Kardio-Med Silesia

Reliable preclinical models that recapitulate human limbal stem cell deficiency (LSCD) and provide objective outcome measures are essential for advancing corneal cell therapies. We induced LSCD in four porcine eyes by total corneal epithelial debridement, tracked healing with serial anterior-segment optical coherence tomography (OCT) (preinjury, 0, 5–11, 17, 23, 28 days), and quantified histological changes via computer-assisted digital pathology. OCT confirmed complete epithelial removal and a reproducible stromal edema peak on days 7–9, followed by incomplete regression by day 28. Histology revealed epithelial hyperplasia, keratinization, inflammatory infiltration and neovascularization, whereas cytokeratin-3 staining revealed patchy loss of the corneal phenotype. The automated analysis revealed significant increases in epithelial roughness (p = 0.049), thickness heterogeneity (p = 0.0001), and stromal cellularity (p = 0.0179) in experimental eyes compared with control eyes. These multimodal, bias-free metrics clearly distinguish healthy corneas from LSCD corneas and provide quantifiable end points for preclinical testing of limbal epithelial, iPSC-derived or mesenchymal stromal cell advanced therapy medicinal products. All the data and analysis codes are provided in the Supplementary Information.

Background Hereditary spherocytosis (HS) is an erythrocytic membranopathy that belongs to a group of rare genetic disorders. Mutations in five genes, including ANK1, cause clinical manifestations of the disease. Identified variations in individual families provide a better understanding of the molecular basis of the disease. Methods In this study, we used two sequencing methods, whole exome sequencing (WES) and Sanger sequencing, analyzing gDNA and cDNA as templates, to detect and verify the variants putatively responsible for the clinical symptoms observed in a Polish family diagnosed with HS. Results We detected two variants that occur in cis in the ANK1 gene, a known missense mutation (NP_000028.3:p.V463I) and a novel frameshift mutation (NP_000028.3: p.V1626fs*64) that appears to be crucial for the probands. As shown by transcriptome studies, the mutant allele is not present at a detectable level. Conclusions We conclude that the molecular basis of this case is related to an unstable transcript of the mutant allele and that the direct cause of the HS is a deficiency of erythrocyte ankyrin leading to a disruption of the AE1-erythrocyte ankyrin-spectrin complex in the erythrocyte membrane.

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease that affects not only the joints but also has significant cardiovascular (CV) manifestations. The mechanistic interplay between RA and cardiovascular complications is not yet well understood due to the lack of relevant in vitro models. In this study, we established RA cardiac microtisses (cMTs) from iPSC-derived cardiomyocytes (CMs), endothelial cells (ECs) and cardiac fibroblasts (CFs) to investigate whether this fully human 3D multicellular system could serve as a platform to elucidate the connection between RA and CV disorders.MethodsPBMC and FLS from healthy and RA donors were reprogrammed to hiPSCs with Sendai vectors. hiPSCs pluripotency was assessed by IF, FACS, spontaneous embryoid bodies formation and teratoma assay. hiPSCs were differentiated to cardiac derivatives such as CMs, ECs and CFs, followed by cell markers characterizations (IF, FACS, qRT-PCR) and functional assessments. 3D cMTs were generated by aggregation of 70% CMs, 15% ECs and 15% CFs. After 21 days in culture, structural and metabolic properties of 3D cMTs were examined by IF, qRT-PCR and Seahorse bioanalyzer.ResultshiPSCs demonstrated typical colony-like morphology, normal karyotype, presence of pluripotency markers, and ability to differentiate into cells originating from all three germ layers. hiPSC-CMs showed spontaneous beating and expression of cardiac markers (cTnT, MYL7, NKX2.5, MYH7). hiPSC-ECs formed sprouting spheres and tubes and expressed CD31 and CD144. hiPSC-CFs presented spindle-shaped morphology and expression of vimentin, collagen 1 and DDR2. Self-aggregation of CMs/ECs/CFs allowed development of contracting 3D cMTs, demonstrating spherical organization of the cells, which partially resembled the cardiac muscle, both in structure and function. IF analysis confirmed the expression of cTnT, CD31, CD144 and DDR2 in generated 3D cMTs. RA cMTs exhibited significantly greater formation of capillary-like structures, mimicking enhanced vascularization—key RA feature—compared to control cMTs. Seahorse examination of cMTs revealed changes in mitochondrial and glycolytic rates in the presence of metabolic substrates and inhibitors.ConclusionsThe cMTs model may represent an advanced human stem cell-based platform for modeling CV complications in RA. The highly developed capillary-like structures observed within RA cMTs highlight a critical feature of inflammation-induced CV dysfunction in chronic inflammatory diseases.

