While specialization plays an essential role in how scientific research is pursued, we understand little about its effects on a researcher’s impact and career. In particular, the extent to which one specializes within their chosen fields likely has complex relationships with productivity, career stage, and eventual impact. Here, we develop a novel and fine-grained approach for measuring a researcher’s level of specialization at each point in their career and apply it to the publication data of almost 30,000 established biomedical researchers to measure the effect that specialization has on the impact of a researcher’s publications.Using a within-researcher, panel-based econometric framework, we arrive at several important results. First, there are significant scientific rewards for specialization—25% more citations per standard deviation increase in specialization. Second, these benefits are much higher early in a researcher’s career—as large as 75% per standard deviation increase in specialization. Third, rewards are higher for researchers who publish few papers relative to their peers. Finally, we find that, all else equal, researchers who make large changes in their research direction see generally increased impact.The extent to which one specializes, particularly at the early stages of a biomedical research career, appears to play a significant role in determining the citation-based impact of their publications. When this measure of impact is, implicitly or explicitly, an input into decision-making processes within the scientific system , these findings lead to some important implications for the system-level organization of scientific research and the incentives that exist therein. We propose several mechanisms within modern scientific systems that likely lead to the scientific rewards we observe and discuss them within the broader context of reward structures in biomedicine and science more generally. No researcher can be an expert in all fields. The entirety of human knowledge, even when considering a single field, is simply too much for a person to accumulate in a lifetime. Faced with this challenge, researchers specialize . ThroughIn addition to reducing the amount of knowledge and skills one must accumulate, specialization plays a key role in the sociology of science literature. In specializing, a researcher becomes a member of a community of researchers working on similar matters, in a similar fashion, with a similar viewpoint. It is within these specialized communities that the so-called invisible college is most visible , 2. The These observations present us with two points of view to consider the specialization of researchers. Viewed through the frontier-of-knowledge lens, specialization is required to reach the frontier of knowledge and start contributing within a human lifetime. Jones  formulatWhile specialization is understood to play an essential role in research and researchers’ careers, most attention has been paid to how specialization emerges  and to tnot interdisciplinarity—working at the intersection of fields is different from publishing work on many different topics—but rather generalization. A researcher working in a highly interdisciplinary space but publishing exclusively on the same small set of topics would be classified as a specialist within our framework. As such, our operationalization of specialization relates to the so-called “balance” dimension that is commonly referred to in the extant literature on interdisciplinarity [This operationalization of specialization is quite different from notions of monodisciplinarity. That is, the opposite of specialization, as defined here, is linarity , 15, tholinarity , 17.and citers’ perception of the author’s social status or ability, rather than solely as indicators of publication quality alone [We use citations as a measure of impact for two primary reasons. First, while undoubtedly noisy and narrow in scope, citations remain a popular indicator of research impact , most pety alone , 26, as Considering the frontier of knowledge point of view, it is unclear what effect the extent of specialization should have upon a researcher’s impact. From the “burden of knowledge” perspective , it is rViewed through the lens of the sociology of science and the invisible college, the expected professional benefits to specialization are more straightforward. Researchers can raise their visibility within a specific community more effectively by specializing to a greater extent, thereby attracting more citations. This line of thinking is consistent with one of the few previous studies using the extent of specialization that we are aware of . In thatA more recent work, using journal classifications to construct a researcher-level measure of interdisciplinarity, found that while more interdisciplinary researchers attracted more citations per paper, this effect was more than offset by lower productivity . Howeverresearch impact, proxied by citations per publication. In addition to measuring whether greater specialization positively or negatively affects a researcher’s impact, we also examine the role of career age, publishing rate, and recent changes in research topics in this relationship. Specifically, we answer four questions. First, does the extent to which a researcher is specialized affect the impact of their research? Second, does the effect of specialization on impact change with career age? Third, does the effect of specialization depend on the number of papers a researcher is producing? Finally, do recent changes in a individual’s research interests affect impact?In this paper, we conduct an exploratory investigation into the scientific rewards associated with specialization using a dataset of 29,197 biomedical researchers. We estimate the extent to which a researcher is specialized using a novel measure that captures how intensely focused they are on specific topics. We measure scientific rewards in terms of To provide answers to these questions, we need to use data that are rich in both longitudinal and cross-sectional dimensions, because we wish to measure the levels of specialization and impact at each point in a researcher’s career. To assess the trends that may be moderated by seniority or experience, we need to repeat these measurements over a long period. These considerations lead us to select a restricted cohort of nearly 30,000 well-published biomedical researchers. Each of these researchers is likely to be considered to be “successful” in the biomedical field by almost any measure. As such, all results are necessarily conditional on long-term success and should be interpreted with this caveat in mind. We discuss the details and implications of this restriction in the following sections. The “Methods” section describes the data, the variables, and the regression models.Table However, care should be taken in interpreting these coefficients: the benefits of specialization decrease as a function of career age and publishing rate, but the overall impact is always positive. That is, hypothetically increasing a researcher’s specialization appears to be associated systematically with a boost in impact. When the same hypothetical increase in specialization is applied to the same researcher later in their career (column 2) or for higher publishing rates (column 3), the boost in impact is less pronounced but always positive during the 35-year period we observe.Lastly, column 4 shows the estimates for the complete interaction model. It includes the interaction between publishing rate and career age and the triple interaction term of specialization, publishing rate, and career age.The variable cosine similarity maintains a negative impact on the average citation counts across all specifications. Thus, a researcher who makes greater changes in research direction from one period to the next achieves greater impact in the next period. This result, which is not causal, indicates that researchers are likely to reorient their research direction toward topics that result in higher future impact. Indeed, at least for our successful cohort, the ability of researchers to identify promising new avenues of research and immerse themselves in those areas may play a large role in the benefits of specialization we observe, adding color to previous work on the evolution of research interests . More imAs our observation periods are 3 years long, it is difficult to assess whether drastic changes in research direction on shorter timescales are rewarded with greater future impact, but recent work examining this question at the individual-paper level indicates that the reverse is likely true . These cFigure To evaluate the validity of these results, we carry out a number of robustness checks, each described in more detail in the Second, we break the 100 or greater, and 75 to 99, publication samples into field-specific subsets and repeat the analysis on each of the eight most frequent fields found in our datasets. These fields include molecular and cell biology, medicine, neuroscience, gastroenterology, infectious diseases, radiology, nephrology, and psychology. In each of the eight fields, for each of the publication thresholds, we find that our main findings are qualitatively similar—however, the statistical significance is weaker for the smaller subfields.Lastly, while our specialization measure is, by definition, conceptually distinct from interdisciplinarity, we also confirm that the measure is also empirically distinct. We split the cohort into monodisciplinary and interdisciplinary researchers (based on whether a researcher has published research in multiple sub-fields within the 3-year window). We then conduct several non-parametric statistical tests to detect any differences between the specialization distributions of these groups. We do not find any significant differences, providing evidence that our specialization measure is not sensitive to the differences in the levels of interdisciplinarity between researchers, at least at the sub-field level.These results have significant implications for how we view and manage academic careers, especially in their fragile early stages. In the context of long-term success in biomedical research, working continuously on a relatively small and stable set of topics is rewarded by citations to the output of this research. Furthermore, there likely exists a complex web of interacting mechanisms that conspire to reinforce this phenomenon in ways that are not necessarily positive for the scientific enterprise. The remainder of this section will discuss these potential mechanisms, the limitations that may reduce the generalizability of this work, and open questions that may add more color to the current findings.These findings also raise a great many questions. A critical one, of course, concerns the mechanisms that generate these rewards. From the frontier-of-knowledge point of view, it would seem that by focusing more intensely, a researcher may be in a better position to push the research frontier and attract more citations (perhaps by establishing priority on new findings). Taking the perspective of the sociology of science, one explanation is that by specializing more narrowly, a researcher may be better positioned to raise their visibility within a specific community. Indeed, the fact that we observe much greater rewards for specialization early in a researcher’s career  suggests that a young researcher’s impact is maximized by focusing on a community that is as specific as possible. However, the degree to which a researcher can “choose” a community at this early stage is unclear. We suggest that the choice of research direction likely comes before any intentional choice of community. The benefits diminish with career age could imply