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To determine whether kinesthetic learning using a simulated pelvic model while learning the uterine manipulation maneuvers of a laparoscopic hysterectomy improves learning compared to an interactive video module alone. Randomized controlled study. Academic medical center. Forty, first-year and second-year medical students who had never performed uterine manipulation A low-cost simulated pelvic model for kinesthetic learning during an interactive video module. Forty, first-year and second-year medical students participated in the study, which included an interactive video module with formative assessment, summative assessment, cadaveric assessment, and qualitative post-participation survey. Participants were randomized to the intervention group (n=21) who used a simulated pelvic model for kinesthetic learning during the video module or the control group (n=19) who only had the video module to learn the uterine manipulation maneuvers of a laparoscopic hysterectomy. There was no difference between the control and intervention groups on final score, number of attempts, or time to completion of the summative assessment. On the cadaveric assessment, participants in the intervention group had fewer unnecessary movements with demonstration of both right pelvic sidewall (control 78.9%, intervention 42.9%, p<0.027) and left pelvic sidewall (control 94.7%, intervention 66.7%, p<0.046). This difference was increased in more novice first-year medical student participants (p<0.009). There was no difference between the control and intervention groups in the amount of force used, need for either verbal or physical cues, or time required to achieve each maneuver. Lastly, the participants in the intervention group reported higher perceived preparedness (100% versus 71.4% in control group, p<0.037). Kinesthetic practice may not be required for learning the uterine manipulation maneuvers of a laparoscopic hysterectomy, but it may be beneficial for more novice learners and to increase learners' perceived preparedness. Our interactive video module alone may be sufficient to prepare learners to perform uterine manipulation maneuvers prior to the operating room. [ABSTRACT FROM AUTHOR]

Study Objective: Several simulation models have been evaluated for gynecologic procedures such as hysterectomy, but there are limited published data for myomectomy. This study aimed to assess the validity of a low-cost robotic myomectomy model for surgical simulation training., Design: Prospective cohort simulation study., Setting: Surgical simulation laboratory., Participants: Twelve obstetrics and gynecology residents and 4 fellowship-trained minimally invasive gynecologic surgeons were recruited for a 3:1 novice-to-expert ratio., Interventions: A robotic myomectomy simulation model was constructed using <$5 worth of materials: a foam cylinder, felt, a stress ball, bandage wrap, and multipurpose sealing wrap. Participants performed a simulation task involving 2 steps: fibroid enucleation and hysterotomy repair. Video-recorded performances were timed and scored by 2 blinded reviewers using the validated Global Evaluative Assessment of Robotic Skills (GEARS) scale (5-25 points) and a modified GEARS scale (5-40 points), which adds 3 novel domains specific to robotic myomectomy. Performance was also scored using predefined task errors. Participants completed a post-task questionnaire assessing the model's realism and utility., Measurements and Main Results: Median task completion time was shorter for experts than novices (9.7 vs 24.6 min, p = .001). Experts scored higher than novices on both the GEARS scale (median 23 vs 12, p = .004) and modified GEARS scale (36 vs 20, p = .004). Experts made fewer task errors than novices (median 15.5 vs 37.5, p = .034). For interrater reliability of scoring, the intraclass correlation coefficient was calculated to be 0.91 for the GEARS assessment, 0.93 for the modified GEARS assessment, and 0.60 for task errors. Using the contrasting groups method, the passing mark for the simulation task was set to a minimum modified GEARS score of 28 and a maximum of 28 errors. Most participants agreed that the model was realistic (62.5%) and useful for training (93.8%)., Conclusion: We have demonstrated evidence supporting the validity of a low-cost robotic myomectomy model. This simulation model and the performance assessments developed in this study provide further educational tools for robotic myomectomy training., (Copyright © 2024 AAGL. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Financial Disclosure The author did not report any potential conflicts of interest.

Background: Hysterectomy is one of the most common gynecologic surgeries, with an increasing proportion of hysterectomies performed by a laparoscopic approach. Uterine manipulation is critical for patient safety and surgical efficiency; however, the most junior member of the surgical team assumes the responsibility of uterine manipulation, commonly without preparation. The objective of our study was to determine whether kinesthetic learning using a low-cost simulated pelvic model while learning the uterine manipulation maneuvers of a laparoscopic hysterectomy improves learning efficacy and application efficiency compared to an interactive video module alone., Methods: Our randomized control trial at an academic medical center included forty first-year and second-year medical students. Participants were randomized to the intervention group that used a low-cost simulated pelvic model for kinesthetic learning during the video module or the control group who only had the interactive video module to learn the uterine manipulation maneuvers of a laparoscopic hysterectomy., Results: Participants in the intervention group were less likely to make unnecessary movements with demonstration of both pelvic side walls (right wall: control 78.9%, intervention 42.9%, p < 0.027; left wall: control 94.7%, intervention 66.7%, p < 0.046), and this was more pronounced in novice first-year participants (p < 0.009). Additionally, participants in the intervention group reported higher perceived preparedness (100% versus 71.4% in control group, p < 0.037). However, there was no difference in verbal or physical cues required, time per task, or force used between the groups., Conclusion: Kinesthetic practice may not be required for learning the uterine manipulation maneuvers of a laparoscopic hysterectomy, but it may be beneficial for more novice learners and to increase learners' perceived preparedness. Our novel interactive video module alone may be sufficient to prepare learners to perform uterine manipulation maneuvers prior to the operating room., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

