Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in dystrophin encoding gene, causing progressive degeneration of cardiac, respiratory, and skeletal muscles leading to premature death due to cardiac and respiratory failure. Currently, there is no cure for DMD. Therefore, novel therapeutic approaches are needed for DMD patients.mdx mouse models of DMD.We have previously reported functional improvements which correlated with increased dystrophin expression following administration of dystrophin expressing chimeric (DEC) cells of myoblast origin to the mdx/scid mouse model of DMD. Functional improvements included maintenance of ejection fraction and fractional shortening levels on echocardiography, reduced enhanced pause and expiration time on plethysmography and improved grip strength and maximum stretch induced contraction of skeletal muscles. Improved function was associated with amelioration of mdx muscle pathology revealed by reduced muscle fibrosis, reduced inflammation and improved muscle morphology confirmed by reduced number of centrally nucleated fibers and normalization of muscle fiber diameters. Our findings confirm the long-term systemic effect of DEC therapy in the most severely affected by DMD organs including heart, diaphragm, and long skeletal muscles.In the current study, we confirmed dose-dependent protective effect of human DEC therapy created from myoblasts of normal and DMD-affected donors, on restoration of dystrophin expression and amelioration of cardiac, respiratory, and skeletal muscle function at 180 days after systemic-intraosseous DEC administration to These encouraging preclinical data introduces human DEC as a novel therapeutic modality of Advanced Therapy Medicinal Product (ATMP) with the potential to improve or halt the progression of DMD and enhance quality of life of DMD patients.mdx muscle pathology after systemic-intraosseous administration of human Dystrophin Expressing Chimeric (DEC) Cells to the mdx/scid mouse model of DMD. 2. Systemic administration of human DEC therapy resulted in amelioration of cardiac, respiratory and skeletal muscle function as confirmed by echocardiography, plethysmography and standard muscle strength tests respectively. 3. We introduce human DEC as a novel Advanced Therapy Medicinal Product (ATMP) for future clinical application in DMD patients.Human DEC as a novel therapeutic modality with the potential to improve or halt progression of the DMD disease and enhance quality of life of DMD patients. Graphical abstract represents manufacturing process of the human DEC therapy for the future clinical applications. 1. We report the long-term efficacy of human DEC therapy resulting in increased dystrophin expression and reduced The online version contains supplementary material available at 10.1007/s12015-022-10384-2. Duchenne Muscular Dystrophy is an X-linked recessive neuromuscular disorder caused by mutations in the dystrophin gene with the incidence of approximately 1 in 3,500–5,000 newborn boys [Due to the lack of dystrophin, a large structural protein that stabilizes the plasma membrane in muscle fibers, progressive weakness of skeletal, cardiac, and respiratory muscles is observed. First symptoms of abnormal gait and walking are observed in the affected boys between 3 and 5 years of age and by age 12, most boys are wheelchair-dependent . Over tiN/MBDMD). Administration of DEC therapy to the mdx mouse models of DMD resulted in restoration of dystrophin expression which correlated with significant improvement of cardiac, respiratory and skeletal muscle function [Moreover, we have applied the chimeric cell therapy concept to muscular dystrophy and created Dystrophin Expressing Chimeric (DEC) Human cells by fusion of human myoblasts (MB) derived from normal and DMD-affected donors  efficacy of human DEC therapy after systemic-intraosseous administration. Our goal was to assess the protective effect of DEC therapy in the most affected by the DMD organs before severe progression of the disease and before development of the overt DMD -related cardiomyopathy or pulmonary failure, to make it more clinically relevant, since once organ failure occurs, treatment with stem-cell based therapies would not reverse damage created by DMD disease.Thus, in this study, the maintenance of long-term engraftment of DEC correlating with restoration of function was confirmed by normalized muscle morphology, reduced pathology, and improved function in the most severely affected by DMD organs including heart, diaphragm, and long skeletal muscles. This encouraging preclinical data introduces human DEC as a novel Advanced Therapy Medicinal Product (ATMP) with the potential to improve or halt progression of the disease and enhance quality of life of DMD patients.mdx/scid mice—the animal model for Duchenne Muscular Dystrophy  with respective background wild type mice  were purchased from Jackson Laboratories. Animals were kept in a pathogen-free environment on a light/dark cycle. Prior to study initiation, aged matched male mdx/scid mice were ear tagged and randomized into following experimental groups: vehicle control injected with 60 µl phosphate-buffered saline, PBS (n = 4), DEC therapy group injected with 1 × 106 DEC cells suspended in 60 µl PBS (n = 5), and DEC therapy group injected with 5 × 106 DEC cells suspended in 60 µl PBS (n = 5).This study was approved by the Institutional Animal Care and Use Committee (IACUC) of University of Illinois at Chicago, which is approved by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). All animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animal Resources’. Six- to eight-week-old male Normal human myoblasts (MB) were purchased from Lonza Bioscience , and DMD-affected myoblasts were purchased from Creative Bioarray Ltd. . Myoblasts (MB) were cultured in Skeletal Muscle Cell Growth Medium-2  supplemented with the human Epidermal Growth Factor (hEGF); Fetal Bovine Serum (FBS); Dexamethasone; Gentamicin/Amphotericin B (GA) . Upon reaching 60–70% confluence, myoblasts were harvested using 0.25% trypsin/EDTA . Enzymatic activity was inhibited with 10% serum-supplemented culture media. Human MBs were harvested between passages 3–7, which is optimal for the ex vivo cell fusion procedure.N and MBDMD) were washed in serum-free media supplemented with antibiotics . Next cell fusion procedure was performed. As previously described [N and MBDMD) were fluorescently labeled using PKH26 or PKH67  membrane dyes respectively, according to manufacturer’s instructions. Parent cells were mixed, washed and fusion was performed using 1.46 g/mL PEG solution  containing 16% DMSO  [mdx/scid mice.After harvesting, staining and viability assessment with 0.4% Trypan Blue , parent myoblasts  –44. FuseMice were anesthetized with 2% isoflurane inhalation along with 1 mL/kg buprenorphine subcutaneous injection. DEC intraosseous administration was performed as previously reported , 41. BriFor immunofluorescence the optimal cutting temperature (OCT) frozen sections of the heart, diaphragm and gastrocnemius muscle were fixed with ice-cold acetone for 10 min and blocked for the unspecific binding with 10% normal goat serum for 60 min in 4 °C. Next, specimens were incubated with mouse monoclonal anti-human dystrophin  and mouse monoclonal anti-human spectrin  primary antibodies, followed by incubation with a goat anti-mouse conjugated AlexaFluor-647 secondary antibody for dystrophin  and goat anti-mouse secondary antibody for spectrin (IgG) Alexa Fluor® 647 . Appropriate positive and negative controls were used. Nuclear counter-staining was performed using 4′,6-diamidino-2-phenylindole (DAPI) . A Zeiss Meta confocal microscope with ZEN software  and Leica DM4000B microscope were used for fluorescence signal detection and analysis.For histological analysis, the heart, diaphragm and gastrocnemius muscles were harvested, fixed in 10% neutral buffered formalin and embedded in paraffin. Next, the paraffin blocks were cut into 5 µm non-consecutive transverse cross-sections. Samples were deparaffinized and subsequently stained with appropriate staining kits, then mounted  to analyze overall muscle morphology, to assess infiltration of inflammatory cells, and to quantify the centrally nucleated fibers (CNF), fibers minimal Feret’s diameter size, and finally to assess the level of fibrosis. All histological assessments were done on 12 ROI (region of interest) acquired from three non-serial cross sections from n = 3 mice (12 ROI/organ/mouse or 36 ROI/organ/group) using ImageJ NIH software. The digital images were acquired  and processed using ImageJ.mdx/scid mice were assessed and compared with the vehicle-injected controls and Wild Type (WT) mice. Inflammation was assessed by counting the total number of small (5–9 inflammatory cells) and large (groups of 10 or more inflammatory cells) foci in the representative images. The numbers of foci counts were normalized to the area unit (number of foci/mm2).H&E-stained sections of target organs of heart, diaphragm and gastrocnemius muscle of DEC injected For analysis of centrally nucleated fibers, H&E-stained sections of diaphragm and gastrocnemius muscle were assessed by counting the number of fibers with centrally located nuclei, which represent pathological changes in the muscle tissue. The number of CNF was compared to the total number of fibers and expressed as a percentage.Fiber diameters were assessed by minimal Feret’s diameter on H&E-stained sections of heart, diaphragm, and gastrocnemius using ImageJ measurement plug-ins. Average number of fibers falling into 5 µm increments was normalized to the total number of fibers and expressed as a percentage.For analysis of cardiac muscle fibrosis, cross sections of heart samples were stained with Picro-Sirius Red kit specific for cardiac muscle . Fibrosis of the diaphragm and gastrocnemius muscle was analyzed in the sections, which were stained with Trichrome Stain kit  for visualization of collagenous fibrotic tissue.Pixels corresponding to the area stained in red (for Picro-Sirius), or blue (for Trichrome) or indicating collagenous areas reflecting fibrosis, were normalized to the total pixel area of the tissue in the assessed image. Results were expressed as a percentage of collagenous area versus total tissue area in the region of interest.Echocardiography was performed as previously described , 41. BriWhole body plethysmography was applied for assessment of respiratory function as described elsewhere , 47. SmaAfter animal euthanasia at the study endpoint of 180 days post-DEC administration, the contractile and passive properties of the gastrocnemius muscle (GM) were measured ex vivo using Aurora Scientific test system as described previously . After dmdx/scid mice motor function after systemic-intraosseous administration of DEC cells was tested by the right hindlimb grip strength test. The function was monitored up to a 180-day endpoint. The tests were performed weekly, for 26 weeks of follow-up. The order of animals for test performance was randomly assigned. A grip meter  was used to measure the GM-specific force. This test allows forelimb force measurements, providing information on the muscle strength. The hook of a grip meter was placed touching the animal’s toes. Once gripped, it was repeatedly pulled 10 times, and the average maximum peak was used for