that it is easier to transfer visibility, accumulated advantage, or reputation across communities than it is to earn such things simultaneously across several communities.It is also likely that current reward systems in contemporary science systems favor scientific specialization. While we draw a conceptual distinction between monodisciplinarity and specialization, there is almost certainly a strong relationship between the disciplinary nature of a particular scientific output and the level of specialization of the authors. The fact that science systems may punish interdisciplinary research , 36, duenegatively associated with impact in prior work, while the other two dimensions are either positively or ambiguously associated with impact. This nuanced point is very pertinent to the current work: the diversity of information sources and the evenness of the distribution of inputs across these sources have distinct and generally opposing relationships with impact. We note that the total number of unique Medical Subject Headings (MeSH) terms, a metric more related to the “variety” dimension, is included as a control in our main model. In sum, while it is not straightforward to directly compare the impact of individual articles with the impact of researchers more broadly, nor to compare operationalizations based on input knowledge with those based on research topics, our results are at least consistent with this prior research, although our researcher-focused approach allows for additional nuance arising from access to the temporal dimension.Our results are in line with prior work in the field of bibliometrics on so-called interdisciplinarity. That body of literature often breaks down interdisciplinarity into various components, most commonly variety, balance, and disparity . While tOur analysis considers the quantity and broad patterns of collaboration by including control variables at the researcher level. However, the role of collaboration and division of labor in these results merits further discussion. An established body of literature shows that papers with a distinct interdisciplinary character , 17 or tSeveral outstanding issues moderate the practical usefulness of the results presented herein, especially in the context of academic careers. The first concerns the extent to which researchers can control their specialization actively. That is to say, how do the concrete day-to-day, project-to-project, and job-to-job decisions a researcher faces map on to specialization? Clearly, some subset of these decisions does affect specialization—for example, the selection of a specific new project. Researchers may perceive a risk of being “left behind” if they spend too much time working on projects outside of their usual specialized research path. These concerns raise an additional question, namely, if researchers do not control their own extent of specialization, who or what does? And in turn, how may those individuals, institutions, or systems craft policy to bring about a better configuration of the academic career in light of these findings? After all, we do not claim that the observed rewards associated with specialization are a good thing for science or society. The patterns we see are, in part, the result of a complex set of incentives and Matthew effects that are embedded in research ecosystems, many aspects of which have been heavily criticized for their various biases and inequitable outcomes –45. Whilboth benefit from lower productivity at the individual output level. A more comprehensive investigation of the relationships between productivity, impact, and a researcher’s precise location on the specialization-disciplinarity plane may be a fruitful avenue for future research on this topic. While necessary for the operationalization of specialization used in this work, the imposed publication threshold implies that several lines of research are closed to us, such as the relationships between early specialization, academic career length, and opportunities outside of academia. For example, it may be the case that less-specialized high-impact early career researchers have a large selection of stable, well-compensated jobs outside of academia due to this breadth. In contrast, their specialized high-impact colleagues may have more choice of fellowships and positions within academia. In other words, funding systems and entrenched departmental structures at research institutions may act as filters in early academic careers, with a bias toward high-impact specialized researchers. Furthermore, it is possible that our results may be driven in part by Matthew effects [A second concern is the inherent selection bias in the above analysis that is necessary to obtain enough data for our method to be effective. The results in Table  effects —a strongFinally, in the analysis presented above, we did not find a point after which increasing one’s extent of specialization becomes significantly deleterious in any discipline we examined . The rewards for specialization are most pronounced early in a researcher’s career and decrease monotonically thereafter. The benefits are greatest for researchers publishing at a lower rate relative to their cohort and decrease monotonically as the publishing rate increases. While the returns to specialization decrease with age and publishing rate, increasing one’s extent of specialization has no beneficial impact only at the longest times we observe, typically 25–35 years after a researcher first publishes. Lastly, changes in research direction at the scales of the time windows considered (3 years) appear to increase (within-researcher) citation-based impact.Author-ity disambiguation of PubMed [Our researcher dataset is drawn from the f PubMed , 59. Eacf PubMed . To be especialization, as well as additional explanatory and control variables, described below. This panel data was then analyzed using a researcher fixed-effect panel regression approach.We broke each researcher’s career into 3-year windows and identified the papers they published during each period. From the papers published in each time window, we calculated our main explanatory variable, Our method for estimating the extent to which a researcher is specialized is based on Medical Subject Headings (MeSH) as outlined in Fig. revealed comparative advantage (RCA) in that MeSH term:i’s papers from time window w on which MeSH term m appears, and m in the PubMed database, and For each MeSH term assigned to a researcher’s papers in a given time window, we calculate the researcher’s z-score transform to the quantity z-score of 1.2 for a specific MeSH term indicates that the MeSH term is assigned to the researcher’s publications 1.2 standard deviations more than would be expected from the global average (within the same time window). Hence, the z-score measures how focused the researcher is on specific topics or concepts at various points in their career. For each window of a researcher’s career, we have a z-score for each MeSH term assigned to their publications in that window. Treating these z-scores as a distribution over MeSH terms for a given individual in a particular time window, our measure of specialization , because the same few MeSH terms appear in many of publications they produce, despite these fields being relatively large. In contrast, a researcher working in the above fields in roughly equal proportions but without much MeSH-term overlap on individual publications would be considered more generalized, even if these publications were relatively monodisciplinary. In this way, a specialist can produce exclusively interdisciplinary work, and a generalist can produce exclusively monodisciplinary work, which distinguishes our measure from those in the extant literature. In reality, while we consider specialization and disciplinarity to be distinct concepts in this work, we expect that there is a strong inverse relationship between the interdisciplinarity of particular publications and the specialization of the authors.With respect to dynamics, because we calculate specialization in 3-year blocks, the mid- to long-term evolution of the MeSH dictionary have minimal impact on our measure. Furthermore, any effects of short-term popularity dynamics of particular MeSH headings are mitigated by considering the RCA rather than considering only the usage of terms by each individual. For example, the measure can differentiate between a particular researcher starting to study a specific topic more frequently and an entire field doing the same.Figure i’s papers published in window w up to 5 years after publication.w), and cosine similarity Next, we estimate the extent to which specialization affects a researcher’s scientific impact using an econometric regression model. As researchers are active over many time windows in the dataset (up to ten time windows covering 30 years), we are able to use variation in each researcher’s specialization across time windows as the source for econometric identification. The specific model we estimate is as follows:mentclass2pt{minimThe interpretation of the specialization parameter is as follows: a one-standard deviation increase in the specialization measure of the researcher in a given time window is associated with a i publishes in window w that are tagged with the jth MeSH term (from the universe of n ordered MeSH terms). This value is maximized  when the vectors Cosine similarity,


Cerebrospinal fluid (CSF) obtained This study aimed to describe the longitudinal profile of routine CSF parameters in patients with SAH and to identify associations with neurological complications.A total of thirty-three patients with spontaneous SAH who required an EVD and had at least three consecutive CSF samples collected over a period of more than 7 days were included in this study.via EVD had no impact on routine CSF parameters.A median of 6 longitudinally collected CSF samples per patient were available within 1–22 days after SAH onset. Overall, red blood cells (RBC) steadily decreased over time, whereas white blood cells (WBC) and total protein (TP) increased until days 6 and 13, respectively, and decreased thereafter. The estimated decay rates of RBC, WBC, and TP were 28, 22, and 6% per day. Distinct CSF patterns over time were linked to known complications after SAH. Patients with rebleeding showed increased RBC, TP, and phagocytosing cells compared to patients without re-bleeding. For ventriculitis, an elevated cell index with a higher proportion of granulocytes was characteristic. CSF of patients with delayed cerebral ischemia showed increased RBC and WBC compared to patients without DCI. Early CSF WBC and cell index were predictive for the occurrence of DCI and ventriculitis later during the disease course. The amount of daily CSF drainage Longitudinal CSF characteristics are associated with SAH-related complications. Due to the blocking of normal cerebrospinal fluid (CSF) circulation resulting from the extravasated blood, a significant proportion of patients develop hydrocephalus and require external ventricular drainage (EVD) . VentricThe aim of this study was to describe the longitudinal profile of routinely analyzed CSF parameters in patients with SAH requiring EVD placement and to describe distinct changes in patients with rebleeding, and in those developing ventriculitis and DCI. Furthermore, a literature search was performed to summarize current evidence on longitudinal CSF changes in patients with SAH.A literature search in PubMed using the search terms “cerebrospinal fluid” AND “subarachnoid hemorrhage” AND “white blood cells” or “red blood cells” or “total protein” or “granulocytes” or “lymphocytes” or “macrophages” or “siderophages” limited to 11 January 2022 returned 7, 43, 17, 4, 26, 42, and 10 references. Abstracts