Deep learning (DL) can accelerate the prediction of prognostic biomarkers from routine pathology slides in colorectal cancer (CRC). However, current approaches rely on convolutional neural networks (CNNs) and have mostly been validated on small patient cohorts. Here, we develop a new transformer-based pipeline for end-to-end biomarker prediction from pathology slides by combining a pre-trained transformer encoder with a transformer network for patch aggregation. Our transformer-based approach substantially improves the performance, generalizability, data efficiency, and interpretability as compared with current state-of-the-art algorithms. After training and evaluating on a large multicenter cohort of over 13,000 patients from 16 colorectal cancer cohorts, we achieve a sensitivity of 0.99 with a negative predictive value of over 0.99 for prediction of microsatellite instability (MSI) on surgical resection specimens. We demonstrate that resection specimen-only training reaches clinical-grade performance on endoscopic biopsy tissue, solving a long-standing diagnostic problem.  •BRAF, and KRAS) using transformersAI-based prediction of biomarkers (MSI, •MSI prediction reaches clinical-grade performance on biopsies of colorectal cancer•Transformer-based biomarker prediction generalizes better and is more data efficient•Large-scale multi-cohort evaluation on over 13,000 patients from 16 cohorts Wagner et al. show that transformer-based prediction of biomarkers from histology substantially improves the performance, generalizability, data efficiency, and interpretability as compared with current state-of-the-art algorithms. The method significantly outperforms existing approaches for microsatellite instability detection in surgical resections and reaches clinical-grade performance on biopsies of colorectal cancer, solving a long-standing diagnostic problem. Thus, tissue sections stained with hematoxylin and eosin (H&E) are routinely available for all patients with CRC. Since 2019, dozens of studies have shown that deep learning (DL) can predict genetic biomarkers directly from digitized H&E-stained CRC tissue sections.feature extractor and the aggregation module.,,The technology underlying these algorithms in literature is based on weakly supervised learning, consisting of two components: the ,,,,,In many non-medical and medical image-processing tasks, transformer neural networks have recently been adopted for computer vision tasks,In this work, we first aim to enhance the performance of DL-based biomarker detection from pathology slides. Thereafter, in order to provide large-scale evidence of the performance on clinically relevant tasks, we investigate the use of a fully transformer-based workflow in CRC. Here, we present a new method derived from a transformer-based feature extractor and a transformer-based aggregation model A-C, whicWe tested our pipeline on MSI prediction in surgical resection cohorts of patients with CRC  in two wSecond, we trained our model on all cohorts of CRC resections except YCR-BCIP and evaluated it on the external cohort YCR-BCIP B. In parThe classical patch-based approach by Echle et al.In summary, these results show that a fully transformer-based approach yields a higher performance for biomarker prediction both on large cohorts  as well as on smaller cohorts (TCGA). Perhaps more importantly from a clinical perspective, the transformer-based approach resulted in a better generalization performance and more reliable results, as the deviation between the external cohorts was smaller. We published all trained models for reuse and further fine-tuning if needed.BRAF and KRAS are determined in addition to MSI. We tested whether and how well these were predictable on the DACHS, QUASAR, MCO, NLCS, TCGA, and Epi700 cohorts, where the Epi700 cohort served as an external test set in the multi-centric run.Next, we investigated whether the fully transformer-based model yields a similar high performance in other biomarker-prediction tasks. Following the experimental setup for MSI prediction, we trained the model first on single cohorts evaluated on all other external cohorts and second on one fully merged multi-center cohort excluding only one cohort from the training set to constitute an external test set. In clinical routine workup for CRC, the biomarkers In the case of the largest cohorts, DACHS and NLCS, single cohort training was already capable of achieving good results, with AUROCs of 0.88 and 0.87, respectively D. The smKRAS as a target .We quantified the morphological patterns occurring in high-attention regions in a small user study, where two pathologists annotated the patterns in 160 patches of 40 patients that the model attributes high attention to. We chose the 10 patients with the lowest and highest classification score for each MSS and MSI-high ground truth. For every patient, we chose the two patches associated with the highest and lowest class scores of the top 100 attention tiles. Our study showed that the majority of tiles belong to the tumor region (0.99% for high and 0.81% for low-classification scores) and cell types that are important for the prediction of MSI-high, such as lymphocytes occur in both tiles with low- and high-classification score in a similar ratio , while the AttentionMIL model exceeded an AUROC of 0.9 only with 4,000 patients A. In a sOur fully transformer-based approach yielded high performance with a small sample size. Compared to an AttentionMIL-based approach, our approach is more data efficient in the regime of small numbers of patients. Looking at larger training numbers, we observed that performance increase is directly proportional to the number of patients for both approaches, but the fully transformer-based approach reaches equivalent performance already with much smaller datasets.,Virtually all previous studies on biomarker prediction in CRC were performed using surgical resection slides. For this reason, commercially available MSI detection algorithms are intended to be used only with resection slides. However, recent clinical evidence shows that MSI-positive patients with CRC require immunotherapy prior to surgeryOur model yielded a mean AUROC score of 0.92 and 0.86 when validated on biopsies from two external cohorts, YCR-BCIP and MAINZ, respectively B. It is BRAF and/or RAS mutation status. In practice, these procedures may take several days to even weeks. However, in low- or middle-income countries, this might not happen at all. Based on the MSI, BRAF, and RAS status, the most suitable treatment approach will be chosen for the patient. For example, in patients with early (non-metastatic) CRC, the presence of MSI could qualify a patient for neoadjuvant immunotherapy followed by surgery with curative intent. Similarly, in the metastatic disease setting, the presence of MSI in the biopsy tissue would qualify a patient for palliative immunotherapy. Because of its high sensitivity, our algorithm could serve as a filtering step followed by affirmative testing for MSI-high predicted cases. Applying AI-based biomarker prediction would reduce the additional testing burden and therefore speed up the step between taking the biopsy and the molecular determination of MSI-high status, thus enabling an earlier treatment with immunotherapy if indicated.Our intended clinical use of this workflow is as follows : First, In summary, to the best of our knowledge, we developed a DL-based MSI-high predictor for biopsies that achieves clinical-grade performance. In particular, this high performance was also observed for external tests and could therefore improve clinical routine and speed up treatment decisions.,,The rollout of precision oncology to patients with CRC promises gains in life expectancy.,,,A key reason for the limited performance of existing DL systems could be the fundamental limitations of the technology employed. Most studies between 2018 and 2020 used convolutional neural networks (CNNs) as their DL backbone,BRAF and KRAS mutational status. Our large-scale evaluation also showed that MIL and in particular transformer-based approaches generalize much better than the existing CNN approaches. We proved this by training the model on single cohorts and testing the generalization on all other cohorts. These experiments showed that the transformer-based approach reduced the drop in AUROC to under 0.09, while the CNN-based approach dropped by more than 0.21 in some cohorts. Most importantly, our approach trained on resections did not only generalize well to external cohorts of resections from geographically distinct regions but also to biopsies with a clinical-grade performance of 0.98 sensitivity on the YCR-BCIP biopsy cohort and 0.91 on the MAINZ cohort.In the present study, we developed a transformer-based approach for biomarker prediction on whole-slide images of H&E-stained CRC tissue sections. Our model consists of a transformer-based feature extractor that was pretrained on histopathology images and a transformer-based aggregation module. In contrast to the state-of-the-art attention-based MIL approaches, the contribution of each patch was not only determined according to its feature embeddings but also contextualized with the feature embeddings of all other patches in the WSI via self-attention layers. Further, we presented a large-scale evaluation of transformers in biomarker prediction on WSIs. We demonstrated that transformer-based approaches learned better from small amounts of data and were therefore more data efficient than attention-based MIL approaches. At the same time, the performance increased proportionally with the number of training samples. Even though the performance seemed to plateau for MSI prediction, this suggests that larger training cohorts could lead to higher performance-approaching clinical application, also for more challenging tasks such as the prediction of the ,POLD1 and POLE mutations with a high-mutation burden that behaves clinically similar to MSI and might have a similar phenotype but are not detected by established MSI detection assays.KRAS/NRAS/BRAF depending on the sensitivity of the technology used and the presence of smaller clonal mutations. Current DL tests are at such high levels of performance that these nuanced subpopulations may be important. Our study has additional limitations: The focus of this study was to investigate the effect of handling the data with fully transformer-based approaches, especially in the context of large-scale multi-institutional data. Therefore, we did not exhaustively optimize every single hyperparameter. Points for optimization in this direction would be finding a fitting positional encoding and tuning the architecture of the transformer network and attention mechanisms. Additionally, collecting biopsy samples from different hospitals, for multi-cohort training directly on biopsy data could potentially improve the performance of our model on biopsy material. This would also hold for the prediction of BRAF mutation status and, in particular, of RAS mutation status, where we observed the largest potential for improvement. In both targets, the performance was higher on the larger cohorts with around 2,000 patients and increased dramatically by training on multiple cohorts. Further, we acknowledge that achieving an even higher specificity would be desirable. Choosing the final classification threshold is always a trade-off between sensitivity and specificity, where clinical application prefers a higher sensitivity, especially for pre-screening test as proposed in this study. Our method’s performance on biopsies is in the same range as current clinically approved assays on resections, but unlike these assays, our method also works on biopsies.A caveat of our observations is that the ground truth might not be perfect. Potentially, the DL model is performing better than stated in the paper because dMMR and MSI only agree around 92% of the time and neither are 100% sensitive.In summary, to the best of our knowledge, we presented a fully transformer-based model to predict MSI on WSI from CRC with an AUROC of 0.97 on resections and 0.92 and 0.86 on biopsies on external validation cohorts. Our model generalized better to unseen cohorts and was more data efficient compared to existing state-of-the-art MIL or CNN approaches. By publishing all trained models, we enable researchers and clinicians to apply the automated MSI prediction tool for research purposes, which we expect to bring the field of DL-based biomarkers a step closer to large-scale integration in the clinical workflow.jakob_nikolas.kather@tu-dresden.de).Further information and requests regarding this manuscript should be sent to and will be fulfilled by the lead investigator, Jakob Nikolas Kather , we have collected over 20,000 H&E tissue sections of 13,689 patients with CRC from 16 patient cohorts in total, including two public databases ,The public database “The Clinical Proteomic Tumor Analysis Consortium”, CPTAC ,3.The DUSSEL  cohort, a case series of CRC tumors resected with curative intent and collected at the Marien-Hospital in Duesseldorf, Germany, between January 1990 and December 19954.,Epi700 ,5.The ERLANGEN cohort, a CRC cohort collected at the Uniklinikum Erlangen in Germany between 2002 and 2010.6.The “Fluoropyrimidine, Oxaliplatin, and Targeted Receptor pre-Operative Therapy for colon cancer cohort” (FOxTROT)7.The GUANGZHOU cohort, a small CRC case series of MSI-high cases collected in The Second Affiliated Hospital of Guangzhou Medical University, China;8.The MAINZ cohort, a small CRC case series of biopsies collected in the University Medical Center Mainz in Germany.9.,,,The Molecular and Cellular Oncology Study (MCO) cohort10.MECC ,11.The MUNICH  CRC series, a case