Since 2016, the European Region has experienced large-scale measles outbreaks. Several measles outbreaks in England during 2017/18 specifically affected Romanian and Romanian Roma communities. In this qualitative interview study, we looked at the effectiveness of outbreak responses and efforts to promote vaccination uptake amongst these underserved communities in three English cities: Birmingham, Leeds and Liverpool. Semi-structured in-depth interviews were conducted with 33 providers involved in vaccination delivery and outbreak management in these cities. Interviews were analysed thematically and factors that influenced the effectiveness of responses were categorised into five themes: (1) the ability to identify the communities, (2) provider knowledge and understanding of the communities, (3) the co-ordination of response efforts and partnership working, (4) links to communities and approaches to community engagement and (5) resource constraints. We found that effective partnership working and community engagement were key to the prevention and management of vaccine-preventable disease outbreaks in the communities. Effective engagement was found to be compromised by cuts to public health spending and services for underserved communities. To increase uptake in under-vaccinated communities, local knowledge and engagement are vital to build trust and relationships. Local partners must work proactively to identify, understand and build connections with communities. Measles is a highly infectious and potentially fatal viral infection which can be prevented by immunisation with the two-dose measles, mumps and rubella (MMR) vaccine . InsuffiSince 2016, the European Region has been affected by large-scale measles outbreaks. There were 83 540 measles cases and 74 measles-related deaths in the World Health Organisation (WHO) European Region in 2018, compared to 5273 measles cases and 13 measles-related deaths in 2016  so much confusion at the start…about whether we were talking about Roma communities or Romanian nationals. They are clearly not the same groups. They have very distinct cultural behaviours. Language issues. Religious influence. Cultural behaviours; and that all drives their engagement in the healthcare system…. there are [also] sub-groups within the populations.’ (Provider#11)At the start of the outbreaks, providers believed that the preferred language of community members would be Romanian. In some instances, this was this case, but many Romanian Roma do not speak Romanian as their first language  and literacy may also be a barrier.Within each city, several providers reported that as the outbreaks unfolded, they knew very little about the Roma and Romanian communities affected. In many instances, providers had not made a distinction between Roma and Romanian and the terms were sometimes used interchangeably.Views from several providers inferred negative attitudes towards the communities. Some interviewees did not comprehend why community members may be distrustful of healthcare professionals and have different beliefs around health. Without having context to the history of discrimination and persecution towards Roma, healthcare professionals instead became frustrated in trying to reach the communities.‘It made a big difference to who was round the table because it meant then that when I started phoning around to get people to come to the outbreak meeting, we got the right people. So, we got the manager of the Children's Centre where the Roma population [go]. We got one of the practice managers from one of the local practices. We got somebody from the local school that's 65% Roma, so we got the right people there.’ (Provider#28)Poor understanding of communities and factors affecting vaccination impeded effective strategies to manage the outbreaks. Understanding the communities was crucial to effective outbreak management.‘Perhaps we need to think about [an integrated] model of working with our local authority partners…. Rather than, “That's your job. That's your job,” Where there's overlap no one's really doing it, no one's coordinating it, and then it becomes very much, “You didn't do this. Why didn't you do that?” rather than, “How can we make this happen?”’ (Provider#16)In a few instances, communication difficulties were reported between PHE teams and frontline healthcare professionals. PHE teams had little power to enforce some of the recommendations as part of the outbreak (e.g. such as requesting busy GPs to support contact tracing for patients sitting in the surgery). In one city, PHE communication with hospitals was difficult due to large numbers of cases being reported, admitted and requiring extensive contact tracing.‘….in hospitals there were issues with communication, certainly, because they weren't notifying us. We ended up with a system where we would contact them every day to ask them, “Do you have anybody who's been tested for measles?” Whereas, it should have been the other way around.’ (Provider#16)Trying to mobilise responses, considering competing pressures on services, was also problematic.‘…. Who's paying for it all? That was our main beef with the GPs. We, as an organisation, would tell them, “We need this Public Health action to happen,” but the GPs would come back and say, “I haven't got the time, I haven't got the staff or resources, …”’ (Provider#15)In one city, efforts between partners were easier to define and co-ordinate the following lesson learnt in a past outbreak, in the same population and postcode area.‘….we were lucky in Leeds actually because we'd done a lot of work last year around roles and responsibilities in response to an outbreak, so actually we had a lot of shared understanding about right, well the CCG's role is this, the PHE's role is this, the local authority role is this. It made life a lot easier’ (Provider#1)In some instances, providers reported that there were challenges in identifying responder roles and responsibilities and promptly co-ordinating efforts.To prevent further outbreak spread, HPTs worked to trace the contacts of measles cases. HPTs in each city communicated with cases over the phone to ascertain the number of contacts, number of children in the household, travel history and length of stay in the country. However, interviewees reported that getting this information was problematic, particularly over the phone, given language barriers and a lack of community member trust in healthcare providers.‘We had minimal [engagement] because the outbreak started around November and as coincidence would have it, the Roma outreach support service that the local authority commissioned from the Red Cross; that ceased in November. I don't think it was a link, but it was just unfortunate timing, so it meant we had no real outreach apart from the [City Council] education team outreach that they do.’ (Provider#11)Providers reported outreach approaches, such as door-to-door knocking, using mobile vaccination vans and offering vaccinations in community centres, as effective in increasing vaccination uptake. Key to the effectiveness of out-reach strategies was being able to take advantage of pre-existing links and established trust with the communities.Though two of the cities reported good links with communities, providers in one city noted some difficulties. In addition, the lack of formal outreach mechanisms impeded the response.part of the community not the establishment’.‘we put on a community meal in the evening in the Children's Centre. Now [the Children's Centre manager] does that regularly so she feeds these kids in school holidays because if they don't get a school dinner, they go short. So that's not unusual…. We just blitzed the area with flyers, that our Romanian Consultant translated… we immunised 80 children on the Monday night in about two-and-a-half hours.’ (Provider#28)Other approaches used by providers, which may well have worked with other communities, such as putting on additional clinics in GP and incentivising GPs to vaccinate, were less successful. This was largely linked to the lack of community engagement with GP.Vaccination in the community centre in Liverpool was particularly effective as the centre was considered ‘In Birmingham, providers had looked to outbreak responses used in Liverpool and Leeds but felt that vaccination through children's centres would not work for their communities, as Romanian and Roma families were reportedly not accessing them.‘…. The biggest problem we think exist is if you send them pre-booked appointments, so if you get them to book an appointment that just does not work; they don't live like that. …. even if they've booked them themselves, it doesn't work, they did not attend (DNA) rates are very high.’ (Provider#12)Some providers discussed using points of engagement with community members for vaccination to also promote registration with GP. This approach was more straightforward in Liverpool when the centre used for vaccination was within easy proximity of a GP.‘…. The evening we did the meal and immunising, we were also literally taking people by the hand to register at the GP practice while we had them.’ (Provider#28)Overall, the most effective approaches to outbreak management were multi-faceted and relied on insight into the communities.‘…. that triple-pronged approach, so doing the schools, the GP practices, and the community at the same time helped sort of bring it all together in some way so you're targeting different parts of the population.’ (Provider#1)It was evident, in speaking with frontline healthcare providers, that in each city there were real attempts being made to engage with Romanian communities, e.g. employing Romanian speaking receptionists, sending letters and SMS messages in Romanian, holding additional clinics and trying outreach approaches. Providers in the three cities highlighted that encouraging communities to access GP was particularly difficult, and that the system of booking future appointments was often ineffective.Face-to-face communication was seen as key to successful engagement. However, participants reported difficulties in sourcing and funding translators and interpreters. Finding Romani speakers was nearly impossible, given the absence of professional Romani interpreters, and prompt access to Romanian speakers could prove difficult. In one of the cities, leaflets had been developed in Romanian but the interpreters stated that the wrong dialect was used in these.don't have time to get an interpreter’, or using Google translate during consultations. This raised concerns that messages would be lost in translation, even with the use of telephone interpreters through LanguageLine.‘…. You sometimes wonder what the Language Line is actually saying, because you're trusting their interpretation. Sometimes the patient looks totally confused with the Language Line.’ (Provider#14)In several instances, participants had appealed to the help of charitable and community organisations and relied on their goodwill to provide translators and interpreters, or ‘fallen lucky’ in having Romanian speakers within their teams.In community and GP settings, several participants discussed using hand gestures to communicate when they ‘Participants working in PHE HPTs also struggled with language barriers, particularly as their work was reliant on over-the-phone communication with people diagnosed with measles and they needed to ask personal questions.‘…. The system is so biased in or towards practices in nice leafy-green areas with English [speaking] people because, you know, that our nice or well-off end but we hit 90 percent vaccination with no trouble at all. We don't have to do anything. Whereas there we spend a huge amount of effort and money and time and we hit about 77 percent.’ (Provider#12)Trying to improve vaccination uptake with underserved communities was considered resource intensive . The focus of the immunisation target payment system on outcome, rather than process, meant that providers felt penalised for not reaching targets even when they ‘work so hard for immunisations’. Providers also felt that this could lead to reduced vaccination call-recall efforts.GPs receive financial payments for administering childhood vaccinations, based on achievement against a 70% or 90% uptake target for children at 2 and 5 years. Practices achieving the 90% target secure the highest financial payment. GPs that served populations with greater barriers to health service found it difficult to achieve the higher immunisation target.