Sistema nervoso enterico e scrapie sperimentale in ovini di razza sarda con diversa suscettibilità genetica nei confronti della malattia

Sebbene il sistema nervoso enterico (“enteric nervous system”, ENS) svolga un ruolo cruciale nella patogenesi della Scrapie ovina, non esistono tuttavia in letteratura dati sulle popolazioni cellulari progressivamente coinvolte nel corso dell’infezione, né sugli eventuali danni morfo-funzionali da esse subiti. Il presente studio è stato condotto sui plessi mienterici e sottomucosi dell’ileo di 46 pecore di razza Sarda, recanti diversi polimorfismi del gene Prnp (ARQ/ARQ, ARQ/AHQ, ARQ/ARR, ARR/ARR). I suddetti animali, infettati per os all’età di 8 mesi con un ceppo di Scrapie precedentemente caratterizzato nel topo, sono stati sacrificati mediante eutanasia a determinati intervalli di tempo post-infezione (p.i.). E’ stata quindi valutata, tramite immunoistochimica ed immunofluorescenza indiretta su sezioni tissutali e su preparati “wholemount”, l’immunoreattività (IR) nei confronti della PrPSc, del “marker” panneuronale Hu C/D, dell’ossido-nitrico sintetasi (nNOS), della calbindina (CALB) e della proteina fibrillare acida gliale (GFAP). In 8 pecore con genotipo ARQ/ARQ, clinicamente sane e sacrificate a 12-24 mesi p.i., nonché in 5 ovini clinicamente affetti (2 con genotipo ARQ/ARQ, 3 con genotipo ARQ/AHQ), questi ultimi sacrificati rispettivamente a 24, 36 e 40 mesi p.i., le indagini immunoistochimiche hanno consentito di dimostrare la presenza di PrPSc a livello sia dell’encefalo (obex), sia dell’ENS, in particolar modo nei plessi mienterici. In tali distretti il deposito della PrPSc risultava pienamente compatibile con un interessamento delle cellule enterogliali (“enteroglial cells”, EGCs), mentre occasionalmente si notava un contestuale coinvolgimento della componente neuronale ivi residente. In conclusione, i dati della presente indagine consentono di ipotizzare un verosimile coinvolgimento delle EGCs e dei neuroni residenti a livello dei plessi dell’ENS nella patogenesi della Scrapie sperimentale realizzata per os in ovini di razza Sarda.

Studio di marcatori immunoistochimici, analisi FISH e ruolo dei microRNA nelle neoplasie gliali di basso e di alto grado

Gliomas are the most common primary brain tumours. Despite advances in surgical techniques, postoperative supportive care, radiation and adjuvant systemic therapy, the life expectancy of patients with high grade glioma has remained essentially poor. Furthermore differential diagnosis among astrocytomas, oligodendrogliomas and oligoastrocytomas is very challenging and subject to inter-observer variability. The purpose of the research was: 1) to investigate a series of high grade and low grade gliomas at gene and protein (immunohistochemistry) levels to disclose possible genetic portraits of malignancy; 2) to verify the utility of Nogo-A, Olig-2 and synaptophysin in providing a correct histological diagnosis of oligodendroglioma and to investigate a possible complementary role in selecting the best areas suitable for detecting 1p/19q codeletion using FISH analysis; 3) to study the role of microRNA in high grade gliomas. In order to obtain these goals large series of brain tumors were studied with DNA microarrays, immunohistochemistry and RT-PCR The results demonstrated that: - Overexpression of IGFBP-2 and CDC20 is highly related to glioblastomas and their immunopositivity can be useful for the identification of glioblastoma in small biopsies. - Nogo-A is the most useful and specific marker in differentiating oigodendrogliomas from other gliomas. Furthermore, using a Nogo-A driven FISH analysis, it is possible to identify a larger number of 1p19q codeletions in gliomas. - microRNAs can be studied in paraffin embedded tissues better than in fresh tissues. A series of six microRNA, significatively deregulated in glioblastomas, may represent a genetic signature with prognostic and predictive value and could constitute candidates for novel anti-cancer therapeutics.

