The inflammatory response in the CF airway begins early in the disease process and becomes persistent through life in most patients. Inflammation, which is predominantly neutrophilic, worsens airway obstruction and plays a critical role in the development of structural lung damage. While cystic fibrosis transmembrane regulator modulators will likely have a dramatic impact on the trajectory of CF lung disease over the coming years, addressing other important aspects of lung disease such as inflammation will nevertheless remain a priority. Considering the central role of neutrophils and their products in the inflammatory response, potential therapies should ultimately affect neutrophils and their products. The ideal anti-inflammatory therapy would exert a dual effect on the pro-inflammatory and pro-resolution arms of the inflammatory cascade, both of which contribute to dysregulated inflammation in CF. This review outlines the key factors to be considered in the design of clinical trials evaluating anti-inflammatory therapies in CF. Important lessons have been learned from previous clinical trials in this area and choosing the right efficacy endpoints is key to the success of any anti-inflammatory drug development program. Identifying and validating non-invasive biomarkers, novel imaging techniques and sensitive lung function tests capable of monitoring disease activity and therapeutic response are important areas of research and will be useful for the design of future anti-inflammatory drug trials. Airway inflammation plays a critical role in the development of bronchiectasis in cystic fibrosis (CF) and contributes to the progressive decline in lung function . InflammThe inflammatory process in CF is predominantly driven by neutrophils . Neutrop1) and reductions in sweat chloride, but there were no significant changes in any sputum inflammatory markers (Cystic fibrosis transmembrane regulator (CFTR) modulators are a new class of therapy that targets the basic genetic defect and could dramatically change the landscape of CF care. These drugs are potentially efficacious for approximately 90% of the CF population . However markers . In cont markers . However markers .Considering that the clinical response to CFTR modulators is variable, they are not universally available and, if bronchiectasis is already established, chronic infection and inflammation persist and lung function continues to decline , developThe complexity of the inflammatory process in CF provides multiple potential targets for intervention. Due to the central role of neutrophils in the inflammatory process, effective anti-inflammatory therapies must target neutrophils or their products .The CF airway contains a broad spectrum of pro-inflammatory mediators, such as TNF-alpha, IL-1beta, IL-6, IL-8, IL-17, IL-33. GM-CSF, G-CSF, and HMGB-1 . The dowAREST CF) surveillance program demonstrated that free neutrophil elastase activity in bronchoalveolar lavage (BAL) fluid at 3 months of age was associated with bronchiectasis on computed tomography (CT) scan at 1 and 3 years of age  is considered the gold standard for quantifying airway inflammation in the CF . However1 ; this inflammatory marker predicted subsequent pulmonary exacerbations and survival during 7 years of follow-up .Despite these strong observational data, it is still not clear what magnitude of biomarker change that could be considered clinically meaningful in the interventional setting. Furthermore, for biomarkers to convincingly reflect the treatment effect of an investigational drug, multiple biomarkers should improve rather than just a single one. Chmiel and colleagues designed a placebo-controlled randomized trial to investigate whether sputum biomarkers could be used to screen candidate anti-inflammatory therapies over a short period of time . ResultsBlood-based markers are relatively non-invasive, are easily standardized and can be obtained from subjects of any age and disease severity. Although the data linking blood-based inflammatory markers to clinical outcomes is less extensive than for sputum and BAL markers, systemic inflammatory biomarkers correlate with important clinical events including pulmonary exacerbations and lung function decline .Pseudomonas aeruginosa investigated the responsiveness of a panel of systemic inflammatory markers to treatment with azithromycin  and suggest that ibuprofen is more efficacious when used in individuals with mild CF lung disease. It is also possible that it is the trajectory of lung function and not just baseline FEV1 that influences response to therapy. Future trials should consider stratifying patients not only based on the severity of lung disease but also on the trajectory of lung function over time.A nd older . The fin1 in the normal range, there is an urgent need for more sensitive outcome measures    up to 2.se alfa) . LCI hasPulmonary exacerbations are important clinical events in the disease process and directly contribute to the progression of lung disease . TherefoThe ideal timing to initiate anti-inflammatory therapies is unclear, whether they would be most efficacious if initiated when a patient is clinically stable or during a pulmonary exacerbation when the inflammatory process is at its peak. A pilot randomized controlled trial investigating the short-term use of oral prednisone in CF patients presenting with pulmonary exacerbations demonstrated a modest improvement in lung function . The PIPVarious types of imaging techniques are now available to determine the presence and extent of lung disease in patients with CF, including CT and chest magnetic resonance imaging (MRI) . StudiesWhile chest CT scans are sensitive at detecting structural changes, the evolution of these changes over time is slow, with bronchiectasis deteriorating at about 1.5% per year on serial CT scans in CF patients. Therefo1/T2 contrast images can be obtained within a single breath‐hold, providing a depiction of structural abnormalities and active inflammation. Hyperpolarized‐gas MRI, increasingly using 129Xe, is now becoming more widespread and has been demonstrated to have high sensitivity to early airway obstruction in CF and could have utility as an endpoint in future clinical trials, particularly in the acute setting of pulmonary exacerbations.Pulmonary MRI can now provide high‐resolution images that are sensitive to early disease and specific to inflammation in cystic fibrosis (CF) lung disease . Unlike Another promising non-invasive imaging technique that provides information about the level of inflammation is positron emission tomography (PET) with [18]fluorodeoxyglucose . 18F-FDG is taken up by activated neutrophils, macrophages, and lymphocytes so in contrast to other imaging techniques, FDG-PET scans assess CF airway inflammation directly. Several observational studies have shown that 18F-FDG uptake can determine the location and intensity of pulmonary inflammation and, combined with CT, PET can be used to assess anatomy and structures. . A studyTime will tell how increasing CFTR modulator access will affect future anti-inflammatory therapeutic development. The positive effects of CFTR modulators on the progression of CF lung disease are undoubtedly positive but this will make the issues of endpoints for anti-inflammatory trials event more challenging.Acebilustat is an inhibitor of leukotriene A4 hydrolase (LTA4H), an enzyme that catalyzes the rate-limiting step in the formation of leukotriene B4 (LTB4), a potent chemoattractant and activator of inflammatory immune cells including neutrophils . This anDesigning trials of anti-inflammatory therapies in CF faces specific challenges different from other drug development programs. Demonstrating safety data from preclinical studies, choosing appropriate and realistic efficacy endpoints and integrating sensitive imaging and lung function outcomes into future trials are important considerations. These measures will increase the likelihood that potentially efficacious therapies are not abandoned prematurely and that efficacy can be conclusively demonstrated in studies that can be completed in reasonable time frames.Both authors conceptualized the content of the review. LP wrote the first draft and FR reviewed and edited the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis mutant plastid-encoded polymerase associated protein 7 (pap7) being defective in the prokaryotic-type plastid RNA polymerase. In all three cases the defect in plastid function has a severe impact on the expression of nuclear genes representing the influence of retrograde signaling pathway(s) from the plastid. We performed a meta-analysis of recently published genome-wide expression studies that investigated the impact of the aforementioned chemical and genetic blocking of chloroplast biogenesis on nuclear gene expression profiles. We identified a core module of 152 genes being affected in all three conditions. These genes were classified according to their function and analyzed with respect to their implication in retrograde signaling and chloroplast biogenesis. Our study uncovers novel genes regulated by retrograde biogenic signals and suggests the action of a common signaling pathway that is used by signals originating from plastid transcription, translation and oxidative stress.Chloroplast biogenesis during seedling development of angiosperms is a rapid and highly dynamic process that parallels the light-dependent photomorphogenic programme. Pre-treatments of dark-grown seedlings with lincomyin or norflurazon prevent chloroplast biogenesis upon illumination yielding albino seedlings. A comparable phenotype was found for the  Chloroplasts are sub-cellular organelles in plants and algae that perform photosynthesis and many other metabolic activities. In angiosperms they develop from undifferentiated precursors called proplastids which are inherited from the mother plant in the cells of the embryo ,2. Upon constitutive photomorphogenesis (cop) mutants from Arabidopsis develop a photomorphogenic phenotype in the dark without chloroplast formation [plastid-encoded RNA polymerase-associated protein (pap) mutants develop a normal photomorphogenic phenotype in the light without chloroplast formation [The molecular steps controlling the biogenesis of chloroplasts are far from understood, mostly because of the rapidity and complexity of the processes involved . Initiatormation . Vice veormation . ChloropThe major steps of chloroplast biogenesis involve the build-up of the internal thylakoid membrane system and the assembly of the photosynthetic apparatus. Because of their endosymbiotic ancestry chloroplasts possess their own genome that encodes central components of the photosynthetic and gene expression machineries . HoweverRetrograde signals from plastids during early steps of chloroplast biogenesis have been named biogenic signals  . These sTwo common approaches for the study of biogenic signals are the treatments of germinating seedlings with norflurazon (NF) and lincomycin (LIN). NF is an inhibitor of the plastid phytoene desaturase and a potent repressor of carotenoid biosynthesis. NF-treated dark-grown seedlings remain completely white and experience a severe oxidative stress from photo-sensitization of protochlorophyllide upon illumination since the quenching properties of the carotenoids are missing. The generated reactive oxygen species block any further steps towards chloroplast biogenesis by oxidative destruction of the internal plastid structures . LIN trepap7-1 mutant of Arabidopsis [Lhc genes contrasting the notion that inhibition of plastid development causes a general repression of PhANGs. Therefore, we were wondering what the “true” impact of biogenic signals on chloroplast biogenesis is. Here, we present a meta-analysis of gene expression profiles obtained from the Arabidopsis pap7-1 mutant and two recent gene expression studies using NF and LIN in order to define commonalities and differences between the three approaches. Our data uncover a core gene module that exhibits common expression profiles in all three conditions and identify potential novel targets of biogenic signals