KLK3 (PSA) gene; AR and KLF4 also colocalized genome-wide. Taken together, our data report an expanded high-confidence proximity network for AR, which will be instrumental to further dissect the molecular mechanisms underlying androgen signaling in PCa cells.Prostate cancer (PCa) is the most frequently diagnosed cancer in men and the third cause of cancer mortality. PCa initiation and growth are driven by the androgen receptor (AR). The AR is activated by androgens such as testosterone and controls prostatic cell proliferation and survival. Here, we report an AR signaling network generated using BioID proximity labeling proteomics in androgen-dependent LAPC4 cells. We identified 31 AR-associated proteins in nonstimulated cells. Strikingly, the AR signaling network increased to 182 and 200 proteins, upon 24 h or 72 h of androgenic stimulation, respectively, for a total of 267 nonredundant AR-associated candidates. Among the latter group, we identified 213 proteins that were not previously reported in databases. Many of these new AR-associated proteins are involved in DNA metabolism, RNA processing, and RNA polymerase II transcription. Moreover, we identified 44 transcription factors, including the Kru¨ppel-like factor 4 (KLF4), which were found interacting in androgen-stimulated cells. Interestingly, KLF4 repressed the well-characterized AR-dependent transcription of the   •BioID proteomics identifies 267 androgen receptor (AR)-associated candidates•Krüppel-like factor 4 (KLF4) is a new AR interaction partner•KLK3 (PSA)AR and KLF4 colocalize genome-wide on >4000 genes, including •KLK3 (PSA)KLF4 acts as a repressor for the AR target gene  PSA (KLK3), for which KLF4 acts as a repressor, without regulating the expression of AR. These results are instrumental to further dissect the molecular mechanisms underlying androgen signaling in prostate cells.A proximity interaction network for the androgen receptor (AR) was obtained from androgen-responsive prostate cancer cells. A total of 267 candidates were identified, most associating following ligand stimulation, including Krüppel-like factor 4 (KLF4). KLF4 and AR were found to colocalize genome-wide on 4097 genes including  Kallikrein-Related Peptidase 3 (KLK3), often referred to as Prostate-Specific Antigen (PSA). Moreover, AR is phosphorylated and activated in a ligand-independent manner by kinases such as MAPK and PI3K/AKT (Prostate cancer (PCa) is the most frequently diagnosed cancer in men and is the third cause of cancer mortality . PCa iniPI3K/AKT , 8. Thuset al. identified a number of associated proteins including RNA-binding protein P54NRB/NONO  complex  to identify 54 and 75 candidates with wild-type AR and the constitutively active ARv567es variant, respectively. Interestingly, they identified the transcription factor Grainyhead-like 2 (GRHL2) and characterized its function as a coregulator of AR, both as an oncogenic enhancer of androgen signaling and as a suppressor of metastasis . Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS on purified AR complexes, Ishitani NRB/NONO . They fu complex . Likewisyme USP7 . More retastasis . In anott models . Hsiao ecription , 16. Pal outcome . While t outcome .et al. developed proximity labeling proteomics, namely BioID  293 cells  and surprisingly act as a repressor for the latter, without regulating the expression of AR itself.Here, we performed BioID proximity labeling proteomics in androgen-dependent LAPC4 cells that express wild-type AR. We delineate an AR proximity network containing 267 proteins, most of which associated following ligand stimulation, providing a large high-confidence interaction network for AR obtained from androgen-responsive cells. Among the AR-associated proteins, we reveal 213 candidates that were not previously reported in the BioGRID, GSP-Prot, or IntAct database and describe the identification of the Krüppel-like factor 4 (KLF4) as a new AR-associated protein. Finally, we show that KLF4 and AR colocalize genome-wide on a number of genes including PSA (Human AR (NCBI clone NP_000035.2) was subcloned into pMSCVpuro (Clontech) in fusion with an N-terminal 3xFLAG epitope tag and a BirA∗ sequence. pEGFP-N1 was obtained from Clontech and human AR was subcloned into pEGFP-C1 (Clontech) in fusion with a N-terminal GFP protein tag. Human KLF4 (NCBI clone NP004226.3) was subcloned into pMSCVpuro (Clontech) with an N-terminal 3xFLAG tag. All inserts were fully sequenced and protein expression was verified by western blot. Adenoviral plasmids for PSEBC-TSTA were previously described  and tranThe DsiRNA catalog numbers and sequences were as follows : DsiAR , DsiKLF4 , DsiGRHL2 , DsiMAML , DsiRBPJ , and DsiTBL1X .Nontargeting DsiRNAs provided by the manufacturer were used as negative controls.mycoplasma using the MycoAlert Mycoplasma Detection kit . Transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific). Stable LAPC4 cells were selected with puromycin (5 μg/ml) for 1 month. Individual clones were picked and selected based on transgene expression. DsiRNAs (5 nM) were transfected using Lipofectamine 2000 (Thermo Fisher) following manufacturer’s guidelines.LNCaP and VCaP cells were obtained from ATCC. LAPC4 cells were kindly provided by Dr C. Sawyers. LNCaP were cultured in RPMI 1640 media containing 10% fetal bovine serum (FBS), LAPC4 and VCaP cells were cultured in DMEM media containing 10% FBS. Cell lines were tested for 3PO4 before digestion with trypsin, as described  for 0 h, 24 h, or 72 h. Biotin (50 μM) was added for the last 24 h prior to cell lysis. Cells were washed twice with PBS, then scrapped in PBS buffer before lysis in ice-cold RIPA buffer followed by benzonase (Sigma-Aldrich) treatment for 1 h and three cycles of sonication. Protein concentration was measured and normalized using a BCA kit (Thermo Fisher Scientific). Lysates were incubated under agitation for 3 h at +4 °C with 30 μl streptavidin agarose beads (Sigma-Aldrich). Beads were washed three times with lysis buffer. For MS experiments, beads were additionally washed twice with 20 mM Tris pH 7.4, and proteins were eluted by incubation with agitation at +4  °C with 50 mM Hescribed . ProteinFor western blotting experiments, cells were washed once with PBS and lysed in 1x Laemmli buffer. Whole-cell lysates were normalized based on their protein concentration, as assessed by BCA assay.6 HEK293T cells were seeded in 10 cm tissue culture dishes and grown for 24 h. Cells were transiently transfected using polyethylenimine (PEI) with pEGFP-N1 or pEGFP-C1-AR to achieve similar levels of purification for GFP or GFP-AR and cotransfected with pMSCV(puro)-3xFLAG-KLF4. Cells were scraped into IP buffer  containing protease  and phosphatase inhibitors . Cell lysates were kept on ice for 15 min and then sonicated 30 s at a low intensity. Cell lysates were cleared by centrifugation at 20,000 g for 1 min, and purifications were performed with 5 μl of GFP-Trap Agarose beads  for 2h at +4oC. Beads were washed twice with IP buffer. Precipitates were recovered after 2 h of incubation on a rotating machine at +4oC and washed three times 10 min in IP buffer. Precipitates and total cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes prior to western blotting with the indicated antibodies.A total of 5 x 10A total of 10–20 μg of whole-cell lysates was resolved by SDS-PAGE and transferred to nitrocellulose membranes . Loading of each track was verified with Ponceau S (Sigma-Aldrich) staining. Antibodies used were as follows: mouse anti-AR , rabbit anti-AR , rabbit anti-KLF4 , mouse anti-tubulin , mouse anti-actin , anti-GFP , rabbit anti-MED1 , rabbit anti-MED12 , rabbit anti-KMT2A , mouse anti-DMAP1 , rabbit anti-POLH , rabbit anti-NIPBL , or mouse H3 . Secondary antibodies were the following: anti-FLAG M2 (Sigma-Aldrich), horse anti-mouse HRP-linked IgG , goat anti-rabbit HRP-linked IgG , or streptavidin-HRP (Life Technologies #434323). Signal was revealed using BioRad Clarity Western ECL substrate and detected either on Hyblot CL autoradiography films (Denville) or with an Amersham Imager 600RGB . Signal quantification was performed using Image J software gel analysis tools (NIH).5 cells/well), LNCaP (1.0 x 105 cells/well), and VCaP (1.6 x 105 cells/well) were seeded in 24-well plates with media containing 5% charcoal stripped FBS and grown for 18 h before PSEBC-TSTA (3.5 MOI) adenovirus infection, DsiRNA transfection, and treatment with vehicle (EtOH) or DHT (10 nM). Seventy-two hours following infection, cells were washed once with HBSS and lysed. Luciferase assay was performed following manufacturer guidelines (Promega) with 20 uL of each lysate. Relative luminescence unit (RLU) was normalized by protein content in each well . Protein concentration was estimated by adding 250 μl of Bradford reagent (ThermoFisher Scientific) to 3 μl of total lysate. Absorbance was measured using an Infinite F50 absorbance microplate reader  at 595 nm.LAPC4 (1.0 x 105 cells/well) cells were seeded in 6-well plates with DMEM medium containing 5% charcoal stripped FBS, transfected with DsiRNAs, and grown 24 h before vehicle/DHT treatment. Cells were washed twice with PBS, and total RNA was extracted using TriPure reagent (Sigma), according to the manufacturer’s recommendations. RNAs were cleaned using the GeneJET RNA purification kit (Thermo Fisher Scientific). Reverse transcription was performed using 2.5 μg of total RNA according to the standard SuperScript VILO Master Mix (Invitrogen). The resulting cDNAs were diluted at 1/24 and then 10 μL was used in a quantitative PCR reaction carried out with the SYBR Select Master Mix (Applied Biosystems). Actin was used for normalization of RT-qPCR data. Fold changes in mRNA expression levels were calculated using the comparative Ct method.LAPC4 . This was followed by three washes with PBS and incubation with the following antibodies diluted in blocking buffer (0.2% BSA (Bioshop), 0.1% Triton X100 (Sigma-Aldrich)): rabbit anti-FLAG , mouse anti-AR , or rabbit anti-KLF4  for 1 h at room temperature. After washes in PBS, coverslips were incubated with Alexa 568-conjugated goat anti-rabbit  or Alexa 488-conjugated goat anti-mouse  antibodies for 1 h at room temperature. They were washed twice with PBS before being mounted on slides using ProLong Gold antifade with DAPI (Thermo Fisher). Pictures were acquired with an Olympus FV1000 using the FluoView 3.0 software or with a Nikon Eclipse E600 imaging system using MetaView.bona fide protein associations , and the peptides were eluted with a linear gradient from 5 to 40% solvent B  in 60 min at 300 nl/min. Mass spectra were acquired using a data-dependent acquisition mode using Thermo XCalibur software version 3.0.63. Full-scan mass spectra (350–1800 m/z) were acquired in the orbitrap using an AGC target of 4e5, a maximum injection time of 50 ms, and a resolution of 120,000. Internal calibration using lock mass on the m/z 445.12003 