Indocyanine green (ICG) fluorescence technology in pediatric robotic surgery.

This study aimed to report our experience in indocyanine green (ICG) fluorescence-guided surgery (FGS) in pediatric robotics. The data of 55 patients (35 boys and 20 girls), who underwent robotic surgery using ICG fluorescence in three institutions over the last 7 years, were retrospectively reviewed. The following robotic procedures were included: pyeloplasty (n = 21), complex Lich-Gregoir ureteral reimplantation (n = 8), varicocelectomy (n = 7), adnexal pathology resection (n = 8), partial nephrectomy (n = 4), nephrectomy (n = 4), renal cyst removal (n = 2), and excision of prostatic utricle (n = 1). The ICG was injected intravenously in all indications except for varicocele where intratesticular injection was done, and prostatic utricle or paraureteral diverticulum where trans-catheter injection was done. The ICG dosage was 0.2-0.3 mg/mL/kg. All the procedures were performed using da Vinci Xi platform. Firefly® allowed to switch form bright light to ICG-NIRF view and vice versa. All the procedures were accomplished in robotics without conversions to laparoscopy or open surgery. No episodes of allergy or anaphylaxis to ICG were recorded. An excellent ICG-NIRF view of target organs was obtained in all procedures. Based on our experience, we believe that application of ICG FGS in pediatric robotics enhances the identification of critical anatomical elements and pathological structures, thereby positively impacting both oncological and functional outcomes. This technique is safe, feasible, and versatile. We advocate the consideration of ICG as the standard of care in certain procedures such as partial nephrectomy, varicocele repair, tumor resection, and ovarian torsion. Nonetheless, further investigations are warranted to explore its potential broader applications in pediatric urology.

Trifecta Outcomes of Robot-Assisted Partial Nephrectomy Using the New Hugo™ RAS System Versus Laparoscopic Partial Nephrectomy.

(1) Background: Laparoscopic partial nephrectomy (LPN) is still performed in many referred urological institutions, representing a valid alternative to robot-assisted partial nephrectomy (RAPN). We aimed to compare trifecta outcomes of LPN and RAPN with the Hugo™ RAS System. (2) Methods: Between October 2022 and September 2023, eighty-nine patients underwent minimally invasive partial nephrectomy (group A, RAPN = 27; group B, Laparoscopic PN = 62) for localized renal tumors at our Institution. Continuous variables were presented as median and IQR and compared by means of the Mann-Whitney U test, while categorical variables were presented as frequencies (%) and compared by means of the χ2 test. (3) Results: Group A showed a higher rate of male patients (81.5% vs. 59.7%, p = 0.04) and a higher trend towards larger clinical tumor size (34 vs. 29 mm, p = 0.14). All the other baseline variables were comparable between the two groups (all p > 0.05). Regarding post-operative data, group A displayed a lower operative time (92 vs. 149.5 min, p = 0.005) and a shorter hospital stay (3 vs. 5, p = 0.002). A higher rate of malignant pathology was evidenced in group A (77.8% vs. 58.1%, p = 0.07) as well as a lower trend towards positive surgical margins (3.7% vs. 4.8%, p = 0.82), even if not statistically significant. (4) Conclusions: The rate of trifecta achievement was 92.6% and 82.3% for group A and B (p = 0.10), respectively. In terms of trifecta outcomes, RAPN using the Hugo™ RAS System showed comparable results to LPN performed by the same experienced surgeon.

Skill Transfer from Laparoscopic Partial Nephrectomy to the Hugo™ RAS System: A Novel Proficiency Score to Assess Surgical Quality during the Learning Curve.

