Indagine sulla presenza di Helicobacter pullorum in allevamenti avicoli italiani

From September 2005 to December 2006, in order to define the prevalence of Helicobacter pullorum in broiler chickens, laying hens and turkey, a total of 365 caecum contents of animals reared in 76 different farms were collected at the slaughterhouse. A caecum content of a ostrich was also sampled. In addition, with the aim of investigating the occurrence of H. pullorum in humans, 151 faeces were collected at the Sant’Orsola-Malpighi University Hospital of Bologna from patients suffering of gastroenteritis. A modified Steele–McDermott membrane filter method was used. Gram-negative curved rod bacteria were preliminary identified as H. pullorum by a PCR assay based on 16S rRNA, then subjected to a RFLP-PCR assay to distinguish between H. pullorum and H. canadensis. One isolate from each farm was randomly selected for phenotypic characterization by biochemical methods and 1D SDSPAGE analysis of whole cell proteins profiles. Minimum Inhibitory Concentration (MIC) for seven different antibiotics were also determined by agar dilution method. Moreover, to examine the intraspecific genomic variability, two strains isolated from 17 different farms were submitted to genotyping by Pulse-Field Gel Electrophoresis (PFGE). In order to assess the molecular basis of fluorquinolone resistance in H. pullorum, gyrA of H. pullorum CIP 104787T was sequenced and nucleotide sequences of the Quinolone Resistance Determining Region (QRDR) of a total of 18 poultry isolates, with different MIC values for ciprofloxacin and nalidixic acid, were compared. According to the PCR and PCR-RFLP results, 306 out of 366 animals examined were positive for H. pullorum (83,6%) and 96,1% of farms resulted infected. All positive samples showed a high number of colonies (>50) phenotipically consistent with H. pullorum on the first isolation media, which suggests that this microrganism, when present, colonizes the poultry caecum at an elevate load. No human sample resulted positive for H. pullorum. The 1D SDS-PAGE whole protein profile analysis showed high similarity among the 74 isolates tested and with the type strain H. pullorum CIP 104787T. Regarding the MIC values, a monomodal distribution was found for ampicillin, chloramphenicol, gentamicin and nalidixic acid, whereas a bimodal trend was noticed for erythromycin, ciprofloxacin and tetracycline (indicating an acquired resistance for these antibiotics). Applying the breakpoints indicated by the CSLI, we may assume that all the H. pullorum tested are sensitive only to gentamicin. The intraspecific genomic variability observed in this study confirm that this species don’t have a clonal population structure, as motioned by other autors. The 2490 bp gyrA gene of H. pullorum CIP104787T with an Open Reading Frame (ORF) encoding a polypeptide of 829 amino acids was for the first time sequenced and characterized. All ciprofloxacin resistant poultry isolates showed ACA®ATA (Thr®Ile) substitution at codon 84 of gyrA corresponding to codons of gyrA 86, 87 and 83 of the Campylobacter jejuni, H. pylori and Escherichia coli, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin resistant phenotype of poultry isolates. This is the first report of isolation of H. pullorum in turkey and in ostrich, indicating that poultry species are the reservoir of this potential zoonotic microorganisms. In order to understand the potential role as food-borne human pathogen of H. pullorum, further studies must be carried on.

Bioimaging of animal embryogenesis: mathematical methods and computational algorithms

Some fundamental biological processes such as embryonic development have been preserved during evolution and are common to species belonging to different phylogenetic positions, but are nowadays largely unknown. The understanding of cell morphodynamics leading to the formation of organized spatial distribution of cells such as tissues and organs can be achieved through the reconstruction of cells shape and position during the development of a live animal embryo. We design in this work a chain of image processing methods to automatically segment and track cells nuclei and membranes during the development of a zebrafish embryo, which has been largely validates as model organism to understand vertebrate development, gene function and healingrepair mechanisms in vertebrates. The embryo is previously labeled through the ubiquitous expression of fluorescent proteins addressed to cells nuclei and membranes, and temporal sequences of volumetric images are acquired with laser scanning microscopy. Cells position is detected by processing nuclei images either through the generalized form of the Hough transform or identifying nuclei position with local maxima after a smoothing preprocessing step. Membranes and nuclei shapes are reconstructed by using PDEs based variational techniques such as the Subjective Surfaces and the Chan Vese method. Cells tracking is performed by combining informations previously detected on cells shape and position with biological regularization constraints. Our results are manually validated and reconstruct the formation of zebrafish brain at 7-8 somite stage with all the cells tracked starting from late sphere stage with less than 2% error for at least 6 hours. Our reconstruction opens the way to a systematic investigation of cellular behaviors, of clonal origin and clonal complexity of brain organs, as well as the contribution of cell proliferation modes and cell movements to the formation of local patterns and morphogenetic fields.

