Laura Vizzotto

Proximity of TPR and NTRK1 rearranging loci in human thyrocytes

Chromosomal rearrangements are frequently associated with cancer; the mechanisms underlying their cell-type specificity are poorly understood. Papillary thyroid carcinomas are marked by a high frequency of chromosome rearrangements involving the RET and NTRK1 tyrosine kinase receptor genes and producing RET and TRK oncogenes. An explanation for the propensity of thyrocytes to undergo gene rearrangements has been recently proposed by Nikiforova and colleagues, who showed that the recombination between RET and H4 is favored by the loci proximity in interphase nuclei. We investigated whether the spatial proximity is a contributing factor also in the generation of the thyroid-specific TRK oncogenes. The distance between NTRK1 and its oncogenic partner TPR was determined by two-color fluorescence in situ hybridization and two-dimensional microscopy. A three-dimensional reconstruction of the data was also done. We show that the two loci in thyrocytes nuclei display a distance reduced with respect to peripheral blood lymphocytes, thus supporting the notion that spatial proximity of translocation-prone gene loci may favor gene rearrangements.

Circadian variations in expression of the trkB receptor in adult rat hippocampus

The expression of brain-derived neurotrophic factor (BDNF) in the central nervous system (CNS) and the expression of its high-affinity trkB receptor on neuron surfaces are known to depend on neuron activity. The expression of BDNF (mRNA and protein) and trkB mRNA shows circadian oscillations in rat hippocampal homogenates. We investigated circadian variations in trkB expression in specific areas of the adult rat hippocampal formation by immunohistochemistry. In sets of two experiments performed in the spring, 39 2-month-old male Wistar rats were accustomed to a 12-h light-12-h dark cycle for 2 weeks. Three animals were then sacrificed every 4 h. Forty-micrometer-thick coronal sections of hippocampal formation were obtained and processed for trkB immunohistochemistry. Cell staining intensity was assessed by image analysis of different hippocampal areas on five sections per animal. Circadian rhythmicity was evaluated by the cosinor method. Statistically significant circadian variations in trkB expression were found in dentate gyrus, entorhinal cortex, and the CA3 and hilar regions of the hippocampus, with highest expression during the first half of the dark (activity) period. These findings suggest a relationship between trkB expression and the physiological neuronal activation of wakefulness. TrkB receptor expression in the hippocampal regions studied was continuous and changes were gradual over the 24-h cycle, suggesting that more complex regulatory mechanisms also intervened.

Three-dimensional nasal morphology in cleft lip and palate operated adult patients

Nasal morphology was assessed three-dimensionally in 18 cleft lip and palate operated patients aged 19 to 27 years, and in 161 sex- and age-matched control subjects. Comparisons were performed by Student t and Watson–Williams’ tests. No differences were found in nasal volume or surface. Nasal width, alar base width, and inferior width of the nostrils were significantly larger in male patients than in the reference men. The nasal bridge was shorter. Similar differences were found in the female patients. In addition, the right nostril was larger and the nasal height shorter. In men, the nasolabial and the nasal tip angles were smaller in the patients than in the reference subjects, whereas the facial convexity angle was larger in the patients. In conclusion, the nose of adult operated cleft lip and palate patients differed from that of normal control subjects. Surgical corrections of the cleft lip and palate failed to provide a completely normal appearance. The methods might be used to indicate where additional procedures might be performed to approximate a reference population.

Position of follicles in normal human ovary during definitive histogenesis

BACKGROUND Follicular growth in the mammalian ovary follows a geographically determined pattern, but no exhaustive data about their spatial localization in the cortex of human ovary exists. AIM The aim of this study is to assess the spatial position of the follicles during human ovarian definitive histogenesis using morphometric methods. SUBJECTS Ovaries removed from four fetuses, five newborns and one 8-month-old child with 46,XX karyotype was used. OUTCOME MEASURES The position of the different follicle categories (primordial, primary, secondary and antral) was estimated as percentage distance of the centroid of the follicle from the cortico-medullary boundary. RESULTS In normal ovaries, during definitive histogenesis, the primordial follicles progressively occupy more peripheral parts of the cortex (on average, 41% of cortical length at 20 weeks of fetal development, 53% at birth, 68% at 8 postnatal months). Primary follicles were crowded in the inner part of the cortex (newborn: 30-40% of cortical length; 8 months: 52% of cortical length). Secondary and antral follicles were sampled in all neonatal specimens and in the 8-month old specimen; they were grouped next to the cortico-medullary boundary. CONCLUSIONS Ovarian development corresponded to a migration of the maximum crowding of follicles from a position next to the medulla towards a more peripheral location. The control of the primordial follicle assembly, recruitment and development are coordinated by locally produced paracrine factors. The action of these factors seems to follow a negative gradient from the cortex toward the medulla.