Background/Objectives: The absence of validated tools to assess the skill transfer from laparoscopy to robotic surgery remains an unsolved issue in the context of robot-assisted partial nephrectomy (RAPN). We aimed to describe and validate a novel proficiency score to critically evaluate the surgical quality of RAPN with the Hugo™ RAS System (Medtronic, Minneapolis, MN, USA). Methods: Between October 2022 and September 2023, 27 consecutive patients underwent off-clamp RAPN for localized renal tumors at our institution. To analyze the learning curve (LC), the cohort was chronologically divided into two phases of 6 months each. Proficiency was defined as the achievement of trifecta while maintaining a comparable intraoperative time in the interquartile range of laparoscopic partial nephrectomy performed by the same surgeon. A logistic binary regression model was built to identify predictors of proficiency achievement. Results: A proficiency score was achieved in 14 patients (74.1%). At univariable analysis, number of consecutive procedures > 12 (OR 13.7; 95%CI 2.05-21.1, p = 0.007), pathological tumor size (OR 0.92; 95%CI 0.89-0.99, p = 0.04) and essential blood hypertension (OR 0.16; 95%CI 0.03-0.82, p = 0.02) were found to be predictors of proficiency score. At multivariable analysis, after adjusting for potential confounding factors, number of consecutive procedures > 12 (OR 8.1; 95%CI 1.44-14.6, p = 0.03) was the only independent predictor of proficiency score achievement. Conclusions: Our results showed that the skills of an experienced laparoscopic surgeon are transferrable to the novel Hugo™ RAS System in the context of nephron-sparing surgery. Improved surgical quality may be expected after completing the first 12 consecutive procedures.

Statins may increase the risk of being diagnosed with prostate cancer.

BACKGROUND: Although statins are known to protect against cardiovascular accidents, their anti-inflammatory features could play a role in preventing tumorigenesis. We investigated the association between statin intake and prostate cancer (PCa) diagnosis and aggressiveness. METHODS: A retrospective analysis was performed. Our dataset on patients undergone systematic prostate biopsy from December 2008 to December 2022 was searched for histopathologic and clinical data. Prognostic Grade Group ≥3 tumors were defined as high-grade (HG). The association between Metabolic Syndrome (MetS), statin use and PCa diagnosis and HG disease was assessed using logistic regression analyses. RESULTS: Data on 1685 patients were collected; MetS affected 344 (20.4%) men and 138 (36.5%) were taking statins at least for 6 months at the time of biopsy. Among the 671 (39.8%) men diagnosed with PCa, 327 (48.7%) presented with a HG disease. Tumor incidence was higher among men taking statins, compared to controls (46.8% vs. 37.8%; P=0.002); also, high grade diseases were more common in the former group, but the difference did not reach statistical significance (49.1% vs. 48.6%; P=0.89). Statin intake (OR 1.44; 95% CI [1.05-1.98]; P=0.02) independently predicted PCa diagnosis but not high-grade disease (P=0.8). CONCLUSIONS: Statin use may be associated with an increased risk of PCa diagnosis.

Laparoscopic versus Ultrasound-Guided Transversus Abdominis Plane Block for Postoperative Analgesia Management after Radical Prostatectomy: Results from a Single Center Study.

(1) Background: Regional anesthesia, achieved through nerve blocks, has gained widespread acceptance as an effective pain management approach. This research aimed to evaluate the efficacy of laparoscopic (LAP) transversus abdominis plane (TAP) block in patients undergoing laparoscopic radical prostatectomy. (2) Methods: From January 2023 to July 2023, 60 consecutive patients undergoing minimally invasive radical prostatectomy were selected. Patients were split into two groups receiving ultrasound-guided (US) or laparoscopic-guided TAP block. The primary outcome was a pain score expressed by a 0-10 visual analog scale (VAS) during the first 72 h after surgery. (3) Results: Both LAP-TAP and US-TAP block groups were associated with lower pain scores postoperatively. No statistically significant differences were observed between the two groups in surgery time, blood loss, time to ambulation, length of stay, and pain after surgery (all p > 0.2). In the LAP-TAP block group, the overall operating room time was significantly shorter than in the US-TAP block group (140 vs. 152 min, p = 0.04). (4) Conclusions: The laparoscopic approach, compared to the US-TAP block, was equally safe and not inferior in reducing analgesic drug use postoperatively. Moreover, the intraoperative LAP-TAP block seems to be a time-sparing procedure that could be recommended when patient-controlled analgesia cannot be delivered.

Robotic Medtronic Hugo™ RAS System Is Now Reality: Introduction to a New Simulation Platform for Training Residents.

