Renato Liguori

Biological role of mannose binding lectin: From newborns to centenarians

Mannose binding lectin (MBL) is a protein of innate immunity that activates the complement and promotes opsonophagocytosis. The deficiency of MBL due to several common gene polymorphisms significantly enhances the risk of severe infections, particularly in the neonatal age and in childhood. On the contrary, the role of the protein in carcinogenesis and atherogenesis is still debated: MBL has a relevant role against neoplastic cells, but some studies described a protective effect of low levels of MBL toward breast cancer and a longer survival of lung cancer patients with a reduced MBL activity. Similarly, some studies concluded on the protective role of low levels of MBL toward cardiovascular diseases while other focused on a higher risk of myocardial infarction in subjects with a deficient activity of the protein. More recently, a role of MBL in the clearance of senescent cells emerged, and a study in two large cohorts of centenarians demonstrated that a high biological activity of the protein enhances the risk of autoimmune diseases. This body of data strongly suggests that the optimal levels of MBL activity depend on the age and on the environmental context of each subject.

A novel polymorphism in the PAI-1 gene promoter enhances gene expression. A novel pro-thrombotic risk factor?

INTRODUCTION Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of tissue-type plasminogen activator in plasma and the most important regulator of the fibrinolytic pathway. The 4G/5G polymorphism (rs1799889) in the PAI-1 promoter is associated with altered PAI-1 transcription. We have identified a new 4G/5G allele, in which a T is inserted near the 4G tract or replaces a G in the 5G tract, forming a T plus 4G (T4G) region. MATERIALS AND METHODS This new variant was first identified in two women, one had experienced juvenile myocardial infarction, the other repeated miscarriage; both had increased PAI-1 plasma activity. In view of the important influence of this promoter region on PAI-1 protein plasma level, we performed in vitro evaluation of the effects of the T4G variant on the transcription activity of the PAI-1 gene promoter. RESULTS AND CONCLUSIONS In silico prediction analysis showed that presence of the T4G allele disrupts the E-Box region upstream of the T4G variant, altering the affinity of the target sequence for E-Box binding factors like upstream stimulatory factor-1 (USF-1). Basal T4G promoter activity was 50% higher compared to 4G and 5G variants, but it was less stimulated by USF-1 overexpression. We also analyzed the effects of IL-1β and IL-6 on the PAI-1 promoter activity of our three constructs and showed that the T4G variant was less affected by IL-1β than the other variants. These findings indicate that the T4G variant may be a novel risk factor for thrombotic events.

Pseudomonas aeruginosa in Swimming Pool Water: Evidences and Perspectives for a New Control Strategy.

Pseudomonas aeruginosa is frequently isolated in swimming pool settings. Nine recreational and rehabilitative swimming pools were monitored according to the local legislation. The presence of P. aeruginosa was correlated to chlorine concentration. The ability of the isolates to form a biofilm on plastic materials was also investigated. In 59.5% of the samples, microbial contamination exceeded the threshold values. P. aeruginosa was isolated in 50.8% of these samples. The presence of P. aeruginosa was not correlated with free or total chlorine amount (R2 < 0.1). All the isolates were moderate- to strong-forming biofilm (Optical Density O.D.570 range 0.7–1.2). To control biofilm formation and P. aeruginosa colonization, Quantum FreeBioEnergy© (QFBE, FreeBioEnergy, Brisighella, Italy), has been applied with encouraging preliminary results. It is a new, promising control strategy based on the change of an electromagnetic field which is responsible for the proliferation of some microorganisms involved in biofilm formation, such as P. aeruginosa.

Genetic Diseases That Predispose to Early Liver Cirrhosis

Inherited liver diseases are a group of metabolic and genetic defects that typically cause early chronic liver involvement. Most are due to a defect of an enzyme/transport protein that alters a metabolic pathway and exerts a pathogenic role mainly in the liver. The prevalence is variable, but most are rare pathologies. We review the pathophysiology of such diseases and the diagnostic contribution of laboratory tests, focusing on the role of molecular genetics. In fact, thanks to recent advances in genetics, molecular analysis permits early and specific diagnosis for most disorders and helps to reduce the invasive approach of liver biopsy.

Impact of 13valent vaccine for prevention of pneumococcal diseases in children and adults at risk: possible sceneries in Campania region

BACKGROUND Pneumonias are the most frequent infectious disease, characterized by a high prevalence especially among children and adults at risk. The socio-economic impact caused by Streptococcus pneumoniae is evaluated in terms of morbidity, death rate and hospitalizations. OBJECTIVE The aim of the study was to demonstrate the potential economic advantages by implementation of an active anti-pneumococcal 13-valent vaccine strategy in Campania region ( Southern Italy) in two different categories of subjects, children (aged 0-12), and adults (aged 50-79) at risk (hypertension, nephropathies, COPD and heart diseases) Methods. Vaccination costs were compared with costs necessary to treat avoidable diseases in presence and absence of a vaccination program. RESULTS Offering anti-pneumococcal 13-valent vaccine to the paediatric population was quantified as saving one million euros for Italian national health service in two years. In addition , offering anti-pneumococcal vaccine to adults at risk would generate a return of around 29 million euros. CONCLUSION In both cases, offering anti-pneumococcal 13-valent vaccine was proven to be a helpful political health strategy, not only in consideration of a reduction of cases, but also in view of the favourable economic impacts.

Supervised physical exercise improves clinical, anthropometric and biochemical parameters in adult cystic fibrosis patients: a three years evaluation

Cystic fibrosis (CF) is the most common inherited, life limiting condition among Caucasians. No healing therapy is currently available for patients with CF. The aim of the study was to define clinical, anthropometric and biochemical effects of regular, supervised physical exercise in a large cohort of patients with CF.

