Atherosclerotic arteries are commonly treated using drug-eluting stents (DES). However, it remains unclear whether and how the properties of atherosclerotic plaque affect drug transport in the arterial wall. A limitation of the currently used atherosclerotic animal models to study arterial drug distribution is the unpredictability of plaque size, composition, and location. In the present study, the aim is to create an artificial atherosclerotic plaque-of reproducible and controllable complexity and implantable at specific locations-to enable systematic studies on transport phenomena of drugs in stented atherosclerosis-mimicking arteries. For this purpose, mixtures of relevant lipids at concentrations mimicking atherosclerotic plaque are incorporated in gelatin/alginate hydrogels. Lipid-free (control) and lipid-rich hydrogels (artificial plaque) are created, mounted on DES and successfully implanted in porcine coronary arteries ex-vivo. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is used to measure local drug distribution in the arterial wall behind the prepared hydrogels, showing that the lipid-rich hydrogel significantly hampers drug transport as compared to the lipid-free hydrogel. This observation confirms the importance of studying drug transport phenomena in the presence of lipids and of having an experimental model in which lipids and other plaque constituents can be precisely controlled and systematically studied.
Abstract Healthy individuals with hybrid immunity, due to a SARS-CoV-2 infection prior to first vaccination, have stronger immune responses compared to those who were exclusively vaccinated. However, little is known about the characteristics of antibody, B- and T-cell responses in kidney disease patients with hybrid immunity. Here, we explored differences between kidney disease patients and controls with hybrid immunity after asymptomatic or mild coronavirus disease-2019 (COVID-19). We studied the kinetics, magnitude, breadth and phenotype of SARS-CoV-2-specific immune responses against primary mRNA-1273 vaccination in patients with chronic kidney disease or on dialysis, kidney transplant recipients, and controls with hybrid immunity. Although vaccination alone is less immunogenic in kidney disease patients, mRNA-1273 induced a robust immune response in patients with prior SARS-CoV-2 infection. In contrast, kidney disease patients with hybrid immunity develop SARS-CoV-2 antibody, B- and T-cell responses that are equally strong or stronger than controls. Phenotypic analysis showed that Spike (S)-specific B-cells varied between groups in lymph node-homing and memory phenotypes, yet S-specific T-cell responses were phenotypically consistent across groups. The heterogeneity amongst immune responses in hybrid immune kidney patients warrants further studies in larger cohorts to unravel markers of long-term protection that can be used for the design of targeted vaccine regimens.
Abstract Kidney transplant recipients (KTRs) are at an increased risk of severe COVID-19 due to compromised immune responses. Although vaccination is critical in preventing severe disease, KTRs have attenuated vaccination-induced immune responses due to underlying kidney disease and immunosuppressive therapies. In this study, the effect of different COVID-19 booster strategies on SARS-CoV-2-specific T-cell responses was assessed in KTRs who showed a poor serological response after the first two mRNA-based primary vaccination doses. In these KTRs, a third vaccination dose led to an increase in antibody levels in the majority of patients. Production of IL-2 and IL-5 by SARS-CoV-2 specific T cells positively correlated with antibody levels, with stronger correlations compared to IFN-γ production, the ‘traditional’ cytokine to measure T-cell responses. Our study underscores the significance a balanced T-cell cytokine response to achieve robust antibody responses in KTRs. Furthermore, we show that multiple cytokines to assess T-cell responses should be explored to identify individuals in need of tailored vaccination strategies.
Cytokines are regulators of the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, the contribution of cytokine-secreting CD4+ and CD8+ memory T cells to the SARS-CoV-2-specific humoral immune response in immunocompromised kidney patients is unknown. Here, we profiled 12 cytokines after stimulation of whole blood obtained 28 days post second 100 μg mRNA-1273 vaccination with peptides covering the SARS-CoV-2 spike (S)-protein from patients with chronic kidney disease (CKD) stage 4/5, on dialysis, kidney transplant recipients (KTR), and healthy controls. Unsupervised hierarchical clustering analysis revealed two distinct vaccine-induced cytokine profiles. The first profile was characterized by high levels of T-helper (Th)1 (IL-2, TNF-α, and IFN-γ) and Th2 (IL-4, IL-5, IL-13) cytokines, and low levels of Th17 (IL-17A, IL-22) and Th9 (IL-9) cytokines. This cluster was dominated by patients with CKD, on dialysis, and healthy controls. In contrast, the second cytokine profile contained predominantly KTRs producing mainly Th1 cytokines upon re-stimulation, with lower levels or absence of Th2, Th17, and Th9 cytokines. Multivariate analyses indicated that a balanced memory T cell response with the production of Th1 and Th2 cytokines was associated with high levels of S1-specific binding and neutralizing antibodies mainly at 6 months after second vaccination. In conclusion, seroconversion is associated with the balanced production of cytokines by memory T cells. This emphasizes the importance of measuring multiple T cell cytokines to understand their influence on seroconversion and potentially gain more information about the protection induced by vaccine-induced memory T cells.
