Immunoadsorption as a method of antibody donation during the COVID-19 pandemic.

BACKGROUND AND OBJECTIVES: Initial therapeutic efforts to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) included the use of plasma from convalescent donors containing anti-SARS-CoV-2 antibodies. High-neutralizing antibody titres are required for therapeutic efficacy. This study aims to show that immunoadsorption followed by tangential flow filtration can be used to obtain antibody concentrates with high-neutralizing capacities. MATERIALS AND METHODS: Eligible donors (n = 10, five males and three females) underwent immunoadsorption using adsorber columns specific for human antibodies. Glycine-washed out eluates of 1.5 L volume were further concentrated by tangential flow filtration using 30 kDa ultrafiltration membranes. The same membranes were applied for diafiltrations to exchange residual glycine for 0.9% normal saline. RESULTS: Antibody concentrates were obtained within 8 h from the start of donation and had, 4.58 ± 1.95, 3.28 ± 1.28 and 2.02 ± 0.92 times higher total IgG, IgA and IgM concentrations, 3.29 ± 1.62 and 3.74 ± 0.6 times higher SARS-CoV-2 N and S antibody concentrations and 3.85 ± 1.71 times higher SARS-CoV-2 S-specific IgG concentrations compared to the donors' peripheral blood. The specific SARS-CoV-2 virus neutralization capacities increased in all but one concentrate. All antibody concentrates (50-70 mL final volume) passed microbiological tests, were free of hazardous glycine levels and could be stored at -80°C and 4°C for 1 year with 20 ± 3% antibody loss. CONCLUSION: Immunoadsorption followed by tangential flow filtration is a feasible procedure to collect IgG, IgA and IgM as well as SARS-CoV-2 N- and S-specific antibody concentrates of low volume, free of albumin and coagulation factors. Whether these concentrates can be used as passive immunisation in infected patients remains to be elucidated.

Effects of Empagliflozin on Fluid Overload, Weight, and Blood Pressure in CKD.

SIGNIFICANCE STATEMENT: SGLT2 inhibitors reduce risk of kidney progression, AKI, and cardiovascular disease, but the mechanisms of benefit are incompletely understood. Bioimpedance spectroscopy can estimate body water and fat mass. One quarter of the EMPA-KIDNEY bioimpedance substudy CKD population had clinically significant levels of bioimpedance-derived "Fluid Overload" at recruitment. Empagliflozin induced a prompt and sustained reduction in "Fluid Overload," irrespective of sex, diabetes, and baseline N-terminal pro B-type natriuretic peptide or eGFR. No significant effect on bioimpedance-derived fat mass was observed. The effects of SGLT2 inhibitors on body water may be one of the contributing mechanisms by which they mediate effects on cardiovascular risk. BACKGROUND: CKD is associated with fluid excess that can be estimated by bioimpedance spectroscopy. We aimed to assess effects of sodium glucose co-transporter 2 inhibition on bioimpedance-derived "Fluid Overload" and adiposity in a CKD population. METHODS: EMPA-KIDNEY was a double-blind placebo-controlled trial of empagliflozin 10 mg once daily in patients with CKD at risk of progression. In a substudy, bioimpedance measurements were added to the main trial procedures at randomization and at 2- and 18-month follow-up visits. The substudy's primary outcome was the study-average difference in absolute "Fluid Overload" (an estimate of excess extracellular water) analyzed using a mixed model repeated measures approach. RESULTS: The 660 substudy participants were broadly representative of the 6609-participant trial population. Substudy mean baseline absolute "Fluid Overload" was 0.4±1.7 L. Compared with placebo, the overall mean absolute "Fluid Overload" difference among those allocated empagliflozin was -0.24 L (95% confidence interval [CI], -0.38 to -0.11), with similar sized differences at 2 and 18 months, and in prespecified subgroups. Total body water differences comprised between-group differences in extracellular water of -0.49 L (95% CI, -0.69 to -0.30, including the -0.24 L "Fluid Overload" difference) and a -0.30 L (95% CI, -0.57 to -0.03) difference in intracellular water. There was no significant effect of empagliflozin on bioimpedance-derived adipose tissue mass (-0.28 kg [95% CI, -1.41 to 0.85]). The between-group difference in weight was -0.7 kg (95% CI, -1.3 to -0.1). CONCLUSIONS: In a broad range of patients with CKD, empagliflozin resulted in a sustained reduction in a bioimpedance-derived estimate of fluid overload, with no statistically significant effect on fat mass. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03594110 ; EuDRACT: 2017-002971-24 ( https://eudract.ema.europa.eu/ ).