as well as regulators of chloroplast biogenesis.Recently we reported a detailed expression profiling of the bidopsis . This mupap7-1 mutant as well as for light-controlled genes in wild-type plants were taken from our own data sets [pap7-1 and LIN data sets increased with time. The 24 h LIN data set, therefore, was used as base for further meta-analysis. Only genes that exhibited a significant relative expression change (repression or induction) in response to plastid dysfunction of at least log2 ≥ 1 and a p-value ≤ 0.05 in all data sets were included in our comparisons. Primary expression data sets for genes being controlled by retrograde signals identified under influence of NF and LIN treatments were taken from previously published studies ,27. Dataata sets . The stuhttp://www.interactivenn.net/index.html) [For all principal comparisons of microarray data sets and the identification of overlapping genes between the three studies we used standard functions of Microsoft EXCEL. The corresponding Venn diagram was generated by using a web-based tool for comparison of large data sets  .https://www.arabidopsis.org) and The Universal Protein Resourcce (UniProt) (https://www.uniprot.org). Information on potential interaction partners was obtained from the Biological General Repository for Interaction Datasets (BioGRID) (https://thebiogrid.org) and the real-time multiple association network integration algorithm for predicting gene function (GeneMANIA) (https://genemania.org) databases. In addition, information on potential localization of gene products was extracted from the Bio-Analytic Resource (BAR) for Plant Biology and the integrated Arabidopsis Cell eFP Browser (https://bar.utoronto.ca) and aligned with the corresponding information in the Arabidopsis thaliana chloroplast protein database AT_CHLORO (http://at-chloro.prabi.fr) for sub-plastidial localization [The initial functional annotation of the identified 152 genes is based on the MapMan Bin categories  and was lization . Genes wArabidopsis seedlings grown for 5–7 days on sugar-supplemented medium in Petri dishes and exposed to light to visualize the effect of blocked chloroplast biogenesis on light-regulated gene expression profiles. In LIN-based experiments seedlings were grown in absence and presence of 0.5 mM LIN under extremely weak blue-red light of 0.5 µE fluence rate for 6 days followed by a shift to 60 µE blue-red light for 24 h [pap7-1 mutant seedlings were grown for 5 days directly under light since the genetic inactivation had become effective already during the establishment of the seeds [Arabidopsis chips providing high technical comparability. We, therefore, regarded the experimental set-ups as sufficiently similar to provide reliable data for a meta-analysis.Recently, we identified a set of retrograde controlled genes that are repressed or induced by biogenic signals in the light when chloroplast biogenesis is blocked at the level of plastid transcription . We werefor 24 h . In the for 24 h . In bothhe seeds . Therefopap7-1 set and 1128 genes for the NF set. Comparison of the three conditions revealed a common gene module of 152 genes being significantly regulated in all three conditions  of genes being stronger expressed than in unaffected wild-type (WT) controls and two third (105) of genes exhibiting a weaker expression . In contpap7-1 mutant were comparable to both LIN and NF profile concerning the direction of expression change; however, the degree of variation was weaker in most cases. Pharmaceutical inhibition of chloroplast development by LIN or NF treatments, thus, appears to have a stronger impact on nuclear gene expression than the genetic block in the pap7-1 mutant, although the overall plant phenotype in all three conditions was largely the same. This suggests that the inhibitor treatments (i) induce stronger inhibitory effects in the plastid than the genetic block and/or (ii) the respectively affected plastid processes contribute to biogenic retrograde signaling with different strength. This suggest that the albino phenotype per se is likely not the cause of the changes in nuclear gene expression arguing for a specific molecular signaling pathway.Except for a few condition-specific expressed genes (see below), the general profiles of genes affected by LIN and NF treatments were found to be highly similar in strength and direction of expression change. The expression profiles in the Because of the high number of genes encoding proteins with unknown or unassigned functions, we curated the functional annotation of each gene in the identified core module manually using current databases and literature and generated an up-dated list that covers detailed information on function, potential interaction partners and intracellular localization . By thisIn total 84 of the 152 genes encode proteins that were predicted to be localized with low, medium or high probability in plastids. The plastid localization of 67 of them was experimentally confirmed by proteomic approaches according to AT_CHLORO. Roughly 45–55% of all genes in the module encode components that have predicted cellular destinations other than plastids . Since prediction is not 100% precise and dual localization may also occur the results must be taken with care. Nevertheless, the data suggest that RB signals affect also other parts of the cell besides plastids. We noted that 80% of the genes with enhanced expression upon blocked chloroplast biogenesis belong to the group of genes encoding non-plastid localized components. Vice versa, 94% of the genes encoding plastid localized components displayed a low expression upon blocked chloroplast biogenesis indicating that the genes within the identified core module are dominated by two opposing expression modes that are largely associated with the potential localization of the affected gene products. In order to test whether or not the opposing expression modes are also associated to specific functions we analyzed the available information in a number of databases and ordered the genes of the core module into functional subsets with at minimum four genes A–F and a6fb-complex and the NAD (P) H-dehydrogenase-like (NDH) complex as well as for enzymes of the Calvin-Benson cycle (with the phosphoribulokinase as only exception) were not identified. This implies a model in which RB signals modulate gene expression of PhANGs in a subset-specific way rather than a common overall control. It appears that especially components of the chlorophyll containing complexes respond to RB signals. Concomitant with this we observed a significant impact of RB signals on genes for key enzymes of Chl and carotenoid biosynthesis suggesting that RB signals may coordinate the syntheses of pigments and pigment-containing complexes.In current literature photosynthesis-associated nuclear genes (PhANGs) are referenced as the “classical” target of biogenic retrograde signals ,35. Our GOLS1  exhibits an opposite expression profile. GOLS1 has been proposed as a negative regulator of seed germination [The direct products of photosynthesis are ATP and NADPH that are primarily used in the Calvin cycle to generate carbohydrates. However, they are also used in many other metabolic pathways that reside either partially or entirely in the plastid. Inhibition or down-regulation of photosynthesis, therefore, has a profound impact on the overall metabolic capacities of plastids. This is reflected in the effect of RB signals on the expression of numerous genes encoding plastid and cytosolic enzymes involved in carbohydrate metabolism that exhibit the same expression profile as the photosynthesis genes. Only the gene mination  as well Many enzymatic reactions in chloroplasts are regulated via the redox state of the corresponding proteins. This control is exerted by a number of redox mediators such as thioredoxins that derive their reduction power from photosynthesis . Many ofAs novel highly interesting targets for RB signals we identified three of the four genes encoding CURT (Curvature thylakoid) proteins within the core module. These proteins are required for the formation of the grana margins in thylakoid membrane system ,41 and, pap8-1, an albino mutant related to pap7-1 [pap mutants overproduce cytokinins [Special interest in retrograde signaling is paid to the identification of nuclear localized transcription factors that mediate the corresponding gene expression responses E. An ofto pap7-1 . The lowo pap7-1 . This obo pap7-1 . Genes fo pap7-1 . Its oppo pap7-1 ,54. A retokinins . We regatokinins ,57,58,59Our meta-analysis was focused on the implications of RB signals on transcript accumulation and cannot account for potential effects on other gene expression levels. However, we observed significant effects of RB signals on genes encoding components involved in protein folding, proteostasis and stress responses, which imply that blocking chloroplast biogenesis affects also these functions. Recent studies revealed a major impact of plastid signals on the unfolded protein response in plastids, endoplasmic reticulum and cytosol as well as on protein accumulation ,61,62. Tpap7-1 profiles. Lipids are major constituents of membranes and are absolutely essential for the build-up of chloroplast. Although blocking of chloroplast biogenesis results in plastids without thylakoid formation we did not find any impact on genes encoding proteins involved in plastid lipid metabolism, but only for non-plastid components. The corresponding gene expression profiles are very complex and difficult to interpret and more analyses will be necessary to understand and to explain the RB impact on them. We also found an impact of RB signals on multiple genes involved in hormone biosynthesis and/or signaling. We identified already in other functional subsets a number of candidate genes that are associated with synthesis and/or the action of several hormones (see above). ABA appears to be an interceptive signal in a few cases , but likInterestingly, we found a strong impact on two genes encoding key enzymes of allene oxide biosynthesis  that produce the precursor for peroxisomal jasmonic acid production, the oxophytodienoic acid (OPDA) . A distiThe core module responsive to RB signals identified in this study is different from retrograde controlled gene modules identified in earlier studies ,66,67, m