(1) Background: Postponing orthodontic treatment in Class III malocclusion until deterioration and growth cessation to perform orthognathic surgery does not seem to be an optimal solution for every patient. This report describes short- and long-term outcomes for nonsurgical treatment of four adolescents with severe Class III malocclusion. (2) Methods: Four patients (aged 13–15 y) with skeletal Class III (Wits appraisal below 7.5 mm) started a three-stage treatment, consisting of a six-month-long phase I and involving orthopedic treatment with an individual chin-cup. Phase II involved orthopedic treatment with a bonded Haas-type expander on acrylic splints, a face-mask, a lower fixed appliance and Class III elastics; phase III involved full fixed appliance, elastics and reuse of the individual chin-cup. Pre and posttreatment cephalograms were analyzed and superimposed. (3) Results: Improved skeletal and dental relationships and facial appearance was achieved in all patients. Wits appraisal, angles ANB, ANPg and lower face height increased; an improvement of overbite was noted. (4) Conclusions: Severe skeletal Class III in adolescents may be successfully treated with combined orthopedic/camouflage treatment with a Haas-type expander on acrylic splints, Class III elastics, fixed appliance and orthopedic devices (individual chin-cup and facemask) with a very good compliance.

Introduction: Atrial fibrillation (AF), apart from non-stenotic supracardiac atherosclerosis and neoplastic disease, is the leading cause of cryptogenic stroke, including embolic stroke of un-determined source (ESUS). The aim of our study was to determine the prevalence of AF in ESUS patients based on 30-day telemetric heart rate monitoring initiated within three months after stroke onset. Another aim was to identify factors that increase the likelihood of detecting subsequent AF among ESUS patients. Material and Methods: patients with first-ever stroke classified as per the ESUS definition were eligible for this study. All patients underwent outpatient 30-day telemetric heart rate monitoring. Results: In the period between 2020 and 2022, 145 patients were included. The mean age of all qualified patients was 54; 40% of eligible patients were female. Six patients (4.14%), mostly male patients (4 vs. 2), were diagnosed with AF within the study period. In each case, the diagnosis related to a patient whose stroke occurred in the course of large vessel occlusion. Episodes of AF were detected between day 1 and 25 after starting ECG monitoring. Out of the analyzed parameters that increase the probability of, A.F.; only supraventricular extrasystoles proved to be an independent factor regarding an increased risk of AF [OR 1.046, CI 95% 1.016-1.071, p-value < 0.01]. Conclusions: The use of telemetry heart rhythm monitoring in an outpatient setting can detect AF in 4% of ESUS patients who have undergone prior diagnostic procedures for cardiogenic embolism. Supraventricular extrasystoles significantly increases the likelihood of AF detection in patients with ESUS within three months following stroke. Comorbid coronary artery disease, diabetes and hypertension, rather than a single-factor clinical burden, increase the likelihood of AF detection in older ESUS patients. ESUS in the course of large vessel occlusion is probably associated with an increased likelihood of cardiogenic embolism.

The aim of this study was to assess the influence of a protective layer of polyvinyl alcohol on the degradation process of artificial leather based on cellulose nitrate. Samples of the artificial leather were obtained from a suitcase dating back to the first half of the 20th century, not considered a historical artifact. The analysis involved Fourier-transform infrared spectroscopy with attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS). Artificial aging was employed for the study. The artificial leather sample with a protective coating of polyvinyl alcohol on a cellulose nitrate base exhibited the lowest degree of degradation due to minimal chemical changes in cellulose esters. The obtained FTIR-ATR spectrum indicated significantly higher nitration of cellulose and, consequently, a lower degree of polymer degradation. The sample without the protective polyvinyl alcohol coating and the sample with the coating removed before artificial aging showed similar reactions.