further analysis.The assessment of p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Data are expressed as mean ± SEM (standard error of the mean). GraphPad Prism (ver. 9.2.1) software was used to perform statistical analysis. Two-tailed Student t-test for group comparisons was used to define statistical significance. Results were considered statistically significant for p < 0.05. The graphs represent mean values with standard error of mean (SEM), statistical significance is marked with asterisks: *mdx/scid mice. At the study baseline, the six to eight-weeks old mdx/scid mice were randomly assigned to two therapy groups of 1 × 106 and 5 × 106 dose of human DEC cells in the respective groups. Following intraosseous administration of DEC, animals were monitored up to the study endpoint of 180 days, when they were assessed for muscle pathology and by the ex vivo and in vivo functional tests.This study assessed the long-term (180 days) effect of DEC therapy after systemic-intraosseous administration to the All animals tolerated well the intraosseous administration of both dosages of human DEC therapy into the femoral bone. The injections were performed under surgical microscope magnification of 20X and the PBS-based DEC cell solution was injected without any spill or leakage. There was no presence of hematoma, inflammation, or infection observed at the surgical site after injection and no side effects were observed during the entire follow-up period up to 180 days.mdx mice [6 or 5 × 106). Immunofluorescence (IF) staining confirmed long-term (180 days) engraftment and increased dystrophin expression in the selected target organs of heart, diaphragm and gastrocnemius muscle (GM) of mdx/scid-injected mice. The IF images of heart sections of both DEC therapy groups  and 5 × 106 (7.52% ± 0.35%) DEC doses when compared to the vehicle-injected controls (2.32% ± 0.21%)  Fig. .Fig. 1Sy6 (5.34% ± 0.36%) and 5 × 106 (7.29% ± 0.52%) DEC cells when compared to the vehicle-injected controls (2.68% ± 0.36%). Moreover, a significant increase in dystrophin positive fibers was observed in 5 × 106 dose when compared to 1 × 106 DEC indicating a dose-dependent effect of DEC therapy on restoration of dystrophin in the diaphragm of mdx/scid mice  and 5 × 106 (8.67% ± 0.50%) DEC cells when compared to the vehicle-injected controls (2.57% ± 0.24%)  of human DEC cells confirms the long-term engraftment of DEC and human origin of the dystrophin positive muscle fibers  Fig. . In addiers Fig. .We have previously confirmed short-term reduction of the inflammatory response at 90 days after systemic DEC administration . Since i6 and 5 × 106) cells to the mdx/scid mouse. In the cardiac muscle sections  of human DEC. The number of small inflammatory foci was significantly decreased after administration of 5 × 106 of human DEC (21.05 ± 4.57 foci/mm2) compared to the vehicle-injected controls (44.80 ± 5.48 foci/mm2). There was a dose-dependent reduction of inflammatory foci observed in higher DEC dose of 5 × 106 (21.05 ± 4.57 foci/mm2) compared to 1 × 106 (35.25 ± 4.04 foci/mm2) dose. Interestingly, there were no large inflammatory foci observed in heart samples of mice injected with 5 × 106 DEC (0.00 ± 0.00) compared to 1 × 106 dose (4.25 ± 1.56 foci/mm2) and vehicle-injected controls (7.45 ± 2.96 foci/mm2)  and the 5 × 106 (21.64 ± 4.68 foci/mm2) cells when compared to the vehicle-injected controls (52.25 ± 6.20 foci/mm2)  and 5 × 106 (26.72 ± 1.48 foci/mm2) when compared to the vehicle-injected controls (140.60 ± 23.92 foci/mm2). Analysis of large foci counts revealed a dose-dependent reduction in the number of inflammatory foci after both DEC doses of 1 × 106 (15.00 ± 2.21 foci/mm2) and 5 × 106 (22.14 ± 1.08 foci/mm2) when compared to the vehicle-injected controls (50.02 ± 11.90 foci/mm2)  and 5 × 106 DEC (48.86 ± 1.52 foci/mm2) compared to the vehicle-injected controls (190.60 ± 31.92 foci/mm2)  compared to the vehicle injected controls (8.42 ± 2.01 foci/mm2)  compared to vehicle-injected controls (29.88 ± 4.19 foci/mm2)  in the H&E sections of heart, diaphragm and gastrocnemius muscle confirmed reduced inflammation at 180 days after intraosseous administration of human DEC  Fig. . Assessm2) Fig. 2 significmdx pathology in the diaphragm and gastrocnemius muscles at 180 days after systemic DEC administration. Histology specimens of diaphragm  and 5 × 106 DEC cells (18.25% ± 4.81%), when compared to vehicle-injected controls (44.90% ± 11.41%). Furthermore, reduction of CNF fibers count was dose-dependent revealing significant drop in 5 × 106 dose compared to 1 × 106 DEC dose  and 5 × 106 DEC (37.51% ± 9.91%), when compared to the vehicle-injected controls (71.16% ± 11.38%) , an indicator of agm Fig. A confirmose Fig. B. Moreovice Fig. C, where Presence of variations in the muscle fiber diameter size represents characteristic features of muscles affected by DMD. We reported short term normalization of fiber size after DEC therapy . Thus, imdx/scid mice injected with both DEC doses, ,15–20 µm (25.12% ± 3.80%) range, 20–25 µm (22.63% ± 1.88%) range and 25–30 µm (12.99% ± 1.35%) (11.73% ± 0.59% at 25–30 µm range)  and 1 × 106 DEC (12.04% ± 0.82%), when compared to the vehicle-injected controls (10.76% ± 1.70%). The presented data demonstrated a trend towards a dose-dependent response, where the higher DEC dose showed a stronger right-ward shift (towards increased mean fiber diameter) in the fiber size distribution over the lower DEC treatment dose  and 5 × 106 DEC cells (1.10% ± 0.12%), when compared to the vehicle-injected controls (2.26% ± 0.24%)  and 5 × 106 DEC cells (20.66% ± 1.16%) when compared to the vehicle-injected (30.43% ± 0.93%) mdx/scid mice.Assessment of fibrosis in the heart sections of ing Fig. A reveale%) Fig. 5. Assessm6 DEC cells  and 5 × 106 DEC cells (2.77% ± 0.21%) when compared to vehicle-injected mdx/scid mice (6.98% ± 0.48%)  on cardiac function monitored by echocardiography over the entire follow-up period from the baseline over and at day 30, day 90 and day 180 after systemic-intraosseous injection of DEC.Since progressive cardiomyopathy is responsible for premature death of DMD patients, evaluation of cardiac function is essential for assessment of disease severity and prognosis. We have reported the protective effect of DEC therapy on cardiac function in stration , 41. In mdx/scid mice  at day 90 and at day 180 (64.40% ± 3.81%) compared with vehicle-injected controls  , and at day 180 a significant EF drop was observed (day 180 – 52.86% ± 0.88%) in the treated mice  in mice injected with 1 × 10y) Fig. 6. In addi6 DEC dose over the entire 180 days follow-up, whereas administration of 5 × 106 DEC resulted in a continuous increase in the FS values observed from the baseline until day 90 . Additionally, at 90 days, mice injected with 5 × 106 DEC dose demonstrated increased FS compared to the vehicle injected controls (28.05% ± 1.70%). The 1 × 106 DEC dose revealed similar trends  compared with the vehicle-injected controls (26.57% ± 0.52%). In contrast, a gradual FS drop was only observed in the vehicle-injected controls over the entire follow-up period and at 180 days, revealed significant decrease when compared with baseline values  confirming progression of the DMD disease  confirmed maintenance of FS values at baseline levels for 1 × 106 DEC dose revealed maintenance of SV at the baseline levels  over the entire 180 days follow-up period. Administration of higher DEC dose (5 × 106 cells) resulted in continuous increase in the SV values from day 30 up to day 180   confirming the long-term dose-dependent protective effect of DEC therapy on cardiac function  for 1 × 10 ± 4.96) ig. 6F anon Fig. 6.6 DEC cells demonstrated maintenance of CO values at baseline levels over the entire 180 days period  and at day 180, was significantly increased compared to vehicle-injected controls (10.38 ± 1.07 ml/min) and 1 × 106 DEC dose (10.38 ± 2.15 ml/min) confirming the long-term, dose-dependent protective effect of DEC therapy on cardiac function  after administration of 1 × 10od Fig. 6. Adminison Fig. 6.6 and 5 × 106) of DEC cells.Since pulmonary fibrosis and respiratory failure is significantly contributing to the premature death of DMD patients, we evaluated pulmonary function via plethysmography at the baseline and at 180 days after systemic administration of two doses  in both DEC therapy groups: 1 × 106 dose (99.60% ± 7.22) and 5 × 106 DEC dose (93.39% ± 3.00), when compared to vehicle-injected controls (107.26% ± 1.65) for which an increase of Penh was observed confirming a natural disease progression in the untreated mice and decreased diaphragm fibrosis after DEC therapy  dose and was significant in 5 × 106 DEC dose (95.31 s ± 0.63) due to reduced diaphragm pathology leading to amelioration of passive exhalation after DEC therapy when compared to the vehicle-injected controls (113.33 s ± 3.96). A significantly lower Te value was observed in 5 × 106 DEC dose compared to 1 × 106 DEC dose confirming a dose-dependent protective effect of DEC therapy on pulmonary function  was reduced in DEC-injected mice in both: 1 × 10ion Fig. B.6 and 5 × 106) to the mdx/scid mice.Deterioration of the skeletal muscle function affects significantly daily activities and overall quality of life of DMD patients; thus, evaluation of muscle function is essential for assessment of disease progression. We have previously reported amelioration of gastrocnemius muscle function at 90 days after DEC therapy . Here, w6 DEC dose (0.23 g ± 0.02) and in 5 × 106 DEC dose (0.25 g ± 0.01), when compared to the vehicle-injected controls (0.18 g ± 0.01) confirming the long-term, dose dependent improvement of gastrocnemius muscle strength after systemic administration of DEC therapy.A significant amelioration of skeletal muscle function was observed for the critical parameters assessed by Aurora ex vivo muscle force test Fig. A, which mdx/scid mice by in vivo grip strength test which revealed improved performance after treatment with both doses of DEC cells when compared with the vehicle-injected controls and at weeks 5–10 and for 1 × 106 DEC dose revealed: . Additionally, mice injected with 1 × 106 DEC dose significantly improved grip strength of the right limb at 9-weeks (24.23 ± 3.44) when compared with the vehicle-injected controls (15.66% ± 3.94%)  Fig. 8.Duchenne Muscular Dystrophy is a severe, genetic disease, caused by the lack of functional dystrophin in the affected muscles. DMD is characterized by progressive course and premature death, usually due to cardiopulmonary complications , 49. Deswww.clinicaltrials.gov/ct2/show/NCT03375164) [Current approaches to treat DMD focus on dystrophin restoration by replacing and/or repairing the mutated dystrophin genes or by application of cell-based therapies , 2. Alth3375164) , 58. How3375164) , 58. The3375164) , however3375164) , 50, 59.In the light of the limitations of gene therapies, the stem cell-based therapies are considered as the most promising therapeutic method for DMD patients. This approach aims for the restoration of dystrophin by distribution of normal, dystrophin expressing cells to the DMD affected muscles. Initial studies confirmed safety of human myoblast and stem cells transplantation, however, a major drawback was the immune response resulting in low efficacy of engraftment and the need for