"Real-world" analysis of battery longevity of implantable cardioverter-defibrillators: an in-depth analysis of a prospective defibrillator database.

BACKGROUND: There is a lack of evidence regarding contemporary implantable cardioverter-defibrillator (ICD) battery longevity. Our aim was to assess battery longevity in ICDs in a real-world setting. METHODS: Retrospective cross-sectional single center study of a prospectively collected database of consecutive patients who underwent ICD implantation from January 2010 to December 2015. Clinical data and battery longevity of all manufacturers were collected. RESULTS: A total of 351 patients (84.6% males, mean age of 61 ± 12 years) were included in the study (292 VVI; 6 VDD; 53 DDD). All manufacturers (Abbott, Biotronik, Boston, Medtronic and Microport) were equally represented in the study (p = 0.110). Median battery longevity was 10.8 years (11 years for VVI and 8.5 for DDD). After a follow-up time of 5 years, 98% of VVI and DDD were still in service (vs. industry-projected longevity of 98%). During this time, 89 patients (25.4%) underwent device replacement - 69 patients (77.5%) due to battery depletion, 6 patients due to infection, 3 patients due to dysfunction and 13 patients due to upgrade to CRT-D. Patients with Medtronic or Biotronik ICDs had a greater probability of being replaced earlier due to battery depletion (Biotronik HR 6.87, 95% CI 2.54-18.58, p < 0.001; Medtronic HR 6.08, 95% CI 2.45-15.06 p < 0.001). CONCLUSIONS: VVI and DDD ICD battery longevity matched industry-projected longevity after 5 years of follow-up. Medtronic and Biotronik ICDs appeared to have an earlier battery depletion. Further randomized studies are required to ensure optimal care.

Prevalence and risk factors for acquired long QT syndrome in the emergency department: a retrospective observational study.

BACKGROUND: Long QT syndrome (LQTS) is a heterogeneous syndrome that may be congenital or, more frequently, acquired. The real-world prevalence of acquired LQTS (aLQTS) in the emergency department (ED) remains to be determined. The aim of this study was to determine prevalence of aLQTS and its impact on symptoms on ED admissions. METHODS: Electrocardiograms (ECG) of 5,056 consecutively patients admitted in the ED of a tertiary hospital between January 28th and March 17th of 2020 were reviewed. All patients with aLQTS were included. Clinical data with a focus on QT prolonging drugs and clinical factors were recorded. Statistical comparison was made between the groups with and without corrected QT (QTc) interval greater than 500 ms (value that is considered severely increased). RESULTS: A total of 383 ECGs with prolonged QTc were recognized, corresponding to a prevalence of aLQTS at admission of 7.82%. Patients with aLQTS were more commonly men (53.3%) with an age of (73.49±14.79) years old and QTc interval of (505.3±32.4) ms. Only 20.4% of these patients with aLQTS were symptomatic. No ventricular arrhythmias were recorded. Patients with QT interval greater than 500 ms were more frequently female (59.5%; P<0.001) and were more frequently on QT prolonging drugs (77.3%; P=0.025). Main contributing factor was intake of antibiotics (odds ratio [OR] 4.680) followed by female gender (OR 2.473) and intake of antipsychotics (OR 1.925). CONCLUSION: aLQTS is particularly prevalent in the ED. Female patients on antibiotics and antipsychotics are at particularly high risk. Efforts must be made to avoid, detect and treat aLQTS as early as possible.

Advanced Manufacturing, Formulation and Microencapsulation of Therapeutic Phages.

Manufacturing and formulation of stable, high purity, and high dose bacteriophage drug products (DPs) suitable for clinical usage would benefit from improved process monitoring and control of critical process parameters that affect product quality attributes. Chemistry, Manufacturing, and Controls (CMC) for both upstream (USP) and downstream processes (DSP) need mapping of critical process parameters (CPP) and linking these to critical quality attributes (CQA) to ensure quality and consistency of phage drug substance (DS) and DPs development. Single-use technologies are increasingly becoming the go-to manufacturing option with benefits both for phage bioprocess development at the engineering run research stage and for final manufacture of the phage DS. Future phage DPs under clinical development will benefit from implementation of process analytical technologies (PAT) for better process monitoring and control. These are increasingly being used to improve process robustness (to reduce batch-to-batch variability) and productivity (yielding high phage titers). Precise delivery of stable phage DPs that are suitably formulated as liquids, gels, solid-oral dosage forms, and so forth, could significantly enhance efficacy of phage therapy outcomes. Pre-clinical development of phage DPs must include at an early stage of development, considerations for their formulation including their characterization of physiochemical properties (size, charge, etc.), buffer pH and osmolality, compatibility with regulatory approved excipients, storage stability (packaging, temperature, humidity, etc.), ease of application, patient compliance, ease of manufacturability using scalable manufacturing unit operations, cost, and regulatory requirements.

