iRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I.

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

Quantitative Proteomics Reveals Changes Induced by TIMP-3 on Cell Membrane Composition and Novel Metalloprotease Substrates.

Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies.

Strategy and validation of a consistent and reproducible nucleic acid technique for mycoplasma detection in advanced therapy medicinal products.

Advanced therapy medicinal products (ATMP) are required to maintain their quality and safety throughout the production cycle, and they must be free of microbial contaminations. Among them, mycoplasma contaminations are difficult to detect and undesirable in ATMP, especially for immunosuppressed patients. Mycoplasma detection tests suggested by European Pharmacopoeia are the "culture method" and "indicator cell culture method" which, despite their effectiveness, are time consuming and laborious. Alternative methods are accepted, provided they are adequate and their results are comparable with those of the standard methods. To validate a novel in-house method, we performed and optimized, a real time PCR protocol, using a commercial kit and an automatic extraction system, in which we tested different volumes of matrix, maximizing the detection sensitivity. The results were compared with those obtained with the gold standard methods. From a volume of 10 ml, we were able to recognize all the mycoplasmas specified by the European Pharmacopoeia, defined as genomic copies per colony forming unit ratio (GC/CFU). Our strategy allows to achieve faster and reproducible results when compared with conventional methods and meets the sensitivity and robustness criteria required for an alternative approach to mycoplasmas detection for in-process and product-release testing of ATMP.

Growth Performance and Recovery of Nosocomial Aspergillus spp. in Blood Culture Bottles.

Theoretically, Aspergillus spp. grow in culture media, but frequently, blood cultures of patients with invasive Aspergillosis are negative, even if until now, the reasons are not clear. This aspect underlines the lack of a good strategy for the cultivation and isolation of Aspergillus spp. In order to develop a complete analytical method to detect Aspergillus in clinical and pharmaceutical samples, we investigated the growth performance of two blood culture systems versus the pharmacopeia standard method. At <72 h, all test systems showed comparable sensitivity, about 1−2 conidia. However, the subculture analysis showed a suboptimal recovery for the methods, despite the positive growth and the visualization of the "Aspergillus balls" in the culture media. To investigate this issue, we studied three different subculture approaches: (i) the use of a sterile subculture unit, (ii) the use of a sterile subculture unit and the collection of a larger aliquot (100 µL), following vigorous agitation of the vials, and (iii) to decapsulate the bottle, withdrawing and centrifuging the sample, and aliquot the pellet onto SDA plates. Our results showed that only the third procedure recovered Aspergillus from all positive culture bottles. This work confirmed that our strategy is a valid and faster method to culture and isolate Aspergillus spp. from blood culture bottles.

Efficacy of Three Commercial Disinfectants in Reducing Microbial Surfaces' Contaminations of Pharmaceuticals Hospital Facilities.

To evaluate and validate the efficacy of disinfectants used in our cleaning procedure, in order to reduce pharmaceutical hospital surfaces' contaminations, we tested the action of three commercial disinfectants on small representative samples of the surfaces present in our hospital cleanrooms. These samples (or coupons) were contaminated with selected microorganisms for the validation of the disinfectants. The coupons were sampled before and after disinfection and the microbial load was assessed to calculate the Log10 reduction index. Subsequently, we developed and validated a disinfection procedure on real surfaces inside the cleanrooms intentionally contaminated with microorganisms, using approximately 107-108 total colony forming units per coupon. Our results showed a bactericidal, fungicidal, and sporicidal efficacy coherent to the acceptance criteria suggested by United States Pharmacopeia 35 <1072>. The correct implementation of our cleaning and disinfection procedure, respecting stipulated concentrations and contact times, led to a reduction of at least 6 Log10 for all microorganisms used. The proposed disinfection procedure reduced the pharmaceutical hospital surfaces' contaminations, limited the propagation of microorganisms in points adjacent to the disinfected area, and ensured high disinfection and safety levels for operators, patients, and treated surfaces.

