Giovanna Barbuti

Helicobacter pylori Neutrophil-Activating Protein Stimulates Tissue Factor and Plasminogen Activator Inhibitor-2 Production by Human Blood Mononuclear Cells

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor that activates phagocytic NADPH-oxidase. The effect of HP-NAP on the production of tissue factor (TF), plasminogen activator inhibitor-2 (PAI-2), and urokinase-type plasminogen activator (u-PA) by human blood mononuclear cells (MNC) was evaluated by using functional and immunological assays and mRNA analysis. HP-NAP induced time- and dose-dependent increases in TF and PAI-2, with a maximal effect at 300 nmol/L (>15-fold increase in antigens). No changes in u-PA were observed. When whole bacteria were used, an H. pylori mutant lacking HP-NAP was significantly less active than the wild-type strain. MNC from a patient with chronic granulomatous disease behaved as do normal cells, which indicates that HP-NAP effects can occur independently of NADPH-oxidase. HP-NAP, by inducing the coordinate expression of cell procoagulant and antifibrinolytic activities, might favor fibrin deposition and contribute to the inflammatory reaction of gastric mucosa elicited by H. pylori.

Legionella spp. and legionellosis in southeastern Italy: disease epidemiology and environmental surveillance in community and health care facilities

BackgroundFollowing the publication of the Italian Guidelines for the control and prevention of legionellosis an environmental and clinical surveillance has been carried out in Southeastern Italy. The aim of the study is to identify the risk factors for the disease, so allowing better programming of the necessary prevention measures.MethodsDuring the period January 2000 - December 2009 the environmental surveillance was carried out by water sampling of 129 health care facilities (73 public and 56 private hospitals) and 533 buildings within the community (63 private apartments, 305 hotels, 19 offices, 4 churches, 116 gyms, 3 swimming pools and 23 schools). Water sampling and microbiological analysis were carried out following the Italian Guidelines. From January 2005, all facilities were subject to risk analysis through the use of a standardized report; the results were classified as good (G), medium (M) and bad (B). As well, all the clinical surveillance forms for legionellosis, which must be compiled by physicians and sent to the Regional Centre for Epidemiology (OER), were analyzed.ResultsLegionella spp. was found in 102 (79.1%) health care facilities and in 238 (44.7%) community buildings. The percentages for the contamination levels < 1,000, 1,000-10,000, > 10,000 cfu/L were respectively 33.1%, 53.4% and 13.5% for samples from health care facilities and 33.5%, 43.3% and 23.2% for samples from the community. Both in hospital and community environments, Legionella pneumophila serogroup (L. pn sg) 2-14 was the most frequently isolate (respectively 54.8% and 40.8% of positive samples), followed by L. pn sg 1 (respectively 31.3% and 33%). The study showed a significant association between M or B score at the risk analysis and Legionella spp. positive microbiological test results (p < 0.001). From clinical surveillance, during the period January 2001 - August 2009, 97 cases of legionellosis were reported to the OER: 88 of community origin and 9 nosocomial. The most frequent symptoms were: fever (93.8%), cough (70.1%), dyspnea (58.8%), shivering (56.7%). Radiological evidence of pneumonia was reported in 68%. The laboratory diagnostic methods used were: urinary antigen (54.3%), single antibody titer (19.8%), only seroconversion (11.1%), other diagnostic methods (14.8%).ConclusionsOur experience suggests that risk analysis and environmental microbiological surveillance should be carried out more frequently to control the environmental spread of Legionella spp. Furthermore, the laboratory diagnosis of legionellosis cannot be excluded only on the basis of a single negative test: some patients were positive to only one of the diagnostic tests.

Occurrence of Aflatoxin M1 in Dairy Products in Southern Italy

A screening survey of the presence of aflatoxin M1 (AFM1) was carried out on 265 samples of cheese made from cow, buffalo, goat, sheep, sheep-goat milk collected in the Apulia region (Southern Italy). Selected samples included unripened, medium and long-term ripened cheeses. AFM1 was found in 16.6% of the analyzed samples. The highest positive incidence was for medium and long-term ripened cheeses, especially those made from sheep-goat milk, while buffalo cheeses tested consistently negative. Our results show that the level of contamination by AFM1 in dairy products from Apulia Region are lower than in other Italian and European regions. Moreover, it is important to underline that a common European norm concerning the AFM1 threshold limits for dairy products is still lacking.

Retinoic acid stimulates plasminogen activator inhibitor 2 production by blood mononuclear cells and inhibits urokinase-induced extracellular proteolysis.

