Introduction: 

Post-acute sequelae of SARS-CoV-2 (PASC) may develop after SARS-CoV-2 infection and cause a wide range of symptoms that can persist for years. Several pathophysiological mechanisms have been proposed, including dysregulation of the complement system.

Methods: 

In this study, we analysed markers of complement activation in a cohort of patients with PASC, up to 33 months after the initial infection. We measured the complement activation markers C3bc, C3bBbP and TCC in 38 PASC patients with an initial mild COVID-19 infection, 10 PASC patients with an initial severe COVID-19 infection and 80 control subjects who had recovered completely after a COVID-19 infection.

Results: 

Although the patients with an acute mild SARS-CoV-2 infection had a trend towards more severe PASC, we could not find any significant differences in complement activation markers between these patients and controls.

Conclusion: 

We could not find convincing evidence of activation of the complement system in PASC patients.

Introduction: This cross-sectional study aimed to determine the prevalence ofRelative Energy Deficiency in Sport (REDs) among Swedish national-teamathletes across multiple sports and to examine selected endocrine andmetabolic markers in relation to within-day energy balance (WDEB) metrics.

Methods: REDs severity was retrospectively classified in 43 athletes (60.5%females) using the IOC REDs Clinical Assessment Tool 2 framework.Assessments included DXA-derived body composition, resting metabolic rate(RMR) by ventilated-hood indirect calorimetry, blood markers, validatedquestionnaires, clinical eating disorder interview. 4-day dietary intake, energyavailability (EA), EB, and WDEB metrics were assessed in a subsample (n = 33;19 females, 14 males).

Results: Twenty athletes (46.5%) were classified as controls (no/low risk; 45.0%female), whereas 23 (53.5%) were classified with REDs (73.9% female). MostREDs cases were mild, however 61% exhibited ≥2 additional potential REDsindicators, a pattern also observed in 50% of controls. Athletes classified withREDs had higher cortisol and cortisol-to-insulin ratio, lower RMR ratio, lowerenergy intake and 24 h EA, and greater exposure to negative WDEB patterns.In unadjusted analyses, insulin was associated with both 24 h EB and EA.Exploratory analyses in females identified associations between cortisol-toinsulinratio and negative WDEB metrics, and between Exercise AddictionInventory score and 24 h EB; however, these associations were no longersignificant after Benjamini-Hochberg false discovery rate correction.

Conclusion: More than half of the elite athletes in this cohort, were classifiedwith REDs, with a higher prevalence among females. REDs classification wasassociated with lower EA, greater exposure to negative WDEB patterns, andadditional endocrine and metabolic alterations consistent with energydeficiency. Notably, a similar pattern was also observed in 50% of controls,supporting a continuum rather than discrete risk categories. Although WDEBand dietary metrics may provide useful contextual information they should beinterpreted cautiously and integrated with comprehensive multi-markerclinical REDs evaluation.

Left ventricular assist device (LVAD) implantation is a life-saving therapy for end-stage heart failure but may compromise immune integrity. Mechanical shear stress and surface-induced innate immune activation can trigger bleeding and thromboembolic complications. While thrombotic mechanisms are well characterized, the associated inflammatory response remains poorly studied. We investigated thromboinflammation in patients with terminal heart failure (n = 8) implanted with the HeartWare ventricular assist device. Blood samples were collected before and immediately after implantation, daily for five days, and three months post-implantation. Ten age- and sex-matched healthy individuals served as controls. Samples were analyzed for a broad panel of thromboinflammatory and cell damage biomarkers. Twenty-eight of 43 biomarkers were significantly elevated (p < 0.05) at patient baseline compared with controls, indicating a pre-existing low-grade inflammatory state prior to LVAD implantation. Complement activation products increased markedly immediately after implantation-C3bc, C3bBbP, and the terminal complement complex C5b-9 rose 2.8-, 8.9-, and 6.6-fold, respectively, compared with baseline (p < 0.0001), but returned toward baseline within 24 h. A similar transient increase was observed for TNF, IL-6, IL-8, IL-10, IFN-gamma, MMP-8, MMP-9, tissue factor, and prothrombin fragment 1.2 (p < 0.05). LVAD implantation with cardiopulmonary bypass induces a strong but transient immune response, including robust complement activation. Targeting upstream complement pathways may help attenuate downstream thromboinflammatory processes during the acute post-implantation period.