Erratum: Complete intra-laboratory validation of a LAL assay for bacterial endotoxin determination in EBV-specific cytotoxic T lymphocytes.

[This corrects the article DOI: 10.1016/j.omtm.2021.05.002.].

Complete intra-laboratory validation of a LAL assay for bacterial endotoxin determination in EBV-specific cytotoxic T lymphocytes.

Endotoxin content is a critical factor that affects the safety of biological pharmaceutical products. International pharmacopoeias describe several reference methods to determine endotoxin levels in advanced therapy medicinal product (ATMP) preparations. Administration of ATMPs must be done as rapidly as possible to ensure complete viability and potency of the cellular product. To evaluate the endotoxin content in the shortest time possible, we chose to validate an alternative method based on the use of the Charles River Portable Testing System (PTS) and FDA-approved cartridges, compliant with the requirements of the European Pharmacopoeia and providing results in <20 min. Here, we describe a unique and complete validation approach for instrument, personnel, and analytical method for assessment of endotoxins in ATMP matrices. The PTS system provides high sensitivity and fast quantitative results and uses less raw material and accessories compared with compendial methods. It is also less time consuming and less prone to operator variability. Our validation approach is suitable for a validated laboratory with trained personnel capable of conducting the ATMP release tests, and with very low intra-laboratory variability, and meets the criteria required for an alternative approach to endotoxin detection for in-process and product-release testing of ATMPs.

Use of 27G needles improves sensitivity and performance of ATCC anaerobe reference microorganism detection in BacT/Alert system.

Effective detection of microbiological contaminations present in medicinal cellular products is a crucial step to ensure patients' safety. In recent decades, several rapid microbiological methods have been developed and validated, but variabilities linked to the use of different resources have led to discordant validation of methods and performance results. Considering this, while developing an in-house BacT/Alert-based method, we evaluated all of the materials used in its validation. Of particular importance, we noticed that the syringe gauge used to inject the samples into the bottles was crucial to obtain robust results. We chose to conduct a comparative test between the BacT/Alert system and the compendial method described in the European Pharmacopoeia, using five dilutions of nine reference microorganism strains and 21G or 27G needles. Our results confirmed that the BacT/Alert system is a valid and faster alternative method to assess sterility of clinical cell therapy products, and that the use of 27G needles increases its sensitivity to detect reference anaerobe microorganisms.

The sea urchin sns5 insulator protects retroviral vectors from chromosomal position effects by maintaining active chromatin structure.

Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders.

Phenotypical and molecular assessment of the virulence potential of KPC-3-producing Klebsiella pneumoniae ST392 clinical isolates.

Klebsiella pneumoniae is a Gram-negative bacterium of clinical importance, due to its resistance to several antibiotic classes. We have identified 4 clinical isolates of K. pneumoniae sequence type (ST) 392 KPC-3-producing strains from patients at the Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (IRCCS-ISMETT), a Southern Italian transplantation health facility, during a routine surveillance for carbapenemase-producing Enterobacterales from in-house clinical samples. Since those were among, to the best of our knowledge, the first KPC-producing K. pneumoniae ST392 isolated in Europe, we assessed their virulence potential, to understand if this particular ST can become an endemic clinical threat. ST392 isolates were investigated to assess their virulence potential, namely resistance to human sera, formation of abiotic biofilms, adhesion to biotic surfaces, exopolysaccharide production and in vivo pathogenesis in the wax moth Galleria mellonella animal model. ST392-belonging strains were highly resistant to human sera. These strains also have a high capacity to form abiotic biofilms and high levels of adhesion to the human epithelial colorectal adenocarcinoma HT-29 cell line. An increase of transcriptional levels of genes involved in serum resistance (aroE and traT) and adhesion (pgaA) was observed when compared with the Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603 reference strain. Infection of G. mellonella larvae with ST392 clinical isolates showed that the latter were not highly pathogenic in this model. Together, our results indicate that ST392 isolates have the potential to become a strain of clinical relevance, especially in health settings where patients are immunosuppressed, e.g., transplant recipients.