apical border of the epithelium. It is believed that the AQP9 is already functional in piglets at only 1 week of age and is more active, playing a pivotal role in the caput and cauda regions of the epididymis. Moreover, the intense AQP9 expression in the apical border of epithelial cells in the cauda region of the boar epididymis suggests a higher performance of AQP9 in this region, where sperm complete their maturation process, stored and concentrated.

Immunolocalization of Aquaporins 1 and 9 in the Ram Efferent Ducts and Epididymis.

Aquaporins (AQPs) are essential membrane protein channels for the transport of water across membranes. Fluid movement in the epididymis is important for modulation of the luminal environment, in which sperm mature and reside. This study was designed to understand the morphology and localization of AQPs in ram efferent ducts (ED) and epididymis. For this purpose, the epididymis of seven animals were removed for histologic and immunohistochemical analyses. AQP1 immunoreactivity was observed in the apex of the ED, and AQP9 was found adjacent to the nuclei of the epithelial cells of the ED. The epithelial lining of ram epididymis is pseudostratified columnar and presents principal, basal, apical and narrow cells. In the initial segment (IS), a moderate reaction for AQP1 was observed in the apical cytoplasm of epithelial cells. An intense reactivity for AQP1 was noted over the microvilli of principal cells and in spermatozoa in the caput. In the corpus and cauda, AQP1 was noted only over the endothelial cells of vascular channels located in intertubular spaces. A weak-to-moderate reaction for AQP9 was observed in the nuclei of epithelial cells in the IS, caput and corpus of the epididymis. In the cauda, an intense reaction to AQP9 was observed in the epithelial border. In the IS, caput and corpus, the reactivity for AQP9 differed from those observed in domestic animals. The cauda showed a pattern similar to that previously described. These results indicate that AQPs 1 and 9 have reversed locations and roles in rams, suggesting activity variations related with fluid and solute absorption throughout the epididymis.

Exercise as a new physiological stimulus for brown adipose tissue activity.

BACKGROUND AND AIM: Brown adipose tissue (BAT) plays a major role in body energy expenditure counteracting obesity and obesity-associated morbidities. BAT activity is sustained by the sympathetic nervous system (SNS). Since a massive activation of the SNS was described during physical activity, we investigated the effect of endurance running training on BAT of young rats to clarify the role of exercise training on the activity and recruitment state of brown cells. METHODS AND RESULTS: Male, 10-week-old Sprague Dawley rats were trained on a motor treadmill (approximately 60% of VO2max), 5 days/week, both for 1 and 6 weeks. The effect of endurance training was valuated using morphological and molecular approaches. Running training affected on the morphology, sympathetic tone and vascularization of BAT, independently of the duration of the stimulus. Functionally, the weak increase in the thermogenesis (no difference in UCP-1), the increased expression of PGC-1α and the membrane localization of MCT-1 suggest a new function of BAT. Visceral fat increased the expression of the FOXC2, 48 h after last training session and some clusters of UCP-1 paucilocular and multilocular adipocytes appeared. CONCLUSION: Exercise seemed a weakly effective stimulus for BAT thermogenesis, but surprisingly, without the supposed metabolically hypoactive effects. The observed browning of the visceral fat, by a supposed white-to-brown transdifferentiation phenomena suggested that exercise could be a new physiological stimulus to counteract obesity by an adrenergic-regulated brown recruitment of adipocytes.

The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.