‘…a few years back [we] had community link workers and their job was to engage with communities and provide them with help and support so that trust develops. So, over the years when the economic crunch happened, they were the first ones, unfortunately, to get their jobs cut. So, when the outbreak happened there were no link workers as such. So, we were stuck, because the local authority had no contact, or very minimal contact with these communities’ (Provider#17)With the cuts, there were frustrations about lost opportunities to discuss and administer vaccinations.‘….we used to run our baby clinic and we used to have health visitors or school nurses in alongside us and that worked well and we got people who just came for weighing who we could then immunise. But they've since moved all of those people out, they say they've not got enough resource to have these people in here. So yet again….the changes and the cuts in the system are actually having the impact’ (Provider#4)Several participants reported cuts to community link services  as a major factor affecting the ability to engage with the communities during the outbreak. This was considered short-sighted and not cost-effective in the long run.We found that improving MMR vaccination uptake and effectively managing measles outbreaks relied on (1) being able to identify and understand undervaccinated communities, (2) having links with undervaccinated communities and using appropriate approaches for community engagement, and (3) working with local partners to co-ordinate efforts. These three factors were found in turn to be largely dependent on available funding and resources, and efficient and creative partnership working. The research has highlighted the importance of having clear financing and working arrangements in place to respond to outbreaks.One of the first challenges that providers faced was being able to identify under-vaccinated communities, particularly those not accessing GP or the school system. In 2017/18, 48.5% of Gypsy/Roma children in England were persistently absent from school . The onlOne resource that could be used by providers to bring a range of existing datasets together to identify under-vaccinated communities is the Strategic Health Asset Planning and Evaluation (SHAPE) Atlas . SHAPE aOnce at-risk communities are identified, it is important that providers use appropriate engagement approaches to promote vaccination . KnowingOutreach approaches appeared to be needed, at least in the short-term, to develop trust and links with communities which can be used to promote access to GP and to other vaccinations. It was noted by providers that although their efforts during the measles outbreaks were focused on improving MMR vaccination rates, there were also concerns about sub-optimal coverage for other vaccinations.We found that providers were severely limited by resource-constraints, including access to translators and interpreters, community link workers and community organisations. Resource constraints can be linked to changes introduced under the Health and Social Care Act 2012 when public health responsibilities shifted from national to local government . Public In general practice, providers felt that their ability to engage with and improve uptake in underserved communities were exacerbated by an immunisation target payment system focused solely on outcomes and not process. Immunisation target payments for GP are currently under review by NHS England, with a focus on trying to reduce inequalities in vaccination , 46. OveChallenges were voiced by general practice providers in tailoring culturally appropriate approaches to an increasingly ethnic diverse population. One recommendation that emerged from the interviews was to try and share more resources, such as translators and interpreters. It was also highlighted that more opportunistic vaccination or reminding about vaccination should take place, e.g. flexibility in giving vaccinations when a child presents in general practice for another reason.Cuts to health visiting and school nursing teams also appear to have had an impact on maintaining links with underserved communities and the capacity to promote access to GP and vaccination. Short-sighted cuts to services have negative economic, societal impacts and public health impacts , and alsThere is a limited evidence base on interventions to improve vaccination amongst potentially underserved groups, including Roma  and migrMaking measles history together – contains a set of prompts to action focused on reaching undervaccinated communities by drawing on existing third sector and local authority resources [The measles outbreaks in 2017/18 served as a wake-up call to reach undervaccinated communities. The GP contract now includes a check of MMR status for 10- and 11-year olds , and lesesources . The resesources . DevelopFindings from our study may be applicable to other underserved communities, particularly those not accessing general practiceor the school system, who may be unfamiliar with vaccination delivery in the UK and experience language and literacy barriers.The quality of the study was enhanced through the use of interviews with providers across three different outbreak settings to corroborate findings and develop a greater understanding of effective approaches to identifying and engaging with underserved communities. However, there were some limitations. Although we recruited a range of providers in each city, the range varied between cities. The study also did not focus on how the outbreak responses were funded. Future research should look at which partners fund activities and how funding decisions are made.Interviews for this study were limited to providers; however, in preceding studies, we have included Romanian and Roma community members to explore factors affecting vaccination uptake , 30.Our study identified partnership working and community engagement as key to the prevention and management of the 2017/18 measles outbreaks that specifically affected Romanian and Roma communities. Without prior understanding and linkage with Romanian and Roma communities, it was difficult for providers to identify and access communities, who may not trust providers. Cuts to public health spending and the provision of services for underserved communities, including under-vaccinated Romanian and Roma communities, have far-reaching implications for reducing inequalities in vaccination.To increase uptake in under-vaccinated communities, such as the Romanian and Roma communities in this research, local knowledge and engagement are vital to build trust and relationships. Local partners must work proactively to identify, understand and build connections with communities. Approaches that appeared to work best in reaching the Romanian and Roma communities in the outbreaks were multi-faceted, involving GP practices, schools and most importantly community representatives.


Several methodologies have been proposed to determine turn switches in alpine skiing. A recent study using inertial measurement units (IMU) was able to accurately detect turn switch points in controlled lab conditions. However, this method has yet to be validated during actual skiing in the field. The aim of this study was to further develop and validate this methodology to accurately detect turns in the field, where factors such as slope conditions, velocity, turn length, and turn style can influence the recorded data. A secondary aim was to identify runs. Different turn styles were performed  and the performance of the turn detection algorithm was assessed using the ratio, precision, and recall. Short carved turns showed values of 0.996 and 0.996, carving long 1.007 and 0.993, drifted 0.833 and 1.000 and snowplow 0.538 and 0.839 for ratio and precision, respectively. The results indicated that the improved system was valid and accurate for detecting runs and carved turns. However, for drifted turns, while all the turns detected were real, some real turns were missing. Further development needs to be done to include snowplow skiing. Turn detection in alpine skiing has been a topic of growing interest over the last 15 years. Turns are the basic unit required for detailed analysis or interpretation of skiing data such as edge angle, symmetry, or turn phases  sensor technology has been proposed as a promising alternative approach  to further develop and validate the algorithm of Martínez et al.  in fieldFor the validation and development of the turn detection algorithm, an experiment was designed where an IMU was attached to each ski boot  volunteered for the study. All participants were expert skiers, with experience as ski racers or ski instructors. Before the measurements were conducted, they were informed in detail about the testing procedure, as well as possible benefits and risks of the investigation prior to signing the consent form approved by the local Ethics Committee (EK-GZ: 11/2018). The experiment was conducted in accordance with the Declaration of Helsinki.An IMU  was placed in the back of the upper cuff of each boot . The X axis of the IMU was aligned with the vertical axis of the boot (yaw) pointing superiorly, the Y with the lateral axis (pitch) pointing to the left, and the Z with the anterior-posterior axis (roll) pointing posteriorly . The IMUIn order to define reference turn switch points and determine the proper detection of turns , all runs were recorded with a camera  with a sampling rate of 25 Hz. The camera was placed on the skiers' chests using an adjustable chest harness, and positioned such that the field of view included both skis, boots and the surrounding area.Video data was analyzed by experienced raters, labeling relevant events or context, such as turn switch point, turn direction, or jump. Turn switch points in the video analysis were defined as the frames where the surfaces of the skis were flat on the ground. Video and sensor data were synchronized using a jump event at the beginning and end of each trial. The jumps were detected in the vertical axis of the accelerometer and synchronized with the frame at landing. For validation, the actual turns obtained by the video analysis and the number of detected turns were used. The detected turns were computed by applying the turn detection algorithm  ratio, (2) turn count precision, and (3) recall (see below). All steps of evaluation  were conducted in a blinded procedure.Ratio: the quotient between the number of turns detected by the algorithm and the number of turns labeled in the video data (see data see . If the Turn count precision (P): the proportion of turns detected by the algorithm that are actual turns (see Equation 1).Recall (R): the proportion of actual turns detected by the turn detection algorithm (see Equation 2).In all metrics a value of 1 means a perfect performance.To calculate the turn count precision and recall values, the following metric were determined :True positives are determined by a detected turn that was an actual turn (based on video). A correct detection is characterized by the correct turn direction (either left or right turn) and by the difference in time between the detected and the actual timestamp. If the time difference is smaller than half the mean duration of actual turns (for a run), it is a true positive.False positives reflect the number of detected turns that are not actual turns.False negatives are the number of actual turns that are not detected by the turn detection algorithm.Where P is the turn count precision, TP the true positives, FP the false positives, R the recall and FN the false negatives.The purpose of the following algorithm was to determinate turn switches and sequences of parallel skiing turns. To keep the algorithm simple, it was assumed that a turn switch is a single point in time where both skis change from the uphill to downhill set of edges simultaneously. The performance metrics and video recordings were used to evaluate the performance of the current algorithm iteration  and detect weaknesses.roll smooths unilateral rotations. In the next step, a fourth-order zero-lag 0.5 Hz low-pass Butterworth (BW)  that describes the rotation rate in the roll axis . The left graph shows a sequence of nine carving turns , the right graph a sequence of 16 short turns (turn duration 1.04 ± 0.41 s). The minus before ωroll flips the positive direction to anterior in agreement with the movement direction. The extrema indicate possible turn switches, whereby the sign of a value in ωds determines