Real-World Effectiveness of Nirmatrelvir/Ritonavir on Coronavirus Disease 2019-Associated Hospitalization Prevention: A Population-based Cohort Study in the Province of Quebec, Canada.

BACKGROUND: Nirmatrelvir/ritonavir has shown to reduce COVID-19 hospitalization and death before Omicron, but updated real-world evidence studies are needed. This study aimed to assess whether nirmatrelvir/ritonavir reduces the risk of COVID-19-associated hospitalization among high-risk outpatients. METHODS: A retrospective cohort study of outpatients with SARS-CoV-2 between March 15 and 15 October 2022, using data from the Quebec clinico-administrative databases. Outpatients treated with nirmatrelvir/ritonavir were compared with infected ones not receiving nirmatrelvir/ritonavir using propensity-score matching. Relative risk (RR) of COVID-19-associated hospitalization within 30 days was assessed using a Poisson regression. RESULTS: A total of 8402 treated outpatients were matched to controls. Regardless of vaccination status, nirmatrelvir/ritonavir treatment was associated with a 69% reduced RR of hospitalization (RR: .31; 95% CI: .28; .36; number needed to treat [NNT] = 13). The effect was more pronounced in outpatients with incomplete primary vaccination (RR: .04; 95% CI: .03; .06; NNT = 8), while no benefit was found in those with a complete primary vaccination (RR: .93; 95% CI: .78; 1.08). Subgroups analysis among high-risk outpatients with a complete primary vaccination showed that nirmatrelvir/ritonavir treatment was associated with a significant decrease in the RR of hospitalization in severely immunocompromised outpatients (RR: .66; 95% CI: .50; .89; NNT = 16) and in high-risk outpatients aged ≥70 years (RR: .50; 95% CI: .34; .74; NNT = 10) when the last dose of the vaccine was received at least 6 months ago. CONCLUSIONS: Nirmatrelvir/ritonavir reduces the risk of COVID-19-associated hospitalization among incompletely vaccinated high-risk outpatients and among some subgroups of completely vaccinated high-risk outpatients.

Altered microRNA Transcriptome in Cultured Human Liver Cells upon Infection with Ebola Virus.

Ebola virus (EBOV) is a virulent pathogen, notorious for inducing life-threatening hemorrhagic fever, that has been responsible for several outbreaks in Africa and remains a public health threat. Yet, its pathogenesis is still not completely understood. Although there have been numerous studies on host transcriptional response to EBOV, with an emphasis on the clinical features, the impact of EBOV infection on post-transcriptional regulatory elements, such as microRNAs (miRNAs), remains largely unexplored. MiRNAs are involved in inflammation and immunity and are believed to be important modulators of the host response to viral infection. Here, we have used small RNA sequencing (sRNA-Seq), qPCR and functional analyses to obtain the first comparative miRNA transcriptome (miRNome) of a human liver cell line (Huh7) infected with one of the following three EBOV strains: Mayinga (responsible for the first Zaire outbreak in 1976), Makona (responsible for the West Africa outbreak in 2013-2016) and the epizootic Reston (presumably innocuous to humans). Our results highlight specific miRNA-based immunity pathways and substantial differences between the strains beyond their clinical manifestation and pathogenicity. These analyses shed new light into the molecular signature of liver cells upon EBOV infection and reveal new insights into miRNA-based virus attack and host defense strategy.

Nonclinical safety assessment of repeated administration and biodistribution of a novel rabies self-amplifying mRNA vaccine in rats.

The novel self-amplifying mRNA (SAM) technology for vaccines consists of an engineered replication-deficient alphavirus genome encoding an RNA-dependent RNA polymerase and the gene of the target antigen. To validate the concept, the rabies glycoprotein G was chosen as antigen. The delivery system for this vaccine was a cationic nanoemulsion. To characterize the local tolerance, potential systemic toxicity and biodistribution of this vaccine, two nonclinical studies were performed. In the repeated dose toxicity study, the SAM vaccine was administered intramuscularly to rats on four occasions at two-week intervals followed by a four-week recovery period. SAM-related changes consisted of a transient increase in neutrophil count, alpha-2-macroglobulin and fibrinogen levels. Transient aspartate aminotransferase and alanine aminotransferase increases were also noted in females only. At necropsy, observations related to the elicited inflammatory reaction, such as enlargement of the draining lymph nodes were observed that were almost fully reversible by the end of the recovery period. In the biodistribution study, rats received a single intramuscular injection of SAM vaccine and then were followed until Day 60. Rabies RNA was found at the injection sites and in the draining lymph nodes one day after administration, then generally decreased in these tissues but remained detectable up to Day 60. Rabies RNA was also transiently found in blood, lungs, spleen and liver. No microscopic changes in the brain and spinal cord were recorded. In conclusion, these results showed that the rabies SAM vaccine was well-tolerated by the animals and supported the clinical development program.

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA.

Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Commercial Dairy Cow Milk microRNAs Resist Digestion under Simulated Gastrointestinal Tract Conditions.

