The relevance of MRI blood-sensitive sequences in the diagnostic assessment of late-onset epilepsy.

OBJECTIVE: Sporadic cerebral amyloid angiopathy (CAA) is a degenerative brain small vessel disease of ageing resulting from progressive amyloid deposition in small arteries and arterioles of the cortex and leptomeninges. CAA may be diagnosed by the mean of Boston criteria, particularly with the use of the blood-sensitive T2* MRI sequences (GRE and SWI). Epileptic seizures have rarely been reported in CAA. PATIENTS AND METHODS: We describe two patients with late-onset unprovoked seizures due to CAA. A short literature review on this topic is presented. RESULTS: In our two patients with late-onset unprovoked seizures as the first manifestation of CAA, only GRE and SWI sequences lead to a correct diagnosis. In literature, only 15 patients with CAA presenting with seizures have been reported. In these subjects, data on seizures semiology and prognosis are scarce. CONCLUSIONS: Our report highlights the importance to perform blood-sensitive sequences in all subjects with LOE of otherwise unknown etiology, not to miss a diagnosis of CAA.

A validation study of a dedicated software for an automated in vivo dosimetry control in radiotherapy.

In vivo dosimetry (IVD) is the last step of a radiotherapy quality control program aimed to ensure that the dose delivered is in agreement with that prescribed. IVD procedures based on single detectors are time-consuming and impossible to use for the modern radiotherapy techniques, based on static or kinetic beams (modulated in intensity fluence); this means that more efficient and practical methods are highly recommended. The practical method SOFTDISO, based on the use of electronic portal image device (EPID), provides two tests (i) the R ratio between the reconstructed and the planned isocenter doses to verify an agreement within 5% and (ii) the γ-analysis of the EPID images, to verify γ% ≥ 90% and γmean ≤ 0.4. This paper reports the results of 11,357 IVD tests carried out for 823 patients treated by three-dimensional conformal radiation therapy and volumetric modulated arc therapy techniques. In particular, the dose disagreements are reported distinguishing two kinds of causes, those of (i) class 1 that includes the errors due to inadequate quality controls and (ii) the class 2, due to patient morphological changes. About the tests out of tolerance, 6% were by VMAT and 21% by 3DCRT, but taking into account the only class 1 of errors, i.e., removing the causes of class 2, only 7% of patients examined presented at least one of the three mean indexes out of tolerance. The workload for IVD on 9 patients/day per linac is about 52 min/day but recently, a new automated SOFTDISO version has been implemented to reduce the time to about 34 min/day.

Interaction between DRD2 variation and sound environment on mood and emotion-related brain activity.

Sounds, like music and noise, are capable of reliably affecting individuals' mood and emotions. However, these effects are highly variable across individuals. A putative source of variability is genetic background. Here we explored the interaction between a functional polymorphism of the dopamine D2 receptor gene (DRD2 rs1076560, G>T, previously associated with the relative expression of D2S/L isoforms) and sound environment on mood and emotion-related brain activity. Thirty-eight healthy subjects were genotyped for DRD2 rs1076560 (G/G=26; G/T=12) and underwent functional magnetic resonance imaging (fMRI) during performance of an implicit emotion-processing task while listening to music or noise. Individual variation in mood induction was assessed before and after the task. Results showed mood improvement after music exposure in DRD2GG subjects and mood deterioration after noise exposure in GT subjects. Moreover, the music, as opposed to noise environment, decreased the striatal activity of GT subjects as well as the prefrontal activity of GG subjects while processing emotional faces. These findings suggest that genetic variability of dopamine receptors affects sound environment modulations of mood and emotion processing.

Ankyrin-3 as a molecular marker of early-life stress and vulnerability to psychiatric disorders.

