Intense antibiotic consumption in Low- and Middle-Income Countries (LMICs) is fueled by critical gaps in laboratory infrastructure and entrenched syndromic management of infectious syndromes. Few data inform the achievability and impact of antimicrobial stewardship interventions, particularly in Sub-Saharan Africa. Our goal was to demonstrate the feasibility of a pharmacist-led laboratory-supported intervention at Tikur Anbessa Specialized Hospital in Addis Ababa, Ethiopia, and report on antimicrobial use and clinical outcomes associated with the intervention.Methods: This was a single-center prospective quasi-experimental study conducted in two phases: (i) an intervention phase (November 2017 to August 2018), during which we implemented weekly audit and immediate  feedback sessions on antibiotic prescriptions of patients admitted in 2 pediatric and 2 adult medicine wards, and (ii) a post-intervention phase (September 2018 to January 2019) during which we audited antibiotic prescriptions but provided no feedback to the treating teams. The intervention was conducted by an AMS team consisting of 4 clinical pharmacists (one trained in AMS) and one ID specialist. Our primary outcome was antimicrobial utilization (measured as days of therapy (DOT) per 1,000 patient-days and duration of antibiotic treatment courses); secondary outcomes were length of hospital stay and in-hospital all-cause mortality. A multivariable logistic regression model was used to explore factors associated with all-cause in-hospital mortality.Results: We collected data on 1,109 individual patients (707 during the intervention and 402 in the post-intervention periods). Ceftriaxone, vancomycin, cefepime, meropenem, and metronidazole were the most commonly prescribed antibiotics; 96% of the recommendations made by the AMS team were accepted. The AMS team recommended to discontinue antibiotic therapy in 54% of cases during the intervention period. Once the intervention ceased, total antimicrobial use increased by 51.6% and mean duration of treatment by 4.1 days/patient. Mean LOS stay as well as crude mortality also increased significantly in the post-intervention phase . The difference in mortality remained significant after adjusting for potential confounders.Conclusions: A pharmacist-led AMS intervention focused on duration of antibiotic treatment was feasible and had good acceptability in our setting. Cessation of audit-feedback activities was associated with immediate and sustained increases in antibiotic consumption reflecting a rapid return to baseline (pre-intervention) prescribing practices, and worse clinical outcomes . Pharmacist-led audit-feedback activities can effectively reduce antimicrobial consumption and result in better-quality care, but require organizational leadership's commitment for sustainable benefits. The association between Antimicrobial Resistance (AMR) and antibiotic consumption has been well documented –3. The gWe recently implemented a laboratory bundle intervention aimed at improving bacteriology services in Ethiopia's largest teaching and referral hospital, which currently contributes a significant proportion of the national AMR surveillance data . In the Studies conducted in LMICs have shown that models of stewardship using non-specialized health care providers such as clinical pharmacists can lead to reductions in antimicrobial consumption –13. HoweWe assessed the feasibility and impact of a pharmacist-led intervention on antibiotic consumption and on clinical outcomes, in a tertiary care referral hospital in Ethiopia with a significant burden of Hospital-associated infections (HAI) and AMR. We used a quasi-experimental study design comparing outcomes during and after the intervention, to determine whether cessation of audit-feedback activities would lead to changes  in outcomes.The study was conducted between November 2017 and January 2019 in Tikur Anbessa Specialized Hospital (TASH), the largest referral and teaching hospital in Ethiopia and the site of an ongoing study on HAI funded by and in collaboration with the Research Institute of the McGill University Health Centre. The hospital has 800 operational beds, of which 191 are dedicated to pediatric services, and provides tertiary medical, surgical and obstetrical care to 20,000 inpatients and 330,000 outpatients per year. Average length of stay is 9.3 days, and in-patient mortality for the year preceding our study was reported as 5.8%. The hospital is staffed with 1059 physicians (residents-in-training and senior faculty) of which 4 had expertise in Infectious Diseases at the time of the study; 80 pharmacists and 7 microbiology laboratory technologists.The study was conducted on 2 general medicine wards  and 2 pediatric wards (66 beds).This was a prospective, quasi-experimental study assessing the impact of a weekly audit-feedback intervention on antibiotic consumption and clinical outcomes. The study population was patients hospitalized in the selected wards and receiving any systemic antibiotics on audit days. The study was approved by the Institutional Review Board (IRB) of the College of Health Sciences, Addis Ababa University (Protocol Number: 045/15/IM), and the Research Institute of the McGill University Health Centre.We developed institutional guidelines for the empiric management of the 4 most common indications for antibiotic therapy in the institution: Sepsis, Febrile Neutropenia, Community-Acquired and Hospital-Associated Pneumonia. Aggregate data collected from the microbiology laboratory over the preceding year were used to develop an institutional cumulative antibiogram. Treatment guidelines and cumulative antibiogram were uploaded into a commercially available customizable stewardship app . Prescribers and pharmacists were given free access to this tool on their mobile devices for a period of 3 years (longer than the duration of the study).duration of treatment for common syndromes. They were further introduced to the microbiology laboratory and given basic training on laboratory report interpretation. We also conducted 30-minute information sessions for clinicians to explain the rationale, benefits, and limitations of stewardship interventions, emphasizing that antimicrobial stewardship (AMS) recommendations were not medical orders and had to be reviewed and approved by treating teams. We further encouraged treating team members to participate in the weekly AMS sessions.We provided 4 half-day case-based interactive training sessions (over a period of 2 weeks) to the clinical pharmacists on basic concepts of antibiotic therapy, with a focus on acceptable The intervention was conducted between November 2017 and August 2018, for a period of 10 months (40 weeks). AMS team rounds were carried out weekly  during the 40 weeks of the intervention period.The AMS team was composed of 4 pharmacists (led by GBG), 3 Ethiopian physicians certified in Infectious Diseases alternating weekly, and one ID physician-in-training. Three Canadian physicians specialized in ID and clinical microbiology provided training of pharmacists, information sessions for physicians, and oversaw the development of the institutional cumulative antibiogram and treatment guidelines. They additionally supervised 4 audit-feedback sessions every 3 months, for a total of 12 sessions.unpublished data), we estimated that 20 audits/week would represent 25% of patients receiving antibiotics on these wards.All systemic antibiotics  prescribed and dispensed in hospital on the selected wards were included; antimicrobials used to treat parasitic, viral, or mycobacterial infections were excluded. We aimed to audit between 8 and 10 charts in medicine and in pediatrics each, for a total of 16–20 charts per week. Given previous point-prevalence estimates that 80% of patients hospitalized in medical and pediatric wards  are on antibiotics on any given day  collect results directly from the laboratory. During the intervention, if results of testing were not yet known to the treating team, pharmacists would go to the laboratory on audit days to check for results.Each case presentation was followed by a short team discussion, centered on findings of the diagnostic workup and on acceptable duration of therapy. Members of the treating team were invited to contribute to the discussion. After reaching a consensus, the AMS team issued a recommendation which was transmitted verbally to the treating team and also documented on a structured form attached to the patient chart . When coRecommendations were broadly categorized into four groups: (1) discontinue antibiotic, (2) change , (3) continue with specified duration, and (4) consult ID service. Pharmacists followed-up within 24 to 48 h to assess acceptance/non-acceptance of recommendations. Reasons for non-acceptance were documented.The post intervention phase was from September 2018 to January 2019. During this period, 2 pharmacists reviewed medical charts of patients receiving any of the targeted antibiotics, at a similar frequency as during the intervention phase, and using similar criteria as during the intervention (patients whose discharge was not anticipated for the next 3 days). They collected audit data but did not provide structured feedback to prescribers and AMS rounds were not held. Only data available on the medical chart was collected by the auditors; if microbiology laboratory results were not known, the auditors did not make specific efforts to collect results from the laboratory. Prescribers continued to have access to treatment guidelines and the cumulative antibiogram on their mobile devices.Data elements for intervention and post-intervention phases included demographic and clinical characteristics (comorbidities and main diagnosis at admission), antibiotic dosage and start dates, and documented indications for antibiotics. When these could not be ascertained from chart review or from discussion with treating teams, they were considered to be “empiric/source of infection undocumented.” During the intervention phase, AMS team recommendations and acceptance or non-acceptance of the recommendations were collected. For both intervention and post-intervention phases, antibiotic end dates, date of discharge from hospital or date of death if occurred in hospital were also collected. Data was entered into an electronic data capture instrument .The primary outcome of our study was antimicrobial utilization, expressed in treatment duration and days of therapy (DOT) per 1,000 patient-days. We defined duration of treatment as the number of consecutive days during which a patient received a specific antibiotic, and DOT as the aggregated sum of all the days during which a patient received any antibiotic. For patients on multiple antibiotics, we calculated the sum of DOTs for each antibiotic received (regardless of dose or route). We compared antibiotic treatment duration and DOT during and post-intervention for our study population.Our secondary outcomes were length of hospital stay (LOS) and all-cause in-hospital mortality during the study periods. These were also compared during and post-intervention.t-test for continuous variables. Data on antibiotic utilization were aggregated into