BACKGROUND: MicroRNAs are small, gene-regulatory noncoding RNA species present in large amounts in milk, where they seem to be protected against degradative conditions, presumably because of their association with exosomes. OBJECTIVE: We monitored the relative stability of commercial dairy cow milk microRNAs during digestion and examined their associations with extracellular vesicles (EVs). METHODS: We used a computer-controlled, in vitro, gastrointestinal model TNO intestinal model-1 (TIM-1) and analyzed, by quantitative polymerase chain reaction, the concentration of 2 microRNAs within gastrointestinal tract compartments at different points in time. EVs within TIM-1 digested and nondigested samples were studied by immunoblotting, dynamic light scattering, quantitative polymerase chain reaction, and density measurements. RESULTS: A large quantity of dairy milk Bos taurus microRNA-223 (bta-miR-223) and bta-miR-125b (∼109-1010 copies/300 mL milk) withstood digestion under simulated gastrointestinal tract conditions, with the stomach causing the most important decrease in microRNA amounts. A large quantity of these 2 microRNAs (∼108-109 copies/300 mL milk) was detected in the upper small intestine compartments, which supports their potential bioaccessibility. A protocol optimized for the enrichment of dairy milk exosomes yielded a 100,000 × g pellet fraction that was positive for the exosomal markers tumor susceptibility gene-101 (TSG101), apoptosis-linked gene 2-interacting protein X (ALIX), and heat shock protein 70 (HSP70) and containing bta-miR-223 and bta-miR-125b. This approach, based on successive ultracentrifugation steps, also revealed the existence of ALIX-, HSP70-/low, and TSG101-/low EVs larger than exosomes and 2-6 times more enriched in bta-miR-223 and bta-miR-125b (P < 0.05). CONCLUSIONS: Our findings indicate that commercial dairy cow milk contains numerous microRNAs that can resist digestion and are associated mostly with ALIX-, HSP70-/low, and TSG101-/low EVs. Our results support the existence of interspecies transfer of microRNAs mediated by milk consumption and challenge our current view of exosomes as the sole carriers of milk-derived microRNAs.

Activated platelets can deliver mRNA regulatory Ago2•microRNA complexes to endothelial cells via microparticles.

Platelets play a crucial role in the maintenance of hemostasis, as well as in thrombosis. Upon activation, platelets release small membrane-bound microparticles (MPs) containing bioactive proteins and genetic materials from their parental cells that may be transferred to, and exert potent biological effects in, recipient cells of the circulatory system. Platelets have been shown to contain an abundant and diverse array of microRNAs, and platelet-derived MPs are the most abundant microvesicles in the circulation. Here we demonstrate that human platelets activated with thrombin preferentially release their miR-223 content in MPs. These MPs can be internalized by human umbilical vein endothelial cells (HUVEC), leading to the accumulation of platelet-derived miR-223. Platelet MPs contain functional Argonaute 2 (Ago2)•miR-223 complexes that are capable of regulating expression of a reporter gene in recipient HUVEC. Moreover, we demonstrate a role for platelet MP-derived miR-223 in the regulation of 2 endogenous endothelial genes, both at the messenger RNA and protein levels. Our results support a scenario by which platelet MPs may act as intercellular carriers of functional Ago2•microRNA complexes that may exert heterotypic regulation of gene expression in endothelial cells, and possibly other recipient cells of the circulatory system.

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.

An Expanded Landscape of Unusually Short RNAs in 11 Samples from Six Eukaryotic Organisms.

Small RNA sequencing (sRNA-Seq) approaches unveiled sequences derived from longer non-coding RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA) fragments, known as tRFs and rRFs, respectively. However, rRNAs and RNAs shorter than 16 nt are often depleted from library preparations/sequencing analyses, although they may be functional. Here, we sought to obtain a complete repertoire of small RNAs by sequencing the total RNA from 11 samples of 6 different eukaryotic organisms, from yeasts to human, in an extended 8- to 30-nt window of RNA length. The 8- to 15-nt window essentially contained fragments of longer non-coding RNAs, such as microRNAs, PIWI-associated RNAs (piRNAs), small nucleolar RNAs (snoRNAs), tRNAs and rRNAs. Notably, unusually short RNAs < 16 nt were more abundant than those >16 nt in bilaterian organisms. A new RT-qPCR method confirmed that two unusually short rRFs of 12 and 13 nt were more overly abundant (~3-log difference) than two microRNAs. We propose to not deplete rRNA and to reduce the lower threshold of RNA length to include unusually short RNAs in sRNA-Seq analyses and datasets, as their abundance and diversity support their potential role and importance as biomarkers of disease and/or mediators of cellular function.

MicroRNAs in the Pathobiology and Therapy of Atherosclerosis.

MicroRNAs are short noncoding RNAs, expressed in humans and involved in sequence-specific post-transcriptional regulation of gene expression. They have emerged as key players in a wide array of biological processes, and changes in their expression and/or function have been associated with plethora of human diseases. Atherosclerosis and its related clinical complications, such as myocardial infarction or stroke, represent the leading cause of death in the Western world. Accumulating experimental evidence has revealed a key role for microRNAs in regulating cellular and molecular processes related to atherosclerosis development, ranging from risk factors, to plaque initiation and progression, up to atherosclerotic plaque rupture. In this review, we focus on how microRNAs can influence atherosclerosis biology, as well as the potential clinical applications of microRNAs, which are being developed as targets as well as therapeutic agents for a growing industry hoping to harness the power of RNA-guided gene regulation to fight disease and infection.

