Identificazione e dosaggio di marcatori molecolari dell'endometriosi nel sangue periferico

Purpose: to quantify the mRNA levels of MMP-3, MMP-9, VEGF and Survivin in peripheral blood and the serum levels of CA-125, Ca19-9 in women with and without endometriosis and to investigate the performance of these markers to differentiate between deep and ovarian endometriosis. Methods: a case controls study enrolled a series of 60 patients. Twenty controls have been matched with 20 cases of ovarian and 20 cases of deep endometriosis. Univariable and multivariable performance of serum CA125 and CA19-9, mRNA for Survivin, MMP9, MMP3 and VEGF genes have been evaluated by means of ROC curves and logistic regression respectively. Results: No difference in markers concentration were detected between ovarian and deep endometriosis. In comparison with controls serum CA19 and CA125 yielded the better sensitivity followed by mRNA for Survivin gene (81.5%, 51.9% and 7.5% at 10% false positive rate respectively). Multivariable estimated odds of endometriosis yielded a sensitivity of 87% at the same false positive rate. Conclusions: A combination of serum and molecular markers could allow a better diagnosis of endometriosis.

Expression profiling und rationale Expressionsoptimierung am Beispiel eines prokaryontischen Wirt-,Vektor-Systems

Durch globale Expressionsprofil-Analysen auf Transkriptom-, Proteom- oder Metabolom-Ebene können biotechnologische Produktionsprozesse besser verstanden und die Erkenntnisse für die zielgerichtete, rationale Optimierung von Expressionssystemen genutzt werden. In der vorliegenden Arbeit wurde die Überexpression einer Glukose-Dehydrogenase (EC 1.1.5.2), die von der Roche Diagnostics GmbH für die diagnostische Anwendung optimiert worden war, in Escherichia coli untersucht. Die Enzymvariante unterscheidet sich in sieben ihrer 455 Aminosäuren vom Wildtyp-Enzym und wird im sonst isogenen Wirt-/Vektor-System in signifikant geringeren Mengen (Faktor 5) gebildet. Das prokaryontische Expressionssystem wurde auf Proteom-Ebene charakterisiert. Die 2-dimensionale differenzielle Gelelektrophorese (DIGE) wurde zuvor unter statistischen Aspekten untersucht. Unter Berücksichtigung von technischen und biologischen Variationen, falsch-positiven (α-) und falsch-negativen (β-) Fehlern sowie einem daraus abgeleiteten Versuchsdesign konnten Expressionsunterschiede als signifikant quantifiziert werden, wenn sie um den Faktor ≥ 1,4 differierten. Durch eine Hauptkomponenten-Analyse wurde gezeigt, dass die DIGE-Technologie für die Expressionsprofil-Analyse des Modellsystems geeignet ist. Der Expressionsstamm für die Enzymvariante zeichnete sich durch eine höhere Variabilität an Enzymen für den Zuckerabbau und die Nukleinsäure-Synthese aus. Im Expressionssystem für das Wildtyp-Enzym wurde eine unerwartet erhöhte Plasmidkopienzahl nachgewiesen. Als potenzieller Engpass in der Expression der rekombinanten Glukose-Dehydrogenase wurde die Löslichkeitsvermittlung identifiziert. Im Expressionsstamm für das Wildtyp-Enzym wurden viele Proteine für die Biogenese der äußeren Membran verstärkt exprimiert. Als Folge dessen wurde ein sog. envelope stress ausgelöst und die Zellen gingen in die stationäre Wuchsphase über. Die Ergebnisse der Proteomanalyse wurden weiterführend dazu genutzt, die Produktionsleistung für die Enzymvariante zu verbessern. Durch den Austausch des Replikationsursprungs im Expressionsvektor wurde die Plasmidkopienzahl erhöht und die zelluläre Expressionsleistung für die diagnostisch interessantere Enzymvariante um Faktor 7 - 9 gesteigert. Um die Löslichkeitsvermittlung während der Expression zu verbessern, wurde die Plasmidkopienzahl gesenkt und die Coexpression von Chaperonen initiiert. Die Ausbeuten aktiver Glukose-Dehydrogenase wurden durch die Renaturierung inaktiven Produkts aus dem optimierten Expressionssystem insgesamt um einen Faktor von 4,5 erhöht. Somit führte im Rahmen dieser Arbeit eine proteombasierte Expressionsprofil-Analyse zur zielgerichteten, rationalen Expressionsoptimierung eines prokaryontischen Modellsystems.

