In the 1970s and 1980s, a multitude of cognitive rehabilitation programs proliferated to facilitate recovery after brain injury. However only a few programs provided a framework for ameliorating disturbances in the cognitive, psychological, and interpersonal spheres of the brain-injured patient. Greatly influenced by Leonard Diller and Yehuda Ben-Yishay’s ideas and methods, George P. Prigatano began, in early 1980, a holistic neuropsychological rehabilitation program at the Presbyterian Hospital in Oklahoma City (Oklahoma). The objective of this paper is to summarize the contributions of George P. Prigatano to neuropsychological rehabilitation and clinical neuropsychology during his 50th year of practice. The main body of the paper is structured in three sections. The first section briefly explains the history of neuropsychological rehabilitation in the twentieth century and the emergence of holistic neuropsychological rehabilitation programs in the 1970s. The second section describes the contributions of George P. Prigatano to neuropsychological rehabilitation and clinical neuropsychology (written by AGM). In the third section, the second author (GPP) prepared an autobiographical statement, which attempts to summarize some of the personal and professional experiences which influenced his work. George P. Prigatano’s contributions to neuropsychological rehabilitation and clinical neuropsychology are essential to understanding the therapeutic approaches currently used in the treatment of brain-injured patients. Neuropsychological rehabilitation has a long history as recently documented by Typically, descriptions of a person’s contributions to a given field of study are written after their death with no input from them concerning what actually influenced their work. It was the absence of this perspective, in other historical accounts, that prompted the writing of this paper.Documents describing techniques and procedures for improving the cognitive functioning of people with brain injuries date back to the seventeenth century . It was Die Psychischen Schädigungen Durch Kopfschuss im Kriege 1914/17  published what is considered the first treatise on neuropsychological rehabilitation:  1914/17 . At the  1914/17 , 1942. Hvia the process of diaschisis (The Second World War (1939–1945) provided a renewed impetus for developments in neuropsychological rehabilitation. Oliver Louis Zangwill (1913–1987) addressed the rehabilitation needs of soldiers in England, while Alexander Romanovich Luria (1902–1977) did the same in Russia. Luria and his colleagues suggested that the restoration higher cortical functions could be partially achieved by “de-inhibition” of a temporary inactivated functional system aschisis . The proAfter the Second World War, the Veterans Administration Hospitals in the United States provided extensive rehabilitation for disabled veterans . Yet holistic programs for neuropsychological rehabilitation had not come into existence.Between 1974 and 1976, Yehuda Ben-Yishay (1933–2021) and Leonard Diller (1924–2019), at the Rush Institute of Rehabilitation Medicine at New York University School of Medicine, in conjunction with the Israeli government, developed a pilot rehabilitation program for brain-injured Israeli war veterans. Many of Goldstein’s ideas, coupled with the rehabilitation technology Diller had developed with stroke patients and Ben-Yishay’s experience living in Israeli community settings, were successfully applied to these patients. Their demonstration of successful outcomes resulted in a funded 5-year clinical research study in the United States and the establishment of the Rusk Institute Brain Injury Day Treatment Program (New York). Based on the successful outcome of this research study, the program became an established clinical service at the Rusk Institute, one which continues to this day . The cenBen-Yishay and Diller’s holistic neuropsychological rehabilitation program provided a framework for ameliorating disturbances in the cognitive, psychological, and interpersonal spheres of the brain-injured patient. The program was organized in a hierarchy of six stages that must be met successfully while undergoing rehabilitation :•Engagement: The goal is to have the trainee/patient become optimally activated to engage in a purposeful and deliberate manner various remedial tasks.•Awareness: The goal is to have the trainee/patient become aware of one’s problems, without being overwhelmed by this knowledge.•Mastery: The goal is to have the trainee/patient learn to compensate for their cognitive deficits.•Control: The goal is to have the trainee/patient learn to concentrate on the idea behind the act and on its ultimate objective, rather than on the “mechanics,” or how the act is to be carried out.•Acceptance: The goal is to have the trainee/patient begin to feel that life, as is, is worth living and that they can still derive some pleasure in his/her present life.•Identity: The goal is to have the trainee/patient learn to reconstitute, to some extent at least, his/her identity.Ben-Yishay and Diller’s holistic program has been the model for several holistic neuropsychological rehabilitation programs over the world. Between 1979 and 1985, for instance, George P. Prigatano adapted that model to develop a holistic neuropsychological rehabilitation program at the Presbyterian Hospital in Oklahoma City (Oklahoma) . In 1985In the 1970s and 1980s, a multitude of cognitive rehabilitation programs proliferated to facilitate recovery after brain injury. However, only a few programs embraced the holistic approach. Greatly influenced by Diller and Ben-Yishay’s ideas and methods, Prigatano began, in early 1980, a holistic neuropsychological rehabilitation program at the Presbyterian Hospital in Oklahoma City (Oklahoma)  as notedPrigatano emphasized that it is artificial to separate cognitive and emotional disturbances in these patients, since how we feel affects how we behave and how we think. He argued that patients’ emotional and motivational reactions to their impairments greatly contribute to their degree of disability. He, like Goldstein and Ben-Yishay before him, stressed the importance of establishing a therapeutic milieu as a key component of the successful brain injury rehabilitation: “Group work alone does not define a holistic approach; rather, it is placing an individual in a therapeutic environment that influences psychosocial outcomes” , p. 497.Principles of Neuropsychological Rehabilitation, one of the seminal books in contemporary neuropsychological rehabilitation  . TherefoPrigatano explicitly stated that psychotherapy could be of value to brain-injury survivors in several areas . It shou•Provide a model that helps the patient understand what has happened to him or her.•Help the patient deal with the meaning of the brain injury in his or her life.•Help the patient achieve a sense of self-acceptance.•Help the patient make realistic commitments to work and interpersonal relations.•Teach the patient how to behave in different social situations.•Provide specific behavioral strategies to compensate for neuropsychological deficits.•Foster a sense of realistic hope.Challenges and opportunities facing holistic approaches to neuropsychological rehabilitation  may provide important insights into mechanisms of “recovery” and “deterioration” after various brain disorders. This has clear implications for treatment and patient management.In bringing attention to the fact that many people with different brain disorders overestimate their abilities, presumably because of their altered awareness of deficits, he also developed, in collaboration with his colleagues in Oklahoma City, the Patient Competence Rating Scale (PCRS), which was designed as a scale to determine awareness of deficits after brain injury . FollowiAmong the best known, and most used, cognitive screening tests are the Mini-Mental State Examination (MMSE)  and the His influence in clinical neuropsychology, therefore, was to expand the scope of a traditional neuropsychological assessment to include assessments of awareness and affect. He also demonstrated that impairments in self-awareness exist in various patient groups even when frank anosognosia is not present. Finally, his research on finger tapping emphasizes the need to have “simple,” direct measures of behavior that can monitor the recovery and deterioration of brain function over time. This perspective expanded the role of neuropsychological assessment from diagnosis to prognosis.I was born on September 13, 1944. My father was the product of the Great Depression and left school in the 8th grade to work to save the family’s home. My mother was born in Sicily and moved to the United States at age 4. One of her brothers became a physician while the other became a chemist trained at Princeton University. This second brother  spent a lot of time with me during my childhood and served as an important role model who emphasized the importance of education, working hard, and speaking one’s mind. My father and his father emphasized the very same values as I was growing up.1 It had a deep personal significance for me. It was a true “hero’s story” that moved me consciously and unconsciously into a clinical profession.Being a product of an Italian-American family, I was raised Catholic and attended Catholic grammar school and high school. At around age 8, I read about the life of “Father Damien.”In college, I was educated by Jesuit priests who emphasized the importance of the individual to think for themselves when making important life decisions. Those teachers also emphasized, in my philosophy courses at Loyola University in Los Angeles (1962–1966), that no theory is “right” or “wrong.” The usefulness of a theory depends on its ability to explain or predict a given phenomenon. This instilled a questioning attitude toward any “dogma” which told clinical neuropsychologists how to practice their profession.During my undergraduate education, both philosophy and psychology were emphasized. I was therefore very much influenced by the debate been Carl Rogers and Burrhus Frederic Skinner at that time. It was true that behavior is a function of its consequences . However it was also true that entering the patient’s subjective experiences, in order to help them clarify how they are experiencing themselves and the world around them, could be very important in helping them find meaning in their lives and reduce their psychological