Retinoids have been shown to modulate several functions of mononuclear phagocytes. We investigated the in vitro effect of all‐trans‐retinoic acid (ATRA) on the production of two major fibrinolytic components, urokinase‐type plasminogen activator (u‐PA) and PA inhibitor 2 (PAI‐2), by human blood mononuclear cells (MNC). ATRA caused a dose‐dependent (range 0.01–10 μm) accumulation of PAI‐2 antigen and activity into the cell culture medium, with a maximal increase (about 5‐fold over control) at a concentration of 1–10 μm. Similarly, a dose‐dependent increase in PAI‐2 antigen was observed in cell extracts upon ATRA stimulation. Northern blot analysis showed a parallel increase in the amount of PAI‐2 mRNA in ATRA‐treated cells. Time‐course experiments with 1 μm ATRA showed enhanced PAI‐2 mRNA expression as early as 2 h, reaching a maximum at 4–6 h and then declining at 18–24 h, and a time‐dependent increase in PAI‐2 antigen in the cell culture medium. At variance with PAI‐2, u‐PA was not influenced by the drug. To establish whether ATRA‐induced changes influenced the fibrinolytic process, we evaluated the effect of MNC stimulated with ATRA on u‐PA‐induced degradation of diluted plasma clots. ATRA‐treated cells markedly inhibited clot lysis induced by low concentrations of u‐PA. The effect was due to enhanced extracellular PAI‐2 accumulation since it was observed with conditioned medium from ATRA‐treated cells; it was abolished by the addition of neutralizing anti‐PAI‐2 antibodies and was negligible when single‐chain t‐PA was used instead of u‐PA. Since monocyte/macrophage‐mediated, plasminogen‐dependent extracellular proteolysis has been proposed as an important mechanism of tissue damage in several inflammatory states, our findings might contribute to better explain the anti‐inflammatory properties of retinoids.

Clinical-environmental surveillance of legionellosis: an experience in Southern Italy.

In Italy, although the number of cases of legionellosis notified to the health authorities has significantly increased in recent years, the incidence is still believed to be underestimated. To verify the true frequency and identify the sources of infection, an active clinical–environmental surveillance program was instituted in three hospital facilities in Southern Italy. Between January 2001 and March 2005, a total of 1000 patients admitted to the three hospitals with a diagnosis of pneumonia were enrolled. The urinary antigen and anti-Legionella antibody titre were assayed in each subject, and direct searches for the microorganism were made in biological specimens. Legionellosis was found to be present in 5.9% of the patients. For each of the cases of legionellosis, microbiological surveys were made of the water supply in the public and/or private facilities involved. Overall, 197 water samples of hospital origin and 218 of community origin were analysed: Legionella spp was isolated in 44.2 and 36.7% of the cases, respectively. Comparison of our data with those of the routine surveillance system for the same area (only 7 cases during the period 1997–2000), showed that the frequency of legionellosis is grossly underestimated in Southern Italy. It is therefore necessary to set up more rigorous controls in both hospital and community facilities, so that timely preventive measures can be taken to avoid any further spread of the disease.

Safety of shellfish and epidemiological pattern of enterically transmitted diseases in Italy

In recent decades in South-Italy, drastic epidemiological changes in the pattern of the enterically transmitted diseases were recorded. This work aims to support the hypothesis that universal routine vaccination against hepatitis A started in Puglia in 1998 could be also effective in reducing contamination in seafood. Three studies on different samples of Mitylus edulis lamellibranch were conducted over 20 years. Microbiological analysis measured both the standard bacteriological indexes (faecal coliforms, Escherichia coli, Salmonella spp.) and the presence of hepatitis A, enterovirus, norovirus and rotavirus. Between 1989 and 2009, the highest number of hepatitis A cases was reported in 1996 and in 1997 (mean incidence rate of 130 per 100,000). Since 1999, the number of cases progressively decreased, reaching 29 cases in 2006 (incidence rate of 0.7 per 100,000). A progressive improvement in vaccination coverage of newborns was recorded ranging from 1997 (38.3%) to 2005 birth-cohorts (63.4%). Vaccination coverage of 12-year-old adolescents was 67.6% (95% CI: 58.4-76.8%). Hepatitis A was detected in 3.7% of samples in the 1987 study, in 18.3% of samples in the 1999-2000 study and it was absent in the 2007 study. The decreasing of HAV circulation in humans and in shellfish provides support for the link between vaccination efforts and the safety of molluscs.

