Retargeting of Viruses to Generate Oncolytic Agents

Oncolytic virus therapy is based on the ability of viruses to effectively infect and kill tumor cells without destroying the normal tissues. While some viruses seem to have a natural preference for tumor cells, most viruses require the modification of their tropism to specifically enter and replicate in such cells. This review aims to describe the transductional targeting strategies currently employed to specifically redirect viruses towards surface receptors on tumor cells. Three major strategies can be distinguished; they involve (i) the incorporation of new targeting specificity into a viral surface protein, (ii) the incorporation of a scaffold into a viral surface protein to allow the attachment of targeting moieties, and (iii) the use of bispecific adapters to mediate targeting of a virus to a specified moiety on a tumor cell. Of each strategy key features, advantages and limitations are discussed and examples are given. Because of their potential to cause sustained, multiround infection—a desirable characteristic for eradicating tumors—particular attention is given to viruses engineered to become self-targeted by the genomic expression of a bispecific adapter protein

Viral protein synthesis in cowpea mosaic virus infected protoplasts

In contrast to the situation concerning bacterial and, to a lesser extent, animal RNA viruses, little is known about the biochemical processes occurring in plant cells due to plant RNA virus infection. Such processes are difficult to study using intact plants or leaves. Great effort has therefore been spent in developing in vitro cultures of plant protoplasts, but the use of these protoplasts has been seriously hampered by various technical problems.It is clear that plant RNA virus infections give rise to a number of specific reactions in infected cells, particularly at the level of RNA and protein synthesis. The multiplication of these viruses implies a manifold copying of their structural components, namely RNA and protein. These activities clearly require the involvement of other RNA and protein species in view of, for instance, the drastic cytopathic effects that are often caused by infection. Thus, detailed knowledge of virus-induced RNA and protein synthesis is a prerequisite for an understanding of the infection process.In this thesis investigations are described concerning the proteins involved in the multiplication of cowpea mosaic virus (CPMV) in cowpea protoplasts.The relevant properties of CPMV are summarized in chapter 2. Research on CPMV is interesting not only in the field of plant virology as the virus is also of general virological significance. Structurally, for example, CPMV shares a number of striking features with the animal picornaviruses like poliovirus and footand-mouth disease virus. From a molecular biological point of view the multiplication of CPMV presents several intriguing specific questions. CPMV is a multicomponent virus: its genome consists of two different single-stranded RNA molecules occurring in separate nucleoprotein particles, both of which are essential for infection. It would be of interest to learn which functions are specified by the two genome pieces, whether the separate viral components can give rise to some virus-specific biochemical expression within the cell and how they complement each other during normal infection. Characteristic cytopathic structures are induced in plant cells upon infection with CPMV in the form of vesicular membranes embedded in amorphous electron-dense material. It is now well established that replication of the viral RNA is localized in these structures. Information about their development and functioning is thus required.Chapter 2 also contains an introduction to the essential features of plant protoplasts and an enumeration of the diverse possibilities they can offer for the investigation of specific virological problems. Finally, the present state of knowledge about the biochemistry of multiplication of plant viruses, as obtained mainly through the use of protoplasts, is reviewed.The isolation of viable