The use of robotic surgery (RS) in urology has grown exponentially in the last decade, but RS training has lagged behind. The launch of new robotic platforms has paved the way for the creation of innovative robotics training systems. The aim of our study is to test the new training system from Hugo™ RAS System-Medtronic. Between July 2020 and September 2022, a total of 44 residents from urology, gynaecology and general surgery at our institution participated in advanced robotic simulation training using the Hugo™ RAS simulator. Information about sex, age, year of residency, hours spent playing video games, laparoscopic or robotic exposure and interest in robotics (90.9% declared an interest in robotics) was collected. The training program involved three robotic exercises, and the residents performed these exercises under the guidance of a robotics tutor. The residents' performance was assessed based on five parameters: timing, range of motion, panoramic view, conflict of instruments and exercise completion. Their performance was evaluated according to an objective Hugo system form and a subjective assessment by the tutor. After completing the training, the residents completed a Likert scale questionnaire to gauge their overall satisfaction. The rate of the residents' improvement in almost all parameters of the three exercises between the first and the last attempts was statistically significant (p < 0.02), indicating significant progress in the residents' robotic surgical skills during the training. The mean overall satisfaction score ± standard deviation (SD) was 9.4 ± 1.2, signifying a high level of satisfaction among the residents with the training program. In conclusion, these findings suggest that the training program utilizing the Hugo™ RAS System is effective in enhancing robotic surgical skills among residents and holds promise for the development of standardized robotics training programs in various surgical specialties.

Robot-Assisted Renal Surgery with the New Hugo Ras System: Trocar Placement and Docking Settings.

The current literature relating to the novel HugoTM RAS System lacks consistent data concerning the bedside features of robot-assisted partial nephrectomy (RAPN). To describe the trocar placement and docking settings for RAPN with a three-arm configuration to streamline the procedure with HugoTM RAS, between October 2022 and April 2023, twenty-five consecutive off-clamp RAPNs for renal tumors with the HugoTM RAS System were performed. We conceived a trouble-free three-arm setting to ease and standardize RAPN trocar placement and docking settings with HugoTM RAS. Perioperative data were collected. Post-operative complications were reported according to the Clavien-Dindo classification. The eGFR was calculated according to the CKD-EPI formula. Continuous variables were presented as the median and IQR, while frequencies were reported as categorical variables. Off-clamp RAPNs were successfully performed in all cases without the need for conversion or additional port placement. The median age and BMI were 69 years (IQR, 60-73) and 27.3 kg/m2 (IQR, 25.7-28.1), respectively. The median tumor size and R.E.N.A.L. score were 32.5 mm (IQR, 26-43.7) and 6 (IQR, 5-7), respectively. Two patients were affected by cT2 renal tumors. The median docking and console time were 5 (IQR, 5-6) and 90 min (IQR, 68-135.75 min), respectively, with slightly progressive improvements in the docking time achieved. No intraoperative complications occurred alongside clashes between instruments or with the bed assistant. In experienced hands, this simplified three-instrument configuration of the HugoTM RAS System for off-clamp RAPN resulted in feasible and safe practice, providing patient-tailored trocar placement and docking with non-inferior peri-perioperative outcomes to other robotic platforms.

Predictors of mid-term functional outcomes for robot-assisted Madigan simple prostatectomy: results of a multicentric series according to the BPH-6 achievement.

BACKGROUND: BPH-6 achievement remains an objective far to be evaluated for every technique currently available for the surgical management of bladder outlet obstruction (BOO) with the goal of preserving ejaculatory function. The aim of this study was to evaluate predictors of BPH-6 achievement of urethral-sparing robot assisted simple prostatectomy (us-RASP) on a large series performed at two tertiary-care centers. METHODS: Two institutional us-RASP datasets were merged, considering eligible all patients with a follow-up >12 months. Baseline, perioperative and functional data according to BPH-6 endpoint were assessed. Descriptive analysis was used. Frequencies and proportions were reported for categorical variables while medians and interquartile ranges (IQRs) were reported for continuously coded variables. A logistic regression model was built to identify predictors of BPH-6 achievement. For all statistical analyses, a two-sided P<0.05 was considered significant. RESULTS: Study cohort consisted of 94 eligible patients. The median follow-up was 40.7 months (IQR 31.3-54.2). Overall BPH-6 achievement was 54.7%. Compared to baseline, reduction of ≥30% in IPSS was observed in 93.6% of patients, reduction of <6 points for SHIM in 95.7% and response to MSHQ-EjD question 3 indicating emission of semen in 72.6%, respectively. On multivariable analysis, prostate volume between 110-180 mL (OR 0.09; 95% CI 0.01-0.92; P=0.043) and higher preoperative SHIM score (OR 1.18; 95% CI 1.05-1.32; P<0.01) were independent predictors of BPH-6 metric achievement. CONCLUSIONS: us-RASP may provide a complete resolution of BOO and preservation of ejaculatory function in sexually active men with a prostate volume ranging 110-180 mL.