Heparin (HP) and dextran sulfate (DS) are well-known for their anti-thrombotic and immunomodulatory properties; however, a direct comparison of their immunological responses when used in drug delivery applications is lacking. This study addresses this gap by evaluating the immunological behavior of superparamagnetic iron oxide nanoparticles (SPIONs) coated with HP or DS in human whole blood, primary immune cells, endothelial cells, and in vivo. Both HP-SPIONs and DS-SPIONs effectively suppressed complement activation, as shown by reduced C3bc, C3bBbP, and TCC levels. Notably, HP-SPIONs activated monocytes (CD11b) and endothelial cells (ICAM-1, CD62P/E), whereas DS-SPIONs suppressed endothelial activation. DS-SPIONs were preferentially internalized by myeloid cells (similar to 50% neutrophils, similar to 42% macrophages, similar to 55% dendritic cells), while HP-SPIONs showed significantly lower uptake (<25% dendritic cells, similar to 5% neutrophils). DS-SPIONs induced an immunosuppressive, pro-healing phenotype in murine and human macrophages, whereas HP-SPIONs drove a pro-inflammatory, M1-like response. In healthy mice, intravenous DS-SPIONs elicited a modest increase in splenic immune cell populations compared to HP-SPIONs, indicating early immune engagement. Collectively, both SPIONs attenuate complement activation, indicating high biocompatibility. Based on the early immunological responses, DS-SPIONs display a pro-healing immune profile suitable for regenerative drug delivery, whereas HP-SPIONs induce pro-inflammatory responses that may be leveraged for anticancer immunotherapy.

Terminal sterilization of hydrogels is challenging because their high-water-content, chemical structures, and cell-friendly properties are often altered by conventional sterilization methods. Hydrogel implants comprising collagen-like peptide (CLP), polyethylene glycol (PEG), and poly (2-methacryloyloxyethyl phosphorylcholine) (MPC) showed promise in promoting corneal regeneration in preclinical animal studies. However, before these CLP-PEG-MPC implants can be used clinically, they must be sterilized effectively to ensure patient safety. Here, we evaluated supercritical carbon dioxide (scCO₂) with peracetic acid (PAA) as a potential sterilization technique for hydrogels. CLP-PEG-MPC was treated with scCO₂ containing PAA and compared against 15 kGy of electron beam irradiation (E-beam) and chloroform (CF) sterilization. Several samples were spiked with 200 colony-forming units (CFU) of either or prior to sterilization to compare the bactericidal efficacy of the three sterilization methods. Only scCO₂-PAA effectively inactivated all the inoculated bioburden. Non-spiked hydrogels were also sterilized and then compared for complement activation, changes to their physical and mechanical properties, and biocompatibility. Optical clarity was unchanged regardless of sterilization mode. scCO₂-PAA did not alter the mechanical properties of the hydrogels but resulted in a modest lowering of corneal epithelial cell proliferation, while E-beamed hydrogels showed reduced mechanical strength. In conclusion, newer scCO₂-PAA terminal sterilization preserves the functional properties of soft hydrogels, such as CLP-PEG-MPC, including transparency, which is needed for corneal implants.

Background Complement activation may promote atherosclerosis. Yet, data on the to which extent complement, and more specifically the alternative complement pathway, is activated in patients with carotid atherosclerosis and related to adverse outcome in these patients, are scarce. Methods and Results We measured, by ELISA, plasma levels of factor D, properdin, C3bBbP (C3 convertase), and factor H in patients with advanced carotid atherosclerosis in a Discovery (n=324) and in a Validation (n=206) cohort in relation to adverse outcome (mean follow-up 7.8 and 6.6 years, respectively). Our major findings were as follows. Compared with healthy controls, patients with carotid atherosclerosis had increased plasma levels of factor D, properdin, and C3bBbP (P<0.001), but not factor H, an inhibitor of the alternative complement pathway, compared with controls. Although patients with carotid atherosclerosis had elevated levels of properdin compared with controls, within these patients, low plasma levels of properdin (ie, Conclusions We show a strong and independent association of low plasma properdin levels with cardiovascular mortality in 2 cohorts. Conversely, the plaque properdin levels linked to features of plaque vulnerability, potentially reflecting increased deposition at the site of inflammation or local production of properdin in the atherosclerotic lesion indicating local enhanced alternative complement pathway activation.

