In vitro and in vivo screening for novel essential cell-envelope proteins in Pseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa represents a prototype of multi-drug resistant opportunistic pathogens for which novel therapeutic options are urgently required. In order to identify new candidates as potential drug targets, we combined large-scale transposon mutagenesis data analysis and bioinformatics predictions to retrieve a set of putative essential genes which are conserved in P. aeruginosa and predicted to encode cell envelope or secreted proteins. By generating unmarked deletion or conditional mutants, we confirmed the in vitro essentiality of two periplasmic proteins, LptH and LolA, responsible for lipopolysaccharide and lipoproteins transport to the outer membrane respectively, and confirmed that they are important for cell envelope stability. LptH was also found to be essential for P. aeruginosa ability to cause infection in different animal models. Conversely, LolA-depleted cells appeared only partially impaired in pathogenicity, indicating that this protein likely plays a less relevant role during bacterial infection. Finally, we ruled out any involvement of the other six proteins under investigation in P. aeruginosa growth, cell envelope stability and virulence. Besides proposing LptH as a very promising drug target in P. aeruginosa, this study confirms the importance of in vitro and in vivo validation of potential essential genes identified through random transposon mutagenesis

Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

Despite intensive antimicrobial and anti-inflammatory therapies, cystic fibrosis (CF) patients are subjected to chronic infections due to opportunistic pathogens, including multidrug resistant (MDR) Pseudomonas aeruginosa. Macrophages from CF patients show many evidences of reduced phagocytosis in terms of internalization capability, phagosome maturation, and intracellular bacterial killing. In this study, we investigated if apoptotic body-like liposomes (ABLs) loaded with phosphatidylinositol 5-phosphate (PI5P), known to regulate actin dynamics and vesicular trafficking, could restore phagocytic machinery while limiting inflammatory response in in vitro and in vivo models of MDR P. aeruginosa infection. Our results show that the in vitro treatment with ABL carrying PI5P (ABL/PI5P) enhances bacterial uptake, ROS production, phagosome acidification, and intracellular bacterial killing in human monocyte-derived macrophages (MDMs) with pharmacologically inhibited cystic fibrosis transmembrane conductance regulator channel (CFTR), and improve uptake and intracellular killing of MDR P. aeruginosa in CF macrophages with impaired bactericidal activity. Moreover, ABL/PI5P stimulation of CFTR-inhibited MDM infected with MDR P. aeruginosa significantly reduces NF-κB activation and the production of TNF-α, IL-1β, and IL-6, while increasing IL-10 and TGF-β levels. The therapeutic efficacy of ABL/PI5P given by pulmonary administration was evaluated in a murine model of chronic infection with MDR P. aeruginosa. The treatment with ABL/PI5P significantly reduces pulmonary neutrophil infiltrate and the levels of KC and MCP-2 cytokines in the lungs, without affecting pulmonary bacterial load. Altogether, these results show that the ABL/PI5P treatment may represent a promising host-directed therapeutic approach to improve the impaired phagocytosis and to limit the potentially tissue-damaging inflammatory response in CF

Contribution of vegetables and cured meat to dietary nitrate and nitrite intake in Italian population: Safe level for cured meat and controversial role of vegetables

Nitrate and nitrite content was determined on a total of 900 samples of vegetables and cured meat and the nitrite and nitrate exposure assessment was evaluated for central Italy population based on the food consumption data reported by the national dietary surveys. The highest average content of nitrate was detected in rocket salad (4415 mg/kg) and radish (3817 mg/kg) and for cured meat in “Bresaola” (188 mg/kg) and in Bacon (178 mg/kg). The nitrite content was negligible both in vegetables than in cured meat. The average consumption among population resulted 3.45 g/kg bw/die and 0.62 g/kg bw/die for vegetables and cured meat respectively. The obtained data confirm that nitrate ADI was higher than the limits of 3.7 mg/kg bw/die for infants and was the highest exposure level for people of all ages. Cured meat consumption did not contribute to nitrate ADI exceedance neither as a mean nor as 99th percentile of exposure

Sepsis promotes splenic production of a protective platelet pool with high CD40 ligand expression.

Platelets have a wide range of functions including critical roles in hemostasis, thrombosis, and immunity. We hypothesized that during acute inflammation, such as in life-threatening sepsis, there are fundamental changes in the sites of platelet production and phenotypes of resultant platelets. Here, we showed during sepsis that the spleen was a major site of megakaryopoiesis and platelet production. Sepsis provoked an adrenergic-dependent mobilization of megakaryocyte-erythrocyte progenitors (MEPs) from the bone marrow to the spleen, where IL-3 induced their differentiation into megakaryocytes (MKs). In the spleen, immune-skewed MKs produced a CD40 ligandhi platelet population with potent immunomodulatory functions. Transfusions of post-sepsis platelets enriched from splenic production enhanced immune responses and reduced overall mortality in sepsis-challenged animals. These findings identify a spleen-derived protective platelet population that may be broadly immunomodulatory in acute inflammatory states such as sepsis.NHS Blood and Transplant European Union's Horizon 2020 (ERC-2014-CoG-648765) MRC-AMED (MR/V005421/1

Road traffic pollution and childhood leukemia: a nationwide case-control study in Italy