Feasibility and diagnostic effectiveness of new capsule endoscopy techniques

Since its approval by FDA in 2001, capsule endoscopy revolutionized the study of small bowel. One of the main limitations of its diffusion has been the high cost. More recently, a new videocapsule system (OMOM CE) has been developed in China and obtained the CE mark. Its cost is approximately half that of other capsule systems. However, there are few studies regarding the clinical experience with this new videocapsule system and none of them has been performed in the western world. Among the limitations of capsule endoscopy, there is also one linked to the diagnostic yield. The rapid transit of the device in the proximal segments implies a high risk of false negatives; an indirect confirmation of this limit is offered by the poor ability to identify the papilla of Vater. In addition, recent studies show that in patients with obscure gastrointestinal bleeding, the negative outcome of capsule endoscopy is correlated to a significant risk of recurrence of anemia in the short term, as well as the presence of small bowel lesions documented by a second capsule endoscopy. It was recently approved the use of a new device called "CapsoCam" (CapsoVision, Inc. Saratoga) characterized by four side cameras that offer a panoramic view of 360 degrees, instead of the front to 160°. Two recent pilot studies showed comparable safety profiles and diagnostic yield with the more standardized capsule. Namely, side vision has made possible a clear visualization of the papilla in 70% of cases. The aim of our study is to evaluate the feasibility and diagnostic yield of these two new devices, which first may allow a reduction in costs. Moreover, their complementary use could lead to a recovery diagnostic in patients with false negative results in an initial investigation.

Tor network forensics and hidden service deanonymization

The cybernetics revolution of the last years improved a lot our lives, having an immediate access to services and a huge amount of information over the Internet. Nowadays the user is increasingly asked to insert his sensitive information on the Internet, leaving its traces everywhere. But there are some categories of people that cannot risk to reveal their identities on the Internet. Even if born to protect U.S. intelligence communications online, nowadays Tor is the most famous low-latency network, that guarantees both anonymity and privacy of its users. The aim of this thesis project is to well understand how the Tor protocol works, not only studying its theory, but also implementing those concepts in practice, having a particular attention for security topics. In order to run a Tor private network, that emulates the real one, a virtual testing environment has been configured. This behavior allows to conduct experiments without putting at risk anonymity and privacy of real users. We used a Tor patch, that stores TLS and circuit keys, to be given as inputs to a Tor dissector for Wireshark, in order to obtain decrypted and decoded traffic. Observing clear traffic allowed us to well check the protocol outline and to have a proof of the format of each cell. Besides, these tools allowed to identify a traffic pattern, used to conduct a traffic correlation attack to passively deanonymize hidden service clients. The attacker, controlling two nodes of the Tor network, is able to link a request for a given hidden server to the client who did it, deanonymizing him. The robustness of the traffic pattern and the statistics, such as the true positive rate, and the false positive rate, of the attack are object of a potential future work.

Multicentre validation study of nucleic acids extraction from FFPE tissues

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Intraobserver and interobserver agreement for results of low-field magnetic resonance imaging in dogs with and without clinical signs of disk-associated wobbler syndrome

OBJECTIVE: To determine interobserver and intraobserver agreement for results of low-field magnetic resonance imaging (MRI) in dogs with and without disk-associated wobbler syndrome (DAWS). DESIGN: Validation study. ANIMALS: 21 dogs with and 23 dogs without clinical signs of DAWS. PROCEDURES: For each dog, MRI of the cervical vertebral column was performed. The MRI studies were presented in a randomized sequence to 4 board-certified radiologists blinded to clinical status. Observers assessed degree of disk degeneration, disk-associated and dorsal compression, alterations in intraspinal signal intensity (ISI), vertebral body abnormalities, and new bone formation and categorized each study as originating from a clinically affected or clinically normal dog. Interobserver agreement was calculated for 44 initial measurements for each observer. Intraobserver agreement was calculated for 11 replicate measurements for each observer. RESULTS: There was good interobserver agreement for ratings of disk degeneration and vertebral body abnormalities and moderate interobserver agreement for ratings of disk-associated compression, dorsal compression, alterations in ISI, new bone formation, and suspected clinical status. There was very good intraobserver agreement for ratings of disk degeneration, disk-associated compression, alterations in ISI, vertebral body abnormalities, and suspected clinical status. There was good intraobserver agreement for ratings of dorsal compression and new bone formation. Two of 21 clinically affected dogs were erroneously categorized as clinically normal, and 4 of 23 clinically normal dogs were erroneously categorized as clinically affected. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that variability exists among observers with regard to results of MRI in dogs with DAWS and that MRI could lead to false-positive and false-negative assessments.