Increase in interleukin-8 production from circulating neutrophils upon antibiotic therapy in cystic fibrosis patients

BACKGROUND It is not known whether antibiotic therapy for lung disease in cystic fibrosis (CF) has an influence on circulating polymorphonuclear neutrophil (PMN) function and apoptosis. PATIENTS AND METHODS Blood PMNs were obtained from 14 CF patients before and after antibiotic treatment for an acute exacerbation, and from 10 healthy controls. PMNs were evaluated for production of reactive oxygen species (ROS) by spectrophotometry, of cytokines in the conditioned medium by ELISA, and apoptotic response by cytofluorimetry. RESULTS ROS and interleukin (IL)-8 were produced at higher levels by CF PMNs pre-therapy than control PMNs under basal conditions. IL-8 levels further increased after therapy. Early apoptotic response was higher in CF PMNs pre-therapy than in control PMNs, and this pattern did not change after antibiotic treatment. CONCLUSIONS Circulating PMNs are primed in CF acute patients. Further studies are needed to consider PMN-produced IL-8 as a biomarker to evaluate response to antibiotic therapy in CF patients.

Streptococcus pneumoniae modulates the respiratory burst response in human neutrophils

The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for high morbidity and mortality worldwide. A major feature of pneumococcal pneumonia is an abundant neutrophil infiltration. In this work we observed that the R6 nonencapsulated S. pneumoniae strain induced a higher oxidative burst in neutrophils compared with its capsulated progenitor D39, by triggering neutrophil NADPH oxidase to produce more reactive oxygen intermediates (ROI) and by interfering with the neutrophil kinase signalling pathway. In addition, we evaluated the possibility that the capsule, lacking in R6 but present in D39, could modulate the S. pneumoniae-induced neutrophil respiratory burst. In this respect, three knock-out isogenic mutants (D39ΔCPS2E, D39ΔCPS-R6 and R6ΔCPS-R6) that were unable to synthesize the capsule, were tested for their capability of inducing the release of neutrophil-ROIs. The results indicate that the mutants behaved similarly to their wild-type parental strains in enhancing respiratory burst activity, suggesting that the capsule itself is not directly involved in modulating the neutrophil oxidative burst induced by S. pneumoniae, but that other genetic differences between D39 and R6 present elsewhere in the genome could be responsible for these mechanisms.

Legionella pneumophila serogroup 5 infection in the presence of multiple environmental contamination. The importance of a bacteriological diagnosis

Legionella pneumophila is a pathogen that causes severe pneumonia in humans; L. pneumophila serogroup 1 accounts for at least 90% of infections. This is not linked to an environmental predominance of Legionella pneumophila 1, but may be due to a greater virulence of the strain. L. pneumophila sg 5 has also been reported, albeit less frequently, to be a cause of the disease. We report a case of L. pneumophila sg 5 occurring in a large hospital in southern Italy (Apulia region), where both L. pneumophila sg 1 and sg 5 were detected in the water supply; the nosocomial origin was demonstrated by molecular subtyping (PFGE). An environmental investigation, performed immediately after diagnosis of the case of legionellosis, identified a ow L. pneumophila sg 5 contamination level. Our experience highlights that in hospital, risk assessment, in rder to institute control measures for Legionella, should be carried out not only in response to a case of the disease and/or in risk wards only, as described in the Italian Guidelines, but periodically in every ward. The present study confirms that, although in the community L. pneumophila sg 1 is the most frequent strain isolated in both outbreaks and isolated cases, in hospital other serogroups and species may often cause infection because of the high susceptibility of the hosts.

Enteric Viruses and Fecal Bacteria Indicators to Assess Groundwater Quality and Suitability for Irrigation

According to Italian Ministerial Decree No. 185 of 12 June 2003, water is considered suitable for irrigation if levels of fecal bacteria (i.e., Escherichia coli and Salmonella) are within certain parameters. The detection of other microorganisms is not required. The aim of this study is to determine the bacteriological quality of groundwater used for irrigation and the occurrence of enteric viruses (Norovirus, Enterovirus, Rotavirus, Hepatovirus A), and to compare the presence of viruses with the fecal bacteria indicators. A total of 182 wells was analyzed. Widespread fecal contamination of Apulian aquifers was detected (141 wells; 77.5%) by the presence of fecal bacteria (i.e., E. coli, Salmonella, total coliforms, and enterococci). Considering bacteria included in Ministerial Decree No. 185, the water from 35 (19.2%) wells was unsuitable for irrigation purposes. Among 147 wells with water considered suitable, Norovirus, Rotavirus, and Enterovirus were detected in 23 (15.6%) wells. No Hepatovirus A was isolated. Consequently, 58 wells (31.9%) posed a potential infectious risk for irrigation use. This study revealed the inadequacy of fecal bacteria indicators to predict the occurrence of viruses in groundwater and it is the first in Italy to describe the presence of human rotaviruses in well water used for irrigation.