that primarily did not deal with the longitudinal evolution of these CSF parameters or their usability to predict clinical disease course were excluded. In addition, articles in reference lists of individual papers were selected if considered appropriate. Only original articles written in English were considered.via the EVD and was usually done at fixed time points (twice per week)  .Demographic and clinical data of patients with SAH were obtained from the medical charts, as well as from the prospective Innsbruck SAH database.The occurrence of ventriculitis was defined according to CDC criteria: (i) detection of pathogens in CSF (by culture), or (ii) fulfillment of at least two clinical criteria , and at least one CSF criteria (increased CSF white blood cells (WBC), elevated CSF protein and decreased CSF glucose; positive CSF gram stain; detection of pathogens in blood by culture or non-culture based microbiologic testing; positive serological result) . The cliRebleeding was defined as a new/ extended hemorrhage on a CT scan associated with a sudden clinical deterioration at any time during the ICU stay. In patients with sedation and analgesia sudden clinical deterioration or unexplainable changes in blood pressure and heart rate, pupil sizes, or increases in intracranial pressure readings provoked a CT scanning to exclude/confirm suspected rebleeding.Delayed cerebral ischemia was defined as clinical deterioration with a new focal neurologic deficit, a decrease of greater than or equal to 2 points on the Glasgow Coma Scale, or new infarction on CT or MRI scan not attributable to other causes .Determination of all CSF parameters was performed at the Neuroimmunology Laboratory of the Department of Neurology, Medical University of Innsbruck, within 2 h after CSF withdrawal . CSF WBCCerebrospinal fluid total protein concentration was measured by spectrophotometry after incubation of centrifuged CSF with 3% trichloroacetic acid .The cytological preparation followed the recommendations of Lehmitz et al.  with sliTo assess the relative distribution of the WBC subpopulation, we counted the number of granulocytes, lymphocytes, monocytes/macrophages, and erythrophages/siderophages in a single spot of each quadrant of the glass slide at × 400 magnification, calculated the mean number of each cell type and finally the relative proportion (in percentage) for each cell type .Data are displayed as mean ± standard deviation (SD) or as median and range or interquartile range as appropriate.To model the decay of RBC, WBC, and CSF total protein over time, we assumed the following functionyt denotes RBC, WBC, or CSF total protein, respectively at time t, y0 is the starting level of the quantity y, λ is the decay parameter due to time (days) and δ is the decay parameter due to cumulative loss of CSF quantity at time t (CSF−quantityt) via the external ventricular drainage (ml).where y for each patient i to consider individual heterogeneity (ci):We estimated the above equation after a logarithmic transformation and used a starting level of it is the remainder noise with the usual ordinary least squares assumptions.where εLogistic regressions were computed to identify influential variables for different clinical outcomes . Repeated measurements were considered by cluster robust standard errors and model assumptions were checked.Regression analyses with the transformation of the dependent variable if needed  and fixed effects for time point and the group at each time point  were used to assess differences of CSF parameters at certain time points between patient groups. Longitudinal measurements were grouped to time points 1–6 according to the following periods: days 1–4, 5–8, 9–11, 12–15, 16–18, and 19–22.Receiver operating characteristic analyses were performed to assess the value of CSF parameters at time point 1 to predict the occurrence of SAH complications later during follow-up. Sensitivities, specificities, and area under the curve (AUC) were provided.The significance level was 5%. All computations were done with R Core Team  and the The conduct of the study was approved by the local ethics committee of the Medical University of Innsbruck . Written informed consent was obtained according to Austrian law and in accordance with the Declaration of Helsinki.Few studies on the longitudinal evolution of routine parameters in ventricular CSF  were identified. The main findings were that RBC and CSF TP decrease with SAH disease duration –20, thatvia the EVD within 1–22 days after SAH onset. A median of 6 (range 3–6) longitudinally collected CSF samples were available per patient. For further detailed clinical characteristics, we refer to A total of 33 patients with a mean age of 57 ± 14 years comprising 70% of women had 177 CSF samples collected P < 0.001), while no evidence for a statistically significant decay parameter due to the cumulative loss of CSF quantity (via the EVD) was found.Overall, CSF RBC count steadily decreased during the disease course, whereas WBC count and CSF total protein first showed an increase reaching the highest values after a median of 6 and 13 days, respectively, before the eventual drop . CSF RBCThe cell index increased until day 13 and declined afterward . WBC subCerebrospinal fluid parameters showed a distinct longitudinal development associated with the clinical disease course.P = 0.236). Patients with re-bleeding showed higher RBC counts, CSF total protein concentration  patients had re-bleeding, in median 2 days after SAH onset. Two patients had re-bleeding before clipping and one patient had insufficient aneurysm coiling. Two patients with re-bleeding showed still sufficiently occluded aneurysms in control digital subtraction angiography. One patient had neither any detectable aneurysm nor any intervention. Overall, the type of intervention was not associated with re-bleeding  had elevated percentages of granulocytes throughout the whole observation period, but lower percentages of phagocytosing cells compared to patients without ventriculitis . CSF total protein showed higher values from day 10 on (P = 0.9).Patients who developed ventriculitis (iculitis . Whereasiculitis . A cell ay 10 on . The typP = 0.039). The WBC subpopulations did not differ between patients with and without DCI (P = 0.757).Cerebrospinal fluid of patients who developed DCI (in median 8 days after SAH onset) had higher RBC counts and contained more WBC compared to those without DCI . WBC couhout DCI . There wCerebrospinal fluid analysis is used in patients with SAH and EVD to detect drain-associated ventriculitis , 4. Besivia the EVD. To overcome these methodical limitations, we applied a longitudinal study design, obtained the whole panel of routine CSF parameters, investigated the associations with SAH complications, and also considered the possible impact of CSF loss via the EVD.Previous studies reported the main CSF findings in patients with SAH and its complications. However, many of these studies are limited by the application of cross-sectional design, varying patient inclusion criteria, and revealed to some extent inconsistent findings. As different complications typically occur at different time points during the SAH disease course, e.g., rebleeding in the first days , DCI witHere, we showed that RBC, WBC, and CSF total protein followed a different temporal evolution. Whereas CSF RBC steadily decreased during the disease course, WBC count and CSF total protein first showed an increase reaching the highest values in the median after 6 and 13 days, respectively, before the eventual drop. These findings largely confirm the findings of previous studies , 20. Forin vivo cell degradation over time. This is relevant, because a significant proportion of patients with thunderclap headaches—at risk of having suffered an intrathecal hemorrhage—present themselves with delay, sometimes several days, and up to 20% of patients experience a “warning leak” before the eventual major SAH onset  SAH and traumatic lumbar puncture. In patients with SAH and available lumbar CSF, the RBC count at the time of the bleeding might be estimated from the RBC count determined at the time of lumbar puncture, using the decay rate and the respective time from symptom onset, i.e., adjusting for the AH onset . This apAH onset  and thusvia the EVD, as different CSF clearance might lead to a different longitudinal CSF profile. We did not observe any impact of CSF quantity loss on CSF parameters; however, we have to admit that this might be confounded by the small number of patients.Furthermore, we addressed the impact of CSF loss With regard to the different SAH complications, we observed distinct CSF patterns. In bacterial ventriculitis, previous studies reported elevated WBC , 29–39 aIn terms of clinical applicability, we fully agree with previous consensus papers stating that routine CSF measures have limited diagnostic value in differentiating between ventriculitis and sterile inflammation in patients with external CSF drains . It is uAseptic inflammation that is caused by the degradation of intracranial blood  plays a crucial role in the pathophysiology of SAH and the occurrence of cerebral vasospasm and DCI, respectively . WBC areThere are several limitations of the present work. First, this was a retrospective study of prospectively collected data with all inherent limitations. Second, the requirement of a minimum number of serial samples at the specified time points per patient reduced the number of subjects eligible for inclusion. Consequently, even clear differences were not statistically significant between groups at every time point. Furthermore, our cohort does not reflect the whole spectrum of patients with SAH, as we included only patients requiring EVD introducing a selection bias toward patients with a more severe disease course, and our results may not be transposed to patients without requiring an EVD. Also, CSF sampling was performed for routine diagnostic purposes , but not on dedicated days with regard to SAH onset. The practice of regular routine CSF sampling might also explain a higher rate of ventriculitis. Due to the small number of patients, multivariate analysis adjusting for between-group differences, such as age, disease severity, the occurrence of more than one complication at the same time, type of intervention (coiling vs. clipping), and differences in CSF parameters, was not feasible.In conclusion, knowledge about the time window for certain complications and the longitudinal evolution of CSF parameters helps interpret CSF results in patients with EVD. As reliable and validated cut-off values for the main CSF parameters, e.g., WBC count or cell index, are still lacking, it is up to clinicians to include several variables ranging from clinical symptoms to certain CSF patterns in order to judge whether e.g., a bacterial ventriculitis is present or not. Although this study is confirmatory to some extent, there are also new findings worth to be considered in clinical decision-making, like the predominance of granulocytes in patients with ventriculitis. A sound and interesting basis for future research provide the applicability of cellular decay rates.