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Abstract The molecular mechanisms that govern differential T cell development from CD4+CD25-conventional T (Tconv) into CD4+CD25+ forkhead-box-P3+ (FoxP3+) inducible regulatory T (iTreg) cells remain unclear. Herein, we investigated the relative contribution of protein kinase A (PKA) in this process. Mechanistically, we found that PKA controlled the efficiency of human iTreg cell generation through the expression of different FoxP3 splicing variants containing or not the exon 2. We found that transient PKA inhibition reduced the recruitment of cAMP-responsive element-binding protein (CREB) on regulatory regions of the FoxP3 gene, a condition that is associated with an impaired acquisition of their suppressive capacity in vitro. To corroborate our findings in a human model of autoimmunity, we measured CREB phosphorylation and FoxP3 levels in iTreg cells from treatment-naïve relapsing–remitting (RR)-multiple sclerosis (MS) subjects. Interestingly, both phospho-CREB and FoxP3 induction directly correlated and were significantly reduced in RR-MS patients, suggesting a previously unknown mechanism involved in the induction and function of human iTreg cells.

Abstract Background Adhesive capsulitis (AC) is a disease of the glenohumeral joint that is characterized by pain and both passive and active global stiffness with a slow and insidious onset. The disease can occur spontaneously (primary AC) or it can be secondary to other comorbidities, surgery, or trauma, such as fracture or dislocation. Multiple treatment approaches have been suggested: intra-articular steroid injection, physical therapy, manipulation under total anesthesia, and arthroscopic or open surgery. Shoulder manipulation under anesthesia is usually proposed to patients that suffer from severe AC and have already undergone several nonoperative treatments without benefit. Different techniques have been proposed. This study presents our manipulation technique and the clinical results we achieved after shoulder mobilization under brachial plexus block in patients with phase III primary AC. Materials and methods A retrospective cohort study was performed on a sample of 110 patients with phase III AC who were treated with this manipulation and followed up for 1 year. Patients underwent two assessments—before the procedure (T0) and 4 months after it (T1)—based on the Numerical Rating Scale, Simple Shoulder Test, and joint range of motion to assess shoulder pain, function, and joint articulation, respectively. Furthermore, the patients had to express their degree of satisfaction with the procedure and the results achieved. Results Positive and statistically significant results were recorded in terms of pain reduction (ΔNPRS = − 5.4; p

Abstract Background KRAS is the undisputed champion of oncogenes, and despite its prominent role in oncogenesis as mutated gene, KRAS mutation appears infrequent in gliomas. Nevertheless, gliomas are considered KRAS-driven cancers due to its essential role in mouse malignant gliomagenesis. Glioblastoma is the most lethal primary brain tumor, often associated with disturbed RAS signaling. For newly diagnosed GBM, the current standard therapy is alkylating agent chemotherapy combined with radiotherapy. Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors (including medulloblastoma and neuroblastoma) and many studies are currently focused on new delivery modalities of effective cisplatin in glioblastoma. Its mechanism of action is mainly based on DNA damage, inducing the formation of DNA adducts, triggering a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis. Methods Long-term cultures of human glioblastoma, U87MG and U251MG, were either treated with cis-diamminedichloroplatinum (cisplatin, CDDP) and/or MEK-inhibitor PD98059. Cytotoxic responses were assessed by cell viability (MTT), protein expression (Western Blot), cell cycle (PI staining) and apoptosis (TUNEL) assays. Further, gain-of-function experiments were performed with cells over-expressing mutated hypervariable region (HVR) KRASG12V plasmids. Results Here, we studied platinum-based chemosensitivity of long-term cultures of human glioblastoma from the perspective of KRAS expression, by using CDDP and MEK-inhibitor. Endogenous high KRAS expression was assessed at transcriptional (qPCR) and translational levels (WB) in a panel of primary and long-term glioblastoma cultures. Firstly, we measured immediate cellular adjustment through direct regulation of protein concentration of K-Ras4B in response to cisplatin treatment. We found increased endogenous protein abundance and involvement of the effector pathway RAF/MEK/ERK mitogen-activated protein kinase (MAPK) cascade. Moreover, as many MEK inhibitors are currently being clinically evaluated for the treatment of high-grade glioma, so we concomitantly tested the effect of the potent and selective non-ATP-competitive MEK1/2 inhibitor (PD98059) on cisplatin-induced chemosensitivity in these cells. Cell-cycle phase distribution was examined using flow cytometry showing a significant cell-cycle arrest in both cultures at different percentage, which is modulated by MEK inhibition. Cisplatin-induced cytotoxicity increased sub-G1 percentage and modulates G2/M checkpoint regulators cyclins D1 and A. Moreover, ectopic expression of a constitutively active KRASG12V rescued CDDP-induced apoptosis and different HVR point mutations (particularly Ala 185) reverted this phenotype. Conclusion These findings warrant further studies of clinical applications of MEK1/2 inhibitors and KRAS as ‘actionable target’ of cisplatin-based chemotherapy for glioblastoma.