immunosuppressive therapy, to prevent myoblasts rejection , 60, 61.mdx/scid mouse model, however, the level of dystrophin expression was low, and amelioration of dystrophic muscle function has not been proven in the in vivo studies [Only a few cell populations with myogenic properties and regenerative potential were investigated as a source for cell-based therapies in DMD and the satellite cells, muscle-derived stem cells and mesenchymal stem cells were found to be the most promising –66. Init studies .One of the most important factors determining efficacy of myoblast-based therapy is the route of cell administration and long-term engraftment. Previous studies testing systemic, therapeutic effect of mesoangioblasts, cardiomyocytes, MSC and iPS, investigated the intravenous, intramuscular, intracardiac and intraarterial routes of delivery with different efficacy results , 67–69.Interestingly, the intraarterial and intravenous delivery of cardiomyocytes representing the striated muscles and cardiosphere-derived cells confirmed improvement of cardiac function as well as elicited the off-cardiac effects confirmed by improvement of the upper limb function, thus supporting the rationale for systemic delivery of cells of muscle tissue origin , 70.We previously confirmed higher engraftment rates and better efficacy of chimeric cells following the systemic-intraosseous cells administration when compared with intravenous route of cell delivery , 71.Moreover, we reported better engraftment of our Dystrophin Expressing Chimeric (DEC) therapy, after systemic intraosseous administration, when compared to the local intramuscular DEC delivery –41 and iUntil now, problems of limited engraftment, low dystrophin expression, limited cell survival and the need for immunosuppression remain unsolved and limit the routine clinical application of cell-based therapies. Thus, we have developed more effective approaches including systemic-intraosseous route of cell delivery for enhancement and maintenance of cell engraftment .N/MBDMD). Intramuscular administration of human DEC tested in preclinical studies in the mdx and mdx/scid mice models confirmed high myogenic potential which correlated with increased dystrophin expression and improved function [Furthermore, to improve engraftment without the need for immunosuppressive therapy, we have created chimeric cells of hematopoietic, mesenchymal and myoblast stem cell lineages of murine and human origin , 39, 41.function . Thus, tmdx/scid mice which were observed up to 26 weeks after intraosseous DEC transplant. When considering the correlation of the age of tested animals and the length of the preclinical study with the future clinical trials in humans, it is estimated that based on this study the conversion of the age of mice to humans corresponds to the human age span from children to adults (7–20 years of age) and covers the age of DMD patients which will be considered in the future clinical trials [In the current study, considering the dynamic progression of DMD, our goal was to assess the long-term systemic effect of human DEC therapy for potential clinical applications. Moreover, considering the fact that clinical reports on assessment of current DMD therapies are tested in children of age 4–7-12 years old , 79 befol trials , 81. Thumdx/scid mice. The increase in dystrophin expression was accompanied by amelioration of muscle pathology revealed by reduced fibrosis and inflammation in the assessed target organs of the DEC-injected mice. Specifically, at the histological level assessment of heart samples confirmed significantly reduced fibrosis, improved midventricular myocardium morphology, less prominent necrosis and decreased number of inflammatory cell infiltrates. Furthermore, amelioration of inflammation and fibrosis as well as normalization of muscle fiber size was observed in the diaphragm, and gastrocnemius muscle further confirming the multiorgan effect of DEC therapy. Moreover, DEC cell migration and long-term engraftment to the heart, diaphragm and gastrocnemius muscle of the mdx host was confirmed by expression of spectrin—the human-specific membrane protein marker, further confirming human origin of the engrafted cells at 180 days after systemic intraosseous DEC administration.Since restoration of dystrophin is the major goal of all current therapies tested for DMD, in this report we have confirmed increased dystrophin expression in the heart, diaphragm and gastrocnemius muscle at 180 days after systemic administration of DEC to the mdx mice [These findings are of clinical importance, since reports on histopathological assessments reveled that the fibro-fatty replacement of cardiomyocytes is a significant pathophysiological mechanism in the development of cardiomyopathy in the mdx mice . Currentmdx mice .In the current study, we confirmed a complimentary, multi-level effect of DEC therapy which was observed at both, the tissue level and the functional level, as confirmed by improvement of cardiac, respiratory and skeletal muscle pathology and function maintained up to 180 days after systemic DEC administration. It should be emphasized that the reported here long-term protective effect was maintained after a single dose of DEC therapy. These observations are clinically relevant, since studies assessing cardiomyopathy linked to DMD in humans, revealed that functional decline in progressive cardiopulmonary is characterized by the thinned wall thickness of the left ventricular (LV) and a progressive decrease in the ejection fraction and fractional shortening . Thus, t6 cells, further confirming long-term protective effect of DEC therapy, since these parameters normally decline with age in the mdx mouse model [In contrast, the vehicle-injected controls showed gradual decline of the left ventricular (LV) function leading to the significant drop in the ejection fraction and fractional shortening at 180-days after DEC transplant. Furthermore, there was a dose-dependent increase of the values of LV functional parameters of the stroke volume and cardiac output between the baseline and 180-day endpoint observed after administration of the higher DEC dose of 5 × 10se model –84.6 and 1 × 106) did not show dose dependent effect on cardiac function.Interestingly, the dose-dependent increase of ejection fraction was observed also at 90 days after DEC administration, which highlights differences between the current study and our previous report , where tImportantly, the long-term improvement in cardiac function correlated with the improvement of respiratory function represented by the reduction of the enhanced pause (Penh) and time of expiration (Te) confirmed by plethysmography at 180-days after DEC transplant.Finally, the analysis of long-term effect of human DEC therapy on the functional outcomes of skeletal muscle revealed increase in the maximum stretch induced contraction of the gastrocnemius muscle at 180-day study endpoint further confirming long-term multi-organ protective effect of DEC therapy after systemic-intraosseous administration.There are several limitations of the study which should be addressed. We have based our study on the literature review of cardiac assessments by echocardiography , 85, 86 mdx mouse model that increased dystrophin expression correlated with significant improvement of skeletal and cardiac muscle function after intramuscular as well as intraosseous DEC administration, without evidence of side effects and without the need for immunosuppression [mdx/scid mouse model of DMD provides opportunity to test human DEC therapy created from normal and DMD affected donors according to the same manufacturing protocol which will be tested in the clinical trials. This fulfills regulatory requirements for the ex-vivo engineered DEC therapy as the novel, Advanced Therapy Medicinal Product (ATMP) [scid mice model in the preclinical studies validating new ATMP of human skin-derived ABCB5 -positive mesenchymal stromal cells for clinical application  [It should be noted that we have tested DEC therapy in the immunocompromised animal model since it allows for assessment of engraftment and efficacy of cells of human origin, but limits evaluation of the potential immune response. However, we have addressed this limitation in our previous work and have proven in the immunocompetent pression –41. Furt4971161) .mdx muscle pathology in the selected DMD-affected organs at 180 days after systemic DEC transplant.It is important to highlight, that functional improvements assessed in heart, diaphragm, and gastrocnemius muscle by standard functional tests of echocardiography, plethysmography and muscle strength tests, correlated with reduced muscle pathology parameters showing evidence of reduced interstitial fibrosis and inflammation, a trend towards normalization of the muscle fiber size, and decreased percentage of the centrally nucleated fibers in diaphragm and GM suggesting normalization of mdx mouse, the proven efficacy and the protective effect of DEC therapy over the long-term follow-up period of this study ranging between 6 and 26 weeks after DEC administration, represents valuable and clinically relevant findings, confirming both, the maintenance of the selected morphological and functional parameters at the baseline levels over the entire study follow-up, indicating the hold on the progression of the DMD disease, as well as the improvement of the important parameters of cardiac, respiratory and skeletal muscle function, confirming the long-term therapeutic effect of DEC therapy on the mdx pathology.It should be emphasized, that despite the natural progression of DMD disease in the aging In this study we have tested the long-term efficacy of DEC therapy by assessment of morphological, pathological and functional changes in the selected DMD-affected organs of heart, diaphragm and skeletal muscles. We confirmed that systemic-intraosseous administration of human DEC therapy restored dystrophin expression, which correlated with improved muscle morphology and pathology and long-term amelioration of function of the heart, respiratory and skeletal muscles as confirmed by standard echocardiography, plethysmography and muscle strength tests. DEC therapy does not require immunosuppression, is not limited to the specific gene mutation, does not cause sensitization and as such is universal for all DMD patients. These attributes make DEC therapy unique among other therapeutic approaches for DMD. Moreover, the long-term (180 days) protective effect of DEC on functional and morphological outcomes assessed in the most severely affected by DMD organs of heart, diaphragm and limb skeletal muscles, supports future clinical application of DEC as a safer and potentially more efficacious therapeutic approach applicable to all DMD patients regardless of gene mutation.To the best of our knowledge, this is the first report assessing the long-term, multi-organ improvement after systemic administration of human DEC therapy.This encouraging preclinical data introduces human DEC as a novel and universal therapeutic modality of the Advanced Therapy Medicinal Product (ATMP) with the potential to improve or halt progression of the disease and enhance quality of life of DMD patients.Suppl Fig. 1 (3.57 MB)High resolution image (TIF 836 KB)Below is the link to the electronic supplementary material.