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The conduct of the study was approved by the local Ethics Committee of the Medical University of Innsbruck . Written informed consent was obtained according to Austrian Law and in accordance with the Declaration of Helsinki.AZ has participated in the acquisition of the data and drafting of the manuscript. GB, FDP, and FD has participated in reviewing the manuscript for intellectual content. VR has participated in the acquisition of the data and reviewing the manuscript for intellectual content. RH has participated in the conception and design of the study and reviewed the manuscript for intellectual content. JW has participated in statistical analysis of the data and reviewed the manuscript for intellectual content. HH has participated in the conception and design of the study, acquisition of the data, statistical analysis of the data, and the drafting of the manuscript. All authors contributed to the article and approved the submitted version.Author AZ has participated in meetings sponsored by, received speaking honoraria or travel funding from Biogen, Merck, Sanofi-Genzyme, and Teva. GB has participated in meetings sponsored by, received speaker honoraria or travel funding from Biogen, Celgene-BMS, Lilly, Merck, Novartis, Sanofi-Genzyme, and Teva and received honoraria for consulting Biogen, Celgene-BMS, Merck, Novartis, Roche, Sanofi-Genzyme, and Teva. FDP has participated in meetings sponsored by, received honoraria  or travel funding from Almirall, Bayer, Biogen, Celgene-BMS, Merck, Novartis, Sanofi-Genzyme, Sandoz, Roche, and Teva. Her institution has received research grants from Roche. FD has participated in meetings sponsored by or received honoraria for acting as an advisor/speaker for Alexion, Almirall, Biogen, Celgene, Genzyme-Sanofi, Merck, Novartis Pharma, Roche, and Teva. His institution has received research grants from Biogen and Genzyme Sanofi. He is section editor of the MSARD Journal (Multiple Sclerosis and Related Disorders). HH has participated in meetings sponsored by, received speaker honoraria or travel funding from Bayer, Biogen, Celgene, Merck, Novartis, Sanofi-Genzyme, Siemens, Teva, and received honoraria for acting as consultant for Biogen, Celgene, Novartis and Teva. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the circulation, all-trans-retinol bound to the RBP4 protein is transported and sequestered into target tissues for long-term storage. Two membrane receptors that facilitate all-trans retinol uptake from RBP4 have been proposed. While it is well established that the membrane receptor, STRA6, binds to circulatory RBP4 for ROL transport into the eye, the second vitamin A receptor, RBPR2, which is expressed in non-ocular tissues, is less characterized. Based on the structural homology between these two RBP4 receptors, published literature, and from our recent work in Rbpr2−/− deficient mice, we hypothesized that RBPR2 might also have high-binding affinity for RBP4 and this mechanism facilitates ROL transport. Herein, we aimed to elucidate the membrane topology and putative RBP4 binding residues on RBPR2 to understand its physiological function for retinoid homeostasis. Using in silico analysis and site-directed mutagenesis, we identified a potential RBP4 binding domain on RBPR2. We employed an in vitro cell-based system and confirmed that mutations of these residues on RBPR2 affected its binding to exogenous RBP4 and subsequently vitamin A uptake. Using Surface Plasmon Resonance assays, we analyzed both the binding affinities and kinetic parameters of wild-type RBPR2 and individual mutants affecting the RBPR2-RBP4 binding domain with its physiological ligand RBP4. These studies not only revealed a putative RBP4 binding domain on RBPR2 but also provided new structural, biochemical, and critical information on its proposed role in RBP4 binding for ROL transport and retinoid homeostasis.The distribution of dietary vitamin A/all- It is essential for normal embryonic development, reproduction, immunity, and is critical for ocular retinoid (trans retinoic acid (RA), an essential ligand for nuclear receptors such as the retinoic acid receptor (RAR), which regulate gene transcription. ROL is also the precursor for 11-cis retinaldehyde (RAL), the essential visual chromophore that isomerizes through exposure to light, and by extension allows photoreceptor cells to detect light  . CurrentRA6 gene . StudiesRA6 gene . To undeRA6 gene . From thRA6 gene .Rbpr2 knockout (Rbpr2−/−) mice showed that under vitamin A deficient diets, Rbpr2−/− mice failed to maintain retinal function and showed decreased systemic and ocular retinoid concentrations, which manifested as photoreceptor phenotypes  protein, also annotated as STRA6like . RBPR2 wenotypes . These R A diets . While t A diets . In thisAll chemicals, unless stated otherwise were purchased from Sigma-Aldrich .http://swissmodel.expasy.org/) was used to generate homology based models of mouse RBPR2, mouse STRA6, and zebrafish Stra6. The model with maximum coverage and lowest Z score for each was selected for further studies. The template selected (by online server SWISS-MODEL) was the cryoEM structure of STRA6, receptor for retinol  which showed nearly 44% sequence identity and Q mean close to −5 for STRA6 identity; and nearly 22% sequence identity and Q mean close to −7 for RBPR2. The structure for RBP4 was obtained from PDB database . The models generated were used for docking studies to analyze the protein-protein interactions employing the online data-driven docking program HADDOCK. HADDOCK requires a set of ambiguous interaction restraints (AIRs) at the binding interface that are divided into “active” and “passive” categories where active residues are those directly implicated in binding from experimental data and passive residues are their near neighbors. The docking process included a rigid body energy minimization step. The residues S294, Y295 and L296 for mouse RBPR2 were assigned as active residues for interaction as per the previous published papers to be essential for binding . The amplified Rbpr2 cDNA product was cloned in frame into the pCDNA3.1 V5/His TOPO vector . Appropriate construction of the wild-type Rbpr2 plasmid in the pCDNA 3.1 V5/His TOPO vector (pRbpr2-V5) was verified by sequence analysis of both strands  and by comparing the sequences to the reference mouse Rbpr2/Stra6like cDNA sequences deposited in Ensembl (www.ensembl.org). The WT-Rbpr2 plasmid was used as a template and mutagenic Rbpr2 primer pairs were used to engineer each of the RBP4 binding residue mutants by in vitro site-directed mutagenesis , as previously achieved  from liver of a 2-month-old wild-type C57/B6 mouse was reverse transcribed using the SuperScript One-Step RT-PCR for LongTemplates system . The full-length mouse achieved . Appropr2. NIH3T3 cells were used in this experiment as they are a well-established cell line to study the in vitro function of vitamin A membrane receptors for RBP4 binding and ROL transport (Mouse NIH3T3 cells obtained from American Type Tissue Culture (ATCC-1658) were maintained in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin sulfate and cultured at 37°C with 5% COransport . To generansport . ApproxiCell lines were grown on coverslips and fixed in a freshly prepared mixture of 4% formaldehyde in 1X PBS  for 30 min at room temperature and processed as previously described . ParentaE.Coli as previously described  (PerkinElmer Life Sciences) and non-radiolabeled ROL (Sigma) at a final concentration of 1 mm. Refolded holo-hRBP4 was dialyzed against 10 mM Tris/HCl buffer, pH 8.0, and loaded onto a DE53 anion exchange chromatography column . Holo-hRBP4 was eluted with linear gradient of NaCl (0–1M) in 10 mM Tris/HCl buffer, pH 8.0. Collected fractions were examined by SDS-PAGE and UV-visible spectroscopy to ensure a proper protein/retinoid ratio. Fractions containing at least 90% holo-hRBP4 were pooled together and concentrated in a Centricon centrifugal filter device   to 5 mg/mL. [3H]ROL was quantified in a scintillation counter . Holo-hRBP4 aliquots were stored at −80°C until used.Human RBP4 expression and purification from eviously . BrieflyTotal proteins from cells were extracted using the M-PER protein lysis buffer  containing protease inhibitors . Approximately 25 μg of total protein was electrophoresed on 4%–12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were probed with primary antibodies against EGFR , HSP90 , V5 , Rbp4 , or β-Actin  in antibody buffer . HRP-conjugated secondary antibodies  were used at 1:10,000 dilution. Protein expression was detected using a LI-COR Odyssey or ChemiDoc Bio-Rad system, and relative intensities of each band were quantified (densitometry) using ImageJ software version 1.49 and normalized to their respective loading controls. Each western blot analysis was repeated thrice.Co-immunoprecipitation (Co-IP) assays were performed with exogenous applied human RBP4 protein in NIH3T3/LRAT cells stably expressing V5-tagged WT-RBPR2 or individual V5-tagged RBPR2-RBP4 mutants. Using a well-established Co-IP protocol that determined extracellular STRA6-RBP4 interactions, we added reduced serum medium (8 mL of OptiMEM) containing 12 μM of purified and crosslinked T7 tagged-RBP4 to the cells and incubate this reaction for 60 min . After bE.coli expression system and extracted in Tris buffer with composition of 50 mM Tris-HCl, 1 M L-Arginine, 10% Glycerol, pH 8.0. The lysate was purified by nickel NTA column. The msRBP4 protein quality was monitored by western blot using anti His-tag antibody. The structural quality of the recombinant RBP4 protein was confirmed with Circular dichroism (CD) spectroscopy  (https://bestsel.elte.hu), (see Supplementary Information Recombinant mouse RBP4 with 6XHis Tag was expressed  Gemini) . The meaPurified RBP4 protein with >90% purity and 0.56 mg/mL concentration was immobilized on Biacore Sensor Chip CM5 (ITDD Biacore S200 Surface Plasmon Resonance instrument at University of Minnesota). The two-flow cell surface activated for using one as blank and other as test. Using Amine Coupling Kit  1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), after surface activation, the purified RBP4 with immobilization buffer 10 mM Sodium acetate, pH 5.0, was immobilized with target of 1,200 Response Unit (RU) for achieving Rmax of 30RU in kinetic study. The reaction stopped and washed with Ethanolamine. The system was re-primed with running buffer PBST . The kinetic assay performed on the two flow cells, the blank was used as reference cell and the active cell with RBP4 was used for the binding study. The mouse and zebrafish RBPR2, mouse RBPR2 mutants affecting the “SYL” binding domain, and mouse STRA6 peptides  were chemically synthesized by Biomatik Corporation, Kitchener, ON, Canada. The peptides were serial diluted in running buffer with range of 0.8–26.6 μM and following parameter was run with contact time: 120 s, flowrate 30 μL/min, Dissociation time 300 s, Regeneration with Glycine-HCl, pH 2.5, contact time 30 s flowrate 30 μL/min and temperature 25°C. The program was run and non-specific binding on the reference cell subtracted bulk refractive index from the active sensorgram and analyzed for the association, dissociation and stabilization of the reads. The plot fitted with 1:1 binding program in Biacore™ Insight Evaluation Software, and the Graph, binding affinity plot, was plotted in GraphPad prism version 9.3.Ms) RBPR2 protein sequences to human (Hs) STRA6 and Ms STRA6 revealed several short amino acid segments with >40% amino acid homology, suggesting analogous roles for these residues in the function or structural integrity of these two proteins  (Comparison of mouse (proteins  (Kawagucproteins . Interes96 (SYL) , which h96 (SYL) .http://swissmodel.expasy.org/), using the cryogenic electron microscopy (cryoEM) structure of zebrafish STRA6    , and humChain E) . The modChain E) . While tith RBP4  and thusTo study the importance of RBP4 binding residues on RBPR2, we used site-directed mutagenesis to individually alter these putative binding residues on mouse RBPR2. Using the wild-type (WT) RBPR2-pCDNA3.1-V5 tagged vector as a template, the polar amino acids (Ser294 and Tyr295) were mutated to hydrophobic amino acids (Ser294Ala and Tyr295Pro), while the hydrophobic amino acid (Leu296) was