protoplasts from the leaf mesophyll tissue of cowpea requires the stringent control of the growth conditions of the plants (chapter 3). Various factors appeared to influence the quality of the protoplasts and their infectability with CPMV. The combination of cowpea mesophyll protoplasts and CPMV appeared to offer a very attractive system. Based on the original study of HIBI et al. (1975) a procedure was developed for the preparation and infection of the protoplasts, the most rapid and simple one so far described (chapter 3). The major reasons for this are:- a short growth period (about 10 days under the conditions used) is required to obtain suitable leaf material- for an efficient enzymatic digestion of the leaves it is sufficient to damage the lower epidermis by means of carborundum powder and paint-brush instead of stripping off the epidermis with forceps- enzymatic one-step isolation of protoplasts can be achieved by simultaneous digestion with pectinase and cellulase- inoculation of cowpea protoplasts with CPMV does not require the use of polycations, making the preincubations of protoplasts and virus superfluous.Using this standard procedure, synchronous infection in virtually all protoplasts could be routinely established.Radioactive precursors of RNA and protein added to the cowpea protoplast incubation medium were taken up and incorporated into macromolecules to high specific activities (chapter 4). This light-dependent metabolic activity increased during the first 30 h after isolation of the protoplasts. It could be influenced by several metabolic inhibitors. RNA synthesis, for instance, was strongly inhibited by actinomycin D and cordycepin, whereas ot-amanitin and rifampicin had only a slight effect. Protein synthesis in the protoplasts could be blocked completely by means of cycloheximide, and was also sensitive to the action of antibiotics such as chloramphenicol, puromycin and, to a lesser degree, lincomycin. These inhibitor studies, as well as the results of analyses of RNAs and proteins synthesized in the protoplasts, indicated that the metabolic activity of the chloroplasts is low and mainly limited to light-dependent energy generation.The multiplication of CPMV in cowpea mesophyll protoplasts relies upon host-coded DNA-dependent RNA synthesis during the earliest stage of infection. As described in chapter 5, propagation of the virus was completely prevented when protoplast DNA transcription and thereby almost all RNA synthesis was inhibited by means of actinomycin D, provided that the drug was applied at the time of inoculation or immediately thereafter. The degree of inhibition of CPMV replication by actinomycin D rapidly decreased when the antibiotic was added later after inoculation; the production of viral nucleoprotein particles became progressively more resistant to the drug, showing complete resistance at about 8 h after inoculation, a time at which infection was still in its latent phase.Surprisingly, synthesis of CPMV antigen was still demonstrable by immunofluorescent antibody staining techniques in protoplasts in which virus replication had been completely blocked by actinomycin D. Under these circumstances de novo production of viral top component (empty capsids) was observed. In addition to the CPMV capsid proteins, these experiments revealed another 3 viral-related polypeptides in infected protoplasts (molecular weights 170,000, 110,000 and 84,000). Since their synthesis also clearly continued during inhibition of host RNA synthesis by actinomycin D it was concluded that they were coded for by the viral genome.A more detailed analysis of the proteins involved in CPMV multiplication is described in chapter 6. At least 11 proteins not present in mock-infected protoplasts or present in much lower amounts were detected in infected protoplasts. Their molecular weights were estimated to be 170,000, 130,000, 112,000, 110,000, 87,000, 84,000, 68,000,37,000, 30,000,24,000 and 23,000. Host-specific protein synthesis appeared to be hardly affected by C~ infection. Virusspecific proteins therefore had to be distinguished from a large variety of