series collected at the Technical University of Munich in Germany.12.,The NLCS 13.,QUASAR, the “Quick and Simple and Reliable” trial investigating survival benefit of adjuvant chemotherapy in patients from the United Kingdom with mostly stage II tumors14.https://portal.gdc.cancer.gov/, USA),The public repository “The Cancer Genome Atlas”, TCGA , a population-based register of bowel cancer patients in Yorkshire, UK,Through coordination by the MSIDETECT consortium  the In the pre-processing pipeline, tissue regions are segmented using RGB thresholding and Canny edge detectionWe extract feature representations of dimension 768 for every tile using the CTransPath model.The final part of the model takes all patches of a WSI as input and predicts one biomarker for all input patches in a weakly supervised manner B. Commonand Assuming that ch block C.After the two transformer layers, the class token of size  version , but we  version .We performed all experiments using 5-fold cross-validation with in-domain validation and testing. In this cross-validation variant, in-domain validation and test set are split off the full dataset on patient level, leaving 3-folds for training. By also cycling the in-domain test set through the complete dataset, we evaluated our model on more representative test sets than when fixing one smaller set for the dataset. During training, the validation set was used to determine the best model, which was finally evaluated on the test set. We further evaluated our models on external cohorts outside the dataset for out-of-domain testing.The transformer models were trained with the AdamWFor comparison, we implemented the attention-based MIL approach from Ilse et al.,The final prediction is retrieved via the class token that is attached to the input sequence. To visualize the contribution of each input patch, we employed attention rollout as introduced by Abnar and Zuidema.To visualize whether a patch contributed toward a positive or negative classification outcome, we fed the patches one-by-one through the transformer and visualized the resulting classification scores of the model. These scores were naturally in the range BRAF, and KRAS (We used the area under the receiver operator curve (AUROC) as our main evaluation metric. Since our data are naturally highly imbalanced with respect to the target variables MSI, and KRAS D and 1E,We acknowledge the support of the Rainbow-TMA Consortium, especially the project group: PA van den Brandt, A zur Hausen, HI Grabsch, M van Engeland, LJ Schouten, J Beckervordersandforth; PHM Peeters, PJ van Diest, HB Bueno de Mesquita; J van Krieken, I Nagtegaal, B Siebers, B Kiemeney; FJ van Kemenade, C Steegers, D Boomsma, GA Meijer; FJ van Kemenade, B Stricker; L Overbeek, A Gijsbers; and Rainbow-TMA collaborating pathologists, among others: A de Bruïne; JC Beckervordersandforth; J van Krieken, I Nagtegaal; W Timens; FJ van Kemenade; MCH Hogenes; PJ van Diest; RE Kibbelaar; AF Hamel; ATMG Tiebosch; C Meijers; R Natté; GA Meijer; JJTH Roelofs; RF Hoedemaeker; S Sastrowijoto; M Nap; HT Shirango; H Doornewaard; JE Boers; JC van der Linden; G Burger; RW Rouse; PC de Bruin; P Drillenburg; C van Krimpen; JF Graadt van Roggen; SAJ Loyson; JD Rupa; H Kliffen; HM Hazelbag; K Schelfout; J Stavast; I van Lijnschoten; and K Duthoi.


Healthy diet and physical activity self-management is important in maintaining weight loss and preventing weight regain after bariatric surgery. We aimed at evaluating covariates of healthy diet and physical activity self-management among patients undergone bariatric surgery using Health Action Process Approach (HAPA) model.In this cross-sectional study, 272 patients with a history of bariatric surgery were selected from the data registry of Tehran Obesity Treatment Study (TOTS). Data were collected using bariatric surgery self-management standard questionnaire (BSSQ), and items based on HAPA model for healthy diet and physical activity self-management. Data were analyzed using Path analysis and AMOS version 24.The mean score of self-management was (32 ± 10SD). Coping planning construct  and risk perception  in dietary self-management and action planning  and risk perception  in physical activity self-management had the highest and lowest effect powers, respectively. Coping planning  and action planning  in diet, and action planning  in physical activity were significantly related to self-management. Also, task-coping self-efficacy , outcome expectancies , risk perception  in diet and coping self-efficacy , outcome expectancies  in physical activity were significantly related to behavioral intention. The values of CFI = 0.939 and RMSEA = 0.052 for diet and CFI = 0.948 and RMSEA = 0.048 for physical activity indicated adequate fit.HAPA was applicable as a framework for interventions promoting healthy diet and physical activity self-management in patients who have undergone bariatric surgery. Severe obesity, a growing health condition worldwide , is assoBariatric surgery is currently the main treatment strategy for severe obesity . ResearcA range of behavioral, dietary, psychological, physical, and medical considerations can play a role in suboptimal long-term weight loss . SeveralThe first step in the planning process of any health education program is selecting an appropriate behavior change theory/model through which the program is directed scientifically and kept on the right path ,28. HealThis cross-sectional study was part of a Ph.D. thesis approved by the Ethics Committee at Tehran University of Medical Sciences (IR.TUMS.SPH.REC.1400.230). The study population consisted of 300 patients registered at Hakim Obesity Clinic in Tehran who had undergone bariatric surgery at least one year ago from November 2021 to March 2021–2022. The inclusion criteria included patients who had undergone bariatric surgery at least one year before and were willing to participate in the study. Research aims were explained to the participants and after obtaining the informed consent, they completed the questionnaires. Confidentiality was ensured.We needed to have at least 217 samples to obtain a minimum precision of 2 for 95% confidence assuming a standard deviation of 15 based on the following formulaThe total score TOTALBSSQ was obtained from the sum of the Likert scores of the questionnaire items, each of which was between 0 and 2.The questionnaire had three parts: 1) demographic items including age, gender, level of education, economic status, and marital status; 2) items based on HAPA for a healthy diet and physical activity; and 3) bariatric surgery self-management standard questionnaire (BSSQ).To design HAPA items, we reviewed the existing questionnaires regarding factors affecting self-management of a healthy diet and physical activity; and also reviewed the existing standard questionnaires on HAPA for other health issues –35. ThenBSSQ was designed and assessed by Welch et al. (2008). This questionnaire includes 32 items with seven behavioral domains of eating, drinking, protein consumption, physical activity, management of dumping syndrome, eating fruits, vegetables, and whole grains, and taking vitamin and mineral supplements . Amini ePath analysis using AMOS 24 was used to evaluate the cause-effect relationship and to determine the strength of the constructs’ effect on a healthy diet, physical activity, and the relationship between the constructs. Also, T-test, analysis of variance, was used to assess the relation of the demographic variables with total BSSQ score and HAPA constructs. To assess the simultaneous effect of this demographic variate on total BSSQ score General Lwasr Model was applied. Also, these relations with HAPA constructs were tested using the MANCOVA . The level of significance in the tests was considered 0.05.When the initial questionnaires were filled, if a question was not answered, the patients were asked to fill it during the follow-up and re-request, so we did not have any data loss in this study.A total of 272 out of 300 participants completed the questionnaires. The mean and standard deviation of the BSSQ total score was 32.2±10.1 (CI: 95% 31.0 TO 33.4). The mean age (SD) of the participants was 32 (6.3) years. Most participants were women (76.1%), had a bachelor’s degree (41.9%), were married (67.3%), and had reported a moderate economic status (75.4%) .The effect of independent variables on a healthy diet and physical activity self-management was assessed using path analysis to identify variables affecting the patients’ self-management regarding a healthy diet and physical activity. The highest and lowest power of significant effect in healthy diet self-management was related to coping planning and risk perception constructs. However, all constructs tended to enhance self-management significantly . Fig 1 staskRegarding physical activity self-management, the highest and lowest power of the effect was related to action planning and risk perception constructs, respectively . Fig 2 sThe results of our study showed that in a healthy diet, self-management, task and coping self-efficacy , outcome expectancies , and risk perception  were significantly related to the behavioral intention of a healthy diet. Task and coping self-efficacy , behavioral intention , and recovery self-efficacy  were significantly related to action planning of diet. Task-coping self-efficacy , behavioral intention , and recovery self-efficacy  were significantly related to coping planning of diet. Coping planning  and action planning  were significantly related to self-management of a healthy diet.In self-management of physical activity, task and coping self-efficacy  and outcome expectancies  were significantly related to physical activity intention. Task and coping self-efficacy  and recovery self-efficacy  were significantly associated with physical activity coping planning. Recovery self-efficacy  was significantly related to action planning in physical activity, and action planning  was significantly associated with self-management of physical activity.There was a significant relationship between gender and self-management (p<0.05); however, this relationship lost its significance in the multivariable analysis .In this cross-sectional study, we aimed to assess healthy diet and physical activity self-management among patients who had undergone bariatric surgery. Previous studies have shown that HAPA was a useful approach for measuring self-management of a healthy diet and physical activity ,31,37,38Patients who underwent bariatric surgery tended not to follow a healthy diet and did not have regular physical activity. Their lack of awareness of the complications and problems of failure to follow a healthy diet necessitates improving their perceived abilities to follow a healthy diet and increasing self-efficacy related to a healthy diet and physical activity. The results of the BSSQ self-management behavior in the Welch study showed that the mean BSSQ total adherence score over time was consistently 60–70%, indicating moderate self-management .Task and coping self-efficacy, outcome expectancies, and risk perception were predictors of healthy diet behavioral intention. Also, task and coping self-efficacy had the highest beta coefficient among different predictors of behavioral intention. These findings were consistent with some previous studies in China  and SwitAction self-efficacy and outcome expectancies were significantly related to the intention of physical activity. This is in line with other studies that showed significant correlations between intention and action self-efficacy, action planning, and psychological consequences of physical activity –43. The Numerous studies have highlighted the prominent role of outcome expectancies in explaining dietary behaviors, which necessitates lifestyle interventions. According to the present study, the outcome expectancies construct significantly predicted behavioral intention of following a healthy diet and physical activity. Many previous studies in the field of healthy lifestyles, including nutritional behaviors and physical activity, considered outcome expectancies as the best predictor of behavioral intention following the action self-efficacy construct ,45. BargThe significant relationship between perceived risk and intention was another finding of the current study, similar to previous studies ,46. PercSimilar to previous studies in nutrition behaviors ,50,51, iOne of the limitations of this study was to measure the participants’ self-management to follow a healthy diet and perform physical activity through a questionnaire, which may not be an accurate picture of the participant’s diet and physical activity self-management. Although HAPA may be part of the solution to the behavior intention gap, the absence of social factors limits it. Since our study population was low average age, half of them had a university degree, and most of them were from medium to high socio-economic status, the results should be generated precautious.The present study showed that the score of healthy diet and physical activity self-management was low in patients who underwent bariatric surgery. Psychological variables associated with HAPA could adequately explain healthy diet behavior and physical activity among the patients. In addition, task and coping self-efficacy, risk perception, and outcome expectancies significantly affected diet and physical activity intention. Using HAPA with special attention to the contribution of the constructs is suggested. Moreover, it is suggested to design interventions with the lens of HAPA constructs to improve healthy diet and physical activity self-management in patients after bariatric surgery.S1 Data(XLSX)Click here for additional data file.