The origin of brown adipocytes arising in white adipose tissue (WAT) after cold acclimatization is unclear. Here, we demonstrate that several UCP1-immunoreactive brown adipocytes occurring in WAT after cold acclimatization have a mixed morphology (paucilocular adipocytes). These cells also had a mixed mitochondrioma with classic "brown" and "white" mitochondria, suggesting intermediate steps in the process of direct transformation of white into brown adipocytes (transdifferentiation). Quantitative electron microscopy disclosed that cold exposure (6 degrees C for 10 days) did not induce an increase in WAT preadipocytes. beta(3)-adrenoceptor-knockout mice had a blunted brown adipocyte occurrence upon cold acclimatization. Administration of the beta(3)-adrenoceptor agonist CL316,243 induced the occurrence of brown adipocytes, with the typical morphological features found after cold acclimatization. In contrast, administration of the beta(1)-adrenoceptor agonist xamoterol increased only the number of preadipocytes. These findings indicate that transdifferentiation depends on beta(3)-adrenoceptor activation, whereas preadipocyte recruitment is mediated by beta(1)-adrenoceptor. RT-qPCR experiments disclosed that cold exposure induced enhanced expression of the thermogenic genes and of genes expressed selectively in brown adipose tissue (iBAT) and in both interscapular BAT and WAT. beta(3)-adrenoceptor suppression blunted their expression only in WAT. Furthermore, cold acclimatization induced an increased WAT expression of the gene coding for C/EBPalpha (an antimitotic protein), whereas Ccna1 expression (related to cell proliferation) was unchanged. Overall, our data strongly suggest that the cold-induced emergence of brown adipocytes in WAT predominantly reflects beta(3)-adrenoceptor-mediated transdifferentiation.

Serum amyloid A: production by human white adipocyte and regulation by obesity and nutrition.

AIMS/HYPOTHESIS: The acute-phase proteins, serum amyloid As (SAA), are precursors of amyloid A, involved in the pathogenesis of AA amyloidosis. This work started with the characterisation of systemic AA amyloidosis concurrent with SAA overexpression in the subcutaneous white adipose tissue (sWAT) of an obese patient with a leptin receptor deficiency. In the present study a series of histopathological, cellular and gene expression studies was performed to assess the importance of SAA in common obesity and its possible production by mature adipocytes. MATERIALS AND METHODS: Gene expression profiling was performed in the sWAT of two extremely obese patients with a leptin receptor deficiency. Levels of the mRNAs of the different SAA isoforms were quantified in sWAT cellular fractions from lean subjects and from obese subjects before and after a very-low-calorie diet. These values were subsequently compared with serum levels of SAA in these individuals. In addition, histopathological analyses of sWAT were performed in lean and obese subjects. RESULTS: In sWAT, the expression of SAA is more than 20-fold higher in mature adipocytes than in the cells of the stroma vascular fraction (p<0.01). Levels of SAA mRNA expression and circulating levels of the protein are sixfold (p<0.001) and 3.5-fold (p<0.01) higher in obese subjects than in lean subjects, respectively. In lean subjects, 5% of adipocytes are immunoreactive for SAA, whereas the corresponding value is greater than 20% in obese subjects. Caloric restriction results in decreases of 45-75% in levels of the transcripts for the SAA isoforms and in circulating levels of the protein. CONCLUSIONS/INTERPRETATION: The results of the present study indicate that SAA is expressed by sWAT, and its production at this site is regulated by nutritional status. If amyloidosis is seen in the context of obesity, it is possible that production of SAA by adipocytes could be a contributory factor.

In vivo leptin expression in cartilage and bone cells of growing rats and adult humans.

The present investigation was carried out to analyse, immunohistochemically, in vivo leptin expression in cartilage and bone cells, the latter restricted to the elements of the osteogenic system (stromal cells, osteoblasts, osteocytes, bone lining cells). Observations were performed on the first lumbar vertebra, tibia and femur of four rats and on the humerus, femur and acromion of four patients. Histological sections of paraffin-embedded bone samples were immunostained using antibody to leptin. The results showed that, in growing rat bone, leptin is expressed in chondrocytes and stromal cells, but not in osteoblasts; bone lining cells were not found in the microscopic fields examined. In adult human bone, leptin is expressed in chondrocytes, stromal cells and bone lining cells; osteoblasts were not found in the microscopic fields examined. Osteocytes were found to be leptin positive only occasionally and focally in both rat and human bone. The in vivo findings reported show, for the first time, that leptin appears to be expressed only in the cells of the osteogenic lineage (stromal cells, bone lining cells, osteocytes) that, with respect to osteoblasts, are permanent and inactive, i.e. in those cells that according to our terminology constitute the bone basic cellular system (BBCS). Because the BBCS seems to be primarily involved in sensing and integrating mechanical strains and biochemical factors and then in triggering and driving bone formation and/or bone resorption, it appears that leptin seems to be mainly involved in modulating the initial phases of bone modelling and remodelling processes.