Robotic Surgery in Urology: History from PROBOT® to HUGOTM.

The advent of robotic surgical systems had a significant impact on every surgical area, especially urology, gynecology, and general and cardiac surgery. The aim of this article is to delineate robotic surgery, particularly focusing on its historical background, its evolution, its present status, and its future perspectives. A comprehensive literature review was conducted upon PubMed/MEDLINE, using the keywords "robotic surgical system", "robotic surgical device", "robotics AND urology". Additionally, the retrieved articles' reference lists were investigated. Analysis concentrated on urological surgical systems for laparoscopic surgery that have been given regulatory approval for use on humans. From the late 1980s, before daVinci® Era in 2000s, ancestor platform as Probot® and PUMA 560 were described to outline historical perspective. Thus, new robotic competitors of Intuitive Surgical such as Senhance®, Revo-I®, Versius®, Avatera®, Hinotori®, and HugoTM RAS were illustrated. Although daVinci® had high level competitiveness, and for many years represented the most plausible option for robotic procedures, several modern platforms are emerging in the surgical market. Growing competition through unique features of the new robotic technologies might extend applications fields, improve diffusion, and increase cost-effectiveness procedures. More experiences are needed to identify the role of these new advancements in surgical branches and in healthcare systems.

State of the Art in Robotic Surgery with Hugo RAS System: Feasibility, Safety and Clinical Applications.

Since its introduction in the early 2000s, robotic surgery has represented a significative innovation within a minimally invasive surgery approach. A variety of robotic platforms have been made available throughout the years, and the outcomes related to those platforms have been described in the literature for many types of surgeries. Medtronic's HugoTM RAS system is one of the newest robotic generations launched, but because of its recent placing on the field, comprehensive clinical data are still lacking. The aim of the present state of the art is to address the current literature concerning the use of the HugoTM RAS robot in order to report its feasibility, safety and clinical applications in different surgical branches. Two reviewers independently conducted a search on the "PubMed" electronic database, using the keywords "Hugo" and "Hugo RAS". After the initial screening of 35 results, a total of 15 articles concerning the Hugo RAS system were selected for the review, including both oncological and benign surgery. Patients' demographic and baseline data were compared including, when available, docking system times, complications and oncological outcomes in the fields of urologic, gynecologic and general surgery. With reference to urological procedures, a total of 156 robot-assisted radical prostatectomies, 10 robot-assisted partial nephrectomies, and 5 robot-assisted adrenalectomies were performed, involving a total of 171 patients. The surgical branch in which the Hugo system found its major application was urology, which was followed by gynecology and general surgery. The Hugo RAS system by Medtronic represents an innovative and safe surgical platform, with excellent perspective for the future and different clinical applications in many surgical branches. More studies are needed to validate the safety and results from this new robotic platform.

Predictors of Urinary Continence Recovery after Laparoscopic-Assisted Radical Prostatectomy: Is Surgical Urethral Length the Only Key Factor?

Several efforts in recent years have been made to predict urinary continence (UC) recovery after radical prostatectomy. The aim of our study was to investigate the impact of surgical urethral length preservation (SULP) on urinary continence after LARP (laparoscopic-assisted radical prostatectomy). We retrospectively queried our datasets from May 2021 to May 2022. After the application of exclusion criteria, a total of 100 patients who underwent LARP for prostate cancer at our institution were enrolled. Through a sterile ruler inserted by a 12 mm trocar, the length of the membranous urethra spared during LARP was assessed intra-operatively. The baseline and peri- and postoperative data of patients were collected, and UC was defined as 0 or 1 on a safety pad. The median SULP was 20.5 mm (IQR, 14.5-25), and the median intraoperative EBL were 150 mL (IQR, 100-200). The Kaplan-Meier curve showed a significant difference at 20 mm, which was used as the cut-off value for SULP (log-rank test, p < 0.001). Multivariate Cox proportional hazards models showed that SULP and EBL < 250 mL were associated with UC recovery (all p < 0.02). Surgical urethral length preservation seemed to improve early UC recovery after LARP. Further multicentric studies are needed to confirm our findings.