the turn direction. As Switch: This kind of local extrema indicates actual turn switches from left to right or right to left.Noise: Local extrema that happen within a turn are called noise. If a skier performs turns with longer duration , the decision signal ωds shows some artifacts as saddle points or small counter oscillations , opposite sign and a time distance >0.3 s and smaller than 5.0 s then label both as switch.Noise: Look at each sequence of four consecutive local extrema: If both outer extrema fulfill rule 1 and both inner local extrema not and if the rotation rates in abs(ωds) of the inner are smaller than the outer then label both inner as noise.Eliminated:switch or noise and if the rotation rate is smaller than a threshold then label as eliminated.Look at each local extrema: If it is not labeled as switch, noise or eliminated and if it has the same sign in ωds as its predecessor then label as eliminated.Look at each local extrema: If it is not labeled as switch, noise, or eliminated apply a decision tree (see below) to label it as eliminated or not.Look at each local extrema: If it is not labeled as switch, noise or eliminated and if the predecessor and successor are eliminated then label as eliminated.Look at each local extrema: If it is not labeled as switch, noise, or eliminated then label as switch.Remaining: Look at each local extrema: If it is not labeled as Decision tree (3c): the goal of the machine learning task was to obtain a simple rule that removes all remaining local extrema outside a turn sequence because these local extrema should have the label eliminated. If we do not have such a rule, rule 4 cannot be applied. A decision tree was trained by means of machine learning. The intent of the decision tree was to discriminate for each local extrema between the classes within a turn sequence or not-eliminated and outside a turn sequence or eliminated. Data were labeled by manually defining the time ranges of the turn sequences, and a cross-validation was used to avoid overfitting the decision tree. Obvious features as roll, ωds, ωds , its absolute values abs(ωds) and t were used to search the most expressive features. The result was the decision tree: abs(ωds) ≤ θ1 ∧ t ≤ θ2 → eliminated. That means that the absolute value ωds and the t  were identified by the machine learning task as most expressive. The variables θ1 and θ2 are the modeling parameters of the machine learning model thresholds.TSds,n, ln))n∈ℕ. Next, the algorithm derives the turns  from the sequence TSds,n. The timestamps tstart and tend are the start and the end of a turn (turn switch points), the value d ∈ {left, right} indicates turn direction and s∈ ℕ is the number of the turn sequence to which the turn belongs )m∈ℕ (m < n), the algorithm iterates through the sequence and merges consecutive extrema with label switch to a turn if and only if they are no interruptions from local extrema with label eliminated. This rule produces a continuous turn sequence with tend,i−1 = tstart,i. Local extrema with label noise (per definition “within turn”) were ignored because they do not interrupt a turn. Eliminated local extrema (per definition “outside turn”) indicate an interruption of a consecutive turn sequence and causes tend,i−1 ≠ tstart,i and si = si−1 + 1. The previous heuristic rules ensure that in a turn sequence each switch is followed by a switch with opposite sign, even in case were local extrema with label noise are between. A strict alternation of the sign is necessary because otherwise there is no alternation of left and right turns, which is not obvious.The result of the previous step is a turn sequence (TS) of labeled local extrema  belongs . The valsi−1 + 1 . The stafc = 3.0 Hz is a proper cut-off frequency to perform such a “fine tuning.” Therefore, a new fine tuning signal is defined by ωft = BW3.0(−ωroll) , li) the global maximum or minimum in ωft in a time window (an asymmetric ε neighborhood) around the turn switch timestamp ti. The algorithm “fine tunes” only local extrema labeled as switch and defines the global maximum or minimum p ∈  defines the size of the asymmetric ε neighborhood (ti − p(ti − ti−1), ti + p(ti+1 − ti)].The decision signal is a strongly smoothed signal. The rotation rates represent the general rotation behavior of  turning during skiing. The degree of smoothing between raw and decision signal can be observed in (−ωroll) . Then thhborhood : [ti − pp = 0.6. Finally, the fine tuned turn switches, turns and turn sequences are based on the sequence t in Tds,m with t′.For the succeeding evaluation we use 60% for the ε neighborhood, thus The results of the turn detection evaluation for each skiing style  are shown in Investigating the turn precision measures of the algorithm for carved turns, almost all detected turns were correctly detected (turn precision was higher than 0.993 for both turn sequences). The recall measures were also high with values >0.990, which indicates that almost all actual turns were detected by the algorithm.The purpose of this study was to develop and validate a turn detection algorithm for parallel alpine skiing turns in the field. The assumption that a turn switch is a single point in time where the edge changes simultaneously for both skis is not too critical if the turns have seamless transitions, and the skier performs parallel styles. Conversely, the algorithm is not able to count all turn styles accurately. For example, snowplow and drifted turns with low dynamics or straight piste crossings cause problems. However, the proposed methodology was aimed at continuous turn sequences of parallel style turns, as carved turns are usually the main subject of interest.For validation, tests were performed by several subjects with diverse techniques, turn lengths, and snow conditions. The instructions for the different turn lengths aimed to define different types of turns  while allowing the participants to perform slightly different turns. We believe that not having an exact turn length adds an extra variability that is beneficial to develop a more robust algorithm. The natural variation in snow conditions across the 4 days of testing provided an additional layer of variability, adding to the robustness of the algorithm. The results of 374 carved turns  prove the validity and robustness of this turn detection algorithm for this particular turning technique. Misdetections during carved turns were only observed at the beginning of the turn sequences; however, they were mostly counted correctly. The three different options observed for the initial turn detected in a turn sequence are shown in Ratio and recall for drifted turns  were lower than for carved turns. The turn detection algorithm worked properly for all participants but four. For those participants, the missing turns were not isolated but consecutive . All those turns had some common characteristics. They were longer slow turns, which produced a less dynamic movement, and consequently were more affected by the snow conditions, in those cases, bumpy surfaces. The algorithm is prepared to recognize and cancel some noise see ; howeverAlthough the algorithm was developed for parallel turns, the performance during snowplow steering was also evaluated. While the turn count precision was acceptable, ratio and recall were unacceptably low . As expected, most of the detected turns were actual turns, but many real turns were not detected. The snowplow turning style is completely different than drifted or carved turns. The legs do not have synchronized rotation rates in roll axis. The main movement does not rotate around the anterior-posterior axis, but around the vertical axis. Consequently, an algorithm that only takes into consideration the anterior-posterior axis might not be the most effective approach. However, snowplow turns are usually turns during learning and they are not used very often in everyday skiing. Thus, the focus on parallel turns is obvious, at least from a practical perspective. Further development, probably including a combination of more than one axis or a turn style detection system, would be needed for the automatic detection of snowplow turns.Over the different iterations of the development process, modifications were included in the algorithm. These modifications were needed in order to cope with signal features that differed between in-lab  in alpine skiing with an accuracy of 95.3%. For carving turns, the algorithm is able to accurately detect 99.7% of actual carving turns. The algorithm is based only on a single axis of gyroscope data. Simple IMUs, securely mounted on both ski boots, provide sufficient data for the segmentation of time series signals into both turn sequences and single turns. Both are very convenient data structures for further analysis tasks. The approach is applicable for in-field studies because the devices do not disturb skiing and the algorithm is fully automated. Further research is needed to assess the performance of the system for skiers with different skill levels, or to include the detection of other turn styles, such as snowplow turns.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee of the University of Salzburg. The participants provided their written informed consent to participate in this study.TS, RJ, and RB: conceptualization and investigation. TS, RJ, RB, MB, and AM: methodology. RB and MB: software. AM, VV, CT, and CS: validation. RB, MB, VV, and AM: formal analysis. TS and RB: resources. RJ, RB, and VV: data curation. AM, RB, and VV: writing-original draft preparation and visualization. CS, RJ, MB, TS, VV, and RB: writing-review and editing. TS: supervision, project administration, and funding acquisition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, we show the synthesis of reduced graphene oxide/hydroxyapatite (rGO/HA) composites using a hydrothermal autoclave with argon-15% hydrogen gas injection. This both increases the hydrothermal pressure and uses hydrogen as a reductive agent in the process. The synthesized powders were then consolidated with spark plasma sintering method. The analysis of the consolidated samples included Vickers Indentation technique and cell viability. The results showed that injected gases in the autoclave produced powders with a higher crystallinity compared to synthesis without the gases. Also, hydrogen gas led to increased reduction of GO. The microscopic analysis confirmed existing graphene sheets with folding and wrinkling in the powders and indicated that various preferential directions played a role in the growth of hydroxyapatite crystals. The results showed that in general, graphene sheets increased the mechanical properties of HA. In the samples synthesized with injected gases, this increase was more significant. Interface analysis results indicate that reduced graphene oxide (rGO)/HA interface is likely coherent. These nanocomposites were biocompatible and showed some hydrophobicity compared to pure HA. The unique properties of HA include excellent bioactivity, osteoconductivity, and biocompatibility. The chemical formula of this ceramic is Ca10(PO4)6(OH)2, in which the ratio of calcium to phosphate is 1.67, and the crystalline structure of HA is similar to the inorganic part of the human skeletal system. HA has a variety of uses other than orthopedic applications5. In recent years, additional researches have been done on calcium phosphates, which indicate the specific value of these ceramic structures9. Despite the excellent biomaterial properties the poor mechanical behavior of HA has limited its application scope. These mechanical properties include brittleness, fracture toughness, and wear resistance, which are important in applications as implants12.Hydroxyapatite (HA), a member of the calcium phosphate family, is one of the most valuable biomaterials used in orthopedics15. But this improvement is not enough for applications as implants, and the mechanical properties of HA still need to be increased by adding a reinforcement phase17. Of all the materials used, carbon nanomaterials such as nanotubes19 and graphene20 have recently received much attention and among the carbon materials, graphene and its derivatives have gained more research because of their unique properties. Graphene has a honeycomb-like structure. Graphene sheets are as thick as a carbon atom. The atoms in the graphene structure are combined with the sp2 hybrid23. The mechanical properties of graphene are very interesting, which include the elastic modulus of 1 TPa and