Role of contrast-enhanced ultrasound to define prognosis and predict response to biotherapy in pancreatic neuroendocrine tumors.

PURPOSE: The incidence of neuroendocrine tumors (NETs) is progressively increasing. Most cases arise from the digestive system, where ileum, rectum and pancreas represent the commonest site of origin. Liver metastases are frequently detected at diagnosis or during the follow-up. Contrast-enhanced ultrasound (CEUS) is used in patients with pancreatic NETs (P-NETs) and liver metastases from P-NET but its role has not been standardized. The aim of this retrospective study was to investigate CEUS in patients with P-NETs and liver metastases from P-NET both as prognostic factor and predictor of response to therapy with somatostatin analogues (SSAs). METHODS: CEUS was performed at the diagnosis of NET and 3, 6 and 12 months after the beginning of SSAs. CEUS pattern was compared with contrast-enhanced computed tomography (CT) pattern. RESULTS: There was a significant association between CEUS and CT pattern (X 2 = 79.0; p < 0.0001). A significant association was found between CEUS pattern and Ki-67 index (X 2 = 24.6; p < 0.0001). The hypervascular homogeneous CEUS typical pattern was associated with low tumor grading (G1 or G2) (X 2 = 24.0; p < 0.0001). CEUS pattern changed from hypervascular homogeneous in baseline to hypovascular/hypervascular inhomogeneous after SSA therapy, with a significant association between tumor response at CT scan and appearance of hypervascular inhomogeneous pattern at CEUS evaluation (6 months: X 2 = 57.0; p < 0.0001; 12 months: X 2 = 49.8; p < 0.0001). CONCLUSIONS: In patients with P-NET, CEUS pattern correlates with tumor grading, being homogeneous in G1-G2 but not in G3 tumors. After therapy with SSAs, CEUS is predictive of response to SSAs. These findings seem to support a role of CEUS as prognostic and predictive factor of response.

Quantification of surgical trauma: comparison of conventional and minimally invasive surgical techniques for pertrochanteric fracture surgery based on markers of inflammation (interleukins).

BACKGROUND: Fixation of pertrochanteric fracture is undoubtedly an additional trauma after the fracture itself. In elderly patients, it might have an important impact on the whole organism. In the literature we find various techniques to perform this type of surgery. Up to now, there are no parameters validated for quantification of the invasiveness of a surgical procedure; it is therefore still not demonstrated that any method is less invasive than any other. In an effort to find a way to quantify the invasiveness of a surgical procedure, inflammatory markers were collected in patients undergoing fixation of trochanteric fracture with gliding hip screw [dynamic hip screw (DHS)] using either a conventional (DHS conv) or minimally invasive (DHS MIO) technique. METHOD: Two groups of patients were investigated prospectively; 36 of them were treated with conventional DHS technique and 32 with minimally invasive technique. Mean age was 84.7 ± 7.20 and 82.78 ± 7.71 years, respectively. Fracture type was classified according to the AO classification. Interleukin (IL)-6, IL-10, IL-8, and tumor necrosis factor (TNF)-α were measured 1 h before and 1 h after surgery. Student's t test, chi-square test, and multivariate logistic regression were used for statistical analysis. RESULTS: Preoperative levels of interleukins showed no significant differences between the two groups. In contrast, the postoperative blood level of IL-6 in patients operated with DHS conv technique (78.41 ± 67.04 pg/ml) was on average higher than in patients operated by DHS MIO technique (39.02 ± 37.36 pg/ml), the mean difference being 39.39 pg/ml [95 % confidence interval (CI) 12.65-66.13 pg/ml; p = 0.0045]. Multivariate logistic regression (backward method with limit of significance 0.05) confirmed that patients operated by conventional technique were significantly more likely to have increased IL-6 after surgery than those operated by MIO technique. IL-8 was measured in only 36 patients (20 for DHS conv, 16 for DHS MIO). No significant differences were found between the two groups; however, there was a drastic decrease postoperatively (p < 0.0001) regardless of the type of surgery performed. IL-10 and TNF-α were tested in all subjects, but did not show significant differences between the two groups. Average length of incision was significantly different (4.61 cm, 95 % CI 3.50-5.71 cm; p < 0.001) between the two groups, being 11.65 ± 2.64 cm for DHS conv and 7.05 ± 1.77 cm for DHS MIO. Similarly, average units of red blood cells (RBCs) transfused [performed for hemoglobin (Hb) <9 g/dl and/or hematocrit (HCT) <27 %] was higher (2.22 ± 0.99) in the DHS conv group compared with the DHS MIO group (1.09 ± 1.20), with average difference of 1.13 (95 % CI 0.59-1.66; p < 0.001). CONCLUSIONS: This attempt to quantify the invasiveness of internal fixation for trochanteric fracture comparing two techniques (DHS conv versus DHS MIO) based on inflammatory markers (IL-6) has given encouraging results. Measurement of systemic inflammatory response to local tissue damage caused by osteosynthesis using IL-6 as marker seems to confirm the lower invasiveness of MIO techniques. These results for trauma cases seem in line with those published for hip prosthesis. Ongoing further studies analyzing the effect of nailing will confirm or invalidate these preliminary results.

Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing.

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen. To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3°C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3°C with dimethylsulfoxide (Me(2)SO), ethylene glycol (EG), 1-2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30min. A first evaluation of freezing rates was made by testing four freezing curves: -1, -3, -6 and -10°C/min. Then, an optimization was made by testing four freezing curves: -2.5, -3.0, -3.5 and -4°C/min. The selected temperature for short term conservation has been 3°C, because only this temperature has allowed good sperm motility conservation after 3h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me(2)SO. EG and MetOH to all concentrations and Me(2)SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate -3°C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.

