Stabilization of cell-cell adhesions prevents symmetry breaking and locks in pluripotency in 3D gastruloids.

3D embryonic stem cell (ESC) aggregates self-organize into embryo-like structures named gastruloids that recapitulate the axial organization of post-implantation embryos. Crucial in this process is the symmetry-breaking event that leads to the emergence of asymmetry and spatially ordered structures from homogeneous cell aggregates. Here, we show that budesonide, a glucocorticoid drug widely used to treat asthma, prevents ESC aggregates to break symmetry. Mechanistically, the effect of budesonide is glucocorticoid receptor independent. RNA sequencing and lineage fate analysis reveal that budesonide counteracts exit from pluripotency and modifies the expression of a large set of genes associated with cell migration, A-P axis formation, and WNT signaling. This correlates with reduced phenotypic and molecular cell heterogeneity, persistence of E-CADHERIN at the cell-cell interface, and cell aggregate compaction. Our findings reveal that cell-cell adhesion properties control symmetry breaking and cell fate transition in 3D gastruloids and suggest a potential adverse effect of budesonide on embryo development.

Daily Intraperitoneal Administration of Rosiglitazone Does Not Improve Lung Function or Alveolarization in Preterm Rabbits Exposed to Hyperoxia.

Thiazolidinediones (TZDs) are potent PPARγ agonists that have been shown to attenuate alveolar simplification after prolonged hyperoxia in term rodent models of bronchopulmonary dysplasia. However, the pulmonary outcomes of postnatal TZDs have not been investigated in preterm animal models. Here, we first investigated the PPARγ selectivity, epithelial permeability, and lung tissue binding of three types of TZDs in vitro (rosiglitazone (RGZ), pioglitazone, and DRF-2546), followed by an in vivo study in preterm rabbits exposed to hyperoxia (95% oxygen) to investigate the pharmacokinetics and the pulmonary outcomes of daily RGZ administration. In addition, blood lipids and a comparative lung proteomics analysis were also performed on Day 7. All TZDs showed high epithelial permeability through Caco-2 monolayers and high plasma and lung tissue binding; however, RGZ showed the highest affinity for PPARγ. The pharmacokinetic profiling of RGZ (1 mg/kg) revealed an equivalent biodistribution after either intratracheal or intraperitoneal administration, with detectable levels in lungs and plasma after 24 h. However, daily RGZ doses of 1 mg/kg did not improve lung function in preterm rabbits exposed to hyperoxia, and daily 10 mg/kg doses were even associated with a significant lung function worsening, which could be partially explained by the upregulation of lung inflammation and lipid metabolism pathways revealed by the proteomic analysis. Notably, daily postnatal RGZ produced an aberrant modulation of serum lipids, particularly in rabbit pups treated with the 10 mg/kg dose. In conclusion, daily postnatal RGZ did not improve lung function and caused dyslipidemia in preterm rabbits exposed to hyperoxia.

The benefits, limitations and opportunities of preclinical models for neonatal drug development.

Increased research to improve preclinical models to inform the development of therapeutics for neonatal diseases is an area of great need. This article reviews five common neonatal diseases - bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, perinatal hypoxic-ischemic encephalopathy and neonatal sepsis - and the available in vivo, in vitro and in silico preclinical models for studying these diseases. Better understanding of the strengths and weaknesses of specialized neonatal disease models will help to improve their utility, may add to the understanding of the mode of action and efficacy of a therapeutic, and/or may improve the understanding of the disease pathology to aid in identification of new therapeutic targets. Although the diseases covered in this article are diverse and require specific approaches, several high-level, overarching key lessons can be learned by evaluating the strengths, weaknesses and gaps in the available models. This Review is intended to help guide current and future researchers toward successful development of therapeutics in these areas of high unmet medical need.