Sodium as an Important Regulator of Immunometabolism.

Salt sensitivity concerns blood pressure alterations after a change in salt intake (sodium chloride). The heart is a pump, and vessels are tubes; sodium can affect both. A high salt intake increases cardiac output, promotes vascular dysfunction and capillary rarefaction, and chronically leads to increased systemic vascular resistance. More recent findings suggest that sodium also acts as an important second messenger regulating energy metabolism and cellular functions. Besides endothelial cells and fibroblasts, sodium also affects innate and adaptive immunometabolism, immune cell function, and influences certain microbes and microbiota-derived metabolites. We propose the idea that the definition of salt sensitivity should be expanded beyond high blood pressure to cellular and molecular salt sensitivity.

Early combination therapy of COVID-19 in high-risk patients.

PURPOSE: Prolonged shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been observed in immunocompromised hosts. Early monotherapy with direct-acting antivirals or monoclonal antibodies, as recommended by the international guidelines, does not prevent this with certainty. Dual therapies may therefore have a synergistic effect. METHODS: This retrospective, multicentre study compared treatment strategies for corona virus disease-19 (COVID-19) with combinations of nirmatrelvir/ritonavir, remdesivir, molnupiravir, and/ or mABs during the Omicron surge. Co-primary endpoints were prolonged viral shedding (≥ 106 copies/ml at day 21 after treatment initiation) and days with SARS-CoV-2 viral load ≥ 106 copies/ml. Therapeutic strategies and risk groups were compared using odds ratios and Fisher's tests or Kaplan-Meier analysis and long-rank tests. Multivariable regression analysis was performed. RESULTS: 144 patients were included with a median duration of SARS-CoV-2 viral load ≥ 106 copies/ml of 8.0 days (IQR 6.0-15.3). Underlying haematological malignancies (HM) (p = 0.03) and treatment initiation later than five days after diagnosis (p < 0.01) were significantly associated with longer viral shedding. Prolonged viral shedding was observed in 14.6% (n = 21/144), particularly in patients with underlying HM (OR 3.5; 95% CI 1.2-9.9; p = 0.02). Clinical courses of COVID-19 were mild to moderate with only few adverse effects potentially related to combination treatment. CONCLUSION: Early combination treatment of COVID-19 effectively prevented prolonged viral shedding in 85.6% of cases. Considering the rapid viral clearance rates and low toxicity, individualized dual therapy approaches may be beneficial in high-risk patients.

CD73 deficiency does not aggravate angiotensin II-induced aortic inflammation in mice.

Vascular inflammation plays a key role in the development of aortic diseases. A potential novel target for treatment might be CD73, an ecto-5'-nucleotidase that generates anti-inflammatory adenosine in the extracellular space. Here, we investigated whether a lack of CD73 results in enhanced aortic inflammation. To this end, angiotensin II was infused into wildtype and CD73-/- mice over 10 days. Before and after infusion, mice were analyzed using magnetic resonance imaging, ultrasound, flow cytometry, and histology. The impact of age and gender was investigated using female and male mice of three and six months of age, respectively. Angiotensin II infusion led to increased immune cell infiltration in both genotypes' aortae, but depletion of CD73 had no impact on immune cell recruitment. These findings were not modified by age or sex. No substantial difference in morphological or functional characteristics could be detected between wildtype and CD73-/- mice. Interestingly, the expression of CD73 on neutrophils decreased significantly in wildtype mice during treatment. In summary, we have found no evidence that CD73 deficiency affects the onset of aortic inflammation. However, as CD73 expression decreased during disease induction, an increase in CD73 by pharmaceutical intervention might result in lower vascular inflammation and less vascular disease.

Red blood cell endothelial nitric oxide synthase: A major player in regulating cardiovascular health.