the fracture strength of 130 GPa25. The high reinforcing properties of graphene are due to the two-dimensional structure with high specific surface area of 2630 m2g−1 26. Graphene, having good biological properties, has been extensively studied in biological applications, including drug delivery27, biosensing28, tissue engineering29, and bioimaging30.With the advent of nanotechnology and advanced synthesis methods, the mechanical properties of HA have improved somewhat by decreasing their particles size and morphologies such as nanorods and nanotubes34. Graphene-HA composites are fabricated in various methods. Some of these methods include hydrothermal process35, electrochemical deposition technique37, sonochemical treatments38, biomineralization39, and self-assembly40. Some methods are more suitable for coating. In some methods, such as hydrothermal, the hybrid powders are first synthesized and subsequently consolidated by spark plasma sintering (SPS)43 or hot pressing method44. Among these methods, the hydrothermal process is very convenient for easier control and the ability to synthesize hybrid powders by in situ method46. When other solvents are used beside water, this process is called solvothermal method47. Graphene oxide is mainly used in this method. Due to the presence of functional agents on the surface of graphene oxide, nucleation and growth of HA crystals are easily accomplished. In the hydrothermal method, high temperature and process time cause the graphene oxide to reduce. This also causes the HA particles to form nanorods on the graphene oxide surface49. With the reduction of graphene oxide, its mechanical properties improve. Different methods are used to increase the reduction rate including adding chemicals such as ethylene glycol33, and injecting hydrogen or nitrogen gas into a hydrothermal autoclave53. Increasing hydrothermal pressure increases the crystallinity of HA54 and increases the rate of graphene oxide reduction45.Several researches have been published on the use of graphene to improve the mechanical and biological properties of HA and the results of these studies have shown that graphene has greatly enhanced the properties of HA2 mixed gases injection condition. Pure HA was previously synthesized under this condition, and increased pressure by injecting this mixture of gases increased the mechanical properties of HA54. The presence of hydrogen gas in this mixture is now exploited to increase the rate of graphene oxide reduction. After the powders were synthesized and characterized , spark plasma sintering (SPS) was performed and the samples were subjected to mechanical and biological assays. It is expected that the combination of the above methods will make the final nano-structured composite suitable for orthopedic applications.In this study, a hydrothermal method was used to synthesize reduced graphene oxide- HA nanocomposite powders under the Ar-15% H+2  was added dropwise to a 20 mL stirred suspension of GO (HA/1.5% rGO) with stirring continued for 1 h. The solution containing phosphate ions (1.56 grams of diammonium hydrogenphosphate in 80 mL of S1) was dropwise added to the solution. The pH of the solutions was adjusted to >10 with ammonium solution. The resulting solution was poured into the Teflon (PTFE) vessel and transferred to the autoclave. The hydrothermal process was carried out for 8 h at 180 °C by injection of nitrogen gas at 0, 5, and 10 bar (The volume of the PTFE container was 340 mL). The powders were dried at oven for 12 h at 60 °C. The resulting powders were sintered after drying and ball milling 49.The primary chemicals used in this study, along with their specifications, are presented in Table 54.Figure 54, at a temperature of 950 °C. Exactly similar to the previous report, the biocompatibility assays were performed54.The powders were consolidated via SPS, as previously reported54.To calculate the relative density of sintered samples the Archimedes method was used. In this method, wet weight, dry weight and immersed weight with a precision of 0.0001 were measured and the Archimedes ratio was used for relative density calculations (ASTM C373-88)55. The modified Antis method was used to evaluate the fracture toughness (K1C) of the samples . λ is a dimensionless constant is close to 0.0498 for Vickers tip. C is the average crack length and P is the applied force. The use of experimental parameters is the major advantage of this method; it is easy to calculate when using instrumented indentation.Instrumented microindentation experiments (Grindosonic tester with a Vickers tip) were conducted on the polished surfaces of samples at a maximum load of 2 N and ramp dwell time of 10 s. Nine tests were performed at different locations of each sample. Elastic modulus and hardness were calculated from the load-displacement curves Fig.  using OlDifferent evaluation methods have been used in this research, some of which are described below. The other instruments used to characterize the samples include inductively coupled plasma (ICP) , X-ray photoelectron spectroscopy , FESEM , and TEM . ImageJ 1.52d and Diamond 3.2 softwares were used in this study.57.(112/300) and I300 are the intensity of the hollow between diffraction peaks of HA in the planes (300) and (112) and the intensity of the peak of HA in the plane (300), respectively. Equation 58.X-ray diffraction  was used to determine the phase constituents of the samples, contained a detector Cu Kα radiation  and 2 theta from 10° up to 80° in steps of 0.02°. Equation }}}_{112/00}}{{{\rIn this equation, d, θ, and λ are grain size, Bragg diffraction angle, and wave length of used X-ray (Cu), respectively. β and ε are full width at half height (FWHM), and crystalline lattice strain, respectively.Field emission scanning electron microscope  and a portable scanning electron microscope  were used to observe the morphology of samples (mounted in an adhesive carbon film and Au coated by sputtering for its observation).−1, spectral region from 400 to 4000 cm−1 using 2 cm−1 steps, scan number of 8). The samples were prepared and mixed with potassium bromide (1 mg powdered samples and 300 mg KBr). The mixture was pressed into discs by applying 200 MPa pressures (1 mm thickness). The spectra were collected at room conditions 49.Fourier transform infrared spectroscopy  was used to identify the functional groups of the samples , which had been excited from an argon ion laser. The samples were subjected on Al foil in order to remove the fluorescence background49.Raman spectroscopy (Renishaw inVia spectrometer) was used in the range of 300–3500 cm48.High-resolution transmission electron microscopy  was used to observe atomic structure of the samples and spatially resolved elemental analysis, with a spatial resolution higher than 2 nm . High angle annular dark field detector (HAADF) was used to obtain STEM images . To study the atomic structure, fast fourier transform (FFT) and inverse fast fourier transform (IFFT) analysis were used54. Accordingly, the HA has a high purity hexagonal structure. In other words, the XRD pattern of the rGO-HA powders is quite similar to pure HA. According to studies, graphene oxide has a peak in the range of 2theta = 1048. After reduction of graphene oxide, this peak is removed and a new one appears around 2theta = 26. This peak is much weaker and wider than the HA (002) peak due to the amorphous structure of rGO. Therefore, the rGO peak is covered by the HA (002) peak which is highly intensified due to its high crystallinity. Table 49.Figure 49.Figure 54.Figure 48.Schematic −1 and 1730 cm−1 are shifted upwards, indicating reduction of GO, since these two peaks are characteristic of GO. As shown in the diagrams, the presence of hydrogen gas in the mixture of gases has led to more reduction. Also, the quality of the peaks obtained for the powders which is synthesized under the gas injection conditions is more accurate and uniform than the other, indicating a better bonds quality49.Figure (GO) ≈ 0.73, ID/IG(P10) ≈ 0.91) which shows the chemical and thermal reduction that has caused structural disorder in the graphene network. It is also clear that the presence of injected gas has led to more reduction of GO49.The Raman spectroscopy has been done to confirm the presence of rGO. Table Density calculations from the Archimedes method showed that the sample synthesized by the classical method (P0) reached to relative density of 96.66% and the samples that were synthesized in the presence of injected gas  reached to relative density of 95.91% and 96.9%, respectively. The temperature of 950 °C has been chosen to reduce the probability of decomposition of HA crystals and degradation of graphene sheets.48.Figure 54.Figure According to Eq. oC. As a result of these findings, HA has retained its composition after high temperature consolidation and has not been decomposed. According to Fig. 48.Figure 54. The only difference is the morphology of the cells that are globular. The presence of rGO sheets, which has become hydrophobic by the loss of its surface agents, may have caused this change. It can be concluded that the leached obtained over a 7-days period showed no significant toxicity to the culture of the osteoblasts.Figure The findings showed that the interface between the two phases in the nanocomposite was coherent and the elements were homogenously distributed. The powders synthesized under the injected gas conditions had higher crystallinity and crystallite size. Also, the presence of hydrogen gas further reduced the graphene oxide. These conditions resulted in higher elastic modulus, hardness and fracture toughness of the nanocomposites synthesized by this method. These composites were biocompatible and showed some hydrophobicity compared to pure HA. These findings will be useful for biomedicine applications of these nanocomposites.


The synthesis of promising nanocomposite materials can always be tricky and depends a lot on the method of synthesis itself. Developing such synthesis routes, which are not only simple but also can effectively catch up the synergy of the compositing material, is definitely a worthy contribution towards nanomaterial science. Carbon-based materials, such as graphene oxide, and conjugative polymers, such as conductive polyaniline, are considered materials of the 21st century. This study involves a simple one pot synthesis route for obtaining a nanocomposite of polyaniline and graphene oxide with synergistic effects. The study was carried out in a systematic way by gradually changing the composition of the ingredients in the reaction bath until the formation of nanocomposite took place at some particular reaction parameters. These nanocomposites were then utilized for the fabrication of electrodes for aqueous symmetric supercapacitor devices utilizing gold or copper as current collectors. The device manifested a good capacitance value of 264 F/g at 1 A/g, magnificent rate performance, and capacitance retention of 84.09% at a high current density (10 A/g) when gold sheet electrodes were used as the current collectors. It also showed a capacitance retention of 79.83% and columbic efficiency of 99.83% after 2000 cycles. Generally, energy storage materials are classified into electrical double layer (EDL) and pseudocapacitive materials . This cl2-hybridized single layer of carbon atoms. This problem has been solved through oxidation of graphite to graphene oxide (GO), which is composed of graphene sheets with large quantities of oxygen containing functional groups, and is synthesized through the oxidation of graphite . T. T71]. TThe synthesis protocol is very important for defining different properties of materials. When properly designed and optimized, materials with valuable properties can be tailored. This is true in the case of the present study as when we optimized the reaction parameters for in situ chemical oxidative synthesis of PANI–GO composites, nanocomposites were obtained in particular conditions. These nanocomposites showed enhanced capacitive and electrochemical properties, an excellent rate capability, and a lower ESR value. EIS analysis showed low Rct, ESR, to, and Rs. A phase angle value close to 90° was observed in certain conditions, which is very close to an ideal capacitor. These nanocomposite materials were active as an electrode material when utilized in the fabrication of symmetric supercapacitor devices with gold or copper as the current collectors, thus indicating their multifunctional abilities.