Exposure to early-life stress (ELS) may heighten the risk for psychopathology at adulthood. Here, in order to identify common genes that may keep the memory of ELS through changes in their methylation status, we intersected methylome analyses performed in different tissues and time points in rats, non-human primates and humans, all characterized by ELS. We identified Ankyrin-3 (Ank3), a scaffolding protein with a strong genetic association for psychiatric disorders, as a gene persistently affected by stress exposure. In rats, Ank3 methylation and mRNA changes displayed a specific temporal profile during the postnatal development. Moreover, exposure to prenatal stress altered the interaction of ankyrin-G, the protein encoded by Ank3 enriched in the post-synaptic compartment, with PSD95. Notably, to model in humans a gene by early stress interplay on brain phenotypes during cognitive performance, we demonstrated an interaction between functional variation in Ank3 gene and obstetric complications on working memory in healthy adult subjects. Our data suggest that alterations of Ank3 expression and function may contribute to the effects of ELS on the development of psychiatric disorders.

Quasi real time in vivo dosimetry for VMAT.

PURPOSE: Results about the feasibility of a method for quasi real time in vivo dosimetry (IVD) at the isocenter point for volumetric modulated arc therapy (VMAT) are here reported. The method is based on correlations between the EPID signal and the dose on the beam central axis. Moreover, the γ-analysis of EPID images was adopted to verify off-axis reproducibility of fractionated plan delivery. METHODS: An algorithm to reconstruct in vivo the isocenter dose, D(iso), for RapidArc treatments has been developed. 20 VMAT plans, optimized with two opposite arcs, for prostate, pancreas, and head treatments have been delivered by a Varian linac both to a conic PMMA phantom with elliptical section and to patients. The ratios R between reconstructed D(iso) and the planned doses were determined for phantom and patient irradiations adopting an acceptance criterion of ±5%. In total, 40 phantom checks and 400 patient checks were analyzed. Moreover, 3% and 3 mm criteria were adopted for portal image γ-analysis to assess patient irradiation reproducibility. RESULTS: The average ratio R, between reconstructed and planned doses for the PMMA phantom irradiations was equal to 1.007 ± 0.024. When the IVD method was applied to the 20 patients, the average R ratio was equal to 1.003 ± 0.017 and 96% of the tests were within the acceptance criteria. The portal image γ-analysis supplied 88% of the tests within the pass rates γ(mean) ≤ 0.4 and P(γ<1) ≥ 98%. All the warnings were understood comparing the CT and the cone beam CT images and in one case a patient's setup error was detected and corrected for the successive fractions. CONCLUSIONS: This preliminary experience suggests that the method is able to detect dosimetric errors in quasi real time at the end of the therapy session. The authors intend to extend this procedure to other pathologies with the integration of in-room imaging verification by cone beam CT.

A practical approach for the detection of DNA nanostructures in single live human cells by fluorescence microscopy.

In the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, cell cycle stage, make the cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population. In this paper, we describe the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with epifluorescence microscopy. We report that HeLa cells internalize these nanostructures spontaneously with a higher efficiency with respect to single-stranded or double-stranded oligonucleotides. Our findings suggest that DNA tetrahedra could serve as a platform for the realization of a series of multifunctional intracellular biosensors for the analysis of single live cells.

An in-vivo dosimetry procedure for Elekta step and shoot IMRT.