Study of two novel streptococcus pneumoniae operons: pilus islet 2 and the lantibiotic operon

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element resembling gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (containing the genes pitA, sipA, pitB, srtG1, and srtG2) codes for a novel functional pilus in pneumococcus. Therefore, there are two pilus islets identified so far in this pathogen (PI-1 and PI-2). Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. PI-2 is associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging in both industrialized and developing countries. Interestingly, strains belonging to clonal complex 271 (CC271) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that may play a role in the initial host cell contact to the respiratory tract. In addition, the pilus proteins are potential antigens for inclusion in a new generation of pneumococcal vaccines. Adherence by pili could represent important factor in bacterial community formation, since it has been demonstrated that bacterial community formation plays an important role in pneumococcal otitis media. In vitro quantification of bacterial community formation by S. pneumoniae was performed in order to investigate the possible role of pneumococcal pili to form communities. By using different growth media we were not able to see clear association between pili and community formation. But our findings revealed that strains belonging to MLST clonal complex CC15 efficiently form bacterial communities in vitro in a glucose dependent manner. We compared the genome of forty-four pneumococcal isolates discovering four open reading frames specifically associated with CC15. These four genes are annotated as members of an operon responsible for the biosynthesis of a putative lanctibiotic peptide, described to be involved in bacterial community formation. Our experiments show that the lanctibiotic operon deletion affects glucose mediated community formation in CC 15 strain INV200. Moreover, since glucose consumption during bacterial growth produce an acidic environment, we tested bacterial community formation at different pH and we showed that the lanctibiotic operon deletion affected pH mediated community formation in CC 15 strain INV200. In conclusion, these data demonstrate that the putative lanctibiotic operon is associated with pneumococcal CC 15 strains in vitro bacterial community formation.

Genetic analysis of tumour initiation and progression in a Drosophila model of epithelial cancer

Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. The molecular crosstalk occurring between precancerous and normal cells strongly influences the early steps of the tumourigenic process as well as later stages of the disease. Precancerous cells are often removed by cell death from normal tissues but the mechanisms responsible for such fundamental safeguard processes remain in part elusive. To gain insight into these phenomena I took advantage of the clonal analysis methods available in Drosophila for studying the phenotypes due to loss of function of the neoplastic tumour suppressor lethal giant larvae (lgl). I found that lgl mutant cells growing in wild-type imaginal wing discs are subject to the phenomenon of cell competition and are eliminated by JNK-dependent cell death because they express very low levels of dMyc oncoprotein compared to those in the surrounding tissue. Indeed, in non-competitive backgrounds lgl mutant clones are able to overgrow and upregulate dMyc, overwhelming the neighbouring tissue and forming tumourous masses that display several cancer hallmarks. These phenotypes are completely abolished by reducing dMyc abundance within mutant cells while increasing it in lgl clones growing in a competitive context re-establishes their tumourigenic potential. Similarly, the neoplastic growth observed upon the oncogenic cooperation between lgl mutation and activated Ras/Raf/MAPK signalling was found to be characterised by and dependent on the ability of cancerous cells to upregulate dMyc with respect to the adjacent normal tissue, through both transcriptional and post-transcriptional mechanisms, thereby confirming its key role in lgl-induced tumourigenesis. These results provide first evidence that the dMyc oncoprotein is required in lgl mutant tissue to promote invasive overgrowth in developing and adult epithelial tissues and that dMyc abundance inside versus outside lgl mutant clones plays a key role in driving neoplastic overgrowth.

Epidemiology and population genetics of Podosphaera fusca and Golovinomyces orontii, causal agents of cucurbit powdery mildew

In 2010, 2011 and 2012 growing seasons, the occurrence of the ascomycetes Podosphaera fusca and Golovinomyces orontii, causal agents of powdery mildew disease, was monitored on cultivated cucurbits located in Bologna and Mantua provinces to determine the epidemiology of the species. To identify the pathogens, both morphological and molecular identifications were performed on infected leaf samples and a Multiplex-PCR was performed to identify the mating type genes of P. fusca isolates. The investigations indicated a temporal succession of the two species with the earlier infections caused by G. orontii, that seems to be the predominant species till the middle of July when it progressively disappears and P. fusca becomes the main species infecting cucurbits till the end of October. The temporal variation is likely due to the different overwintering strategies of the two species instead of climatic conditions. Only chasmothecia of P. fusca were recorded and mating type alleles ratio tended to be 1:1. Considering that only chasmothecia of P. fusca were found, molecular-genetic analysis were carried out to find some evidence of recombination within this species by MLST and AFLP methods. Surprisingly, no variations were observed within isolates for the 8 MLST markers used. According to this result, AFLP analysis showed a high similarity within isolates, with SM similarity coefficient ranging between 0.91-1.00 and also, sequencing of 12 polymorphic bands revealed identity to some gene involved in mutation and selection. The results suggest that populations of P. fusca are likely to be a clonal population with some differences among isolates probably due to agricultural practices such as fungicides treatments and cultivated hosts. Therefore, asexual reproduction, producing a lot of fungal biomass that can be easily transported by wind, is the most common and useful way to the spread and colonization of the pathogen.

