Clonal spread of a vancomycin-resistant Enterococcus faecium strain among bloodstream-infecting isolates in Italy

Recent data indicated that the rate of vancomycin resistance in bloodstream-infecting enterococcal isolates in Italy is one of the highest in Europe. The aims of this study were to characterize bloodstream-infecting vancomycin-resistant enterococci (VRE) obtained from various Italian hospitals and to establish whether the isolates were clonally related. During the years 2001 to 2003, a total of 39 VRE isolates were obtained from 19 hospital laboratories in various areas of Italy. Species identification and resistance genotypes of the isolates were obtained by multiplex PCR. Further characterization included antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, detection of virulence genes (esp and hyl), and multilocus sequence typing (MLST) of selected isolates. VRE were identified as 31 Enterococcus faecium (VREfm) isolates and 8 E. faecalis isolates. All but one isolate carried the vanA gene; one VREfm isolate carried the vanB gene. Analysis of the PFGE profiles showed that 28 VREfm isolates shared a similar electrophoretic profile, designed type 1, and were clonally related. All type 1 isolates were resistant to ampicillin, streptomycin, gentamicin, and rifampin and were positive for the esp gene. MLST identified an allelic profile (ST78) comprising purK allele 1, belonging to the C1 clonal lineage, characteristic of human infection and hospital outbreak isolates. The vanB-carrying VREfm isolate, of PFGE type 2, was shown to be a single-locus variant of ST78. Our data indicate that the recent increase in the number of bloodstream infections caused by VRE in Italy is due to the spread of a hospital-adapted, multidrug-resistant VREfm clone belonging to an internationally disseminated lineage.     

Analysis of the binding of peanut agglutinin (PNA) to leukaemic cells and its relationship to T-cell differentiation.

Several leukaemias have been screened with a panel of monoclonal antibodies as well as fluoresceinated peanut lectin (FITC-PNA). Approximately 25% of T-acute lymphoblastic leukaemias (T-ALLs) were strongly positive with FITC-PNA. The staining distribution pattern did not correlate with any other monoclonal antibody used, although the phenotypes of the PNA+ T-ALLs were similar to those found on cortical thymocytes and probably reflect a more mature cellular phenotype within the T-ALL group. Some myeloid leukaemias were also PNA+ although the staining was generally weak. Several T-cell lines were examined and generally the TdT- lines showed strongest fluorescence after incubation with FITC-PNA. If these lines were induced to differentiate with 12-O-tetradecanoyl phorbol 13-acetate (TPA) they became PNA-. This was accompanied by an increase in cellular sialyl transferase activity, suggesting that one step in the differentiation process of "early' T cells is the terminal sialylation of existing oligosaccharide chains. Metabolic labelling of PNA+ T-cell lines with [35S]-methionine followed by detergent lysis and affinity chromatography on PNA-agarose showed that several bands of molecular weights 40-100,000 were bound to the column when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. If TPA-treated cells were examined these bands were absent.     

Selected RD1 Peptides for Active Tuberculosis Diagnosis: Comparison of a Gamma Interferon Whole-Blood Enzyme-Linked Immunosorbent Assay and an Enzyme-Linked Immunospot Assay

We recently set up a gamma interferon (IFN-γ) enzyme-linked immunospot assay (ELISPOT), using selected early secreted antigenic target 6 (ESAT-6) peptides, that appears specific for active tuberculosis (A-TB). However, ELISPOT is difficult to automate. Thus, the objective of this study was to determine if the same selected peptides may be used in a technique more suitable for routine work in clinical laboratories, such as whole-blood enzyme-linked immunosorbent assay (WBE). For this purpose, 27 patients with A-TB and 41 control patients were enrolled. Our WBE, using the already described selected peptides from ESAT-6 plus three new ones from culture filtrate protein 10, was performed, and data were compared with those obtained by ELISPOT. Using our selected peptides, IFN-γ production, evaluated by both WBE and ELISPOT, was significantly higher in patients with A-TB than in controls (P < 0.0001). Statistical analysis showed a good correlation between the results obtained by WBE and ELISPOT (r = 0.80, P < 0.001). To substantiate our data, we compared our WBE results with those obtained by QuantiFERON-TB Gold, a whole-blood assay based on region of difference 1 (RD1) overlapping peptides approved for TB infection diagnosis. We observed a slightly higher sensitivity with QuantiFERON-TB Gold than with our WBE (89% versus 81%); however, our test provided a better specificity result (90% versus 68%). In conclusion, results obtained by WBE based on selected RD1 peptides significantly correlate with those generated by ELISPOT. Moreover, our assay appears more specific for A-TB diagnosis than QuantiFERON-TB Gold, and thus it may represent a complementary tool for A-TB diagnosis for routine use in clinical laboratories.     