As our understanding of chronic pain conditions, including endometriosis-related pain and chronic pelvic pain evolves, the evaluation and management of patients should reflect our increasing appreciation of the role of central sensitization, comorbid conditions and biopsychosocial factors on the pain experience and treatment outcomes. This review provides a systematic approach to persistent pain in patients with endometriosis. Expanding the evaluation and treatment of endometriosis-related pain by all health care providers could limit unnecessary surgical interventions and best meet our patient's needs., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Simulation is an important adjunct to traditional surgical training, allowing for repetitive practice of new skills without compromising patient safety. Although several simulation models have been described and evaluated for gynecologic procedures, there is a lack of such models for laparoscopic myomectomy., Objective: This study aimed to design a low-cost, low-fidelity laparoscopic myomectomy simulation model and to assess the model's validity as a training tool., Study Design: The model was constructed using a "cup turner" foam cylinder, felt, a 2-inch stress ball, self-adhesive bandage wrap, multipurpose sealing wrap, red marker, and hook-and-loop fastener. Participants were recruited at a quaternary care academic center and at the Society for Gynecologic Surgeons Annual Scientific Meeting. The simulation task involved the following 2 steps: fibroid enucleation and hysterotomy repair. Validity evidence was collected by comparing expert and novice simulation task performances. Video recordings were scored by 2 blinded reviewers using the Global Operative Assessment of Laparoscopic Skills scale (5-20 points) and a modified Global Operative Assessment of Laparoscopic Skills scale (5-35 points), incorporating 3 novel domains specific to laparoscopic myomectomy. The Mann-Whitney U test was used to compare the task completion times and performance scores. Interrater reliability of scoring was assessed using the interclass correlation coefficient. Validity was also assessed with a post-task survey regarding the model's realism, utility, and educational effect., Results: The total cost to construct each model was under $5. A 3:1 ratio was used to recruit 15 novices and 5 experts. The median time to task completion was shorter for experts than for novices (11.8 vs 20.1 minutes; P=.004). The experts scored higher than the novices on both the Global Operative Assessment of Laparoscopic Skills scale (median 19 [range 13-20] vs 10 [6-17.5]; P=.007) and the modified Global Operative Assessment of Laparoscopic Skills scale (31.5 [21.5-33.5] vs 18.5 [13.5-32]; P=.009). The interclass correlation coefficient was 0.95 for the Global Operative Assessment of Laparoscopic Skills scores and 0.96 for the modified Global Operative Assessment of Laparoscopic Skills scores. Most of the participants agreed that the model closely approximated the feel of fibroid enucleation (70% [14/20]) and suturing the uterus (80% [16/20]). All the participants agreed that the model was useful for learning or teaching laparoscopic myomectomy., Conclusion: This study demonstrates evidence supporting the validity of a novel, low-cost laparoscopic myomectomy model and a novel assessment scale for laparoscopic myomectomy training. This simulation model provides a targeted training tool that allows learners to focus on the key aspects of laparoscopic myomectomy and may improve readiness for the operating room., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose of Review: Endometriosis is a complex benign gynaecologic condition with heterogenous presentations and a large impact on the global healthcare system and on the quality of life for millions of women. Currently, the gold standard for diagnosis involves direct visualization of lesions during surgery confirmed by histopathological diagnosis, resulting in an average delay in its initial diagnosis of 8-10 years. Therefore, the search for noninvasive diagnostic testing options has been subject to a large body of research., Recent Findings: Multiple potential biomarkers have been explored for noninvasive testing for endometriosis, including glycoproteins, inflammatory cytokines, immunological molecules, angiogenesis markers, hormones, micro RNAs (miRNAs), proteomics, metabolomics, genomics and the microbiome., Summary: Although there are challenges to consider, areas for real promise and advancement in the noninvasive diagnosis of endometriosis are currently being explored with real promise in the area of miRNAs, proteomics, metabolomics, genomics and the microbiome., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Study Objective: To gather validity evidence for and determine acceptability of Surgical Science-Simbionix Hysterectomy Modules for the DaVinci Xi console simulation system (software; 3D Systems by Simbionix [now Surgical Science-Simbionix], Littleton, CO, and hardware; Intuitive Surgical, Inc., Sunnyvale, CA) and evaluate performance benchmarks between novice and experienced or expert surgeons., Design: Prospective education study (Messick validity framework)., Setting: Multicenter, academic medical institutions., Participants: Residents, fellows, and faculty in obstetrics and gynecology were invited to participate at 3 institutions. Participants were categorized by experience level: fewer than 10 hysterectomies (novice), 10 to 50 hysterectomies (experienced), and more than 50 hysterectomies (expert). A total of 10 novice, 10 experienced, and 14 expert surgeons were included., Interventions: Participants completed 4 simulator modules (ureter identification, bladder flap development, colpotomy, complete hysterectomy) and a qualitative survey. Simulator recordings were reviewed in duplicate by educators in minimally invasive gynecologic surgery using the Modified Global Evaluative Assessment of Robotic Skills (GEARS) rating scale., Measurements and Main Results: Most participants felt that the simulator realistically simulated robotic hysterectomy (64.7%) and that feedback provided by the simulator was as or more helpful than feedback from previous simulators (88.2%) but less helpful than feedback provided in the operating room (73.5%). Participants felt that this simulator would be helpful for teaching junior residents. Simulator-generated metrics correlated with GEARS performance for the bladder flap and ureter identification modules in multiple domains including total movements and total time for completion. GEARS performance for the bladder flap module correlated with experience level (novice vs experienced/expert) in the domains of interest and total score but did not consistently correlate for the other procedural modules. Performance benchmarks were evaluated for the bladder flap module for each GEARS domain and total score., Conclusion: The modules were well received by participants of all experience levels. Individual simulation modules appear to better discriminate between novice and experienced/expert users than overall simulator performance. Based on these data and participant feedback, the use of individual modules in early residency education may be helpful for providing feedback and may ultimately serve as 1 component of determining readiness to perform robotic hysterectomy., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 AAGL. Published by Elsevier Inc. All rights reserved.)

Objective: To review the current status of robotic training and the impact of various training platforms on the performance of robotic surgical trainees., Data Sources: Literature review of Google Scholar and PubMed. The search terms included a combination of the following: "robotic training," "simulation," "robotic curriculum," "obgyn residency robotic training," "virtual reality robotic training," "DaVinci training," "surgical simulation," "gyn surgical training." The sources considered for inclusion included peer-reviewed articles, literature reviews, textbook chapters, and statements from various institutions involved in resident training., Methods of Study Selection: A literature search of Google Scholar and PubMed using terms related to robotic surgery and robotics training, as mentioned in the "Data Sources" section., Results: Multiple novel platforms that use machine learning and real-time video feedback to teach and evaluate robotic surgical skills have been developed over recent years. Various training curricula, virtual reality simulators, and other robotic training tools have been shown to enhance robotic surgical education and improve surgical skills. The integration of didactic learning, simulation, and intraoperative teaching into more comprehensive training curricula shows positive effects on robotic skills proficiency. Few robotic surgery training curricula have been validated through peer-reviewed study, and there is more work to be completed in this area. In addition, there is a lack of information about how the skills obtained through robotics curricula and simulation translate into operating room performance and patient outcomes., Conclusion: Data collected to date show promising advances in the training of robotic surgeons. A diverse array of curricula for training robotic surgeons continue to emerge, and existing teaching modalities are evolving to keep up with the rapidly growing demand for proficient robotic surgeons. Futures areas of growth include establishing competency benchmarks for existing training tools, validating existing curricula, and determining how to translate the acquired skills in simulation into performance in the operating room and patient outcomes. Many surgical training platforms are beginning to expand beyond discrete robotic skills training to procedure-specific and team training. There is still a wealth of research to be done to understand how to create an effective training experience for gynecologic surgical trainees and robotics teams., (Copyright © 2020. Published by Elsevier Inc.)

