Paola Valentini

Down regulation of NM23.H1, NM23.H2 and c-myc genes during differentiation induced by 1,25 dihydroxyvitamin D3.

The NM23 gene, involved in the negative regulation of metastatic progression, has been found to be highly homologous to developmentally regulated genes such as the awd gene in Drosophila melanogaster and the Gip17 gene in Dyctiostelium discoideum. To ascertain whether the NM23 genes are involved in the differentiation processes of human cell lines, the NM23.H1 and NM23.H2 expression level has been determined during the monocyte-macrophage differentiation of HL-60 and U-937 cell lines induced by vitamin D3. In both lines, vitamin D3 produced induction of differentiative markers, inhibition of cell proliferation and a decrease of the NM23.H1, NM23.H2 and c-myc genes, behaving both as a differentiative and an antiproliferative agent. The fact that the c-myc transcriptional factor PuF is identical to the NM23.H2 gene and that NM23 protein could be a transcriptional factor suggests that the regulatory action exerted by vitamin D3 on c-myc transcription is mediated by NM23.H2.

Long-Term Effects of Hospital Water Network Disinfection on Legionella and Other Waterborne Bacteria in an Italian University Hospital

OBJECTIVE AND DESIGN Legionella control still remains a critical issue in healthcare settings where the preferred approach to health risk assessment and management is to develop a water safety plan. We report the experience of a university hospital, where a water safety plan has been applied since 2002, and the results obtained with the application of different methods for disinfecting hot water distribution systems in order to provide guidance for the management of water risk. INTERVENTIONS The disinfection procedures included continuous chlorination with chlorine dioxide (0.4-0.6 mg/L in recirculation loops) reinforced by endpoint filtration in critical areas and a water treatment based on monochloramine (2-3 mg/L). Real-time polymerase chain reaction and a new immunoseparation and adenosine triphosphate bioluminescence analysis were applied in environmental monitoring. RESULTS After 9 years, the integrated disinfection-filtration strategy significantly reduced positive sites by 55% and the mean count by 78% (P < .05); however, the high costs and the occurrence of a chlorine-tolerant clone belonging to Legionella pneumophila ST269 prompted us to test a new disinfectant. The shift to monochloramine allowed us to eliminate planktonic Legionella and did not require additional endpoint filtration; however, nontuberculous mycobacteria were isolated more frequently as long as the monochloramine concentration was 2 mg/L; their cultivability was never regained by increasing the concentration up to 3 mg/L. CONCLUSIONS Any disinfection method needs to be adjusted/fine-tuned in individual hospitals in order to maintain satisfactory results over time, and only a locally adapted evidence-based approach allows assessment of the efficacy and disadvantages of the control measures.

Idarubicin is active on MDR cells : evaluation of DNA synthesis inhibition on P388 cell lines

SummaryMultidrug resistance is frequently found in patients affected by hematological malignancies and has been related to a poor prognosis of acute leukemia. In the present paper we report results concerning the activity of idarubicin, an anthracycline derivative, on the leukemic P388 and P388 doxorubicin-resistant cell lines. The results clearly show that idarubicin inhibits DNA synthesis in the resistant cell line more actively than doxorubicin.

GST-7T and P-170 co-expression in multiple myeloma

Summary. Bone marrow samples from 40 patients affected by multiple myeloma either treated or untreated were examined for expression of glutathione‐S‐transferase n (GST‐TT), P‐glycoprotein and the protein product of ras oncogenes family, p‐21, on plasma cells, by immuno‐cytochemical detection. 72% of evaluated samples were positive for P‐170 and 82% for GST‐7T without any correlation with clinical or prognostic parameters. A significant relationship between GST‐7T expression and P‐l 70 positivity was found and co‐expression was observed in 91% of evaluated samples.

Comparison of two molecular methods used for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply.

The results of the pulsed-field gel electrophoresis and the sequence-based typing (using the loci flaA, pilE, asd, mip, mompS and proA) were compared for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply. Molecular typing was carried out on 61 isolates (38% of the positive samples) selected on space and temporal criteria in order to follow the evolution of the water-system colonization. For all the 61 isolates, the sequence of the amplified mip gene fragment identified Legionella pneumophila strain Wadsworth. Genotype testing by PFGE analysis showed three different patterns, correspondent to three SBT types according to the allelic profiles. Both PFGE and SBT indicated the circulation and the persistence in the hospital potable water-system of three types randomly distributed in space and time. The two molecular methods adopted showed a 100% concordance, although a low degree of genetic heterogeneity characterized the isolates. The electrophoretic patterns were sufficiently unambiguous to consider PFGE a highly discriminatory typing method, but the SBT technique besides accurately characterizing isolates, was able to identify Legionella strains through analysis of the mip gene. A typing method with this level of discriminatory power has great potential for assisting in epidemiological studies.

Binding of GC (VDBP) to membranes of human B lymphocytes following stripping of extant protein.

The presence of Gc (vitamin D binding protein) has been consistently demonstrated on the membrane of B lymphocytes. This protein appears to be spatially associated with surface immunoglobulins. The origin of this surface protein has not yet been determined and the purpose of the present paper was to investigate if Gc may bind to human lymphocytes after immunoglobulin (Ig) capping. For this purpose the presence of Gc on B lymphocytes was examined by three different approaches. First, when cells were examined by immunofluorescence and quantified by flow cytometry, membrane Ig capping was followed by a dramatic decrease in positivity for Gc when compared to native cells. In addition, incubation of capped cells with purified Gc was followed by a significant increase in fluorescence, indicating that this protein had been able to bind again. Second, analysis of solubilized lymphocytes by Western blotting showed that native lymphocytes and capped cells incubated with purified Gc contained a large quantity of a 56kDa protein which was immunoreactive with anti Gc antibodies. This protein band was much weaker on blots from capped cells not treated with Gc. Third, radiobinding assays indicated that, following capping, cells were able to bind Gc in saturable fashion. These results suggest that membrane Gc could play a role in the entry of vitamin D metabolites into lymphocytes.

