2642. Development of Human Intestinal Organoids as an Antiviral Evaluation Platform for Enteroviruses

Abstract Background Enteroviruses are non-enveloped, single-stranded positive-sense RNA viruses belonging to the family Picornaviridae. Enterovirus A71 (EV-A71) has caused recurrent outbreaks of hand, foot, and mouth disease especially among children in Asia. Some patients develop severe complications, such as meningitis, encephalitis, myocarditis, and pulmonary edema. A major hurdle for the development of antivirals for EV-A71 infection is the lack of robust antiviral platforms that closely mimic the in vivo setting. Organoids are laboratory-adapted miniaturized organs with preserved three-dimensional micro-anatomical architecture. In recent years, organoid cultures have been increasingly used for studying the pathogenesis of and evaluating antiviral treatment options for viral infections. In this study, we developed human intestinal organoids as a robust platform for evaluating antiviral options for EV-A71. Methods An epidemic strain of EV-A71 isolated from a patient with laboratory-confirmed EV-A71 infection was used. We compared the performance of multiple antiviral evaluation assays (virus yield reduction, plaque reduction, and cell protection assays) between human intestinal organoids and Caco-2 cells, using itraconazole (an antifungal previously shown to exhibit potent anti-enteroviral effects) and DMSO as positive and negative controls, respectively. Results The antiviral effect of itraconazole was comparable between human intestinal organoids and Caco-2 cells in the virus yield reduction and plaque reduction assays. In the cell protection assay, Caco-2 cells failed to demonstrate significant differences between the itraconazole-treated and DMSO-treated groups. In contrast, cell protection effects were easily observed and quantified in human intestinal organoids. Moreover, the human intestinal organoids allowed the characterization of the different cell types affected in EV-A71 infection with or without itraconazole treatment. Conclusion Human intestinal organoids support the replication of EV-A71 and provides a robust platform for antiviral evaluation for EV-A71 infection. Disclosures All authors: No reported disclosures.

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Therapeutic targets in COVID-19 coagulopathy

Тромбоз, гемостаз и реология ◽

10.25555/thr.2021.1.0956 ◽

2021 ◽

Author(s):

М.В. Кондашевская

Keyword(s):

Viral Infections ◽

Cell Types ◽

Infectious Agents ◽

Therapeutic Goals ◽

Extracellular Traps ◽

Activated Neutrophils ◽

Life Threatening ◽

Tight Connection ◽

New Type

В обзоре представлены сведения о некоторых биохимических и клеточных механизмах патогенеза потенциально смертельного вирусного заболевания, получившего название COVID-19, провоцируемого вирусами из семейства коронавирусов SARS-CoV-2. Наибольшую опасность для жизни и здоровья пациентов представляет коагулопатия, обусловленная ответной реакцией на инфекцию — чрезмерной генерацией клетками пациента внеклеточных мембранных нановезикул (ВМН), которые могут спровоцировать активизацию гемостаза, приводящую к разрушительной полиорганной недостаточности. Вирусы также способны генерировать внеклеточные везикулы, называемые виросомами. Встраивая вирусные компоненты в свои виросомы, вирусы повышают персистентность за счет маскировки своих геномов, умножая вирусную инфекцию. В статье охарактеризованы свойства нейтрофильных гранулоцитов, активирующихся при инфицировании, и представлены сведения о молекулярных механизмах действия их компонентов при попадании во внеклеточное пространство. ВМН уже имеют практическое применение в качестве вакцинных носителей для иммунизации человека и животных против многих инфекционных заболеваний. В настоящее время актуальнейшей темой, обуславливающей большой практический интерес, стала роль ВМН в индуцировании иммунитета против коронавирусной инфекции. Охарактеризованы другие возможные терапевтические мишени. Virus-induced coagulopathy is a typical example of the tight connection between inflammation and thrombosis. These two reactions are linked by pro-inflammatory agents, generated by activated neutrophils and their neutrophil extracellular traps (NETs). Extracellular membrane nanobubbles (EMNs), formed by a wide variety of cell types, have recently been identified as new entrants that play a key role in coagulopathy. EMNs directly and indirectly activate coagulation systems that lead to the further upregulation of inflammation and life-threatening organ dysfunction and thrombosis. EMNs are known to be responsible for the secretion, exchange, and transmission of important active biomolecules in COVID-19. Indeed, EMNs represent an essential mechanism in intercellular communication, and the roles of EMNs in infection and thrombosis have been increasingly recognized. The extracellular microvesicles of viruses, virosomes, represent a new type of infectious agents, which determines new therapeutic goals in solving the problems of controlling viral infections. Understanding the biological nature of all these microvesicles when studying them in vivo is of paramount importance for the development of diagnostic and therapeutic methods.

