Earlier studies indicate a correlation between disability claims experience and return to work outcomes. Thus, the insurer’s role and actions may affect the self-rated health of the disabled worker and the outcomes of occupational rehabilitation. This study diversifies the existing empirical evidence on the role of the insurer in the rehabilitation process and reveals the critical actions that best promote success.n = 661) who had undergone an occupational rehabilitation within an earnings-related pension insurance system in Finland.Explorative factor analysis (EFA) and confirmatory factor analysis (CFA), followed by binary regression, were used to analyse survey data of disabled workers (The claimant’s perceptions of the insurer’s (1) high-quality informing and guidance, (2) customer orientation, (3) smooth process flow and (4) positive service attitude had substantial and statistically significant effects on the success of occupational rehabilitation after adjusting for all likely confounding variables.The insurer’s actions are significant predictors of the outcome of occupational rehabilitation. The insurer can promote the health of rehabilitees most effectively by ensuring a smooth process flow and adopting a customer-oriented approach.The online version contains supplementary material available at 10.1186/s12889-021-11758-w. In most developed countries, public compensation systems providing support for those unable to work because of illness or injury are usually governed by public institutions or private insurance providers. Many disability insurance systems only cover injuries or illnesses if they are due to a specific cause, including workers’ compensation, automobile insurance and crime victim’s insurance. These systems are referred to as cause-based systems. However, there are also disability-based systems that provide sickness and disability insurance for all forms of work disability, regardless of the cause .While there are huge disparities between and within nations when it comes to public disability insurance systems, they still have common features regarding the compensation process and the role of the administrative body . This organisation is usually responsible for the compensations and it interacts with all the stakeholders involved in the rehabilitation and return to work process –3.Today, the return to work (RTW) process after an illness or injury is understood as a biopsychosocial phenomenon . This meCurrent theories suggest that more positive relationships between the insurer and claimants may improve the worker’s chance of recovery , 10 and The results are similar between sick-listed individuals and social insurance officials in disability-based insurance schemes. Negative encounters with social insurance officers are negatively correlated with self-rated health, self-rated ability to work and mood, whereas positive encounters with social insurers facilitate RTW , 19–21.H1: Claimant’s perceptions of insurer actions during occupational rehabilitation are related to the outcome of rehabilitation.H2: Claimant’s perceptions of positive insurer actions, including high-quality informing, supportive service attitude and individual approach, are positively related to successful occupational rehabilitation.H3: The positive relationship between the claimant’s perceptions of positive insurer actions and successful occupational rehabilitation remains even if an adjustment is made for all likely confounding variables.As shown above, there is some evidence indicating a connection between insurer actions and the probability of RTW. However, there is a need for further evidence and a more comprehensive understanding of the insurer’s role and the specific actions that best support the health of disabled workers and promote RTW. While the earlier quantitative studies have concentrated on sick-listed patients or work injury victims, this article focuses on occupational rehabilitees, thus enlarging the scope and providing useful information to all rehabilitation stakeholders. Here, we do not collapse groups of stakeholders together , buThis section presents the key elements of the study design, describes the research setting and participants, defines the used variables, their handling and measurement and presents the statistical methods. Here, we follow the STROBE statement .We used a cross-sectional survey design to investigate the connection between the claimant’s perceptions of insurer actions and the outcome of occupational rehabilitation. The study population included all private sector wage earnersThe cross-sectional data were collected in 2017, about 2 years after the claimant’s rehabilitation case had been closed in the web service. This point of time was chosen because the occupational rehabilitation would probably be over by then for most claimants, and its outcome would be known. On the other hand, not too much time would have passed after the rehabilitation so that the claimants could still remember the insurer’s actions during the process.The study sample was obtained from KuntoutuNET, a Finnish online service for the processing and maintenance of customer data about occupational rehabilitation. This service is used as a communication tool between the subscribers  and producers of rehabilitation services . The insurers may choose to use service providers to assist in the planning and execution of occupational rehabilitation within an earnings-related pension scheme in Finland. According to the Service Network of Occupational Rehabilitation (2016), service providers are used to assist in the settling and planning of occupational rehabilitation when the application for rehabilitation compensation lacks a rehabilitation plan, employment or work trials in a former or current workplace are not possible or when the rehabilitee needs close guidance. The service providers are also used to assess the rehabilitee’s abilities and opportunities for employment or studying .The sample consisted of 2264 individuals and included all rehabilitees whose cases were closed in the KuntoutuNET online service in 2015. The data acquired from KuntoutuNET included the rehabilitee’s identification number, age, gender, and contact details (phone number and/or email-address). An online questionnaire was sent to the respondents on June 6, 2017, and reminder messages were delivered on June 13 and June 20, 2017. The data were collected using a text message and an e-mail survey. The survey was sent by e-mail to a total of 836 people whose e-mail addresses were stored in the KuntoutuNET online service. In addition, the survey was sent by text message to 1428 people, for whom only a telephone number was available.We took into account earlier empirical findings and considered potential effects of insurer actions on RTW outcomes when designing the questionnaire. In addition to questions related to sociodemographic variables and the process and outcomes of occupational rehabilitation, the questionnaire included altogether 26 propositions concerning the actions of the insurers and rehabilitation service providers. These propositions were formed on the basis of earlier studies , 12–22, To isolate the effect of insurer actions, we included several potential confounding factors in our regression models, for example age, employment situation before rehabilitation, level of education, occupation, monthly net income and cause of rehabilitation. Age was included as a continuous variable and employment situation before rehabilitation as a dichotomic variable. The level of education was grouped into six categories: primary school, secondary school, vocational college, general upper secondary school, and university. The occupation had also six categories: employee, lower official, senior officer, leading position, entrepreneur, and agricultural entrepreneur. The cause of rehabilitation had three categories: musculoskeletal diseases, mental disorders and other causes; and monthly net income had six categories: below 500 euros, 500–999 euros, 1000–1499 euros, 1500–2499 euros, 2500–3999 euros, over 4000 euros.Furthermore, we included the respondent’s motivation and the objectives of rehabilitation as possible confounding factors in our regression models. Motivation was measured on a 5-point Likert scale, the response alternatives ranging from 1  to 5 (extremely motivated). In addition, the questionnaire included six pre-formulated objectives for occupational rehabilitation: (1) return to work, (2) more suitable profession, (3) new job in earlier workplace, (4) improvement of professional capabilities, (5) improvement of mental well-being and (6) receiving a disability pension. The respondents were once more asked to use a 5-point Likert scale ranging from 1  to 5 (extremely important) to rate how important these objectives were to them before starting the process. Similar classifications regarding the motivation and objectives of rehabilitation have been used, for example, by Gould et al. .The most frequently used measures of rehabilitation outcomes are self-rated health or self-rated ability to return to work e.g. , 19, 20), 2019, 2The employment status after rehabilitation was not an ideal measure of rehabilitation success in our study, since the RTW rate was only 30% and a significant amount of respondents were still in rehabilitation  or unemployed. Therefore, we formed a binary indicator of rehabilitation success by combining the self-rated ability to work and return to work variables. The rehabilitation was regarded as successful if the respondent had fully returned to work successfully or if the self-rated ability to work was 7 (satisfactory) or higher. We considered the objective of occupational rehabilitation  to have been achieved in these situations.Since our aim was to explore the connections between insurer actions and successful rehabilitation, we decided to use factor analysis to determine the dimensions of the insurer’s role, and binary regression to explain the result of rehabilitation by factor scores. There were 661 observations which had responses in the relevant variables, and in 108 cases some of the variables had missing values. To include these partially missing observations, we used the full information maximum likelihood (FIML) method.The factor analysis was conducted in two steps: First, exploratory factor analysis (EFA) was used to determine the constructs or factors which best explain the observed correlations structure of the indicator variables, and, second, confirmatory factor analysis (CFA) was used to check the adequacy of the adopted factor model.In the explorative analysis we used principal-axis factoring (PAF) since the data were not normally distributed. According to Fabrigar, Wegener, MacCallum and Strahan , the priFurther, since the sample size was fairly large (549 observations after listwise deletion of missing data) and the results of EFA were almost identical to those obtained by the maximum likelihood estimation (MLE) method, we are confident that the results of CFA, which are based on the assumption of multivariate normality, are sufficiently accurate. Further, since the factors were obviously correlated, we used the promax rotation, which allows for such correlation. For EFA, we provide the factor loadings, means, standard deviations, Cronbach’s alphas, and average variance extracted (AVE) and construct reliability (CR) statistics for each measured item in Table 1 to F4) in order