siloxane ion was used. Each MS scan was followed by acquisition of fragmentation MSMS spectra of the most intense ions and with a minimum intensity threshold of 5000 for a total cycle time of 3 s (top speed mode). The selected ions were isolated using the quadrupole analyzer in a window of 1.6 m/z and fragmented by higher-energy collision-induced dissociation (HCD) with 35% collision energy. The resulting fragments were detected by the linear ion trap in rapid scan rate with an AGC target of 1e4 and a maximum injection time of 50 ms. Dynamic exclusion of previously fragmented peptides was set for a period of 20 s and a tolerance of 10 ppm.Samples were analyzed by nanoLC/MSMS. For each injection, 1 μg of peptide samples was injected and separated by online reversed-phase (RP) nanoscale capillary liquid chromatography (nanoLC) and analyzed by electrospray mass spectrometry (ESI MS/MS). Experiments were performed with a Dionex UltiMate 3000 nanoRSLC chromatography system (Thermo Fisher Scientific/Dionex Softron GmbH) connected to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) driving with Orbitrap Fusion Tune Application 2.0 and equipped with a nanoelectrospray ion source. Peptides were trapped at 20 μl/min in loading solvent  on a 5 mm x 300 μm C18 pepmap cartridge precolumn (Thermo Fisher Scientific/Dionex Softron GmbH) during 5 min. Then, the precolumn was switched online to a house-made 50 cm x 75 μm internal diameter separation column packed with ReproSil-Pur Homo sapiens database  assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.6 Da and a parent ion tolerance of 10 ppm. Carbamidomethylation of cysteines was set as fixed modification and oxidation of methionine, deamidation of asparagine and glutamine, and phosphorylation on serine, threonine, and tyrosine were specified as a variable modification. Two missed cleavages were allowed.All MS/MS peak lists (MGF files) were generated using Thermo Proteome Discoverer software . MGF sample files were then analyzed using Mascot . Mascot was set up to search the Uniprot Complete Proteome Scaffold (version 4.8.1), Proteome Software Inc, Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Proteins/peptides FDR rate was set to 1% or less based on decoy database searching. Protein probabilities were assigned by the Protein Prophet algorithm . Proteinhttp://broadinstitute.github.io/picard/). Narrow peaks were called using MACS2 callpeak . To determine whether the BirA∗-AR fusion was functional, we analyzed its subcellular compartmentalization following stimulation with its ligand, DHT. We found that DHT stimulation led to nuclear accumulation of 3xFLAG-BirA∗-AR, as it did for wild-type AR, in LNCaP cells . To further support this, we tested if 3xFLAG-BirA∗-AR could substitute endogenous AR function in LAPC4 cells. Using a luciferase PSA reporter assay (C-D). In addition, we validated that addition of biotin to LAPC4 cells expressing 3xFLAG-BirA∗-AR induced a strong biotinylation pattern . Together, these observations suggest that 3xFLAG-BirA∗-AR recapitulates endogenous AR functions.To establish cell lines relevant to AR function, we selected LAPC4 prostate cells, which express a wild-type AR , 44. We Fig. 1er assay , 46, we via SAINTexpress (A). We identified 31 proteins associated with AR in the absence of agonist (B–G). Therefore, this data set meaningfully confirms and expands the number of reported AR-associated proteins according to the BioGRID, GSP-Prot, and IntAct databases.To delineate the AR proximity network, we affinity-purified biotinylated proteins following the addition of biotin to 3xFLAG-BirA∗-AR and 3xFLAG-BirA∗ LAPC4 cell cultures and identified them using MS. We treated cells with DHT or vehicle for 24h or 72h , 48 and Texpress , using 3Texpress . Only hiTexpress A. We ide agonist . Strikin agonist , GSP-Pro agonist  and IntA agonist , SMARCD1 agonist , TCF20/S agonist  and RNF2 agonist . Interes agonist , B–G. ThB–C) (G); BAF, NuRD complexes), transcription initiation (TFIID), cell cycle regulation (LIN complex), or RNA processing (spliceosome)  , 56. In B–C) G; BAF, Nceosome) . Togethei.e., GRHL2, KLF4, MAML1, RBPJ, TBL1X) based on SAINT score and gene expression in prostate , thus confirming the validity of our assay (A). KLF4 depletion surprisingly led to a stronger activation of the AR-driven luciferase reporter in DHT-stimulated LAPC4 cells (A). While the depletion of the four other AR-associated candidates that we tested did not lead to statistically significant changes, it did induce in most cases a slight decrease in AR reporter activation. We confirmed by coexpression and affinity purification that KLF4 associates with AR (B), reinforcing our BioID proximity labeling results and supporting a role for KLF4 in regulating PSA transcription, as suggested by our luciferase PSA reporter assays (A). We further found that in KLF4-depleted cells, DHT stimulation increased AR-driven luciferase reporter signal by at least 1.7-fold, while cells depleted of AR displayed a 3.7-fold decrease (p ≤ 0.0001) (C and D). Interestingly, KLF4 was previously reported to directly bind to the AR promoter and KLF4 depletion to decrease AR protein levels in LNCaP cells, which express a mutated AR . We also observed that AR protein expression was unaffected (C). Consistent with this observation, ectopic 3xFLAG-KLF4 expression did not lead to increased AR levels (G). We detected a slight increase in KLF4 expression in AR-depleted cells with one of the two siRNAs targeting AR (F); however, this was not confirmed at the protein level (C).To investigate the function of these new AR-associated proteins, we selected five candidates (prostate  57). Us. Usi.e.,ur assay A. KLF4 dC4 cells A. While  with AR B, reinfor assays A. We fur 0.0001) , C and Dtated AR . To deteed cells E. We alsaffected C. ConsisR levels G. We deteting AR F; howevein level C.Fig. 3KPSA reporter repression was due to KLF4 loss-of-function, we re-expressed 3xFLAG-tagged KLF4 that is not affected by siRNAs, which both target the 3’UTR sequence. Re-expression of 3xFLAG-KLF4, but not 3xFLAG-BirA∗, decreased luciferase levels to those of control LAPC4 cells in the presence of DHT (G and H). Cells transfected with siCtrl or siAR did not display a change in luciferase levels following 3xFLAG-KLF4 re-expression compared with controls. To corroborate our findings in another prostate cell line that expresses wild-type AR, we opted for VCaP cells (I and J). Taken together, our results suggest that AR and KLF4 are functional partners.To confirm that the e of DHT , G and HaP cells . We confaP cells , I and JPSA in an endogenous context, we performed RT-qPCR analyses of mRNA levels of PSA. Following DHT stimulation, PSA mRNA levels increased 45-fold in LAPC4 cells. This activation was hindered in AR-depleted cells, as PSA messengers decreased by 2.5-fold  (A and B). As observed in the luciferase reporter assay, PSA mRNA increased significantly by an average of 1.5-fold , following KLF4 knockdown with two independent siRNA sequences tested (A and C). This latter increase in PSA expression was not due to an accumulation of AR in the nucleus  , A and Bs tested , A and C nucleus . TogetheD). Interestingly, for regions co-occupied by KLF4 and AR, the signal densities for both transcription factors were correlated, thus supporting co-occupancy. In addition, as expected, signal density for AR increased following DHT stimulation while KLF4 was relatively stable. While many regions occupied by the AR following the DHT treatment were also occupied by KLF4 in control conditions, a similar fraction was not occupied (KLK3) was occupied by both KLF4 and AR . Similar results were observed for KLK2, SGK1, and IDH1. Therefore, the ChIP-seq experiments support subnuclear colocalization of KLF4 and AR, suggesting a functional association.To determine if AR and KLF4 shared cis-regulatory regions genome-wide, we surveyed their recruitment with/without a 1h DHT treatment using chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) in LAPC4 cells. As expected, the number of regions occupied by AR increased from 24,379 in control conditions to 85,312 when cells were stimulated with DHT. For KLF4, 26,891 regions were observed in control cells compared with 59,806 in DHT-stimulated cells . OverallFig. 4occupied , suggestoccupied . Closer et al. reported an AR proximity network comprised of 32 preys in HEK293 cells . Among those, we identified KLF4 as a new AR-associated protein. Interestingly, Siu et al. previously discovered the existence in LNCaP cells of a reciprocal feedback loop between KLF4 and AR, in which each protein binds to the promoter of each other’s gene (AR expression or protein levels upon KLF4 knockdown, neither a change in KLF4 expression or protein levels in cells depleted of AR. Therefore, it remains possible that the self-reinforcing loop between KLF4 and AR is cell-type specific. However, we did observe a significant overlap genome-wide between regions occupied by KLF4 and AR. These observations underline the importance of further examining AR-associated proteins in several PCa cell lines harboring different genetic and proteomic landscapes to possibly extend findings to primary PCa.Using an affinity purification approach, we confirmed the previously reported association between AR and MED1 or KMT2A, but we also validated new interaction partners for AR (Fig. 3r’s gene . This obPSA, but exclusively yet consistently when cells are stimulated with DHT to activate AR. Interestingly, KLF4 was previously reported to directly associate with the DNA-binding region of ERα, thereby inhibiting the transcriptional activity of this nuclear receptor, in an estrogen-dependent manner (PSA gene regulation by KLF4.KLF4 function in transcriptional regulation is considered to be context-specific , 61. Fort manner . Our datet al. reported a genome-wide CRISPR/Cas9 knockout screen to identify essential genes in LNCaP PCa cells (Recently, Fei Ca cells . Among tCa cells . InteresCa cells , includiKLF4 transcription and KLF4 protein levels were decreased in metastases, while its re-expression inhibited prostate cancer cell migration and invasion (KLF4 has been associated with either oncogenic properties, such as in osteosarcoma , either invasion . This suPSA.Together, we provide a large, high-confidence proximity interaction network for AR obtained from androgen-responsive cells. We further demonstrate the relevance of our data by characterizing the codistribution of KLF4 and AR across the genome, as well as a key repressive function for KLF4 in the regulation of the AR target gene via the PRIDE partner repository with the data set identifiers PXD011974 and 10.6019/PXD011974 (The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium XD011974 .The sequencing data generated for this publication is available on GEO , under aThis article contains The authors declare no competing interests.