Background and Objective: Patients with abnormal uterine bleeding (AUB) often require hysteroscopy as part of the diagnostic workup. The purpose of this study was to improve efficiency by shortening the time to patient appointment for office hysteroscopy., Interventions: Preintervention, nurse practitioners (NPs) and gynecologists saw patients with abnormal uterine bleeding (AUB) separately. This created inefficiency for the patients, frequently requiring second visits for hysteroscopy. A new hysteroscopy clinic was designed to increase practice efficiency. A collaborative team model was created including consecutive visits with NPs and gynecologists. Each patient with AUB was first evaluated by an NP, followed immediately by a shorter visit with a gynecologist for office hysteroscopy as indicated. NPs managed other diagnostic evaluation and bleeding treatment if hysteroscopy was not warranted. Collaborative clinic staffing consisted of two NPs seeing patients with AUB paired with one gynecologist for procedural support., Measurements and Results: Electronic records of 393 patients scheduled for AUB visits from January to June 2015 were evaluated for preintervention data. Postintervention, 647 patient records were reviewed from January to June of 2016. During the preintervention period, 30% of patients had a hysteroscopy appointment scheduled within 0-13 days from the initial visit for AUB. Postintervention, the wait time for appointments decreased, with 63% of patients scheduled within 0-13 days. Clinic redesign also resulted in an increase of 57.5% in appointment slots. No-show rates and appointment fill rates were not adversely affected., Conclusions: A collaborative team-based care model using NPs and gynecologists improved efficiency and access to office hysteroscopy services.

Objective: The primary objective is to assess psychomotor aptitude of medical students interested in pursuing a procedural career. Secondary objectives include exploring the relationship between actual and perceived aptitude, and identifying predictors of superior aptitude., Design: This is a cross-sectional, multisite study in which participants completed a paper survey, four visuospatial aptitude assessments, and a laparoscopic simulation modeled after the Fundamentals of Laparoscopic Surgery (FLS) peg transfer test (used as a proxy for psychomotor aptitude)., Setting: Johns Hopkins University School of Medicine and Mayo Clinic School of Medicine., Participants: All second-year medical students who had not yet initiated clinical rotations were eligible. Sixty-four students participated., Results: Students interested in a procedural career exhibited superior psychomotor aptitude (faster FLS task completion time), and a majority of these students correctly identified themselves as having above-average aptitude compared with peers. However, over one quarter of all students, regardless of career interest, incorrectly over- or under-rated their psychomotor aptitude. Upon completing their preclinical curriculum, a minority of students felt prepared to participate or assist in their surgical clinical rotations., Conclusions: Prior to embarking on their clinical rotations, over one quarter of medical students lack awareness of their psychomotor aptitude and many do not feel prepared to participate in the next phase of their training. Early aptitude testing and introduction to laparoscopic training may assist in career selection, preparedness, and success., (Copyright © 2019 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.)

Introduction and Hypothesis: The objective was to discuss the importance of apical suspension following vaginal hysterectomy and demonstrate a surgical model to aide in educating learners on a variety of apical suspension procedures., Methods: Rates of pelvic organ prolapse are not insignificant following hysterectomy. Re-support of the vaginal apex should be performed at the time of hysterectomy in those with or without a diagnosis of prolapse. Exposure to vaginal apical support procedures may be limited owing to declining rates of vaginal hysterectomy and limited trainee work hours. Surgical models are increasingly being used to supplement operating room experience. The model we present was originally developed for hysterectomy, although its design allows for teaching a variety of apical support procedures that incorporate the uterosacral ligament (USL) for support. We demonstrate performing a USL suspension, internal McCall suture, and modified McCall suture using the model., Results: The model is constructed from readily available supplies, is multi-use, and inexpensive. It allows learners to identify relevant anatomy, understand/visualize surgical steps, and practice suturing technique., Conclusion: Pelvic organ prolapse is common in women, although opportunities to teach apical suspension procedures may be limited. The proposed vaginal surgery simulator can be used to supplement the experience of gynecological surgery trainees with apical suspension procedures.

Objective: To assess the effect of using of asynchronous video interviewing as a screening tool for obstetrics and gynecology residency selection., Methods: This project was part of a quality-improvement effort to enhance the resident application process. Applications to a single obstetrics and gynecology residency program were scored using standardized criteria. In the 2018 Match, top-scored applicants were invited to in-person interviews, and second-tier applicants were asked to complete a three-question asynchronous video interview. Video interviews were scored and used to invite the remaining applicants for in-person interviews. In the 2019 Match, video interviewing was expanded to all applicants with top application scores, and the video score was used to determine in-person interview invitations. Applicants for 2019 were surveyed on their views regarding video interviewing., Results: Half of the candidates interviewed in person in the 2018 season were screened by the video interview process compared with 82% in the 2019 season. The mean in-person interview score increased from 59.0 in 2017, before screening with asynchronous video interviews, to 62.2 in 2018 (effect size 0.50; 95% CI 0.09-0.90) In 2018, a nonsignificant correlation was seen between the video interview score and rank list percentile (r=0.22, P=.15, n=27) and in-person interview score (r=0.18, P=.12, n=46). United States Medical Licensing Examination step 1 and 2 scores were not correlated with video interview scores in either season. Most (58%) candidates indicated that the asynchronous video interview was an effective way to tell their story; however, only 42% were comfortable with the interview process., Conclusion: Video interviewing may have promise as a tool for program directors to use to select candidates from a competitive applicant pool by measuring important noncognitive skills. Acceptance by obstetrics and gynecology applicants was mixed.

Purpose: Caring for women with chronic pelvic pain (CPP) is challenging. There have been few studies on what factors patients consider to be important when being treated for their pelvic pain. This study sought to identify the key factors of the health care visit that contribute to patient's overall satisfaction with their care in a CPP clinic. Materials and methods: Between January 2015 and December 2016, new patients visiting a tertiary care CPP clinic were recruited to complete a patient satisfaction survey. Inductive thematic analysis was performed on response data regarding important factors that impact patient satisfaction with their visit/care. Results: Five themes of patient satisfaction identified included: providers with a compassionate and caring attitude, being listened to, clear communication with collaboration when needed, quality time spent with patient, and having a plan of care with recommendations. The theme regarding provider's compassion and listening skills was the most frequently identified. Pain relief was seldom mentioned as a source of patient satisfaction. Conclusions: The data suggest that a focus on empathic communication may make a meaningful difference in meeting the needs of women with CPP as well as strengthening the provider/patient relationship.