Anaerobutyricum spp.  was found to be associated with improved insulin-sensitivity in subjects with the metabolic syndrome. After the preclinical testing with Anaerobutyricum soehngenii in mice models was completed, the strain was produced under controlled conditions and several clinical studies evaluating its safety and efficacy in humans were performed. Here, we describe and reflect on the development of A. soehngenii for clinical use, providing practical guidance for the development and testing of NGPs and reflecting on the current regulatory framework.The commensal gut microbiota is important for human health and well-being whereas deviations of the gut microbiota have been associated with a multitude of diseases. Restoration of a balanced and diverse microbiota by fecal microbiota transplantation (FMT) has emerged as a potential treatment strategy and promising tool to study causality of the microbiota in disease pathogenesis. However, FMT comes with logistical challenges and potential safety risks, such as the transfer of pathogenic microorganisms, undesired phenotypes or an increased risk of developing disease later in life. Therefore, a more controlled, personalized mixture of cultured beneficial microbes might prove a better alternative. Most of these beneficial microbes will be endogenous commensals to the host without a long history of safe and beneficial use and are therefore commonly referred to as next-generation probiotics (NGP) or live biotherapeutic products (LBP). Following a previous FMT study within our group, the commensal butyrate producer  Clostridioides difficile infections -producing bacteria  was obtained from collaborators in the UK  in 100 μl. Purity was assessed by 16S rRNA sequencing and microscopic evaluation of cellular morphology. Viability was assessed by most probable number (MPN) analysis and confirmed by microscopic analysis. Samples were directly stored at −80°C and used within 6 months of production, during which time viability was stable. In addition, some of these samples were tested for stability during 2 years to support the product development for the clinical trial.After escribed . In shorA. soehngenii on insulin sensitivity and lipid metabolism (A. soehngenii or placebo (10% glycerol) for up to 4 weeks, during which time no adverse events were observed . A significant improvement on insulin sensitivity was observed during the insulin tolerance test, which was strongest for the 108 CFU dose. This was accompanied by a decrease in hepatic fat and a reduced expression of the Fasn and Acc1 genes, both involved in lipogenesis.Next, we performed a dose-finding study in male diabetic (db/db) mice to test the safety and efficacy of orally administered tabolism . Mice weA. soehngenii, a second study with db/db mice was performed independently by the lab of prof. Bäckhed (Gothenburg)  . Mice weA. soehngenii CH106, a tetracycline-sensitive derivative from A. soehngenii type strain L2-7T, has been performed to show that the intake at the recommended dosages is safe  exceeding human recommended daily intake more than 100-fold  principles for Good Laboratory Practice (GLP). However, due to the need for innovative methods and models  which may not be validated nor at GLP level, this might prove difficult . However, safety and toxicity studies with NGPs are challenging. Since the product generally does not reach the systemic circulation, but its metabolites or its activity could directly or indirectly influence physiological functions in the body, efficacy and toxicity are not necessarily related to the dosage. In addition, other factors such as the human physiology and microbiota composition might influence the safety and efficacy. Furthermore, since most NGPs have coevolved with the human host, the holobiont concept, it is difficult to translate the results from animal studies to the human setting –70. Therifficult .A. soehngenii CH106 , A. soehngenii was regarded as a probiotic and had to comply with the Dutch “Warenwet”  of the culture was used to determine the fermentation time (between 14 and 18 h). After 16 h of fermentation in the large fermenter, A. soehngenii grew to an OD of approximately 10.Fermentation was performed in four sequential steps, which are depicted in Using hollow fiber membranes  and diafiltration with PBS, the cells were concentrated and washed. The fermentate was cooled to 10°C, pumped through the anaerobic membrane unit and concentrated to 40–50 L within 3 h. During the second phase diafiltration was performed to reduce the levels of medium components and fermentation products. Wash buffer was sterilized using ultra-high temperature, de-aerated and directly added to the returning cell flow into the fermenter. After 6 h, the cells were concentrated about 20-fold to 15 L and 99.8% of medium compounds were discarded to waste, leaving solely 2.9% of medium components in the final concentrate. Finally, 9 L of product could be harvested from the system into a sterile, N2-flushed container of 10 L.A. soehngenii in concentrations of 106, 108, and 1010 CFU/ml in PBS + 10% glycerol and one placebo batch with only 10% glycerol in PBS. For every batch 7 L bottles were prepared with glycerol and PBS for further dilution, which were autoclaved, cooled and flushed with N2. From the 9 L harvested concentrate, the necessary volume was added to these bottles to obtain the correct concentration. Bottles were placed on ice, under continuous stirring and N2 flush. The 10 mL tubes were first filled with N2, followed by 10 mL of product using a dosing-tube-pump. Tubes were immediately closed, labeled and placed in a freezer at −30°C within 10 min of filling. All filling was performed inside a disinfected laminar flow cabinet.Four different batches were produced for the clinical study, consisting of 600 tubes with 10 mL During the manufacturing, there was a continuous monitoring of temperature, pH and oxygen level. In addition, the cell count and OD were determined at every step during the process, as well as the absence of any contaminants. Since anaerobes are hard to enumerate quantitatively on agar plates, an MPN analysis was performed under anaerobic conditions to obtain the number of viable cells and cell morphology was assessed microscopically. All above quality controls were performed for the packaged vials, which complied with the standards for human consumption. A. soehngenii during a 3-year time period.Subsequently, the stability of the produced vials was tested every 6 months. After production, the vials were given a “best before” date of 6 months, which is required by law for food products in the Netherlands. This gave us the opportunity to extend the expiration date of the vials if the viability and purity criteria were met. Producing a strain at industrial scale sets different requirements for strains and culture media than laboratory scale culturing . TherefoFor medicinal products or LBPs, production according to Good Manufacturing Practices (GMP) is required . For fooA. soehngenii in obese, insulin-resistant subjects  A. soehngenii abundance was transient and mostly gone 2 weeks after cessation. The viability of the administered strain was negatively affected by stomach acid and oxygen. However, A. soehngenii was partially able to survive the gastrointestinal passage as indicated by the highest replication signal in the feces of subjects that received the highest dose. The viability (and therapeutic efficacy) could be further improved by protecting the strain better from the acidic and oxygenic environment through encapsulation and/or freeze-drying.Treatment with e events . When alA. soehngenii in humans, a randomized placebo-controlled crossover trial was performed in which the strain was directly administered in the duodenum, thereby circumventing the stomach acid and reducing the exposure to oxygen (A. soehngenii could further enhance the therapeutic effect (N = 12) were included and randomized to a single nasoduodenal infusion with the highest dose of A. soehngenii (1011 cells) or placebo (10% glycerol in PBS). After 6 h, a duodenal biopsy and mixed meal test was performed. In addition, subject monitored their 24-h glucose and collected several fecal samples. After a 4-week washout period subjects switched to the other treatment arm, which was determined long enough to lose the strain during the first trial.To further elucidate the mode of action of o oxygen . Since tc effect . Again, A. soehngenii was safe and well-tolerated. Treatment with the strain increased postprandial excursion of insulinotropic hormone glucagon-like peptide 1 (GLP-1), which was accompanied by a reduced glucose variability  and its target gene OSTa, which may also account for an increased GLP-1 availability  directions. Above we described our experience with the development of A. soehngenii as example. Identified as potential beneficial microbe after an FMT intervention, this microbe showed promising results in both preclinical in vitro and in vivo studies as well as in humans. Treatment with A. soehngenii was found to be safe and well tolerated. It showed promising effects on improving insulin sensitivity, increased GLP-1 secretion and reduced glucose variability. These effects are potentially mediated through the production of butyrate and secondary bile acids. By protecting the strain better from the acidic and oxygenic environment, e.g., through lyophilization and encapsulation, the viability and thereby therapeutic efficacy could potentially be increased. This NGP is currently being further developed as a food supplement.We illustrated the development of NGPs with the strict anaerobe KW wrote the first draft of the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted version.