to separate the effects of the factors and to have a parsimonious correlation structure.In CFA, we first fitted a model corresponding to that from EFA. The resulting factor scores were used as independent variables in a logistic regression model explaining the success of rehabilitation. However, since the factor effects were ambiguous due to high factor correlation, we fitted another confirmatory factor model with independent factors  to account for the correlations between the variable groups representing the independent factors. Factor G would represent the individual effect of the respondents, that is, their tendency to answer in a similar way to all questions. The factor loadings of G on all indicators were set to be equal. Further, since factor FIn the next step, factor scores were computed using the regression method. It turned out that the distribution of the scores of factor G had a long-left tail Fig. . HoweverWe also studied whether the connection between the insurer’s actions and the outcome of occupational rehabilitation could be explained by confounding variables. We added several likely confounding factors in our logistic regression model, including gender, age, level of education, employment situation, occupation, monthly net income, cause of rehabilitation, motivation and rehabilitation objectives. The demographics were computed and the explorative factor analyses conducted using the SPSS Version 26.0 for Windows; the CFA was run using the package lavaan  of the R software , and the logistic regression using the R function glm.n = 661). Table The response rate of the survey was 29% n = 66. Table 1n = 395) of the respondents.Table We conducted an explorative factor analysis (EFA) with four common factors for the 17 insurer related propositions. All these items exhibited sufficient factor loadings and communalities (> .50) and were retained for further analysis. The four factors accounted for 93.37% of the total variance, and each factor explained at least 4.34% of the total variance, fulfilling the minimal requirements presented by Netemeyer et al. . Table 3The first factor is theorized to represent informing and guidance during the rehabilitation process (variables IG1–IG6), the second factor is related to customer oriented approach (variables CO1–CO5), the third factor describes the process flow (variables PF1– PF4), and the fourth factor indicates the service attitude of the insurance personnel (variables SA1–SA2). Altogether, the sizes of the factor loadings , AVE measurements , and CR scores  lend support to the convergent validity of each scale.2 = 310.8, df = 130, p = .000). When the resulting factor scores were used as independent variables in a logistic regression model explaining the success of rehabilitation, we could find support for hypothesis H1, which states that the claimant’s perceptions of insurer actions during occupational rehabilitation are related to the outcome of rehabilitation.Next, we conducted a confirmatory factor analysis (CFA) based on the results above. We first fitted a model with a free factor correlation structure and zero restrictions on factor loadings as indicated by EFA. The model fit was good  although the hypothesis of perfect fit was rejected . Finally, we fitted a factor model, with four independent factors as indicated by EFA, and one further independent factor accounting for the individual effect and correlation between the variable groups. The fit was almost as good as in the first case . The items’ squared multiple correlations (SMCs) ranged from 0.853 to 0.945.However, since it was not possible to separate the effects of the factors due to high factor correlations (ranging from 0.548 to 0.746), we fitted another model with uncorrelated factors. The model had an inferior and inadequate fit  all indicate excellent model fit. Obviously, this result supports our research hypothesis H2, which states that the claimant’s perceptions of positive insurer actions are positively related to successful occupational rehabilitation.Then, factor scores were computed using the regression method. These scores, excluding the individual effect score, were used as independent variables in a logistic regression model explaining the success of rehabilitation. Figure 2 values improved . The number of included observations was 608. Thus, we find support for the research hypothesis H3, according to which the positive relationship between the claimant’s perceptions of positive insurer actions and successful occupational rehabilitation remains even if an adjustment is made for all likely confounding variables.We also studied whether these results could be explained by confounding variables , which are background variables affecting both the independent variables and the outcome variable. We found that age, self-rated ability to work before rehabilitation, motivation, and the objectives of either professional development or receiving disability pension, along with the insurer factors, were statistically significant predictors of the rehabilitation outcome. Table Our purpose was to explore the connection between the insurer’s actions and the outcome of occupational rehabilitation. Factor scores measuring the dimensions of the insurer’s role were used as independent variables in a binary regression model explaining the success of rehabilitation. According to earlier studies, positive encounters with insurers improve the worker’s chance of recovery after an illness or injury and vice versa , 10. Our1), the claimant’s perceptions of positive insurer actions would be positively related to the success (H2) and the positive relationship would remain even if an adjustment was made for all likely confounding variables (H3). All these hypotheses were supported. The second objective of the study was to explore which insurer actions best promote success.Our results illustrate the multidimensional nature of the insurer’s role. Exploratory factor analysis suggested four dimensions for insurer actions. This was tested using confirmatory factor analysis, and factor scores from our final confirmatory model were used as input in a binary regression model. The first objective of the study was to examine the possible connection between insurer actions and the outcome of occupational rehabilitation. According to the research hypotheses, this type of connection would exist  informing and guidance, (2) customer orientation, (3) process flow and (4) service attitude. Second, we used binary regression to explain the result of rehabilitation by factor scores. The results showed that sufficient, adequate, accessible, understandable, and professional informing and guidance is one significant factor promoting a positive outcome of rehabilitation. The importance of high-quality information and professional expertise has been brought up by various scholars , 32, andThe insurer’s customer-oriented approach, which includes paying attention to customer needs and involving customers in the planning and decision-making processes, had a significant positive effect on the outcome of rehabilitation. While the importance of customer-oriented approach has been highlighted in previous studies , 27, 32,Several authors report friction between injured workers and insurers caused by delays in claims handling or claims manipulation , 33, 34,Earlier studies have reported both negative , 33 and We also tested whether confounding variables could explain the connection between insurer actions and the rehabilitation outcome. The results showed that besides insurer actions, also age, self-rated ability to work before rehabilitation, motivation and rehabilitation objectives were statistically significant predictors of the rehabilitation outcome. When adjusting for potential confounding variables, all insurer factors remained statistically significant and the changes in the regression coefficients were minor.The questionnaire was created purely for this study. It was built and the hypotheses were formulated on the basis of previous literature. However, there were limitations concerning the development and testing of models. First, the questionnaire was part of a larger survey related to the functioning of occupational rehabilitation. Thus, the focus of the researchers or respondents was not limited to this instrument. Second, the questionnaire had not been used or tested before. However, it was reviewed by external researchers and the steering group of the rehabilitation service network. After receiving feedback, the comments were evaluated, and the questionnaire was revised.The empirical testing was based on cross-sectional data gathered after occupational rehabilitation. Thus, memory errors and opinion changes are possible, which should be considered when interpreting the results. Furthermore, the Likert scale-based answer options of the survey questions might have been interpreted differently by the respondents. This tendency of giving systematically better or worse assessments on every question was taken into account in our final model by adding an individual effect factor (G). All criteria set for the validity of the factor model were met and all the hypotheses were supported by the data. In addition, the developed model helped verify the results of the earlier studies.The other factors undermining the reliability of the research are related to the representativeness and suitability of the research data and the reliability of the used methods. The final data set included 661 respondents and the response rate was 29%, which is quite typical of an online survey. In surveys, non-response always occurs, and it is difficult to set unambiguous criteria for acceptable loss. Yet, non-response bias and representation of the population of interest should be evaluated. Table Regarding the representation of the population of interest, we compared our actual sample to the population of all rehabilitees within an earnings-related pension in Finland in 2015 . The disHowever, the respondents could vary from these populations on an innumerable number of other factors that have not been tested. One major consideration is that our study sample only included rehabilitees who had a contact with a rehabilitation service provider. This choice is not random: a service provider is not usually involved in cases which are straightforward or easy to carry through. Therefore, our results may not fully generalize to a wider population of disabled workers.Regarding the actual sample size, it can be considered sufficient for the methods used. Musil et al.  suggesteFinally, the path coefficients in our two-stage model in Fig.  should bOur results suggest that the insurer’s actions are substantially associated with the outcome of occupational rehabilitation. These findings support and confirm previous research results and help assess the relative importance of different actions by providing effect estimates for them. As for practical implications, the study reveals that the insurer would benefit most from ensuring a smooth process flow and adopting a customer-oriented approach, since they are the most important insurer-related determinants of successful occupational rehabilitation.Additional file 1.


