Search

Abstract Introduction Acquired factor VIII deficiency is a rare entity that can lead to severe and life-threatening bleeding. We describe a case of severe bleeding from the tongue secondary to acquired hemophilia and discuss treatment options, including aminocaproic acid and recombinant factor VIII, which have not been widely reported in the literature for the management of such patients. Case presentation A 94-year-old Caucasian man presented to our institution with diffuse bruising and extensive bleeding from the tongue secondary to mechanical trauma. He had no prior history of bleeding and his medical history was unremarkable except for dementia and hypertension. Coagulation studies revealed a prolonged activated partial thromboplastin time and a mixing study was consistent with the presence of an inhibitor. Quantitative assays revealed a reduced level of factor VIII activity (1%) and the presence of a factor VIII inhibitor, measured at seven Bethesda units, in the serum. Oral prednisone therapy (60mg/day) was given. He also received intravenous aminocaproic acid and human concentrate of factor VIII (Humate-P) and topical anti-thrombolytic agents (100 units of topical thrombin cream). His hospital course was prolonged because of persistent bleeding and the development of profuse melena. He required eight units of packed red blood cells for transfusion. Hospitalization was also complicated by bradycardia of unclear etiology, which started after infusion of aminocaproic acid. His activated partial thromboplastin time gradually normalized. He was discharged to a rehabilitation facility three weeks later with improving symptoms, stable hematocrit and resolving bruises. Conclusions Clinicians should suspect a diagnosis of acquired hemophilia in older patients with unexplained persistent and profound bleeding from uncommon soft tissues, including the tongue. Use of factor VIII (Humate-P) and aminocaproic acid can be useful in this coagulopathy but clinicians should be aware of possible life-threatening side effects in older patients, including bradycardia.

Pathogens trigger activation of sensors of the innate immune system that initiate molecular signaling enabling appropriate host defense programs. Although recognition of pathogen-specific moieties or PAMPs by specialized receptors of the immune system is well defined for a great number of pathogens, the mechanisms of sensing of pathogen-induced functional perturbations to the host cell remain poorly understood. Here we show that the disruption of endosomal compartments in macrophages by a bacterium or fully synthetic nanoparticles activates stress-response p38MAPK kinase, which triggers execution of cell death of a necrotic type. p38MAPK-mediated necrosis occurs in cells with a compound homozygous deletion of pyroptosis-inducing caspases-1 and -11, apoptotic caspase-8, and necroptosis-inducing receptor-interacting protein kinase-3 (RIPK3), indicating that all of these principal cell death mediators are dispensable for p38MAPK-induced necrosis in response to endosome rupture. p38MAPK-mediated necrosis is suppressed by the receptor-interacting protein kinase 1, RIPK1, and degradation of RIPK1 sensitizes macrophages to necrotic death. Since pathogen-induced cell death of necrotic types is implicated in host defense against infection, our results indicate that functional perturbations in host cells are sensed as a component of the innate immune system., (© 2022. The Author(s).)

Cas13a has been used to target RNA viruses in cell culture, but efficacy has not been demonstrated in animal models. In this study, we used messenger RNA (mRNA)-encoded Cas13a for mitigating influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mice and hamsters, respectively. We designed CRISPR RNAs (crRNAs) specific for PB1 and highly conserved regions of PB2 of influenza virus, and against the replicase and nucleocapsid genes of SARS-CoV-2, and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. We delivered polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract using a nebulizer. In mice, Cas13a degraded influenza RNA in lung tissue efficiently when delivered after infection, whereas in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and reduced symptoms. Our findings suggest that Cas13a-mediated targeting of pathogenic viruses can mitigate respiratory infections.

Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants and the elderly. RSV is a non-segmented negative strand RNA virus. The viral M2-1 protein plays a key role in viral transcription, serving as an elongation factor to enable synthesis of full-length mRNAs. M2-1 contains an unusual CCCH zinc-finger motif that is conserved in the related human metapneumovirus M2-1 protein and filovirus VP30 proteins. Previous biochemical studies have suggested that RSV M2-1 might bind to specific virus RNA sequences, such as the transcription gene end signals or poly A tails, but there was no clear consensus on what RSV sequences it binds. To determine if M2-1 binds to specific RSV RNA sequences during infection, we mapped points of M2-1:RNA interactions in RSV-infected cells at 8 and 18 hours post infection using crosslinking immunoprecipitation with RNA sequencing (CLIP-Seq). This analysis revealed that M2-1 interacts specifically with positive sense RSV RNA, but not negative sense genome RNA. It also showed that M2-1 makes contacts along the length of each viral mRNA, indicating that M2-1 functions as a component of the transcriptase complex, transiently associating with nascent mRNA being extruded from the polymerase. In addition, we found that M2-1 binds specific cellular mRNAs. In contrast to the situation with RSV mRNA, M2-1 binds discrete sites within cellular mRNAs, with a preference for A/U rich sequences. These results suggest that in addition to its previously described role in transcription elongation, M2-1 might have an additional role involving cellular RNA interactions., Competing Interests: The authors have declared that no competing interests exist.

Innate immune responses to pathogens are driven by co-presentation of multiple pathogen-associated molecular patterns (PAMPs). Combinations of PAMPs can trigger synergistic immune responses, but the underlying molecular mechanisms of synergy are poorly understood. Here, we used synthetic particulate carriers co-loaded with monophosphoryl lipid A (MPLA) and CpG as pathogen-like particles (PLPs) to dissect the signaling pathways responsible for dual adjuvant immune responses. PLP-based co-delivery of MPLA and CpG to GM-CSF-driven mouse bone marrow-derived antigen-presenting cells (BM-APCs) elicited synergistic interferon-β (IFN-β) and interleukin-12p70 (IL-12p70) responses, which were strongly influenced by the biophysical properties of PLPs. Mechanistically, we found that MyD88 and interferon regulatory factor 5 (IRF5) were necessary for IFN-β and IL-12p70 production, while TRIF signaling was required for the synergistic response. Both the kinetics and magnitude of downstream TRAF6 and IRF5 signaling drove the synergy. These results identify the key mechanisms of synergistic Toll-like receptor 4 (TLR4)-TLR9 co-signaling in mouse BM-APCs and underscore the critical role of signaling kinetics and biophysical properties on the integrated response to combination adjuvants., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 from in vitro transcribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection., Competing Interests: The authors have declared that no competing interests exist.

The biological pathways that affect drug delivery in vivo remain poorly understood. We hypothesized that altering cell metabolism with phosphatidylinositol (3,4,5)-triphosphate (PIP3), a bioactive lipid upstream of the metabolic pathway PI3K (phosphatidylinositol 3-kinase)/AKT/ mTOR (mammalian target of rapamycin) would transiently increase protein translated by nanoparticle-delivered messenger RNA (mRNA) since these pathways increase growth and proliferation. Instead, we found that PIP3 blocked delivery of clinically-relevant lipid nanoparticles (LNPs) across multiple cell types in vitro and in vivo. PIP3-driven reductions in LNP delivery were not caused by toxicity, cell uptake, or endosomal escape. Interestingly, RNA sequencing and metabolomics analyses suggested an increase in basal metabolic rate. Higher transcriptional activity and mitochondrial expansion led us to formulate two competing hypotheses that explain the reductions in LNP-mediated mRNA delivery. First, PIP3 induced consumption of limited cellular resources, "drowning out" exogenously-delivered mRNA. Second, PIP3 triggers a catabolic response that leads to protein degradation and decreased translation., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