suffering in its many forms  as Carl Rogers quietly but persuasively stated.During pre-doctoral academic training as a clinical psychologist, my teachers emphasized the role of learning-based techniques for reducing behavioral problems. I was taught such procedures as systematic desensitization to reduce certain types of anxiety or avoidance behaviors in college students seeking services from the Department of Psychology’s Psychological Clinic. The theories of Freud, Jung and Adler were glossed over in my academic doctoral training program and considered having no scientific and little clinical value.Later, during my pre-doctoral clinical internship at the University of Oklahoma Health Science Center, I was confronted with the real problems of psychiatric patients . Listening to these patients’ life stories and current struggles, it became clear that the teachings of Rogers and Skinner were only partially helpful. The teachings of Freud, Jung, and Adler suddenly seemed to be very relevant to these psychiatric problems.I was fortunate to obtain excellent supervision from experienced psychiatrists particularly influenced by Freudian and Neo-Freudian theories during this time. The role of unconscious influences on behavior now became clearer to me; working with these influences seemed to produce meaningful behavioral changes and a reduction of personal  suffering in some of the patients I helped treat.During my pre-doctoral clinical psychology internship, I was also introduced to the field of neuropsychology by Oscar Parsons, who emphasized the importance of writing a clear neuropsychological report and publishing one’s findings in a concise manner. This influence persists in my professional life as a diagnostician, a psychotherapist, and a researcher.Many individuals had a substantial impacted my clinical and research work, as acknowledged in previous publications , 2019. II also found the writings of Karl Pribram especially interesting since he discussed Freudian concepts within the context of the neurosciences . After pDenial of Illness: Symbolic and Physiological Aspects. It was the only book that seem to address this important issue. I asked Edwin Weinstein to participate in an American Psychological Association symposium I had organized on neuropsychological rehabilitation. Weinstein accepted the invitation, and we began several dialogues about denial phenomenon. Weinstein’s insights helped clarify an important clinical and theoretical question. Was it possible for a brain dysfunctional person to have altered self-awareness of a neuropsychological disturbance due to the direct effect of a brain disorder  and at the same time minimize the presence or extent of a neuropsychological impairment as a method of coping with anxiety over the presence of that impairment ? That question lead to a long series of studies done with colleagues to help arrive at a reasonable answer. The answer was “yes.”In my initial efforts at holistic neuropsychological rehabilitation with post-acute young adults with severe traumatic brain injury, I became impressed that many of these patients seemed to not be aware of or were denying the presence and severity of various neuropsychological impairments. Reviewing the existing literature at the time, I came across Undoubtedly, however, the biggest influence on my work in neuropsychological rehabilitation came from my professional interactions with Leonard Diller and Yehuda Ben-Yishay. The development of a holistic approach that including both cognitive rehabilitation and psychotherapy now became a passion for me. Dr. Ben-Yishay played a very special role in helping me organize a day treatment program and provided me with a methodology for approaching the cognitive rehabilitation of patients with chronic brain injuries who had not benefited from traditional forms of rehabilitation.Being impressed with the importance of impairments of self-awareness in the rehabilitation process, I worked with patients to determine if their emotional state could be improved. However, it was unclear to me as to the best way of approaching this domain of the patients’ lives. Early in our rehabilitation work, a young man presented me with a spontaneous drawing, which reflected his phenomenological experience of his brain injury on his life and how he was attempting to come to grips with the consequences of his brain injury in the most productive manner. A picture of the drawing is found in The Spontaneous Paintings of Severely ill Patients: A Contribution to Psychosomatic Medicine, I sought out a personal consultation with Dr. Bach, a Jungian analyst living in London. The discussion left an indelible mark on how I thought about the unconscious and how it influences various art expressions that can inform the psychotherapist about what the patient is experiencing. These art forms often point to the next step in their psychological development as clearly described in Sandplay.Many individuals influenced my thinking about psychotherapy. They are listed by name in the Preface of my most recent book . By and These professional dialogues lead to several papers on the importance of symbolism in the neuropsychological rehabilitation process. Helping patients relate to their symbols of work, love and play guided psychotherapeutic efforts when supporting their return to a productive lifestyle and re-establishing meaning in their lives. Symbols involving the hero’s journal now became an important prototype of the psychotherapeutic process for many individuals . It contThe intent of this paper was to first summarize the contributions of George P. Prigatano during his 50th year of practice from the perspective of a part-time amateur historian and to then to combine that perspective with Prigatano’s own views of the primary influences on his work. Normally, the person’s professional career is reviewed by others, and the person’s own view of what influenced their work is not present. We believe both sources of information can be useful in historical accounts. It is our hope that this paper has achieved that goal.At the conclusion of this paper, we include an Addendum that summarizes George P. Prigatano’s professional career to date.George P. Prigatano obtained his Ph.D. in Clinical Psychology on August 31, 1972 from Bowling Green University in Ohio. He attended Bowling Green University because it offered a joint Ph.D. degree in both Industrial and Clinical Psychology, but soon his interests turned to Clinical Psychology. During his pre-doctoral clinical psychology internship at the University of Oklahoma Health Sciences Center, he was introduced to a new field of study called neuropsychology. After completing his Clinical Psychology Internship, he became an Assistant Professor of Psychiatry and Behavioral Sciences and was the Director of the Neuropsychology Testing Laboratory. In 1978, he was promoted to Associate Professor (Tenured), but left his academic position to study neuropsychology with Karl Pribram, M.D. at Stanford University. Following that time, he developed a Neuropsychology Rehabilitation Program at Presbyterian Hospital in Oklahoma City (1979–1985). He was recruited to the BNI in Phoenix Arizona in 1985 to begin the Department of Clinical Neuropsychology and to establish a Holistically Oriented Neuropsychological Rehabilitation Program like the one he developed at Presbyterian Hospital in Oklahoma City. That program was influenced and supported of two key figures -Yehuda Ben-Yishay, Ph.D. and R. Barton Carl, M.D.-. He was the Founder and first Chairman of the Department of Clinical Neuropsychology at the BNI from 1985 to 2014. From 2014 to this date, he has been the Emeritus Chairman and has remained clinically and academically active. He served as the President of the National Academy of Neuropsychology in 1998. In conjunction with Neil Pliskin edited the first book on the importance of cost outcome research for the future of clinical neuropsychology .Over the course of his career, Dr. Prigatano has lectured nationally and internationally on the various topics listed above. His most recent workshop was held at the National Academy of Neuropsychology meeting in December of 2021. He spoke on the important role of providing psychological care for persons with various brain disorders. The lecture was devoted to the memory of Leonard Diller and Yehuda Ben-Yishay.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


KLK3 and FKBP5 mRNAs. The reduced AR binding to the KLK3 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay. In addition, some indoles significantly decreased AR protein and mRNA level. By using CRISPR/Cas9 AhR knockout technology, no relationship between AhR and AR, measured as target gene expression, was observed. In conclusion, some indoles that activate AhR possess AR-inhibiting activity, which seems to be related to the downregulation of AR expression rather than to AR degradation alone. Moreover, there does not seem to be a clear relationship that would connect AhR activation with AR activity suppression in 22Rv1 cells.The activation of the aryl hydrocarbon receptor (AhR) by xenobiotic compounds was demonstrated to result in the degradation of the androgen receptor (AR). Since prostate cancer is often dependent on AR, it has become a significant therapeutic target. As a result of the emerging concept of bacterial mimicry, we tested whether compounds with indole scaffolds capable of AhR activation have the potential to restrict AR activity in prostate cancer cells. Altogether, 22 indolic compounds were tested, and all of them activated AhR. However, only eight decreased DHT-induced AR luciferase activity. All indoles, which met the AhR-activating and AR-suppressing criteria, decreased the expression of DHT-inducible AR target genes, specifically  Prostate cancer is one of the most common types of cancer in men, and the risk of developing it increases with advanced age. More than 75% of patients with prostate cancer are men over the age of 65 . AccordiAR belongs to the nuclear receptor superfamily. Upon androgen (ligand) binding, AR translocates to the nucleus and binds to specific responsive elements in DNA. The most vital androgens are testosterone (T), dihydrotestosterone (DHT), and dehydroepiandrosterone (DHEA) . TestostMore than a decade ago, a different prostate cancer therapeutic strategy was suggested, assuming suppression of AR function through activation of the aryl hydrocarbon receptor (AhR), i.e., an indirect approach. Similarl to AR, upon ligand binding, AhR translocates into the nucleus, where it binds to a specific xenobiotic-responsive element in DNA, with a consequent increase in the expression of the target genes ,14. AhR +/+AhR, +/−AhR, and −/−AhR, and studied the impact on prostate cancer development [+/+ < AhR+/−AhR < −/−AhR, where +/+AhR mice had the lowest incidence of prostate cancer development compared to −/−AhR mice (16% vs. 60%) [An in vivo connection between AhR and AR in relation to prostate cancer was suggested in the study by Fritz et al. 2007, who used three different genotypes of mice, namely elopment . The obtvs. 