Analyse genomischer Imbalancen in hepatozellulären Karzinomen und präneoplastischen Leberläsionen

Das hepatozelluläre Karzinom (HCC) ist mit ungefähr 1,000,000 neuen Fällen pro Jahr einer der häufigsten malignen Tumore weltweit. Es ist hauptsächlich in Südost-Asien und im südlichen Afrika verbreitet. Risikofaktoren sind chronische Infektionen mit Hepatitis Viren (HBV, HCV), Aflatoxin B1-Belastung und chronischer Alkoholkonsum. Um Veränderungen auf genomischer Ebene in HCCs zu untersuchen, wurden in der vorliegenden Untersuchung Frischgewebeproben von 21 Patienten mit HCCs und formalin-fixiertes, paraffineingebettetes Material von 6 Dysplastischen Knoten mittels Comparativer genomischer Hybridisierung (CGH) analysiert. In den untersuchten HCCs konnte Zugewinne auf 1q (12/21), 6p ( 6/21), 8q (11/21), 17q (6/21), 20q (6/21), sowie Verluste auf 4q (7/21), 6q (4/21), 10q (3/21), 13q (4/21), 16q (3/21) identifiziert werden. Die Validität der mit diesem Ansatz erzielten Ergebnisse konnte anhand von unabhängigen Kontrollexperimenten mit Interphase-FISH-Analyse nachgewiesen werden. Die in Dysplastische Knoten identifizierten Veränderungen sind Gewinne auf 1q (50% ) sowie Verluste auf 8p und 17p. Daher stellt 1q eine Kandidatenregion für die Identifizierung jener Gene dar, die bereits im frühem Stadium der Hepatokarzinogenese aktiviert sind. Die Gen-Expressionsanalyse eines HCCs mit Gewinnen auf 1q, 8q, und Xq zeigte die Überexpression von einigen Genen, die in den amplifizierten Regionen liegen. Daher kann spekuliert werden, dass die DNA-Amplifikation in der Hepatokarzinogenese bei einigen Genen ein Mechanismus der Aktivierung sein kann. Zusammengefasst konnte somit durch CGH-Analyse charakteristische, genomische Imbalances des HCC ermittelt werden. Der Vergleich mit Veränderungen bei prämalignen Läsionen erlaubt die Unterscheidung früher (prämaligner) und später (progressionsassoziierter) Veränderungen

Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT

1) Background: The most common methods to evaluate clarithromycin resistance is the E-Test, but is time consuming. Resistance of Hp to clarithromycin is due to point mutations in the 23S rRNA. Eight different point mutations have been related to CH resistance, but the large majority of the clarithromycin resistance depends on three point mutations (A2142C, A2142G and A2143G). A novel PCR-based clarithromycin resistance assays, even on paraffin-embedded biopsy specimens, have been proposed. Aims: to assess clarithromycin resistance detecting these point mutation (E-Test as a reference method);secondly, to investigate relation with MIC values. Methods: Paraffin-embedded biopsies of patients Hp-positive were retrieved. The A2142C, A2142G and A2143G point mutations were detected by molecular analysis after DNA extraction by using a TaqMan real-time PCR. Results: The study enrolled 86 patients: 46 resistant and 40 sensible to CH. The Hp status was evaluated at endoscopy, by rapid urease test (RUT), histology and hp culture. According to real-time PCR, 37 specimens were susceptible to clarithromycin (wild type dna) whilst the remaining 49 specimens (57%) were resistant. A2143G is the most frequent mutation. A2142C always express a resistant phenotype and A2142G leads to a resitant phenotype only if homozigous. 2) Background: Colonoscopy work-load for endoscopy services is increasing due to colorectal cancer prevention. We tested a combination of faecal tests to improve accuracy and prioritize the access to colonoscopy. Methods: we tested a combination of fecal tests (FOBT, M2-PK and calprotectin) in a group of 280 patients requiring colonoscopy. Results: 47 patients had CRC and 85 had advanced adenoma/s at colonoscopy/histology. In case of single test, for CRC detection FOBT was the test with the highest specificity and PPV, M2-PK had the highest sensitivity and higher NPV. Combination was more interesting in term of PPV. And the best combination of tests was i-FOBT + M2-PK.