protoplast proteins. This appeared to be greatly facilitated by radioactive labeling of the protoplasts for short periods during a suitable phase of the infection cycle and by subcellular fractionation of the protoplasts. The treatment of the particulate fractions with the mild detergent digitonin yielded extracts in which proteins could be detected by polyacrylamide gel electrophoretic analysis that otherwise remained hidden by a general high background of radioactivity upon direct analysis of the untreated particulate fractions.All of the proteins apparently concerned in CPMV infection were most clearly observed late in infection. None of them was detectable during the actinomycin D-sensitive latent period. The 170 and 30 kilodalton proteins were the first detectable, namely from about 10 h after inoculation. All others became apparent about 6 h later.The induction of the characteristic cytopathic structures in CPMV-infected cells suggests the induction and/or stimulation of synthesis of a large number of proteins by the virus. Upon fractionation these proteins should appear in the particulate fractions of the protoplasts. However, only 2 virus-induced polypeptides (112 and 68 kilodalton) were demonstrated exclusively in these fractions. Other components of the cytopathic structures presumably remained in the extracted pellet residues.In chapter 8 further characterization of the viral-related proteins found in CPMV-infected protoplasts is described. The 37, 24 and 23 kilodalton proteins were identified as viral capsid proteins by comparing their electrophoretic mobility in polyacrylamide gels with those of proteins from purified CPMV and by immunoprecipitation with anti-CPMV serum. They occurred in the soluble fraction of protoplasts only in viral particles, not as free proteins. The 170 and 30 kilodalton polypeptides also appeared to be virus-specific and to be coded for by CPMV bottom component RNA: they comigrated in polyacrylamide slabgels with the main products of in vitro translation of this RNA in a messengerdependent lysate of rabbit reticulocytes. These polypeptides were also induced in protoplasts after inoculation with purified CPMV bottom component. Under these conditions some other viral-related proteins were also synthesized, but not the viral capsid proteins. In contrast, inoculation with purified CPMV middle component did not delectably affect protoplast protein synthesis. These experiments strongly suggest that CPMV B-RNA specifies one or more early functions one of which being the replicase function, while M-RNA specifies late functions, particularly the viral capsid proteins.Since the total size of the viral-related proteins by far exceeded the theoretical coding potential of the CPMV genome, the occurrence of precursor-product relations was investigated by means of pulse-chase experiments and by attempting to accumulate possible precursors by blocking their proteolytic processing. The results of the latter method were negative. Upon chasing the 35 S-methionine label with unlabeled amino acid the 87,000 dalton polypeptide appeared to be unstable, as was a protein with the same electrophoretic mobility synthesized in uninfected protoplasts. This protein thus probably represents a host-specific protein the synthesis of which is stimulated by CPMV infection. Furthermore, these experiments revealed that several discrete oligopeptides were cleaved from the smaller CPMV coat protein after assembly into a virion. Although the involvement of precursor- product relations could not be definitely excluded on the basis of the methods used, another possible mechanism of viral gene expression was also investigated, namely the occurrence of subgenomic viral messengers. Upon analysis of 32 P-labeled RNA from CPMV-infected protoplasts by polyacrylamide gel electrophoresis, 2 RNAs of genome length were the only detectable virus-specific polynucleotides. These results suggest a mechanism for the translation of the CPMV genome using internal initiation sites. Further research is, however, required to unequivocally elucidate the strategy of the viral gene expression.<p/