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Intranasal Borna Disease Virus (BoDV-1) Infection: Insights into Initial Steps and Potential Contagiosity

International Journal of Molecular Sciences ◽

10.3390/ijms20061318 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1318 ◽

Cited By ~ 6

Author(s):

Alexandra Kupke ◽

Sabrina Becker ◽

Konstantin Wewetzer ◽

Barbara Ahlemeyer ◽

Markus Eickmann ◽

...

Keyword(s):

Viral Infections ◽

Cell Types ◽

Nerve Fibers ◽

Olfactory Pathway ◽

Adult Rats ◽

The Central Nervous System ◽

Receptor Neurons

Mammalian Bornavirus (BoDV-1) typically causes a fatal neurologic disorder in horses and sheep, and was recently shown to cause fatal encephalitis in humans with and without transplant reception. It has been suggested that BoDV-1 enters the central nervous system (CNS) via the olfactory pathway. However, (I) susceptible cell types that replicate the virus for successful spread, and (II) the role of olfactory ensheathing cells (OECs), remained unclear. To address this, we studied the intranasal infection of adult rats with BoDV-1 in vivo and in vitro, using olfactory mucosal (OM) cell cultures and the cultures of purified OECs. Strikingly, in vitro and in vivo, viral antigen and mRNA were present from four days post infection (dpi) onwards in the olfactory receptor neurons (ORNs), but also in all other cell types of the OM, and constantly in the OECs. In contrast, in vivo, BoDV-1 genomic RNA was only detectable in adult and juvenile ORNs, nerve fibers, and in OECs from 7 dpi on. In vitro, the rate of infection of OECs was significantly higher than that of the OM cells, pointing to a crucial role of OECs for infection via the olfactory pathway. Thus, this study provides important insights into the transmission of neurotropic viral infections with a zoonotic potential.

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Anti CMV and/or Anti Adenovirus IFN-g-Positive CD4+ CD8+ T Lymphocytes for Treatment of Viral Infections After Allogeneic HSC Transplantation: First Results

Blood ◽

10.1182/blood.v120.21.1906.1906 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1906-1906

Author(s):

Liliane Dal Cortivo ◽

RITA Creidy ◽

Aurélie Gabrion ◽

Marianne Leruez-Ville ◽

Sebastien Heritier ◽

...

Keyword(s):