Red blood cells (RBCs) have traditionally been seen as simple carriers of gases and nutrients in the body. One important non-canonical function of RBCs in the cardiovascular system is the regulation of nitric oxide (NO) metabolism. It has been shown that RBCs can scavenge NO, transport NO metabolites and produce NO in hypoxic conditions, thereby inducing hypoxic vasodilation. RBCs also express endothelial nitric oxide synthase (eNOS). However, the physiological significance of RBC eNOS has been controversial for many years. This review article provides a comprehensive overview of the experimental research on RBC eNOS signalling in vivo. The data show that RBC eNOS signalling modulates intracellular NO production and NO-haem levels, as well as participating in extracellular paracrine NO metabolite signalling, which contributes to regulating peripheral vascular resistance, blood pressure and cardioprotection. Additionally, this article explores the molecular mechanisms of sytemic regulation mediated by RBC eNOS and the implications of RBC eNOS in cardiovascular health and disease.

Contrast medium free selective adrenal vein sampling in the management of primary aldosteronism.

BACKGROUND: To analyze contrast free adrenal vein sampling (AVS) for differentiating unilateral from bilateral disease in patients diagnosed with hypertension due to primary aldosteronism (PA). METHODS: Consecutive patients with PA and subsequent contrast medium free AVS between April 2015 and March 2020 were retrospectively included. Cross-sectional imaging (CSI), AVS and clinical data were analyzed regarding diagnostic performance. In addition, patients with lateralisation receiving adrenalectomy were compared to a control group treated with mineralocorticoid antagonists. RESULTS: In total 186 patients with AVS were included. The success rate for bilateral catheterization was 88% (median effective dose 2.8 mSv). CSI had an accuracy of 60% (CI: 0.52-0.67) in the detection of lateralization compared to AVS. Patients with bilateral adrenal hyperplasia and those with aldosterone-producing adenoma did not differ in systolic blood pressure (sBP) (p = 0.63) or number of antihypertensive drugs (NAD) (p = 0.11). After adrenalectomy, 28 patients were cured (51%; sBP ≤130 mmHg, NAD = 0), 18 were improved (33%; decrease of sBP ≥20 mmHg and NAD), and 8 were unchanged (15%). Serum renin increased significantly after treatment (p < 0.01). CONCLUSION: Contrast medium free AVS is a reliable procedure in the diagnostic management of patients with PA with high technical success rate. The accordance between CSI and results from AVS was only moderate indicating the central role of AVS in the diagnostic work-up of patients with PA. Patients with predominant disease diagnosed with AVS had a high cure rate and/or significant improvement after adrenalectomy.

The Effect of Renal Denervation on T Cells in Patients with Resistant Hypertension.

(1) Background: Sympathetic overactivity is a major contributor to resistant hypertension (RH). According to animal studies, sympathetic overactivity increases immune responses, thereby aggravating hypertension and cardiovascular outcomes. Renal denervation (RDN) reduces sympathetic nerve activity in RH. Here, we investigate the effect of RDN on T-cell signatures in RH. (2) Methods: Systemic inflammation and T-cell subsets were analyzed in 17 healthy individuals and 30 patients with RH at baseline and 6 months after RDN. (3) Results: The patients with RH demonstrated higher levels of pro-inflammatory cytokines and higher frequencies of CD4+ effector memory (TEM), CD4+ effector memory residential (TEMRA) and CD8+ central memory (TCM) cells than the controls. After RDN, systolic automated office blood pressure (BP) decreased by -17.6 ± 18.9 mmHg. Greater BP reductions were associated with higher CD4+ TEM (r -0.421, p = 0.02) and CD8+ TCM (r -0.424, p = 0.02) frequencies at baseline. The RDN responders, that is, the patients with ≥10mmHg systolic BP reduction, showed reduced pro-inflammatory cytokine levels, whereas the non-responders had unchanged inflammatory activity and higher CD8+ TEMRA frequencies with increased cellular cytokine production. (4) Conclusions: The pro-inflammatory state of patients with RH is characterized by altered T-cell signatures, especially in non-responders. A detailed analysis of T cells might be useful in selecting patients for RDN.

Sodium perturbs mitochondrial respiration and induces dysfunctional Tregs.