mutated to a polar amino acid (Leu296Ser) . WT-RBPRp < 0.005)  to uptake [3H]ROL-RBP4, indicating that the amino acids Ser294, Tyr295, and Leu296 likely encompass the RBP4 binding residues on RBPR2 that would be crucial for ROL transport  . To conf; < 0.005Counting . This an4 uptake . Howeversed LRAT . In contransport . Based oransport , but witransport  and [3H]bilities , indicatKd , the site-specific binding fitting model described below was used.max represents the maximum number of binding sites (Response Unit/RU), X represents the analyte concentration, and Y represents the binding affinity (Response Unit). Interestingly, the binding affinity of mouse RBPR2 to RBP4 approximates to the affinity of mouse STRA6 peptides to RBP4. The Kd of WT-msRBPR2 peptide with msRBP4 was 25.42 ± 6.01 μM, and Bmax was 183.17 ± 25.66 μM (mean ± S.E). The Kd of msSTRA6 peptide with msRBP4 was 26.73 ± 5.67 μM, and Bmax was 178.45 ± 22.26 (mean ± S.E) (Koff) of msRBPR2 and msSTRA6 to msRBP4 was not statistically significant (p < 0.99) in an unpaired t-test, suggesting similar Kd values and binding affinities of these two proteins for its extracellular ligand  had a lower Kd value of 21.30 μM, compared to Kd value of 25.42 μM for WT-RBPR2, dissociation rate (Koff) for mutant msRBPR2 was significantly lower, suggesting a tighter bond formation between the RBP4 protein and mutant peptides (Surface Plasmon Resonance (SPR) is a common technique used to study protein-ligand interactions. SPR can measure the binding affinities and association/dissociation kinetics of protein to ligand complexes in real-time. The interaction levels measured in Response Units (RU), and real-time plot sensorgram displays the dynamics of the analysis. The purified mouse Retinol Binding Protein, RBP4 , was immn ± S.E) , 6. The r ligand . SPR anapeptides .trans retinol in the mammalian system, where retinoids are not a product of de-novo synthesis and thus require an active transport mechanism/membrane receptor to reach their target organs  selection and distribution in a tissue-specific manner provides an advantage in the proper transport, storage, and utilization of all-t organs  , and on the binding of RBP4 to STRA6, retinol is transported into the cell without internalization of RBP4  receptor, RBPR2, is necessary for the systemic facilitation of dietary retinoid uptake, storage, and transport to targeted organs, specifically the liver and eye . We base of RBP4 . To undein vitro and CRISPR mutant zebrafish model (s), and are critical for RBP4-ROL binding and retinol uptake and transport, in supporting visual function  of how the membrane expressed vitamin A receptor STRA6 facilitates retinol transport from its carrier protein, RBP4, into cells, where they used an elegant large-scale mutagenesis approach and identified three essential residues on STRA6 that might be essential for RBP4 binding and subsequent ROL transport into target tissues . These rfunction .Kd) of RBP4 for RBPR2 peptides (encompassing the SYL domain) was 25.43 μM for mouse RBPR2 and 33.25 μM for zebrafish RBPR2. The calculated binding affinity (Kd) of RBP4 for mouse STRA6 peptide encompassing the previously reported Tyr336, Gly340, and Gly342 residues was 26.73 μM and this was comparable to mouse RBPR2. The binding affinity (Kd) values for RBPR2 binding for its ligand RBP4 were comparable to previously published values for STRA6 for its physiological ligand RBP4 (Kd = 22.4 μM). Interestingly, we observed a reduced binding affinity (Kd) and stronger dissociation rate (Koff) for the RBPR2 mutants to its ligand RBP4, compared to the WT. In our docking analysis, we observed Ser294 and Tyr295 on RBPR2 to interact with Arg167 on RBP4, while Leu296 RBPR2 was not directly involved in the interaction. Leu296 might play an essential role in structure stabilization for the interaction; if we compare its observed Kd values to the other RBPR2 mutants and WT-RBPR2, it becomes evident that structural stabilization is equally important. The Kd values for RBPR2 peptides interaction with RBP4, S294A = 89.33 μM, Y295P = 34.91 μM, and L296S = 21.30 μM, indicating that the S294A and Y295P mutant peptides required a higher concentration of RBP4 for binding saturation, while the L296S mutant retained a comparable Kd value when compared to WT-RBPR2 (Rbpr2-KO mice (Based on the work of the Sun Lab, we propose that a similar SYL binding motif might also be found in RBPR2, given its similar capability in binding RBP4-ROL  Radhakr. ThroughWT-RBPR2 –7. The SWT-RBPR2 . Based o-KO mice , to dete-KO mice . Therefo


Facial discoid dermatosis (FDD) is a recently described condition comprising discrete facial papulo‐squamous lesions. We report three cases that clinically and histologically resemble FDD and demonstrate its resistance to treatment. Awareness of this new clinical entity will allow early diagnosis and the ability to make patients aware that there is unlikely to be a successful treatment. However, our study suggests that although FDD can persist for many years, it appears to remain stable. Facial discoid dermatosis (FDD) is a recently described condition comprising discrete facial papulo‐squamous lesions. We report three cases that clinically and histologically resemble FDD, and demonstrate its resistance to treatment. The entity facial discoid dermatosis (FDD) has been suggested for this group of disorders.3Case 1A 19‐year‐old male with Fitzpatrick Skin Type 2 presented with an 18‐month history of an asymptomatic facial rash. Antecedent treatments included coal tar, hydrocortisone and allantoin cream and Clotrimazole 1% cream. On examination he had discrete orange‐pink slightly scaly discoid plaques on the cheeks and forehead Figure . He was Case 2A 40‐year‐old female with Fitzpatrick Skin Type 4 presented with a 7‐year history of multiple asymptomatic facial erythematous plaques, which had been treated with miconazole nitrate with hydrocortisone cream with no benefit. On examination, she had multiple small discoid erythematous plaques on her face Figure . DiffereCase 3A 44‐year‐old female with Fitzpatrick Skin Type 3 presented with asymptomatic facial plaques which had gradually increased in number and had been unresponsive to mildly potent topical corticosteroids. She had a history of depression treated with fluoxetine. Examination revealed 4 mildly scaly erythematous discoid plaques on the face Figure . Skin sc4FDD was first reported in 2010. It is an under recognised condition characterised by persistent orange‐pink annular plaques, with variable surface scale, that localise to the face and neck.Multiple therapies have been trialled without success in the reports published to date.Histologically, FDD demonstrates hyperkeratosis, parakeratosis, acanthosis and follicular plugging. A lymphocytic infiltrate in the superficial dermis is also commonly seen.None to declare.


Organ transplantation remains the treatment of last resort in case of failure of a vital organ  or non-vital organ  for which supplementary treatments exist. It remains the best alternative both in terms of quality-of-life and life expectancy for patients and of public health expenditure. Unfortunately, organ shortage remains a widespread issue, as on average only about 25% of patients waiting for an organ are transplanted each year. This situation has led to the consideration of recent donor populations (deceased by brain death with extended criteria or deceased after circulatory arrest). These organs are sensitive to the conditions of conservation during the ischemia phase, which have an impact on the graft’s short- and long-term fate. This evolution necessitates a more adapted management of organ donation and the optimization of preservation conditions. In this general review, the different aspects of preservation will be considered. Initially done by hypothermia with the help of specific solutions, preservation is evolving with oxygenated perfusion, in hypothermia or normothermia, aiming at maintaining tissue metabolism. Preservation time is also becoming a unique evaluation window to predict organ quality, allowing repair and/or optimization of recipient choice.  Transplantation remains the best therapeutic alternative in the event of terminal organ failure, both in terms of quality-of-life and life expectancy, as well as in terms of public health expenditure. The number of candidates for transplantation is constantly increasing and there is now a very important organ shortage  living donors ; (ii) brain-dead donors (DBDs) ; and (iii) deceased donors after circulatory arrest (DCDs) . In recent years, the contraindications to donation have been revised in order to better meet the demand for organs. Indeed, in order to increase the number of organs available for transplantation, there has been a renewed interest in donation from DCDs , which iThe main purpose of organ preservation is to maintain the viability of individual organs ex vivo for a period of time that is commensurate with the type of organ being considered. It must be sufficient for the eventual transport of the organs from the procurement center to the transplant center(s) in order to be allocated according to the degree of priority and/or best tissue compatibility. The management of these organs is very different depending on whether it is a vital organ such as the heart, liver or lung, or the kidney and pancreas for which a delay in the recovery of the graft function is “tolerable” thanks to the availability of substitution therapeutics . Preservation also aims at reducing IRI. Importantly, tolerance to IRI varies according to the type of organ and the type of donor.Organ transplant teams determines three categories of donors: living donors, donation after brain death donors (DBDs) and donation after circulatory death donors (DCDs).The first category is the living donor (kidney and liver). These transplants are not without morbidity and mortality, but their evolution is very favorable and superior in terms of short- and long-term outcomes for the graft and the patient . The devBecause the number of standard criteria donors (SCDs) is not sufficient to meet the demand, it was necessary to resort to alternative sources of organs, such as expanded criteria donor (ECDs) ,7. Thus,DCDs are categorized according to the Maastricht classification . Their hThe practice of solid organ transplantation from DCDs poses certain ethical, and pathophysiological problems in relation to cardiac arrest . It is aThe use of lower-quality organs, including those from DCDs, has become an established routine but increases the risk of graft dysfunction. This risk is further compounded by IRI, which is unavoidable during the transplantation process ,15,16. GThus, one of the current challenges for the transplant community is to improve the quality of preservation in order to reduce complications related to IR. The use of cold storage conditions for flushing and static cold storage (CS) has been the standard storage technique for many years. However, new donor demographics, such as expanded criteria donors (ECDs) and DCDs, have led to the development of more diverse preservation techniques . We refeStatic storage in hypothermia without oxygen supply has been the chosen solution for decades. The purpose of hypothermia (between 4 and 6 °C) was to limit metabolism and oxygen requirements . Many soHowever, clinical evidence is still lacking ,32,33. IToday, given the evolution of donors, hypothermic machine perfusion (HMP) is recommended for DCDs . Our groHypothermia is currently being reevaluated as it is suspected of having deleterious effects. For instance, ex vivo normothermic perfusion (EVNP) maintains cellular processes at physiological temperature. This technology has been widely tested in the UK, Canada, The