The purpose of this investigation was to describe the lived experience of aging in communities of faith, more specifically how older adults experienced social support. Semi-structured interviews were conducted with 15 older adults ranging in age from 63 to 84 years of age. Grounded Theory coding techniques were applied to identify themes. Findings include people feeling supported by community, connections, and beliefs, but also failed regarding opportunities for elder ministry or other types of worship. Participants expressed wanting more intentional opportunities for engagement. Many participants described the desire for an “expert” in their communities of faith. We consider the culture of support and how communities of faith may need to reconsider traditional ways of meeting the needs of people as they age. Furthermore, we explore the creation of educational tools that may allow for the emergence of an aging specialist in communities of faith.


Aging is an inevitable and complex natural phenomenon due to the increase in age. Cellular senescence means a non-proliferative but viable cellular physiological state. It is the basis of aging, and it exists in the body at any time point. Idiopathic pulmonary fibrosis (IPF) is an interstitial fibrous lung disease with unknown etiology, characterized by irreversible destruction of lung structure and function. Aging is one of the most critical risk factors for IPF, and extensive epidemiological data confirms IPF as an aging-related disease. Senescent fibroblasts in IPF show abnormal activation, telomere shortening, metabolic reprogramming, mitochondrial dysfunction, apoptosis resistance, autophagy deficiency, and senescence-associated secretory phenotypes (SASP). These characteristics of senescent fibroblasts establish a close link between cellular senescence and IPF. The treatment of senescence-related molecules and pathways is continually emerging, and using senolytics eliminating senescent fibroblasts is also actively tried as a new therapy for IPF. In this review, we discuss the roles of aging and cellular senescence in IPF. In particular, we summarize the signaling pathways through which senescent fibroblasts influence the occurrence and development of IPF. On this basis, we further talk about the current treatment ideas, hoping this paper can be used as a helpful reference for future researches. Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease of unknown etiology, characterized by massive deposition of extracellular matrix (ECM) in the lung interstitium, leading to the irreversible and slowly progressive destruction of lung structure and function . Most paThe risk of interstitial lung disease increases with age. The risk of IPF illness in the elderly over 70 is 6.9 times higher than in the people aged over 40 . With thResearchers have been trying to figure out how the connections between IPF and aging were established. For one thing, the respiratory system itself shows signs of aging with years. Significant increases in peribronchial collagen and progressive fibrosis are observed in the lungs of natural aging mice . In asymWhen compared with the age-matched control group, the primary fibroblasts isolated from the lung of IPF patients showed more senescent characteristics, which indicates that cellular senescence is persistent and intense in IPF patients . Under nUnlike lung epithelial cells, which are more like the sensor to external injury and stimulation, pulmonary fibroblasts act as the fibrosis process’s direct executor during the disease. Senescent fibroblasts in IPF are abnormally activated, accompanied by telomere shortening, metabolic reprogramming, mitochondrial dysfunction, apoptosis resistance, insufficient autophagy, and senescence-associated secretory phenotypes (SASP) . Most ofin vitro, demonstrating the declined proliferation capacity , telomerase synergistic protein 1 (hTP1), and telomerase reverse transcriptase (hTRT). Telomerase can make catalytic reverse transcription and provide the needed RNA template. Both functions are essential guarantees of normal telomere length. Multiple mutations of hTRT and hTR gene have been found in IPF patients, and they all lead the way to telomere shortening . Among tIn primary lung fibroblasts obtained from aged mice, glucose transporter protein 1 (GLUT1) dependent glycolysis is activated. By down-regulating the activity of the AMP-activated protein kinase (AMPK), this glycolytic pathway promotes the protein expression of α-SMA in cells . And in The increased glycolysis is also the result of multiple signal transduction and interaction within the senescent fibroblasts. Hypoxia resulted from the progress of IPF disease will lead to the disorder of cell energy metabolism, but on the other hand, hypoxia also up-regulates the expression of hypoxia-inducible factor-1α (HIF-1α) protein and mRNA. HIF-1α mediates the overexpression of pyruvate dehydrogenase kinase 1 (PDK1) gene, which in turn activated glycolysis and increased the activation of lung fibroblasts . ConsistApart from glycolysis, metabolic heterogeneity in IPF fibroblasts also includes changes in bile acid, heme, amino acid, and lipid metabolism . TGF-β1 The mitochondrial dysfunction triggers include transmembrane potential loss in the mitochondrial inner membrane, down-regulation of electron transport chain (ETC) function, and reduction of key metabolites entering mitochondria from the cytoplasm . These aMitophagy, which occurs in normal cells to maintain mitochondrial homeostasis, is down-regulated in senescent fibroblasts, leading to decreased PTEN-induced putative kinase 1 (PINK1) . InhibitIn addition, signal transduction and transcriptional activator factor 3 (STAT3) is necessary for the ETC of mitochondrial complexes I and II to remain activation and is involved in coordinating intracellular homeostasis . The eleApoptosis is an autonomously ordered death controlled by genes and can maintain homeostasis. In the restoration process of lung injury, the activated fibroblasts show changes corresponding to apoptosis and gradually disappear at the end . ApoptosReduced pro-apoptotic proteins Bak and Bax and increased anti-apoptotic protein Bcl-2 family proteins are found in IPF senescent fibroblasts . The accAlso, senescent IPF lung fibroblasts are found to be highly resistant to Fas ligand-induced (FasL) and TNF-associated apoptotic ligand-induced (TRAIL) apoptosis. FasL, TRAIL, and Caveolin-1 (Cav-1) protein abundance is decreased, and AKT activity is increased in these cells . TGF-β1 Autophagy is responsible for the retrieval of various components by degrading organelles and macromolecules to provide the raw material for reconstructing cellular structures, carefully maintaining the usual fate of fibroblasts . The cysAutophagy-related biomarkers in lung fibroblasts consist of apoptotic effector protein Beclin1, LC3, p62, and the autophagosome number . All of Autophagy is also involved in the regulation of ECM formation. Studies have shown that increasing the autophagy clearance rate to type 1 collagen by lung fibroblasts could reduce the aggressiveness of IPF fibroblasts . In IPF,Senescent cells typically secrete a complex set of factors known as senescence-associated secretory phenotype (SASP), a unique cellular senescence feature. Elevated transcription levels of these factors can be synchronously detected in the cells . SASP inThe SASP activated in senescent cells is a self-amplified secretory network . In IPF Senescent fibroblasts also influence the local microenvironment and the senescence of adjacent cells through SASP in a paracrine manner and finally aggravate IPF disease . When thFibroblast senescence is an important therapeutic target of IPF . In the Given that fibroblast senescence in IPF cannot be entirely attributed to a single cause, these targeted drugs are probably incapable of achieving satisfactory results due to their limited effectiveness. Concomitant administration of several drugs is likely to fall into the dilemma between the chaos caused by drug interactions and the inability to cover every discovered signal pathways. Thus, researchers turn to drugs with multiple effects. Pirfenidone is a pleiotropic molecule approved to be one of the antifibrotic drugs to IPF. It inhibits the MUC1 phosphorylation and β-catenin activation induced by TGF-β1 and prevents SMAD binding elements from forming or activating, thereby combating the fibroblast senescence in IPF . NintedaIn addition to these drugs mentioned above, there are also other treatments for senescent fibroblasts in IPF. Stem cell therapy is one of them. The exocrine body derived from human amniotic epithelial cells contains many soluble factors that can regulate inflammation and fibrosis pathways and is considered a potential treatment for IPF. These isolated stem-cell-derived extracellular vesicles reduce lung inflammation and fibrosis in mice by nasal drip . Gene thCellular senescence is characterized by cell cycle arrest, macromolecular damage, metabolic disorders, and SASP, which are important aging pathways. IPF is an aging-related interstitial lung disease of unknown etiology. The pathogenesis of IPF itself is difficult to be directly and clearly summarized with several abnormalities. Otherwise, it cannot be called “idiopathic.” Although the source of the disease is the key to solve the problem, for IPF patients, intervention in the process of their onset or progression may be a better choice, at least at this stage. We must be clear about the role of cellular senescence in IPF. The related experiments in IPF patients have the problem that the control group is not suitable, while the IPF mice model induced by bleomycin also has the problem of heterogeneity and self-remission. There is no better way to solve the experimental control group’s problems, but the improvement in animal model construction can provide more opportunities for senescence-related research in IPF.Just as cellular senescence is implicitly multifaceted in IPF, how senescent fibroblasts function is also multifaceted. Senescent fibroblasts exhibit abnormal activation, telomere shortening, metabolic reprogramming, mitochondrial dysfunction, apoptosis resistance, autophagy deficiency, and SASP secretion, involving a variety of molecular signaling pathways. However, we have not yet found a sufficiently unique and universal biomarker for cellular senescence. The signaling pathways in senescent fibroblasts are like a dense web, which tightly controls the IPF disease’s severity. It may not be enough only to untie a knot on the web.Under these conditions, the idea of using drugs that precisely clear senescent cells are gaining ground. In the selection of senolytics, compounds from nature undoubtedly perform better for their pleiotropic effects as well as security. When we attempt to discover new senolytics from a vast array of natural and human-made compounds, it is also a beneficial idea in our opinion to find appropriate answers from drugs already on the market or in clinical trials. As the old saying goes, the best way to discover a new drug is to start with an old one. Do drugs that can act on multiple senescence-related pathways at the same time also hide anti-aging properties? In the future, more cell experiments and animal experiments will give us the answer. In addition, just as new pathways are constantly confirmed in IPF, the signaling network in senescent fibroblasts we map will also be enriched. We believe that cellular senescence and senolytics will become new lights in the treatment of IPF.Both authors read and approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Keloids are dermal fibroproliferative tumors that arise beyond the boundary of the original wound edges and invades adjacent tissue. Keloids are characterized by the extensive production of extracellular matrix (ECM) and abnormal fibroblast proliferation. Chondroitin sulfate (CS) is one of the major structural components of cartilage and