FOXP3+ regulatory T cells (Tregs) are central for peripheral tolerance, and their deregulation is associated with autoimmunity. Dysfunctional autoimmune Tregs display pro-inflammatory features and altered mitochondrial metabolism, but contributing factors remain elusive. High salt (HS) has been identified to alter immune function and to promote autoimmunity. By investigating longitudinal transcriptional changes of human Tregs, we identified that HS induces metabolic reprogramming, recapitulating features of autoimmune Tregs. Mechanistically, extracellular HS raises intracellular Na+, perturbing mitochondrial respiration by interfering with the electron transport chain (ETC). Metabolic disturbance by a temporary HS encounter or complex III blockade rapidly induces a pro-inflammatory signature and FOXP3 downregulation, leading to long-term dysfunction in vitro and in vivo. The HS-induced effect could be reversed by inhibition of mitochondrial Na+/Ca2+ exchanger (NCLX). Our results indicate that salt could contribute to metabolic reprogramming and that short-term HS encounter perturb metabolic fitness and long-term function of human Tregs with important implications for autoimmunity.

Factors Associated With Vaccine-Induced T-Cell Immune Responses Against Severe Acute Respiratory Syndrome Coronavirus 2 in Kidney Transplant Recipients.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important prophylactic measure in kidney transplant recipients (KTRs), but the immune response is often impaired. Here, we examined the T-cell immune response against SARS-CoV-2 in 148 KTRs after 3 or 4 vaccine doses, including 35 KTRs with subsequent SARS-CoV-2 infection. The frequency of spike-specific T cells was lower in KTRs than in immunocompetent controls and was correlated with the level of spike-specific antibodies. Positive predictors for detection of vaccine-induced T cells were detection of spike-specific antibodies, heterologous immunization with messenger RNA and a vector vaccine, and longer time after transplantation. In vaccinated KTRs with subsequent SARS-CoV-2 infection, the T-cell response was greatly enhanced and was significantly higher than in vaccinated KTRs without SARS-CoV-2 infection. Overall, the data show a correlation between impaired humoral and T-cell immunity to SARS-CoV-2 vaccination and provide evidence for greater robustness of hybrid immunity in KTRs.

Cardiomyocyte p38 MAPKα suppresses a heart-adipose tissue-neutrophil crosstalk in heart failure development.

Although p38 MAP Kinase α (p38 MAPKα) is generally accepted to play a central role in the cardiac stress response, to date its function in maladaptive cardiac hypertrophy is still not unambiguously defined. To induce a pathological type of cardiac hypertrophy we infused angiotensin II (AngII) for 2 days via osmotic mini pumps in control and tamoxifen-inducible, cardiomyocyte (CM)-specific p38 MAPKα KO mice (iCMp38αKO) and assessed cardiac function by echocardiography, complemented by transcriptomic, histological, and immune cell analysis. AngII treatment after inactivation of p38 MAPKα in CM results in left ventricular (LV) dilatation within 48 h (EDV: BL: 83.8 ± 22.5 µl, 48 h AngII: 109.7 ± 14.6 µl) and an ectopic lipid deposition in cardiomyocytes, reflecting a metabolic dysfunction in pressure overload (PO). This was accompanied by a concerted downregulation of transcripts for oxidative phosphorylation, TCA cycle, and fatty acid metabolism. Cardiac inflammation involving neutrophils, macrophages, B- and T-cells was significantly enhanced. Inhibition of adipose tissue lipolysis by the small molecule inhibitor of adipocytetriglyceride lipase (ATGL) Atglistatin reduced cardiac lipid accumulation by 70% and neutrophil infiltration by 30% and went along with an improved cardiac function. Direct targeting of neutrophils by means of anti Ly6G-antibody administration in vivo led to a reduced LV dilation in iCMp38αKO mice and an improved systolic function (EF: 39.27 ± 14%). Thus, adipose tissue lipolysis and CM lipid accumulation augmented cardiac inflammation in iCMp38αKO mice. Neutrophils, in particular, triggered the rapid left ventricular dilatation. We provide the first evidence that p38 MAPKα acts as an essential switch in cardiac adaptation to PO by mitigating metabolic dysfunction and inflammation. Moreover, we identified a heart-adipose tissue-immune cell crosstalk, which might serve as new therapeutic target in cardiac pathologies.

Development and validation of multivariable prediction models of serological response to SARS-CoV-2 vaccination in kidney transplant recipients.