Introduction: The Inspiris Resilia aortic valve® (INSPIRIS) is a pericardial bio-prosthesis with a new sterilization procedure that shows promising results in terms of reduced calcification. Methods: The 30-day mortality and morbidity were analyzed, comparing the INSPIRIS implanted between May 2017 and the end of January 2019, with its "predecessor", the Carpentier-Edwards Perimound Magna Ease (ME). Echocardiography was performed one-week after surgery. 125consecutively operated patients were included .Results: One patient in the ME group died and one patient in the INSPIRIS group had a complicated postoperative course due to right heart failure. Two patients  suffered a perioperative stroke. The hemodynamic evaluation shows an effective reduction of mean transvalvular pressure gradients after surgery in both groups. INSPIRIS tended to have lower trans-prosthetic pressure gradients , reduced trans-prosthetic blood flow acceleration  and increased permeability indices .Conclusion: There are only few clinical data available from INSPIRIS, and the present analysis confirms good results initial postoperatively with a tendency towards possibly improved hemodynamics compared to ME. Current research focuses on new preservation technologies for xeno-pericardial prostheses with the aim of improving long-term durability, lowering the age threshold for bio-prosthetic valve implantation and thus reducing the side effects of long-lasting anticoagulation.4The Inspiris Resilia aortic valve® (INSPIRIS)  is a new bovine pericardial prosthesis for SAVR. The functional group capping of aldehydes  to prevent oxidation and calcification, glycerolization to reduce the amount of residual chemicals and the new sterilization process in which the water of the pericardial tissue is displaced and replaced by glycerol  are the key innovations that promise lasting tissue integrity. In vivo animal models confirmed significantly reduced calcifications and initial clinical studies showed promising follow-up results.The use of INSPIRIS for SAVR, especially in younger patients, can combine the advantages of a durable bio-prosthesis with the potential future option of a simplified valve-in-valve procedure for late structural valve deterioration by incorporating an extendable stent for smaller sized annular diameters. The INSPIRIS has been available on the European market since the spring of 2017 after receiving the CE mark. Post-operative clinical data are therefore rare.We present a small series of early postoperative results  of the INSPIRIS and compare them with those of its “predecessor”, the Carpentier-Edwards Perimound Magna Ease (ME)  aortic bio-prosthesis, with a special emphasis on a comparative evaluation of their postoperative hemodynamic performance.A single database of two surgeons was reviewed, retrospectively searching for either isolated or combined aortic valve replacement with INSPIRIS. The data collection started in February 2019 and covered the period from the beginning of the INSPIRIS implantation in May 2017 until the end of January 2019. According to these 21 months of observation, a comparison group was formed. This control group consisted of patients who had been operated on by the same team during the 21 preceding months (August 2015 to April 2017) and who had thus received the predecessor model, the ME.Included were elective and urgent, isolated or in combination with coronary artery bypass graft (CABG) performed aortic valve replacements. Patients with concomitant mitral valve or aortic root surgery as well as patients with severe pre-existing left ventricular (LV) dysfunction were excluded with the aim of at least partially reducing possible factors that might influence the postoperative hemodynamic performance of the prostheses to allow for better comparability of the groups.All patients had a postoperative echocardiography after one week. In addition to the left ventricular ejection fraction (LVEF), the mean pressure gradients (mm Hg), blood flow accelerations (cm/s) and permeability indices (%) of the bio-prostheses were analyzed. Clinical follow-up after 30 days was completed for all patients and early postoperative mortality and morbidity were documented. The latter was classified into major complications, i.e. non-fatal myocardial infarction, early re-operation , endocarditis and stroke, or into minor complications including new postoperative arrhythmias requiring permanent pacemaker implantation (PPM), acute renal failure and blood transfusions. All-cause mortality, postoperative morbidity and postoperative hemodynamic parameters of the two different prostheses were compared. The primary endpoint was to demonstrate the non-inferiority of the INSPIRIS.t test or the Mann-Whitney U test was used to compare the results. A P value <0.05 was considered significant. The authors had full access to the data and take responsibility for its integrity.The SPSS 22.0 software package for Windows  was used. Categorical variables are presented as numbers and proportions (%). Continuous normally distributed variables are summarized as mean ± one standard deviation (SD), while non-normally distributed variables (according to the Kolmogorov-Smirnovand the Shapiro-Wilk test) are described by their median and interquartile range (IQR). Accordingly, either the 125 patients, 59 in the INSPIRIS group and 66 in the ME group, were identified. All were operated on during the observation period and met the inclusion criteria. The main demographic parameters , risk factors and existing comorbidities as well as the logistic EuroSCORE 2 risk calculations were comparable , the patient had a cardiac arrest a few hours later, probably due to a severe right ventricular dysfunction , he developed severe right ventricular (RV) dysfunction requiring urgent reopening of the sternum. A coronary angiography confirmed patent grafts. The patient remained unstable and started bleeding again from the sternum due to a beginning disseminated intravascular coagulation. After blood transfusions and sternal wound revisions with a “chest packing”, the patient gradually stabilized and improved his RV function. The sternum was closed 10 days later and the patient left the hospital after one-month. 17 months later he was still alive .In two patients from the INSPIRIS and one from the ME group, epicardial PPMs were implanted simultaneouslyduring surgery, which were indicated due to pre-operative rhythmic disorders. Nobody required PPM implantation due to new, procedure-related conduction disorders. Deep sternal wound infections requiring reopening of the sternum with subsequent vacuum-assisted closure and rewiring occurred in two patients from the INSPIRIS group and one patient from the ME group. No postoperative endocarditis  was observed.A symptomatic perioperative stroke occurred in one INSPIRIS and one ME patient. Both showed a new right hemiparesis immediately after surgery with confirmed acute cerebral ischemic lesions at MRI. The clinical course was favorable in both patients, with a progressive recovery.P = 0.17) of the stenotic aortic valves was achieved.Echocardiography showed slightly reduced mean trans-prosthetic pressure gradients, reduced trans-valvular flow accelerations and increased permeability indices in favor of INSPIRIS , reduced trans-prosthetic blood flow acceleration  and increased permeability indices .To obtain a reliable comparison, only patients with severe native valve stenosis were compared. INSPIRIS still showed lower mean trans-prosthetic pressure gradients  showed significantly lower mean gradients in favor of 21 and 25, reduced flow acceleration in favor of 25 and increased permeability indices in favor of all small and medium INSPIRIS compared to ME .Based on these early postoperative results, comparing INSPIRIS with its predecessor model, the ME, it can be concluded the prosthesis is safe. Postoperative mortality and morbidity were low in both successively operated comparable groups. By including high-risk patients and patients with combined procedures, a cross-section of the patient population currently assigned to the SAVR was obtained.4 Based on their findings, the authors concluded that Inspiris was safe due to the observed low complication rate and had acceptable hemodynamic performance.4 This last point was supported by indirect comparison with the hemodynamic properties of various predecessormodels evaluated in previous studies.4Currently, only few postoperative data are available from INSPIRIS. Bartuś et al reported their experience with 133 INSPIRIS patients, the vast majority of whom (≈86%) had isolated SAVR, with full follow-up after one year being about 90%. The study was designed as a prospective safety study and was sponsored by the manufacturer before INSPIRIS was launched on the European market.In contrast, the present analysis focuses on a direct comparison of consecutive patients operated on by the same surgeons. A good postoperative hemodynamic performance with significantly reduced mean trans-aortic gradients was confirmed for both valve types. In addition, a tendency towards improved hemodynamic flow behavior in favor of INSPIRIS was observed, which was also at least partially confirmed by the direct comparison of the different implanted prosthesis sizes. Although this small difference in trans-valvular gradients  between the two study groups was statistically significant, the resulting potential clinical effects may be negligible.Major limitations result from the non-randomized, retrospective approach, the limited number of patients and the short follow-up time. On the other hand, the fact that these (selected) patients were operated on by the same two surgeons using the same technique and within the same hospital during an equivalent timespan speaks for a certain comparability of the data. In this sense, the present results should be seen as an incentive to initiate further comparable and larger studies to analyze whether a potential hemodynamic advantage of INSPIRIS is reproducible and maintained during a longer follow-up.None declared.Ethics approval was guaranteed according to international recommendations by the institutional review board of the investigator center (ethics no. 2018-02182).None.


Fractional killing illustrates the cell propensity to display a heterogeneous fate response over a wide range of stimuli. The interplay between the nonlinear and stochastic dynamics of biochemical networks plays a fundamental role in shaping this probabilistic response and in reconciling requirements for heterogeneity and controllability of cell-fate decisions. The stress-induced fate choice between life and death depends on an early adaptation response which may contribute to fractional killing by amplifying small differences between cells. To test this hypothesis, we consider a stochastic modeling framework suited for comprehensive sensitivity analysis of dose response curve through the computation of a fractionality index. Combining bifurcation analysis and Langevin simulation, we show that adaptation dynamics enhances noise-induced cell-fate heterogeneity by shifting from a saddle-node to a saddle-collision transition scenario. The generality of this result is further assessed by a computational analysis of a detailed regulatory network model of apoptosis initiation and by a theoretical analysis of stochastic bifurcation mechanisms. Overall, the present study identifies a cooperative interplay between stochastic, adaptation and decision intracellular processes that could promote cell-fate heterogeneity in many contexts. Cell-fate heterogeneity is indeed a key feature of microbial adaptation to adverse environments2, of the development and homeostasis of tissues and organs3 or of tumor resistance to drug therapy4. The differential fate outcome of isogenic cells exposed to the same environmental stimuli involves the interplay of stochastic and deterministic mechanisms7, where regulatory mechanisms can determine both the statistics and dynamics of stochastic events and the effect of those stochastic events on molecular trajectories dictating cell fate choices. Several experimental studies have shown that cell-fate decisions are often preceded by a highly fluctuating intracellular dynamics. Pulsatile or oscillatory activities have been observed in signaling pathways operating during the stochastic choice of various differentiation or proliferation fates13. The profile characteristics of those dynamic signaling activities have been proposed to either direct decision outcomes or promote cell-fate heterogeneity15. Transient dynamics occurring at epigenetics, transcriptome-wide or multicellular levels18 have also been proposed to regulate cell-fate heterogeneity and plasticity. All these examples support a key role of transient dynamics in orchestrating fate decisions from diverse signaling and stochastic cues.In many adaptation and developmental contexts, isogenic cells make stochastic fate decisions to generate diversified cell types and subpopulations19. On this issue, several modelling studies have been devoted to identify which sources of fluctuations and which parts of the apoptotic network could contribute the most to the variability of decision time and outcomes23, while the impact of the transient dynamics has been seldomly addressed24. Yet, singe-cell analysis of the temporal trajectories of caspase 8 activity in response to TRAIL has revealed a signature of adaptation dynamics whose transient kinetics determines whether a cell survives or dies25. Caspase 8 is likely to be part of negative feedback regulation involving for instance the formation of inactive heterodimers of procaspase-826. The importance of transient dynamics of apoptotic inducers has been also emphasized in the case of cisplatin drug exposure27. The proposed mechanism involves a competition between positive and negative regulation of caspase 8-dependent