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Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene. PKD is characterized by non-spherocytic hemolytic anemia of variable severity and may be fatal in some cases during early childhood. Although not considered the standard of care, allogeneic stem cell transplantation has been shown as a potentially curative treatment, limited by donor availability, toxicity, and incomplete engraftment. Preclinical studies were conducted to define conditions to enable consistent therapeutic reversal, which were based on our previous data on lentiviral gene therapy for PKD. Improvement of erythroid parameters was identified by the presence of 20%–30% healthy donor cells. A minimum vector copy number (VCN) of 0.2−0.3 was required to correct PKD when corrected cells were transplanted in a mouse model for PKD. Biodistribution and pharmacokinetics studies, with the aim of conducting a global gene therapy clinical trial for PKD patients (RP-L301-0119), demonstrated that genetically corrected cells do not confer additional side effects. Moreover, a clinically compatible transduction protocol with mobilized peripheral blood CD34+ cells was optimized, thus facilitating the efficient transduction on human cells capable of repopulating the hematopoiesis of immunodeficient mice. We established conditions for a curative lentiviral vector gene therapy protocol for PKD., Graphical abstract, We have established safe transduction conditions of human hematopoietic stem and progenitors cells (HSPCs) using a therapeutic lentiviral vector to correct pyruvate kinase deficiency (PKD). Lentiviral transduction protocol efficiently corrects HSPCs without affecting engraftment properties. This protocol is being applied to a gene therapy clinical trial for PKD patients.

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials. ispartof: Molecular Therapy vol:29 issue:12 pages:3383-3397 ispartof: location:United States status: published

Soil to plant Transfer Factors (TFs) are commonly used to estimate the food chain transfer of chemical elements. The entry of trace contaminants, such as potentially toxic trace elements (PTEs), from soil, into human food chains, is controlled in the long term by their uptake by plant roots. Some PTEs in the soil-plant system were studied through the TFs in an olive orchard in southern Italy. Soil samples and olive leaves were collected and analysed for Cd, Co, Cr, Cu, Mn, Ni, Pb, Zn by using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). In addition, pH, electrical conductivity, organic matter, clay content and cation exchange capacity were measured in soil samples. Factor of trace elements from soil to olive plants showed average value

Negli ultimi anni l'attenzione di molti gruppi di ricerca è stata rivolta allo studio delle microalghe come potenziale materia prima per la produzione di biocarburanti. Infatti l'ottimizzazione del processo produttivo a partire dalle microalghe potrebbe fornire una valida alternativa all'utilizzo dei combustibili fossili risultando al tempo stesso economica ed ecosostenibile. Nell'ottica di progettazione di un impianto dedicato allo sfruttamento di biomassa algale, devono essere necessariamente valutati gli impatti ambientali legati alle fasi di coltivazione, raccolta, trasformazione, distribuzione, impiego e smaltimento della biomassa stessa. In linea di massima la produzione di biodiesel da oli vegetali richiede grande dispendio di energia ed estese superfici agricole. Da ciò derivano costi molto elevati; infatti è stato calcolato che il costo delle materie prime inciderebbe per circa il 70 - 85% dell'intero costo di produzione (Miao & Wu, 2006). Nel caso dell'impianto di Marina di Gioiosa Jonica, gli impatti sono stati determinati in relazione alle effettive caratteristiche e condizioni operative del sistema prototipale stesso. I principali impatti analizzati sono riconducibili alle risorse idriche, all'uso del suolo, all'uso di fertilizzanti e nutrienti in genere, alla fertilizzazione carbonica, all'uso di combustibili fossili, all'eutrofizzazione, alla problematica delle alghe geneticamente modificate, ai fenomeni di tossicità delle alghe (Wijffels e Barbosa, 2010; Acquafuels, 2010) ed ai ceppi di microalghe utilizzate.