Actin cytoskeleton remodeling at the cancer cell side of the immunological synapse: good, bad, or both?

Cytotoxic lymphocytes (CLs), specifically cytotoxic T lymphocytes and natural killer cells, are indispensable guardians of the immune system and orchestrate the recognition and elimination of cancer cells. Upon encountering a cancer cell, CLs establish a specialized cellular junction, known as the immunological synapse that stands as a pivotal determinant for effective cell killing. Extensive research has focused on the presynaptic side of the immunological synapse and elucidated the multiple functions of the CL actin cytoskeleton in synapse formation, organization, regulatory signaling, and lytic activity. In contrast, the postsynaptic (cancer cell) counterpart has remained relatively unexplored. Nevertheless, both indirect and direct evidence has begun to illuminate the significant and profound consequences of cytoskeletal changes within cancer cells on the outcome of the lytic immunological synapse. Here, we explore the understudied role of the cancer cell actin cytoskeleton in modulating the immune response within the immunological synapse. We shed light on the intricate interplay between actin dynamics and the evasion mechanisms employed by cancer cells, thus providing potential routes for future research and envisioning therapeutic interventions targeting the postsynaptic side of the immunological synapse in the realm of cancer immunotherapy. This review article highlights the importance of actin dynamics within the immunological synapse between cytotoxic lymphocytes and cancer cells focusing on the less-explored postsynaptic side of the synapse. It presents emerging evidence that actin dynamics in cancer cells can critically influence the outcome of cytotoxic lymphocyte interactions with cancer cells.

Cutting-edge advances in modeling the blood-brain barrier and tools for its reversible permeabilization for enhanced drug delivery into the brain.

The blood-brain barrier (BBB) is a sophisticated structure whose full functionality is required for maintaining the executive functions of the central nervous system (CNS). Tight control of transport across the barrier means that most drugs, particularly large size, which includes powerful biologicals, cannot reach their targets in the brain. Notwithstanding the remarkable advances in characterizing the cellular nature of the BBB and consequences of BBB dysfunction in pathology (brain metastasis, neurological diseases), it remains challenging to deliver drugs to the CNS. Herein, we outline the basic architecture and key molecular constituents of the BBB. In addition, we review the current status of approaches that are being explored to temporarily open the BBB in order to allow accumulation of therapeutics in the CNS. Undoubtedly, the major concern in field is whether it is possible to open the BBB in a meaningful way without causing negative consequences. In this context, we have also listed few other important key considerations that can improve our understanding about the dynamics of the BBB.

Cirrhosis is associated with lower serological responses to COVID-19 vaccines in patients with chronic liver disease.

