Proliferation of psychrotrophic bacteria in cold-stored platelet concentrates.

BACKGROUND AND OBJECTIVES: Platelet concentrates (PC) are stored at 20-24°C to maintain platelet functionality, which may promote growth of contaminant bacteria. Alternatively, cold storage of PC limits bacterial growth; however, data related to proliferation of psychotrophic species in cold-stored PC (CSP) are scarce, which is addressed in this study. MATERIALS AND METHODS: Eight laboratories participated in this study with a pool/split approach. Two split PC units were spiked with ~25 colony forming units (CFU)/PC of Staphylococcus aureus, Klebsiella pneumoniae, Serratia liquefaciens, Pseudomonas fluorescens and Listeria monocytogenes. One unit was stored under agitation at 20-24°C/7 days while the second was stored at 1-6°C/no agitation for 21 days. PC were sampled periodically to determine bacterial loads. Five laboratories repeated the study with PC inoculated with lyophilized inocula (~30 CFU/mL) of S. aureus and K. pneumoniae. RESULTS: All species proliferated in PC stored at 20-24°C, reaching concentrations of ≤109 CFU/mL by day 7. Psychrotrophic P. fluorescens and S. liquefaciens proliferated in CSP to ~106 CFU/mL and ~105 CFU/mL on days 10 and 17 of storage, respectively, followed by L. monocytogenes, which reached ~102 CFU/mL on day 21. S. aureus and K. pneumoniae did not grow in CSP. CONCLUSION: Psychrotrophic bacteria, which are relatively rare contaminants in PC, proliferated in CSP, with P. fluorescens reaching clinically significant levels (≥105 CFU/mL) before day 14 of storage. Cold storage reduces bacterial risk of PC to levels comparable with RBC units. Safety of CSP could be further improved by implementing bacterial detection systems or pathogen reduction technologies if storage is beyond 10 days.

Monte-Carlo simulation and tissue-phantom model for validation of ocular oximetry.

Ocular oximetry, in which blood oxygen saturation is evaluated in retinal tissues, is a promising technique for the prevention, diagnosis and management of many diseases and conditions. However, the development of new tools for evaluating oxygen saturation in the eye fundus has often been limited by the lack of reference tools or techniques for such measurements. In this study, we describe a two-step validation method. The impact of scattering, blood volume fraction and lens yellowing on the oximetry model is investigated using a tissue phantom, while a Monte Carlo model of the light propagation in the eye fundus is used to study the effect of the fundus layered-structure. With this method, we were able to assess the performance of an ocular oximetry technique in the presence of confounding factors and to quantify the impact of the choroidal circulation on the accuracy of the measurements. The presented strategy will be useful to anyone involved in studies based on the eye fundus diffuse reflectance.

Comparison of Neutrophil Function in Granulocyte Concentrates From Prednisone- and G-CSF-Treated Donors: Effect of Stimulant, Leukapheresis and Storage.

Transfusion of granulocyte concentrates (GC) is an alternative therapy for neutropenic patients with life-threatening infections. While neutrophils are the main source of antimicrobial activity, only neutrophil numbers are used to certify GCs. The objective of this study was thus to functionally characterize neutrophils in GCs prepared by leukapheresis from G-CSF-stimulated donors and compare to the less characterized prednisone GCs. GCs prepared from healthy donors stimulated with prednisone and then G-CSF after a 6-month washout period were analyzed prior to and after leukapheresis, and after storage. Leukocyte composition, neutrophil viability, calcium mobilization, chemotaxis, phagocytosis, reactive oxygen species, cytokine production and metabolites were determined. G-CSF GCs contained significantly more neutrophils than prednisone GCs of which 40% were immature. In comparison to non-stimulated healthy donor neutrophils, prednisone GC neutrophils exhibited enhanced phagocytosis and G-CSF GC neutrophils showed decreased chemotaxis but increased IL-8 production. Leukapheresis altered prednisone GC neutrophil responses. Storage had a significant, negative impact on G-CSF GC neutrophils compared to prednisone GC neutrophils. G-CSF and prednisone GC neutrophils thus differ in maturity and function, and G-CSF GC neutrophils are more sensitive to storage. Functional testing of GC neutrophils and better storage conditions would improve the quality of this blood product.