A modified test for small-study effects in meta-analyses of controlled trials with binary endpoints

Publication bias and related bias in meta-analysis is often examined by visually checking for asymmetry in funnel plots of treatment effect against its standard error. Formal statistical tests of funnel plot asymmetry have been proposed, but when applied to binary outcome data these can give false-positive rates that are higher than the nominal level in some situations (large treatment effects, or few events per trial, or all trials of similar sizes). We develop a modified linear regression test for funnel plot asymmetry based on the efficient score and its variance, Fisher's information. The performance of this test is compared to the other proposed tests in simulation analyses based on the characteristics of published controlled trials. When there is little or no between-trial heterogeneity, this modified test has a false-positive rate close to the nominal level while maintaining similar power to the original linear regression test ('Egger' test). When the degree of between-trial heterogeneity is large, none of the tests that have been proposed has uniformly good properties.

Multiple Testing With an Empirical Alternative Hypothesis

An optimal multiple testing procedure is identified for linear hypotheses under the general linear model, maximizing the expected number of false null hypotheses rejected at any significance level. The optimal procedure depends on the unknown data-generating distribution, but can be consistently estimated. Drawing information together across many hypotheses, the estimated optimal procedure provides an empirical alternative hypothesis by adapting to underlying patterns of departure from the null. Proposed multiple testing procedures based on the empirical alternative are evaluated through simulations and an application to gene expression microarray data. Compared to a standard multiple testing procedure, it is not unusual for use of an empirical alternative hypothesis to increase by 50% or more the number of true positives identified at a given significance level.

MODIFIED TEST STATISTICS BY INTER-VOXEL VARIANCE SHRINKAGE WITH AN APPLICATION TO fMRI

Functional Magnetic Resonance Imaging (fMRI) is a non-invasive technique which is commonly used to quantify changes in blood oxygenation and flow coupled to neuronal activation. One of the primary goals of fMRI studies is to identify localized brain regions where neuronal activation levels vary between groups. Single voxel t-tests have been commonly used to determine whether activation related to the protocol differs across groups. Due to the generally limited number of subjects within each study, accurate estimation of variance at each voxel is difficult. Thus, combining information across voxels in the statistical analysis of fMRI data is desirable in order to improve efficiency. Here we construct a hierarchical model and apply an Empirical Bayes framework on the analysis of group fMRI data, employing techniques used in high throughput genomic studies. The key idea is to shrink residual variances by combining information across voxels, and subsequently to construct an improved test statistic in lieu of the classical t-statistic. This hierarchical model results in a shrinkage of voxel-wise residual sample variances towards a common value. The shrunken estimator for voxelspecific variance components on the group analyses outperforms the classical residual error estimator in terms of mean squared error. Moreover, the shrunken test-statistic decreases false positive rate when testing differences in brain contrast maps across a wide range of simulation studies. This methodology was also applied to experimental data regarding a cognitive activation task.

Ultrasound diagnosis of spontaneous carotid dissection with isolated Horner syndrome

BACKGROUND AND PURPOSE: Isolated Horner syndrome without associated cranial nerve palsies or ischemic symptoms is an important presentation of spontaneous internal carotid artery dissection (sICAD). Ultrasound is often used as a screening method in these patients because cervical MRI is not always available on an emergency basis. Current knowledge on ultrasound findings in patients with sICAD presenting with isolated Horner syndrome is limited. METHODS: Patients were recruited from prospective cervical artery dissection databases of 3 tertiary care centers. Diagnosis of sICAD was confirmed by cervical MRI and MR angiography or digital subtraction angiography in all patients. Data on Doppler sonography and color duplex sonography examinations performed within 30 days of symptom onset were analyzed. RESULTS: We identified 88 patients with Horner syndrome as the only sign of sICAD. Initial ultrasound examination was performed in 72 patients after a mean time interval from symptom onset to examination of 11 (SD 8) days. The overall frequency of false-negative ultrasound findings was 31% (22 of 72 patients). It showed stenosis >or=80% or occlusion in 34 (47%) patients, and stenosis