T Cells ◽

Viral Load ◽

T Lymphocytes ◽

Viral Infections ◽

Clinical Symptoms ◽

Antiviral Treatment ◽

Specific Cell ◽

Low Grade ◽

First Results

Abstract Abstract 1906 Reactivation of latent viruses such as cytomegalovirus (CMV) and adenovirus (AdV) is responsible for infections which may be life-threatening in HSCT recipients. In the post-transplantation period, severity and frequency of these infections depend on (a) the degree of donor-recipient HLA incompatibility and (b) the intensity of immunosuppressive therapy used to prevent immunological complications. Antiviral drugs may be partially effective, often toxic and cannot always control those viral infections.T cell immunity plays a major role in the control of viral infections. It has been demonstrated that the transfer of donor T lymphocytes specifically directed against viral antigens is capable of preventing, controlling and clearing viral infection (Feuchtinger T et al., 2004 and 2010). The present project aimed the evaluation of specific, cell-based immunity against CMV and AdV by injection of IFN-g-positive CD4+and CD8+ donor T lymphocytes isolated ex vivo after stimulation with viral peptides. Methods: Our protocol was designed for pediatric or adult patients treated by allogeneic HSCT and matching the following inclusion criteria: (1) biological and/or clinical symptoms of CMV and/or AdV infection 2) no response or contraindication to conventional antiviral treatment and (3) no or low grade pre-existing aGvHD at inclusion (≤ grade II) controlled by corticoids (<1 mg/kg). Antiviral treatments are allowed during the inclusion period. Donor IFN-g-positive T lymphocytes are isolated with the CliniMACS Cytokine Capture System (Miltenyi Biotech) after incubation with viral peptide pools. Primary evaluation criterion is the efficacy of the treatment on CMV viral load 21 days after the first injection. In the event of a negative or partial response and the absence of aGvHD, a second injection may be scheduled. Secondary evaluation criteria are (1) the occurrence of de novo aGvHD or aggravation of existing aGvHD, (2) the evolution of clinical symptoms potentially related to the infection, (3) the demonstration of biological in vivo expansion of injected T lymphocytes (as evidenced by the IFN-g secretion capacity and specific tetramer assays) and (4) for AdV infection, evaluation of efficacy (viral load, in vivo expansion of transfused lymphocytes, clinical symptoms) and the safety (occurrence of aGvHD) of this immunotherapy. Results: From September 2010 to July 2012, 9 patients were included: 3 male adults (46–54 years, 1 CLL, 1 CML and 1 AA, 2 geno- and 1 pheno-identical transplantation) and 6 children (age: 7–25 months, sex ratio F/M: 4/2, 4 FLH, 1 SCID and 1AA, 4 haplo, 1 geno- and 1 pheno-id transplantation). 4/9 patients were treated for CMV, 3/9 for AdV and 2/9 for CMV and AdV reactivation. 5/9 patients received 2 cytotoxic T lymphocytes (CTL) injections. Mean number of CD3 IFN-g positive cells injected was 4206/kg (1167–6000/kg) with 55% and 69% of CD4 and CD8 anti CMV-T cells and 56% and 61% of CD4 and CD8 anti AdV T cells respectively. Mean delay of first immunotherapy was 109 days (28–270) after transplantation. 2/9 patients were not evaluable due to early death (<21 days post injection) and 1/9 patient died of graft failure 43 days after CTL injection without efficacy on infectious evolution. 6 patients are still alive: 4 with complete, 1 with partial remission of virus replication and 1 recently included, is still under evaluation. An in vivo expansion of transfused CTL was observed (mean expansion was 33 and 35 fold for CD8-IFN-g and CD4-IFN-g positive cells respectively 42 days after injection) in parallel with the decrease of viral load in all alive patients. No aGvHD was detected in the 5/6 evaluated patients. One of 6 presenting cGvH at inclusion need increase of corticotherapy 3 months after second injection of CTL One patient presenting with CMV retinitis received 2 CTL injections without worsening of retina lesions which healed. Conclusion: The CliniMACS Cytokine Capture System allows the isolation of virus-specific T cells in a brief delay (24 hours) with a satisfactory enrichment of both CD4 and CD8 T cells. First results show efficacy of virus-specific T cells injection on viral load without signs of aGvHD in 5/6 evaluable patients. More patients need to be included in this trial in order to confirm these encouraging results. Disclosures: Cambouris: Miltenyi Biotec: Employment.

