Pharmo Institute

In cartilage tissue engineering, there is a growing interest in dynamic hydrogels that promote spheroid fusion and cartilaginous matrix deposition, while maintaining sufficient stability for long-term construct maturation. In this study, Diels-Alder click chemistry is employed as a dynamic-covalent crosslinking method to create hydrogels composed of hyaluronic acid, gelatin, and PEG. By adjusting the pH during crosslinking, the tuneability of hydrogel stiffness and stability at pH values around the physiological pH of native cartilage is demonstrated. This pH modulation does not compromise hydrogel functionality, as encapsulated equine articular cartilage progenitor cell spheroids remain viable and functional for a culture period of 28 days. The hydrogel environment supports the deposition of cartilaginous extracellular matrix components, including collagens and sulphated glycosaminoglycans. Enhanced chondrogenesis and deposition of collagen type II are observed at higher spheroid concentrations, corresponding to inter-spheroid distances of 100-150µm following hydrogel swelling, compared to lower concentrations at a distance of >500µm. To further improve construct robustness, the hydrogel constructs can be reinforced on day 1 with a melt electrowritten scaffold, increasing the compressive modulus 100-fold by day 28 compared to non-reinforced constructs, highlighting the potential of this system for engineering cartilage implants.

Background/Objectives: Administrative and clinical databases are increasingly used for research, but their value depends on coding accuracy. The Spanish National Hospital Discharge Database (CMBD) is a standardised registry widely applied in epidemiology. Type 1 diabetes mellitus (T1DM) is an autoimmune disease with early onset and long-term complications. This study aimed to validate the accuracy of T1DM diagnoses recorded in the CMBD. Methods: A cross-sectional validation study was conducted at Hospital Clínico San Carlos (Madrid, Spain) including discharges from 2016-2023. Two age- and sex-matched samples of 384 admissions each (with and without T1DM coding, ICD-10 E10) were randomly selected. The gold standard was the confirmation of T1DM based on the diagnostic criteria established by the 2016 American Diabetes Association (ADA) consensus, which remained valid through 2025, verified by a detailed review of electronic health records (EHRs). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated with 95% confidence intervals (CIs), and interobserver concordance was assessed with Cohen's kappa. Results: Of the 245,206 discharges, 1324 (0.54%) included a T1DM diagnosis. Validation showed a sensitivity of 100% (95% CI: 98.7-100), specificity of 80.2% (95% CI: 76.4-83.5), PPV of 75.3% (95% CI: 70.7-79.3), and NPV of 100% (95% CI: 99.0-100). Interobserver agreement was excellent (κ = 0.869). Specificity declined with age, from 100% in patients < 30 years to 60% in those ≥ 80 years, mainly due to misclassification with insulin-treated type 2 diabetes. Conclusions: T1DM diagnoses in the CMBD show very high validity and reliability in younger patients, supporting their use in epidemiological and clinical research, while complementary verification is advisable in older adults.

Abstract Kdnases have been reported in a variety of organisms, including marine species such as trout and oysters, the opportunistic Gram-negative bacterium Sphingobacterium multivorum , and several fungal species of the genus Aspergillus , including Aspergillus terreus and Aspergillus fumigatus .. In particular, the Kdnase from the opportunistic airborne pathogen Aspergillus fumigatus ( Af Kdnase) plays an important role in fungal cell wall integrity and virulence, although the underlying mechanisms remain unclear. To better understand this class of enzymes, selective and sensitive tools are required for discovery, detection and visualization of active Kdnases in complex biological samples. In this work, we report the development of difluoro-Kdn mechanism-based probes functionalized with azide and biotin tags for labeling and detection of Kdnases. We show that the probes exhibit selectivity for Kdnase over the neuraminidases tested and efficiently label recombinantly expressed Af Kdnase at micromolar concentrations. In addition, using the azide-bearing probe and click chemistry, we successfully visualized native Kdnases in A. fumigatus mycelia, demonstrating their utility for studying these enzymes in crude biological samples and highlighting their potential for discovering Kdnases in other organisms including fungal and bacterial species.

PurposeApplying standardized Brighton Collaboration (BC) case definitions retrospectively to heterogeneous real-world data (RWD) is challenging due to inconsistent clinical detail and data structures across settings. To address this, the Vaccine Monitoring Collaboration for Europe (VAC4EU) developed a structured validation pipeline that operationalizes BC case definitions for vaccine safety outcomes in RWD in a harmonized, scalable, and reusable manner.MethodsVAC4EU developed a systematic, stepwise approach to validate vaccine safety outcomes. BC case definitions were utilized when available, adapted for RWD as needed, and newly developed for outcomes without an existing BC definition. The approach involves: 1) Critical review and adaptation of BC definition by clinical and RWD experts; 2) Creation of dummy cases based on published reports; 3) Creation of REDCap electronic data collection forms (eDCF) incorporating decision logic to assigned levels of certainty (LOC); 4) Iterative testing of decision logic; and 5) comprehensive training of abstractors with real-time feedback. A dedicated task force assigned reference LOCs for dummy cases. Inter-rater reliability was measured using Fleiss’ kappa (κ) by comparing abstractor LOCs to the reference standard.ResultsThe pipeline was applied to 16 COVID-19 vaccine safety outcomes, of which 13 had existing BC definition. In total, 78 dummy case descriptions were developed across the outcomes for training of abstractors, and 15 REDCap eDCFs were created. Myocarditis and pericarditis shared an eDCF. Across 33 abstractors, 747 dummy case abstractions were completed. Agreement analysis showed 93 discrepancies (12.4%) and moderate overall concordance (κ = 0.55, across all outcomes), with the lowest for thrombosis with thrombocytopenia syndrome (κ = –0.05).ConclusionThe VAC4EU validation pipeline provides a standardized framework for training and validating vaccine safety outcome in RWD. By adapting BC case definitions and dedicated training of abstractors, we will reduce variability in outcome validation in post-authorization safety studies.