Netherlands and the United States, and several studies have demonstrated the advantages of EVNP over hypothermia ,40,41,42An international comparative study has shown that DCD donor livers maintained under oxygenated hypothermia perfusion (HOPE) or NRP have post-transplant survival rates comparable to those of standard liver . The useFor the lung, several clinical trials (NCT01190059 and NCT01963780) have shown that EVNP is a real alternative for high-risk donor lungs in transplantation, with similar results to those obtained with conventionally selected lungs ,50. In aAnother strategy is investigated: controlled oxygen rewarming (COR). This technique was performed in humans, using a kidney graft from an extended criteria donor. The kidney was preserved during ex vivo perfusion by a machine from hypothermia to normothermic conditions (35 °C with 100% oxygen), and the results seem promising . Several reviews complement the data on the value of temperature and perfusion machines, especially in marginal donors ,58,59,60Several works emphasize the provision of oxygen to improve hypothermic perfusion ,62. SeveAnother alternative is the use of oxygen carriers during organ preservation. A recent review on the use of oxygen carriers has been published . DiffereThese preclinical studies led the molecule to be accepted for clinical trials, with the results of the safety trial (OXYOP), published in June 2020, showing that the use of marine worm hemoglobin M101 is safe and has promising efficacy data . The authors reported less delay in recovery of renal graft function, fewer dialysis sessions per patient in the first month, a decrease in creatinine levels over the first 7 days, and a better time to reach creatinine <250 μmol/L in the M101 group compared with the contralateral group without M101 . These rInterestingly, a group in Guangzhou evaluated a procedure, called ischemia-free organ transplantation (IFOT), to optimize organ transplantation in humans. They demonstrated that the liver and kidney can be collected, preserved and implanted under continuous normothermic oxygen machine perfusion to limit ischemia. Under these conditions, the liver did not suffer in the post-reperfusion period, with minimal injury, little inflammation and no complications , and norA recent review offers an in-depth exploration of the impact of oxygen in organ preservation . AdditioEx vivo preservation is also a window to evaluate organs and implement therapeutic strategies exerting antioxidant, anti-inflammatory and anti-apoptotic activities, siRNA delivery or even cell therapy. The combination of different perfusion methods is also explored, which implies a tailor-made organ preservation in connection with the evaluation of the organs but also the donor in order to best match the recipient ,69,86. IThe preservation period is a very interesting therapeutic window allowing the use of targeted pharmacological agents. One of the difficulties is to have agents that can act in static or perfusion conditions and in particular in hypothermia. The development of normothermia and the evolution of perfusion media should allow the use of molecules that can act in a more physiological manner. Several recent reviews have presented the different pharmacological agents that can be used ,59,87.Among these, two strategies stand out the most:Mitonchondrial protection targeting the factor eIF5A (eukaryotic initiation factor 5A) and its enzymatic activation step (hypusination) using a spermidine-like compound (GC7). This molecule induces a drastic fall of oxygen consumption associated with a reduction of the mitochondrial membrane potential and a decreased expression of mitochondrial complexes . We testMineralocorticoid receptor modulation, which can lead to a better management of kidney function, improved protection against inflammation and maintenance of the integrity of the vascular bed, a key target of ischemia reperfusion injury ,90. In aNumerous other candidates have been studied over the past few decades but with very few clinical outcomes. Moreover, there is a need to target the primary pathophysiological mechanisms involved during preservation without ignoring the donor and recipient.Given the diversity of organ quality in DCD, and the fact that donor inclusion criteria will need to be expanded to address the growing organ shortage, new quality classification tools are needed. There is an increasing need for pre-transplant prognostic models of recipient outcomes based on accurate surrogates of organ quality, not only for health care organizations responsible for transplant allocation, but also for physicians and patients. This would increase the organ pool of deceased donors, improve donor-recipient matching, and facilitate decision making for living donors , top. One such tool is the Kidney Donor Risk Index (KDRI), which takes into account demographic and clinical characteristics related to the donor and transplantation ,93. AnotOther means such as the metabolic, transcriptomic or metabolic approach are interesting perspectives to optimize the organ donation process. A better knowledge of the physiopathology specific to each type of donation, as well as the implementation and application of specific protocols, would eventually allow a personalized management of the grafts from the selection of the donor to their conservation and transplantation.There is a need for teams involved in this area to consider networking in order to have coordinated protocols. There is also a need to establish international registries and to target key questions in unified international efforts. The metabolic aspect is emerging for both the tissues that make up the organs and the inflammatory cells involved. A recent study indicated that the liver and kidney have a markedly different metabolic profile in deceased organ donors, with a preference for alanine production in the liver and lactate in the kidney . ExpressThe final point to emphasize, given the complexity of organ assessment and management, with the multiplicity of investigative tools and preservation techniques, would be the creation of Organ Hubs , bottom,


Covalent organic frameworks (COFs) with donor–acceptor (D–A) units are credible photocatalysts for their per-designed structure, inherent porosity, large surface area, splendid stability and so forth. Developing COFs with an excellent photocatalytic efficiency for hydrogen evolution is of a great significance in alleviating the energy crisis. Herein, a D–A type imine-linked crystalline Zn-Por-TT COF was fabricated successfully via the co-polymerization of electron-deficient Zinc (II) 5,10,15,20-tetrakis(para-aminophenyl) porphyrin (Zn-TAPP), and electron-rich thienothiophene-2,5-dicarbaldehyde (TT). Profiting from the D–A complex structure, the obtained Zn-Por-TT COF showcases an excellent photocatalytic activity with a hydrogen evolution rate of 8200 μmol/g/h, while the Zn-TAPP monomer presents practically no capacity for the generation of hydrogen under identical conditions. In addition, the counterparts Por-TT COF and COF-366-Zn were employed to illustrate the enhancement of the photocatalytic performance by metal catalytic sites and D–A structures. In addition, the counterparts Por-TT COF and COF-366-Zn were employed to illustrate the enhancement of metal catalytic sites and D–A structures for the photocatalytic performance. A promising and sustainable path for hydrogen generation is photocatalytic water splitting under visible light, which is propelled by ubiquitous solar energy thiophene-2,5-dicarbaldehyde (TT) as the donor to fabricate a metalloporphyrin-based D–A COF by the solvothermal method. The obtained Zn-Por-TT COF presented an excellent crystallinity, a large surface area, a good thermal/chemical stability, and a narrow band gap. As expected, the Zn-Por-TT COF demonstrated a good activity of photocatalytic hydrogen production and the hydrogen evolution rate of the Zn-Por-TT COF could attain to 8200 μmol/g/h in the presence of Pt co-catalyst and ascorbic acid sacrificial agent, which is above the average level among the reported COF photocatalysts. More importantly, a metal-free Por-TT COF and COF-366-Zn were synthesized and employed as counterparts to confirm that Zno-dichlorobenzene/benzyl alcohol/6M AcOH at 120 °C for 3 days and what was obtained was a dark purple powder with a yield of 75% (2 (~3352 and 3316 cm−1) and -CHO (~1660 cm−1) were dramatically decreased in Zn-Por-TT COF, and a new peak was able to be observed at ~1612 cm−1, which corresponded to the stretching vibration of C=N linkages. In addition, the single peak which appeared at ~150.4 ppm in the 13C solid-state NMR spectrum  combined with the theoretical simulation a. There 2 sorption experiment was proceeded at 77 K and the samples were degassed at 150 °C for 8 h under a vacuum (10−5 bar) before their analysis  surface area of the Zn-Por-TT COF was 632 m2/g, calculated from the absorption curve. The pore size distribution was analyzed by the nonlocal density functional theory (NLDFT) method and afforded an average pore size of 2.6 nm, which is approximated with the theoretical value (2.5 nm). Moreover, the morphology feature of the Zn-Por-TT COF was assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As revealed by the SEM images  measurement under Ncollapse a. The chcollapse b. Furthecollapse a. The Mocollapse b. Subseq2 evolution under a visible light illumination. To demonstrate this hypothesis, the photocatalytic hydrogen production experiments were performed to evaluate the photocatalytic performance of a Zn-Por-TT COF, and the general H2 evaluation procedure is described as below. Primarily, 5 mg of the Zn-Por-TT COF powder was dispersed in 50 mL of ultrapure water, a few amounts of ascorbic acid (AA), and K2PtCl6 were added into the system which functions as an electron sacrificial agent and co-catalyst to produce hydrogen, respectively. The visible light  was employed as the optical source and the photocatalytic system should be kept at 4 °C during the process of a H2 evolution. In order to identify the optimal conditions for a H2 evolution catalyzed by Zn-Por-TT COF, the effects of the pH and co-catalyst ratio were investigated unambiguously. The optimal pH value was revealed to be three  coordination center on the photocatalytic performance, COF-366-Zn without the TT donor units and a Por-TT COF free of Zn (II) coordination center were constructed and employed to function as contrasted photocatalysts for the HPS, etc. . Both COPS, etc. a. Howeve reduced b and the reduced c, which 2 generation. As our hypothesis, the Zn-Por-TT COF exhibits a satisfactory H2 evolution rate which can be stabilized at ~8200 μmol/g/h eventually. Moreover, the formed D–A structures and abundant metal catalytic active sites synergistically promote the H2 production performance of the Zn-Por-TT COF, as demonstrated by the controlled experiments. This work provides an available path for the construction of highly efficient photosensitizers for solar energy harvesting and conversion. We envision that COF photocatalysts with desirable properties will be constructed via incorporating stronger D–A complex structures, and related works are in progress in our laboratory.In summary, a Zn(II)-porphyrin built-in COF was successfully fabricated via a reasonable design and the obtained Zn-Por-TT COF exhibited a high crystallinity, a good stability, and a broad absorption capacity of visible light so that can be employed as an effective photocatalyst for a H