In this paper, we explore a new approach in which understanding is interpreted as a set representation. We prove that understanding/representation, finding the appropriate coordination of data, is equivalent to finding the minimum of the representational entropy. For the control of the search for the correct representation, we propose a loss function as a combination of the representational entropy, type one and type two errors. Computational complexity estimates are presented for the process of understanding and using the representation found. Intelligence in general is the ability to respond quickly and adequately to challenges of the external world. Animals need to be able to recognise enemies and predators quickly. This can be considered as a one-sided classification, one class versus all the others. In  the authThe authors in  argue, fThere is a subtle philosophical difference between understanding and learning, although they may seem technically similar and are part of our cognitive work. Classic AI/ML tasks such as classification, clustering, encoding, etc. go hand in hand with understanding, “model building”. Before we are able build a proper model, we try to classify objects based on their characteristics, features, or try to cluster them. If we find a strikingly good arrangement, it can lead to a model idea. After some trials we find a model that fits the data well (after adjusting a few parameters) and meets our expectations of the model. We stress here that model building always involves some preliminary assumptions (which can be good or bad). Once you have a suitable model in hand, the task of classification or clustering is easy. As an example, let us consider a classification problem. If we have to classify a new item, we have to compare its features with those of the possible classes. If we have a model, a few relations between some features should be checked to identify the right class. This is the bird tweet phenomenon. Without a model, it is difficult  to find the solution, while the model plays the role of the bird and tells us the correct answer. Imagine that we have sample points from several polynomials of order one, two and three. If we are given a new set of sample points from an unknown polynomial, even if we know the three classes, we need to compare several class elements with the new one until we find a class element that is convincingly close, similar, to the new one. Contrary, if we know the laws of the three classes, a quick model fitting yields the answer. We will see that this quick solution comes at a price. While supervised or unsupervised learning algorithms have relatively low complexity, finding a model that well characterizes the classes, the relationship between the features, is a very difficult task with much higher complexity. The difference is typically exponential, or even hyper-exponential, depending on the task.Let us take a closer look at the learning and model building processes. We talk about training when we have a feature set of data and we want to fine-tune a combination of these features (pick out a few key ones) to optimise some behaviour . The task of understanding, on the other hand, is to find the best data model that reveals the relationships  between features and best describes a problem that is set prior the whole investigation. We call this the context of model building. Training always requires an explicit loss function that decides whether the trained system behaves correctly or not. This may be rather implicit, as in the case of auto-encoders or reinforcement learning. In the process of understanding, we consider only the input data set and try to figure out the feature functions that separate our data set from the rest of the world.More formally, in the task of understanding the representative elements of the system (sub-universe) we want to understand, are presented one by one to the model-building apparatus. It has separating and descriptive coordinates  or do we need further investigation, sample elements? To facilitate this, it was suggested in  to assocIn the present paper, we take a closer look at the practical understanding process. First, we recall the formal model of understanding. We then show that representation entropy is minimal if and only if the representation is canonical, i.e., a representation that separates the subset and describes the elements in terms of “independent” coordinates. We propose a loss function for representations and provide theoretical and practical calculation of the type one and type two errors of representations. Finally, an estimate of the cost/complexity of the understanding procedure is given.P is uniform over We assume that For simplicity, we use sets with cardinality Definition 1.Ω is a bijection A coordination of For continuous variables see the discussion in .Definition 2.A representation of Definition 3.Ω).A representation of Let us denote the descriptive coordinates by It is shown in  that theDefinition 4.The representation entropy is defined as follows: let For convenience, let Lemma 1.The representation has minimal representational entropy if and only if the coordinates are independent .Proof. At first, let us note that the entropy s  but noz is not independent. In that case, there are x and y are not independent andIf the family of random variables For convenience let us reorder x. Without loss of generality, we can assume that the first Let us assume that we have a given coordination The loss function is then defined asOne should take into consideration when choosing the meta-parameters The theoretical values of the error probabilities are cumbersome, but interesting on their own. Their detailed calculation is given in The representation cost can also be a factor in the design of the coordination. There are different costs.1.Creation of the coordinates that include the presentation of 2.Storage of the coordinate functions.3.Calculation of the representation entropy, type one and type two errors.4.Cost of decision if N inputs: . The cost of learning Let s: (c.f. )lxi=lN∼The storage and recall of the representation needs at leastThe representation entropy for a complete representation is fixed  that is minimal for the correct data representation. In this work, we have proven with mathematical rigour that the representation entropy defined in Definition 4 has exactly this property.Algorithm 1 Representation Entropy from bitwise probability distributions1:procedureS as follThis solution, however, is not feasible in practice, since we do not have complete information. Then we shall use the given pseudo-code, with two modifications. The first is that we can not sample the whole The other reason is that usually we do not know the complete Even with the best strategy, if we do not have complete information, the fixed values of the separation coordinates determine an


Health-related quality of life (HRQoL) is severely impaired in persons receiving dialysis. Malnutrition has been associated with some measures of poor HRQoL in cross-sectional analyses in dialysis populations, but no studies have assessed the impact of malnutrition and dietary intake on change in multiple measures of HRQoL over time. We investigated the most important determinants of poor HRQoL and the predictors of change in HRQoL over time using several measures of HRQoL. We enrolled 119 haemodialysis and thirty-one peritoneal dialysis patients in this prospective study. Nutritional assessments , anthropometry and 24-h dietary recalls) and HRQoL questionnaires (Short Form-36 (SF-36) mental (MCS) and physical component scores (PCS) and European QoL-5 Dimensions (EQ5D) health state (HSS) and visual analogue scores (VAS)) were performed at baseline, 6 and 12 months. Mean age was 64 (14) years. Malnutrition was present in 37 % of the population. At baseline, malnutrition assessed by SGA was the only factor independently (and negatively) associated with all four measures of HRQoL. No single factor was independently associated with decrease in all measures of HRQoL over 1 year. However, prevalence/development of malnutrition over 1 year was an independent predictor of 1-year decrease in EQ5D HSS, and 1-year decrease in fat intake independently predicted the 1-year decline in SF-36 MCS and PCS, and EQ5D VAS. These findings strengthen the importance of monitoring for malnutrition and providing nutritional advice to all persons on dialysis. Future studies are needed to evaluate the impact of nutritional interventions on HRQoL and other long-term outcomes. HRQoL is severely impaired in persons receiving dialysis compared with the general population, and decreased HRQoL has been associated with increased number of hospitalisations and poor survival in persons receiving haemodialysis (HD) and performing peritoneal dialysis (PD),4. Several factors have been identified as important determinants of poor HRQoL in persons on dialysis, including older age, female sex, unemployment, lack of educational qualifications, anaemia, presence of diabetes and other co-morbidities, lack of sleep, depression and poor nutritional status,5–7.Health-related quality of life (HRQoL) is one of the most important and widely used patient-centred outcome measures in renal research and clinical settings that provides information about an individual’s well-being with respect to physical, mental, social and somatic domains of health. Several terms for referring to malnutrition have been used in both the renal dietetic practice and research. However, in 2008, the International Society of Renal Nutrition and Metabolism suggested a single term, ‘Protein-energy wasting’, which has improved communication and clarified thinking across renal multidisciplinary care teams. For the purpose of this study, the term ‘malnutrition’ will be used as a synonymous with ‘Protein-energy wasting’. The pathogenesis of malnutrition is complex and results from the interaction of several factors such as loss of appetite causing poor nutritional intake, loss of protein and micronutrients during dialysis, increased inflammation and oxidative stress, presence of co-morbidities and decreased physical activity. Previous cross-sectional analyses have reported that HRQoL, as assessed by the thirty-six-Item Short Form Health Survey (SF-36), Kidney Disease Quality of Life Short Form or the European Quality of Life 5-Dimensions (EQ5D) questionnaire, was significantly lower in malnourished persons on dialysis compared with those who were well-nourished,6,11–15. However, none of these studies included a comprehensive assessment of dietary intake, and all used a single instrument to assess HRQoL. Hence, further evidence is needed regarding the impact of malnutrition and dietary intake on HRQoL.Malnutrition is a common and major complication, as well as an independent risk factor for increased mortality in the dialysis population,17, but factors that contribute to changes in HRQoL over time, in particular measures of nutritional status, have not been adequately investigated. We therefore sought to determine the most important determinants of poor HRQoL, as well as the predictors of change in HRQoL over time in persons receiving dialysis in a prospective study, with a particular focus on dietary intake and malnutrition.It has been previously reported that HRQoL declines over time in persons receiving dialysisOne hundred nineteen HD and thirty-one PD patients who were ≥18 years of age, had a dialysis vintage >3 months or were starting either HD or PD treatment, and were able to give written informed consent were enrolled in this 1-year single-centre prospective observational study conducted in the Department of Renal Medicine, Royal Derby Hospital. Recruitment was from September 2016 to August 2017. Persons receiving HD used high-flux polysulphone, polyarylethersulfone or polyvinylpyrrolidone dialyzers and were dialyzed at least three times per week for 3–4 h. Persons performing PD used lactate/bicarbonate-buffered 1·36 and 3·86 % glucose , 7·5 % icodextrin  and/or 1·1 % amino acid-containing solutions . The exclusion criteria were pregnancy or intending pregnancy, breast-feeding and hospitalisation at the time of recruitment. This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving patients were approved by the local Research Ethics Committee (East Midlands – Nottingham 1. REC reference: 16/EM/0243). Written informed consent was obtained from all patients.Baseline sociodemographic characteristics including chronological