Observational Study of Repeat Immunoadsorption (RIA) in Post-COVID ME/CFS Patients with Elevated ß2-Adrenergic Receptor Autoantibodies-An Interim Report.

There is increasing evidence for an autoimmune aetiology in post-infectious Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). SARS-CoV-2 has now become the main trigger for ME/CFS. We have already conducted two small proof-of-concept studies on IgG depletion by immunoadsorption (IA) in post-infectious ME/CFS, which showed efficacy in most patients. This observational study aims to evaluate the efficacy of IA in patients with post-COVID-19 ME/CFS. The primary objective was to assess the improvement in functional ability. Due to the urgency of finding therapies for post-COVID-Syndrome (PCS), we report here the interim results of the first ten patients, with seven responders defined by an increase of between 10 and 35 points in the Short-Form 36 Physical Function (SF36-PF) at week four after IA. The results of this observational study will provide the basis for patient selection for a randomised controlled trial (RCT), including sham apheresis, and for an RCT combining IA with B-cell depletion therapy. Trial registration number: NCT05629988.

Biomechanical Properties of the Aortic Wall: Changes during Vascular Calcification.

Medial vascular calcification (MAC) is characterized by the deposition of hydroxyapatite (HAP) in the medial layer of the vessel wall, leading to disruption of vessel integrity and vascular stiffness. Because currently no direct therapeutic interventions for MAC are available, studying the MAC pathogenesis is of high research interest. Several methods exist to measure and describe the pathophysiological processes in the vessel wall, such as histological staining and gene expression. However, no method describing the physiological properties of the arterial wall is currently available. This study aims to close that gap and validate a method to measure the biomechanical properties of the arterial wall during vascular calcification. Therefore, a stress-stretch curve is monitored using small-vessel-myography upon ex vivo calcification of rat aortic tissue. The measurement of biomechanical properties could help to gain further insights into vessel integrity during calcification progression.

Impact of first-line use of caplacizumab on treatment outcomes in immune thrombotic thrombocytopenic purpura.

BACKGROUND: The von Willebrand factor-directed nanobody caplacizumab has greatly changed the treatment of immune thrombotic thrombocytopenic purpura (iTTP) in recent years. Data from randomized controlled trials established efficacy and safety. OBJECTIVES: This study aims to address open questions regarding patient selection, tailoring of therapy duration, obstacles in prescribing caplacizumab in iTTP, effect on adjunct treatment, and outcomes in the real-world setting. METHODS: We report retrospective, observational cohorts of 113 iTTP episodes treated with caplacizumab and 119 historical control episodes treated without caplacizumab. We aggregated data from the caplacizumab phase II/III trials and real-world data from France, the United Kingdom, Germany, and Austria (846 episodes, 396 treated with caplacizumab, and 450 historical controls). RESULTS: Caplacizumab was efficacious in iTTP, independent of the timing of therapy initiation, but curtailed the time of active iTTP only when used in the first-line therapy within 72 hours after diagnosis and until at least partial ADAMTS13-activity remission. Aggregated data from multiple study populations showed that caplacizumab use resulted in significant absolute risk reduction of 2.87% for iTTP-related mortality (number needed to treat 35) and a relative risk reduction of 59%. CONCLUSION: Caplacizumab should be used in first line and until ADAMTS13-remission, lowers iTTP-related mortality and refractoriness, and decreases the number of daily plasma exchange and hospital stay. This trial is registered at www. CLINICALTRIALS: gov as #NCT04985318.

Molecular Imaging and Quantification of Smooth Muscle Cell and Aortic Tissue Calcification In Vitro and Ex Vivo with a Fluorescent Hydroxyapatite-Specific Probe.

Vessel calcification is characterized by the precipitation of hydroxyapatite (HAP) in the vasculature. Currently, no causal therapy exists to reduce or prevent vessel calcification. Studying the underlying pathways within vascular smooth muscle cells and testing pharmacological intervention is a major challenge in the vascular research field. This study aims to establish a rapid and efficient working protocol for specific HAP detection in cells and tissue using the synthetic bisphosphonate fluorescence dye OsteoSense™. This protocol facilitates especially early quantification of the fluorescence signal and permits co-staining with other markers of interest, enabling smaller experimental set-ups with lesser primary cells consumption and fast workflows. The fluorescence-based detection of vascular calcification with OsteoSense™ combines a high specificity with improved sensitivity. Therefore, this methodology can improve research of the pathogenesis of vascular calcification, especially for testing the therapeutic benefit of inhibitors in the case of in vitro and ex vivo settings.