optomotor-blind and the horizontal and vertical system cells of the drosophila optic lobes

The horizontal and vertical system neurons (HS and VS cells) are part of a conserved set of lobula plate giant neurons (LPGNs) in the optic lobes of the adult brain. Structure and physiology of these cells are well known, predominantly from studies in larger Dipteran flies. Our knowledge about the ontogeny of these cells is limited and stems predominantly from laser ablation studies in larvae of the house fly Musca domestica. These studies suggested that the HS and VS cells stem from a single precursor, which, at least in Musca, has not yet divided in the second larval instar. A regulatory mutation (In(1)omb[H31]) in the Drosophila gene optomotor-blind (omb) leads to the selective loss of the adult HS and VS cells. This mutation causes a transient reduction in omb expression in what appears to be the entire optic lobe anlage (OLA) late in embryogenesis. Here, I have reinitiated the laser approach with the goal of identifying the presumptive embryonic HS/VS precursor cell in Drosophila. The usefulness of the laser ablation approach which has not been applied, so far, to cells lying deep within the Drosophila embryo, was first tested on two well defined embryonic sensory structures, the olfactory antenno-maxillary complex (AMC) and the light-sensitive Bolwing´s organ (BO). In the case of the AMC, the efficiency of the ablation procedure was demonstrated with a behavioral assay. When both AMCs were ablated, the response to an attractive odour (n-butanol) was clearly reduced. Interestingly, the larvae were not completely unresponsive but had a delayed response kinetics, indicating the existence of a second odour system. BO will be a useful test system for the selectivity of laser ablation when used at higher spatial resolution. An omb-Gal4 enhancer trap line was used to visualize the embryonic OLA by GFP fluorescence. This fluorescence allowed to guide the laser beam to the relevant structure within the embryo. The success of the ablations was monitored in the adult brain via the enhancer trap insertion A122 which selectively visualizes the HS and VS cell bodies. Due to their tight clustering, individual cells could not be identified in the embryonic OLA by conventional fluorescence microscopy. Nonetheless, systematic ablation of subdomains of the OLA allowed to localize the presumptive HS/VS precursor to a small area within the OLA, encompassing around 10 cells. Future studies at higher resolution should be able to identify the precursor as (an) individual cell(s). Most known lethal omb alleles do not complement the HS/VS phenotype of the In(1)omb[H31] allele. This is the expected behaviour of null alleles. Two lethal omb alleles that had been isolated previously by non-complementation of the omb hypomorphic allele bifid, have been reported, however, to complement In(1)omb[H31]. This report was based on low resolution paraffin histology of adult heads. Four mutations from this mutagenesis were characterized here in more detail (l(1)omb[11], l(1)omb[12], l(1)omb[13], and l(1)omb[15]). Using A122 as marker for the adult HS and VS cells, I could show, that only l(1)omb[11] can partly complement the HS/VS cell phenotype of In(1)omb[H31]. In order to identify the molecular lesions in these mutants, the exons and exon/intron junctions were sequenced in PCR-amplified material from heterozygous flies. Only in two mutants could the molecular cause for loss of omb function be identified: in l(1)omb[13]), a missense mutation causes the exchange of a highly conserved residue within the DNA-binding T-domain; in l(1)omb[15]), a nonsense mutation causes a C-terminal truncation. In the other two mutants apparently regulatory regions or not yet identified alternative exons are affected. To see whether mutant OMB protein in the missense mutant l(1)omb[13] is affected in DNA binding, electrophoretic shift assays on wildtype and mutant T-domains were performed. They revealed that the mutant no longer is able to bind the consensus palindromic T-box element.