Patients suffering from connective tissue disorders like Ehlers–Danlos syndrome hypermobility type/joint hypermobility syndrome (EDS-HT/JHS) may be affected by craniocervical instability (CCI). These patients experience myalgic encephalomyelitis, chronic fatigue, depression, extreme occipital-cervical pain, and severe widespread pain that is difficult to relieve with opioids. This complex and painful condition can be explained by the development of chronic neuroinflammation, opioid-induced hyperalgesia, and central sensitization. Given the challenges in treating such severe physical pain, we evaluated all the analgesic methods previously used in the perioperative setting, and updated information was presented. It covers important physiopathological aspects for the perioperative care of patients with EDS-HT/JHS and CCI undergoing occipital-cervical/thoracic fixation/fusion. Moreover, a change of paradigm from the current opioid-based management of anesthesia/analgesia in these patients to the perioperative opioid minimization strategies used by the authors was analyzed and proposed as follow-up considerations from our previous case series. These strategies are based on total-intravenous opioid-free anesthesia, multimodal analgesia, and a postoperative combination of anti-hyperalgesic coadjuvants  with an opioid-sparing effect. Chronic neuroinflammation, neuronal damage and central sensitization are common pathways that cause severe widespread pain in patients with Ehlers–Danlos syndrome hypermobility type/joint hypermobility syndrome (EDS-HT/JHS) and craniocervical instability.The use of opioid-free anesthesia with propofol, ketamine, lidocaine, and dexmedetomidine is feasible in patients with EDS-HT/JHS who are to undergo occipital/cervical fixation, and the support of a service specialized in postoperative acute pain is essential for the viability of this management.Opioid-free anesthesia plus perioperative opioid-minimization management may reduce the postoperative pain in patients with EDS-HT/JHS that undergo occipital/cervical/thoracic fixation..Postoperative infusions of anti-hyperalgesic coadjuvants  along with multimodal analgesia provide a significant opioid-sparing effect. However, more studies are needed to justify their use for more than 72 h in this type of patientThe perioperative opioid-minimization approach is a reasonable option for patients with EDS-HT/JHS suffering from gastrointestinal dysfunction, opioid-induced hyperalgesia, central sensitization and severe widespread pain.In the context of the perioperative opioid-minimization approach, methadone is the most suitable opioid for use as a rescue analgesic for severe pain owing to its anti-NMDA effect and its effect of reducing the reuptake of serotonin and norepinephrine. Some patients with Ehlers–Danlos syndrome-hypermobility type/joint hypermobility syndrome (EDS-HT/JHS) may suffer from Craniocervical Instability (CCI). CCI can result in continuous microtraumas and inflammation on craniocervical joint surfaces. It causes repetitive peripheral sensitization, which eventually leads to the phenomenon of central sensitization (CS) and hyperalgesia , 2. ThesThe current perioperative anesthesia and pain management protocols followed with patients with EDS-HT/JHS and CCI who were programmed for occipital-cervical fixation/fusion (OCF) call for opioid-based approaches. Using opioid-based protocols seems to be a contradiction, considering the proposed nociceptive mechanisms and the high percentage of patients with an intolerance to opioids.Owing to the complex clinical features of these patients and the difficulty encountered in managing their physical pain, updated information is included in this paper which covers important physiopathological considerations for the perioperative care of patients with EDS-HT/JHS and CCI that undergo OCF. Moreover, a change of paradigm from the current opioid-based management of anesthesia/analgesia in these patients to the perioperative opioid-minimization strategies used by the authors was analyzed and proposed as follow-up considerations from our previous case series. These strategies are based on total-intravenous opioid-free anesthesia (OFA-TIVA), multimodal analgesia, and a postoperative combination of anti-hyperalgesic coadjuvants  with an opioid-sparing effect.Due to the complex variety of symptoms, clinical signs, and additional syndromes that coexist in patients with EDS-HT/JHS and CCI who are going to undergo OCF, it is important that the perioperative team understand the proposed physiopathology that may explain the cause of all symptomatic features. Likewise, it is particularly important that anesthesiologists know that these patients frequently take a wide variety of chronic medications, some of which might provoke drug interactions with anesthetic medications and postoperative treatment for pain management Table .Table 1CPatients with connective tissue diseases may suffer from craniocervical instability (CCI). These diseases include systemic lupus, rheumatoid arthritis, and genetic disorders such as Ehlers–Danlos syndrome-hypermobility type/joint hypermobility syndrome (EDS-HT/JHS), Marfan syndrome, Loeys–Dietz syndrome, Stickler syndrome, Cleidocranial dysostosis, Morquio syndrome, osteogenesis imperfecta, and Down’s syndrome. This broad group of genetic diseases characterized by generalized joint hypermobility can bring on laxity of the spinal ligaments that provoke severe symptoms due to CCI , 6.Both CCI and EDS-HT/JHS can lead to the adaptation and compensation of movement patterns. Consequently, they cause overloading in other areas along with additional constant microtraumas to other joints, ligaments, and tendons of the movement apparatus, all of which are characterized by generalized soft tissue laxity. These conditions produce widespread chronic pain, which is triggered by complex noxious mechanisms like CS –11. AnotCMS may explain some of the neurological and ancillary symptoms in patients with CCI and EDS-HT/JHS, particularly when a Chiari malformation is present. An unstable cervical spine may cause functional brainstem compression, which may be influenced by neck movements and axonal damage due to deformative stress . CervicaOn the other hand, the presence of migraines, temporo-mandibular joint (TMJ) dysfunction syndrome and myofascial neck pain in patients with JHS are factors that make the diagnosis of craniocervical pain produced by CCI difficult , 18 whicUndoubtedly, some overlap exists among all hereditary diseases of the connective tissue when considering the multi-systemic nature of generalized joint hypermobility. Interestingly, the clinical characteristics of most patients who have undergone OCF by our surgical team present with EDS-HT/JHS accompanied by CCI, chronic fatigue, severe occipital-cervical pain, severe widespread pain with a poor response to opioids, functional gastrointestinal disturbances, MCAS and autonomic symptoms like POTS .On the other hand, genetic conditions in the encoding of α-tryptase presented in some patients with EDS-HT/JHS may be involved in the coexistence of symptoms that affect the skin, gastrointestinal tract, and circulatory and musculoskeletal systems , 20.may partially explain the coexistence of these multi-systemic symptoms that affect the skin, gastrointestinal tract, cardiovascular and musculoskeletal systems. This might also be related to the coexistence of MCAS, POTS, gastrointestinal disorders and EDS-HT/JHS. This coexistence is not a constant phenomenon (less than 15%) and it can occur with different levels of severity [Studies suggest that there is a common genetic condition related to excessive germline duplications and triplications in the allelic TPSAB1 gene encoding α-tryptase that provokes an increase in serum basal tryptase levels from mast cell activity (tryptase ≥ 8.0 ng/ml). It is a common autosomal dominant inheritance that severity , 20. We There are some opioids that are considered to have a high risk of histamine release, such as morphine, pethidine (meperidine) and codeine. These opioids should be avoided, particularly in patients with MCAS and POTS. On the other hand, there is evidence that tramadol, oxycodone, fentanyl, sufentanil and methadone have a low risk of histamine release. These opioids may be an alternative for occasional use, such as rescue analgesia . In factRelative to joint laxness, the symptoms of patients overlapped in the EDS-HT and JHS groups. Experts have recently considered these two disorders as indistinguishable at the clinical level , 22, 23.In patients with EDS-HT/JHS who develop CCI, both severe craniocervical pain and widespread pain have multifactorial causes . Pain isMoreover, many patients with CCI, JHS, chronic fatigue, and severe chronic pain receive distinct types of opioids, which further complicate pain due to opioid-induced hyperalgesia (OIH) , 24, 25.As noted in other publications, it is possible that the diagnosis of withdrawal-associated hyperalgesia (WAH) was erroneously included in our data as part of the prevalence of OIH, which may be biased. Therefore, it is advisable not to extrapolate our data to the general and the entire EDS-HT/JHS population under chronic opioid medication . An advaTaking into consideration the probable mechanisms of chronic severe pain that affect some our patients with EDS-HT/JHS and CCI, and their frequent association with gastrointestinal dysfunction, the use of opioids was stopped in total intravenous anesthesia (TIVA) during OCF.The differences were clearly visible. Therefore, we believe that the opioid-based protocol for anesthesia and pain management may be an improper way to manage EDS-HT/JHS patients with CCI and severe chronic pain who are going to have OCF.Intraoperative opioid-based analgesia (remifentanil or sufentanil infusions) has been replaced with infusions of lidocaine, ketamine, magnesium, and dexmedetomidine. They are coadjuvants with known analgesic and antihyperalgesic properties. This anesthetic protocol is directed toward enhancing postoperative pain control, decreasing postoperative opioid rescues, and reducing preoperative opioid doses in patients who have been prescribed these medications over a prolonged period. The infusions of lidocaine, ketamine, and dexmedetomidine were continued at lower doses up to 72 h postoperatively as part of a multimodal analgesia plan Tables , 5. ReceIn certain cases of EDS-HT/JHS patients underwent OCF, these infusions were used for more than 72 h without any complication (5 days). These cases suffered from severe widespread pain with significant neuropathic symptoms, and the use of opioids was completely contraindicated for severe intestinal disorders and MCAS. These cases represented less than 2% of our case series. It is important to mention that postoperative infusion of lidocaine, ketamine and dexmedetomidine for more than 72 h is not yet supported by the literature. In our cases, the prolongation of these infusions was justified by compassionate use.An exhaustive search for evidence related to opioid-free anesthesia and perioperative opioid-minimization management in patients with EDS-HT/JHS undergoing OCF has been carried out. Upon completion of this research, two independent reviewers (C.R-P and C.RC) screened the titles and abstracts using the Cochrane Library, Ovid Medline, PubMed Central, and Google Scholar databases according to PRISMA guidelines. Any arising differences were settled by a discussion with a third and fourth party (A.CB and A.SS). Only publications written in English since 2010 to 2022 with the following terms were applied to the search strategy: (((((hypermobile syndrome) OR (EDS-HT)) AND (Opioid-Free Anesthesia)) AND (opioid-free anaesthesia)) AND (perioperative opioid minimizing approach)) AND ) AND ) AND (OCF)) OR . Randomized clinical trials, case series, case reports, narrative review and editorials were equally included to complete the current information available on OFA plus perioperative opioid-minimization management in patients with EDS-HT/JHS undergoing OCF.Patients diagnosed with EDS-HT/JHS and CCI.Patients with EDS-HT/JHS and CCI undergoing OCF with opioid free-total intravenous anesthesia.Patients with an opioid intolerance due to gastrointestinal disorders, nauseas and vomiting, and/or OIH, and/or MCAS triggers.Patients under OFA-TIVA and postoperative use of lidocaine, ketamine, and dexmedetomidine infusions.The inclusion criteria were as follows:Patients with CCI without EDS-HT/JHS.Patients with EDS-HT/JHS and CCI who underwent any other surgery than OCF or OCF plus thoracic fixation.Patients with EDS-HT/JHS and CCI who underwent OCF under opioid-based anesthesia/analgesia, with or without medication for multimodal analgesia.The exclusion criteria were as follows:The database search identified 328 records. After removing duplicate records, 324 articles were screened for relevance of titles and abstracts. Four relevant articles were reviewed for eligibility, resulting in a full-text review. Two articles were excluded. One of them is a systematic review focused solely on the surgical treatment of craneocervical instability in patients with EDS-HT/JHS. The other article deals with the use of OFA plus opioid-minimization management in various clinical settings other than OCF, including EDS-HT/JHS , 35. Fi.Fig. 1FlTwo remaining articles were included in the final qualitative analysis, a case series study of 42 patients, and a narrative review , 37. The2O in their groups and the patients do not suffer from EDS-HT/JHS or any other collagen disorder.Information related to the use of opioid-free anesthesia plus postoperative infusions of lidocaine, ketamine, and dexmedetomidine as part of opioid-minimization management in patients with EDS-HT/JHS for any type of surgery is lacking. Only one previously published study by the authors of this paper can be found, registered in ClinicalTrials.gov Identifier: NCT04437589 by Ramirez-Paesano et al. , entitleA recently published review referred to OFA techniques followed by opioid-free analgesia such as OFAA, whose name and acronym could be synonymous with our OFA-plus protocol .p < 0.001). VAS at hospital-discharge was lower in the “OFA-plus” group (p < 0.001).For many years, our team has managed patients with EDS-HT/JHS who underwent OCF with TIVA using opioids like fentanyl, sufentanyl, or remifentanil as intraoperative analgesics. In the past, postoperative pain management was based on morphine, hydromorphone, nonsteroidal anti-inflammatory drugs, and benzodiazepines. The postoperative period used to be a challenging time with poor outcomes in terms of pain management, gastrointestinal side effects, and patient satisfaction. Based upon a better comprehension of nociceptive physiopathology, a decision was taken to change the paradigm around the use of opioids as mandatory analgesics in the perioperative period. The new paradigm for opioid minimization management includes infusions of lidocaine, ketamine, and dexmedetomidine as coadjuvant painkillers with significant anti-hyperalgesic and opioid sparing effects. Reducing the use of opioids by managing these patients with the “OFA-plus” protocol has resulted in a reduction in Visual Analogue Scale (VAS) in terms of postoperative time (p < 0.001). Among patients in the OFA-plus group, 78% (95% CI) required no methadone rescue, 8.7% required 10–15 mg/day, and 4.3% of patients required more than 15 mg/day.The methadone requirement was lower in the OFA-plus group (p < 0.001). In the OFA-plus group, 60.9% of patients had decreased opioid requirements at hospital discharge compared with the preoperative values. However, in the OP group, 26.3% of patients maintained the same opioid requirement, 63.2% increased, and no patients showed a decrease. A 77% reduction in anxiolytic requirements was observed [.In contrast, 95% (95% CI) of the OP group (group with opioid-based management) required methadone rescue at high doses. Thirty-six percent required 10–15 mg/day and 42.8% required more than 15 mg/day of methadone rescue. The OFA-plus group showed decreased ileus, nausea, and vomiting , an important part of the OFA-plus protocol, was provided following the protocol described in our recently published article . Since tAlso, the preventive use of haloperidol to reduce the incidence of ketamine-induced hallucinations have been included in patients who suffer from severe anxiety and important psychological disorders. The literature suggests that low-dose ketamine has a low potential for hallucinations. However, in our case series, the incidence of hallucinations was 17.4% despite all the patients received midazolam at 0.1 mg/kg intraoperatively. In addition, many of them were chronically medicated with benzodiazepines and antidepressants to treat severe anxiety, mood disorders, panic attacks, and depression.This incidence of hallucinations was considered to be very high, and this fact motivated us to search for the mechanisms of ketamine-induced hallucinations. We do not have a definitive explanation for this result. We hypothesized that potential causes such as genetic, psychologic or neuropsychiatric susceptibilities may be the explaination, but it is only a speculative reasoning. In any case, we needed an optional medication to reduce the ketamine-induced hallucinations. A recent meta-analysis and systematic review suggested that the phycomimetic effects of ketamine may involve the dopaminergic system, and a mechanism of neuronal firing by dopamine release was proposed . Acute aTable To decrease the requirement of opioids during postoperative period, we believe that it is helpful to keep the postoperative infusions of lidocaine, ketamine, and dexmedetomidine at lower doses. The goal is to attenuate the inflammatory response resulting from surgical trauma and its effect on pain, CS, and gastrointestinal function , 49, 50.Many of these patients present involuntary cervical and thoracic muscle contractures and spasticity leading to exacerbation of postoperative pain. Tizanidine or baclofen are used when muscle contractures and spasticity are identified as an additional component of pain. Tizanidine is a muscle relaxant with alpha-2-agonist analgesic mechanism with anti-nociceptive effect in neuropathic pain through inhibition of pro-inflammatory cytokines production via suppression of Toll-like receptor 4/Nuclear Factor-ƙB (TLR4/NF- ƙB) activation as well as postoperative opioid-sparing effect , 52.Baclofen, a gamma-aminobutyric acid type B receptor agonist, blocks the release of excitatory neurotransmitter in neurons and glial cells (glutamate and aspartate) by interfering with voltage-gate calcium channels. Baclofen decreases muscle tone and prevents reflex muscle contraction and spasticity .An improvement in those patients with “uncontrolled pain” by using oral baclofen (25 mg e/8 h) has been noticed. This fact led us to consider cervical-dorsal muscular spasticity or muscle contracture as an additional component of pain crisis. , 55.Methadone, an opioid with anti-NMDA effect, is used as a painkiller rescue to treat postoperative breakthrough pain . So far,Later, the administration of oral ketamine may be continued in patients with widespread pain that is difficult to control. This is also the case if there is a preoperative history of treatment with opioids, OIH, an important neuropathic component, or a propensity to receive high opioid rescue doses.Many articles on the advantages of OFA have been published. However, studies of OFA in patients that have undergone spinal surgery have revealed controversial findings relative to postoperative outcomes –59. The . On the contrary, the nociceptive flexion reflex threshold (NFRT) may be more fitting to monitor nociception in patients under opioid-free total intravenous anesthesia (OFA-TIVA) without neuromuscular blockers (NMB). NFRT, a method based on electromyography (EMG) and bispectral index (BIS), is able to predict movement as a response to surgical pain under propofol monoanesthesia. When using OFA-TIVA, NFRT might be useful in predicting movement due to surgical pain [A balanced combination of lidocaine, dexmedetomidine, ketamine and MgSO4 has antinociceptive and anti-hyperalgesic effects. However, there are still doubts about the analgesic efficacy of its use in OFA when compared to opioids. Doubts have also arisen as to the most appropriate combination of these coadjuvants to replace opioids. The absence of dependable nociceptive monitors has been an obstacle to changing the paradigm in spine surgery . Monitorcal pain .This article deals with the topic of a propofol-based OFA in a group of specific patients (EDS-HT/JHS) with widespread pain and SC that have undergone major spinal surgery to treat CCI. Intraoperative analgesia is delivered by means of a balanced combination of lidocaine, ketamine, MgSO4 and dexmedetomidine. These "opioid substitutes" have anti-hyperalgesic and anti-inflammatory properties as well as multiple mechanisms of analgesic action. In addition, anesthesia is provided without neuromuscular blockers (NMB) because our surgical team requires continuous evaluation with neurophysiologic monitoring that includes motor evoked potentials (MEPs) and electromyography (EMG). Somatosensory evoked potentials (SSEPs) are also monitored. In our practice, EMG recordings have confirmed the absence of movements related to the surgical stimulus in any patient. The dosage of lidocaine, ketamine, MgSO4 and dexmedetomidine used in our OFA-plus protocol  has been significantly reduced and methadone rescue has been reduced. Moreover, the postoperative comfort of the patients, gastrointestinal side effects and the need for anxiolytics drugs decreased ..Therefore, we believe that we are on the right pathway to providing better management of these patientsSome resistance to the LA effect has been noted in regional anesthesia techniques for dental, orthopedic, and obstetric procedures. However, there is no evidence that peripheral nerve blocks or spinal/epidural anesthesia are ineffective. On the contrary, regional anesthesia may be the better choice for some orthopedic and obstetric procedures in patients with EDS-HT/JHS. On the other hand, there are some case reports of resistance to certain LA with local infiltration –71. ResiAt the moment, our OFA-plus protocol does not currently include regional techniques with local anesthetic for postoperative analgesia. However, we believe that in the future the use of some techniques such as ultrasound-guided interfascial blocks would be possible and beneficial to further reduce methadone rescue. Further study will be necessary to test this hypothesis.So far, any sign of resistance or the absence of an analgesic effect from intravenous lidocaine has not been observed. This may be because the mechanisms of intravenous lidocaine analgesia are mediated by a strong systemic anti-inflammatory effect and multiple antinociceptive pathways other than Na+ channel blockade-mediated analgesia at therapeutic plasma concentrations , 66, 74.The coexistence of widespread chronic pain, CS, opioid intolerance, OIH, gastrointestinal disturbances, and the potential presence of MCAS has been the reason for the use of OFA-plus in these patients , 24, 25.If necessary, we use methadone to relieve severe breakthrough pain in the postoperative period. However, including the use of non-opioid options that affect different nociceptive pathways  remains important to reduce the use of postoperative opioids –78. BecaMoreover, at present, no analgesic can completely replace opioids during the postoperative period