PURPOSE: The aim of this work was to extend an in-vivo dosimetry (IVD) method, previously developed by the authors for 3D-conformal radiotherapy, to step and shoot IMRT treatments for pelvic tumors delivered by Elekta linacs. MATERIALS AND METHODS: The algorithm is based on correlation functions to convert EPID transit signals into in-vivo dose values at the isocenter point, Diso. The EPID images were obtained by the so-called "IMRT Dosimetric Weighting" mode as a superposition of many segment fields. This way each integral dosimetric image could be acquired in about 10 s after the end of beam delivery and could be processed while delivering the successive IMRT beams. A specific algorithm for Diso reconstruction especially featured for step and shoot IMRT was implemented using a fluence inhomogeneity index, FI, introduced to describe the degree of beam modulation with respect to open beams. A γ-analysis of 2D-EPID images obtained day to day, resulted rapid enough to verify the plan delivery reproducibility. RESULTS: Fifty clinical IMRT beams, planned for patients undergoing radiotherapy of pelvic tumors, were used to irradiate a homogeneous phantom. For each beam the agreement between the reconstructed dose, Diso, and the TPS computed dose, Diso,TPS, was well within 5%, while the mean ratio R = Diso/Diso,TPS resulted for 250 tests equal to 1.006 ± 0.036. The same beams were checked in vivo, i.e. during patient treatment delivery, obtaining 500 tests whose average R ratio resulted equal to 1.011 ± 0.042. The γ-analysis of the EPID images with 5% 3 mm criteria supplied 85% of the tests with pass rates γ(mean) ≤ 0.5 and P(γ<1) ≥ 90%.

Gamma rays induce a p53-independent mitochondrial biogenesis that is counter-regulated by HIF1α.

Mitochondrial biogenesis is an orchestrated process that presides to the regulation of the organelles homeostasis within a cell. We show that γ-rays, at doses commonly used in the radiation therapy for cancer treatment, induce an increase in mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence, in the presence of a functional p53. Although the main effector of the response to γ-rays is the p53-p21 axis, we demonstrated that mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a murine double minute 2 (MDM2)-mediated hypoxia-inducible factor 1α (HIF1α) degradation, leading to the release of peroxisome-proliferator activated receptor gamma co-activator 1ß inhibition by HIF1α, thus promoting mitochondrial biogenesis. Mimicking hypoxia by HIF1α stabilization, in fact, blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally, we also show in vivo that post-radiotherapy mitochondrial DNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of cell senescence.

Real-time dose reconstruction for wedged photon beams: a generalized procedure.

A practical and accurate generalized procedure to reconstruct the isocenter dose D(iso) for 3D conformal radiotherapy (3DCRT) has been developed for X-ray open beams supplied by linacs of different manufacturers and equipped with aSi electronic portal imaging devices (aSi EPIDs). This paper reports an extension of the method, to be applied at the wedged X-ray beams characterized by the wedge attenuation factor W(AF). Using water-equivalent solid phantoms (SPs) of different thicknesses, w, and photon square fields of sizes, L, the generalized midplane doses D(0)(W(AF), w/2,L) and generalized transit signals s(t)(0)(W(AF),w,L) by 38 beams of six different linacs were determined. The generalized data were fitted by surface equations and used together with the information of the 'record & verify' network of the centers. In this manner, for every beam, the D(iso) reconstruction was obtained in about 25 seconds after the treatment. To test the in vivo dosimetric procedure, six pelvic treatments that used conformed wedged beams were carried out with three linacs of different manufacturers. For every beam, the comparison between the reconstructed D(iso) and the D(iso,TPS) computed by the TPS, resulted in an acceptable tolerance level of ±5%, estimated for this kind of treatment. Generally the in vivo dosimetry methods that use EPIDs require: (i) a special effort for the dosimetric commissioning with SPs of different thicknesses, and (ii) extra time for the analysis of the EPID signals. The proposed procedure simplifies the commissioning step and supplies for Varian, Elekta, and Siemens linacs equipped with the aSi EPIDs a quasi-real time in vivo dosimetry for open and wedged 3DCRT fields.

The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I.

Human thyroid carcinoma XTC.UC1 cells harbor a homoplasmic frameshift mutation in the MT-ND1 subunit of respiratory complex I. When forced to use exclusively oxidative phosphorylation for energy production by inhibiting glycolysis, these cells triggered a caspase-independent cell death pathway, which was associated to a significant imbalance in glutathione homeostasis and a cleavage of the actin cytoskeleton. Overexpression of the anti-apoptotic Bcl-2 protein significantly increased the level of endogenous reduced glutathione, thus preventing its oxidation after the metabolic stress. Furthermore, Bcl-2 completely inhibited actin cleavage and increased cell adhesion, but was unable to improve cellular viability. Similar effects were obtained when XTC.UC1 cells were incubated with exogenous glutathione. We hence propose that Bcl-2 can safeguard cytoskeletal stability through an antioxidant function.