60%) . Interestingly, only a limited number of studies have dealt with possible crosstalk between AhR and AR in human prostate cells. One such cross-talk can represent AhR-mediated AR degradation, since AhR has been demonstrated as a ligand-dependent E3 ubiquitin ligase that induces proteasomal degradation of AR in the androgen-sensitive prostate cancer cell line LNCaP . HoweverAR, KLK2, TMPRSS2, and PSA), PSA protein levels, and DHT levels were evaluated as the end points. The study confirmed that FICZ induced CYP1A1 activity, which is a marker of AhR activation. In addition, a significant decrease in AR-target gene mRNA expression was observed with the combination of FICZ (50 nM) and testosterone (100 nM). PSA protein and DHT levels were reduced after treatment with FICZ + T. However, no decrease was observed in the absence of testosterone. The obtained results indicated that AhR plays an important role in AR signaling and that FICZ has anti-androgenic effects through the AhR/AR pathway [CYP1A1 [The study of Arabnezhad et al. 2020 considered the effects of endogenously activated AhR on AR . The pro pathway . Moreove pathway . Using a [CYP1A1 .Epimedium [Interestingly, the concept of AhR-mediated degradation of AR was described for other compounds, namely Icaritin  and Carbpimedium . Carbidopimedium . Both ofpimedium ,25. Howepimedium ,25.Therefore, available studies strongly indicate the presence of AhR/AR crosstalk in prostate cancer cells, as various AhR ligands were reported to inhibit prostate cancer development, probably through several different mechanisms . PublishRecently, our research team observed that the group of mostly synthetic compounds with an indole scaffold has the ability to activate AhR in hepatocarcinoma cells   with effTherefore, the objective of this study was to determine whether these indoles have the ability to suppress AR function through AhR activation and thus slow the proliferation of cancer cells.Firstly, the new reporter cell line 22AhRv1 was constructed, characterized , and subIn the 22AhRv1 reporter cell line, the ability of tested indoles to activate AhR was measured after 4 h and 24 h. Significant AhR activation was observed for 1MI, 2MI, 4MI, 5MI, 6MI, 7MI, 2,5DMI, 6MeO, 4MeO1MI, and 7MeO4MI after 4 h A. Intere50 approx. 10 µM).In the AIZ-AR reporter cell line, the ability of the tested indoles to affect AR was measured after 24 h. DHT-inducible AR-dependent luciferase activity reached approximately 7-fold induction next to UT A. True A50~5 µM) and 4MI (IC50~50 µM). Some compounds, such as 2,5DMI, 2,3,3TMI, 7MeO, or 5MeO2MI, were able to coactivate AR in combination with DHT (10 nM), but this was consistent with their activity observed in the agonist mode  have been described as nontoxic by both assays, since the decrease in cell viability was not less than 20% . HoweverCYP1A1 mRNA expression, which is a marker of AhR activation [CYP1A1 expression by 106-fold  and controls in antagonist mode  for 90 min. The positive control DHT (10 nM) enriched the 4.9 fold A. The pr50 approx. 10 µM), followed by 4MI, 2,3DMI, 2,3,7TMI, and 7MeO4MI  on AR activity and cell viability. Skatole is naturally a product of the intestinal microbiota and can be found in wide range of concentrations in the serum at least of patients with hepatic encephalopathy . UnfortuAhRR mRNA . The expression of AhRR and AhR is regulated by NF-kB [Furthermore, we observed a visible morphological change in 22Rv1 cells at the end of skatole treatment . Thus, it is likely that skatole triggers some sort of apoptotic event and apparently has more molecular targets inside the cells. Our assumption is based on the observation of different skatole action in relation to other indoles as it reduced rather than induced hRR mRNA B and conby NF-kB , and theby NF-kB  . Human prostate carcinoma epithelial cell lines 22Rv1 (ECACC No. 105092802) and AIZ-AR  were culThe tested indoles were selected based on previous research  in our l4 cells per well in a volume of 200 µL and stabilized overnight. 22AhRv1 cells were incubated with tested indoles for 4 h and 24 h. AIZ-AR cells were treated with indoles for 24 h in agonist and antagonist mode (in the presence of DHT 10 nM). DMSO  was used as the vehicle/negative control, TCDD (10 nM) was used as the positive control in 22AhRv1 and DHT (10 nM) was used as the AIZ-AR positive control. Furthermore, anti-androgen ENZ (10 µM) was used as AR suppression control in AIZ-AR cells, and the effects of TCDD (10 nM) and FICZ (10 µM) in the presence or absence of DHT (10 nM) were also evaluated. After treatments, cells were lysed with 1x Reporter Lysis Buffer  and luciferase activity was measured with the Infinite M200.Initially, a cellular system was established to monitor AhR transcription activity in the 22Rv1 cell line . AIZ-AR 6 cells per well in a volume of 1.5 mL and stabilized overnight. Cells were treated with 8 selected indoles in 4 concentrations  in antagonist conditions (with DHT at 10 nM). After 24 h of treatment, cells were washed with PBS, and total mRNA was isolated using TRI Reagent® , according to protocol. Quality was monitored in the A260/A280 ratio. cDNA was synthesized from 1000 ng of total RNA using M-MuLV Reverse Transctiptase  at 42 °C for 60 min in the presence of Random Primer 6 . Subsequently, enzyme activity was inactivated when incubated at 65 °C for 10 min in Dry Bath Incubator . The mRNA expression was evaluated with KiCqStart® Probe Assays (Sigma-Aldrich) or with SYBR® Green  according to the manufacturer’s recommendations. The expression of genes related to the AhR pathway  and AR-target genes  was monitored. The list of used probes and primers is given in ® Probe Assays analysis are shown in ® Green analysis was performed according to the manufacturer´s recommendations. Sample measurements, including the non-template control (NTC), were performed in triplicate, and gene expression was normalized to the housekeeping gene GAPDH. The data obtained were evaluated by the delta-delta method.Only indoles capable of activating AhR and simultaneously suppressing AR activity were selected for PCR analysis. Cells were seeded in 6-well plates at a density of 1.5 × 106 cells per well in a 1.5 mL volume and stabilized. The treatment was similar to that for RT-qPCR. Total protein extracts were isolated for each sample after 24 h of treatment. Cells were washed with 1× PBS, scraped into 1 mL of PBS (cold) and centrifuged . Pellet was re-suspended in 80 µL protein lysis buffer  and centrifuged at 13 000 rpm , 15 min, 4 °C. The supernatant was collected, and the protein concentration in each sample was determined by 1x Bradford reagent (Sigma Aldrich). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis  in the BioRad Miniprotean system , according to the manufacturer’s recommendations. SDS-PAGE was followed by semi-dry protein transfer onto a polyvinylidene difluoride (PVDF) membrane. The membranes were stained with Ponceau S Rouge solution (0.1% w/v in 5% acetic acid), washed with 1xTBS/Tween, and incubated with 5% non-fat dried milk for 1 h at room temperature (RT). Membranes were incubated overnight with appropriate antibody diluted in 5% solution of bovine serum albumin (BSA) in 1xTBS/Tween at 4 °C. After incubation, the membranes were washed with 1xTBS/Tween and incubated with secondary horseradish peroxidase-conjugated antibody diluted in 5% non-fat dried milk for 1 h at RT. List of used antibodies and their concentrations is given in ® PREMIUM Chemiluminiscent Substrate  and the C-DiGit Chemiluminiscence Western Blot Scanner (Li-Cor). Obtained results were processed by Image Studio 5.0 for C-DiGit Scanner software. Protein expression was normalized per β-actin (43 kDa).Cells were seeded in 6-well plates at a density of 1.5 × 106 cells per plate in a 4 mL volume and stabilized overnight. The following day, cells were incubated with selected indoles in antagonist mode (only 10 µM concentration with DHT 10 nM) and controls . Furthermore, the effect of some other compounds  in combination with DHT 10 nM was evaluated. Cells were incubated with the tested compounds for 90 min at 37 °C. Subsequently, ChIP was performed according to the manufacturer’s recommendations in the manual for the Simple ChIP Plus Enzymatic Chromatin IP Kit  as previously described, with minor modifications [2O (Roche) and 1 µL of KLK3 Promoter Primers . The setting of this reaction is given in ® nucleic acid stain . The result was visualized with the Syngene G:BOX gel documentation system .22Rv1 cells were seeded in a 60-mm plate at a density of 4 × 10ications . Briefly5 cells and 3 mL of medium per well) and stabilized for 24 h in a humidified incubator (80% confluence). Subsequently, cells were transfected with the CRISPR/Cas9 AhR KO plasmid or with the control CRISPR/Cas9 plasmid, as a negative control. Solution A, 1 µg of plasmid DNA in Plasmid Transfection Medium in a total volume of 150 µL, and solution B, 5 µL of UltraCruz® Transfection Reagent in Plasmid Transfection Medium in a total volume of 150 µL, were prepared for each well and let stand for 5 min at RT. Subsequently, solution A was added to solution B, vortexed, and incubated for 20 min at RT. To verify the relationship between AhR and AR, 22Rv1 cells were transiently transfected with an AhR CRISPR/Cas9 KO plasmid according to the manufacturer’s protocol , with minor modifications. Briefly, 22Rv1 cells were seeded in a 6-well plate  was evaluated as described earlier . Subsequently, transiently transfected cells were exposed to the tested compounds  for 24 h. Total mRNA was isolated, and the expression of genes . Significant values in all experiments were determined by two-way ANOVA (symbol * in the charts). Western blot results were analysed using Image Studio 5.0 for C-DiGit Scanner software .