Whey protein isolate (WPI) is a by-product from the dairy industry, whose main component is β-lactoglobulin. Upon heating, WPI forms a hydrogel which can both support controlled drug delivery and enhance the proliferation and osteogenic differentiation of bone-forming cells. This study makes a novel contribution by evaluating the ability of WPI hydrogels to support the growth of endothelial cells, which are essential for vascularization, which in turn is a pre-requisite for bone regeneration. In this study, the proliferation and antioxidant levels in human umbilical vascular endothelial cells (HUVECs) cultured with WPI supplementation were evaluated using real-time cell analysis and flow cytometry. Further, the attachment and growth of HUVECs seeded on WPI-based hydrogels with different concentrations of WPI (15%, 20%, 30%, 40%) were investigated. Supplementation with WPI did not affect the viability or proliferation of HUVECs monitored with real-time cell analysis. At the highest used concentration of WPI (500 µg/mL), a slight induction of ROS production in HUVECs was detected as compared with control samples, but it was not accompanied by alterations in cellular thiol levels. Regarding WPI-based hydrogels, HUVEC adhered and spread on all samples, showing good metabolic activity. Notably, cell number was highest on samples containing 20% and 30% WPI. The demonstration of the good compatibility of WPI hydrogels with endothelial cells in these experiments is an important step towards promoting the vascularization of hydrogels upon implantation in vivo, which is expected to improve implant outcomes in the future.

Diabetes mellitus (DM) is a well-known risk factor for atrial fibrillation (AF), but the mechanism(s) by which DM affects AF prevalence remains unclear. This study aims to evaluate the impact of diabetes mellitus severity (expressed as its known duration), antihyperglycemic treatment regimen and glycaemic control on AF prevalence. From the representative sample of 3014 participants (mean age 77.5, 49.1% female) from the cross-sectional NOMED-AF study, 881 participants (mean age 77.6 ± 0.25, 46.4% female) with concomitant DM were involved in the analysis. AF was screened using a telemonitoring vest for a mean of 21.9 ± 9.1 days. The mean DM duration was 12 ± 0.35 years, but no significant impact of DM timespan on AF prevalence was observed. No differences in the treatment pattern (oral medication vs insulin vs both oral + insulin) among the study population with and without AF were shown (p = 0.106). Metabolic control reflected by HbA1c levels showed no significant association with AF and silent AF prevalence (p = 0.635; p = 0.094). On multivariate analyses, age (Odds Ratio (OR) 1.35, 95%CI: 1.18–1.53, p < 0.001), p = 0.042), body mass index (BMI; OR 1.043, 95%CI: 1.01–1.08, p = 0.027) and LDL < 100 mg/dl (OR 0.64, 95%CI: 0.42–0.97, p = 0.037) were independent risk factors for AF prevalence, while age (OR 1.45, 95%CI: 1.20–1.75, p < 0.001), LDL < 100 mg/dl (OR 0.43, 95%CI 0.23–0.82, p = 0.011), use of statins (OR 0.51, 95%CI: 0.28–0.94, p = 0.031) and HbA1c ≤ 6.5 (OR 0.46, 95%CI: 0.25–0.85, p = 0.013) were associated with silent AF prevalence. Diabetes duration, diabetic treatment pattern or metabolic control per se did not significantly impact the prevalence of AF, including silent AF detected by prospective continuous monitoring. Independent predictors of AF were age, BMI and low LDL levels, with statins and HbA1c ≤ 6.5 being additional independent predictors for silent AF.Trial registration: NCT03243474.

