Sequence-specific modification of a β-thalassemia locus by small DNA fragments in human erythroid progenitor cells

Gene therapy has been proposed as a definitive cure of beta-thalassemia. We applied a gene targeting approach, based on the introduction of small DNA fragments (SDF) into erythroid progenitor cells, to specifically modify the beta-globin gene sequence at codon 39. The strategy was first tested in normal individuals by delivering mutant SDF that were able to produce the beta-39 (C-T) mutation. Secondly, wild-type SDF were electroporated into target cells of beta-3i9/beta-39 b-thalassemic patients to correct the endogenous mutation. In both cases, gene modification was assayed by allele-specific polymerase chain reaction of DNA and mRNA, by restriction fragment length polymorphism analysis and by direct sequencing

Cell therapy: cGMP facilities and manufacturing

Advanced therapies constitute one of the most complex, organizational, and regulatory areas currently approached by clinical researchers in order to explore new therapeutic applications. Basic scientists and clinicians trying to implement cell therapies into clinical practice, may feel overwhelmed by the apparently endless regulatory requirements that apply. However, regulatory agencies have primary responsibility on patient safety and law enforcement are, and should be, their main considerations. Cell- and tissuebased therapies have the potential to treat many conditions, where present conventional treatments are inadequate. The current approach to cell- and tissue-based therapy development requires using good manufacturing production facilities through master and working cell banks. Facilities need to be purpose-designed and accredited by their national medicinal regulatory body and production scientists need to work in close tandem with quality assurances and ethics committees to absolutely ensure the safety of this cellular product

Development and evaluation of diagnostic tests for the serological diagnosis of brucellosis in swine

A competitive enzyme-linked immunosorbent assay (c-ELISA), an indirect ELISA (i-ELISA) and a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) were developed to test for antibodies to Brucella suis in pig and wild boar sera. An anti-Brucella-LPS monoclonal antibody (MAb 4B5A) (c-ELISA and DELFIA) and an anti-swine IgG monoclonal antibody (MAb 10C2G5) (i-ELISA) were used for the three assays. The specificity (Sp) and sensitivity (Se) of the assays gave the following results: Se and Sp = 100% at a cut-off value of 61.0% (B/B0%) for c-ELISA; Sp = 99.1% and Se = 100% at a cut-off value of 21.7% (percentage positivity: PP%) for i-ELISA; Sp = 91.0% and Se = 75% at a cut-off value of 37.0% (B/B0%) for DELFIA. In addition, the performance of a commercial fluorescence polarisation assay (FPA), standardised for bovine sera, was evaluated in swine sera. The specificity and sensitivity obtained were both 100% at a cut-off value of 99.5 (millipolarisation unit values). These results suggest that the combination of c-ELISA, i-ELISA and FPA can be used to improve the serological diagnosis of swine brucellosis

Sviluppo e valutazione di test diagnostici per la sierodiagnosi di brucellosi suina

Sono stati sviluppati una ELISA competitiva (c-ELISA), una ELISA indiretta (i-ELISA) e un test immunologico DELFIA (Dissociation-Enhanced Lanthanide Fluorescence Immunoassay) per la ricerca di anticorpi verso Brucella suis in sieri di maiale e cinghiale. I tre test prevedono l’utilizzo di un anticorpo monoclonale (MAb 4B5A) verso l’LPS di Brucella (c-ELISA e DELFIA) e di un anticorpo monoclonale (MAb 10C2G5) verso le IgG suine (i-ELISA). La specificità (Sp) e la sensibilità (Se) dei tre test sono le seguenti: per la c-ELISA Se e Sp = 100% con un valore di cut-off pari al 61.0% (B/B0%); per la i-ELISA Sp = 99.1% e Se = 100% con un valore di cut-off di 21.7% (PP%); per il DELFIA Sp = 91.0% e Se = 75% ponendo il valore di cut-off al 37.0% (B/B0%). Inoltre sono state valutate le performance, nei confronti di sieri suini, di un test FPA (Fluorescence Polarization Assay) commerciale sviluppato per la ricerca di anticorpi anti-Brucella in sieri bovini; la specificità e la sensibilità ottenute sono entrambe del 100% al valore di cut-off di 99.5 (mP). Questi risultati suggeriscono che la combinazione di c-ELISA, i-ELISA e FPA può essere utilizzata per migliorare la diagnosi di brucellosi suina

A Nosocomial Cluster of Candida inconspicua Infections in Patients with Hematological Malignancies

Candida inconspicua was recovered from three patients with hematological malignancies. Two patients had intravenous-catheter-associated fungemia, whereas the third had fungal hepatitis. The three cases of infection occurred over a period of 1 month in patients staying in adjacent single rooms. In vitro susceptibility testing of fungal strains showed all isolates to be resistant to fluconazole, with MICs greater than 32 μg/ml. All of the strains had identical DNA restriction profiles and randomly amplified polymorphic DNA fingerprints. These data suggest a nosocomially acquired infection emanating from a common source within the hospital environment

Megadoses of Sodium Ascorbate Efficiently Kill HL60 Cells <i>in Vitro</i>: Comparison with Arsenic Trioxide

Arsenic Trioxide (ATO) is widely acknowledged as the treatment of choice for Acute Promyelocytic Leukemia (APL). It is a “two-sided” drug since it can induce differentiation or kill APL and other tumor cells according to the dosage. Part of the cytotoxic effects of ATO on APL cells is due to its pro-oxidant activity, a characteristic which ATO shares with a number of other compounds, including high doses of ascorbate (ASC). In a comparative investigation on the cy-totoxic effects of both ATO and ASC on HL60 (APL) cell lines, in vitro, we have been able to confirm the known cy-totoxic effects of ATO, but, more importantly, we have demonstrated that ASC is significantly more effective than ATO, in killing these cancer cells in vitro, when the concentrations are maintained within the millimolar (mM) range, i.e. the range of plasma concentrations at which ASC induces oxidative damage to tumor cells. Since these plasma lev- els can be reached only by the intravenous administration of high doses of ASC, we propose that intravenous high doses of ASC may represent a potentially revolutionary new approach in the management of APL

Intravenous Infection of Small Ruminants Suggests a Goat-Restricted Host Tropism and Weak Humoral Immune Response for an Atypical Bluetongue Virus Isolate

Bluetongue virus (BTV) is the etiologic agent of bluetongue (BT), a viral WOAH-listed disease affecting wild and domestic ruminants, primarily sheep. The outermost capsid protein VP2, encoded by S2, is the virion’s most variable protein, and the ability of reference sera to neutralize an isolate has so far dictated the differentiation of 24 classical BTV serotypes. Since 2008, additional novel BTV serotypes, often referred to as “atypical” BTVs, have been documented and, currently, the full list includes 36 putative serotypes. In March 2015, a novel atypical BTV strain was detected in the blood of asymptomatic goats in Sardinia (Italy) and named BTV-X ITL2015. The strain re-emerged in the same region in 2021 (BTV-X ITL2021). In this study, we investigated the pathogenicity and kinetics of infection of BTV-X ITL2021 following subcutaneous and intravenous infection of small ruminants. We demonstrated that, in our experimental settings, BTV-X ITL2021 induced a long-lasting viraemia only when administered by the intravenous route in goats, though the animals remained healthy and, apparently, did not develop a neutralizing immune response. Sheep were shown to be refractory to the infection by either route. Our findings suggest a restricted host tropism of BTV-X and point out goats as reservoirs for this virus in the field