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Lassa viral dynamics in non-human primates treated with favipiravir or ribavirin

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008535 ◽

2021 ◽

Vol 17 (1) ◽

pp. e1008535

Author(s):

Guillaume Lingas ◽

Kyle Rosenke ◽

David Safronetz ◽

Jérémie Guedj

Keyword(s):

Mechanism Of Action ◽

Ebola Virus ◽

Antiviral Treatment ◽

Antiviral Effect ◽

Lassa Fever ◽

Pharmacokinetic Data ◽

Virus Infectivity ◽

Lassa Virus ◽

Viral Dynamics

Lassa fever is an haemorrhagic fever caused by Lassa virus (LASV). There is no vaccine approved against LASV and the only recommended antiviral treatment relies on ribavirin, despite limited evidence of efficacy. Recently, the nucleotide analogue favipiravir showed a high antiviral efficacy, with 100% survival obtained in an otherwise fully lethal non-human primate (NHP) model of Lassa fever. However the mechanism of action of the drug is not known and the absence of pharmacokinetic data limits the translation of these results to the human setting. Here we aimed to better understand the antiviral effect of favipiravir by developping the first mathematical model recapitulating Lassa viral dynamics and treatment. We analyzed the viral dynamics in 24 NHPs left untreated or treated with ribavirin or favipiravir, and we put the results in perspective with those obtained with the same drugs in the context of Ebola infection. Our model estimates favipiravir EC50 in vivo to 2.89 μg.mL-1, which is much lower than what was found against Ebola virus. The main mechanism of action of favipiravir was to decrease virus infectivity, with an efficacy of 91% at the highest dose. Based on our knowledge acquired on the drug pharmacokinetics in humans, our model predicts that favipiravir doses larger than 1200 mg twice a day should have the capability to strongly reduce the production infectious virus and provide a milestone towards a future use in humans.

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Recent Advances in Extracellular Vesicles as Drug Delivery Systems and Their Potential in Precision Medicine

Pharmaceutics ◽

10.3390/pharmaceutics12111006 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1006 ◽

Cited By ~ 2

Author(s):

Bart de Jong ◽

Eric Raul Barros ◽

Joost G. J. Hoenderop ◽

Juan Pablo Rigalli

Keyword(s):

Drug Delivery ◽

Extracellular Vesicles ◽

Current Knowledge ◽

Early Stage ◽

Treatment Options ◽

Cell Types ◽

Administration Routes ◽

The Individual ◽

Key Steps

Extracellular vesicles (EVs) are membrane-bilayered nanoparticles released by most cell types. Recently, an enormous number of studies have been published on the potential of EVs as carriers of therapeutic agents. In contrast to systems such as liposomes, EVs exhibit less immunogenicity and higher engineering potential. Here, we review the most relevant publications addressing the potential and use of EVs as a drug delivery system (DDS). The information is divided based on the key steps for designing an EV-mediated delivery strategy. We discuss possible sources and isolation methods of EVs. We address the administration routes that have been tested in vivo and the tissue distribution observed. We describe the current knowledge on EV clearance, a significant challenge towards enhancing bioavailability. Also, EV-engineering approaches are described as alternatives to improve tissue and cell-specificity. Finally, a summary of the ongoing clinical trials is performed. Although the application of EVs in the clinical practice is still at an early stage, a high number of studies in animals support their potential as DDS. Thus, better treatment options could be designed to precisely increase target specificity and therapeutic efficacy while reducing off-target effects and toxicity according to the individual requirements of each patient.

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Regenerative Models for the Integration and Regeneration of Head Skeletal Tissues

International Journal of Molecular Sciences ◽

10.3390/ijms19123752 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3752 ◽

Cited By ~ 1

Author(s):

Warren Vieira ◽

Catherine McCusker

Keyword(s):