Design-Based Stereology of the Lung in the Hyperoxic Preterm Rabbit Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a complex condition frequently occurring in preterm newborns, and different animal models are currently used to mimic the pathophysiology of BPD. The comparability of animal models depends on the availability of quantitative data obtained by minimally biased methods. Therefore, the aim of this study was to provide the first design-based stereological analysis of the lungs in the hyperoxia-based model of BPD in the preterm rabbit. Rabbit pups were obtained on gestation day 28 (three days before term) by cesarean section and exposed to normoxic (21% O2, n = 8) or hyperoxic (95% O2, n = 8) conditions. After seven days of exposure, lung function testing was performed, and lungs were taken for stereological analysis. In addition, the ratio between pulmonary arterial acceleration and ejection time (PAAT/PAET) was measured. Inspiratory capacity and static compliance were reduced whereas tissue elastance and resistance were increased in hyperoxic animals compared with normoxic controls. Hyperoxic animals showed signs of pulmonary hypertension indicated by the decreased PAAT/PAET ratio. In hyperoxic animals, the number of alveoli and the alveolar surface area were reduced by one-third or by approximately 50% of control values, respectively. However, neither the mean linear intercept length nor the mean alveolar volume was significantly different between both groups. Hyperoxic pups had thickened alveolar septa and intra-alveolar accumulation of edema fluid and inflammatory cells. Nonparenchymal blood vessels had thickened walls, enlarged perivascular space, and smaller lumen in hyperoxic rabbits in comparison with normoxic ones. In conclusion, the findings are in line with the pathological features of human BPD. The stereological data may serve as a reference to compare this model with BPD models in other species or future therapeutic interventions.

Lung ultrasound features and relationships with respiratory mechanics of evolving BPD in preterm rabbits and human neonates.

Evolving bronchopulmonary dysplasia (BPD) is characterized by impaired alveolarization leading to lung aeration inhomogeneities. Hyperoxia-exposed preterm rabbits have been proposed to mimic evolving BPD; therefore, we aimed to verify if this model has the same lung ultrasound and mechanical features of evolving BPD in human neonates. Semiquantitative lung ultrasound and lung mechanics measurement was performed in 25 preterm rabbits (28 days of gestation) and 25 neonates (mean gestational age ≈ 26 wk) with evolving BPD. A modified rabbit lung ultrasound score (rLUS) and a validated neonatal lung ultrasound score (LUS) were used. Lung ultrasound images were recorded and evaluated by two independent observers blinded to each other's evaluation. Lung ultrasound findings were equally heterogeneous both in rabbits as in human neonates and encompassed all the classical lung ultrasound semiology. Lung ultrasound and histology examination were also performed in 13 term rabbits kept under normoxia as further control and showed the absence of ultrasound and histology abnormalities compared with hyperoxia-exposed preterm rabbits. The interrater absolute agreement for the evaluation of lung ultrasound images in rabbits was very high [ICC: 0.989 (95%CI: 0.975-0.995); P < 0.0001], and there was no difference between the two observers. Lung mechanics parameters were similarly altered in both rabbits and human neonates. There were moderately significant correlations between airway resistances and lung ultrasound scores in rabbits (ρ = 0.519; P = 0.008) and in neonates (ρ = 0.409; P = 0.042). In conclusion, the preterm rabbit model fairly reproduces the lung ultrasound and mechanical characteristics of preterm neonates with evolving BPD.NEW & NOTEWORTHY We have reported that hyperoxia-exposed preterm rabbits and human preterm neonates with evolving BPD have the same lung ultrasound appearance, and that lung ultrasound can be fruitfully applied on this model with a brief training. The animal model and human neonates also presented the same relationship between semiquantitative ultrasound-assessed lung aeration and airway resistances. In conclusion, this animal model fairly reproduce evolving BPD as it is seen in clinical practice.

Surfactant lung delivery with LISA and InSurE in adult rabbits with respiratory distress.