We investigated clinical equipoise across surgical members of the British Elbow and Shoulder Society (BESS) in relation to rehabilitation following rotator cuff repair.An online survey explored clinical equipoise regarding early patient-directed versus standard rehabilitation after rotator cuff repair to inform the design of a national randomised controlled trial (RCT). It described different clinical scenarios relating to patient age, tear size, location and whether other patient-related and intra-operative factors would influence equipoise.76 surgeons completed the survey. 81% agreed/ strongly agreed that early mobilisation might benefit recovery; 57% were neutral/ disagreed that this approach risks re-tear. 87% agreed/ strongly agreed that there is clinical uncertainty about the effectiveness of different approaches to rehabilitation. As age of the patient and tear size increased, the proportion of respondents who would agree to recruit and accept the outcome of randomisation reduced, and this was compounded if subscapularis was torn. Other factors that influenced equipoise were diabetes and non-secure repair.Surgical members of BESS recognise uncertainty about the effectiveness of different approaches to rehabilitation following rotator cuff repair. We identified a range of factors that influence clinical equipoise that will be considered in the design of a new RCT. After surgery, patients undergo rehabilitation and despite its major role in a patient's recovery, there is clinical uncertainty regarding the optimal approach to rehabilitation after rotator cuff repair. Cautious approaches, where patients use a sling for over a month and only passive movements are allowed in the initial postoperative stages are common in current clinical practice. A recent survey with physiotherapists reported that the most frequent length of immobilisation after rotator cuff repair is four to six weeks. The rationale for more conservative approaches is the perceived risk of tendon re-tear if early mobilisation is introduced.Rotator cuff repair surgery is a common intervention indicated for patients with symptomatic rotator cuff tears. The pilot and feasibility RCT met the predefined success criteria and suggested that a fully powered RCT is feasible with minor amendments to the research design. One of the main issues observed in the pilot RCT was the withdrawal of 5/37(13.5%) patients randomised to early patient-directed rehabilitation due to surgeons perceived risk of re-tear. Treatment preference and lack of clinical equipoise may hinder patient recruitment to an RCT, adherence to the allocated treatment and even early closure of a trial.6 The concept of equipoise has been defined as, based on current knowledge, the patient being neither advantaged nor disadvantaged if they were to receive any of the treatments being tested. Equipoise can be sub-divided into individual equipoise, where there is uncertainty at the level of the individual clinician, and community or clinical equipoise, where there is uncertainty in the clinical community about which treatment is best. It is typically the latter, community equipoise, upon which recruitment to RCTs is grounded.We have recently completed a National Institute for Health Research funded pilot and feasibility randomised controlled trial (RCT) that recruited 73 participants across five NHS hospitals comparing early patient-directed rehabilitation  with standard rehabilitation (sling use for four weeks following surgery).To help with refining the design of a future fully powered RCT, in this current study our objective was to explore clinical equipoise among surgical members of the British Elbow and Shoulder Society (BESS) in relation to early mobilisation of the shoulder following rotator cuff repair, in the context of different clinical scenarios and patient-related and surgical factors.https://www.onlinesurveys.ac.uk). The survey was designed with reference to different clinical scenarios relating to the age of the patient, size and location of the rotator cuff tear and whether other factors, e.g. smoking status, diabetes, non-secure surgical repair, would influence a surgeon's decision to recruit or withdraw participants following randomisation . For the clinical questions, surgeons were asked to specify their agreement on a 5-point Likert scale  or as Yes/No/Unsure. The survey was developed and piloted across the study team. Based on the pilot testing, the survey took less than 10 min to complete. It was distributed by email to BESS members with a reminder sent after two weeks. The survey was open for four weeks (11th March 2021 to 8th April 2021). A favourable ethical opinion was gained from the Manchester Metropolitan University Research Ethics Committee (Ref: 28603).An online survey was hosted by Online Surveys  reported less than 10 years, 13.2% reported between (10/76) 10 to 15 years and 43.4% (33/76) reported more than 15 years of clinical experience. The reported number of rotator cuff repairs performed monthly was mean = 3.7 (SD = 3), median = 3 and mode = 3.The following sections report the responses to the statements and questions posed in the survey.It is possible that early patient-directed rehabilitation might benefit recovery after rotator cuff repair.A total of 75 responses were obtained. 61 respondents (81.3%) strongly agreed/agreed that early rehabilitation might be beneficial for patients recovery after rotator cuff repair .Early patient-directed rehabilitation risks re-tear following rotator cuff repair.A total of 75 responses were obtained. 42 respondents (56%) were neutral or disagreed that early rehabilitation risks re-tear .There is clinical uncertainty about the effectiveness of different approaches to rehabilitation following rotator cuff repair surgery.A total of 75 responses were obtained. 65 respondents (86.7%) strongly agreed/agreed that there is clinical uncertainty regarding the effectiveness of different approaches to rehabilitation after surgery .n = 600) comparing early patient-directed versus standard rehabilitation following rotator cuff repair is feasible within the UK NHS.A large randomised controlled trial  strongly agreed/agreed that a large RCT is feasible within the UK NHS .I would be interested in taking part in a fully powered randomised controlled trial.A total of 75 responses were obtained. 54 respondents (72%) strongly agreed/agreed that they would be interested in taking part in the RCT . For a small tear and intact subscapularis, the percentage of surgeons who strongly agreed/agreed with recruiting and accepting the outcome of randomisation in the trial decreased from 89.6% for a 55-years old to 82.9% for a 70-years old. For a large tear and intact subscapularis, the percentage of surgeons who strongly agreed/agreed decreased from 47.3% for a 55-years old to 35.5% for a 70-years old.A total of 74 responses were obtained. 29 respondents (39.2%) strongly agreed/agreed that if the patient been a regular smoker it would influence their decision to recruit and accept the outcome of the randomisation .A total of 74 responses were obtained. 28 respondents (37.8%) strongly agreed/agreed that the patient reporting alcohol intake over recommended limits would influence their decision to recruit and accept the outcome of the randomisation .A total of 74 responses were obtained. 34 respondents (46%) strongly agreed/agreed that the patient having diabetes would influence their decision to recruit and accept the outcome of the randomisation .A total of 73 responses were obtained. 28 respondents (38.3%) strongly agreed/agreed that expectation of high functional demand post-surgery would influence their decision to recruit and accept the outcome of the randomisation 33 respondents (45.2%) were neutral .The three main intra-operative findings that would influence surgeons’ decision to withdraw patients from the RCT were, in order, a repair that would not be regarded as secure, poor tissue quality and poor bone quality .Reliability is an important property of a survey instrument. We did not evaluate the reliability of this current instrument and so do not know if surgeons would respond differently if repeating this survey. We also received a limited number of responses from surgical members of BESS (76/583 = 13%) which means the findings might not be generalisable to the wider community of surgeons performing rotator cuff repair'.We completed an online survey to investigate clinical equipoise among surgical members of BESS with regard to rehabilitation following rotator cuff repair. We found that there is clinical equipoise across the surgical community reflected in the range of different opinions, and that there is willingness to participate in a future national trial.The majority of respondents strongly agreed or agreed that early patient-directed rehabilitation might be beneficial to a patient's recovery. In terms of trial design, this suggests that a superiority trial is plausible from a clinical perspective. When asked whether early patient-directed rehabilitation risks re-tear, we found a spread in opinion suggesting that there is community equipoise regarding the most effective approach to postoperative rehabilitation. However, only a minority of the RCTs that were included in the systematic review recruited patients with large tears. The RCT from Sheps, Silveira, the largest completed to date (n = 206), included patients with large tears in their sample. They compared early rehabilitation (sling for comfort and pain-free shoulder movement for six weeks) with standard rehabilitation (sling for six and only passive and active-assisted shoulder exercises for six weeks). Their results showed that early rehabilitation was not superior to standard rehabilitation for range of movement, pain, strength and quality of life on the long-term. They also found that the risk of re-tear was higher for patients with large tears, but this was not influenced by the type of postoperative rehabilitation protocol; both early and standard rehabilitation groups had a similar number of re-tear events at 12 months. However, definitive recommendations for patients with a large tear cannot be made based only on the findings from Sheps, Silveira. An RCT with a representative sample of all tear sizes is needed to determine the safety, clinical and cost-effectiveness of early rehabilitation following rotator cuff repair. The tear size should also be considered a stratification factor for randomisation.Regarding tear characteristics, we found that the size of the tear and subscapularis involvement influenced respondents’ decisions as to whether they would recruit and accept the outcome of the randomisation in the RCT. Almost 90% of surgeons strongly agreed/agreed that they would recruit and accept the outcome of the randomisation for a 55-year old patient with a small tear and intact subscapularis; however, this percentage decreased by 42.3% if a large tear was present. Our survey findings in relation to tear size reflect the current body of evidence. A recent systematic review suggested that early rehabilitation may be beneficial to patients with no additional risk imposed to the repair integrity. Patients who were eligible but were excluded tended to be older and with less severe problems. Our survey also suggests that other factors affecting rotator cuff repair healing rates commonly reported in the literature, i.e. diabetes, unsecure repair and tissue quality, would be the most common reasons for surgeons not recruiting and accepting the outcome of randomisation. The survey findings also suggest that there is a readiness for a definitive trial. 