IntroductionPaliperidone palmitate 6-month (PP6M), administered twice-yearly, demonstrated non-inferiority to paliperidone palmitate 3-month (PP3M) in preventing relapse in patients with schizophrenia in a phase-3 randomized, double-blind (DB) global study.1 We report results of a 2-year single-arm, open-label extension (OLE) of this study (NCT04072575).ObjectivesTo assess long-term efficacy and safety of PP6M in patients with schizophrenia.MethodsPatients who completed DB study without relapse were enrolled and followed up every 3 months for up to 2 years. Patients received 4 PP6M injections (700/1000 mg eq.) at baseline, 6-month, 12-month, and 18-month visits. Efficacy endpoints included relapse rate, Positive and Negative Syndrome Scale (PANSS) total score, Personal and Social Performance (PSP) score, and Clinical Global Impression-Severity (CGI-S) scale change from baseline. Safety was assessed by treatment-emergent adverse events (TEAEs), physical examinations and laboratory tests.ResultsOf 178 patients, 154 (86.5%) completed the study; mean age: 40.4 years; 70.8% were men. Mean duration of PP6M exposure was 682.1 days. Overall, 7/178 (3.9%) patients relapsed between 20 to 703 days after enrolment. Mean (SD) change from baseline to endpoint: PANSS total score, 0.7 (8.22); CGI-S, 0.0 (0.51); PSP Scale, 0.5 (7.47). Overall, 111/178 patients (62.4%) reported ≥1 TEAE; most common (&gt;10%) TEAEs were headache (13.5%) and blood prolactin increased (10.7%). Total, 7/24 patients withdrew due to TEAEs, and 8/178 (4.5%) patients experienced serious TEAEs; no deaths were reported.ConclusionsRelapse rate with PP6M was very low (&lt;4%). Clinical improvements in PANSS, CGI-S, and PSP scales demonstrated in DB study were maintained during this 2-year OLE study and no new safety concerns were identified.Reference1. D. Najarian et al. Int J Neuropsychopharmacol. 2022 Mar 17;25(3):238-251.Disclosure of InterestD. Najarian Shareolder of: Johnson &amp; Johnson, Employee of: Janssen Research &amp; Development, LLC, I. Turkoz Shareolder of: Johnson &amp; Johnson, Employee of: Janssen Research &amp; Development, LLC, S. Galderisi Consultant of: Janssen, Gedeon Richter-Recordati, Angelini, Speakers bureau of: Angelini, Gedeon Richter-Recordati, Janssen, Lundbeck, Sunovion, Recordati, H. Lamaison Grant / Research support from: Novartis, Eli Lilly, Lundbeck, Servier, AstraZeneca, Wyeth, Pfizer, Otsuka, Takeda, Sunovion, Roche, Janssen Pharmaceutical, Speakers bureau of: Servier, Abbot, Raymonds, Raffo, Temis Lostalo and Janssen Pharmaceutical, P. Zalitacz: None Declared, S. Aravind Shareolder of: Johnson &amp; Johnson, Employee of: Advarra, Inc. USA, U. Richarz Shareolder of: Johnson &amp; Johnson, Employee of: Janssen Research &amp; Development-Cilag, Switzerland.

Although it is well‑known that longer electrocardiographic (ECG) monitoring allows for detection of paroxysmal silent atrial fibrillation (SAF), it is still unknown how long the ECG monitoring should last to increase the probability of SAF diagnosis. Our aim was to analyze ECG acquisition parameters and timing to detect SAF during the Noninvasive Monitoring for Early Detection of Atrial Fibrillation study. The protocol assumed up to 30 days of ECG telemonitoring of each participant in order to reveal AF / atrial flutter (AFL) episodes lasting at least 30 seconds. SAF was defined as AF detected and confirmed by cardiologists in asymptomatic individuals. The ECG signal analysis was based on the results of 2974 participants (98.67%). AF/AFL episodes were registered and confirmed by cardiologists in 515 individuals, that is, 75.7% of all patients (n = 680) in whom AF/AFL diagnosis was established. The median monitoring time to detect the first SAF episode was 6 days (interquartile range [IQR], 1-13). Fifty percent of the patients with this type of arrhythmia were identified by 6th day (IQR, 1-13) of the monitoring, and 75% by the 13th day of the study. Paroxysmal AF was registered on average on 4th day (IQR, 1-10). The ECG monitoring time to detect the first episode of SAF in at least 75% of patients at risk of this arrhythmia was 14 days. Detection of 1 patient with de novo AF, SAF, or de novo SAF, required monitoring of, respectively, 17, 11, and 23 patients.