Umberto Moscato

Phenylbutyrate increases SMN gene expression in spinal muscular atrophy patients

Spinal muscular atrophy (SMA) is caused by insufficient levels of survival motor neuron (SMN) protein. Recently, we found that sodium 4-phenylbutyrate (PB), a well-tolerated FDA approved drug, enhances SMN gene expression in vitro. We provide here the first evidence that oral administration of PB (triButyrate®) significantly increases SMN expression in leukocytes of SMA patients. This finding provides a strong rationale to further investigate the effects of PB as also supported by preliminary clinical data.

Topical use of tranexamic acid in coronary artery bypass operations: A double-blind, prospective, randomized, placebo-controlled study

OBJECTIVES We sought to investigate the effect of topical application of tranexamic acid into the pericardial cavity in reducing postoperative blood loss in coronary artery surgery. METHODS A prospective, randomized, double-blind investigation with parallel groups was performed. Forty consecutive patients undergoing primary coronary surgery were randomly assigned to group 1 (tranexamic acid group) or group 2 (placebo group). Tranexamic acid (1 g in 100 mL of saline solution) or placebo was poured into the pericardial cavity and over the mediastinal tissues before sternal closure. The drainage of mediastinal blood was measured hourly. RESULTS Chest tube drainage in the first 24 hours was 485 +/- 166 mL in the tranexamic acid group and 641 +/- 184 mL in the placebo group (P =.01). Total postoperative blood loss was 573 +/- 164 mL and 739 +/- 228 mL, respectively (P =.01). The use of banked donor blood products was not significantly different between the two groups. Tranexamic acid could not be detected in any of the blood samples blindly collected from 24 patients to verify whether any systemic absorption of the drug occurred. There were no deaths in either group. None of the patients required reoperation for bleeding. CONCLUSIONS Topical application of tranexamic acid into the pericardial cavity after cardiopulmonary bypass in patients undergoing primary coronary bypass operations significantly reduces postoperative bleeding. Further studies must be carried out to clarify whether a more pronounced effect on both bleeding and blood products requirement might be seen in procedures with a higher risk of bleeding.

Differential epigenetic modifications in the FMR1 gene of the fragile X syndrome after reactivating pharmacological treatments.

The fragile X syndrome is caused by a >200 CGG repeat expansion within the FMR1 gene promoter, with consequent DNA hypermethylation and inactivation of its expression. To further clarify the mechanisms that suppress the activity of the mutant gene and the conditions that may permit its reactivation, we investigated the acetylation and methylation status of three different regions of the FMR1 gene (promoter, exon 1 and exon 16) of three fragile X cell lines, using a chromatin immunoprecipitation (ChIP) assay with antibodies against acetylated-H3/H4 histones and against dimethylated lysine residues K4 and K9 of histone H3 (H3-K4 and H3-K9). We then coupled the ChIP assay with real-time PCR, obtaining absolute quantification of immunoprecipitated chromatin. Basal levels of histone acetylation and H3-K4 methylation were much higher in transcriptionally active wild-type controls than in inactive fragile X cell lines. Treatment of fragile X cell lines with the DNA demethylating drug 5-aza-2-deoxycytidine (5-azadC), known to reactivate the FMR1 gene, induced a decrease of H3-K9 methylation, an increase of H3 and H4 acetylation and an increase of H3-K4 methylation. Treatment with acetyl-L-carnitine (ALC), a compound that reduces the in vitro expression of the FRAXA fragile site without affecting DNA methylation, caused an increase of H3 and H4 acetylation. However, H3-K4 methylation remained extremely low, in accordance with the observation that ALC alone does not reactivate the FMR1 gene. Our experiments indicate that H3-K4 methylation and DNA demethylation are the main epigenetic switches activating the expression of the FMR1 gene, with histone acetylation playing an ancillary role.