86.7% of surgeons strongly agreed/agreed that a large RCT is feasible within the UK NHS and 72% strongly agreed/agreed that they would be interested in taking part in a fully powered RCT. This is encouraging given that the expected sample size needed for the fully powered RCT is over 600 patients.Patients’ age seems to have less influence on surgeons equipoise than tear characteristics. The percentage of surgeons who strongly agreed/agreed with recruiting and accepting the outcome of randomisation decreased only by 6.7% from a 55-years old to a 70-years old patient with a small tear and intact subscapularis but decreased by almost 12% from a 55-years old to a 70-years old patient with a large tear and intact subscapularis. In terms of research design, this is an important factor to consider. Another RCT on shoulder surgery reported that surgeons who were not in equipoise did not recruit patients who were eligible.There is uncertainty about the optimal approach to rehabilitation following rotator cuff repair. This was recognised by surgical members of BESS, who are also willing to participate in a future RCT to answer the research question of whether an early patient-directed rehabilitation is clinically effective compared to standard rehabilitation. We have identified a range of factors that influence clinical equipoise, and a range of different opinions across the community. This information will be factored into the design and in the training of clinicians taking part in a fully powered RCT to be undertaken in the UK NHS.Click here for additional data file.Supplemental material, sj-docx-1-sel-10.1177_17585732211059804 for Rehabilitation following rotator cuff repair: A survey exploring clinical equipoise among surgical members of the British Elbow and Shoulder Society by Bruno Mazuquin, Marcus Bateman, Alba Realpe, Steve Drew, Jonathan Rees and Chris Littlewood in Shoulder & Elbow


Augmented reality- (AR-) based interventions have shown potential benefits for lower limb rehabilitation. However, current literature has not revealed these benefits as a whole. The main purposes of this systematic review were to determine the efficacy of AR-based interventions on lower limb recovery of the larger population based on the current process that has been made in this regard. Relevant studies were retrieved from five electronic databases  using “augmented reality” OR “AR” AND “lower limb” OR “lower extremity” AND “intervention” OR “treatment”. Sixteen studies that met the eligibility criteria were included in this review, and they were further grouped into three categories based on the participant types. Seven studies focused on the elderly adults, six on the stroke patients, and the last three on Parkinson patients. Based on the findings of these trials, the significant effects of AR-based interventions on lower limb rehabilitation  have been initially confirmed. Favorable results were achieved at least the same as the interventions without AR except for the turning and timing in the freezing of gait of Parkinson patients. However, given the infancy of this technology in clinical practices, more robust trials with larger sample sizes and greater homogeneity in terms of devices and treatment settings are warranted for further verification. Nowadays, advanced technologies are becoming increasingly common as rehabilitation therapeutic tools in the healthcare setting –6. AmongDue to the above limitations, the requirement for a safer and automatic rehabilitation training tool has accumulated. In recent years, the introduction of augmented reality (AR) into clinical applications has been proposed and verified –19. AR cAs AR technology becomes much more accessible, AR-based treatments could be widely used not only in clinical practices but also in home settings for function rehabilitation. Therefore, this review summarized and analyzed the efficacy of AR-based interventions on lower limb recovery based on the current process that has been made in this regard, aiming to determine whether it could contribute significant benefits to the larger population and further add help to guide future utilization of AR technology.Five English databases including Web of Science, PubMed, ScienceDirect, Scopus, and Cochrane Library were searched independently by two authors to identify relevant studies published from the very beginning until 1 June 2022. The searching keywords include “augmented reality” OR “AR” AND “lower limb” OR “lower extremity” AND “intervention” OR “treatment”. In addition, both the reference lists of the included studies and the retrieved reviews were further searched to identify other relevant articles.In this review, two investigators assessed the retrieved studies independently, and the third investigator would be consulted if any inclusion disagreements happened. The literature exclusion was initially conducted by screening the titles and abstracts, and then, the investigators would screen the full texts of the papers for further confirmation. The eligibility criteria were formed based on participants, experimental design, and outcomes; (1) subjects: studies that reported the effects of AR-based interventions/applications on lower limb rehabilitation of both healthy participants and patients without age limit were included; (2) experimental design: this review focused on the effectiveness of AR for lower limb rehabilitation, and therefore, any randomized controlled trials or observational studies related to this topic were all included; (3) outcomes: the study results should include variables related to lower limb rehabilitation, such as gait kinematics, muscle strength, and balance. In addition, only English original researches published on peer-reviewed journals were included in this study, while reviews, conference proceedings, and study protocols were excluded.The following data from included studies were further extracted and summarized by two investigators and verified by the third one: (1) author, including the name of the first author and published year; (2) study purposes; (3) participant, including the number of participants, age, gender, and physical conditions; (4) intervention, experimental design, and intervention protocols ; (5) comparison and comparison protocols ; and (6) outcome, the primary findings of these studies. In addition, the Mendeley Desktop Reference Management Software  was applied for organizing articles and generating citations.n = 7), and the rest of the studies were performed on stroke patients (n = 6) and Parkinson patients (n = 3). The detailed study characteristics are summarized in The flow chart of the search procedure of this review is presented in Most of the AR rehabilitation systems utilized in the included studies consist of a server computer with a web camera and an optical see-through HMD connected to a personal computer –37, 39. The first group of studies investigated the effects of AR interventions for lower limb rehabilitation of healthy elderly adults. Yoo et al.  first stTwo subsequent studies determined the effects of a 3D interactive AR program on the balance and mobility rehabilitation of elderly adults. Im et al.  conducteThe last three studies were all published in 2020, and they all provided positive results in terms of the effects of AR-based intervention on lower limb rehabilitation of elderly adults. Chen et al.  investigThe second set of identified studies focused on the effects of AR intervention for lower limb rehabilitation of stroke patients. The first research was performed to compare the effects of an obstacle training program with or without an AR environment on gait parameters of stroke patients . SubjectThe remaining three studies all investigated the effects of AR-based interventions on gait functions of Parkinson patients. Espay et al.  investigThis systematic review summarized and analyzed previous studies that explored the effects of AR-based interventions on lower limb rehabilitation, with the aim to determine whether it could contribute significant benefits to the larger population and further reveal its potential applications to further enhance lower limb rehabilitation.According to the results of this study, the significant effects of AR-based interventions on lower limb rehabilitation have been initially confirmed. To be specific, the AR-based interventions have currently been conducted for elderly adults, stroke patients, and Parkinson patients. Favorable results were achieved in dynamic balance, gait spatiotemporal variables, gait kinematics, muscle mass, muscle activation, muscle force, physical performance, and fall efficacy. Nevertheless, one study that investigated the effects of an AR-based system on gait function and turning in place of Parkinson patients did not come out with any positive results when compared to the preintervention; they demonstrated that the insufficient cues, influences of AR glasses, and subjects' unfamiliarity with the training could be the main reasons for this controversy . In addiThere is great variability in AR-based training programs evaluated in these included studies, which on one hand makes it hard to determine a general intervention that could contribute benefits to the larger population, while on the other hand, it is supported that AR can provide the opportunities to extend beyond rote rehabilitation training by adding more exercises that is highly related to daily living. Based on the needs and actual motor functions of the patients, an appropriate AR intervention mode could be further designed . In termTwo potential limitations that existed should be noted here. Firstly, it is in some cases difficult to compare these findings statistically such as performing a meta-analysis since the homogeneity of instruments, measurement scales, and units applied in these included studies was relatively limited , 41. MorBased on the results of this literature review, it was found that AR-based interventions have been applied for the lower limb rehabilitation of balance, gait, muscle, physical performance, and fall efficacy of the elderly, stroke, and Parkinson patients. Favorable results were achieved at least the same as the interventions without AR except for the turning and timing in the freezing of gait of Parkinson patients. However, given the infancy of this technology in clinical practices, more robust trials with larger sample sizes and greater homogeneity in terms of devices and treatment settings are warranted for further verification. On the other hand, it is proposed that the future development of AR systems should focus on the following several aspects to make it widespread in the motor rehabilitation field. First, some high technologies such as artificial intelligence should be integrated into the AR rehabilitation systems as they can accurately track patients' progress and intelligently adapt the training programs based on their feedback. Moreover, the development of an AR-based system on mobile devices could contribute to more benefits because it can realize the remote monitoring of patients' recovery and provide real-time attention with lower costs. Finally, more game-based rehabilitation content targeted at different populations would also be of interest for AR systems as it may further improve the users' motivation.