months (10 months intervention and 5 months post-intervention); differences in mean duration of treatment and DOT per 1,000 patient-days during the 2 periods were compared using t-test.Patients baseline and hospital stay characteristics during and post-intervention periods were compared using Fisher's exact or χ2 tests for categorical variables, and We performed univariate and multivariable logistic regressions to assess the association of certain parameters on the pre-specified secondary outcome, in-hospital mortality. Adjustments were made for age, known comorbidities, febrile neutropenia, and suspected HAI. Further subgroup mortality analyses were performed for those with specific comorbidities .All tests were conducted at 2-sided 0.05 level of significance. All statistical analyses were performed on SPSS .We audited a total of 1264 prescriptions from 707 individual patients during the intervention phase, and 1,138 prescriptions from 402 patients in the post-intervention phase, for an average of 18 chart reviews per week over the duration of the study. The most commonly reported indications for antibiotics during both periods were infections of respiratory source . A greatOf the 1,264 prescriptions audited during the intervention phase, the AMS team recommended to discontinue antibiotic in 685 (54.2%). The most common justification (52% of cases) for this recommendation was sufficient treatment duration. The majority (96.4%) of recommendations were accepted by the treating teams .The most commonly prescribed antibiotics were third-generation cephalosporins (3GC) followed by vancomycin and cefepime during the intervention phase. Once the intervention ceased, meropenem became the third most commonly prescribed antibiotics after 3GC and vancomycin. Narrow-spectrum antibiotics particularly those targeting gram-positive organisms  were rarely prescribed . The AMSp = 0.002). The increase in duration was noted for all antibiotics with the exception of azithromycin and combination beta-lactamase inhibitor antibiotics, both of which were prescribed only in a small number of cases  during the 10 months of the intervention period, rose very sharply once the intervention ceased and reached a plateau .There was a 2-fold increase in DOT/1000 patient days once the intervention ceased, from a mean DOT of 754 ± 99.8/1000 patient-days in the intervention phase to 1549 ± 175.2/1000 patient-days during the following 5 months. Trends of DOT/1000 patient-days over time showed a similar pattern to durations of treatment: stable rates during the intervention, followed by a sharp increase and a new plateau .p < 0.001). Additionally, we note that all-cause in-hospital mortality increased from 6.9% during the intervention to 14.7% post-intervention (p < 0.01). Variables associated with in-hospital death for our study population are shown in Length of stay in hospital increased from 19.8 ± 12.0 days during the intervention period, to 24.1 ± 13.9 days once the intervention ceased  was well received and beneficial. We embedded audit-feedback activities within existing structures and involved members of the treating team (housestaff). We ensured treating teams maintained prescription autonomy. We provided modest structural and educational support through an academic collaboration with McGill University, leveraging external clinical microbiology expertise to build local capacity. The presence of one infectious diseases specialist per audit-feedback session to supervise and approve recommendations facilitated implementation and acceptability.The majority of study patients were receiving antibiotics for a suspected HAI. By focusing most of our feedback on duration of treatment, we were able to recommend discontinuing antibiotics in over 50% of cases. Since the wards we targeted were the main consumers of costly antibiotics, an intervention limited to only 25% of patients on just 2 wards resulted in substantial cost savings for the entire institution: antibiotic costs decreased by an amount equivalent to 35,000 USD (19% reduction compared to the year preceding the intervention)—equivalent to the total cost of laboratory reagents invested in support of the bacteriology laboratory .One of the strengths of our study is that we analyzed DOT rather than Daily Defined Dose (DDD); the latter would have been difficult to interpret in a population that includes children, and would have provided a biased view of antibiotic consumption in a setting where combination therapy is very popular (the average patient is prescribed 2 antibiotics simultaneously). Cessation of audit-feedback activities resulted in an immediate 2-fold increase in antibiotic utilization (duration of treatment and DOT/1000 patient days), and was most marked for meropenem (74% increase post intervention), vancomycin (61%), and cefepime (53%). This suggests that clinicians had acquired better knowledge of the burden of drug-resistance, through access to the cumulative institutional antibiogram which revealed widespread resistance of gram-negative organisms to 3rd generation cephalosporins. It is therefore not suprising they prescribed more “last resort” drugs. In the absence of immediate feedback, they would opt for prolonged treatment durations as the “safer” option.to receive intravenous antibiotics. It is therefore reasonable to suggest that cessation of audit-feedback activities had a direct impact on length of stay, highlighting the need for sustained efforts.Cessation of weekly feedback sessions was also associated with an increase in hospital length of stay compared to the intervention period  – a meaningful difference for the targeted wards, which at baseline have substantially longer lengths of stay than the institutional average of 9 days. Audit-feedback interventions in high-resource settings have reported an average reduction in length of stay of 1.12 days  analysis, a stronger design for causal inference in quasi-experimental studies (Assessing outcomes during 3 time periods, namely pre- during and post-intervention, would have provided a more optimal assessment of the intervention's impact over time. This was not possible in the context we were operating under, notably the risk that overly restrictive policies would be implemented before we could demonstrate feasibility and effectiveness of “ studies , 29. ITSFinally, this was a single center study conducted in a tertiary teaching hospital; hence our specific approach may not be applicable to smaller hospitals in LRS with even fewer infection specialists. In our experience the infection specialists played a key role in terms of providing credibility to the AMS team, interpreting available clinical data and assessing the safety of discontinuing antibiotics based on patients' clinical course. Nevertheless, the main lesson applicable to most LRS settings is that non-specialized health care providers can directly contribute to quality improvement in clinical care through specific antimicrobial stewardship activities.In conclusion, we demonstrate that an enabling intervention that is tailored to existing systems is feasible with modest external support and training. Audit-feedback activities, with attention paid to bacteriology results, lead to significant decreases in antibiotic consumption and cost savings, and resulted in better outcomes than standard medical care in our setting. These findings should catalyze the commitment of the organizational leadership to sustain enabling antimicrobial stewardship activities and from health authorities to scale up similar programs in LRS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by institutional review board, College of Health Sciences, Addis Ababa University. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.GG, MS, DH, and TG: conceptualization. MS and CY: funding acquisition. GG and AM: data collection. MS and WAb: project administration. GG and MS: data analysis and writing of first draft. DH, WAb, AM, WAm, TA, TB, ML, and CY: review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Haemophilia is a congenital bleeding disorder mainly affecting males. To prevent bleeding, patients need to perform regular intravenous injections (prophylaxis) throughout life. Non-adherence often occurs. Problems with acceptance or self-management appear to be the main reasons for non-adherence in haemophilia. The aim of this study was to test the feasibility and effects of two interventions focussed on acceptance (face-to-face) and self-management (online).N = 8 participants). The online intervention was based on a successful online programme in rheumatoid arthritis . Both interventions were designed according to the MRC framework in collaboration with the patient society and experts. We compared adherence , quality of life  and illness perception  before start (T0) and after 2 months (T2). Feasibility criteria were as follows: completion of training by > 50% of participants and ability to collect at least 80% of outcome parameters.Patients with severe haemophilia and acceptance or self-management problems were eligible. The face-to-face group intervention was based on Acceptance and Commitment Therapy (ACT) . One hundred percent of the outcome parameters was collected. Results were promising: although adherence (VERITAS-Pro) was stable (from 64 to 62 points), quality of life (SF-36) showed a clinically relevant improvement (> 5 points) in five of eight domains. Illness perception (BIPQ) showed a clinically relevant increase from 47 to 39 points. Patient evaluation was positive.The online intervention, however, was infeasible: enrolment was only 20% (6/30). Only three patients signed informed consent (recruitment 10%), and none completed more than one module (retention 0%). Consequently, the online intervention was terminated.The face-to-face acceptance intervention was considered feasible with promising results. Unfortunately, the online intervention was infeasible and therefore terminated. These findings suggest that adapting effective interventions to other settings does not guarantee success, despite the use of established methodology and patient participation. Population differences  could be an explanation for failure of the online intervention in haemophilia despite success in rheumatoid arthritis.NL55883.041.16 What uncertainties existed regarding the feasibility?Uncertainty regarding recruitment of patients struggling with illness acceptance and self-management. Uncertainty regarding the ability of patients to complete the programmes.What are the key feasibility findings?The face-to-face group intervention regarding illness acceptance was feasible and showed promising results on quality of life and illness acceptance with stable adherence. The online intervention was infeasible due to recruitment problems.What are the implications of the feasibility findings for the design of the main study?The face-to-face intervention will be evaluated in a larger group. The online training was removed from the study.The introduction of intravenous clotting factor replacement therapy has enabled the substitution of the missing clotting factor in haemophilia . This thP = < 0.01 and Pettersson score: 16 vs 5 points P = < 