Uremic mouse model to study vascular calcification and "inflamm-aging".

Calcification and chronic inflammation of the vascular wall is a high-risk factor for cardiovascular mortality, especially in patients with chronic uremia. For the reduction or prevention of rapid disease progression, no specific treatment options are currently available. This study aimed to evaluate an adenine-based uremic mouse model for studying medial vessel calcification and senescence-associated secretory phenotype (SASP) changes of aortic tissue to unravel molecular pathogenesis and provide a model for therapy testing. The dietary adenine administration induced a stable and similar degree of chronic uremia in DBA2/N mice with an increase of uremia blood markers such as blood urea nitrogen, calcium, creatinine, alkaline phosphatase, and parathyroid hormone. Also, renal fibrosis and crystal deposits were detected upon adenine feeding. The uremic condition is related to a moderate to severe medial vessel calcification and subsequent elastin disorganization. In addition, expression of osteogenic markers as Bmp-2 and its transcription factor Sox-9 as well as p21 as senescence marker were increased in uremic mice compared to controls. Pro-inflammatory uremic proteins such as serum amyloid A, interleukin (Il)-1ß, and Il-6 increased. This novel model of chronic uremia provides a simple method for investigation of signaling pathways in vascular inflammation and calcification and therefore offers an experimental basis for the development of potential therapeutic intervention studies.

Vascular Calcification in Rodent Models-Keeping Track with an Extented Method Assortment.

Vascular calcification is a multifaceted disease and a significant contributor to cardiovascular morbidity and mortality. The calcification deposits in the vessel wall can vary in size and localization. Various pathophysiological pathways may be involved in disease progression. With respect to the calcification diversity, a great number of research models and detection methods have been established in basic research, relying mostly on rodent models. The aim of this review is to provide an overview of the currently available rodent models and quantification methods for vascular calcification, emphasizing animal burden and assessing prospects to use available methods in a way to address the 3R principles of Russel and Burch: "Replace, Reduce and Refine".

Long-Term Treatment of Azathioprine in Rats Induces Vessel Mineralization.

Medial vascular calcification (mVC) is closely related to cardiovascular disease, especially in patients suffering from chronic kidney disease (CKD). Even after successful kidney transplantation, cardiovascular mortality remains increased. There is evidence that immunosuppressive drugs might influence pathophysiological mechanisms in the vessel wall. Previously, we have shown in vitro that mVC is induced in vascular smooth muscle cells (VSMCs) upon treatment with azathioprine (AZA). This effect was confirmed in the current study in an in vivo rat model treated with AZA for 24 weeks. The calcium content increased in the aortic tissue upon AZA treatment. The pathophysiologic mechanisms involve AZA catabolism to 6-thiouracil via xanthine oxidase (XO) with subsequent induction of oxidative stress. Proinflammatory cytokines, such as interleukin (IL)-1ß and IL-6, increase upon AZA treatment, both systemically and in the aortic tissue. Further, VSMCs show an increased expression of core-binding factor α-1, alkaline phosphatase and osteopontin. As the AZA effect could be decreased in NLRP3-/- aortic rings in an ex vivo experiment, the signaling pathway might be, at least in part, dependent on the NLRP3 inflammasome. Although human studies are necessary to confirm the harmful effects of AZA on vascular stiffening, these results provide further evidence of induction of VSMC calcification under AZA treatment and its effects on vessel structure.

Acid sphingomyelinase promotes SGK1-dependent vascular calcification.

