The data project: a shared approach between stakeholders of the healthcare system in definition of a therapeutic algorithm for inflammatory arthritis.

Rheumatic musculoskeletal diseases or RMD [rheumatoid arthritis (RA) and spondyloarthritis (SpA)] are systemic inflammatory diseases for which there are no biomarkers capable of predicting treatments with a higher likelihood of response in naive patients. In addition, the expiration of the anti-TNF blocking drugs' patents has resulted in the availability of anti-TNF biosimilar drugs with the same efficacy and safety than originators but at significantly reduced prices. To guarantee a personalized therapeutic approach to RMD treatment, a board of rheumatologists and stakeholders from the Campania region, Italy, developed a clinically applicable arthritis therapeutic algorithm to guide rheumatologists (DATA project). The general methodology relied on a Delphi technique forecast to produce a set of statements that summarized the experts' consensus. Selected clinical scenarios were discussed in light of the available evidence, and there were two rounds of voting on the therapeutic approaches. Separate discussions were held regarding rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. The decision-making factors for each disease were clinical presentation, demographics, and comorbidities. In this paper, we describe a virtuous process between rheumatologists and healthcare system stakeholders that resulted in the development of a shared therapeutic algorithm for RMD patients naive to bDMARDs.

Regular football training down-regulates miR-1303 muscle expression in veterans.

PURPOSE: Regular exercise affects the expression of several genes, proteins and microRNAs (miRNAs) in time- and intensity-dependent manner promoting longevity. We previously identified from GeneChip Array analysis several differentially expressed genes and miRNAs in muscle from veteran football players (VPG) compared to active untrained elderly subjects (CG); here we focussed on miRNA-1303 (miR-1303). The aims of the present research were: to analyse the effects of football training on the expression of miR-1303 and to identify its putative target involved in the longevity pathways in skeletal muscle from VPG compared to CG. METHODS: RNA samples from 12 VPG and 12 CG muscle biopsies were used to validate miR-1303 expression. Crossing four different bioinformatic algorithms, we identified 16 putative targets of miR-1303; from these, BAG-2, KLHL7 and KBTBD6 were chosen for further validation by Western blot analysis in LHCN-M2 human myoblasts transiently transfected with miR-1303. RESULTS: Football training down-regulates miR-1303 expression in muscle from VPG compared to CG and the expression of BAG-2, a chaperon protein involved in the autophagy pathway, inversely correlated to overexpression of miR-1303 in a time-dependent manner, indicating that it is a miR-1303 potential target. CONCLUSIONS: This is the first report, to our knowledge, describing miR-1303 regulation in skeletal muscle by football training and the identification of a target protein, BAG-2, involved in the autophagy pathway. This result contributes to the enlargement of knowledge on the molecular mechanisms linking football training, autophagy and longevity.

Serum from differently exercised subjects induces myogenic differentiation in LHCN-M2 human myoblasts.

Myogenesis is the formation of muscle tissue from muscle precursor cells. Physical exercise induces satellite cell activation in muscle. Currently, C2C12 murine myoblast cells are used to study myogenic differentiation. Herein, we evaluated whether human LHCN-M2 myoblasts can differentiate into mature myotubes and express early (myotube formation, creatine kinase activity and myogenin) and late (MyHC-ß) muscle-specific markers when cultured in differentiation medium (DM) for 2, 4 and 7 days. We demonstrate that treatment of LHCN-M2 cells with DM supplemented with 0.5% serum from long-term (3 years) differently exercised subjects for 4 days induced myotube formation and significantly increased the early (creatine kinase activity and myogenin) and late (MyHC-ß expression) differentiation markers versus cells treated with serum from untrained subjects. Interestingly, serum from aerobic exercised subjects (swimming) had a greater positive effect on late-differentiation marker (MyHC-ß) expression than serum from anaerobic (body building) or from mixed exercised (soccer and volleyball) subjects. Moreover, p62and anti-apoptotic Bcl-2 protein expression was lower in LHCN-M2 cells cultured with human sera from differently exercised subjectst han in cells cultured with DM. In conclusion, LHCN-M2 human myoblasts represent a species-specific system with which to study human myogenic differentiation induced by serum from differently exercised subjects.

Effect of lifelong football training on the expression of muscle molecular markers involved in healthy longevity.