0.01) [Non-adherence to prophylaxis  . This jo < 0.01) , 11, whiIn a previous qualitative study, illness acceptance problems and lack of self-management skills were identified as important reasons for non-adherence . PatientTherefore, two tailored interventions were developed. The first intervention was focussed on improvement of illness acceptance using a haemophilia-adapted version of Acceptance and Commitment Therapy (ACT). The second intervention was focussed on improvement of self-management through an online programme including peer support. The aim of this study was to test the feasibility and effectiveness of both interventions (1) acceptance programme (face-to-face) and (2) self-management programme (online) in patients with severe haemophilia using prophylaxis.The study design of this feasibility study  is shownThe face-to-face group training ‘Living with haemophilia’ was focused on improving illness acceptance, which could lead to a higher adherence to prophylaxis. This face-to-face group training was based on the Acceptance and Commitment Therapy (ACT) approach . ACT is N = 5) peer trainers. The peer trainers received a formal peer trainers’ training from an external specialized agency. Each module took approximately 45 min; training had to be completed within 2 months. Details of the modules are shown in Table The online training ‘Challenging your haemophilia’ was aimed at improving self-management, which could lead to a higher adherence to prophylaxis. This online training was based on a successful comparable online training developed to improve self-management in rheumatic arthritis  which waFor both interventions, male adults with haemophilia who were prescribed prophylactic treatment were eligible. Ability to understand written language and speak Dutch was a prerequisite. Patients diagnosed with a serious psychiatric disorder interfering with the training were not eligible. For the online training, access to internet was a prerequisite. Based on the need to follow the conversations at the online platform and to perform an oral evaluation with each participant, the maximum number of participants per group was set at eight. At the beginning of each week, all patients visiting the clinic were discussed (multidisciplinary) and eligibility was considered. Patients who were eligible were informed about this study (and both interventions) by their health care provider or through various digital platforms . Patients received information about both interventions and could choose between both trainings based on their personal preference and their opinion regarding the reason for non-adherence: struggle with acceptance of haemophilia or with self-management. Informed consent was signed prior to the study.For both interventions, data was collected before start and directly after the intervention. The primary outcome of both interventions was adherence and secondary outcome was quality of life. Additionally, each intervention included an intervention-specific outcome measurement: participants in the face-to-face intervention completed an illness perception questionnaire and those in the online intervention completed a health education impact questionnaire.Adherence was measured by assessment of three domains  based on a pre-specified definition  and the We considered an intervention feasible if more than 50% of the patients completed the training and if more than 80% of the data for outcome parameters could be adequately collected. If not, the study team will consider early determination of adaptation.Additionally, according to the definitions proposed by Craig et al., feasibility was expressed by a comparison of the number of patients on prophylaxis having problems with acceptance or self-management (eligible) with the number of patients willing to participate and signed informed consent (enrolment and recruitment), number of patients that followed and completed the training (retention) and time spent on the training practicability . PatientN = 8 patients in each intervention.This study was performed at the Van Creveldkliniek of the University Medical Center, Utrecht, the Netherlands. This clinic was established in 1964 and has always provided multidisciplinary treatment including designated and specialized physicians, nurses, physiotherapists and a social worker. The Van Creveldkliniek treats 250 adults with severe haemophilia, of which ± 50% has adherence problems, resulting in ± 125 eligible patients overall. For this feasibility study, our aim was to include N = 8) completed all seven training sessions. Consequently, enrolment and recruitment was 88% (8/9), and retention was 100% (8/8).Figure On two occasions, a participant could not attend a session because of work or illness. In these cases, the continuity of the training was covered by seeing the participant individually before the start of the next session. The eventual participation retention for this intervention was 100%. All participants joined the free dinner at the start of each session. The trainers evaluated the training as practical and achievable, and all outcome parameters were collected. The trainer’s preparation time before every session took approximately 15–30 min. This was considered achievable in a daily healthcare setting.Patient and treatment characteristics are shown in Table p = 0.92). Quality of life (SF-36) showed clinically relevant improvements on five of the nine domains. Two mental domains showed a large improvement: role-emotional (83 points), role-physical (63 points) and social functioning (13 points) due to emotional problems and social functioning related to work, daily activities and social relations. But surprisingly, domains that are considered related to ‘physical’ health such as general health (10 points) and pain (5.5 points) improved too. Furthermore, illness perception (BIPQ) improved from baseline 47 points to 39 points (P = 0.46) indicating a clinically relevant improvement in illness acceptance, without reaching statistical significance in this small sample.Baseline and follow-up assessments are shown in Fig. N = 8) described that they were very satisfied and valued the training highly. All participants considered all sessions valuable, some preferred even more sessions. Participants felt more accepting towards haemophilia and felt less frustration regarding haemophilia-related limitations. They recognized themselves in the stories from others. After the training, most participants reported experiencing more joy in daily life activities and felt more comfortable to ask for help. They also mentioned that their partners experienced positive changes.All participants  had severe haemophilia A and had a median age of 24 years. Table These three participants N =  had seveParticipants only completed the baseline questionnaire. One completed only 10/40 questions of the HEIQ. Before starting, adherence  median score was 56 points indicating moderate adherence. Quality of life (SF-36) domain scores varied between 62 and 100 points indicating average quality of life. Self-management (HEIQ) varied between 3.0 and 3.8 indicating moderate self-management. No follow-up data are available because of early discontinuation.N = 3) were also contacted to evaluate the training. In retrospect, they suggested that the format was a bit too scholastic for adult participants. All peer trainers wondered if participants preferred face-to-face peer contact instead of an online training. He suggested that participants with haemophilia may not be aware about their own gap in knowledge, because they have been living with this disease their whole life. All trainers were disappointed, yet understood the early termination of the intervention.Two participants were still prepared to evaluate the training. Both were positive about the modules they completed. In their opinion, the format was easy to use, had a nice layout and design and included relevant information. Both were surprised about the lack of interest of other potential participants and the fact that the training was discontinued. One suggested to use this training for a younger population because of the bit scholastic format. The peer trainers (This study aimed to test the feasibility and primary effect of two interventions to improve adherence to prophylactic replacement therapy in haemophilia. The interventions consisted of a (1) face-to-face training focussed on acceptance and an (2) online programme focussed on self-management. The face-to-face training was evaluated as feasible; the preliminary results were promising as adherence, quality of life and illness perception all improved. Both participants and the trainers evaluated the training very positively. The online training was evaluated as not feasible because of difficulties with the enrolment, recruitment and retention. A possible explanation could be a lack of perceived need to improve self-management in persons with haemophilia or disease-specific aspects. This result was unexpected as compared to rheumatic arthritis, where the online intervention was very successful.Both interventions were based on the systematic and extensive research, based on the van Meijel model  and MRC Concerning the face-to-face group training, this is the first ACT-based training in haemophilia. However, there is extensive experience with ACT in other chronic conditions. A systematic review showed that ACT is effective in several chronic conditions  , 33, witWhy did the online training fail? Several potential reasons for failure were identified: age, gender and disease-specific differences. The online training was based on the comparable training for rheumatic arthritis (RA) , 32. TheThis study has clinical and research implications. In our opinion, the results of the present study provide sufficient reasons  for continuation of the face-to-face group training into a national pre-post-test study. To evaluate the long-term effects of this intervention, data collection will be extended to 12 months follow-up in the main study. If the face-to-face group training is effective, it could be applied in daily healthcare for both severe and non-severe haemophilia patients struggling with acceptance. For clinical practice, the positive feedback from both patients and their relatives concerning the face-to-face group training has inspired us to continue. We now believe that this kind of acceptance intervention could improve daily healthcare. This acceptance and commitment therapy training is not a country- and language-specific training , 4. In oThis paper described the feasibility of two tailored interventions concerning acceptance (face-to-face) and self-management (online) in people with severe haemophilia. Both interventions were adapted from already existing and effective interventions. The face-to-face training was evaluated as feasible with promising preliminary results . The online training was evaluated as not feasible because of recruitment problems, and therefore, it was stopped. The failure of the online training may be attributed to age, gender and disease-specific differences between the already effective intervention and this tailored intervention in patients with severe haemophilia.