Evaluation of a modified cleaning procedure in the prevention of carbapenem-resistant Acinetobacter baumannii clonal spread in a burn intensive care unit using a high-sensitivity luminometer

BACKGROUND Enhanced environmental cleaning practices are among the most accepted measures for controlling the spread of carbapenem-resistant Acinetobacter baumannii (CR-Ab). AIM To evaluate the impact of heightened cleaning on an ongoing CR-Ab outbreak in a burn intensive care unit (BICU) of an Italian teaching hospital, where chlorhexidine-60% isopropyl alcohol was applied as a complementary disinfectant on high-touch surfaces. METHODS Compliance with the microbial limit proposed for the BICU by AFNOR-NF-S90-351 (20 colony-forming units/100cm2) was assessed by plate count, and compared with the results obtained with intracellular adenosine triphosphate (ATP) detection. Genotyping was performed using pulsed-field gel electrophoresis. FINDINGS During the standard cleaning regimen, three out of 23 samples (13%) gave results over the AFNOR limit and five (21.7%) showed unacceptable ATP levels with 100 relative light units/100cm2 as the benchmark limit (sensibility 86.4%, specificity 92.2%). Following improvement of the cleaning procedure, only two samples out of 50 (4%) did not satisfy the microbiological criteria and seven (14%) exceeded the ATP limit. In a successive phase, eight of 30 samples collected showed unacceptable results (27%). CONCLUSIONS Adding chlorhexidine-60% isopropyl alcohol as complementary disinfectant proved to be effective for reducing environmental microbial contamination, ATP levels and CR-Ab infection/colonization in patients admitted to the BICU. Real-time monitoring by ATP assay was useful for managing the cleaning schedule and reducing hospital infections, although the calculated values must be interpreted as cleanliness indicators rather than risk indicators.

Colonization by Legionella spp. of water networks in residential buildings of the Province of Pisa, Italy

BACKGROUND Despite the increase of community acquired cases of legionellosis in Italy over the last years, the Italian guidelines do not give indications for prevention and control of Legionella in the hot water networks (or centralized conditioning systems) of residential buildings. We performed a survey on eight medium sized apartment buildings in the Pisa district to assess the prevalence of Legionella spp. in the water network and the respondance to drinking water requisites at the point of use, according to the Italian norms. METHODS For each building two hot water and three cold water samples (located at water entrance from the aqueduct network into the building pipework, at the exit from pressure autoclave, and at a remote tap) were collected. RESULTS Legionella was detected in 20% of residential buildings, mostly in those with a central hot water production system. CONCLUSIONS The study highlights a condition of potential risk for susceptible population subgroups and supports the need for measures of risk assessment and control.

Is binding of vitamin D binding protein related to cell differentiation

Vitamin D binding protein (Gc) is present on the surface of several blood cells and may interfere with the activity of 1,25(OH)2D3. It has previously been reported that Gc may bind to the U-937 line which is known to differentiate upon exposition to 1,25(OH)2D3. In the present paper, we evaluate the expression of Gc on the surface of U-937 and HL-60 lines. Both cell lines did not express Gc on their surface but U-937 cells were able to bind human purified Gc added to the medium whereas HL-60 were not. After culturing with 1,25(OH)2D3, HL-60 became able to bind Gc. This property seems to be related to the monocytic differentiation induced by 1,25(OH)2D3. Conversely, when present together, 1,25(OH)2D3 reduces binding on U-937.

Application of Hydrogen Peroxide as an Innovative Method of Treatment for Legionella Control in a Hospital Water Network

Objectives: To evaluate the effectiveness of hydrogen peroxide (HP) use as a disinfectant in the hospital water network for the control of Legionella spp. colonization. Methods: Following the detection of high levels of Legionella contamination in a 136-bed general hospital water network, an HP treatment of the hot water supply (25 mg/L) was adopted. During a period of 34 months, the effectiveness of HP on Legionella colonization was assessed. Legionella was isolated in accordance with ISO-11731 and identification was carried out by sequencing of the mip gene. Results: Before HP treatment, L. pneumophila sg 2–15 was isolated in all sites with a mean count of 9950 ± 8279 cfu/L. After one-month of HP treatment, we observed the disappearance of L. pneumophila 2–15, however other Legionella species previously not seen were found; Legionella pneumophila 1 was isolated in one out of four sampling sites (2000 cfu/L) and other non-pneumophila species were present in all sites (mean load 3000 ± 2887 cfu/L). Starting from September 2013, HP treatment was modified by adding food-grade polyphosphates, and in the following months, we observed a progressive reduction of the mean load of all species (p < 0.05), resulting in substantial disappearance of Legionella colonization. Conclusion: Hydrogen peroxide demonstrated good efficacy in controlling Legionella. Although in the initial phases of treatment it appeared unable to eliminate all Legionella species, by maintaining HP levels at 25 mg/L and adding food-grade polyphosphates, a progressive and complete control of colonization was obtained.