Introduction: Drug eluting stents (DES) used to treat acute myocardial infarction, are often placed on thrombus. Mathematical modelling indicates that thrombus acts as a barrier for local drug deli...
Abstract Protein arginine methyltransferase 3 (PRMT3) is a co-activator of liver X receptor capable of selectively modulating hepatic triglyceride synthesis. Here we investigated whether pharmacological PRMT3 inhibition can diminish the hepatic steatosis extent and lower plasma lipid levels and atherosclerosis susceptibility. Hereto, male hyperlipidemic low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet and injected 3 times per week intraperitoneally with PRMT3 inhibitor SGC707 or solvent control. Three weeks into the study, SGC707-treated mice developed severe pruritus and scratching-associated skin lesions, leading to early study termination. SGC707-treated mice exhibited 50% lower liver triglyceride stores as well as 32% lower plasma triglyceride levels. Atherosclerotic lesions were virtually absent in all experimental mice. Plasma metabolite analysis revealed that levels of taurine-conjugated bile acids were ~ threefold increased ( P < 0.001) in response to SGC707 treatment, which was paralleled by systemically higher bile acid receptor TGR5 signalling. In conclusion, we have shown that SGC707 treatment reduces hepatic steatosis and plasma triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice. These findings suggest that pharmacological PRMT3 inhibition can serve as therapeutic approach to treat non-alcoholic fatty liver disease and dyslipidemia/atherosclerosis, when unwanted effects on cholesterol and bile acid metabolism can be effectively tackled.
Introduction: In transplantation, the endothelial lining is the first barrier between the donor organ and recipient immune system. Damaged endothelium exposes extracellular matrix (ECM) molecules that can aggravate inflammation and cause graft rejection. Preservation and restoration of the endothelial barrier function is thus crucial for the normal performance of the kidney vascular system after transplantation. Here we prove that re-endothelialization of acellular blood vessels using patient derived kidney-vein endothelial cells (EC) restores both the vascular barrier function and innate immune function of the endothelium. Methods: Human common iliac veins (CIV) (n=19) from deceased healthy donors were decellularized by submersion in Triton X-100 (4%), ammonia (1%) and DNase. Efficacy of the process was evaluated via histological analysis and quantification of DNA. The ECM protein makeup preservation was assessed via collagen and sGAG content. Decellularized CIV were subsequently repopulated with human umbilical vein endothelial cells (HUVEC) or patient derived kidney-vein EC. The re-endothelialized veins were analysed using confocal microscopy for EC confluency. Functionality of the EC barrier was analyzed using trans-endothelial electrical resistance (TEER), dextran (4 and 70 kDa) permeability and nitric oxide production (eNOS). The innate immune barrier function of recellularized scaffolds was assessed by co-culture with THP-1 monocytic cells (5:1 ratio) in a home built transmigration system. Results: The CIV were fully decellularized, demonstrated by the complete removal of cellular components, and the removal of dsDNA (before: 83.8±29.0, after:13.0±6.5 ng/mg). Histological integrity was preserved, as well as ECM polysaccharides and collagen. Confocal microscopy showed the formation of a confluent monolayer of cells as soon as 24 hours after seeding. After 28 days of culture repopulated CIV scaffolds remained confluent and cells expressed the proliferation marker Ki-67 and PECAM-1. At day 10, the constructs had TEER measurements above background of 15.1±12.2 Ω·cm2 (n=4); reduced dextran permeability compared to decellularized CIV (2.3-fold for 4kDa and 4.7-fold for 70kDa; n=3); and showed higher nitrate and nitrite concentration compared to plastic cultures (n=4). These results indicated the restoration of a functional EC barrier. The innate immune barrier function was demonstrated by THP-1 cell adhesion (only on recellularized scaffolds) and transmigration through the EC monolayer. THP-1 differentiation into M1 inflammatory macrophages and M2 anti-inflammatory macrophages was confirmed via flow cytometry and immunohistochemistry with representative markers (CD14, CD16, CD80 and CD163). Conclusion: We used an in-vitro ECM blood vessel model to prove functionality of recellularized human vein tissue with patient-derived kidney vein EC. Repopulated scaffolds also showed immune innate function, paving the way to future translation into clinical practice.