age, sex, ethnicity, educational level and employment status, as well as present co-morbidities, history of CVD, blood results and time since first dialysis treatment  were collected from direct interview and/or electronic medical records.At baseline, 6 and 12 months, we conducted the following detailed nutritional assessments:Twenty-four-hour dietary recalls were used for dietary intake assessment. From each participant, an experienced dietitian collected precise and comprehensive information regarding food and drink intake during a 24-h period. In persons receiving HD, 24-h dietary recalls included information from a dialysis day, a non-dialysis day and a weekend day, while in persons performing PD, dietary recalls obtained information from two weekdays and one weekend day. We used the software Dietplan 7 (Forestfield Software Limited) to calculate the average intake of energy content, protein and fat. Average energy and protein intake were then expressed in daily kcal and g, respectively, per kg of ideal body weight. were followed to measure post-dialysis weight, height, mid-arm circumference and triceps skinfold thickness. Weight and height were used to calculate BMI (reported in kg/m2). Mid-arm muscle circumference was calculated using the following equation: mid-arm muscle circumference (cm2) = mid-arm circumference – (3.14 × triceps skinfold thickness), where mid-arm circumference and triceps skinfold thickness were measured in cm.International standards for anthropometric assessment.We used the Takei 5401 handgrip digital dynamometer (Takei Scientific Instruments Co. Ltd) to measure handgrip strength (HGS) within the first hour of HD treatment or during PD clinic visits. HGS measurement was conducted in the non-fistula arm or the dominant arm if this did not have a fistula as previously described,21 for the assessment of nutritional status. The seven-point scale SGA is composed of six elements , which are scored between 1 and 7 in order to determine the overall SGA score. The lower the overall SGA score, the more severe the degree of malnutrition. For baseline analysis, participants were classified as being well-nourished (SGA scores 6–7) or malnourished (SGA score ≤ 5). For further analysis, participants who completed 12 months of follow-up were classified according to their nutritional status over 1 year into two groups: (a) ‘stayed or became well-nourished’ – participants who were well-nourished throughout the 1 year or became well-nourished at either 6 or 12 months ; (b) ‘stayed or became malnourished’ – participants who were malnourished throughout the 1 year or who became malnourished at either 6 or 12 months . As part of their routine clinical care, all malnourished patients received dietetic advice by their usual renal dietitian, which may have included the use of nutritional supplements; however, we did not assess the impact of specific nutritional supplements in our analyses.An experienced dietitian conducted the validated seven-point scale Subjective Global Assessment (SGA),22–24.HRQoL was assessed at baseline, 6 and 12 months using the SF-36 survey and the EQ5D questionnaire, which are validated and standardised instruments that have been widely used to assess HRQoL in the general and dialysis populations. Both the PCS and MCS were calculated according to well-defined guidelines–27. In brief, ten questions of the SF-36 survey were first recoded so that a higher score represented a better health state . Next, raw scores for each health state domain were calculated by summing across items in the same health state domain , and then raw scores were transformed to a 0–100 scale. Each of the eight SF-36 transformed scales was then standardised using a z-score transformation and the means and standard deviations from the general UK population. Then, the PCS and MCS were calculated by multiplying each scale z-score by their respective physical and mental factor score coefficients and summing the eight products. Finally, both the PCS and MCS were standardised to a T-score by multiplying by 10 and adding the resultant product to 50. A PCS or MCS score above or below 50 is therefore above or below the average for the general population.The SF-36 survey comprises thirty-six questions that assess eight health state domains: physical functioning, role physical, bodily pain, general health, vitality, social functioning, role emotional and mental health. These eight domains are then summarised into two scores: the physical component score (PCS) and the mental component score (MCS), and it ranges from −0·285  to 1 . The VAS uses a thermometer-like scale numbered from 0 to 100 to grade the current health status of individuals; the higher the VAS the better the health state.The EQ5D questionnaire consists of a health state score (HSS) and a visual analogue score (VAS). The HSS comprises five dimensions  with five available response levels . The HSS is calculated using specific coefficients for the five dimensions and response levels as described elsewheren 7 and HGS, n 6). Paired t test and Wilcoxon test were used for intragroup comparisons in the case of continuous variables. Student’s t test and Mann–Whitney U test were used for intergroup comparisons for continuous variables and χ2 test or Fisher’s exact test for categorical variables. To determine the significance and strength of associations between continuous variables, we used Pearson’s and Spearman’s correlation coefficients. Multivariable linear regression analyses were performed to identify the independent determinants associated with HRQoL at baseline. Adjusted R2, unstandardised (B) and standardised (Beta) coefficients were reported.All statistical analyses were conducted using the statistical software SPSS version 25.0 (IBM Corporation). Continuous variables are presented as mean (standard deviation) or median (interquartile range), while categorical variables are presented as percentages. Missing data were omitted , though these are in fact similar–31. For statistical analysis, participants were grouped into those with an increase in or stable HRQoL scores over time v. a decrease in HRQoL scores. Multivariable logistic regression analyses were conducted to identify the independent predictors of increased/stable HRQoL v. decreased HRQoL over 1 year. Nagelkerke R2 for the models and Hosmer and Lemeshow test P-value were reported.Change in HRQoL over 1 year was defined as a five-point change (increase or decrease) in the SF-36 MCS, SF-36 PCS and EQ5D VAS, and a 0·037 change (improvement or deterioration) in the EQ5D HSS. These thresholds represent the Minimally Important Difference defined as the smallest change in the HRQoL score of interest which a patient perceives as meaningful or beneficialP-value <0·05 was considered to have statistical significance.Independent variables included in the multivariable linear and logistic regression analyses were selected on the basis of significant associations in univariable analyses or biological plausibility . For all statistical analyses, a . However, for the purpose of this analysis, we conducted a retrospective sample size calculation with decrease in MCS, PCS, HSS and VAS as the primary outcomes. With a sample size of 117 participants split into two groups , the analysis would hypothetically have had 80 % power to detect OR of 3·45, 3·47, 3·57 and 3·51 for the decrease in MCS, PCS, HSS and VAS, respectively .Our original sample size determination was performed for an observational study with mortality as the primary outcome. EQ5D HSS  and VAS  were also lower than that reported for the general UK population , VAS 82·5 (16·9)),34.Baseline characteristics of 119 HD and thirty-one PD participants are summarised in 1).In multivariable linear regression analyses , nutritiv. 46·5 (12·9), 25·7 (12·5) v. 24·1 (13·5), 60 (50 to 77·5) v. 55 (40 to 75); P > 0·05 for all comparisons); however, median EQ5D HSS decreased significantly at 1 year in comparison with baseline (0·751 (0·539 to 0·879) v. 0·718 (0·390 to 0·877); P = 0·02).During follow-up, eighteen participants died, twelve received a kidney transplant, two withdrew their consent and one recovered kidney function sufficiently to discontinue dialysis. Thus, 117 participants completed 1 year of follow-up . There wn 27) were more likely to evidence a decrease in EQ5D HSS  at 12 months compared with those who stayed or became well-nourished during 1 year (n 90). Univariable analysis also showed that those participants who had a decrease in energy and fat intake over 1 year had a decrease in three of the four HRQoL measures at 12 months compared with those who had an increase in energy and fat intake over 1 year. Additionally, 1-year decrease in serum total protein and Hb was associated with the 1-year decline in PCS. Furthermore, participants with CHD evidenced a greater proportion with the 1-year decrease in MCS and EQ5D VAS, while lack of educational qualifications was associated with the 1-year decline in EQ5D VAS. No associations were observed with other potential risk factors, including chronological age, sex, employment status, presence of diabetes and dialysis modality (online Supplementary Table 2).Univariable analysis showed that participants who stayed or became malnourished during 1 year  independently predicted decreases in MCS, PCS and EQ5D VAS.. The relationship between malnutrition and decreased HRQoL in the dialysis population has been previously investigated only in cross-sectional analyses. Gunalay et al. observed that malnourished persons on HD and performing PD had significantly lower EQ5D scores (both HSS and VAS) compared with those who were well-nourished. A cross-sectional analysis of the Convective Transport Study reported that a higher SGA score was independently associated with higher SF-36 PCS and MCS, after adjusting for covariates. Several other studies have also reported that a low SGA score and/or a high Malnutrition Inflammation Score (a modified version of the SGA) correlate with lower SF-36 PCS and MCS,6,12,14. Our study adds to published data by showing that malnutrition at baseline was an independent determinant of decreased HRQoL across all domains and using two different measures (SF-36 and EQ5D), whereas previous studies have used only one measure or have reported associations with some but not all measures. Moreover, our analysis was adjusted for other important determinants of HRQoL including chronological age, sex, presence of diabetes, employment status, dialysis modality, dialysis vintage and HGS. Additionally, we have confirmed an association between the severity of malnutrition and HRQoL as shown by the strong and independent positive correlation between SGA score and all HRQoL scores.Malnutrition is one of the major and most frequent complications observed in persons receiving dialysis that is also often underrecognised and neglected. It is clinically important because it is associated with poor survival and decreased HRQoL,17; however, they did not explore the factors associated with this decrease, particularly those related to dietary intake and malnutrition. Additionally, previous prospective studies have observed an independent association between malnutrition and decreased PCS and MCS only at baseline,14 but did not assess the impact of malnutrition on change in HRQoL over time. We have now helped to fill this knowledge gap by showing that prevalence/development of malnutrition over 1 year was an independent predictor of the 1-year decrease in EQ5D HSS, and the 1-year decrease in fat intake  was independently associated with the 1-year decline of MCS, PCS and EQ5D VAS. Inadequate dietary intake is an important marker of malnutrition and is associated with poor outcomes. We observed that energy and protein intake were low at baseline compared with the recommended intake for persons receiving dialysis. Lower protein intake correlated with lower PCS and EQ5D HSS at baseline and a decrease in energy intake over 1 year also correlated with the 1-year decrease in MCS and both EQ5D scores in univariable analyses but change in energy and protein intake did not enter the final multivariable models. These observations reinforce the need to conduct comprehensive nutritional screening and monitoring to identify those persons on dialysis at nutritional risk or already malnourished and then implement appropriate nutritional interventions to prevent malnutrition or improve nutritional status. This approach would be expected to improve HRQoL and clinical