Identificazione di un profilo molecolare di rischio nei pazienti pediatrici affetti da Linfoma di Hodgkin

Obiettivi: nonostante i miglioramenti nel trattamento, circa il 30% dei pazienti pediatrici affetti da Linfoma di Hodgkin (LH) in stadio avanzato recidiva o muore per progressione di malattia e i correnti metodi predittivi biologico-clinici non consentono di individuare tali pazienti. L’obiettivo dello studio è stato quello di definire un profilo molecolare di rischio che correli con l’outcome in questi pazienti. Materiali e metodi: studio retrospettico condotto su pazienti pediatrici affetti da LH omogeneamente trattati dal 2004 in poi. Su tali pazienti è stato intrapreso uno studio di validazione di marcatori molecolari già identificati in studi esplorativi precedenti. 27 geni sono stati analizzati in RT PCR su campioni di tessuto istologico prelevato alla diagnosi fissato in formalina e processato in paraffina relativi a una coorte di 37 pazienti, 12 ad outcome sfavorevole e 25 ad outcome favorevole. Risultati: dall’analisi univariata è emerso che solo l’espressione di CASP3 e CYCS, appartenenti al pathway apoptotico, è in grado di influenzare l’EFS in modo significativo nella nostra coorte di pazienti. Lo studio delle possibili combinazioni di questi geni ha mostrato l’esistenza di 3 gruppi di rischio che correlano con l’EFS: alto rischio (down regolazione di entrambi i geni), rischio intermedio (down regolazione di uno solo dei 2 geni), basso rischio (up regolazione di entrambi i geni). Dall’analisi multivariata è emerso che CASP3 è l’unica variabile che mantiene la sua indipendenza nell’influenzare la prognosi con un rischio di eventi di oltre il doppio di chi ha un’espressione bassa di questo gene. Conclusioni: i risultati ottenuti sulla nostra coorte di pazienti pediatrici affetti da LH confermano l’impatto sulla prognosi di due marcatori molecolari CASP3 e CYCS coinvolti nel patwhay apoptotico. La valutazione del profilo di espressione di tali geni, potrebbe pertanto essere utilizzata in corso di stadiazione, come criterio di predittività.

High sensitivity analysis of BRAF mutations in neoplastic and non-neoplastic thyroid lesions

The clonal distribution of BRAFV600E in papillary thyroid carcinoma (PTC) has been recently debated. No information is currently available about precursor lesions of PTCs. My first aim was to establish whether the BRAFV600E mutation occurs as a subclonal event in PTCs. My second aim was to screen BRAF mutations in histologically benign tissue of cases with BRAFV600E or BRAFwt PTCs in order to identify putative precursor lesions of PTCs. Highly sensitive semi-quantitative methods were used: Allele Specific LNA quantitative PCR (ASLNAqPCR) and 454 Next-Generation Sequencing (NGS). For the first aim 155 consecutive formalin-fixed and paraffin-embedded (FFPE) specimens of PTCs were analyzed. The percentage of mutated cells obtained was normalized to the estimated number of neoplastic cells. Three groups of tumors were identified: a first had a percentage of BRAF mutated neoplastic cells > 80%; a second group showed a number of BRAF mutated neoplastic cells < 30%; a third group had a distribution of BRAFV600E between 30-80%. The large presence of BRAFV600E mutated neoplastic cell sub-populations suggests that BRAFV600E may be acquired early during tumorigenesis: therefore, BRAFV600E can be heterogeneously distributed in PTC. For the second aim, two groups were studied: one consisted of 20 cases with BRAFV600E mutated PTC, the other of 9 BRAFwt PTCs. Seventy-five and 23 histologically benign FFPE thyroid specimens were analyzed from the BRAFV600E mutated and BRAFwt PTC groups, respectively. The screening of BRAF mutations identified BRAFV600E in “atypical” cell foci from both groups of patients. “Unusual” BRAF substitutions were observed in histologically benign thyroid associated with BRAFV600E PTCs. These mutations were very uncommon in the group with BRAFwt PTCs and in BRAFV600E PTCs. Therefore, lesions carrying BRAF mutations may represent “abortive” attempts at cancer development: only BRAFV600E boosts neoplastic transformation to PTC. BRAFV600E mutated “atypical foci” may represent precursor lesions of BRAFV600E mutated PTCs.