Comparison of tendency-oriented perimetry and dynamic strategy in octopus perimetry as a screening tool in a clinical setting: a prospective study

OBJECTIVE: The aim of this study was to compare the results of tendency-oriented perimetry (TOP) and a dynamic strategy in octopus perimetry as screening methods in clinical practice. DESIGN: A prospective single centre observational case series was performed. PARTICIPANTS AND METHODS: In a newly opened general ophthalmologic practice 89 consecutive patients (171 eyes) with a clinical indication for octopus static perimetry testing (ocular hypertension or suspicious optic nerve cupping) were examined prospectively with TOP and a dynamic strategy. The visual fields were graded by 3 masked observers as normal, borderline or abnormal without any further clinical information. RESULTS: 83% eyes showed the same result for both strategies. In 14% there was a small difference (with one visual field being abnormal or normal, the other being borderline). In only 2.9% of the eyes (5 cases) was there a contradictory result. In 4 out of 5 cases the dynamic visual field was abnormal and TOP was normal. 4 of these cases came back for a second examination. In all 4 the follow-up examination showed a normal second dynamic visual field. CONCLUSIONS: Octopus static perimetry using a TOP strategy is a fast, patient-friendly and very reliable screening tool for the general ophthalmological practice. We found no false-negative results in our series.

Laryngeal and hypopharyngeal carcinomas after (chemo)radiotherapy: a diagnostic dilemma

PURPOSE OF REVIEW: During recent years, (chemo)radiotherapy has evolved into a primary treatment modality for both early and advanced laryngeal and hypopharyngeal carcinomas. Head and neck surgeons will be concerned more frequently with patients presenting symptoms and signs suggesting recurrent tumor or complications of (chemo)radiotherapy. RECENT FINDINGS: Analysis of histologic characteristics and tumor spread of recurrent carcinomas on whole-organ slices of salvage laryngectomy specimens showed that recurrent laryngeal carcinomas are often present with multiple tumor foci dispersed in different regions; furthermore, they may develop beneath an intact mucosa. Only a few articles analyze the reliability of laryngoscopy and biopsy in detecting recurrences after (chemo)radiotherapy: the number of false negative biopsies is relatively high. The differentiation between radionecrosis and tumor recurrence is difficult by computed tomography scan and magnetic resonance imaging in many cases. Positron emission tomography-computed tomography and diffusion-weighted magnetic resonance imaging are promising diagnostic modalities to detect or exclude persistent or recurrent disease after (chemo)radiotherapy. SUMMARY: Endoscopy with biopsy, computed tomography scan and conventional magnetic resonance imaging present several deficiencies in diagnosing recurrent disease after (chemo)radiotherapy. New imaging modalities such as positron emission tomography-computed tomography and diffusion-weighted magnetic resonance imaging show promising results, increasing the diagnostic efficacy.

The accuracy of FAST in relation to grade of solid organ injuries: a retrospective analysis of 226 trauma patients with liver or splenic lesion

BACKGROUND: This study investigated the role of a negative FAST in the diagnostic and therapeutic algorithm of multiply injured patients with liver or splenic lesions. METHODS: A retrospective analysis of 226 multiply injured patients with liver or splenic lesions treated at Bern University Hospital, Switzerland. RESULTS: FAST failed to detect free fluid or organ lesions in 45 of 226 patients with spleen or liver injuries (sensitivity 80.1%). Overall specificity was 99.5%. The positive and negative predictive values were 99.4% and 83.3%. The overall likelihood ratios for a positive and negative FAST were 160.2 and 0.2. Grade III-V organ lesions were detected more frequently than grade I and II lesions. Without the additional diagnostic accuracy of a CT scan, the mean ISS of the FAST-false-negative patients would be significantly underestimated and 7 previously unsuspected intra-abdominal injuries would have been missed. CONCLUSION: FAST is an expedient tool for the primary assessment of polytraumatized patients to rule out high grade intra-abdominal injuries. However, the low overall diagnostic sensitivity of FAST may lead to underestimated injury patterns and delayed complications may occur. Hence, in hemodynamically stable patients with abdominal trauma, an early CT scan should be considered and one must be aware of the potential shortcomings of a "negative FAST".