Background & Aims: The response of patients with chronic liver disease (CLD) to COVID-19 vaccines remains unclear. Our aim was to assess the humoral immune response and efficacy of two-dose COVID-19 vaccines among patients with CLD of different aetiologies and disease stages. Methods: A total of 357 patients were recruited in clinical centres from six European countries, and 132 healthy volunteers served as controls. Serum IgG (nM), IgM (nM), and neutralising antibodies (%) against the Wuhan-Hu-1, B.1.617, and B.1.1.529 SARS-CoV-2 spike proteins were determined before vaccination (T0) and 14 days (T2) and 6 months (T3) after the second-dose vaccination. Patients fulfilling inclusion criteria at T2 (n = 212) were stratified into 'low' or 'high' responders according to IgG levels. Infection rates and severity were collected throughout the study. Results: Wuhan-Hu-1 IgG, IgM, and neutralisation levels significantly increased from T0 to T2 in patients vaccinated with BNT162b2 (70.3%), mRNA-1273 (18.9%), or ChAdOx1 (10.8%). In multivariate analysis, age, cirrhosis, and type of vaccine (ChAdOx1 > BNT162b2 > mRNA-1273) predicted 'low' humoral response, whereas viral hepatitis and antiviral therapy predicted 'high' humoral response. Compared with Wuhan-Hu-1, B.1.617 and, further, B.1.1.529 IgG levels were significantly lower at both T2 and T3. Compared with healthy individuals, patients with CLD presented with lower B.1.1.529 IgGs at T2 with no additional key differences. No major clinical or immune IgG parameters associated with SARS-CoV-2 infection rates or vaccine efficacy. Conclusions: Patients with CLD and cirrhosis exhibit lower immune responses to COVID-19 vaccination, irrespective of disease aetiology. The type of vaccine leads to different antibody responses that appear not to associate with distinct efficacy, although this needs validation in larger cohorts with a more balanced representation of all vaccines. Impact and Implications: In patients with CLD vaccinated with two-dose vaccines, age, cirrhosis, and type of vaccine (Vaxzevria > Pfizer BioNTech > Moderna) predict a 'lower' humoral response, whereas viral hepatitis aetiology and prior antiviral therapy predict a 'higher' humoral response. This differential response appears not to associate with SARS-CoV-2 infection incidence or vaccine efficacy. However, compared with Wuhan-Hu-1, humoral immunity was lower for the Delta and Omicron variants, and all decreased after 6 months. As such, patients with CLD, particularly those older and with cirrhosis, should be prioritised for receiving booster doses and/or recently approved adapted vaccines.

Comparison of the Barrett Universal II, Kane and VRF-G formulas with existing intraocular lens calculation formulas in eyes with short axial lengths.

BACKGROUND: To compare the accuracy of recently developed modern intraocular lens (IOL) power formulas (Barrett Universal II, Kane and VRF-G) with existing IOL power formulas in eyes with an axial length (AL) ≤ 22 mm. METHODS: This analysis comprised 172 eyes of 172 patients operated on by one surgeon (LT) with one IQ SN60WF (Alcon Labs, Fort Worth, TX, USA) hydrophobic lens. Ten IOL formulas were evaluated: Barrett Universal II (BUII), Haigis, Hoffer Q, Holladay 1, Holladay 2, Kane, SRK/T, T2, VRF and VRF-G. The median absolute error (MedAE), mean absolute error (MAE), standard deviation (SD) and all descriptive statistics were evaluated. Percentages of eyes with a prediction error within ±0.25 D, ±0.50 D, ±0.75 D and ±1.00 D were calculated using standard optimised constants for the entire range of axial lengths. RESULTS: The VRF-G, Haigis and Kane produced the smallest MedAE among all formulas (0.242 D, 0.247 D and 0.263 D, respectively) and had the highest percentage of eyes with a PE within ±0.50 D (75.67%, 73.84% and 75.16%, respectively). The Barrett was less accurate (0.298 D and 68.02%, respectively). Statistically significant differences were found predominantly between the VRF-G (P < 0.05), Kane (P < 0.05) and Haigis (P < 0.05) and all other formulas. The percentage of eyes with a PE within ±0.50 D ranged from 66.28% to 75.67%. CONCLUSIONS: In eyes with AL ≤ 22.0 mm, the VRF-G, Haigis and Kane were the most accurate predictors of postoperative refraction, and the Barrett formula was less predictable.

Severe Ascites in Common Variable Immunodeficiency.

Liver disease is one of the possible clinical manifestations of common variable immunodeficiency and can range from mild hepatomegaly and persistent elevation of liver enzymes to cirrhosis, portal hypertension, and nodular regenerative hyperplasia. The last one is the most common histologic presentation of liver involvement by common variable immunodeficiency and its clinical spectrum can range from asymptomatic to cholestasis, liver cirrhosis, or idiopathic non-cirrhotic portal hypertension, with the severe manifestations being less recognised. We present a case of a 48-year-old woman who was referred for an internal medicine consultation for evaluation of rapidly progressing (span of three months) large-volume ascites and marked asthenia. The patient had a past medical history of common variable immunodeficiency and a recent episode of severe haemolytic anaemia. Peritoneal fluid analyses identified portal hypertension as the cause of the ascites. Abdominal Doppler ultrasound and contrasted abdominal computed tomography confirmed the presence of permeable hepatic and portal veins. Liver biopsy revealed regenerative nodular hyperplasia without cirrhosis. A diagnosis of idiopathic non-cirrhotic portal hypertension secondary to common variable immunodeficiency was made. Treatment was adjusted with considerable improvement in ascites. In conclusion, idiopathic non-cirrhotic portal hypertension is a possible and often overlooked complication in patients with common variable immunodeficiency and is an exclusion diagnosis that requires a high level of suspicion, especially in patients with ascites.