For decades, cationic polymer nanoparticles have been investigated for nucleic acid delivery. Despite promising in vitro transfection results, most formulations have failed to translate into the clinic due to significant in vivo toxicity - especially when delivered intravenously. To address this significant problem, we investigated the detailed mechanisms that govern the complex in vivo systemic toxicity response to common polymeric nanoparticles. We determined that the toxicity response is material dependent. For branched polyethylenimine (bPEI) nanoparticles - toxicity is a function of multiple pathophysiological responses - triggering of innate immune sensors, induction of hepatic toxicity, and significant alteration of hematological properties. In contrast, for chitosan-based nanoparticles - systemic toxicity is primarily driven through innate immune activation. We further identified that modification of primary amines to secondary and tertiary amines using the small molecule imidazole-acetic-acid (IAA) ameliorates in vivo toxicity from both nanocarriers by different, material-specific mechanisms related to Toll-like receptor 4 activation (for bPEI) and complement activation driven neutrophil infiltration (for chitosan), respectively. Our results provide a detailed roadmap for evaluating in vivo toxicity of nanocarriers and identifies potential opportunities to reduce toxicity for eventual clinical translation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Abnormal post-transcriptional regulation induced by alterations of mRNA-protein interactions is critical during tumorigenesis and cancer progression and is a hallmark of cancer cells. A more thorough understanding is needed to develop treatments and foresee outcomes. Cellular and mouse tumor models are insufficient for vigorous investigation as they lack consistency and translatability to humans. Moreover, to date, studies in human tumor tissue are predominately limited to expression analysis of proteins and mRNA, which do not necessarily provide information about the frequency of mRNA-protein interactions. Here, we demonstrate novel optimization of a method that is based on FISH and proximity ligation techniques to quantify mRNA interactions with RNA-binding proteins relevant for tumorigenesis and cancer progression in archival patient-derived tumor tissue. This method was validated for multiple mRNA-protein pairs in several cellular models and in multiple types of archival human tumor samples. Furthermore, this approach allowed high-throughput analysis of mRNA-protein interactions across a wide range of tumor types and stages through tumor microarrays. This method is quantitative, specific, and sensitive for detecting interactions and their localization at both the individual cell and whole-tissue scales with single interaction sensitivity. This work presents an important tool in investigating post-transcriptional regulation in cancer on a high-throughput scale, with great potential for translatability into any applications where mRNA-protein interactions are of interest. SIGNIFICANCE: This work presents an approach to sensitively, specifically, and quantitatively detect and localize native mRNA and protein interactions for analysis of abnormal post-transcriptional regulation in patient-derived archival tumor samples., (©2019 American Association for Cancer Research.)

The characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile, and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can be manufactured rapidly at large scale to meet emerging pathogens. However, its performance as a robust vaccine is strengthened by supplemental immune stimulation, which is typically provided by adjuvant formulations that facilitate delivery and stimulate immune responses. Here, we present a strategy for increasing translation of a model IVT mRNA vaccine while simultaneously modulating its immune-stimulatory properties in a programmable fashion, without relying on delivery vehicle formulations. Substitution of uridine with the modified base N1-methylpseudouridine reduces the intrinsic immune stimulation of the IVT mRNA and enhances antigen translation. Tethering adjuvants to naked IVT mRNA through antisense nucleotides boosts the immunostimulatory properties of adjuvants in vitro, without impairing transgene production or adjuvant activity. In vivo, intramuscular injection of tethered IVT mRNA-TLR7 agonists leads to enhanced local immune responses, and to antigen-specific cell-mediated and humoral responses. We believe this system represents a potential platform compatible with any adjuvant of interest to enable specific programmable stimulation of immune responses.