as rescue to control severe breakthrough pain.We are in complete agreement with some authors that OFA should not be taken as a popular (“fashionable”) trend to be followed in any type of surgery under general anesthesia . We firmRegarding pain management in patient with OIH and CS, we believe that methadone is the most suitable rescue opioid to treat severe pain owing to its anti-MNDA effect. Methadone decreases OIH and attenuates central sensitization. It also reduces the reuptake of serotonin and norepinephrine. Furthermore, the use of methadone with ketamine (both anti-NMDA) shows a synergy that enhances the opioid-sparing effect .Patients that have undergone major surgical procedures frequently require postoperative opioids to relieve breakthrough pain control. The OFA-plus protocol used in our hospital includes methadone only as a rescue agent for severe postoperative pain that is difficult to control with non-opioid analgesics .Recent literature has recommended the use of methadone (0.15–0.2 mg/kg bolus) at the beginning of anesthetic induction in complex spinal surgery . MethadoA recent meta-analysis confirmed the benefits of methadone use at the onset of anesthesia in extensive and painful surgeries , 84.If methadone is needed as a postoperative rescue, nausea and vomiting may be a problem. At some point, fosaprepitant may be an option for the management of intractable nausea and vomiting. Currently, our protocol does not include the use of methadone in the intraoperative period, which is consistent with the OFA concept. If a single dose of methadone is administered at the beginning of our OFA protocol, the anesthetic technique should be called modified OFA-plus, or simply not called OFA.Patients with EDS-HT/JHS and CCI experience severe chronic widespread pain and hyperalgesia, which are strongly related to the central sensitization phenomenon. The use of the “OFA-plus” protocol is feasible and safe to use in patients who are to undergo OCF with EDS-HT/JHS associated with severe pain, POTS, MCAS, and gastrointestinal dysfunction.The infusions of lidocaine, ketamine, and dexmedetomidine in combination with propofol for TIVA without NMB seem to provide adequate analgesia. The neuromonitoring  records suggest that this management is appropriate.Postoperative pain management in patients who are to undergo OCF is difficult and complex. The use of lidocaine, ketamine, and dexmedetomidine infusions during the postoperative period may be helpful in improving pain management while reducing the total amount of opioids used during the hospital stay.To date, it has been difficult to eliminate the use of opioid rescues to control severe postoperative breakthrough pain in its entirety. It is important to continue studying non-opioid therapeutic options to reduce gastrointestinal side effects, OIH, and opioid tolerance and dependence.Does OFA offer short-term benefits in the postoperative period?: In response to this it should be said that there are no randomized studies to give a definitive answer. However, at present we could say that OFA plus perioperative opioid-minimization approach reduce the total amount of opioid and the gastrointestinal side-effects in this patients. (2) Does “OFA-plus” offer long-term benefits regarding the pain?: To this question it is important to state that there are no studies that support it. (3) In these types of patients undergoing OCF, Could the use of a single dose of methadone be recommended at the beginning of the anesthetic induction? This could be possible. However, there are no studies using “the modified-OFA” in this context.Finally, the last questions we should answer are: (1)


An association between SMK and renal arteriolar wall thickening might be prevalent only among patients with HU and in patients with IgA nephropathy. Further prospective studies are needed to determine whether patients with HU and SMK history exhibit rapid eGFR deterioration.The combination effects of smoking (SMK) and hyperuricemia (HU) on renal arteriolosclerosis in patients with IgA nephropathy remain unknown. We examined the cross-sectional association between smoking (current or former) and renal arteriolar hyalinosis and wall thickening with or without HU [uric acid (UA) level ≥ 7 and ≥5 mg/dL in men and women] in 87 patients with IgA nephropathy who underwent renal biopsy. Arteriolar hyalinosis and wall thickening were assessed by the semiquantitative grading of arterioles. The SMK/HU subgroup showed the highest indices for hyalinosis and wall thickening, followed by the non-SMK/HU, SMK/non-HU, and non-SMK/non-HU subgroups. Multiple logistic analysis showed that SMK/HU, but not SMK/non-HU, was significantly associated with an increased risk of higher-grade renal arteriolar wall thickening. However, this did not occur with hyalinosis compared to non-SMK/non-HU. The adjusted odds ratio (95% confidence interval,  A meta-analysis of observational studies has suggested that smoking (SMK) has adverse effects on chronic kidney disease (CKD) with considerable heterogeneity . PreviouAlthough an association between SMK and CKD progression has not been clearly demonstrated, hemodynamic and nonhemodynamic factors may play important roles in their underlying mechanism . SMK is A total of 93 consecutive patients who were newly diagnosed with IgA nephropathy at the University of the Ryukyus Hospital between 1 January 2003 and 31 December 2006 were considered for inclusion in this study. The six patients using urate-lowering drugs were excluded from the analysis. Finally, 87 patients were included in this study.Using previously described methods , we asse2) = 194 × serum creatinine−1.094 × age−0.287  [We identified sex-specific differences in serum uric acid (UA) cut-off values, which were 5 mg/dL for women and 7 mg/dL for men, which indicated an increased risk of renal arteriolar hyalinosis according to our previous study. Therefore, we defined UA levels as ≥5 mg/dL in women and ≥7 mg/dL in men. The smokers were defined as current or former smokers. Body mass index (BMI) was calculated by dividing the weight in kilograms by the height in meters squared. Dyslipidemia was defined as hyper-low-density lipoprotein (LDL) cholesterolemia, hypertriglyceridemia, hypo-high-density lipoprotein (HDL) cholesterolemia, and/or the use of antidyslipidemia. In addition to a medical history of diabetes, diabetes mellitus was determined by fasting and a postprandial glucose level measurement or by a 75 g oral glucose tolerance test. Urinary protein levels were measured in first-spot morning urine samples. The estimated glomerular filtration rate (eGFR) was calculated using a revised equation for Japanese individuals: eGFR  .p-value of <0.05.A one-way analysis of variance (ANOVA) and the chi-square test were used to analyze differences in discrete variables between the groups. A one-way ANOVA was also used to analyze the interaction effect of SMK and HU on arteriolosclerosis indices. Higher-grade arteriolar hyalinosis and wall thickening were defined as hyalinosis or wall thickening with values equal to or greater than the third tertial hyalinosis index (0.41) and the wall thickening index (0.316). Multivariate logistic regression analysis was employed to determine the relationships between SMK/HU subgroups and arteriolar hyalinosis and wall thickening. We adjusted for age, sex, and potential confounding factors such as hypertension. The use of renin-angiotensin system (RAS) inhibitors was also included because they reversed arteriolar hypertrophy independent of their blood pressure-lowering effect ,24. The The SMK/HU subgroup was characterized by older age, a higher prevalence of hypertension, hyperlipidemia, a higher BMI, and lower eGFR .We analyzed the indices of hyalinosis for each SMK/HU subgroup. The hyalinosis index in the SMK/HU subgroup was the highest among the groups . The ratWe analyzed the indices of wall thickening for each SMK/HU subgroup. The wall thickening index in the SMK/HU subgroup was the highest among the groups . The ratWe analyzed the odds ratios (ORs) of various risk factors for higher-grade arteriolar hyalinosis. Age, the history of hypertension or diabetes mellitus or dyslipidemia, eGFR (high to low), SMK/HU, and non-SMK/HU (non-SMK/non-HU was used as a reference) were significantly associated with a higher grade of arteriolar hyalinosis in the non-adjusted model. The association between non-SMK/HU and arteriolar hyalinosis was significant in multivariate model 3, which included age, sex, potential risk factors such as hypertension, and the use of RAS inhibitors .We also analyzed the ORs of different risk factors for higher-grade arteriolar wall thickening. Age, BMI > 25, history of hypertension or diabetes mellitus or dyslipidemia, eGFR (high to low), and SMK/HU (non-SMK/non-HU was used as a reference) were significantly associated with a higher grade of arteriolar hyalinosis in the non-adjusted model. The relationship between SMK/HU and arteriolar wall thickening remained significant even in multivariate model 3, which included age, sex, and potential risk factors such as hypertension and RAS inhibitors .The findings of this study demonstrate the associations of a combination of SMK and HU with renal arteriolar wall thickening but not hyalinosis in IgA nephropathy. In this study, SMK was associated with arteriolar wall thickening only for the existence of HU. Although the confounding effects of advanced age and a history of hypertension or diabetes might be relevant, the relationship between SMK/HU and arteriolar wall thickening was substantial even after adjusting for these confounding factors. Although non-SMK/HU was significantly associated with renal arteriolar hyalinosis, we could not confirm a singular association between SMK and renal arteriolar hyalinosis and wall thickening. With regard to renal outcomes, current SMK was reportedly associated with CKD progression ,26 and eSeveral mechanisms could underlie the significant association of the combination of HU and SMK with renal arteriolar wall thickening. By activating RAS and the sympathetic nervous system, SMK is linked to hypertension . HU can Regarding the clinical relevance of the association of the combination of SMK and HU with renal arteriolar wall thickening, the coexistence of HU and SMK could have a great effect on CKD progression. Arteriolar wall thickening, which is a typical finding of nephrosclerosis that can cause renal ischemia . VasoconThis study has several limitations. First, we could not confirm whether the combination of SMK and HU induced renal arteriolar hyalinosis because of the cross-sectional design. Second, the results of this study cannot be applied to CKD of any etiology. Renal arteriolosclerosis is commonly observed in patients with diabetes mellitus and/or hypertension. Therefore, the additional effects of SMK and HU on renal arteriolar hyalinosis in patients with CKD remain to be determined. Third, the limited number of renal biopsy samples raises a concern regarding sampling bias. However, we certified a linear correlation between the hyalinosis index and age or blood pressure in this study in accordance with the results of a previous autopsy-based study . TherefoWe found that SMK affected renal arteriolar wall thickening in the presence of HU but not in the absence of HU in patients with CKD. These associations were independent of age, the history of hypertension or diabetes mellitus, and antihypertensive medication. These findings suggest that the combination of SMK and HU could promote CKD progression through the potentiation of renal arteriolosclerosis. Therefore, large prospective trials are needed to determine the efficacy of interventions such as SMK cessation combined with urate-lowering drugs to retard CKD progression when complicated with HU.