Soft Tissues ◽

Treatment Options ◽

Positional Information ◽

Cell Types ◽

Model Organisms ◽

Biological Targets ◽

Tissue Integration ◽

Repair Mechanisms

Disease of, or trauma to, the human jaw account for thousands of reconstructive surgeries performed every year. One of the most popular and successful treatment options in this context involves the transplantation of bone tissue from a different anatomical region into the affected jaw. Although, this method has been largely successful, the integration of the new bone into the existing bone is often imperfect, and the integration of the host soft tissues with the transplanted bone can be inconsistent, resulting in impaired function. Unlike humans, several vertebrate species, including fish and amphibians, demonstrate remarkable regenerative capabilities in response to jaw injury. Therefore, with the objective of identifying biological targets to promote and engineer improved outcomes in the context of jaw reconstructive surgery, we explore, compare and contrast the natural mechanisms of endogenous jaw and limb repair and regeneration in regenerative model organisms. We focus on the role of different cell types as they contribute to the regenerating structure; how mature cells acquire plasticity in vivo; the role of positional information in pattern formation and tissue integration, and limitations to endogenous regenerative and repair mechanisms.

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Antiherpes Effect of Dextran Sulphate Combined with Acyclovir in vitro and in vivo

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029300400309 ◽

1993 ◽

Vol 4 (3) ◽

pp. 189-191 ◽

Cited By ~ 1

Author(s):

S. N. Pancheva

Keyword(s):

Herpes Simplex ◽

Pseudorabies Virus ◽

Antiviral Effect ◽

Yield Reduction ◽

Ocular Infection ◽

Dextran Sulphate ◽

Virus Shedding ◽

Hsv 1

The combined antiviral effect of dextran sulphate (DS, MW 40000) and acyclovir (ACV) on herpes simplex virus type 1 (HSV-1) and Pseudorabies virus (PRV) in vitro and on experimental HSV-1 keratitis in rabbits, was studied. Dextran sulphate in combination with ACV results in synergistic effect, as measured by yield reduction assay and on herpes simplex keratitis in rabbits. The better therapeutic effect of the combination was proved by the decreased severity of ocular infection and the reduction of the virus shedding in tear film.

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Modeling Intestinal Epithelial Response to Interferon-γ in Induced Pluripotent Stem Cell-Derived Human Intestinal Organoids

International Journal of Molecular Sciences ◽

10.3390/ijms22010288 ◽

2020 ◽

Vol 22 (1) ◽

pp. 288

Author(s):

Michael J. Workman ◽

Elissa Troisi ◽

Stephan R. Targan ◽

Clive N. Svendsen ◽

Robert J. Barrett

Keyword(s):

Interferon Gamma ◽

Adherens Junction ◽

Induced Pluripotent Stem Cell ◽

Enrichment Analysis ◽

Cell Types ◽

Gene Set Enrichment Analysis ◽

Intestinal Organoids ◽

Ifn Γ ◽

Induced Pluripotent

Human intestinal organoids (HIOs) are increasingly being used to model intestinal responses to various stimuli, yet few studies have confirmed the fidelity of this modeling system. Given that the interferon-gamma (IFN-γ) response has been well characterized in various other cell types, our goal was to characterize the response to IFN-γ in HIOs derived from induced pluripotent stem cells (iPSCs). To achieve this, iPSCs were directed to form HIOs and subsequently treated with IFN-γ. Our results demonstrate that IFN-γ phosphorylates STAT1 but has little effect on the expression or localization of tight and adherens junction proteins in HIOs. However, transcriptomic profiling by microarray revealed numerous upregulated genes such as IDO1, GBP1, CXCL9, CXCL10 and CXCL11, which have previously been shown to be upregulated in other cell types in response to IFN-γ. Notably, “Response to Interferon Gamma” was determined to be one of the most significantly upregulated gene sets in IFN-γ-treated HIOs using gene set enrichment analysis. Interestingly, similar genes and pathways were upregulated in publicly available datasets contrasting the gene expression of in vivo biopsy tissue from patients with IBD against healthy controls. These data confirm that the iPSC-derived HIO modeling system represents an appropriate platform to evaluate the effects of various stimuli and specific environmental factors responsible for the alterations in the intestinal epithelium seen in various gastrointestinal conditions such as inflammatory bowel disease.