Synthesis and characterization of artificial aggregates and their potential applications in road construction

L’obiettivo del progetto è stato quello di realizzare ed analizzare aggregati artificiali creati attraverso geopolimerizzazione e macro-incapsulazione di paraffina in aggregati leggeri espansi, discutendo i loro possibili impieghi nelle pavimentazioni stradali. Dopo un'accurata calibrazione delle miscele geopolimeriche, sono stati realizzati degli aggregati artificiali, in seguito caratterizzati in accordo con la norma UNI EN 10343, con l'intento di sostituire materiali stradali vergini. Contemporaneamente, sono stati prodotti aggregati leggeri impregnati di paraffina (PCM), in grado di cambiare fase una volta raggiunti all'incirca i 3 °C, e successivamente rivestiti da due strati di resina poliestere e polvere di granito, denominati PLA: sfruttandone le proprietà termiche, si è valutato il loro possibile utilizzo come soluzione anti-icing. L’ultima fase della ricerca è stata incentrata nella realizzazione di aggregati geopolimerici espansi e molto porosi che potessero contenere una elevata quantità di PCM, sostituendo l'argilla espansa utilizzata nella produzione degli PLA.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue

The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months.

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.

Primer extension based quantitative polymerase chain reaction reveals consistent differences in the methylation status of the MGMT promoter in diffusely infiltrating gliomas (WHO grade II-IV) of adults

Diffusely infiltrating gliomas (WHO grade II-IV) are the most common primary brain tumours in adults. These tumours are not amenable to cure by surgery alone, so suitable biomarkers for adjuvant modalities are required to guide therapeutic decision-making. Epigenetic silencing of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene by promoter methylation has been associated with longer survival of patients with high-grade gliomas who receive alkylating chemotherapy; and molecular testing for the methylation status of the MGMT promoter sequence is regarded as among the most relevant of such markers. We have developed a primer extension-based assay adapted to formalin-fixed paraffin-embedded tissues that enables quantitative assessment of the methylation status of the MGMT promoter. The assay is very sensitive, highly reproducible, and provides valid test results in nearly 100% of cases. Our results indicate that oligodendrogliomas, empirically known to have a relatively favourable prognosis, are also the most homogeneous entities in terms of MGMT promoter methylation. Conversely, astrocytomas, which are more prone to spontaneous progression to higher grade malignancy, are significantly more heterogeneous. In addition, we show that the degree of promoter methylation correlates with the prevalence of loss of heterozygosity on chromosome arm 1p in the oligodendroglioma group, but not the astrocytoma group. Our results may have potentially important implications for clinical molecular diagnosis.

Immunohistochemical localization of osteoclastogenic cell mediators in feline tooth resorption and healthy teeth

