First Results of an External Quality Assessment (EQA) Scheme for Molecular, Serological and Antigenic Diagnostic Test for SARS-CoV-2 Detection in Lombardy Region (Northern Italy), 2020-2022.

For diagnosing SARS-CoV-2 infection and for monitoring its spread, the implementation of external quality assessment (EQA) schemes is mandatory to assess and ensure a standard quality according to national and international guidelines. Here, we present the results of the 2020, 2021, 2022 EQA schemes in Lombardy region for assessing the quality of the diagnostic laboratories involved in SARS-CoV-2 diagnosis. In the framework of the Quality Assurance Programs (QAPs), the routinely EQA schemes are managed by the regional reference centre for diagnostic laboratories quality (RRC-EQA) of the Lombardy region and are carried out by all the diagnostic laboratories. Three EQA programs were organized: (1) EQA of SARS-CoV-2 nucleic acid detection; (2) EQA of anti-SARS-CoV-2-antibody testing; (3) EQA of SARS-CoV-2 direct antigens detection. The percentage of concordance of 1938 molecular tests carried out within the SARS-CoV-2 nucleic acid detection EQA was 97.7%. The overall concordance of 1875 tests carried out within the anti-SARS-CoV-2 antibody EQA was 93.9% (79.6% for IgM). The overall concordance of 1495 tests carried out within the SARS-CoV-2 direct antigens detection EQA was 85% and it was negatively impacted by the results obtained by the analysis of weak positive samples. In conclusion, the EQA schemes for assessing the accuracy of SARS-CoV-2 diagnosis in the Lombardy region highlighted a suitable reproducibility and reliability of diagnostic assays, despite the heterogeneous landscape of SARS-CoV-2 tests and methods. Laboratory testing based on the detection of viral RNA in respiratory samples can be considered the gold standard for SARS-CoV-2 diagnosis.

Glutamine Synthetase 1 Increases Autophagy Lysosomal Degradation of Mutant Huntingtin Aggregates in Neurons, Ameliorating Motility in a Drosophila Model for Huntington's Disease.

Glutamine Synthetase 1 (GS1) is a key enzyme that catalyzes the ATP-dependent synthesis of l-glutamine from l-glutamate and is also member of the Glutamate Glutamine Cycle, a complex physiological process between glia and neurons that controls glutamate homeostasis and is often found compromised in neurodegenerative diseases including Huntington's disease (HD). Here we report that the expression of GS1 in neurons ameliorates the motility defects induced by the expression of the mutant Htt, using a Drosophila model for HD. This phenotype is associated with the ability of GS1 to favor the autophagy that we associate with the presence of reduced Htt toxic protein aggregates in neurons expressing mutant Htt. Expression of GS1 prevents the TOR activation and phosphorylation of S6K, a mechanism that we associate with the reduced levels of essential amino acids, particularly of arginine and asparagine important for TOR activation. This study reveals a novel function for GS1 to ameliorate neuronal survival by changing amino acids' levels that induce a "starvation-like" condition responsible to induce autophagy. The identification of novel targets that inhibit TOR in neurons is of particular interest for the beneficial role that autophagy has in preserving physiological neuronal health and in the mechanisms that eliminate the formation of toxic aggregates in proteinopathies.

Human CD14+ cells loaded with Paclitaxel inhibit in vitro cell proliferation of glioblastoma.