Platelet microparticles reprogram macrophage gene expression and function.

Platelet microparticles (MPs) represent the most abundant MPs subtype in the circulation, and can mediate intercellular communication through delivery of bioactives molecules, such as cytokines, proteins, lipids and RNAs. Here, we show that platelet MPs can be internalised by primary human macrophages and deliver functional miR-126-3p. The increase in macrophage miR-126-3p levels was not prevented by actinomycin D, suggesting that it was not due to de novo gene transcription. Platelet MPs dose-dependently downregulated expression of four predicted mRNA targets of miR-126-3p, two of which were confirmed also at the protein level. The mRNA downregulatory effects of platelet MPs were abrogated by expression of a neutralising miR-126-3p sponge, implying the involvement of miR-126-3p. Transcriptome-wide, microarray analyses revealed that as many as 66 microRNAs and 653 additional RNAs were significantly and differentially expressed in macrophages upon exposure to platelet MPs. More specifically, platelet MPs induced an upregulation of 34 microRNAs and a concomitant downregulation of 367 RNAs, including mRNAs encoding for cytokines/chemokines CCL4, CSF1 and TNF. These changes were associated with reduced CCL4, CSF1 and TNF cytokine/chemokine release by macrophages, and accompanied by a marked increase in their phagocytic capacity. These findings demonstrate that platelet MPs can modify the transcriptome of macrophages, and reprogram their function towards a phagocytic phenotype.

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.

Elevated Abundance, Size, and MicroRNA Content of Plasma Extracellular Vesicles in Viremic HIV-1+ Patients: Correlations With Known Markers of Disease Progression.

BACKGROUND: Because of factors only partly understood, the generalized elevated immune activation and inflammation characterizing HIV-1-infected patients are corrected incompletely with antiretroviral therapy (ART). Extracellular vesicles (EVs) including exosomes and microvesicles released by several cell types may contribute to immune activation and dysfunction. EV size, abundance, and content appear to differ according to infection phase, disease progression, and ART. METHODS: We examined whether the size of EVs and the abundance of exosomes in plasma are associated with cell and tissue activation as well as with viral production. Acetylcholinesterase-bearing (AChE+) exosomes in plasma were quantified using an AChE assay. EV size was analyzed using dynamic light scattering. Proteins and microRNAs present in EVs were detected by Western blot and real-time polymerase chain reaction, respectively. RESULTS: Exosomes were found more abundant in the plasma of ART-naive patients. EV size was larger in ART-naive than in ART-suppressed patients, elite controllers, or healthy control subjects. Both exosome abundance and EV sizes were inversely correlated with CD4/CD8 T-cell ratio and neutrophil, platelet, and CD4 T-cell counts and positively correlated with CD8 T-cell counts. A negative correlation was found between CD4 T-cell nadir and exosome abundance, but not EV size. Levels of miR-155 and miR-223 but not miR-92 were strongly correlated negatively with EV abundance and size in ART-naive patients. CONCLUSIONS: Monitoring of circulating EVs and EV-borne microRNA is possible and may provide new insight into HIV-1 pathogenesis, disease progression, and the associated inflammatory state, as well as the efficacy of ART and the treatments intended to reduce immune activation.

Microvascular decompression of cochlear nerve for tinnitus incapacity: pre-surgical data, surgical analyses and long-term follow-up of 15 patients.

The level of success of neurovascular decompression in ponto-cerebellar angle for hemifacial spasm and trigeminal neuralgia has already established the reality of the pathology to explain such symptoms. However, cochlear nerve compression syndrome by vascular loop is still a controversial topic. We have performed a retrospective cases review with long-term follow-up (5-7 years) concerning the results of microvascular decompression surgery of the cochlear nerve via an endoscopy assisted retrosigmoid approach on 15 patients suffering from unilateral incapacitating tinnitus with abnormal auditory brainstem response and an offending vessel on magnetic resonance imaging. During the surgery, a vascular compression was found on every patient. In a long-term follow-up, 53.3% (8 cases) of our tinnitus cases improved and 20% (3 cases) of them were completely cured. The ABR returned to normal in all patients who had good clinical results (diminished or disappeared tinnitus). When a vertebral artery loop (5 cases) was concerned we obtained 80% of good clinical results. No one showed amelioration or sudden aggravation of their hearing. Three cases required surgical correction of cerebrospinal fluid leak and one case developed spontaneously regressive swallowing problems. Such microvascular decompression surgery of the cochlear nerve appears to be successful in treating incapaciting tinnitus in particular when a vertebral artery loop is observed. Therefore, in such a case, one might recommend neurovascular decompression surgery, keeping in mind that the complications of this surgery should be minimized by a careful closure of the retrosigmoid approach. In order to ensure a better selection of patient more accurate cochlear nerve monitoring and functional MRI should be a promising assessment.