As a result of increasing numbers of studies most recently, mitophagy plays a vital function in the genesis of cancer. However, research on the predictive potential and clinical importance of mitophagy-related genes (MRGs) in hepatocellular carcinoma (HCC) is currently lacking. This study aimed to uncover and analyze the mitophagy-related diagnostic biomarkers in HCC using machine learning (ML), as well as to investigate its biological role, immune infiltration, and clinical significance.In our research, by using Least absolute shrinkage and selection operator (LASSO) regression and support vector machine- (SVM-) recursive feature elimination (RFE) algorithm, six mitophagy genes  were identified from twenty-nine mitophagy genes, next, the algorithm of non-negative matrix factorization (NMF) was used to separate the HCC patients into cluster A and B based on the six mitophagy genes. And there was evidence from multi-analysis that cluster A and B were associated with tumor immune microenvironment (TIME), clinicopathological features, and prognosis. After then, based on the DEGs  between cluster A and cluster B, the prognostic model (riskScore) of mitophagy was constructed, including ten mitophagy-related genes . This study uncovered and analyzed the mitophagy-related diagnostic biomarkers in HCC using machine learning (ML), as well as to investigate its biological role, immune infiltration, and clinical significance. Based on the mitophagy-related diagnostic biomarkers, we constructed a prognostic model(riskScore). Furthermore, we discovered that the riskScore was associated with somatic mutation, TIME, chemotherapy efficacy, TACE and immunotherapy effectiveness in HCC patients.Mitophagy may play an important role in the development of HCC, and further research on this issue is necessary. Furthermore, the riskScore performed well as a standalone prognostic marker in terms of accuracy and stability. It can provide some guidance for the diagnosis and treatment of HCC patients. HCC is the world’s fifth most prevalent malignancy, with the third highest fatality rate . EffectiThe intracellular mitochondria are responsible for transmitting several complex signals, including those associated with cell growth, energy consumption, cell differentiation, cell repair, and cell death . A changModern technologies, including ML algorithms, have been created to cope with the increasing volume and complexity of cancer and other multi-omics data , 15. A cWe used an ML algorithm to select six diagnostic biomarkers among 29 mitophagy genes for future investigation. Next, a cluster of 113 HCC patients from the GEO database and 367 HCC patients from the TCGA database was formed based on the expression of six mitophagy genes. The variations between mitophagy-related patterns were examined using multi-omics analysis, which included clinical relevance, survival analysis, and TIME. We developed the prognostic model (riskScore), which was demonstrated to be an independent prognostic marker. The riskScore can predict the overall survival rate of HCC patients. Furthermore, we discovered a relationship between riskScore and somatic mutation, TIME, chemotherapy efficacy, TACE and immunotherapy effectiveness in HCC patients.The HCC and paracancerous tissues (n = 40 for both) were procured for patients in the Department of Hepatobiliary Pancreatic Surgery, Northern Jiangsu People’s Hospital, and surgical therapy was provided to forty patients with primary HCC. The goals of the Declaration of Helsinki were followed when conducting the current investigation. The people who donated the samples gave their written, informed consent.https://www.ncbi.nlm.nih.gov/gds) and Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov/) platform. The GEO data come from the GSE76427 microarray data. We also handled the clinical data of patients. In the TCGA, GEO, and ICGC cohorts, we retrieved baseline information, survival information, and pathological information for analysis. The TCGA database patients serve as a training set for constructing the prognosis model. In contrast, the International Cancer Genome Consortium (ICGC) database patients (https://dcc.icgc.org/projects/LIRI-JP) serve as a validation set for establishing and validating the prognosis model. To assess TACE sensitivity, we employed the GSE104580 in the research. From GeneCards (https://pathcards.genecards.org/), the 29 mitophagy genes were retrieved.All patients in this investigation had transcriptome and clinical data collected using the Gene Expression Omnibus (GEO) databases  gathered information from GEO and Array Express to create a single-cell RNA-seq (scRNA-seq) atlas. By displaying gene expression across many data sets at the single-cell or cluster level, TISCH compares various patients, therapy and response groups, tissue origins, cell types, and even cancer sorts. In this work, we used TISCH datasets to investigate the TIME heterogeneity of six mitophagy genes at the single-cell level in GSE140228  score system.The non-parametric, unsupervised GSEA method may transform a segregated gene expression matrix into a characteristic expression matrix for a particular gene set. The “enrichplot,” DOSE,” and “clusterProfiler” R packages were used to implement the technique. We looked at the statistical changes in the modified expression matrix using the “limma” package.To establish TIME’s characteristics, the percentages of immune cell types in each sample were determined using the MCP-counter approach. Furthermore, we examined the association between the number of immune cells and riskScore using the Wilcoxon rank-sum test. To investigate the immunological state, we performed an ssGSEA analysis. The superior algorithm  was usedUsing T statistics and p values (p < 0.001), the “limma” tool was used to assess changes in gene expression following NMF clustering to identify DEGs between two distinct phenotypes. Next, we examined DEGs between mitophagy-related patterns using the Metascape web-based platform’s enrichment of the Gene Ontology (GO) and KEGG pathways .The relevant prognostic genes, which consisted of ten DEGs , were discovered by applying the LASSO regression to the prognosis-related DEGs in the model of univariate Cox regression. Finally, we got the riskScore formula:The R function “surv cutpoint” was used to divide the TCGA cohort into high- and low-risk groups after determining the appropriate riskScore cutoff. We assessed the predicted reliability of predictive models using Kaplan-Meier analysis  and the receiver operating characteristic (ROC) curve (package of “timeROC”). The area under the curve (AUC) was used to quantify the ROC curve. We employed identical analytic methodologies, riskScore algorithm, and cutoff value to verify the signature in the ICGC cohort.in vivo drug responses in cancer patients, OncoPredict compares the gene expression profiles of tissues and cancer cell lines from the Broad Institute Cancer Cell Line Encyclopedia Genomics of Drug Sensitivity in Cancer  and  based on the half-maximal inhibitory concentration (IC50) of drugs in cancer cell lines. Unpaired t-tests were used to compare the sensitivity of the drugs (between the high- and low-risk groups) in 198 medicines. The significance level was chosen at P-value < 0.05.Maeser et al. created the OncoPredict R package . To predTRIzol Reagent  was used to extract total RNA for assay from 40 matched HCC tissues and paracancerous tissues following the manufacturer’s instructions. Each sample’s total RNA was converted into cDNA using the PrimeScriptTM RT reagent Kit from Takara Bio Inc. in Japan. Real-time PCR was carried out in a Rotor-Gene 3000 device using an SYBR-Green PCR kit from Takara in Osaka, Japan . The statistical analysis for this study was carried out using R (version 4.0.5) software. To validate a clear difference between the two groups, we used the paired samples t-test or the Wilcoxon rank-sum test. The Kruskal-Wallis test was done to see if there was a significant difference between more than two groups. Spearman’s correlation analysis was used to determine the relevant coefficients between immune checkpoint gene expression, TMB, and riskScore. To show the frequency of gene mutations, we used the “maftools” software to create waterfall charts and established a statistical difference criterion of P-value < 0.05.From the GeneCards, a total of twenty-nine mitophagy genes were obtained for this investigation. Then, to find putative HCC diagnostic biomarkers, we employed two different ML algorithm, LASSO regression and SVM-RFE, in the TCGA dataset. By narrowing the range of DEGs using the LASSO regression method, seventeen genes were identified as diagnostic indications of HCC  mutations were found in six mitophagy genes studied. It has been demonstrated that mitophagy affects immune cells’ infiltration of the tumor microenvironment . Thus, wWe used the TCGA and GEO datasets to evaluate these six mitophagy genes to improve the analysis’s precision and reliability. We conducted consensus clustering on the mRNA expression patterns of six mitophagy genes in combined TCGA-HCC and GSE76427 samples. We found two molecular clusters : high-risk (n = 76) and low-risk (n = 155). And as the figure exhibition, the trend of the TCGA-HCC dataset and the ICGC-HCC dataset were similar in the heatmaps of ten mitophagy-related genes expression profiles, the riskScore distribution, and the status of survival . In 40 HCC and paracancerous tissues, the results showed that the relative mRNA expression of G6PD, KIF20A, TPX2, TRNP1, and SPP1 was higher in HCC tissues. In comparison, the relative mRNA expression of ANXA10, ADH4, CYP2C9, and CFHR3 was higher in paracancerous tissues  as diagnostic feature biomarkers for HCC according to a combination of ML algorithms and classical bioinformatics. Existing research shows that during carcinogenesis, mitophagy activates an adaptive immune response . And ourThe DEGs between cluster A and cluster B patterns were then investigated. Using LASSO regression, we built a prognostic model in the TCGA-HCC cohort, which included ten mitophagy-related genes . We demonstrated that the model was applicable in clinical. HCC patients were split into high- and low-risk groups based on their riskScore, and several analyses were done. Patients in the high-risk group had a lower OS than those in the low-risk group, demonstrating that the riskScore is associated with tumor progression or poor prognostic events. It was also shown in another external ICGC-HCC cohort. We found that the riskScore was a valuable independent prognostic factor in both the TCGA-HCC and the ICGC-HCC cohorts using univariate and multivariate Cox regression.via the TIME.Since riskScore was constructed based on mitophagy genes, and as mentioned earlier, mitophagy genes were closely related to immune cell infiltration, we further explored the relationship between riskScore and immune cell infiltration and validated it by single cell sequencing. Interestingly, patients in high-risk