Repeated vaccination against SARS-CoV-2 increases serological response in kidney transplant recipients (KTR) with high interindividual variability. No decision support tool exists to predict SARS-CoV-2 vaccination response to third or fourth vaccination in KTR. We developed, internally and externally validated five different multivariable prediction models of serological response after the third and fourth vaccine dose against SARS-CoV-2 in previously seronegative, COVID-19-naïve KTR. Using 20 candidate predictor variables, we applied statistical and machine learning approaches including logistic regression (LR), least absolute shrinkage and selection operator (LASSO)-regularized LR, random forest, and gradient boosted regression trees. For development and internal validation, data from 590 vaccinations were used. External validation was performed in four independent, international validation cohorts comprising 191, 184, 254, and 323 vaccinations, respectively. LASSO-regularized LR performed on the whole development dataset yielded a 20- and 10-variable model, respectively. External validation showed AUC-ROC of 0.840, 0.741, 0.816, and 0.783 for the sparser 10-variable model, yielding an overall performance 0.812. A 10-variable LASSO-regularized LR model predicts vaccination response in KTR with good overall accuracy. Implemented as an online tool, it can guide decisions whether to modulate immunosuppressive therapy before additional active vaccination, or to perform passive immunization to improve protection against COVID-19 in previously seronegative, COVID-19-naïve KTR.

Immune response to third SARS-CoV-2 vaccination in seronegative kidney transplant recipients: Possible improvement by mycophenolate mofetil reduction.

Modification of vaccination strategies is necessary to improve the immune response to SARS-CoV-2 vaccination in kidney transplant recipients (KTRs). This multicenter observational study analyzed the effects of the third SARS-CoV-2 vaccination in previously seronegative KTRs with the focus on temporary mycophenolate mofetil (MMF) dose reduction within propensity matched KTRs. 56 out of 174 (32%) previously seronegative KTRs became seropositive after the third vaccination with only three KTRs developing neutralizing antibodies against the omicron variant. Multivariate logistic regression revealed that initial antibody levels, graft function, time after transplantation and MMF trough levels had an influence on seroconversion (P < .05). After controlling for confounders, the effect of MMF dose reduction before the third vaccination was calculated using propensity score matching. KTRs with a dose reduction of ≥33% showed a significant decrease in MMF trough levels to 1.8 (1.2-2.5) µg/ml and were more likely to seroconvert than matched controls (P = .02). Therefore, a MMF dose reduction of 33% or more before vaccination is a promising approach to improve success of SARS-CoV-2 vaccination in KTRs.

Inhibition of p38 MAPK decreases hyperglycemia-induced nephrin endocytosis and attenuates albuminuria.

Chronic hyperglycemia, as in diabetes mellitus, may cause glomerular damage with microalbuminuria as an early sign. Noteworthy, even acute hyperglycemia can increase glomerular permeability before structural damage of the glomerular filter can be detected. Despite intensive research, specific antiproteinuric therapy is not available so far. Thus, a deeper understanding of the molecular mechanisms of albuminuria is desirable. P38 MAPK signaling is involved in the development of hyperglycemia-induced albuminuria. However, the mechanism of increased p38 MAPK activity leading to increased permeability and albuminuria remained unclear. Recently, we demonstrated that acute hyperglycemia triggers endocytosis of nephrin, the key molecule of the slit diaphragm, and induces albuminuria. Here, we identify p38 MAPK as a pivotal regulator of hyperglycemia-induced nephrin endocytosis. Activated p38 MAPK phosphorylates the nephrin c-terminus at serine 1146, facilitating the interaction of PKCα with nephrin. PKCα phosphorylates nephrin at threonine residues 1120 and 1125, mediating the binding of ß-arrestin2 to nephrin. ß-arrestin2 triggers endocytosis of nephrin by coupling it to the endocytic machinery, leading to increased glomerular permeability. Pharmacological inhibition of p38 MAPK preserves nephrin surface expression and significantly attenuates albuminuria. KEY MESSAGES: Acute hyperglycemia triggers endocytosis of nephrin. Activated p38 MAPK phosphorylates the nephrin c-terminus at serine 1146, facilitating the interaction of PKCα with nephrin. PKCα phosphorylates nephrin at threonine residues 1120 and 1125, mediating the binding of ß-arrestin2 to nephrin. ß-arrestin2 triggers endocytosis of nephrin by coupling it to the endocytic machinery, leading to a leaky glomerular filter. Pharmacological inhibition of p38 MAPK preserves nephrin surface expression and significantly attenuates albuminuria under hyperglycemic conditions.

Rapid shear stress-dependent ENaC membrane insertion is mediated by the endothelial glycocalyx and the mineralocorticoid receptor.