BACKGROUND: In preterm infants, InSurE (Intubation-Surfactant-Extubation) and LISA (less invasive surfactant administration) techniques allow for exogenous surfactant administration while reducing lung injury associated with mechanical ventilation. We compared the acute pulmonary response and lung deposition of surfactant by LISA and InSurE in surfactant-depleted adult rabbits. METHODS: Twenty-six spontaneously breathing surfactant-depleted adult rabbits (6-7 weeks old) with moderate RDS and managed with nasal continuous positive airway pressure were randomized to 3 groups: (1) 200 mg/kg of surfactant by InSurE; (2) 200 mg/kg of surfactant by LISA; (3) no surfactant treatment (Control). Gas exchange and lung mechanics were monitored for 180 min. After that, surfactant lung deposition and distribution were evaluated monitoring disaturated-phosphatidylcholine (DSPC) and surfactant protein C (SP-C), respectively. RESULTS: No signs of recovery were found in the untreated animals. After InSurE, oxygenation improved more rapidly compared to LISA. However, at 180' LISA and InSurE showed comparable outcomes in terms of gas exchange, ventilation parameters, and lung mechanics. Neither DSPC in the alveolar pool nor SP-C signal distributions in a frontal lung section were significantly different between InSurE and LISA groups. CONCLUSIONS: In an acute setting, LISA demonstrated efficacy and surfactant lung delivery similar to that of InSurE in surfactant-depleted adult rabbits. IMPACT: Although LISA technique is gaining popularity, there are still several questions to address. This is the first study comparing LISA and InSurE in terms of gas exchange, ventilation parameters, and lung mechanics as well as surfactant deposition and distribution. In our animal study, three hours post-treatment, LISA method seems to be as effective as InSurE and showed similar surfactant lung delivery. Our findings provide some clarifications on a fair comparison between LISA and InSurE techniques, particularly in terms of surfactant delivery. They should reassure some of the concerns raised by the clinical community on LISA adoption in neonatal units.

Intratracheal budesonide/surfactant attenuates hyperoxia-induced lung injury in preterm rabbits.

Recent clinical trials have shown improvements in neonatal outcomes after intratracheal administration of a combination of budesonide/surfactant (ITBS) in infants at risk of bronchopulmonary dysplasia. However, the effect of ITBS on lung function and alveolar structure is not known. We aimed to determine the effect of ITBS on lung function, parenchymal structure, and inflammatory cytokine expression in a relevant preterm animal model for bronchopulmonary dysplasia. Premature neonatal rabbits were administered a single dose of ITBS on the day of delivery and exposed to 95% oxygen. Following 7 days of hyperoxia, in vivo forced oscillation and pressure-volume maneuvers were performed to examine pulmonary function. Histological and molecular analysis was performed to assess alveolar and extracellular matrix (ECM) morphology, along with gene expression of connective tissue growth factor (CTGF), IL-8, and CCL-2. ITBS attenuated the functional effect of hyperoxia-induced lung injury and limited the change to respiratory system impedance, measured using the forced oscillation technique. Treatment effects were most obvious in the small airways, with significant effects on small airway resistance and small airway reactance. In addition, ITBS mitigated the decrease in inspiratory capacity and static compliance. ITBS restricted alveolar septal thickening without altering the mean linear intercept and mitigated hyperoxia-induced remodeling of the ECM. These structural changes were associated with improved inspiratory capacity and lung compliance. Gene expression of CTGF, IL-8, and CCL-2 was significantly downregulated in the lung. Treatment with ITBS shortly after delivery attenuated the functional and structural consequences of hyperoxia-induced lung injury to day 7 of life in the preterm rabbit.

Small Molecule Inhibitor Adjuvant Surfactant Therapy Attenuates Ventilator- and Hyperoxia-Induced Lung Injury in Preterm Rabbits.

BACKGROUND: Invasive mechanical ventilation (IMV) has become one of the mainstays of therapy in NICUs worldwide, as a result of which premature babies with extremely low birth weight have been able to survive. Although lifesaving, IMV can result in lung inflammation and injury. Surfactant therapy is considered a standard of care in preterm infants with immature lungs. Recently, small molecule inhibitors like siRNAs and miRNAs have been used for therapeutic purposes. Ddit3 (CHOP), Ang2 and miR34a are known to be upregulated in experimental lung injury. We wanted to test whether inhibitors for these molecules (CHOP siRNA, Ang2 siRNA, and miR34a antagomir) if used alone or with a combination with surfactant (Curosurf®) would help in reducing ventilation and hyperoxia-induced injury in an experimental lung injury model. METHODS: Preterm rabbits born by cesarean section were intratracheally instilled with the three small molecule inhibitors with or without Curosurf® prior to IMV and hyperoxia exposure. Prior to testing the inhibitors in rabbits, these small molecule inhibitors were transfected in mouse lung epithelial cells (MLE12 and AECII) and delivered to neonatal mouse pups intranasally as a proof of concept that surfactant (Curosurf®) could be used as an effective vehicle for administration of such drugs. Survival, pulmonary function tests, histopathology, immunostaining, quantitative PCR and western blotting were done to see the adjuvant effect of surfactant with these three small molecule inhibitors. RESULTS: Our data shows that Curosurf® can facilitate transfection of small molecules in MLE12 cells with the same and/or increased efficiency as Lipofectamine. Surfactant given alone or as an adjuvant with small molecule inhibitors increases survival, decreases IMV and hyperoxia-induced inflammation, improves pulmonary function and lung injury scores in preterm rabbit kits. CONCLUSION: Our study shows that Curosurf® can be used successfully as an adjuvant therapy with small molecule inhibitors for CHOP/Ang2/miR34a. In this study, of the three inhibitors used, miR34a inhibitor seemed to be the most promising compound to combat IMV and hyperoxia-induced lung injury in preterm rabbits.