Cortical neurodegeneration-induced non-fluent/agrammatic variant of primary progressive aphasia (nfvPPA) is a clinical syndrome characterized by non-fluent speech, such as apraxia of speech or agrammatism. We describe the case of an 80-year-old right-handed woman who exhibited nfvPPA. Atypically, our patient also presented with generalized auditory agnosia. Brain magnetic resonance imaging revealed left-sided predominant atrophy of the bilateral perisylvian area, including the inferior frontal and superior temporal lobes. In a series of auditory tasks assessing generalized auditory agnosia, our patient was unable to accurately identify verbal sounds, environmental sounds, or familiar Japanese songs that she could sing. In the context of recent studies, our study indicates the existence of a clinical syndrome characterized by progressive speech disorder with auditory agnosia. This case report thus provides novel insights into the spectrum of language impairment induced by neurodegenerative disease. Primary progressive aphasia (PPA) is a collective term for neurodegenerative diseases that present with language impairment as the most salient feature. Consensus criteria were proposed in 2011 for three clinical syndromic variants of PPA: non-fluent/agrammatic (nfvPPA), semantic, and logopenic , indicatN-Iso-propyl-p-[123I] iodoamphetamine single-photon emission computed tomography (SPECT) revealed predominant left-sided hypoperfusion of the bilateral frontal and temporal lobes  / . We used a Z score of 2 as the cutoff value in each voxel, and voxels with a Z score ≤ 2 were considered voxels without significantly decreased regional cerebral blood flow. Brain SPECT data analyzed with 3D SSP revealed relative hypoperfusion, mainly in the left superior temporal and inferior frontal gyri  with a standard pure tone threshold audiometry test . On the To examine the temporal resolution of the auditory system, click fusion, and counting tests were performed following the method used by Albert and Bear . In the We assessed our patient's ability to recognize non-verbal sounds. Twenty environmental audio recordings consisting of the following four sound categories were presented to both ears: human non-verbal , manmade inanimate , non-human animate , and natural inanimate  , 13. AftThe patient was asked to sing 20 familiar Japanese songs without accompaniment but with the provision of the song title and lyrics in writing; a correct response was noted when the patient's singing preserved most of the original melody. The singing score of four age-matched healthy controls from our hospital was 16.0 ± 1.6. Our patient provided correct responses for 17 out of 20 (85%) songs; her memory of the 20 songs thus seemed to have been intact. We then presented each of the songs, and the patient was asked to name the song's title or artist. While the mean naming score of the four healthy controls was 14.0 ± 1.4, our patient could identify the title and artist for only 1 out of the 20 (5%) songs. Finally, each of the 20 songs was presented to the patient, and she was asked to match the album cover, written song title, and artist name with the song in a four-alternative forced-choice paradigm. The controls were easily able to choose the correct answers and achieved a common score of 20.0. Our patient provided correct responses for 11 out of the 20 (55%) songs . AlthougHerein, we present a case of unclassifiable PPA: a combination of nfvPPA and generalized auditory agnosia. The patient's speech fluency was impaired due to AOS and agrammatism, the core features of nfvPPA. She did not exhibit any problem with object knowledge as indicated by the WAIS-III score, which further supported a diagnosis of nfvPPA. In addition, neuropsychological examination revealed that she did not exhibit any problems other than conversation. Hence, except for the generalized auditory agnosia, this patient met all the criteria for nfvPPA .The anterior components of the language network, including the inferior frontal lobe, and the anterior opercular and perisylvian areas, including the anterior insula and superior temporal gyrus, have been implicated as the neuroanatomical substrates of nfvPPA . The lesGeneralized auditory agnosia refers to a rare impairment in the ability to recognize sounds despite adequate hearing ability, as measured using standard audiometry , 16. On 18F-labeled 2-fluoro-2- deoxyglucose  and non-verbal auditory agnosia have been reported in the stroke literature  but are The present study has several limitations. First, although we believed that the auditory agnosia was in the context of language deficits , the preThe current study describes a rare case of unclassifiable PPA: a combination of nfvPPA and generalized auditory agnosia caused by neurodegenerative disease. In extensive auditory examinations assessing generalized auditory agnosia, our patient was unable to accurately identify both speech and non-speech sounds. Our results provide novel insights into the spectrum of language impairment induced by neurodegenerative disease.All data generated in this study are included in the article/supplementary material.Written informed consent was obtained from the patient and her family members for publication of this case report and any accompanying images.HW acquired case data, designed the study, and drafted the manuscript. MI and EM supervised the study and helped to draft the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


When comparing two extreme Kubios filters, these statistical differences could be clinically relevant, e.g. more than 10 ms in the standard deviation of all normal R-R intervals (SDNN). In conclusion, the results of the present study suggest that the application of different Kubios filters had a significant impact on HRV derived parameters obtained from short-term recordings in both time and frequency-domains.Heart rate variability (HRV) is a non-invasive indicator of autonomic nervous system function. HRV recordings show artefacts due to technical and/or biological issues. The Kubios software is one of the most used software to process HRV recordings, offering different levels of threshold-based artefact correction . The aim of the study was to analyze the impact of different Kubios filters on the quantification of HRV derived parameters from short-term recordings in three independent human cohorts. A total of 312 participants were included: 107 children with overweight/obesity , 132 young adults  and 73 middle-aged adults . HRV was assessed using a heart rate monitor during 10–15 min, and the Kubios software was used for HRV data processing using all the Kubios filters available . Repeated-measures analysis of variance indicated significant differences in HRV derived parameters in the time-domain (all  Heart rate variability (HRV) refers to the variation of the time interval between R peaks  registered in an electrocardiogram (ECG) . PreviouHRV recordings using heart rate monitors are affected by technical  and/or biological  artefacts. These artefacts could contaminate the HRV recordings, making it difficult to obtain HRV derived parameters from long-term (≈24 h) and especially short-term (≈5 min) recordings ,7,8. ArtDifferent commercially available software have been developed to process HRV raw data . The KubThe present study included baseline data from the ActiveBrains , the ACTServicio Andaluz de Salud  and the Human Research Ethics Committee of the Junta de Andalucia (0838-N-2017) for the FIT-AGEING study. All of them were performed in accordance with the Declaration of Helsinki (revision of 2013) and registered in a specific clinical trial database . Both, oral and written informed consent were obtained from all the participants and the children parents before their enrollment. Although the participants were enrolled in the aforementioned main studies, all of them agreed to transfer their scientific data for other scientific purposes or research studies.All studies were conducted according to the Committee for Research Involving Human Subjects at the University of Granada , Participants came in the morning to the research centre by car or by bus, avoiding any physical activity before the HRV assessment. Measurements took place between 8 AM and 12 PM for the ActiveBrains study, and between 8 and 9 AM for both, the ACTIBATE and the FIT-AGEING studies. Once in the research centre, participants lay on a bed or a stretcher in supine position equipped with a Polar RS800CX heart rate monitor  in a quiet room with dim lighting , and controlled ambient temperature and humidity (for the ACTIBATE and the FIT-AGEING studies). Heart rhythm was recorded during 10–15 min (sampling frequency of 1000 Hz). Before such a recording, participants were instructed to breathe normally, and not to talk, fidget, or sleep while measurements were being taken . Of noteFor the HRV data processing we used the Kubios software  ,12. The All Kubios filter levels . The LF/HF ratio was also calculated.In the present study, we considered the most-frequently used HRV derived parameters from short-term recordings in both time and frequency domains. In the time-domain, we computed (1) the standard deviation of all normal R-R intervals (SDNN) in milliseconds (ms), (2) the squared root of the mean of the sum of the squares of successive normal R–R interval differences (RMSSD) in ms, and (3) the number of pairs of adjacent normal R-R intervals differing by more than 50 ms in the entire recording (pNN50) expressed as percentage. Regarding the frequency-domain, we derived (1) the power in the high frequency (HF: 0.15–0.4 Hertz (Hz)), (2) the power in the low frequency (LF: 0.04–0.15 Hz), and (3) the power in the very low frequency (VLF: 0–0.04 Hz) using the fast Fourier transformation algorithm (FFT), all expressed in absolute units (ms2). Children were classified as children with overweight/obesity accordingly to the World Obesity Federation sex-and-age specific international BMI standards [We measured the participants’ height and body weight without shoes and with light clothing using a Seca scale  and a stadiometer. Body mass index (BMI) was calculated as body weight (kg) divided by square height . The significance level was set at 0.05.Repeated-measure analyses of variance (ANOVA) were used to test differences in HRV derived parameters in time- and frequency-domains across the different Kubios filters . Analyses were replicated with the non-parametric Friedman test and similar results were found, and therefore, only ANOVA results are reported. Bonferroni corrections were used for The descriptive characteristics of the participants are presented in p < 0.001). Significant differences were observed after post-hoc Bonferroni corrections in both children and young adults  than middle-aged