The contribution of the shear stress-sensitive epithelial Na+ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to laminar (LSS) or non-laminar shear stress (NLSS). ENaC membrane insertion was quantified using Quantum-dot-based immunofluorescence staining and the mechanical properties of the cell surface were probed with the Atomic Force Microscope (AFM) in vitro and ex vivo in isolated aortae of C57BL/6 and ApoE/LDLR-/- mice. Flow- and acetylcholine-mediated vasodilation was measured in vivo using magnetic resonance imaging. Acute LSS led to a rapid mineralocorticoid receptor (MR)-dependent membrane insertion of ENaC and subsequent stiffening of the endothelial cortex caused by actin polymerization. Of note, NLSS stress further augmented the cortical stiffness of the cells. These effects strongly depend on the presence of the endothelial glycocalyx (eGC) and could be prevented by functional inhibition of ENaC and MR in vitro endothelial cells and ex vivo endothelial cells derived from C57BL/6, but not ApoE/LDLR-/- vessel. In vivo In C57BL/6 vessels, ENaC- and MR inhibition blunted flow- and acetylcholine-mediated vasodilation, while in the dysfunctional ApoE/LDLR-/- vessels, this effect was absent. In conclusion, under physiological conditions, endothelial ENaC, together with the glycocalyx, was identified as an important shear stress sensor and mediator of endothelium-dependent vasodilation. In contrast, in pathophysiological conditions, ENaC-mediated mechanotransduction and endothelium-dependent vasodilation were lost, contributing to sustained endothelial stiffening and dysfunction.

Serum Calcification Propensity and Calcification of the Abdominal Aorta in Patients With Primary Aldosteronism.

Patients with primary aldosteronism (PA) are more susceptible to cardiovascular disease and mortality than patients with primary hypertension. This is mostly attributed to excess production of aldosterone and its effects on the development of vascular injury. A novel functional test (T50) measures serum calcification propensity. Lower T50-values predict higher cardiovascular risk. We investigated serum calcification propensity and vascular calcification in PA and resistant hypertension (RH). T50 measurement was performed in patients with PA (n = 66) and RH (n = 28) at baseline and after 403 (279-640) and 389 (277-527) days of treatment. No significant differences in T50-values were observed between the groups (371 ± 65 and 382 ± 44 min, in PA and RH group, respectively, p > 0.05). However, higher aldosterone-to-renin ratios were associated with lower T50-values in PA-patients (r -0.282, p < 0.05). Furthermore, lower T50-values were associated with increased abdominal aortic calcification measured by Agatston score in PA (r -0.534, p < 0.05). In both, PA and RH, higher atherosclerotic cardiovascular disease (ACSVD) scores (r -0.403, p < 0.05) and lower HDL (r 0.469, p < 0.05) was related to lower T50-values in a linear regression model. Adrenalectomy or medical treatment did not increase T50-values. In comparison to patients with stable T50-values, PA patients with a decrease in T50 after intervention had higher serum calcium concentrations at baseline (2.24 ± 0.11 vs. 2.37 ± 0.10 mmol/l, p < 0.05). This decline of T50-values at follow-up was also associated with a decrease in serum magnesium (-0.03 ± 0.03 mmol/l, p < 0.05) and an increase in phosphate concentrations (0.11 ± 0.11 mmol/l, p < 0.05). Resistant hypertension patients with a decrease in T50-values at follow-up had a significantly lower eGFR at baseline. In summary, these data demonstrate an association between a high aldosterone-to-renin ratio and low T50-values in PA. Moreover, lower T50-values are associated with higher ACSVD scores and more pronounced vascular calcification in PA. Thus, serum calcification propensity may be a novel modifiable risk factor in PA.

Intensity of mycophenolate mofetil treatment is associated with an impaired immune response to SARS-CoV-2 vaccination in kidney transplant recipients.