apoptosis, thereby defining an incoherent feedforward loop. More generally, environmental stressors are prone to upregulate both pro-survival and pro-death pathways31 through negative feedback or incoherent feedforward loop motifs which ultimately lead to a dynamical adaptation response34. These stochastic and deterministic features associated with fractional killing raise the more general question of the role of adaptation dynamics in shaping the timing and probabilities of stochastic fate decisions.An attractive case study is the stochastic fate decision between life and death, commonly termed fractional killing, for which the systematic measure of probabilistic dose-response curves coupled with single-cell analysis of stochastic and dynamical signatures are possible39. In the present study, we use the joint framework of chemical Langevin equations40 and bifurcation theory to address the interplay of stochasticity, transient adaptation and bistable switching. The deterministic and stochastic analysis of a simple model combining adaptation and bistability deciphers how the adaptation overshoot dynamics modulate, concomitantly, the nonlinear decision-making properties and the probabilistic fate-response properties. The biological relevance of this behavior is assessed by simulating a more detailed model of programmed death pathways. Finally, the generality of the proposed noise-amplification mechanism is addressed within the theoretical framework of stochastic nonlinear dynamics.Diverse modelling approaches have proved useful to study stochastic switching in regulatory networks, ranging from the discrete chemical master equations and stochastic simulation algorithms to the continuous Fokker-Planck and Langevin equations. Those diverse tools and their refinements have been broadly used to study the interplay between noise properties and network topologies in shaping the steady-state bimodal distribution and transition rates associated with two phenotypic statesH(t) and 41, chemical Langevin equation approach is nevertheless convenient to study asymptotic cases of small noise, large size or separated timescales and, thus, to relate with noise-free dynamical properties42.Fractional killing can be defined as the population-level property by which isogenic cells exposed to increasing doses of death-inducing stimuli will tend to display a fraction of surviving cells and dying cells, although with increasing probability of death. This stochastic decision process can be studied in a general theoretical framework that applies to cases of fate decisions other than survival and death. Probabilistic cell-fate response commonly involves the interplay between intracellular mechanisms of fate decision and intracellular sources of cell-to-cell variability. Without loss of generality, a possible framework to study such probabilistic fate response to a step stimulus consists in a Langevin differential equation description of the stochastic dynamics of a biochemical reaction network  can be minimally implemented in the nonlinear dynamics of the biochemical network by the presence of a saddle-node bifurcation mechanism at a critical stimulus level mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minim43, whose interactions between three coarse-grained variables implement a basic setting of a negative feedback-driven adaptation and a positive feedback-driven decision switch  than Before considering a source of variability, we first need to investigate the main effect of transient adaptation dynamics on the fate decision properties. In this case, probabilistic response and fractional killing do not occur, and the system response to a step stimulus is essentially determined by a threshold entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaIn summary, while varying Based on our comprehensive analysis of the deterministic decision dynamics in the coarse-grained model combining adaptation and bistability, we aim to investigate how adaptation influences cell-fate heterogeneity in a population of noisy cells. We therefore apply the general stochastic modeling framework to this biochemical network model see "" sectionels Fig. .Figure 3To begin with, we consider the case of cell-to-cell variability arising from molecular noise solely  Fig. g. Again,45 depending on the involved multi-protein signaling complexes, transcriptional factors and other signaling and metabolic cues .We have shown that the sensitivity of cell-fate decision to molecular noise depends on the state-space paths taken to reach a saddle instability, along, either, its stable manifold or its unstable manifold Fig. a. In ordned Fig. d. Scalin48.In the case of a saddle-collision scenario, perturbed trajectories evolve in the neighborhood of the deterministic trajectory entclass1pt{minimac) Figs. d and, th(t) Fig. b. Rewritentclass1pt{minima∝σ Figs. d and 5d 50. For this decision-making regime, an effective potential U, a potential barrier 51 (Fig. The sensitivity to noise is very different in the other scenario of noise-induced escape from a stable state (d in Eq. c, the paTo conclude, these very distinct formulas for 32 and which is subjected to tradeoffs associated with homeostatic or sensory process53. In addition, we propose that this transient overshoot dynamics can also significantly impact fate-switching behaviors, so as to extend the stimulus range of fate heterogeneity and to allow for tunable fate probability. This adaptation-dependent fate stochasticity relies on the manner how the overshoot of some intracellular species drive cell state in the neighborhoohd of a saddle instability, rather than a saddle-node instability, along a path where molecular noise are more prone to promote divergent decisions. This noise-amplification behavior illustrates how molecular noise and instability mechanisms can cooperate to shape cellular dynamics, like genetic timers54, boundary formation55 or versatile sensory processing56.The present modeling study deciphers the role of adaptation dynamics in promoting cell-fate heterogeneity associated for instance with the fractional killing behavior. A common property of adaptation is the transient overshoot of some cellular variables above its steady state value, which can be implemented by diverse circuit topologies58 which is of the range of magnitude of the initiator caspase rise time and death onset timing. Moreover, noise-induced fate heterogeneity is the most effective when fluctuating variables are those involved in the positive feedback that triggers death initiation. This requirement is consistent with modeling evidences that variability in diverse regulatory molecules can contribute in very different ways to variability in cell death outcomes20 and that the main contributions seem to occur in the initial decision commitement phase, whether it is at the level of the fluctuations of short-lived antiapoptotic proteins22 or the stochastic assembly of DISC/RIPoptosome platform23. The manner how cell fate is determined by the impact of these fluctuations at the level of concentration trajectories has been also investigated27. In relation to these studies, our study presents a broad and comprehensive view of this cooperative process and, thus, provides strategies, by monitoring transient characteristics, to either increase or reduce fractional killing.The biological relevance of the proposed mechanism is most likely in a context of fractional killing for which the choice between life and death depends on adaptation processes. The timescales of those adaptation responses range from half an hour to few hours depending on stress type and regulatory mechanisms59, the osmotic stress response of yeast60 or ethanol stress in Bacillus61. In case of stress-induced fate response, monitoring the stress stimulus profile would therefore be expected to modulate not only threshold stimulus level (The profile characteristics of adaptation dynamics, such as the ratio between its maximal and steady-state values, are highly sensitive to the temporal profile of the stimulus. Ramp increase of a stimulus or a preconditioning stimuli are known to reduce the transient overshoot behavior. This feature has been exploited to test the role of adaptation in oxidative stress response of yeast32, but also in excitable or pulsatile systems combining negative and positive feedback loops. For the latter case, numerous signaling pathways, such as P53, Erk or NF-15. In relation to our result, the transient and stochastic characteristics of these signaling dynamics may also suggest a role for promoting cell-fate heterogeneity. This is supported by some experimental evidences that have mapped the cell-cell variability of the pulsing dynamics of Erk11, p5363, 13 and NF-12 with the heterogeneity of cell-fate outcomes. Data-driven and fine-grained modeling of specific dynamic signaling and fate-regulatory pathways64 are definitively the step further to evaluate on a case-by-case basis to which extent transient adaptation or pulsing dynamics may contribute, fortuitously or functionally, to cell-fate heterogeneity.It is tempting to extrapolate the biological relevance of such adaptation-dependent mechanism beyond the scope of fractional killing and transient adaptation dynamics. The mechanism itself only requires a regulatory network featured with an upsteam overshoot response and a downstream switching response, which could be implemented by diverse network topologies and in diverse cell-fate contexts. For instance, overshoot dynamics can also occur in regulatory systems comprising incoherent feedforward loopss and defined rescaling variables (t again for simplicity. The chemical Langevin equation associated to Eq. (The set of regulatory reactions depicted in Fig. t of Eq. , we perfmentclasspt{minimalify Eq.  where did to Eq.  is also 24. From the original model, the equations for CIAP, p53 and Mdm2 have been removed and the equation for Bh3T has been changed to incorporate activation by Equations and parameters of the biochemical reaction model of apoptosis initiation have been taken frommentclass2pt{minimFor both models, numerical integation of Langevin equations are performed with 4th-order Runge-Kutta method and probability distribution mentclass2pt{minim


Perinatal hypoxic-ischemic (HI)-related brain injury is an important cause of morbidity and long-standing disability in newborns. The only currently approved therapeutic strategy available to reduce brain injury in the newborn is hypothermia. Therapeutic hypothermia can only be used to treat HI encephalopathy in full term infants and survivors remain at high risk for a wide spectrum of neurodevelopmental abnormalities as a result of residual brain injury. Therefore, there is an urgent need for adjunctive therapeutic strategies. Inflammation and neurovascular damage are important factors that contribute to the pathophysiology of HI-related brain injury and represent exciting potential targets for therapeutic intervention. In this review, we address the role of each component of the neurovascular unit (NVU) in the pathophysiology of HI-related injury in the neonatal brain. Disruption of the blood-brain barrier (BBB) observed in the early hours after an HI-related event is associated with a response at the basal lamina level, which comprises astrocytes, pericytes, and immune cells, all of which could affect BBB function to further exacerbate parenchymal injury. Future research is required to determine potential drugs that could prevent or attenuate neurovascular damage and/or augment repair. However, some studies have reported beneficial effects of hypothermia, erythropoietin, stem cell therapy, anti-cytokine therapy and metformin in ameliorating several different facets of damage to the NVU after HI-related brain injury in the perinatal period. The brain endothelial cells (BECs) of the BBB supply nutrients and other essential molecules from blood to brain cells and eliminate waste products to provide an optimal environment for brain development. The BECs are also important for signal transduction including numerous immune responses. For example, immune blood born cell