Single-Setting 3D MRI/US-Guided Frozen Sectioning and Cryoablation of the Index Lesion: Mid-Term Oncologic and Functional Outcomes from a Pilot Study.

Our study explored frozen section reliability in prostate cancer (PCa) diagnoses and described surgical steps of a 3D magnetic resonance imaging (MRI)-ultrasound (US)-guided prostate biopsy (PB) and focal cryoablation of the index lesion (IL) in a single-setting procedure. Patients with a suspicious prostatic specific antigen (PSA) value, with a PIRADS 4 or 5 single lesion, were enrolled for trans perineal 3D MRI-US-guided PB and TRUS-guided focal cryoablation. Three cores were taken from the IL, three cores from the surrounding area, while systematic sampling was performed for the rest of the gland. After confirmation of PCa in frozen sections, focal cryoablation was performed. The 1st-year follow-up schedule included a PSA test at a 3-month interval, MRI 3 months and 1 year postoperatively and PB of the treated area at 1 year. Following the follow-up schedule, an involved PSA test at a 3-month interval and yearly MRI were performed. The PCa diagnosis was histologically confirmed in all three patients with frozen sections. At final histology, a single Gleason score upgrade from 6 (3 + 3) to 7 (3 + 4) was observed. All patients were discharged on postoperative day 1. At the 3-month evaluation, mean PSA values decreased from 12.54 (baseline) to 1.73 ng/mL and MRI images showed complete ablation of the IL in all patients. Urinary continence and potency were preserved in all patients. At the 1-year follow-up, one patient had suspicious ipsilateral recurrence on MRI and underwent a new analogous procedure. Post follow-up was uneventful and PSA remained stable in all patients. Three-dimensional MRI-US-guided frozen sectioning and focal cryoablation of the IL is a step forward towards a "patient-tailored" minimally invasive approach to the diagnosis and cure of PCa.

Video Consensus and Radical Prostatectomy: The Way to Chase the Future?

In the age of information, new platforms are consulted by patients to acquire consciousness about medical treatments. The aim of this study was to assess the level of understanding and feasibility of video consensus (VC) administration in patients scheduled for radical prostatectomy (RP), comparing it with standard informed consensus (SIC). According to the European Association of Urology Patient Information, we set up a video content for RP that was translated in Italian and implemented with information about possible perioperative and postoperative complications, days of hospitalization etc. From 2021 to 2022, all patients undergoing RP at our institution were prospectively included in this study. Patients received an SIC and after that, a VC about RP. After two consensuses were administered, patients received a preformed Likert 10 scale and STAI questionnaires. On the RP dataset, 276 patients were selected and 552 questionnaires for both SIC and VC were evaluated. Out of these, the median age was 62 years (IQR 60-65). Patients reported a higher overall satisfaction for VC (8.8/10) compared to the traditional informed consent (6.9/10). Therefore, VC may play a role in the future of surgery, improving the consciousness and satisfaction of patients and reducing preoperative anxiety.

Impact of Robotic Technologies on Prostate Cancer Patients' Choice for Radical Treatment.

