The biocompatibility of porous vs non-porous bone cements: a new methodological approach.

Composite cements have been shown to be biocompatible, bioactive, with good mechanical properties and capability to bind to the bone. Despite these interesting characteristic, in vivo studies on animal models are still incomplete and ultrastructural data are lacking. The acquisition of new ultrastructural data is hampered by uncertainties in the methods of preparation of histological samples due to the use of resins that melt methacrylate present in bone cement composition. A new porous acrylic cement composed of polymethylmetacrylate (PMMA) and ß-tricalciumphosphate (ß-TCP) was developed and tested on an animal model. The cement was implanted in femurs of 8 New Zealand White rabbits, which were observed for 8 weeks before their sacrifice. Histological samples were prepared with an infiltration process of LR white resin and then the specimens were studied by X-rays, histology and scanning electron microscopy (SEM). As a control, an acrylic standard cement, commonly used in clinical procedures, was chosen. Radiographic ultrastructural and histological exams have allowed finding an excellent biocompatibility of the new porous cement. The high degree of osteointegration was demonstrated by growth of neo-created bone tissue inside the cement sample. Local or systemic toxicity signs were not detected. The present work shows that the proposed procedure for the evaluation of biocompatibility, based on the use of LR white resin allows to make a thorough and objective assessment of the biocompatibility of porous and non-porous bone cements.

[The role of eggs in the diet: nutraceutical and epigenetic aspects]. / Il ruolo delle uova nella dieta: aspetti nutraceutici ed epigenetici.

The use of eggs in human diet has been object of many prejudices which are not yet completely disappeared The evolution of knowledge in the field of nutrition has, partially, countered these prejudices by highlighting the biological importance of several compounds present in the eggs. The nutritional and commercial revaluation of the eggs are passed through the enrichment of the lipid fraction in omega3 polyunsaturated fatty acids (PUFA omega3) which, have shown positive effects against cardiovascular diseases and development of the central nervous system and retina. The enrichment of eggs lipid with omega3 fatty acids is carried out by the integration of feeding hens with oils rich in omega3 fatty acids such as plant or marine oils. The results showed that the accumulation of omega3 in the egg yolk lipids is strongly affected by the type of oil used as supplement and by the amounts of oils administrated to the hens.

Molecular pathology of differentiated thyroid cancer.

Thyroid cancer is the most common endocrine malignancy; it accounts for approximately 1% of all new case of cancer each year, and its incidence has increased significantly over the last few decades. The majority of thyroid tumors originate from follicular epithelial cells. Among them, papillary (PTC) and follicular carcinomas (FTC) represent the most common forms of differentiated thyroid cancer and account for approximately 80% and 15% of all cases, respectively. Specific genetic lesions are associated to each thyroid tumor histotype: BRAF mutations and RET/PTC and TRK oncogenes have been detected in PTC, whereas FTC is characterized by PAX8/PPARgamma rearrangements and RAS mutations. In this review we summarize studies on the molecular biology of the differentiated thyroid tumors, with particular interest in the associated genetic lesions and their role in thyroid carcinogenesis. We also report recent findings on gene expression and miRNA profiles of PTC and FTC.

Gestational diabetes insipidus: a morphological study of the placenta.

Gestational diabetes insipidus (GDI) refers to the state of excessive water intake and hypotonic polyuria. Those cases manifesting in pregnancy and referred to as GDI may persist thereafter or may be a transient latent form that resolves after delivery. Microscopic examination of affected subjects has not been previously reported. In the literature, there are various case reports and case series on diabetes insipidus in pregnancy. In this study, we present a case that had transient diabetes insipidus during pregnancy in which the placenta was examined.

O6-Methylguanine-DNA-methyltransferase in recurring anaplastic ependymomas: PCR and immunohistochemistry.