Numerical and experimental simulation of a dynamic-rotational 3D cell culture for stratified living tissue models.

Human tissues and organs are inherently heterogeneous, and their functionality is determined by the interplay between different cell types, their secondary architecture, and gradients of signalling molecules and metabolites. To mimic the dynamics of native tissues, perfusion bioreactors and microfluidic devices are widely used in tissue engineering (TE) applications for enhancing cell culture viability in the core of 3D constructs. Still, mostin vitroscreening methods for compound efficacy and toxicity assessment include cell or tissue exposure to constant and homogeneous compound concentrations over a defined testing period. Moreover, a prevalent issue inhibiting the large-scale adoption of microfluidics and bioreactor is the tubing dependence to induce a perfusion regime. Here, we propose a compartmentalized rotational (CR) 3D cell culture platform for a stable control over gradient tissue culture conditions. Using the CR bioreactor, adjacent lanes of constructs are patterned by controlled flow dynamics to enable tissue stratification. Numerical and experimental simulations demonstrate cell seeding dynamics, as well as culture media rotational perfusion and gradient formations. Additionally, the developed system induces vertical and horizontal rotations, which increase medium exchange and homogeneous construct maturation, allowing both perfused tubing-based and tubing-free approaches. As a proof-of-concept, experiments and accompanying simulation of cellular inoculation and growth in 3D scaffold and hydrogel were performed, before the examination of a blood-brain-barrier model, demonstrating the impact of a heterotypic culture on molecular permeability under mimetic dynamic conditions. Briefly, the present work discloses the simulation of 3D dynamic cultures, and a semi-automated platform for heterotypic tissuesin vitromodelling, for broad TE and drug discovery/screening applications.

Impact of the COVID-19 lockdown in urgent pacemaker implantations: A cross-sectional study.

AIMS: The COVID-19 pandemic resulted in a decrease in patients' follow-up and interventions with cardiovascular disease. In Portugal, the consequences on emergent pacemaker implantation rates are largely unknown. We sought to analyze the impact of the COVID-19 pandemic on emergent pacemaker implantation rate and patient profile. METHODS: We retrospectively reviewed the clinical profile of the 180 patients who had pacemakers implanted in our hospital in an emergent setting from March 18, 2020, to May 17, 2020 ("lockdown") and May 19 to July 17, 2020 ("postlockdown"). This data was then directly compared to the homologous periods from the year before. RESULTS: Urgent pacemaker implantation rates during "lockdown" was lower than its homologous period (-23.7%), and cases in "postlockdown" were significantly increased (+106.9% vs. "lockdown"; +13.2% vs. May-July 2019).When comparing "lockdown" and "postlockdown," there was a tendency for a higher number of temporary pacemaker use (p = .076). Patients during "lockdown" were 7.57 times more likely to present with hypotension/shock (odds ratio 7.57; p = .013). We also noted a higher tendency for hypotension on presentation during "lockdown" (p = .054) in comparison to 2019. In comparison to its homologous 2019 period, "postlockdown" saw more patients presenting with bradycardia (p = .026). No patients were admitted to the emergency department during "lockdown" for anomalies detected on ambulatory tests. CONCLUSION: Our data show that the COVID-19 pandemic had a real impact on urgent pacemaker implantation. Patients with bradyarrhythmias are at particular risk for severe complications and should seek medical care regardless of the pandemic.

Anterior chamber depth, lens thickness and intraocular lens calculation formula accuracy: nine formulas comparison.