Complement analysis necessitates strict control of pre-analytical blood handling, including time, temperature, and additives. Here, we compared complement function and activation status across five different serum preparations and two plasma preparations. Serum was collected from ten healthy volunteers using glass tubes without additives, tubes with a silica clot activator (with or without a gel separator), and tubes containing thrombin (with or without a gel separator). Plasma was collected in the presence of EDTA or the thrombin inhibitor lepirudin. Serum and plasma aliquots were snap-frozen in liquid nitrogen and stored at −80 °C. Complement functional analysis was performed using Wieslab and Hycult Biotech pathway-specific assays. Complement activation was determined by quantifying specific activation markers: C1s/C1-INH, MASP-1/C1-INH, C3bc, C3bBbP, and sC5b-9. All serum samples exhibited increased complement activation compared to EDTA and lepirudin plasma, with serum tubes containing thrombin and gel separators showing the highest levels of complement activation. However, normal complement function was observed in all serum preparations, indicating that the complement activation and consumption that occurred did not affect complement functional analysis. While all tested serum tubes provided accurate functional activity, the type of tube and the presence of additives like thrombin and gel separators significantly influenced the degree of complement activation. We recommend preparing functionally active serum either in glass tubes or in silica clot activator tubes, and avoiding gel separators. For complement activation studies, lepirudin plasma is preferable over serum due to its complement functional capacity, low level of complement activation, and lack of excessive hemostatic activation.

Introduction The functionalization of nanoparticles (NPs) with an antiCD19 targeting mechanism represents a promising approach for the selective delivery of drugs and nucleic acids into normal and tumor B cells. This strategy has the advantage of minimizing off-target effects by restricting gene delivery to the desired cell population. However, the nanoplatform must guarantee both the local production of the protein and the safety of the treatment to allow an effective therapy with reduced systemic toxicity.Methods In order to ensure a selective delivery of nucleic acids, we developed poly(lactic-co-glycolic acid) (PLGA)-poly(vinyl alcohol) (PVA) NPs loaded with an Enhanced Green Fluorescent Protein (EGFP)-coding plasmid and covalently coated with antiCD19 recombinant antibody as a targeting mechanism. To assess the functionality of the NPs, physicochemical characterization, safety tests, and transfection assay were employed to evaluate the NPs' behavior in vitro and in vivo, in a human/zebrafish lymphoma xenograft model.Results The results demonstrated that the PLGA-PVA nanoplatform was capable of efficiently encapsulating and releasing the payload. These nanostructures demonstrated a favorable safety profile, as evidenced by the absence of significant cell cytotoxicity, coagulation activation, complement system activation, and the slight activation of endothelial cells and leukocytes. The targeting mechanism facilitated the interaction of NPs with target cells, thereby enhancing their internalization and subsequent exogenous plasmid DNA (pDNA) translation and protein expression. In the human/zebrafish lymphoma xenograft model, no evidence of toxicity was observed, and targeted NPs demonstrated the capacity to enhance exogenous pDNA expression.Conclusion Our findings provide a rationale for the use of targeted NPs as a DNA delivery system for the local expression of therapeutic proteins.

Purpose: To determine predictors of success and failure events during treatment of neovascular AMD (nAMD) using real-world data.