Ependymomas are the third most common brain tumor in children. The post surgical management is controversial. There are no convincing data on an effective role for chemotherapy. O(6)-Methylguanine-DNA-Methyltransferase (MGMT) is a DNA repair protein considered to be a chemosensitivity predictor. Hypermethylation of the MGMT gene promoter is an important cause of MGMT inactivation. We evaluated the MGMT gene promoter methylation and the immunohistochemical MGMT protein expression in 12 recurrent anaplastic ependymomas affecting children. Our purpose was to investigate the molecular rationale of the administration of alkylating agents to children affected by recurrent anaplastic ependymomas. All ependymomas lacked MGMT promoter hypermethylation and 9 (75%) showed high MGMT protein expression (>50% tumoral cells). Differences between different recurrences in the same patient were not observed. These results may indicate MGMT as a factor of chemoresistance to alkylating drugs in anaplastic ependymomas and support the uncertainties regarding the actual benefit of chemotherapy for patients with anaplastic ependymomas.

Placental findings in pregnancy-associated haemolytic uremic syndrome: clinicomorphological study of three cases.

Acute renal failure occurring in pregnancy or postpartum is often associated with preeclampsia, HELLP syndrome or haemolytic uremic syndrome: differential diagnosis may be difficult due to the overlapping symptoms of these syndromes. We report our experience on diagnosis, management and outcome of women with pregnancy associated haemolytic uremic syndrome, focusing on placental features.

SH2B1beta adaptor is a key enhancer of RET tyrosine kinase signaling.

The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.

Could gamma/delta T cells influence the mother's rejection in recurrent aborted embryos?

The possibility that the investigation of aborted material may identify aetiologies not easily detectable from even a careful clinical investigation, suggested a study of the T-cell receptors (TCRs) of decidual-infiltrating T-lymphocytes in recurrent spontaneous miscarriage (RSM). From 33 cases of RSM (>3 previous consecutive miscarriages, range 3-5, mean 3.7), PCR products were analysed by 15% acrylamide gel electrophoresis and visualised under UV illumination after ethidium bromide staining. A broad band obtained suggests the presence of a monoclonal T-lymphocyte proliferation. A PCR not showing bands means that the tissue does not contain reactive T cells. A total of 11 samples (33.3%) revealed the presence of receptor TCRgamma with the presence of a specific band. T-cell receptors in RSM were identified in one-third of cases. These data underline the importance of a maternal immune host response to the embryo and the need to study the immune mechanisms with the hope of modulating therapeutic treatment of recurrent abortion.

Microvessel density in ovarian carcinoma: computer image analysis in patients with shorter and longer survival.

We previously reported that tumor microvessel density (MVD) may have prognostic significance in ovarian carcinoma. The aim of this study was to compare the intratumoral microvessels using a computer-aided image analysis system between FIGO stage IIIC, serous, G3, ovarian carcinomas obtained from living patients who had no evident disease 5 years after primary treatment and ovarian carcinomas, matched for stage, histopathology, grade of differentiation, and treatment, obtained from patients who had died of progression of disease no later than 1 year after primary treatment. We observed that MVD is statistically correlated, according to the logistic regression in univariate and multivariate ways, with the survival (P= 0.03 and P= 0.05, respectively) and with the progression of the disease during first-line chemotherapy (P= 0.009 and P= 0.012, respectively). In the past years, the modulation of first-line chemotherapeutic treatment has been a question of discussion, because the oncologist observes extremely unpredictable behaviors with surprisingly long survivals and also short survivals. Pathologists may give clinicians some additional prognostic information useful in the management of ovarian carcinoma patients. The results of this study support the hypothesis that the evaluation of MVD with computer image analysis can help clinicians in the choice of the tailored treatment of the single case.

Tumour-infiltrating gamma/delta T-lymphocytes are correlated with a brief disease-free interval in advanced ovarian serous carcinoma.

BACKGROUND: Significant progress has been made in understanding the molecular biology of ovarian carcinoma. Along with the molecular characteristics of cancer, the patient's response to the tumour may also contribute to survival; in particular, the effect of the immune system may play an important role on survival of cancer patients. PATIENTS AND METHODS: We analysed the CD3 positive tumour-infiltrating T cells and direct molecular assessment of T cell receptors (TCRs) gamma and beta in 95 advanced ovarian carcinomas. RESULTS: Gamma/delta T cells are statistically correlated with a brief disease-free interval (P=0.036). CD3 positive tumour-infiltrating T cells are correlated with a brief disease-free interval and with survival (P=0.004 and P=0.0001, respectively). CD3 positive tumour-infiltrating T cells are associated with clinical responsiveness to chemotherapy (P=0.003). CONCLUSIONS: Further studies are required to better understand the role of gamma/delta T cells in ovarian carcinoma, yet these data underline the importance of host immune response to cancer and the need to better study immune mechanisms to modulate the therapeutic treatment of cancer.

The role of apoptosis in the pathogenesis of dermatitis herpetiformis.

Apoptosis is a form of cell death that is claimed to be involved in a number of chronic inflammatory and malignant skin diseases. The aim of this study was to investigate whether apoptosis may contribute to the pathogenesis of epidermal changes in dermatitis herpetiformis (DH) and, in particular, whether certain apoptosis-related markers such as Bax, Bcl-2, Fas and Fas ligand (FasL) take part in this process. For the detection of apoptotic nuclei, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling technique (TUNEL) was employed on cryostat sections. Skin lesions from six and perilesional skin from four DH patients were stained with monoclonal antibodies to Bax, Bcl-2, Fas and FasL. The same evaluation was also performed on three patients affected by bullous pemphigoid (BP) and in two healthy donors. Using TUNEL technique, a remarkable increase in the apoptotic rate within the epidermal compartment was observed in DH and BP patients in comparison with normal controls. In our immunohistochemical analysis, Bax/Bcl-2 ratio was almost the same in the epidermis of perilesional/lesional DH, BP and healthy skin specimens. In DH and BP specimens both Bax and Bcl-2 proteins were increased in the dermal perivascular compartment. Fas showed a prevalently epidermal staining, both in DH and BP lesions, while FasL was distributed in perivascular and subjunctional dermis; some FasL+ cells infiltrated the DEJ and the basal layer of epidermis. This study allowed us to highlight conspicuous apoptotic phenomena in basal and suprabasal keratinocytes within lesional and perilesional skin of DH. We conclude that in DH, as well as in BP, apoptosis plays a role in the pathogenesis of cutaneous lesions in concert with other pathogenetic mechanisms.

[Bcl-2 in ovarian carcinoma: a clinicopathologic, immunohistochemical and molecular study]. / bcl-2 nel carcinoma ovarico: studio clinicopatologico, immunoistochimico e molecolare.

We investigated the immunohistochemical espression of bcl-2 and the genetic assessment of the bcl-2 gene in relation to responsiveness to first line chemotherapy and to the clinical outcome in advanced ovarian carcinoma patients. We have compared 17 patients, with FIGO stage III C, ovarian serous carcinomas, G3, living with no evident disease five years after primary surgical treatment; to 19 patients who had died of progression of disease no later than two years after primary surgical treatment. The correlation of bcl-2 expression with the survival and the clinical responsiveness to chemotherapy, were analysed with the logistic regression. We observed a bcl-2 expression in tumor cells in 25% of the cases. Molecular genetic analysis of the bcl-2 gene was performed for all the bcl-2 immunohistochimical positive cases. No traslocation t(14;18)(q32;q21) of the gene bcl-2 were found. The bcl-2 over-expression was found to be a significant independent predictor of responsiveness to chemotherapy (p = 0.04), but it was not correlated with the overall survival of the ovarian cancer patients. The prognostic value of bcl-2 espression may help in the management of ovarian cancer patients permitting the selection of more aggressive first line chemotherapy. In addition, the knowledge of the molecular mechanism, which is responsible of the over-expression of bcl-2, may help in the understanding of mechanisms responsible for chemoresistance. Further studies in this area will help clarify this therapeutic possibility.

[BCL-2 in primary central nervous system lymphomas. Immunohistochemistry and molecular biology]. / La BCL-2 nei linfomi primitivi del sistema nervoso centrale. Immunoistochimica e biologia molecolare.

BCL-2 is a membrane protein known to be an apoptosis inhibitor. It is the product of the bcl-2 gene located on chromosome 18. Several different tumors show BCL-2 over-expression as result of a translocation or independently from it. More than 85% of follicular lymphomas and a smaller number of diffuse large cell B lymphomas contain t(14;18) (q32;q21). The aim of this study was to investigate the immunohistochemical expression of the BCL-2 protein and to ascertain, by means of traditional PCR (Polimerase Chain Reaction), its possible dependence from t(14;18) (q32;q21) in 9 primary central nervous system lymphomas. Six cases (67%) shoved immunohistochemical BCL-2 over-expression and 3 cases (33%) had t(14;18). Precisely: 2 cases (22%) had immunohistochemical BCL-2 over-expression and t(14;18) (q32;q21); 4 cases (44%) had BCL-2 over-expression without translocation; 1 case (11%) did not show diffuse BCL-2 over-expression in presence of the traslocation; the remaining 2 cases (22%) did not demonstrate BCL-2 over-expression or t(14;18) (q32;q21). In conclusion, our results indicate primary central nervous system lymphomas frequently show BCL-2 over-expression that in some case may be related to t(14;18) (q32;q21). Nevertheless, t(14;18) (q32;q21), as evaluated by traditional PCR, may not correspond to diffuse immunohistochemical BCL-2 positivity.

The role of H2O2 in the platelet-derived growth factor-induced transcription of the gamma-glutamylcysteine synthetase heavy subunit.

This study demonstrates that platelet-derived growth factor (PDGF) increases transcription of the gamma-glutamylcysteine synthetase (GCS) heavy subunit (GCS-HS) in NIH 3T3 fibroblasts via H2O2 and activation of protein kinase C (PKC). The data obtained using catalase, H2O2, phorbol-12-myristate 13-acetate (PMA) or a specific inhibitor of PKC demonstrate the possibility of a PDGF up-regulation pathway of GCS synthesis. Moreover, since PDGF mitogenic activity takes place through PKC activation and sphingosine-1-phosphate (S1P) production, the involvement of sphingosine kinase activity in the PDGF effect was also investigated. No clear direct relationship emerged between S1P production and any PDGF- or H2O2-induced increase in the GCS-HS mRNA level. However, for the first time, in S1P-stimulated NIH 3T3 cells, increased levels of GCS-HS mRNA were shown to be related to increases in the reduced glutathione synthesis rate similar to those obtained after PMA and PDGF stimulation.

Interaction between acylphosphatase and SERCA in SH-SY5Y cells.

Ca2+ transport by sarco/endoplasmic reticulum, tightly coupled with the enzymatic activity of Ca2+ -dependent ATPase, controls the cell cycle through the regulation of genes operating in the critical G, to S checkpoint. Experimental studies demonstrated that acylphosphatase actively hydrolyses the phosphorylated intermediate of sarco/endoplasmic reticulum calcium ATPase (SERCA) and therefore enhances the activity of Ca2+ pump. In this study we found that SH-SY5Y neuroblastoma cell division was blocked by entry into a quiescent G0-like state by thapsigargin, a high specific SERCA inhibitor, highlighting the regulatory role of SERCA in cell cycle progression. Addition of physiological amounts of acylphosphatase to SY5Y membranes resulted in a significant increase in the rate of ATP hydrolysis of SERCA. In synchronized cells a concomitant variation of the level of acylphosphatase isoenzymes opposite to that of intracellular free calcium during the G1 and S phases occurs. Particularly, during G1 phase progression the isoenzymes content declined steadily and hit the lowest level after 6 h from G0 to G1 transition with a concomitant significant increase of calcium levels. No changes in free calcium and acylphosphatase levels upon thapsigargin inhibition were observed. Moreover, a specific binding between acylphosphatase and SERCA was demonstrated. No significant change in SERCA-2 expression was found. These findings suggest that the hydrolytic activity of acylphosphatase increase the turnover of the phosphoenzyme intermediate with the consequences of an enhanced efficiency of calcium transport across endoplasmic reticulum and a subsequent decrease in cytoplasmic calcium levels. A hypothesis about the modulation of SERCA activity by acylphosphatase during cell cycle in SY5Y cells in discussed.

Increased glutathione synthesis associated with platelet-derived growth factor stimulation of NIH3T3 fibroblasts.

Previous data show a relation between GSH content and proliferation of normal and tumour cells. We recently demonstrated a specific involvement of GSH in the autophosphorylation activity of the platelet-derived growth factor (PDGF) receptor in NIH3T3 fibroblasts. In this study we demonstrate that the stimulation by PDGF of serum-starved NIH3T3 cells increases cellular GSH content, while no change in oxidized GSH content was measured. Experiments performed with actinomycin, cycloheximide and buthionine sulfoximide, a specific inhibitor of the rate-limiting enzyme of the de novo synthesis of GSH gamma-glutamylcysteine synthetase (gamma-GCS), confirm PDGF induction of GSH synthesis. These results provide the first demonstration that PDGF mediated transduction signals seem strictly related to mechanisms involved in the increase of gamma-GCS activity associated with increased gamma-GCS heavy subunit mRNA levels. In fact, serum and epidermal growth factor (EGF) stimulation of quiescent NIH3T3 and NIH3T3, which overexpress EGF receptor, does not affect GSH content or its synthesis. These data may be related to a possible GSH role in the redox regulation of cell proliferation mediated by PDGF.

GSH system in relation to redox state in dystrophic skin fibroblasts.

Glutathione and GSH-related enzymes were determined in human Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) skin fibroblasts in order to relate muscular dystrophy to the redox state of the cell. The analysis of GSH, GSSG and total GSH levels in normal and dystrophic-cultured fibroblasts shows no differences in normal growth condition. However, the specific activity of two GSH-related enzymes, glutathione S-transferases (GST) and gamma-glutamylcysteine synthetase (gamma-GCS), shows significant variations between normal and both types of dystrophic skin fibroblasts. These results suggest that even in normal growth condition some components of GSH metabolism may be altered. A condition of sublethal oxidation obtained by H(2)O(2) treatment was able to show a difference in the cellular response of GSH system components between normal and dystrophic cells. While in DMD cells there is a decrease of roughly 55% in GSH and of 30% in total GSH concentration, no changes are measured in normal and BMD cells. The remarkable increase in glutathione peroxidase (GPx) activity and decrease in GSH-reductase (GR) activity measured in DMD cells can in part explain these changes. These results indicate a different capacity of DMD cells to support oxidative stress with respect to BMD and normal cells, and suggest a possible role of the GSH-antioxidant system in dystrophic pathology.

Acylphosphatase expression during macrophage differentiation and activation of U-937 cell line.

The two acylphosphatase isoenzymes (muscle type and common type) are differently involved in cell differentiation processes. In this paper we investigate the expression of the two isoenzymes during macrophage differentiation and activation. The U-937 human promonocytic cell line is a model for cell differentiation induced by the tumor promoter phorbol myristic acetate (PMA). Here we show that only the expression of the muscle type acylphosphatase increases during U-937 differentiation and macrophage activation, confirming that the two isoenzymes are differently regulated. Moreover, we determined, in the same conditions, the level of specific mRNA. Results show that after an initial two-fold decrease during PMA stimulation, the muscle type acylphosphatase mRNA levels remain constant also after the treatment with lipopolysaccharide and gamma-interferon, treatments that lead to macrophage activation. It is possible that post-transcription regulation is responsible for the regulation of muscle type acylphosphatase in the cell during differentiation and macrophage activation.

Preferential accumulation of muscle type acylphosphatase in the nucleus during differentiation.

In a previous paper we observed a direct involvement of acylphosphatase in differentiation, associated with enhanced levels of the enzyme in the cell. We have here investigated the subcellular localization of the two known acylphosphatase isoforms during this process. We show that in C2C12 myoblast cells, muscle type acylphosphatase accumulates in the nucleus during differentiation. The same pattern of accumulation is observed also in K562 erythroleukemia cells, although at a lower extent: this fact indicates that this phenomenon is not restricted to muscular cells but rather it could be of general importance in the differentiative process. The common type acylphosphatase, showing an 8-fold increase in the cytoplasm during differentiation, does not accumulate in the nucleus, suggesting distinct roles of the two isoenzymes in this process.