BACKGROUND/AIMS: To investigate the influence of anterior chamber depth (ACD) and lens thickness (LT) on 9 intraocular lens (IOL) power calculation formulas accuracy, in patients with normal axial lengths. METHODS: Retrospective case series, including patients having uncomplicated cataract surgery with insertion of a single IOL model, divided into three groups according to preoperative ACD. Each group was further subdivided into three subgroups, according to the LT. Using optimised constants, refraction prediction error was calculated for Barrett Universal II, Emmetropia Verifying Optical (EVO) V.2.0, Haigis, Hill-RBF V.2.0, Hoffer Q, Holladay 1, Kane, PEARL-DGS and SRK/T formulas. Mean prediction error, mean and median absolute error (MedAE) and the percentage of eyes within ±0.25D, ±0.50D and ±1.00D were also calculated. RESULTS: The study included 695 eyes from 695 patients. For ACD ≤3.0 mm and ≥3.5 mm, mean prediction error of SRK/T, Hoffer Q and Holladay 1 was significantly different from 0 (p<0.05). PEARL-DGS, Kane, EVO V.2.0 and Barrett Universal II were more accurate than the Hoffer Q in ACD ≤3.0 mm (p<0.05). Kane, PEARL-DGS, EVO V.2.0 and Barrett Universal II revealed the lowest variance of mean and MedAE by ACD and LT subgroup. Haigis and Hill-RBF V.2.0 were significantly influenced by LT, independently of the ACD, with a myopic shift with thin lenses and a hyperopic shift with thick lenses (p<0.05). CONCLUSION: New generation formulas, particularly Kane, PEARL-DGS and EVO V.2.0, seem to be more reliable and stable even in eyes with extreme ACD-LT combinations.

Bioengineered Nanoparticles Loaded-Hydrogels to Target TNF Alpha in Inflammatory Diseases.

Rheumatoid Arthritis (RA) is an incurable autoimmune disease that promotes the chronic impairment of patients' mobility. For this reason, it is vital to develop therapies that target early inflammatory symptoms and act before permanent articular damage. The present study offers two novel therapies based in advanced drug delivery systems for RA treatment: encapsulated chondroitin sulfate modified poly(amidoamine) dendrimer nanoparticles (NPs) covalently bonded to monoclonal anti-TNF α antibody in both Tyramine-Gellan Gum and Tyramine-Gellan Gum/Silk Fibroin hydrogels. Using pro-inflammatory THP-1 (i.e., human monocytic cell line), the therapy was tested in an inflammation in vitro model under both static and dynamic conditions. Firstly, we demonstrated effective NP-antibody functionalization and TNF-α capture. Upon encapsulation, the NPs were released steadily over 21 days. Moreover, in static conditions, the approaches presented good anti-inflammatory activity over time, enabling the retainment of a high percentage of TNF α. To mimic the physiological conditions of the human body, the hydrogels were evaluated in a dual-chamber bioreactor. Dynamic in vitro studies showed absent cytotoxicity in THP-1 cells and a significant reduction of TNF-α in suspension over 14 days for both hydrogels. Thus, the developed approach showed potential for use as personalized medicine to obtain better therapeutic outcomes and decreased adverse effects.

Intraocular lens power calculation formulas accuracy in combined phacovitrectomy: an 8-formulas comparison study.

BACKGROUND: Our study aimed to assess and compare the accuracy of 8 intraocular lens (IOL) power calculation formulas (Barrett Universal II, EVO 2.0, Haigis, Hoffer Q, Holladay 1, Kane and PEARL-DGS) in patients submitted to combined phacovitrectomy for vitreomacular (VM) interface disorders. METHODS: Retrospective chart review study including axial-length matched patients submitted to phacoemulsification alone (Group 1) and combined phacovitrectomy (Group 2). Using optimized constants in both groups, refraction prediction error of each formula was calculated for each eye. The optimised constants from Group 1 were also applied to patients of Group 2 - Group 3. Outcome measures included the mean prediction error (ME) and its standard deviation (SD), mean (MAE) and median (MedAE) absolute errors, in diopters (D), and the percentage of eyes within ± 0.25D, ± 0.50D and ± 1.00D. RESULTS: A total of 220 eyes were included (Group 1: 100; Group 2: 120). In Group 1, the difference in formulas absolute error was significative (p = 0.005). The Kane Formula had the lowest MAE (0.306) and MedAE (0.264). In Group 2, Kane had the overall best performance, followed by PEARL-DGS, EVO 2.0 and Barrett Universal II. The ME of all formulas in both Groups 1 and 2 were 0.000 (p = 0.934; p = 0.971, respectively). In Group 3, a statistically significant myopic shift was observed for each formula (p < 0.001). CONCLUSION: Surgeons must be careful regarding IOL power selection in phacovitrectomy considering the systematic myopic shift evidenced-constant optimization may help eliminating such error. Moreover, newly introduced formulas and calculation methods may help us achieving increasingly better refractive outcomes both in cataract surgery alone and phacovitrectomy.