Background The association of childhood leukemia with traffic pollution was considered in a number of studies from 1989 onwards, with results not entirely consistent and little information regarding subtypes. Aim of the study We used the data of the Italian SETIL case-control on childhood leukemia to explore the risk by leukemia subtypes associated to exposure to vehicular traffic. Methods We included in the analyses 648 cases of childhood leukemia (565 Acute lymphoblastic–ALL and 80 Acute non lymphoblastic-AnLL) and 980 controls. Information on traffic exposure was collected from questionnaire interviews and from the geocoding of house addresses, for all periods of life of the children. Results We observed an increase in risk for AnLL, and at a lower extent for ALL, with indicators of exposure to traffic pollutants. In particular, the risk was associated to the report of closeness of the house to traffic lights and to the passage of trucks (OR: 1.76; 95% CI 1.03–3.01 for ALL and 6.35; 95% CI 2.59–15.6 for AnLL). The association was shown also in the analyses limited to AML and in the stratified analyses and in respect to the house in different period of life. Conclusions Results from the SETIL study provide some support to the association of traffic related exposure and risk for AnLL, but at a lesser extent for ALL. Our conclusion highlights the need for leukemia type specific analyses in future studies. Results support the need of controlling exposure from traffic pollution, even if knowledge is not complete

ECMO for COVID-19 patients in Europe and Israel

Since March 15th, 2020, 177 centres from Europe and Israel have joined the study, routinely reporting on the ECMO support they provide to COVID-19 patients. The mean annual number of cases treated with ECMO in the participating centres before the pandemic (2019) was 55. The number of COVID-19 patients has increased rapidly each week reaching 1531 treated patients as of September 14th. The greatest number of cases has been reported from France (n = 385), UK (n = 193), Germany (n = 176), Spain (n = 166), and Italy (n = 136) .The mean age of treated patients was 52.6 years (range 16–80), 79% were male. The ECMO configuration used was VV in 91% of cases, VA in 5% and other in 4%. The mean PaO2 before ECMO implantation was 65 mmHg. The mean duration of ECMO support thus far has been 18 days and the mean ICU length of stay of these patients was 33 days. As of the 14th September, overall 841 patients have been weaned from ECMO support, 601 died during ECMO support, 71 died after withdrawal of ECMO, 79 are still receiving ECMO support and for 10 patients status n.a. . Our preliminary data suggest that patients placed on ECMO with severe refractory respiratory or cardiac failure secondary to COVID-19 have a reasonable (55%) chance of survival. Further extensive data analysis is expected to provide invaluable information on the demographics, severity of illness, indications and different ECMO management strategies in these patients

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Study of acrylamide level in food from vending machines

Acrylamide is a by-product of the Maillard reaction and is potentially carcinogenic to humans. It is found in a number of foods with higher concentrations in carbohydrate-rich foods and moderate levels of protein-rich foods such as meat, fish and seafood. Acrylamide levels in food distributed in vending machines placed in public areas of the city of Perugia were analysed by high-performance liquid chromatography. Samples included five different categories, depending on the characteristics of the products: i) potato chips; ii) salted bakery products; iii) biscuits and wafers; iv) sweet bakery products; v) sandwiches. A high variability in acrylamide level among different foods and within the same category was detected. Potato chips showed the highest amount of acrylamide (1781±637 μg/kg) followed by salted bakery products (211±245 μg/kg), biscuits and wafers (184±254 μg/kg), sweet bakery products (100±72 μg/kg) and sandwiches (42±10 μg/kg). In the potato chips and sandwiches categories, all of the samples revealed the presence of acrylamide, while different prevalence was registered in the other foods considered. The data of this study highlight the presence of acrylamide in different foods sold in vending machines and this data could be useful to understand the contribution of this type of consumption to human exposure to this compound

Contribution of street food on dietary acrylamide exposure by youth aged nineteen to thirty in Perugia, Italy

Acrylamide dietary intakes from selected street foods in youth population are estimated. The intake evaluation was carried out by combining levels of acrylamide in food, analytically determined by high performance liquid chromatography, with individual consumption data recorded using a questionnaire applied to a group of 200 students aged 19 to 30. The mean value of acrylamide exposure was recorded to be 0.452 μg/kg bw/day, while the average intakes at 50th and 95th percentile were 0.350 μg/kg bw/day and 1.539 μg/kg bw/day, respectively. The street food categories that contributed the most to acrylamide intake are pizza and French fries. The margins of exposure, based on benchmark dose limits defined for neoplastic effects and peripheral neuropathy, are within the range of values that indicate a concern for public health as defined by European Authority for Food Safety (EFSA), confirming the needed effort to reduce acrylamide dietary exposure

Continuous Erector Spinae Plane Block for Pain Management in a Pediatric Kidney Transplant Recipient: A Case Report and Review of the Current Literature

Pain management in patients undergoing kidney transplantation requires careful consideration due to their altered physiology, and potential risks associated with certain analgesic options. In recent years, personalized and multimodal approaches have proven to be pivotal in perioperative pain management, as well as in children. Implementing regional analgesia methods offers a valuable solution in many pediatric surgical settings and the erector spinae plane block (ESPB) could represent a possible analgesic strategy in pediatric patients undergoing renal transplantation. Here, we report the case of a 13-year-old child who underwent living-donor kidney transplantation (LDKx) and received continuous erector spinae plane block (ESPB) for perioperative pain management. This multimodal approach with continuous ESPB resulted in optimal pain control without the need for opioids, allowing for early mobilization and for an optimal postoperative course