Effects of air pollution on cup a 3 allergen in Cupressus arizonica pollen grains.

BACKGROUND: Cupressaceae is a family of plants resistant to airborne contamination, and its pollen is the main cause of winter allergic respiratory diseases, especially in North America, Japan, and Mediterranean countries. Recently, a major allergen from Cupressus arizonica pollen grains, Cup a 3, was cloned and expressed. OBJECTIVE: To study the effects of air pollution on the expression of Cup a 3, a thaumatinlike protein, in C. arizonica pollen grains using a combination of transmission electron microscopy and immunocytochemical techniques. METHODS: Observations were made in mature and hydrated C. arizonica pollen grains from various regions in Spain with different degrees of air pollution. Specimens were fixed using freezing protocols, and ultrathin sections were incubated with anti-rCup a 3 rabbit polyclonal antibodies. RESULTS: Labeling of Cup a 3 was detected in mature and hydrated C. arizonica pollen grains. It was more intense in pollen from polluted air regions, and abundant gold particles were observed as they were released through the pollen grain walls. Furthermore, gold particles remained abundant in the pollen cytoplasm. The labeling was noticeably lower in pollen grains from unpolluted air regions. CONCLUSIONS: Cup a 3 is present in the cytoplasm and walls of cypress pollen grains during the air dispersion and hydration stages. The abundance of Cup a 3 in pollen grains under polluted air conditions indicates that these cypresses intensify their activity as a defense from environmental pollution, thus strengthening their allergenicity.     

Antibody for detecting p53 protein by immunohistochemistry in normal tissues.

AIMS--To establish whether PAb248 recognises human p53 as well as murine p53 and if so, to determine its distribution in normal tissues. METHODS--The ability of PAb248 to recognise human p53 was established by analysis of the human osteosarcoma derived Saos-2 cell line, which lacks the p53 gene, before and after transfection with p53 cDNA, using western blotting and immunoprecipitation. Immunostaining on normal tissues and cell lines was carried out using an immunoperoxidase technique. The two anti-p53 antibodies PAb 240 and DO-7 were used as controls. RESULTS--The anti-p53 PAb248 monoclonal antibody stained the Saos-2 cell line after, but not before, transfection with p53 cDNA. Both western blots and immunoprecipitations performed with this antibody revealed a 53,000 molecular weight band. With immunostaining, this antibody detects p53 protein in most lymphoid and human epithelial cells in a cytoplasmic-perinuclear localisation that has not been described before. In the same tissues nuclear staining could be seen in a few scattered cells using the PAb240 antibody. The topographical distribution of wild type p53 was not related to proliferating areas but, rather, to short-lived populations of cells. CONCLUSIONS--Immunostaining of wild type p53 is demonstrable not only in its nuclear form using antibody PAb240 but also in it common cytoplasmic-perinuclear localisation in normal tissues using the PAb248 monoclonal antibody. This opens up new possibilities for its study in both physiological and pathological conditions.     