ECM. Recently, we reported the over-accumulation of CS in keloid lesions. Keloid-derived fibroblasts (KFs) and normal dermal fibroblasts (NFs) were incubated with CS. The fibroblast proliferation rate was analyzed using a tetrazolium salt colorimetric assay. The activation of the intracellular signaling pathway was analyzed by Western blotting. Wortmannin, a PI3K inhibitor, and anti-integrin antibodies were tested to investigate the mechanism of the CS-induced cell proliferation. CS strongly stimulated the proliferation of KFs, but not NFs. The analysis of the intracellular signal transduction pathway revealed that the stimulation effect of CS on KF proliferation was due to the activation of the protein kinase B (AKT) pathway and that integrin α1 was responsible for this phenomenon. We revealed that CS probably activates the AKT pathway through integrin to induce KF proliferation. CS may be a novel clinical therapeutic target in keloids. Keloids are benign dermal fibroproliferative tumors that grow beyond the boundary of the original wound edge and invade the adjacent tissue due to the extensive production of extracellular matrix and the abnormal proliferation of fibroblasts . KeloidsMultimodal treatments, including surgical excision, radiotherapy, corticosteroid injection, physical therapy, and combinations of these treatments, have been applied. Still, there is no satisfactory treatment. The mechanism underlying keloid formation remains unclear. Thus, revealing the etiology of keloids is a fundamental step in the development of clinical treatments for the condition.Recently, the formation of keloids has been recognized to occur due to the excess proliferation of fibroblasts with the over-accumulation of the extracellular matrix. Fibroblasts cultured from keloids (KFs) show altered behavior from normal dermal fibroblasts (NFs), including rapid proliferation, excessive extracellular matrix production, and increased migration activity, which may be a significant etiological factor in keloid development.To understand the behavior of KFs, we studied molecular signal transduction pathways, including growth factor-induced cell growth, apoptosis, and intracellular signal transduction ,3,4,5. KChondroitin sulfate (CS) is a glycosaminoglycan (GAG) composed of alternating, differently-sulfated residues of β-D-glucuronate and β-D-N-acetylgalactosamine . CS is oOur previous study showed that Chondroitin sulfate was over-accumulated in keloids  and thatWe explored the novel roles of CS in the positive regulation of KF proliferation by focusing on the promotion of the CS-mediated signaling pathway. This was directed by our previous study, which suggested that the over-accumulation of CS in keloid lesions may be the deteriorating etiological factor ,15. ReceThe following assays were conducted to evaluate CS-mediated cell proliferation. We used culture plates precoated with poly-D-Lysine (PDL), which is used as a substrate to support the maintenance of negatively charged CSs. Fibroblasts were harvested from tissue specimens using the explant method. We seeded fibroblasts onto PDL plates and incubated the plates for 24 h in serum-free media before CS stimulation. We analyzed five KFs and five NFs in total. A colorimetric tetrazolium salt assay revealed that CS significantly stimulated KF proliferation in comparison to the control (no CS) group . CS did To elucidate the effects of CS on KFs in cell proliferation, we next identified the canonical pathways that have been shown to alter the activation of KFs from recent studies ,20, suchWe then checked AKT activation. In KFs, incubation with CS elevated the phosphorylation level of AKT, in a time-dependent manner . In NFs,Next, we focused on the AKT pathway change caused by the CS stimulation. We examined downstream signal proteins related to the cell cycle. The cell cycle progression from G0 to S phase requires several cell-cycle regulating molecules in specific states, such as decreased p21, activated CDK2, and increased cyclin D. p21 is a cyclin-dependent kinase inhibitor (CKI) that decreases when the cell cycle progresses. A few hours after CS stimulation, we discovered the time-dependent phosphorylation of AKT, decreased p21, and CDK2 phosphorylation . In contTo reinforce our theory, we used wortmannin, a selective and irreversible PI3K/Akt inhibitor. We incubated KFs with wortmannin and obtained protein samples. Wortmannin successfully blocked CS-mediated activation of AKT and blocked p21 decrease , and inhFrom recent research  and our To determine which subtype of integrin was responsible for this mechanism, we selected and investigated several subtypes of integrin based on the relevant literature: integrin α1, integrin α2, integrin α4, integrin α5, integrin β1, and integrin Av. We measured the expression levels of these subtypes by RT-PCR. Integrin α1 (ITGA1) was highly expressed in KFs, relative to NFs . We also conducted an integrin expression assay to measure the integrin expression in tissue specimens. The expression of ITGA1 in keloids was three times greater than that in normal skin tissue . The ITGTo further investigate the role of integrin α1 in CS-induced KF proliferation, we used an anti-integrin α1 antibody and mouse anti-human IgG as a control. When incubated with anti-integrin α1 antibodies, the stimulation effect of CS on KF proliferation was successfully blocked . FurtherIn the present study, we revealed that the cell-proliferative effect of chondroitin sulfate (CS) in KFs in comparison to NFs. A tetrazolium salt assay and intracellular signaling pathway analysis revealed that the cellular proliferation of starved KFs was actively promoted. In keloids, growth factors, such as IGF-1 and TGFβ1, are closely associated with the pathogenesis of keloids, and fibroproliferation has been reported to be regulated—in part—by the ERK pathway, which is signaled by receptor tyrosine kinase phosphorylation by IGF-1. The PI3K-Akt signaling pathway was also found to be synchronously activated ,20,22. TWe propose that CS induces the activation of AKT in KFs via integrins . CS has Integrins are transmembrane receptors that activate intracellular signal transduction pathways, such as cell cycle regulation ,27, orgaFAK is a 125 kDa non-receptor protein kinase that directly binds to the cytoplasmic tail of β-integrin and which plays a significant role in integrin-mediated signaling. The biological importance of FAK-mediated signal transduction is understood by the fact that it plays a fundamental role in embryonic development, control of cell migration, and cell cycle progression . Recent Marlene et al. reported  that KFsSpecific types of CS have been reported to be responsible for the pathogenesis of diseases in neural cells ,39,40,41These observations suggest that CS plays a fundamental role in KF proliferation as a ligand of integrins. We showed that integrin α1 is overexpressed in keloids, which was consistent with the previous report . IntegriRather than as a growth factor reservoir or a scaffold of cell attachment, CS itself had an inductive role in the cellular proliferation of KF. Although we demonstrated that CS played a role in the proliferation of KFs, we could not rule out the possibility that another pathway (besides the integrins) produces intracellular proliferative signals, like contactin-1 in neural cells . The difRecently, Shida et al.  reportedIn conclusion, our data presented the mechanism responsible for the efficacy of CS degradation in keloid treatment . The actFive patients with keloids  and seven unrelated patients  undergoing surgical treatment between September 2014 and July 2016 were enrolled in this study. The present study was performed with approval from the Institutional Reviewing Committee in Kyoto University Faculty of Medicine , which adhered to the ethical standards as formulated in the Helsinki Declaration. Written informed consent was obtained from all patients. The skin tissue samples were obtained as surplus skin in plastic surgery. The sample information is shown in 3 pieces, placed in plastic tissue culture dishes, and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS), 10,000 U/mL penicillin G, and 10 mg/mL streptomycin sulfate. Cells were propagated at 37 °C, and semi-confluent cultures of fibroblasts were passaged by trypsinization up to two times before the analysis [Keloid fibroblasts and normal fibroblasts were extracted from surgical specimens by the explant method. Briefly, tissues were cut into 1 mm® Blot module (Thermo Fisher Scientific). The membrane was blocked with 5% non-fat dry milk  in Tris-based saline containing 0.05% Tween 20 (TBST) for 1 h at room temperature and then incubated with primary antibodies (at the dilution described in Trypsinized and counted cells were seeded in 6-well poly-D-Lysine (PDL)-coated cells at a density of 400,000 cells per well. After incubation with CS, fibroblasts were washed twice with ice-cold phosphate-buffered saline (PBS), and then harvested with RIPA buffer  containing phosphatase and protease inhibitors. The cell lysate was sonicated and cleared by centrifugation . The protein concentration of cell extracts was determined using a BCA protein assay kit . Cell lysate samples were separated by SDS-PAGE  and then blotted onto 0.20 µm nitrocellulose membranes  using the XcellTotal RNA was extracted from cells using RNeasy Micro Kits . First-strand cDNA was synthesized using a Prime Script RT reagent kit with a gDNA eraser . RT-PCR was performed with cDNA using a TaqMan Probe Assay (Thermo Fisher Scientific). Taqman Assay IDs Cyclin D1: Hs0076553_m1, integrin α 1: Hs00235006_m1, integrin α 2: Hs00158127_m1, integrin α 4: Hs00168433_m1, integrin α 5: Hs01547673_m1, integrin α v: Hs00233808_m1, integrin β 1: Hs01127536_m1, GAPDH: Hs02758991_g were used. The relative gene expression was normalized against an internal control, GAPDH. The expression of each gene was compared between depots using the ΔΔCt method.Serum-free DMEM containing KFs prepared as described above was added to 96-well PDL-coated plates. After 24 h of incubation, an anti-integrin α1 antibody  was added to the wells. Mouse anti-human IgG  was used as a control.Chondroitin sulfate  was obtained from Seikagaku Biobusiness Corporation  under a Material Transfer Agreement between Kyoto University and Seikagaku Biobusiness Corporation. We applied CS as a mixture of equal amounts of CS-A, CS-B, and CS-C, which was a similar composition to that observed in keloids, as we previously reported . All otht-test were used to analyze the results. P values of < 0.05 were considered to indicate statistical significance.Data were expressed as the mean ± SEM of at least three independent experiments. Unless otherwise stated, an ANOVA or two-tailed Student’s The present study showed that CS strongly stimulates the signaling to induce KF proliferation via the AKT pathway, which was successfully inhibited by blocking PI3K. Anti-integrin α1 antibodies blocked the proliferative effect of CS on KF. These findings provide a reasonable explanation for the rapid proliferation of KFs in vivo, where accumulated CS is abundant. We conclude that the excessive accumulation of CS and the elevated expression of α1 bata1 integrin and IGF-1R have a synergistic effect on KF proliferation and the overproduction of ECM components, such as CS, which plays an essential role in the etiology of keloids. The CS-enriched extracellular milieu may interact with KF and vice versa resulting in the deposition of CS into ECM. We consider CS to be a key molecule in the etiology of keloids. Further study is required because our experiment only used a 2-D in vitro model. The positive effect on fibroblast proliferation may increase in 3-D cultures.In conclusion, the KF-specific role of CS revealed in the present study represents a significant advance in the understanding of ECM-triggered proliferation, and has the potential to be applied in the development of treatments for keloids.