Per limitare l'utilizzo di carburanti derivanti dal petrolio, che contribuiscono al riscaldamento globale e sono disponibili in numero limitato, è necessario studiare ed individuare fonti alternative di approvvigionamento, green e rinnovabili, da destinare alla produzione di biocarburanti. Lo studio effettuato nel presente lavoro ha aperto una nuova strada verso la produzione di microalghe all'interno di un impianto dimostrativo su scala pseudo-industriale da destinare alla produzione di bio-olio e altri output finali quali energia e prodotti a maggiore valore aggiunto. Le microalghe rappresentano una valida alternativa non solo alla produzione di carburanti di origine fossile, ma anche ai biocarburanti di prima generazione che utilizzano piante superiori prodotte tramite i normali metodi di coltivazione. Però, al momento non esistono processi produttivi in grado di fornire quantitativi di biodiesel adeguati ad una scala produttiva elevata e che garantiscano un bilancio positivo dal punto di vista economico ed energetico. I punti critici sono rappresentati soprattutto dai processi per la separazione della biomassa microalgale dal mezzo acqueo di coltura e dall'estrazione dell'olio contenuto nelle microalghe, ancora molto costosi per grandi quantitativi. Il sistema studiato ha tentato di superare queste criticità, focalizzandosi sull'individuazione di un metodo semplice da utilizzare, con il minimo impiego di risorse energetiche e con un'alta riproducibilità ed esportabilità. Quest'ultimo aspetto diventa fondamentale per poter replicare le metodologie di coltivazione studiate anche in altre realtà, soprattutto quelle del bacino del Mediterraneo che presentano caratteristiche climatiche ed ambientali molto simili al sito di installazione dell'impianto prototipale. I risultati raggiunti sono stati soddisfacenti; le soluzioni individuate, seppure rispondono positivamente in particolare ai problemi di dewatering e di estrazione degli olii, per poter essere effettivamente implementate richiedono ancora un ulteriore step di ottimizzazione.

Objectives To determine whether anti-GM 1 antibodies are increased in Parkinson's disease (PD). Methods Serum immunoglobulin M (IgM) and IgG anti-GM1 antibodies were detected by enzyme-linked immunosorbent assay (ELISA) in 147 patients with PD and in 186 age-matched normal control subjects. Sera were assayed at initial dilution of 1:800 for IgM and 1:200 for IgG and were considered positive at absorbance values exceeding the value of 0.05 for IgM and 0.1 for IgG. Results Forty patients with PD (27.2%) had sera positive for IgM anti-GM 1 antibodies, whereas only five normal controls (2.7%) resulted positive (P < 0.0001). Most of patients (75%) with positive sera had a tremor-dominant form of PD. Only two patients with PD (1.4%) and none of normal controls had sera positive for IgG anti-GM 1 antibodies. Conclusion -A consistent portion of parkinsonians, mainly with a tremor-dominant form of PD, may have increased circulating IgM anti-GM 1 antibodies.

Il progetto, in questo secondo stato di avanzamento intermedio, ha condotto attività realizzative strumentali e connesse agli OO.RR. che avrebbero dovuto essere perseguiti, in toto ovvero in parte, già da questa fase intermedia di prosecuzione dell'esecuzione progettuale. Infatti, nel secondo semestre di esecuzione, la pianificazione temporale di progetto prevedeva lo svolgimento di attività realizzative funzionali a tutti gli OO.RR., con l'eccezione del solo O.R.6 («Estrazione di lipidi dalla biomassa algale»). Dunque, nel rispetto dell'impianto progettuale condiviso e concertato, è stato regolarmente avviato il perseguimento di tutti i previsti OO.RR. salvo che del predetto O.R.6, il cui conseguimento graduale inizierà e si compirà in periodi successivi.