Germinal matrix hemorrhage (GMH) is defined by the rupture of immature blood vessels in the germinal matrix, where subsequent hemorrhage enters the subependymal zone and the cerebral lateral ventricles. The consequent blood clot has been identified as the causative factor of secondary brain injury, which triggers a series of complex parallel and sequential harmful mechanisms, including neuroinflammation. The orphan G-protein-coupled receptor 40 (GPR40), a free fatty acid (FFA) receptor 1, has been shown to exert anti-inflammatory effects when activated and improved outcomes in animal models of stroke. We aimed to investigate the anti-inflammatory effects of GPR40 and its underlying mechanisms after GMH.GMH model was induced in 7-day-old rat pups by an intraparenchymal injection of bacterial collagenase. GPR40 agonist, GW9508, was administered intranasally 1 h, 25 h, and 49 h after GMH induction. CRISPR targeting GPR40, PAK4, and KDM6B were administered through intracerebroventricular injection 48 h before GMH induction. Neurologic scores, microglia polarization, and brain morphology were evaluated by negative geotaxis, right reflex, rotarod test, foot fault test, Morris water maze, immunofluorescence staining, Western blots, and nissl staining respectfully.The results demonstrated that GW9508 improved neurological and morphological outcomes after GMH in the short  and long-term (days 21–27). However, the neuroprotective effects of treatment were abolished by GW1100, a selective GPR40 antagonist. GW9508 treatment increased populations of M2 microglia and decreased M1 microglia in periventricular areas 24 h after GMH induction. GW9508 upregulated the phosphorylation of PAK4, CREB, and protein level of KDM6B, CD206, IL-10, which was also met with the downregulation of inflammatory markers IL-1β and TNF-α. The mechanism study demonstrated that the knockdown of GPR40, PAK4, and KDM6B reversed the neuroprotective effects brought on by GW9508. This evidence suggests that GPR40/PAK4/CREB/KDM6B signaling pathway in microglia plays a role in the attenuation of neuroinflammation after GMH.In conclusion, the present study demonstrates that the activation of GPR40 attenuated GMH-induced neuroinflammation through the activation of the PAK4/CREB/KDM6B signaling pathway, and M2 microglia may be a major mediator of this effect. Thus, GPR40 may serve as a potential target in the reduction of the inflammatory response following GMH, thereby improving neurological outcomes in the short- and long-term. Germinal matrix hemorrhage (GMH) is the most common neurological disorder of preterm infants, which is associated with a high rate of disability and mortality . GMH resMicroglia are resident macrophages of the central nervous system, which have been shown to contribute to the acute inflammatory response after GMH and other hemorrhagic brain diseases , 5. AfteG-protein-coupled receptor 40 (GPR40), also known as free fatty acid (FFA) receptor 1, belongs to class A G-protein-coupled receptors (GPCRs) and has a high affinity for medium- and long-chain saturated or unsaturated free fatty acid. GPR40 is located in various diverse organs such as the brain, pancreas, spinal cord, and heart –18. SeveIn this study, we demonstrated that GPR40 agonism attenuated pro-inflammatory cytokines, through the promotion M2-like microglia which improved short- and long-term neurological deficits in neonatal rat models of GMH through the PAK4/CREB/KDM6B signaling pathway.All experiments were approved by the Institutional Animal Care and Use Committee at Loma Linda University and in compliance with the NIH Guidelines for the use of animals in neuroscience research. Two hundred and thirty-five P7 Sprague-Dawley neonatal pups  were used.The GMH model was induced by bacterial collagenase fusion as previously described in  . P7 neonThe current study was performed in four separate experiments.Forty-two P7 rat pups were randomly divided into seven groups (n = 6 per group): Sham and GMH . The whole brains of six rats from each group were collected for western blotting to determine the protein levels of GPR40, p-PAK4, PAK4, KDM6B, CD16, and CD206. The double immunofluorescence staining of GPR40 or KDM6B were used in the sham group and GMH (24 h) group (n = 3 per group) to evaluate the cellular localization of GPR40 or KDM6B in microglia, neurons, and astrocytes.Thirty P7 neonatal rat pups were randomly assigned to five groups (n = 6 per group): Sham; GMH + vehicle (10% dimethyl sulfide), GMH + GW9508 (0.84 mg/kg), GMH+ GW9508 (2.5 mg/kg), and GMH+ GW9508 (7.5 mg/kg). GW9508 was administrated intranasally (i.n.) 1 h, 25 h and 49 h after GMH. Neurological behavior tests were measured at 24 h, 48 h, and 72 h after GMH. Randomly, fifteen more rat pups were divided into three experimental groups with n = 5 for each group: Sham, GMH + vehicle (10% dimethyl sulfide), and GMH + GW9508 (best dose). These three groups were evaluated for microglia polarization by double immunofluorescence staining. The best dose of GW9508 was chosen based on the short-term neurological outcomes for the long-term outcome and mechanism experiments.Sixty animals were randomly subjected to four groups (n = 15 per group): Sham, GMH + vehicle (10% dimethyl sulfide), GMH + GW9508, and GMH + GW9508 + GW1100 for evaluation of long-term neurological scores and histological result. GW9508 was administrated intranasally (i.n.) 1 h, 25 h, and 49 h after GMH. GW1100 was administrated intranasally (i.n.) 1 h before, and 23 h, 47 h after GMH. The rotarod and foot fault tests were performed on day 21 post-GMH. Morris water maze test was carried out on days 22–27 following GMH. Brain tissues were then obtained for Nissl staining to evaluate morphological changes.To investigate the potential molecular mechanism of GPR40 activation, 42 rats were randomly divided to seven groups (n = 6 per group): Sham, GMH + vehicle , GMH + GW9508, GMH + GW9508 + scrambled CRISPR, GMH + GW9508 + GPR40 K.O. CRISPR, GMH + GW9508 + PAK4 K.O. CRISPR, GMH + GW9508 + KDM6B K.O. CRISPR. Furthermore, to determine the CRISPR/Cas9 editing efficiency, another 40 rats (n = 5 for each group) were randomly allocated into eight groups: naive + Scrambled CRISPR, naive + GPR40 K.O. CRISPR, naive + PAK4 K.O. CRISPR, naive + KDM6B K.O. CRISPR, GMH + scrambled CRISPR, GMH + GPR40 K.O. CRISPR, GMH + PAK4 K.O. CRISPR, GMH + KDM6B K.O. CRISPR. The whole brain was used for WB detection after neurological test.Intranasal drug administration was conducted at 1 h, 25 h, and 49 h after GMH induction as described in  . A volumIntracerebroventricular (ICV) drug administration was delivered as previously described in  . BrieflyNegative geotaxis and right reflex were performed at 24 h, 48 h, and 72 h post-GMH as previously reported in  . NegativRotarod test2.Foot fault testTwenty-eight days post-ictus, rotarod test was conducted to assess the motor impairment as previously described in  . The dur3.Morris water mazeFoot fault test was employed to assess changes in locomotor function . The numThe Morris water maze was used to evaluate spatial learning and memory at days 23–27 after GMH . The watImmunofluorescence was conducted as previously reported in  , 38. AniWestern blot was conducted as previously reported –43. The micrometer-thick brain sections were stained with 0.5% cresyl violet (Sigma-Aldrich), and the volume of ventricular and cortical thickness was calculated using ImageJ . The volume was calculated with the following formula: average  × section interval × number of sections as described in  [At 28 days post-ictus, the rats were euthanized and whole brains were extracted. Nissl staining was conducted and analyzed as previously reported in . Sixten-., 2016) .p < 0.05 was considered to be statistically significant.All data are shown as the mean ± SD. GraphPad Prism  was applied to analyze the data as previously described in  . One-wayA time-course study was conducted to measure the endogenous expression levels of GPR40, p-PAK4, PAK4, and KDM6B after GMH via western blot. The expression of endogenous GPR40 decreased at 3 h and reached its lowest level at 24 h after GMH Fig. a, b. HowFurthermore, double immunofluorescence staining of GPR40 or KDM6B with Iba-1 (microglia marker), GFAP (astrocytes), or NeuN (neurons) indicated that GPR40 and KDM6B were expressed in microglia Figs. a, b, neuEndogenous CD16 expression initially peaked at 3 h after GMH  demonstrated significant neurobehavioral deficits in the GMH + vehicle group in the first 3 days after GMH induction when compared to the Sham group Fig. a–c. GW95+/CD11b+ cells) in the GMH + vehicle group was significantly increased when compared with the sham group at 24 h after GMH  the expression of GPR40, p-PAK4, and KDM6B decreased at 3 h and reached the lowest level of expression at 24 h after GMH; the expression of M1 marker CD16 was significantly upregulated at 3 h after GMH, while M2 marker CD206 expression had a delayed increase in expression after GMH; (2) GPR40 and KDM6B were found to be expressed in neurons, astrocytes, and microglia at 24 h after GMH; (3) GW9508 at a dose of 2.5 mg/kg improved neurological outcomes at 24 h after GMH; (4) GW9508 attenuated neuroinflammation and decreased M1 and increased M2 microglia populations at 24 h after GMH; (5) GW9508 improved long-term neurobehavior impairments and preserved brain morphology brought on by GMH; (6) and lastly, GPR40 agonism attenuated neuroinflammation and regulated microglia polarization via the PAK4/CREB/KDM6B signaling pathway.GPR40, also known as free fatty acid receptor 1 (FFAR1), has been shown to be highly expressed in microglia, astrocytes, and neurons in the CNS . ActivatKDM6B (JMJD3) is one of two epigenetic modifiers responsible for the enzymatic removal of the repressive chromatin mark histone H3 lysine 27 trimethylation (H3K27me3). The demethylation of H3K27me3 is catalyzed by KDM6B which acts as a response gene to inflammatory cytokines and regulates macrophage polarization . Suppres+ cells and decreased CD16+ cells in GW9508-treated GMH pups compared to the vehicle group. Our data suggest that the anti-inflammatory effects of GPR40 activation are attributed through microglia polarization.Microglia are a unique cell type of immune/myeloid cells in the central nervous system . StudiesWe further explored the molecular mechanisms underlying the anti-inflammatory effects of GPR40 activation after GMH. The p21-activated kinases (PAKs) are a family of protein serine/threonine kinases . InitialThe present study has several limitations. First, previous studies and our results revealed that GPR40 was also found to be localized in astrocytes and neurons. Yet this manuscript only focused on the mechanism of action of GPR40 in microglia cells, and further studies need to be conducted to establish the role of GPR40 on other cell types. Second, we verified a novel mechanism of the PAK4/CREB/KDM6B signaling pathway in the anti-inflammatory effect of GPR40 activation after GMH. However, other signaling pathways involved in the anti-inflammatory process such as AMPK/PLC/IP3 and TLR4/NF-κB as well as ARRB2/NLRP3 need to be elucidated in future studies after GMH –59. ThirIn conclusion, this study is the first to demonstrate that GPR40 agonism via GW9508 ameliorated neurobehavioral impairments by attenuating neuroinflammation and regulating microglia polarization after GMH in neonatal rats. The anti-inflammatory and microglia polarization effects of GPR40 agonism were mediated, at least in part, through the PAK4/CREB/KDM6B signaling pathway. Therefore, GPR40 demonstrates promise in being a potential non-invasive target to attenuate neuroinflammation after GMH.