BACKGROUND AIMS: In attempting to develop new strategies to circumvent the immunosuppression associated with glioblastoma (GB), novel approaches have been designed using dendritic cell (DC)-based vaccination, which is considered a promising strategy to attack high-grade glioma. In previous studies, we demonstrated that human mesenchymal stromal cells without genetic manipulation but primed with Paclitaxel (PTX) acquire a potent anti-tumor activity, providing an interesting new biological approach for drug delivery. On the basis of these results, we here investigated whether both CD14+ and their derived DCs may behave like mesenchymal stromal cells acquiring anti-tumor activity on priming with PTX. METHODS: Human CD14+ cells were isolated from peripheral blood. Fluorescence-activated cell sorter analysis was performed to determine the purity of CD14+ and their differentiation into mature DCs. Cells were primed by incubation with 1 µg/mL of PTX for 24 h, and the PTX released by cells was assessed by mass spectrometry analysis. Anti-tumor activity was checked by testing the conditioned medium (CM) on the proliferation of U87 MG, a GB cell line. RESULTS: Both CD14+ and DCs were able to incorporate PTX and release the drug in the CM in a time-dependent manner (maximal release over 24 h). The addition of CM from CD14+ and DCs loaded with PTX strongly inhibits proliferation of U87 MG cells. CONCLUSIONS: Our results are the first demonstration that peripheral blood-derived CD14+ and DCs, in addition to their application for immunotherapy for GB, could also be used to delivery anti-cancer drugs, such as PTX, to kill GB cells.

Mesenchymal stromal cells uptake and release paclitaxel without reducing its anticancer activity.

To improve the drug delivery efficiency on target cells, many strategies have been developed including Mesenchymal Stromal Cells (MSCs) approaches. In a previous study, we found that bone-marrow-derived MSCs (BM-MSCs) were able to incorporate and release the anti-tumor and anti-angiogenic drug, Paclitaxel (PTX). In this study, we evaluated the stability of PTX in standard cell culture conditions by analyzing the metabolites produced by MSCs after their incorporation of the drug. We are able to show that MSCs do not release either 3-OH-PTX or 6-OH-PTX metabolites (having a lower anticancer activity) but release an active PTX molecule together with the isomer 7-Epitaxol, is known to maintain the whole biological activity. This confirms that the simple procedure of MSCs priming with a drug (without any genetic cell manipulation), in our case PTX, does not modify the activity of the molecule and provides a new biological-device to carry and deliver PTX in tumor sites, by contributing to improve drug efficacy and target selectivity in cancer therapy.

Hyperargininemia: 7-month follow-up under sodium benzoate therapy in an Italian child presenting progressive spastic paraparesis, cognitive decline, and novel mutation in ARG1 gene.

BACKGROUND: Hyperargininemia due to mutations in ARG1 gene is an autosomal recessive inborn error of metabolism caused by a defect in the final step of the urea cycle. Common clinical presentation is a variable association of progressive spastic paraparesis, epilepsy, and cognitive deficits. METHODS: We describe the clinical history of an Italian child presenting progressive spastic paraparesis, carrying a new homozygous missense mutation in the ARG1 gene. A detailed clinical, biochemical, and neurophysiological follow-up after 7 months of sodium benzoate therapy is reported. RESULTS: Laboratory findings, gait abnormalities, spastic paraparesis, and electroencephalographic and neurophysiological abnormalities remained quite stable over the follow-up. Conversely, a mild cognitive deterioration has been detected by means of the neuropsychologic assessment. CONCLUSIONS: Further longitudinal studies by means of longer follow-up and using clinical, biochemical, radiological, and neurophysiological assessments are needed in such patients to describe natural history and monitor the effects of treatments.

Riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency with unknown genetic defect.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare autosomal recessively inherited disorder of fatty acid metabolism due to ETFA, ETFB or ETFDH mutations. Riboflavin treatment ameliorates symptoms and metabolic profile in ETFDH-related MADD patients. We report on a 20-year-old boy with an 8-year history of progressive difficulty in walking, running and climbing stairs. Muscle biopsy showed a lipid myopathy and the acylcarnitine profile analysis was suggestive of MADD. Nevertheless, no evidence of molecular defects in the ETFA, ETFB and ETFDH exons or intron-exon boundaries was found. Treatment with riboflavin for 1 year resulted in a clear improvement in motor functions. Our report shows that some cases of MADD are not linked to ETFA, ETFB and ETFDH exon or intron-exon boundary changes. They could be due to quite rare promoter or deep intronic mutations or, most likely, to some unknown genetic defect. We therefore suggest to extend in these cases molecular studies to cDNA analysis and indicate the need of extensive pedigree studies to identify other possible disease-related loci. Most important, treatment of these cases with riboflavin can also be effective. Therefore, early diagnosis is essential to achieve the best treatment response.