Involvement of the European Medicines Agency in multi-stakeholder regulatory science research projects: experiences of staff members and project coordinators.

Background: The European Medicines Agency (EMA) interacts with many different stakeholders involved in the development of drugs, including academic researchers. In recent years, EMA has collaborated more closely with academia, inter alia by taking part in external research projects such as those set up under the Horizon 2020 program in general and the Innovative Medicines Initiative in particular. The aim of this study was to evaluate the perceived added value of EMA's involvement in these projects, both from the perspective of the Agency's participating Scientific Officers and of the coordinators of the consortia that undertook them. Methods: Semi-structured interviews were conducted with the coordinators of 21 ongoing or recently finalized projects in which EMA has participated, as well as with the Agency experts contributing to them. Results: In total, 40 individuals were interviewed, of whom 23 were project coordinators and 17 were EMA staff members. While most of the projects were reported to suffer from delays due to the SARS-CoV-2 pandemic, the consortia adapted to the circumstances and their members still expected to deliver on their objectives. EMA's input into the projects ranged from providing guidance by reviewing documents and attending meetings to creating project materials and disseminating them. The frequency of communication between EMA and the consortia varied widely. The projects generated a diverse set of outputs, which encompassed new or improved medicinal products, methodological standards, research infrastructures, and educational tools. All of the coordinators expressed that EMA's contributions to their projects had increased the scientific relevance of their consortium's work, and the EMA experts found that the knowledge and the deliverables produced by the projects were valuable, taking into consideration the time they had invested into them. In addition, interviewees highlighted some actions which could be taken to increase the regulatory significance of the project outcomes. Conclusion: EMA's engagement in external research projects benefits the consortia conducting them and supports the Agency's mission to foster scientific excellence and advance regulatory science.

Cells and mediators of inflammation as effectors of epithelial repair in the inflamed intestine.

Mucosal and histological healing have become the gold standards for assessing the efficacy of therapy in patients living with inflammatory bowel diseases (IBD). Despite these being the accepted goals in therapy, the mechanisms that underlie the healing of the mucosa after an inflammatory insult are not well understood, and many patients fail to meet this therapeutic endpoint. Here we review the emerging evidence that mediators (e.g., prostaglandins, cytokines, proteases, reactive oxygen, and nitrogen species) and innate immune cells (e.g., neutrophils and monocytes/macrophages), that are involved in the initiation of the inflammatory response, are also key players in the mechanisms underlying mucosal healing to resolve chronic inflammation in the colon. The dual function mediators comprise an inflammation/repair program that returns damaged tissue to homeostasis. Understanding details of the dual mechanisms of these mediators and cells may provide the basis for the development of drugs that can help to stimulate epithelial repair in patients affected by IBD.

The Shc protein Rai enhances T-cell survival under hypoxia.

Hypoxia occurs in physiological and pathological conditions. T cells experience hypoxia in pathological and physiological conditions as well as in lymphoid organs. Indeed, hypoxia-inducible factor 1α (HIF-1α) affects T cell survival and functions. Rai, an Shc family protein member, exerts pro-survival effects in hypoxic neuroblastoma cells. Since Rai is also expressed in T cells, we here investigated its role in hypoxic T cells. In this work, hypoxia differently affected cell survival, proapoptotic, and metabolic programs in T cells, depending upon Rai expression. By using Jurkat cells stably expressing Rai and splenocytes from Rai-/- mice, we demonstrated that Rai promotes T cell survival and affects cell metabolism under hypoxia. Upon exposure to hypoxia, Jurkat T cells expressing Rai show (a) higher HIF-1α protein levels; (b) a decreased cell death and increased Akt/extracellular-signal-regulated kinase phosphorylation; (c) a decreased expression of proapoptotic markers, including caspase activities and poly(ADP-ribose) polymerase cleavage; (d) an increased glucose and lactate metabolism; (e) an increased activation of nuclear factor-kB pathway. The opposite effects were observed in hypoxic splenocytes from Rai-/- mice. Thus, Rai plays an important role in hypoxic signaling and may be relevant in the protection of T cells against hypoxia.

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an extracellular opportunistic bacterial pathogen commonly associated with infectious complications in susceptible individuals, such as those with underlying diseases including HIV/AIDS and cystic fibrosis. Antibiotic resistance in multiple strains of P. aeruginosa is a rapidly developing clinical problem. We have previously demonstrated that the oxygen levels at the site of P. aeruginosa infection can strongly influence virulence and antibiotic resistance in this pathogen, although the oxygen-sensing and -signaling mechanisms underpinning this response have remained unknown. In this study, we investigated the potential role of the putative oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) in the control of virulence and antibiotic resistance in P. aeruginosa We found that a P. aeruginosa strain lacking PPHD (PAO310) exhibits increased virulence associated with increased bacterial motility. Furthermore, PPHD-deficient P. aeruginosa displayed enhanced antibiotic resistance against tetracycline through increased expression of the xenobiotic transporters mexEF-oprN and MexXY. Of note, the effect of the PPHD knockout on antibiotic resistance was phenocopied in bacteria exposed to atmospheric hypoxia. We conclude that PPHD is a putative bacterial oxygen sensor that may link microenvironmental oxygen levels to virulence and antibiotic resistance in P. aeruginosa.

Hydroxylase Inhibition Selectively Induces Cell Death in Monocytes.

