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Parents are often appointed a passive role in the care for their hospitalised child. In the family-integrated care (FICare) model, parental involvement in neonatal care is emulated. Parental participation in medical rounds, or family-centred rounds (FCR), forms a key element. A paucity remains of randomised trials assessing the outcomes of FCR (embedded in FICare) in families and neonates, and outcomes on an organisational level are relatively unexplored. Likewise, biological mechanisms through which a potential effect may be exerted are lacking robust evidence. Ten level two Dutch neonatal wards are involved in this stepped-wedge cluster-randomised trial FCR (embedded in FICare) by one common implementation strategy. Parents of infants hospitalised for at least 7 days are eligible for inclusion. The primary outcome is parental stress (PSS:NICU) at discharge. Secondary outcomes include parental, neonatal, healthcare professional and organisational outcomes. Biomarkers of stress will be analysed in parent–infant dyads. With a practical approach and broad outcome set, this study aims to obtain evidence on the possible (mechanistic) effect of FCR (as part of FICare) on parents, infants, healthcare professionals and organisations. The practical approach provides (experiences of) FICare material adjusted to the Dutch setting, available for other hospitals after the study.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance. Oncolytic viruses have emerged as a new treatment approach and convey their antitumor activity through lysis of cancer cells. The therapeutic efficacy of oncolytic viruses is largely dependent on the induction of immunogenic cell death (ICD) and the subsequent antitumor immune responses. However, the concurrent generation of antiviral immune responses may also limit the a virus’ therapeutic window. GoraVir is a new oncolytic adenovirus derived from the Human Adenovirus B (HAdV-B) isolate AdV-lumc007 which was isolated from a gorilla and has demonstrated excellent lytic activity in both in vitro and in vivo models of PDAC. In this study, we characterized the immunostimulatory profile of cancer cell death induced by GoraVir and the concerted cellular antiviral responses in three conventional pancreatic cancer cell lines. While GoraVir was shown to induce late apoptotic/necrotic cell death at earlier time points post infection than the human adenovirus type 5 (HAdV-C5), similar levels of ICD markers were expressed. Moreover, GoraVir was shown to induce ICD not dependent on STING expression and regardless of subsequent antiviral responses. Together, these data demonstrate that GoraVir is an excellent candidate for use in oncolytic virotherapy.

Reoviruses are used as oncolytic viruses to destroy tumor cells. The concomitant induction of anti-tumor immune responses enhances the efficacy of therapy in tumors with low amounts of immune infiltrates before treatment. The reoviruses should provoke immunogenic cell death (ICD) to stimulate a tumor cell-directed immune response. Necroptosis is considered a major form of ICD, and involves receptor-interacting protein kinase 1 (RIPK1), RIPK3 and phosphorylation of mixed-lineage kinase domain-like protein (MLKL). This leads to cell membrane disintegration and the release of damage-associated molecular patterns that can activate immune responses. Reovirus Type 3 Dearing (T3D) can induce necroptosis in mouse L929 fibroblast cells and mouse embryonic fibroblasts. Most human tumor cell lines have a defect in RIPK3 expression and consequently fail to induce necroptosis as measured by MLKL phosphorylation. We used the human colorectal adenocarcinoma HT29 cell line as a model to study necroptosis in human cells since this cell line has frequently been described in necroptosis-related studies. To stimulate MLKL phosphorylation and induce necroptosis, HT29 cells were treated with a cocktail consisting of TNFα, the SMAC mimetic BV6, and the caspase inhibitor Z-VAD-FMK. While this treatment induced necroptosis, three different reovirus T3D variants, i.e., the plasmid-based reverse genetics generated virus (T3DK), the wild-type reovirus T3D isolate R124, and the junction adhesion molecule-A-independent reovirus mutant (jin-1) failed to induce necroptosis in HT29 cells. In contrast, these viruses induced MLKL phosphorylation in murine L929 cells, albeit with varying efficiencies. Our study shows that while reoviruses efficiently induce necroptosis in L929 cells, this is not a common phenotype in human cell lines. This study emphasizes the difficulties of translating the results of ICD studies from murine cells to human cells.

The adenoviruses (AdVs) isolated from humans are taxonomically grouped in seven different species in the Mastadenovirus genus (HAdV-A through G). AdVs isolated from apes are often included in one of the human AdV species. Here we describe the sequence analyses of ten new AdVs that are related to the HAdV-C species and that were isolated from healthy western lowland gorillas, bonobos, chimpanzees, and orangutans kept in Dutch zoos. We analyzed these viruses and compared their genome sequences to those of human- and ape-derived AdV sequences in the NCBI GenBank database. Our data demonstrated that the ape-derived viruses clustering to HAdV-C are markedly distinct from the human HAdV-C species in the size and nucleotide composition (%GC) of their genome, differ in the amino-acid sequence of AdV proteins, and have longer RGD-loops in their penton-base proteins. The viruses form three well-separated clades (the human, the gorilla, and the combined group of the bonobo and chimpanzee viruses), and we propose that these should each be given species-level ranks. The Ad-lumc005 AdV isolated from orangutans was found to be very similar to the gorilla AdVs, and bootstrap inference provided evidence of recombination between the orangutan AdV and the gorilla AdVs. This suggests that this virus may not be a genuine orangutan AdV but may have been transferred from a gorilla to an orangutan host.