trafficking is regulated in part by the expression of adhesion molecules on BECs. It is important to emphasize that BECs by themselves are not capable of intrinsically forming a fully functional BBB. The relatively new concept of neurovascular unit (NVU) underscores the requirement for coordinated cell interactions that include BECs, the basal lamina, the surrounding glial limitans, pericytes, microglia and neurons . The funBECs possess the most characteristic features of the barrier in the brain, which include reduced paracellular transport as a result of tight junctions (TJ) between neighboring BECs and the expression of efflux and influx transporters that regulate the brain microenvironment . TJs areA number of brain related injuries observed in neonates could also be caused by genetic-related and other factors that occur during antenatal brain development . SometimNormal brain development is highly dependent on a sufficient blood, oxygen and energy supply. HI-related brain injury is a result of an insufficient blood flow to the brain combined with lower-than-normal concentrations of oxygen in the arterial blood. These events in the immature and developing brain cause significant mortality and can result in long-term neurological deficits such as cerebral palsy, epilepsy and seizure disorders, developmental delay, severe learning disabilities, cognitive, motor and behavioral abnormalities . Randomi2 |2.1 |Animal models used for the study of HI-related brain injury in neonates have been reviewed in detail by others . These rThere is now sufficient evidence to justify the use of 7-days old rats because the brain maturation is thought to be roughly comparable to that of the third trimester human fetus . Nonethe2.2 |BECs represent the fundamental cell type of the BBB because they are the first cells to encounter substances in the systemic circulation. The BECs differ considerably from classic endothelial cells in peripheral vessels because they contain a large quantity of cytosolic mitochondria, low pinocytotic activity and lack fenestrations. The tight junctions (TJ) between neighboring cells form the physical barrier separating blood stream and brain parenchyma and resulting in very limited paracellular passage. Various transport systems are present on abluminal and luminal membranes also regulate the transcellular traffic. These polarized transporters are responsible for the selective ‘transport barrier’ in order to meet the high nutritional and energy requirements of the brain and to exclude potentially harmful substances such as exogenous toxins circulating in blood.2.2.1 |Disturbances in endothelial permeability have been evaluated by several methods and described in multiple models of HI-related injury in the fetal and neonatal brain . Many ofFindings in human neonates appear to be consistent with these observations because albumin concentrations in cerebro-spinal fluid compared with plasma levels are increased in infants that had been exposed to hypoxic-ischemic encephalopathy (HIE) . The impThe tightness of the BBB endothelium is a result of TJ proteins that seal neighboring BECs and restrict the diffusion from the lumen of the vessel into the brain parenchyma. The TJs are composed of a combination of transmembrane (claudins family and occludin) and cytoplasmic (i.e. zonula occludens (ZO) family) proteins that interact with the actin cytoskeleton. Decreases in the expression of TJ proteins have been reported after HI-related injury in the rodent brain  and afteIn addition, there is increasing evidence of sexual dimorphism in response to neonatal HI-related injury that remains to be explained . Immune 2.2.2 |The transport systems at the BBB have two main purposes: To carry essential molecules  and to limit the entry of xenobiotics into the brain via the efflux system. In this regard, the ATP binding cassette (ABC) transporters P-glycoprotein (Pgp) , Breast in utero (Radiolabeled interleukin-1 β (IL-1 β) transfer across the BBB has been shown to be increased at 4 h and 24 h after exposure to ischemic brain injury in fetal sheep in utero . The incin utero . Even thGlucose, an essential substrate for brain oxidative metabolism, is transported across the BBB into glia and neurons by a family of structurally related membrane spanning glycoproteins called facilitative glucose transporters (glut) . GlucoseThe expression of major efflux and influx pumps, such as the ABC or SLC transporters, have not been investigated in the neonatal brain after HI injury, even though they have a major role in BBB function. However, studies in adults suggest that ischemic stroke can modify BBB transporter function. For example, P-glycoprotein (P-gp/ MDR1/ ABCB1), a particularly important ABC transporter that is responsible for handling a wide variety of endogenous substrates and xenobiotics, is upregulated after ischemic stroke in rodents . The breIn summary, there is a paucity of information regarding the regulation of transport activity across the BBB after HI-related injury in the neonatal brain. It is likely that there is an age dependent response of the transporters to HI in the neonatal brain because ontogenic modulations in transporters have been reported at the BBB during development. Transporters (i.e. ABC and SLC) provide a considerable opportunity to protect the BBB and/or promote BBB repair by facilitating endothelial uptake of drugs with cytoprotective/antioxidant properties or by preventing cellular loss of critical endogenous substances. However, further work is required to provide information regarding the interplay of cell-to-cell interactions, transporters, and signaling pathways at the BBB endothelium to understand how these systems could be effectively targeted. Furthermore, these systems are extremely important and require further investigation because infants with HIE are currently exposed to numerous medications for which these transporters could have important effects on brain drug concentrations and deposition.2.2.3 |BECs express several enzymes responsible for the metabolism of neurotransmitters. For example, they express monoamine estherases, cholinesterases, GABA transaminases, aminopeptidases and endopeptidases. BECs also express different systems responsible for the metabolism of drugs typically found in the liver, such as the cytochromes P450 (CYP450 or phase I enzymes) and phase II enzymes of metabolism . Toxin aHI-related brain damage is mediated through numerous overlapping mechanisms resulting in cell death: excitotoxicity, oxidative stress and inflammation . The mec2.2.4 |The basal lamina membrane is composed of ECM proteins  . BECs anECM glycoprotein staining can be used to examine vasculature density after HI injury in the neonatal brain. Laminin was used as maker to show decreased vessel density after umbilical cord occlusion fetal sheep. Decreases in vessel density was observed in the caudate nucleus along with shifts in the frequency of smaller to larger blood vessels in periventricular and subcortical white matter. In contrast, blood vessel density and morphology did not change after ischemia in the cerebral cortex . Blood vVascular endothelial growth factor (VEGF) is a key mediator of vessel proliferation that is up regulated after neonatal HI. VEGF signaling pathways have been shown to stimulate angiogenesis after recovery from HI-related brain injury in neonatal rodents . Signifia nuclear factor that modulates many Hl-related processes including neovascularization by regulating the transcription of its downstream elements such as VEGF. Increases in cerebral cortical FGF-2 was also associated with increases in neovascularization in the ovine cerebral cortex after ischemia (Basic fibroblast growth factor (FGF-2) and hypoxia-inducible factor-1 α (HIF-1 α) are two other growth factors that have been shown to augment angiogenesis after FI I in neonatal rodents . HIF-1α ischemia .Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases. They are responsible for remodeling the extracellular matrix including the basal lamina of the BBB, regulating cell receptors and cleaving their ligand binding domains in the brain . Tissue 2.3 |Pericytes surround the BBB providing additional separation of the blood components from neural tissue. They are evenly spaced along the walls of capillaries and share a common basal lamina with BECs. Pericytes are also termed vascular smooth muscle cells or myofibroblasts of blood vessels because of their ability to contract and, consequently, regulate local microvascular blood flow . They alin vitro during exposure to hypoxia  is a major water channel in the mammalian brain that is highly expressed on the astrocytes end-foot processes . Increas2.5 |Perivascular microglia/macrophages are in intimate contact with the BBB, particularly surrounding penetrating vessels and venules, and have a role as the first immune defense against pathogens invading the CNS. Perivascular microglia extend processes that surround brain microvessels and regulate the uptake of some macromolecules . In addiThe NVU is a complex multicellular system. Each component of the system is interdependent upon the other constituents and, consequently, the function and regulation of the entire system are dependent upon all of its components. Many variables of the NVU remain to be investigated in the developing brain such as metabolic activities. The most widely reported effects of HI-related insults on the NVU are the increases in the permeability of the BBB. However, the effect of HI injury upon consequences in the later life as well as sex differences remains to be investigated. The effects of HI-related injury upon potential alterations of the NVU in the neonatal brain are graphically summarized in 3 |3.1 |in vitro and in adult subjects.The only strategy approved to treat HIE is therapeutic hypothermia. This strategy is only approved to treat full term newborns and is unfortunately only partially protective primarily after exposure to moderate HIE . Minimalin vitro oxygen-glucose deprivation on BECs, astrocytes and neurons are also temperature-dependent  is an attractive anti-inflammatory neuroprotective agent. EPO binds to its receptor expressed on the neuronal membranes, astrocytes and microglial cells to accomplish its anti-inflammatory effects. Numerous studies have shown that EPO attenuates the effects of HI-related injury in neonatal subjects . Howeverult mice . Pretreault mice . Treatmeult mice . Therefo3.3 |3.3.1 |Stem cell therapy ameliorates neurological deficits after HI-related injury in neonatal subjects . Cell-ba3.3.2. |Targeting pro-inflammatory cytokines could represent a potentially significant therapeutic strategy to treat perinatal HI injury. Recently, we demonstrated encouraging neuroprotective capacities of anti-cytokine monoclonal antibodies (mAbs) after ischemic-reperfusion (I/R) brain injury in the ovine fetus . The mos3.3.3 |Metformin is a widely prescribed drug to treat type 2 diabetes mellitus and metabolic syndrome. It has anti-inflammatory and anti-oxidative effects potentially beneficial after exposure to HI in neonates. In adults, metformin has been shown to promote neurogenesis and protect BBB integrity in experimental stroke . Treatme3.3.4 |Other studies have attempted to attenuate HI related brain injury . Xenon, 3 |Exposure to HI initiates a wide variety of deleterious effects on the neonatal brain resulting in long-term consequences later in life. The NVU represents an important component in the pathophysiology of neonatal HI-related brain injury. However, many characteristics of the NVU remain to be elucidated after exposure to HI. Although BBB permeability increases immediately after the onset of HI in perinatal brain, there is a paucity of knowledge regarding its effects on transport function and metabolism in BECs. Similarly, studies are still required to understand the impact of HI on other components of NVU as well as their interactions and role of this system during development. Prevention of neurovascular damage or enhancement of repair represents a potentially important therapeutic strategy to attenuate brain injury.