Background: Vaginal diazepam is frequently used to treat pelvic floor tension myalgia and pelvic pain despite limited knowledge of systemic absorption., Aim: To determine the pharmacokinetic and adverse event profile of diazepam vaginal suppositories., Methods: We used a prospective pharmacokinetic design with repeated assessments of diazepam levels. Eight healthy volunteers were administered a 10-mg compounded vaginal diazepam suppository in the outpatient gynecologic clinic. Serum samples were collected at 0, 45, 90, 120, and 180 minutes; 8, 24, and 72 hours; and 1 week following administration of a 10-mg vaginal suppository. The occurrence of adverse events was assessed using the alternate step and tandem walk tests, the Brief Confusion Assessment Method, and numerical ratings. Plasma concentrations of diazepam and active long-acting metabolites were measured. Pharmacokinetic parameters were calculated by standard noncompartmental methods., Results: The mean peak diazepam concentration (C max ) of 31.0 ng/mL was detected at a mean time (T max ) of 3.1 hours after suppository placement. The bioavailability was found to be 70.5%, and the mean terminal elimination half-life was 82 hours. The plasma levels of temazepam and nordiazepam peaked at 0.8 ng/mL at 29 hours and 6.4 ng/mL at 132 hours, respectively. Fatigue was reported by 3 of 8 participants., Clinical Implications: Serum plasma concentrations of vaginally administered diazepam are low; however the half-life is prolonged., Strengths & Limitations: Strengths include use of inclusion and exclusion criteria aimed at mitigating clinical factors that could adversely impact diazepam absorption and metabolism, and the use of an ultrasensitive LC-MS/MS assay. Limitations included the lack of addressing the efficacy of vaginal diazepam in lieu of performing a pure pharmacokinetic study with healthy participants., Conclusion: Vaginal administration of diazepam results in lower peak serum plasma concentration, longer time to peak concentration, and lower bioavailability than standard oral use. Providers should be aware that with diazepam's long half-life, accumulating levels would occur with chronic daily doses, and steady-state levels would not be reached for up to 1 week. This profile would favor intermittent use to allow participation in physical therapy and intimacy. Larish AM, Dickson RR, Kudgus RA, et al. Vaginal Diazepam for Nonrelaxing Pelvic Floor Dysfunction: The Pharmacokinetic Profile. J Sex Med 2019;16;763-766., (Copyright © 2019. Published by Elsevier Inc.)

Background: In 2005, in response to a decline in residency applications in obstetrics and gynecology (OB GYN), the American College of Obstetrics and Gynecology Presidential Task Force outlined strategies for attracting medical students to OB GYN. Application rates have increased since then, but little is known about which interventions are effective. We aimed to identify modifiable and nonmodifiable variables that may contribute to students choosing OB GYN for their careers; this information could be used to inform curriculum design, faculty development, and innovative exposures to women's health., Methods: This qualitative study received institutional review board approval. Eligible participants were students who applied or recently matched into OB GYN residency programs from the class of 2014-2016 at our institution. Students were interviewed with open-ended questions and a Likert-type survey. Thematic analysis was performed., Results: Ten qualitative interviews were completed and analyzed. Intrinsic themes such as the potential for a meaningful job in women's health, advocacy for women, or empowerment of women were identified as factors contributing to participant career choice. Extrinsic themes such as positive impressions during the clinical clerkship and welcoming teams were also identified. Most students indicated that the clerkship was the most influential experience., Conclusions: Participants identified important events, including some that even preceded medical school that guided them toward OB GYN. The data guide us to consider the importance of emphasizing the unique combination of characteristics in OB GYN and improving the learning environment in the clerkship as a way to encourage student recruitment.

This systematic review and meta-analysis compares hysteroscopic morcellation with electrosurgical resection to treat uterine cavitary lesions. A search of Ovid MEDLINE, Ovid Embase, Scopus, and Web of Science was conducted through August 18, 2015, for randomized controlled trials (RCTs) and prospective and retrospective studies, regardless of surgical indication and study language or sample size. Seven studies were eventually included (4 RCTs and 3 retrospective observational studies), enrolling 650 women. The meta-analysis showed that the total procedure time was significantly shorter for morcellation than for resection (weighted mean difference = 9.36 minutes; 95% confidence interval [CI], -15.08 to -3.64). When reviewing RCTs only, intrauterine morcellation was associated with a smaller fluid deficit and lower odds of incomplete lesion removal. This difference was not statistically significant in observational studies. There was no significant difference in the odds of surgical complications (odds ratio = 0.72; 95% CI, 0.20-2.57) or the number of insertions (weighted mean difference = -3.04; 95% CI, -7.86-1.78). In conclusion, compared with hysteroscopic resection, hysteroscopic morcellation is associated with a shorter operative time and possibly lower odds of incomplete lesion removal. The certainty in evidence was limited by heterogeneity and the small sample size., (Copyright © 2016 AAGL. Published by Elsevier Inc. All rights reserved.)

The Robotic Objective Structured Assessment of Technical Skills (R-OSATS) is a previously validated assessment tool that is used to assess 5 standardized inanimate robotic surgery drills. R-OSATS is used to evaluate performance on surgical drills, with scores of 0 to 20 for each drill. Our objective was to establish the minimum threshold score that denotes competence on these drills. Thus, we performed a standard setting study using data from surgeons and trainees in 8 academic medical centers. Cutoff scores for the minimal level of competence using R-OSATS were established using 2 techniques: the modified Angoff and the contrasting groups methods. For the modified Angoff method, 8 content experts met and, in an iterative process, derived the scores that a minimally competent trainee should receive. After 2 iterative rounds of scoring and discussion with the modified Angoff method, we established a minimum competence score per drill with high agreement (rWG range, 0.92-0.98). There was unanimous consensus that a trainee needs to achieve competence on each independent drill. A second method, the contrasting groups method, was used to verify our results. In this method, we compared R-OSATS scores from "inexperienced" (34 postgraduate year 1 and 2 trainees) with "experienced" (22 faculty and fellow) robotic surgeons. The distributions of scores from both groups were plotted, and a cutoff score for each drill was determined from the intersection of the 2 curves. Using this method, the minimum score for competence would be 14 per drill, which is slightly more stringent but confirms the results obtained from the modified Angoff approach. In conclusion, using 2 well-described standard setting techniques, we have established minimum benchmarks designating trainee competence for 5 dry lab robotic surgery drills., (Copyright © 2016 AAGL. Published by Elsevier Inc. All rights reserved.)

Objective: Objective Structured Assessments of Technical Skills have been developed to measure the skill of surgical trainees. Our aim was to develop an Objective Structured Assessments of Technical Skills specifically for trainees learning robotic surgery., Methods: This is a multiinstitutional study conducted in eight academic training programs. We created an assessment form to evaluate robotic surgical skill through five inanimate exercises. Gynecology, general surgery, and urology residents, Fellows, and faculty completed five robotic exercises on a standard training model. Study sessions were recorded and randomly assigned to three blinded judges who scored performance using the assessment form. Construct validity was evaluated by comparing scores between participants with different levels of surgical experience; interrater and intrarater reliability were also assessed., Results: We evaluated 83 residents, nine Fellows, and 13 faculty totaling 105 participants; 88 (84%) were from gynecology. Our assessment form demonstrated construct validity with faculty and Fellows performing significantly better than residents (mean scores 89±8 faculty, 74±17 Fellows, 59±22 residents; P<.01). In addition, participants with more robotic console experience scored significantly higher than those with fewer prior console surgeries (P<.01). Robotic Objective Structured Assessments of Technical Skills demonstrated good interrater reliability across all five drills (mean Cronbach's α 0.79±0.02). Intrarater reliability was also high (mean Spearman's correlation 0.91±0.11)., Conclusion: We developed a valid and reliable assessment form for robotic surgical skill. When paired with standardized robotic skill drills, this form may be useful to distinguish between levels of trainee performance., Level of Evidence: II.