We investigated the clinical relevance of urinary cytokines/chemokines reflecting intrarenal immunologic micromilieu as prognostic markers and the optimal measurement timing after living donor kidney transplantation (LDKT). This prospective cohort study included 77 LDKT patients who were followed for ≥ 5 years. Patients were divided into control (n = 42) or acute rejection  group. Early AR was defined as AR occurring within 3 months. Serum and urine cytokines/chemokines were measured serially as follows: intraoperative, 8/24/72 h, 1 week, 3 months, and 1 year after LDKT. Intrarenal total leukocytes, T cells, and B cells were analyzed with immunohistochemistry followed by tissueFAXS. Urinary MCP-1 and fractalkine were also analyzed in a validation cohort. Urinary MCP-1 after one week was higher in the AR group. Urinary MCP-1, fractalkine, TNF-α, RANTES, and IL-6 after one week were significantly higher in the early AR group. Intrarenal total leukocytes and T cells were elevated in the AR group compared with the control group. Urinary fractalkine, MCP-1, and IL-10 showed positive correlation with intrarenal leukocyte infiltration. Post-KT 1 week urinary MCP-1 showed predictive value in the validation cohort. One-week post-KT urinary MCP-1 may be used as a noninvasive diagnostic marker for predicting AR after LDKT. Therefore, non-invasive surrogate markers for early diagnosis and treatment of AR are required.Kidney transplantation (KT) is the final and definitive treatment option for end-stage kidney disease (ESKD) patients. Early prediction of acute rejection (AR) and overall renal allograft function is crucial for appropriate management considering long-term outcome of KT. Although kidney biopsy is the gold standard to diagnose rejection in KT patients4. Urinary cytokines and chemokines have been reported as promising biomarkers substituting kidney biopsy because urine was expected to contain inflammatory mediators involved in alloimmune responses leading to AR or interstitial fibrosis/tubular atrophy after KT8. Since the association of pretransplant serum levels of CXCL9 and AR was reported9, urinary CXCL9 at 6 months post-KT was suggested as a promising biomarker in KT patients10. If specific urine cytokines/chemokines can be used as surrogate markers substituting kidney biopsy, especially during the early postoperative period, it would be very helpful for both KT patients and physicians.With the advance of molecular biology, a number of biomarkers have been evaluated for prediction, diagnosis, and risk stratification in KT patients. However, there is no consensus regarding specific types of noninvasive biomarkers and timing of sample acquisition in KT patientsIn this study, we aimed to evaluate the diagnostic and prognostic potential of urinary cytokines/chemokines at different time points after KT and to identify specific cytokines/chemokines reflecting intrarenal immunologic micromilieu as well as the optimal sample acquisition time for early diagnosis of AR.Table Changes in renal function were assessed as serial changes in eGFR over 5 years. Renal function of the AR group was comparable with the control group until 3 months after KT. The mean eGFR from 2 years after KT was significantly lower in the AR group compared with the control group. (P < 0.05) Fig. .Figure 1Urinary level of MCP-1 at 1 week after KT was higher in the AR group Fig. a. To comSerial changes in serum cytokines/chemokines according to AR are shown in Fig. Intrarenal infiltration of total leukocytes, T cells, and B cells was analyzed in renal allograft biopsy tissues using immunohistochemistry with anti-CD45, anti-CD3, and anti-CD20. Total leukocytes, T cells, and B cells were significantly elevated in the AR group . The area under the receiver operating characteristic (AUROC) of urinary MCP-1 predicting early AR and AR was 0.795  0.659–0.931) and 0.691 (95% CI 0.563–0.818), respectively  and intraclass correlation coefficient (ICC), respectively. MCP-1 and fractalkine showed low variability (CV = 0.181 and 0.091), and high agreement  In this study, the diagnostic and prognostic potentials of several serum and urinary cytokines/chemokines were analyzed with serially collected urine samples after LDKT. Urinary MCP-1 at 1 week after KT was identified as an important predictive factor of AR development. Urinary fractalkine, TNF-α, RANTES, and IL-6 at 1 week after KT showed predictive potential for early AR within 3 months post-KT. Urinary fractalkine, MCP-1, and IL-10 exhibited positive correlation with intrarenal total leukocytes, T cells, and B cells. These results suggest clinical usefulness of urinary cytokines/chemokines for early diagnosis of AR in LDKT patients and support the potential role of urinary cytokines/chemokines as surrogate markers of intrarenal immunologic micromilieu.14. Therefore, new biomarkers for predicting changes in intrarenal immunologic micromilieu after KT are required15. Ideal biomarkers should be measured reproducibly in noninvasively collected samples. Urine biomarkers are ideal for immune monitoring after KT because they are directly produced by the allograft and can be collected noninvasively and repeatedly. Cytokines and chemokines have been reported to play crucial roles in controlling immune responses within renal allografts16. After KT, proinflammatory cytokines are released from allografts and subsequently induce local chemokine secretion18. Based on urinary cytokines/chemokines can reflect overall changes in intrarenal cytokines/chemokines, our study aimed to find the most appropriate timing and types of urinary cytokines/chemokines during the early postoperative period for predicting AR.AR is a serious problem after KT and exerts significant impacts on both allograft and patient survival. Early diagnosis and treatment of AR are crucial for long-term allograft survival. Although kidney biopsy is the gold standard for assessing kidney injury, timely detection of early alterations in allograft immunologic micromilieu has not been feasible with current techniques20. MCP-1 and RANTES, important members of the C–C chemokine family, are known to substantially contribute to glomerular and tubulointerstitial damage in diabetic nephropathy through a positive feedback loop of inflammation21. Blocking of the MCP-1/CCR2 chemokine pathway was reported to improve survival of islet allografts22. Intrarenal tissue expression of RANTES and MCP-1 was elevated in KT patients with chronic allograft dysfunction23. Urine MCP-1 was also reported to be elevated in patients with AR25 or polyomavirus nephropathy26. However, most previous studies investigated these cytokines/chemokines using a cross-sectional study design. Our results support these previous findings and further reveal the diagnostic potential of urinary MCP-1 as a surrogate biomarker for predicting AR via serial measurement.In our study, urinary fractalkine, MCP-1, RANTES, TNF-α, and IL-6 at 1 week after KT were significantly higher in patients who developed early AR within 3 months post-KT. Urinary MCP-1 at 1 week post-KT showed the most prominent increment in early AR patients compared with the control group. Elevation of serum proinflammatory cytokines and chemokines depending on allograft status was previously reported in KT patients27. Fractalkine expression was significantly higher in the tubular epithelium and endothelium of the allografts in AR patients28. Increased excretion of urinary TNF-α and IL-6 was reported in patients with AR31. Although our findings were consistent with previous reports and further revealed that excretion of these proinflammatory cytokines/chemokines increased early 1 week after KT in patients at risk for AR, external validation of urinary fractalkine using an independent cohort did not show reproducible results. Further studies in a larger cohort may be required to evaluate the clinical value of urinary fractalkine as a biomarker in LDKT patients.Urinary fractalkine at 1 week after KT showed not only diagnostic potential for predicting early AR, but also a positive correlation with intrarenal T cells and B cells in protocol biopsy. Fractalkine and its interaction with receptors on leukocytes mediate chemoattraction and play an important role in recruiting NK cells into cardiac allografts33. Increased infiltrating leukocytes into renal allografts and activated intrarenal T cells were reported in AR34. In our study, the AR group showed more enhanced infiltration of total leukocytes, T cells, and B cells into renal allografts. Urinary fractalkine, MCP-1, and IL-10 showed significant correlation with intrarenal infiltration of total leukocytes, T cells, and B cells. A previous study analyzing the mechanisms of AR in a baboon KT model reported a significant correlation between urinary excretion and intrarenal expression of interferon-γ inducible protein-10 and monokine35. Our study supports the clinical usefulness of urinary cytokines/chemokines, especially urinary fractalkine and MCP-1, as surrogate biomarkers that reflect the intrarenal immunologic micromilieu.The activity and subsets of intrarenal leukocytes are known to change actively within renal allografts and create a specific intrarenal immune environment such as renal tissue memory T cells39, and dividing the raw concentration of urinary cytokines/chemokines by the urinary protein-to-creatinine ratio might be a more appropriate method40. However, correction for urinary concentration differences using urinary creatinine concentration has been widely used in previous studies investigating urine biomarkers42, and using the urinary protein-to-creatinine ratio as a denominator can cause other confounding effects because the total amount of urinary protein excretion per se already includes urinary cytokines/chemokines. We also protocolized the procedures for urine collection, storage, and analysis since standardized management of urine samples is critical for consistent results. Third, some discrepancies in the types of cytokines/chemokines showing correlations with AR or intrarenal leukocytes were found. Further studies including a large cohort of KT patients are required to confirm the types of urinary cytokines/chemokines that more precisely reflect both cellular and humoral factors of intrarenal immunologic micromilieu. Fourth, a power study estimation could not be performed in our original cohort because of the prospective design and serial measurements of multiple serum and urine samples. The validation cohort was designed with a power study estimation to overcome this limitation.A few limitations of this study require consideration. First, there were some time differences in the collection of urine samples and kidney biopsies, ranging from 5 days to 1 month, which was inevitable given the prospective study design and serial collection of urine samples. Second, dividing the raw concentration of urinary cytokines/chemokines by urinary creatinine concentration might not be ideal for correcting for differences in urine concentration. Diverse ranges of proteinuria have been reported in KT patientsIn conclusion, urinary MCP-1 at 1 week after KT can be used as a noninvasive surrogate marker for predicting early AR in LDKT patients. Moreover, positive correlation with intrarenal leukocytes infiltration and urinary fractalkine, MCP-1, and IL-10 suggests the clinical usefulness of these urinary cytokines/chemokines as biomarkers that reflect the intrarenal immunologic micromilieu.This prospective cohort study recruited 77 adult patients who underwent living donor KT (LDKT) from April 2011 to August 2013 at Samsung Medical Center and were followed-up for more than 5 years. Adult (≥ 18 years) LDKT patients with no history of cancer whose urine samples were collected at each time point were included in the study. Exclusion criteria were (1) serum samples missing at more than 3 time points, (2) patients with autosomal-dominant polycystic kidney disease who received simultaneous bilateral nephrectomy of native kidneys with LDKT, and (3) significant bleeding or infection during the immediate postoperative period.11.Validation cohort for post-KT 1 week urinary MCP-1 and fractalkine included 79 adult patients who underwent LDKT from September 2013 to December 2015. Sample size was determined to achieve 80% power at a 0.05 significance level considering the incidence of AR and early AR This study was approved by the Institutional Review Board of Samsung Medical Center in compliance with the Declaration of Helsinki (IRB number: 2011-06-117). Informed consent was obtained from all participants before enrollment.Clinical data including baseline characteristics, induction therapy for KT, immunologic risk factors, and serial changes in renal function were collected from the electronic medical record system. Immunologic risk factors included the presence of ABO incompatibility, panel reactive antibodies (PRA), and donor specific antibody (DSA).2. There were no cases of antibody-mediated rejection. Early AR was defined as AR within 3 months. Renal function was compared with the estimated glomerular filtrate rate (eGFR), which was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.Patients who did not experience AR were classified into the control group and those who developed acute cellular rejection were classified as the AR group. Renal allograft biopsy was used as the diagnostic standard and AR was scored according to the Banff criteriaBoth urine and serum samples were serially collected at the following time points to measure cytokines/chemokines: intraoperative (during KT) and postoperative 8 h, 24 h, 72 h, 1 week, 3 months, and 1 year. The collected urine samples were centrifuged at 2500 rpm for 15 min under − 4 °C. The supernatant of each sample was aliquoted with 1.4 mL of urine and 1.0 mL of serum. After adding 1/100 volume of protease inhibitor and 0.1 M PMSF  to each sample, all samples were stored at − 70 °C until analysis. A total of 8 cytokines/chemokines were measured in each sample as follows: regulated on activation, normal T cell expressed and secreted (RANTES), fractalkine, interleukin (IL)-4, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF). These 8 cytokines/chemokines were measured using a Milliplex MAP human cytokine/chemokine kit  according to the manufacturer’s instructions. In the validation cohort, urinary MCP-1 and fractalkine were measured using Quantikine ELISA Kits  according to the manufacturer’s instructions. The concentrations of urinary cytokines/chemokines (pg/mL) were normalized by dividing the raw value by urinary creatinine concentration (mg/dL). Therefore, final values of urinary cytokines/chemokines are expressed as pg/mg.45.Renal allograft biopsy was performed between 12 and 14 days after KT as protocol biopsies in 37 patients or when AR was suspected in 9 patients. Immunohistochemical staining with CD45, CD3, and CD20 was performed on formalin-fixed kidney tissues. Sections (4 μm) were deparaffinized with xylene, rehydrated in a graded alcohol series, and then placed in citrate buffer solution (pH 6.0). Slides were placed in a pressure cooker and heated with microwaves for 10 min to enhance antigen retrieval. After cooling, sections were immersed in hydrogen peroxide solution  for 30 min to block endogenous peroxidase and then treated with serum-free protein block (DAKO) at 4 °C overnight. The next day, slides were incubated for 1 h at room temperature with a 1:100 dilution of monoclonal antibody to CD45, CD3, and CD20. After incubation of slides with a mixed solution containing dextran coupled with peroxidase molecules and goat secondary antibody molecules (DAKO) for 30 min at room temperature, 3,3′-diaminobenzidine tetrahydrochloride (DAKO) was applied to the slides to develop a brown color. Then, slides were counterstained with Mayer’s hematoxylin (DAKO). To calculate the percentage of CD45-, CD3-, and CD20-positive cells among total nucleated cells in renal allografts, the whole field of the slide was scanned and analyzed with TissueFAXS. All results were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM) as appropriate. SPSS 24.0 statistical software  and GraphPad Prism 9  were used for statistical analyses. Group means were compared using the Mann–Whitney test. The Chi-square test or Fisher's exact test was used for comparing baseline characteristics between the control and AR groups. The correlations between each cytokine/chemokine and intrarenal leukocytes, and post-KT 1 week urinary and serum MCP-1 or fractalkine were assessed using the Pearson R correlation. Internal validation of post-KT urinary MCP-1 and fractalkine was performed using bootstrapping (bootstrap B = 1000) by calculating AUROC.11. Reliability of measurements was evaluated with coefficient of variation (CV) and intraclass correlation coefficient (ICC)13.Power analysis was performed using PASS software Statistical significance was determined when the P value was < 0.05.