Ciclesonide versus other inhaled corticosteroids for chronic asthma in children

Background Inhaled corticosteroids (ICS) are the cornerstone of asthma maintenance treatment in children. Particularly among parents, there is concern about the safety of ICS as studies in children have shown reduced growth. Small-particle-size ICS targeting the smaller airways have improved lung deposition and effective asthma control might be achieved at lower daily doses. Ciclesonide is a relatively new ICS. This small-particle ICS is a pro-drug that is converted in the airways to an active metabolite and therefore with potentially less local (throat infection) and systemic (reduced growth) side effects. It can be inhaled once daily, thereby possibly improving adherence. Objectives To assess the efficacy and adverse effects of ciclesonide compared to other ICS in the management of chronic asthma in children. Search methods We searched the Cochrane Airways Group Register of trials with pre-defined terms. Additional searches of MEDLINE (via PubMed), EMBASE and Clinicalstudyresults. org were undertaken. Searches are up to date to 7 November 2012. Selection criteria Randomised controlled parallel or cross-over studies were eligible for the review. We included studies comparing ciclesonide with other corticosteroids both at nominally equivalent doses or lower doses of ciclesonide. Data collection and analysis Two review authors independently assessed trial quality and extracted data. Study authors were contacted for additional information. Adverse effects information was collected from the trials. Main results Six studies were included in this review (3256 children, 4 to 17 years of age). Two studies were published as conference abstracts only. Ciclesonide was compared to budesonide and fluticasone. Ciclesonide compared to budesonide (dose ratio 1: 2): asthma symptoms and adverse effect were similar in both groups. Pooled results showed no significant difference in children who experience an exacerbation (risk ratio (RR) 2.20, 95% confidence interval (CI) 0.75 to 6.43). Both studies reported that 24-hour urine cortisol levels showed a statistically significant decrease in the budesonide group compared to the ciclesonide group. Ciclesonide compared to fluticasone (dose ratio 1: 1): no significant differences were found for the outcome asthma symptoms. Pooled results showed no significant differences in number of patients with exacerbations (RR 1.37, 95% CI 0.58 to 3.21) and data from a study that could not be pooled in the meta-analysis reported similar numbers of patients with exacerbations in both groups. None of the studies found a difference in adverse effects. No significant difference was found for 24-hour urine cortisol levels between the groups (mean difference 0.54 nmol/mmol, 95% CI -5.92 to 7.00). Ciclesonide versus fluticasone (dose ratio 1: 2) was assessed in one study and showed similar results between the two corticosteroids for asthma symptoms. The number of children with exacerbations was significantly higher in the ciclesonide group (RR 3.57, 95% CI 1.35 to 9.47). No significant differences were found in adverse effects (RR 0.98, 95% CI 0.81 to 1.14) and 24-hour urine cortisol levels (mean difference 1.15 nmol/mmol, 95% CI 0.07 to 2.23). The quality of evidence was judged 'low' for the outcomes asthma symptoms and adverse events and 'very low' for the outcome exacerbations for ciclesonide versus budesonide (dose ratio 1: 1). The quality of evidence was graded 'moderate' for the outcome asthma symptoms, 'very low' for the outcome exacerbations and 'low' for the outcome adverse events for ciclesonide versus fluticasone (dose ratio 1: 1). For ciclesonide versus fluticasone (dose ratio 1: 2) the quality was rated 'low' for the outcome asthma symptoms and 'very low' for exacerbations and adverse events (dose ratio 1: 2). Authors' conclusions An improvement in asthma symptoms, exacerbations and side effects of ciclesonide versus budesonide and fluticasone could be neither demonstrated nor refuted and the trade-off between benefits and harms of using ciclesonide instead of budesonide or fluticasone is unclear. The resource use or costs of different ICS should therefore also be considered in final decision making. Longer-term superiority trials are needed to identify the usefulness and safety of ciclesonide compared to other ICS. Additionally these studies should be powered for patient relevant outcomes (exacerbations, asthma symptoms, quality of life and side effects). There is a need for studies comparing ciclesonide once daily with other ICS twice daily to assess the advantages of ciclesonide being a pro-drug that can be administered once daily with possibly increased adherence leading to increased control of asthma and fewer side effects

Retargeting of viruses to generate oncolytic agents

Oncolytic virus therapy is based on the ability of viruses to effectively infect and kill tumor cells without destroying the normal tissues. While some viruses seem to have a natural preference for tumor cells, most viruses require the modification of their tropism to specifically enter and replicate in such cells. This review aims to describe the transductional targeting strategies currently employed to specifically redirect viruses towards surface receptors on tumor cells. Three major strategies can be distinguished; they involve (i) the incorporation of new targeting specificity into a viral surface protein, (ii) the incorporation of a scaffold into a viral surface protein to allow the attachment of targeting moieties, and (iii) the use of bispecific adapters to mediate targeting of a virus to a specified moiety on a tumor cell. Of each strategy key features, advantages and limitations are discussed and examples are given. Because of their potential to cause sustained, multiround infection-a desirable characteristic for eradicating tumorsparticular attention is given to viruses engineered to become self-targeted by the genomic expression of a bispecific adapter protein

Effect of oxygen on the expression of hypoxia-inducible factors in human fetal lung explants