Tracing exogenous surfactant in vivo in rabbits by the natural variation of 13C.

BACKGROUND: Respiratory Distress Syndrome (RDS) is a prematurity-related breathing disorder caused by a quantitative deficiency of pulmonary surfactant. Surfactant replacement therapy is effective for RDS newborns, although treatment failure has been reported. The aim of this study is to trace exogenous surfactant by 13C variation and estimate the amount reaching the lungs at different doses of the drug. METHODS: Forty-four surfactant-depleted rabbits were obtained by serial bronchoalveolar lavages (BALs), that were merged into a pool (BAL pool) for each animal. Rabbits were in nasal continuous positive airway pressure and treated with 0, 25, 50, 100 or 200 mg/kg of poractant alfa by InSurE. After 90 min, rabbits were depleted again and a new pool (BAL end experiment) was collected. Disaturated-phosphatidylcholine (DSPC) was measured by gas chromatography. DSPC-Palmitic acid (PA) 13C/12C was analyzed by isotope ratio mass spectrometry. One-way non-parametric ANOVA and post-hoc Dunn's multiple comparison were used to assess differences among experimental groups. RESULTS: Based on DSPC-PA 13C/12C in BAL pool and BAL end experiment, the estimated amount of exogenous surfactant ranged from 61 to 87% in dose-dependent way (p < 0.0001) in animals treated with 25 up to 200 mg/kg. Surfactant administration stimulated endogenous surfactant secretion. The percentage of drug recovered from lungs did not depend on the administered dose and accounted for 31% [24-40] of dose. CONCLUSIONS: We reported a risk-free method to trace exogenous surfactant in vivo. It could be a valuable tool for assessing, alongside the physiological response, the delivery efficiency of surfactant administration techniques.

Local pulmonary drug delivery in the preterm rabbit: feasibility and efficacy of daily intratracheal injections.

Recent clinical trials in newborns have successfully used surfactant as a drug carrier for an active compound, to minimize systemic exposure. To investigate the translational potential of surfactant-compound mixtures and other local therapeutics, a relevant animal model is required in which intratracheal administration for maximal local deposition is technically possible and well tolerated. Preterm rabbit pups (born at 28 days of gestation) were exposed to either hyperoxia or normoxia and randomized to receive daily intratracheal surfactant, daily intratracheal saline, or no injections for 7 days. At day 7, the overall lung function and morphology were assessed. Efficacy in terms of distribution was assessed by micro-PET-CT on both day 0 and day 7. Lung function as well as parenchymal and vascular structure were altered by hyperoxia, thereby reproducing a phenotype reminiscent of bronchopulmonary dysplasia (BPD). Neither intratracheal surfactant nor saline affected the survival or the hyperoxia-induced BPD phenotype of the pups. Using PET-CT, we demonstrate that 82.5% of the injected radioactive tracer goes and remains in the lungs, with a decrease of only 4% after 150 min. Surfactant and saline can safely and effectively be administered in spontaneously breathing preterm rabbits. The described model and method enable researchers to evaluate intratracheal pharmacological interventions for the treatment of BPD.

A fully-virulent retargeted oncolytic HSV armed with IL-12 elicits local immunity and vaccine therapy towards distant tumors.