Distal radial fractures (DRF) are often encountered in upper limb fractures globally, and their associated complications affect the functional independence of the individual following the injury. The potential of gamification in applied rehabilitation is expanding its horizons in the rehabilitation of conditions ranging from neuromotor deficits to cognitive impairments. However, the synthesis of the literature is aimed at analyzing and summarizing the evolution of gamification in DRF rehabilitation. A comprehensive search and extraction of relevant literature were conducted and reviewed for the applicability of population analysis, interventional methodology, comparative factors, outcome measures, and the type of study. Thirteen studies were included and evaluated, including randomized controlled trials (RCTs), literature reviews, systematic reviews, meta-analyses, and bibliometric analyses. The conclusions demonstrated an improvement with gamification and addressed it as an effective rehabilitation method. Based on the analysis of the data that was extracted, the conclusion supports the use of gamification in the rehabilitation of DRF and looks into how it can help improve the person's functional capacity. A distal radial fracture (DRF) is the most common upper limb fracture that limits the functional activities of daily living (ADLs) and requires precise attention during rehabilitation for an effective return to functional independence . PatientVirtual reality (VR), augmented reality (AR), and mixed reality (MR) provide mature access to rehabilitation technology and are versatile fields for evidence-based clinical practice . The OcuResearch methodologyA combination of words was used for searching the literature in all databases, which included "distal radius fracture" AND "physiotherapy" AND "rehabilitation" AND "virtual reality" AND "gamification." The articles published since 2013 were included in the rapid synthesis of the literature. Articles were searched from PubMed, Emerald, ScienceDirect, SAGE Publications, Wiley, and Google Scholar. The inclusion criteria included the peer-reviewed literature having immersive VR or gamification as an intervention in rehabilitation, a rehabilitative approach to DRF, randomized controlled trials (RCTs), systematic reviews and meta-analyses, and articles published since 2013. The exclusion criteria included non-peer-reviewed literature, non-immersive VR as an intervention, gamification used as a diagnostic method, and articles published prior to 2013. The studies exploring the effectiveness of gamification as an intervention in the rehabilitation of different conditions were given preference.The literature was extracted from relevant studies and reviewed for the applicability of population analysis, interventional methodology, comparative factors, outcome measures, and the type of study as shown in Table A study on the epidemiology of DRF predicted risk factors and prognosis and found the responsive factors of chronic disability and pain, which lead to an imperative perspective for therapists to implement more extensive assessment, interventional strategies, and customized educational programs. This leads to the conclusion that DRF is the most common fracture worldwide with a need for rehabilitation to return to functional ADLs . The phyA bibliometric analysis analyzed two-decade data on VR-aided therapy for health. A blended examination strategy was embraced for this exploration, utilizing a bibliometric investigation where a quantitative technique led to an overview outline of VR-assisted treatment, along with a literature review putting an impact on the qualitative insights of the application. The data cost is added to design further healthcare administrations and associations between healthcare counterparts utilizing elements of the game and presenting new exploration programs . Again, A review of RCTs concentrating on physical activity (PA) improvement in adolescents found that exergaming can be a successful tool to further develop PA in adolescents that could be more adequate and maintainable than numerous established procedures following its potential and interests. The resilience of this review lies in its incorporation of RCTs making it a higher level of evidence. In any case, involving exergaming to advance and impact adherence to PA or elements that obstruct or urge adolescents to play active video games has not yet been determined . GamificSystematic review and meta-analysis of randomized clinical trials of VR-based treatments for persistent low backache indicated that VR-based rehabilitation could significantly reduce pain intensity and kinesiophobia in chronic low backache patients. However, the application of VR-based rehabilitation is highly heterogeneous and can affect the outcomes. VR-based rehabilitation can be incorporated into clinical platforms with reference to its impact on the intensity of pain and kinesiophobia following short- and mid-term follow-ups in chronic low backache patients . A systeThe implication of the principle of motor learning in VR-based rehabilitation of patients with cerebral palsy was analyzed in a systematic review of 26 studies, and it was concluded that tailored VR-based rehabilitation programs customized for individual needs and requirements lead to a better functionally independent life. VR-based rehabilitation involves motor learning, including serious gaming as a rehabilitative platform to improve the functional capacity of the upper limbs . It boosA single-blinded prospective RCT was conducted on 51 Parkinson's patients to compare the efficacy of VR-based rehabilitation programs concluding with an improvement in upper limb function, gait adaptation, complex ambulation skills, and quality of life . The utiAn analysis of ambiguity applied as concepts for serious games and gamification in healthcare reviewed the literature systematically and also developed a guideline for gaming, i.e., Game-Based Intervention Reporting Guidelines. It involved an explicit analysis of 206 studies leading to a result of the application of gaming concepts as serious gaming in healthcare elevation. The outcomes highlighted that explicit definition is lacking more than half of the concept application of serious gaming. It is heterogenous in nature, followed by the ambiguity of different perspectives and concepts .The analysis of the literature builds an evolutionary process in the development of gamification in rehabilitation. The impact of the integration of gamification has an imperative effect on the physical activity of adolescents, as well as in patients with chronic low back pain, lower limb amputation, cerebral palsy, and traumatic brain injuries along with clinical practice. The expanded horizon of the application of gamification in rehabilitation is expanding on the basis of its efficacy in improving fine and gross motor skills. The immersive environment providing real-time feedback proves promising in improving the compliance and adaptability of long-term patient care. An effective response time, movement velocity, and precision of movement develop using gamification creating a powerful determination in a diseased condition. Integrating the heterogenous, cost-effective, and creative ways of the pertinence, the ambiguity of the application of gamification can be expanded and explored in a wide range, explaining its evolution in different variants in rehabilitation.


The pathophysiology of allergic rhinitis (AR), one of the most common diseases in the world, is still not sufficiently understood. Extracellular vesicles (EVs), which are secreted by host and bacteria cells and take part in near and distant intracellular communication, can provide information about AR. Recently, attention has been drawn to the potential use of EVs as biomarkers, vaccines, or transporters for drug delivery. In this review, we present an up-to-date literature overview on EVs in AR to reveal their potential clinical significance in this condition. A comprehensive and systematic literature search was conducted following PRISMA statement guidelines for original, completed articles, available in English concerning EVs and AR. For this purpose, PubMed/MEDLINE, Scopus, Web of Science, and Cochrane, were searched up until 10 Novenmber 2022. From 275 records, 18 articles were included for analysis. The risk of bias was assessed for all studies as low or moderate risk of overall bias using the Office and Health Assessment and Translation Risk of Bias Rating Tool for Human and Animal Studies. We presented the role of exosomes in the pathophysiology of AR and highlighted the possibility of using exosomes as biomarkers and treatment in this disease. Allergic rhinitis (AR) is one of the most common diseases, affecting 10–20% of people worldwide, and there is an upward trend in the incidence of the disease . AlthougExtracellular vesicles (EVs) play an important role in the intercellular transport of proteins, lipids, and nucleic acids, thus controlling various cellular processes ,7. In adIn this review, we aimed to comprehensively search the literature for studies that have identified exosomal cargo present in AR patients, the utility of exosomes as biomarkers, and the treatment of AR. It allowed us to specify the current state of knowledge of the role of EVs in AR and present clinical relevance and potential for further investigations.This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines .A comprehensive systematic literature search was conducted to retrieve studies from the electronic bibliographic databases PubMed, Scopus, Web of Science, and Cochrane according to a systematic predefined search strategy that included terms such as “allergic rhinitis,” “exosomes,” “exosome,” “extracellular vesicles,” “exosomal,” and “liquid biopsy.” The last search was conducted on 10 November 2022, for all databases. A detailed description of the search strategy for each database is shown in All citations identified for further evaluation were imported into the EndNote 20 reference management software package . Duplicates were found by the EndNote software and manually by two reviewers (KC and WM). The records were then screened sequentially by title, abstract, and full text by two authors independently (KC and WM) to find good-quality articles exploring exosomes in AR. Any disagreements were resolved after discussion by a third investigator (KD). The papers that were included in the data extraction met the inclusion and exclusion criteria . No restTwo investigators (KC and WM) independently filtered included studies considering the following study data: first author, year of publication, country of origin, study type, purpose, main study steps, main results, source of exosomes, methods of their isolation and characterization, exosomal markers, and cargo. Additionally, in studies involving human subjects, information was collected on who constituted the study group and who constituted the control group, the sizes of these groups, and the inclusion and exclusion criteria of the study group.https://ntp.niehs.nih.gov/ntp/ohat/pubs/riskofbiastool_508.pdf, accessed on 26 November 2022) by two independent investigators (KC and WM) and disagreements were resolved by discussion by the third researcher (KD). The summarised quality assessment for every single study is reported in The risk of bias in each study was assessed according to the modified Office and Health Assessment and Translation (OHAT) Risk of Bias Rating Tool for Human and Animal Studies  ,26,30,33Data extracted from people came from more than 800 participants (of which 460 were AR patients). Information on the participants of each included study, as well as on the inclusion and exclusion criteria of the study groups, are presented in A detailed assessment of the risk of bias for each study is shown in In recent years, there has been a surge in exosome