The translational efficiency of an in vitro transcribed (IVT) mRNA was measured upon delivery to primary skeletal muscle cells and to a mouse model system, towards the development of a predictive in vitro assay for the screening and validation of intramuscular mRNA-based vaccines. When IVT mRNA was delivered either naked or complexed with novel aminoglycoside-based delivery vehicles, significant differences in protein expression in vitro and in vivo were observed. We hypothesized that this previously anticipated discrepancy was due to differences in the mechanism of IVT mRNA endosomal entry and release following delivery. To address this, IVT mRNA was fluorescently labeled prior to delivery, to visualize its distribution. Colocalization with endosomal markers indicated that different entry pathways were utilized in vivo and in vitro, depending on the delivery vehicle, resulting in variations in protein expression levels. Since extracellular matrix stiffness (ECM) influences mRNA entry, trafficking and release, the effect of mechanotransduction on mRNA expression was investigated in vitro upon delivery of IVT mRNA alone, and complexed with delivery vehicles to skeletal muscle cells grown on ∼10 kPa hydrogels. This in vitro hydrogel model more accurately recapitulated the results obtained in vivo upon IM injection, indicating that this approach may assist in the characterization of mRNA based vaccines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The human respiratory syncytial virus G protein plays an important role in the entry and assembly of filamentous virions. Here, we report the use of fluorescently labeled soybean agglutinin to selectively label the respiratory syncytial virus G protein in living cells without disrupting respiratory syncytial virus infectivity or filament formation and allowing for interrogations of respiratory syncytial virus virion assembly. Using this approach, we discovered that plasma membrane-bound respiratory syncytial virus G rapidly recycles from the membrane via clathrin-mediated endocytosis. This event is then followed by the dynamic formation of filamentous and branched respiratory syncytial virus particles, and assembly with genomic ribonucleoproteins and caveolae-associated vesicles prior to re-insertion into the plasma membrane. We demonstrate that these processes are halted by the disruption of microtubules and inhibition of molecular motors. Collectively, our results show that for respiratory syncytial virus assembly, viral filaments are produced and loaded with genomic RNA prior to insertion into the plasma membrane.Assembly of filamentous RSV particles is incompletely understood due to a lack of techniques suitable for live-cell imaging. Here Vanover et al. use labeled soybean agglutinin to selectively label RSV G protein and show how filamentous RSV assembly, initiated in the cytoplasm, uses G protein recycled from the plasma membrane.

The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA binding proteins (RBP) and small RNAs regulate the editing, localization, stabilization, translation, and degradation of ribonucleic acids (RNAs) through their interactions with specific cis-acting elements within target RNAs. Here, we describe a novel method to detect protein-mRNA interactions, which combines FLAG-peptide modified, multiply-labeled tetravalent RNA imaging probes (FMTRIPs) with proximity ligation (PLA), and rolling circle amplification (RCA). This assay detects native RNA in a sequence specific and single RNA sensitive manner, and PLA allows for the quantification and localization of protein-mRNA interactions with single-interaction sensitivity.

The detection of viral dynamics and localization in the context of controlled HIV infection remains a challenge and is limited to blood and biopsies. We developed a method to capture total-body simian immunodeficiency virus (SIV) replication using immunoPET (antibody-targeted positron emission tomography). The administration of a poly(ethylene glycol)-modified, (64)Cu-labeled SIV Gp120-specific antibody led to readily detectable signals in the gastrointestinal and respiratory tract, lymphoid tissues and reproductive organs of viremic monkeys. Viral signals were reduced in aviremic antiretroviral-treated monkeys but detectable in colon, select lymph nodes, small bowel, nasal turbinates, the genital tract and lung. In elite controllers, virus was detected primarily in foci in the small bowel, select lymphoid areas and the male reproductive tract, as confirmed by quantitative reverse-transcription PCR (qRT-PCR) and immunohistochemistry. This real-time, in vivo viral imaging method has broad applications to the study of immunodeficiency virus pathogenesis, drug and vaccine development, and the potential for clinical translation.

This psychological autopsy of the suicidal death of an adolescent Indian boy includes a brief family background, a history of his difficulties, and a report of his psychological evaluation. There is an attempt to understand the societal and familial factors that seem to have prediposed him to commit suicide. The report includes an analysis of the resources currently available in Indian communities and a recommendation for the kinds of resources that might prevent suicide in this setting.