PURPOSE: We investigated whether lifelong football training affects the expression of healthy longevity-related muscle molecular markers. METHODS: Biopsies were collected from the vastus lateralis muscle of 10 lifelong football-trained men (68.2 ± 3.0 years) and of 10 active untrained healthy men (66.7 ± 1.3 years). Gene and protein expression was measured by RTqPCR on RNA and by western blotting on protein extracts from muscle biopsies, respectively. RESULTS: The expression of AMPKα1/α2, NAMPT, TFAM and PGC1α, which are markers of oxidative metabolism, and MyHC ß isoform expression was higher in the muscle of football-trained men vs untrained men. Also citrate synthase activity was higher in trained than in untrained men (109.3 ± 9.2 vs 75.1 ± 9.2 mU/mg). These findings were associated with a healthier body composition in trained than in untrained men [body weight: 78.2 ± 6.5 vs 91.2 ± 11.2 kg; body mass index BMI: 24.4 ± 1.6 vs 28.8 ± 4.0 kg m-2; fat%: 22.6 ± 8.0 vs 31.4 ± 5.0%)] and with a higher maximal oxygen uptake (VO2max: 34.7 ± 3.8 vs 27.3 ± 4.0 ml/min/kg). Also the expression of proteins involved in DNA repair and in senescence suppression (Erk1/2, Akt and FoxM1) was higher in trained than in untrained men. At BMI- and age-adjusted multiple linear regression analysis, fat percentage was independently associated with Akt protein expression, and VO2max was independently associated with TFAM mRNA and with Erk1/2 protein expression. CONCLUSIONS: Lifelong football training increases the expression of key markers involved in muscle oxidative metabolism, and in the DNA repair and senescence suppression pathways, thus providing the molecular basis for healthy longevity.

Effects of long-term football training on the expression profile of genes involved in muscle oxidative metabolism.

We investigated whether long-term recreational football training affects the expression of health-related biochemical and molecular markers in healthy untrained subjects. Five untrained healthy men trained for 1 h 2.4 times/week for 12 weeks and 1.3 times/week for another 52 weeks. Blood samples and a muscle biopsy from the vastus lateralis were collected at T0 (pre intervention) and at T1 (post intervention). Gene expression was measured by RTqPCR on RNA extracted from muscle biopsies. The expression levels of the genes principally involved in energy metabolism (PPARγ, adiponectin, AMPKα1/α2, TFAM, NAMPT, PGC1α and SIRT1) were measured at T0 and T1. Up-regulation of PPARγ (p < 0.0005), AMPKα1 (p < 0.01), AMPKα2 (p < 0.0005) and adiponectin was observed at T1 vs T0. Increases were also found in the expression of TFAM (p < 0.001), NAMPT (p < 0.01), PGC1α (p < 0.01) and SIRT1 (p < 0.01), which are directly or indirectly involved in the glucose and lipid oxidative metabolism. Multiple linear regression analysis revealed that fat percentage was independently associated with NAMPT, PPARγ and adiponectin expression. In conclusion, long-term recreational football training could be a useful tool to improve the expression of muscle molecular biomarkers that are correlated to oxidative metabolism in healthy males.

Functional foods and cardiometabolic diseases* International Task Force for Prevention of Cardiometabolic Diseases.

Mounting evidence supports the hypothesis that functional foods containing physiologically-active components may be healthful. Longitudinal cohort studies have shown that some food classes and dietary patterns are beneficial in primary prevention, and this has led to the identification of putative functional foods. This field, however, is at its very beginning, and additional research is necessary to substantiate the potential health benefit of foods for which the diet-health relationships are not yet scientifically validated. It appears essential, however, that before health claims are made for particular foods, in vivo randomized, double-blind, placebo controlled trials of clinical end-points are necessary to establish clinical efficacy. Since there is need for research work aimed at devising personalized diet based on genetic make-up, it seems more than reasonable the latter be modeled, at present, on the Mediterranean diet, given the large body of evidence of its healthful effects. The Mediterranean diet is a nutritional model whose origins go back to the traditional dietadopted in European countries bordering the Mediterranean sea, namely central and southern Italy, Greece and Spain; these populations have a lower incidence of cardiovascular diseases than the North American ones, whose diet is characterized by high intake of animal fat. The meeting in Naples and this document both aim to focus on the changes in time in these two different models of dietary habits and their fall out on public health.

DHT and IGF-1 in peripheral blood lymphocytes: new markers for the biological passport of athletes.

We performed a pilot study using human peripheral blood lymphocytes (PBL) as a novel system to identify new biomarkers of dihydrotestosterone (DHT) and insulin-like growth factor-1 (IGF-1) abuse in sport. First, to obtain a gene signature, we treated cultures of lymphocytes from sedentary males with three doses of 0.237 microg/ml DHT, each of which is 80-fold the physiological concentration in young adult male serum, at days 0, 2 and 4, or with a single dose of 1.25 microg/ml IGF-1, which is 5-fold the physiological concentration in young adult male serum. We then used the Human Genome U133 Plus 2.0 microarray to identify a gene signature related to DHT or IGF-1 administration. Gene expression was evaluated after 7 and 21 days of DHT treatment, and after 24 h, 72 h and 7 days of IGF-1 treatment. Microarray analysis yielded a list of genes whose expression was altered after DHT or IGF-1 treatment. Among these we selected the genes that are most representative of the pathways associated with skeletal and muscular disorders using the IPA bioinformatics tool. We identified six (IDO1, CXCL13, CCL1, GZMB, VDR and IL2RA) and two (FN1 and RAB31) genes that were up-regulated in lymphocytes from sedentary subjects after 7 days of DHT and IGF-1 treatment, respectively. The expression of these genes in lymphocytes from differently trained athletes was either down-regulated or similar to that in lymphocytes from sedentary subjects. This finding suggests that up-regulation was due to the drug and not to physical exercise. In conclusion, we demonstrate that PBL can be useful in anti-doping checks, and we describe new biomarkers of DHT and IGF-1 abuse which can be included in the Athlete's Biological Passport.