Hydrogels in the treatment of rheumatoid arthritis: drug delivery systems and artificial matrices for dynamic in vitro models.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disorder that mostly affects the synovial joints and can promote both cartilage and bone tissue destruction. Several conservative treatments are available to relieve pain and control the inflammation; however, traditional drugs administration are not fully effective and present severe undesired side effects. Hydrogels are a very attractive platform as a drug delivery system to guarantee these handicaps are reduced, and the therapeutic effect from the drugs is maximized. Furthermore, hydrogels can mimic the physiological microenvironment and have the mechanical behavior needed for use as cartilage in vitro model. The testing of these advanced delivery systems is still bound to animal disease models that have shown low predictability. Alternatively, hydrogel-based human dynamic in vitro systems can be used to model diseases, bypassing some of the animal testing problems. RA dynamic disease models are still in an embryonary stage since advances regarding healthy and inflamed cartilage models are currently giving the first steps regarding complexity increase. Herein, recent studies using hydrogels in the treatment of RA, featuring different hydrogel formulations are discussed. Besides, their use as artificial extracellular matrices in dynamic in vitro articular cartilage is also reviewed.

Clinical Accuracy of 18 IOL Power Formulas in 241 Short Eyes.

PURPOSE: To analyze the accuracy of 18 intraocular lens (IOL) power calculation formulas in eyes with axial length (AL) ≤ 22 mm. METHODS: We analyzed 241 eyes of 241 patients. Eighteen formulas were evaluated: Barrett Universal II (BUII), EVO 2.0, Haigis, Hoffer Q, Holladay 1 and 2, Cooke K6, Kane, LadasSuperFormula AI, Naeser 2, Olsen, Panacea, Pearl-DGS, RBF 2.0, SRK/T, T2, VRF and VRF-G. Optical biometry was performed with an IOLMaster 700 (Carl Zeiss Meditec, Jena, Germany). With lens constants optimized for the whole range of AL, the mean prediction error (PE) and its standard deviation (SD), the median absolute error (MedAE), the mean absolute error (MAE) and the percentage of eyes with PEs within ±0.25 D, ±0.50 D and <±1.00 D were calculated. RESULTS: Post-hoc analysis of the absolute PE revealed statistically significant differences (P < .05) between some of the newer formulas (K6, Kane, Naeser 2, Olsen and VRF-G), which obtained the lowest MedAE (respectively, 0.308, 0.300, 0.277, 0.310 and 0.276 D) and the remaining ones. These formulas yielded also the highest percentage of eyes with a PE within ±0.50 D (70.54%, 72.20%, 71.37%, 70.95% and 73.03%, respectively), whereas Panacea and SRK/T yielded the lowest percentage (62.24%), with a stastically significant difference (P < .05) with respect to most formulas. CONCLUSION: In eyes with AL ≤22.0 mm, new formulas (K6, Kane, Naeser 2, Olsen and VRF-G) offer the most accurate predictions of postoperative refraction.

A rich gallery of carbon dots based photoluminescent suspensions and powders derived by citric acid/urea.

In this study we demonstrate simple guidelines to generate a diverse range of fluorescent materials in both liquid and solid state by focusing on the most popular C-dots precursors, i.e. the binary systems of citric acid and urea. The pyrolytic treatment of those precursors combined with standard size separation techniques (dialysis and filtration), leads to four distinct families of photoluminescent materials in which the emissive signal predominantly arises from C-dots with embedded fluorophores, cyanuric acid-rich C-dots, a blend of molecular fluorophores and a mixture of C-dots with unbound molecular fluorophores, respectively. Within each one of those families the chemical composition and the optical properties of their members can be fine-tuned by adjusting the molar ratio of the reactants. Apart from generating a variety of aqueous dispersions, our approach leads to highly fluorescent powders derived from precursors comprising excessive amounts of urea that is consumed for the build-up of the carbogenic cores, the molecular fluorophores and the solid diluent matrix that suppresses self-quenching effects.

Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques.

Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC-MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011-8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011-7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3-8% and 10-37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC-MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

Corneal Subbasal Nerve Plexus Evaluation by in Vivo Confocal Microscopy in Multiple Sclerosis: A Potential New Biomarker.