outcomes, though prospective clinical trials are warranted to test this hypothesis.We observed that EQ5D HSS  decreased over 1 year in the whole cohort, though no change in mean MCS and PCS or median EQ5D VAS was observed. This may be in part because participants with decreasing HRQoL may have been more likely to die during the observation period. Previous prospective studies have reported that persons on dialysis experience a decline in the physical and mental components of HRQoL over time,24,36,37, we observed that diabetes, being on HD and unemployment status were independently associated with lower HRQoL scores at baseline. Also as reported in previous studies,39, older age was an independent determinant of better MCS, PCS and EQ5D HSS. One possible explanation may be that older people are more accepting of the limitations caused by illness and have lower expectations of HRQoL, but this requires further investigation. Similar to our findings, previous studies have confirmed that low educational level and presence of CVD are independently associated with lower HRQoL scores,41.Similar to other studies conducted in dialysis populationsR2 values in the multivariable analyses, suggesting the presence of residual confounding, and may have also resulted in a failure to detect associations between some variables and decrease in HRQoL scores. As this was a single centre study, our results cannot necessarily be extrapolated to other dialysis populations. Thus, larger multicentre studies are needed to confirm these findings. We did not use the Kidney Disease Quality of Life survey and therefore could not assess the impact of malnutrition on the kidney-specific QoL domains. However, the SF-36 questionnaire, which is included in the Kidney Disease Quality of Life survey as a generic chronic disease core component, is a widely used HRQoL instrument that has been validated in multicultural environments with large general population samples, as well as in persons receiving dialysis. We acknowledge the use of multiple comparisons in our statistical analyses, and thus the borderline ‘significant’ associations that we observed could be due to chance. We have not adjusted P-values (e.g. Bonferroni correction)–45 but have interpreted our results with caution in the light of multiple testing.Several limitations need to be considered when interpreting our results. Owing to the observational nature of this study, we cannot infer a causal relationship between malnutrition and HRQoL. Prospective clinical trials will be needed to investigate this further. The relatively small sample size prevented us from including more potential determinants of HRQoL in multivariable analyses. This may in part account for the relatively low adjusted In conclusion, these findings strengthen the importance of undertaking nutritional screening and monitoring in all persons on dialysis to identify malnutrition, and providing specialised, individualised nutritional advice in order to prevent malnutrition and/or improve nutritional status. Further prospective clinical trials with larger sample sizes and longer follow-up are needed to evaluate the impact of dietetic interventions on HRQoL and other clinical outcomes.


Video consultations are a rapidly growing service model, particularly in secondary care.Studies, mainly using trials and post-hoc surveys, have routinely documented that they canbe a safe and effective means to deliver care at a distance. While video offers newopportunities to provide health services, it also constrains how patients and clinicianscan interact, raising questions about feasibility, quality, and safety—questions thatcannot be adequately addressed with prevailing methods and approaches. To supportsuccessful and appropriate implementation, use and spread of video consultations, we needto investigate how video changes the interaction. In this article, we use two workedexamples to demonstrate how Linguistic Ethnography, a methodological approach combiningethnographic with linguistic analysis, enables a detailed understanding of howcommunication in video consultations works, providing an evidence base to support patientsand clinicians with using this service model. Video consulting  is a rapidly growing service modelin healthcare. The COVID-19 pandemic has brought requirements for social distancing toreduce the spread of a highly infectious disease that have forced clinicians to limitin-person consultations whenever possible. This has led to video consulting, previously aniche activity, having a bigger role in health care delivery ,especiaIntegrating video consultations in routine clinical practice is not easily achieved. Inaddition to organizational and infrastructure challenges , video caffordances  patients and professionals canuse it, however the technology changes the interactional dynamics. Video technology, likeany communication medium, has ordances : it provordances , that viordances .how they affect theconsultation, that is, how patients and professionals communicate over video and howparticipants do and do not successfully achieve their goals for a particular consultation linguistic analyses of recorded video consultations is critical forunderstanding video consulting, for informing appropriate communication strategies forparticipants, and for supporting appropriate spread of video consulting. We argue thatLinguistic Ethnography (LE) is one potential methodological framework for achieving thisgoal: it facilitates focus on the interplay between social life and the inner workings ofcommunication. This enables researchers to simultaneously gain a fine-grained appreciationof how participants accomplish technology-mediated interaction and how this shapes and isshaped by the wider context in which video consulting is rapidly evolving.We first discuss the current state of research on video consultations, including the use ofdeductive approaches to study interaction. We argue that such studies only provide a partialpicture of video consultations. We then set out how linguistic ethnography can enable anin-depth understanding of video consulting and providing two worked examples from our recentresearch. We close by discussing the practical challenges of using LE to study videoconsulting and setting out an agenda for future research.There is now a vast body of work investigating the feasibility, acceptability andpracticalities of video consultations in a range of clinical settings. Findings havelargely been positive, reporting that video consultations are (on the whole) safe,effective and acceptable to both patients and clinicians, serving as an adequatecomplement or even alternative to in-person consultations or other forms of telemedicine .These studies have provided valuable insights into the work involved in videoconsultations. However, the majority typically focus on testing the technology, usingquantitative methods such as clinical trials and surveys. In this section, we review thedominant methods used to date and what the data they produce tell us about videoconsulting.To date, the majority of research on video consultations has explored patient andclinician perceptions of feasibility and acceptability. Studies generally involvesmall-scale trials that investigate whether and under what conditions video consultationsare acceptable in a specific clinical setting. After conducting a video consultation,patients and clinicians complete a survey with closed questions and open-ended questions. In this way, they document their experiences and what they seeas the benefits and challenges of video. In some studies, researchers use semi-structuredinterviews to develop a richer understanding of participants’ perspectives e.g., .These studies have been valuable in demonstrating widespread feasibility andacceptability of video consultations across specialties—for example, in diabetes  or orthoSome studies have indicated the potential of video consulting to change the interactionbetween patients and clinicians and to enable a high quality consultation . To studHuman social actions are constructed through a complex combination of semiotic resources. To gainResearch on interaction and communication in video consulting does exist. Publishedstudies have typically used deductive coding systems  . We usedFirst, deductive coding systems only allow researchers to see data within the constraintsof that coding system. New or unexpected actions or structures of action have to be codedaccording to the existing system, limiting researchers to what they already (presume to)know about the working of social interaction. This is a salient issue for videoconsultations. Deductive coding systems were designed for analyzing in-personconsultations , but tecSecond, deductive coding systems do not adequately account for non-verbal interactionor the wIn sum, current evidence on video consulting draws largely on  feasibilitytrials and indirect methods  to understand the experiences andexpectations around video consulting; and on deductive coding systems to analyzeinteraction. These methods are limited. They provide little (if any) opportunity fordetailed understanding of the interactions, norms and routines that make up a videoconsultation.Linguistic Ethnography is a methodological approach that uses mainly qualitativeapproaches to enable intensive analysis of language and communication in ways that shedlight on small, but consequential, aspects of social practice . LE has This set of European circumstances prompted the foundation of Linguistic EthnographyForum in 2001, through which “a number of key scholars and lines of enquiry were pushedtogether by circumstance, open to the recognition of new affinities, and sufficientlyfamiliar with one another to treat differences with equanimity” , p. 585.LE researchers typically unite ethnographic methods  withclose inspection of interactional data. This enables understanding of both howinteractions work and the socio-cultural and organizational contexts in which they takeplace . InrelaThe first way in which LE can contribute is by demonstrating how the context in whicheach video consultation takes place affects the way patients and clinicians communicate ina video consultation. This is characteristic of LE, with ethnography used to “open up” theanalysis and linguistic analysis used to “tie it down” . EthnogrSecond, linguistic ethnography encourages investigation of the mechanics of socialinteraction , including video-mediatedinteraction . As suchThird, linguistic ethnography applies inductive and data-driven approaches focused oneveryday use and experience that can reveal aspects of video consultations and theircontext that researchers may not expect to find or think worthy of study. People structuresocial interaction in ways that are generally “seen but unnoticed” , meaningFourth, by grounding analysis in an understanding of how social interaction actuallyworks, instead of common-sense assumptions about how interaction supposedly works,linguistic ethnography can generate beneficial societal impact . In the In this section, we present two worked examples from our research on video consulting toillustrate the potential of linguistic ethnography. We draw on secondary analysis of videoconsultation data from two studies   and examines mmunity) . The latmmunity) . It takemmunity) . The driEthnography of Communication and Conversation Analysis, have similar ontological andepistemological roots: they were developed in parallel in the 1960s and 1970s asconstructivist perspectives to study language-in-use, with the aim of understanding howlanguage and the context of interaction matter for participants. In spite of the frictionthat exists in their approaches to context , combiniSecond, to develop useful guidance or policy it is critical to understand the broadersocial and cultural context: for example, procurement of different video technologyplatforms, booking systems for patient appointments, professional indemnity. Ethnographycan help us to assess the challenges and benefits of video consultations in regards tothese wider social issues, with CA aiding appreciation of how participants attend (or not)to these contextual features.Studies from which data were drawn received ethical approval from the National ResearchEthics Committee London-City Road and Hampstead in December 2014 (14/LO/1883) and by theSouth Central-Berkshire Research Ethics Committee in September 2015 (15/SC/0553). Allparticipating staff and patients provided informed consent for audio