adults. Thus, potential artefacts could be corrected or interpolated with Kubios filters of lower intensity in children and in young adults. The Very Strong filter should be used with caution, given that the interpolated R-R intervals were 39% (from 1% to 77.2%) in children, 33% (from 0 to 75.5%) in young adults, and 17% (from 0 to 60.8%) in middle-aged adults in the selected best 5 min period, which means that most of the selected period is “artificial”  in younger populations. Furthermore, after the visual inspection of the R-R signals in children, young adults and middle-aged adults we did not visually find any premature contractions based on the observer criteria. However, we do not have ECG tracings to verify whether “real” artefacts happened or not. Therefore, we cannot exclude that the high number of interpolated R-R intervals using the Very Strong filter could be related to either the Very Strong filter has some problem of excessive sensitivity or the heart rate monitor used for HRV assessment creates an excess of artefacts that are detected only by the Very Strong filter. Nevertheless, the former option is very likely since children and young adults usually present high sinus rhythm fluctuations resulting in big differences between consecutive R-R intervals which may be “overfiltered” by the Kubios filters. This is even more possible because the visual inspection before HRV analysis did not detect artefacts or premature contractions in the most of the R-R interval signals visually inspected.Several studies have reported the importance of artefact correction on the quantification of HRV derived parameters from short-term recordings ,28,29. HTo the best of our knowledge, there is only one study testing the impact of the application of different Kubios filters on the quantification of the HRV derived parameters obtained by short-term recordings . Aranda The clinical relevance of the observed differences on the HRV derived parameters should be considered. SDNN in resting conditions is considered a good indicator of cardiovascular and mortality risk . Indeed,The Kubios user’s guide recommends a percentage of interpolated R-R intervals lower than 5%. The software developers argue that this percentage should be enough to remove artefacts avoiding an excessive interpolation of R-R intervals. To our knowledge, this recommendation has not been tested in large and/or different cohorts. Moreover, they suggested that the Kubios filter should be individually selected to account for inter-individual variability in the HRV. Based on our results, we suggested the use a lower intensity Kubios filter  in younger populations, while a stronger Kubios filter could be used in older populations (with caution when using the Very Strong Kubios filter) to achieve the manufacturer’ recommendation of less than 5% of R-R intervals interpolated to remove HRV recordings artefacts see . We alsoX” on the HRV derived parameters. Thus, the Kubios software users might have a higher confident level in their results because are not “filter dependent” .The design of the current study precludes providing definitive thresholds and future studies are needed testing those recommendations. Moreover, we do not know whether Kubios filters remove “real” artefacts or large prolongations of R-R interval which may appear in healthy young individuals. Therefore, for Kubios software users, it is important to choose a reasonable filtering level , and hence, this study may have practical implications. Indeed, the kind of filter should correspond to the quality of the data, e.g., data collected during exercises or daily activity will need stronger filters than those recorded at rest. In addition, according to the results of our study, one should also consider the age of subjects before choosing the appropriate filter. Furthermore, we would recommend to the Kubios software users to apply different filters and to perform sensitivity analyses in order to study the effect of a treatment “We are aware of some limitations in our study: (1) the children cohort included in the present study was a sample of sedentary children with overweight/obesity, so we cannot extrapolate our findings to normal-weight children, (2) the experimental conditions during the HRV assessment were not exactly the same in the three different studies , (3) the HRV assessment was performed once per participant during a relatively short period (10–15 min), and (4) we did not employ ECG tracings to verify whether “real” artefacts happened or not, thus our results could be influenced by the fact that we have used a polar RS800CX. Therefore, future studies should be performed using other alternatives tools  to confirm whether the impact of the different Kubios filters remained independently of the tool employed for recording the heart rhythm. On the other hand, the strengths of our study were: (1) a large sample size; (2) the impact of different Kubios filters was tested in three different and independent human cohorts; (3) all R-R interval signals have been visually inspected to detect obvious artefacts; and, (4) the HRV derived parameters were obtained from short-term recordings, which are increasing their popularity in both clinical and research context due their relative ease in data collection and management.The results of the present study suggest that the application of different Kubios filters had a significant impact on the quantification of HRV derived parameters obtained from short-term recordings in both time and frequency-domains in children, young and middle-aged adults. Moreover, reporting the filter employed should be mandatory to allow comparisons across studies. Although the design of the current study precludes providing definitive thresholds, we suggested to use a Very Low, Low or Medium filter in children and young adults, whereas any Kubios filter (with caution when using the Very Strong Kubios filter) may be employed for middle-aged adults, when the processing of the short-terms HRV recordings is performed using the commercially available Kubios software. Further studies are guaranteed in order to elucidate which are the best Kubios filters to obtain valid data of HRV derived parameters.


Protein drugs own a large share in the market and hold great prospects for the treatment of many diseases. However, the available protein drugs are limited to the extracellular target, owing to the inefficient transduction and activity modulation of proteins targeting intracellular environment. In this study, we constructed ATP-charged platforms to overcome the above-mentioned barriers for cancer theranostics. The phenylboronic acid-modified polycations (PCD) were synthesized to assemble with enzymes and shield its activity in the blood circulation. When the PCD nanoclusters reached tumor site, they effectively transported the enzymes into the cells, followed by recovering its catalytic activity after being charged with ATP. Importantly, the cascaded enzyme systems (GOx&HRPA) selectively induced starvation therapy as well as photoacoustic imaging of tumor. Our results revealed that the intelligent nanoclusters were broadly applicable for protein transduction and enzyme activity modulation, which could accelerate the clinical translation of protein drugs toward intracellular target.  •ATP-charged nanoclusters capable of enzyme activity modulation•Universal platform for intracellular protein delivery•Selective cellular cascaded catalysis for cancer theranostics•Photoacoustic (PA) imaging and starvation therapy of breast cancer Medical Imaging; Catalysis; Cancer; Nanostructure Proteins drugs have been widely developed for the diagnosis and treatment of various diseases  owing to their high bioactivity and target selectivity . HoweverIn recent decades, covalent modification or assembly with carriers has been applied to improve the intracellular delivery of proteins . UnfortuIntelligent dynamic assemblies have attracted great attention because they can achieve temporal and spatial controlled activation of therapeutic payloads by predefined program . Here, w2O2, which acts as the substrate of HRPA to obtain active ABT·+ for cascaded amplifying photoacoustic (PA) imaging for diagnosis. The ATP-charged nanoclusters are believed to significantly improve the cytosolic transduction of proteins, especially for the enzyme activity modulation and tumor selective catalysis, and hence promote the diagnosis and therapeutic efficacy of cancers. Moreover, the Shield-Transport-Recover (defined as STR) intelligent cluster is a universal platform that can not only deliver the current enzymes but also be adapted to other enzyme systems.To achieve these goals, a polycationic polymer mPEG-b-poly(2-[(2-aminoethyl)amino]ethylaspatamide) (pDET) was synthesized, and modified with phenylboronic acid (PBA) , to assemble with enzymes such as GOx and ABTS )-loaded HRP (horseradish peroxidase) (named as HRPA) by electrostatic interaction, and to form enzyme nanoclusters. The assembled PCD/enzyme nanoclusters have a relative low enzyme activity (low-power state) to avoid unspecific catalysis in blood circulation. Besides, the formation of relatively large-sized nanoparticles could extend the blood circulation time and enhance the tumor accumulation of enzymes. When the nanoclusters reached tumor site, the assembly with carriers could promote the intracellular transduction of enzymes. Once in the cell, the diols on the pentose ring of ATP were able to form dynamic chemical bonds with PBA for accelerating the charge and hydrophobic property reversal of PCD, resulting in disassembly of the nanoclusters and release of the toxic enzymes   . The ABTThus, the above-mentioned data demonstrated that the ATP-charged nanoclusters capable of activity modulation were generally applicable for most proteins with different MWs or PIs.2O2 and series concentrations of ATP was measured by LAZR PA Imaging System. In accordance with the UV-vis absorbance result, the PA signal of PCD/HRPA increased gradually with increasing amount of ATP added, whereas the PA signal of non-responsive pDET/HRPA did not change with the increasing ATP  for amplifying the PA imaging signal  and MCF-7 cells (human breast cancer cells). As displayed in Subsequently, to further evaluate whether the PCD nanoclusters were broadly applicable for most of the proteins and could achieve enhanced tumor cell killing we took two representative bioactive proteins of RNase A and GOx for uptake and cell growth inhibition study. The uptake of PCD/RNase A-FITC shown in 2O2 and glucuronic acid for starvation and oxidation therapy. The improved cell transduction of GOx by PCD was validated in 2O2 in the medium environment even if it was not internalized by the cells, leading to the slight cell toxicity. Excitingly, the assembly