Abstract Background Obstetric brachial plexus injury (OBPI) is a weakening or paralysis of the upper arm caused by brachial plexus injury followed by a muscle paralysis with severe repercussions on the movement of the shoulder joint following a progressive glenohumeral joint deformity. This case series analyzes the clinical and radiological outcomes of reverse total shoulder arthroplasty (RSA) in OBPI patients with a follow-up of 2 years. Materials and methods OBPI patients with secondary end-stage glenohumeral arthritis were enrolled in the study and they were treated with RSA. Patient demographics and clinical outcomes [Range of Motion (ROM), Visual Analog Scale (VAS), Oxford Shoulder Score (OSS)] were evaluated. A novel Shoulder motion analysis was carried out to investigate specific movement patterns of scapulothoracic movements in these patients. This study is a prospective cohort study. Results Four Patients (M: F = 1:3) were enrolled in the study, the mean age was 49.3 years (+ 2.75), the mean OSS (Oxford Shoulder Score) decreased from 48.8 (± 2.5) preoperatively to 18.30 (± 2.78), the mean VAS (Visual Analog Scale) decreased from 7.25 (± 0.5) to 1.7 (± 0.3) in the follow up (∆% relative pain reduction:− 76.5%), Shoulder ROM obtained an improvement (p

The intricate parallels in structure and function between the human retina and the central nervous system designate the retina as a prospective avenue for understanding brain-related processes. This review extensively explores the shared physiopathological mechanisms connecting age-related macular degeneration (AMD) and proteinopathies, with a specific focus on tauopathies. The pivotal involvement of oxidative stress and cellular senescence emerges as key drivers of pathogenesis in both conditions. Uncovering these shared elements not only has the potential to enhance our understanding of intricate neurodegenerative diseases but also sets the stage for pioneering therapeutic approaches in AMD.

Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand (CD40LG) gene with a median survival of 25 years, potentially treatable with in situ CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of CD40LG in edited healthy donor and patient cells recapitulating the physiological CD40LG regulation. We then documented the preserved fitness of edited cells by xenotransplantation into immunodeficient mice. Finally, we transitioned to large-scale manufacturing, and developed a panel of quality control assays. Overall, our GMP-compliant process takes long-range gene editing one step closer to clinical application with a reassuring safety profile.

Abstract Background The functional results of total elbow arthroplasty (TEA) are controversial and the medium- to long-term revision rates are relatively high. The aim of the present study was to analyze the stresses of TEA in its classic configuration, identify the areas of greatest stress in the prosthesis–bone–cement interface, and evaluate the most wearing working conditions. Materials and methods By means of a reverse engineering process and using a 3D laser scanner, CAD (computer-aided drafting) models of a constrained elbow prosthesis were acquired. These CAD models were developed and their elastic properties, resistance, and stresses were studied through finite element analysis (finite element method—FEM). The obtained 3D elbow-prosthesis model was then evaluated in cyclic flexion–extension movements (> 10 million cycles). We highlighted the configuration of the angle at which the highest stresses and the areas most at risk of implant mobilization develop. Finally, we performed a quantitative study of the stress state after varying the positioning of the stem of the ulnar component in the sagittal plane by ± 3°. Results The greatest von Mises stress state in the bone component for the 90° working configuration was 3.1635 MPa, which occurred in the most proximal portion of the humeral blade and in the proximal middle third of the shaft. At the ulnar level, peaks of 4.1763 MPa were recorded at the proximal coronoid/metaepiphysis level. The minimum elastic resistance and therefore the greatest stress states were recorded in the bone region at the apex of the ulnar stem (0.001967 MPa). The results of the analysis for the working configurations at 0° and 145° showed significant reductions in the stress states for both prosthetic components; similarly, varying the positioning of the ulnar component at 90° (− 3° in the sagittal plane, 0° in the frontal plane) resulted in better working conditions with a greater resulting developed force and a lower stress peak in the ulnar cement. Conclusion The areas of greatest stress occur in specific regions of the ulnar and humeral components at the bone–cement–prosthesis interface. The heaviest configuration in terms of stresses was when the elbow was flexed at 90°. Variations in the positioning in the sagittal plane can mechanically affect the movement, possibly resulting in longer survival of the implant. Level of evidence: 5