Comparison of the Effect of Holder Pasteurization and High-Pressure Processing on Human Milk Bacterial Load and Bioactive Factors Preservation.

OBJECTIVES: This project aims at comparing the impact of Holder pasteurization (HoP) and high-pressure processing (HPP) on bacterial load and retention of immunological components in human milk. METHODS: Human milk samples discarded by the Public Mothers' milk bank (Montreal, Canada) for bacterial purpose were pooled (n = 6) and pasteurized either by heating in a water bath (62.5°C, 30 minutes) or by HPP treatment (425 MPa, four cycles of 6 minutes, initial milk temperature of 4°C or 37°C). Bacterial load, lysozyme activity, and levels of immunoglobulins, lactoferrin, lipase, and 26 cytokines were analyzed. Untreated milk samples from same pools served as control. RESULTS: HPP treatment of milk allows a similar elimination of bacteria than HoP; bacterial counts were under the detection limit [<3 colony-forming units (CFU)/mL] in 50% of milk pools after HPP treatment, compared to 17% for HoP. With initial heating of samples to 37°C before HPP treatment, inactivation to an extent under the detection limit was reached in 67% of pools. There is no significant difference in IgA, lysozyme, and cytokines concentrations between untreated milk and all treatment methods. While no significant difference was observed in the amount of lipase (P > 0.07) and IgG (P > 0.11) between untreated milk and HPP-treated milk samples, HoP seems to be damaging for these factors (P < 0.04). IgM is well preserved in HPP-4°C samples compared to untreated milk (P = 0.07) whereas a decrease is observed for this immunoglobulin levels in HPP-37°C and HoP samples (P < 0.01). Lactoferrin activity, is well maintained in HPP-37°C milk samples in comparison to untreated milk samples (P = 0.52). A decrease in activity of this molecule is noted for samples treated with HPP at 4°C (P = 0.02) and this decrease is even more pronounced for HoP samples (P = 0.004). CONCLUSIONS: HPP is a promising alternative to HoP for treatment of human milk intended to preterm babies. Our results demonstrate that HPP treatment of human milk provides safe milk with less detrimental effects on the biochemically and immunologically active milk components than HoP.

Major role of IgM in the neutralizing activity of convalescent plasma against SARS-CoV-2.

Characterization of the humoral response to SARS-CoV-2, the etiological agent of COVID-19, is essential to help control the infection. The neutralization activity of plasma from patients with COVID-19 decreases rapidly during the first weeks after recovery. However, the specific role of each immunoglobulin isotype in the overall neutralizing capacity is still not well understood. In this study, we select plasma from a cohort of convalescent patients with COVID-19 and selectively deplete immunoglobulin A, M, or G before testing the remaining neutralizing capacity of the depleted plasma. We find that depletion of immunoglobulin M is associated with the most substantial loss of virus neutralization, followed by immunoglobulin G. This observation may help design efficient antibody-based COVID-19 therapies and may also explain the increased susceptibility to SARS-CoV-2 of autoimmune patients receiving therapies that impair the production of immunoglobulin M (IgM).

TWEAK Negatively Regulates Human Dicer.

The ribonuclease Dicer plays a central role in the microRNA pathway by processing microRNA precursors (pre-microRNAs) into microRNAs, a class of 19- to 24-nucleotide non-coding RNAs that regulate expression of ≈60% of the genes in humans. To gain further insights into the function and regulation of Dicer in human cells, we performed a yeast two-hybrid (Y2HB) screen using human Dicer double-stranded RNA-binding domain (dsRBD) as bait. This approach identified tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) as a Dicer-interacting protein candidate. Confocal immunofluorescence microscopy revealed the colocalization of Dicer and TWEAK proteins at the perinuclear region of HeLa cells. The Dicer-TWEAK protein interaction was confirmed by coimmunoprecipitation and found not likely to be mediated by RNA. TWEAK dose-dependently reduced pre-microRNA conversion into mature microRNA in Dicer activity assays using extracts of transfected human HEK 293 cells. TWEAK expression also impaired microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. These findings suggest a role for TWEAK-a pro-inflammatory cytokine-in regulating Dicer function and microRNA biogenesis, and its possible involvement in regulating gene expression during inflammatory processes and diseases.

Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation.

Platelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3'UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of proteinmRNA complexes rather than disassembly of Ago2microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation.

Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function.

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen+ ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin+ UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.

The repertoire and features of human platelet microRNAs.

Playing a central role in the maintenance of hemostasis as well as in thrombotic disorders, platelets contain a relatively diverse messenger RNA (mRNA) transcriptome as well as functional mRNA-regulatory microRNAs, suggesting that platelet mRNAs may be regulated by microRNAs. Here, we elucidated the complete repertoire and features of human platelet microRNAs by high-throughput sequencing. More than 492 different mature microRNAs were detected in human platelets, whereas the list of known human microRNAs was expanded further by the discovery of 40 novel microRNA sequences. As in nucleated cells, platelet microRNAs bear signs of post-transcriptional modifications, mainly terminal adenylation and uridylation. In vitro enzymatic assays demonstrated the ability of human platelets to uridylate microRNAs, which correlated with the presence of the uridyltransferase enzyme TUT4. We also detected numerous microRNA isoforms (isomiRs) resulting from imprecise Drosha and/or Dicer processing, in some cases more frequently than the reference microRNA sequence, including 5' shifted isomiRs with redirected mRNA targeting abilities. This study unveils the existence of a relatively diverse and complex microRNA repertoire in human platelets, and represents a mandatory step towards elucidating the intraplatelet and extraplatelet role, function and importance of platelet microRNAs.

Modulation of microRNA Activity by Semi-microRNAs.

The ribonuclease Dicer plays a central role in the microRNA pathway by catalyzing the formation of 19-24-nucleotide (nt) long microRNAs. Subsequently incorporated into Argonaute 2 (Ago2) effector complexes, microRNAs are known to regulate messenger RNA (mRNA) translation. Whether shorter RNA species derived from microRNAs exist and play a role in mRNA regulation remains unknown. Here, we report the serendipitous discovery of a 12-nt long RNA species corresponding to the 5' region of the microRNA let-7, and tentatively termed semi-microRNA, or smiRNA. Using a smiRNA derived from the precursor of miR-223 as a model, we show that 12-nt long smiRNA species are devoid of any direct mRNA regulatory activity, as assessed in a reporter gene activity assay in transfected cultured human cells. However, smiR-223 was found to modulate the ability of the microRNA from which it derives to mediate translational repression or cleavage of reporter mRNAs. Our findings suggest that the 12-nt RNA species, generated along the microRNA pathway, may participate to the control of gene expression by regulating the activity of the related full-length mature microRNA in vivo.

Detection of human Dicer and Argonaute 2 catalytic activity.

The microRNA (miRNA)-guided RNA silencing pathway is a central and well-defined cellular process involved in messenger RNA (mRNA) translational control. This complex regulatory process is achieved by a well orchestrated machinery composed of a relatively few protein components, among which the ribonuclease III (RNase III) Dicer and Argonaute 2 (Ago2) play a central role. These two proteins are essential and it is of particular interest to measure and detect their catalytic activity under various situations and/or conditions. In this chapter, we describe different protocols that aim to study and determine the catalytic activity of Dicer and Ago2 in cell extracts, immune complexes, and size-fractionated cell extracts. Another protocol aimed at assessing miRNA binding to Ago2 is also described. These experimental approaches are likely to be useful to researchers investigating the main steps of miRNA biogenesis and function in human health and diseases.

An innovative noninvasive respiratory stress test indicates significant coronary artery disease.