Gliomas are the most frequent primary tumors of the brain. They can be divided into grade II-IV astrocytomas and grade II-III oligodendrogliomas, based on their histomolecular profile. The prognosis and treatment is highly dependent on grade and well-identified prognostic and/or predictive molecular markers. Multi-parametric MRI, including diffusion weighted imaging, perfusion, and MR spectroscopy, showed increasing value in the non-invasive characterization of specific molecular subsets of gliomas. Radiolabeled amino-acid analogues, such as 18F-FET, have also been proven valuable in glioma imaging. These tracers not only contribute in the diagnostic process by detecting areas of dedifferentiation in diffuse gliomas, but this technique is also valuable in the follow-up of gliomas, as it can differentiate pseudo-progression from real tumor progression. Since multi-parametric MRI and 18F-FET PET are complementary imaging techniques, there may be a synergistic role for PET-MRI imaging in the neuro-oncological imaging of primary brain tumors. This could be of value for both primary staging, as well as during treatment and follow-up.

The maintenance of pancreatic islet architecture is crucial for proper β-cell function. We previously reported that disruption of human islet integrity could result in altered β-cell identity. Here we combine β-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of β-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and β-cell phenotype, and strengthen the concept of altered β-cell identity as a mechanism underlying the loss of functional β-cell mass.

Oncolytic adenovirus therapy is believed to be a promising way to treat cancer patients. To be able to target tumor cells with an oncolytic adenovirus, expression of the adenovirus receptor on the tumor cell is essential. Different adenovirus types bind to different receptors on the cell, of which the expression can vary between tumor types. Pre-existing neutralizing immunity to human adenovirus species C type 5 (HAdV-C5) has hampered its therapeutic efficacy in clinical trials, hence several adenoviral vectors from different species are currently being developed as a means to evade pre-existing immunity. Therefore, knowledge on the expression of appropriate adenovirus receptors on tumor cells is important. This could aid in determining which tumor types would benefit most from treatment with a certain oncolytic adenovirus type. This review provides an overview of the known receptors for human adenoviruses and how their expression on tumor cells might be differentially regulated compared to healthy tissue, before and after standardized anticancer treatments. Mechanisms behind the up- or downregulation of adenovirus receptor expression are discussed, which could be used to find new targets for combination therapy to enhance the efficacy of oncolytic adenovirus therapy. Additionally, the utility of the adenovirus receptors in oncolytic virotherapy is examined, including their role in viral spread, which might even surpass their function as primary entry receptors. Finally, future directions are offered regarding the selection of adenovirus types to be used in oncolytic adenovirus therapy in the fight against cancer.

Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models.

Non-human primate (NHP)-derived adenoviruses have formed a valuable alternative for the use of human adenoviruses in vaccine development and gene therapy strategies by virtue of the low seroprevalence of neutralizing immunity in the human population. The more recent use of several human adenoviruses as oncolytic agents has exhibited excellent safety profiles and firm evidence of clinical efficacy. This proffers the question whether NHP-derived adenoviruses could also be employed for viral oncolysis in human patients. While vaccine vectors are conventionally made as replication-defective vectors, in oncolytic applications replication-competent viruses are used. The data on NHP-derived adenoviral vectors obtained from vaccination studies can only partially support the suitability of NHP-derived adenoviruses for use in oncolytic virus therapy. In addition, the use of NHP-derived adenoviruses in humans might be received warily given the recent zoonotic infections with influenza viruses and coronaviruses. In this review, we discuss the similarities and differences between human- and NHP-derived adenoviruses in view of their use as oncolytic agents. These include their genome organization, receptor use, replication and cell lysis, modulation of the host’s immune responses, as well as their pathogenicity in humans. Together, the data should facilitate a rational and data-supported decision on the suitability of NHP-derived adenoviruses for prospective use in oncolytic virus therapy.