In chronic kidney disease (CKD), hyperphosphatemia is a key factor promoting medial vascular calcification, a common complication associated with cardiovascular events and high mortality. Vascular calcification involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs), but the complex signaling events inducing pro-calcific pathways are incompletely understood. The present study investigated the role of acid sphingomyelinase (ASM)/ceramide as regulator of VSMC calcification. In vitro, both, bacterial sphingomyelinase and phosphate increased ceramide levels in VSMCs. Bacterial sphingomyelinase as well as ceramide supplementation stimulated osteo-/chondrogenic transdifferentiation during control and high phosphate conditions and augmented phosphate-induced calcification of VSMCs. Silencing of serum- and glucocorticoid-inducible kinase 1 (SGK1) blunted the pro-calcific effects of bacterial sphingomyelinase or ceramide. Asm deficiency blunted vascular calcification in a cholecalciferol-overload mouse model and ex vivo isolated-perfused arteries. In addition, Asm deficiency suppressed phosphate-induced osteo-/chondrogenic signaling and calcification of cultured VSMCs. Treatment with the functional ASM inhibitors amitriptyline or fendiline strongly blunted pro-calcific signaling pathways in vitro and in vivo. In conclusion, ASM/ceramide is a critical upstream regulator of vascular calcification, at least partly, through SGK1-dependent signaling. Thus, ASM inhibition by repurposing functional ASM inhibitors to reduce the progression of vascular calcification during CKD warrants further study.

Stressor-Induced "Inflammaging" of Vascular Smooth Muscle Cells via Nlrp3-Mediated Pro-inflammatory Auto-Loop.

Calcification of the vessel wall as one structural pathology of aged vessels is associated with high cardiovascular mortality of elderly patients. Aging is linked to chronic sterile inflammation and high burden of reactive oxygen species (ROS), leading to activation of pattern recognition receptors (PRRs) such as Nlrp3 in vascular cells. The current study investigates the role of PRR activation in the calcification of vascular smooth muscle cells (VSMCs). Therefore, in vitro cell culture of primary rat VSMCs and ex vivo aortic stimulations were used to analyze osteogenic, senescence and inflammatory markers via real-time PCR, in situ RNA hybridization, Western Blot, photometric assays and histological staining. Induction of ROS and DNA-damage by doxorubicin induces a shift of VSMC phenotype toward the expression of osteogenic, senescence and inflammatory proteins. Induction of calcification is dependent on Nlrp3 activity. Il-1ß as a downstream target of Nlrp3 induces the synthetic, pro-calcifying VSMC phenotype. Inhibition of PRR with subsequent reduction of chronic inflammation might be an interesting target for reduction of calcification of VSMCs, with subsequent reduction of cardiovascular mortality of patients suffering from vessel stiffness.

Would Oscillometry be Able to Solve the Dilemma of Blood Pressure Independent Pulse Wave Velocity - A Novel Approach Based on Long-Term Pulse Wave Analysis?

The utility of pulse wave velocity (PWV) as a surrogate parameter of arterial vessel damage (AVD) beyond the traditional brachial blood pressure (BP) measurement may be questioned as changes in BP are often accompanied by the corresponding changes in PWV. We sought to establish a new way for BP-independent estimation of AVD with PWV. We retrospectively analyzed data from 507 subjects with at least one available 24 h ambulatory BP- and pulse wave analysis, performed with Mobil-O-Graph (I.E.M., Stolberg, Germany). Individual relationship between eaPWV and central systolic BP (cSBP) was analyzed for every 24 h recording. The analysis revealed linear relation between eaPWV and cSBP in all subjects, which is described by equation eaPWV = a∗cSBP + b. We termed "a" as PWVslope and "b" as PWVbaseline. All available demographic parameters and clinical data were correlated with eaPWV, PWVslope and PWVbaseline. 108 subjects had repeated 24 h recordings. Mean age was 60.7 years and 48.7% were female. 92.5% had hypertension, 22.9% were smoker, 20.5% had diabetes mellitus and 29.6% eGFR < 60 ml/min/1,73 m2. Direct correlation was observed between age, SBP and eaPWV, while diastolic BP (DBP) and eGFR correlated inversely with eaPWV. PWVbaseline correlated directly with age and inversely with DBP, while PWVslope didn't correlate with any inputted parameter. Using simple mathematical approach by plotting eaPWV and cSBP values obtained during ABPM, it is possible to visualize unique course of individual PWV related to BP. Using PWVslope and PWVbaseline as novel parameters could be a feasible way to approach BP-independent PWV, though their clinical relevance should be tested in future studies. Our data underline the importance of BP-independent expression of PWV, when we use it as a clinical surrogate parameter for the vascular damage.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Efficacy of Repeat Immunoadsorption.