Immunohistochemical identification of the beta(3)-adrenoceptor in intact human adipocytes and ventricular myocardium: effect of obesity and treatment with ephedrine and caffeine.

OBJECTIVES: To investigate whether the beta(3)-adrenoceptor could be identified by immunohistochemistry in intact human white and brown adipocytes and other human tissues, and to investigate the influence of obesity and its treatment with ephedrine and caffeine on the expression of the beta(3)-adrenoceptor in adipocytes. METHODS: Morbidly obese patients were given a hypoenergetic diet (70% of energy expenditure) and some were also treated with ephedrine and caffeine (20/200 mg, three times daily) for 4 weeks. Adipose tissue and other tissues were taken during surgery. Immunohistochemistry was carried out using a monoclonal antibody raised against the human beta(3)-adrenoceptor. RESULTS: Staining was localized to the periphery of cells. All white adipocytes were stained. Those from lean subjects and obese subjects treated with ephedrine and caffeine showed more intense staining than those from untreated obese subjects. Staining was more intense in brown than in white adipocytes in perirenal adipose tissue from phaeochromocytoma patients. Staining was also seen in ventricular myocardium, and in smooth muscle of the prostate, ileum, colon and gall bladder. DISCUSSION: The tissue and subcellular distribution of staining was consistent with it being due to binding of the antibody to the human beta(3)-adrenoceptor. The presence of the beta(3)-adrenoceptor in human white adipocytes is consistent with evidence that it can mediate lipolysis in human white adipocytes. The increased expression of the beta(3)-adrenoceptor in obese subjects treated with caffeine and ephedrine supports the potential of beta(3)-adrenoceptor agonists in the treatment of obesity and type 2 diabetes. Its expression in ventricular myocardium is consistent with evidence that the beta(3)-adrenoceptor mediates a negative inotropic effect in this tissue.

Intralobular ducts of human major salivary glands contain leptin and its receptor.

Leptin, a 16-kDa hormone, plays an important role in the control of food intake and in energy homeostasis both in rodents and in man. Leptin is mainly produced and secreted by adipocytes, but other tissues and gastric glands have also recently been shown to produce it in a dual (endocrine and exocrine) mode. In addition, a leptin receptor has been detected in taste cells of mouse circumvallate papillae and in rat intestinal epithelium. These data prompted us to carry out a detailed study of human salivary glands as potential leptin-producing organs. Biopsies of salivary glands (submandibular and parotid) obtained from male and female patients during surgery for different clinical indications were subjected to immunohistochemical study for the presence of leptin, its functional receptor, insulin and glucagon. The presence and cellular distribution of glucocorticoid receptor in leptin-secreting cells were also investigated. Double immunohistochemical staining (silver-gold intensification and avidin-biotin-peroxidase) was used for the visualization of glucocorticoid receptor and leptin labelling, respectively. The results show that intralobular duct cells of submandibular and parotid glands are immunoreactive for leptin, leptin receptor and glucagon but not for insulin. Leptin was also detected in some microglobules in whole saliva obtained from four healthy volunteers. Co-localization for leptin, leptin receptor and glucocorticoid receptor in the same cell type suggested a functional relationship between glucocorticoid hormone and leptin secretion also at the level of the salivary glands.

Oleoyl-estrone does not have direct estrogenic effects on rats.

The estrogenic effects of oleoyl-estrone (OE) administration, either though continuous i.v. infusion with osmotic minipumps or administered by daily oral gavage, were studied. Binding of OE to human recombinant purified alpha receptors was negligible, and that of estrone (E1) was only a fraction of 17beta-estradiol (E2) binding. Intravenous--but not oral--OE administration resulted in marked increases of both E1 and E2 in rat plasma, but oral OE did not induce significant changes in either plasma hormone in Wistar or Zucker rats. The weight of uteri and ovaries increased with time of administration in Zucker rats treated with i.v. OE, but inguinal mammary gland proliferation between subcutaneous adipose tissue was even more marked. Oral administration of OE, however, did not increase either uterine weight or mammary gland proliferation, even at doses (10 micromol/kg x d) higher than those given i.v. (3.5 micromol/kg x d). The results indicate that i.v. administration of OE resulted in limited estrogenic effects mainly due to the high accumulation of E1 giving rise to significant increases in E2. On the other hand, oral administration of OE, even at higher daily doses, did not increase the circulating levels of either estrogen and, therefore, there were no significant effects on mammary gland proliferation or uterine weight. The oral administration of OE as a slimming drug, then, do not result in estrogenic side effects over a wide range of daily doses.