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Epithelial cells of the intestine acquire cell-intrinsic inflammation signatures during ageing

10.1101/2021.12.19.473357 ◽

2021 ◽

Author(s):

Maja C Funk ◽

Jan G Gleixner ◽

Florian Heigwer ◽

Erica Valentini ◽

Zeynep Aydin ◽

...

Keyword(s):

Epithelial Cells ◽

Mhc Class Ii ◽

Ex Vivo ◽

Cell Types ◽

Chromatin Accessibility ◽

Extrinsic Factors ◽

Intestinal Organoids ◽

Persistent Inflammation ◽

A Cell

During ageing, cell-intrinsic and extrinsic factors lead to the decline of tissue function and organismal health. Disentangling these factors is important for developing effective strategies to prolong organismal healthspan. Here, we addressed this question in the mouse intestinal epithelium, which forms a dynamic interface with its microenvironment and receives extrinsic signals affecting its homeostasis and tissue ageing. We systematically compared transcriptional profiles of young and aged epithelial cells in vivo and ex vivo in cultured intestinal organoids. We found that all cell types of the aged epithelium exhibit an inflammation phenotype, which is marked by MHC class II upregulation and most pronounced in enterocytes. This was accompanied by elevated levels of the immune tolerance markers PD-1 and PD-L1 in the aged tissue microenvironment, indicating dysregulation of immunological homeostasis. Intestinal organoids from aged mice still showed an inflammation signature after weeks in culture, which was concurrent with increased chromatin accessibility of inflammation-associated loci. Our results reveal a cell-intrinsic, persistent inflammation phenotype in aged epithelial cells, which might contribute to systemic inflammation observed during ageing.

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The ESCRT-Related ATPase Vps4 Is Modulated by Interferon during Herpes Simplex Virus 1 Infection

mBio ◽

10.1128/mbio.02567-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 2

Author(s):

Jorge Ruben Cabrera ◽

Richard Manivanh ◽

Brian J. North ◽

David A. Leib

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Viral Infections ◽

Cell Types ◽

Primary Neurons ◽

Herpes Simplex Virus 1 ◽

Escrt Machinery ◽

Simplex Virus ◽

Hsv 1

ABSTRACTInterferons (IFNs) and autophagy are critical neuronal defenses against viral infection. IFNs alter neuronal autophagy by promoting the accumulation of IFN-dependent LC3-decorated autophagic structures, termed LC3 clusters. Here, we analyzed LC3 clusters in sensory ganglia following herpes simplex virus 1 (HSV-1) infection. In the vicinity of acutely infected neurons, antigen-negative neurons contained structures resembling accumulated autophagosomes and autolysosomes that culminated in LC3 clusters. This accumulation reflects a delayed completion of autophagy. Theendosomalsortingcomplexesrequired fortransport (ESCRT) machinery participates in autophagosome closure and is also required for HSV-1 replication. In this study, our results showed that HSV-1 infectionin vivoand in primary neurons caused a decrease in Vps4 (a key ESCRT pathway ATPase) RNA and protein with concomitant Stat1 activation and LC3 cluster induction. We also observed that IFNs were sufficient to decrease RNA and protein levels of Vps4 in primary neurons and in other cell types. The accumulation of ubiquitin was also observed at the LC3 cluster sites. Together, our results show that IFNs modulate the ESCRT machinery in neurons in response to HSV-1 infections.IMPORTANCENeurons rely on IFNs and autophagy as major defenses against viral infections, and HSV must overcome such defenses in order to replicate. In addition to controlling host immunity, HSV must also control host membranes in order to complete its life cycle. HSV uses the host ESCRT membrane scission machinery for viral production and transport. Here we present evidence of a new IFN-dependent mechanism used by the host to prevent ESCRT subversion by HSV. This activity also impacts the dynamics of autophagy, possibly explaining the presence of recently described LC3 clusters in the HSV-infected nervous system. The induced accumulations of ubiquitin observed in these LC3 clusters resembled those observed in certain neurodegenerative diseases, suggesting possible mechanistic parallels between these conditions.

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