of PCD nanoclusters dramatically amplifies the cell killing effect of GOx on 4T1 cell lines. The IC50 of PCD/GOx was pretty low, about 0.15 μg/mL. Furthermore, the tumor-specific catalysis of GOx-loaded nanoclusters was also validated by detecting the toxicity on L02 cells  technology. The BSA was labeled with FITC as the donor, and the PCD was labeled with RhB as the acceptor. First, the generation of FRET signal was checked by scanning the fluorescence spectrum of PCD-RhB/BSA-FITC. As shown in In addition, the release behavior of PCD/BSA-FITC nanoclusters was also evaluated on normal cells (L02 cells). Without IAA treatment, there was still clear red fluorescence signal of the clusters, indicating the inefficient release of loaded enzymes in normal cells . These r2O2 (3 (HRP inhibitor), the red fluorescence intensity dramatically decreased, validating that the generation of red fluorescence was attributed to the HRP catalysis. Moreover, the fluorescence intensity of IAA-treated cells also weakened, demonstrating the idea of ATP-dependent enzyme activation. Furthermore, to further validate the role of ATP-triggered enzyme release in activity recovery, ATP-insensitive pDET/enzyme clusters and ATP-sensitive PCD/enzyme clusters were both incubated in 4T1 cells with different enzyme concentrations to obtain the same uptake level as analyzed by flow cytometry  staining. As shown in Stability of the nanoclusters was very important for successful enzyme delivery, including in the presence of serum, under different temperature or in the tumor acidic microenvironment. Here, PCD/GOx was chosen as model cluster for evaluation, where the GOx activity was indicated by detecting the intracellular Hin vivo biodistribution and tumor targeting of the PCD/GOx nanoclusters were evaluated by labeling GOx with NIR fluorescence probe Cy5.5 to make it visible under in vivo imaging system. As depicted in in vivo clearance behavior of the free GOx-Cy5.5 and PCD/Cy5.5, mice were injected with the enzyme solutions and the fluorescence intensity in the blood sample was monitored. As shown in 1/2) was only 45 min. Expectantly, the nanoclusters dramatically extended the t1/2 of GOx-Cy5.5 to around 4 h. Generally, the size of the nanoparticles was one of the key factors that affected the in vivo circulation and biodistribution. Hence, we concluded that the PCD/GOx nanoclusters significantly extended the blood circulation time of GOx and increased the tumor accumulation of the enzymes.The in vitro experiment had demonstrated that ATP was able to activate the catalysis of HRPA or GOx&HRPA and the generation of PA signal. Thus the in vivo ATP-lighted tumor PA imaging ability of the nanoclusters was further investigated. The imaging effect of free GOx&HRPA, PCD/HRPA, and the cascade enzyme nanoclusters PCD/GOx&HRPA were compared here. For PA imaging, the background of the mice was first obtained, shown as weak PA signal at tumor site of 0 h , which may need further explorations.PCD with ATP-responsive behavior was successfully applied to modulate the protein activity and enable the intracellular delivery of protein drugs. However, the types of the protein-loading materials should not be ignored. In this study, we only chose the specific linear polypeptide (pDET) as the model material for protein delivery research. The shapes , dimensions, or molecular weights of the polymers may also have influence on the protein loading and activity modulation. Besides, it is not clear whether this strategy is applicable for inorganic materials (silicon, FeIn summary, we demonstrated an ATP-charged nanocluster for controlled intracellular catalysis and cascaded amplification of PA imaging-guided cancer therapy via modulation of the enzyme activity. The PCD nanoclusters shielded the enzyme activity in the blood circulation to reduce the side effects while selectively activating it in the tumoral ATP-abundant environment to obtain highly efficient intracellular catalysis. In addition, the size-switchable strategy among small free enzymes and the relatively larger PCD/enzyme nanoclusters significantly increased the blood circulation time and tumor accumulation. Notably, the preparation of nanoclusters dramatically augmented the PA imaging signal as well as the breast cancer therapeutic effect after loading with HRPA and GOx as the cascaded enzymes. Meanwhile, the PCD/GOx nanoclusters were biocompatible with the mice compared with the toxic free GOx. Thus, the ATP-charged nanoclusters gave an example of bio-responsive enzyme catalysis modulator and could inspire the fabrication of site-specific treatment strategy based on physiological signals. It could also promote the clinical development of protein drugs toward intracellular target by enhancing the therapeutic efficiency and reducing the side effects.All methods can be found in the accompanying


Osteoarthritis (OA) is a significant cause of pain in both humans and horses with a high socio-economic impact. The horse is recognized as a pertinent model for human OA. In both species, regenerative therapy with allogeneic mesenchymal stem cells (MSCs) appears to be a promising treatment but, to date, no in vivo studies have attempted to compare the effects of different cell sources on the same individuals. The objective of this study is to evaluate the ability of a single blinded intra-articular injection of allogeneic bone-marrow (BM) derived MSCs and umbilical cord blood (UCB) derived MSC to limit the development of OA-associated pathological changes compared to placebo in a post-traumatic OA model applied to all four fetlock joints of eight horses. The effect of the tissue source (BM vs. UCB) is also assessed on the same individuals. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analysis of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints until Week 12. A significant reduction in the progression of OA-associated changes measured with imaging techniques, especially radiography, was observed after injection of bone-marrow derived mesenchymal stem cells (BM-MSCs) compared to contralateral placebo injections. These results indicate that allogeneic BM-MSCs are a promising treatment for OA in horses and reinforce the importance of continuing research to validate these results and find innovative strategies that will optimize the therapeutic potential of these cells. However, they should be considered with caution given the low number of units per group. Degenerative joint disease also known as osteoarthritis (OA) is the most common joint disease found in both humans and horses with high socio-economic repercussions ,2,3. ThiWhile some joint tissues such as bone have spontaneous healing properties, others such as articular cartilage do not: this highly specialized connective tissue is non-innervated and avascular and therefore has a very low repair capacity. As a result, traumatic and degenerative damage to articular cartilage inevitably progresses to OA and no treatment can currently stop this progression. In this context, cartilage regenerative medicine strategies based on intra-articular injection of stem cells represent a promising therapeutic tool thanks to their anti-inflammatory and pro-regenerative properties. Among the available sources of stem cells, adult mesenchymal stem cells (MSCs) are the most studied: they are easy to isolate from a multitude of tissues  and are not subjected to the ethical concerns of embryonic stem cells . AllogenTo date, few randomized controlled blinded equine studies have evaluated the effectiveness of intra-articular MSCs in the treatment of OA. Only one, published in 2009, has evaluated the effects of MSCs injected alone in carpal joints compared to a placebo in an OA model. This study compared two different cell products, namely autologous adipose-derived stromal vascular fractions and autologous BM-MSCs ,22,23. TThe objective of this study is to evaluate the ability of a single intra-articular injection of allogeneic MSCs of controlled quality and viability, derived from two different tissue sources, namely BM-MSCs and UCB-MSCs, to limit the development of OA-associated pathological changes compared to placebo, in a post-traumatic osteoarthritis model applied to all four fetlock joints of eight horses. The hypothesis is that clinical, imaging, biochemical, macroscopic, and histologic OA-associated pathological changes develop less in MSC treated fetlocks than in placebo-treated fetlocks over a 12-week period. The secondary goal of this study is to compare treatment effects of BM-MSCs and UCB-MSCs with the hypothesis that BM-MSCs have a better ability to decrease the progression of OA than UCB-MSCs, according to a previous study indicating a better chondrogenic potential for BM-MSCs . After the surgical procedure , no signp = 0.006) (p = 0.049) in the development of osteophytes on radiographs between BM-MSC treated fetlocks and placebo treated fetlocks  with less severe imaging signs of OA developing in the BM-MSC treated fetlocks than in placebo treated fetlocks, when considering all four imaging parameters polled together (= 0.006) . When cofetlocks . Indeed,fetlocks .No significant differences in the evolution of synovial fluid total protein concentration and total nucleated cell counts were observed between treatments. Similarly, no significant treatment effects were seen on the concentration of synovial fluid biomarkers of inflammation and cartilage turnover .p = 0.07)  . There w = 0.07) .The current study used a model of OA that was proven to successfully induce mild osteoarthritis-associated changes in the four fetlock joints of each horse . OA-assoThese results suggest that intra-articular injection of a 10 million dose of allogeneic BM-MSCs into fetlock joints, 3 weeks after surgical induction of OA, has a beneficial effect on limiting the progression of imaging signs of OA. To our knowledge, this is the first time that a difference in the monitored imaging parameters has been demonstrated between placebo and treated joints during a short-term study (12 weeks) in a large animal model of OA. The results of the present study must however be examined with caution, considering the low number of units per group, especially for the group which received the UCB-MSCs, but also considering the absence of correction for multiple testing. In addition, only short-term effects (9 weeks) of these therapeutic MSCs were assessed. A lower number of treated joints per group had already shown significant results in a previous study evaluating the efficacy of blood derived chondrogenically induced stem cells combined to allogeneic equine plasma, for which there was also no correction for multiple testing or repeated measures . In thisThe first published randomized blinded controlled experimental equine study