Background: Fetal lung development requires proper coordination between lung epithelial and vascular morphogenesis. A major determinant in lung vascular development is vascular endothelial growth factor (VEGF), which is regulated by hypoxia-inducible factors (HIFs). VEGF is expressed in the airway epithelium, while its receptors (VEGFRs) are expressed in the pulmonary mesenchyme. The hypoxic environment in utero is beneficial for fetal organogenesis, especially vascular development. However, little is known about the expression of HIFs and VEGFR-2 in the human fetal lung in vitro. Objectives: The purpose of this study was to investigate the effects of hypoxia on fetal lung morphology and mRNA expression of VEGF, VEGFR-2, HIF-2α, and HIF-3α. Methods: An explant culture technique was used to study the effects of normoxic and hypoxic conditions on human fetal lung. Results: The morphology remained largely unchanged in explants cultured under hypoxic or normoxic conditions. Quantitative RT-PCR showed that the mRNA expression of VEGF-A, but not VEGFR-2 is upregulated in explants cultured at 1.5% compared with 21% oxygen. We observed a nonsignificant increase in HIF-2α and HIF-3α mRNA expression in explants cultured at 1.5% oxygen. These data suggest that the mRNA expression of VEGF, and possibly HIF-2α and HIF-3α, is regulated by hypoxia in the developing human lung. Conclusion: This lung explant culture model appears to be a valuable model to unravel the molecular mechanisms of human lung development

Combating pan-coronavirus infection by indomethacin through simultaneously inhibiting viral replication and inflammatory response

Severe infections with coronaviruses are often accompanied with hyperinflammation, requiring therapeutic strategies to simultaneously tackle the virus and inflammation. By screening a safe-in-human broad-spectrum antiviral agents library, we identified that indomethacin can inhibit pan-coronavirus infection in human cell and airway organoids models. Combining indomethacin with oral antiviral drugs authorized for treating COVID-19 results in synergistic anti-coronavirus activity. Coincidentally, screening a library of FDA-approved drugs identified indomethacin as the most potent potentiator of interferon response through increasing STAT1 phosphorylation. Combining indomethacin with interferon-alpha exerted synergistic antiviral effects against multiple coronaviruses. The anti-coronavirus activity of indomethacin is associated with activating interferon response. In a co-culture system of lung epithelial cells with macrophages, indomethacin inhibited both viral replication and inflammatory response. Collectively, indomethacin is a pan-coronavirus inhibitor that can simultaneously inhibit virus-triggered inflammatory response. The therapeutic potential of indomethacin can be further augmented by combining it with oral antiviral drugs or interferon-alpha.</p

Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability

Background and aims: Obese and diabetic mice display enhanced intestinal permeability and metabolic endotoxaemia that participate in the occurrence of metabolic disorders. Our recent data support the idea that a selective increase of Bifidobacterium spp. reduces the impact of high-fat diet-induced metabolic endotoxaemia and inflammatory disorders. Here, we hypothesised that prebiotic modulation of gut microbiota lowers intestinal permeability, by a mechanism involving glucagon-like peptide-2 (GLP-2) thereby improving inflammation and metabolic disorders during obesity and diabetes. Methods: Study 1: ob/ob mice (Ob-CT) were treated with either prebiotic (Ob-Pre) or non-prebiotic carbohydrates as control (Ob-Cell). Study 2: Ob-CT and Ob-Pre mice were treated with GLP-2 antagonist or saline. Study 3: Ob-CT mice were treated with a GLP-2 agonist or saline. We assessed changes in the gut microbiota, intestinal permeability, gut peptides, intestinal epithelial tight-junction proteins ZO-1 and occludin (qPCR and immunohistochemistry), hepatic and systemic inflammation. Results: Prebiotic-treated mice exhibited a lower plasma lipopolysaccharide (LPS) and cytokines, and a decreased hepatic expression of inflammatory and oxidative stress markers. This decreased inflammatory tone was associated with a lower intestinal permeability and improved tight-junction integrity compared to controls. Prebiotic increased the endogenous intestinotrophic proglucagon-derived peptide (GLP-2) production whereas the GLP-2 antagonist abolished most of the prebiotic effects. Finally, pharmacological GLP-2 treatment decreased gut permeability, systemic and hepatic inflammatory phenotype associated with obesity to a similar extent as that observed following prebiotic-induced changes in gut microbiota. Conclusion: We found that a selective gut microbiota change controls and increases endogenous GLP-2 production, and consequently improves gut barrier functions by a GLP-2-dependent mechanism, contributing to the improvement of gut barrier functions during obesity and diabetes