Sarcoidosis is a systemic, granulomatous, and noninfectious disease of unknown etiology. The clinical heterogeneity of the disease (targeted tissue(s), course of the disease, and therapy response) supports the idea that a multiplicity of trigger antigens may be involved. The pathogenesis of sarcoidosis is not yet completely understood, although in recent years, considerable efforts were put to develop novel experimental research models of sarcoidosis. In particular, sarcoidosis patient cells were used within in vitro 3D models to study their characteristics compared to control patients. Likewise, a series of transgenic mouse models were developed to highlight the role of particular signaling pathways in granuloma formation and persistence. The purpose of this review is to put in perspective the contributions of the most recent models in the understanding of sarcoidosis. NOD2 mutations were involved in the pathogenesis of sarcoidosis, as well as in Blau syndrome (BS) and early onset sarcoidosis (EOS) [FCRL1, IL23R) that could distinguish patients with or without Löfgren’s syndrome [Sarcoidosis is a systemic disease of unknown etiology that is characterized by the formation of immune granulomas in different organs, mainly the lungs, the lymphatic system, the skin, the eye, and the heart . The diais (EOS) ,5,6,7. Nis (EOS) . In a Swis (EOS) . Likewisis (EOS) . A gene–syndrome .The epidemiology and phenotype of sarcoidosis varies highly around the world across age, sex, ethnicity groups, environmental exposure, and socioeconomic status ,13,14,15Even if sarcoidosis is considered as a benign disease, it still represents an important issue considering that mortality is higher in patients with sarcoidosis than in the general population with an average age of death of 70.4 versus 76.2, respectively ,24. MortSarcoidosis presents a highly variable and unpredictable course. It is estimated that half of patients progress to spontaneous regression within a few years of diagnosis . The treSarcoidosis is distinct from other granulomatosis by the peculiarity of the granulomas location and distribution . LymphocM. tuberculosis, M. bovis, M. leprae, and M. avium [beryllium disease [Research models aim to be isomorphic , homologous (identification of the pathogenic mechanisms), and predictive . In sarcoidosis, the development of research models was initiated several years ago, with various degrees of success. Mainly in vitro and in vivo models brought some responses at the isomorphic and homologous levels, but they were not predictive yet. Generating sarcoidosis models is crucial considering excess mortality in sarcoidosis and the necessity to understand molecular mechanisms involved in sarcoidosis to develop alternatives therapies to corticotherapy. Developing experimental models is challenging for multiple reasons, including the following reasons: (1) causative agent(s) of the disease are unknown; (2) it is a systemic disease with different targeted organs for each patient and highly diverse presentations; (3) the multiplicity of cellular actors and their interactions; (4) genetic variability; and (5) the course of disease resolution is unpredictable amongst patients, indicating the influence of both environmental and genetic factors. Research models were developed using putative sarcoidosis causing agents, either antigens of infectious origin or inorganic particles. Several species of mycobacteria have been implicated as the origin of the immune reaction, notably M. avium . Exposur disease ,40,41,42 disease . Nonethe disease ,48,49,50 disease ,52,53,54The present review is not an exhaustive retrospective of all the literature on experimental granuloma formation, but it aims to put in perspective the contributions of the most recent models in the understanding of sarcoidosis.So far, in vitro models used peripheral blood mononuclear cells (PBMCs) i.e., a mixed population of peripheral monocytes and lymphocytes reproducing an immune milieu or isolated monocytes derived into differentiated macrophages. + monocytes, CD163+ mature macrophages, multinucleated giant cells (CD11b+), and T and B lymphocytes  generated granulomas-like structures composed of a mix of CD14iewed in ). AlthouThe multi-walled carbon nanotubes (MWCT) model was tested using non-adherent murine macrophages in 3D cultures to determine whether granulomas would arise independently of an immune context . Indeed,M. bovis bacillus Calmette–Guérin (BCG) [high Treg lymphocytes in granuloma formation [high FoxP3+ cells exhibited powerful antiproliferative activity, without completely inhibiting TNF-α production, indicating a role for Tregs in suppressing the early stages of autologous granuloma formation. However, functional differences were observed in CD4+CD25high cells from healthy subjects compared to those of patients with sarcoidosis. While healthy subjects’ CD4+CD25high cells accelerated granuloma formation, no changes in the time course of granuloma growth were observed with sarcoidosis patient cells, indicating a defect in the suppressive function of Tregs in sarcoidosis.Very few in vitro models were developed with PBMCs from patients with sarcoidosis. Taflin et al. cultured PBMCs from sarcoidosis patients with sepharose beads coated with in (BCG) . This stormation ,58. CD4+Epithelioid cells and multi-nucleated giant cells (MGC) derived from the monocyte–macrophage lineage are the main component cells intervening in the architecture of sarcoidosis granulomas. The differentiation of monocytes into MGC can be induced by various stimuli, including cytokines , growth factors, lectins, and muramyl dipeptide. Monocytes from patients with sarcoidosis stimulated by the supernatant of concanavalin A-stimulated mononuclear cells showed an enhanced potential to form MGCs compared to monocytes from healthy controls or patients with other granulomatous diseases . In the Mycobacterium tuberculosis (PPD)-coated polystyrene beads, or microparticles generated from Mycobacterium abscessus (MAB) cell walls. After 7 days of culture with the PPD model, granuloma-like multicellular aggregates were formed with the presence of macrophages in the center and peripheral lymphocytes. Crouser et al. showed that macrophages from sarcoidosis patients maintained a M2 phenotype in the presence of PPD beads compared to non-coated beads, compared to control patients for whom macrophages acquired M1 characteristics [IL13 gene expression profile in sarcoidosis patients compared to control patients in response to PPD beads [Recently, new in vitro models were developed using either PBMCs exposed to purified protein derivative of eristics . LikewisPD beads . In the PD beads . hi cells) were increased in sarcoidosis when compared with other ILD, whereas M2 macrophages (CD163 expressing cells) were similar [The polarization state of macrophages varies in various lung diseases and could depend on signals present within the microenvironment. While macrophage states are globally divided into classically (M1) and alternatively (M2) activated macrophages, their polarization appears as transient and reversible, depending on the milieu ,64. Data similar . On the  similar . Immunoh similar . Similar similar . Recentl similar . ObviousIn vivo models were developed in mice exposed to putative sarcoidosis-causing agents, either antigens of infectious origin or inorganic particles. In addition, two murine models of transgenic mice showed a spontaneous apparition of granulomatous with sarcoidosis-like aspects, indicating the potential role for particular genes/signaling pathways in sarcoidosis. (C. acnes) , which is a commensal strain of bacteria. Since sarcoidosis patients can be reactive to some mycobacterial proteins  [M. tuberculosis catalase-peroxidase (mKatG) and M. tuberculosis superoxide dismutase A (mSodA) induced pulmonary granulomatosis in mice [+ T lymphocytes with Th1 cytokine (IL-2 and IFNγ) production, and rare B lymphocytes. The presence of serum amyloid A (SAA), a highly inducible acute-phase reactant and amyloid precursor protein, was readily detected in CD68+ macrophages and multi-nucleated giant cells from sarcoidosis patient granulomas in various organs  and in mycobacterial KatG–bead–induced granulomas in mice [+ lymphocyte in part through a toll-like receptor 2 TLR2 signaling and correlated with tissue chronic inflammation and fibrosis. Huppertz et al. showed an increased expression of activated NLRP3 (NOD-like receptor (NLR) pyrin domain-containing protein 3) inflammasome components (caspase-1 and IL-1β) in lung granuloma, and increased IL-1β release of BAL cells from sarcoid patients [Mycobacterium tuberculosis, granuloma formation was decreased in -/-Nlrp3 mice and increased in -/-miR-223 (micro-RNA downregulating NLRP3) mice compared to wild-type mice. The two main in vivo models based on infectious agents followed different strategies: the use of mycobacterial antigens without provoking tuberculosis and the exposure to Cutibacterium acnes  or HSP)  and that or HSP) ,76,77,78 in mice ,80. Gran in mice . SAA exppatients . In a moC. acnes based on its detection in the lung and lymph nodes of patients [C. acnes-induced lung granulomatosis were published, each of them with differences in their experimental protocols [+T and CD45/B220+ B lymphocytes promoted by Th1 cytokines and chemokines  [C. acnes to induce pulmonary granulomatosis showed great variability concerning the mode of administration , the presence or not of adjuvant and the type of adjuvant used , the dose of C. acnes, the number of challenges, and the date of sacrifice. Several types of bacterial strains used in these experiments were either commercially provided (ATCC® 6919 and ATCC® 11828) [C. acnes in mice before immunization decreases pulmonary granulomatosis [C. acnes model, Werner et al. showed that the deletion of Myd88 or Cybb in mice increased the persistence of alive bacteria in the lung and enhanced granuloma formation, indicating that a defect of bacterial clearance through the impairment phagosome activity