Vincenzo Falbo

Cytoskeletal changes induced in HEp-2 cells by the cytotoxic necrotizing factor of Escherichia coli

The effect of the cytotoxic necrotizing factor of Escherichia coli on HEp-2 cells was studied by fluorescence and scanning electron microscopy. This cytotoxin, known for inducing the formation of giant multinucleated cells in several cell lines, caused changes in actin and tubulin organization. The presence of membrane ruffles at the cell border and of numerous thick bundles of actin crossing the cell body, suggests that the factor promotes cell spreading; this probably interferes with cytokinesis, ultimately leading to the formation of very large flattened multinucleated cells. Moreover, the nuclear segmentation observed in treated cells seems to be associated with a rearrangement of actin in the perinuclear region and with the presence of tubulin bundles in proximity to nuclear clefts. Although the primary target is still unknown, these findings suggest that the cytoskeleton is affected accounting for the multinucleation process induced by the factor.

A two-year study of enteric infections associated with diarrhoeal diseases in children in urban Somalia.

A hospital-based systematic sample of 1667 children with severe diarrhoeal disease was studied in Mogadishu, Somalia, throughout 1983 and 1984. One or more enteric pathogens were found in 61% of the patients. Rotavirus (25%), enterotoxigenic Escherichia coli (11%), Shigella spp. (9%), Aeromonas hydrophila (9%), Giardia lamblia trophozoites (8%), Campylobacter jejuni (8%), and Vibrio cholerae non-O1 (6%) were the most frequently identified pathogens. Age-specific detection rates of enteric pathogens and helminths, seasonal patterns, and relationship of some specific infections with feeding status and main clinical features have been defined for all the sample examined.

A cell division-active protein from E. coli

A purification procedure for a protein obtained from an pathogenic strain of E. coli is described. The protein-called CNF-is active in inhibiting the duplication of cultured mammalian cells. Since nuclei division is apparently normal, treatment of cultured cells with CNF leads to the formation of gigantic, polynucleated cells. The purified protein is chromatographically and electrophoretically homogeneous. A partial characterization of CNF protein is also given.

Detection of enteroadherent Escherichia coli associated with diarrhoea in Italy

One hundred and sixty-eight isolates of Escherichia coli obtained in Italy from 112 children with diarrhoea and 56 age-matched controls were examined by the HEp-2 cell adhesion assay. Sixteen strains showed localised adherence (LA), 29 showed diffuse adherence (DA) and eight strains showed aggregative adherence (AA). No adhesion pattern was significantly associated with disease. Strains that showed LA or AA were further characterised by serotyping, fluorescent actin staining (FAS) test and hybridisation with the EPEC adherence factor (EAF), E. coli attaching and effacing (eae) and enteroaggregative (EAgg) DNA probes. Strains that showed poor LA were FAS-negative, did not belong to EPEC serotypes and did not hybridise with EPEC probes. Conversely, the two strains that showed a good LA pattern belonged to serotype O128:H2, were FAS positive and hybridised with the eae probe. No isolate hybridised with the EAF probe. Only three of the eight strains with the AA pattern hybridised with the EAgg probe. Probe positivity correlated with the ability to produce clumps at the surface of the liquid culture and to agglutinate rat erythrocytes. In two of these EAgg probe-positive strains, electronmicroscopy revealed the presence of fibrillar bundles which seem to mediate bacterial aggregation.

Quality assessment in cytogenetic and molecular genetic testing: the experience of the Italian Project on Standardisation and Quality Assurance.

Abstract The first Italian national trial of external quality assessment in genetic testing was organised within the framework of the “Italian National Project for Standardisation and Quality Assurance of Genetic Tests”. Sixty-eight Public Health Service laboratories volunteered for the trial, which involved molecular genetic tests (cystic fibrosis, β-thalassaemia, familial adenomatous polyposis coli and fragile-X syndrome) and cytogenetic tests (prenatal and postnatal, the latter included cancer cytogenetics). The response rate was high (88.2%). The level of analytical accuracy was good, i.e., the percentage of laboratories that correctly genotyped all samples was 89.3% for cystic fibrosis, 90.9% for β-thalassaemia, 100% for familial adenomatous polyposis coli (despite two laboratories did not complete the analysis because the amount of DNA was considered insufficient), and 90.5% for fragile-X syndrome. Written reports differed widely and were judged “inadequate” in over 50% of cases. Most laboratories from the present study already have experience in previous European external quality assessments for at least one genetic test; this can explain the higher analytical accuracy in the Italian external quality assessment with respect to quality control programmes in other countries. Collaborative networks are strongly suggested to improve the quality of the reports.