evaluating effects of MSCs in the treatment of OA  had failOur study has the advantage of using quality-controlled cells, but other factors related to the injection should be considered in order to improve the efficacy of the MSC-based therapies evaluated in this study. First, the dosage of injected cells might be lowered. Indeed, significant effects were obtained in the study of Broeckx et al. (2019)  when usiBoth MSC sources consisting of BM-MSCs and UCB-MSCs injected in the present study had already been evaluated in a previous safety study . In thatEight clinically sound French Standardbred horses owned by the Center of Imaging and Research on the Equine Locomotor Affections  were included in this blinded, controlled, randomized study. There were 4 geldings and 4 mares, median age was 3 years (ranging from 3 to 4), and median weight was 440.5 kg (ranging from 402 to 483 kg). None of the horses had a history of pregnancy, had received a blood transfusion before recruitment, or had kin relationships with MSCs donors. All horses had been trained for racing before being retired because of failure to trot at racing speeds. All were treadmill conditioned before the beginning of the study. The eight horses were divided into two groups. The first was composed of five horses whose fetlocks were assigned to one of three treatments groups: (1) allogeneic BM-MSCs, (2) allogeneic UCB-MSCs or (3) placebo control. The second was composed of three horses whose fetlocks were assigned to only two treatments groups: (1) BM-MSCs or (2) placebo control, in order to obtain more data on the cell source expected to have more favorable results according to the literature ,24, and All horses received a single intra-articular treatment in all 4 fetlock joints, i.e., in both metacarpo-phalangeal and both metatarso-phalangeal joints, 3 weeks after surgical induction of OA lesions (W3) . An indeAll procedures described in this study were approved by the ComEth Anses/ENVA/UPEC Ethical Committee . Surgeries were performed under general anesthesia and all efforts were made to minimize suffering and stress.One week before surgical induction of the lesions (W-1), each horse was evaluated clinically for lameness and underwent full radiographic and ultrasonographic examination of its four fetlock joints to rule out the presence of pre-existing OA before being included in the study . InductiAll MSC lines used in the present study were cryopreserved MSCs that had been previously isolated, cultured and characterized for the needs of a previously published safety study . BrieflyTwo lines of BM-MSCs and 2 lines of UCB-MSCs from the safety study  were ranAll horses were treated on W3 . PlaceboThe protocol used was the same as the one previously described for the characterization of OA-associated changes in placebo treated fetlocks . To contClinical evaluations consisted in fetlock joints circumference measurements, sensitivity to static digital flexion tests (grade 0–4), and joint effusion (grade 0–4) . Degree Images from radiographic and ultrasonographic evaluation of the fetlock joints were evaluated for osteophyte formation on the dorsal, lateral and medial aspects of the joint and scored based on osteophyte size  as previously reported ,63. The 2 (PGE2) and C-terminal of type II collagen (CTX-II) as markers of inflammation and cartilage turnover respectively, were estimated by use of commercially available high-sensitivity enzyme immunoassay kits  EIA, Immunodiagnostic Systems Holdings PLC, Boldon Business Park, UK) [Synovial fluid samples were assessed for total protein concentration using a refractometer and for total nucleated cell counts using an automatic analysis system  (cells/µL). The remaining fluid was stored at −80 °C for ELISA analysis. Synovial fluid concentration of Prostaglandin Eark, UK) ,66.Horses were subjected to euthanasia on W12  with an Osteochondral sections were harvested from the medial part of the distal aspect of the metacarpal/tarsal condyle and processed as previously described  for hist2 concentration were normally distributed. When the outcome was not normally distributed, the non-parametric Wilcoxon signed-rank test was performed while paired Student’s t-test were used when the outcome was normally distributed. In addition, in order to compare treatment effects between the 3 treatment groups, values resulting from the difference between W12 and W-1 were analyzed using a one-way repeated measure analysis of variance (ANOVA) with the horse as a random effect for normally distributed outcomes, or a Kruskal-Wallis test for the others. The variables were finally presented descriptively, stratified by study date and treatment group. The mean and standard deviation of normally distributed outcomes and the median and quartiles of the others were calculated at each time point for each treatment groups. All statistical analyses were performed using R software  or Excel 2016 for Windows . A p-value of 0.05 was considered to be significant.Development of OA-associated changes was compared between BM-MSC and placebo treated fetlocks pairs, and between UCB-MSC and placebo treated fetlock pairs, respectively. An overall evaluation of changes from the pre-osteoarthritis state to the post-osteoarthritis state (surgery combined to exercise) was made by comparing values resulting from the difference between W12 and W-1 values. An exception was made for the post-mortem scores that were analyzed on W12 only. For the imaging parameters, the 4 outcomes were analyzed separately but also as a group where all imaging scores were polled together by treatment category and analyzed as an additional outcome. Data was visually assessed for normality, and two different tests depending on the distribution of the variables were used. Differences in fetlock joint circumference, total nucleated cell counts and PGEIn the absence of available data on the efficacy of the studied cell-based therapies, an exact sample size could not be determined. Nevertheless, based on previous studies using MSCs  a MSC trTo conclude, in an inflammatory environment, intra-articular injection of a 10 million dose of allogeneic BM-MSCs into fetlock joints of experimental horses, 3 weeks after surgical induction of OA, significantly reduces the progression of imaging signs of OA compared to contralateral placebo treated fetlocks, in particular regarding the formation of osteophytes on radiographs. No significant differences in the development of OA-associated pathological changes were observed between UCB-MSC treated fetlocks and their contralateral placebo treated joints. Therefore, allogeneic BM-MSCs could potentially be a promising treatment of OA in horses. These results are however to be tempered given the small number of horses and the lack of correction for multiple testing. In addition, the experimental design of this study does not rule out a potential systemic effect of the MSCs on placebo treated joints. As a step toward improvement of the efficacy of stem-cell based therapies in the treatment of OA in the future, either used alone or combined to biomaterials as vehicles, or even through injection of MSC extra-cellular vesicles, this study highlights the need to pursue investigation on the best modalities of administration and on strategies to potentiate their therapeutic effects.


Caenorhabditis elegans larvae can undergo developmental arrest upon entry into the dauer stage in response to suboptimal growth conditions. Dauer larvae can exit this stage in replete conditions with no reproductive consequence. During this diapause stage, the metabolic regulator AMP-activated protein kinase (AMPK) ensures that the germ line becomes quiescent to maintain germ cell integrity. Animals that lack all AMPK signalling undergo germline hyperplasia upon entering dauer, while those that recover from this stage become sterile. Neuronal AMPK expression in otherwise AMPK-deficient animals is sufficient for germline quiescence and germ cell integrity and its effects are likely mediated through an endogenous small RNA pathway. Upon impairing small RNA biosynthesis, the post-dauer fertility is restored in AMPK mutants. These data suggest that AMPK may function in neurons to relay a message through small RNAs to the germ cells to alter their quiescence in the dauer stage, thus challenging the permeability of the Weismann barrier. Caenorhabditis elegans (C. elegans), reduced insulin-like signalling induces physiological and morphological changes that can ultimately result in lifespan extension, reproductive delay, and developmental quiescence [In iescence . The devC. elegans orthologues of AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) cooperate to maintain cell cycle quiescence in the germline stem cell population [aak(0)) develop normally; they exhibit normal somatic and germline development similar to wild-type animals and grow into fertile adults. However, if these mutant animals are stressed, they show multiple phenotypes, many of which become apparent during the dauer stage [In a genetic screen conducted to identify factors that are required to maintain germline stem cell quiescence, we found that the pulation . The lospulation . Quite ser stage ,3,4.aak(0) mutants do not die immediately as dauer larvae, they are unable to adjust to the energy stress of this diapause stage and this has dramatic effects following recovery. While wild-type animals recover from dauer with little phenotypic consequence, a significant number of AMPK mutants die during dauer exit. Those that survive exhibit highly penetrant sterility and a range of vulval defects [Although the  defects . The vulC. elegans, AMPK is composed of two catalytic α subunits, aak-1 and aak-2; two β regulatory subunits, aakb-1 and aakb-2; and five γ regulatory subunits called aakg-1, aakg-2, aakg-3, aakg-4, and aakg-5 [C. elegans and human α subunits share 30% identity and 78% similarity between protein residues [C. elegans [C. elegans and humans, and mutants that completely lack AMPK signalling are viable, our laboratory uses genetic analysis in C. elegans to identify novel physiological targets of AMPK by exploring its role in regulating developmental plasticity in response to environmental cues.In residues . The β sresidues . As withC. elegans hermaphrodite, the larval gonadal primordium consists of four cells: Z1 and Z4 are progenitor cells that will undergo several rounds of mitotic divisions to give rise to all the cells of the somatic gonad. The Z2 and Z3 progenitor cells are referred to as the primordial germ cells and will generate the germline stem cells. These cells will continue to divide throughout the life of the animal to populate the adult bi-lobed gonad with germ cells that will first produce sperm (~300) and then continue to produce oocytes