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Background Although the protooncogenes small GTPases Ras are redox-sensitive proteins, how they are regulated by redox signaling in the central nervous system (CNS) is still poorly understood. Alteration in redox-sensitive targets by redox signaling may have myriad effects on Ras stability, activity and localization. Redox-mediated changes in astrocytic RAS may contribute to the control of redox homeostasis in the CNS that is connected to the pathogenesis of many diseases.Results and Methods Here, we investigated the transient physiological induction, at both transcriptional and translational levels, of small GTPases Ras in response to redox stimulation. Cultured astrocytes were treated with hydrogen peroxide as in bolus addition and relative mRNA levels of murine hras and kras genes were detected by qRT-PCR. We found that de novo transcription of hras mRNA in reactive astrocytes is redox-sensitive and mimics the prototypical redox-sensitive gene iNOS. Protein abundance in combination with protein turnover measurements by cycloheximide-chase experiments revealed distinct translation efficiency, GTP-bound enrichment, and protein turnover rates between the two isoforms H-Ras and K-Ras.Conclusion Reports from recent years support a significant role of H-Ras in driving redox processes. Beyond its canonical functions, Ras may impact on the core astrocytic cellular machinery that operates during redox stimulation.

Abstract Background Proximal humeral fractures are commonly observed in elderly patients. Management of these injuries is controversial. Literature comparing locking plate fixation, arthroplasty, and conservative treatments show no clear advantages for any of these management strategies. Thus far, no study has considered anatomically reduced fractures obtained after locking plate treatment. To clarify the best surgical procedure in middle-aged patients, we considered outcomes and major complications leading to surgical revision following an anatomically reduced fracture fixed with locking plate and reverse shoulder arthroplasty (RSA) in the treatment of type B/C fractures in patients between 50 and 75 years of age. Methods This is a retrospective study including 59 patients between 50 and 75 years of age with type B/C proximal humeral fracture treated with RSA or with locking plate fixation (resulting in an anatomical reduction) between January 2010 and December 2018. Preoperative radiographs and computed tomography (CT) were evaluated in all patients. Clinical and radiologic follow-up was performed using range of motion (ROM), the Constant–Murley Score (CMS), the Oxford Shoulder Score (OSS), the Simple Shoulder Test (SST), the Subjective Shoulder Value (SSV), and visual analog scale (VAS). Major complications were considered. Results In the plate fixation group, ROM, CMS, SST, and VAS were higher than in the RSA group. Lower complication rates compared with the literature were observed in both groups. Anatomically reduced fracture fixed with plate and screw could outperform RSA in terms of outcome. In second-level centers where traumatology is performed by surgeons with great expertise in upper limb trauma, the choice between plate fixation and reverse arthroplasty should be made during surgery. Conclusion Anatomically reduced fractures showed better outcomes compared with RSA in type B/C fractures. Surgeons should always try to perform a reduction of the fracture in order to understand if a plate fixation could be feasible. If it is impossible to perform an anatomical reduction, we suggest to consider RSA. This is a retrospective observational study.

Glaucoma is a chronic neurodegenerative disorder affecting the visual system which can result in vision loss and blindness. The pathogenetic mechanisms underlying glaucomatous optic neuropathy are ultimately enigmatic, prompting ongoing investigations into its potential shared pathogenesis with other neurodegenerative neurological disorders. Tauopathies represent a subclass of neurodegenerative diseases characterized by the abnormal deposition of tau protein within the brain and consequent microtubule destabilization. The extended spectrum of tauopathies includes conditions such as frontotemporal dementias, progressive supranuclear palsy, chronic traumatic encephalopathy, and Alzheimer’s disease. Notably, recent decades have witnessed emerging documentation of tau inclusion among glaucoma patients, providing substantiation that this ocular disease may similarly manifest features of tauopathies. These studies found that: (i) aggregated tau inclusions are present in the somatodendritic compartment of RGCs in glaucoma patients; (ii) the etiology of the disease may affect tau splicing, phosphorylation, oligomerization, and subcellular localization; and (iii) short interfering RNA against tau, administered intraocularly, significantly decreased retinal tau accumulation and enhanced RGC somas and axon survival, demonstrating a crucial role for tau modifications in ocular hypertension-induced neuronal injury. Here, we examine the most recent evidence surrounding the interplay between tau protein dysregulation and glaucomatous neurodegeneration. We explore the novel perspective of glaucoma as a tau-associated disorder and open avenues for cross-disciplinary collaboration and new treatment strategies.