and video recordingand for data to be used for research.Video consultations may seem straightforward, with clinicians simply using videotechnology to call their patients in much the same way as they use the telephone. But aswe documented in previous ethnographic research , video cThe audio was not working properly again. SB and SR called me in to help out, butthere was little I could do to rectify it. The video quality was fine but there was noaudio – neither side could hear. I felt powerless to help, as their audio (microphoneand speaker) was on and full volume. The call was shut down. They decide to run Skype(for video) and SR’s mobile phone on loudspeaker (for audio) simultaneouslyThe use of video consultations—and interaction in video consultations—is shaped by thisevolving, and highly challenging, context. For example, in our research staff at twoclinical sites (part of the same NHS Trust), used Skype to conduct video consultations.While seemingly straightforward , staff faced repeated material, technological andoperational issues that were not easily overcome, and were frequently compounded by widerorganizational constraints . In our research this was identifiable, not only in our ethnographic data, but alsoour interactional data, with latency making it interactionally challenging forparticipants to maintain the conversational flow or turn-taking, that is, how theydetermine whose turn it is to talk  is unremarkable. Response pursuits such as the one producedby the patient occur routinely in in-person conversations. However, if we consider thenurse’s end of the call, a different perspective emerges. In excerpt (1b), we see the samestretch of talk, but notice now that there is less than 200ms silence between thepatient’s repeat “inhaling” in line 5 and the nurse’s response in line 7. In other words,the nurse responds “on time” . We alsoIt’s getting used to it, isn’t it. Because you’re saying something, and he’s sayingsomething, and then you think he’s finished, and he hasn’t finished. And of coursewhen you’re together, you talk like that, don’t you. Because you wanna move it eitheron a bit or you want to clarify a bit that they just said and stuff. But whereas ofcourse with it- when you’re not together, it’s harder, isn’t it. Cause you’re not surewho’s picking up what really.The combination of interactional and ethnographic data was key in enabling us, not onlyto see how problems with turn-taking arise and are resolved, but also how they areexplained and accounted for through the mechanics of social interaction. Interviewsfollowing the consultation, for instance, showed that participants do recognizeturn-taking problems (even if they do not articulate it as such) and that interactionalproblems matter for them. Take the following example from a community heart failure nurseexplaining her concern that latency in the video connection means she is not sure if thepatient is picking up what she’s saying (and vice versa).It is through such interview and observation data that we began to grasp how the problemsof turn-taking are noticed by participants.We know from our ethnographic data and other studies e.g.,  that parlonger silences when the patient stoppedtalking . Clinicians therefore found ithard to make visual assessments of the patient’s body, and had to rely on the patient’s orcarer’s assessment instead. This was neatly articulated by one community heart failurenurse, describing how she felt the remote physical examination went:As the above extract illustrates, interview data helped us to understand where clinicianssaw as a discrepancy between what the patient told them and what they could see. Byzooming in on the interaction, we then got an in-depth understanding of how these problemsemerge. Consider excerpt (2) . The patThis excerpt reveals a problem with video examinations where participants attempt toovercome the contextual restriction that clinicians cannot lay hands on the patients. Thepatient and carer need to perform the examination instead, and they need to show theclinician what they are doing—only the clinician has the training and expertise to make aclinical assessment. However, they cannot adequately monitor what the clinician can seeand instead have to rely on instructions and feedback from the clinician . In a video call, a small screen-in-screen shows people what their co-participant cansee. Once the carer aims the camera away from herself, she loses track of that screen. Shehas the tablet at a slight angle, forming a sort of triangle with herself and thepatient’s leg. In this way, she can still see what the camera is recording, but she aimsthe camera at the patient’s hip in #screengrab1 and above the patient’s leg in#screengrab2.The carer continues to struggle to show the patient’s leg to the nurse. The carereventually manages to show the patient’s leg after about a minute of maneuvering, but shedoes not manage to conduct the examination while simultaneously showing it to the nurse.As a result, the nurse has to rely on the carer’s “lay” assessment that the patient doesnot have edema.But it’s partly, because I’m holding it, and I can’t see what I’m looking at. Whereasif you put it down, and you see, oh well yeah that’s a way off, now I can, you know,so, you can see immediately.One could conclude after this consultation that these physical examinations for edema arenot feasible by video, even with the support of a carer and the flexibility of a tablet.And while the carer reported in the interview after the consultation that she was“fannying about” to get a picture of the patient’s leg, she did offer a solution:By conducting a data-driven analysis, we see how other patients and carers use differentstrategies to overcome this problem, and some of those are in fact successful.Consider the screengrabs in In sum, linguistic ethnography proved crucial in allowing us to (i) show how thevideo-mediated context changes how physical examinations can be done, and that despiteconcerns raised in interview and survey studies, in some cases and for some patients theycan be done; (ii) draw on foundational work on multimodality e.g.,  to make With video consulting becoming an important service model in response to the COVID-19pandemic it is crucial that clinicians and patients have appropriate evidence and support tohelp them use it. This requires a comprehensive understanding of video consulting.Traditional research approaches using trials, surveys and interviews have demonstratedfeasibility and acceptability. A wider range of research designs and methods is urgentlyneeded in order to access the social, organizational and clinical context in which videoconsultations take place, and the interactional routines through which clinicians andpatients accomplish them. This requires studies that examine the real-life complexities ofimplementing and using video consultation services. In this article we have (i) set out astrong argument for the value of Linguistic Ethnography as an overarching methodologicalapproach to studying video consulting that focuses on the context and structuralorganization of social interaction, both in and outside healthcare; (ii) used that frameworkto provide illustrative examples combining ethnographic methods with a fine-grained analysisof communication in video consultations; (iii) demonstrated the criticality of this approachfor generating understanding about how the use of video technology shapes the clinicalencounter and how and when it is feasible and appropriate; and (iv) shown how detailedobservation and rigorous analysis of video consultations, guided by linguistic ethnography,can inform practical guidance to support video consulting.There is a range of other methodological approaches to thinking about and analyzinginteraction, each with strengths and weaknesses. This includes, for instance, LinguisticAnthropology (LA), situated in North-American anthropology and focused on the culturalsettings and activities in which people use language . LA combStudies using linguistic ethnography and similar approaches are rarely straightforward.Conducting a qualitative analysis using observations, interviews and video-recordings ofconsultations is labor intense, provides a range of practical and ethical challenges andrequires specific methodological and analytical expertise from researchers. As with anyobservational study of healthcare interaction, the work is even more challenging during apandemic . We often need to rely on remote recording techniques. Such methods have practical and ethical implications, not least given theadditional burden on patients and clinicians. Even in post-pandemic times, visiting sitesmay not always be feasible. Close inspection of interaction requires researchers to makevideo recordings of the communication setting. As discussed by There is already a small but growing body of research using interactional and ethnographicapproaches to understand video mediated communication within and outside of healthcare. Given tFirst, research on how interaction in video consultation works must consider how it isshaped by the affordances of the technology—its opportunities for new ways of interacting aswell as its restrictions for achieving the goals of a video consultation . ParticiSecond, interaction research needs to pay greater attention to how communication in videoconsultation is contextually situated . The rapid spread of video consulting has led to use across health carespecialties , with thecondition and clinical focus shaping wide-ranging use cases. These range from one-offprimary care consultations between clinician and patient for a minor ailment  through to repeated consultations between hospital-based clinicians, patients andcarers regarding management of a long term condition , and use of groupvideo consultations with multiple patients and clinicians . Use of video also varies by setting with differences, for instance, acrossprimary and secondary care and in low- and middle-income countries compared to high incomecountries where technology, data and video-based services are more available. Finally, attention is needed on the immediate environment in which videoconsultations take place , and allied resources thatmay shape the interaction . Each context (and combination ofcontexts) will have its own social and technological benefits and challenges that shapeinteraction, the quality of the service, and the experiences of the participants. Approaches like linguistic ethnography that can connect the evolution oftechnological, infrastructural and social developments with the back and forth ofcommunication between clinicians and patients have a critical role to play in understandingthis dynamic.Third, research will also need to take account of the evolving nature of video consulting.The COVID-19 pandemic has led to rapid implementation of video consultations by healthcareproviders, most of whom had little to no previous experience with this service model. Asclinicians and patients gain experience, they will inevitably develop new routines and waysof working. These will be shaped by technological developments and wider infrastructure, aswell as societal constraints, such as the push by national policymakers for a “digitalfirst” or “remote-by-default” service model .2 ApprFinally, and relevant to all of the above, there is a need for mixed methods andinterdisciplinary approaches. It is unlikely that individual studies alone can capture therichness needed to study the unfolding interaction of video consulting in situ as well asthe social contexts shaping that interaction (and vice versa). This not only involvesnaturalistic observation of video consultations , but also direct observation of clinical routines and the settings inwhich consultations take place, as well as interviews with service users and policymakersthat capture post-hoc reflections. It might also involve, for instance, access to patientrecords or other artefacts or technologies  and thereadouts and data they provide. It may well be that individual studies focus on one or twoof these, with researchers then striving to integrate data and findings within and acrossstudies. Interdisciplinary working is essential, enabling expertise, exchange and synthesisacross, for instance, linguistics and language use, multimodality, ethnography,human-computer interaction and health services research.Successful (and sustainable) spread and scale up of video consulting depends on adequatesupport for patients and clinicians who use the service . Methodo