These distinctive genera were significantly associated with the change of AR clinical indices and the predicted functional pathways such as PPAR signaling pathway, peroxisome, and citrate cycle (TCA cycle) (p<0.05), indicating that they may be important bacterial signatures involving in the sustained remission in AR (p<0.05). Besides, lower Firmicutes/Bacteroidetes (F/B) ratio at 6 months follow-up may also contribute to the long-term remission of AR. No seriously adverse events and safety concerns were observed in this study. In conclusion, XQLD is a meaningful, long-term efficient and safe medication for AR treatment. The underlying mechanisms of sustained remission in AR after XQLD treatment may be associated with the dynamic alteration of featured gut bacteria taxa.A mixed Chinese herbal formula, Xiao-Qing-Long-Decoction (XQLD), may contribute to sustained remission in allergic rhinitis (AR), but it is unknown which factors determine such long-term effect. Here, we aimed to identify bacterial signatures associated with sustained remission. To this end, samples from AR patients at four different times were analyzed to compare the dynamic bacterial community and structure shifts. Diversity indices Chao1 showed significant difference across different time ( The complex ecosystem of the commensal microbiota and its interactions with the host are fundamental in determining health . Gut micvia modulating the gut microbiota , also known as So-Chenog-Ryong-Tang in Korea and Sho-seiryu-to in Japan, is a mixed Chinese herbal formula that has been commonly used for AR in China . The useThe study was a case series study with 10-week treatment and 24-week follow-up that was approved by the Ethics Committee of Xiamen Hospital of Traditional Chinese Medicine (Permit Number ID: 2019-K020-01), and later it was registered in Chinese Clinical Trial Registry (ChiCTR1900028613). Participants were recruited by Xiamen Hospital of Traditional Chinese Medicine  or Xiamen University Hospital  from December 2019 to September 2020. All participants signed informed consent forms before beginning the study. The study was conducted in accordance with the principles of the Declaration of Helsinki (via screening patients who randomly visit the authors’ (LZ) TCM clinic or (LT and BY) otorhinolaryngology clinic. The inclusion and exclusion criteria for AR subjects were the same as the previous study  Baillon Table S2The following primary efficacy outcomes were used: changes in Total Nasal Symptom Score (TNSS) and gut microbiota. Secondary efficacy outcomes included changes in Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ), eosinophil (EO) counts, cytokines , and tIgE. The TNSS is calculated based on the sum up of nasal obstruction, rhinorrhea, nasal itching, and sneezing, which represents perspectives of AR symptom, that is, the higher score means the worse symptom condition. RQLQ score, which consists of activity limitation, sleep disorders, non-eye/nasal symptoms, practical problem, nasal symptom, eye symptoms, and emotions, reflects the life quality status that was associated with AR. The value for each score and RQLQ positively correlated with the extent of the AR impact on the life quality. The safety evaluation included the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine, routine urine test, and complete blood counts (CBC). All study assessments were performed at 0, 10, 14, and 34 weeks. Registered TCM Practitioner (LZ) asked participants questions regarding adverse events (AEs) during every visit and recorded AE details in case report forms.Biochemical measurements of AST, ALT, BUN, creatinine, routine urine test, and CBC were performed in the laboratory of Xiamen University Hospital. Cytokines including IL-2, TNF-α, IFN-γ, IL-4, IL-6, IL-10 were performed in the laboratory of hematology department ; tIgE was performed in the laboratory of rheumatology and immunology department . Cytokines were measured by immunofluorescence technique . ELISA Kits were used to measure tIgE . Automatic hematology analyzer was used to measure CBC . The urine was detected and analyzed by dry chemical test strip . AST, ALT, BUN, and creatinine were measured by automatic biochemical analyzer .Fecal samples from AR patients were collected on the day of the medical examination and immediately frozen at −80°C before use. Fecal DNA (from approximately 0.25 g subsamples) was extracted using the QIAamp Fast DNA Stool Mini Kit . The purity and concentration of the isolated DNAs were assessed using spectrophotometry . The V3-V4 hypervariable region of the bacterial 16S ribosomal RNA (rRNA) gene was amplified from the DNA samples with the barcoded forward primers 341F (5’-CCTACGGGNBGCASCAG-3’) and the reverse primers 806R (5’-GGACTACNVGGGTWTCTAAT-3’) using KAPAHIFI HotStart ReadyMix . The raw paired-end reads were assembled using the flash . The chip<0.05). Bacterial alpha diversity, community ordination based on Bray-Curtis distance, PERMANOVA and ANOSIM analysis were based on the R package vegan (version 2.5.5)  . Non-metn 2.5.5) , 27. Prin 2.5.5) . Spearman 2.5.5) .From December 2019 to September 2020, a total of 53 patients were screened and 33 patients completed the whole study. Among these participants, 25 completed the pretreatment assessment and post-treatment assessment, of which 11 participants completed 1 month follow-up assessment; 8 participants completed the pretreatment assessment and 6 months follow-up assessment. We didn’t schedule the post-treatment assessment for these eight participants until the COVID-19 in China was well controlled, which was 6 months later after the end of treatment (p<0.05). The TNSS decreased from 8.30 ± 2.32 at week 0 to 1.79 ± 2.32 at week 10 to 2.64 ± 1.86 at week 14, and 5.25 ± 3.15 at week 34 (p<0.05) (p<0.05)  (p<0.05) (p<0.05) , while not for Shannon and Pielou’s evenness indices (J) (p>0.05) (p<0.05), while it rose again at week 34 (p<0.05) , average of 70,796 OTU sequence/sample, and an average 374 OTUs were obtained from 77 samples Table S32 = 0.094, p<0.05) and PERMANOVA analyses  and further graphed by Bray-Curtis distance based PCoA and NMDS plot  at different time points. Dialister (OTU_31), Roseburia (OTU_36), Alloprevotella (OTU_62), Alistipes (OTU_19), Bacteroides (OTU_30), Prevotella_2 (OTU_80), Prevotella_9 (OTU_68), Bacteroides (OTU_2040), Bacteroides (OTU_10), and Prevotella_9 (OTU_5) were the top 10 ranked distinguished genera (relative abundance≥0.1)  decreased to the lowest after treatment, but gradually increased over time (p<0.05). In contrast, the relative abundance of Roseburia (OTU_36), Bacteroides (OTU_2040), and Bacteroides (OTU_22) increased to the highest after treatment, but gradually decreased over time (p<0.05). The abundance of Alloprevotella (OTU_62) and Bacteroides (OTU_30) decreased until week 14, but raised again at week 34 (p<0.05). The lowest abundance of Alistipes (OTU_19) was observed at week 14 (p<0.05), and no significant difference was observed at other time points. The relative abundance of Prevotella_2 (OTU_80), Bacteroides (OTU_10), and Prevotella_9 (OTU_5) gradually decreased over time (p<0.05). The relative abundance of Prevotella_9 (OTU_68) increased at week 10 and then decreased at week 14 and increased again at week 34 (p<0.05)  Table S6p<0.05) (p<0.05) , cytochrome P450, PPAR signaling pathway, adipocytokine signaling pathway, peroxisome, and amoebiasis. Then the Kruskal-Wallis test was made to compare the significantly distinguished features, and nine of them were screened out (p<0.05) Table S7Prevotella_9 (OTU_5) was negatively related with meiosis-yeast, alpha-linolenic acid metabolism, PPAR signaling pathway, adipocytokine signaling pathway, peroxisome, and amoebiasis, which was the opposite condition for Bacteroides (OTU_22) (p<0.05). Roseburia (OTU_36), Bacteroides (OTU_2040), Bacteroides (OTU_2), and Bacteroides (OTU_10) were positively related with citrate cycle (TCA cycle), PPAR signaling pathway, adipocytokine signaling pathway, peroxisome, and amoebiasis (p<0.05) (The correlationship between the nine distinguished pathway and the 23 significantly differed genera indicated that p>0.05)  was negatively related with RQLQ at week 14 as well as TNF-α at week 0, while positively related with IL-2 at week 14 and IFN-γ at week 34 (p<0.05). Roseburia (OTU_36) was positively related with IL-2, IL-4, and IFN-γ at week 14 as well as TNF-α at week 0 (p<0.05). Prevotella_2 (OTU_80) was negatively related with IL-2 and IL-4 at week 0 (p<0.05). Prevotella_9 (OTU_68) was negatively related with RQLQ and IFN-γ at week 10 (p<0.05). Bacteroides (OTU_10) was negatively related with RQLQ at week 10 and IFN-γ at week 34 (p<0.05). Prevotella_9 (OTU_5) was negatively related with RQLQ at week 14 and IFN-γ at week 0 and week 10, but positively related with IFN-γ at week 34 (p<0.05). Bacteroides (OTU_22) and Bacteroides (OTU_2) were positively related with TNSS score and RQLQ at week 34, which was opposite for Fusobacterium (OTU_6) (p<0.05). Butyricimonas (OTU_93) was negatively related with RQLQ at week 34 and IFN-γ at week 0 (p<0.05). Dialister (OTU_24) was positively related with TNSS score and RQLQ at week 10 and negatively correlated with IL-4 at week 0 (p<0.05). Prevotellaceae_UCG-001 (OTU_77) was positively related with IL-2, IL-4, TNF-α, and IFN-γ at week 10 (p<0.05). Ruminococcus_1 (OTU_69) was negatively related with IL-2 at week 10 and positively correlated with TNF-α at week 34 (p<0.05).There was no significant correlation between Chao1 index and TNSS, RQLQ, or cytokines across different times (p>0.05) Table S8There were no instances of serious AEs observed or reported during the study. The only minor AEs observed during treatment were nausea (n=7), xerostomia (n=8), poor appetite (n=8), loose stools (n=2), and night sweating (n=1), but these events disappeared during the follow-up period. There were no significant differences at different time points in the safety evaluation Table 1.p<0.05), and such differences were maintained at a 6 months follow-up visit (p<0.05), indicating the long-term remission of AR after XQLD treatment.The current first-line therapy for AR was almost short-term effect. Once these medications are discontinued, most AR patients will relapse within a brief period . The unsBacteroides is more abundant in healthy people as compared with AR patients, while Firmicutes is more abundant in AR patients  and the release of immunomodulatory molecule such as polysaccharide A , which may be in association with the prepathologic state.The gut microbiota is hypothesized to have a critical role in allergic disease, including allergic rhinitis (AR) . Our prepatients . This stharide A . Howeverharide A , 32. Theharide A . The incFusobacteria.Our previous study showed that the bacterial diversity in AR patients was significantly higher than that of healthy participants, which might be due to the expansion of bacteria taxa within Firmicutes . This stDialister (OTU_31), Roseburia (OTU_36), Bacteroides (OTU_22), Bacteroides (OTU_2040), and Prevotella_9 (OTU_5), which altered significantly across different time points after XQLD treatment, were closely related with the change of AR clinical indices. Dialister, Roseburia, and Bacteroides were reported to produce short-chain fatty acids (SCFAs) to bring benefits for the human host  and