Oncolytic herpes simplex viruses (oHSVs) showed efficacy in clinical trials and practice. Most of them gain cancer-specificity from deletions/mutations in genes that counteract the host response, and grow selectively in cancer cells defective in anti-viral response. Because of the deletions/mutations, they are frequently attenuated or over-attenuated. We developed next-generation oHSVs, which carry no deletion/mutation, gain cancer-specificity from specific retargeting to tumor cell receptors-e.g. HER2 (human epidermal growth factor receptor 2)-hence are fully-virulent in the targeted cancer cells. The type of immunotherapy they elicit was not predictable, since non-attenuated HSVs induce and then dampen the innate response, whereas deleted/attenuated viruses fail to contrast it, and since the retargeted oHSVs replicate efficiently in tumor cells, but spare other cells in the tumor. We report on the first efficacy study of HER2-retargeted, fully-virulent oHSVs in immunocompetent mice. Their safety profile was very high. Both the unarmed R-LM113 and the IL-12-armed R-115 inhibited the growth of the primary HER2-Lewis lung carcinoma-1 (HER2-LLC1) tumor, R-115 being constantly more efficacious. All the mice that did not die because of the primary treated tumors, were protected from the growth of contralateral untreated tumors. The long-term survivors were protected from a second contralateral tumor, providing additional evidence for an abscopal immunotherapeutic effect. Analysis of the local response highlighted that particularly R-115 unleashed the immunosuppressive tumor microenvironment, i.e. induced immunomodulatory cytokines, including IFNγ, T-bet which promoted Th1 polarization. Some of the tumor infiltrating cells, e.g. CD4+, CD335+ cells were increased in the tumors of all responders mice, irrespective of which virus was employed, whereas CD8+, Foxp3+, CD141+ were increased and CD11b+ cells were decreased preferentially in R-115-treated mice. The durable response included a breakage of tolerance towards both HER2 and the wt tumor cells, and underscored a systemic immunotherapeutic vaccine response.

Surfactant replacement therapy in combination with different non-invasive ventilation techniques in spontaneously-breathing, surfactant-depleted adult rabbits.

Nasal intermittent positive pressure ventilation (NIPPV) holds great potential as a primary ventilation support method for Respiratory Distress Syndrome (RDS). The use of NIPPV may also be of great value combined with minimally invasive surfactant delivery. Our aim was to implement an in vivo model of RDS, which can be managed with different non-invasive ventilation (NIV) strategies, including non-synchronized NIPPV, synchronized NIPPV (SNIPPV), and nasal continuous positive airway pressure (NCPAP). Forty-two surfactant-depleted adult rabbits were allocated in six different groups: three groups of animals were treated with only NIV for three hours (NIPPV, SNIPPV, and NCPAP groups), while three other groups were treated with surfactant (SF) followed by NIV (NIPPV+SF, SNIPPV+SF, and NCPAP+SF groups). Arterial gas exchange, ventilation indices, and dynamic compliance were assessed. Post-mortem the lungs were sampled for histological evaluation. Surfactant depletion was successfully achieved by repeated broncho-alveolar lavages (BALs). After BALs, all animals developed a moderate respiratory distress, which could not be reverted by merely applying NIV. Conversely, surfactant administration followed by NIV induced a rapid improvement of arterial oxygenation in all surfactant-treated groups. Breath synchronization was associated with a significantly better response in terms of gas exchange and dynamic compliance compared to non-synchronized NIPPV, showing also the lowest injury scores after histological assessment. The proposed in vivo model of surfactant deficiency was successfully managed with NCPAP, NIPPV, or SNIPPV; this model resembles a moderate respiratory distress and it is suitable for the preclinical testing of less invasive surfactant administration techniques.

Estimating the contribution of surfactant replacement therapy to the alveolar pool: An in vivo study based on 13 C natural abundance in rabbits.