The objectives of this review were to analyze the literature describing the benefits of minimally invasive gynecologic surgery in obese women, to examine the physiologic considerations associated with obesity, and to describe surgical techniques that will enable surgeons to perform laparoscopy and robotic surgery successfully in obese patients. The Medline database was reviewed for all articles published in the English language between 1993 and 2013 containing the search terms "gynecologic laparoscopy" "laparoscopy," "minimally invasive surgery and obesity," "obesity," and "robotic surgery." The incidence of obesity is increasing in the United States, and in particular morbid obesity in women. Obesity is associated with a wide range of comorbid conditions that may affect perioperative outcomes including hypertension, atherosclerosis, angina, obstructive sleep apnea, and diabetes mellitus. In obese patients, laparoscopy or robotic surgery, compared with laparotomy, is associated with a shorter hospital stay, less postoperative pain, and fewer wound complications. Specific intra-abdominal access and trocar positioning techniques, as well as anesthetic maneuvers, improve the likelihood of success of laparoscopy in women with central adiposity. Performing gynecologic laparoscopy in the morbidly obese is no longer rare. Increases in the heaviest weight categories involve changes in clinical practice patterns. With comprehensive and thoughtful preoperative and surgical planning, minimally invasive gynecologic surgery may be performed safely and is of particular benefit in obese patients., (Copyright © 2014 AAGL. Published by Elsevier Inc. All rights reserved.)

Study Objective: To analyze the learning curves of gynecology trainees on several virtual reality da Vinci Skills Simulator exercises., Design: Prospective cohort pilot study., Setting: Academic hospital-based gynecology training program., Participants: Novice robotic surgeons from a gynecology training program., Methods: Novice robotic surgeons from an academic gynecology training program completed 10 repetitions of 4 exercises on the da Vinci Skills Simulator: matchboard, ring and rail, suture sponge, and energy switching. Performance metrics measured included time to completion, economy of instrument movement, excessive force, collisions, master workspace range, missed targets, misapplied energy, critical errors, and overall score. Statistical analyses were conducted to define the learning curve for trainees and the optimal number of repetitions for each exercise., Results: A total of 34 participants were enrolled, of which 9 were medical students, 22 were residents, and 3 were fellows. There was a significant improvement in performance between the 1st and 10th repetitions across multiple metrics for all exercises. Senior trainees performed the suture exercise significantly faster than the junior trainees during the first and last repetitions (p = 0.004 and p = 0.003, respectively). However, the performance gap between seniors and juniors narrowed significantly by the 10th repetition. The mean number of repetitions required to achieve performance plateau ranged from 6.4 to 9.3., Conclusion: Virtual reality robotic simulation improves ability through repetition at all levels of training. Further, a performance plateau may exist during a single training session. Larger studies are needed to further define the most high-yield simulator exercises, the ideal number of repetitions, and recommended intervals between training sessions to improve operative performance., (Copyright © 2014 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.)

Introduction and Hypothesis: Our aim was to assess the impact of immediate preoperative laparoscopic warm-up using a simulator on intraoperative laparoscopic performance by gynecologic residents., Methods: Eligible laparoscopic cases performed for benign, gynecologic indications were randomized to be performed with or without immediate preoperative warm-up. Residents randomized to warm-up performed a brief set of standardized exercises on a laparoscopic trainer immediately before surgery. Intraoperative performance was scored using previously validated global rating scales. Assessment was made immediately after surgery by attending faculty who were blinded to the warm-up randomization., Results: We randomized 237 residents to 47 minor laparoscopic cases (adnexal/ tubal surgery) and 44 to major laparoscopic cases (hysterectomy). Overall, attendings rated upper-level resident performances (postgraduate year [PGY-3, 4]) significantly higher on global rating scales than lower-level resident performances (PGY-1, 2). Residents who performed warm-up exercises prior to surgery were rated significantly higher on all subscales within each global rating scale, irrespective of the difficulty of the surgery. Most residents felt that performing warm-up exercises helped their intraoperative performances., Conclusion: Performing a brief warm-up exercise before a major or minor laparoscopic procedure significantly improved the intraoperative performance of residents irrespective of the difficulty of the case.

Background and Objectives: Our objectives were to compare the utility of learning a suturing task on the virtual reality da Vinci Skills Simulator versus the da Vinci Surgical System dry laboratory platform and to assess user satisfaction among novice robotic surgeons., Methods: Medical trainees were enrolled prospectively; one group trained on the virtual reality simulator, and the other group trained on the da Vinci dry laboratory platform. Trainees received pretesting and post-testing on the dry laboratory platform. Participants then completed an anonymous online user experience and satisfaction survey., Results: We enrolled 20 participants. Mean pretest completion times did not significantly differ between the 2 groups. Training with either platform was associated with a similar decrease in mean time to completion (simulator platform group, 64.9 seconds [P = .04]; dry laboratory platform group, 63.9 seconds [P < .01]). Most participants (58%) preferred the virtual reality platform. The majority found the training "definitely useful" in improving robotic surgical skills (mean, 4.6) and would attend future training sessions (mean, 4.5)., Conclusion: Training on the virtual reality robotic simulator or the dry laboratory robotic surgery platform resulted in significant improvements in time to completion and economy of motion for novice robotic surgeons. Although there was a perception that both simulators improved performance, there was a preference for the virtual reality simulator. Benefits unique to the simulator platform include autonomy of use, computerized performance feedback, and ease of setup. These features may facilitate more efficient and sophisticated simulation training above that of the conventional dry laboratory platform, without loss of efficacy.

Hyperlipidemia in the general population has been linked to the development of chronic kidney disease with both oxidative and endoplasmic reticulum stress implicated. Physiological levels (50-300 micromol/L) of saturated fatty acids such as palmitic acid (PA) cause cytotoxicity in vitro. We investigated cell type- and stimulus-specific signaling pathways induced by PA in renal proximal tubular cells and whether oxidative stress leads to ER stress or vice versa and which pathways predominate in signaling for PA-induced apoptosis and necrosis. NRK-52E cells were incubated with PA or hydrogen peroxide (H(2)O(2)) combined with SP600125 which blocks c-Jun N-terminal kinase (JNK) activation; salubrinal, which maintains eukaryotic initiation factor 2 alpha in its phosphorylated state and the antioxidant EUK-134 - a superoxide dismutase mimetic with catalase activity. We found that (i) PA causes both oxidative and ER stress leading to apoptosis which is mediated by phosphorylated JNK; (ii) oxidant-induced apoptosis generated by H(2)O(2) involves ER stress signaling and CHOP expression; (iii) the ER stress mediated by PA is largely independent of oxidative stress; (iv) in contrast, the apoptosis produced by PA is mediated partly via oxidative stress. PA-mediated cell signaling in renal NRK-52E cells therefore differs from that identified in neuronal, hepatic and pancreatic beta cells., ((c) 2010 Elsevier Inc. All rights reserved.)