Biblical references aside, restoring vision to the blind has proven to be a major technical challenge. In recent years, considerable advances have been made towards this end, especially when retinal degeneration underlies the vision loss such as occurs with retinitis pigmentosa. Under these conditions, optogenetic therapies are a particularly promising line of inquiry where remaining retinal cells are made into “artificial photoreceptors”. However, this strategy is not without its challenges and a model system using human retinal explants would aid its continued development and refinement. Here, we cultured post-mortem human retinas and show that explants remain viable for around 7 days. Within this period, the cones lose their outer segments and thus their light sensitivity but remain electrophysiologically intact, displaying all the major ionic conductances one would expect for a vertebrate cone. We optogenetically restored light responses to these quiescent cones using a lentivirus vector constructed to express enhanced halorhodopsin under the control of the human arrestin promotor. In these ‘reactivated’ retinas, we show a light-induced horizontal cell to cone feedback signal in cones, indicating that transduced cones were able to transmit their light response across the synapse to horizontal cells, which generated a large enough response to send a signal back to the cones. Furthermore, we show ganglion cell light responses, suggesting the cultured explant’s condition is still good enough to support transmission of the transduced cone signal over the intermediate retinal layers to the final retinal output level. Together, these results show that cultured human retinas are an appropriate model system to test optogenetic vision restoration approaches and that cones which have lost their outer segment, a condition occurring during the early stages of retinitis pigmentosa, are appropriate targets for optogenetic vision restoration therapies.

Oncolytic adenoviral vectors are a promising alternative for the treatment of glioblastoma. Recent publications have demonstrated the advantages of shielding viral particles within cellular vehicles (CVs), which can be targeted towards the tumor microenvironment. Here, we studied T-cells, often having a natural capacity to target tumors, for their feasibility as a CV to deliver the oncolytic adenovirus, Delta24-RGD, to glioblastoma. The Jurkat T-cell line was assessed in co-culture with the glioblastoma stem cell (GSC) line, MGG8, for the optimal transfer conditions of Delta24-RGD in vitro. The effect of intraparenchymal and tail vein injections on intratumoral virus distribution and overall survival was addressed in an orthotopic glioma stem cell (GSC)-based xenograft model. Jurkat T-cells were demonstrated to facilitate the amplification and transfer of Delta24-RGD onto GSCs. Delta24-RGD dosing and incubation time were found to influence the migratory ability of T-cells towards GSCs. Injection of Delta24-RGD-loaded T-cells into the brains of GSC-bearing mice led to migration towards the tumor and dispersion of the virus within the tumor core and infiltrative zones. This occurred after injection into the ipsilateral hemisphere, as well as into the non-tumor-bearing hemisphere. We found that T-cell-mediated delivery of Delta24-RGD led to the inhibition of tumor growth compared to non-treated controls, resulting in prolonged survival (p = 0.007). Systemic administration of virus-loaded T-cells resulted in intratumoral viral delivery, albeit at low levels. Based on these findings, we conclude that T-cell-based CVs are a feasible approach to local Delta24-RGD delivery in glioblastoma, although efficient systemic targeting requires further improvement.

Mammalian reovirus is a double-stranded RNA virus that selectively infects and lyses transformed cells, making it an attractive oncolytic agent. Despite clinical evidence for anti-tumor activity, its efficacy as a stand-alone therapy remains to be improved. The success of future trials can be greatly influenced by the identification and the regulation of the cellular pathways that are important for reovirus replication and oncolysis. Here, we demonstrate that reovirus induces autophagy in several cell lines, evident from the formation of Atg5-Atg12 complexes, microtubule-associated protein 1 light chain 3 (LC3) lipidation, p62 degradation, the appearance of acidic vesicular organelles, and LC3 puncta. Furthermore, in electron microscopic images of reovirus-infected cells, autophagosomes were observed without evident association with viral factories. Using UV-inactivated reovirus, we demonstrate that a productive reovirus infection facilitates the induction of autophagy. Importantly, knock-out cell lines for specific autophagy-related genes revealed that the expression of Atg3 and Atg5 but not Atg13 facilitates reovirus replication. These findings highlight a central and Atg13-independent role for the autophagy machinery in facilitating reovirus infection and contribute to a better understanding of reovirus-host interactions.

Reoviruses are non-enveloped viruses with a segmented double stranded RNA genome. In humans, they are not associated with serious disease. Human reoviruses exhibit an inherent preference to replicate in tumor cells, which makes them ideally suited for use in oncolytic virotherapies. Their use as anti-cancer agent has been evaluated in several clinical trials, which revealed that intra-tumoral and systemic delivery of reoviruses are well tolerated. Despite evidence of anti-tumor effects, the efficacy of reovirus in anti-cancer monotherapy needs to be further enhanced. The opportunity to treat both the primary tumor as well as metastases makes systemic delivery a preferred administration route. Several pre-clinical studies have been conducted to address the various hurdles connected to systemic delivery of reoviruses. The majority of those studies have been done in tumor-bearing immune-deficient murine models. This thwarts studies on the impact of the contribution of the immune system to the tumor cell eradication. This review focuses on key aspects of the reovirus/host-cell interactions and the methods that are available to modify the virus to alter these interactions. These aspects are discussed with a focus on improving the reovirus’ antitumor efficacy.