A large number of patients need critical physical rehabilitation after the stroke. This study aimed to review and report the result of published studies, in which newly emerged games were employed for physical rehabilitating in poststroke patients.  This systematic review study was performed based on the PRISMA method. A comprehensive search of PubMed, Scopus, IEEE Xplore Digital Library, and ISI Web of Science was conducted from January 1, 2014, to November 9, 2020, to identify related articles. Studies have been entered in this review based on inclusion and exclusion criteria, in which new games have been used for physical rehabilitation.  Of the 1326 retrieved studies, 60 of them met our inclusion criteria. Virtual reality-oriented games were the most popular type of physical rehabilitation approach for poststroke patients. “The Nintendo Wii Fit” game was used more than other games. The reviewed games were mostly operated to balance training and limb mobilization. Based on the evaluation results of the utilized games, only in three studies, applied games were not effective. In other studies, games had effective outcomes for target body members.  The results indicate that modern games are efficient in poststroke patients' physical rehabilitation and can be used alongside conventional methods. Stroke is one of the diseases that can lead to disability and affect people's daily activities and lead to reduced performance . AccordiHowever, patients may not be able to attend these treatment sessions for rehabilitation fully. Several factors may lead to limited access to these treatment sessions, including the following: difficulty accessing a physiotherapist by the patient, high cost of attending the treatment session, patient's age and disability, the long distance that the patient has to travel, or poor patient compliance . One of Which type of games is the most used?Which gamification approaches have been used to improve the performance of poststroke patients?What was the most common type of physical rehabilitation in stroke survivors?What are the evaluation results of games used in poststroke patients?As it turns out, various studies have been performed to determine the effectiveness of the mentioned games. A systematic review study by Primack et al. found that games improved 69% of psychological therapy outcomes, 46% of clinician skills outcomes, 42% of health education outcomes, and 37% of disease self-management outcomes . AnotherThis systematic review was conducted based on Preferred Reporting Items for Systematic Reviews and Meta-Analyzes (PRISMA) checklist to ensure inclusion of relevant studies .In this review, a systematic search of scientific databases including ISI Web of Science, PubMed, IEEE Xplore Digital Library, and Scopus was performed from January 1, 2014, to November 9, 2020. The comprehensive search strategy comprised a set of main keywords from PubMed Mesh terms and Emtree related to “game,” “rehabilitation,” and “poststroke” patients. The specific detail of main applied keywords for each database is presented in The extracted studies were included if they fulfilled the following criteria: (1) original articles and proceedings, (2) the focus of this review was on only physical rehabilitation innovative game-based solutions for poststroke physical disabilities, (3) one of the gamification techniques was employed for physical rehabilitation or disability treatment, (4) in this study, the result of using different games and outcomes of video games or immersive-oriented approaches on physical rehabilitation were reviewed, and (5) studies were limited to those published in the English language. Besides, the studies were excluded if they met the following criteria: (1) the title, abstract, or full text of the article that did not relate to video games, virtual, or mixed reality-based games, (2) studies which were review or meta-analysis, book chapters, letter, reports, and technical reports, (3) articles in which the result of applying games was not reported quantitatively, (4) articles about cognitive rehabilitation, and (4) non-English published ones.In comprehensive and scientific databases searching, 833 studies were retrieved after duplicate removal. Some inclusion and exclusion criteria were set for the study selecting phase. In the first phase, two independent reviewers (SS and SR) specified the primary classification of included studies; then, they synthesized selected citations' critical features. MG validated the primarily determined classifications. All titles and abstracts of extracted studies were investigated and screened based on the research questions and unique aims to select relevant ones by two reviewers under MG's supervision. In the last phase, citations that met inclusion and exclusion criteria were selected to enter the full-text review phase. The full-texts of relevant studies were screened by SS and SR thoroughly. Key characteristics were entered into a spreadsheet in Excel for each study. Two authors (SR and SS) independently extracted the study characteristics based on the predefined classifications. For reaching an agreement, the information was examined again by two authors. The next reviewer (MG) evaluated and validated all of the obtained results. EndNote X9 was used for resource management, and all qualitative analyses were performed in SPSS v20. The main classifications of screened citations are shown in The flow of screening articles based on the PRISMA method is shown in The reviewed studies in this study were published in 53 journals and 7 international conferences. All the names of journals and conferences are listed in Based on analysis, virtual reality-oriented games and video games are the most popular physical rehabilitation types for poststroke patients. The distribution of reviewed literature based on the type of games is shown in n = 28, 46.66%) was Nintendo and Microsoft Xbox 360 Kinect.The deployment platform for most of the studies included in this review  is shown in According to the results of reviewed different studies, numerous indicators and tests have been applied to evaluate physical rehabilitation outcomes in poststroke patients. In other words, according to the type of rehabilitation treatment provided to patients, different indicators and metrics have been calculated to assess the condition of the rehabilitated organs of the body (before and after the intervention). For this reason, we were unable to compare the assessment scores calculated during the intervention. However, considering how many indicators and metrics in each study were affected by physical rehabilitation during the intervention treatment, we added a brief assessment from the authors' perspective. At the end of the intervention, if all indicators of functional or physical appraisal of patients are affected by game-based rehabilitation and a significant difference is seen, then we have labeled this rehabilitation approach “Effective.” If only one or two of the several evaluation metrics are not affected by game-based rehabilitation, then we label them “Partly effective.” Finally, if there is no significant difference in all evaluation measures before and after the intervention, we label them “No effective.” In the following, the distribution of the reviewed studies based on effectiveness is shown in This survey's main objective was to review the studies in which games were applied for improving the physical functions and rehabilitation of poststroke patients. According to results, emerging games possess the capacity and potential to rehabilitate physical aspects in poststroke patients; furthermore, these games can help patients improve their independence. According to surveys, virtual reality-based approaches and “the Nintendo Wii Fit” games were used more than other games. The most common use of games in poststroke survivors' rehabilitation was related to limb movement and balance training.Due to the included studies' results, different indicators and scales have been calculated and statistically analyzed to evaluate and test game-based physical rehabilitation therapies for poststroke patients. These statistical analyses demonstrated the positive effect of innovative rehabilitation is provided in the form of games for these patients. Even in many studies, applied games in different environments  have led to a great improvement in patients' physical problems such as balance disorder, upper extremity spasticity, and limbs' immobility and muscular weakness , 74–77. The reason for the ineffectiveness of newly emerged games for motor rehabilitation of stroke patients can have different reasons as follows: insufficient session times and training duration to generate consistent improvements in all patients, the insufficient number of participants in the experimental studies (randomized trails would require at least 25 participants in each group) , 76, theAccording to this study's results, the most popular type of game for physical rehabilitation of poststroke patients was virtual reality games. Virtual reality-based games allow patients to interact with a virtual environment while performing rehabilitation exercises and simulating real functions. These games increase patients' motivation to perform rehabilitation exercises and provide a pleasant environment for patients, which can lead to more repetition of rehabilitation exercises in these patients . People Results have shown that “the Nintendo Wii Fit” games are used more than other games to rehabilitate poststroke patients. Several factors can lead to the most use of this game. Among these factors, we can mention the price of these games, which are relatively inexpensive. These games are widely available to people, and studies have shown that providing an attractive environment increases patients' enjoyment and more repetition of rehabilitation exercises –84. FeatStudies showed that the most common use of games in the rehabilitation of stroke survivors was related to limb movement and balance training. In other words, the results of studies that were run to examine the effect of games on people after a stroke had shown these games were effective in improving the balance of people and strengthening the muscles of the limbs . In a syThis systematic review had several strengths and limitations. One of the strengths was the use of broad keywords to search in 4 crucial databases. Another strength of this survey was the inclusion of studies presented at conferences. The limitations were the exclusion of articles in non-English language and the time limit imposed on searching databases (from 2014 onwards). Another limitation of this review was the different scales used to measure people's performance, and this factor made it difficult to compare the results of different surveys.Game-based approaches lead to patients being able to smoothly perform their rehabilitation movement techniques without going to the treatment centers. These games can immerse the person in the environment by providing virtual or augmented reality capabilities and multiplying the effectiveness of the treatment. Therefore, the use of appropriate technology-based gaming solutions can improve patients' treatment and minimize the waste of time and cost of providing traditional motor rehabilitation. Consequently, these game-based treatments are considered complementary to traditional ones and can reduce the workload of therapists and accelerate the rehabilitation process. Future research should focus on how task-specific game-oriented systems can improve function after stroke, and statistical studies can show this effect more.