Purpose/Aim: Our study aims to evaluate corneal subbasal nerve plexus morphology by in vivo corneal confocal microscopy (CCM) in Multiple Sclerosis (MS) patients and to explore its potential ability to distinguish between MS patients and healthy subjects.Materials and methods: Cross-sectional study, including 60 MS patients and 22 healthy subjects. Expanded Disability Status Scale (EDSS) was used to assess neurological disability. All participants underwent full ophthalmology evaluation, CCM and optical coherence tomography (OCT). Corneal nerve fibre density (CNFD), branch density (CNBD), fibre length (CNFL) and fibre tortuosity (CNFT) were analysed. Generalized additive regression models were used to analyse the data.Results: Compared to controls, MS patients had lower CNFD, CNBD and CNFL (p < .001) and higher CNFT (p = .002). The area under the ROC curve to distinguish MS patients from healthy controls with CNFD and CNBD was 0.84 (95%CI: 0.75 to 0.93; 95%CI: 0.75 to 0.92, respectively). A nonlinear association between EDSS and CNFD was found, with an initial density increase followed by a significant decrease until more severe disability status. EDSS was associated with CNFL and CNBD, with values being significantly lower for patients with an EDSS > 2.5 (-2.06 mm/mm2; 95%CI: -3.84 to -0.28; p = .027 and -8.70 branches/mm2; 95%CI: -14.69 to -2.71; p = .006, respectively). An optic neuritis (ON) history did not influence CCM parameters.Conclusions: Our results confirm CCM parameters' potential to differentiate MS patients from healthy subjects, not being influenced by a previous ON history. A significant relationship between patient's disability and corneal nerve morphology was also found.

Corneal sub-basal nerve plexus assessment and its association with phenotypic features and lymphocyte subsets in Sjögren's Syndrome.

PURPOSE: To assess and compare corneal sub-basal nerve plexus morphology with circulating lymphocyte subsets, immunologic status and disease activity in Sjögren syndrome (SjS) patients. METHODS: Fifty-five SjS patients, 63 Sicca patients (not fulfilling SjS criteria), 18 rheumatoid arthritis (RA) patients and 20 healthy controls (HC) were included. Systemic disease activity in SjS was assessed with the ESSDAI score. Lymphocyte subpopulations were studied with flow cytometry. Corneal confocal microscopy and ImageJ software were used to characterize corneal sub-basal nerve plexus in terms of nerve density (CNFD), length (CNFL) and tortuosity (CNFT). Conventional dry eye tests were also performed. RESULTS: CNFL and CNFD were lower in SjS, Sicca and RA groups, compared to HC (p < 0.001 for both SjS and Sicca); CNFL p = 0.005, CNFD p = 0.018 in RA). CNFT was higher in SjS, followed by Sicca, RA and HC. A negative correlation was found between ESSDAI score and CNFL (r=-0.735, p = 0.012). CNFL correlated negatively with IL21+ CD8+ T cells (r=-0.279, p = 0.039) and a positively with total memory (r = 0.299, p = 0.027), unswitched memory (r = 0.281, p = 0.038) and CD24Hi CD27+ (r = 0.278, p = 0.040) B cells. CNFD showed a tendency to significance in its negative correlation with ESSDAI (r=-0.592, p = 0.071) and in its positive correlation with switched memory B cells (r = 0.644, p = 0.068). CONCLUSIONS: This is the first study aiming to correlate ocular findings with lymphocyte subsets in SjS. The associations founded between CNFL and CNFD and disease activity, IL21+ follicular T cells and some B-cell subsets suggest that corneal nerve damage may parallel systemic disease activity and inflammatory cells' dynamics.

Non-central serous chorioretinopathy in a patient with systemic lupus erythematosus and hydroxychloroquine retinopathy.

Subretinal fluid accumulation in a patient with systemic lupus erythematosus (SLE) may represent a diagnostic challenge. We present a case of a 43-year-old man with baseline diagnosis of SLE and hydroxychloroquine-associated maculopathy who reported progressive vision loss on the right eye, associated with corticosteroids use for an arthritic crisis. Ophthalmological examination did not reveal any acute finding. On optical coherence tomography, subretinal fluid in the perifoveal area was visible on the right eye, with corresponding enlargement of the visual field defect. An increased choroidal thickness was also visible. Fluorescein angiography revealed, on the right eye, two pinpoint areas of leakage and indocyanine green angiography signs of choroidal vascular hyperpermeability. Considering a diagnosis of a non-central central serous chorioretinopathy, corticosteroids use was interrupted, with resolution of the subretinal fluid. This case illustrates the relevance of a multimodal imaging approach to guide the diagnosis of patient with an SLE with subretinal fluid.