(1) Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmunological disease. There is evidence for an autoimmune mechanism for ME/CFS with an infection-triggered onset and dysfunction of ß2-adrenoreceptor antibodies (ß2AR-AB). In a first proof-of-concept study, we could show that IA was effective to reduce ß2AR-AB and led to improvement of various symptoms. (2) Five of the ME/CFS patients who had clinical improvement following treatment with a five-day IA were retreated in the current study about two years later with a modified IA protocol. The severity of symptoms was assessed by disease specific scores during a follow-up period of 12 months. The antibodies were determined by ELISA. (3) The modified IA treatment protocol resulted in a remarkable similar clinical response. The treatment was well tolerated and 80-90% decline of total IgG and ß2AR-AB was achieved. Four patients showed a rapid improvement in several clinical symptoms during IA therapy, lasting for six to 12 months. One patient had no improvement. (4) We could provide further evidence that IA has clinical efficacy in patients with ME/CFS. Data from our pilot trial warrant further controlled studies in ME/CFS.

Real-world data confirm the effectiveness of caplacizumab in acquired thrombotic thrombocytopenic purpura.

Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare but life-threatening condition. In 2018, the nanobody caplacizumab was approved for the treatment of adults experiencing an acute episode of aTTP, in conjunction with plasma exchange (PEX) and immunosuppression for a minimum of 30 days after stopping daily PEX. We performed a retrospective, observational analysis on the use of caplacizumab in 60 patients from 29 medical centers in Germany during acute disease management. Caplacizumab led to a rapid normalization of the platelet count (median, 3 days; mean 3.78 days). One patient died after late treatment initiation due to aTTP-associated complications. In 2 patients with initial disease presentation and in 4 additional patients with laboratory signs of an exacerbation or relapse after the initial therapy, PEX-free treatment regimens could be established with overall favorable outcome. Caplacizumab is efficacious in the treatment of aTTP independent of timing and ancillary treatment modalities. Based on this real-world experience and published literature, we propose to administer caplacizumab immediately to all patients with an acute episode of aTTP. Treatment decisions regarding the use of PEX should be based on the severity of the clinical presentation and known risk factors. PEX might be dispensable in some patients.

ADAMTS13 and VWF activities guide individualized caplacizumab treatment in patients with aTTP.

Introduction of the nanobody caplacizumab was shown to be effective in the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in the acute setting. The official recommendations include plasma exchange (PEX), immunosuppression, and the use of caplacizumab for a minimum of 30 days after stopping daily PEX. This study was a retrospective, observational analysis of the use of caplacizumab in 60 patients from 29 medical centers in Germany. Immunosuppressive treatment led to a rapid normalization of ADAMTS13 activities (calculated median, 21 days). In 35 of 60 patients, ADAMTS13 activities started to normalize before day 30 after PEX; in 11 of 60 patients, the treatment was extended beyond day 30; and in 5 patients, it was extended even beyond day 58 due to persistent autoimmune activity. In 34 of 60 instances, caplacizumab was stopped before day 30 with a favorable outcome whenever ADAMTS13 activities were >10%. In contrast, 11 of 34 patients with ADAMTS13 activities <10% at the time of stopping caplacizumab treatment developed a nonfavorable outcome (disease exacerbation or relapse). In some cases, prolongation of the treatment interval to every other day was feasible and resulted in a sustained reduction of von Willebrand factor activity. ADAMTS13 activity measurements are central for a rapid diagnosis in the acute setting but also to tailor disease management. An ADAMTS13 activity-guided approach seems safe for identifying the individual time point when to stop caplacizumab to prevent overtreatment and undertreatment; this approach will result in significant cost savings without jeopardizing the well-being of patients. In addition, von Willebrand factor activity may serve as a biomarker for drug monitoring.

A Novel Protocol for Detection of Senescence and Calcification Markers by Fluorescence Microscopy.

