Routine techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) include density centrifugation with Ficoll-Paque and isolation by cell preparation tubes (CPTs) and SepMate tubes with Lymphoprep. In a series of experiments, these three PBMC isolation techniques were compared for cell recovery and viability, PBMC population composition, and cell functionality, aiming to provide a starting basis for the selection of the most appropriate method of PBMC isolation for a specific downstream application. PBMCs were freshly isolated from venous blood of healthy male donors, applying the different techniques in parallel. Cell recovery and viability were assessed using a hemacytometer and trypan blue. Immunophenotyping was performed by flow cytometry. Cell functionality was assessed in stimulated (100 ng/mL staphylococcal enterotoxin B [SEB]) and unstimulated 24 hours PBMC cultures, with cytokine production and lactate dehydrogenase (LDH) release as readout measures. PBMC isolation by SepMate and CPT resulted in a 70% higher recovery than Ficoll isolation. CPT-isolated populations contained more erythrocyte contamination. Cell viability, assessed by trypan blue exclusion, was 100% for all three isolation techniques. SepMate and CPT isolation gave higher SEB-induced cytokine responses in cell cultures, for IFNγ and for secondary cytokines. IL-6 and IL-8 release in unstimulated cultures was higher for CPT-isolated PBMCs compared to Ficoll- and SepMate-isolated PBMCs. LDH release did not differ between cell isolation techniques. In addition to criteria such as cost and application practicalities, these data may support selection of a specific PBMC isolation technique for downstream analysis.

Receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1) regulates inflammation, cytokine release, and necroptotic cell death and is implicated in pathogenic cellular pathways in amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and multiple sclerosis. Inhibition of RIPK1 activity protects against inflammation and cell death in multiple animal models. DNL104 is a selective, brain‐penetrant inhibitor of RIPK1 phosphorylation in clinical development for AD and ALS. DNL104 was tested in 68 healthy volunteers to investigate safety and tolerability, pharmacokinetic profile in plasma and cerebrospinal fluid, and pharmacodynamic effects of RIPK1 inhibition in peripheral blood mononuclear cells in a first‐in‐human, placebo‐controlled, double‐blind, randomized single‐ascending dose (SAD) and multiple‐ascending dose (MAD) study. DNL104 was well‐tolerated in the SAD group and during the dosing period of the MAD group. However, postdose liver toxicity in 37.5% of subjects was observed in the MAD, and assessed to be drug related. We demonstrate that DNL104 leads to RIP1 kinase inhibition, and this is not associated with central nervous system (CNS) toxicities, supporting future development of CNS penetrant RIPK1 inhibitors.

The safety, tolerability, immunogenicity, and pharmacokinetic (PK) profile of an anti‐OX40L monoclonal antibody (KY1005, currently amlitelimab) were evaluated. Pharmacodynamic (PD) effects were explored using keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT) immunizations. Sixty‐four healthy male subjects (26.5 ± 6.0 years) were randomized to single doses of 0.006, 0.018, or 0.05 mg/kg, or multiple doses of 0.15, 0.45, 1.35, 4, or 12 mg/kg KY1005, or placebo (6:2). Serum KY1005 concentrations were measured. Antibody responses upon KLH and TT immunizations and skin response upon intradermal KLH administration were performed. PD data were analyzed using repeated measures analysis of covariances (ANCOVAs) and post hoc exposure‐response modeling. No serious adverse events occurred and all adverse events were temporary and of mild or moderate severity. A nonlinear increase in mean serum KY1005 concentrations was observed (median time to maximum concentration (Tmax) ~ 4 hours, geometric mean terminal half‐life (t½) ~ 24 days). Cutaneous blood perfusion (estimated difference (ED) −13.4 arbitrary unit (AU), 95% confidence interval (CI) −23.0 AU to −3.8 AU) and erythema quantified as average redness (ED −0.23 AU, 95% CI −0.35 AU to −0.11 AU) decreased after KY1005 treatment at doses of 0.45 mg/kg and above. Exposure‐response analysis displayed a statistically significant treatment effect on anti‐KLH antibody titers (IgG maximum effect (Emax) −0.58 AU, 95% CI −1.10 AU to −0.06 AU) and skin response (erythema Emax −0.20 AU, 95% CI −0.29 AU to −0.11 AU). Administration of KY1005 demonstrated an acceptable safety and tolerability profile and PK analyses displayed a nonlinear profile of KY1005. Despite the observed variability, skin challenge response after KY1005 treatment indicated pharmacological activity of KY1005. Therefore, KY1005 shows potential as a novel pharmacological treatment in immune‐mediated disorders.