Inferring the potential success of pneumococcal vaccination in Italy: serotypes and antibiotic resistance of Streptococcus pneumoniae isolates from invasive diseases

To evaluate the potential impact of antipneumococcal vaccination in Italy, Streptococcus pneumoniae isolates from invasive disease were collected from 65 laboratories in the years 1997-2000. Of the 503 isolates examined, 15% were from children <5 years and 34% from adults > or = 65 years. The most frequent serogroups were, in ranking order, 14, 19, 6, and 23. Overall, 93.8% of the isolates belonged to serogroups enclosed in the 23-valent polysaccharide vaccine. Among children isolates, serotypes 14, 6B, and 23F comprised 60% of the isolates; overall, 72% of the isolates belonged to serotypes included in the heptavalent conjugate vaccine. Penicillin nonsusceptible isolates (10%) belonged to a limited number of serogroups, being more common in serogroups 19 and 9 and in the nonvaccine serogroups 24 and 35. Erythromycin-resistant isolates (29%) belonged to several serogroups, more frequently to serogroups 14, 6, and 19. Both vaccines are potentially able to prevent the majority of resistant infections in the respective age groups in Italy.     

MRE11 mutations and impaired ATM-dependent responses in an Italian family with ataxia-telangiectasia-like disorder

Hypomorphic mutations of the MRE11 gene are the hallmark of the radiosensitive ataxia-telangiectasia-like disorder (ATLD). Here, we describe a new family with two affected siblings, ATLD5 and ATLD6, now aged 37 and 36, respectively. They presented with late onset cerebellar degeneration slowly progressing until puberty and absence of telangiectasias, and were cancer-free. Both patients were wild-type for ATM and NBS1, but compound heterozygotes for MRE11 gene mutations [1422C-->A, T481K; 1714C-->T, R571X]. The 1422C-->A allele was inherited from the mother, whereas the 1714C-->T, allele paternally inherited, was apparently null as a result of nonsense-mediated mRNA decay (NMD). Interestingly, the 1714C-->T mutation is the same as previously identified in an unrelated English ATLD family (probands ATLD3 and ATLD4), suggesting an important role for NMD in saving potentially lethal mutations. Lymphoblastoid cell lines (LCLs) derived from ATLD5 and ATLD6 were normal for ATM, but defective for Mre11, Rad50 and Nbs1 (the MRN complex) protein expression. Their response to gamma-radiation was abnormal, as evidenced by the enhanced radiosensitivity, attenuated autophosphorylation of ATM-S1981 and phosphorylation of the ATM targets p53-S15 and Smc1-S966, failure to form Mre11 nuclear foci and defective G1 checkpoint arrest. The fibroblasts, but not LCLs, from ATLD5 and ATLD6 showed an impaired ATM-dependent Chk2 phosphorylation. These findings further underscore the interconnection between ATM activity and MRN function, which rationalizes the clinical similarity between ataxia-telangiectasia (A-T) and ATLD.     

Allergenic proteins in Urtica dioica, a member of the Urticaceae allergenic family.

BACKGROUND: Allergy to the pollen of flowering plant species significantly affects the health of people in many parts of the world. Pollens of related genera usually share common antigens and are often, but not always, cross-reactive. Several studies have shown that Parietaria pollen is one of the most common causes of pollinosis in the Mediterranean area, whereas Urtica has no allergenic significance. OBJECTIVES: To report on the localization of Parietaria judaica major allergen in Urtica dioica pollen grains and on the detection of allergenic proteins in U. dioica pollen grains during the hydration-activation process. METHODS: A combination of transmission electron microscopy and immunocytochemical methods was used to locate allergenic proteins in U. dioica pollen grains after different periods of hydration-activation using the anti-Par j 1 (4.1.3.) monoclonal antibody and serum samples from allergic patients. RESULTS: No significant labeling was noted for Parj 1 allergen after 10, 15, and 20 minutes in the walls and cytoplasm. Slight labeling was observed for allergic proteins in the walls of U. dioica after 10 minutes of hydration, and no significant labeling was found after 15 and 20 minutes of hydration. CONCLUSIONS: Immunocytochemical methods confirmed the absence of cross-reactivity between 2 related genera, Parietaria and Urtica, and the lowest allergenic potential of U. dioica.