Variation of the isotopic abundance of selected nutrients and molecules has been used for pharmacological and kinetics studies under the premise that the administered molecule has a different isotopic enrichment from the isotopic background of the recipient subject. The aim of this study is to test the feasibility of assessing the contribution of exogenous surfactant phospholipids to the endogenous alveolar pool in vivo after exogenous surfactant replacement therapy in rabbits. The study consisted in measuring the consistency of 13 C/12 C ratio of disaturated-phosphatidylcholine palmitate (DSPC-PA) in 7 lots of poractant alfa, produced over a year, and among bronchoalveolar lavages of 20 rabbits fed with a standard chow. A pilot study was performed in a rabbit model of lavage-induced surfactant deficiency: 7 control rabbits and 4 treated with exogenous surfactant. The contribution of exogenous surfactant to the alveolar pool was assessed after intra-tracheal administration of 200 mg/kg of poractant alfa. The 13 C content of DSPC-PA was measured by isotope ratio mass spectrometry. The mean DSPC-PA 13 C/12 C ratio of the 7 lots of poractant alfa was -18.8‰ with a SD of 0.1‰ (range: -18.9‰; -18.6‰). The mean 13 C/12 C ratio of surfactant DSPC recovered from the lung lavage of 20 rabbits was -28.8 ± 1.2‰ (range: -31.7‰; -25.7‰). The contribution of exogenous surfactant to the total alveolar surfactant could be calculated in the treated rabbits, and it ranged from 83.9% to 89.6%. This pilot study describes a novel method to measure the contribution of the exogenous surfactant to the alveolar pool. This method is based on the natural variation of 13 C, and therefore it does not require the use of chemically synthetized tracers. This method could be useful in human research and especially in surfactant replacement studies in preterm infants.

High-avidity IgA protects the intestine by enchaining growing bacteria.

Vaccine-induced high-avidity IgA can protect against bacterial enteropathogens by directly neutralizing virulence factors or by poorly defined mechanisms that physically impede bacterial interactions with the gut tissues ('immune exclusion'). IgA-mediated cross-linking clumps bacteria in the gut lumen and is critical for protection against infection by non-typhoidal Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium). However, classical agglutination, which was thought to drive this process, is efficient only at high pathogen densities (≥108 non-motile bacteria per gram). In typical infections, much lower densities (100-107 colony-forming units per gram) of rapidly dividing bacteria are present in the gut lumen. Here we show that a different physical process drives formation of clumps in vivo: IgA-mediated cross-linking enchains daughter cells, preventing their separation after division, and clumping is therefore dependent on growth. Enchained growth is effective at all realistic pathogen densities, and accelerates pathogen clearance from the gut lumen. Furthermore, IgA enchains plasmid-donor and -recipient clones into separate clumps, impeding conjugative plasmid transfer in vivo. Enchained growth is therefore a mechanism by which IgA can disarm and clear potentially invasive species from the intestinal lumen without requiring high pathogen densities, inflammation or bacterial killing. Furthermore, our results reveal an untapped potential for oral vaccines in combating the spread of antimicrobial resistance.

A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells.

The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene γ134.5 and additional genes deleted. One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 (human epidermal growth factor receptor 2), which is present in breast, ovary, and other cancers, and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical-grade viruses destined for humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-amino-acid (aa) short peptide (named GCN4) in the gH of R-LM113; this recombinant was retargeted to HER2 through insertion in gD of a single-chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R) whose N terminus consists of an scFv to GCN4 and therefore is capable of interacting with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R cells, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected for an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSVs in non-cancer cells.IMPORTANCE There is growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immune checkpoint inhibitors. The oncolytic HSV approved for clinical practice and those in clinical trials are attenuated viruses. An alternative to attenuation is a cancer specificity achieved by tropism retargeting to selected cancer receptors. However, the retargeted oncolytic HSVs strictly depend on cancer receptors for infection. Here, we devised a strategy for in vitro cultivation of retargeted HSVs in non-cancer cells. The strategy envisions a double-retargeting approach: one retargeting is via gD to the cancer receptor, and the second retargeting is via gH to an artificial receptor expressed in Vero cells. The double-retargeted HSV uses alternatively the two receptors to infect cancer cells or producer cells. A universal non-cancer cell line for growth of clinical-grade retargeted HSVs represents a step forward in the translational phase.

Retargeting Strategies for Oncolytic Herpes Simplex Viruses.