Multiple studies have attempted to increase the rate of intravenous thrombolysis for ischemic stroke using interventions to promote adherence to guidelines. Still, many of them did not measure individual-level impact. This study aimed to make a posthoc comparison of the clinical outcomes of patients in the “Thrombolysis ImPlementation in Stroke (TIPS)” study, which aimed to improve rates of intravenous thrombolysis in Australia.A posthoc analysis was conducted using individual-level patient data. Excellent (Three-month post treatment modified Rankin Score 0–2) and poor clinical outcome (Three-month post treatment modified Rankin Score 5–6) and post treatment parenchymal haematoma were the three main outcomes, and a mixed logistic regression model was used to assess the difference between the intervention and control groups.There was a non-significant higher odds of having an excellent clinical outcome of 57%  and 33%  during the active-and post-intervention period respectively, for the intervention compared to the control group. A non-significant lower odds of having a poor clinical outcome was also found in the intervention, relative to control group of 4%  and higher odds of having poor outcome of 44% (odds ratio: 1.44 95% CI: 0.61–3.41) during both active and post-intervention period respectively. Similarly, a non-significant lower odds of parenchymal haematoma was also found for the intervention group during the both active-  and post-intervention period .The TIPS multi-component implementation approach was not effective in reducing the odds of post-treatment severe disability at 90 days, or post-thrombolysis hemorrhage.http://www.anzctr.org.au/ Unique Identifier: ACTRN12613000939796.Clinical Trial Registration-URL:  Internationally and in Australia, stroke is a leading cause of death and disability , 2. ImprThe Thrombolysis Implementation in Stroke (TIPS) study, was a cluster-randomized trial which aimed to improve thrombolysis rates through a multi-level, multi-component, in-hospital intervention which was implemented in 10 of 20 study hospitals across Australia . The triThis posthoc analysis of the TIPS data tested the hypothesis that the three-month clinical outcomes of disability  and post-thrombolysis hemorrhage would show more favourable profiles in patients managed at the hospitals exposed to the TIPS intervention, relative to control hospitals.TIPS was a clustered randomized controlled trial that involved 20 hospitals from three Australian states: Victoria, New South Wales, and Queensland. All hospitals that participated in the TIPS study had either a Stroke Care Unit or staffing equivalent to a stroke physician and a nurse, and an emergency department. Ethical approval for the TIPS study was obtained from relevant human research ethics committees in each state, from each participating hospital, and The University of Newcastle Human Research Ethics Committee. The study adheres to CONSORT guidelines  was defined as small petechiae along the margins of the infarct; haemorrhagic infarction 2 (HI2) as confluent petechiae within the infarcted area but no space-occupying effect; parenchymal haemorrhage (PH1) defined as blood clots in 30% or less of the infarcted area with some slight space-occupying effect; and parenchymal haemorrhage (PH2) defined as blood clots in more than 30% of the infarcted area with substantial space-occupying effect . For ourOther data of interest included age, gender, pre-stroke mRS, pre- and post-thrombolysis National Institutes of Health Stroke Scale (NIHSS) which measures the severity of stroke; and pre-thrombolysis systolic blood pressure (SBP) on admission which is a risk factor for poor outcome.The intervention was a multi-component, collaborative intervention that was based on a knowledge translation approach and the behavioural change wheel  Fig. 1)1). The in = 1559) had 80% power to detect absolute differences in these key secondary outcomes between intervention and control groups of between 22 and 33% for outcomes with prevalence’s ranging from 50 to 75%. The analysis population is all those that were treated with intravenous thrombolysis. Data was not available on patients that were considered for intravenous thrombolysis but not thrombolysed. Primary analyses compared outcomes between intervention and control group at the active intervention and post-intervention phases. Three mixed effects logistic regression models were used to assess the difference in the study outcomes, proportion of patients with excellent and poor clinical outcome and with PH, between the intervention and control arm during the both active and post intervention period separately. These three models included fixed effects for baseline thrombolysis rates (site-level), pre-morbid mRS, baseline NIHSS; treatment group. Another three mixed-effects logistic regression models were used to determine the effect of the educational intervention on changes in clinical outcomes from pre to active and pre to post-intervention period. These three models included fixed effects for baseline thrombolysis rates (site-level), pre-morbid mRS, baseline NIHSS; treatment group, period (pre vs post) and the interaction between treatment and period. All the above mentioned models included a random intercept for hospital site to account for correlations of individuals within the same site. Due to missing data on mRS outcome, multiple imputation analyses using the chained regression equations method were performed. The missing data was imputed based on hospital site, pre-morbid mRS, NIHSS, age and gender. The imputation process (n = 200 imputations) was conducted assuming the data were missing at random and combined using Rubin’s method. Statistical significance was defined as a two-tailed p-value of < 0.05. Statistical analyses were programmed using SAS v9.4 .The main study was powered to detect a difference in thrombolysis rates between the two groups . Assuminn = 393) of patients reported no pre-stroke disability (mRS = 0) during the pre-intervention phase, with the corresponding proportions being 64% (n = 318) in the active-intervention period and 55% (n = 188) in the post-intervention period. A detailed description of patient characteristics has shown in Table From January 2011 to December 2015, data on 1559 patients who received intravenous thrombolysis were recorded from the 20 hospitals that participated in the TIPS study. This included pre-intervention period data from 599 38%) patients, active-intervention period data from 538 (35%) patients and post-intervention period data from 422 (27%) patients. Finally, a total of 1184 (76%) patient’s data were included. The pattern of missing data for 3 month post treatment mRS between intervention and control groups is reported in Fig. 8% patienThere was a non-significantly higher odds of having an excellent clinical outcome of 57%  and 4% , during active and post-intervention period respectively  during the initial 16-month active intervention period , this poA second factor is the trend throughout the trial for increasingly favourable outcome rates in the control group. The control group demonstrated an increase in the proportion of patients with excellent outcomes in both the active and post-intervention periods. Such results could have several explanations. In Australia, from 2010, several national-level health policy initiatives were released with the intent to improve the management of stroke broadly including intravenous thrombolysis . The revFurther interpretation of the negative results of this individual patient analysis aligns with the well-recognised difficulty in shifting clinician behaviour and the neutral outcome of the main trial, where a comprehensive and multifaceted package of support and education aimed at knowledge translation failed to substantially or sustained oblique change practice. This difficulty is mirrored in several previous studies such as the PRomoting ACute Thrombolysis in Ischemic StrokE (PRACTISE) trial. PRACTICE was a cluster randomised trial in The Netherlands which used the breakthrough approach aiming to improve the thrombolysis rates. PRACTISE intervention failed to achieve a significant overall increase in thrombolysis rates. The study also evaluated the rate of symptomatic intracranial bleeding complications, the good clinical outcome at 3 months (mRS < 3) and mortality, and the effect of PRACTICE intervention on clinical outcome was non-significant. Finally, the study concluded that unregistered comorbidities and risk factors might have contaminated the findings . The INcIt should also note that there is also uncertainty around the choice of clinical outcome of relevance in health systems trials such as TIPS. The limitations of the modified Rankin score are well recognised , and it The TIPS multi-component implementation approach was not effective in improving the odds of the 90-day excellent outcome or reducing the odds of having 90 days post-treatment poor outcome and severe post-treatment haemorrhage. Further study of the clinical impact of efforts to improve thrombolysis rate is warranted.Additional file 1 : Supplement 1: CONSORT 2010 checklist of information to include when reporting a randomised trial. Supplement 2: Number and percentage of missing values, n (%), for each patient characteristics. Supplement 3: Table comparing the clinical and demographic features of patients with vs without mRS at 90 day. Supplement 4: Odds ratio after multiple imputation between intervention vs control during active and post intervention period. Supplement 5: Within group and between group change after multiple imputation between pre vs. Active and pre vs. post intervention period for the both intervention and control.