Corticosteroid-binding globulin synthesis and distribution in rat white adipose tissue.

Corticosterone binding (CB) capacity was determined in visceral and subcutaneous white adipose tissue (WAT), as well as in plasma of lean Zucker rats. Perfusion of rats with saline eliminated most liver and kidney corticosterone binding but did not affect CB in WAT. The cytosol extracts of isolated cells, however, did not bind corticosterone in detectable amounts. By means of a RT-PCR procedure it was found that corticosterone-binding globulin (CBG) was expressed in WAT. By immunohistochemical detection in WAT sections, CBG was seen in a thin layer surrounding the cells near the plasma membrane. These data suggest that the CBG layer surrounding the cells may act as a protective barrier limiting the access of glucocorticoids to adipocytes.

Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion.

Leptin is a circulating hormone which plays an important role in the regulation of energy balance, haemopoiesis and reproduction. Leptin and its receptor (leptin-R) are localized in human placental tissue but their function is not known. In this study we have investigated the expression of leptin and leptin-R in the human placenta with particular attention to extravillous cytotrophoblastic cell islands and cell columns which play a pivotal role in trophoblast invasion and placental growth. We demonstrate that leptin-R immunoreactivity shows a strong expression in the distal extravillous cytotrophoblastic cells of cell columns invading the basal plate, whereas leptin expression is homogeneously expressed in all the cellular components of cell columns. Since the invasive ability of the distally located extravillous cytotrophoblast of cell columns is known to be regulated by a variety of proteases and some extracellular matrix molecules, we tested the influence of leptin on the in-vitro production of matrix metalloproteinase (MMP)-2, MMP-9 and fetal fibronectin (fFN) by cytotrophoblastic cells. We demonstrate that leptin increases, in a dose-dependent manner, the secretion of immunoreactive MMP-2 and fFN and enhances the activity of MMP-9 in cultured cytotrophoblastic cells. Our results suggest that leptin and leptin-R could have a role in the invasive processes of the extravillous cytotrophoblastic cells by modulating the expression of MMPs. In addition, these results provide a foundation for studying pathological conditions characterized by insufficient or excessive trophoblast invasion.

Ultrastructural immunolocalization of leptin receptor in mouse brain.

Antibodies directed to amino acids 877-894 (M-18) and 32-51 (K-20) were used to localize leptin receptor by immunocytochemistry in mouse brain. Both antibodies stained several hypothalamic nuclei (paraventricular nucleus, supraoptic nucleus, supraoptic retrochiasmatic nucleus, suprachiasmatic nucleus, preoptic area, ventromedial nucleus, dorsomedial nucleus, lateral hypothalamus, arcuate nucleus, ventral and dorsal premammillary nuclei), the thalamic and amygdaloid nuclei, neurons of the neocortex and archicortex and the epithelial cells of the choroid plexus. While M-18 staining was concentrated in the Golgi area, with K-20 it was dispersed in the cytoplasm. Glial cells were stained only by K-20. These results suggest that the trans-membrane forms of the receptor are concentrated at the membrane level of the Golgi complex of neurons and in epithelial cells of the choroid plexus while the soluble form is dispersed in their cytoplasm. Glial cells express only the soluble form.

TH-, NPY-, SP-, and CGRP-immunoreactive nerves in interscapular brown adipose tissue of adult rats acclimated at different temperatures: an immunohistochemical study.