Tooth resorption is among the most common and most challenging problems in feline dentistry It is a progressive disease eventually leading to tooth loss and often root replacement. The etiology of moth resorption remains obscure and to date no effective therapeutic approach is known. The present study is aimed at assessing the reliability of radiographic imaging and addressing the possible involvement of receptor activator of NF kappa B (RANK), its ligand (RANKL), and osteoprotegerin (OPG) in the process of tooth resorption. Teeth from 8 cats were investigated by means of radiographs and paraffin sections followed by immunolabeling. Six cats were diagnosed with tooth resorption based on histopathologic and radiographic findings. Samples were classified according to a four-stage diagnostic system. Radiologic assessment of tooth resorption correlated very strongly with histopathologic findings. Tooth resorption was accompanied by a strong staining with all three antibodies used, especially with anti-RANK and anti-RANKL antibodies. The presence of OPG and RANKL at the resorption site is indicative of repair attempts by fibroblasts and stromal cells. These findings should be extended by further investigations in order to elucidate the pathophysiologic processes underlying tooth resorption that might lead to prophylactic and/or therapeutic measures. J Vet Dent 27(2); 75 - 83, 2010

PTCH promoter methylation at low level in sporadic basal cell carcinoma analysed by three different approaches

Please cite this paper as: PTCH promoter methylation at low level in sporadic basal cell carcinoma analysed by three different approaches. Experimental Dermatology 2010. Abstract: Basal cell carcinoma (BCC) is the most common form of skin cancer. Mutations of the PTCH hallmark gene are detected in about 50-60% of BCCs, which raises the question whether other mechanisms such as promoter methylation can inactivate PTCH. Therefore, we performed methylation analysis of the PTCH promoter in a total of 56 BCCs. The sensitivity of three different methods, including direct bisulphite sequencing PCR, MethyLight and high-resolution melting (HRM), was applied and compared. We found that HRM analysis of DNA from fresh tissue [rather than formalin-fixed and paraffin-embedded tissue (FFPE)] was the most sensitive method to detect methylation. Low-level methylation of the PTCH promoter was detected in five out of 16 analysed BCCs (31%) on DNA from fresh tissue but only in two (13%) samples on short-time stored FFPE DNA from the very same tumors. In contrast, we were unable to detect methylation by HRM on long-time stored DNA in any of the remaining 40 BCC samples. Our data suggest that (i) HRM on DNA extracted from fresh tissue is the most sensitive method to detect methylation and (ii) methylation of the PTCH promoter may only play a minor role in BCC carcinogenesis.

Accidental infection of veterinary personnel with Mycobacterium tuberculosis at necropsy: a case study

Mycobacterium tuberculosis is the main cause of human tuberculosis. Infection in companion animals is mainly acquired from close contact to a diseased human patient and hence rarely diagnosed in countries with low tuberculosis incidence rates. Therefore the general awareness of the disease might be low. Here we report the potential risk of infection for veterinary personnel with M. tuberculosis during the clinical and pathological examination of a dog with unexpected disseminated tuberculosis. The dog had presented with symptoms of a central nervous system disease; rapid deterioration prevented a complete clinical workup, however. Post-mortem examination revealed systemic mycobacteriosis, and M. tuberculosis was identified by PCR amplification of DNA extracts from paraffin-embedded tissue sections and spoligotyping. Contact investigations among the owners and veterinary personnel using an IFN-? release assay indicated that the index dog did not infect humans during its lifetime. Serological and IFN-? release assay results of one of two cats in direct contact with the index dog, however, suggested that transmission of M. tuberculosis might have occurred. Importantly, all three pathologists performing the necropsy on the dog tested positive. Accidental infection was most likely due to inhalation of M. tuberculosis containing aerosols created by using an electric saw to open the brain cavity. As a consequence routine necropsy procedures have been adapted and a disease surveillance program, including tuberculosis, has been initiated. Our results highlight the importance of disease awareness and timely diagnosis of zoonotic infectious agents in optimizing work safety for veterinary personnel.

PRO_10--a new tissue-based prognostic multigene marker in patients with early estrogen receptor-positive breast cancer

Clinicopathological and molecular factors determine the prognosis of breast cancer. PRO_10 is a prognostic score based on quantitative RT-PCR of 10 proliferation-associated genes obtained from formalin-fixed, paraffin-embedded breast cancer tissues. We revalidated PRO_10 in patients treated in a non-trial setting.