A mutation in the GBA1 gene is the most common genetic risk factor for developing Parkinson's disease. GBA1 encodes the lysosomal enzyme glucosylceramidase beta (glucocerebrosidase, GCase) and mutations decrease enzyme activity. LTI-291 is an allosteric modulator of GCase, enhancing its activity. These first-in-human studies evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics of single and multiple ascending doses of LTI-291 in healthy volunteers.Methods: In the single ascending dose (SAD) study, 40 healthy volunteers were randomly assigned to LTI-291 (n = 8 per dose level) or placebo (n = 2 per dose level).Single doses of 3, 10, 30 and 90 mg LTI-291 were investigated. In the multiple ascending dose (MAD) study, 40 healthy middle-aged or elderly volunteers were randomly assigned to LTI-291 (n = 8 per dose level) or placebo (n = 2 per dose level).Fourteen consecutive daily doses of 3, 10, 30 and 60 mg LTI-291 or placebo were administered. In both the SAD and MAD studies, glycosphingolipid levels were measured and a test battery of neurocognitive tasks was performed.Results: LTI-291 was generally well tolerated and no deaths or treatment-related SAEs occurred and no subject withdrew from a study due to AEs. C max , AUC 0-24 and AUC 0-inf increased in a dose proportional manner. The median half-life was 28.0 hours after multiple dosing. No dose-dependent glycosphingolipid changes occurred. No neurocognitive adverse effects were detected.Conclusions: These first-in-human studies demonstrated that LTI-291 was well tolerated when given orally once daily for 14 consecutive days. This supports the continued clinical development and the exploration of LTI-291 effects in a GBA1-mutated Parkinson population.The authors confirm that the Principal Investigator for this paper is Geert Jan Groeneveld and that he had direct clinical responsibility for participants.

Processing of pro-interleukin (IL)-1β and IL-18 is regulated by multiprotein complexes, known as inflammasomes. Inflammasome activation results in generation of bioactive IL-1β and IL-18, which can exert potent pro-inflammatory effects. Our aim was to develop a whole blood-based assay to study the inflammasome in vitro and that also can be used as an assay in clinical studies. We show whole blood is a suitable milieu to study inflammasome activation in primary human monocytes. We demonstrated that unprocessed human blood cells can be stimulated to activate the inflammasome by the addition of adenosine 5’-triphosphate (ATP) within a narrow timeframe following lipopolysaccharide (LPS) priming. Stimulation with LPS resulted in IL-1β release; however, addition of ATP is necessary for “full-blown” inflammasome stimulation resulting in high IL-1β and IL-18 release. Intracellular cytokine staining demonstrated monocytes are the major producers of IL-1β in human whole blood cultures, and this was associated with activation of caspase-1/4/5, as detected by a fluorescently labelled caspase-1/4/5 probe. By applying caspase inhibitors, we show that both the canonical inflammasome pathway (via caspase-1) as well as the non-canonical inflammasome pathway (via caspases-4 and 5) can be studied using this whole blood-based model.