It is now well-recognized that the tumor microenvironment (TME) is not only a key regulator of cancer progression but also plays a crucial role in cancer treatment responses. Recently, several high-profile publications have demonstrated the importance of particular immune parameters and cell types that dictate responsiveness to immunotherapies. With this increased understanding of TME-mediated therapy, approaches that increase therapeutic efficacy by remodeling the TME are actively being pursued. A classic example of this, in practice by urologists for over 40 years, is the manipulation of the bladder microenvironment for the treatment of non-muscle invasive bladder cancer (NMIBC) by instillation of intravesical bacillus Calmette-Guerin (BCG). The success of BCG treatment is thought to be due to its ability to induce a massive influx of Th1-polarized inflammatory cells, production of Th1 inflammatory cytokines and the generation of tumor-targeted Th1-mediated cytotoxic responses. Whilst BCG immunotherapy is currently the best treatment for NMIBC, ~30% of patients show no response to this treatment. Here we present a review highlighting a variety of promising alternative immunotherapies being developed that remodel the bladder tumor microenvironment. These include (1) the use of oncolytic viruses which selectively replicate within cancer cells whilst also modifying the immunological components of the TME, (2) manipulation of the bladder microbiome to augment the response to BCG or other immunotherapies (3) utilizing Toll-like Receptor agonists as anti-tumor agents due to their potent stimulation of innate and adaptive immunity and (4) the growing recognition that immunotherapeutic strategies that will have the largest impact on patients may require multiple therapeutic approaches combined together. The accumulating knowledge on TME remodeling holds promise for providing an alternative therapy for patients with BCG-unresponsive NMIBC. In addition to malignant cells the TME is also made up of other non-transformed cells and secreted extracellular components. The interactions between the tumor cells and the tissue microenvironment are such that they regulate tumor progression but also determine cancer treatment responses. In particular, the tumor microenvironment often limits the infiltration and function of effector T cells into the tumor, recruiting myeloid derived suppressor cells (MDSCs), tumor associated macrophages (TAMs) and regulatory T cells (Tregs), providing an immunosuppressive niche to help cancer cells escape from immune surveillance , 2. UndeOne of the oldest such immunomodulatory approaches and the gold standard treatment for non-muscle invasive bladder cancer is serial intravesical instillations with bacillus Calmette-Guerin (BCG) , 4. EffeAs is the case for many solid tumors, the tumor microenvironment of non-muscle invasive bladder cancer is characterized by the presence of pro-inflammatory cells  and cytokines (such as tumor necrosis factor-α and interleukins) both in the supporting stroma and in tumor areas. Many of these cell types have been studied to investigate their influence on outcomes following BCG immunotherapy. Several published reports have provided evidence that a predominance of immunosuppressive cell types are associated with BCG immunotherapy failure. In particular, the presence of M2-like tumor associated macrophages (TAMs) has consistently been associated with shorter recurrence-free survival and thus a poor response to BCG , 9–11. Iin vitro with BCG could shift ILCs toward the ILC2 phenotype producing the Th-2 cytokine IL-13 which allowed the recruitment and immunosuppressive function of monocytic cells.Whilst the TAMs are localized in the stroma-tumor margin of NMIBC, infiltrating the tumor area in high-grade tumors, another immunosuppressive cell type the Tregs localize in the stroma around the cancer lesion regardless of tumor stage and grade . High Trin vitro and in vivo studies have suggested a role for NK cells in BCG-induced cytotoxicity (in vitro that BCG-activated killer (BAK) cells, of which NK cells were the major effector cell population, displayed substantial cytotoxicity against bladder tumor cells. Furthermore, using a syngeneic orthotopic murine bladder cancer model they demonstrated in NK-deficient beige mice and in mice treated with anti-NK1.1 monoclonal antibody that BCG therapy was completely ineffective, suggesting a key role for NK cells during BCG immunotherapy  with higher numbers of IL-17+ mast cells showed significantly longer event-free survival after intravesical BCG therapy than patients with less IL-17+ mast cells. This significant effect was only observed in patients who underwent intravesical BCG treatment suggesting that BCG amplifies the beneficial effects associated with increased numbers of IL-17+ mast cells.As well as detecting a diverse infiltrate of innate and adaptive immune cells including the above mentioned cell types, another study reported a significant role for IL-17 positive mast cells in influencing the outcomes from BCG therapy . PatientClearly the interactions between cancer and immunity are highly complex and multifactorial and in particular we still need to have a better understanding of the mechanisms hindering efficient constitutive and/or treatment-induced immune responses to tumors. Currently, in NMIBC, there are no reliable biomarkers which allow prediction of the efficacy of the BCG induced anti-tumor response despite many attempts to look at the immune response in bladder tissue before and after BCG treatment. This may be reflective of the fact that many of the studies to date looking at the immune response to BCG treatment have focused on individual cell types using traditional single immunohistochemical stains rather than performing a multiplex comprehensive analysis of both cellular and non-cellular immune components. With the introduction of new technologies that allow for a more global analysis of complex disease states a more comprehensive picture of the roles and interactions between these different components and their influence on therapies should be revealed. This will allow for more informed treatment strategies on how best to immunomodulate particular bladder cancer microenvironments to achieve the optimal therapeutic outcomes.Lactobacillus iners, one of the prevalent genera detected in the human urinary microbiome (n = 6) versus urothelial carcinoma patients (n = 8) using 16S sequencing found that the abundance of the genus Streptococcus was most often significantly elevated in urothelial carcinoma patients  and decrease of other bacterial genera  in the cancer group when compared to the non-cancer group. A further finding was the enrichment of Herbaspirillum, Porphyrobacter, and Bacteroides observed in cancer patients with high risk of recurrence and progression indicating that these genera may be potential biomarkers for risk stratification  . OVs leain vivo studies with CG0070 demonstrated strong anti-tumor activity of the virus in bladder transitional cell carcinoma xenograft tumor models and showed significant anti-tumor synergy when combined with the chemotherapeutic agent docetaxel  designed to preferentially replicate in and kill retinoblastoma (Rb) pathway defective cells using the E2F-1 promotor . To enhaocetaxel . In a phocetaxel . High leocetaxel . Unfortuocetaxel . Importain vitro and in vivo  of pembrolizumab (antibody which targets and blocks PD-1) in combination with BCG for patients with high-risk NMIBC  and Atez in vivo . Wang et in vivo .Another promising immunotherapy agent currently being trialed in different immunotherapy combination strategies is an IL-15 superagonist, ALT-803, that has a proven potent ability to expand and functionally activate both NK cells and T-cells . This ILThe use of low-dose chemotherapy that elicits immune-potentiating effects by either inducing immunogenicity or relieving tumor-induced immunosuppression is another strategy being pursued to immunomodulate the tumor microenvironment. Certain chemotherapeutic agents not only have direct anti-tumor effects but have also been shown to play a role in depleting regulatory T cells , upregulSalmonella enterica typhi Ty21a live vaccine-strain against typhoid fever), but not CpG, after tumor antigen vaccination efficiently recruits vaccine-specific CD8 T cells to the bladder, resulting in tumor regression and 90% survival of the mice. Salmonella can provide TLR-4  agonists that are able to modify the expression of selectins, integrins, chemokines, and chemokine receptors, thus enhancing T-cell attraction to the tumor site , 62. Domde TLR-4  and TLR-de TLR-4  and may de TLR-4 . In a mode TLR-4 .in-situ reported results from 12 patients, of which half (6/12) had received ≥2 prior induction courses of BCG (In a phase 1 clinical trial the activity of TMX-101, a liquid formulation of the toll-like receptor 7 agonist, imiquimod, was analyzed in low-grade NMIBC . Whilst s of BCG . TMX-101s of BCG . PerhapsThe tumor microenvironment has a profound impact on the success or failure of treatments for NMIBC. There is a growing realization that remodeling the tumor microenvironment to achieve optimal therapeutic effects will require multiple complementary therapeutic approaches. At the core to achieving this is a critical understanding of the bladder tumor microenvironment including the influence of the bladder microbiome in NMIBC both during tumor progression and in response to treatment. Only with this knowledge can we optimally make use of novel emerging immunotherapies and how they can complement existing therapies to achieve alternative bladder-sparing options critically needed in patients with BCG unresponsive NMIBC.NA wrote the main body of the text. GS contributed to the oncolytic virus section. HP reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This review provides a comprehensive overview of immune cells involved in microenvironment, available immunotherapies and imaging agents to highlight the importance of new therapeutic biomarkers and their in vivo evaluation to improve the management of cancer patients.Tumor-infiltrating immune cells play a key role against cancer. However, malignant cells are able to evade the immune response and establish a very complex balance in which different immune subtypes may drive tumor progression, metastatization and resistance to therapy. New immunotherapeutic approaches aim at restoring the natural balance and increase immune response against cancer by different mechanisms. The complexity of these interactions and the heterogeneity of immune cell subpopulations are a real challenge when trying to develop new immunotherapeutics and evaluate or predict their efficacy in vivo. To this purpose, molecular imaging can offer non-invasive diagnostic tools like radiopharmaceuticals, contrast agents or fluorescent dyes. These agents can be useful for preclinical and clinical purposes and can overcome [ Immunotherapy is the most appealing anti-cancer approach of the modern era and researchers are continuously exploring new ways to reprogram immune cells of the host against cancer . DespiteMoreover, because of the complex and dynamic nature of the interactions between cancer and immune cells, a high inter- and intra-patient heterogeneity is observed, sometimes leading to failure of the treatment . That isIn this scenario, molecular medicine imaging offers plenty of tools to specifically follow immune cell subtypes in a non-invasive manner . This isCancers are not a mass of transformed cells but rather a new organ composed of various non-malignant cells comprising a large portion of the tumor mass, which have become wayward and lost the ability to maintain a dialogue enabling homeostasis of the tissue architecture . These cT lymphocytes are the most potent mediators of adaptive anti-tumor immune response. The cytotoxic CD8+ T cell population, supported by CD4+ T helper (Th1) cells through the production of IL2 and IFNγ, generates the final effector mechanism leading to tumor elimination and are associated with a good prognosis , 11. WheThe successful control of tumor progression, mediated by T lymphocytes, firstly requires that they infiltrate the tumors. In fact, the immune contexture and the T cell abundance, functional activity and spatial distribution in the TME are crucial prognostic and predictive factors , as receCompartmentation of the immune response into three major phenotypes - inflamed, immune-excluded and immune-desert phenotypes - has been proposed as the major predictor of response to different cancer treatments in the new era of immune inhibitory receptor blockades , 18. TheThe above-mentioned suppressive cells, accompanied by the hindrance of lymphocyte infiltration and trafficking, may be modulated in multiple ways. Indeed, soluble molecules, such as vascular endothelial growth factor (VEGF) and the consequent abnormal neovasculature  as well The lack of an endogenous anti-tumor response in TME described as the third immune-desert phenotype, may be due to insufficient T cell priming, immunological ignorance or induction of tolerance. This immune contexture is characterized by the presence of Treg, MDSC and macrophages, which wire a circuit inhibiting dendritic cell (DC) maturation and hamper T cell expansion and activation .Recent findings have assessed a role for B cells in the anti-tumor immune