Therefore, we studied FR-β expression in vitro in macrophage subtypes and determined their response to triamcinolone acetonide (TA), a clinically often-used corticosteroid.Folate receptor beta (FR-β) has been used as a clinical marker and target in multiple inflammatory diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). However, the conditions under which FR-βHuman monocyte–derived macrophages were differentiated to the known M0, M1, or M2 macrophage phenotypes. The phenotype and FR-β expression and plasticity of the macrophage subtypes were determined using flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA).+ phenotype.FR-β expression was low in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated (M1-like) macrophages and high in macrophage colony-stimulating factor (M-CSF)-generated (M0 and M2-like) macrophages. FR-β expression remained high once the M0 or M2 macrophages were stimulated with pro-inflammatory stimuli (interferon-γ plus lipopolysaccharide) to induce M1-like macrophages. On the contrary, anti-inflammatory TA treatment skewed GM-CSF macrophage differentiation toward an M2 and FR-β+ macrophages (exposed to M-CSF) are observed in arthritis patients and correlate with disease severity.As corticosteroids skewed monocytes toward an FR-β-expressing, anti-inflammatory phenotype, even in an M1 priming GM-CSF environment, FR-β has potential as a biomarker to monitor success of treatment with corticosteroids. Without corticosteroid treatment, M-CSF alone induces high FR-β expression which remains high under pro-inflammatory conditions. This explains why pro-inflammatory FR-β Within the field of (osteo)arthritis, macrophages play an important role in inflammatory processes in the joints.5 Their role is strongly dependent on their phenotype. Distinct macrophage phenotypes have been identified in vitro based on their surface markers, each with their own function in inflammation, wound healing, and immune regulation.7 Classically activated pro-inflammatory macrophages are known as M1. They can produce many pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12 and are hallmarked by the expression of surface marker CD80. Alternatively activated anti-inflammatory macrophages are known as M2 and are generally identified by expression of CD163, arginase 1, and/or CD206, depending on their subtype. Macrophages can be generated in vitro from monocytes by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), which are often used to prime toward M1 or M2 phenotypes, respectively. Although the M-CSF-generated macrophage primarily differentiates into an M2-like phenotypic and functional properties, they remain very plastic and can be skewed to an M1-like phenotype by stimulation with interferon (IFN)-γ and lipopolysaccharide (LPS). Therefore, the M-CSF-generated macrophage is often referred to as M0 phenotype. Further subtypes for M2 macrophages have been described that are induced in vitro by stimulation of M0 macrophages, such as M2a (tissue repair) macrophages that are induced by stimulation with IL-4, IL-10, and IL-13. M2b (regulatory) macrophages are induced by immune complexes (ICs) in combination with LPS, which results in the production of IL-10 and IL-6. Finally, the M2c (immunosuppressive) subtype is induced by corticosteroids, IL-10, and transforming growth factor (TGF)-β.8 However, it is becoming clear that in vivo macrophages are not dichotomously classifiable in terms of M1 and M2, but are more plastic, and can even simultaneously express M1 and M2 markers.9 In addition, therapeutic agents can affect macrophages directly and alter their phenotype. Corticosteroids, which are commonly used in inflammatory joint diseases, can drive macrophages toward an M2 phenotype based on upregulated CD163 surface expression, production of anti-inflammatory cytokine TGF-β, and increased IL-10 gene expression.1012Macrophages are heterogeneous immune cells that play an important role in tissue homeostasis and innate immunity. Most macrophages develop from peripheral blood monocytes that have migrated to local tissues, although tissue-resident macrophages have also been identified.1317 FR-α/β-specific tracers have been developed for gamma scintigraphy and SPECT (EC20 FolateScan),1821 PET,23 and fluorescence imaging, and are being assessed for their possible diagnostic use in RA and OA.25 FR-based agents are already widely used to monitor macrophage activation in animal models for RA and OA.27 The specific surface expression of FR-β on macrophages also provides a potential target in these diseases for the use of FR-linked drugs or FR-β-directed immunotherapy.2831Macrophages have been found to specifically express folate receptor beta (FR-β), a member of a family of receptors with high affinity for physiological folates. Because of this, folate receptor–based imaging and therapeutic approaches  have been evaluated for their use in macrophage-mediated diseases.The clinical implementation of FR-targeted imaging and therapies is, however, hampered by the fact that it remains unclear which macrophage phenotypes express FR-β. Furthermore, accurate monitoring of disease activity and therapeutic responses in patients requires knowledge about the possible interactions with clinically commonly applied drugs, such as corticosteroids. Interaction between corticosteroids and FR-β expression may have significant implications for the use of FR-based agents in (osteo)arthritis or other inflammatory and autoimmune diseases, in which corticosteroid use is part of standard of care. Therefore, this study aimed to identify FR-β expression in currently recognized macrophage subtypes and to assess the effects thereon of triamcinolone acetonide (TA), a clinically commonly used corticosteroid.n = 15) was supplied by the Dutch blood bank  in accordance with applying laws and ethical guidelines and under legal agreement (number 150731). Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Ficoll-Paque  density centrifugation. Monocytes were positively selected by magnetic-activated cell sorting (MACS) with anti-CD14-labeled microbeads  according to manufacturers’ instructions using the autoMACS (Miltenyi Biotec). Purity of the monocytes was evaluated on flow cytometry by assessing the percentage of CD14+-stained cells, which was on average 92.4% ± 4.5%.Blood from healthy human donors . For the M2 macrophage subtypes, M-CSF-generated macrophages were stimulated with 10 ng/ml IL-4 and 10 ng/ml IL-10 (M2a), 50 μg/ml ICs and 10 ng/ml LPS (M2b), or 10 ng/ml TGF-β (M2c), further referred to as and M2a(IL4+IL10), M2b(LPS+ICs), and M2c(TGF-β), respectively.3234 IFN-γ, IL-4, IL-10, and TGF-β were all from PeproTech , LPS from Sigma-Aldrich . M-CSF- and GM-CSF-generated macrophages where no stimulation was added during the additional 24 hours of culture are referred to as M-CSF control and GM-CSF control. ICs were produced according to the method of Ottonello et al. by incubating human albumin (15 μg/ml) with rabbit anti-human albumin (150 μg/ml) at 37 °C for 1 hour and then overnight at 4 °C. The precipitated ICs were washed twice in phosphate-buffered saline by centrifugation  and then stored at 4 °C.Monocytes were cultured in RPMI/glutamax  with additional pen/strep , and 10% v/v fetal calf serum  at a cell density of 500,000/well in 24-well plates . Monocyte-derived macrophages were generated by culturing monocytes for 7 days in the presence of 800 U/ml human recombinant GM-CSF (M1 priming cytokine) or 25 ng/ml human recombinant M-CSF .The effect of an inflammatory stimulus on M-CSF-generated macrophages was studied by stimulating with 10 ng/ml IFN-γ and 10 ng/ml LPS or 10 ng/ml LPS only for 24 hours. To study the effect of the anti-inflammatory drug TA, 100 nM TA  was added to the medium on day 0, day 3, or day 7 in the cell culture with M-CSF or GM-CSF, no additional stimulators were added at day 7. On day 8, supernatants and cells were harvested for further analysis. The culture medium was refreshed after 3 to 4 days. Macrophage subtypes were analyzed with the aid of surface markers via flow cytometry, gene expression analysis, and cytokine measurement on protein level. followed by incubation with a fluorescein (FITC)-labeled streptavidin , CD80-PE , CD163-PerCP-Cy5.5 , CD14 APC-AF750 , CD206-PC-7 , and CD16-APC . Alternatively, CD68-FITC  or CD86-PE  was included. Dead cells were excluded using viability marker SYTOX . More details on the antibodies and gating strategy that were used are shown in Suppl. Table S1 and Suppl. Figure S1. Flow cytometry was performed on a FACSCanto II cytometer (Becton Dickinson) according to the manufacturer’s protocols. Fluorescence minus one controls were used to confirm there were no false-positive populations. Values were expressed as mean fluorescence intensity (MFI); at least 6 donors were used for all flow cytometry experiments.Membrane markers to characterize the macrophage subtypes and FR-β were analyzed using immunofluorescent flow cytometry by incubating the cells with specific fluorescent antibodies diluted in phosphate-buffered saline + 1% w/v bovine serum albumin + 0.1% v/v sodium azide + 1% v/v normal rabbit serum . Cells were detached from the culture plate by scraping, no trypsin was used. Macrophages were first incubated at 4 °C for 30 minutes with biotinylated M909, a biotin-labeled human monoclonal antibody that specifically binds human FR-β,TBP and YWHAZ using the delta Ct method. At least 6 donors were used in all qPCR measurements. Details on the primers that were used for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) are shown in Suppl. Table S2.Messenger RNA (mRNA) was isolated using RNeasy Mini Kit . After on-column DNase-I treatment (Qiagen RNAse-free DNase kit), RNA was quantified using Nanodrop ND-1000  and reverse transcribed into cDNA using the iScript cDNA Synthesis Kit . Gene expression was determined using the CFX384 Real-Time PCR Detection System (Biorad). The quantitative polymerase chain reaction (qPCR) reactions were performed in duplicate in 384-well plates in a final volume of 10 μl, using IQ SYBR Green Supermix (Biorad). The mRNA levels were normalized to those of the reference genes Cytokine production was measured in supernatants after 24 hours stimulation. TNF-α, IL-6, IL-10, and IL-12 (p40/p70) were measured using novex cytoset enzyme-linked immunosorbent assay (ELISA) kits (ThermoFisher) according to the manufacturer’s recommendations. Analysis was performed using an iMARK microplate absorbance reader and Microplate Manager 6 software (Bio-Rad). At least 5 donors were used in all cytokine measurements.P values <0.05 were considered statistically significant. Confidence intervals (CI) are given at the 95% level. Results are shown as mean ± standard deviation.Data were analyzed using GraphPad Prism version 7.04  and SPSS Statistics for Windows, version 25.0 . A generalized linear mixed model was used to compare differences between groups; depending on the distribution of the data, either a normal distribution with identity link or a gamma distribution with log link was used. The Bonferroni method was used to correct for multiple comparisons and  and P < 0.05 for all groups). Although the M2b(LPS+ICs) macrophages showed lower FR-β surface expression compared with the other M-CSF groups , it was still significantly higher than in GM-CSF-generated macrophages (P < 0.05). Phenotypes of the macrophage subtypes were confirmed with the aid of surface marker flow cytometry analysis. M-CSF-generated macrophages displayed higher CD163 expression compared with GM-CSF-generated macrophages  and here too CD163 expression by the M-CSF-generated macrophages was lowest upon stimulation with LPS and ICs to induce an M2b subtype (P < 0.05 compared with M-CSF control). These results indicate that, in M1 and M2 in vitro macrophage subtypes, the FR-β receptor expression pattern is similar to the expression of traditional M2-like marker CD163. GM-CSF-generated macrophages displayed higher surface expression of CD80 after stimulation with