Chronic pelvic pain (CPP) has many potential causes and is often a complex disorder with multiple contributing etiologies. The evaluation and treatment of women with CPP often requires a multidimensional approach. The treatment of CPP consists of two approaches: 1) treatment of pain itself or global treatment, and 2) treatment of disease-specific etiologies. Most often, treatment requires a combination of both approaches. This article reviews recent literature in the global treatment of CPP, including pharmacologic, psychotherapy, and neuroablative, as well as specific interventions for endometriosis, interstitial cystitis, pelvic adhesive disease, adenomyosis, and pelvic venous congestion.

Unsaturated fatty acids may counteract the lipotoxicity associated with saturated fatty acids. Palmitic acid induced endoplasmic reticulum (ER) stress and caused apoptotic and necrotic cell death in the renal proximal tubular cell line, NRK-52E. We investigated whether alpha-linolenic acid, an unsaturated fatty acid, protected against ER stress and cell death induced by palmitic acid or by other non-nutrient ER stress generators. Incubation of NRK-52E cells for 24h with palmitic acid produced a significant increase in apoptosis and necrosis. Palmitic acid also increased levels of three indicators of ER stress - the phosphorylated form of the eukaryotic initiation factor 2alpha (eIF2alpha), C/EBP homologous protein (CHOP), and glucose regulated protein 78 (GRP78). alpha-Linolenic acid dramatically reduced cell death and levels of all three indicators of ER stress brought about by palmitic acid. Tunicamycin, which induces ER stress by glycosylation of proteins, produced similar effects to those obtained using palmitic acid; its effects were partially reversed by alpha-linolenic acid. Salubrinal (a phosphatase inhibitor) causes increased levels of the phosphorylated form of eIF2alpha - this effect was partially reversed by alpha-linolenic acid. Palmitoleate, a monosaturated fatty acid, had similar effects to those of alpha-linolenic acid. These results suggest that part of the mechanism of protection of the kidney by unsaturated fatty acids is through inhibition of ER stress, eIF2alpha phosphorylation and consequential reduction of CHOP protein expression and apoptotic renal cell death.

The use of laparoscopy to perform all or part of hysterectomy has become widely accepted, with laparoscopic hysterectomy accounting for up to 15% of all hysterectomies performed in the United States. A recent Cochrane analysis has clearly shown that laparoscopic hysterectomy is associated with decreased length of stay and faster recovery time compared with laparotomy. There is no evidence to support a supracervical hysterectomy over a total hysterectomy in terms of frequency of pelvic support disorders or sexual function. This does not preclude the use of a supracervical hysterectomy in some clinical situations.

We sought to compare the complications and efficacy of tension-free vaginal tape (TVT) and Pelvilace. Chart reviews and telephone questionnaires documented complications, urinary symptoms [Urogenital Distress Inventory short form (UDI-6) and Patient Global Impression of Improvement (PGII)], and re-operations. Failure was defined as PGII > or = 4 (no change or worse) or reoperation for urinary incontinence. There were 91 TVT and 22 Pelvilace slings. The TVT group had lower rates of urinary retention beyond 1 week (8% vs. 43%, p < 0.001) and reoperation for retention (1% vs. 19%, p < 0.001). Median changes in UDI-6 scores were -33.3 for the TVT group and -29.6 for the Pelvilace group (p = 0.21). Median PGII scores were 1 for the TVT group and 2 for the Pelvilace group (p = 0.024). Ten percent of the TVT group and 24% of the Pelvilace group (p = 0.095) were failures. Urinary retention was more common after Pelvilace than TVT slings. Symptomatic outcomes and failure rates were similar.

We have previously shown that saturated fatty acids induce DNA damage and cause apoptotic cell death in insulin-producing beta-cells. Here we examine further the effects of single or combined dietary fatty acids on RINm5F survival or cell death signalling. Palmitate and stearate, but not linoleate, oleate or palmitoylmethyl ester, induced growth inhibition and increased apoptosis in RINm5F cells following 24 h exposure. Co-incubation with inhibitors of ceramide synthesis, myriocin or fumonisin B(1), did not improve viability of palmitic acid treated RINm5F cells. The inhibitor of inducible nitric oxide synthase, 1400 W, similarly had no protective effect. However, linoleic acid protected against palmitic acid-induced apoptotic and necrotic cell death. The specific pharmacological inhibitors of phosphatidylinositol 3-kinase, LY294002 and wortmannin, abolished the protective effect of linoleic acid on apoptosis but not on necrosis. These data show that the growth inhibitory and apoptosis-inducing effect of the saturated fatty acid palmitate on RINm5F cells is prevented by co-incubation with the polyunsaturated fatty acid linoleate but not inhibitors of ceramide or nitric oxide generation. A key role for phosphatidylinositol 3-kinase in mediating the linoleic-acid reduction in apoptosis is suggested.

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1beta (IL-1beta), interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during,the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-beta-D-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.

It has been proposed that low activities of antioxidant enzymes in pancreatic beta cells may increase their susceptibility to autoimmune attack. We have therefore used the spontaneously diabetic BB/S rat model of type 1 diabetes to compare islet catalase and superoxide dismutase activities in diabetes-prone and diabetes-resistant animals. In parallel studies, we employed the RINm5F beta cell line as a model system (previously validated) to investigate whether regulation of antioxidant enzyme activity by inflammatory mediators (cytokines, nitric oxide) occurs at the gene or protein expression level. Diabetes-prone rat islets had high insulin content at the age used (58-65 days) but showed increased amounts of DNA damage when subjected to cytokine or hydrogen peroxide treatments. There was clear evidence of oxidative damage in freshly isolated rat islets from diabetes-prone animals and significantly lower catalase and superoxide dismutase activities than in islets from age-matched diabetes-resistant BB/S and control Wistar rats. The mRNA expression of antioxidant enzymes in islets from diabetes-prone and diabetes-resistant BB/S rats and in RINm5F cells, treated with a combination of cytokines or a nitric oxide donor, DETA-NO, was analysed semi-quantitatively by real time PCR. The mRNA expression of catalase was lower, whereas MnSOD expression was higher, in diabetes-prone compared to diabetes-resistant BB/S rat islets, suggesting regulation at the level of gene expression as well as of the activities of these enzymes in diabetes. The protein expression of catalase, CuZnSOD and MnSOD was assessed by Western blotting and found to be unchanged in DETA-NO treated cells. Protein expression of MnSOD was increased by cytokines in RINm5F cells whereas the expression of CuZnSOD was slightly decreased and the level of catalase protein was unchanged. We conclude that there are some changes, mostly upregulation, in protein expression but no decreases in the mRNA expression of catalase, CuZnSOD or MnSOD enzymes in beta cells treated with either cytokines or DETA-NO. The lower antioxidant enzyme activities observed in islets from diabetes-prone BB/S rats could be a factor in the development of disease and in susceptibility to DNA damage in vitro and could reflect islet alterations prior to immune attack or inherent differences in the islets of diabetes-prone animals, but are not likely to result from cytokine or nitric oxide exposure in vivo at that stage.