Their costs in comparison to regular protein-containing (n = 182) and ‘free-from’ products (n = 135) were also analysed. Similar foods were grouped into subgroups (n = 40). The number of units and costs of SLPFs prescribed in total and per subgroup from January to December 2020 were calculated using National Health Service (NHS) Business Service Authority (NHSBSA) ePACT2  for England. Monthly patient SLPF units prescribed were calculated using patient numbers with PKU and non-PKU inherited metabolic disorders (IMD) consuming SLPFs. This was compared to the National Society for PKU (NSPKU) prescribing guidance. Ninety-eight percent of SLPF subgroups (n = 39/40) were more expensive than regular and ‘free-from’ food subgroups. However, costs to prescribe SLPFs are significantly less than theoretical calculations. From January to December 2020, 208,932 units of SLPFs were prescribed (excluding milk replacers), costing the NHS £2,151,973 (including milk replacers). This equates to £962 per patient annually, and prescribed amounts are well below the upper limits suggested by the NSPKU, indicating under prescribing of SLPFs. It is recommended that a simpler and improved system should be implemented. Ideally, specialist metabolic dietitians should have responsibility for prescribing SLPFs. This would ensure that patients with PKU have the necessary access to their essential dietary treatment, which, in turn, should help promote dietary adherence and improve metabolic control.Patients with phenylketonuria (PKU) are reliant on special low protein foods (SLPFs) as part of their dietary treatment. In England, several issues regarding the accessibility of SLPFs through the national prescribing system have been highlighted. Therefore, prescribing patterns and expenditure on all SLPFs available on prescription in England ( Phenylketonuria (PKU), an inborn error of amino acid metabolism, is caused by phenylalanine hydroxylase deficiency, an enzyme that converts phenylalanine to tyrosine . This leThe cost of SLPFs to patients in England is reimbursed by the National Health Service (NHS), as these foods are considered borderline substances and are available on NHS prescription ,12,13. BGeneral Practitioners (GPs) issue prescriptions for SLPFs monthly on request, which are then dispensed through local pharmacists or specialist home delivery companies linked to the suppliers of SLPFs . The NHSn = 25/58) of caregivers and parents said they needed more SLPFs for their children than they had been prescribed .,20.19,20n = 26 NHS England hospitals/centres who care for patients with PKU. These data were used to calculate how many units of SLPFs were being prescribed per patient per month and the cost to the NHS per patient per month in England. This was then compared to NSPKU prescribing guidance.In order to estimate the number of patients with PKU cared for by NHS centres in England, all NHS centres known to treat and monitor PKU patients were contacted in order to determine the number of patients with PKU (paediatric and adult), the number on dietary treatment , the number of shared care patients and the number of non-PKU inherited metabolic disorders (IMD) patients accessing SLPFs. Information was supplied by dietitians working in free-from’ comparators for four SLPFs  were unavailable. Thus, 142 SLPFs were available for comparison with 182 regular products and 135 ‘free-from’ products. free-from’ food subgroups (n = 40), the mean cost per kg of products within each subgroup and % differences between costs.One hundred and forty-six SLPFs were identified as being available on ACBS prescription in England, with these products grouped and further subcategorised for comparison with at least two regular food products per subgroup. Regular and ‘Sixty-eight of 142 SLPFs (48%) were unavailable on BNF resources at the time of data collection (August to October 2020), and therefore, their costs had to be obtained directly from the manufacturer or supplier’s website or through email contact with the manufacturer/supplier.free-from’ foods, except for regular eggs and ‘free-from’ flavour puddings, where their cost per kg was comparable to low protein equivalents.When analysed by subgroup, all SLPFs were more expensive than regular foods and ‘‘free-from’ foods, low protein flour, bread mix and egg whites had the highest percentage differences (575% to 825%) in costs. In contrast, low protein milk powder, fish substitute and jelly were only 27% to 61% more expensive than their ‘free-from’ food comparators. Basic SLPFs, including bread, pasta, rice, noodles and milk replacers (liquid), were 76% to 451% more expensive than ‘free-from’ equivalent foods.Low protein crispbread crackers, Xpots  and milk replacements (liquid) had the highest percentage cost difference, being 1117% to 1143% more expensive than the regular food comparator. When compared to In total, 208,932 units of SLPFs  were prescribed from January to December 2020. This equated to a total actual cost of £2,151,973 . The most frequently prescribed subgroups were bread, pasta/rice and flour, in total equating to 54.6% of all SLPFs prescribed. Milk replacers accounted for the highest percentage (30.5%) of the total actual cost of these products. There is not a definition for a unit of milk replacer, as the amount prescribed should be determined on an individual patient basis  19,20].,20.19,20Other expenses included payment for containers, consumables and out of pocket expenses, contributing 4.4%  of the annual SLPFs costs to the NHS in England. Out of pocket expenses reimbursed to the pharmacy may include: postage and packaging costs; handling costs; and the cost of phone calls to manufacturers or suppliers to order products . Paymentn = 1814 (77%) on dietary treatment , with reatment . There wreatment . Actual n = 175), 20% 4–6 years (n = 175), 20% 7–10 years (n = 175) and 40% 11–18 years (n = 352). Therefore, if all of these children, combined with adults with PKU on a full or partial diet (n = 937) were receiving the maximum number of low protein items on prescription each month, as per NSPKU guidance  prescribed in England was minimal, with each subgroup only accounting for 0.1–5.9% of all SLPFs prescribed and contributing just 0.1–3.0% of the total NHS expenditure on SLPFs from January to December 2020. This is consistent with research reporting that special low protein cakes, biscuits and chocolate provide minimal contributions to daily energy intake in children with PKU [Over half 54.6%) of the units of SLPFs prescribed from January to December 2020 were basic foods such as bread, flour/mixes and pasta/rice. This accounted for just over one-third (35.6%) of the total annual costs. Just under a third (30.5%) of the costs were attributed to prescribing special low protein milks (liquid). It is likely that it is primarily children accessing SLPFs, as recent research suggested that it is mainly children aged <10 years with PKU who use prescribed special low protein milks % of the . In ScotOverall, very little is known about SLPFs usage by adults with PKU in England. Our study suggests that 35% of adults with PKU were not following a phenylalanine restricted diet . Althoug‘free-from’ foods. The demand for SLPFs is small in a limited global market. Few companies manufacture or distribute SLPFs in the UK [It is understandable that SLPFs cost more than regular and n the UK . ProductThe availability, accessibility and cost of SLPFs vary between countries ,24,25,34The overall use of SLPFs is affected by the national access system and any consequential economic burden ,24,25,26For patients with PKU to have better access to SLPFs through the NHS, several recommendations should be implemented. Consistent with previous suggestions by MacDonald et al. and Ford et al. ,22, spec‘free-from’ foods as comparators, and how the products were grouped, was subjective. Certain powdered/dried SLPF products e.g., burger mix, had to be compared to a prepared regular protein-containing or ‘free-from’ product e.g., cooked burger; therefore, the cost of the SLPF in its prepared form per kg was estimated. This study only examined products accessible on prescription in England compared with protein-containing products and ‘free-from’ foods available from supermarket websites in England. Also, NHS prescribing and costing data were only available for England and not the whole of the UK, and were only collected from January to December 2020. From March 2020 onwards, England experienced multiple ‘lockdowns’ due to the coronavirus pandemic, and it is possible that this may have affected food behaviours and, consequently, the number and/or types of SLPFs that patients were requesting on prescription. However, there was no evidence from clinical practice that use or supplies of SLPFs were affected in England.This study has some limitations. When obtaining the cost of each SLPF in August–October 2020, 68 products were not visible on any BNF resource, and therefore, prices were obtained directly from the manufacturer or supplier of SLPFs. The selection of protein-containing foods and When calculating the number of units of SLPF and the costs per person with PKU in England, the numbers of patients on dietary treatment were estimated. However, dietetic colleagues throughout England provided representative and recent data from their clinics. It is difficult to state exactly how many patients were requesting SLPFs, as we did not examine individual prescribing data for each patient. On ePACT2, there were nine occasions in 2020 where a SLPF appeared on a prescription, but the quantity prescribed was unclear. Consequently, these data were removed from our spreadsheet. It is possible that there may be under-reporting of SLPFs by the NHSBSA ePACT2. The NHSBSA ePACT2 trainers/help team stated that there was a small possibility that data can be incorrectly processed, but that data is scanned from each prescription form directly, so the NHSBSA ePACT2 should accurately reflect all the prescriptions issued in England.The annual cost to the NHS in England to prescribe SLPFs is £962 per patient with PKU and non-PKU IMD conditions. Surveys have repeatedly shown that patients or caregivers have access difficulties with current systems. If patients with PKU are expected to adhere to their dietary treatment for life, they must be able to easily access all SLPFs on prescription in a timely manner via the NHS. Given how little is currently being spent on prescribing SLPFs in England in comparison to the upper NSPKU guidance, cost should not be given as a reason to restrict a patient’s access to their essential dietary treatment. A review of how SLPFs are prescribed, supplied and controlled is warranted to improve the system, which, in turn, could lead to increased dietary adherence and improved patient outcomes.


During the COVID-19 pandemic, the higher susceptibility of post-stroke patients to infection calls for extra safety precautions. Despite the imposed restrictions, early neurorehabilitation cannot be postponed due to its paramount importance for improving motor and functional recovery chances. Utilizing accessible state-of-the-art technologies, home-based rehabilitation devices are proposed as a sustainable solution in the current crisis. In this paper, a comprehensive review on developed home-based rehabilitation technologies of the last 10 years (2011–2020), categorizing them into upper and lower limb devices and considering both commercialized and state-of-the-art realms. Mechatronic, control, and software aspects of the system are discussed to provide a classified roadmap for home-based systems development. Subsequently, a conceptual framework on the development of smart and intelligent community-based home rehabilitation systems based on novel mechatronic technologies is proposed. In this framework, each rehabilitation device acts as an agent in the network, using the internet of things (IoT) technologies, which facilitates learning from the recorded data of the other agents, as well as the tele-supervision of the treatment by an expert. The presented design paradigm based on the above-mentioned leading technologies could lead to the development of promising home rehabilitation systems, which encourage stroke survivors to engage in under-supervised or unsupervised therapeutic activities. Coronavirus disease 2019 (COVID-19) is an infectious disease with serious public health risk declared a global pandemic by WHO on March 11, 2020. In the present time of the COVID-19 pandemic, the lives of individuals have been drastically affected due to the imposed restrictions such as social distancing, curfews, and travel restrictions. This situation has had a considerable impact on the lives of certain more vulnerable groups on a larger scale, namely people living with chronic diseases such as stroke. In a pooled data analysis published in the International Journal of Stroke (IJS), Aggarwal et al. emphasized that COVID-19 puts post-stroke patients at a greater risk of developing complications and death. In fact, the odds of severe COVID-19 infection increase by 2.5 times in patients with a history of cerebrovascular disease . This caStroke is accounted one of the dominant causes of severe and long-term disability. It leads to a total or partial loss of aptitude to trigger muscle activation to perform any activity . The broRobot-mediated therapy for post-stroke rehabilitation offers highly repetitive, high-dosage, and high-intensity alternatives, while reducing labor intensity and the manual burden on therapists. Hence, myriads of studies have focused on exploring robotics technologies for post-stroke rehabilitation. An increasing amount of research has investigated the efficiency of different types of robotic rehabilitation systems and found that these interventions can effectively complement conventional physical therapy, e.g., Mehrholz et al. and Bertani et al. investigated the effects of robot-assisted gait and upper-limb training, respectively, and both concluded that using robotic technologies positively affects post-stroke recovery . To tackHome-based rehabilitation systems can be considered viable options capable of promoting care delivery while adhering to physical distancing measures and reducing the potential exposure to the infectious virus along with protecting vulnerable stroke survivors. Besides, even prior to this pandemic, the demand for home-based rehabilitation far exceeded its availability, further emphasizing the importance of this form of rehabilitation as a sustainable solution. According to WHO, demand for rehabilitation is approximately ten times that of the capacity of the service that the current healthcare system can provide, in terms of both rehabilitation professionals and rehabilitative tools . This poHome-based systems offer a platform for unsupervised or under-supervised therapy, in which the need for the physical presence of a therapist is reduced. Rehabilitative treatments need to be intensive with long duration to improve functional outcomes and motor recovery . CompareHowever, deployment in the unsupervised context of home-based rehabilitation poses risks to patients. Therapists perceive risks to patients regarding the training/acquisition of harmful movements when unsupervised at home—abnormal movement can be damaging or slow recovery . CarefulDue to the benefits mentioned above, robot-mediated home therapy has gained attraction in recent years. Its feasibility has been evaluated through several studies using state-of-the-art home robots. The literature surveys indicated the feasibility of self-administered treatment at home using rehabilitation robots in terms of functional outputs, training duration, user acceptance, motivation, and safety concerns. Remotely supervised participants of these studies exhibited increased motivation and autonomy in completing the prescribed task with no adverse events or edema. They also self-reported increased mobility, improved mood, and an outlet for physical and mental tension and anxiety . CatalanThis article is intended to be used as a general guideline for developing robotic home-based rehabilitation systems. For this purpose, first, the authors conducted a comprehensive review on developed home-based rehabilitation technologies of the last 10 years (2011–2020), categorizing them into upper and lower limb devices and considering both commercialized and state-of-the-art realms. The literature review analyzes and synthesizes the current knowledge of home-based robotic systems. This aims to provide a categorized comparison among reviewed literature leading to a classified roadmap to help guide current research and propose recommendations for advancing research development in this field. By addressing current challenges and shortcomings, three main aspects are considered in the proposed design paradigm, i.e., mechatronics, control, and user interface. While existing reviews take a generalized approach on home-based rehabilitation solutions—e.g., Chen et al. provided a systematic review based on the utilized technology types —we focusOver the last decade, researchers have been addressing existing challenges and requirements to design and develop rehabilitation robots suitable for home therapy. As a result, many at-home rehabilitation devices have been designed within the research realm, and some have been commercialized –4. In thWe conducted a literature review in the PubMed search engine. The search included the following terms: “Rehabilitation Robotics,” “Home-Based Rehabilitation,” and “Stroke Rehabilitation.” Secondary references and citations of the resultant articles were checked to further identify relevant literature and other available sources providing information on commercially available solutions. Reviewers screened the abstracts of the collected articles for extracting those satisfying the eligibility criteria. Studies were excluded if they were not implemented and/or did not demonstrate implementation potential in home settings, based on the criteria introduced by authors in the following subsections.Approximately 70% of stroke patients experience impaired arm function . HemiparDue to the significant results of active participation between patients and robots in functional improvement, the majority of current rehabilitative robots are equipped with electrical or pneumatic motors . HoweverThe Hand Extension Robot Orthosis (HERO) Glove is another wearable rehabilitation system that provides mechanical assistance to the index and middle fingers and thumb . Linear Targeting individuals with functional grasp pathologies, Delph et al. developed an sEMG-based cable-driven soft robotic glove that can independently actuate all five fingers to any desired position between open and closed grip using position or force control and simultaneously regulate grip force using motor current . Coffey The majority of upper-limb devices are dedicated to wrist rehabilitation due to its importance for peoples’ daily work and life . By combOne of the first projects dedicated to enabling home rehabilitation is the SCRIPT project. SCRIPT project—Supervised Care, and Rehabilitation Involving Personal Tele-robotics—is based on designing a passive finger, thumb, and wrist orthosis for stroke rehabilitation . SPO-F, Liu et al. integrated a powered variable-stiffness elbow exoskeleton device with an sEMG-based real-time joint stiffness control to offer bilateral rehabilitation to patients suffering from hemiparesis . For a pMotivated by their prior research with HandSOME, Chen et al. attempted to target another population of patients with arm weakness instead of grasping impairment and developed a spring-operated wearable upper limb exoskeleton, called SpringWear, for potential at-home arm rehabilitation . With a As the end-effector of the human body, the hand takes the lead of ADL . Hence, A novel home-based End-effector-based Cable-articulated Parallel Robot, PACER—Parallel articulated-cable exercise robot—, was developed by Alamdari et al. for post-stroke rehabilitation . The devArmAssist  is a portable, modular, easy-to-use, low-cost robotic system consisting of a tabletop module using omni-wheels, an arm, and hand gravity compensator orthosis aimed at post-stroke shoulder and elbow rehabilitation. Also, at the University of Idaho, add-on modules for wrist prono-supination and hand grasping training have been presented. Over the years, the device has been iteratively redesigned based on the updated requirements gained from clinical interviews, expert focus groups, and pilot tests with patients and therapists .As an intermediate step between high-power active and passive assistive robots, Westerveld et al. developed a low-power three-dimensional damper-driven end-point robotic manipulator, called active therapeutic device (ATD) . ATD proBased on the premise that the best way to reacquire the capability to perform a task is to practice that task repeatedly, Saebo Inc. proposed SaeboVR, a non-immersive VR rehabilitation system incorporating motivating games. These games are designed to simulate activities of daily living (ADL) and engage patient’s impaired arm in meaningful tasks aiming to evoke functional movements . The goaTo target a specific group of patients with various treatment options, this device can be upgraded with additional technologies, e.g., SaeboMas, SaeboRejoyce, or SaeboGlove, which can be integrated into the virtual environment. SaeboGlove, a functional hand orthosis, combined with electrical stimulation, has been shown to be beneficial for functional use of moderately to severely impaired hands in sub-acute stroke patients . SaeboMAAnother home rehabilitation device for post-stroke patients is WeReha, which is intended to be used for hand impairments with the possible remote supervision of physiotherapists . The priThe soft extra muscle (SEM) Glove by Bioservo Technologies AB, Sweden , was devIn an attempt to transfer Gloreha—Hand Rehabilitation Glove—Professional, a wearable hand rehabilitation hospital device, to a home setting, Gloreha Lite has been miniaturized and specifically designed for home use in a safe and feasible way for hand rehabilitation . Glorehahttps://motusnova.com/hand), previously known as Hand Mentor Pro, is a portable robotic device designed to enhance active flexion and extension movements of wrist and fingers along with motor control of the distal upper limb. The device deploys pneumatic artificial muscles for simulating dorsal muscle contraction and relaxation. The Motus Hand has been classified as an FDA class 1 device presenting non-significant risk (NSR). Several clinical trials investigated and supported the clinical efficiency, feasibility, and user-friendliness of the Motus Hand for in-home telerehabilitation among subacute to chronic post-stroke  . DrawingMany developed lower-limb robotic systems offer rehabilitation in sitting/lying positions for stroke patients who cannot stand or walk safely . In thisSpasticity of the limbs is one of the most common impairments ensuing onset of stroke. It puts patients at a high risk of developing foot deformity. Hence, treating the spasticity of lower extremities to prevent any deformity and facilitate ankle muscle activities during the acute phase and even after the long bedridden period is of utmost importance. In order to reduce or prevent the occurrence of spasticity at later stages, Low et al. developed a soft robotic sock, which can provide compliant actuation to simulate natural ankle movements in the early stage of stroke recovery. The soft robotic sock controls the internal pneumatic pressure of the soft extension actuators to assist the patient in ankle dorsiflexion and plantarflexion . For theAs one of the first home-based EMG-controlled systems, Another example of lower-limb devices is a multi-posture electric wheelchair developed