Bacteroides (OTU_2040) increased after XQLD treatment but decreased at the follow-up periods, of which Bacteroides (OTU_22) was negatively related with TNSS and RQLQ at week 10, while it was positively related with TNSS and RQLQ at week 34. Prevotella_9 produces sulfatases that degrade mucus polysaccharide, which could disrupt intestinal mucosal barrier and increase the production of pro-inflammatory cytokines in the body , and Bacteroides (OTU_2040) together with the decreasing abundance of Prevotella_9 may serve as microbial signature of long-term remission in AR after XQLD treatment. However, these findings need further verification for the true role of them in AR.The distinguished genera including man host . Dialistof atopy . Dialistllergies . The higuria OTU_, Bacterothe body . The relPrevotella_9 was negatively related with PPAR signaling pathway, which significantly reduced after XQLD treatment, while Bacteroides (OTU_22) and Bacteroides (OTU_2040) were positively related with PPAR signaling pathway and significantly enriched after XQLD treatment. The significantly higher expression of PPAR signaling pathway after XQLD treatment contributed to the reduction in inflammation of the nasal mucosa. Peroxisome proliferator–activated receptor γ (PPARγ) plays a role in the regulation of intestinal inflammation , and Bacteroides (OTU_2040) were positively related with peroxisome, which was the opposite condition in Prevotella_9. Higher expression of peroxisome pathway reshaping the microbiota balance is helpful to the long-term remission of AR. Citrate cycle (TCA cycle) is a common metabolic pathway in aerobic organisms. In a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis in rats, sulfasalazine (SASP), a sulfonamide antibiotic clinically used to treat IBD, was used to treat these experimental colitis rats. As a result, SASP not only restores the TNBS-induced gut dysbiosis but also modulates the dysregulated function of the TNBS-induced colitis, including an increased capacity for basic citrate cycle metabolism. This study also showed an upregulation of citrate cycle metabolism pathway after XQLD treatment , and Bacteroides (OTU_2040) were positively correlated with citrate cycle metabolism. Therefore, the future research of probiotics which focuses on regulating the activity of PPAR signaling pathway, peroxisome, or citrate cycle (TCA cycle) can be used as a new target for the treatment of AR.PPAR signaling pathway, which could be related to the reduction in inflammation and degradation of air pollutants, was higher in healthy people as compared with asthmatic patients . In thisammation . Microbiammation . SCFAs-pes OTU_20 were posTCM formula has been developed and advocated for use in the treatment of many diseases for over 2,500 years in China. However, the complexities and unknown mechanisms of TCMs prevent the active chemical components from being identified. Our study suggests that gut microbiota might be involved in the effect of a widely used TCM formula, XQLD. This opens an avenue for identifying chemical components in TCMs, which can modulate gut microbiota structure as a potential mechanism for disease alleviation. Glycyrrhizin, paeoniflorin, ephedrine, and catechin were identified as the four most abundant chemical components in XQLD . GlycyrrThere were several limitations to this study. Firstly, we included a limited number of subjects in a cases series study, which reminds of us that more samples included in a RCT should be considered to make more robust conclusion. Secondly, each time point’s bacterial metabolites should be detected because metabolites of gut microbiota may be a bridge to link the host and bacteria. Thirdly, each week’s fecal sample should be collected for 16S rRNA test so that we can judge whether the gut microbiota alterations occurred before or after the significant improvement of AR symptoms. Overall, further studies of larger sample size, RCT, host-microbiome interaction are needed to validate these findings, which will make us clearly understand the precise roles of gut microbiota in the sustained remission of XQLD treatment for AR.Dialister (OTU_31), Roseburia (OTU_36), Bacteroides (OTU_22), and Bacteroides (OTU_2040) and low in Prevotella_9 (OTU_5) could be predictive of good sustained response to XQLD treatment. SCFA producers mainly dominate this favorable signature, and durable response is associated with a restoration of the significantly high expression of PPAR signaling pathway, peroxisome, or citrate cycle (TCA cycle) found in AR patients. The relative high abundance of Fusobacteria can be used as a biomarker species predictive of a prepathologic state in AR.In summary, AR patients with a microbiota signature high in The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Xiamen hospital of Traditional Chinese Medicine (Permit Number ID: 2019-K020-01). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.Conceived and designed the study: LZ, WW, YD and WL. Participated in investigation: LT, BY, WW, YD, and JX. Performed formal analysis: LZ, WL, and YW. Collected the resources: LZ, LT, BY, CL, ZC, and JX. Curated the data: LZ, YW, ZC, CL, and WL. Wrote the manuscript: LZ, YW, and WL. Supervised the study: LZ, ZC, and YW. All authors approved the final version of the manuscript and agreed for publication once accepted. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We report the first clinical use of Endosight, a new guidance system for percutaneous interventional procedures based on augmented reality, to guide percutaneous thermal ablations. The new system was demonstrated to be precise and reliable, with a targeting accuracy of 3.4 mm. Clinically acceptable, rapid setup and procedural times can be achieved.Background: Over the last two decades, augmented reality (AR) has been used as a visualization tool in many medical fields in order to increase precision, limit the radiation dose, and decrease the variability among operators. Here, we report the first in vivo study of a novel AR system for the guidance of percutaneous interventional oncology procedures. Methods: Eight patients with 15 liver tumors  underwent percutaneous thermal ablations using AR guidance . Prior to the intervention, the patients were evaluated with US and CT. The targeted nodules were segmented and three-dimensionally (3D) reconstructed from CT images, and the probe trajectory to the target was defined. The procedures were guided solely by AR, with the position of the probe tip was subsequently confirmed by conventional imaging. The primary endpoints were the targeting accuracy, the system setup time, and targeting time . The technical success was also evaluated and validated by co-registration software. Upon completion, the operators were assessed for cybersickness or other symptoms related to the use of AR. Results: Rapid system setup and procedural targeting times were noted . The high targeting accuracy  was accompanied by technical success in all 15 lesions . No intra/periprocedural complications or operator cybersickness were observed. Conclusions: AR guidance is highly accurate, and allows for the confident performance of percutaneous thermal ablations. Precision and targeting accuracy are key for the success of all image-guided interventional procedures. Over the last 20 years, several new navigational tools have been added to conventional imaging modalities  with the purpose of increasing precision, favouring dose reduction, decreasing variability among operators, and thus promoting the diffusion of diagnostic and therapeutic interventional procedures based on ever-increasing reliability. Image fusion platforms based on electromagnetic or optical devices ,2,3,4, CThis study was performed at two tertiary referral centres for liver diseases , with the approval of the local Institutional Ethics Committees. Written informed consent was obtained from all of the subjects involved in the study.Fifteen hepatic malignancies (9 hepatocellular carcinomas (HCCs), 3 metastases from breast carcinoma, and 3 from pancreatic adenocarcinoma) in eight patients  underwent AR-guided percutaneous thermal ablation. The treated nodule size ranged from 0.7 to 3.0 cm (mean 1.56 + 0.55).n = 4), V (n = 2), II (n = 2) and VI (n = 1); the sizes ranged from 1.2 to 3.0 cm (mean 1.69 + 0.53). One patient had four HCCs, and one two HCCs. All of the nodules were treated in the same session. The other three patients had only one HCC. All of the HCCs were diagnosed through a non-invasive radiological work-up, following the European Association of the Study of the Liver (EASL) 2018 clinical practice guidelines [For all of the cases, the treatment decision was determined by the consensus of an Institutional Multidisciplinary Liver Team. According to the BCLC classification, the nine HCCs in five patients were either very early (8/9 cases) or early stage (1/9), in a subset of HCV-related early stage cirrhosis  . These widelines .The six metastases in the three patients ranged from 0.7 to 2.1 cm (mean 1.35 cm + 0.56) in size, and were diagnosed by percutaneous US-guided biopsies using 20 G Menghini-modified needles .All of the patients were initially evaluated with a baseline ultrasound of the liver which included contrast enhanced ultrasound (CEUS) after the intravenous administration of 2.4 to 4.8 mL second-generation contrast agent  A, and anThe AR set-up comprised a proprietary augmented reality system  that features a 27” medical display , a laptop  with installed proprietary image processing and augmented reality software, and a commercially available head-mounted display (HMD)  paired with a binocular camera  .The binocular camera viewed the patient from two different angles in order to register the patient model in the camera frame using the markers visible in both video images while tracking the ablation applicator. The software enabled the 3D reconstruction (from CT scans to 3D volumes), co-registration, and AR intervention. Specifically, after uploading the CECT scans into the system, followed by the automatic segmentation and 3D reconstruction of the liver, spleen, bones, liver blood vessels and radiopaque markers, the semi-automatic segmentation of the target lesions occurred using proprietary reconstruction algorithms. In addition, the most suitable trajectory path from the skin to the target was defined. Subsequently, by moving the HMD around the patient, the system software co-registered (matched) all of the radiopaque markers segmented on the CT scans with all of the real markers applied to the patient’s skin. This allowed for the simultaneous visualization of the patient’s surface and internal anatomy, the target lesion, and the trajectory path to the target in 3D, by superimposing—in real-time—virtual images on the operator’s real field of sight D. Next, All of the procedures were performed by three interventional radiologists with more than 15 years of experience in percutaneous thermal ablations. In 14 of the 15 patients, the ablations were performed in the CT room coupled with real-time ultrasound, under assisted ventilation, during short-acting anaesthesia using propofol  (10 mg/mL) and alfentanil  (0.5 mg/mL), with continuous hemodynamic monitoring throughout the procedure. In the remaining patient, the ablation was performed under direct CT control (Lightspeed VCT 64) after local