thereafter [In the ereafter . Under rereafter .During optimal conditions, these cell divisions continue throughout larval development uninterrupted. However, they arrest upon entry into the developmentally-arrested dauer diapause that is specialized for long-term survival and dispersal . This stDuring the dauer stage, the animal is sealed off from its external environment by a highly resistant cuticle, occluding the buccal cavity and making it impossible to feed. Energy is consequently rationed, and metabolism is altered to favour catabolic reactions. AMPK is presumably activated by the reduced food intake typical of the diapause. In response, AMPK activates a number of catabolic enzymes and inhibits anabolic enzymes, such as protein translation and cell proliferation, by inactivating the target of rapamycin (TOR) pathway to reduce energy expenditure .C. elegans, but germ cell mutations are problematic as they can be amplified in the germ line and can be inherited throughout subsequent generations.In addition, several cellular processes, such as cell division, and other developmental events are arrested. Germline proliferation is slowed followed by the establishment of a G2 phase cell cycle arrest ,13,14. MC. elegans. On the other hand, one of the key effectors of the insulin signalling pathway, the phosphatase and tensin homolog (PTEN) orthologue in C. elegans (daf-18), can work with AMPK in some contexts, but in the dauer germ line, it appears to function in parallel to AMPK to suppress the germ cell divisions in the dauer stage [Both PAR-4 and AMPK are critical to maintain germline quiescence by halting the cell cycle in the germline stem cell population upon entering the dauer stage ,4, and ter stage ,4.aak(0) mutants, the germ cells enter pachytene but fail to progress to diplotene, where the paired homologous chromosomes normally become apparent [aak(0) mutants fail to exit pachytene and do not undergo diakinesis to produce mature functional gametes. These data indicate that the loss of AMPK during the dauer stage results in a post-dauer meiotic defect that could account for at least some portion of the observed sterility.In AMPK mutant dauer larvae, the integrity of the germ cells is compromised and therefore, they do not produce functional sperm or oocytes, rendering the post-dauer mutants sterile . The morapparent . There wapparent . These faak(0) dauer larvae is not sufficient to correct the highly penetrant sterility in the post-dauer aak(0) adult [glp)-1, the resulting reduction of germ cell divisions in the AMPK mutant dauer larvae had little to no effect on the frequency of sterility in the post-dauer animals [Interestingly, reducing the germline hyperplasia in the 0) adult . By comp animals ,15. ThesC. elegans larvae that transit through the dauer stage exhibit distinct gene expression profiles that correlate with changes in chromatin marks compared to animals that undergo reproductive development [elopment ,17. Theselopment ,17. In telopment ,17.C. elegans, where AMPK signalling also plays a role in modulating the chromatin landscape during periods of stress, namely during the L1 diapause and the dauer arrest [In mammalian cells, AMPK has been shown to regulate gene expression by directly interacting with chromatin and phosphorylating histone H2B in promoter and transcribed regions . Therefor arrest .During the dauer stage, AMPK activity is required to ensure that transcriptional activity is modified in accordance with this state of stress in the germ cells, thereby maintaining a state of quiescence during periods when energy must be conserved . In the aak(0) adults is unknown, but the chromatin marks persist into the post-dauer aak(0) adults, where their levels are globally increased compared to the control post-dauer animals [Moreover, the distribution of the chromatin marks in each individual germ cell nucleus was dramatically different in animals that lacked AMPK signalling . Whether animals . AMPK isppk-2, pmk-1, spe-26, pro-2, mek-2) are significantly altered in the dauer and post-dauer animals compared to those that never transited through the dauer stage [The aberrant deposition of the chromatin marks that occurs in the germ cells in the absence of AMPK manifests as changes in gene expression in the dauer larvae and in post-dauer adults . The traer stage ,17. Furter stage . The misC. elegans, endo-siRNAs impinge upon specific regions of the chromatin to control which genes will be expressed and which genes will be silenced [C. elegans, these small RNAs likely respond in an AMPK-dependent manner to adjust the small RNA repertoire in order to remodel the germ line chromatin landscape and couple gene expression to the energetic needs typical of the dauer stage. Not surprisingly, compromise of various components involved in microRNA, germline/nuclear RNAi, or small RNA biogenesis pathways also has consequences on diverse aspects of dauer development, and potentially on the sterility of post-dauer aak(0) adults, although not all of them are necessarily associated with AMPK function [Small RNAs control many aspects of gene expression within eukaryotic cells. In silenced ,21,22. Isilenced ,24. In Cfunction ,17.aak(0) adults and the germline hyperplasia in the aak(0) dauer larvae [aak(0) mutants [The compromise of the RNase III-like dicer (DCR-1), its accessory factor RNAi-defective (RDE)-4, and the primary Argonaute protein endogenous-RNAi-deficient Argonaute (ERGO)-1 partially rescues the sterility and vulval defects of the post-dauer r larvae . On the  mutants .These findings were surprising considering that the effects of AMPK compromise impact nuclear events, namely the state of the chromatin. Following analysis with genetic mutants and by varying the RNAi strength, it was later determined that the RNAi experiments revealed a specific dosage threshold that resulted in a correction of the sterile phenotype that occurs in the AMPK mutant dauer larvae. If the RNAi was too strong, or alternatively if it was too weak, no effect on the sterility was observed. Moreover, timing of the RNAi application was also important. These findings suggest that the small RNA pathway may be used repeatedly during dauer formation and during recovery. It is critical for the remodelling that must occur first to convert the germ cell chromatin to a quiescent state, and then it may be further required to reactivate germ cell function in response to favourable recovery signals following the diapause . Currentdcr-1 or rde-4 using RNAi corrects the inappropriate increase and distribution of chromatin marks in the dauer germ line of AMPK mutants [dcr-1 function [Consistent with this, the compromise of  mutants . Moreovefunction . CurrentC. elegans can cell nonautonomously regulate a number of developmental and physiological processes, including lifespan and behaviour [Neuronal activity in ehaviour ,26. AMPKehaviour . Neuronaehaviour . AMPK phehaviour . When AMehaviour .C. elegans [rde-4 results in a chemotaxis defect, where mutants no longer respond to chemical attractants [saeg-2, which was transgenerationally downregulated in the germline, was shown to rescue the chemotaxis defect of rde-4 mutants [Neurons are also at the centre of small RNA-mediated effects that alter behaviour in a transgenerational manner in  elegans . Recent  elegans ,27. Endo elegans . The tra elegans . The losractants . The rem mutants . Thus, n mutants .aak-2) in the soma, restores the fertility of post-dauer AMPK mutants while also rescuing the dauer germline hyperplasia [aak(0) mutants, it was found that aak-2 expression in the neurons and the excretory system was sufficient to rescue the sterility associated with post-dauer animals that lack all AMPK signalling [Recent findings suggest that the soma may regulate changes in the germ line in response to environmental cues through an AMPK-mediated pathway. The ubiquitous expression of one of the two catalytic subunits ; this process is highly dependent on the dsRNA-selective importer systemic RNA-deficient (SID)-1 ,30. Sincsid-1, if other import channels that could transport the small RNAs, namely other members of the rme gene superfamily, remained intact.Alternatively, there may also be additional SID-1-independent mechanisms involved in regulating the movement of small RNAs in the AMPK mutants. Studies have shown that the accumulation of the fluorescently-labelled dsRNA resembles the accumulation of yolk proteins (vitellogenins) in proximal oocytes . Yolk prMultiple approaches could be employed to further test the purported permeability of the Weismann barrier in this context. To demonstrate that molecular information generated within the neurons must leave these cells to confer changes in the germ line, the exit of information-containing vesicles could be disrupted genetically by compromising key gene products required for exocytosis. The role of these gene products could then be assessed by quantifying the impact of these genetic changes on the sterility of post-dauer AMPK mutant adults. These same genetic backgrounds could also be used to evaluate how disruption of exocytosis affects the sufficiency of AMPK re-activation in the neurons to correct the germ line integrity of post-dauer AMPK mutants.Although very informative, these approaches would not provide direct evidence of the movement of information from the soma to the germ line. Molecular labelling of small RNAs in the somatic tissues, such as the neurons or the excretory system, may provide a useful means of following these molecules and identifying their final target tissues. So far, however, this has proven quite challenging due to technical issues with specificity and subsequent tracking. Alternatively, new proximity-labelling techniques might offer a more efficient approach that could facilitate the identification of the molecular nature of this information and the gene products involved in its transmission .AMPK modulates cellular energy metabolism while transiting through the dauer stage in part by regulating the chromatin landscape and consequent gene expression . In the Although we have identified an important link between this protein kinase and the chromatin remodelling that occurs concurrent with dauer entry and exit, the precise targets that are involved and that mediate these changes remain elusive. In addition, the major changes that must occur to the small RNA pathways and the small RNAs themselves must also be under some AMPK control, and this may occur in a collection of neurons that respond to the stress of the dauer stage. Our future work will focus on identifying these key hinge pins, in order to better understand how environmental change can be transduced by AMPK to bring about adaptive, or sometimes even maladaptive, epigenetic responses in the germ line.