Twelve Novel Mutations in the slc26a3 Gene in 17 Sporadic Cases of Congenital Chloride Diarrhea

Objectives: We aimed to improve the knowledge of pathogenic mutations in sporadic cases of congenital chloride diarrhea (CCD) and emphasize the importance of functional studies to define the effect of novel mutations. Methods: All member 3 of solute carrier family 26 (SLC26A3) coding regions were sequenced in 17 sporadic patients with CCD. Moreover, the minigene system was used to analyze the effect of 2 novel splicing mutations. Results: We defined the SLC26A3 genotype of all 17 patients with CCD and identified 12 novel mutations. Using the minigene system, we confirmed the in silico prediction of a complete disruption of splicing pattern caused by 2 of these novel mutations: the c.971+3_971+4delAA and c.735+4_c.735+7delAGTA. Moreover, several prediction tools and a structure-function prediction defined the pathogenic role of 6 novel missense mutations. Conclusions: We confirm the molecular heterogeneity of sporadic CCD adding 12 novel mutations to the list of known pathogenic mutations. Moreover, we underline the importance, for laboratories that offer molecular diagnosis and genetic counseling, to perform fast functional analysis of novel mutations.

Potential testing of reprocessing procedures by real-time polymerase chain reaction: A multicenter study of colonoscopy devices

Background: Reprocessing of endoscopes is key to preventing cross‐infection after colonoscopy. Culture‐based methods are recommended for monitoring, but alternative and rapid approaches are needed to improve surveillance and reduce turnover times. A molecular strategy based on detection of residual traces from gut microbiota was developed and tested using a multicenter survey. Methods: A simplified sampling and DNA extraction protocol using nylon‐tipped flocked swabs was optimized. A multiplex real‐time polymerase chain reaction (PCR) test was developed that targeted 6 bacteria genes that were amplified in 3 mixes. The method was validated by interlaboratory tests involving 5 reference laboratories. Colonoscopy devices (n = 111) were sampled in 10 Italian hospitals. Culture‐based microbiology and metagenomic tests were performed to verify PCR data. Results: The sampling method was easily applied in all 10 endoscopy units and the optimized DNA extraction and amplification protocol was successfully performed by all of the involved laboratories. This PCR‐based method allowed identification of both contaminated (n = 59) and fully reprocessed endoscopes (n = 52) with high sensibility (98%) and specificity (98%), within 3‐4 hours, in contrast to the 24‐72 hours needed for a classic microbiology test. Results were confirmed by next‐generation sequencing and classic microbiology. Conclusions: A novel approach for monitoring reprocessing of colonoscopy devices was developed and successfully applied in a multicenter survey. The general principle of tracing biological fluids through microflora DNA amplification was successfully applied and may represent a promising approach for hospital hygiene.

Cystic fibrosis, molecular genetics for all life

Cystic fibrosis (CF) is the most frequent lethal autosomal recessive disorder among Caucasians (incidence: 1:2,500 newborn). In the last two decades CF prognosis considerably improved and many patients well survive into their adulthood. Furthermore, milder CF with a late onset was described. CF is a challenge for laboratory of molecular genetics that greatly contributes to the natural history of the disease since fetal age. Carrier screening and prenatal diagnosis, also by non-invasive analysis of maternal blood fetal DNA, are now available, and many labs offer preimplantation diagnosis. The major criticism in prenatal medicine is the lack of an effective multidisciplinary counseling that helps the couples to plan their reasoned reproductive choice. Most countries offer newborn screening that significantly reduce CF morbidity but different protocols based on blood trypsin, molecular analysis and sweat chloride cause a variable efficiency of the screening programs. Again, laboratory is crucial for CF diagnosis in symptomatic patients: sweat chloride is the diagnostic golden standard, but different methodologies and the lack of quality control in most labs reduce its effectiveness. Molecular analysis contributes to confirm diagnosis in symptomatic subjects; furthermore, it helps to predict the disease outcome on the basis of the mutation (genotype-phenotype correlation) and mutations in a myriad of genes, inherited independently by CF transmembrane conductance regulator (CFTR), which may modulate the clinical expression of the disease in each single patient (modifier genes). More recently, the search of the CFTR mutations gained a role in selecting CF patients that may benefit from biological therapy based on correctors and potentiators that are effective in patients bearing specific mutations (personalized therapy). All such applications of molecular diagnostics confirm the “uniqueness” of each CF patient, offering to laboratory medicine the opportunity to reposition the patient in the “core” of the medical process. Proceedings of the 11 th International Workshop on Neonatology and Satellite Meetings · Cagliari (Italy) · October 26 th -31 st , 2015 · From the womb to the adult Guest Editors: Vassilios Fanos (Cagliari, Italy), Michele Mussap (Genoa, Italy), Antonio Del Vecchio (Bari, Italy), Bo Sun (Shanghai, China), Dorret I. Boomsma (Amsterdam, the Netherlands), Gavino Faa (Cagliari, Italy), Antonio Giordano (Philadelphia, USA)

Omics in laboratory medicine

Abstract In the last years, molecular diagnosis of human genetic diseases has greatly improved thanks to the knowledge of more than 7,000 disease genes (genomics). However, the study of such diseases revealed the very complex relationships between the phenotype of each disease and the molecular alterations responsible. The analysis of proteins (proteomics) revealed that most proteins are subjected to post-translational changes or to alternative splicing; the study of gene expression identified a series of mechanisms that modulate gene expression (epigenomics) which include microRNA regulation, histone acetylation and gene methylation. The alteration of all these mechanisms may contribute to the pathogenesis or to the phenotypic expression of most human genetic diseases. Molecular analysis became more and more complex, but “omics” studies revealed that each single individual is “unique”.