research; therefore, guidelines have been established to introduce consistent principles for the isolation, purification, and characterization of EVs . Highlig9 vs. 9.58 × 108 per 106 MSCs), shorter isolation time (3 h vs. 15 h to process 600 mL of supernatant), lower cost, higher reproducibility, scalable possibility, no specialized equipment needed, and less damage to EVs [Isolation and purification methods were heterogeneous, but the most frequent was differential ultracentrifugation, proposed by Thery , to whice to EVs .Methods for analyzing exosomes also varied, but transmission electron microscopy (TEM) ,29,30,34Information on the storing method of exosomes was provided only in three included studies ,23,28, aThe researchers described EVs as spheroid, ovoid, or small cup-shaped structures ,21,22 wiThe effect of AR-Tfh-derived exosomes was explored in an in vivo study on a murine model . Four grDermatophagoides pteronyssinus protease) and the second was HLA-DR (Human Leukocyte Antigen—DR isotype)—a representative marker of APCs. Derp 1 levels on plasma EVs were strongly associated with symptom scores and type 2 cytokines (IL-13). Additionally, Derp 1 was also presented on EVs in nasal secretions of AR patients [Similar conclusions come from human studies . Studyinpatients .Studying the effect of EVs on the development of AR, the authors hypothesized that APCs may release EVs into circulation and mediate allergic reactions. To support this hypothesis, the authors investigated the expression of Derp 1 and molecules associated with EV antigen presentation in the plasma of house-dust-mite (HDM) -sensitive AR patients. As expected, Derp 1 was significantly higher on moderate–severe AR-EVs than on mild AR-EVs and EVs collected from healthy controls. Additionally, there was also detected the expression of costimulatory and antigen-presentation-related molecules . To sum Furthermore, exosomes derived from nasal epithelial cells, collected from patients diagnosed with atypical AR, were found to promote antigen-specific differentiation of CD8+ cells . Upon exIn turn, it was indicated that the imbalance of helper T cell differentiation is involved in the development of AR . Recent To sum up, most researchers confirmed that exosomes derived from AR patients influence Th1/Th2 differentiation, promote Th2-type inflammation, and are strongly associated with AR symptoms. Various miRNAs have been investigated as biomarkers or treatment targets in AR, such as miR-124-3p ,58, miR-In nasal-mucus-derived EVs, exactly 35 miRNAs were found differentially expressed in AR patients compared to healthy subjects (HS), of which 21 were upregulated and 14 were downregulated . SubsequOn the other hand, RNA was extracted from Tfhs-derived exosomes, and miR-142b and miR-142-5p were downregulated in exosomes from the AR group, compared to the control group . The autIn turn, the effect of miR-146a-5p in MSC-EVs was examined on allergic airway inflammation in mice . The autIn conclusion, recent research indicates that miR-146a can induce IL-10-producing monocytes to suppress the skewed Th2 polarization, suggesting that miR-146a has potential in the treatment of allergic disorders, such as AR .Long non-coding RNAs (LncRNAs) are non-coding transcripts more than 200 nucleotides long, with one or more short open reading frames, which encode functional micropeptides . The funRecently, LncRNAs were investigated as biomarkers in AR , becauseIt was demonstrated that fragments of non-coding RNA-Lnc00632—carried by some exosomes  are responsible for the suppression of Th2 differentiation in CD4+ T cells . TransfeThe role of other exosomal LncRNAs in AR pathogenesis was observed as well. A close correlation was noticed between LncRNA NEAT1 (Nuclear Paraspeckle Assembly Transcript 1) and the severity of AR and its overexpression in this group . UpregulOn the other hand, it was demonstrated that the next LncRNA–LncGAS5 was upregulated in AR epithelial samples and exosomes isolated from nasal mucus samples of AR patients and OVA-induced RPMI 2650 cells. They showed that LncGAS5 suppresses Th1 differentiation and promotes Th2 differentiation via the regulation of EZH2 and T-bet .Tfhs are a specialized CD4+ T cells subpopulation that play an important role in humoral immunity in allergic diseases by controlling immune responses to allergens ,79. SomeIn turn, in another study  it was iIn line with the studies above, the exosomes impact on DCs differentiation was confirmed based on adipose-stem-cell-derived EVs .In recent times, the therapeutic potential of MSCs in many diseases is being extensively investigated ,84,85,86Taking into consideration the potential MSC-EV therapy, the biggest challenges seem to be a huge number of homogeneous MSCs and a scalable isolation protocol for a large preparation of MSC-EVs . StandarIt has been confirmed that some immune-competent cells such as DCs and B lymphocytes secrete exosomes that contain antigens, enabling them to contact T cells ,91. BaseAllergen-specific immunotherapy (AIT) is the only treatment for AR that targets the underlying pathophysiology and has a disease-modifying effect ,93. Its Furthermore, an AR mouse model treated intranasally with CpG-OVA-EVs showed a statistically significant reduction in serum IgE levels and allergic symptoms such as rubbing and sneezing. Moreover, the mouse nasal sections showed less infiltration of inflammatory cells and thinner respiratory epithelium in the CpG-OVA-EV-treated group . Other researchers also investigated nasal drops of exosomes in AR therapy . Luo et AIT may be carried out subcutaneously (SCIT) or sublingually (SLIT) and is recommended in both seasonal and perennial AR . SCIT isThe miRNAs in serum-derived exosomes of AR children were investigated and it was noticed that hsa-miR-4669 was significantly downregulated in the effective group . MoreoveIn recent years, special attention has been paid to the role of the microbiome in allergic diseases, as it is believed that it can modulate the immune response  and coulUrinary EVs are relatively easy to obtain and have potential applications for determining biomarkers not only related to the urinary tract, but also neurological diseases such as Parkinson’s disease . In a study  where DNEscherichia-Shigella, Klebsiella, and Haemophilus) were significantly more abundant and positively correlated with total IgE and percentage of eosinophils [In another study , urinaryinophils . The above studies demonstrate the use of EVs from accessible, easily collected biological material such as urine in uncovering the relationship between microbiota and allergic diseases. To the best of our knowledge, this is the first systematic review describing the role of exosomes in AR. In recent years, interest has grown significantly regarding the mechanisms and role of EVs in various diseases, including AR, because EVs play a key role in intercellular communication. In this systematic review, we presented the current knowledge of the participation of exosomes in the pathophysiology of AR, summarized the underlying mechanisms, and highlighted the possibility of using exosomes as biomarkers in this disease and their potential use in treatment. We also introduced information about research related to the role of microbiota in AR, studied in both nasal and urine-derived exosomes. The main strength of this review is the systematic approach to identifying all research on the topic of EVs and AR. We have also included an assessment of the quality of the included studies. Concurrently, we also note the limitations of this systematic review. The included studies examined exosomes of various origins, both human and bacterial, which were isolated from various body fluids, secretions, and cell lines. Different methods of exosome isolation and various bioinformatics tools were used to analyze the resulting data. Due to the wide variety of included studies, we could only provide a narrative summary of the obtained data. The results of in vitro studies or in vivo studies on animals must be interpreted with caution as they do not always coincide with clinical trials on humans. Additionally, the cross-sectional character of some of the included studies ,27,31,32In the studies ,31, it wIn conclusion, in recent years, knowledge of EVs has expanded significantly to also include AR topics. A growing body of evidence suggests that various exosome-dependent mechanisms play an important role in the pathogenesis of AR and can be used as biomarkers in this disease. This review provides information on the latest developments from preclinical studies of exosome therapy in AR, which may lay the groundwork for future research and further development of AR treatment.


To demonstrate that surgical sperm retrieval (SSR) and spermatogonial stem cell retrieval (SSCR) in an oncological context are safe and successful.This a retrospective study in a tertiary hospital in the UK. Patients requiring fertility preservation from December 2017 to January 2020 were included. Data were analysed with Microsoft Excel 2016 and the Statistical Package for the Social Sciences (version 20).P = 0.07). However, age, hormonal profile and type of cancer did not predict SSR outcome.Among 1264 patients referred to the Reproductive Medical Unit at the University College of London Hospitals for cryopreservation prior to gonadotoxic treatment, 39 chose to go forward with SSR/SSCR because they presented as azoo‐/cryptozoospermic or an inability to masturbate/ejaculate. Interventions were testicular sperm extraction (23 patients) or aspiration (one), electroejaculation (one), and testicular wedge biopsy for SSCR (14). The median (range) age was 15.0 (10–65) years and the median testosterone level was 4.4 nmoL/L. Primary diagnoses were sarcoma in 11 patients, leukaemia in nine, lymphoma in eight, testicular tumour in five, other oncological haematological entities in two, other solid cancers in two, while two patients had non‐oncological haematological diseases. SSR/SSCR could be offered within 7.5 days on average. Chemotherapy could follow within 2 days from SSR/SSCR, and bone marrow transplant occurred within 19.5 days . The success rate for SSR was 68.0% . The mean (SD) Johnsen score of testicular biopsies was 5.23 (2.25) with a trend towards positive correlation with SSR success (We show that SSR and SSCR in an oncological context are valid treatment options with a high success rate for patients in which sperm cryopreservation from semen is impossible. By providing an effective pathway, fertility preservation is possible with minimal delay to oncological treatment. Fertility preservation (FP) in men and boys is increasingly recognised as a vital part of oncological treatment, with guidelines now recommending that FP is discussed with all patients about to undergo cancer or medical treatment that may affect their fertility , 2, 3.Currently, the only established clinical option for FP in these male patients is cryopreservation of sperm after masturbation. However, it is not always possible to obtain a semen specimen as a result of physical, psychological, or cultural factors or azoospermia at presentation.Traditionally, oncologists or haematologists have undertaken these initial discussions and organised for sperm cryopreservation, where deemed appropriate. However, if the patient is unable to ejaculate or found to be azoospermic this is frequently the end of the line for FP. There is often a concern that cancer treatment must be started as soon as possible, and any form of surgical sperm retrieval (SSR) will result in unacceptable delays. However, this does need to be weighed up with the excellent long‐term outcomes particularly for paediatric cancers .Men and adolescents who are at risk of future infertility but unable to produce a semen sample can be referred to a urologist for investigation and consideration of urgent SSR. Formal