anesthesia and deep sedation with 0.2 mg Fentanyl  without additional ultrasound guidance. Using AR guidance alone, the coaxial needle was inserted following the predefined trajectory line planned during the setup F. This wTM, R.A.W. Srl, Milan, Italy) [The CECT was performed immediately after withdrawing the ablation device I. A prop, Italy) —which en, Italy) ,38. AfteThe primary endpoints evaluated included the time required to set up the system and to position the antenna tip inside the nodule, the mean depth of the target centre from the needle entry point on the skin, and the deployment accuracy, defined as the mean distance between the geometric center of the target and the ablation device tip measured on unenhanced CT or US. Secondary endpoints included the technical success, i.e., the complete ablation of the entire tumor and the achievement of an >90% 5-mm periablational margin ablation , complicThe time required to set up the system ranged from 12.0 to 17.2 min (12.3 ± 2.1 min), and the time required to perform each insertion and tumor targeting ranged from 3.2 to 5.7 min (4.3 ± 0.9 min). In 7 of the 15 (46.7%) cases, the target nodule was visible on the US, and the real location of the target nodule and the position of the coaxial needle tip in respect to the target centre were verified using real-time US. In the remaining 8 of the 15 (53.3%) cases, unenhanced CT was employed for verification. The mean depth of the target centre from the needle entry point on the skin was 76.0 ± 28.2 mm. The distance between the geometric center of the target and the ablation device tip measured on unenhanced CT or US ranged from 2.1 to 4.5 mm (3.2 ± 0.7 mm). TM software, the complete ablation of the tumors  was found. The residual 5-mm ablative margin percentage ranged from 0 to 14.1 % (5.5 ± 4.3%), with 13 of the 15 (86.7%) patients showing >90% ablation of this margin. For the MWA, the power delivered ranged from 50 to 60 W, with a treatment duration of 5 min in four HCCs, and 6 min in the remaining five HCCs and the five metastases. For the case of radiofrequency ablation (RFA), the power delivered was 1500 mA for 12 min. A single ablation device insertion was performed for each target tumor. Technical success was achieved in each case. After the automatic coregistration of the 3D volumes of the pre-ablation tumors and post-ablation necrotic changes, achieved with the Ablation-fitNo intra- or periprocedural adverse events occurred. No user-dependent calibration and adjustment for the HMD was needed, and no significant eye fatigue or “cybersickness” was reported by any of the users.Modern imaging modalities enable the visualization of increasingly small target lesions, often in difficult-to-target locations, which is particularly suitable for local, image-guided treatments (IGTs). Consequently, the requests for image-guided therapy, accompanied by expectations of favorable outcomes, are constantly increasing. However, some problems still remain unsolved. First of all, the learning curve for the use of these technologies is often long, and this limits the diffusion of interventional procedures, particularly among young operators and/or in low-referral centers. The lack of the real, live, 3D visualization of targets, and the poor working ergonomics  are additional important limitations. The mental registration of the target position seen in the reference image  with the corresponding position in patients is often challenging, particularly for liver dome lesions requiring non-orthogonal or out-of-plane approaches, even when CT guidance is used. The difficulty and subjectivity of this process may also increase the risks for patients. Thus, the need for a technically easy combination of “real-world” visualization with virtual objects precisely superimposed upon the scene is increasingly desired. This can be achieved with AR technology in the actual interventional field, where the operator can visualize and interact simultaneously with the real world  and virtual objects  based on the superimposition of the “two worlds”, as displayed on HMD, smartphones, tablets, screens or videoprojectors. Moreover, HMDs can be relatively advantageous compared to the direct line of sight through the lens display .The most critical issue for the use of AR in medical applications is the superimposition precision, i.e., the registration accuracy. Multiple studies on phantoms, animal models, and human cadavers have primarily focused upon the assessment of registration accuracy, either for AR navigation  or the AUsing the same system employed for the present study, Solbiati et al. recently published a proof-of-concept study on phantoms, animal models, and human cadavers targeted with AR guidance. In the rigid phantom, sub–5-mm accuracy (2.0 + 1.5 mm) (mean + standard deviation) was achieved. In a porcine model with small (2 × 1 mm) metallic targets embedded, the accuracy was 3.9 + 0.4 mm when the targeting was performed with respiration suspended at maximum expiration, as in the initial CT scan, and 8.0 + 0.5 mm when the procedure was performed without breathing control. In a human cadaver attached to a ventilator to induce simulated respirations, two liver metastases (1.8 cm and 3.0 cm) were targeted with an accuracy of 2.5 mm and 2.8 mm, respectively .Here, we note the similar accuracy of 3.4 mm in living, breathing patients. Regarding AR-guided needle insertions in human patients, De Paolis et al.  reportedThe AR system used for our current report is specifically designed to guide percutaneous biopsies and ablation procedures. It is based on disposable markers with no repetitive pattern affixed to the patient’s abdominal skin before performing the CT scans. The associated software enables us to visualize and segment the markers on the patient  and the target tumor, to automatically register and superimpose virtual and real images in real-time, to define the safe and accurate trajectory line to the target center, to depict the guided movements of the interventional device without the need for additional imaging, and to show the whole procedure on a display, HMD, or screen ,44. The In recent years, two issues have been raised regarding the technology of HMDs used for AR, i.e., the field of view (FOV) and the need for calibration . The binThe patient’s respiratory movement and motion remain one of the largest technical and practical hurdles, as AR guidance systems are currently unable to follow respiratory excursions in mobile organs with real-time corrections, bearing a risk of the shifting of the intended target relative to the expected location. Other target-related limitations arise from the abilities of lesions to warp or move within their environments. Respiratory motion tracking and the monitoring of respiration during deep sedation or general anesthesia seem to offer the best solutions to date. The guiding information is provided regularly at the point of the breathing that matches the respiratory phase during which the preoperative CT image was acquired (the middle respiratory or expiratory phase). In this time interval window, the operator can move the needle toward the target as rapidly as possible. In our study, the insertion was conducted during the patient’s free breathing (as in the pre-ablation acquisition of the CT scans) in order to minimize the organ displacement caused by breathing. Given that this was the initial study of AR-guided thermal ablation, we selected only tumors which were visible on US or on CT in order to be able to check the position of the device tip after its insertion, before starting the ablation. Probe repositioning was never required, as the position achieved with AR guidance was always sufficiently accurate. Nevertheless, we acknowledge that this will not always be invariable, and note that—should minor placement corrections be needed—the virtual system will potentially save a substantial amount of radiation exposure compared to fully CT-guided procedures, be they CT-guided freehand, cone-beam CT, or CT fluoroscopy guidance . Indeed,An additional potential challenge of AR-guided interventional procedures is needle bending during the insertion, exacerbated by increased applied pressure or the use of thinner needles. The solution we successfully utilized was the use of a rigid coaxial needle to maintain the interventional device fixed in 3D space during its advancement, minimizing the bending of the ablation device inserted into the coaxial needle. We further demonstrated that the attachment of a clip with markers with no repetitive pattern to the coaxial needle permits precise monitoring by AR of the probe advancement towards the target, and the interventional device subsequently inserted into the coaxial needle can easily hit the target center. Coaxial needles have been used for interventional procedures for decades, and do not appreciably increase the risk of bleeding because their construction is engineered to result in an ultimate size only 1–2 G larger than that of ablation devices or biopsy needles.With respect to other navigation systems, AR guidance offers an ergonomic advantage that the overlay of treatment information  is shown directly in the procedural environment, and not on a display screen away from the patient on a monitor, as occurs with CT- or CBCT-guided fluoroscopy. Additional advantages of AR guidance are the ease of use, the reduced procedural time compared to more traditional guidance systems, and the short learning curve (compared to that of CT-guided procedures), which is particularly useful for young operators with limited experience, who perform equally or even better than senior operators with long experience. Furthermore, AR guidance systems are significantly less expensive than all of the other needle guidance systems. This may favour the diffusion of AR, and consequently of image-guided procedures in small centers, and in developing countries that cannot afford to buy complex and expensive guidance technologies .With AR, the same images seen by the operator can also be visualized on monitors inside and outside the interventional room, and can be broadcast on a larger scale, allowing interventional radiologists to visualize live or recorded procedures performed by experts. AR can provide not only an excellent opportunity for physician training and education but also a very useful tool to exchange collaborative experiences among various centers for remote real-time instruction or expert assistance ,45.We acknowledge that this study has some limitations, most notably the small number of patients within the cohort, and the non-randomized type of lesions treated, all of which visible on both US and CT despite their small size. Nonetheless, we believe that it will encourage new prospective studies, and will work as the basis for the development of AR technology in the clinical field.In this retrospective study, we obtained high standards of targeting accuracy, technical efficacy, procedural time, and radiation dose reduction using AR as the sole guidance method for percutaneous thermal ablation, without encountering any complications. In spite of the small cohort analyzed, we propose that our preliminary data demonstrate the potential for AR, with further validation, to become a leading and low-cost modality for the guidance of interventional procedures worldwide.