Whereas intravenous administration of Toll-like receptor 4 ligand lipopolysaccharide (LPS) to human volunteers is frequently used in clinical pharmacology studies, systemic use of LPS has practical limitations. We aimed to characterize the intradermal LPS response in healthy volunteers, and as such qualify the method as local inflammation model for clinical pharmacology studies.Methods: Eighteen healthy male volunteers received 2 or 4 intradermal 10 ng LPS injections and 1 saline injection on the forearms. The LPS response was evaluated by noninvasive (perfusion, skin temperature and erythema) and invasive assessments (cellular and cytokine responses) in skin biopsy and blister exudate.Results: LPS elicited a visible response and returned to baseline at 48 hours.Erythema, perfusion and temperature were statistically significant (P < .0001) over a 24-hour time course compared to saline. The protein response was dominated by an acute interleukin (IL)-6, IL-8 and tumour necrosis factor response followed by IL-1β, IL-10 and interferon-γ. The cellular response consisted of an acute neutrophil influx followed by different monocyte subsets and dendritic cells.Discussion: Intradermal LPS administration in humans causes an acute, localized and transient inflammatory reaction that is well-tolerated by healthy volunteers. This may be a valuable inflammation model for evaluating the pharmacological activity of anti-inflammatory investigational compounds in proof of pharmacology studies.

Aims Aging is a dominant driver of atherosclerosis and induces a series of immunological alterations, called immunosenescence. Given the demographic shift towards elderly, elucidating the unknown impact of aging on the immunological landscape in atherosclerosis is highly relevant. While the young Western diet-fed Ldlr-deficient (Ldlr-/-) mouse is a widely used model to study atherosclerosis, it does not reflect the gradual plaque progression in the context of an aging immune system as occurs in humans. Methods and Results Here, we show that aging promotes advanced atherosclerosis in chow diet-fed Ldlr-/- mice, with increased incidence of calcification and cholesterol crystals. We observed systemic immunosenescence, including myeloid skewing and T-cells with more extreme effector phenotypes. Using a combination of single-cell RNA-sequencing and flow cytometry on aortic leukocytes of young versus aged Ldlr-/- mice, we show age-related shifts in expression of genes involved in atherogenic processes, such as cellular activation and cytokine production. We identified age-associated cells with pro-inflammatory features, including GzmK+CD8+ T-cells and previously in atherosclerosis undefined CD11b+CD11c+T-bet+ age-associated B-cells (ABCs). ABCs of Ldlr-/- mice showed high expression of genes involved in plasma cell differentiation, co-stimulation, and antigen presentation. In vitro studies supported that ABCs are highly potent antigen-presenting cells. In cardiovascular disease patients, we confirmed the presence of these age-associated T- and B-cells in atherosclerotic plaques and blood. Conclusions Collectively, we are the first to provide comprehensive profiling of aged immunity in atherosclerotic mice and reveal the emergence of age-associated T- and B-cells in the atherosclerotic aorta. Further research into age-associated immunity may contribute to novel diagnostic and therapeutic tools to combat cardiovascular disease.

LL‐37 is a cationic antimicrobial peptide and the sole human member of cathelicidins. Besides its bactericidal properties, LL‐37 is known to have direct immunomodulatory effects, among which enhancement of antiviral responses via endosomal toll‐like receptors (TLRs). Omiganan pentahydrochloride is a synthetic cationic peptide in clinical development. Previously, omiganan was primarily known for its direct bactericidal and antifungal properties. We investigated whether omiganan enhances endosomal TLR responses, similar to LL‐37. Human peripheral blood mononuclear cells were treated with endosomal TLR3, −7, −8, and −9 ligands in the presence of omiganan. Omiganan enhanced TLR‐mediated interferon‐α release. Subsequent experiments with TLR9 ligands showed that plasmacytoid dendritic cells were main contributors to omiganan‐enhanced IFN production. Based on this type I interferon‐enhancing effect, omiganan may qualify as potential treatment modality for virus‐driven diseases. The molecular mechanism by which omiganan enhances endosomal TLR responses remains to be elucidated.