Large idiopathic unilateral adrenal hematoma in a young woman.

This is a case report on a young woman with a large idiopathic unilateral adrenal hematoma (AH). Only few cases of AH which were not associated with any trauma, previous surgery, coagulative or any other systemic disorders have been described. The mass was discovered by abdominal ultrasound which was performed for a recent flank pain. Magnetic resonance imaging (MRI) confirmed the presence of a 13-cm sized lesion in the right hemi-abdomen; T1 and T2 weighed imaging was compatible with subacute-to-chronic adrenal hematoma. The lesion dislocated the liver and right kidney. Positron emission tomography (PET) did not show any significant radiotracer uptake by the mass. Serum cortisol, aldosterone, renin activity and DHEA-S were normal. Urinary catecholamines and free cortisol excretion were within the normal range too. The lesion was removed by transabdominal laparoscopic adrenalectomy without any complication. The histological exam confirmed a large subacute- to-chronic organized AH. In conclusion, in the absence of known risk factors, differential diagnosis of a large AH may not be easy. The possibility of an underlying pheochromocytoma, malignant adrenal or metastatic tumor must always be considered. In our patient, computed tomography (CT) scan and MRI suggested the presence of a large subacute-to-chronic AH, and PET excluded metabolic activity of the mass. Laparoscopic adrenalectomy can be the surgical treatment of choice in organized symptomatic AH. The correct diagnosis, early recognition and treatment of complications including adrenal insufficiency may decrease patient morbidity and mortality.

Caspase-independent death of Leber's hereditary optic neuropathy cybrids is driven by energetic failure and mediated by AIF and Endonuclease G.

Leber's hereditary optic neuropathy (LHON) is associated with mitochondrial DNA point mutations affecting different subunits of complex I. By replacing glucose with galactose in the medium, cybrids harboring each of the three LHON pathogenic mutations (11778/ND4, 3460/ND1, 14484/ND6) suffered a profound ATP depletion over a few hours and underwent apoptotic cell death, which was caspase-independent. Control cybrids were unaffected. In addition to cytochrome c, apoptosis inducing factor (AIF) and endonuclease G (EndoG) were also released from the mitochondria into the cytosol in LHON cybrids, but not in control cells. Exposure of isolated nuclei to cytosolic fractions from LHON cybrids maintained in galactose medium caused nuclear fragmentation, which was strongly reduced by immuno-depletion with anti-AIF and anti-EndoG antibodies. In conclusion, the caspase-independent death of LHON cybrids incubated in galactose medium is triggered by rapid ATP depletion and mediated by AIF and EndoG.

Apoptosis induced by staurosporine in ECV304 cells requires cell shrinkage and upregulation of Cl- conductance.

We show that dysregulation of the Cl- homeostasis mediates the staurosporine-induced apoptotic cell death in human ECV304 cells. A pronounced apoptotic volume decrease (AVD), and an increase in plasma membrane Cl- conductance were early (<1 h) events following staurosporine challenge. Both processes were involved in apoptotic death, as demonstrated by the observation that the Cl- channel blocker phloretin inhibited both the staurosporine-evoked Cl- current and AVD, and preserved cell viability. Prolonged incubation (>2 h) with staurosporine caused a decrease in intracellular pH, which, however, was not required for the progression of the apoptotic process, because inhibitors of proton extrusion pathways, which lowered cytoplasmic pH, failed to inhibit both caspase-3 activation and DNA laddering. Moreover, clamping the cytosolic pH to an alkaline value did not prevent the apoptotic cell death. Collectively, these data demonstrate that staurosporine-mediated apoptosis of ECV304 cells is caused by the upregulation of Cl- channel activity and subsequent AVD, but is independent of intracellular acidification.