Four novel UCP3 gene variants associated with childhood obesity: effect on fatty acid oxidation and on prevention of triglyceride storage.

OBJECTIVE: The objective of the study was to look for uncoupling protein 3 (UCP3) gene variants in early-onset severe childhood obesity and to determine their effect on long-chain fatty acid oxidation and triglyceride storage. METHODS AND RESULTS: We identified four novel mutations in the UCP3 gene (V56M, A111V, V192I and Q252X) in 200 children with severe, early-onset obesity (body mass index-standard deviation score >2.5; onset: <4 years) living in Southern Italy. We evaluated the role of wild-type (wt) and mutant UCP3 proteins in palmitate oxidation and in triglyceride storage in human embryonic kidney cells (HEK293). Palmitate oxidation was ∼60% lower (P<0.05; P<0.01) and triglyceride storage was higher in HEK293 cells expressing the four UCP3 mutants than in cells expressing wt UCP3. Moreover, mutants V56M and Q252X exerted a dominant-negative effect on wt protein activity (P<0.01 and P<0.05, respectively). Telmisartan, an angiotensin II receptor antagonist used in the management of hypertension, significantly (P<0.05) increased palmitate oxidation in HEK293 cells expressing wt and mutant proteins (P<0.05; P<0.01), including the dominant-negative mutants. CONCLUSIONS: These data indicate that protein UCP3 affects long-chain fatty acid metabolism and can prevent cytosolic triglyceride storage. Our results also suggest that telmisartan, which increases fatty acid oxidation in rat skeletal muscle, also improves UCP3 wt and mutant protein activity, including the dominant-negative UCP3 mutants.

An Approach to Prevent Frailty in Community Dwelling Older Adults: a pilot study performed in Campania region in the framework of the PERSSILAA project.

We developed and tested an innovative physical training method in older adults that embeds the gym program into everyday life in the most conservative way possible. Physical training was included in the activities of local parishes where older women from Southern Italy spend most of their free time and was delivered by trained physical therapists with the support of an ICT tool known as CoCo. 113 older women (aged 72.0 [69.0-75.0] years) noncompliant to conventional exercise programs participated to the study. 57 of them underwent the final anthropometric assessment and 50 the final physical tests. In study completers handgrip strength and physical performance evaluated with the chair-stand, the two minutes step and the chair-sit and -reach tests significantly improved. Quality of life as evaluated with the EuroQol-5dimension (EQ-5D) questionnaire improved as well. In conclusion, a training program designed to minimally impact on life habits of older people is effective in improving fitness in patients noncompliant to other to physical exercise programs.

Molecular analysis of the adiponectin gene in severely obese patients from southern Italy.

BACKGROUND: Severe obesity is a major worldwide public health concern affecting 0.5-5% of the adult population. Adiponectin (Acpr30), an adipokine secreted from adipocytes, shows pleiotropic beneficial effects on obesity and related disorders. In this study, sequence analysis of Acpr30 gene (ACDC) was performed in a highly selected population of severely obese young adult patients from Southern Italy to investigate the associations between polymorphisms in the ACDC gene and the development of severe obesity concomitantly with other features of the metabolic syndrome. METHODS: The ACDC gene was analyzed by direct sequencing in the severely obese patients (n=220) and compared to healthy controls (n=116). The associations between the ACDC gene single-nucleotide polymorphisms (SNPs) and the levels of serum Acpr30 as well as the correlation with the presence of severe obesity jointly associated with other features of the metabolic syndrome were also investigated. Total serum Acpr30 concentrations were measured by the ELISA method. RESULTS: ACDC gene molecular screening revealed the presence of previously described SNPs and a new nucleotide alteration, c.355T>G, leading to a protein variant, p.L119V. Measurement of serum concentration of Acpr30 demonstrated lower levels of Acpr30 in the obese population compared to controls (30.5+/-28.3 vs. 43.9+/-35.7 microg/ml, p<0.01); in particular, significantly lower Acpr30 concentrations were observed in obese patients bearing c.-11377C>G SNP CG+GG genotypes than in those with CC genotype (22.9+/-20.5 vs. 33.1+/-29.4 microg/ml, p<0.05). CONCLUSIONS: Our results confirmed that low serum levels of Acpr30 are related to severe obesity and a difference in protein expression is associated with variants in ACDC gene promoter region.