Epigenetic analysis reveals a euchromatic configuration in the FMR1 unmethylated full mutations

Fragile X syndrome (FXS) is caused by the expansion of a CGG repeat in the 5′UTR of the FMR1 gene and the subsequent methylation of all CpG sites in the promoter region. We recently identified, in unrelated FXS families, two rare males with an unmethylated full mutation, that is, with an expanded CGG repeat (>200 triplets) lacking the typical CpG methylation in the FMR1 promoter. These individuals are not mentally retarded and do not appear to be mosaic for premutation or methylated full mutation alleles. We established lymphoblastoid and fibroblast cell lines that showed essentially normal levels of the FMR1-mRNA but reduced translational efficiency of the corresponding mRNA. Epigenetic analysis of the FMR1 gene demonstrated the lack of DNA methylation and a methylation pattern of lysines 4 and 27 on histone H3 similar to that of normal controls, in accordance with normal transcription levels and consistent with a euchromatic configuration. On the other hand, histone H3/H4 acetylation and lysine 9 methylation on histone H3 were similar to those of typical FXS cell lines, suggesting that these epigenetic changes are not sufficient for FMR1 gene inactivation. These findings demonstrate remarkable consistency and suggest a common genetic mechanism causing this rare FMR1 epigenotype. The discovery of such a mechanism may be important in view of therapeutic attempts to convert methylated into unmethylated full mutations, restoring the expression of the FMR1 gene.

Prospective 3-year surveillance for nosocomial and environmental Legionella pneumophila: implications for infection control.

OBJECTIVES To perform a 3-year, prospective surveillance program for legionnaires disease (LD) in a large university hospital in Rome, and to assess the usefulness of the hospital water monitoring program in predicting the risk of nosocomial LD. METHODS Samples from patients with new cases of nosocomial pneumonia were sent for legionella laboratory investigations. Meanwhile, water samples for bacteriological analysis were collected every 6 months from high- and medium-risk hospital wards (10 in total). Legionella pneumophila isolates collected were serotyped and analyzed by pulsed-field gel electrophoresis. RESULTS From June 2001 through May 2004, the pneumonia surveillance identified one case of nosocomial LD among 43 cases of nosocomial pneumonia (2.3%). Environmental investigations detected L. pneumophila in 12 (18.7%) of the 64 water samples, of which 50% belonged to serogroup 1. The L. pneumophila count and the percentage of positive locations never exceeded 10(2) colony-forming units/L and 20%, respectively, except when the LD nosocomial case occurred (positive water samples, 40%; L. pneumophila count, <10(2) colony-forming units/L). Genotyping showed 3 prevalent clones of L. pneumophila in the water distribution network, of which one persisted over the 3 years. One clone contained 3 different L. pneumophila serogroups (2, 4, and 6). CONCLUSIONS The low incidence of nosocomial cases of LD appears to be associated with a low percentage (<20%) of positive water samples per semester and with a low contamination level (<10(2) colony-forming units/L). An infection control system for nosocomial LD should, therefore, be based on both environmental and clinical surveillance, together with the appropriate maintenance of the hospital water distribution system.

Modest reactivation of the mutant FMR1 gene by valproic acid is accompanied by histone modifications but not DNA demethylation

Fragile X syndrome (FXS), the leading cause of inherited mental retardation, is due to expansion and methylation of a CGG sequence in the FMR1 gene, which result in its silencing. We previously demonstrated a reactivation of FMR1 in FXS cells treated with the DNA demethylating drug 5-azadeoxycytidine, and, to a lesser extent, with the histone deacetylating drug butyrate. To identify other reactivating drugs, we now treated three FXS lymphoblastoid cell lines with valproic acid (VPA), a well-known antiepileptic drug, causing histone deacetylase inhibition and, possibly, DNA demethylation. After VPA treatment, FMR1-mRNA levels were low and FMRP protein was undetectable. The gene remained methylated, whereas histones were acetylated and a modest variation of histone methylation was observed. These results confirm the histone hyperacetylating effect of VPA but do not support its putative DNA demethylation activity. The primary role of DNA demethylation in the reactivation of the FMR1 gene was confirmed.

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.