Galectin-3 and CD44v6 isoforms in the preoperative evaluation of thyroid nodules.

PURPOSE: Thyroid cancer is the most frequently occurring endocrine malignancy; however, preoperative diagnosis of some lesions, in particular those with follicular histology, is difficult, and a consistent number of not otherwise-specified "follicular nodules" are surgically resected more for diagnosis than therapeutic purposes. In this study, we investigated whether the lectin-related molecules CD44v6 and galectin-3, the expression of which is altered during deregulated cell growth and malignant transformation, could be potential markers for improving the diagnostic accuracy of conventional cytology. MATERIALS AND METHODS: A comparative immuno-chemical and molecular analysis was performed on 157 thyroid specimens representative of normal, benign, and malignant tissues, and on 36 cytologic samples obtained preoperatively by fine-needle aspiration biopsy from nonselected patients with palpable thyroid nodules. RESULTS: Normal thyrocytes did not express galectin-3 nor CD44v6. Although the expression of CD44v6 isnegligible in thyroiditis, these molecules are variably detected in benign and malignant proliferative lesions. Interestingly, galectin-3 is never expressed in benign lesions, but it is invariably detected in cancers. A comparative evaluation of CD44v6 and galectin-3 expression in thyroid malignancies demonstrated that these molecules are coexpressed at the messenger RNA and protein level in almost all lesions. CONCLUSION: Our findings suggest that CD44v6 and galectin-3 could be potential markers to preoperatively identify malignant transformed thyrocytes. Immunodetection of these molecules on cytologic specimens obtained by fine-needle aspiration biopsy is an accurate and improved method for selecting, on a molecular basis, those nodular lesions of the thyroid gland that need to be surgically resected.

CD44s adhesive function spontaneous and PMA-inducible CD44 cleavage are regulated at post-translational level in cells of melanocytic lineage.

Adhesion between the CD44s receptor and hyaluronic acid plays an important role in cell migration, tumour growth and progression. Although the alternative splicing of CD44 variant exons represents the principal regulatory mechanism of CD44-mediated functions, CD44v spliced variants are scantily expressed in melanoma cells. For this reason, we have investigated the possibility that post-translational modifications of the CD44 standard receptor could play a pivotal role in regulating CD44-mediated functions in melanoma. Using metabolic inhibitors of N- and O-glycosylation, as well as melanoma transfectants expressing CD44s O-glycosylation site-specific mutants, we performed structural and functional analysis of N- and O-deglycosylated CD44s molecules expressed in melanoma cells. We discovered that complete N- and O-glycosylation is not required by CD44s to be correctly expressed on the melanoma cell surface. Indeed, variably glycosylated and functionally different CD44s molecules were constitutively expressed in primary and metastatic lesions. Furthermore, we observed that changes in N- and O-glycosylation of CD44s could modulate its cleavage. In fact, spontaneous CD44s shedding was dependent on the presence of partial or complete O-glycosylation of four serine-glycine motifs localized in the membrane-proximal CD44 ectodomain. Mutation of these serine residues, as well as an extensive metabolic O-deglycosylation, strongly impaired spontaneous CD44 shedding. Furthermore, an O-glycosylation-independent mechanism of CD44 cleavage has been identified. This alternative mechanism of receptor cleavage is phorbol 12-myristate-13-acetate (PMA) inducible, mediated by metalloproteinase and requires the presence of N-linked sugar residues. Our findings demonstrate that the post-translational modification of CD44s represents the principal regulatory mechanism of CD44s-mediated functions in melanoma.

Extra-articular synovial chondromatosis. Description of a rare localization in the arm.

The author presents a rare instance of extra-articular chondromatosis located around the tendon of the long head of the biceps brachii. The tendon subsequently tore and retracted, resulting in the formation of numerous cartilaginous loose bodies which collected in a mid-brachial cavity communicating with the canal which had previously housed the tendon. The author comments on the peculiar mechanism of production of this rare case of synovial chondromatosis as well as possible interpretations.

Structural variability of CD44v molecules and reliability of immunodetection of CD44 isoforms using mAbs specific for CD44 variant exon products.

CD44 can be considered structurally and functionally one of the most variable surface molecules. Alternative splicing of variant exons as well as posttranslational modifications of the molecule (differences in glycosylation) generate a rich repertoire of CD44 isoforms (CD44v), some of which seem to play a key role in tumor growth and progression. Immunodetection of CD44 isoforms in vivo, using mAbs specific for CD44 variant exon products, is largely used to identify those CD44 molecules involved in tumor growth and progression and to interfere with CD44-mediated processes. In the present work we demonstrate that the immunoreactivity of some mAbs directed to CD44 exon-specific epitopes can be impaired by the structural variability of the molecule. Our findings demonstrate that (1) specific exon assortment and/or posttranslational modifications of CD44v molecules can mask CD44 exon-specific epitopes; (2) glycosaminoglycan side chains, carried by some CD44v isoforms of high molecular weight, may play a critical role in determining the exact conformation of the molecule, which is necessary for the detection of CD44 variant epitopes by specific mAbs; and (3) in a panel of stable transfectants expressing CD44 N-glycosylation site-specific mutants, generated in the constant region of CD44 extracellular domain, asparagine-isoleucine substitution is sufficient per se to impair the immunoreactivity of several mAbs to pan-CD44. Thus, conformational changes due to the alternative splicing of CD44 variant exons and/or posttranslational modifications of the molecule (different degree of glycosylation), which are cell type-specific, are likely to generate CD44 variants that elude immunodetection. These findings strongly suggest that immunohistochemical analysis of CD44 expression in vitro and in vivo, using mAbs specific for CD44 variant exon epitopes, can potentially be impaired by a large number of false negative results.