Colistin dry powder inhalation with the Twincer (TM):An effective and more patient friendly alternative to nebulization

BACKGROUND: Nebulization of antimicrobial drugs such as tobramycin and colistin is a cornerstone in the treatment of patients with cystic fibrosis (CF) infected with Pseudomonas aeruginosa. However, nebulization has a high treatment burden. The Twincer™ is a dry powder inhaler specifically developed for the inhalation of antibiotics such as colistin. The aim of this study was to compare patient outcomes and experience with colistin dry powder by the Twincer with nebulization of colistin or tobramycin in adult CF patients in a real-life setting. METHODS: This was a retrospective study from 01-01-2015 until 01-07-2018. Effectiveness was evaluated by comparing FEV1 decline and exacerbation rate during a mean of 4.1 years of nebulization therapy prior to the initiation of the Twincer against the same values during a mean of 1.7 years of treatment with the Twincer. RESULTS: Twenty-one patients were evaluated, of whom twelve could be included in the effectiveness analysis, with a total of twenty patient years. Of all patients 71.4% preferred therapy with the Twincer over nebulization. Twincer use resulted in high treatment adherence with an average adherence rate of 92.5%. There was no significant difference in annual decline in FEV1%pred prior to and after start changing from nebulization to the use of the Twincer powder inhaler (median decline -1.56 [-5.57-5.31] and 1.35 [-8.45-6.36]) respectively, p = 0.45 (linear mixed effect model)). No significant difference was found in the number of intravenous or combined total intravenous and oral antibiotic courses during Twincer therapy compared to when using nebulization (1.68 and 2.49 courses during Twincer therapy versus 1.51 and 2.94 courses during nebulization, p = 0.88 and p = 0.63). CONCLUSION: Colistin dry powder inhalation with the Twincer is a more patient friendly alternative to nebulization, and we did not observe significant differences in the clinical outcome, regarding lung function and exacerbation rates

Modulation of airway epithelial innate immunity and wound repair by M(GM-CSF) and M(M-CSF) macrophages

Airway epithelial cells and macrophages participate in inflammatory responses to external noxious stimuli, which can cause epithelial injury. Upon injury, epithelial cells and macrophages act in concert to ensure rapid restoration of epithelial integrity. The nature of the interactions between these cell types during epithelial repair is incompletely understood. We used an in vitro human coculture model of primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC) and polarized primary monocyte-derived macrophages. Using this coculture, we studied the contribution of macrophages to epithelial innate immunity, wound healing capacity, and epithelial exposure to whole cigarette smoke (WCS). Coculture of ALI-PBEC with lipopolysaccharide (LPS)-activated M(GM-CSF) macrophages increased the expression ofDEFB4A,CXCL8, andIL6at 24 h in the ALI-PBEC, whereas LPS-activated M(M-CSF) macrophages only increased epithelialIL6expression. Furthermore, wound repair was accelerated by coculture with both activated M(GM-CSF) and M(M-CSF) macrophages, also following WCS exposure. Coculture of ALI-PBEC and M(GM-CSF) macrophages resulted in increasedCAMPexpression in M(GM-CSF) macrophages, which was absent in M(M-CSF) macrophages.CAMPencodes LL-37, an antimicrobial peptide with immune-modulating and repair-enhancing activities. In conclusion, dynamic crosstalk between ALI-PBEC and macrophages enhances epithelial innate immunity and wound repair, even upon concomitant cigarette smoke exposure.Pathogenesis and treatment of chronic pulmonary disease