pathways and a fast referral service are uniquely offered at our hospital, whereby we aim to review any oncology patient within 24 h of referral and if needed a SSR is organised, which is expedited and carried out as a matter of urgency.This study aimed to evaluate the success rate for SSR and quantify the delay in cancer treatment for all male patients requiring FP and referred to the andrology team. In addition, the options available for younger boys who are either pre‐ or peri‐pubertal will also be discussed in this article.We conducted a retrospective cohort study involving patients treated at a single‐centre tertiary referral hospital, namely University College of London Hospital (UCLH), from December 2017 to January 2020. All patients who had been referred for semen cryopreservation prior to undergoing treatment with a high risk of subsequent infertility, including orchidectomy, chemotherapy and/or bone marrow transplant were included.Azoospermic (no sperm) or cryptozoospermic  patients (according to WHO standards) as well as patients unable to ejaculate, either because they were too unwell or actually suffering from anejaculation (inability to orgasm) were referred to a urologist for assessment and FP counselling. After referral, an initial meeting with the patient would be organised, usually within 24 h.Referrers were asked to organise urgent hormonal profiles , which ideally should be taken first thing in the morning and with the patient fasting. Sex hormone levels can be very deranged in some of the very unwell patients and can give an indication of poor spermatogenesis thus directing potential surgical treatments.In younger patients, clinical parameters, including Tanner stage and biochemical hormonal profile levels, as well as sensitive discussions on sexual function and masturbation, were used to assess the likelihood of spermarche and the chance of finding sperm at the time of the SSR. The Tanner stage is an objective classification system, which is a scale of physical development based on external secondary sexual characteristics, that aims to identify the stage of puberty that an adolescent is in. Biochemical hormonal levels may be useful to indicate spermatogenesis in these young patients .Prepubertal boys, in whom spermatogenesis would not yet be present, can be listed for a testicular biopsy in order to take a small amount of tissue to cryopreserve immature testicular tissue containing spermatogonial stem cells (SSCs), an approach that remains experimental.In a small number of cases where the urgency to start chemotherapy prevented FP being carried out, the collection of testicular tissue for SSC preservation between the first few treatment cycles of treatment may be considered. SSCs should still be present, and storage of the tissue will then give patients options for fertility restoration at some future time point, although it must be made clear that established clinical options for this are not yet available. Mature sperm in contrast should not be stored after commencing chemotherapy as this exposure could lead to mutagenic changes in the mature sperm . Whilst Fertility preservation options offered included electroejaculation (EEj), percutaneous epididymal sperm aspiration (PESA), testicular sperm aspiration (TESA), testicular sperm extraction (TESE), microTESE  or SSC Electroejaculation was performed under general anaesthesia for patients unable to ejaculate using the Seager Model 14 Electroejaculator . An anal electrode is inserted into the rectum and comes into contact with the region of the prostate and seminal vesicles. Electrical stimulations are administered in order to produce ejaculation as previously described . An embrIn those patients who had been able to ejaculate but found to have azoospermia, they would undergo a SSR. This usually involved a small 3‐cm incision on the scrotum and was carried out under a general anaesthetic, or local anaesthetic with sedation if the patient was considered unfit for a general anaesthetic.The least invasive procedure possible would always be carried out but ensuring enough sperm could be cryopreserved for multiple assisted reproductive cycles in the future. The secondary aim would be to limit surgical risks of haematoma or wound issues that might delay oncological treatment.The least invasive surgical procedure, a PESA was reserved for obstructive azoospermia cases, which is uncommon in this patient group. In cases of non‐obstructive azoospermia and cryptozoospermia, a small unilateral TESE was performed and then depending on the results relayed by the embryologists in theatre, a decision would be made on the necessity of using the operating microscope and carrying out a microTESE and if a bilateral testicle procedure was needed. Whatever the decision, this could all be carried out through the same, small scrotal incision. For testicular cancers, oncological TESE (onco‐TESE) was performed as previously described .Success rate for SSR was defined as enough sperm frozen for at least one intra‐cytoplasmic sperm injection (ICSI) cycle; that is at least one stored vial or straw cryopreserved.Boys who were clearly prepubertal may be offered SSCR by testicular biopsy and storage to preserve SSCs, although this remains experimental. The procedure involves a small scrotal incision and testicular wedge biopsy.In peri‐pubertal cases where spermatogenesis was uncertain, both the embryologist and the Oxford Cell and Tissue Biobank (OCTB) team would be present in theatre. A small testicle biopsy would be analysed intraoperatively and depending on the findings of mature spermatozoa present under the microscope, a decision would be made on suitability for either sperm cryopreservation or SSCR.Hospitals offering SSCR need to have an arrangement with a Biobank that holds a Human Tissue Authority (HTA) licence for the procurement, storage, and distribution of testicular tissue. The UCLH have such an agreement with the OCTB. The OCTB arrange for consent for tissue storage and attendance in theatre to collect and transport tissue to the OCTB for processing and storage. Cryopreservation of the SSC involves a controlled slow‐freezing protocol.P < 0.05) with inclusion following univariate multinomial logistic regression (significance level P < 0.20).Data were analysed with Microsoft Excel 2016 and Statistical Package for the Social Sciences (SPSS®), version 20.0 . The latter was used for step‐by‐step descending multinomial logistic regression  under HTA Human Application Licence, as part of the Future Fertility Programme Oxford (FFPO). The FFPO is a clinical programme and does not require research and development and ethical approval. The programme has been approved by OUHFT Technical Advisory Group and Clinical Ethics Group. The PanCareLIFE Consortium and the International Late Effects of Childhood Cancer Guideline Harmonisation Group  publisheFrom December 2017 to January 2020 1264 patients were referred for sperm cryopreservation prior to gonadotoxic treatment, of which 1140 attended. Among those patients, 58 (5.1%) were unsuccessful: 32 (2.8%) were azoospermic, nine (0.8%) had cryptozoospermia without viable sperm and 17 (1.5%) were unable to provide a semen sample on the day. Out of the 58 eligible patients, 39 (67.2%) chose to go forward with SSR/SSCR. The median (range) age was 15.0 (10–65) years and the median testosterone level was 4.4 nmoL/L, while the overall mean (SD) Johnsen score of testicular biopsies was 5.23 (2.25). Tanner stage of puberty, cancer type, and hormone levels are described in Tables The SSR or SSCR surgery was offered within a median (range) duration of 5.0 (0–19) days from referral. Chemotherapy was started within a median (range) of 2.0 (−115 to 41) days from the date of surgery.Five patients underwent their stem cell harvesting after starting chemotherapy, between the first and third cycles.When applicable, bone marrow transplant occurred within a median (range) of 19.5 (3–154) days from SSR or SSCR, while chemotherapy prior to bone marrow transplant could be started 1 or 2 days after surgery. There was no delay for chemotherapy treatment for any patient secondary to a surgical complication.Oncological primary diagnoses are summarised in Fig. Among patients undergoing SSR, 23 patients had TESE: including microTESE (13), onco‐TESE (five), unsuccessful EEj followed by standard TESE (four), and PESA followed by standard TESE (one). Other forms of SSR were TESA (one patient) and EEj only (one). The success rate for SSR overall was 68.0% (17/25) with a mean (SD) of 5.2 (4.7) vials/straws stored. The success rates were 60.0% for EEj ± TESE, 60.0% for onco‐TESE and 69.2% for microTESE. Failed SSR comprises failed EEj and failed microTESE. In all, 14 pre‐ or peripubertal boys had testicular wedge biopsy for SSCR after failure to observe sperm intraoperatively from a single biopsy (microTESEwas not attempted in this subset of patients). The detail of both SSR and SSCR is depicted in Fig. In our data set, the success rate of SSR did not correlate with either age or hormonal levels whether LH, FSH or testosterone but numbers are small to comment on significance. As for men initially found to be azoospermic, SSR was successful in two of five compared with three of four cryptozoospermic men, two of two anejaculating patients, and seven of nine unable to provide a sample.P = 0.07).Moreover, the underlying oncological diagnosis was not significantly correlated with success of SSR. Mean Johnsen score as expected showed a positive trend of correlation with SSR success , this is not possible for prepubertal boys. Currently, there are no proven clinical options for fertility preservation or restoration in this patient group . Over reWhilst survival of SSCs has been demonstrated within frozen–thawed tissues , functioAn alternative approach that has the added advantage of restoring natural fertility is SSC transplantation. This approach requires dissociation of the cryopreserved testicular tissue and isolation of SSCs. The SSCs can be injected directly into the seminiferous tubules after completion of treatment. Animal studies have shown that this is feasible and the SSCs re‐colonise the testis resulting in restoration of spermatogenesis and fertility  of males who had failed semen cryopreservation were able to have a successful SSR, giving themselves the potential for parenthood in the future. Emergency procedures were carried out within 7 days in 75% of men and all patients were able to commence further oncology treatment within 16 days (excluding the two deaths unrelated to the FP procedure). There was no delay to any cancer treatment secondary to surgical complications.This paper reinforces the need for collaboration between oncologists and urologists to improve access to male FP services. It also demonstrates that these FP options can be undertaken without significant delay for cancer treatment.Lionel A. Micol’s institution receives consulting fees from Coloplast AG and Debiopharm International SA, payment for expert testimony from Dialectica Ltd. and meeting support from Debiopharm International. Lionel A. Micol has stock options in Cabinet d'Urologie and Andrologie, Dr Lionel Micol, Sàrl. Rod T. Mitchell received payment from Novo Nordisk as a guest speaker; is a MHRA Expert Working Group Member, and a Society for Reproduction and Fertility – Council Member.EEjelectroejaculationFFPOFuture Fertility Programme OxfordFPfertility preservationHTAHuman Tissue AuthorityICSIintra‐cytoplasmic sperm injectionIVFin vitro fertilisationLHluteinising hormoneMicroTESEmicroscope‐assisted testicular sperm extractionOCTBOxford Cell and Tissue Biobank(onco‐)TESE testicular sperm extractionOUHFTOxford University Hospitals NHS foundation TrustPESApercutaneous sperm aspirationSSC(R)spermatogonial stem cell SSRsurgical sperm retrievalTESAtesticular sperm aspirationUCLHUniversity College of London Hospital