RNA interference has been used to silence gene expression and evaluate the contribution of a gene product to cell function. Here, we investigated conditions under which expression of an inducible protein, nitric oxide synthase 2 (NOS2), is decreased by RNA interference. Cytokine treatment of insulin-producing RINm5F cells results in NOS2 induction and cell death. Conditions used here favoured cytokine-induced apoptosis, for the first time--rather than necrosis, previously recorded. In RINm5F cells, transfected with NOS2-specific small interfering RNA followed by a 12 h exposure to cytokines, there was a significant reduction in NOS2 protein, nitrite, and apoptosis. There were no changes in these three parameters when experiments were carried out with unrelated vimentin siRNA. To interpret the NOS2-siRNA result further, we compared it with complete pharmacological inhibition of nitric oxide (NO) production by the NOS2 competitive inhibitor, 1400W, which lowered apoptosis by only 50% in the RINm5F cells. We conclude that the use of NOS2-specific siRNA has resulted in the subsequent lowering of expression of a cytokine-inducible protein whose function can be quantified. siRNA results have compared favourably with use of a pharmacological inhibitor of NOS2, in revealing the subtle, partial contribution of cytokine-induced NO to apoptosis induction in these insulin-producing cells., (Copyright 2004 Elsevier Inc.)

Pancreatic beta-cells have low activities of the antioxidant enzyme catalase. Nitric oxide interacts with the haem group of catalase inhibiting its activity. We have studied the activity of catalase in beta-cells under conditions mimicking prediabetes and in which nitric oxide is generated from cytokine treatment in vitro. We also studied whether there is regulation of catalase enzyme activity by nitric oxide at the protein or gene expression level. RINm5F insulin-producing cells, treated for 24 h with cytokines, showed increased medium nitrite production (17+/-2.2 vs 0.3+/-0.2 pmol/ micro g protein) and significantly decreased cellular catalase activity (42.4+/-4.5%) compared with control cells. A similar reduction was seen in catalase-overexpressing RIN-CAT cells and in rat or human pancreatic islets of Langerhans. Catalase activity was also suppressed by the long-acting nitric oxide donor diethylenetriamine/nitric oxide adduct (Deta-NO) and this inhibition was reversible. The inhibition of catalase activity by cytokines in RINm5F cells was significantly reversed by the addition of the nitric oxide synthase 2 (NOS2) inhibitors nitro monomethylarginine or N-(3-(aminomethyl)benzyl)acetamidine (1400W). Protein expression was found to be unchanged in cytokine- or Deta-NO-treated RINm5F cells, while mRNA expression was marginally increased. We have shown that inhibition of catalase activity by cytokines is nitric oxide dependent and propose that this inhibition may confer increased susceptibility to cytokine- or nitric oxide-induced cell killing.

Polyunsaturated fats are the main target for lipid peroxidation and subsequent formation of mutagenic metabolites, but diets high in saturated fats are more strongly associated with adverse health effects. We show that the common saturated fatty acid, palmitic acid, is a potent inducer of DNA damage in an insulin-secreting cell line, and in primary human fibroblasts. Damage is not associated with upregulation of inducible nitric oxide synthase, but is prevented by two different antioxidants, alpha-lipoic acid and 3,3'-methoxysalenMn(III) (EUK134), which also partly prevent palmitic acid-induced apoptosis and growth inhibition. Since mutagenic metabolites can be formed from peroxidation of polyunsaturated fatty acids, co-administration of palmitic and a polyunsaturated fatty acid might be particularly harmful. Palmitic acid-induced DNA damage is instead prevented by linoleic acid, which is acting here as a protective agent against oxidative stress, rather than as a source of mutagenic metabolites. These results illustrate the complexity of the relationship of dietary fat intake to genotoxicity.

The expression of 47 genes involved in the biosynthesis and secretion of insulin, apoptosis, and cellular stress was evaluated in isolated human islets using cDNA probes arrayed on nitrocellulose membranes. Isolated human islets were cultured for four days, or one month, with glucose present at a concentration of either 5.5 or 16.7 mmol/L. Extracted islet total RNA was used to generate [32P]dATP-labelled complex cDNA targets and hybridised with immobilised cDNA arrays. The positive expression of 45 mRNA transcripts in isolated human islets was documented. The coefficient of variance for relative levels of expression of transcripts was <25% for 9, 25-50% for 22, and 50-100% for 10, indicating good reproducibility between islet preparations from five different human pancreas donors. This study demonstrates the utility of nitrocellulose-based cDNA arrays for a focused reproducible analysis of gene expression changes in human islets of Langerhans.

Interleukin-1beta (IL-1beta) treatment of neonatal rat islets for 24 h induces changes in the expression of 105 of 2,200 proteins, as determined previously by two-dimensional (2D) gel electrophoresis. Nitric oxide (NO) has been implicated as one of the mediators of IL-1beta effects in insulin-containing cell lines and rat islets. The aims of this study were 1) to determine the involvement of NO in IL-1beta-induced alterations in protein expression and 2) to investigate the effects of chemically generated NO on protein expression by 2D gel electrophoresis of neonatal rat islet samples. IL-1beta-induced NO production was prevented by incubation of islets in arginine-free medium supplemented with the arginine analog NG-nitro-L-arginine. [35S]methionine-labeled islet proteins were separated using 2D gel electrophoresis and analyzed using the BioImage computer program. Analysis revealed that the expression levels of 23 protein spots of the 105 protein spots, altered by prior treatment with IL-1beta (60 U/ml) alone, were significantly affected (P < 0.01 [n = 4] and P < 0.05 [n = 19]) when NO production was prevented. The effects of chemically generated NO were investigated by exposing islets to the NO donor GSNO (100 micromol/l) for 24 h before labeling with [35S]methionine and 2D gel electrophoresis. Computer-based analysis identified alterations in the expression of 19 of a total of 1,600 detectable proteins in GSNO-treated islets (P < 0.01). We conclude 1) that the expression of up to 42 proteins is altered by cytokine-induced or chemically generated NO in the precise experimental conditions chosen and 2) that the majority of proteins altered by prior treatment with IL-1beta may be the result of NO-independent IL-1beta-mediated regulation of gene expression. This study demonstrates that the combination of 2D gel electrophoresis and mass spectrometry is a powerful tool in the identification of beta-cell proteins involved in the response to toxic mediators.