Most of the oncolytic herpes simplex viruses (HSVs) exhibit a high safety profile achieved through attenuation. They carry defects in virulence proteins that antagonize host cell response to the virus, including innate response, apoptosis, authophagy, and depend on tumor cell proliferation. They grow robustly in cancer cells, provided that these are deficient in host cell responses, which is often the case. To overcome the attenuation limits, a strategy is to render the virus highly cancer-specific, e.g., by retargeting their tropism to cancer-specific receptors, and detargeting from natural receptors. The target we selected is HER-2, overexpressed in breast, ovarian and other cancers. Entry of wt-HSV requires the essential glycoproteins gD, gH/gL and gB. Here, we reviewed that oncolytic HSV retargeting was achieved through modifications in gD: the addition of a single-chain antibody (scFv) to HER-2 coupled with appropriate deletions to remove part of the natural receptors' binding sites. Recently, we showed that also gH/gL can be a retargeting tool. The insertion of an scFv to HER-2 at the gH N-terminus, coupled with deletions in gD, led to a recombinant capable to use HER-2 as the sole receptor. The retargeted oncolytic HSVs can be administered systemically by means of carrier cells-forcedly-infected mesenchymal stem cells. Altogether, the retargeted oncolytic HSVs are highly cancer-specific and their replication is not dependent on intrinsic defects of the tumor cells. They might be further modified to express immunomodulatory molecules.

αvß3 Integrin Boosts the Innate Immune Response Elicited in Epithelial Cells through Plasma Membrane and Endosomal Toll-Like Receptors.

We report that αvß3 integrin strongly affects the innate immune response in epithelial cells. αvß3 integrin greatly increased the response elicited via plasma membrane Toll-like receptors (TLRs) by herpes simplex virus or bacterial ligands. The endosomal TLR3, not the cytosolic sensor interferon gamma-inducible protein 16 (IFI16), was also boosted by αvß3 integrin. The boosting was exerted specifically by αvß3 integrin but not by αvß6 or αvß8 integrin. Current and previous work indicates that integrin-TLR cooperation occurs in epithelial and monocytic cells. The TLR response should be considered an integrin-TLR response.

Latent virus infection upregulates CD40 expression facilitating enhanced autoimmunity in a model of multiple sclerosis.

Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of multiple sclerosis (MS) by multiple groups working worldwide. Previously, we reported that when experimental autoimmune encephalomyelitis (EAE) was induced in mice latently infected with murine γ-herpesvirus 68 (γHV-68), the murine homolog to EBV, a disease more reminiscent of MS developed. Specifically, MS-like lesions developed in the brain that included equal numbers of IFN-γ producing CD4(+) and CD8(+) T cells and demyelination, none of which is observed in MOG induced EAE. Herein, we demonstrate that this enhanced disease was dependent on the γHV-68 latent life cycle and was associated with STAT1 and CD40 upregulation on uninfected dendritic cells. Importantly, we also show that, during viral latency, the frequency of regulatory T cells is reduced via a CD40 dependent mechanism and this contributes towards a strong T helper 1 response that resolves in severe EAE disease pathology. Latent γ-herpesvirus infection established a long-lasting impact that enhances subsequent adaptive autoimmune responses.

Gammaherpesvirus latency accentuates EAE pathogenesis: relevance to Epstein-Barr virus and multiple sclerosis.

Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of multiple sclerosis (MS), yet EBV's role in MS remains elusive. We utilized murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, to examine how infection by a virus like EBV could enhance CNS autoimmunity. Mice latently infected with γHV-68 developed more severe EAE including heightened paralysis and mortality. Similar to MS, γHV-68EAE mice developed lesions composed of CD4 and CD8 T cells, macrophages and loss of myelin in the brain and spinal cord. Further, T cells from the CNS of γHV-68 EAE mice were primarily Th1, producing heightened levels of IFN-γ and T-bet accompanied by IL-17 suppression, whereas a Th17 response was observed in uninfected EAE mice. Clearly, γHV-68 latency polarizes the adaptive immune response, directs a heightened CNS pathology following EAE induction reminiscent of human MS and portrays a novel mechanism by which EBV likely influences MS and other autoimmune diseases.

Epstein-Barr virus infection of human brain microvessel endothelial cells: a novel role in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory neurological disease that is widely regarded as the outcome of complex interactions between a genetic predisposition and an environmental trigger. Epstein-Barr virus (EBV) has recently been associated with the onset of MS, yet understanding how it elicits autoimmunity remains elusive. Neuroinflammation, including the entry of autoreactive T cells, likely follows a breach of the blood-brain barrier (BBB) leading to CNS lesions in MS. We show that EBV can infect human BBB cells leading to increased production of pro-inflammatory mediators that result in immune cell adherence thus modeling a key step in MS pathogenesis.