Methods: We analysed data from one eye of 54 patients treated for nAMD, diagnosed up to 36 months from the date of participation and loading phase completed. Exclusion criteria: missing pre-treatment optical coherence tomography (OCT) scan or visual acuity (VA). Longitudinal morphological measures were obtained from the central 1mm of macular cube scans using the artificial intelligence (AI) based software RetinAI OCT Segmentation in Discovery (Ikerian AG). Measurements performed: intraretinal fluid (IRF), sub-retinal fluid (SRF), posterior epithelial detachment all in nanolitres, and retinal thickness in micrometres. VA and OCT scans were available from the treatment visits; plasma cytokines were measured at the time of participation in the current study. Definition of events – success: IRF+SRF reduced 50% or more, VA improved 5 letters or both; failure: IRF+SRF reduced by less than 50%, VA reduced 5 letters, or both. The reference for comparisons were pre-treatment OCT and VA measures. Time-to-event was modulated using Cox regression, that provides the hazard ratio of the event (HR).

Results: The mean age of the participants was 78.04 years (SD=7.69), 75% females, baseline VA mean=59.6 letters (SD=13.7). The median time for the first treatment success event was 125 days (IQR=108-199), 73% of the eyes experienced a treatment success event during follow-up. Higher levels of cytokine MCP-1 had a negative association, HR=0.92 (CI=0.84-1.00, p=0.046), and start VA less than 70 letters had a (marginally sig.) positive association, HR=2.52 (CI=0.98-6.44, p=0.057) with success events. The median time for the first treatment failure event was 196 days (IQR=122-405), 47% of the eyes experienced a failure event during the follow-up. Higher levels of cytokine MCP-1 had a positive association, HR=1.16 (CI=1.03-1.31, p=0.018), and start VA less than 70 letters had a negative association, HR=0.18 (CI=0.05-0.53, p=0.007) with failure events.

Conclusions: Most eyes experienced at least one treatment success event, close to half experienced a treatment failure event during the follow-up time. Chemokine MCP-1 was consistently associated with both types of events. Further work is warranted to confirm the robustness of the current findings.

A series of nanostructured polysulfobetaine (PSB) hydrogel-coated surfaces were fabricated and tested for hemocompatibility in contact with human blood. PSB films were grafted onto SiO2-coated silicon wafers or Au/quartz via photochemically induced polymerization of a sulfobetaine-based monomer (SBMA, [2-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide). An anodized aluminum oxide (AAO) membrane and latex beads (LB) were used as sacrificial template structures to synthesize polysulfobetaine nanowires (PSBAAO) and hyperporous (PSBLB) networks, respectively. Two soft sacrificial templates, a liquid crystalline medium (LC) and amide-based non-ionic deep eutectic solvent (ni-DESs) providing one-dimensional ordered arrays and flickering clusters, respectively, were utilized to grow nanofibrous (PSBLC) and mesoporous (PSBDES) polysulfobetaine film. Selective dissolution of the sacrificial templates affords the transposed pattern of the template with long-range periodicity from nano to micro scale (20 to 400 nm). Electron micrograph studies revealed nanostructured materials in the form of wires (198 +/- 5 nm), cavities (300 nm) and fibers (20 +/- 2 nm) when AAO, LB and LC-medium were used as templates, while the polymer films prepared from ni-DESs (PSBDES), water (PSBWAT) and methanol (PSBMeOH) were devoid of any noticeable topographical features. PSB-coated surfaces (except for PSBLB) inhibited non-specific adhesion of protein and biomolecules when presented with purified human proteins, i.e., albumin, fibrinogen, hemoglobin, or human plasma, down to 20-125 ng cm(-2) as shown by the QCM studies. Interestingly, the hierarchical nanostructures in polymer films (PSBAAO and PSBLC) resisted the adsorption of albumin and hemoglobin (_20 ng cm(-2)), even at 50 mg mL(-1) concentration. The hemocompatibility of the PSB nanostructures, analyzed after contact with human whole blood for one hour on the PSBAAO and PSBLC, revealed reduced complement activation, quantified as the generation of C3bc fragments and terminal complement sC5b-9 complex formation, in comparison to acrylate glass. The nanowires of PSBAAO showed significantly lower MPO release than the PSBWAT-onto surface, whereas no difference in platelet activation was seen between the surfaces. Compactly organized nanowires and fibers increase the water of hydration layers to strengthen the antifouling and hemocompatibility features, demonstrating the bio-inert nature of the PSB nanostructures. The inherent gelation (hydrophilicity) afforded by the PSB has substantial implications in designing bio-inert surfaces for hemocompatible devices.