response  B cell iTumor stroma may be infiltrated by innate cytotoxic lymphocytes, the natural killer (NK) cells. NK cells not only recognize and kill cancer cells through the release of cytolytic granules, but also greatly impact the adaptive anti-tumor immune response by producing chemokines and cytokines. NK cells are highly heterogeneous, and the availability of different combination markers has allowed researchers to identify distinct subpopulations with definite functionality . OtherwiDCs are antigen-presenting cells (APC) able to capture antigens in the form of peptide-major histocompatibility (MHC) molecule complexes and present them to the T cells . They arTLS are lymphoid aggregates induced postnatally in non-lymphoid tissues that resemble the organization of lymph nodes, characterized by clusters of mature DCs and T cells juxtaposing B-cell follicles and high endothelial venules without encapsulation. Similar to lymph nodes, they are assumed to provide the main lymphocytic functional environments for both cellular and humoral immunity . The TLSMost of the immune cell populations within the tumor stroma are made up of tumor-associated macrophages (TAMs), major players in orchestrating cancer-related inflammation. Pre-clinical and clinical evidences demonstrated that an abundance of TAMs in the TME is associated with a poor prognosis . The bi-Neutrophils constitute 50-70% of all circulating leukocytes and representing the traditional front line of defense against infection. Inside the TME, a number of key molecular mechanisms can promote neutrophil polarization in two opposite subpopulations of anti-tumorigenic (N1) and pro-tumorigenic (N2) tumor-associated neutrophils (TANs) . In partMyeloid-derived suppressor cells MDSCs are classed as one of the major sub-populations of inhibitory immune cells that are frequently found in several mouse and human cancers and show a plastic phenotype , 61 rendAmong the non-neoplastic cells in the TME, CAFs are the most prominent stromal component and key players in cancer progression . CAFs seThe complex mixture of immune cells, non-cancerous cells and cancer cells are embedded in the extracellular matrix. The dysregulation of ECM composition, structure, stiffness and quantity, by regulating mechanical and biochemical cues in the TME, is crucial in cancer progression, invasion and immunosuppression . A recenECM deposition and remodelling are strictly linked to a reduction of the oxygen level, known as hypoxia. Rapid growth and poor vasculature development frequently lead to hypoxic microenvironments within the tumor. The association between hypoxia and ECM remodelling is mediated by hypoxia inducible factor-1 and 2, which regulate the expression of enzymes related to bio-synthesis fibres in collagen degradation . To survThe overview of this amazing complexity surely justifies a great multidisciplinary effort in cancer research and new methodologies to track the immune cells in the highly trafficked highways and byways of the cancer road map.The ability of the host immune system to identify and eradicate malignant cells with minimal systemic toxicity remains the holy grail of cancer immunotherapy . The firThe discovery and characterization of dendritic cells by Ralph Steinman in 1973, the description of MHC restriction in 1974 by Zinkernagel and Doherty’s, the documentation of NK cell activity in 1975 by Eva Klein’s, the investigation in large-scale of cytokines in breast cancer, renal cell cancer (RCC), glioblastoma, lymphoma, and melanoma in the 1980s, initiated the modern immune-based cancer treatments in clinical medicine , 87.During the past 20 years, mAbs have been a major component of treatment for many cancers, including breast, lymphoma, and colo-rectal cancer malignancies . The proThe challenge of identifying antigen-specific cells and expanding them to numbers that enabled researchers to overcome the barrier of low fusion efficiency would, however, require several more decades of investigation. The principal advantage of the use of human hybridoma technology for mAb generation is that this approach preserves the authentic sequence and pairing of antibody DNA from a natural B cell for the expression of a naturally occurring full-length human mAb . TherapeImmune checkpoint inhibitors constitute an important breakthrough positively influencing treatment outcomes in cancer patients . TreatmeBrunet and colleagues in 1987 described for the first time CTLA-4, also known as CD152, a co-inhibitory molecule that functions to regulate T cell activation and its effect in melanoma were described by Jim Allison’s group in 1995; fourteen years later the FDA approved the revolutionary checkpoint inhibitor ipilimumab a mAb for the treatment of stage IV melanoma.More recently, the PD-L1 interaction was described as a major pathway used by tumors to suppress immune control . PD-1 reAnti PD-1 or anti PD-L1 antibodies are currently registered by the FDA for metastatic malignant melanoma, non-small cell lung cancer (NSCLC), renal cell cancer, head and neck cancer, urothelial carcinoma and Hodgkin’s lymphoma in various stages of the respective disease and in the context of varying treatment histories . Many otAnti-CTLA-4 antibodies (ipilimumab and tremelimumab), anti-PD-1 antibodies (nivolumab and pembrolizumab), and anti-PD-L1 antibodies  have produced remarkable results regarding tumor control in many malignancies; however, response is often followed by relapse and disease progression.In this context, potential antitumor targets are regulatory T cells (Treg cells). It was proposed that they impair activation, survival and expansion of antitumor T cells through the production of immunosuppressive cytokines, such as transforming growth factor-β (TGFβ) and interleukin-10 (IL-10), and the CTLA4 . DepletiInflammatory infiltrates in tumors are considered to be a host attempt at the detection of emerging tumor cells and their elimination, for this reason researchers are trying to identify new drugs to increase this immunological infiltrate .For this purpose, viruses have been used based on the observation that some of them could infect and kill leukemic peripheral blood cells in vitro ; while mCytokines, such as interferons, interleukins, chemokines, and growth factors, are immune modulators that are produced naturally by numerous cell types . CertainAlthough IFN-α and IL-2 have been best characterized and used for cancer treatment, many additional cytokines are being investigated for use in cancer immunotherapy ; the disIL-2 is one of the key cytokines with pleiotropic effects on the immune system and it was an early candidate for cancer immunotherapy, approved for the treatment of metastatic renal cell carcinoma (1992) and later for metastatic melanoma (1998) by FDA. Although high doses of IL-2 showed promising results in metastatic renal cell carcinoma and melanoma, the toxicity and cost limited its application in a large population . Thus, sInterferons are agents with antiviral, antiproliferative, and immunomodulatory properties. IFN-α has shown antitumor and antiviral efficacy and FDA approval was granted for the treatment of patients with hairy cell leukaemia, acquired immune deficiency syndrome-related Kaposi's sarcoma, and condylomata acuminata. Although IFNs are effective as single agents in certain clinical pathologic entities, increasing experience with these cytokines suggests that their greatest therapeutic potential may be realized in combination with other biological response modifiers, cytotoxic, or antiviral agents . While ITherapeutic vaccines represent a viable option for active immunotherapy of cancers that aim to treat late stage disease by using a patient's own immune system. The promising results from clinical trials recently led to the approval by the FDA of sipuleucel-T, a dendritic cell vaccine, for the treatment of stage IV metastatic but asymptomatic castrate-resistant prostate, the first therapeutic cancer vaccine . Based oOne goal of cancer vaccines is to stimulate the immune system to attack and eradicate cancer cells. To this end, cancer vaccines contain whole cancer cells, parts of cancer cells, or purified antigens that enhance the immune response against cancer cells. In this context, cancer vaccines exhibit high specificity and low toxicity, but their therapeutic efficacy had been very low with a reported overall objective response rate of only 3.3%; tumor eradication has been achieved in models of cancer by intratumoral or peritumoral application of cytokines or by implantation of tumor cells expressing cytokines .Autologous tumor vaccines prepared using patient-derived tumor cells represent one of the first types of cancer vaccines to be tested . These tAllogeneic whole tumor cell vaccines typically contain two or three established human tumor cell lines, may be used to overcome many limitations of autologous tumor cell vaccines . These iP = 0.03), longer progression-free survival  and improved median overall survival  compared with the IL-2 group  Fluorodeoxyglucose ([18F]FDG) trying to exploit glucose transporters were not successful because of slow accumulation of cells in the tumors, leakage of the radiopharmaceutical and high accumulation of injected cells in the lungs at early time points FDG has been attempted to follow NK infiltration in patients undergoing immunotherapy or in pre-clinical models, but with limited success. Issues related to these techniques like poor sensitivity or altered biodistribution are amplified by the low number of NK cells and the cumbersome purification procedure prior their labelling and injection. This has been confirmed by Meller et al. that analysed NK cell number after 3 d from their administration in patients with renal cell carcinoma that received 111In-oxine-labelled and unlabelled NK cells from allogeneic donors FDG. However, in a study by Quillien et al., 111In-oxine-labelled macrophages, after in vitro expansion, accumulated in only 1 lesion out of 15 patients studied by SPECT imaging. They analysed cell’s phenotype after culturing them ex vivo and hypothesized that culturing conditions might have influenced their homing properties -(R)PK11195 that binds the translocator protein (TSPO) expressed at high grade in the mitochondrial membrane of macrophages and microglial cells. This has been mainly studied to image neuroinflammation, but may have application in imaging tumor-associated macrophages (TAMs). In vivo studies in mice that were not able to correlate the radiopharmaceutical uptake with TSPO expression, revealed by immunohistochemistry -SFB-counterpart that showed higher sensitivity and better biodistribution. A more recent approach exploits the use of 3′-Aza-2′-[18F]-fluoro-folic acid, also known as [18F]-AzaFol, which was previously used to image macrophages in various diseases 3-(hydroxymethyl)butyl] guanine ([18F]FHBG), 2-deoxy-2-[18F]5-ethyl-1-D-arabinofuranosyluracil ([18F]FEAU) or 2-deoxy-2-[18F]5-iodo-1-D-arabino-furanosyluracil ([18F]FIAU). These compounds are taken up by nucleoside transporters and then are phosphorylated by the enzyme remaining trapped in the cytoplasm [The HSV1-tk reporter gene is a common technique that exploit the specificity of this enzyme for 9-[4-[ytoplasm . This al18F]FDG is observed. This limits the use of current available criteria and new ones are under definition with limited success. That is why we need new non-invasive tools to rely on and molecular imaging offers the most suitable approach.In the present review, we wanted to give an overview of the immune cells that are involved in tumor microenvironment infiltration to highlight why imaging of their trafficking is so crucial with so many new immunotherapies entering the clinical practice. The main issue when evaluating tumor response to cancer immunotherapy is the enlargement due to infiltrating immune cells that eventually leads to tumor shrinkage and death. The same enlargement occurs in case of tumor progression due to cancer cell growth and in both situations increased uptake of [Unfortunately, to fully achieve this goal we still have to face many open challenges like the many immune cell subtypes, small number and dynamic behaviour. This implies that radiopharmaceuticals of choice should be highly specific for biomarkers expressed by different immune cells. Molecular imaging can guide basic research, drug development and clinical follow up. It can also help researchers to elucidate mechanisms of pathology, effect of new drugs and predict efficacy of immunotherapies. There is still a long way to go, but many tools, summarized in Table Radiolabelled immune checkpoint inhibitor mAbs showed great results in vivo, but to date they are still limited to pre-clinical studies. Also, cytokines like IL2 showed great potential and pilot human studies have already been performed with interesting results. The next step, for each of these radiopharmaceuticals, would be to increase the number of enrolled patients and define patterns of uptake to define new criteria for therapy decision-making and follow-up. This is particularly important, since inter- and intra-patient tumor heterogeneity is a real concern that may even require a “lesionalised therapy”.This will require multimodal approaches as interactions in tumor microenvironment and different mechanisms of evasion from immune response are too complex to be unfolded by a single imaging tool.