The search for Multiple Myeloma Stem cells: molecular characterization and self-renewal mechanisms involved in the disease persistence

The existence of Multiple Myeloma Stem cells (MMSCs)is supposed to be one of the major causes of MM drug-resistance. However, very little is known about the molecular characteristics of MMSCs, even if some studies suggested that these cells resembles the memory B cells. In order to molecularly characterize MMSCs, we isolated the 138+138- population. For each cell fraction we performed a VDJ rearrangement analysis. The complete set of aberrations were performed by SNP Array 6.0 and HG-U133 Plus 2.0 microarray analyses (Affymetrix). The VDJ rearrangement analyses confirmed the clonal relationship between the 138+ clone and the immature clone. Both BM and PBL 138+ clones showed exactly the same genomic macroalterations. In the BM and PBL 138-19+27+ cell fractions several micro-alterations (range: 1-350 Kb) unique of the memory B cells clone were highlighted. Any micro-alterations detected were located out of any genomic variants region and are presumably associated to the MM pathogenesis, as confirmed by the presence of KRAS, WWOX and XIAP genes among the amplified regions. To get insight into the biology of the clonotypic B cell population, we compared the gene expression profile of 8 MM B cells samples 5 donor B cells vs, thus showing a differential expression of 11480 probes (p-value: <0,05). Among the self-renewal mechanisms, we observed the down-regulation of Hedgehog pathway and the iperactivation of Notch and Wnt signaling. Moreover, these immature cells showed a particular phenotype correlated to resistance to proteasome inhibitors (IRE1α-XBP1: -18.0; -19.96. P<0,05). Data suggested that the MM 138+ clone might resume the end of the complex process of myelomagenesis, whereas the memory B cells have some intriguing micro-alterations and a specific transcriptional program, supporting the idea that these post germinal center cells might be involved in the transforming event that originate and sustain the neoplastic clone.

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.

Next-Generation Sequencing-Based Mutations Scanning Strategy of the BCR-ABL Kinase Domain in Patients with PhiladelPhia-Chromosome Positive Leukemias Treated with Tyrosine Kinase Inhibitors

In chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL kinase domain mutation status is an essential component of the therapeutic decision algorithm. The recent development of Ultra-Deep Sequencing approach (UDS) has opened the way to a more accurate characterization of the mutant clones surviving TKIs conjugating assay sensitivity and throughput. We decided to set-up and validated an UDS-based for BCR-ABL KD mutation screening in order to i) resolve qualitatively and quantitatively the complexity and the clonal structure of mutated populations surviving TKIs, ii) study the dynamic of expansion of mutated clones in relation to TKIs therapy, iii) assess whether UDS may allow more sensitive detection of emerging clones, harboring critical 2GTKIs-resistant mutations predicting for an impending relapse, earlier than SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II (Interlaboratory Robustness of Next-Generation Sequencing) International consortium.Samples from CML and Ph+ ALL patients who had developed resistance to one or multiple TKIs and collected at regular time-points during treatment were selected for this study. Our results indicate the technical feasibility, accuracy and robustness of our UDS-based BCR-ABL KD mutation screening approach. UDS was found to provide a more accurate picture of BCR-ABL KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity and showed that BCR-ABL KD mutations detected by SS are only the “tip of iceberg”. In addition UDS may reliably pick 2GTKIs-resistant mutations earlier than SS in a significantly greater proportion of patients.The enhanced sensitivity as well as the possibility to identify low level mutations point the UDS-based approach as an ideal alternative to conventional sequencing for BCR-ABL KD mutation screening in TKIs-resistant Ph+ leukemia patients

The impact of intraclonal heterogeneity on the outcomes of Multiple Myeloma patients treated with new drugs

Understanding the biology of Multiple Myeloma (MM) is of primary importance in the struggle to achieve a cure for this yet incurable neoplasm. A better knowledge of the mechanism underlying the development of MM can guide us in the development of new treatment strategies. Studies both on solid and haematological tumours have shown that cancer comprises a collection of related but subtly different clones, a feature that has been termed “intra-clonal heterogeneity”. This intra-clonal heterogeneity is likely, from a “Darwinian” natural selection perspective, to be the essential substrate for cancer evolution, disease progression and relapse. In this context the critical mechanism for tumour progression is competition between individual clones (and cancer stem cells) for the same microenvironmental “niche”, combined with the process of adaptation and natural selection. The Darwinian behavioural characteristics of cancer stem cells are applicable to MM. The knowledge that intra-clonal heterogeneity is an important feature of tumours’ biology has changed our way to addressing cancer, now considered as a composite mixture of clones and not as a linear evolving disease. In this variable therapeutic landscape it is important for clinicians and researchers to consider the impact that evolutionary biology and intra-clonal heterogeneity have on the treatment of myeloma and the emergence of treatment resistance. It is clear that if we want to effectively cure myeloma it is of primarily importance to understand disease biology and evolution. Only by doing so will we be able to effectively use all of the new tools we have at our disposal to cure myeloma and to use treatment in the most effective way possible. The aim of the present research project was to investigate at different levels the presence of intra-clonal heterogeneity in MM patients, and to evaluate the impact of treatment on clonal evolution and on patients’ outcomes.