Forget the blood, not the stone! Microhaematuria in acute urolithiasis and the role of early CT scanning

BACKGROUND: Although urinalysis is simple and inexpensive to perform, the finding of microhaematuria on urinalysis may be unreliable for diagnosing urolithiasis. OBJECTIVE: To evaluate microhaematuria as a diagnostic marker for urolithiasis compared with low-dose unenhanced multidetector computed tomography (MDCT) as the "gold standard". SETTING: A level 1 emergency department in a tertiary referral university teaching hospital. DESIGN: Retrospective analysis. METHODS: A study was undertaken to assess whether the finding of microhaematuria was diagnostic for urolithiasis using a low-dose unenhanced MDCT-based diagnosis as the reference standard by reviewing the records of all patients who presented to the emergency department with colicky flank pain and underwent a CT scan between January 2003 and December 2005. RESULTS: Urolithiasis was present (as defined by low-dose unenhanced MDCT) in 507/638 patients (79%); 341/638 (53%) were true positive for urolithiasis, 76 (12%) were true negative, 55 (9%) were false positive and 166 (26%) were false negative. Microhaematuria as a test for urolithiasis in patients presenting to the emergency department therefore has a sensitivity, specificity, positive predictive value and negative predictive value of 67%, 58%, 86% and 31%, respectively. 58% of the urinalysis results were negative for haematuria in the subset of patients with significant alternative diagnoses. CONCLUSIONS: The sensitivity, specificity and negative predictive value of microhaematuria on urinalysis for urolithiasis using unenhanced MDCT as the reference standard were low. This suggests that, when urolithiasis is clinically suspected, unenhanced MDCT is indicated without urinalysis being a prerequisite.

Pelvic radiography in ATLS algorithms: A diminishing role?

ABSTRACT: BACKGROUND: Pelvic x-ray is a routine part of the primary survey of polytraumatized patients according to Advanced Trauma Life Support (ATLS) guidelines. However, pelvic CT is the gold standard imaging technique in the diagnosis of pelvic fractures. This study was conducted to confirm the safety of a modified ATLS algorithm omitting pelvic x-ray in hemodynamically stable polytraumatized patients with clinically stable pelvis in favour of later pelvic examination by CT scan. METHODS: We conducted a retrospective analysis of all polytraumatized patients in our emergency room between 01.07.2004 and 31.01.2006. Inclusion criteria were blunt abdominal trauma, initial hemodynamic stability and a stable pelvis on clinical examination. We excluded patients requiring immediate intervention because of hemodynamic instability. RESULTS: We reviewed the records of n = 452 polytraumatized patients, of which n = 91 fulfilled inclusion criteria (56% male, mean age = 45 years). The mechanism of trauma included 43% road traffic accidents, 47% falls. In 68/91 (75%) patients, both a pelvic x-ray and a CT examination were performed; the remainder had only pelvic CT. In 6/68 (9%) patients, pelvic fracture was diagnosed by pelvic x-ray. None of these 6 patients was found having a false positive pelvic x-ray, i.e. there was no fracture on pelvic CT scan. In 3/68 (4%) cases a fracture was missed in the pelvic x-ray, but confirmed on CT (false negative on x-ray). None of the diagnosed fractures needed an immediate therapeutic intervention. 5 (56%) were classified type A fractures, and another 4 (44%) B 2.1 in computed tomography (AO classification). One A 2.1 fracture was found in a clinically stable patient who only received CT scan (1/23). CONCLUSION: While pelvic x-ray is an integral part of ATLS assessment, this retrospective study suggests that in hemodynamically stable patients with clinically stable pevis, its sensitivity is only 67% and it may safely be omitted in favor of a pelvic CT examination if such is planned in adjunct assessment and available. The results support the safety and utility of our modified ATLS algorithm. A randomized controlled trial using the algorithm can safely be conducted to confirm the results.