Innate immune factors may restrict hematopoietic stem cell (HSC) genetic engineering and contribute to broad individual variability in gene therapy outcomes. Here, we show that HSCs harbor an early, ...constitutively active innate immune block to lentiviral transduction that can be efficiently overcome by cyclosporine H (CsH). CsH potently enhances gene transfer and editing in human long-term repopulating HSCs by inhibiting interferon-induced transmembrane protein 3 (IFITM3), which potently restricts VSV glycoprotein-mediated vector entry. Importantly, individual variability in endogenous IFITM3 levels correlated with permissiveness of HSCs to lentiviral transduction, suggesting that CsH treatment will be useful for improving ex vivo gene therapy and standardizing HSC transduction across patients. Overall, our work unravels the involvement of innate pathogen recognition molecules in immune blocks to gene correction in primary human HSCs and highlights how these roadblocks can be overcome to develop innovative cell and gene therapies.
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•The innate immune factor IFITM3 blocks VSV-G-mediated lentiviral (LV) entry into HSCs•CsH treatment causes rapid and transient IFITM3 degradation•CsH potently enhances LV gene transfer and editing in human long-term repopulating HSCs•CsH negates IFITM3-mediated variability in LV transduction efficiency across donors
Petrillo et al. demonstrate that the innate antiviral factor IFITM3 constitutively inhibits entry of lentiviral vectors into hematopoietic stem cells. CsH efficiently overcomes this block by degrading IFITM3, significantly improving HSC gene transfer and editing efficiency. These findings will improve lentiviral gene therapy in IFITM3-positive stem cells.
Pentraxins like C-reactive protein are key components of the innate immune system. Recently, pentraxin-3 (PTX3) has been proposed to be a specific marker of vascular inflammation, yet its association ...with atherosclerosis is still unclear.
PTX3 serum levels were measured in three independent studies of 132 young men (ARMY Study), 205 young women (ARFY Study) and 562 individuals 55 to 94 years old (Bruneck Study). In contrast to C-reactive protein, PTX3 showed little relationships with classic vascular risk factors and pro-inflammatory conditions. In the population based Bruneck Study, PTX3 level was independently associated with prevalent cardiovascular diseases (multivariable odds ratio 95%CI 3.09 1.65-5.79; P<0.001). Moreover, PTX3 level correlated with the severity of carotid and femoral atherosclerosis and was highest in individuals with multiple vascular territories affected. In contrast, there was no association with elevated intima-media thickness, a precursor lesion of atherosclerosis, in any of the three populations investigated.
Level of PTX3 is independently associated with atherosclerosis and manifest cardiovascular disease but not early vessel pathology. Unlike C-reactive protein, PTX3 is not a component of the classic acute phase response (systemic inflammation) but appears to be more specific for vascular inflammation.
Purpose
Pentraxin 3 (PTX3) is an inflammatory mediator produced by neutrophils, macrophages, myeloid dendritic and endothelial cells. During sepsis a massive inflammatory activation and ...coagulation/fibrinolysis dysfunction occur. PTX3, as a mediator of inflammation, may represent an early marker of severity and outcome in sepsis.
Methods
This study is based on a prospective trial regarding the impact of glycemic control on coagulation in sepsis. Ninety patients admitted to three general intensive care units were enrolled when severe sepsis or septic shock was diagnosed. At enrollment, we recorded sepsis signs, disease severity, coagulation activation prothrombin fragments 1 + 2 (F
1+2
) and fibrinolysis inhibition plasminogen activator inhibitor-1 (PAI-1). We measured plasma PTX3 levels at enrollment, everyday until day 7, then at days 9, 11, 13, 18, 23 and 28. Mortality was recorded at day 90.
Results
Although not different on day 1, PTX3 remained significantly higher in non-survivors than in survivors over the first 5 days (
p
= 0.002 by general linear model). On day 1, PTX3 levels were higher in septic shock than in severely septic patients (
p
= 0.029). Day 1 PTX3 was significantly correlated with platelet count (
p
< 0.001), SAPS II score (
p
= 0.006) and SOFA score (
p
< 0.001). Day 1 PTX3 was correlated with F
1+2
concentration and with PAI-1 activity and concentration (
p
< 0.05 for all).
Conclusions
Persisting high levels of circulating PTX3 over the first days from sepsis onset may be associated with mortality. PTX3 correlates with severity of sepsis and with sepsis-associated coagulation/fibrinolysis dysfunction.
Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can ...interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental studies and preclinical development of ex vivo and in vivo gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 109 transducing unit (TU)/mL and 5 × 104 TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA, and protein impurities, resulting in higher gene transfer and editing efficiency in severe combined immunodeficiency (SCID)-repopulating hematopoietic stem and progenitor cells (HSPCs) ex vivo, as well as reduced activation of inflammatory responses ex vivo and in vivo as compared to TU-matched, laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and preclinical testing.
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Lentiviral vectors (LVs) are powerful gene-transfer tools routinely exploited for distinct research and clinical applications. LVs produced in most research laboratories contain contaminants that can generate confounding effects in experimental studies. Soldi et al. describe a laboratory-scale workflow for purified LV production, highlighting enhanced gene-editing efficiency and diminished inflammatory responses.
Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to lentiviral vector (LV) transduction without compromising their biological properties remains critical for broad-range ...implementation of gene therapy as a treatment option for several inherited diseases. This study demonstrates that the use of one-hit
LV transduction protocols based on either cyclosporin A (CsA) or rapamycin enable as efficient gene transfer as the current two-hit clinical standard into bone marrow-derived CD34
cells while better preserving their engraftment capacity
. CsA was additive with another enhancer of transduction, prostaglandin E2, suggesting that tailored enhancer combinations may be applied to overcome multiple blocks to transduction simultaneously in HSPC. Interestingly, besides enhancing LV transduction, CsA also significantly reduced HSPC proliferation, preserving the quiescent G
fraction and the more primitive multipotent progenitors, thereby yielding the highest engraftment levels
. Importantly, no alterations in the vector integration profiles could be detected between CsA and control transduced HSPC. Overall, the present findings contribute to the development of more efficient and sustainable LV gene therapy protocols, underscoring the benefits of scaling down required vector doses, as well as shortening the HSPC
culture time.
Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited ...extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.
The interferon‐induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of ...pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co‐factors for endosomal antiviral inhibition through high‐throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)‐localized IFITM restriction that targets infectious SARS‐CoV2 and other PM‐fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5‐trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon‐inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS‐CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell‐compartment‐specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.
Synopsis
The interferon‐induced transmembrane proteins (IFITM) can restrict entry of multiple viruses through mechanisms that are still elusive. This work shows that IFITM3‐dependent endosomal restriction of viral entry requires phosphatidylinositol (3,4,5)‐trisphosphate (PIP3) binding.
Inhibition of endosomal viral entry depends on the interaction of lysines within the conserved IFITM3 intracellular loop with PIP3.
PIP3 is an interferon‐inducible phospholipid that acts as a rheostat for endosomal innate immunity.
An interferon‐dependent increase in PIP3 is regulated by the PI3K/Akt/mTOR pathway in a cell‐type dependent manner.
Exogenous PIP3 delivery enhances inhibition of endocytic viral entry, including that of the recent SARS‐CoV2 Omicron variant.
Phosphatidylinositol (3,4,5)‐trisphosphate enhances inhibition of endocytic viral entry via binding to the intracellular loop of interferon‐induced transmembrane protein 3.
In this paper we report methane gas photonic sensors exploiting the principle of absorption-induced redirection of light propagation in coupled resonant cavities. In particular, an example of ...implemented architecture consists of a Fabry-Pérot (FP) resonator coupled to a fibre ring resonator, operating in the near IR. By changing the concentration of the methane gas in the FP region, the absorption coefficient of the FP changes. In turn, the variation of the methane gas concentration allows the redirection of the light propagation in the fibre ring resonator. Then, the methane gas concentration can be evaluated by analysing the ratio between the powers of two resonant modes, counter-propagating in the fibre ring resonator. In this way, a self-referenced read-out scheme, immune to the power fluctuations of the source, has been conceived. Moreover, a sensitivity of 0.37 ± 0.04 dB/%, defined as the ratio between resonant modes at different outputs, in a range of methane concentration included between the 0% and 5%, has been achieved. These results allow a detection limit below the lower explosive limit (LEL) to be reached with a cost-effective sensor system.
In this paper, we propose a new method for AAC encoding detection and bitrate estimation from PCM material. The algorithm is based on a Convolutional Neural Network that can distinguish between eight ...different bitrates. It achieves an average accuracy of 94.65% by analysis of only 116.10 ms of content.
Combinations of different molecules usually co-exuded by plant roots in soil can significantly affect the mobilization of mineral elements from soil. The flavonoids rutin and quercetin appeared to be ...highly efficient in Fe and Mn mobilization from soil, being rutin about 25 times more effective than citrate in extracting Fe from an alkaline calcareous soil, and 15 times in mobilizing Mn from a slight acidic agricultural soil. Quercetin was particularly effective in mobilizing Mn from the acidic soil and 50 times more efficient than citrate. A significant synergistic effect was detected when either quercetin or rutin was combined with citrate, extracting, respectively, 1.7 and 1.5 times more Mn than what is expected by a simple combination of the two. Sorption processes on soil particles were more relevant for flavonoids than for organic acids for which microbial degradation often prevails, with the only exception of oxalate. Citrate was usually the most efficient organic acid in mobilizing major and trace elements from soil even if oxalate was a better extractant for Cu, Zn, and Ni from the alkaline soil. In the acid soil, a synergistic effect among organic acids was observed only for Mn while in alkaline soil it was observed for Si. The mechanism by which Fe is extracted by rutin in the alkaline soil is a reductive one, with one molecule of rutin being capable of mobilizing two atoms of Fe. Also for Mn in the acid soil, quercetin and rutin solubilize this element by a reductive process. However, when quercetin and rutin are combined with citrate, a complex-forming dissolution mechanism also occurs which increases the mobilization of Mn over the expected rates.
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