group exhibited greater levels of infiltration of immune cells than low-risk group. What’s more, we further analyzed the differences between the components of immune cells in the high- and low-risk group. And, the ratio of immune cell composition differs significantly between the two groups. However, immune cells are widely established to be directly associated with tumor growth –35 or anCancer patients with increased TMB had a higher likelihood of long-lasting and efficient treatment responses . The lowFor advanced HCC, chemotherapy, immunocompetent individuals (ICIs), and TACE are all viable therapeutic options. In this investigation, we confirmed that the IC50 values of Cisplatin, Gemcitabine, Oxaliplatin, and Sorafenib were lower in the low-risk group than in the high-risk group, whereas 5-Fluorouracil and Afatinib were lower in the high-risk group. The previous analysis showed riskScore is a useful indicator for determining immunological status. As a result, we intended to investigate riskScore’s response rate to ICI treatment based on the TIDE score further. Interestingly, the low-risk group had a higher TIDE score, which suggests that they may respond worse to ICI therapy. In addition, we find that TACE treatment regimens may be more beneficial for low-risk patients. According to the analysis above, including immunotherapy, chemotherapy, and TACE, riskScore has significant guiding relevance for HCC treatment.Collectively, we uncovered the mitophagy-related diagnostic biomarkers in HCC using ML algorithms and found they are closely related to biological function, immune infiltration, clinical prognosis. This suggests that mitophagy may play an important role in the development of HCC, and further research on this issue is necessary. Furthermore, we constructed a reliable prognostic model (riskScore). The phenotype of HCC patients may be quantified and individualized using riskScore by performing a complete review of the cellular, molecular, and clinical aspects of HCC patients. RiskScore is an important independent prognostic measure for choosing chemotherapy, TACE treatments and immunotherapy for HCC patients. Yet, our analysis includes limitations that need to be addressed further. Although the 29 genes in the mitophagy pathway we obtained from GeneCards can reflect the important role of mitophagy in HCC through analysis, based on the complexity of mitophagy function and the diversity of genetic phenotypes, these 29 genes cannot fully reflect the significance of mitophagy in HCC, and further experimental research is required to deepen our understanding of the link between mitophagy and HCC. Moreover, multicenter, high sample size, and prospective investigations, mainly as retrospective research, are necessary to corroborate the reliability of our constructed prognostic model.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Northern Jiangsu People’s Hospital. The patients/participants provided their written informed consent to participate in this study.D-YT, JC, JZ, D-SB, and RP designed and implemented the research. G-QJ and S-JJ collated and analyzed the data. D-YT, JC, JZ, D-SB, and RP provided technical support. RP and JZ provided the language polishing for this article. D-YT wrote the manuscript. D-SB and JC revised the manuscript. All authors contributed to the article and approved the submitted version.


Depressive symptoms the most prevalent clinical condition in the field of mood disorders in older populations. Depressive symptoms are associated to poorer morbidity and mortality, and is considered a component of frailty and intrinsic capacity. Dementia could overlap with DS in clinical and brain abnormalities. Moreover, there are sex-differences in the field of Neuro- and Gero-science. To date, no review has addressed the neuro-anatomical basis of DS in older adults using magnetic resonance imaging (MRI), neither has investigated the discrimination of dementia nor sex-differences. This narrative review investigated studies about older adults; depressive symptoms evaluation via MRI, and published in English or Spanish over the past 7 years. Moreover, it evaluated dementia discrimination and sex-related differences. The most accurate evidence showed cerebral small vessel disease as a predictor of depressive symptoms worsening. Most studies were cross-sectional, with a coarse dementia screening and sex-unrepresentative samples. Cingulate cortex and hippocampus showed a negative association to depressive symptoms, and Precuneus cortex a positive association; although these inferences require further investigation. Additional research is needed to identify the brain imaging signature of depressive symptoms in older population (if any), and if this would be associated with sex and individuals’level of frailty and intrinsic capacity. ACCanterior cingulate cortexADaxial diffusivityAPOEε4apolipoprotein Eε4CA1first region of hippocampal circuitCCcingulate cortexCES-Dcentre for epidemiologic studies depression scaleCSVDcerebral small vessel diseaseCTcortical thicknessDSdepressive symptomsDSpdepressive symptomatic participantsDTIdiffusion tensor imagingFAfractional anisotropyGDSgeriatric depression scaleGMgrey matterICintrinsic capacityMDmean diffusivityMDDmajor depressive disorderMRImagnetic resonance imagingPCCposterior cingulate cortexRDradial diffusivitySSsomatic symptomsTBSStract-based spatial statisticsTBVtotal brain volumeWBAwhole-brain analysisWMwhite matterWMHwhite matter hyperintensityAt older ages, clinically relevant depressive symptoms (DS) frequently occur. The prevalence ranges from 10 to 14%, which for some cohorts represents 3-fold more than the observed for Major Depression Disorder (MDD) . DS are In the past years, a growing body of literature has showed the neuro-anatomical basis of DS using magnetic resonance imaging (MRI). The areas which have received the most attention are those embedded in the neurobiological models of MDD. The frontal lobe, the hippocampus, and cingulate cortex (CC) are involved in the fronto-limbic network. The CC is divided into four functionally distinct regions: the anterior cingulate cortex (ACC), mid-cingulate cortex, posterior cingulate cortex (PCC), and retrosplenial cortex, which comprises the isthmus . Also thAnother target of DS and brain-MRI studies, is white matter hyperintensities (WMH): hyper-intense areas, diffuse or patchy, in deep or periventricular distribution (i.e. in T2 MRI). Furthermore, white matter microstructure have been investigated through MRI-diffusion tensor imaging (DTI). DTI-MRI measures the diffusion of water molecules in neural fibers. Several DTI-indices can be measured, such as fractional anisotropy (FA); mean, radial, and axial diffusivity . These measures can be locally assessed in pre-defined regions of interest, tracts of interest using tract-based spatial statistics (TBSS), or globally, with voxel-wise whole-brain analysis (WBA) .These investigations help to understand the biological mechanisms of DS, and evidence suggest that they are similar to those found in MDD in older adults . DespiteThe current work aims at reviewing the main results of studies on MRI markers associated to DS in older adult populations. It also evaluates how sex-related differences and dementia discrimination are accounted.This is a narrative review. We looked for original articles on (i) older population (≥62 years old), (ii) measuring DS, (iii) neuroimaging assessment through brain-MRI. Electronic searches were performed in January 2023 by one reviewer using different databases . We focused on recent literature (i.e. past 7 years) published in English and Spanish. Studies related to a specific neurological disease, or other psychiatric disorders, or depression treatment distinct from antidepressants (i.e: electroconvulsive therapy) were excluded. We extracted information on study design, sample size , mean age of subjects, cognitive performance screening, DS variable definition , DS scoring tool, MRI methods and focus, main outcomes and limitations.MRI studies have been focused on grey matter volume and thickness, and white matter abnormalities. Five broad anatomical categories can be distinguished in GM studies.A cross sectional study reported that DS were negatively associated to the frontal cortex thickness, globally, and in regions of the prefrontal cortex: dorso-lateral prefrontal cortex, ventro-lateral prefrontal cortex and orbito-frontal cortex. In contA correlation was found for DS and temporal cortex thinning and this was also observed in the Entorhinal cortex .Two studIn the parietal lobe, only the precuneus has been studied in relation to DS.A cross-sectional study observed that more somatic symptoms (SS)  were associated with greater right precuneus cortical thickness, nonetheless, when testing age and DS-subscales interaction, found that younger ages were associated with greater cortical thickness, whilst older ages were associated with less cortical thickness . MoreoveThe ACC showed a negative association with DS in one study  whereas CT has been found to be a better indicator of brain abnormalities related to DS than surface area . NonetheOne study, which presumed lower TBV as a marker of cerebral small vessel disease (CSVD), found that a decrease in TBV over time was associated with higher incident DS . SmallerAll associations investigated in every grey matter study are depicted in White matter studies could be classified into two main categories according to a macro or micro-scope.One longitudinal study found that, baseline and incident subcortical infarcts, incident Virchow-Robin spaces and increased WMH volume over time, were associated to incident DS . This association was stronger in subcortical areas and frontal region . Other sInterestingly, one longitudinal study based on the evidence of non-proportional association for WMH volume and DS, investigated the modification effect of apolipoprotein Eε4 (APOEε4) genotype in this context. Authors found that severe deep WMH (i.e. according to size of cap and band) among homo- and heterozygote APOEε4 carriers predicted progression of DS, compared to mild WMH, and this was not seen among APOEε4 non-carriers . On the Regarding a micro-scope, three studies investigated MRI-DTI parameters. When focusing on overall measures (WBA), lower FA was associated to DSp , even whCerebral small vessels disease markers showed the clearest evidence. Higher number of subclinical infarcts, subcortical infarcts and white matter hyperintensities are associated to more depressive symptoms and predicted its progression. Interestingly, one study found that for APOEε4 genotype carriers, severe WMH was a predictor of DS worsening , but thiDTI studies also found consistent results: lower fractional anisotropy; and higher mean, radial, and axial diffusivity, overall, and in specific tracts, were associated to greater DS. To note, DTI changes can only be interpreted as damage in the WM micro-structure in the context of aging and disease-related studies, thus