IFN-γ and LPS compared with GM-CSF control  and compared with all M-CSF-generated subtypes . When looking at the CD14 and CD16 surface expression, only CD16 showed an increase in expression in the M2a(IL4+IL10) group (P = 0.0002) and a decrease in expression in the M2b(LPS+ICs) group .FR-β surface expression was markedly higher in all M-CSF (M2-like) generated macrophage subtypes compared with GM-CSF (M1-like) generated macrophages (FOLR2) relative gene expression was also significantly higher in M-CSF compared with GM-CSF-generated macrophages , with no difference between the M-CSF subtypes. To confirm the M2, anti-inflammatory, phenotype in M-CSF-generated macrophages, IL10 and TGF-β-Induced Protein (TGFBI) gene expression were quantified. The IL10 gene expression was significantly increased in all M-CSF-generated macrophages compared with GM-CSF control  and particularly high in M2b(LPS+ICs) macrophages. The same was true for the TGFBI expression, with the exception of the M2b(LPS+ICs) macrophages, where the expression level was comparable with that of the GM-CSF control and significantly lower than that of the other M-CSF macrophage subtypes .In accordance with the FR-β surface marker, the FR-β . As expected, M2b(LPS+ICs) macrophages produced high levels of TNF-α, IL-10, and IL-6, compared with the M-CSF control . TNF-α concentration was also increased in M1(IFN+LPS), whereas it was decreased for M2a(IL4+IL10) macrophages compared with their controls . In addition, the IL-10 concentration was increased in M2a(IL4+IL10) macrophages . The IL-6 concentration was also higher in M1(IFN+LPS) macrophages compared with GM-CSF control .To unequivocally confirm the phenotype of the M2b(LPS+ICs) macrophages , we measured protein (ELISA) levels of TNF-α, IL-10, and IL-6 in the culture supernatant as a confirmation of M2b functionality , but significantly after exposure to IFN+LPS . In addition, CD80 expression was increased in M-CSF-generated macrophages after LPS and IFN+LPS stimulation . Altogether, these findings suggested that in M-CSF-generated macrophages both CD163 and CD80 expression changed to a more M1-like phenotype, as would be expected upon stimulation with LPS and IFN-γ, whereas FR-β surface expression remained high.It is known that FOLR2 expression remained at the same high level as the M-CSF control group . TGFBI expression decreased after LPS and IFN-γ stimulation (P < 0.01 for both groups) but was still higher compared with GM-CSF-generated macrophages . IL10 gene expression and IL-10 cytokine concentration were elevated in the M-CSF-generated LPS and IFN+LPS groups, compared with the M-CSF control group , but also compared higher relative to the M1(IFN+LPS) macrophages . The levels of IL10 gene expression and IL-10 cytokine concentration were similar to what was observed previously for the M2b(LPS+ICs) subtype ( and ), pointing toward an LPS-induced IL-10 release in all these M-CSF-generated macrophages. TNF-a and IL-6 concentrations were increased in the media of the M-CSF-generated LPS and IFN+LPS groups compared with the M-CSF control and reached comparable levels to those detected for GM-CSF-generated macrophages cultured under the same conditions ( and ). Only the IL-12 level in the M-CSF-cultured LPS and LPS+IFN groups was still clearly lower than M1(IFN+LPS) macrophages . Overall, of all measured markers only FR-β surface expression and FOLR2 gene expression did not change in M-CSF-generated macrophages after addition of LPS and IFN-γ. In fact, these M-CSF-generated macrophages, when subjected to treatment with LPS and IFN-γ, obtained an M1-like phenotype, with the exception of both IL-10 and FR-β at gene and protein level.A similar tendency was observed at the gene expression level in the M-CSF-cultured LPS and LPS+IFN groups, where To study the effects of an anti-inflammatory environment on plasticity and FR-β expression in M-CSF- and GM-CSF-generated macrophages, the corticosteroid TA was used. TA was supplemented at day 7, for 24 hours, similar to the stimulations in previous culture experiments with IFN and LPS, but also at earlier time points (day 0 and day 3) during the macrophage generation, to examine the effects of early stimulation. Surface marker and gene expression were always analyzed on day 8.; day 0: P = 0.07, CI = 0.2-1.9 and day 3: P = 0.043, CI = 0.02-2.1). In accordance with our previous observations, M-CSF-generated macrophages had high FR-β surface expression, and this did not increase further upon supplementation of TA. Further analysis confirmed that TA addition on day 0 or day 3 increased CD163 surface expression in GM-CSF-generated macrophages . For M-CSF-generated macrophages, an increase in CD163 surface expression was observed when TA was added at day 0 . The fold changes in the surface marker profiles relative to the GM-CSF control without TA  and relative to M-CSF control without TA  indicate that for most surface markers the change in expression was largest when TA was added on day 0 and somewhat limited when TA was added on day 3, whereas it remained unaltered when TA was added on day 7. This suggests that there is more plasticity in response to the anti-inflammatory stimulus in early maturation stages at day 0 and day 3 of culture.TA supplementation at day 7 to GM-CSF-generated macrophages did not affect FR-β expression. However, TA supplementation on day 0 or day 3 resulted in almost a 2-fold increase in FR-β expression on day 8 in GM-CSF-generated macrophages . In contrast to what was observed for FR-β surface marker in M-CSF-generated macrophages, TA addition at day 0 resulted in an increase in FOLR2 gene expression compared with the M-CSF control . TA treatment on day 0 increased gene expression of IL10 and TGFBI in GM-CSF-generated macrophages . Furthermore, TA addition decreased TNF-α production of all macrophages (P < 0.01); the effect was most prominent when TA was added at day 0 .The in vitro. In addition, we show that FR-β is induced by TA treatment during macrophage generation, resulting in a CD163+ FR-β+ (M2-like) anti-inflammatory macrophage phenotype, even in an M1 priming environment with GM-CSF and no M-CSF.In this study, we show that FR-β expression is strongly induced by M-CSF in monocyte-derived anti-inflammatory M2-like macrophages 35 We observed that FR-β expression was lower in IC-induced M2b-like macrophages, as was their expression of CD163 and CD16. In agreement with previous findings,3638 ICs induced production of TNF-α, IL-6, and IL-10 in these macrophages. To the best of our knowledge, this study is the first to investigate FR-β expression in M2b macrophages stimulated with LPS and ICs.In agreement with our findings, FR-β expression has previously been shown to be higher in anti-inflammatory macrophages and is also high in tumor-associated macrophages (TAM) that have a similar M2-like phenotype.). Remarkably, we observed that FR-β expression remained high when M-CSF-generated macrophages were switched to a pro-inflammatory M1-like phenotype, unlike the other M2 markers such as CD163. The fact that FR-β remains highly expressed in these M-CSF-generated pro-inflammatory macrophages indicates that FR-β expression is not limited to an anti-inflammatory phenotype. This may be more in line with the complex in vivo situation in inflammatory joint diseases, where a monocyte can be exposed to M-CSF during maturation but may also encounter an inflammatory environment in the joint, skewing the macrophage to a pro-inflammatory phenotype that (being exposed to M-CSF before) still remains a high expression of FR-β+.Macrophages that are generated by M-CSF are known to be plastic and a pro-inflammatory phenotype can be induced in these macrophages by further stimulation with LPS and IFN-γ. For this reason they are also often referred to as M0  macrophages. At the same time, FR-β can also be expressed on pro-inflammatory macrophages with an M2 (M-CSF) history, as we observed that M-CSF-generated macrophages maintain their FR-β expression levels after stimulation with LPS and IFN-γ. This could explain why pro-inflammatory FR-β+ macrophages are observed in the synovium of OA and RA patients and why FR-β+ macrophages are associated with disease symptoms such as pain, joint space narrowing, and the presence of osteophytes.42 Likewise, in animal models, FR-β+ macrophages accumulate at the site of inflammation, relate to OA and RA disease severity, and can successfully be used as a target for therapy.4346 However, when TA treatment is administered in a rat OA model, osteophyte formation is prevented, but at the same time FR-β expression is extremely elevated. This suggests that FR-β expression in the latter model does not reflect disease severity, but rather a corticosteroid induced shift toward FR-β+ anti-inflammatory macrophages. This is in line with our observation that TA induces FR-β+ GM-CSF-generated macrophages, despite the M1 priming environment with GM-CSF. Apparently, macrophages with an M1 history can become FR-β positive and anti-inflammatory M2-like upon stimulation with corticosteroids. The latter finding could provide new opportunities to monitor the macrophage response to corticosteroid treatment in patients using FR-β-based imaging techniques. In addition, these findings reveal corticosteroid use as an important exclusion criterion for experimental treatment of OA and RA with FR-β-based therapeutic approaches, as targeting of FR-β+ anti-inflammatory (M2) macrophages could be detrimental. As long as corticosteroid use is taken into account, FR-β will remain an exciting potential target in clinical applications. On one hand, as shown in many previous studies to monitor disease status and as a target for treatment or, alternatively, to monitor treatment response to corticosteroid therapy.FR-β is under evaluation as a clinical marker and target in OA and RA, but ambiguous findings regarding the phenotype of FR-β+ (M1-like) pro-inflammatory macrophage population. When TA is added during macrophage generation, FR-β+ (M2-like) anti-inflammatory macrophages will form, despite the absence of M-CSF and the presence of GM-CSF, which would normally result in an M1 priming environment. This effect of corticosteroids on FR-β expression and previous observations made in vivo indicates that the purpose of FR-β-based imaging can be expanded to monitor patient response to corticosteroid therapy. In OA and RA patients who do not receive corticosteroids, FR-β can remain a potential marker for disease severity and treatment target. This is an important observation in view of further developments in FR-β-based diagnostic and therapeutic approaches in inflammatory joint diseases and warrants further investigation.In conclusion, we show that in an environment without corticosteroids, FR-β is induced by M-CSF and highly expressed by M2-like anti-inflammatory macrophages. In these macrophages, FR-β expression remains high after stimulation with IFN-γ and LPS, resulting in an FR-βClick here for additional data file.Supplemental material, sj-docx-1-car-10.1177_19476035221081469 for Folate Receptor Expression by Human Monocyte–Derived Macrophage Subtypes and Effects of Corticosteroids by Kelly Warmink, Michiel Siebelt, Philip S. Low, Frank M. Riemers, Bingbing Wang, Saskia G. M. Plomp, Marianna A. Tryfonidou, P. René van Weeren, Harrie Weinans and Nicoline M. Korthagen in CARTILAGEClick here for additional data file.Supplemental material, sj-docx-2-car-10.1177_19476035221081469 for Folate Receptor Expression by Human Monocyte–Derived Macrophage Subtypes and Effects of Corticosteroids by Kelly Warmink, Michiel Siebelt, Philip S. Low, Frank M. Riemers, Bingbing Wang, Saskia G. M. Plomp, Marianna A. Tryfonidou, P. René van Weeren, Harrie Weinans and Nicoline M. Korthagen in CARTILAGE