by Chen et al. with a lower-limb training function. Combined with virtual reality games and a tele-doctor–patient interaction, this device forms an intelligent rehabilitation system suitable for home therapy . Apart fOne of the most prevalent lower-limb impairments following hemiplegia in post-stroke patients is asymmetric gait patterns and balance dysfunction. These impairments can adversely affect the quality of life as they lead to compensatory movement patterns, slowed gait speed, limited functional mobility, which results in reduced performance of the activities of daily living and increased risk of experiencing falls. Therefore, regaining autonomous gait and improving independent walking ability should be among the top priorities of rehabilitation interventions post-stroke. Aimed at improving stability and walking capacity, Morone et al. introduced i-Walker, a robotic walker for overground training with embedded intelligence that provides asymmetrical assistance as needed by detecting the imposed force by the user to adjust the amount of help to the impaired side .Developing wearable robots for lower-limb treatments has increasingly gained traction for their capability to facilitate ambulatory rehabilitation delivery. Among them, Gait Exercise Assist Robot (GEAR), proposed by Hirano et al. in collaboration with Toyota Motor Corporation, is a wearable knee-ankle-foot robot  integrated with a low floor treadmill and a safety suspending device . The LokWalking Assist for Hemiplegia (WA-H) is a portable, lightweight, modular, and wearable exoskeletal robot supporting the hip and knee joint movements that, by providing customized gait training, can be used in various environments depending on the degree of impairment in patients. WA-H has an inherently safe design in which all robot joints mechanically limit the movements occurring beyond the natural range of motion. The device features a passive joint simulating the weight shift occurring during walking in the hip joint in the coronal plane .® is a wear robot that assists hip and knee joints in both impaired and unaffected legs simultaneously with no rigid connection between joint frames, resulting in a higher degree of freedom. Prioritizing user-friendliness in terms of ease in don/doffing and minimizing the restraining stress against the natural human movement, Mizukami et al. adopted a non-exoskeletal structure coupled with a synchronization-based control system, introducing the ability to feel what natural movement would be like. Due to the absence of any rigid connection between joint frames, the device provides a high degree of freedom for patient movement  is developed and available for purchase (https://eksobionics.com/eksonr/).There are several commercially available lower-extremity rehabilitation robots, and those exhibiting potential for home therapy are presented. The wear overground robotic Stride Management Assist  a mechatronic device delivering rehabilitation intervention, 2) a control system ensuring proper performance of the system, 3) interactive interfaces for patients and medical professionals who provide remotely supervised therapy, 4) a communication system gluing the whole system together.To provide a classified roadmap for assisting researchers who aim at further developing this field, a design paradigm is proposed to form a guideline on developing each component based on the engineering design process.Among different post-stroke symptoms, motor deficits are the most commonly recognized impairments that affect the face, arm, and leg motor functions. These impairments result in various manifestations, including impaired motor control, muscle weakness or contracture, changes in muscle tone, joint laxity, spasticity, increased reflexes, loss of coordination, and apraxia . To recoEngineers should develop the rehabilitation system based on multiple contributing factors, including the part of the limb being trained, the targeted stage of recovery, the severity of initial motor deficit, range of movements in the paretic limb, grade of spasticity, age, and individual patient’s characteristics. Depending on the patient, it is known that motor impairments can induce disabilities in several functions, such as range of motion, speed, coordination, cadence (steps/minute), balance, precision, the ability to regulate forces, muscle strength, and energy efficiency . PhysiolRecovery would benefit if scientific principles behind post-stroke motor learning were incorporated into the design of the rehabilitation device. Under the assumption that performance improvement is dependent on the amount of practice, most current mechatronic devices for post-stroke therapy are solely based on the repetition of a single task, termed “massed practice” . It is iFor rehabilitation interventions to be meaningful, learned tasks must generalize to new tasks or contexts, especially real-world tasks. Introducing variability to training sessions, though worsening the patients’ performance in the short term, improves their performance in retention sessions and also increases generalization by representing each task as a problem to be solved rather than just memorized and repeated . ContextOnce the target group and treatment plan have been identified, the design criteria, including requirements and constraints, need to be established and prioritized to fit the need. Since the device is being designed for the home setting, certain factors are introduced, and some others become more prominent—safety, adaptability to the home setting, the autonomy of patients, aesthetic appeal, affordability, to name a few .In addition to the general criteria, the adoption of each home rehabilitation solution requests specific features of the device itself. For example, in the case of exoskeletons, besides absolute safety when worn, lightness, wearing ease, comfortability, and smoothness, there should be an absence of friction and allergenic factors as it is in contact with the skin. So the device should guarantee a high tunability and reliability . ConsideHaving design criteria and target functionality in mind, the designer has to decide the mechanism type. The type of the mechanism and the treatment options are correlated; for example, additional movement protocols can be utilized based on the number of arms. In general, human limb rehabilitation robots are divided into two groups based on the target limbs: upper-limb rehabilitation devices and lower-limb rehabilitation devices, each divided into several subgroups. Based on motion systems, upper-limbs are categorized into exoskeletons or end-effector devices. Based on the patient’s posture, lower-limb devices can be designed to be used in sitting/lying positions or standing positions with the help of body and robot weight support.In his study in 2019, Aggogeri et al. categorize robotic rehabilitation technologies into end-effector or exoskeleton devices based on design concepts . End-effComparing these two different approaches, end-effector robots are more flexible than exoskeleton devices in fitting the different sizes, require less setup time, and increase the usability for new patients. Besides, end-effector mechanisms are also generally ambidextrous. On the contrary, exoskeletons should be fully user-adjustable and therefore require more complex control systems. While both distal and proximal joints are constrained in exoskeleton devices, end-effector robots merely constrain the distal joints . TherefoIn conventional exoskeleton mechanisms, the rigidity of the frames and fixed straps poses an issue on their wearability and usability. The heaviness and bulkiness of such frames result in high energy cost and also affects the natural gait dynamic and kinematics of the patient. Hence, soft orthotic systems have been developed as an alternative to traditional rigid exoskeletons . In thisIn rehabilitation devices, it is essential to improve physical human-robot interaction (pHRI). For each type of rehabilitation device, the recruitment of different engineering methods is required for such improvements. This interaction is fundamentally affected by the mechanisms that should be designed by taking sophisticated biological features and activities into account. By considering the compliance/stiffness factor, modes of actuation and transmission need to be selected in a systematic way. Control methods also affect pHRI .The number of active and passive DOFs determines the system’s functionality. They condition the workspace in which the joints are capable of moving, indicating the assistance/rehabilitation, which is needed to be delivered to each joint . PatientIn the lower extremity, the hip is simplified as three rotary joints to achieve flexion/extension, abduction/extension, and internal/external rotation. The knee achieves pure sagittal rotation and flexion/extension. And finally, the ankle, simplified into three rotation joints, achieves plantar/dorsiflexion, eversion/inversion, and internal/external rotation . The kinModeling tools are of paramount importance in the design of rehabilitation devices for both robot and musculoskeletal modeling. Rigid body simulation programs or general-purpose simulation software such as Adams, Matlab, and Modelica can be used to evaluate the mechanics and control aspects. Also, computer-aided design software, such as CATIA and SOLIDWORKS, could be used to design, simulate, and analyze these robotic mechanisms. To simulate and control soft robots, SOFA, an open-source framework, can be used. It provides an interactive simulation of the mechanical behavior of the robot and its interactive control. It is also possible to model a robot’s environment to be able to simulate their mechanical interaction.On the other hand, the heavy dependence of design parameters upon the targeted application requires careful analysis of the human body anatomy to design the device by considering the end-user application . In turnTwo commonly used dynamic modeling methods are Newton-Euler and Lagrange’s methods. In the Newton Euler method, by solving the Newton-Euler equation, the robot’s internal and external forces are extracted. In Lagrange’s method, which is based on the system’s energy, the external driving force/torque of the system can be calculated. In 2019 Zhang et al. drew a comparison between these two methods and provided a table to demonstrate the differences between these methods. Derivation analysis in the Newton-Euler method is more complicated than the Lagrange method, but calculations in the Newton-Euler method are large and heavier to compile, while in the Lagrange method, are easily compiled. In the Newton-Euler method, in addition to the driving force/moment, internal forces can be obtained . DynamicThe design of the device, further completed and integrated with the detailed design of chosen actuators, the transmission system, and the sensing system, should be presented at the next step. As there are myriad options to choose from, the designer has to weigh his/her options against the design criteria and their allocated importance and priority.Efficient actuator design is important for home-based rehabilitation systems since these systems should be compact. Therefore, as the main powering elements, small-sized actuators that have a high power-to-weight ratio are required as they are capable of producing high torques with precise movement . ActuatoElectric motors are used for their quick responses and capability of providing high controllability and controlled precision. However, they have a low power-to-mass ratio and are usually expensive. Pneumatic actuators can yield high torques but could save self-weight. Nevertheless, by using these types of actuators, the portability of the system is compromised due to the accompanying inherent components such as pump, regulators, valves, and reservoirs. Another factor that makes these types of actuators unsuitable for home-based systems is that they require maintenance since lubricant/oil leakages could be problematic for users. In hydraulic and pneumatic actuators, control is less precise, and hence the safety cannot be ensured. According to Shen et al. and Gul et al., these actuators are not suitable for providing assistance or therapy or for rehabilitation purposes due to their possessing high impedances; however, some studies utilize them because of their ability to provide high power. Ultimately, based on the studies done by Gull et al., Series Elastic Actuators (SEAs) by reducing inertia and user interface offer a safe pHRI and can achieve stable force control . Tuning On the other hand, passive robots must be equipped with passive actuators to enable scalable resistance and assistance based on the patient’s mobility status. There are various types of passive actuators such as friction brakes, viscous dampers, and elastic springs to name a few .Power transmission may be realized through the utilization of direct drive, gear, linkages, or cable-driven methods. Cable-driven transmission means allow for a more lightweight and compliant