Toxin production and haemagglutination in strains of Escherichia coli from diarrhoea in Brescia, Italy

Two hundred and ninety-nine different strains of Escherichia coli, isolated from 172 patients with diarrhoea and 113 healthy subjects, were examined for enterotoxin, cytotoxin and haemolysin (Hly) production and for mannose-resistant haemagglutination (MRHA) and invasive properties. Three strains proved enterotoxigenic, none enteroinvasive; cytotoxin and Hly production was shown in 25 strains from patients and in 3 from controls. Ten strains produced the cytotoxic necrotizing factor (CNF), 6 released other factors which kill cell cultures. Hly production was shown in 21 strains, 9 of which were also positive for CNF. MRHA was detected in 26% of strains from diarrhoea compared with 14% of strains from healthy people. A strong association between toxin production and MRHA was demonstrated. Serotyping results showed that the strains exhibiting virulence traits mostly belonged to serogroups commonly involved in extra-intestinal infections. The possible role of strains of E. coli showing one or more virulence factors as opportunistic pathogens in diarrhoeal diseases is discussed.

Detection of Clostridial Toxins in Stools from Children with Diarrhoea

A cell-culture assay was used to detect toxins directly in stools from sporadic cases of infantile diarrhoea. Cytotoxins were revealed in 11 out of 58 samples from children with diarrhoea, nine of whom had no common enteric pathogens in their stools. A preliminary characterisation of the cytotoxins was obtained by neutralisation tests with clostridial antitoxins.

Organization and integration sites in the human genome of endogenous retroviral sequences belonging to HERV-E family

Abstract. The present paper reports the characterization of HERV-E endogenous retroviral sequences in the human genome by using three complementary approaches. Firstly, we identified genomic clones containing HERV-E by BLAST screening of human DNA databases by using the entire sequence of a characterized HERV-E clone (M10976) as a query. The genomic structure and integration sites of the HERV-E elements were characterized. Secondly, new integration sites of HERV-E elements were revealed by a retroviral LTR-arbitrary primer-PCR (RELAP-PCR) technique. BLAST analysis of the PCR products identified a subgroup that shows low identity (75%) to the original clone M10976 and slightly higher identity (80%) to a closely related HERV-E (Ac. n. K02166). Finally, we performed FISH mapping, which revealed sites of integration of HERV-E not previously identified at the cytogenetic level. A preliminary analysis of genes mapping in the same bands as HERV-E integration sites was performed: several loci relevant to physiological and/or pathological processes were detected. Our findings may provide clues to identify HERV-E integration sites adjacent to genes with important biological roles.

The Italian External Quality Assessment Scheme for Fragile X Syndrome: The Results of a 5-Year Survey

The Italian External Quality Assessment scheme for fragile X syndrome started in 2001 as an activity funded by the National Health System and coordinated by the National Institute of Public Health. The aim of this work is to present the data of 5 years (2001--2004 and 2006) of survey. The External Quality Assessment scheme was designed to cover the following points: (a) genotyping and (b) interpretation and reporting of results. Overall, the scheme covered about 65% of all Italian public laboratories. The average reporting of results was 91.6%, with an overall success rate of 76%. The rate of diagnostic errors observed was on average 5%. Inaccuracy in sizing of CGG repeats of normal and premutated alleles was reported. During the survey the proportion of laboratories using a Southern blotting, polymerase chain reaction, and ABI sizing kit in combination rose from 36.8% to 70.6%. The reports from laboratories showed incompleteness and considerable variations in expected outcomes. For this reason, in 2004 a model for written reports was introduced. In conclusion, these data underscore the need to participate in External Quality Assessment schemes as an educational resource to ensure quality in molecular genetic testing.

Three cases of rare salivary gland tumours: a molecular study of TP53, CDKN2A/ARF, RAS, BRAF, PTEN, MAPK2 and EGFR genes.

Salivary gland tumours are rare tumours characterized by histopathologic complexity and a wide variety of morphologic features. Studies on genetic changes in different histological subtypes of salivary gland tumours are important to better understand molecular pathogenetic mechanisms and to identify diagnostic and prognostic markers. Data are even more scanty dealing with unusual subtypes of these tumours. The aim of the present study was to analyse two high grade transformation adenoid cystic carcinomas (hgACC) and one hybrid tumour in order to identify, by mutational and microsatellite analysis, genetic alterations in TP53, CDKN2A/ARF, RAS, BRAF, PTEN, MAPK2 and EGFR genes. The two hgACCs showed snps missense in RAS genes and alterations with allelic instability in CDKN2A/ARF; moreover, a double mutation in TP53 was detected in one case. The hybrid tumour showed alterations in CDKN2A/ARF gene and snps missense in NRAS genes. Our data suggest that CDKN2A/ARF pathway might be involved in pathogenesis of the salivary gland tumours analysed. Further molecular analyses of these very rare tumours are necessary to better understand the role of other genetic alterations detected in our study.