BACKGROUND: Respiratory maneuvers can uncover manifestations of myocardial ischemia. Some pulse wave characteristics are strongly associated with significant coronary artery disease (S-CAD). An innovative test using the respiratory stress response (RSR) has been developed for the detection of S-CAD. It is based on spectral analysis of finger pulse wave oscillations measured by photoplethysmography during deep, paced breathing at a rate of six breaths per minute (0.1 Hz) over 70 s. METHODS: RSR was assessed, prior to the procedure, in 150 consecutive patients referred for coronary angiography. It was calculated by analyzing the relative spectral power of the respiratory peak area at 0.1 Hz, using proprietary software. The coronary angiograms were analyzed by quantitative coronary angiography by 1 cardiologist who was blinded to the RSR results. S-CAD was defined as luminal stenosis >70% of > or = 1 coronary artery with a diameter > or = 2 mm, or left main stenosis >50%. A valid RSR was obtained in 150 of 153 patients (98%) with a mean age of 58.7 + or - 10.6 years (67% males). RESULTS: S-CAD was found in 36 patients (24%). S-CAD patients had significantly lower RSR compared to patients without S-CAD (6.7% + or - 5.1 vs. 17.4% + or - 10.6; P<.001, respectively). Multivariate logistic regression analysis, adjusted for known CAD risk factors, showed that RSR is a strong independent indicator of S-CAD (odds ratio 41.2, 95% CI 12.2-139.3; P<.001). CONCLUSION: The innovative RSR test is a simple, noninvasive bedside or office-based tool for the detection of S-CAD.

Existence of a microRNA pathway in anucleate platelets.

Platelets have a crucial role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion, which underlie stroke and acute coronary syndromes. Anucleate platelets contain mRNAs and are capable of protein synthesis, raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs), which are known as key regulators of mRNA translation in other cell types. Further analyses revealed that platelets contain the Dicer and Argonaute 2 (Ago2) complexes, which function in the processing of exogenous miRNA precursors and the control of specific reporter transcripts, respectively. Detection of the receptor P2Y(12) mRNA in Ago2 immunoprecipitates suggests that P2Y(12) expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system.

Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element.

The interaction between human immunodeficiency virus type 1 (HIV-1) and RNA silencing pathways is complex and multifaceted. Essential for efficient viral transcription and supporting Tat-mediated transactivation of viral gene expression, the trans-activation responsive (TAR) element is a structured RNA located at the 5' end of all transcripts derived from HIV-1. Here, we report that this element is a source of microRNAs (miRNAs) in cultured HIV-1-infected cell lines and in HIV-1-infected human CD4+ T lymphocytes. Using primer extension and ribonuclease (RNase) protection assays, we delineated both strands of the TAR miRNA duplex deriving from a model HIV-1 transcript, namely miR-TAR-5p and miR-TAR-3p. In vitro RNase assays indicate that the lack of a free 3' extremity at the base of TAR may contribute to its low processing reactivity in vivo. Both miR-TAR-5p and miR-TAR-3p down-regulated TAR miRNA sensor activity in a process that required an integral miRNA-guided RNA silencing machinery. miR-TAR-3p exerted superior gene downregulatory effects, probably due to its preferential release from HIV-1 TAR RNA by the RNase III Dicer. Our study suggests that the TAR element of HIV-1 transcripts releases functionally competent miRNAs upon asymmetrical processing by Dicer, thereby providing novel insights into viral miRNA biogenesis.

Identification of a peach latent mosaic viroid hairpin able to act as a Dicer-like substrate.

The ability of several viroids to induce posttranscriptional gene silencing has been demonstrated; however, the structure recognized by the Dicer enzyme(s) responsible for the initiation of this mechanism remains a mystery. Here, we show that the hairpin known to be implicated in the replication of peach latent mosaic viroid has the ability to trigger the Dicer enzyme(s). This domain, which is composed of a succession of several small stems separated by symmetrical bulges, is reminiscent of the precursor micro-RNAs.