Vascular calcification and stiffening of the arterial wall is a systemic phenomenon that is associated with aging and it can be increased by several risk factors. The underlying mechanisms, especially the pathways of cellular senescence, are under current investigation. Easily manageable in vitro settings help to study the signaling pathways. The experimental setting presented here is based on an in vitro model using rat vascular smooth muscle cells and the detection of senescence and osteoblastic markers via immunofluorescence and RNAscope™. Co-staining of the senescence marker p21, the osteoblastic marker osteopontin, detection of senescence-associated heterochromatin foci, and senescence-associated ß-galactosidase is possible within one test approach requiring fewer cells. The protocol is a fast and reliable evaluation method for multiplexing of calcifying and senescence markers with fluorescence microscopy detection. The experimental setting enables analysis on single cell basis and allows detection of intra-individual variances of cultured cells.

Research Models for Studying Vascular Calcification.

Calcification of the vessel wall contributes to high cardiovascular morbidity and mortality. Vascular calcification (VC) is a systemic disease with multifaceted contributing and inhibiting factors in an actively regulated process. The exact underlying mechanisms are not fully elucidated and reliable treatment options are lacking. Due to the complex pathophysiology, various research models exist evaluating different aspects of VC. This review aims to give an overview of the cell and animal models used so far to study the molecular processes of VC. Here, in vitro cell culture models of different origins, ex vivo settings using aortic tissue and various in vivo disease-induced animal models are summarized. They reflect different aspects and depict the (patho)physiologic mechanisms within the VC process.

True Arterial Stiffness Does Not Change between Dialysis Sessions during 1 Week in Outpatients on Intermitted Hemodialysis.

INTRODUCTION: End-stage renal disease (ESRD) is associated with exponentially elevated cardiovascular mortality. Arterial stiffness (AS) - usually expressed with pulse wave velocity (PWV) - is an established independent predictor of cardiovascular risk beyond the traditional risk factors. Higher PWV values are frequently observed in patients with ESRD. Due to the intrinsic physiologic relationship between PWV and prevailing arterial pressure, PWV can change without relevant changes in the arterial wall structure, and thus an individual pressure-independent expression of PWV is essential. METHODS: The study is a single-center observational study. Repeated measurements of blood pressure (BP) and pulse wave analysis were performed during each dialysis session of 1 week. Aortic PWV was then adjusted to 120 mm Hg central systolic BP (PWV120) based on individually determined relationship. PWV120 values were compared between single sessions. Calculation of the PWV120 was performed retrospectively. RESULTS: Fifty-four subjects were included, 61.1% of whom were male. The median age was 75.5 years, and median dialysis vintage was 33.1 months. Mean systolic/diastolic BP was 121.4/70.5 mm Hg, and the median heart rate was 64.6 beats/min. Mean PWV was 10.9 m/s, and mean PWV120 was 11.3 m/s. PWV120 did not change across single dialysis session during 1 week, while systolic, diastolic BP, PWV, and ultrafiltration volume differed significantly. DISCUSSION/CONCLUSIONS: Our data suggest that true AS does not change in the short-term course in dialysis patients. The observed changes in PWV are rather associated with BP change due to intrinsic pressure dependence. Our analytical approach represents a novel method for this purpose, which is easy in performance and also applicable for large interventional trials and clinical practice.

A Novel Long-Term ex vivo Model for Studying Vascular Calcification Pathogenesis: The Rat Isolated-Perfused Aorta.

The investigation of vascular calcification and its underlying cellular and molecular pathways is of great interest in current research efforts. Therefore, suitable assays are needed to allow examination of the complex calcification process under controlled conditions. The current study describes a new ex vivo model of isolated-perfused rat aortic tissue with subsequent quantification and vessel staining to analyze the calcium content of the aortic wall. A rat aorta was perfused ex vivo with control and calcification media for 14 days, respectively. The calcification medium was luminally perfused and induced a significant increase in calcium deposition within the media of the vessel wall detected alongside the elastic laminae. Perfusion with control medium induced no calcification. In addition, the mRNA expression of the osteogenic marker bone morphogenetic protein 2 (BMP-2) increased in aortic tissue after perfusion, while SM22α as smooth muscle marker decreased. This newly developed ex vivo model of isolated-perfused rat aorta is suitable for vascular calcification studies testing inducers and inhibitors of vessel calcification and studying signaling pathways within calcification progression.