Interscapular brown adipose tissue (IBAT), a site of nonshivering thermogenesis in mammals, is neurally controlled. The co-existence of sympathetic and peptidergic innervation has been demonstrated in different brown adipose depots. We studied the morphological profile of IBAT innervation and tested by immunohistochemical methods whether cold and warm stimulation are accompanied by modifications in the density of parenchymal noradrenergic nerve fibers. We also studied the immunoreactivity of afferent fibers--which contain calcitonin gene-related peptide (CGRP) and substance P (SP)--in different functional conditions. IBAT was obtained from adult rats (6 weeks old) acclimated at different temperatures (4 degrees, 20 degrees, and 28 degrees C). Tissue activity was evaluated by studying the immunolocalization of uncoupling protein (UCP-1), a specific marker of brown adipose tissue. Noradrenergic and peptidergic innervation were seen to arise from morphologically different nerves. Fibers staining for tyrosine hydroxylase (TH) were thin, unmyelinated hilar nerves, and CGRP- and SP-positive fibers were in thick nerves containing both myelinated and unmyelinated fibers. Under cold stimulation, noradrenergic neurons produce greater amounts of TH, and their axons branch, resulting in increased parenchymal nerve fibers density. Neuropeptide Y (NPY) probably co-localizes with TH in noradrenergic neurons, but only in the perivascular nerve fiber network. The parenchymal distribution of NPY to interlobular arterioles and capillaries suggests that this peptide must have other functions besides that of innervating arteriovenous anastomoses, as hypothesized by other researchers. The different distribution of CGRP and SP suggests the existence of different sensory neuronal populations. The detection of CGRP at the parenchymal level is in line with the hypothesis of a trophic action of this peptide.

Immunohistochemical localization of leptin and uncoupling protein in white and brown adipose tissue.

Leptin is synthesized exclusively by adipocytes and acts on the hypothalamus to regulate energy balance. Previous messenger RNA expression studies demonstrated that leptin is expressed in white adipocytes and also in brown adipose tissue, however expression in brown fat is markedly lower than in white fat. This suggests the possibility that leptin expression in brown adipose tissue is due to the presence of white adipocytes that reside within brown adipose tissue, and that brown adipocytes actually do not express leptin. To address this point, we performed immunohistochemistry on paraffin sections and studied leptin protein expression in different depots of white and brown fat of lean and obese (db/db) mice. To establish the cell type expressing leptin, we also assessed the size and organization of lipid droplets, the ultrastructural features of mitochondria, and the presence or absence of uncoupling protein, a brown fat-specific marker. In white adipose tissue of lean and obese (db/db) mice, leptin protein was expressed in adipocytes of various sizes (range examined: 19.67-200 microns), including adipocytes at the multilocular stage of differentiation. Leptin staining was more intense in some depots (retroperitoneal), and appeared to decrease with fasting. In brown adipose tissue of lean animals, multilocular uncoupling protein (UCP)-positive brown adipocytes had typical brown mitochondria and were leptin-negative, both in fed and fasted conditions. At the periphery of the interscapular brown adipose tissue depot, unilocular, UCP-negative adipocytes (mean diameter: 41.55 microns) with white-type mitochondria were observed, and these cells were leptin-positive. In obese (db/db) animals, brown fat was composed mainly of small unilocular, UCP-positive. adipocytes (mean diameter: 40.08 microns), which were also leptin-positive. At the periphery of the organ, numerous large, unilocular, UCP-negative adipocytes (mean diameter: 73.65 microns) with white-like mitochondria were present. As expected, these cells were also leptin-positive. In summary, classical brown adipocytes differ from white adipocytes, not only by their morphology and UCP expression, but also by their apparent lack of detectable leptin expression. db/db brown adipocytes, however, were unilocular and leptin-positive. The molecular mechanisms mediating expression of leptin in white but not brown adipocytes of lean animals, and the significant expression of leptin in brown adipocytes of db/db mice will be the focus of future studies.

Vitamin E affects quantitative age changes in lumbar motoneurons and in their peripheral projections.

Vitamin E deficiency was previously found to induce plastic changes in the number of primary sensory neurons and in motoneuron peripheral field projections. In this work, quantitative changes in motoneurons of lumbar segments, in nerve fibres constituting ventral roots and in innervating leg motor fibres were studied in normal and vitamin E deficient rats from 1 to 5 months of age. The number of lumbar motoneurons was found to decrease, while there were no changes in the number of ventral root fibres. An increase in the number of innervating leg motor fibres was observed during ageing in control rats; in vitamin E deficient rats the number of fibres in the ventral roots did not change, as occurred in controls, but the decrease in the number of motoneurons was smaller and the number of innervating leg motor fibres increased further in comparison to the controls. The findings are consistent with the idea that vitamin E deficiency causes a decrease in motoneuron death or, alternatively, that it induces some process partially compensating naturally occurring motoneuron death.