design. Backlash and transmission losses render the control of such systems challenging . SanjuanIn order to provide proper guidance for the device’s movement to execute the required tasks, the system should utilize sensing methods as input signals. Generally, four main sensors are used in rehabilitation devices, namely Motion and Position sensors, Force/Torque sensors, Electromyograms (EMG), and Electroencephalogram (EEG) . Since tThe spatial configuration of the device is needed in order to analyze its kinematics and dynamics. For this purpose, position sensors are used to measure and indicate this spatial configuration. Among the sensors used for this purpose are: encoders, potentiometers, flex sensors, and transducers. For haptic applications like rehabilitation in VR, force/torque sensors are required. Usually, extra force/torque sensors are added to the system to provide additional safety levels. Gyro and acceleration sensors can be mounted on the mechanical structure for measuring the patient’s posture, for example, in HAL .Moreover, EMG and EEG could be used for measurements in noninvasive ways. The received signals in these two methods are often noisy and thus require further processing. Also, sensor fusion, in which the data from multiple sensors are merged, could provide a safer and more stable intention detection for the system.Last but not least, in the unsupervised context of home-based rehabilitation, safety is the most crucial criterion to be considered. Multiple redundant safety features must be incorporated in different modules of the device. In designing the mechanism stage, the device could be designed to offer intrinsic mechanical safety so as not to transfer excessive force to the patient’s body. Also, abnormal reactions of the patient should not be transferred to the actuator. For example, in Gloreha, these eventual reactions are absorbed by the mechanical transmission . In “DesUltimately, at the end of each step, following the suit of the engineering design process, iterative optimization is needed to redefine the design process based on the predefined criteria. All of these design steps are highly interconnected and, therefore, should be considered from different aspects . The HERAfter considering the mechatronic aspects, the control system has to be developed to ensure the proper behavior of the device . From thDuring the last two decades, many solutions have been proposed to provide motion tasks suitable for post-stroke motor recovery. Priotteti et al. categorized existing global strategies for robotic-mediated rehabilitation into three groups of assistive, corrective, and resistive controllers .Passive assistance, where the device moves a muscle rigidly along the desired path by adopting different techniques, is the most common treatment strategy for acute patients due to the unresponsiveness of the paretic limb at early post-stroke stages. As soon as patients regain some degrees of mobilization, switching to other assistive solutions, such as triggered passive, where the patient initiates the assistance, or partial assistance, where the device assists the patient as needed (AAN), as utilized by Díaz et al. in HomoRehab , may leaThe corrective mode is linked to the rehabilitation situation in which the patient is not performing the movement correctly, and the robot intervenes by forcing the impaired limb to the correct orthogonal direction. However, it is not easy to clearly differentiate assistive and corrective techniques. Hence, combined assistive-corrective controllers are mostly recurrent among existing controllers .® employed a synchronization-based assistance control strategy using neural oscillators based on a central pattern generator network. The synchronization-based controller detects the user’s movement and measures the mutual gap between the user and the device, and then achieves a desired joint angle trajectory to be synchronized with human movements  provides access for displaying and editing stored data. This provides a base for therapists upon which they can make more educated decisions and choose the best treatment plan for their patients. Also, it is possible to add reminders to the system for notifying the patient to do his/her daily practices. In the end, the system uses this information to adapt the treatment program focusing on the parts that require more recovery to increase therapeutic value.There are some other crucial principles to be considered for offering software interfaces that assist home-based systems in providing structured training sessions. A user-friendly interface in terms of environment and layout of features and components is the starting point of having a well-established interaction between patients and the device. This friendliness arises from patients’, as well as their caregivers’, ability to interact easily with the device in a familiar environment and to have quick and straightforward access to the training and monitoring components . EnablinVirtual/Augmented Reality  and gaming present an alternative world in which mundane rehabilitation exercises are disguised in the shape of appealing fantasies. Integrating these methods into home rehabilitation is a common practice aimed at encouraging motivation in patients. The score-based nature of gaming further motivates patients—by rewarding points, coins, and badges as immediate feedback—and reinforces confidence and positivity through achievements  algorithms such as artificial neural networks. Although training of such systems can be computationally very demanding for these processors, the training process is performed mostly offline. Applying the trained AI feature and even its required recursive update does not involve significant computation, and can be easily handled on the processor of a smartphone, PC, or any other available digital processor.Acquired data from agents should be comparable with each other, and therefore it is critical to bring the collected data into a common format which occurs through data standardization. By providing internally consistent data with the same content and format, any confusion and ambiguity are avoided. This ensures that all parties involved have mutual understanding, and view the results in the same way which leads to reliable measurements and decision plans. Standardized quantitative and qualitative data should be collected for reliable and educated assessment of patients’ performance through evaluation forms and outcome measurements. From a therapist’s point of view, the prescribed tasks, reports, and evaluations should be offered in a standard format. To do so, there needs to be a predetermined data extraction on which the community of healthcare professionals has reached a consensus. However, in case of any ambiguity or to provide further details and explanations, therapists should have the option of attaching notes to the data .The proposed community-based rehabilitation framework facilitates knowledge sharing and provides comprehensive data to ensure educated judgment regarding the best treatment regimen yielding the best recovery outcome. This massive pool of information—both from recorded data and also normalized datasets from already existing medical center repositories—generates big data. By applying machine learning, meaningful information from these large data sets can be extracted. This extensive database can be beneficial for various applications, one of which could be providing a research base enabling further studies in fields of neurorehabilitation, engineering, and occupational/physical therapists. Thus, in this IoT-enabled rehabilitation system, data analysis is performed by intelligent algorithms and knowledge-based methods to provide intelligent suggestions to the therapists, creating a basis for the best treatment plan assignment.In the framework, the capability of communication among agents of each community is also feasibly provided. The interaction of healthcare professionals gives them the opportunity to clarify any issues or ambiguities, gain additional expertise, discuss possible treatments, and share their knowledge. The provided platform can also enable socialization among patients. Social restrictions imposed by stroke and exacerbated by the COVID-19 pandemic, have adversely affected patients’ lives. Therefore, any means of social interaction could potentially improve their quality of life, which in turn could further motivate them to adhere to the therapy . This coWe suggest an alternative approach in which the purpose of the framework goes beyond rehabilitation to contribution to society and household chores. The virtual environment tasks should be designed so that, along with rehabilitation, they can offer patients the opportunity to learn real-life and daily living skills—cooking, gardening, and housekeeping, to name a few. For example, learning the cooking skill takes place in a virtual kitchen where all of the ingredients and utensils are available along with cooking instructions in the form of video and audio to enable the patient to learn and practice simultaneously. The theme-based free world gameplay would be one of the best options as it can be flexible enough to reflect various aspects of daily life. For example, Saebo Inc. has developed a virtual reality environment in which in the cooking scenario, should the virtual kitchen lack any of the ingredients or utensils required to complete the task, the game enables the patient to go grocery shopping; this way through a comprehensive experience, patient not only goes through therapy but gets to enhance his/her life skills. Integrating this free world environment with our suggested community-based framework adds an additional social layer where patients can teach and learn these skills from each other. For instance, if a patient knows a particular recipe, she/he can teach and share it with the patient community. This provides the patients with a sense of control over their treatment and fosters a sense of usefulness, purpose, and contribution to society. Also, in this time of COVID-19, where everybody, especially elders, feels disconnected from the world, this will excite the feelings of inclusion and connection, which in turn will further motivate the patients to partake more frequently and actively in their treatment sessions.To go one step further, this virtual environment could be linked to existing online shops allowing the patient to contribute to household chores by purchasing the required and essential items for the house and facilitating the acquisition of his/her own necessities. According to Finally, the framework has the capacity to incorporate an ambient assisted living ecosystem that can provide patients, specifically elders, with personalized options. These options include remote monitoring, assessment, and support based on their unique profiles and surrounding context. The purpose of such options is to enhance the independence of elderly or disabled individuals in their own secure and convenient space of living .Rehabilitation programs, even in the clinical setting, are often not fully supported by public health systems, and hence not all post-stroke patients can afford this option, and due to the enormous socioeconomic impact and high costs, not just on the patient but also on their families, patients opt to drop out . AdditioEarly-stage post-stroke rehabilitation, a crucial phase of rehabilitation, could be a direct indicator of the possibility of motor recovery. In the current restrictive climate of the COVID-19, due to the restrictions associated with social distancing and limitations for commuting, it has become more difficult for patients to use this sensitive window of time for rehabilitation treatments. In this context, home-based rehabilitation can be a solution.Several home-based rehabilitation devices have been developed in recent years, both at the research and commercial levels. Although there are some challenges in the research and development of these devices, based on a technical evaluation of aspects such as mechatronics, the control system, and software, these devices can be tuned to be suitable for home-based rehabilitation therapy. Eventually, such systems can be utilized in a framework aiming at creating a comprehensive network for therapists, patients, engineers, and researchers. To this end, standardization in every aspect of home-based systems, from design criteria to performance evaluation, is required.The COVID-19 pandemic has proved the necessity of further deploying the power of the internet and computers, not only for the purpose of communication but also for big data analysis. Such technologies can be feasibly integrated with mechatronic systems such as rehabilitation robots to enable new applications such as remote home-based therapies. The outcome of such an interconnected framework would be significantly vital for the recovery of post-stroke patients, promoting the quality of their lives, and eventually reducing the associated burdens on the healthcare system in the long term.


Allergic rhinitis (AR) and allergic contact dermatitis (ACD) are prevalent allergic diseases and have significant impacts on patients’ daily life. Despite many studies on AR or ACD have been conducted separately, little is known about the immune responses in patients of AR combined with ACD and the interplay between AR and ACD. Our study compared various aspects of immune elements in patients with AR or/and ACD, aiming to characterize the immune responses in AR, ACD, and AR combined with ACD.A total of 57 patients diagnosed with AR or/and ACD and 28 healthy volunteers were included. AR patients were further divided into seasonal AR (SAR) and perennial AR (PAR). All subjects’ blood samples were taken to assess the concentration of immunoglobulins, complement C3, C4, autoantibodies and cytokines in serum by immunoturbidimetry, ELISA or Luminex200 platform. Peripheral blood mononuclear cells (PBMCs) were subjected to the analysis of lymphocyte subpopulations by flow cytometry.It indicated that AR disease caused elevated levels of IgE, IgA, IgG, IgG4, as well as IL-4, IL-15, IL-8 and IL-6 in serum. AR patients possessed a decreased CD4/CD8 ratio and an increased proportion of memory CD4 + T-cell subset, with a skewed Th2 response and an enhanced CD8 + T-cell activation. Compared with patients with sole AR or ACD condition, AR + ACD patients presented with a significantly increased proportion of memory CD8 + T-cell subset and were prone to autoimmune disorders as indicated by the increased autoantibodies. The immune elements in patients with ACD only were least affected compared with those in other conditions. Additionally, seasonal or perennial AR patients exhibited different cytokine profiles and proportions of memory T-cell subsets.In this study, we illuminated the respective characteristics of immune responses in AR, ACD, and AR combined with ACD. Meanwhile, we discovered that the PAR and SAR patients possessed different cytokine profiles and T-cell compartments. It suggested that these allergic conditions belong to different disease entities. Characterizing the detailed immune changes in these allergic diseases would help to develop proper treatments targeting particular immune elements in different allergic diseases.The online version contains supplementary material available at 10.1186/s13223-022-00646-6. Allergic rhinitis (AR) and allergic contact dermatitis (ACD) are common allergic diseases worldwide characteristic of high IgE and allergic inflammation readily triggered by allergens , 2. AR iAR is an IgE-mediated type I hypersensitivity process that involves several types of immune cells and cytokines , 9. DepeBy contrast, ACD is a delayed type IV hypersensitivity reaction resulting from the activation of allergen-specific T cells . ACD occThe present study recruited 57 patients diagnosed with AR and/or ACD and 28 healthy volunteers aged from 19 to 35 years. The information of the patients and healthy controls was displayed in Table Dermatophagoides. pteronyssionus, Dermatophagoides. farinae), trees pollen , and weeds pollen . All extracts were prepared from the Allergen Manufacturing and Research Center . Histamine phosphate (0.01 mg/ml) was used as the positive control, and an allergen diluent  as the negative control. The results were evaluated 15 min after application. A wheal diameter ≥ 5 mm after subtracting the negative control for each of the allergens tested was considered as a positive response. The results were listed in Table Skin prick tests were performed for the common inhalant allergens in China, including dust mite  tests were performed on the upper back and evaluated after 48 and 72 h. The test reactions were graded from no reaction to grade + , +  +, and +  +  +, depending on the intensity of the reaction following the recommendations of the International Contact Dermatitis Research Group . The resFor surface marker labeling, 50 μl EDTA anticoagulated blood of each sample was incubated with fluorochrome-conjugated antibodies for 20 min in dark. After that, 450 μl OptiLyse C  was added for erythrocytes lysis. Following 10 min incubation in dark, 50 μl absolute count beads  were added before events of stained cells were acquired with a FACS CantoII flow cytometer .g for 20 min at room temperature, the interphase cell layer was carefully transferred into a 15 ml tube. The cell pellet was washed with 10 ml PBS containing 5% FBS and centrifuged at 600g for 5 min. For cytokine detection, 2 × 106 cells of each sample were cultured in 10% FBS RPMI-1640 medium  with 50 ng/ml of PAM , 1 μg/ml Ionomycin  in the presence of 1 μg/ml Brefeldin A  at 37 °C cell incubator with 5% CO2. Then cells were permeabilized using Fixation/Permeabilization Solution Kit  according to the manufacturer’s protocol after surface marker labeling. Cell events were acquired with FACS CantoII flow cytometer  and data were analyzed with FlowJo . Following antibodies were used: For surface marker labeling: CD3 APC-H7, CD4 BV421, CD8 APC-R700, CD19 PE-Cy7, CD16 V500, CD56 PerCP-Cy5.5, CD45RA APC, CD45RO FITC. For intracellular labeling: CD4 PerCP-Cy5.5, CD8 PE-Cy7, IL-4 PE, IFN-γ APC, granzyme B FITC. All antibodies were purchased from BioLegend.For intracellular labeling, PBMCs were isolated with Ficoll-Hypaque density gradients. After centrifuging at 1000According to the manufacturer’s instructions of the Human Cytokine/Chemokine Panel MILLIPLEX® MAP kits , IL-15, IL-17A, TNF-α) , serum concentrations of multiple cytokines were measured by a Luminex200 platform . In brief, the plate was pretreated with 200 μl wash buffer for10 min. Next, 25 μl serum of each sample, 25 μl assay buffer and 25 μl mixed beads were added to sample wells. The background wells, QC wells and standard wells were added with corresponding reagents following the instructions. The plate was sealed and incubated with shaking at 4 °C overnight. The next day, the plate was washed twice with wash buffer and 25 μl detection antibodies were added for a 1 h incubation at room temperature. After washed twice with wash buffer, each well was incubated with 25 μl Streptavidin Phycoerythrin for 30 min. After two wash steps, 150 μl sheath fluid was added and the data were collected by the Luminex200 platform. All samples were measured in duplicate.Serum IgG, IgA, IgM and C3, C4 were measured by immunoturbidimetry using an automated Beckman Image 800 Immunochemistry System  according to the manufacturer’s instructions. Serum IgE was measured by ELISA using EUROIMMUN Analyzer I  according to the manufacturer’s instructions. Serum IgG4 was measured by ELISA . Serum autoantibodies  were measured by EUROBlotMater and the data were analyzed by EUROBlotCamera .t-tests (unpaired) in Prism 7.0 . Significant differences between groups were represented by *p < 0.05, **p < 0.01, and ***p < 0.001.The scatter plots data were represented as the mean ± SEM. Statistical analyses were performed using one-way ANOVA and Tukey’s multiple comparisons test or two-tailed Student A total of 57 patients diagnosed with AR or/ and ACD aged from 19 to 35 years were included in the study. AR combined with ACD patients were defined as the AR + ACD group. Results for skin prick tests and epicutaneous patch tests were listed in Tables We firstly investigated the serum levels of immunoglobulin and complement C3, C4. IgE played a key role in type I hypersensitivity mediating various atopic diseases . Indeed,Next, we examined the levels of serum cytokines , IL-15, IL-17A, TNF-α) in each group by Luminex200 platform. Our data revealed that serum IL-6 remarkably increased in both AR group and AR + ACD group, compared to that in HCs  as well as a reduced CD4/8 ratio in addition to an augmented memory CD4 + T-cell compartment in the peripheral circulation. AR + ACD group was featured with a moderate change in serum cytokines (increased in IL6 expression only) and an increased ratio in peripheral memory CD8 + T cells. Based on our data, it was perceivable that cytokines and T-cell compartment in patients with ACD only were least affected in comparison with other allergic conditions.To dissect the T-cell function in these patient groups, intracellular staining for Th1-related cytokine (IFN-γ), Th2-related cytokine (IL-4) and lytic granule (granzyme B) in T-cell subsets were performed. A reduction of IFN-γ + CD4 + subset was observed in AR group Fig. A. AlthouPrevious research has established that autoantibodies were involved in the pathogenesis of autoimmune diseases . To inveThe results showed that the autoantibody positive rate in AR and ACD groups was 26.7% and 25%, respectively Fig. A. StrikiConsidering AR group exhibited significant differences in several cytokines and T-cell subsets compared with HC group, a more detailed analysis was made. AR patients were divided into SAR n = 27) or PAR n = 16) depending on the reactivity of skin prick tests to cyclic pollens or year-round allergens (mite) (Table 7 or PAR  dependinIn this study, we investigated the immune responses in peripheral blood from AR, ACD and AR combined with ACD patients, including serum levels of immunoglobulin, cytokines, the proportion of lymphocytes subsets, as well as autoantibody profile. It expanded our understanding on the pathogenesis of allergic disease and clarified the different characteristics of immune responses among patients diagnosed with AR or/and ACD.In the serological examination, AR patients and AR + ACD patients exhibited an elevated level of IgE, IgG and IgA compared with the HC group, while patients with ACD only showed an increase in IgE level but not IgA and IgG. There is consensus that antigen-specific IgE is crucial for the development of these allergic disorders , 26. ConIn cytokines/chemokines determination, AR patients expressed elevated levels of IL-4, IL-15, IL-8 and IL-6, while AR + ACD patients only showed an increase in IL-6. However, patients with ACD did not show any significant change in these cytokines. IL-4 is a characteristic Th2 cytokine critical for IgE-mediated inflammatory response . IL-6 isThe presence of autoantibodies in serum reflects leakiness of central and/or peripheral tolerance and may lead to the manifestation of autoimmune diseases . A plethIn lymphocytes subsets analysis, a significant decrease of CD4 + /CD8 + ratio in AR patients was observed. Several previous studies showed no significant difference in CD4 + T cells in AR patients, but some others found increased CD4 + T cells –59. It mIn conclusion, our study characterized the immune responses in AR, ACD and AR combined with ACD. AR patients showed elevated serum levels of IgE, IgA, IgG, IgG4, as well as IL-4, IL-15, IL-8 and IL-6. Furthermore, IL-4 and IL-6 were elevated in SAR patients, while IL-6 and IL-8 were elevated in PAR patients, which hinted the different tendencies of immune responses. What’s more, AR disease caused a decreased CD4/CD8 ratio and an imbalance in T cell subsets, including the increased proportion of memory CD4 + T cells, skewed Th2 response, and enhanced CD 8 + T cells activation. SAR but not PAR caused intense changes in T cell subsets. Despite not intensifying the immune responses, comorbidity of AR with ACD presented with an increased proportion of memory CD8 + T cells and an increased propensity for autoimmune disorders.Additional file 1: Fig. S1. The serum levels of IgM, C3, C4 and cytokines. Fig. S2. Flow cytometry analysis of B cell and NK cell subsets in peripheral blood.