Two types of zinc fingers are required for dimerization of the serendipity delta transcriptional activator.

The serendipity (sry) delta zinc finger protein controls bicoid gene expression during Drosophila melanogaster oogenesis. In addition, sry delta mutants display various zygotic phenotypes, ranging from abnormal embryogenesis to sex-biased adult lethality. We report here that sry delta is a sequence-specific transcriptional activator. A single sry delta consensus binding site (SDCS), in either orientation, is sufficient to promote transcription activation in cell culture, and multiple SDCSs mediate a strong synergistic activation, reflecting the cooperativity of sry delta binding to DNA. Further, several lines of evidence strongly suggest that sry delta binds to DNA as a dimer. While each of three point mutations located in the third zinc finger of sry delta drastically reduces its DNA binding affinity, a fourth mutation, located in the N-terminal region of the protein, specifically affects the cooperativity of DNA binding. This mutation reveals the functional importance of a putative Cys2/Cys2 zinc finger motif of a novel type, located outside the DNA binding domain. A systematic deletion analysis shows that interaction between this proposed Cys2/Cys2 motif and a classical Cys2/His2 zinc finger mediates homodimerization, which is required for DNA binding cooperativity.

Metabolic syndrome and ADRB3 gene polymorphism in severely obese patients from South Italy.

OBJECTIVE: To evaluate the prevalence of beta(3)-adrenergic receptor (ADRB3) Trp64Arg polymorphism and its relationship with the metabolic syndrome in severe obesity. DESIGN: Cross-sectional outpatients study. PATIENTS AND METHODS: In 265 (100 men) severely obese non-diabetic subjects and 78 (25 men) healthy volunteers, genomic DNA was isolated from peripheral leukocytes. In obese patients, plasma concentrations of leptin, lipids, glucose and insulin, the homeostasis model assessment index and blood pressure have been measured. The Trp64Arg mutation was identified with the real-time TaqMan method. RESULTS: Neither genotype distribution nor allele frequency differed between the two groups. The metabolic syndrome prevalence was 59% in obese subjects, and was higher in men than in women (65 vs 55%: P=0.03). The body mass index (BMI) was related to age tertiles (beta=0.08; P<0.001; multiple linear regression) in Trp64Arg-positive obese subjects. CONCLUSION: We confirm the high prevalence of the metabolic syndrome among severely obese subjects. ADRB3 polymorphism was significantly related to insulin resistance only in obese male subjects. Moreover, increased BMI was related to age in obese subjects with the ADRB3 polymorphism.

Cis-acting elements in the promoter region of the human aldolase C gene.

We investigated the cis-acting sequences involved in the expression of the human aldolase C gene by transient transfections into human neuroblastoma cells (SKNBE). We demonstrate that 420 bp of the 5'-flanking DNA direct at high efficiency the transcription of the CAT reporter gene. A deletion between -420 bp and -164 bp causes a 60% decrease of CAT activity. Gel shift and DNase I footprinting analyses revealed four protected elements: A, B, C and D. Competition analyses indicate that Sp1 or factors sharing a similar sequence specificity bind to elements A and B, but not to elements C and D. Sequence analysis shows a half palindromic ERE motif (GGTCA), in elements B and D. Region D binds a transactivating factor which appears also essential to stabilize the initiation complex.

Genetic typing of Corallium rubrum.

Corallium rubrum taxonomy is based on morphologic criteria; little is known about its genome. We set up a rapid, easy method based on amplified fragment length polymorphism to characterize the genetic patterns of C. rubrum in an attempt to understand better the evolutionary relations between species from diverse geographic areas and to help define migration patterns. Applying this procedure to C. rubrum specimens from Spain and Italy, we identified 6 AFLP amplification fragments common to the 4 coral populations studied and 4 fragments that differentiated between these populations. Using this characterization we were able to plot a "genetic identity card" of this commercially harvested species, which is also a marker of pollution.

Adrenocortical tumor in a boy: final height is not impaired despite a severe advancement of bone age.

The long-term sequelae on the growth pattern in successfully resected virilizing adrenal tumors (ACT) have not been clearly defined. We report on 10 years follow-up of a boy with virilizing ACT until the attainment of final height. This is the first clinical description in a boy with a marked advancement of bone age, indicating that despite advanced physical and skeletal maturity the prognosis on growth is good, provided that regression of virilization is obtained.

Lichen nitidus of the palms.

A case is presented of lichen nitidus that evolved into an acquired palmar keratoderma in a fifty year old woman; this is a rare manifestation of the disease. Lichen nitidus must be considered in the differential diagnosis of keratoderma palmaris.