Changes in the number of primary sensory neurons in normal and vitamin-E-deficient rats during aging.

In the dorsal root ganglia (DRGs) of vitamin-E-deficient rats, we previously found an increase in the number of neurons during the first 5 months of life (Cecchini et al., 1993, 1994). This neurogenetic event seems to bring forward in time the increase in the number of primary sensory neurons that Devor et al. (1985) found in normal rats aged more than 1 year, but that other authors have not confirmed. The present study had two aims: first, to verify whether neurogenesis spontaneously occurs in DRGs of 14-month-old Sprague-Dawley rats; and, second, to determine whether the neurogenesis enhanced by vitamin E deficiency continues further in the long run, or whether it stops or reverses into neuron loss. A quantitative and morphometric analysis was performed on neurons of L3-L6 DRGs in 14-month-old normal and vitamin-E-deficient rats: the results obtained were compared to those previously obtained in 1-month-old and 5-month-old animals of both dietetic treatment groups, in order to observe the effects of aging on these neuronal populations. The total number of DRG neurons in the control group was higher in older than in younger animals, whereas the value in the vitamin-E-deficient group was lower in older than in younger animals. The present data confirm that neurogenesis occurs in DRGs of normal rats during adult life. Moreover, they show that once the premature neurogenesis in the deficient rats is completed, no further increase in the number of neurons takes place.

Increased number of sciatic sensory neurons in vitamin-E-deficient rats.

The number and morphometric characteristics of sciatic sensory neurons were studied in Vitamin-E-deficient rats. Horseradish peroxidase (HRP) was injected into the sciatic nerves of normal and vitamin-E-deficient rats of the same age, and retrogradely labeled sensory neurons were counted and measured. The study was also carried out in rats that had previously undergone sciatic nerve crush, in order to observe the effects of axotomy on primary sensory neurons. In vitamin-E-deficient rats the number of sciatic sensory neurons was significantly higher than normal, with an increase of about 30%, in agreement with a previous finding concerning total population of primary sensory neurons in lumbar dorsal root ganglia (DRGs) of vitamin-E-deficient rats. The increase involved the small cell classes in particular. Axotomy induced similar percentages of neuron loss in normal and in vitamin-E-deficient rats (about 40%). In the latter, death affected small cell classes in particular--that is, the same classes that had increased in number in vitamin-E-deficient rats by comparison with controls. These results, together with previous findings, suggest that neurogenesis may occur in DRGs of vitamin-E-deficient rats.

[Critical analysis of the diagnostic imaging of focal hepatic lesions]. / Analisi critica della diagnostica per immagini delle lesioni epatiche focali.

The aim of this paper is to evaluate the number and type of consecutive initial diagnoses of focal hepatic lesions obtained by abdominal US and CT. The diagnoses were coded according to the Index for Radiological Diagnoses (ACR). From January 1990 through April 1991, US and CT diagnosed focal hepatic lesion in 16.4% and 9.1% of cases, respectively. A lower number of focal hepatic lesions was diagnosed by CT, which however was more accurate as to the nature of the lesion itself. This is due to the fact that CT is often used to stage hepatic neoplasms already confirmed by US-guided fine-needle biopsy (FBN). The correlation between the initial diagnosis and actual clinical status demonstrates a high rate of occasional findings, especially with US. The rate of questionable diagnoses relative to primary or secondary malignancies was very high. This could be explained by caution in making a severe diagnosis, by awareness of the limits of macroscopic diagnostic techniques and by the immediate availability of US-guided FNB. In conclusion, the coding of consecutive initial diagnoses, by US and CT, could contribute to a practical evaluation of diagnostic imaging techniques, especially when correlated with the respective anamnestic and clinical data. Further studies are necessary to confirm this hypothesis.