Prostate cancer (PCa) surgery has a strong impact on men's social and sexual lives. For this reason, many patients ask for robotic surgery. To assess the rate of lost patients due to the lack of a robotic platform (RPl) at our center, we retrospectively selected 577 patients who underwent prostate biopsy between 2020 and 2021 who were eligible for radical prostatectomy (RP) (ISUP ≥ 2; age ≤ 70 yr). Patients eligible for surgery who decided to be operated received a phone call interview asking the reason for their choice. Overall, 230 patients (31.7%) underwent laparoscopic-assisted radical prostatectomy (LaRP) at our center, while 494 patients (68.3%) were not treated in our hospital. Finally, 347 patients were included: 87 patients (25.1%) underwent radiotherapy; 59 patients (17%) were already under another urologist's care; 113 patients (32.5%) underwent robotic surgery elsewhere; and 88 patients (25.4%) followed the suggestion of friends or relatives based on their surgical experience. Despite no surgical technique for RP having shown superiority in terms of oncological or functional outcomes, patients eligible for PCa treatment decided to be operated on elsewhere because of the lack of an RPl. Our results show how the presence of an RPl may increase the case volume of RP by 49% at our center.

Complement activation predicts negative outcomes in COVID-19: The experience from Northen Italian patients.

Coronavirus disease 19 (COVID-19) may present as a multi-organ disease with a hyperinflammatory and prothrombotic response (immunothrombosis) in addition to upper and lower airway involvement. Previous data showed that complement activation plays a role in immunothrombosis mainly in severe forms. The study aimed to investigate whether complement involvement is present in the early phases of the disease and can be predictive of a negative outcome. We enrolled 97 symptomatic patients with a positive RT-PCR for SARS-CoV-2 presenting to the emergency room. The patients with mild symptoms/lung involvement at CT-scan were discharged and the remaining were hospitalized. All the patients were evaluated after a 4-week follow-up and classified as mild (n. 54), moderate (n. 17) or severe COVID-19 (n. 26). Blood samples collected before starting any anti-inflammatory/immunosuppressive therapy were assessed for soluble C5b-9 (sC5b-9) and C5a plasma levels by ELISA, and for the following serum mediators by ELLA: IL-1ß, IL-6, IL-8, TNFα, IL-4, IL-10, IL-12p70, IFNγ, IFNα, VEGF-A, VEGF-B, GM-CSF, IL-2, IL-17A, VEGFR2, BLyS. Additional routine laboratory parameters were measured (fibrin fragment D-dimer, C-reactive protein, ferritin, white blood cells, neutrophils, lymphocytes, monocytes, platelets, prothrombin time, activated partial thromboplastin time, and fibrinogen). Fifty age and sex-matched healthy controls were also evaluated. SC5b-9 and C5a plasma levels were significantly increased in the hospitalized patients (moderate and severe) in comparison with the non-hospitalized mild group. SC5b9 and C5a plasma levels were predictive of the disease severity evaluated one month later. IL-6, IL-8, TNFα, IL-10 and complement split products were higher in moderate/severe versus non-hospitalized mild COVID-19 patients and healthy controls but with a huge heterogeneity. SC5b-9 and C5a plasma levels correlated positively with CRP, ferritin values and the neutrophil/lymphocyte ratio. Complement can be activated in the very early phases of the disease, even in mild non-hospitalized patients. Complement activation can be observed even when pro-inflammatory cytokines are not increased, and predicts a negative outcome.

Investigating the role of dystrophin isoform deficiency in motor function in Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full-length Dp427 is the primary dystrophin isoform expressed in muscle and is also expressed in the central nervous system (CNS). Two shorter isoforms, Dp140 and Dp71, are highly expressed in the CNS. While a role for Dp140 and Dp71 on DMD CNS comorbidities is well known, relationships between mutations expected to disrupt Dp140 and Dp71 and motor outcomes are not. METHODS: Functional outcome data from 387 DMD boys aged 4-15 years were subdivided by DMD mutation expected effects on dystrophin isoform expression; Group 1 (Dp427 absent, Dp140/Dp71 present, n = 201); Group 2 (Dp427/Dp140 absent, Dp71 present, n = 152); and Group 3 (Dp427/Dp140/Dp71 absent, n = 34). Relationships between isoform group and North Star ambulatory assessment (NSAA) scores, 10 m walk/run velocities and rise time velocities were explored using regression analysis. Western blot analysis was used to study Dp427, Dp140 and Dp71 production in myogenic cells (control and DMD human), control skeletal muscle, DMD skeletal muscle from the three isoform groups and cerebral cortex from mice (wild-type and DMD models). Grip strength and rotarod running test were studied in wild-type mice and DMD mouse models. DMD mouse models were mdx (Dp427 absent, Dp140/Dp71 present), mdx52 (Dp427/Dp140 absent, Dp71 present) and DMD-null (lacking all isoforms). RESULTS: In DMD boys, mean NSAA scores at 5 years of age were 6.1 points lower in Group 3 than Group 1 (P < 0.01) and 4.9 points lower in Group 3 than Group 2 (P = 0.05). Mean peak NSAA scores were 4.0 points lower in Group 3 than Group 1 (P < 0.01) and 1.6 points lower in Group 2 than Group 1 (P = 0.04). Mean four-limb grip strength was 1.5 g/g lower in mdx52 than mdx mice (P = 0.003) and 1.5 g/g lower in DMD-null than mdx mice (P = 0.002). Dp71 was produced in myogenic cells (control and DMD human) and skeletal muscle from humans in Groups 1 and 2 and mdx mice, but not skeletal muscle from human controls, myogenic cells and skeletal muscle from humans in Group 3 or skeletal muscle from wild-type, mdx52 or DMD-null mice. CONCLUSIONS: Our results highlight the importance of considering expected effects of DMD mutations on dystrophin isoform production when considering patterns of DMD motor impairment and the implications for clinical practice and clinical trials. Our results suggest a complex relationship between dystrophin isoforms expressed in the brain and DMD motor function.