it cannot be attributed to a particular aspect of the brain micro-structure (e.g: myelinisation) . AdditioRegarding grey matter studies, the majority found reduced volumes, thickness and surface associated with depressive symptoms. The most examined structures were the cingulate cortex and the hippocampus volume. This seems expected, since the CC and hippocampus are part of the fronto-limbic networks, nevertheless the frontal lobe has been scarcely studied and conclusions are inconsistent. Most of findings showed decreased hippocampal volume associated to greater DS. Nonetheless one study found that age lowering-effects on hippocampal volume were counteracted by DS in the hippocampus ; what asThese neural correlations should not endorse a discrete and organic health scope which favours diagnosis, treatment, and disease monitoring, based on complementary tests. On the contrary, they herald the embracement of cognitive-neural-affective dimensions, and thereby, the importance of DS as a proxy of clinical global health. Furthermore, these results stand against those who argue that DS is an age-related condition. Not only because of the proven evidence of its association to brain abnormalities independently of age, but also to the fact that changes identified in both conditions are different .Due to the heterogeneity of the methodology studies, we acknowledge some limitations when making conclusions out of the selected studies. These challenges deserve further commentaries.The majority of studies have been limited to a cross-sectional design. Cross-sectional studies are particularly affected by uncertain theoretical basis of causality , what isDementia and DS discrimination is crucial, given that both share clinical and brain abnormalities. A cross-sectional study showed that the differences found for FA in DSp compared to non-DSp disappeared after adjustment for a cognitive index . FactorsAfter applying the guidelines for reporting of Sex and Gender Equity from the European Association of Science Editors , we founAnother concern is the lack of consensus on how to measure DS. There are diverse screening tools and different cut-offs to categorize participants, thus it is difficult to generalize findings (see table 1 and 2). The CES-D was the most used scale, and the cut-offs varied (Some studies considered the prescription of antidepressants as a synonymous of presence of DS, what riThis review concludes that the most reliable evidence shows cerebral small vessel disease as a predictor of DS worsening. Also, cross-sectional associations related: lower FA, higher RD, higher AD, thinner frontal and temporal cortex, lower hippocampal volume, and increased Precuneus thickness to higher DS. Results related to cingulate cortex, are still controversial and demand further investigation. Dementia discrimination was inaccurate in some of studies and none of them followed it up over time. Moreover, sex-related differences were mostly overlooked.Taking into account that MDD is the most common psychiatric disorder seen in community-dwelling older adults, and that DS largely outnumbers it, DS in older people deserve research efforts from the translational standpoint. Moreover, disentangling the interdependent links in between DS and the intrinsic capacity domains is an underpinning pathway for achieving an integrated care for older people , which f


There is ongoing debate as to whether abacavir (ABC) increases the risk for cardiovascular disease(CVD) in people living with HIV (PLHIV) and the mechanisms underlying this possible association. We recently showed that the use of an ABC-containing regimen was independently associated with increased thrombin generation (TG). In the present study, we aim to explore these findings further, by studying the mechanistical processes that underly the global thrombin generation test via thrombin dynamics analysis. Thrombin dynamics analysis can pinpoint the cause of increased thrombin generation associated with ABC-use either to the procoagulant prothrombin conversion pathway or the anticoagulant thrombin inactivation pathway. In this cross-sectional study, 208 virally suppressed PLHIV were included, of whom 94 were on a ABC-containing regimen, 92 on a tenofovir disoproxil fumarate (TDF)-containing regimen, and the remainder on other regimens. We used Calibrated Automated Thrombinography to measure thrombin generation and perform thrombin dynamics analysis. The total amount of prothrombin conversion, as well as the maximum rate of prothrombin conversion were significantly increased in PLHIV on an ABC containing regimen compared to other treatment regimens. The levels of pro- and anticoagulant factors were comparable, indicating that the ABC-induced changes affect the kinetics of prothrombin conversion rather than procoagulant factor levels. Moreover, Von Willebrand Factor (VWF), active VWF and VWF pro-peptide levels were significantly higher in PLHIV than controls without HIV. However, they did not differ between ABC and non-ABC treated participants. AdditioThe coagulation system can be assessed by the thrombin generation (TG) test by applying Calibrated Automated Thrombinography (CAT). The CAT assay is sensitive to small alterations in coagulation . An incrtot) and the maximum prothrombin conversion rate (PCmax). In previous studies, we have shown that prothrombin conversion is increased in conditions associated with an increased risk of thrombotic events, such as the antiphospholipid syndrome . The generated TG curves were used in thrombin dynamics analysis, as described below.Thrombin generation was measured in PPP using the CAT assay , as described in more detail previously . Thrombi2.3tot) during the TG test, and the peak height of the prothrombin conversion curve, which is defined as the maximum rate of prothrombin conversion (PCmax). Additionally, the amount of thrombin-antithrombin (T-AT) and thrombin-α2Macroglobulin (T-α2M) complexes formed during the experiment are quantified . Antithrombin levels were measured chromogenically on the automated coagulation analyzer STA-R max using STA-Chrom ATIII reagents, according to manufacturer’s specifications  . Plasma s et al. .Prothrombin levels were determined with an in-house sandwich ELISA assay  using a 2.52 test. P-values <0.05 were considered statistically significant.Statistical analysis was performed using IBM SPSS version 27 GraphPad Prism version 8. Normality of the data was assessed using the Shapiro-Wilk test. Data are presented as median with interquartile range (IQR). Comparison between groups was performed by either the Mann-Whitney test or Chi3vs. 60.7%, p<0.001), and older (52 (46-59) vs. 30 (26-53) years, p<0.001) compared to uninfected controls , p =0.015) and active VWF were significantly lower in PLHIV (144 (115-183) vs. 164 (134-204), p=0.014). Nevertheless, VWF levels, active VWF levels and VWF pro-peptide levels did not differ significantly in PLHIV receiving ABC-based or non-ABC based treatment.Furthermore, endothelial damage could contribute to the prothrombotic phenotype as associated with ABC-based treatment in PLHIV. We quantified Von Willebrand factor (VWF) in its native and activated form, and the pro-peptide of VWF form as biomarkers of endothelial damage Figure 2We previously described an increase of thrombin generation in PLHIV on ABC-based treatment in the same cohort . The thrtot) and the peak height of the prothrombin conversion curve was quantified as the maximum rate of prothrombin conversion (PCmax) throughout the measurement  and maximum rate of prothrombin conversion  were significantly higher in PLHIV on ABC-based treatment compared to other treatments , which is a main inhibitor of thrombin. The formation of thrombin-α2-macroglobulin (T-α2M) complexes did not differ between participants with ABC-based and non-ABC-based treatments (The individual quantification of prothrombin conversion parameters is shown in Moreover, the thrombin decay capacity was determined based on the levels of the natural anticoagulants and the modulating effect of fibrinogen Figure 54Debate remains on whether specific antiretroviral drugs, such as ABC, increase the risk for CVD, including stroke and myocardial infarction –6, 31. Iin vitro addition of ABC or its active metabolite carbovir diphosphate to platelet rich plasma does not cause and increase of thrombin generation (In this cohort of PLHIV, thrombin inactivation, as quantified by the thrombin decay capacity, did not differ between uninfected controls and PLHIV, irrespective of their treatment regime. Together, these findings indicate that the balance between pro- and anticoagulant processes shifts towards the procoagulant prothrombin conversion process, leading to the increase of thrombin generation in PLHIV on ABC-based treatment, as thrombin inactivation was unaltered. The elevation of prothrombin conversion could explain the increased risk of thrombotic events associated with ABC , 34. Wheneration . This suin vitro (The cross-sectional design of the study prevents the investigation of causal interference of thrombin generation and thrombin dynamics parameters and thrombotic risk in PLHIV, as the primary aim of the study was to investigate the association between thrombin dynamics and cART. A limitation of the study is that we were unable to study these associations in platelet rich plasma or whole blood, to include the effect of platelets. However, it has been previously reported that ABC treatment has no prothrombotic effect on platelets in vitro . AnotherIn conclusion, we found that the use of ABC is associated with increased prothrombin conversion without changes in thrombin inactivation. The net result of these changes is an increase in the TG potential. This finding explains the reported increase in thrombin generation ETP in PLHIV on ABC-based treatment compared to PLHIV on non-ABC-based treatments. Moreover, this increase in prothrombin conversion and subsequently ETP may contribute to the increased risk of thrombotic events PLHIV receiving ABC-based treatment.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by CMO Arnhem-Nijmegen. The patients/participants provided their written informed consent to participate in this study.QY performed experiments, analyzed the data, and drafted the manuscript. SH performed experiments, analyzed the data, and drafted the manuscript. WH collected the samples, included the participants and co-wrote the manuscript. LW collected the samples, included the participants and co-wrote the manuscript. MN supervised the data collection and co-wrote the manuscript. RL-K performed thrombin dynamics analysis, performed analyses, supervised the data collection, and co-wrote the manuscript. AV designed the study, supervised the project and co-wrote the manuscript. BL designed the study, supervised the project, and co-wrote the manuscript. QM designed the study, supervised the project, and co-wrote the manuscript. All authors contributed to the article and approved the submitted version.