Agricultural soil geochemistry is an important research issue because soil pollution by potentially toxic trace elements (PTEs) is of concern worldwide. High concentrations of PTEs in soil pose a threat for human health because they can be taken up into the trophic chain via assimilation by plants. Mapping the PTEs in soil is essential to delineate contamination. Geostatistical methods provide us a valuable tool to study spatial structure of the PTEs. They take into account spatial autocorrelation of data to create mathematical models of spatial correlation structures commonly expressed by variograms. The aims of this study were to quantify the spatial structure and the relationships of some PTEs (Ag, Be, Bi, Cd, Co, Cr, Cu, Ni, Pb, Sn, Tl, Zn): To do that a multivariate geostatistical approach was used. The experimental area (100 m x 100 m) was an olive orchard located in southern Italy (Calabria) where at 100 locations topsoil (0-0.20 m) samples were collected. Soil samples were ground, dried, weighed, digested in aqua regia and analyzed in laboratory for Ag, Be, Bi, Cd, Co, Cr, Cu, Ni, Pb, Sn, Tl, and Zn by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Four elements exceeded the regulatory threshold value (Limit A) in analysed soils: Be, Sn, Zn, and V, while Cr, Pb, Cu, Tl, Co, Ni and Cd were within the tolerability limits. A variographic analysis was carried out to quantify the spatial structure of PTEs and no significant difference as a function of direction was found. Mapping the spatial distribution of PTEs allowed defining their spatial relationships and delineating the potentially risky areas. Finally, stochastic images generated by the sequential Gaussian simulation were jointly combined to calculate the probability of exceeding the regulatory threshold values that might cause concern for human health.

The unlikely high frequency of G6PD deficiency which we found in a mountain village (San Giovanni in Fiore: province of Cosenza — Calabria, Southern Italy), with respect to the mean frequency of the other mountain villages in the same province, led us to carry out some investigations on the population of this village to understand this difference. The historical analysis, together with the social-demographic, endogamic and isonymyc study revealed the isolation of this village; while, the analysis of the allele frequencies of AK, ADA, 6PGD, PGM1, ABO and Rh markers revealed no significant differences if compared to the rest of the province. But the AK*5 variant which we found in ten unrelated subjects with a frequency of 0.0104, allows us to hypothesise together with the high G6PD frequency a random genetic drift effect on this village.

Complex diseases such as cardiovascular disease are likely due to the effects of high-order interactions among multiple genes and demographic factors. Therefore, in order to understand their underlying biological mechanisms, we need to consider simultaneously the effects of genotypes across multiple loci. Statistical methods such as multifactor dimensionality reduction (MDR), the combinatorial partitioning method (CPM), recursive partitioning (RP), and patterning and recursive partitioning (PRP) are designed to uncover complex relationships without relying on a specific model for the interaction, and are therefore well-suited to this data setting. However, the theoretical overlap among these methods and their relative merits have not been well characterized. In this paper we demonstrate mathematically that MDR is a special case of RP in which (1) patterns are used as predictors (PRP), (2) tree growth is restricted to a single split, and (3) misclassification error is used as the measure of impurity. Both approaches are applied to a case-control study assessing the effect of eleven single nucleotide polymorphisms on coronary artery calcification in people at risk for cardiovascular disease.

Amyotrophic lateral sclerosis (ALS) occurs in both sporadic (SALS) and, less commonly, familial (FALS) forms. In familial cases, inheritance is usually autosomal dominant, but recessive transmission of the disease also occurs.6 In a subset (15-20%) of FALS cases the disease is caused by mutations in the gene coding Cu-Zn superoxide dismutase (Cu-Zn SOD1) on chromosome 21.5 In SALS cases, mutations have rarely been found. Variability in disease duration exists within families with the same SOD1 mutation.

We conducted a pharmacokinetic study in 164 patients with sporadic Parkinson's disease (PD) to address the relationship between body weight and levodopa pharmacokinetics. Patients underwent an oral acute levodopa test with 250 mg levodopa and pharmacokinetic variables were further assessed. Plasmatic levodopa area under the curve (AUC-l) and body weight were significantly and inversely correlated. Women were significantly lighter and more dyskinetic than men, and had greater AUC-l values. Our data suggest that during long-term treatment, lighter PD patients, especially women, may receive a greater cumulative dosage of levodopa per kilogram of body weight. This could explain gender differences for the development of levodopa-induced peak-dose dyskinesias observed during the course of the disease.