An allosteric redox switch in domain V of ß2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition.

ß2-glycoprotein I (ß2GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that ß2GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to ß2GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate ß2GPI's structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of ß2GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating ß2GPI's physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.

ß2 glycoprotein I participates in phagocytosis of apoptotic neurons and in vascular injury in experimental brain stroke.

Beta-2 Glycoprotein I (ß2-GPI) is the main target of anti-phospholipid antibodies (aPL) in the autoimmune anti-phospholipid syndrome, characterized by increased risk of stroke. We here investigated the antibody independent role of ß2-GPI after ischemia/reperfusion, modeled in vivo by transient middle cerebral artery occlusion (tMCAo) in male C57Bl/6J mice; in vitro by subjecting immortalized human brain microvascular endothelial cells (ihBMEC) to 16 h hypoxia and 4 h re-oxygenation. ApoH (coding for ß2-GPI) was upregulated selectively in the liver at 48 h after tMCAo. At the same time ß2-GPI circulating levels increased. ß2-GPI was detectable in brain parenchyma and endothelium at all time points after tMCAo. Parenchymal ß2-GPI recognized apoptotic neurons (positive for annexin V, C3 and TUNEL) cleared by CD68+ brain macrophages. Hypoxic ihBMEC showed increased release of IL-6, over-expression of thrombomodulin and IL-1α after re-oxygenation with ß2-GPI alone. ß2-GPI interacted with mannose-binding lectin in mouse plasma and ihBMEC medium, potentially involved in formation of thrombi. We show for the first time that brain ischemia triggers the hepatic production of ß2-GPI. ß2-GPI is present in the ischemic endothelium, enhancing vascular inflammation, and extravasates binding stressed neurons before their clearance by phagocytosis. Thus ß2-GPI may be a new mediator of brain injury following ischemic stroke.

Cellular dynamics of myogenic cell migration: molecular mechanisms and implications for skeletal muscle cell therapies.

Directional cell migration is a critical process underlying morphogenesis and post-natal tissue regeneration. During embryonic myogenesis, migration of skeletal myogenic progenitors is essential to generate the anlagen of limbs, diaphragm and tongue, whereas in post-natal skeletal muscles, migration of muscle satellite (stem) cells towards regions of injury is necessary for repair and regeneration of muscle fibres. Additionally, safe and efficient migration of transplanted cells is critical in cell therapies, both allogeneic and autologous. Although various myogenic cell types have been administered intramuscularly or intravascularly, functional restoration has not been achieved yet in patients with degenerative diseases affecting multiple large muscles. One of the key reasons for this negative outcome is the limited migration of donor cells, which hinders the overall cell engraftment potential. Here, we review mechanisms of myogenic stem/progenitor cell migration during skeletal muscle development and post-natal regeneration. Furthermore, strategies utilised to improve migratory capacity of myogenic cells are examined in order to identify potential treatments that may be applied to future transplantation protocols.