Introduction: Alterations of scapular kinematics are generically reported as scapular dyskinesis (SD), and are a nonspecific response to various shoulder pathologies. The most widely used classification is Kibler's (K), which is, however, characterized by poor sensitivity. To overcome this limit, using a 3D motion analysis system, we identified a specific pattern for each type of SD according to Kibler. Materials and methods: We analyzed 34 patients with a total of 68 shoulders who came to our observation for shoulder pain. All patients underwent clinical examination, video-recording and motion analysis with SHoW Motion 3D kinematic tracking system (SM). Three independent observers classified SD into K types I, II and III. Only patients with concordant classification among the 3 operators were studied to identify a characteristic graphic pattern by type of SD. Results: Typical patterns emerged from the examination with SM. K. type 1 consists of decreased or reversed posterior tilt and increased protraction in flexion–extension (FE) in early degrees of motion. K. type 2 consists of increased protraction and marked reversal of lateral rotation in abduction–adduction (Ab–Ad) in early degrees of movement. K. type 3 has been subdivided into two subgroups: K. type 3-A, composed of patients with massive rotator cuff lesions, shows an increase in all scapular movements in both FE and Ab–Ad. K. type 3-B, composed of patients with scapular stiffness and/or impingement, presents a slight increase in posterior tilt and lateral rotation in the final grades of FE and Ab–Ad. Conclusions: The SM system allows reproducible dynamic analyses with low intra- and intra- operator variability. In our study, we demonstrated its applicability in the classification of SD. It also provides an objective and quantitative assessment of motor pattern alteration that is essential in the follow-up of patients to evaluate the effectiveness of rehabilitation and/or surgical treatment. Level of Evidence 3: According to "The Oxford 2011 Levels of Evidence". [ABSTRACT FROM AUTHOR]

Abstract Background Elbow arthroscopy is becoming increasingly important for the treatment of a wide range of acute and chronic elbow pathologies. Even if elbow arthroscopy is technically demanding, in the pediatric population this minimally invasive technique is preferred by many surgeons for the treatment of pathologies such as osteochondritis dissecans (OCD), posttraumatic stiffness (PTS), or elbow posterior impingement (PI). The aim of this study is to evaluate outcomes and safety of elbow arthroscopy in the pediatric and adolescent population after long-term follow-up. Materials and methods In this retrospective study, 26 patients younger than 18 years old undergoing elbow arthroscopy were evaluated. All surgeries were performed by a single senior surgeon. Patients were divided into three subgroups based on preoperative diagnosis: OCD, PTS, and PI. After at least 60 months follow-up, several outcome measures, including range of motion (ROM), Mayo Elbow Performance Score (MEPS), and visual analog scale (VAS) were evaluated in relation to preoperative values. The level of patient satisfaction on a five-level Likert scale, any limitation or change in sport activity, and the onset of any possible complications were also evaluated. Results In the study population, we found an improvement in ROM (flexion of 14.4 ± 13.6°, extension of 19.5 ± 13.9°, pronation of 5.8 ± 5.7°, and supination of 8.5 ± 11.6°) and in validated outcome measures (MEPS of 21.0 ± 13.5 points and VAS of 3.8 ± 2.2 points). The satisfaction rate was 4.5, with no dissatisfaction. Eighty-seven percent of patients fully recovered their performance levels, 9% changed sport, and 4% were unable to return to sport. We identified one major and one minor complication, with an overall complication rate of 7.7%. No neurovascular injuries were detected. Conclusions Elbow arthroscopy in a pediatric population can be considered an effective and safe procedure for selected pathologies when performed by an experienced surgeon. At long-term follow-up, we reported excellent clinical outcomes (both objective and subjective), with a relatively low complication rate without permanent injuries. Level of evidence Level IV—case series.

Metastasis is responsible for the majority of cancer‐related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin‐fixed paraffin‐embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions.