DHEA supplementation improves follicular microenviroment in poor responder patients

Objective: To analyze the effect of dehydroepiandrosterone (DHEA) supplementation on follicular microenvironment and on in vitro fertilization (IVF) outcomes among poor responder patients. Study design: We enrolled 24 patients diagnosed as poor responders based on ESHRE consensus criteria. One group received 25?mg/die three times daily of DHEA supplementation for 3 months previous to IVF cycle, while the other did not receive any treatment. COH was performed with rFSH and hMG, and a GnRH antagonist was administered according to a flexible protocol. We evaluated perifollicular vascularization of recruited follicles through power Doppler blood flow analysis and follicles were graded as described by Chui et al. Follicular fluids (FF) from F3-F4 follicles were collected, and FF levels of vascular endothelial growth factor (VEGF) and hypoxic inducible factor1 (HIF1) were measured. Results: FF levels of HIF1 were statistically significant lower in women treated with DHEA (14.76 ± 51.13 vs. 270.03 ± 262.18 pg/ml; p = 0.002). On the contrary, VEGF levels did not differ between the two groups. Concerning COH, in the DHEA-group the mean duration of treatment was significantly shorter (9.83 ± 1.85 vs. 12.09?±?2.81; p = 0.023). Total numbers of oocytes retrieved, fertilized oocytes, good quality embryos, number of transferred embryos and clinical pregnancies tended to be higher in study group, but the results were not significant. On the other hand, considering the oocytes retrieved in selected F3-F4 follicles, there was a relation between HIF1 levels and oocytes quality. In fact, mature oocytes retrieved in selected follicles were significantly more numerous in DHEA-group (0.50 ± 0.52 vs. 0.08?±?0.29; p = 0.018). Conclusions: The improvement of reproductive parameters after DHEA supplementation in poor responders may be explained through the effect that this pro-hormone exerts on follicular microenvironment.     

Bone marrow-infiltrating human neuroblastoma cells express high levels of calprotectin and HLA-G proteins

Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5 y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.     

Q-Elastography in the Presurgical Diagnosis of Thyroid Nodules with Indeterminate Cytology

Quantitative ultrasound (US) elastography (Q-USE), able to evaluate tissue stiffness has been indicated as a new diagnostic tool to differentiate benign from malignant thyroid lesions. Aim of this prospective study, conducted at the Department of Surgical Sciences, of the “Sapienza” University of Rome, was to evaluate the diagnostic accuracy of Q-USE, compared with US parameters, in thyroid nodules with indeterminate cytology (Thy3).The case study included 140 nodules from 140 consecutive patients. Patient’s thyroid nodules were evaluated by Q-USE, measuring the strain ratio (SR) of stiffness between nodular and surrounding normal thyroid tissue, and conventional US parameters prior fine-needle aspiration cytology. Those with Thy3 diagnosis were included in the study. Forty of the nodules analyzed harbored a malignant lesion. Q-USE demonstrated that malignant nodules have a significant higher stiffness with respect to benign one and an optimun SR cut-off value of 2.05 was individuated following ROC analysis. Univariate analysis showed that hypoechogenicity, irregular margins and SR >2.05 associated with malignancy, with an accuracy of 67.2%, 81,0% and 89.8%, respectively. Data were unaffected by nodule size or thyroiditis. These findings were confirmed in multivariate analysis demonstrating a significant association of the SR and the irregular margins with thyroid nodule’s malignancy. In conclusion, we demonstrated the diagnostic utility of Q-USE in the differential diagnosis of thyroid nodules with indeterminate cytology that, if confirmed, could be of major clinical utility in patients’ presurgical selection.     

Electronic properties of some nitrobenzo[<Emphasis Type="Italic">a</Emphasis>]pyrene isomers: a possible relationship to mutagenic activity

Ionization potential (IP), electron affinity (EA), dipole moment (μ) and electronic polarizability (α) of 1-, 3- and 6-nitrobenzo[a]pyrene isomers (1-NBaP, 3-NBaP, 6-NBaP) were determined by using density functional theory (DFT) and recent semiempirical PM6 methods. Calculated IP value remains almost constant along the series of isomers, while EA value depends on the nitro group position, increasing by ca. 0.2 eV on passing from 6- to 1-NBaP (or 3-NBaP) isomer. Stability, μ and α values decrease in the order 6-NBaP < 1-NBa ∼ 3-NBaP, the largest μ variation being predicted to be 1.5 D (30%) by DFT computations. The results obtained herein are consistent with the observed greater mutagenic activity of 3- and 1-NBaP in comparison to 6-NBaP isomer, suggesting that both binding to enzyme, which depends on electric properties, and reduction process, which is related to EA value may be crucial steps in the mutagenic mechanism of this series of isomers. Figure Structure and dipole moment vector of nitrobenzo[a]pyrene isomers     

Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase

Background

There is growing awareness that tumour cells build up a “self-advantageous” microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP.

Methodology/Principal Findings

Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours.

Conclusions/Significance

Our results show that ATP in the tumour interstitium is in the hundrends micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.     

Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues

As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.     

Autophagy is involved in nutritional stress response and differentiation in Trypanosoma cruzi

Autophagy is the major mechanism used by eukaryotic cells to degrade and recycle proteins and organelles. Bioinformatics analysis of the genome of the protozoan parasite Trypanosoma cruzi revealed the presence of all components of the Atg8 conjugation system, whereas Atg12, Atg5, and Atg10 as the major components of the Atg12 pathway could not be identified. The two TcATG4 (autophagin) homologs present in the genome were found to correctly process the two ATG8 homologs after the conserved Gly residue. Functional studies revealed that both ATG4 homologues but only one T. cruzi ATG8 homolog (TcATG8.1) complemented yeast deletion strains. During starvation of the parasite, TcAtg8.1, but not TcAtg8.2, was found by immunofluorescence to be located in autophagosome-like vesicles. This confirms its function as an Atg8/LC3 homolog and its potential to be used as an autophagosomal marker. Most importantly, autophagy is involved in differentiation between developmental stages of T. cruzi, a process that is essential for parasite maintenance and survival. These findings suggest that the autophagy pathway could represent a target for a novel chemotherapeutic strategy against Chagas disease.