could participate in granuloma development [C. acnes [C. acnes induction of lung granulomas was reduced in both IL-17A gene knockout mice and mice receiving an IL-17A neutralizing antibody. To conclude, the difficulties in efficiently reproducing the C. acnes-induced pulmonary granulomatosis is probably dependent on the mechanism of repeated antigen recognition and the activation of the CD4 + lymphocyte toward a TH1 phenotype. This mechanism calls upon many factors, which are difficult to control despite the standardization of the conditions of animal facilities. In this sense, this model also responds to the random, multifactorial character described in sarcoidosis. The second model used patients ,83. Severotocols ,91,92,93IL12p70) ,91,92,93® 11828) ,90,91,92elopment , the TLRC. acnes . InteresC. acnes . C. acneBased on the possible association between carbon nanoparticles and sarcoid-like pulmonary granulomas, rodent models of chronic granulomatous inflammation were generated by the administration of MWCTs. After pulmonary exposure to MWCTs, a marked inflammatory response was present and associated with the development of granulomas and/or fibrosis ,96. DepeIn the pathology of sarcoidosis, alterations in circulating lipid levels are observed, notably the reduction in HDL-cholesterol levels and ApoA1 concentrations ,105,106.Peroxisome Proliferator-activated Receptor gamma) that controls the transcription of numerous genes implicated in differentiation, proliferation, and apoptosis. PPARγ was reduced in BAL lysates and nuclear PPARγ content in macrophages, suggesting that PPARγ could be an important negative regulator of inflammation in macrophages in sarcoidosis [Pparg specifically in the myeloid compartment. The loss of PPARγ expression in macrophages promoted a more pronounced granuloma formation and an elevated pro-inflammatory cytokine expression in mice [Pparg deletion in the myeloid compartment favored an increase in the Th1 cell population , and no changes in the Th2 population in BAL cells [EBF1) that could bind and activate the promoter of master regulators of adipocyte, including CCAAT-enhancer-binding proteins C/EBPα and PPARγ [IFNγ is a key mediator of granuloma formation in sarcoidosis. Barna et al. showed that this cytokine altered the expression of the transcription factor PPARγ  familial cases of sarcoidosis [de novo sarcoidosis after liver transplantation, the use of rapamune, an immunosuppressor that binds to the specific cytosolic protein FKPB-12 inhibiting mTOR activation, improved dramatically the patient status with both normal chest imaging and liver biopsy [A second model of transgenic mice spontaneously developing sarcoid-like granulomas was generated with the deletion of the inhibitor tuberous sclerosis complex 2 (Tsc2) in the myeloid lineage in mice. This model demonstrated a major role of a Tsc2 downstream target, rapamycin (mTOR) complex 1 (mTORC1) and its signaling pathway in the initiation and persistence of granulomas . Mice expatients  and an ecoidosis . Interesr biopsy .So far, the multiplicity of research model tried to replicate both pathological and histopathological features of sarcoidosis. However, very few were used as a predictive tool to test the toxicity/efficacy of potential new pharmaceutical molecules or combination of existing treatments.A major predictive model would be to test corticosteroids (CS) resistance. Failure to respond to CS therapy is a common feature of chronical inflammatory diseases, including sarcoidosis, asthma, inflammatory bowel diseases, rheumatoid arthritis, organ transplant rejection, and chemotherapy ,130,131.Recently, Zhang et al. evaluated the anti-inflammatory properties of α-melanocyte stimulating hormone (α-MSH) in the MAB model . α-MSH iRosiglitazone is a selective nuclear receptor agonist of PPARγ and is an antidiabetic agent of the class of thiazolidinediones. McPeek et al. used rosiglitazone to maintain high alveolar macrophage PPARγ levels in MWCNT-induced pulmonary granulomas. Mice receiving rosiglitazone had lower MWCNT-induced pulmonary granulomas in association with a reduced pro-inflammatory response, and sustained ABCG1 expression in macrophages, supporting the concept that PPARγ deficiency could play a role in sarcoidosis pathogenesis .In a different model of granulomatosis induced by Schistosoma Mansoni infection, Soliman et al. used resveratrol (RSV), a polyphenolic stilbenoid that is present in several plants . ResveraFinally, new perspectives of treatment should be considered in regard to the potential role of the NLRP3 inflammasome pathway in sarcoidosis. The use of MCC950, a potent and selective inhibitor of the NLRP3 inflammasome or anti-IL-1β antibody, reduced granuloma formation . Actual in vitro and in vivo models seek to understand the pathophysiology of the complex disease that is sarcoidosis. The need for models concerns in particular patients with poor outcomes. Future models should focus on the identification and the understanding of the mechanisms influencing the switch between spontaneous sarcoidosis resolution and treatments necessity as well as resistance/insensitivity to treatments.To date, research models allowed identifying and dissecting some potential interesting actors and their molecular pathways involved in antigen-driven cell aggregation/granuloma formation . We have-No spontaneous in vitro model of granuloma formation exists, and antigenic stimulation is required. Granuloma formation (in vitro and in vivo) is induced in response to agents  that are probably implicated in sarcoidosis, without certainty. In addition, certain models are more representative of foreign body granulomas (nanotube model) than sarcoidosis granulomas.-No experimental study was done on sarcoidosis patient cells that takes into consideration the epidemiological characteristics of sarcoidosis  to form distinct experimental sub-groups inside the “sarcoidosis group”. Exploration of the late stages of the disease is also missing, notably the comparison of different stages of sarcoidosis . The generation of cell lines and/or transgenic mice expressing polymorphisms identified in sarcoidosis patients may represent great tools to understand the influence of genetic variants in the pathology.-In vitro models are short-term studies due to the limited life span of certain cell types.-Granuloma dynamics cannot be studied in vitro as new cell recruitment from systemic origin does not occur.-Influence of the local environment  remains unknown.-Murine models of “sarcoidosis-like” granulomatosis present slightly different characteristics in terms of temporality and fibrotic evolution. It is interesting to note that exposure to sarcoidosis-causing agents generates only lung phenotypes, while genetically modified mice developed multiple sites of granulomatosis . In mice, local pulmonary exposure is not sufficient to spread the disease in other physiologic systems, suggesting that either the murine immune system is more efficient than humans to neutralize and eliminate pathogens, or that sarcoidosis patients are chronically exposed to one or multiple triggers, promoting granuloma development and persistence.-Most in vivo models are restricted to a pulmonary phenotype that does not recapitulate neither the human lung disease (no PAH has been observed) nor certain extrapulmonary lesions . Notably, no models of cardiac sarcoidosis or neurosarcoidosis exist, although they represent the two more severe organ complications after pulmonary damages.-Fibrotic lesions were rarely observed in the various in vivo models. In sarcoidosis, fibrosis begins at the periphery of sarcoid active granulomas. Chronic inflammation is probably responsible for fibrosis extension, resulting in larger collagen deposition, and fibroblasts/myofibroblasts differentiation causing parenchymal destruction. Recently, studies have identified several MMPs in BAL fluid and the granulomas of sarcoid patients ,158,159,-In silico experimentation involves the combination of biological data and mathematical and computer-based representations to granuloma models. Often, biological data are not sufficient or precise enough to establish proper mathematical models. Carrying computer-based experiments in combination with in vitro/in vivo research could facilitate not only the understanding of the disease but also the testing of new therapeutics. Humanized mice  are potential preclinical animal models for the study of human diseases. The engraftment of hematopoietic stem cells from sarcoidosis patients would address not only patient uniqueness  but also experimental differences between human and murine immune systems. The engraftment of microdissected human granulomas would allow studying granuloma biodynamics in an in vivo context and potentially drug testing. Ultimately, the use of humanized mice may lead to the achievement of personalized medicine. Very few in silico models were proposed so far ,165. In However, the generation of a valid and widely accepted experimental model that responds to all expectations is probably impossible. Models described so far have a number of weaknesses that would need to be addressed in futures studies:Although a great effort has been put into the development of various sarcoidosis models, there is still a lack of in vitro/in vivo models of sarcoidosis that recapitulate the human disease. A financing program was initiated by the Foundation of Sarcoidosis Research (FSR) to fund research projects developing novel experimental models to explore sarcoidosis pathogenesis and potentially the development of new therapies. Ideally, the molecular features of model granulomas would be conformed with human sarcoidosis tissue granulomas. In addition, the model should be relatively inexpensive, high-throughput, and easily manipulated experimentally. This type of sarcoidosis research model should benefit the testing of new pharmaceutical strategies intended to target a large number of patients. Nowadays, three lines of therapy are sequentially considered ,167. Cor