Kidney transplant recipients (KTRs) are extremely vulnerable to SARS-CoV-2 infection and show an impaired immune response to SARS-CoV-2 vaccination. We analyzed factors related to vaccination efficiency in KTRs. In a multicenter prospective observational study (NCT04743947), IgG antibodies levels against SARS-CoV-2 spike S1 subunit and their neutralization capacity after SARS-CoV-2 vaccination were analyzed in 225 KTRs and compared to 176 controls. After the vaccination, 56 (24.9%) KTRs became seropositive of whom 68% had neutralizing antibodies. This immune response was significantly lower compared to controls (239 [78-519] BAU/ml versus 1826 [560-3180] BAU/ml for KTRs and controls, p < .0001). The strongest predictor for an impaired response was mycophenolate mofetil (MMF) treatment. Multivariate regression analysis revealed that MMF-free regimen was highly associated with seroconversion (OR 13.25, 95% CI 3.22-54.6; p < .001). In contrast, other immunosuppressive drugs had no significant influence. 187 out of 225 KTRs were treated with MMF of whom 26 (13.9%) developed antibodies. 23 of these seropositive KTRs had a daily MMF dose ≤1 g. Furthermore, higher trough MMF concentrations correlated with lower antibody titers (R -0.354, p < .001) supporting a dose-dependent unfavorable effect of MMF. Our data indicate that MMF dose modification could lead to an improved immune response.

Beyond Vessel Diameters: Non-invasive Monitoring of Flow Patterns and Immune Cell Recruitment in Murine Abdominal Aortic Disorders by Multiparametric MRI.

The pathophysiology of the initiation and progression of abdominal aortic aneurysms (AAAs) and aortic dissections (AADs) is still unclear. However, there is strong evidence that monocytes and macrophages are of crucial importance in these processes. Here, we utilized a molecular imaging approach based on background-free 19F MRI and employed perfluorocarbon nanoemulsions (PFCs) for in situ 19F labeling of monocytes/macrophages to monitor vascular inflammation and AAA/AAD formation in angiotensin II (angII)-treated apolipoproteinE-deficient (apoE-/-) mice. In parallel, we used conventional 1H MRI for the characterization of aortic flow patterns and morphology. AngII (1 µg/kg/min) was infused into apoE-/- mice via osmotic minipumps for 10 days and mice were monitored by multiparametric 1H/19F MRI. PFCs were intravenously injected directly after pump implantation followed by additional applications on day 2 and 4 to allow an efficient 19F loading of circulating monocytes. The combination of angiographic, hemodynamic, and anatomical measurements allowed an unequivocal classification of mice in groups with developing AAAs, AADs or without any obvious aortic vessel alterations despite the exposure to angII. Maximal luminal and external diameters of the aorta were enlarged in AAAs, whereas AADs showed either a slight decrease of the luminal diameter or no alteration. 1H/19F MRI after intravenous PFC application demonstrated significantly higher 19F signals in aortae of mice that developed AAAs or AADs as compared to mice in which no aortic disorders were detected. High resolution 1H/19F MRI of excised aortae revealed a patchy pattern of the 19F signals predominantly in the adventitia of the aorta. Histological analysis confirmed the presence of macrophages in this area and flow cytometry revealed higher numbers of immune cells in aortae of mice that have developed AAA/AAD. Importantly, there was a linear correlation of the 19F signal with the total number of infiltrated macrophages. In conclusion, our approach enables a precise differentiation between AAA and AAD as well as visualization and quantitative assessment of inflammatory active vascular lesions, and therefore may help to unravel the complex interplay between macrophage accumulation, vascular inflammation, and the development and progression of AAAs and AADs.

Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure.

BACKGROUND: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. METHODS: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. RESULTS: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. CONCLUSIONS: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

COVID-19 antibody donation using immunoadsorption: Report of two cases.

For more than a year the whole world is suffering from the COVID-19 pandemic with no treatment option in sight. Administration of plasma from convalescent donors containing anti-SARS-CoV-2 antibodies, though promising according to case reports, failed to show a clear benefit in a greater number of trials. One reason could be varying and low antibody contents in a majority of plasma units hampering standardization and clinical efficacy. Besides, other plasma components unnecessarily transfused like coagulation factors might promote hypercoagulation seen in severe COVID-19 etiopathology. We therefore hypothesized that instead of collecting whole plasma units, convalescent donors could donate solely immunoglobulins by undergoing immunoadsorption, a mode of therapy regularly applied in autoimmune diseases. Here, we report the results of the first two antibody donations performed at the University Hospital Düsseldorf. In both cases, immunoadsorptions were very well tolerated with no side effects. Collected and neutralized eluates were concentrated using tangential flow filtration increasing the concentration of immunoglobulins 10fold as compared to peripheral blood and leading to probably eight times more neutralizing antibodies than in one plasma unit. Therefore, immunoadsorption can be used as a method of antibody donation. Whether these donated antibodies can be used as passive immunization in acutely infected patients remains to be elucidated.