Staurosporine induces apoptotic volume decrease (AVD) in ECV304 cells.

Incubation of ECV304 cells with 1 micro M staurosporine (STS) causes apoptotic cell death. In the present study, we investigate whether a significant apoptotic volume decrease (AVD) was apparent during the very early times (1 h) of the apoptotic process. Our data suggest that upregulation of Cl(-) (and possibly K(+)) channels by STS may be a very early primary event required for the subsequent onset of AVD, which results in apoptosis.

[Minilaparoscopy and open laparoscopy: prevention of laparoscopic complications]. / Minilaparoscopie e "open-laparoscopy". Prevenzione delle complicanze laparoscopiche.

BACKGROUND: The study aimed to compare laparoscopy, open-laparoscopy and mini-laparoscopy and to correlate the results of each technique with the respective percentages of laparotomic conversion. METHODS: A total of 101 laparoscopies were performed between November 1997 and April 1999: 18.8% were diagnostic and 81.2% operative. The latter included 54 traditional laparoscopies (65.9%), 18 open-laparoscopies (21.9%) and 10 minilaparoscopies (12.2%). RESULTS: Laparotomic conversion was required in 5.5% of laparoscopies. No laparotomic conversion was necessary for the open-laparoscoples and for mini-laparoscopies. CONCLUSIONS: The possibility of resorting to open-laparoscopy and mini-laparoscopy may represent a valid tool when the patient has previously undergone laparoscopic or laparotomic surgery, with the supposition of pelvo-abdominal adherences that would increase the risk of traditional laparoscopy.

[Role of capacitation in intrauterine insemination as a treatment of male infertility]. / Ruolo della capacitazione nella inseminazione intrauterina quale trattamento della sterilità da fattore maschile.

BACKGROUND: Although FIVET and ICSI efficacy and efficiency are continuously increasing, intrauterine insemination (IUI) is a very used technique for many different types of sterility. It is also used in male sterility for its cost/benefit positive rate. Pregnancies frequency obtained after IUI in male factor infertility cases, with or without ovarian stimulation, shows the value of this work. METHODS: We evaluated 149 insemination cycles in 34 couples with a male infertility diagnosis confirmed after at least 2 semen analysis, according to OMS criteria. All the 34 patients had at least one Fallopian tube open and some spontaneous ovulatory cycle to enter in this study. The patients without biochemical pregnancy signs after 6 intrauterine insemination cycles were induced to multiple ovulations through oral administration of 50 mg of Clomifene citrate from the 5th to the 9th cycle-day or through intramuscular administration of 75 I.U. of FSH from the 5th cycle-day. A luteal support with 200 mg of vaginal progesterone, 2 times for day, was reserved for all the patients with endometrium <8 mm or with low progesterone serum levels (<20 nmol/l). RESULTS: All patients made 149 IUI cycles, with a total medium pregnancy rate for cycle of 6.0% and for patient of 26.4%. We didn't observe statistically significant differences related to spermatozoa total number for ml in native sperm, in terms of pregnancy rate in men with 100-150 millions of spermatozoa. We observed a lower pregnancy rate in men with less than 5 millions moving spermatozoa. Under 1 million of total spermatozoa, pregnancy rate is very low (2.3%): it is established for higher values, obtaining the best results between 10 and 20 millions of total spermatozoa (10.5%) and a little reduction over 20 millions (8.3%). CONCLUSIONS: The evaluation of spermatozoa total number and also their motility does not present any prognostical significance as to the pregnancy rate in intrauterine insemination for male factor infertility.

The phorbol ester PMA and cyclic AMP activate different Cl(-) and HCO3(-) fluxes in C127 cells expressing CFTR.