Background: Breast cancer survivors report negative impacts of cancer, augmented by specific vulnerabilities to body changes, negative self-assessment, and quality-of-life concerns. The main objective of our work was to test the effect of a rehabilitation program on breast cancer patients by evaluating the change in their physical well-being during an outpatient rehabilitation setting and, subsequently, in a home rehabilitation setting, considering the individual personality profile. Methods: Patients who underwent total mastectomy with breast prostheses or tissue expanders were enrolled. Outcome assessments  before treatment (T0), at the end of the rehabilitative treatment , and after two months of follow-up (T2) were performed. Results: The data of 38 included patients were analyzed. The quadratic trend of the Visual Analogue Scale can be explained by the fact that patients have a strong reduction in the perceived pain immediately after rehabilitation in the clinic. This reduction remains constant for the home period of the rehabilitation. The personality profiles of all the participants were substantially valid. Only three patients obtained scores higher than 65 points. Conclusions: The study evidenced that in the initial phase of the rehabilitation, psychological traits such as anxiety, depression, and preoccupation could have a strong association especially with the autonomous functions and the perceived physical symptoms. However, during the therapeutic process, this association decreased and these decrements were higher when patients performed their rehabilitation at home, in a more familiar and comfortable setting. Breast cancer (BC) is the most common cancer among women in developed countries, causing premature mortality, also presenting several disabling complications related to treatment . BC survA clinical study (July–September 2019) took place at the rehabilitation out-patient clinic of University Hospital Umberto I, Sapienza University of Rome . The study protocol was approved by the Ethics Committee of Sapienza University of Rome (N° 4985).Patients were enrolled in the study after an evaluation by a rehabilitation physician considering the following inclusion criteria: total mastectomy with breast prostheses or tissue expanders; age 18–60 years; body mass index (BMI) < 30; no cognitive dysfunctions (MMSE > 24); no lymphedema. The exclusion criteria were: lymphangitis, metastasis, surgical complications of the intervention; neurological deficits and complications; important shoulder problems before the BC-surgery; web axillary syndrome; other or previous physiotherapy; psychiatric disorders.All patients signed an informed consent, after receiving detailed information about the study’s aim and procedures for the Declaration of Helsinki. The rights of human subjects involved in the study were protected. This study protocol was developed in accordance with the Trend Guidelines .Outcome assessments were performed before treatment (T0 = baseline), at the end of the rehabilitative treatment , and after two months of follow-up (T2). Because the first part of the rehabilitative therapy was conducted in the clinic, and the second part continued at home, we defined two time periods for the rehabilitation: (a) the clinical period (T0–T1) and the (b) home period (T1–T2).Age, height, weight, and BMI were collected by the rehabilitation physician at the first evaluation. A clinical evaluation of the shoulder Range of Motion (ROM) on the operated side was performed. Patients were referred, by the oncologist, or by the surgeon or general practitioner, after the surgery (time from intervention 9.67 ± 5.12) to the psychiatric examination, which did not have the possibility to carry out a pre-surgical evaluation.The Visual Analogue Scale (VAS) was administered as a simple, robust, sensitive, and reproducible instrument, which enables the patients to express their pain intensity as numerical values from 0 to 10 cm ,22.The European Organization for Research and Treatment of Cancer Quality of Life core questionnaire (EORTCQLQ-C30) ,24,25 isThe MMPI-2 is one of the most widely used personality questionnaires used for assessing personality traits or psychological syndromes. Each item of the MMPI-2 describes a typical sensation, thought, or behavior associated with a specific personality trait or syndrome, and patients responded to each item using “true” or “false” if they had or not, respectively, that specific characteristic. The questionnaire is composed of 567 items and it comprises 10 basic clinical scales  and three basic validity scales . Validity scales estimated the tendency of participants to improve their characteristics for social desirability (Lie), to exaggerate their symptoms or traits (Frequency), or to hide some specific problems (Correction). In addition, we included in the analysis some content scales that are the Anxiety (ANX), Fears (FRS), Obsessiveness (OBS), Depression (DEP), Health Concerns (HEA), and Negative Treatment Indicators (TRT) scales to have further confirmation of the probable connection between the personality traits and the measure of pain, level of functioning, and severity of physical symptoms. The TRT scale is normally used to test the existence of a possible resistance against a psychotherapeutic treatment. In this case, we used this scale to test the possible resistance against the rehabilitative procedure. We used the Italian adaption of the MMPI-2 ,27. MMPIThe patients underwent ten single rehabilitation treatment sessions . Then, during the rehabilitation process, the patients were taught active recovery exercises for the articulation of the shoulder on the operated side, which they could repeat independently at home at the end of the cycle of ten sessions with the physiotherapist. For greater protection, patients were given an illustrative pamphlet with the exercises to continue to be performed at home at least twice a week. The physiotherapists tailored the rehabilitation to the patients’ functional problems. A previously validated rehabilitation protocol was considered ,29.Exercises carried out with the physiotherapist in the first phase of outpatient rehabilitation: in supine position for relaxation and diaphragmatic breathing; exercises in the supine position of postural re-alignment with the use of a small cylinder on the line of the vertebral spines; exercises of decoaptation and co-activation of the glenohumeral joint; exercises to stretch the pectoral muscles, subscapularis muscle; exercises to mobilize the scapulo-thoracic joint; cervical pumping; active exercises in the mirror in a sitting position, for the recovery of the range of motion of the shoulder ; isometric strengthening exercises in flexion, abduction, and adduction of the arms also with the use of rubber bands; Codman’s pendulum; balance exercises standing in front of the mirror. All proposed exercises were repeated, starting from 10 repetitions for 3 times . Before starting the session, the patients performed a short 10–15 min warm-up.At the end of the rehabilitation treatment, patients were given an illustrative pamphlet with the exercises to be performed at home and with the indication to walk as much as possible at least 30 min almost every day. Exercises during the first month of observation/home-based exercise were given twice a week and continued in the second month with once a week. The exercises indicated aimed at the active mobilization of the shoulder such as flexion and extension with the use of the stick, or favor abduction by going up the wall with the hand in a lateral position; exercises of rotation of the arms in 90° abduction; exercises in adduction; twisting exercises for the trunk in sitting and standing position; mirror active cervical mobilization exercises. The patients had a rehabilitation diary available to record adherence to treatment.Sample size was not predefined but determined by the period of observation of the study, that is, 1 year.A self-report questionnaire was used to analyze the evolution of pain and QoL, and the severity of physical symptoms in patients. In addition, we measured personality trait to analyze their mediation effect on the evolution of the pain, functioning, and physical symptoms during the rehabilitation.Frequencies and descriptive statistics were provided for demographic and biometric data. Descriptive data were mean, standard deviation, minimum and maximum values, skewness, and kurtosis. Skewness and kurtosis values between −2 and 2 indicate a good distribution of the data .t-test was applied to determine whether the linear or quadratic trend was significant. Positive (negative) t-values for the linear trend indicated a constant increasing (decreasing) effect from T0 to T2, and positive (negative) t values for the quadratic trend indicated a maximum (minimum) effect in the middle (T1) of the time series [We analyzed the trend of the rehabilitation effects on the measures of pain, functioning, and severity of physical symptoms  using the regression analysis with orthogonal polynomial coefficients. The polynomial coefficients for the linear trend were −1, 0, and 1, and the coefficients for the quadratic trend were −1, 2, and −1. The e series . We alsoe series ,33.Ti is the variable  measured in the i-th period of time  or the criterion, βTi is the coefficient of the slope associated with XTi, β0 is the intercept, and ej the random error. The variation in βTi indicates the strength of the relationship between M and XTi. With the mediation analysis, it is possible to analyze the variation in this strength from T0 to T2. In particular, if the strength increases, then we should have βT0 < βT1 < βT2. If the strength decreases, then we should have βT0 > βT1 > βT2. If the slopes vary for each time period, then there is an interaction between M and XTi or, in other words, there is a moderation of Mj on XTi. The moderation effects were tested using regression models with the following equation:During a period of 12 months, 58 patients were observed. In addition, 41 patients were enrolled into the study and assigned to the intervention. During treatment, 3 patients dropped out for personal problems. None complained about pain. The data of 38 included patients were analyzed .p < 0.001), while only the linear components of QL2, GIF, and GSI were significant (p < 0.001) (The linear and quadratic components of the VAS were significant (< 0.001) .For the QL2, GIF, and GSI, there was a prolonged positive effect of the rehabilitation, both for the clinic and the home period. T1–βT0) and home period (βT2–βT1). The t-values and relative probabilities p(t) for each β differences are reported in The aim of our research was to test the influence of a rehabilitation program on BC survivors with respect to their QoL considering the individual personality profile. Usually, it is not possible to perform prolonged treatments in the rehabilitation outpatient setting; therefore, an early discharge is required with an acceptable achievement of the rehabilitation goal, and it is not possible to renew the rehabilitation cycle, hence the importance of giving the patient instructions to be able to continue the treatment in the home environment independently and safely, with “remote” support from the physiotherapist. Our results appeared to be encouraging; the rehabilitation plan reduced the perceived pain and increased the perceived quality and the level of functioning, and these effects were persistent also when patients must perform their rehabilitation at home. The quadratic trend of the VAS can be explained by the fact that patients have a strong reduction in the perceived pain immediately after the rehabilitation in clinic; this reduction remained constant for the home period of the rehabilitation.The literature indicates that rehabilitation exercise is effective in improving the QoL in patients who have survived cancer ,36; in aIn conclusion, our study evidenced that the rehabilitation in BCs has a positive effect, because patients showed a reduction in pain and an increment in the QoL, with an increment in autonomous functioning and a decrement in physical symptoms. The study evidenced that in the initial phase of the rehabilitation, psychological traits such as anxiety, depression, preoccupation, and, in some cases, hypochondria and hysteria could have a strong association, especially with the autonomous functions and the perceived physical symptoms. Further studies are needed, with a larger sample and a longer follow-up, in order to better highlight the effects of rehabilitation intervention on the quality of life in breast cancer survivors.