Changes in body weight may induce substantial variations in peripheral pharmacokinetics of drugs, but the relation between body weight and levodopa (LD) pharmacokinetics has never been investigated in Parkinson's disease. To address this issue, we conducted a pharmacokinetic study with 164 patients with sporadic Parkinson's disease. Patients underwent an oral acute LD test with 250 mg of LD, and pharmacokinetic variables were assessed at baseline and at 30, 60, 120, and 240 minutes after LD administration. Plasmatic-LD areas under the curve and body weight were significantly and inversely correlated as well as the elimination of the half-life of LD and body weight. In our sample, women were significantly lighter and had a significantly greater area under the curve than men. Moreover, a greater percentage of women showed LD peak-dose dyskinesias compared with men. Our findings suggest that lighter patients with Parkinson's disease probably receive a greater cumulative dosage of LD per kilogram of body weight during long-term treatment, because in clinical practice, LD is administered without any adjustment of the dose to body weight. This could explain gender differences for the development of LD- induced peak-dose dyskinesias observed during the course of the disease.

Objectives The aim of this study was to correlate the brain atrophy with serum levels of anti-GM1 antibodies in patients with relapsing–remitting multiple sclerosis (RRMS). Methods Plasma sample from 52 patients with RRMS and 65 healthy controls were examined for anti-GM1 antibodies. Patients with RRMS underwent to MRI study with automated method called SIENAX that calculated an estimate of gray matter (GMV) and white matter (WMV) volumes. Results The percentage of RRMS patients with increased anti-GM1 was 37.8%. Elevated levels of anti-GM1 antibodies did not correlate with brain atrophy. Conclusions Anti-GM1 antibodies do not represent a marker of axonal damage in patients with RRMS.

Objective: To determine the modifications of the long-duration response to levodopa in PD over a 1-year period.Background: The development of predictable motor fluctuations in PD has been attributed mainly to modifications over time of the short-duration response to levodopa, whereas the role of the long-duration response has not been widely investigated.Methods: In 17 patients with PD the authors examined prospectively both the short-duration response and the long-duration response to levodopa under standardized conditions on two different occasions separated by a period of approximately 1 year (11.7 ± 3.6 months).Results: At the end of the follow-up period, the short-duration response increased in magnitude but did not change significantly in duration. A total of 24% of patients lost the long-duration response 1 year after their first examination, but a sustained long-duration response could be reestablished by shortening the interdose interval for levodopa intake. Moreover, the duration of the long-duration response after discontinuation of treatment became significantly shorter during 1 year.Conclusion: Modifications of the long-duration response may have a pivotal role in generating a fluctuating response, and suggest that therapeutic strategies based on maintenance of the long-duration response should be sought to avoid the appearance of motor fluctuations.

1315 school-boys of the Cosenza province (Calabria - southern Italy) were subtyped for the PGM1 locus. Three subsamples were examined, two from the Tyrrhenian and the Ionian coasts, respectively, and one from the internal part of this province. The PGM1*1S, PGM1*2S and PGM1*2F gene frequency estimates obtained for the population of the Ionian coast were significantly different from those of the tyrrhenian coast. Since the three subsamples were of similar size, estimates weighted also with the respective population sizes were calculated.

Oxidative stress has been implicated as a major contributor to selective neuronal death in Parkinson's disease (PD). Vitamin E is an antioxidant that may protect the brain from free radical-induced oxidative damage. It is, therefore, reasonable to hypothesize that low levels of vitamin E concentrations may increase the risk of developing PD. To elucidate the possible role of vitamin E in the pathogenesis of PD, we assessed the plasma levels of vitamin E, measured by high-performance liquid chromatography (HPLC), in 54 patients with PD. Vitamin E concentrations were also assessed in 93 age and sex matched normal individuals. The mean plasma levels of vitamin E did not differ significantly between these two groups (22.5±8.15 μmol/l for PD patients and 21.0±7.9 μmol/l for controls). The results of our study suggest that plasma vitamin E concentrations do not play a major role in the pathogenesis of PD.