Hypoxia is a common and prominent feature of the microenvironment at sites of bacteria-associated inflammation in inflammatory bowel disease. The prolyl-hydroxylases (PHD1/2/3) and the asparaginyl-hydroxylase factor-inhibiting HIF are oxygen-sensing enzymes that regulate adaptive responses to hypoxia through controlling the activity of HIF and NF-κB-dependent transcriptional pathways. Previous studies have demonstrated that the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) is effective in the alleviation of inflammation in preclinical models of inflammatory bowel disease, at least in part, through suppression of IL-1ß-induced NF-κB activity. TLR-dependent signaling in immune cells, such as monocytes, which is important in bacteria-driven inflammation, shares a signaling pathway with IL-1ß. In studies into the effect of pharmacologic hydroxylase inhibition on TLR-induced inflammation in monocytes, we found that DMOG selectively triggers cell death in cultured THP-1 cells and primary human monocytes at concentrations well tolerated in other cell types. DMOG-induced apoptosis was independent of increased caspase-3/7 activity but was accompanied by reduced expression of the inhibitor of apoptosis protein 1 (cIAP1). Based on these data, we hypothesize that pharmacologic inhibition of the HIF-hydroxylases selectively targets monocytes for cell death and that this may contribute to the anti-inflammatory activity of HIF-hydroxylase inhibitors.

Pharmacologic inhibition of hypoxia-inducible factor (HIF)-hydroxylases ameliorates allergic contact dermatitis.

BACKGROUND: When an immune cell migrates from the bloodstream to a site of chronic inflammation, it experiences a profound decrease in microenvironmental oxygen levels leading to a state of cellular hypoxia. The hypoxia-inducible factor-1α (HIF-1α) promotes an adaptive transcriptional response to hypoxia and as such is a major regulator of immune cell survival and function. HIF hydroxylases are the family of oxygen-sensing enzymes primarily responsible for conferring oxygen dependence upon the HIF pathway. METHODS: Using a mouse model of allergic contact dermatitis (ACD), we tested the effects of treatment with the pharmacologic hydroxylase inhibitor DMOG, which mimics hypoxia, on disease development. RESULTS: Re-exposure of sensitized mice to 2,4-dinitrofluorobenzene (DNFB) elicited inflammation, edema, chemokine synthesis (including CXCL1 and CCL5) and the recruitment of neutrophils and eosinophils. Intraperitoneal or topical application of the pharmacologic hydroxylase inhibitors dymethyloxalylglycine (DMOG) or JNJ1935 attenuated this inflammatory response. Reduced inflammation was associated with diminished recruitment of neutrophils and eosinophils but not lymphocytes. Finally, hydroxylase inhibition reduced cytokine-induced chemokine production in cultured primary keratinocytes through attenuation of the JNK pathway. CONCLUSION: These data demonstrate that hydroxylase inhibition attenuates the recruitment of neutrophils to inflamed skin through reduction of chemokine production and increased neutrophilic apoptosis. Thus, pharmacologic inhibition of HIF hydroxylases may be an effective new therapeutic approach in allergic skin inflammation.

Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors.

The factors influencing the virulence of P. aeruginosa in the development of invasive infection remain poorly understood. Here, we investigated the role of the host microenvironment in shaping pathogen virulence and investigated the mechanisms involved. Comparing seven paired genetically indistinguishable clinical bloodstream and peripheral isolates of P. aeruginosa, we demonstrate that isolates derived from bloodstream infections are more virulent than their peripheral counterparts (p = 0.025). Bloodstream and peripheral isolates elicited similar NF-kB responses in a THP-1 monocyte NF-kappaB reporter cell line implicating similar immunogenicity. Proteomic analysis by mass spectrometry identified multiple virulence and virulence-related factors including LecA and RpoN in significantly greater abundance in the bacterial supernatant from the bloodstream isolate in comparison to that from the corresponding peripheral isolate. Investigation by qPCR revealed that control of expression of these virulence factors was not due to altered levels of transcription. Based on these data, we hypothesize a post-transcriptional mechanism of virulence regulation in P. aeruginosa bloodstream infections influenced by surrounding microenvironmental conditions.

REST is a hypoxia-responsive transcriptional repressor.

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.

FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1.

The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.

Crosstalk between toll-like receptors and hypoxia-dependent pathways in health and disease.

Toll-like receptors (TLRs) play an important role in shaping the host immune response to infection and inflammation. Tissue hypoxia is a common microenvironmental feature of infected and inflamed tissues. Furthermore, hypoxia significantly impacts the development of immune and inflammatory responses through the regulation of host innate and adaptive immunity. Here, we will discuss current knowledge in relation to the crosstalk that exists between toll-like receptor- and hypoxia-dependent signaling pathways in health and disease.

REST mediates resolution of HIF-dependent gene expression in prolonged hypoxia.

The hypoxia-inducible factor (HIF) is a key regulator of the cellular response to hypoxia which promotes oxygen delivery and metabolic adaptation to oxygen deprivation. However, the degree and duration of HIF-1α expression in hypoxia must be carefully balanced within cells in order to avoid unwanted side effects associated with excessive activity. The expression of HIF-1α mRNA is suppressed in prolonged hypoxia, suggesting that the control of HIF1A gene transcription is tightly regulated by negative feedback mechanisms. Little is known about the resolution of the HIF-1α protein response and the suppression of HIF-1α mRNA in prolonged hypoxia. Here, we demonstrate that the Repressor Element 1-Silencing Transcription factor (REST) binds to the HIF-1α promoter in a hypoxia-dependent manner. Knockdown of REST using RNAi increases the expression of HIF-1α mRNA, protein and transcriptional activity. Furthermore REST knockdown increases glucose consumption and lactate production in a HIF-1α- (but not HIF-2α-) dependent manner. Finally, REST promotes the resolution of HIF-1α protein expression in prolonged hypoxia. In conclusion, we hypothesize that REST represses transcription of HIF-1α in prolonged hypoxia, thus contributing to the resolution of the HIF-1α response.

Binding of carbonic anhydrase IX to 45S rDNA genes is prevented by exportin-1 in hypoxic cells.

Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45 S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45 S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45 S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45 S rDNA gene promoters.