Highly pathogenic H5Ny influenza A viruses are causing unprecedented, season-independent outbreaks across avian and mammalian species, including dairy cattle, a novel reservoir. The sialoside-binding properties of influenza A hemagglutinin (HA) are strongly related to its ability to infect and transmit between hosts. Mucin-like O-glycans, omnipresent in respiratory tracts, have been understudied as viral receptors due to their complexity. To address this, we synthesized 25 O-linked glycans with diverse sialosides, including modifications by fucosides and sulfates. Our findings reveal that H5Ny 2.3.4.4b viruses bind core 3 sialyl-Lewisx and Sia-Gal-β3GalNAc, O-linked glycans not recognized by classical H5 or other avian viruses. By determining crystal structures, we resolved the structural features of four glycans in an H5 hemagglutinin (HA) from a 2016 2.3.4.4b virus. While these viruses do not bind human-type receptors, their broad receptor specificity enhances binding to human tracheal tissues, suggesting that O-glycan recognition could contribute to the continues spillover of this clade.

Medication use complexity is associated with non-adherence, hospitalizations, and lower quality of life. It is most frequently measured with the Medication Regimen Complexity Index (MRCI). This instrument estimates the complexity of a patient's medication regimen, taking into account the pharmaceutical dosage forms and routes, dosage frequency, and instructions for use. This instrument was, however, developed without patient experience data and may overlook patient perspectives. Therefore, this study aimed to identify older patients' perspectives on medication use complexity. This qualitative study used semi-structured interviews among patients aged ≥ 70 years using ≥ 5 chronic medications, recruited at community pharmacies and a geriatric outpatient clinic. After medication reconciliation, open-ended questions about the patient's medication use and four hypothetical medication regimens with similar MRCI scores but different schemes were discussed. Interviews were transcribed verbatim and coded independently by two researchers. Inductive and deductive thematic analysis using NVivo was applied to explore themes influencing medication use complexity according to patients. Sixteen patients were included (median age, 76; 56% female; median number of medications including OTC, 14). Four themes were identified, of which three are medication-related themes: medication characteristics, preparation for administration, and administration regimen. Factors that reduced medication use complexity included identification by medication appearance, use of medication aids or multidose drug dispensing systems, and development of routines. Factors that increased complexity included alterations in medication appearance or routine. The fourth theme was user-related and focused on the complexity of medication use in the context of an individual patient's attitudes and beliefs regarding medication and healthcare. This study identified many themes that can influence medication use complexity for older patients, including new themes beyond those measured by the MRCI. Individualized assessments may better address patient-specific challenges in medication use, ultimately enhancing the potential effectiveness of complexity-reducing interventions.

ObjectivesThe early development of humoral immunity is important for long‐term protection of the newborn. Here, we set out to discern these infant‐produced antibodies from the vast background of maternal antibodies, which is challenging but essential to shed light on the infant‐produced repertoire.MethodsUsing IgA1 and IgG1 antibody clonal repertoire analysis by mass spectrometry, we compared matched maternal serum, maternal milk, and infant serum samples, both at birth (T1) and at 7–11 weeks after delivery (T2) in four mother–infant dyads.ResultsWe observed for both IgA1 and IgG1 unique infant‐produced antibody repertoires at T2. For IgA1 at T2, no substantial clonal overlap was found between infant serum and breastmilk. The serum IgG1 clonal repertoires were highly alike at birth for mother and infant, but at T2, the contribution of the maternal clonal population in the infant had been drastically reduced, and a large portion of the T2 IgG1 repertoire originated from the infant.ConclusionsNewborns produce their own antibody repertoires as early as a few months after birth. From this small study, no convincing evidence is found for transfer of milk antibodies into the infant circulation.

Adequate immune fitness, i.e. the body’s capacity to respond to health challenges (such as infections) by activating an appropriate immune response is vital to maintain health and prevent or resolve disease. Aim of the current survey among n = 450 Turkish pharmacy visitors was to investigate the sociodemographic, health, and lifestyle factors that are related to experiencing reduced immune fitness. The analyses revealed that individuals with reduced immune fitness reported significantly poorer general health and immune fitness, significantly poorer sleep, and significantly poorer mood, and a significantly lower quality of life. The analyses revealed significantly poorer immune fitness among individuals with a chronic disease, and those with a lower education level. A regression analysis evaluated possible predictors of reduced immune fitness. The strongest predictor of reduced immune fitness was having a chronic disease. Other significant contributing factors were older age, lower BMI, poorer sleep quality, greater anxiety, greater fatigue, lower hostility, and attaining a less healthy daily diet. In conclusion, reduced immune fitness is associated with having chronic diseases, and poorer physical and mental health.