Aims/hypothesis: The aim of this study was to develop immunomagnetic purification by the Dynabead system to separate insulin-containing beta cells from a mixed rat islet cell population. Functional studies on insulin secretion and a test of the susceptibility of Dynabead-separated beta cells to DNA damage following cytokine exposure were carried out., Methods: Dynabeads are uniform, paramagnetic particles coated with specific antibodies. Single rat islet cells were initially incubated with the beta-cell surface specific antibody (K14D10 mouse IgG) for 20-60 min. A suspension of Dynabeads coated with a secondary antibody (anti-mouse IgG) was added for a further 15 min, after which the Dynabead-coated cells were instantaneously pelleted by contact between the tube and a magnet (Dynal MPC). Immunocytochemistry was used to confirm that the Dynabead-coated cells contained insulin and to quantify the efficiency of the method. Dynabead-coated and non-coated cells were stained for insulin and glucagon., Results: Dynabead immunopurification yielded 95% pure insulin-containing beta cells, which released insulin in response to isobutylmethylxanthine and glucagon-like polypeptide 1. The insulin content of Dynabead-coated beta cells was significantly higher than that of non-coated cells. Successful separation was achieved using as few as 30 islets as starting material. Using the comet assay, we found that Dynabead-coated beta cells showed equal susceptibility to cytokine-induced DNA damage as non-coated cells., Conclusion/interpretation: We conclude that Dynabead separation of beta cells is simple, rapid, applicable to large or small numbers of islets and can be used to study beta-cell specific function and responsiveness.

Nitric oxide synthase, induced by cytokines in insulin-containing cells, produces nitric oxide which inhibits function and may promote cell killing. Since glucagon was shown to prevent inducible nitric oxide synthase (iNOS) expression in rat hepatocytes it was of interest to examine the action of glucagon (and cyclic AMP) on iNOS induction in insulin-producing cells. Cultured RIN5F cells and primary rat and human islets of Langerhans were treated with interleukin 1beta (IL-1beta) or a combination of cytokines, and were co-treated or pre-treated with glucagon. In RIN5F cells, the activity of iNOS induced by IL-1beta (10 pM, 24 h), was significantly reduced by glucagon (1000 nM), which raises cyclic AMP, and by forskolin (1-10 microM), a non specific activator of adenylate cyclase. Glucagon and forskolin also decreased iNOS expression in RIN5F cells, and rat and human islets, as shown by Western blotting. The inhibitory action of IL-1beta (100 pM, 24 h) on rat islet insulin secretion was partially reversed by 1-h pre-treatment with glucagon (10-1000 nM), while the contrasting stimulatory effect of 48-h treatment with cytokines on insulin secretion from human islets was similarly prevented by glucagon (1000 nM) pre-treatment. These results suggest that glucagon inhibits iNOS expression in insulin-containing cells and imply that glucagon could modulate the inhibitory effects of cytokines., (Copyright 1999 Academic Press.)

Treatment of neonatal rat islets of Langerhans with combined cytokines (interleukin-1beta 10(-10) M, tumour necrosis factor-alpha 10(-10) M, interferon-gamma 5 U/ml) led to extensive cell death, which was potentiated by Fas activation with the anti-Fas cytolytic antibody JO2. Pre-treatment with insulin (25 ng/ml) or insulin-like growth factor-1 (10(-8)M) gave only partial protection against cell killing, but prevented the Fas-mediated component. In the absence of cytokine treatment, Fas-mediated killing was not observed.

Nitric oxide and superoxide are formed endogenously and can react with each other and with other molecules to form a range of secondary and tertiary products. Some of these (e.g., peroxynitrite) are potent DNA-damaging agents and others (e.g., S-nitrosoglutathione) can act as reservoirs of the reactive species. Although the chemistry of these processes is now becoming understood, the question of which products are significant in vivo is not necessarily clear. To investigate these processes we have developed a cell-free version of the Comet assay, where the DNA from isolated nuclei is treated in agar on a microscope slide, following lysis. This offers an exceptionally sensitive assay for strand breakage in free DNA. Despite being present as a scavenger in the cell at millimolar levels, glutathione can act as a DNA-damaging pro-oxidant. Under appropriate conditions, glutathione-mediated damage is suppressed by superoxide dismutase and we suggest that superoxide may be a direct damaging agent, whose activity can be masked because of the involvement of superoxide in indirect mediation of damage or because of concomitant presence of hydroxyl radical., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Although glutathione is an important scavenging molecule within the cell, it can also act as a pro-oxidant and at biological concentrations (1 mM) can induce DNA damage. We have used a sensitive cell-free Comet assay for DNA strand breakage to investigate this damage and to try to determine the active species involved. We show a substantial protection against glutathione-mediated DNA damage by superoxide dismutase (200 U/ml) and complete protection by combined superoxide dismutase and catalase. Damage is also prevented by EDTA but only at 100 mM and is not prevented by the chelating agent diethylenetriamine-pentaacetic acid (100 microM). Although superoxide is known to potentiate DNA damage by other reactive species, none of these indirect mechanisms seem to account for our results and it is possible that superoxide may damage DNA directly. Under the same experimental conditions, S-nitrosoglutathione requires ultraviolet A photolysis to cause DNA strand breakage and superoxide dismutase increases the level of this damage. When intact human lymphocytes are incubated with glutathione (1 mM) in phosphate buffer, DNA damage is also observed, but in this case it is completely preventable by catalase, with no protective effect of superoxide dismutase. Since cellular scavenging systems are not completely protective against reactive species formed from autooxidation of extracellular glutathione and since glutathione and oxygen are ubiquitously present within cells, our results imply that cells may have a mechanism of preventing autooxidation, rather than simply relying on scavenging the reactive species formula.

Cytokine-induced damage may contribute to destruction of insulin-secreting beta-cells in islets of Langerhans during autoimmune diabetes. There is considerable controversy (i) whether human and rat islets respond differently to cytokines, (ii) the extent to which cytokine damage is mediated by induction of nitric oxide formation, and (iii) whether the effects of nitric oxide on islets can be distinguished from those of reactive oxygen species or peroxynitrite. We have analyzed rat and human islet responses in parallel, 48 h after exposure to the nitric oxide donor S-nitrosoglutathione, the mixed donor 3-morpholinosydnonimine, hypoxanthine/xanthine oxidase, peroxynitrite, and combined cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma). Insulin secretory response to glucose, insulin content, DNA strand breakage, and early-to-late stage apoptosis were recorded in each experiment. Rat islet insulin secretion was reduced by S-nitrosoglutathione or combined cytokines, but unexpectedly increased by peroxynitrite or hypoxanthine/xanthine oxidase. Effects on human islet insulin secretion were small; cytokines and S-nitrosoglutathione decreased insulin content. Both rat and human islets showed significant and similar levels of DNA damage following all treatments. Apoptosis in neonatal rat islets was increased by every treatment, but was at a low rate in adult rat or human islets and only achieved significance with cytokine treatment of human islets. All cytokine responses were blocked by an arginine analogue. We conclude: (i) Reactive oxygen species increased and nitric oxide decreased insulin secretory responsiveness in rat islets. (ii) Species differences lie mainly in responses to cytokines, applied at a lower dose and shorter time than in most studies of human islets. (iii) Cytokine effects were nitric oxide driven; neither reactive oxygen species nor peroxynitrite reproduced cytokine effects. (iv) Rat and human islets showed equal susceptibility to DNA damage. (v) Apoptosis was not the preferred death pathway in adult islets. (vi) We have found no evidence of human donor variation in the pattern of response to these treatments.