In mouse mammary epithelial C127 cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR), chloride efflux, measured with the Cl(-)-sensitive dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), was stimulated by activation of protein kinase A with cyclic AMP elevating agents forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) and, to a less extent, by activation of protein kinase C with the phorbol 12-myristate 13-acetate (PMA). Conversely, bicarbonate influx, determined by intracellular alkalinization of cells incubated with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluoresceintetraacetoxymethyl ester (BCECF-AM), was stimulated by cyclic AMP elevation, but not by PMA. Patch clamp analysis revealed that PMA activated a Cl(-) current with the typical biophysical characteristics of swelling-activated current and not of CFTR.

[Ureaplasma urealyticum vaginosis and premature rupture of membranes. What is its role?]. / Vaginosi da ureaplasma urealyticum e rottura prematura delle membrane. Quale ruolo?

BACKGROUND: The aim of this study was to evaluate a correlation between PROM and genital infections. METHODS: A total of 308 vaginal swabs were made in randomized study group composed by 184 pregnant women aged between 26 and 32 years with an extreme age of 19 (one) and 40 years (one). Three vaginal swabs and one cervical swab (searching for Chlamydia) were made for every patient. Sixteen patients were excluded during this study, because they decided to have their babies in other hospitals. Therefore, the patients totally included in the study were 166: 109 at the first pregnancy, 33 at the second pregnancy and 5 at the third pregnancy. No one of them had any spontaneous abortion in the past. All possible other factor which ca be considered responsible and/or inductive of premature ruptures, such as cervical incontinence, cigarette smoke and coitus were excluded. RESULTS: 280 vaginal swabs were made in this study: 134 were positive, with a global positivity percentage of 47.85%. Premature rupture of membranes (PROM) was observed in 38 cases with an incidence of 23.03%: 26 were PROM and 12 were pPROM. The extreme pPROMs occurred, respectively, at the 21st and at 34th gestational week; 29 of the 38 cases of PROM, were associated with positive cultures. The results obtained show an evident correlation between PROM and Ureaplasma urealyticum vaginosis: this fact is improved by the high incidence percentage of this mycobacter in pregnant women and also by an absolute predomination of Ureplasma urealyticum in PROM cases (72.41%). CONCLUSIONS: These data obtained confirm the importance of this microorganism in PROM genesis, according to some recent studies. It is suggested that Ureaplasma urealyticum infection can contribute to a premature start of labour.

Sphingosine-1-phosphate activates phospholipase D in human airway epithelial cells via a G protein-coupled receptor.

Sphingosine-1-phosphate (SPP) acts as a first messenger in immortalized human airway epithelial cells (CFNPE9o(-)), possibly interacting with an Edg family receptor. Expression of the SPP receptors Edg-1 and Edg-3, as well as a low level of Edg-5/H218, was detected in these cells, in agreement with their ability to specifically bind SPP. The related lipids, lysophosphatidic acid and sphingosylphosphorylcholine, were unable to displace SPP from its high affinity binding sites, suggesting that the biological responses to these different lysolipids are mediated by distinct receptors. SPP markedly inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner and caused a remarkable elevation of intracellular calcium, both effects being sensitive to pertussis toxin treatment. Most importantly, SPP stimulated phosphatidic acid formation, which was maximal after 2 min and decreased within 8-10 min. In the presence of butan-1-ol, suppression of SPP-induced phosphatidic acid formation and production of phosphatidylbutanol were found, clearly indicating activation of phospholipase D (PLD). This finding was also confirmed by analysis of the fatty acid composition of phosphatidic acid, showing an increase in the monounsaturated oleic acid only. The decrease of phosphatidic acid level after 8-10 min incubation with SPP was accompanied by a parallel increase of diacylglycerol production, which was abolished in the presence of butan-1-ol. This result indicates that activation of phospholipase D is followed by stimulation of phosphatidate phosphohydrolase activity. Phosphatidic acid formation was insensitive to protein kinase C inhibitors and almost completely inhibited by pertussis toxin treatment, suggesting that SPP activates phospholipase D via a G(i/o) protein-coupled receptor.