Role of CTCF protein in regulating FMR1 locus transcription.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the FMR1 gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (FMR1-AS1), starting from both promoter and intron 2 of the FMR1 gene, was demonstrated in transcriptionally active alleles, but not in silent FXS alleles. Moreover, a DNA methylation boundary, which is lost in FXS, was recently identified upstream of the FMR1 gene. Several nuclear proteins bind to this region, like the insulator protein CTCF. Here we demonstrate for the first time that rare unmethylated full mutation (UFM) alleles present the same boundary described in wild type (WT) alleles and that CTCF binds to this region, as well as to the FMR1 gene promoter, exon 1 and intron 2 binding sites. Contrariwise, DNA methylation prevents CTCF binding to FXS alleles. Drug-induced CpGs demethylation does not restore this binding. CTCF knock-down experiments clearly established that CTCF does not act as insulator at the active FMR1 locus, despite the presence of a CGG expansion. CTCF depletion induces heterochromatinic histone configuration of the FMR1 locus and results in reduction of FMR1 transcription, which however is not accompanied by spreading of DNA methylation towards the FMR1 promoter. CTCF depletion is also associated with FMR1-AS1 mRNA reduction. Antisense RNA, like sense transcript, is upregulated in UFM and absent in FXS cells and its splicing is correlated to that of the FMR1-mRNA. We conclude that CTCF has a complex role in regulating FMR1 expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis.

The impact of aerators on water contamination by emerging gram-negative opportunists in at-risk hospital departments

OBJECTIVE Our aim was to evaluate the impact of aerators on water microbiological contamination in at-risk hospital departments, with a view to quantifying the possible risk of patient exposure to waterborne microorganisms. DESIGN We analyzed the microbiological and chemical-physical characteristics of hot and cold water in some critical hospital departments. SETTING Two hospitals in northern Italy. METHODS We took 304 water samples over a 1-year period, at 3-month intervals, from taps used by healthcare personnel for handwashing, surgical washing, and the washing of medical equipment. We analyzed heterotrophic plate counts (HPCs) at 36°C and 22°C, nonfastidious gram-negative bacteria (GNB-NE), and Legionella pneumophila. RESULTS The percentages of positivity and mean values of HPCs at 22°C, HPCs at 36°C, and GNB-NE loads were significantly higher at outlet points than in the plumbing system. In particular, GNB-NE positivity was higher at outlet points than in the plumbing system in both the cold water (31.58% vs 6.58% of samples were positive) and hot water (21.05% vs 3.95%) supplies. Our results also revealed contamination by L. pneumophila both in the plumbing system and at outlet points, with percentages of positive samples varying according to the serogroup examined (serogroups 1 and 2-14). The mean concentrations displayed statistically significant (P < .001) differences between the outlet points (27,382.89 ± 42,245.33 colony-forming units [cfu]/L) and the plumbing system (19,461.84 ± 29,982.11 cfu/L). CONCLUSIONS These results reveal a high level of contamination of aerators by various species of gram-negative opportunists that are potentially very dangerous for immunocompromised patients and, therefore, the need to improve the management of these devices.

Effects of acute hypoventilation and hyperventilation on exhaled carbon monoxide measurement in healthy volunteers

BackgroundHigh levels of exhaled carbon monoxide (eCO) are a marker of airway or lung inflammation. We investigated whether hypo- or hyperventilation can affect measured values.MethodsTen healthy volunteers were trained to achieve sustained end-tidal CO2 (etCO2) concentrations of 30 (hyperventilation), 40 (normoventilation), and 50 mmHg (hypoventilation). As soon as target etCO2 values were achieved for 120 sec, exhaled breath was analyzed for eCO with a photoacoustic spectrometer. At etCO2 values of 30 and 40 mmHg exhaled breath was sampled both after a deep inspiration and after a normal one. All measurements were performed in two different environmental conditions: A) ambient CO concentration = 0.8 ppm and B) ambient CO concentration = 1.7 ppm.ResultsDuring normoventilation, eCO mean (standard deviation) was 11.5 (0.8) ppm; it decreased to 10.3 (0.8) ppm during hyperventilation (p < 0.01) and increased to 11.9 (0.8) ppm during hypoventilation (p < 0.01). eCO changes were less pronounced than the correspondent etCO2 changes (hyperventilation: 10% Vs 25% decrease; hypoventilation 3% Vs 25% increase). Taking a deep inspiration before breath sampling was associated with lower eCO values (p < 0.01), while environmental CO levels did not affect eCO measurement.ConclusionseCO measurements should not be performed during marked acute hyperventilation, like that induced in this study, but the influence of less pronounced hyperventilation or of hypoventilation is probably negligible in clinical practice