Analysis of Histological and Immunohistochemical Patterns of the Liver in Posthepatitic and Alcoholic Cirrhosis by Computerized Morphometry

To assess the degree of fibrosis and the structural changes affecting parenchymal and extraparenchymal components in liver cirrhosis, a computerized morphometric model has been applied to liver specimens from patients with posthepatitic and alcoholic cirrhosis. All specimens have been stained with chromotrope-aniline blue method and monoclonal antibodies against cytokeratin 7, CD31, and VIII factor. Volume fractions of parenchymal compartment and fibrosis have been determined stereologically on CAB slices; moreover, volume fractions of portal bile ducts and proliferated bile ductules, hepatocytes with biliary metaplasia, capillary units, and vascular structures have been measured. Volume fraction of fibrosis was higher in alcoholic cirrhosis when compared with the case of posthepatitic cirrhosis. Volume fractions describing parenchymal compartment showed a similar trend in both viral groups. The main differences were related to immunohistochemical stainings. Volume fraction of hepatocytes with biliary metaplasia was higher in hepatitis C virus-related cirrhosis, whereas volume fractions of biliary structures were more prominent in hepatitis B virus-related cirrhosis. Capillary units were more prominent in posthepatitic cirrhosis than in alcoholic cirrhosis. Interestingly, both forms of posthepatitic cirrhosis show similar features when compared with alcoholic cirrhosis. Our computerized morphometric model well describes and quantifies the morphological alterations of the liver, and it could represent an adjunctive tool to evaluate the degree of dysplastic phenomena involving parenchymal and extraparenchymal components.

Variations in facial soft tissues of italian individuals with ectodermal dysplasia.

Objective To supply quantitative information about the facial soft tissues of patients with ectodermal dysplasia (ED). Design, Setting, and Patients The three-dimensional coordinates of soft tissue facial landmarks were obtained using an electromagnetic digitizer in 20 Caucasian patients with ED (11 male and 9 female patients aged 7 to 41 years) and 391 healthy controls (163 female and 228 male subjects of comparable ages). From the landmarks, 15 facial dimensions and two angles were calculated. Data were compared with those collected in healthy individuals by computing z-scores. Two summary anthropometric measurements for quantifying craniofacial variations were assessed in both the patients and reference subjects: the mean z-score (an index of overall facial size) and its standard deviation, called the craniofacial variability index (an index of facial harmony). Results In patients with ED, a large variability was found for both the mean z-score and the craniofacial variability index. On average, the patients had a somewhat smaller facial size than the normal individuals, with a global disharmonious appearance. Overall, 70% of patients fell outside the quantitative definitions for normal facial size, harmony or both. When examining the distribution of the single z-scores of the 17 selected measurements, variable patterns were found, without consistent effects of age or sex. Conclusions The facial soft tissue structures of patients with ED differed both in size and shape from those of normal controls of the same age, sex, and ethnic group.

Bronchial Branching Correlates with Specific Glycosidase Activity, Extracellular Glycosaminoglycan Accumulation, TGFβ2, and IL-1 Localization During Chick Embryo Lung Development

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), β-N-acetyl-d-glucosaminidase activity, the presence of transforming growth factor β2 (TGFβ2), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of β-N-acetyl-d-glucosaminidase with biochemical methods. Our data show that β-N-acetyl-d-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that β-N-acetyl-d-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.

Liver transplantation in man: morphometric analysis of the parenchymal alterations following cold ischaemia and warm ischaemia/reperfusion

Ischaemia and reperfusion phases represent critical events during liver transplantation. The purpose of this study was to describe morphological alterations of both vascular and parenchymal compartments after ischaemia and reperfusion and to evaluate the possible relationship between morphometric parameters and biochemical/clinical data. Three needle biopsies were drawn from 20 patients who underwent orthotopic liver transplantation. The first biopsy was taken before flushing with preservation solution, and the second and the third to evaluate respectively the effects of cold ischaemia and of warm ischaemia/reperfusion. Biopsies were examined by an image analyser and morphometric parameters related to the liver parenchyma were evaluated. At the second biopsy we observed a decrease of the endothelium volume fraction while the same parameter referred to the sinusoidal lumen achieved a peak value. The hepatocytes showed a lower surface parenchymal/vascular sides ratio. This parameter was reversed at the end of the reperfusion phase; furthermore the third biopsy revealed endothelial swelling and a decreased volume fraction of the sinusoidal lumen. The results quantify the damage to the sinusoidal bed which, as already known, is one of the main targets of cold ischaemia; warm ischaemia and reperfusion accentuate endothelial damage. The end of transplantation is characterised by damage chiefly to parenchymal cells. Hepatocytes show a rearrangement of their surface sides, probably related to the alterations of the sinusoidal bed. In addition, the fluctuations of morphometric parameters during ischaemia/reperfusion correlate positively with biochemical data and clinical course of the patients.

Morphometric analysis of hepatocellular nodular lesions in HCV cirrhosis.

We generated a computerized morphometric model to evaluate and quantify the morphological features in large regenerative nodules (LRN), high-grade dysplastic nodules (HGDN) and hepatocellular carcinoma (HCC). Sixteen LRN, 10 HGDN and 16 HCC in HCV-cirrhotic livers were stained with H&E, smooth muscle actin, CD34, CD31 and reticulin to evaluate volume and surface fractions. On H&E stains, the most discriminatory features between LRN, HGDN and HCC were volume fraction and the number of hepatocyte nuclei in unit volume and hepatocyte nuclear/cytoplasmic ratio. On immunohistochemistry, volume fractions of capillarised sinusoids, capillary units and isolated arteries were significantly different among all groups and highest in HCC; surface fraction of reticulin was markedly decreased in HCC. Our morphometric model is an objective method for quantification of the morphological changes of the nodular lesions, and it could be applied to studies involving histological evaluation of the spectrum of nodular lesions arising in the cirrhotic liver.

Morphometric analysis of primary graft non-function in liver transplantation

Aims : Primary graft non‐function (PNF) is a life‐threatening condition that is thought to be the consequence of microcirculation injury. The aim of the present study was to assess, with a computerized morphometric model, the morphological changes at reperfusion in liver biopsy specimens from patients who developed PNF after liver transplantation.